Process for purification of 9-(2,6-dihalobenzyl)adenines

An efficient method has been found for the removal of the 3-isomer present in crude 9-(2-chloro-6-fluorobenzyl)adenine. Crude 9-(2-chloro-6-fluorobenzyl)adenine is transalkylated in sulfuric acid-toluene to reduce the 3-isomer to undetectable levels (<100 ppm). Alternately, crude 9-(3-chloro-6-fluorobenzyl)adenine is first treated with dilute nitric acid to remove the bulk of the 3-isomer followed by transalkylation to sulfuric acid-toluene to reduce the 3-isomer to undetectable levels (<100 ppm). The overall yield for this purification is about 95% based on the 9-(2-chloro-6-fluorobenzyl)adenine content of the crude material. The resulting pure 9-(2-chloro-6-fluorobenzyl)adenine has anticoccidial activity and is useful in controlling cecal and/or intestinal coccidiosis when administered in minor quantities to animals, in particular to poultry usually in admixture with animal sustenance.

BACKGROUND OF THE INVENTION 
This invention relates to a novel process for obtaining pure 
9-(2,6-dihalobenzyl)adenines from crude reaction mixtures containing 
substantial amounts of 3-(2,6-dihalobenzyl)adenines. Said crude reaction 
mixtures containing 9-(2,6-dihalobenzyl)adenines can be obtained by 
alkylating adenine by the process described in U.S. Ser. No. 766,326 filed 
Feb. 7, 1977 or U.S. Pat. No. 3,846,426. Said alkylated adenines are 
described in U.S. Pat. No. 3,846,426 as being useful in the treatment and 
prevention of coccidiosis. 
Coccidiosis is a widespread poultry disease which is produced by infections 
of protozoa of the genus Eimeria which causes severe disorders in the 
intestines and ceca of poultry. Some of the most significant of these 
species are E. tenella, E. acervulina, E. necatrix, E. brunetti and E. 
maxima. This disease is generally spread by the birds picking up the 
infectious organism in droppings on contaminated litter or ground, or by 
way of contaminated food or drinking water. The disease is manifested by 
hemorrhage, accumulation of blood in the ceca, passage of blood in the 
droppings, weakness and digestive disturbances. The disease often 
terminates in the death of the animals, but the flow which survive severe 
infections have had their market value substantially reduced as a result 
of the infection. Coccidiosis is, therefore, a disease of great economic 
importance and extensive work has been done to find new and improved 
methods for controlling and treating coccidial infections in poultry. 
9-(2,6-Dihalobenzyl)adenines, useful for the control and treatment of 
coccidial infections, are prepared by alkylation of adenine. Alkylation of 
adenine results primarily at the 9-position, but substantial amounts of 
the undesired 3-isomer and traces of the 7-isomer are also formed. The 
alkylation reaction and the structure of the main products i.e., the 
3-isomer and 9-isomer are illustrated by the following equation: 
##STR1## 
wherein X and X' are independently fluorine, chlorine or bromine. 
After the reactants, adenine and .alpha.-chloro-2,6-dihalotoluene, are 
contacted with each other in the reaction medium, the product of the 
reaction, i.e., the 9-(dihalobenzyl)adenine, along with the other isomers 
that form, (e.g. the 3-isomer and to a lesser extent the 7-isomer) will 
either remain entirely in solution or precipitate on standing, depending 
upon the quantity of solvent utilized. Upon completion of the reaction, 
the reaction mixture may be cooled, e.g., to a temperature of about room 
temperature to precipitate a solid product or to precipitate a further 
quantity of this product. The product is then isolated in the usual 
manner, such as by filtration, and, if desired, purified by conventional 
methods such as washing with ethanol or water and recrystallizing from a 
suitable solvent such as acetic acid, aqueous acetic acid, 
dimethylformamide or dimethylsulfoxide. Swishing the product with dilute 
nitric acid or tetrafluoroboric acid (HBF.sub.4) also results in some 
purification. 
The conventional purification methods recited above such as acetic acid 
recrystallization of crude 9-(2,6-dihalobenzyl)adenine produces a 99% pure 
product containing about 1% of the 3-isomer. Since it was learned that the 
3-isomer gives a weak positive Ames test, an objective was set aimed at 
its virtual complete removal to levels less than 100 ppm. The process of 
the present invention is directed to methods for 3-isomer removal which 
involves a transalkylation reaction wherein the 2,6-dihalobenzyl group at 
the 3-position of adenine is removed as a carbenium ion by sulfuric acid 
and the carbenium ion allowed to react with a suitable carbenium ion trap. 
