Organic derivatives of tellurium and selenium and their use to stimulate cytokine production

Certain tellurium compounds have been found to have the ability to stimulate the in vivo and in vitro production of cytokines and their receptors. These compounds may be utilized in the treatment of certain tumors, autoimmune diseases, immune diseases and infectious diseases.

BACKGROUND OF THE INVENTION 
It is well known that the growth of normal lymphocytes is dependent not 
only on contact with an antigenic substance or a mitogen, but also on the 
presence of certain growth factors known as lymphokines. One of these 
growth factors is known as T-cell growth factor (TCGF) better known as 
interleukin-2 (IL-2). The discovery of this growth factor (Gillis, et al., 
Nature, 268; 154 (1977) and Ruscetti, et al., J. Immunol, 119; 131 (1977)) 
resulted in the large scale growth and cloning of T-lymphocytes as sources 
for IL-2. 
The lymphocytes or white blood cells in the animal body come in two types, 
B-cells and T-cells. The B-cells produce antibodies in the form of 
immunoglobulins that bind onto invading organisms while the T-cells 
produce the lymphokines which are responsible for turning B-cells on or 
off. See for example Cell. Immunol. 36: 15 (1978); J. Cell Physiol. 96: 53 
(1978); Eur. J. Immunol. 8: 681 (1978); Immunol. Rev. 54: 188 (1981); 
Immunol. Rev. 54: 158 (1981); J. Exp. Med. 154: 1500 (1981); National 
Cancer Institute Mon. 60: 211 (1982); Int. J. Cancer 28: 157 (1981); The 
Potential Role of T-Cell Subpopulations in Cancer Therapy, Eds. A. Fefer & 
A. Goldstein, Raven Press, N.Y. pp 173 et seq. (1982); J. Immunol, 128: 
(258) 1982. 
The known types of lymphokines include, in addition to IL-2, B-cell 
factors, macrophage activation factor (MAF), Interleukin-3 (IL-3), Colony 
Stimulating Factor (CSF), Tumor Necrosis Factor, and other factors 
produced by monocytes such as Interleukin-1 (IL-1) and Gamma Interferon. 
All of these factors are secreted by white blood cells and are 
collectively known as cytokines. Great attention has been given to using 
various recombinant DNA techniques and other methodologies for cloning 
normal T and B cell lines that can produce these materials. See for 
example Nature 259: 130 (1976); Immunology 32: 319 (1977); Exp. Hemat, 8: 
494 (1980); Nature 283: 581 (1980); Proc. Natl. Acad. Sci. U.S.A. 78: 1858 
(1981); J. Immunol. Methods 49: 1 (1982); Nature 29424/31: 697-699 (1981), 
all of which are incorporated by reference. 
The present invention is based on the discovery of a class of synthetic 
organic derivatives of tellurium or selenium that are capable of 
stimulating cytokine producing cells to produce significant quantities of 
cytokines both in vivo and in vitro. This discovery makes possible a novel 
therapeutic approach in the treatment of cancer, immune deficiencies, 
autoimmune diseases and infectious diseases. 
Accordingly, it is an object of the invention to provide novel compounds 
based on tellurium or selenium that are useful as therapeutic agents. 
It is also an object of the invention to provide a novel method for 
producing in vitro cytokines such as lymphokines and induce receptors to 
these cytokines. 
It is also an object of the invention to produce in vivo cytokines such as 
lymphokines and and induce receptors to cytokines for the treatment of 
diseases such as cancer, immune deficiencies, autoimmune diseases and 
infectious diseases. 
It is also an object of the invention to provide novel pharmaceutical 
compositions that are based on tellurium compounds that produce cytokines 
in vivo and in vitro.

SUMMARY OF THE INVENTION 
The derivatives of tellurium or selenium that are useful in the present 
invention include those compounds of the following general formulas which 
stimulate cells to produce lymphokines: 
##STR1## 
wherein Q is Te or Se; t is 1 or 0; u is 1 or 0; v is 1 or 0; R, R.sub.1, 
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 
are the same or different and are independently selected from the group 
consisting of hydrogen, hydroxyalkyl of 1 to 5 carbons, hydroxy, alkyl or 
from 1 to 5 carbon atoms, halogen, haloalkyl of 1 to 5 carbon atoms, 
carboxy, alkylcarbonylalkyl of 2 to 10 carbons, alkanoyloxy of 1 to 5 
carbon atoms, carboxyalkyl of 1 to 5 carbon atoms, acyl, amido, cyano, 
amidoalkyl of 1 to 5 carbons, N-monoalkylamidoalkyl of 2 to 10 carbons, 
N,N-dialkylamidoalkyl of 4 to 10 carbons, cyanoalkyl of 1 to 5 carbons, 
alkoxy of 1 to 5 carbon atoms, alkoxyalkyl of 2 to 10 carbon atoms and 
--COR.sub.10 wherein R.sub.10 is alkyl of from 1 to 5 carbons; and X is 
halogen; while the ammonium salt is illustrated, it is understood that 
other pharmaceutically acceptable salts are within the scope of the 
invention. The compounds with the five membered rings are preferred. 
As used herein and in the appended claims, the term alkyl of 1 to 5 carbon 
atoms includes straight and branched chain alkyl groups such as methyl; 
ethyl; n-propyl; n-butyl, and the like; the term hydroxyalkyl of 1 to 5 
carbon atoms includes hydroxymethyl; hydroxyethyl; hydroxy-n-butyl; the 
term haloalkyl of 1 to 5 carbon atoms includes chloromethyl; 2-iodoethyl; 
4-bromo-n-butyle; iodoethyl; 4-bromo-n-pentyl and the like; the term 
alkanoyloxy of 1 to 5 carbon atoms includes acetyl, propionyl, butanoyl 
and the like; the term carboxyalkyl includes carboxymethyl, carboxyethyl, 
ethylenecarboxy and the like; the term alkylcarbonylalkyl includes 
methanoylmethyl, ethanoylethyl and the like; the term amidoalkyl includes 
--CH.sub.2 CONH.sub.2 ; --CH.sub.2 CH.sub.2 CONH.sub.2 ; --CH.sub.2 
CH.sub.2 CH.sub.2 CONH.sub.2 and the like; the term cyanoalkyl includes 
--CH.sub.2 CN; --CH.sub.2 CH.sub.2 CN; --CH.sub.2 CH.sub.2 CH.sub.2 CN and 
the like; the term alkoxy of 1 to 5 carbon atoms includes methoxy, ethoxy, 
n-propoxy, n-pentoxy and the like; the terms halo and halogen are used to 
signify chloro, bromo, iodo and fluoro; the term acyl includes R.sub.16 CO 
wherein R.sub.16 is H, or alkyl of 1 to 5 carbons such as methanoyl, 
ethanoyl and the like; the term aryl includes phenyl, alkylphenyl and 
naphthyl; the term N-monoalkylamidoalkyl includes --CH.sub.2 CH.sub.2 
CONHCH.sub.3, --CH.sub.2 CONHCH.sub.2 CH.sub.3 ; the term 
N,N-dialkylamidoalkyl includes --CH.sub.2 CON(CH.sub.3).sub.2 ; CH.sub.2 
CH.sub.2 CON(CH.sub.2 CH.sub.3). Compounds which are based on tellurium 
are the presently preferred compounds of the invention. The tellurium 
based compounds that are preferred include those of the formula: 
##STR2## 
wherein X is halogen. The preferred halogen species is chloro. These 
compounds are capable of inducing IL-2 formation as well as the 
proliferation of IL-2 producer cells and the activation of IL-2 receptor 
sites. 
