AU544990B2 - Antiprotozal composition containing amino compounds - Google Patents

Description

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.D -lH1654/81 !B~7654&tH 2 N-CHI--(Cl 2 2 and H2N-.CH 2 CH'~cf; with the proviso

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AUSRALIA

Patents .4ct VIN~ST11umT ()Insert "ConyA (bi) Delete one APPLICAfON FOR A STANDARD/WTViY (b) -it applicable b) 7naert FULL of apsuIntt(

I/We (c) 7 16 54/81 Insert FULL MSERRELL DOW PHIARM4ACEUTICALS INC--- namne(s) of (c)Insert "of ad .applicant(s) ()it applcable (8srt IIITU12

I invention

Insert FULL Of (d) 2110 E. Galbraith Road address(es) of appiicant(s) Cincinnati, Ohio 45215 inlert PL'LL United States of America AND addressi dedarant(s) (See hesdnoti

Me Delete one hereby apply "or the gran~t of a Standard/lkaMPatent for an invention entitled Mf Insert TITLL (0 METHOD OF INH-IBITING THE GROWTH OF PROTOZOA uw invention

1 2(glfInsert "comnplete" which is described in the accompanying (g) complete specification. 4 OR "provisional" QR "petty patent" M ess( ~t~1invento (Note. The foic, .'hzg applies Only to Convention applications) Details of basic application(s) Recite low a] cant s) dezive( title fomactl Insert number, inventr) cot'ntry and Application No. Country I Filing Date (Se Ijeadootel filing date for tle/r EACH 4,4, ~bscapplication 44q' 159,973 United States of June 16, 1980 4,

America

PT ~i 1.~ -basic -appilcati

4, 4' '.4,4,4*4 4,44,4,4". 4 C 5 4' 4, 4 Address for Serv ce: PHILLIPS ORMONDE AND FITZPATRIC 4' 4,a~ 94' *4~a a a 4' 4' a C Patent and Trade Mark Attorneys 367 Collins Street Melbourne. Australia 3000 Isert DATE of signing. O~inqert 'PLACE Dated A 1.21 1/f P/ Insert 1ATLE

(in) Signature(s) of declarssnts)

Note-, No legalizati other witness regul

PI17/78

PHILLIPS ORMONLX~ND FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne,. Australia STUART TAYLOR

AUSTRALIA

Sproviso Patent Act

N. K'

AUSIRALIA Parents Act DECLARATION FOR A PATENT APPLICATION

4 Insert "Convention In support of the Convention application made by .if applicable (b) Jntert ULL uatn C(s). of applicant(s) 14ERRELL DOW PHARMACEUTICALS INC. 71654/81 T(c) Inscrt "of addition (hereinafter called "applicant(s) for a patent (c) It 2Pplcable for an Insert 'ITI! of invention entitled invention

METHOD OF INHIBITING THE GROWTH OF PROTOZOA insert PL nes ANDP address(es) of I/We (el Joseph G. Temple, Jr. hdnte*) (Set of MERRELL DOW PHARMACEUTICALS INC. Q, 2110 E. Galbraith Road Cincinnati, Ohio 45215, United States of Amrica do solemnly and sincerely declare as follows: Axiwr mxtapca~) (or,_in the case of an application by a body corporate) 1. 1 am/Win;*e authorized to n.alce this declaration on behalf of the applicant(s). 2 (or,_where the applicant(s) !s/are not the actual inventor(s)) lnset4LL(es) 2. Albert Sjoerdsma Peter P. McCann ;tt 01 inventor(s) 5475 Waring Drive 4405 Williamsburg Road, N.W. Cincinnati, Ohio 45215 Cincinnati, Ohio 45215 United States of America United States of Ainerics' (W)Recite 'how aspa, cbnt(s)derive(s) 4&/are the actual inventor(s) of the invention and the facts upon which the applicant(s) Inventr) 5 ::j$are entitled to make the application are as follows: =St beante Applicant .s the assignee of the invention from the actual inventors by way of an Assignmlent dated May 7, 1981.