Alternatively, the crude 9-(2,6-dihalobenzyl)adenine may be subjected to a 
first step preliminary purification by extraction with dilute mineral acid 
before treatment with sulfuric acid as a second step. Furthermore, any 
conventional purification method recited above may be used as a 
preliminary first step to obtain partial purification before treatment 
with sulfuric acid. The sulfuric acid treatment process of the present 
invention produces 9-(2,6-dihalobenzyl)adenine containing &lt;100 ppm of 
3-isomer in 92-94% yield over the two steps or in 96% yield in one step. 
SUMMARY OF THE INVENTION 
The teachings set forth herein with respect to 
9-(2-chloro-6-fluorobenzyl)adenine are generally equally applicable to 
9-(2,6-dihalobenzyl)adenines. 
There are two chemical differences between the 9-isomer and the 3-isomer 
which form the basis for the purification of the present invention: (1) 
the 3-isomer (pK.sub.a 5.6) is 40 times more basic than 
9-(2-chloro-6-fluorobenzyl)adenine (pK.sub.a 4.0) and (2) the 3-isomer is 
chemically less stable than 9-(2-chloro-6-fluorobenzyl)adenine in strong 
acid solutions. 
Advantage is taken of the pK.sub.a difference to effect substantial 
reduction of the 3-isomer in the crude reaction mixture by simply 
extracting the solid crude with dilute mineral acid solution. Thus 
extraction of crude 9-(2-chloro-6-fluorobenzyl)adenine containing 20% of 
the 3-isomer with a dilute aqueous solution of nitric acid produces 
9-(2-chloro-6-fluorobenzyl)adenine containing 3-4% of the 3-isomer with a 
96-97% recovery of 9-(2-chloro-6-fluorobenzyladenine. Repeating this 
procedure on the enriched 9-(2-chloro-6-fluorobenzyl)adenine sample fails 
to reduce the 3-isomer level below about 0.3-0.5% (3000-5000 ppm). This is 
due to the pronounced tendency toward solid solutions involving the 
9-isomer and 3-isomer. The same problem is encountered when 3-isomer 
removal is attempted using two acetic acid recrystallizations. The 
3-isomer level reaches the range of 0.05-0.1% (500-1000 ppm) even though 
the liquid phase is not saturated in the 3-isomer. 
Very low levels of the 3-isomer (&lt;100 ppm) have been achieved by the 
process of the present invention by selective chemical degradation of the 
3-isomer, taking advantage of its inherent lower thermodynamic stability. 
The 3-isomer can be totally degraded to adenine and a benzyl polymer by 
treatment with 96% sulfuric acid without affecting the 9-isomer according 
to the following equation: 
##STR2## 
Under the same conditions 9-(2-chloro-6-fluorobenzyl)adenine is 
unreactive. Thus, treatment of 9-(2-chloro-6-fluorobenzyl)adenine 
containing 3-4% of 3-isomer with 96% sulfuric acid results in a 96% 
recovery of 9-(2-chloro-6-fluorobenzyl)adenine containing &lt;100 ppm of the 
3-isomer. It has proved very difficult to completely separate the polymer 
from the 9-(2-chloro-6-fluorobenzyl)adenine. This problem has been 
overcome by the process of the present invention by carrying out the 
sulfuric acid treatment in the presence of a suitable carbenium ion trap. 
Suitable carbenium ion traps are those well known in the art such as 
dialkyl sulfide, wherein the alkyl group contains 1 to 5 carbon atoms; 
diaryl sulfide, wherein the aryl groups contain 6 to 18 carbon atoms; 
benzene, toluene, xylene, mixed xylenes, mesitylene, alkoxybenzene wherein 
the alkyl group contains 1 to 3 carbon atoms such as anisole; thiophene, 
iodobenzene, naphthalene or triphenylphosphine. Out of several substances 
examined, toluene and mixed xylenes were found to be the most suitable for 
this purpose. Toluene reacts rapidly with the intermediate carbenium ion 
to form the transalkylation product, according to the following equation: 
##STR3## 
thus preventing the polymerization. Utilizing the method of sulfuric acid 
transalkylation in the presence of toluene, 
9-(2-chloro-6-fluorobenzyl)adenine containing 3-4% of 3-isomer provides 
high purity 9-(2-chloro-6-fluorobenzyl)adenine containing no polymer and 
&lt;100 ppm of the 3-isomer in 97-98% yield. 