Other compounds which are based on tellurium and may be used in the 
practice of the invention include Ph Te Cl.sub.3, Te O.sub.2 and (C.sub.6 
H.sub.5).sub.4 P+ (TeCl.sub.3 (O.sub.2 C.sub.2 H.sub.4).sup.- (Z. 
Naturforsh, 36B, 307-312 (1981). The compound hereinafter described in 
Example 2 has the following structure: 
##STR3## 
Other compounds useful for the practice of invention include: 
##STR4## 
Wherein R.sub.11, R.sub.12, R.sub.13 and R.sub.14 are independently 
selected from the group consisting of hydrogen, hydroxyalkyl of 1-5 carbon 
atoms, hydroxy and alkyl of 1-5 carbon atoms. 
Useful dihydroxy compounds for use in the preparation of compounds of 
structure A or B, include those of formula I wherein R, R.sub.1, R.sub.4 
and R.sub.5 are as shown in the Table: 
TABLE 
______________________________________ 
##STR5## (I) 
R R.sub.1 R.sub.4 R.sub.5 
______________________________________ 
H H H H 
H Cl H H 
H OCH.sub.3 H H 
H COOCH.sub.3 H H 
H H CN H 
H CHO H H 
H H COOH H 
H CH.sub.2 COOH 
H H 
H H CH.sub.2 COOCH.sub.3 
H 
H I H H 
H H Br H 
H H CONH.sub.2 H 
H H CH.sub.2 OH H 
H COOH H H 
______________________________________ 
Other dihydroxy compounds for use in the preparation of compounds A and B 
include those of formula II wherein R, R.sub.1, R.sub.2, R.sub.3, R.sub.4 
and R.sub.5 are as shown in the Table: 
______________________________________ 
##STR6## (II) 
R R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5 
______________________________________ 
H H H H H H 
H H Cl H H H 
H CH.sub.2 OH 
H H H H 
H H OH H H H 
H H H CH.sub.3 
H H 
H H H CH.sub.2 Cl 
H H 
H H H COOH H H 
H H H CH.sub.2 COOH 
H H 
H H H CHO H H 
H H H H H CH.sub.2 CHO 
H H CONH.sub.2 
H H.sub.2 CH.sub.3 
H H H CN H H 
H H H H CH.sub.2 CONH.sub.2 
H 
H H H COOCH.sub.3 
H.sub.3 H 
H H.sub.3 OCH.sub.3 
H H H 
______________________________________ 
Other dihydroxy compounds for use in making compound of formula A and B 
include those of formula III wherein R, R.sub.1, R.sub.2, R.sub.3, R.sub.4 
and R.sub.5 are as shown in the Table. 
______________________________________ 
##STR7## (III) 
R R.sub.1 R.sub.2 R.sub.3 
R.sub.4 R.sub.5 
R.sub.8 
R.sub.9 
______________________________________ 
H H H H H H H H 
H H Cl H H H H H 
H H H H Br H H H 
H H OCH.sub.3 
H H H H H 
H H CONH.sub.2 
H H H H H 
H Br H H Br H H H 
H H H H CH.sub.2 COOH 
H H H 
H H Cl Cl H H H H 
H CH.sub.2 COOH 
H H H H H H 
H H CH.sub.3 H H H H H 
H CH.sub.3 H H H H H H 
H CH.sub.2 Cl 
H H H H H H 
H H H I H H H H 
H CH.sub.2 CN 
H H H H H H 
H H H H CH.sub.2 CH.sub.2 OH 
H H H 
______________________________________ 
Additional dihydroxy compounds include those of formula IV wherein R, 
R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are as shown in the Table. 
__________________________________________________________________________ 
##STR8## (IV) 
R R.sub.1 R.sub.2 
R.sub.3 
R.sub.4 
R.sub.5 
R.sub.6 
R.sub.7 
R.sub.8 
R.sub.9 
__________________________________________________________________________ 
H H H H H H H H H H 
H H Cl H H H Cl H H H 
H H Cl Cl H H H H H H 
H H CONCH.sub.3 
H H H Br H H H 
H H Br H H H CON(CH.sub.3).sub.2 
H H H 
H H H OCH.sub.3 
H H H H H H 
H H H H OCH.sub.3 
H H H H H 
H H H H CH.sub.2 COOH 
H H H H H 
H H COOH H H H H H H H 
H CH.sub.3 
H H H H H H H H 
CH.sub.3 
H H H H CH.sub.3 
H H H H 
H CH.sub.2 CH.sub.3 
H H H H H Cl 
H H 
H CH.sub.2 CN 
H H CH.sub.2 OH 
H H H H H 
H H H I H H H H CN H 
H CH.sub.2 CH.sub.2 COOH 
H H H H H H H H 
H H CHO H H H H H H H 
H H H F H H H H H H 
__________________________________________________________________________ 
The compounds of the formula: 
##STR9## 
wherein R.sub.11, R.sub.12 and R.sub.15 are as defined hereinabove; may be 
made by reacting the appropriate di, tri or tetrahaloselenide or telluride 
with the appropriate hydroxy compound which may be of the formula: 
EQU HO--R.sub.16 
R.sub.16 is alkyl of 1 to 5 carbons, haloalkyl of 1 to 5 carbons, aryl, 
alkylaryl, alkylamido of 1 to 5 carbons, alkylcarbonyl of 1 to 5 carbons, 
cyanoalkyl of 1 to 5 carbons, cyanoalkyl of 1 to 5 carbons, and an 
alkoxyalkyl of 2 to 10 carbons. Specific examples of R.sub.16 include 
methyl, ethyl, n-propyl, phenyl, tolyl, amidoethyl, cyanomethyl, 
methyloxymethyl and CH.sub.2 CH.sub.2 COOH. 
The compounds are made by combining substantially equimolar amounts of the 
reactants in a suitable reactor at room temperature or at elevated 
temperatures up to the reflux temperature. It is preferred to utilize a 
solvent that is capable of dissolving the reactants such as acetonitrile, 
benzene, toluene, xylene, dimethylsulfoxide, mixtures thereof and the 
like. Compounds of structure (A) are only obtained in acetonitrile. The 
preferred method requires heating the reaction mixture to the reflux 
temperature of the solvent while stirring the reaction mixture with a 
suitable magnetic or mechanical stirrer. The reaction may be carried out 
for a sufficient period of time to ensure complete reaction of the 
reactants. This time will vary with the reaction conditions, the 
particular compound being made and the nature of the solvents. The 
reaction may be run at atmospheric pressure but if desired may be carried 
out at reduced or elevated pressure. The reaction is practically carried 
out in the presence of an oxygen containing atmosphere such as air but 
inert atmospheres such as nitrogen, argon, helium or mixtures thereof may 
be utilized if desired. Reaction times of 1 minute to 168 hours may be 
used although reaction times of 6-16 hours are preferred. 
The reactor should be of glass construction or lined with glass or other 
ceramic material that is inert with respect to the reactants. 
Usually the compounds produced in the process will precipitate as the 
reaction mixture is cooled to room temperature. Precipitation may also be 
effected by adding a suitable precipitant such as a liquid alkane such as 
hexane or by concentration of the reaction mixture by solvent removal by 
evaporation with or without the aid of vacuum. The product may be 
collected in a sintered glass filter, washed with a cold solvent and dried 
using conventional techniques. The product is stored in a suitable 
container, protected from light, and preferably at reduced temperature to 
avoid decomposition. 
