IL40324A - 3-tert.butyl-6-methyl-5-nitrosalicyl-anilides methods of preparation and anthelmintic compositions thereof - Google Patents

Description

-Taj .Btttyl~6-Methyl- -Hitroaalicylanllide3t Methods of Preparation and An helmintic Compositions thereof οηικ m>»aan o*y¾nn ni3 manvm- The present Invention pertains to novel specifically substituted sal 1cylan1l Ides, methods of their preparation and anthel mintic compositions for their use. More specifically 1t pertains to 3-tert. butyl -6-methy1-5-n1trosal 1cy1an1l 1des and sub classes of such compounds having specific 2* .4' -substitution.

A large number of substituted salicylic adds and their derivatives are known and have been described in the literature. Prior known acids of related structure to the add of this Invention are: 3-tert . utyl -6-methy 1 sal 1 cyl 1 c acid [see A. Lespagnol et al . , Chemical Abstracts, 33; 1295 (1939)] and 3-tert . but l -5 -chl oro-6-methyl sal 1 cyl 1 c add (see J.D. Early et. al . , U. S. Patent Nos. 3,325,353; 3,375,756; 3,388,163) . 3-tert . Butyl -5-n1 tro-2 ' ,5 ' -d1 hal o anH ldes appear to be described 1n the l iterature as larvaddes for chewing Insects such as Lepldoptera, British Patent No. 1 ,252,087. But this patent does not describe the compounds of this Invention, their novel anthelmintic compositions or the specific advantages of the 3-tert. butyl -6-methyl-S-nltro^ ' ^'-dlsubstltuted-sal lcylanlHdes which are the preferred compounds of this Invention.

Patent 25918 describes sal lcyl anil Ides having a necessary nltro or cyano group at the 4' -position and a nonspecific arrangement of subsfrltuents in the salicylate portion of the structure. These compounds are not as active as those claimed herein.

The present Invention derives much of Its novelty from the discovery that the subst1tu jents on both the salicylic and an1l 1de moieties of the compounds , while Indi vidually known in the prior art, have unusually and unexpected useful properties when simultaneously present In a single chemical structure. The properties are used as active Ingredients 1n veterinary preparations for anthelmintic activity especial ly against Intestinal worms and fluxes in ruminant animals.

- The new compounds of this nvention are represented by the following formula is hydrogen, halogen, trl f 1 uoromethy 1 or methyl . 1s trlfl uoromethy 1 or halogen.

Compounds of the Invention that exhibit particularly powerful and broad spectrum of anthelmintic properties are those of formula I 1n which R Is 2 , -chloro, methyl or trlfluoromethyl and R-j 1s 4, -chloro.

The objects are further accompl ished by the preparation of anthelmintic compositions 1n the form of tablets, l iquid suspension, powders, or solutions suitable to the prevailing mode of commercial util ization.

The objects are also accomplished, 1n part, by nitration of 3-tejrt. butyl -6-methyl sal icyl ic add, 1n an aromatic solvent using aqueous nitric add at temperatures below 80°C. to produce 3-tert. butyl -6-methyl -5-n1trosal1 cyl 1c add. The final step of the reaction of this invention is the condensation of the salicylic acid with the critically substituted aniline in a suitable solvent such as benzene, chlorobenzene, toluene in the presence of a halogenating condensation agent such as phosphorus trichloride, pentachloride, oxytrichloride or thionylchloride. Other equivalent condensation reactions will be apparent to those skilled in the art.

It has been found particularly advantageous first to produce 3-tert»butyl-6-methyl-5rnitrosalicylic acid. This compound is then condensed with an appropriately substituted aniline to give the desired salicylanilide. Other alternate synthetic sequences will also be apparent to organic chemists skilled in the art of synthesis,, However the preparation of the compounds of this invention as described herein provides the greatest flexibility in subsequent syntheses, and generally provides the best overall yield and most economical approach to the desired products.

Preparation of 3-tert.butyl-6-methyl-5- nitrosalicylic acid.