Crude 9-(2-chloro-6-fluorobenzyl)adenine containing 3-isomer in the range 
of 20% may be extracted with a solution of dilute mineral acid to achieve 
a partial removal of the undesired 3-isomer and particularly to remove 
traces of the 7-isomer, if desired, prior to treatment with sulfuric acid. 
The mineral acid used is not critical provided it does not react with the 
9-isomer. Suitable mineral acids are hydrochloric, phosphoric or nitric 
acid. Nitric acid is preferred. To avoid excessive loss of the 9-isomer 
during the extraction, best results are obtained if the quantity of the 
3-isomer is determined by L.C. assay and an equimolar (or a slight excess) 
of acid with respect to 3-isomer is added. The extraction may be carried 
out between room temperature and about 100.degree. C. The preferred 
conditions are reflux temperature with vigorous stirring. Extraction with 
vigorous stirring for about 1 hour to about 5 hours is sufficient. An 
optimal time is about 2 hours. The hot mixture is collected by filtration 
and washed with hot water, with base to remove excess acid and finally 
with hot water again. The resulting partially purified material enriched 
in the 9-isomer is subjected to treatment with sulfuric acid in the 
presence of a carbenium trap to obtain pure 
9-(2-chloro-6-fluorobenzyl)adenine. 
According to the preferred process of the present invention, crude 
9-(2-chloro-6-fluorobenzyl)adenine is treated with concentrated sulfuric 
acid (96% assay) to selectively dealkylate the 3-isomer and regenerate 
adenine. At least a twice molar excess of sulfuric acid with respect to 
the 3-isomer are required for the dealkylation. The concentration of the 
3-isomer is determined by liquid chromatography assay (L.C. assay) as 
described below under the heading Assay. The amount of excess sulfuric 
acid used is not critical. For example, an equal weight of crude 
9-(2-chloro-6-fluorobenzyl)adenine to volume of sulfuric acid or up to 
10.times. volume sulfuric acid with respect to weight of crude 
9-(2-chloro-6-fluorobenzyl)adenine may be used. A preferred ratio is 1 g. 
of crude 9-(2-chloro-6-fluorobenzyl)adenine for each 2 ml. of sulfuric 
acid. 
The quantity of carbenium ion trap used is not critical, provided there is 
at least an equimolar amount with respect to the 3-isomer. However, a 
large excess is preferred because it acts as both a reagent and a solvent. 
The crude 9-(2-chloro-6-fluorobenzyl)adenine is treated with concentrated 
sulfuric acid at a temperature range of from room temperature to about 
90.degree. C. A preferred temperature is room temperature with an 
approximately 10 minute final heating period at about 80.degree. C. to 
insure complete reaction. The reaction time is not critical provided a 
minimum of 2 hours has elapsed. After the initial 2 hours, the reaction is 
essentially complete and may be terminated when convenient. No deleterious 
effects are observed even if the reaction is allowed to proceed for 36 
hours. A convenient optimal time is about 5 hours. 
The aqueous layer is separated from the reaction. Warming may be necessary 
up to a temperature of 50.degree. C. to 100.degree. C. depending on the 
amount of solvent and acid present to facilitate the separation of the 
aqueous phase. The aqueous phase is made basic by the addition of base. 
Any base is suitable provided only that it forms a water soluble sulfate. 
Suitable bases are sodium hydroxide, potassium hydroxide, ammonium 
hydroxide, sodium carbonate, and potassium carbonate. The preferred base 
is sodium hydroxide. After the aqueous phase is made strongly basic the 
pure product precipitates and is collected by filtration and washed with 
water or an aqueous alcohol solution and dried in vacuo. 
An added advantage of the present process is that the costly adenine can be 
recovered from the alkaline filtrate by neutralizing the filtrate and 
collecting the precipitated adenine by filtration. 
ASSAY 
A. Assay for weight % of 9-isomer and for 3-isomer when 3-isomer is present 
in excess of 1% 
The weight % of 9-isomer, 3-isomer and 7-isomer in the product is 
determined by high pressure liquid chromatography (L.C.) and U.V., 
respectively. The weight % of 9-isomer is determined by a high pressure 
liquid chromatography assay (L.C.) using a 15 cm., 5 micron totally porous 
silica column (DuPont, Zorbax-SIL) eluted with CHCl.sub.3 :MeOH (95:5) and 
measuring the absorbance of each component at 254 nm, 9-benzyladenine was 
used as an internal standard (accurate to .+-.0.3%). The weight % of the 
3-isomer is determined by U.V. assay. The sample is assayed in 0.1N 
methanolic base at 310 nm, the 3-isomer has an .epsilon. of 2300 at this 
wave-length and the 9-isomer does not absorb (accurate to .+-.0.1%). 