The solvent system for administraton of the compounds of the invention may 
be based on dimethylsulfoxide or lower alkanols such as ethanol and 
propanol, glycols such as ethylene glycol, glycerol, propylene glycol and 
the like. The preferred solvent system is a phosphate buffered saline 
solution which contains an amount of sodium acid phosphate and sodium 
phosphate in water to give a pH of 7.1-7.2 (PBS). 
Those skilled in the art will appreciate that the presence of a reactive 
group that will interfere with the synthesis of a particular compound will 
require the use of a protective group that is removable using known 
methods. 
The compounds of the invention may be administered to mammals for treatment 
of cancer, immune deficiencies, autoimmune diseases and infectious 
diseases using amounts that are effective in each condition. The treatment 
will alleviate the symptoms of these diseases by causing the mammalian 
body to produce increased amounts of lymphokines. The invention also 
includes the in vitro production of increased amounts of cytokines such as 
lymphokines and or their receptors and the use of these materials and/or 
as therapeutic agents to be administered to mammals for the alleviation of 
cancer, immune deficiences and infectious diseases. It is contemplated 
that the composition of the invention may be used in combination with 
other anti-cancer chemotherapeutic agents such as cyclophosphamide. 
The term cancer is used to include leukemia and solid tumors that arise 
spontaneously, by contact with a carcinogenic agent, by irradiation or by 
oncoviruses. These conditions are well known to those who are skilled in 
the art and include such conditions as adrenal tumors, bone tumors, 
gastrointestinal tumors, brain tumors, breast tumors, skin tumors, lung 
tumors, ovarian tumors, genitourinary tumors and the like. The Merck 
Manual 13th Edition, Merck & Co. (1977) describes many of these 
conditions. Pages 647-650; 828-831; 917-920; 966; 970-974; 1273, 1277, 
1371-1376; 1436-1441; 1563; 1612-1615 of the publication are incorporated 
herein by reference. 
The term immunodeficiency diseases is used to describe a diverse group of 
conditions such as Acquired Immunedeficiency Syndrome (AIDS) characterized 
chiefly by an increased susceptibility to various infections with 
consequent severe acute, recurrent and chronic disease which result from 
one or more defects in the specific or nonspecific immune systems. Pages 
205-220 of the Merck Manual 13th Edition describe many of these conditions 
and they are incorporated herein by reference. 
The term autoimmune diseases includes disorders in which the immune system 
produces autoantibodies to an endogenous antigen, with consequent injury 
to tissues. Pages 241-243 of the Merck Manual 13th Edition describe these 
conditions and they are incorporated herein by reference. 
The term infectious diseases includes those pathologic conditions that 
arise from bacterial, viral or fungus organisims that invade and disrupt 
the normal function of the mammalian body. Pages 3-149 of the Merck Manual 
13th Edition describe these conditions and they are incorporated herein by 
reference. 
The compounds may be administered orally, parenterally, transcutaneously, 
topically or by contacting mucous membranes. The compounds may be 
administered orally in capsules or tablets that may be prepared using 
conventional excipients, binders, disintegrating agents and the like. The 
parenteral route is presently preferred and compositions may be prepared 
by dissolving the compound in a suitable solvent such as an aqueous buffer 
and dimethyl sulfoxide or glycerol. The parenteral route may be 
intramuscular, intravenous, intradermal using a sustained release carrier 
or subcutaneous. The concentration of the compounds in combination with a 
pharmaceutical carrier is not critical and is a matter of choice. 
Remingtons Practice of Pharmacy, 9th, 10th and 11th Ed. describe various 
pharmaceutical carriers and is incorporated herein by reference. 
It has been found that a number of the tellurium compounds useful in the 
practice of the invention will hydrolyze in the presence of water. These 
hydrolyzed compositions are active in vivo and in vitro although the 
hydrolyzed compositions eventually decompose and lose their ability to 
induce lymphokine secretion. For this reason, the compositions should be 
freshly prepared. If the compounds are administered orally in dry form, 
they are active in inducing the production of lymphokines. Preferably, the 
compounds should be kept under anhydrous conditions until just prior to 
being used. 
It has been found that certain compounds such as TeO.sub.2 alone will 
induce lymphokine production in producer T-cell lymphocytes in vitro and 
in vivo but it will not cause proliferation of IL-2 producer cells or 
activate the receptor site in responder T-cell lymphocytes. Thus the 
invention also contemplates the use alone of TeO.sub.2 and tellurium 
compounds that are active as lymphokine inducers. 
Topical compositions may be prepared by dispersing the compounds in 
hydrophillic or hydrophobic cosmetic base. Petroleum jelly or commercial 
preparations such as Oil of Olay may be used. The concentration may be 
from 0.0001-5% on a weight/weight basis. 
The dosage of the compounds of the invention used to stimulate lymphokine 
production or treat the specific disease condition described herein may be 
varied depending on the particular disease and the stage of the disease. 
Generally an amount of the compound may be administered which will range 
from 0.05.times.10.sup.-3 to 1.times.10.sup.-3 g/Kg of body weight and 
preferably from 0.1.times.10.sup.-3 to 0.5.times.10.sup.-3 g/Kg of body 
weight. For example a dosage of 1-3 mg per day for a 75 Kg mammal is 
contemplated as a sufficient amount to induce lymphokines production but 
the dosage may be adjusted according to the individual response and the 
particular condition that is being treated. 
In addition to treating the mammalian disorders described hereinabove, the 
compounds may be utilized for veterinary purposes in the treatment of 
viral and immune diseases that afflict horses, ungulates and fowl. These 
disorders may be treated using quantities of the compound that may be used 
in treating the mammalian disorders described hereinabove. 
For in vitro use, cells may be stimulated to produce lymphokines by use of 
1.times.10.sup.-8 to 1.times.10.sup.-4, preferably 1.times.10.sup.-7 to 
1.times.10.sup.-5 g of compound per 10.sup.6 cells/ml. 
Preliminary toxicity studies in mice have established an LD.sub.50 of 300 
ug./25 g of body weight in 6 week old mice for the compound of Example 1. 
The compounds may be used as anti-bacterial or anti-viral agents in plants 
or in animals. Virus infections such as West Nile virus infections in mice 
are susceptible to the compound of the Example 1 at a dose of 10 
.mu.g/day/mouse. Plant bacterial infections such as crown gall caused by 
Agrobacterium tumefaciens may be treated or prevented by the application 
of a 0.1% solution of compounds of the invention. 
The invention also contemplates a method for dissolving the compounds of 
the invention in an aqueous vehicle. This method comprises the use of 
ultrasound or mechanical agitation for an extended period of time which 
will dissolve the compound. Generally ultrasound is produced by a 
transformer which transforms 50/60 hertz, line voltage AC into high 
frequency electrical energy which is coupled to a transducer. By using 
piezoelectric ceramics, electrical frequency is converted into mechanical 
vibration. Typical amplitudes of 0.0003 for 40 k Hz equipment and 0.00007 
to 0.001 for 20 k Hz equipment are useful. The transducer may be provided 
with a booster that is connected to a horn that has means for conducting 
the ultrasound to a container that holds the liquid for dissolving the 
compounds of the invention. Useful devices include small scale ultrasonic 
cleaners such as the Bronson instrument. It has been found that solutions 
containing about 5 mg/100 ml of the compound of the invention may be 
prepared by applying ultrasound for a sufficient period of time to provide 
an aqueous liquid containing the compound. The time required for this is 
usually 3 hours to 24 hours. High speed mechanical shakers such as a 
Tutenhauer shaker or waring blenders may be used for this purpose. The use 
of an electically operated agitator will cause the compounds to form a 
solution or dispersion after about 3 to 4 hours of agitation. 