In a glass reaction vessel equipped with internal stirrer, 104 parts of 3-tert.butyl-6-methylsalicylic acid were mixed with 520 parts of toluene at 60°C„ Next, 108 parts of aqueous nitric acid (35% HNO3) were added dropwise over the course of one hour with constant stirring. With the temperature held by external heating at 65-70°C. and stirring continued, the reaction was continued for another hour. The mixture was then cooled to 0-10°C. and the precipitated product was separated from supernatant liquor by filtration. After the product was washed with heptane, a yield of 68.1 parts of 3-tert.butyl-6-methyl-5-nitrosalicylic acid was obtained (n p. 205-6°C., By analgous procedures, using appropriately substituted an111 the following compounds of the Invention were prepared: 3-tert. but l -4' -bromo-6-methyl - 5-n1 trosal 1 cyl an111 de m.p. 133-7°C. 3-tert. butyl -4*· -chloro-6 methyl - 5-nTtros al i cyl an111 de m.p. 150-152°C. 3-tert. butyl -6-methyl -5-ni tro- 4" -tr fluoromethyl sail cyl an1l1de m.p. 175-176°C. 3-tert. butyl -4" -1odo-6-methyl-5- nltrosallcylanillde m.p. 143-145°C. 3-tert. butyl -4" -chloro-2»,6- d1 methyl -5-n1trosal1cylan1l1de m.p. 167-168°C. 3-tert. utyl -2! ' ,4» -d1 chl oro-6- methyl -5-ni trosal 1 cyl anilide m.p. 150-152°C. 3-tert. butyl -4' -fluro-6-methyl- 5n1 trosal cyl anil 1 de m.p. 205-206°C. 3-tert. butyl -4* -chloro-6 -meth l - 5-n1tro-2' -trlfluoromethyl- sal1cylan1l1de m.p. 152.5-153.5°C. 40324/2 3-¾oyt»bu yl 6 methyl 5-n1 trosal 1oy1 ani l 1 do m.p. 1GG-1 G0PC. 3 tort. butyl 6 methyl 6 nitro-2 ' -tnl f 1 uoromothy sal i oylani 11 do m.p. 205-9 °C. 3-tert . butyl -41 -bromo-6-meth 1 -5-nl trosal 1 cyl anil 1 de m.p. 133-7°C. 3-tert. butyl - 1 -chl oro-6-methy 1 -5-nl trosal 1 cyl an111 de m.p. 150-152°C. 3-tert. butyl -6-methyl -5-n1tro-4 '-trifl uoromethyl sal 1 cyl an111 de m.p. 175-176°C. 3-tert. butyl - · -1 odo-6-methyl -5-n1 trosal 1 cyl an111 de m.p. 143-145°C. 3-tert. butyl^'-chloro^' ,6-d1 methyl -5-n1 trosal 1 cyl an 111 de m.p. 167-168°C. 3-tert. butyl -21 ,4 ' -d chl oro-6-methyl -5 -ni trosal 1 cyl an 11 de m.p. 150-152°C. 3-tert. butyl -41 -f 1 uoro-6-methyl -5-nl trosal 1 cyl an 111 de m.p. 205-206eC. 3-tert. butyl -4 * -chloro-6-methyl -5-n1tro-21 -tr1 f 1 uoromethyl -sal lcylanlUde m.p. 152.5-153.5°C. 4o324/2 Anilides can be similarly prepared from; 4-fluoroaniline 4-bromo=»2°chloroaniline -jiic hylmilll-iu The preparation of 3°tert ebutyl°6°methyl° 5~nitro° salicylic acid requires controlled conditions of nitration if a satisfactory yield is to be obtained.

Temperatures between 40 and 90°C. and particularly 50=80°C. should be usede If the temperature is too lour, the reaction proceeds too slowly; if it is too high the yield is reduced. Concentration of HNO^ and its ratio to the acid reactant are also significant.