B. Assay for weight % of trace amounts of 3-isomer after sulfuric acid 
treatment 
The weight % of 3-isomer in the product is determined by high pressure 
liquid chromatography (L.C.) using a 10 micron, 30 cm., microporous, 
octadecylsilane bonded phase column (Waters' Associates Micro Bondapak 
C-18 No. 27324) with a methanol, aqueous phosphate mobile phase at 
35.degree. C. The mobile phase is prepared from 30 parts methanol plus 70 
parts 0.01M Na.sub.2 HPO.sub.4 adjusted to pH 7 with H.sub.3 PO.sub.4. 
Detection is by U.V. at 280 nm. The limit of detection is 100 ppm.

The following non-limiting Examples will serve to further illustrate the 
instant invention. 
EXAMPLE 1 
Process for Alkylating Adenine with .alpha.,2-Dichloro-6-fluorotoluene in 
the Presence of Aliquat 336 in Acetone (Solid-liquid Reaction Mixture) 
Step A--Alkylation 
A 250ml. round bottom flask was sequentially charged with 100 ml. of 
acetone, 6.95 g. of adenine (97% pure, 50 mmole) and 4.0 g. of sodium 
hydroxide solution (assay 50%, 50 mmole) and the suspension refluxed for 
one and one-half hours. The suspension was charged with a solution of 9.8 
g. of .alpha.,2-dichloro-6-fluorotoluene (91.4% pure, 50 mmole) and 1.25 
g. of Aliquat 336 (Aliquat 336 is a mixture of tetraalkylammonium salts in 
which the alkyl groups are primarily caprylyl (C.sub.8), manufactured by 
General Mills, Inc., Chemical Division, 4320 West 77th Street, 
Minneapolis, Minn.) (2.5 mmole, 5 mole %) in 10 ml. of acetone and 
refluxed, with rapid stirring, for six hours. (In the absence of the 
"phase transfer catalyst", Aliquat 336, the reaction is about 5.times. 
slower). The reaction mixture was cooled to room temperature and the 
solids collected by filtration. The solids were washed twice with 15 ml. 
of acetone and then swished with 50 ml. of 0.1N sodium hydroxide solution 
for 15 minutes (this removes any unreacted adenine and the NaCl formed 
during the alkylation). The solids were collected by filtration, washed 
twice with 20 ml. of water and dried in vacuo (100.degree. C. for 4.5 
hours) to give 13.12 g. of 2-chloro-6-fluorobenzylated adenines (94.4%). 
U.V. (N/10 HCl).lambda.max=262, E%=534; L.C. wt. % 9-isomer=77.4; U.V. wt. 
% 3-isomer=20.4. 
STEP B--Purification 
Ten g. of the above material was added to 18 ml. of hot glacial acetic acid 
(.about.60.degree. C.). The mixture was heated to 110.degree. C. (solution 
occurred between 60.degree. and 90.degree. C.), filtered and the filtrate 
added to 80 ml. of hot water (95.degree. C.) over a five minute period 
with rapid stirring (2 more ml. of acetic acid were used for rinses). When 
the temperature dropped to 37.degree. C. the suspended solids were 
collected by filtration, washed once with 10 ml. of 4:1 H.sub.2 O:HOAc and 
twice with 15 ml. of water. Vacuum oven drying (100.degree. C. for 6 
hours) gave 7.64 g. of 9-(2-chloro-6-fluorobenzyl)adenine, 76.4%. U.V. 
(N/10 HCl).about.max=260, E% 570; DSC=0.5 mole % impurity, m.p. 
(uncor)=244.5.degree.-246.degree. C.; tlc on silic gel in CHCl.sub.3 : 
MeOH (10:1) showed one minor impurity at R.sub.f =0.57 and a main spot at 
R.sub.f =0.86. 