It has been discovered that glycerol may be used in the preparation of 
aqueous liquids that contain the compound. These preparations are then 
diluted with an aqueous injectable diluent such as water, saline solution 
etc. The preferred diluent is PBS. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The following examples are given to illustrate the invention and it is 
understood that they do not limit the scope of the invention. 
EXAMPLE 1 
0.01 mol of ethylene glycol and 0.01 mol of tellurium tetrachloride were 
dissolved in 35 ml of dry acetonitrile and placed in a flask fitted with a 
reflux condenser and a magnetic stirrer. The reaction mixture was refluxed 
for six hours. The solution was filtered while hot through a sintered 
glass filter. The filtrate was collected and allowed to reach room 
temperature which resulted in the formation of a white precipitate. The 
precipitate was filtered and collected on a sintered glass filter and 
washed with cold acetonitrile. It was dried for 10 hours under vacuum of 
0.05 mm/Hg. The mp(d) was ca. 200.degree. C. The calculated elemental 
analysis was %C=7.70; H=2.57; N=4.49; Cl=34.06; O=10.26; Te=40.92 found 
C=7.87; H=2.5; N=4.5; Cl=33.38; O=10.13; Te=41.12 The P.M.R. spectra 
(deuterated DMF).delta.(ppm): 4.43(4H, S); 7.7(4H, t); The mass spec. was 
m/e: 130, 160, 165, 180, 190, 200, 222, 224, 247, 251, 258, 260; the 13C 
NMR spectrum confirms the presence of CH.sub.2 groups; the .sup.125 Te NMR 
spectrum confirms the presence of one Te atom in the molecule. 
The difference in the Cl analysis may be due to the presence of a small 
quantity of the compounds of formula II in the sample. 
EXAMPLE 2 
0.01 mol of ethylene glycol was added to 0.01 mol of tellurium 
tetrachloride in 50 ml of dry benzene in flask fitted with a reflux 
condenser and a magnetic stirrer. The reaction mixture was refluxed for 16 
hours and filtered while hot through a sintered glass filter and worked up 
as in Example 1 using benzene as a wash liquid to give the compound of 
formula II. The mp(d) was ca. 250.degree. C. 
EXAMPLE 3 
A solution of the compound of Example 1 was prepared as follows: 5 mg was 
placed in a volumetric flask to which was added 100 ml of a solutin of 40% 
dimethyl sulfoxide (DMSO) and 60% phosphate buffer saline (PBS) solution 
resulting in a concentration of 10 .mu.g/0.2 ml. If the solution becomes 
turbid, it is centrifuged at 2000 rpm for ten minutes and the clear 
supernatant portion is used. 
The test animals were Balb-c, male, mice, 6 to 8 weeks of age. All 
injections were made intraperitoneally using 0.2 ml of the solution of the 
compound of Example 1 using 25 gauge 5/8" hypodermic needle. 
The animals received the following injections: 
(a) Control (no injection) 
(b) Control (0.2 ml DMSO) 
(c) 1 .mu.g of compound of Example 1 (in 0.2 ml DMSO/PBS solution) 
(d) 10 .mu.g of compound of Example 1 (in 0.2 ml of DMSO/PBS solution) 
Each of groups a, b, c and d consisted of 21 animals. The animals were 
sacrificed daily from 24 h to 7 days after injection. 
On each day, spleen cells from three of the animals from each control group 
were pooled together and processed by passing the spleen cells through a 
60 mesh stainless steel net in a 5 mm Petri dish containing PBS in order 
to separate the cells. The cells were collected and centrifuged at 1000 
rpm for 10 minutes. The supernatant was discarded and the cells were 
treated for two minutes with 5 ml of hypotonic buffer (0.15M NH.sub.4 CL, 
0.01M KHCO.sub.3 dissolved in double distilled water, pH 7.2) to kill the 
erythrocytes. Thereafter, PBS was added to the cells and the cells were 
centrifuged for 10 minutes at 1000 rpm. The cells were washed twice with 
PBS and counted in a haemocytometer using trypan blue to test for 
viability. The cells were brought to a concentration of 10.sup.7 cells/ml 
using enriched RPMI containing 10% fetal calf serum (Ser Lab, Sussex, 
England); 5.times.10.sup.-5 M 2-mercaptoethanol and 3% of d-glutamine (Bio 
Lab Israel) (stock solution 2 mM.times.1000 nonessential amino acids) (Bio 
Lab, Israel) (stock solution.times.100) and sodium pyruvate (Bio Lab, 
Israel) (stock solution 1 mM.times.100). 
An additional three animals from each of the experimental groups were 
sacrificed and each of the spleens was processed separately using the same 
procedure. 
The cell mixture was divided into two groups. 
(a) Cells at a concentration of 10.sup.7 cells/ml enriched RPNI to which 
was added concanavalin-A (CON A) (DiFCO, Batch 352) 2 .mu.g./ml. These 
cells were incubated in 5 mm Petri dishes (NUNC) for 24 hours at 
37.degree. C., 7.5% CO.sub.2. Supernatants were collected, centrifuged at 
1,600 rpm for 10 minutes and stored at 4.degree. C. until used. These 
supernatants were assayed for IL-2 and CSF activity. 
(b) Cells at a concentration of 10.sup.7 cells/ml enriched RPMI which were 
incubated at 37.degree. C., 7.5% CO.sub.2 for 96 h., without addition of 
CON A. Supernatants were collected, centrifuged and stored at 4.degree. C. 
until used. These supernatants were assayed for CSF activity. 
Prior to incubation of the cells, samples were removed from culture plates 
and smears of the cultures were made by cytocentrifugation. Slides were 
stained with May-Grunwald-Giemsa (1:10) solution and evaluated 
morphologically. A radioactive thymidine assay was used to determine IL-2 
activity. 
ASSAY FOR IL-2 ACTIVITY 
1. Supernatants were tested for IL-2 activity by the proliferation of the 
IL-2 dependent cell line CTLD. The IL-2 assay is based on the growth 
dependence of these cultured T-cell lines on IL-2. T cells harvested from 
IL-2 dependent culture, washed and placed back in culture in the absence 
of IL-2 invariably die within 24 hr. By using tritiated thymidine 
incorporation (.sup.3 H-TdR) as an index of cultured T-cell replication, 
the IL-2 microassay provides a highly reproducible and quantitive 
indication of the amount of IL-2 activity in the supernatant prepared 
hereinabove. 
2. To assay a condition medium for IL-2 activity, a sample containing 
5.times.10.sup.4 CTLD cells, 10% fetal calf serum and 50% of supernatant 
in question, all were suspended in a final volume of 1 ml RPMI. Aliquots 
of 0.2 ml from each sample were placed in four replicate wells of 96 
microwell tissue culture plates (NUNC). Conditioned medium was obtained 
from cultures of Charles River rat spleen cells stimulated with Con A that 
contained a known amount of IL-2 as a reference in all assays. 
3. The microwells were incubated fo 24 hr. at 37 degrees C. after which 1 
microcurie/well of .sup.3 H-methylthymidine was added. Cells were then 
further incubated overnight, harvested with a cell harvester, and counted 
in a beta scintillator. 
The results, in counts per minute (CPM) were as follows and indicate the 
relative quantity of IL-2 that is present in the supernatants. 