Aqueous nitric acid containin 20-50% H O^ and mol ratios of about 0.9 to 1.2 for H 03: acid reactant have been found most effective. Since the reaction is exothermic, the nitric acid is added at a rate consistent with the heat dissipation" capabilities of the equipment, to a mixture of the salicylic acid reactant and an inert organic liquid, preferably a liqui having a boiling point above about 100°Co, e.g., toluene, xylene, decane, chlorobenzene, and the like. It is not necessary that all or even that most of the solid reactant di solve in the reaction medium. Reaction times of about 30-150 minutes and nitric acid addition times of about 10-100 minute are generally advantageous.

Recovery of the 3-tert.butyl-6-methyl-5-nitro-salicylic acid may be effected by filtration or contrifugatio or other means of solid-liquid separation. The recovered aci may be used directly as a chemical intermediate or may be further purified before such use.

Formula in which X is OH or a reactive ester derivative thereof, and R-| and are as des cri bed .

The anilides of 3-tert.butyl-5-nitro-6-methyl-salicylic acid of Formula I are preferably formed by the chemical reaction of this invention, namely by reaction of th parent acid with an amine, advantageously in the presence of catalyst, dehydration agent or the like or by reaction of a reactive ester of the parent acid with an amine. Exemplary of the active ester are the esters v?ith inorganic acids, such as the acid halides or mixed esters with organic acids, such or acid. Anilides of particular importance are those repre- ¾ sented by Formula I wherein ¾_is 2' -methyl, chloro or tri-fluoro; Rj "s ^'-chloro and R2 is hydrogen. Another preferred class of the anilides are the 41 -nitro-substituted anilides.

The new method of controlling helminths or parasitic worms by this invention comprises administration to a host animal such as a ruminant animal in need of such treatment orally, combined with a pharmaceutical or a feed carrier in the form of a veterinary composition, an effective anthelmintic but nontoxic quantity of a compound of Formula I. In certain cases such as extragastrointestinal infestation of flukes as in sheep, the compounds may be administered parenterally, i.e., in a sterile micronized suspension or solution.

The administration is in quantities nontoxic but effective either for curative or prophylactic purposes and has broad range of activity on gastrointestinal parasites of warm blooded animals especially sheep and cattle. The helminths most effectively treated with the new compounds are' the Trematodes, Cestodes or, and especially, Nematodes. Activity against flukes such as Fasciola gigantica or Fasciola hepatica is also particularly pronounced. More specific parasitic infestations in which this invention may be applied are found in the Merck Veterinary Manual, Third Edition, pages 699-806, as are general methods of control of internal parasites; see also Great Britain Patent No. Ijl83,641. Generally effective doses range from about 1 up to about 50 mg./kg. of body weight, preferably about 2 to 25 mg./kg. Effective doses in sheep with.-out significant side effects have been found to be about -1 to 50 mg./kg. Most usually the dosage unit compositions are administered in veterinary medicine from 1 to 5 times Veterinary compositions containing sufficient quantities of the compounds of Formula I to reach the dose levels mentioned above are prepared as known to the art by preparing tablets, capsules, boluses, liquid suspensions, powders, drenches or solutions for injection in packaged form. Alternatively, especially for prophylaxis, premix or feed compositions containing effective but nontoxic quantities of the active salicylanilide are used. For these purposes particulate carriers, inert powders or, especially, feed carriers such as soybean meal, corn oil, vermiculite, diatomaceous earth, barley or wheat are used. In dosage unit or premix fe@d compositions the compound can comprise from about 5 to 75% of the final composition as is convenient for the farmer or veterinarian. As an example, a 5% salicylanilide-vermiculite or soybean meal premix can be used which will be uniformly mixed with the animal feedstuff. Alternatively, a lick or pasture block can be used for field animals.

Specific examples of veterinary compositions of this invention are as follows: Sheep Drench Parts by Weight 3-tert.Butyl-2' ,4' -dichloro-6- methyl-5-nitrosalicylanilide 20 Terra alba 75.5 Tragacanth 3.0 Sodium lauryl sulfate 1.5 Water The above solid components are mixed to give a water-dispersible powder to be used on concentrations of 5 g. of powder to 5 ml. of water. The drench is used orally as necessary and practical to control gastrointestinal infections.