EXAMPLE 2 
One Step Purification of Crude 9-(2-Chloro-6-fluorobenzyl)adenine by 
Treatment with Sulfuric Acid 
To a stirred suspension of crude 2-chloro-6-fluorobenzylated adenines (2.0 
g., L.C. assay wt.% of 9-/3-/7-isomer=79.7/17.8/1.2) in xylene (4 ml.) was 
added dropwise concentrated sulfuric acid (96%, 4 ml.) at room 
temperature. The mixture was stirred for 12 hours at room temperature and 
then for an additional 10 minutes at 80.degree. C. After the reaction 
mixture was cooled to room temperature, the mixture was poured into ice 
water (25 ml.) containing xylene (10 ml.). The resulting mixture was 
transferred to a steam-jacketed separatory funnel and heated to 85.degree. 
C. in order to redissolve the pecipitate. The aqueous layer was separated 
and made basic by addition of concentrated ammonium hydroxide. The 
precipitated solids were filtered and washed with hot water (2.times.10 
ml.). Yield, 1.53 g. (95.6% based on available 9-isomer). L.C. assay 
9-isomer 100.07%; 3-isomer, none detectable (&lt;100 ppm); 7-isomer 
.about.0.8%. 
EXAMPLE 3 
Two Step Purification of Crude 9-(2-Chloro-6-fluorobenzyl)adenine by 
Extraction with Dilute Nitric Acid and Treatment with Sulfuric Acid 
Step A.--Extraction with Dilute Nitric Acid 
A suspension of 40.0 g. (0.144 mole) crude 2-chloro-6-fluorobenzylated 
adenines (wt. % of 9-3-isomers=79.1/19.3 determined by L.C. assay) 
representing 31.64 g. of 9-isomer and 7.72 g. of 3-isomer in 440 ml. water 
containing 19.0 ml. (0.0285 mole) of 1.5N nitric acid was heated under 
reflux for 2 hours with vigorous stirring. The mixture was filtered while 
hot onto a pre-heated funnel, washed with hot water (3.times.50 ml. 
slurries), conc. NH.sub.4 OH (2.times.25 ml.) and hot water (3.times.50 
ml.). The product was sucked damp-dry and afinally dried in vacuo at 
65.degree.-70.degree. C. overnight to yield 31.67 g. (97.1% yield) of 
9-isomer enriched 2-chloro-6-fluorobenzylated adenines. This yield is 
based on available 9-isomer and is corrected for purity. L.C. assay 
9-isomer 97.2% and 3-isomer 3.0%. 
Step B.--Dealkylation of 3-(2-Chloro-6-fluorobenzyl)adenine with Sulfuric 
Acid 
To a vigorously stirred suspension of 50.0 g. (0.18 mole) of 9-isomer 
enriched 2-chloro-6-fluorobenzylated adenines (wt. % of 
9-/3-isomers=96.8/3.2 by L.C. assay) representing 48.4 g. of 9-isomer and 
1.6 g. of 3-isomer in 100 ml. of toluene (reagent grade) was added 
dropwise 100 ml. of concentrated sulfuric acid (assay=96.02%) with 
ice/water cooling as required to maintain a temperature of 
50.degree.-60.degree. C. The mixture was heated with stirring at 
50.degree.-60.degree. C. for 18 hours (all the solid dissolved in the acid 
to give a two-phase system). The batch was cooled to room temperature and 
poured into ice/water (300 ml.) whereupon the product precipitated as the 
sulfate salt. The mixture was transferred to a steam-jacketed separatory 
funnel using hot water rinses and heated to 80.degree.-85.degree. C. in 
order to redissolve the precipitate and effect separation of the aqueous 
layer from the toluene layer. The aqueous layer (650 ml.) was separated 
and washed with 50 ml. of hot toluene using the same apparatus. The 
aqueous layer (while still warm) was made basic (pH 10) by the cautious 
addition of concentrated ammonium hydroxide solution. The precipitated 
white solid was aged with stirring for 1 hour and collected while the 
batch was still warm. The product was washed with hot water (3.times.100 
ml.) and 50% aqueous methanol (2.times.100 ml.). The batch was sucked 
damp-dry and finally dried in vacuo at 70.degree. C. overnight to give 
pure 9-(2-chloro-6-fluorobenzyl)adenine. The yield was 47.8 g. (98.8% 
based on available 9-isomer), m.p. 247.degree.-248.degree. C.; tlc on 
silica gel in CHCl.sub.3 :MeOH (9:1 ) showed essentially a single spot, 
R.sub.f =0.48. No polymer or other impurity was detected. L.C. assay 
showed 9-isomer=100.68%; 3-isomer, none detected. Overall yield=95.9%.