(Note: Paragraphs 3 and 4 apply only to Convention applications) (h)4et count,. 3. The bas-c applicationW) for patent or similar protection on which the application is based basic aplan) is/tm& identified by country, filing date, and basic applicant(s) as follows: or the/o fbasic EC a plcatio United States of America June 16, 1980 By; Albert Sjoerdsma and Peter P. McCann

S C 4. The basic apphicationN referred to in paragraph 3 hereof was/woe the first application( made in a Convention country in respect of( the invention the subject of flie application.

I ietPLACE of Declared at Cincinnati, Ohio Thsert DATE of

declarairtts)

Noe~Io legalization or other witness required To: The Commissioner of Patents

P19/7/78 PHILLIPS ORMONDE ZPAT1R1CK V Patent and Trade Mark'Attorneys 367 Collins Street

K r% 7 Melbourne. Australia J j a ii

(12) AUSTRLAN PATENT ABRIDGMENT (19) AU (11) AUB-71654/81

(54) ANTI PROTOZAL COMPOSITION CONTAINING AMINO COMPOUNDS (71) MERRELL DOW PHARMACEUTICALS INC 71654/81 544990 (22) 11.6 .81 (24) 16.6.80 (31) 159973 (32) 16.6.80 (33) US (43) 24.12.81 (44) 27.6.85 (51)3 A61K 31/13 A61K 31/195 A61K 31/505 A61K 31/155 A61K 31/55 A61K 31/71 A61K 37/02

(72) ALBERT SJOERDSMA AND PETER P. MCCANN (74) PO (56) 87245/82 C07C 87/26, A61K 31/13 87244/82 C07C 87/26, 101/28, A61K 231/13 87243/82 C07C 87/822, A61K 31/195

(57) This invention re'-ates to certain a-substituted amines and a-substitutedi-amino acids which are useful in inhibiting the growth of protozoa in animals and particularly in poultry.

CLAIM 1. A method of inhibiting the growth of protozoa in animals which comprises administering a protozoal inhibiting amount of an a-substituted amino acid or an a-substituted amine having the formula

Y Z-C-Ri I

NH 2 wherein R1 is hydrogen or carboxy; Y is selected from the group consisting of CH2F, CHF 2 CF 3 and C=CH; Z is selected from the group consisting of H 2 N-(CH 2 3

120, 371, 369 22 7

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butvl ketonp- anA i^i ka IR

.2N-CI-(CH2 2 2 CH=CH; with the proviso and H2N-CHf

that when'R 1 is hydrogen, Y cannot be CF 3 and Z must be H 2 N--C:C-(CH 2 2 and CH 3 the salts and individual optical isomers thereof.

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When Ri and RA toqether form an alkvlene arntin in

STUART TAYLOR

4

AUSTRALIA

Patents Act3 44a COMPLETE SPECIFICATION (ORIGINAL)

Class Int. Class Application Number: Lodged:

Complete Specification Lodged.:71 Accepted: Published: lThis docutment cOntainls arasndments rn~de uiud-r Section 49. the

Priority:

Related Art: rnl aad

APPLICANT'S REF.: M-107U6' AU Name(s) of Applicant(s): MtERPRELL DOW PHARMACEUTICALS INC.

Address~es) or Applicant(s): 2 110 E. Gaibraith Road Cincinnati, Ohio 45215 United States of America

Actual Inventor(s): Albert Sjoerdsma and Peter P. McCann

Address for Service is: PHILLIPS, ()RMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne, Australia, 3000 Complete Specification for the invention entitled:

"METOD OF INHIBITING THE GROWTH OF PROTOZOA'!

The following statement is a full description of this invention, including the best method of perforning it known to applicant(s):

FP19/11/77 ~2

367 Collins Street Melbourne Australia :t

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I STechnical Field This invention relates to certain a-substituted S: amines and a-substituted-amino acids which are useful in inhibiting the growth of protozoa in animals and

S 10 particularly in poultry.