Ruminant Bolus Grams 3-tert.Butyl-4'-chloro-2l ,6- dimethyl-5-nitrosalicylanilide 0,5 Calcium phosphate ' 4,0 Maize starch 0.54 Talcum 0.14 Gum arable 0.15 Magnesium stearate 0.05 The phosphate and salicylanilide are mixed and screened, then granulated using one-half the starch. The screened and dried granules are mixed with the remaining ingredients, blended thoroughly and compressed on a bolus press.

Similarly, tablets can be prepared with reduced fillers.

Anthelmintic activity of compounds of this invention was measured in several ways. While variations in chemical procedure occur from laboratory to laboratory, the following method is typical of those used to obtain the data on cestocidal (an itapeworm) activity in mice infected with Hymenolepis nana, For these tests, mice were infected by stomach gavage with 200 Hymenolepis nana ova per mouse. Twelve days later, a single dose of 50 mg. /kg. body weight of the test compound was orally administered to each of five mice. Four days after administration of the test compound, the mice were sacrificed and the number of scolices in the lower half of the small intestine of each mouse were counted. The counts were compared with those of control groups which had not been treated with the test compounds. The results were recorded (average for 5 mice) as % efficiency of tapeworm removal, i.e., 100 (Sc-St)/Sc where Sc is the number of scolices for the control St is the number of scolices for the test compound. The data are reported as PETR (% efficiency of tapeworm 40324/2 removal) and where more than one set of tests 1s performed under comparable conditions, the average of the PETR of the sets may be reported.

Using the test procedure against Hymenolepis nana described above, 3-tert. butyl -2 ' ,4 ' -di chloro-5-ni tro-6-methyl - salicylanilide (A), a compound of this invention was evaluated. To compare 1t with related compounds outside the invention, similar tests were run using 3-tert .butyl -5-chloro-6-methyl -4 ' -ni trosal icyl anilide (B), 3,6-dimethyl-4' ,5-d1nitrosal1cylan1lide (C), 3-tert. • butyl -4', 5-d1ni trosal icyl anlUde (D), 2' ,5-dichloro-4'-nitro- sal1cylan1lide (E), and 2'-chloro-4' ,5-din1trosal1cylan1lide (F).

The following results were obtained: PETR A 99 (three sets of tests) B 24 C 20 D 38 E 69 F 0 Compound A is thus unexpectedly efficacious. The unexpectedness of these results 1s emphasized by the fact that the replacemnt of 5-Cl by B- Og, in proceeding from E to F, completely destroys utility, while the corresponding replacement in going from B to A enormously enhances activity.

It was discovered that the addition of methyl or chloro groups of the 2 '-position of A causes a notable increase in toxicity to the host animals, so that the dosage must be reduced below 50 mg./kg. to insure the safety of the host animal. Thus the specificity of the salicylic acid portion of the compounds of Formula I is noticeable.

To show the relative effect of compounds of the present invention d-jffeif ng in the R and oubotitucnto of Formula I when is hydrogen, as well as to show the relative effect of those compounds with respect to still other related present compounds which are not of the preferred aspect of the present, invention, that is, in which R and R-^ are monovalent substit-uents other than those preferred., a series of tests were carried out on living animals as follows.

Two groups of sheep were used in the tests. One group was used as a nonmedicated control, while the other group was treated with the compound under investigation. Helminth-free young lambs were infected with about 10,000 filariform larvae of Haemonchus contortus. In three weeks when the in-fection became apparent, egg counts were made to determine the density of the worm burden.

Tests for activity against live flukes were conducted as follows.

Eggs of Fasciola hepatica were collected from the bile of donor sheep„ The eggs were embryonated and snails were infected (the genus Lymnaea serves as intermediate host) to produce Metacarcariae which are the infective forms for sheep.

Each sheep was infected with 250 metacarcariae intrarurainally.