(a) Control (no injection) 
(b) Control (0.2 ml DMSO) 
(c) 1 .mu.g of compound of Example 1 (in 0.2 ml DMSO/PBS solution) 
(d) 10 .mu.g of compound of Example 1 (in 0.2 ml of DMSO/PBS solution) 
______________________________________ 
day 1 day 2 day 3 day 4 day 6 day 7 
______________________________________ 
(a)* 37,695 32,055 24,758 
45,029 25,065 
36,775 
(b)* 16,323 30,824 24,861 
30,555 48,921 
38,626 
(c) 25,919 21,398 10,130 
31,999 41,261 
66,854 
(c) 34,326 22,050 13,235 
14,226 80,314 
58,094 
(c) 16,718 9,338 2,176 
17,228 42,485 
51,268 
(d) 24,335 31,901 20,316 
26,644 22,040 
85,216 
(d) 21,193 36,390 18,288 
18,051 74,043 
6,299 
(d) 25,381 22,066 12,126 
65,963 43,838 
-- 
______________________________________ 
*spleens from control animals were pooled after removal from animal. 
EXAMPLE 4 
This example describes the stimulation of IL-2 production from human 
mononuclear cells by the use of the compound of Example 1. 
Venous whole blood (with heparin, Evans: 10 IU/ml blood) was diluted with 
RPMI in a ratio of 1:1. The diluted blood was gently placed on Lymphoprep 
(Nylgard & Co., Oslo, Norway, density 1.077 g/ml) two parts of diluted 
blood on one part of Lymphoprep. Each tube was provided with 3 ml 
Lymphoprep and 6 to 7 ml diluted blood. The tubes were centrifuged 30 
minutes at 1600 rpm at room temperature. After the centrifugation, 
mononuclear cells were collected from the interphase fraction and washed 
with RPMI three times. The cells were resuspended in RPMI, counted on a 
hemocytometer, using trypan blue to test for viability and brought to a 
concentration of 1.times.10.sup.-6 cells per ml in enriched RPMI. Varying 
concentrations of the compound of Example 1 ranging from 50 .mu.g/ml to 5 
pg/ml were added in a volume of 10% of cell mixture. Aliquots of 0.2 ml 
from each sample were placed in wells of microplates (NUNC) (triplicates). 
The microplates were incubated for 72 hours at 37.degree. C. after which 
.sup.3 H-methylthymidine, 1 u Ci/well (Nuclear Research Center, Israel) 
was added to the cultures. Cells were further incubated overnight and 
harvested with a cell harvester. Proliferation of human mononuclear cells 
was increased by 5 to 6 fold in the range of 1 to 10 ng/ml of cells of the 
compound of Example 1 thus suggesting that the compound of Example 1 
either induced the production of IL-2 in a subset of the mononuclear cells 
resulting in the observed proliferation and/or the induced receptor 
formation in a given population which would also result in proliferation. 
EXAMPLE 5 
This example illustrates the in vivo effect of the compound of Example 1 on 
an experimentally induced tumor. 
A solution of 0.2% of methylcholanthrene (MCA, Sigma, USA) was prepared by 
dissolving 2 mg of the carcinogen in 1.0 ml of olive oil (Ref: Petra, et 
al., Cancer 19: 302, 1961) with continuous shaking at 37.degree. C. for 30 
minutes. Six to eight week old C.sub.3 Heb mice were injected with 0.6 mg 
MCA/0.3 ml of solvent/mouse subcutaneously in the rear right thigh. 
After 21-30 days the induced tumor was surgically removed and pushed 
through 60 mesh stainless nets to obtain isolated cells. These cells were 
then further injected subcutaneously into the rear right thigh of C.sub.3 
Heb mice 5 to 8 weeks of age, at a concentration of 10.sup.6 cells/0.3 ml 
PBS/mouse/hypodermic needle 25 gauge, 5/8" to further induce tumor 
formation. 
Five days after injection of the tumor cells, a palpable tumor was induced. 
The animals were thereafter treated as follows: 
(a) control (0.2 ml 40% DMSO 60% PBS, IP 1 day after the induced tumor was 
palpable) 
(b) 10 .mu.g of compound of Example 1, IP to 5 mice (in 0.2 ml 40% DMSO 60% 
PBS, 3 days after the induced tumor was palpable and a second injection of 
10 .mu.g of compound in the same solvent was administered 5 days after the 
first injection to 3 of the 5 mice. 
The tumors were excised after 13 days and the volume was determined and is 
reported in the Table. All animals expired 35 to 38 days after the initial 
innoculation. 
TABLE 
______________________________________ 
Compound of Ex. 1 
Vol. of Tumor 
Group Mouse Administration 
(13 days) 
______________________________________ 
a* 1 -- 4.01 
a* 2 -- 3.7 
a* 3 -- 3.9 
b 7 day 3 0.77 
b 8 day 3 1.66 
b 4 day 3 and 5 0.7 
b 5 day 3 and 5 0.52 
b 6 day 3 and 5 0.31 
______________________________________ 
*Control 
EXAMPLE 6 
Balb-c mice, age 7 weeks were injected with methylcholanthrene to induce 
the formation of fibrosarcoma cells according to the procedure of Example 
5. The test animals were divided into two groups. 
(a) control (0.2 IP of 40% DMSO and 60% PBS) 
(b) 10 .mu.g of compound of Example 1 (in 0.2 ml 40% DMSO 60% PBS, IP at 
intervals shown in Table 2) 
TABLE 2 
______________________________________ 
Days After Inno- 
Days after Injection 
culation With With Compound Of 
Tumor Cells Example 1 % Survival 
______________________________________ 
(a)* 
24 -- 100% 
25 -- 63% 
34 -- 55% 
46 -- 45% 
60 -- 35% 
67 -- 18% 
69 -- 0% 
(b) 
4 4 100% 
9 9 100% 
23 23 100% 
30 30 100% 
37 37 100% 
.sup. 39.sup.1 
39 100% 
41 41 100% 
43 43 100% 
46 46 80% 
48 48 80% 
50 50 80% 
53 53 60% 
60 60 40% 
67 -- 20% 
68 -- 0% 
______________________________________ 
*Control 
.sup.1 Day 39 marked the start of an increased dosage regimen to determin 
the toxicity of the compound of Example 1. The mortality results for grou 
(b) were 0% until just after the increased dosage regimen. 
EXAMPLE 7 
This example describes the in vitro making of IL-2 and CSF from mouse 
spleen cells using the compound of Example 1 as the extrinsic stimulating 
agent. 
Spleens were removed from 15, male Balb-c mice 6 to 8 weeks of age. The 
spleen cells were pushed through stainless steel 60 mesh (U.S. Standard) 
nets resting in 5 mm Petri dishes containing PBS in order to separate the 
cells. The cells were then collected into centrifuge tubes and spun at 
1000 rpm for 10 minutes. The supernatant was discarded and cells were 
treated with 5 ml of hypotonic buffer (0.15 MNH.sub.4 Cl; 0.01M KHCO.sub.3 
dissolved in double distilled water, pH 7.2) for exactly two minutes. 
Thereafter, PBS was added to the cells and the test tubes were centrifuged 
for 10 minutes at 1000 rpm. The cells were rinsed twice and counted in a 
heamocytometer using trypan blue to test for viability. The cells were 
brought to a concentration of 10.sup.7 viable cells/ml. The cells were 
contacted with varying amounts of the compound of Example 1 in 1 ml of 40% 
DMSO 60% PBS. Table 3 shows the induction of IL-2 activity and colony 
stimulating factor that was obtained with varying amounts of the compound 
of Example 1. 
