Derivative of T-2 toxin and anguidine

3-ACYL AND 3-ALKYL TRICHOTHECENE DERIVATIVES HAVING THE GENERAL FORMULA ##STR1## in which R.sub.1 is hydrogen or ##STR2## and R is an acyl group ##STR3## in which R' is a branched or straight-chained aliphatic, cycloaliphatic or aromatic group having from 2 to 12 carbon atoms or a carbamate group ##STR4## in which R' has the same meaning as above or an alkyl group, either branched or straight-chained, or an aromatic group having from 1 to 12 carbon atoms.

The invention described herein was made in the course of work under a grant 
or award from the Department of Health, Education, and Welfare. 
This invention relates to 3-acyl and 3-alky trichothecene derivatives and 
to the method for preparation of such 3-acyl and 3-alkyl derivatives 
having the general formula 
##STR5## 
When R is hydrogen and R.sub.1 is hydrogen, the compound is known as 
3-hydroxy-4,15-diacetoxy-12,13-epoxy-.DELTA..sup.9 -trichothecene, and is 
generally referred to as diacetoxyscirpenol or anguidine. When R is 
hydrogen and R.sub.1 is 
##STR6## 
the compound is identified as 3-hydroxy-4,15-diacetoxy-8-(3-methyl 
butyryloxy)-12,13-epoxy-.DELTA..sup.9 -trichothecene, or more generally 
referred to as T-2 toxin. 
Both anguidine and T-2 toxin are produced naturally by several species of 
fungi of the genus Fusarium on such food products as corn or grain. Both 
of these compounds are very toxic and find use as cytotoxic agents which 
destroy cell cultures. A characteristic feature of this family of 
sesquiterpenes is the pronounced cytotoxicity of most of its members. In 
tests with pigeons, both T-2 toxin and anguidine also cause vomiting. 
The oral LD.sub.50 (dose at which 50% of the pigeons are killed) is 2.7 
mg/kg and the oral TD.sub.50 (level which makes 50% of the pigeons vomit) 
is 0.75 mg/kg. 
It has been found that acylation to substitute an acyl group for the 
hydrogen at R on the C.sub.3 position greatly increases the activity of 
the derivative while making it much less toxic to animals. For example, 
when R is 
##STR7## 
hereinafter referred to as AT-2 toxin, the oral LD.sub.50 with pigeons 
becomes at least 18 mg/kg, or less than 1/6 the toxicity of T-2 toxin, 
while vomiting was virtually completely eliminated. In mice, wherein the 
compounds were tested by injection into the intraperitoneal cavity as 
compared to oral administration with pigeons, the difference in toxicity 
was not as great, although substantial, in that the AT-2 toxin was less 
than 1/2 as toxic as T-2 toxin. 
In protocol 1.6 of the NCI anti-cancer screen, (a KB cell culture assay -- 
a human epidermal carcinoma), AT-2 toxin was about 5,000 times more active 
than T-2 toxin. 
It has been found further that the compound is more stable to hydrolysis 
and that the toxicity of the compound is further desirably reduced when 
the acyl group applied to the C.sub.3 position contains at least 3 carbon 
atoms, but not more than 12 carbon atoms in the aliphatic or 
cycloaliphatic group, branched or straight chained, and when the R group 
preferably contains from 3 to 5 carbon atoms. 
The following illustrates the growth of KB cells in nutrient solution (NCI 
protocol 1.6) affected by compounds which, except for acetoxy, are 
representative of the practice of this invention: 
______________________________________ 
R Activity in .mu.g/ml* 
Common name 
______________________________________ 
##STR8## 10.sup.-8 AT-2 
##STR9## 10.sup.-8 PT-2 
##STR10## 10.sup.-9 BT-2 
##STR11## 10.sup.-8 iBT-2 
##STR12## 10 BzT-2 
f) CH.sub.3 10.sup.-2 MT-2 
______________________________________ 
*ED.sub.50 = concentration causing 50% inhibition of growth 
It is believed that the larger acyl groups are hydrolyzed more slowly which 
has the effect of preventing or slowing the production of the more toxic 
T-2 toxin and further that the larger acyl groups also render the compound 
intrinsically less toxic. The alky derivatives, although less cytotoxic, 
completely prevent hydrolysis. Thus, an important concept of this 
invention resides in the acyl and alkyl derivatives of T-2 toxin and 
anguidine wherein the acyl group has from 2 to 12 carbon atoms and 
preferably 3 to 5 carbon atoms and the alkyl group has from 1 to 12 carbon 
atoms. 
Included also within the scope of this invention are the urethane 
derivatives represented by the group 
##STR13## 
wherein R" is an alkyl or cycloalkyl group (branched or straight chained) 
containing from 1 to 11 carbon atoms, as previously defined, including 
ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, cyclopentyl, and 
the like. The derivatives are characterized by greater water solubility 
which enables use by oral administration instead of by injection into the 
intraperitoneal cavity, but they are less active as cytotoxic agents than 
the described acyl derivatives. 
The derivatives of this invention are prepared using T-2 toxin or anguidine 
as the starting material, both of which are available from fermentation 
using a number of strains of the fungal genus Fusarium. For the 
preparation of the acyl derivatives, T-2 toxin, for example, is reacted 
with 
##STR14## 
or the anhydride 
##STR15## 
in which R" signifies a straight or branched chain aliphatic, 
cycloaliphatic or aromatic group having from 1 to 11 carbon atoms. For the 
preparation of the carbamate (the urethane derivative), T-2 toxin is 
reacted with O.dbd.C.dbd.N--R" wherein R" has the same meaning as above. 
The alkyl ethers are prepared by reacting T-2 toxin with I-R" where R" has 
the same meaning as above and from 1 to 12 carbon atoms. 
The following are specific examples of the preparation of derivatives of 
this invention:

EXAMPLE 1 
Preparation of BT-2 in which 
##STR16## 
A mixture of T-2 toxin (200 mg, 0.428 m mol), butyric anhydride (4.0 g, 25 
m mol) and pyridine (5 drops) is stirred overnight at room temperature 
under anhydrous conditions. The excess anhydride and pyridine are removed 
by high vacuum distillation (&lt; 1mm, 75.degree.). The oily residue is 
dissolved in ether and washed once with 5% aqueous sodium bicarbonate 
followed by two washings with water. The ether layer is separated, dried 
over anhydrous sodium sulfate and evaporated to yield 3-n-butyryloxy-T-2 
toxin as a white solid (187mg). This may be further purified by column 
chromatography (silica gel, ethyl acetate -- Skellysolve B, 5.7:1). 
EXAMPLE 2 
Instead of butyric anhydride, others of the C.sub.3 to C.sub.12 aliphatic 
or cycloaliphatic, branched or stright chained anhydrides are substituted 
in equimolecular amounts for butyric anhydride to produce the 
corresponding derivatives. 
EXAMPLE 3 
Preparation of iBT-2 in which 
##STR17## 
To a solution of T-2 toxin (25 mg, 0.054 m mol) in ether (1 ml) was added 
pyridine (5 drops) and isobutyryl chloride (5 drops). The reaction mixture 
is stirred overnight at room temperature under anhydrous conditions. The 
mixture is then diluted with chloroform (10 ml) and washed sucessively 
with ice-cold water (10 ml), cold 5% aqueous sodium bicarbonate and then 
cold water (10 ml). The organic layer is separated, dried over anhydrous 
sodium sulfate and evaporated to afford 3-isobutyryloxy T-2 toxin as a 
white solid. This compound may be further purified by column 
chromatography as in Example 1. 
EXAMPLE 4 
The isobutyryl chloride of Example 3 is replaced by equimolecular amounts 
of 
##STR18## 
in which R" is a branched or straight chained alkyl, cycloalkyl or 
aromatic group having from 2 to 11 carbon atoms to yield the corresponding 
3-acyl derivative of T-2 toxin, or the corresponding 3-acyl derivative of 
anguidine, when anguidine is substituted for the T-2 toxin, in 
equimolecular amounts, in Example 3. 
EXAMPLE 5 
Preparation of T-2 toxin-3-(N-isopropyl) carbamate 
A mixture of 20 mg T-2 toxin (0.044 mmol) and 1 ml isopropyl isocyanate is 
refluxed in a dry, round bottomed flask fitted with a dry ice condenser. 
After 24 hours, the excess isocyanate was removed by distillation under 
high vacuum. The residue was dissolved in chloroform (10 mg) and extracted 
once with 5% aqueous sodium bicarbonate and then with 10 ml of water. The 
organic layer was dried with sodium sulphate and evaporated in vacuo to 
yield 38 mg of a white solid identified as T-2 toxin-3-(N-isopropyl) 
carbamate. 
EXAMPLE 6 
The isopropyl isocyanate in Example 4 is replaced with equimolecular 
amounts of others of the R" isocyanates described in which the R" group is 
a C.sub.1 to C.sub.11 branched or straight chained aliphatic or 
cycloaliphatic group, to produce the corresponding derivatives. 
EXAMPLE 7 
Preparation of MT-2 (R .dbd. CH.sub.3) 
A solution of T-2 toxin (40 mg, 0.088 mmol) in dry tetrahydrofuran (5 ml) 
was slowly added to a suspension of sodium hydride (2.11 mg, 0.088 mmol) 
in dry tetrahydrofuran (10 ml). After 15 minutes, iodomethane (2 drops) 
was added and the mixture stirred at room temperature under a dry 
atmosphere. After 3 hours the reaction was diluted with ether (15 ml) and 
washed three times with cold water (10 ml). The ether layer was separated, 
dried over anhydrous sodium sulphate and evaporated to yield a yellow 
residue (35 mg). Purification using preparative thin layer chromatography 
(silica gel plates, 2 mm; ethyl acetate -- Skelly B, 5.7:1) yielded the 
methyl ether of T-2 toxin (8 mg) as a white solid. 
EXAMPLE 8 
This example is the same as Example 7 except that iodoethane, iodopropane, 
iodobutane, benzyl iodide; iodoisobutane or other branched or straight 
chained C.sub.2 -C.sub.12 alkanes are substituted in equal molecular 
amounts for iodomethane in Example 7 to produce the corresponding ether 
derivatives. 
EXAMPLE 9 
When the T-2 toxin in Example 5 is replaced with anguidine, the 
corresponding 3-acyl and 3-alkyl derivatives are produced.