When the infection became patent in about 80 days, egg counts were carried out to determine the degree of worm burden. The When administered an oral dosage of 5 milligrams p ia^ kilogram of body weight of sheep infected with liver flukes of the genus Fasciola. 3-tert.butyl-2' ,4' -dichloro-6-methyl-5-nitrosalicylanilide completely destroyed all flukes within three days. Against immature fluke infestations in sheep similar activity was found at 15 mg./kg. The same effects against mature flukes were observed when 3-tert.butyl-4f -chloro-2f ,6-dimethyl-5-nitrosalicylanilide was administered at 5 milligrams per kilogram of body weight. The 4'-cyano congener was active against mature flukes at 2.5 and 5 mg./kg.; the 4'-bromo was active at 15 mg./kg. By contrast the isomeric compound, 3-tert.butyl-3 ' -chloro-2 ' ,6-dimethyl-5-nitrosalicyl-anilide is ineffective against liver flukes even when the sheep receive 15 milligrams per kilogram of body weight.

Other compounds of this invention which were tested against flukes and found efficacious are 3-tert.butyl-6-methyl-5-nitro-4'-trifluoromethylsalicylanilide (15 mg./kg.); 3-tert.butyl-4' -chloro-6-methyl-5-nitrosalicylanilide (15 mg./ kg.); 3" ort*bu yl - ' j5-dinitro-6-mothyloalioylonilidg (1 ma./kfii)l 3-tort.butyl-2 ' ,5' -dimethyl 4' ^5 dinitgo 6 methyl salicvlanilida (10 mg./kg.) ; and 3 tort» utyl 2 ' miti oAy |5-dinitro«6«mathy1igfllir'yl ληί 1 ίήρι ( , 1 S mg. /kg.) .

In this respect, compounds of this invention differ from compounds currently in commercial use which are not effective against both gastrointestinal worms and liver flukes. Since the simultaneous occurrences of both forms of infections is common in commercial ruminant husbandry, the value of a single form of medication to cure both types of infection is self-evident.

The results of Haemonchus tests on sheep in a manner "Haemonchus (15 mg. /kg.)" refer to the percent of parasites destroyed or expelled when the indicated compound was introduced into the rumen of sheep at a dosage of 15 milligrams per kilogram of body weight. The values under "Tolerance (mg./kg.)" refer to the highest test dosage in the milligrams of anthelmintic per kilogram of body weight at which the sheep developed no toxic symptoms.

Table 1 Substituents Haemonchus Haemonchus Haemonchus Tolerance -21 4' 2 mfi./kfi. 5 mfi./kfi. 15 mg./kg. mg./kg.

In contrast to the good activity of the 2 ' ,4 ' -dichloro congener, 100% at 5 mg./kg. and tolerance at 50 and 100 mg./kg., an isomeric compound of the prior art,, namely the 2' ,5' -dichloro See Monsanto British Patent No. 1,252,087 published November 3, 1971, first compound on page 5) has only 61% activity at 5 mg./kg. and shows toxic effects at 50 mg./kg. It will be appreciated that only a 60% reduction in worm burden is not a practical or commercial objective for a new anthelmintic. The Monsanto patent also discloses the V ,51 -dihalosalicylanilides only as larvicidal compounds against Lepidoptera or chewing insects. It discloses no activity against internal parasites such as pin worms, round worms, flukes, etc., where a principal use of this invention lies.