TABLE 3 
______________________________________ 
Compound of 
Example 1 
IL-2 (cpm) 
CSF (colonies/dish) 
______________________________________ 
50 .mu.g 5,000 2 
5 .mu.g 5,000 5 
500 ng 5,000 25 
50 ng 6,000 75 
5 ng 15,000 120 
500 pg 30,000 175 
50 pg 38,000 260 
5 pg 12,000 140 
______________________________________ 
.mu.g = micrograms 
ng = nanograms 
pg = picograms 
Control animals injected with the DMSO solvent were found to have a IL-2 
baseline of 4,000-5,000 CPM and a CSF of 70-80/colonies/dish. 
EXAMPLE 8 
Human mononuclear cells were obtained as described above and cultured for 
72 hours at a concentration of 10.sup.6 cells/ml enriched RPMI, in the 
presence of varying concentrations of the compound of Example 1. Culture 
supernatants were collected, centrifuged and tested for IL-2 activity by 
using 50% of the volume of the supernatant assaying their ability to 
support the proliferation of the IL-2 dependent cell line CTLD. Table 4 
reports the results of this assay. 
TABLE 4 
______________________________________ 
Counts per minute 
______________________________________ 
Compound of Example 1 
1 .mu.g 250 
100 .mu.g 280 
10 ng 1,500 
1 ng 11,000 
Control: 
CTLD cells 3,500 
______________________________________ 
EXAMPLE 9 
This example describes the stimulation of IL-2 production from human 
mononuclear cells by the use of the compound of Example 2. 
Venous whole blood (with heparin, Evans: 10IU/ml blood) was diluted with 
RPMI in a ratio of 1:1. The diluted blood was gently placed on Lymphoprep 
(Nylgard & Co., Oslo, Norway, density 1.077 g/ml) two parts of diluted 
blood on one part of Lymphoprep. Each tube was provided with 3 ml 
Lymphoprep and 6 to 7 ml diluted blood. The tubes were centrifuged 30 
minutes at 1600 rpm at room temperature. After the centrifugation, 
mononuclear cells were collected from the interphase fraction and washed 
with RPMI three times. The cells were resuspended in RPMI, counted on a 
hemocytometer, using trypan blue to test for viability and brought to an 
enriched RPMI. Varying concentrations of the compound of Example 2 ranging 
from 50 .mu.g/ml to 1 ng/ml were added in a volume of 10% of cell mixture. 
Aliquots of 0.2 ml from each sample were placed in triplicate wells of 
microplates (NUNC). The microplates were incubated for 72 hours at 
37.degree. C. after with .sup.3 H-methylthyrmidine, 1 u Ci/well (Nuclear 
Research Center, Israel) was added to the cultures. Cells were further 
incubated overnight and harvested with a cell harvester. Proliferation of 
human mononuclear cells was incresed by 10 fold in the range of 1 to 10 ng 
of the compound of Example 2/ml cells thus suggesting that the compound of 
Example 2 either induced the production of IL-2 in a subset of the 
mononuclear cells resulting in the observed proliferation and/or the 
induced receptor formation in a given population which would also result 
in proliferation. 
EXAMPLE 10 
To a 100 ml solution of PBS* is added 5.0 mg of the compound of Example 1 
using sterile conditions. The mixture is placed in a sonicator, and is 
sonicated for 4 hours. After the 4 hour period, the compound is dissolved 
to give a concentration of 10 .mu.g/0.2 ml. 
______________________________________ 
*NaCl 8.0 g 
KCl 200 mg 
Na.sub.2 HPO.sub.4 1150 mg 
KH.sub.2 PO.sub.4 200 mg 
CaCl.sub.2 (anhyd.) 100 mg/L 
Mg Cl.sub.2 6H.sub.2 O 
100 mg/L 
H.sub.2 O sufficient to 
make 1 liter 
______________________________________ 
EXAMPLE 11 
Using 100 ml of the PBS of Example 10, 5.0 mg of the compound of Example 1 
is dissolved by shaking in an electrically operated shaker for 4 hours, 
using sterile conditions to obtain a 10 .mu.g/0.2 ml solution. 
EXAMPLE 12 
Using stirring 5.0 mg of the compound of Example 1 is dissolved under 
sterile conditions in 20 ml of glycerol. Thereafter 80 ml of PBS is added 
to form a solution containing 10 .mu.g/0.2 ml. 
It has been determined that the compound of Example 1 will dissolve as 
follows: 
6.0 g/L in 40% glycerol/60% PBS 
1.3 g/L in 20% glycerol/80% PBS 
1.0 g/L in 10% glycerol/90% PBS 
EXAMPLE 13 
This example demonstrates the effect of oral administration of the 
compounds of Example 1 and 2 on the induction of lymphokines. 
The compounds were administered to male Balb-C mice, 6-8 weeks of age by 
the administration of the compounds of Examples 1 and 2 in drinking water 
at a concentration of 10 .mu.g/ml of water and 1 .mu.g/ml of water for the 
compound of example 1 and 25 .mu.g, 10 .mu.g and 1 .mu.g/ml of water for 
the compound of example 2. The water solution was prepared by dissolving 
the compounds in PBS at a concentration of 50 .mu.g/ml PBS and diluting 
the resulting solution to the desired concentration. The compounds were 
administered over a 4 day period. The exact amount of liquid intake was 
recorded daily. After 4 days the mice were sacrificed and spleens removed 
and processed as described in Example 3. The cells were incubated at a 
concentration of 10.sup.7 cells/ml in enriched RPMI containing 2 .mu.g/ml 
of con-A for 24 hours at 37.degree. C. The supernatants were collected and 
tested for IL-2 content. 
______________________________________ 
Compound ug/ml H.sub.2 O 
Intake/animal 
cpm 
______________________________________ 
Ex 1 10 .mu.g/ml 
248 .mu.g 
Ex 1 1 .mu.g/ml 
23 .mu.g 49179 [+50%]* 
Ex 2 25 .mu.g/ml 
406 .mu.g 44500 [+35%]* 
Ex 2 10 .mu.g/ml 
123 .mu.g 36815 
Ex 2 1 .mu.g/ml 
17 .mu.g 42500 [+30%]* 
Control 32843 
______________________________________ 
*Percent increase as compared to control 
The compound of Example 1 was thoroughly mixed with powdered sucrose and 10 
.mu.g of the compound of example 1 in 25 mg of powdered sucrose was placed 
in the mouth of each mouse. 
After 24 hours the mice were sacrificed and the spleens were processed as 
described above. The IL-2 content was as follows: 
______________________________________ 
cpm 
______________________________________ 
Test 1 
A 250,175 [+117%]* 
B 166,475 [+50%]* 
Control 110,853 
Test 2 
A 55,382 [+69%]* 
B 41,938 [+28%]* 
C 29,172 [0] 
Control 32,980 
______________________________________ 
*Calculated as percent increase as compared to control. 
This experiment shows that the compound of example 1 is active for inducing 
lymphokine production when given in dry form or when hydrolyzed in an 
aqueous diluent. 
EXAMPLE 14 
Tellurium dioxide (0.5 mole) was suspended in 250 ml 1,2-ethanediol 
(excess) and the mixture was heated under reflux at 90.degree. C. under a 
slight vacuum for 16 hours. A white crystalline product was obtained. The 
material was separated by filtration, dried and then purified by 
sublimation at 150.degree. C. (0.25 mm Hg) mp 206.degree.-210.degree. C. 
Cf. JACS 103, 2340-2347 (1981) Anal. calc. for C.sub.4 H.sub.8 O.sub.4 Te: 
C, 19.2; H, 3.3; O, 25.07; Te 51.2. Found: C, 19.91; H, 3.12; O, 24.98; 
Te, 49.99. MS m/e 250. This compound has the following structure: 
##STR10## 
EXAMPLE 15 
To a stirred solution of TeCl.sub.4 (0.015 mol) and 1,2-propanediol (0.03 
mol) in tetrahydrofuran (70 ml) at -40.degree. C. was added dropwise 
triethylamine (0.06) in 30 ml of tetrahydrofuran. The white precipitate of 
triethylamine hydrochloride was removed by filtration. The filtrate was 
concentrated at room temperature and white oily crystals were obtained and 
purified by sublimation at 120.degree. C. (0.25 mm Hg) M.S. m/e=204,278. 
This compound has the following structure: 
##STR11## 
EXAMPLE 16 
Using the procedure of Example 1, the following compounds were made using 
tellurium tetrachloride and the corresponding diol: 
##STR12## 
M.S.: 165; 200; 239. 
##STR13## 
M.S.: 165; 200; 253. 
##STR14## 
M.S.: 165; 200; 239. 
##STR15## 
EXAMPLE 17 
The compounds listed below were tested in vitro at various concentrations 
that ranged from 10 .mu.g/ml cells to 1.times.10.sup.-6 .mu.g/ml of cell 
mixtures. Cells were obtained from spleens of male Balb/c mice 6-8 weeks 
of age. Spleens were pushed through a stainless steel 60 mesh strainer to 
separate cells. The erythrocytes were lysed by treatment with hypotonic 
buffer (0.15M NH.sub.4 CL, 0.001M KHCO.sub.3 dissolved in double distilled 
water, pH 7.21 for two minutes. The remaining cells were rinsed twice and 
brought to a concentration of 10.sup.7 cells/ml enriched RPMI. The cells 
were then innoculated with the test compound and incubated for 24 hours at 
37.degree. C. in the presence of 7.5% CO.sub.2. The supernatants were 
collected thereafter and were tested for IL-2 activity by the 
proliferation of the IL-2 dependent cell line CTLD. These experiments 
showed that at 1 .mu.g/ml of cell mixture most of the compounds exerted a 
maximum effect on IL-2 secretion. 
______________________________________ 
Compound Spleen Cells (cpm) 
______________________________________ 
Example 1 14,600 (+472%) 
Example 14 10,300 (+304%) 
Example 2 18,000 (+605%) 
Ph.sub.2 Te (OCOCH.sub.3).sub.2 * 
3,500 
Ph.sub.2 Te Cl.sub.2 * 
2,300 
Ph.sub.2 Te (OCOPh).sub.2 * 
4,300 
Ph.sub.2 Te Cl* 2,100 
Ph Te Cl.sub.3 18,000 (+605%) 
(pCH.sub.3 OPh).sub.2 TeCl.sub.2 
2,148 
Example 16(a) 13,540 (+435%) 
Example 16(b) 10,650 (+318%) 
Example 16(c) 15,720 (+516%) 
Example 16(d) 18,075 (+608%) 
TeO.sub.2 17,055 (+508%) 
Control 2,550 
______________________________________ 
*no significant activity at level tested 
EXAMPLE 18 
This Example shows the stimulative effect of the compounds of Example 1, 
Example 16(a) and TeO.sub.2 on the induction of IL-2 receptors of human 
mononuclear cells. 
Human MNC were brought to a concentration of 10.sup.6 cells/ml. RPMI+10% 
FCS. Aliquots of 0.2 ml. were placed in duplicate wells of microdishes and 
plates were incubated at 37 C. for 24 hrs. Thereafter wells were rinsed 
twice with RPMI and cells were resuspended with 20 I.U./ml recombinant 
IL-2 in RPMI and 10% FCS. Plates were further incubated for 48 hrs. and 
labelled with 3H thymidine 24 hrs. before harvesting. 
The proliferation was measured by .sup.3 HT uptake as described by Gillis 
et al, J. Immunol. 120, 2027 (1978). The results are expressed in counts 
per minute. 
______________________________________ 
Com- Com- Com- 
Test A pound pound pound of Compound 
(.mu.g/ml) 
of Ex. 1 of Ex. 1 Ex. 16(a) 
TeO.sub.2 
of Ex. 16(b) 
______________________________________ 
1 28625 27593 21910 1563 1018 
5 .times. 10.sup.-1 
120755 105943 145208 4667 1195 
7 .times. 10.sup.-1 
164538 115195 130845 2475 2102 
5 .times. 10.sup.-2 
20022 5702 8752 2515 1602 
1 .times. 10.sup.-2 
1952 3963 9543 3108 5883 
1 .times. 10.sup.-3 
3652 5055 6685 2867 1093 
1 .times. 10.sup.-4 
3558 4047 6447 5540 1138 
1 .times. 10.sup.-5 
2474 4063 8177 3442 2146 
______________________________________ 
(a) Control -2338 (no chemical) 
(b) Cell plus recombinant IL2 (human) Biogen 1.5 .times. 10.sup.-6 units 
-3260 
(c) Phytohemagglutinin M (Difco) 195,432 
______________________________________ 
Test B Compound Compound Compound 
(.mu.g/ml) 
of Ex. 1 of Ex. 2 TeO.sub.2 
of Ex. 14 
______________________________________ 
1 23488 9315 3275 2620 
5 .times. 10.sup.-1 
66910 8688 5405 2402 
7 .times. 10.sup.-1 
17620 5250 4302 2000 
5 .times. 10.sup.-2 
5390 5538 4280 3290 
1 .times. 10.sup.-2 
6057 6418 3077 3928 
1 .times. 10.sup.-3 
5865 5167 3800 3007 
1 .times. 10.sup.-4 
4960 5372 2925 2327 
1 .times. 10.sup.-5 
7155 6397 3645 2242 
______________________________________ 
(a) Control 5573 (no chemical) 
(b) Cell plus recombinant IL2 (human) Biogen 1.5 .times. 10.sup.-6 units 
6858 
(c) Phytohemagglutinin M (Difo) 125,272 
EXAMPLE 19 
This example shows the stimulative effect of the compounds of the invention 
on the proliferation of human mononuclear cells. 
Human mononuclear cells were obtained by layering heparinized blood over a 
Ficoll/Hypaque gradient. The mononuclear cells were resuspended in 
enriched RPMI, rinsed three times and brought to a concentrations of 
5.times.10.sup.5 cells/ml encriched RPMI. Varying concentrations of the 
compounds of Ex. 1 and Ex. 2, ranging from 0.005 .mu.g to 5 .mu.g/cell 
mixture were added to the cells. Aliquots of 0.2 ml of each sample were 
placed in wells of microplates (triplicates). Microplates were incubated 
for 72 hours at 37.degree. C. after which they were labelled with H.sup.3 
methyl-thymidine 1 uCi/well for an additional 24 hours. Cells were then 
harvested with a cell harvester. 