The 2'-mothoKy-4'-nitrooalioylanilido exemplifies - - Preparation of 8 3-Cer o butyl-41 -bromo-6-methyl-5-nitro-2 ' =trifluororaethylsalicylenili and its biological activity,, 50o6 go ('Jo 2 nio) of 3-t „ -Butyl-5°nitro=6=iaethyl salicylic acid, 48 0 (0o2 m0) of 2-amino-5=bromobenzotri= fluoride and 300 ml0 of chlorobenzene were added to a 110. flask and HCl gas bubbled into the reaction mixture for about 10 raimutes0 was :., The reaction mixtureAthen heated to reflux and 2 03 g0 (0o205 m0) of thionyl chloride was added dropwise over a period of 1»5 ■ was hourso The resulting reaction mixture Arefluxed for h^ee . hours and allowed to cool overnighto The resulting solid was collected and the filtrate evaporated to give additiox¾al product0 he solids were combined, dissolved in ethyl ether and the ether solution washed with 6N hydrochloric acid solution^ saturated sodium bicarbonate solution, then water, and driedo Evaporation of the ether gave a yellow solid which was triturated w t tot heptane0 After cooling 6505 go (69%) of solid product, m„p. 161°163°Co was obtained,, Anal0. calc0 for C^H^gB F^NgO^ C,48o02; H, 3o82; , 5.89; Found C, 48o10; H, 3o99| N, 5077o Activity against F. Eepatica (flukes) - (97% at Q05 mgo/kg0)s Haemonchus - (99,8% at 2 mgo/kg0); nontoxic at 20 mgo/kg0

Claims (6)

1. 40324/2 WHAT IS CLAIMED IS; Ί. A 3-tert. butyl -6-methyl -5-nltrosal 1 cyl an 11 Ide of the general formula wherein: R-j 1s hydrogen, halogen, tr1 f 1 uoromethyl , or methyl j 2 Is halogen or tr1 f 1 oromethyl .

2. A sail cyl an1l1de of Claim 1 1n which R-j Is 2'-methyl, chloro or tr1 fl uoromethyl and R21s 4'-chloro.

3. A sallcylanlllde of Claim 1 In which R^ 1s 2 ' -tr1 fl uoromethyl and ¾ 1s 4'-bromo.

4. The sallcylanll de of Claim 1 1n which R1 1s 2'-chloro and R-j 1s 4' -chloro.

5. The sallcylanlllde of Claim 1 In which R1 is 2'-tr1fluoro- methyl and R2 1s 4* -chloro.

6. The sallcylanlllde of Claim 1 1n which R-j 1s 2*-methyl and ¾ 1s 4'-chloro.

7. The sallcylanlllde of Claim 1 1n which R1 1s -CH3 and R2 1s -CF3.

8. An anthelmintic composition comprising a salicylanilide of any of Claims 1-7 in admixture with a suitable extending agent.

9. An anthelmintic composition comprising a salicylanilide of Claim 2 in admixture with a suitable < extending agent.

10. An anthelmintic composition comprising a salicylanilide of Claim 3 in admixture with a suitable extending agen o

11. An anthelmintic composition comprising a salicylanilide of Claim 4 in admixture with a suitable ex-tending agent.

12. An anthelmintic composition comprising a salicylanilide of Claim 5 in admixture with a suitable ex-tending agent.

13. An anthelmintic composition comprising a salicylanilide of Claim 6 in admixture with a suitable extending agent.

14. An anthelmintic composition comprising a salicylanilide of Claim 7 in admixture with a suitable extending agent.

15. An anthelmintic composition having activity against Haemonchus and flukes comprising a salicylanilide of Claims 2, 4, 5, or 6 in admixture with a solid veterinary extender.

16. A ρ¾¾1^!Ί¾br treating or preventing infestations of helminths comprising adminiateiping to an animal 1 subjects- an effective but nontoxic quantity of a salicylanilide of any of Claims 1*7. composition 17 o A pireeess for treating or preventing infesta∞ 40324/2 tlons of helminths comprising an effective but nontoxic quantity of a sa11 cy1an111de of Claim 2.

18. A composition for treating or preventing Infestations of helminths comprising an effective but nontoxi c quantity of a saH cylanlU de of Claim 4.

19. A composition ifor treating or preventing Infestations of helminths comprising an effective but nontoxic quantity of a sal1 cy1an1 l1de of Claim 5.

20. A composi tion for treating or preventing Infestations of helminths comprising an effective but nontoxic quantity of a sa11cy1an1 l1de of Claim 6. Attorneys or pp cant