______________________________________ 
Compound Compound 
(.mu.g/ml) of Ex. 1 of Ex. 1 
______________________________________ 
1 873 4700 
5 .times. 10.sup.-1 
18515* 33700* 
1 .times. 10.sup.-1 
2735 708 
5 .times. 10.sup.-2 
3865 910 
1 .times. 10.sup.-2 
3235 1362 
1 .times. 10.sup.-3 
2553 2180 
1 .times. 10.sup.-4 
3838 2387 
1 .times. 10.sup.-5 
3218 2442 
control 2700 2943 
______________________________________ 
Compound Compound Compound 
(.mu.g/ml) 
of Ex. 1 of Ex. 1 of Ex. 16(a) 
TeO.sub.2 
______________________________________ 
5 .mu.g/ml 
5008 5182 4118 3028 
1 .mu.g/ml 
6000 5600 4557 2842 
5 .times. 10.sup.-1 
20488* 13600* 18415* 4773 
1 .times. 10.sup.-1 
7037 10382* 12435* 4825 
5 .times. 10.sup.-2 
5520 5765 5953 5802 
1 .times. 10.sup.-2 
6898 5712 6000 4730 
1 .times. 10.sup.-3 
5800 6000 6300 6168 
1 .times. 10.sup.-4 
5513 6212 4587 5331 
Control 3600 6117 6113 4912 
______________________________________ 
Compound Compound 
.mu.g/ml of Ex. 2 of Ex. 2 
______________________________________ 
4557 943 
5 .times. 10.sup.-1 
18415 23957* 
1 .times. 10.sup.-1 
12435 31424* 
5 .times. 10.sup.-2 
5953 5532 
1 .times. 10.sup.-2 
6000 2987 
1 .times. 10.sup.-3 
6300 1510 
1 .times. 10.sup.-4 
4321 2332 
1 .times. 10.sup.-5 
5118 2481 
Control 4587 2018 
______________________________________ 
*The concentrations that induced proliferation range from 5 .times. 
10.sup.-1 to 1 .times. 10.sup.-1 .mu.g. No significant effect was found 
for TeO.sub.2 at any concentration that was tested. 
EXAMPLE 20 
Using the procedure of Example 4, human mononuclear cells were tested for 
their ability to produce IL-2 after induction with PHA or in unstimulated 
cells from normal donors and from patients suffering from systemic lupus 
erthyrematous. The IL-2 content was tested according to the procedure of 
Example 3 using the CTLD IL-2 dependent cell line. The results are 
reported in Table 5. 
TABLE 5 
______________________________________ 
IL-2 PRODUCTION BY 
COMPOUND OF EXAMPLE 1 
5 .mu.g 
0.2 
SUBJECTS PHA mlPBS 1:50PBS* 
1:100PBS* 
1:200PBS* 
______________________________________ 
NORMAL 
1 - 2.3** 43.6 38.4 30.1 
+ 36.4 48.2 46.2 38.6 
2 - 2.1 52.2 50.3 47.1 
+ 30.4 38.9 54.2 49.8 
3 - 2.1 50.8 46.1 31.3 
+ 38.9 53.5 44.8 38.3 
SLE 
1 - 2.0 37.3 32.1 26.1 
+ 6.3 24.2 19.7 15.4 
2 - 2.4 43.6 35.1 30.3 
+ 8.2 28.1 24.0 18.6 
3 - 2.4 19.2 18.1 14.4 
+ 4.1 23.8 20.2 16.8 
______________________________________ 
*Dilution of 1 part of 0.2 ml of PBS containing 5 .mu.g of compound of Ex 
1 in 50, 100 or 200 parts of PBS. 
**CPM .times. 10.sup.-3 of .sup.3 H Thymidine of 5 .times. 10.sup.4 CTLD 
cells in presence of 1:2 dilution of the supernatants as in Ex. 3. 
EXAMPLE 21 
This example provides an assay to detect the presence of receptor sites for 
IL-2. Human mononuclear cells were incubated for 24 hours in the presence 
of the compound of Example 1 and TeO.sub.2. The cells were washed twice 
with PBS and then incubated with a specific fluoresceinated antibody 
against IL-2 receptors as described in Uchiyama et al, J. Immunol. 126, 
1398 (1981) The results were that in the control 2% of the cells were 
positive; in the presence of PHA 80% of the cells were positive and with 1 
.mu.g/ml of the compounds of Example 1, 20% of the cells were found to be 
positive. It was found that TeO.sub.2 gave 5% positive cells at a level of 
1 .mu.g/ml. 
EXAMPLE 22 
The effect of the compound of Example 1 on an infection with West Nile 
virus (WNV) was determined. WNV is a toga virus of the flavivirus group, a 
positive single stranded RNA virus, which when injected IP to mice usually 
kills them within 5-8 days as a result of extensive damage to the central 
nervous system. For this study ICR mice (3 wks of age) were injected IP 
with the virus at the concentration of 10.sup.3 or 10.sup.4 LD 50 
units/mouse. Injections of 10 .mu.g/0.2 ml PBS/mouse of the compound of 
Ex. 1 were given on day -1 (one day prior to injection of virus) and 6 
days after injection of virus. Table A shows preliminary results of one 
such experiment. As can be seen, after 8 days all animals injected with 
the virus alone died, whereas three out of five animals receiving 
treatment with the compound of Ex. 1 remained alive. 
TABLE A 
______________________________________ 
Treatment 10.sup.3 LD.sub.50 
10.sup.3 LD.sub.50 
10.sup.4 LD.sub.50 
10.sup.4 LD.sub.50 
0 
Comp. of 
Ex. 1 injected 
NO YES NO YES YES 
# Alive/ 0/6 3/5 0/6 3/5 6/6 
Total Animals 
______________________________________ 
In a second experiment, mice were injected with 10.sup.3 IPLD.sub.50 virus 
and received injections of the compound of Example 1 (1 .mu.g/0.2 ml 
PBS/mouse) on days -1, 1, 2 and 4. Preliminary results of one such 
experiment on day 8 after injection are shown in Table B. As seen on day 8 
all animals injected with virus alone died whereas 3 out of 5 receiving 
the compound of Ex. 1 remained alive. Two out of the remaining three 
survived an additional 8 days, whereas the third remained alive without 
any manifestations of clinical symptoms. 
TABLE B 
______________________________________ 
Treat- 10.sup.3 IPLD.sub.50 
10.sup.3 IPLD.sub.50 
Compound of Ex. 1 
ment + 
Compound of Ex. 1 
# Alive/ 
0/8 3/5 5/5 
Total 
______________________________________ 
EXAMPLE 23 
This example shows that the interaction of WNV with cultures of ICR mouse 
macrophage results in a productive infection. Varying amounts of the 
compound of Example 1 (5 .mu.g, 1 .mu.g, 0.1 .mu.g) were incubated with a 
monolayer of mouse macrophages for 24 h. After 24 h macrophages incubated 
with 5 ug of the compound of Example 1 died whereas others remained 
unaffected. All cultures were then infected with 10.sup.4 PFU/plate. After 
72 h incubation the supernatants were collected and the virus was titrated 
against Vera cells. Table A shows preliminary results of one such 
experiment. As can be seen, incubation of macrophage cultures with 1 ug of 
the compound of Example 1 resulted in a 40-fold reduction of virus yield, 
whereas incubation with 0.1 .mu.g of the compound of Ex. 1 plate resulted 
in a ten-fold reduction in virus yield. 
TABLE A 
______________________________________ 
Treatment Virus Yield (PFU/ml) 
______________________________________ 
Control (virus alone) 
2 .times. 10.sup.4 /ml 
Compound of Example 1 .mu.g/plate 
5 .times. 10.sup.2 /ml 
Compound of Example 1 0.1 .mu.g/plate 
2 .times. 10.sup.3 /ml 
______________________________________