Background Art e: Polyamines have been implicated in many aspects of cell division. Impairment of the biosynthesis of poly- amines by means of enzyme inhibitors is believed t6 cause a decrease in cell proliferation in mammals. Although the i Sphysiological role of polyamines has not been clearly delineated, there is evidence to suggest their Sinvolvement with cell division and growth, H.G. Williams- Ashman et al., The Italian J. Biochem. 25, 5-32 (1976), iM-1076-C1

-2-

A. Raina and J. Janne, Med. Biol. 53, 121-147 (1975) and D.H. Russell, Life Sciences 13, 1635-1647 (1973). Polyamines are also known to be essential growth factors for certain microorganisms, as for example E. coli, Enterobacter, Klebsiella, Staphylococcus aureus, C. cadaveris, Salmonella typhosa and Haemophilus parainfluenza. There is evidence to suggest that poly- amines are associated with both normal and neoplastic mammalian cell growth, there being an increase in both the synthesis and accumulation of polyamines following a stimulus causing cellular proliferation. It is also known that there is a correlation between polyamine formation and the activity of the decarboxylase enzymes of ornithine, S-adenosylmethionine, arginine and lysine. The term polyamine is taken to include the diamine putrescine and the polyamines spermidine and spermine. Putrescine is the decarboxylation product of ornithine, catalyzed by ornithine decarboxylase. Putrescine forma- tion may also occur by decarboxylation of arginine to 20 form agmatine, which is hydrolyzed to give putrescine and urea. Arginine is also involved in ornithine formation by action of the enzyme arginase. Activation of methio- S nine by the enzyme S-adenosylmethionine synthetase forms S-adenosylmethionine which is decarboxylated. The propylamine moiety of the activated methionine may then be transferred to putrescine to form spermidine. Alter- natively, the propylamine moiety may be transferred to spermidine to form spermine. Hence, putrescine serves as a precursor to spermidine and spermine. Additionally, 30 putrescine has been shown to have a marked regulatory effect upon the polyamine biosynthetic pathway. Also an increased synthesis of putrescine has been shown to be an early indication that a tissue will undergo renewed *growth processes Cadaverine, which is the decarboxyl- 35 ation product of lysine, has been shown to stimulate the iactivity of S-adenosylmethionine decarboxylase and is i known to be essential to the growth processes of many

S' M-1076-C1

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microorganisms, for example, H. parainfluenza. The rationale of polyamine metabolism has been suggested by Cohen, Science 205, 964 (1979). The apparent unique role of polyamine metabolism in trysano- somes and the dependence of trysanosomes upon ornithine decarboxylasc as a source of putrescine further supports our observaticas that certain specific ornithine de- carboxylase inhibitors of polyamine synthesis are highly effective in inhibiting the growth of protozoa.

SUMMARY OF THE INVENTION

'We have discovered that certain compounds that belong to a class of irreversible inhibitors of ornithine de- PI carboxylase are useful in inhibiting tie growth of protozoa. Moreover, this inhibition occurs throughout a wide spectrum of protozoa such as with members of the subpbylum Sarcomastigophora and Sporozoa. More particularly, the class of compounds hereinafter described are particularly useful in inhibiting the growth of members of the super- class of Mastigophora, specifically Trypanosoma brucei brucei and members of the class of Telosporea, specifically Eimeria tenella the organism which causes cocidiosis in poultry. The compounds useful in the practice of this invention are a-substituted amines o a-substituted-a-amino acids 25 having the 'eneral formula o

Y I Z-C-RI NH2

(I)

wherein R1 is hydrogen or carboxy; Y is selected from the group consisting of CH 2 F, CHF 2 CF 3 and C=CH; Z is

1j Jf A- t- (A

M-1076-C1

~i B B

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-4-

selected from the group consisting of H 2 N-(CH 2 )3r, H 2 N-CH-(CH 2 2 and H 2 N-CH 2 CH=CH; with the provisc that CH3 when R 1 is hydrogen, Y cannot be CF 3 and Z must be H2N-CH-(CH2) 2 and the salts and individual optical CH3 isomers thereof. When administered in vivo to animals containing active protozoal infections, the compounds of formula (I) can be utilized to treat such animals by inhibiting the further growth of the protozoal infections. Alternatively, the compounds described above can be administered prophylactically to prevent such protozoal infections from occurring.

DETAILED DESCRIPTION OF THE INVENTION

In general formula above the symbol R 1 is represented either by hydrogen or a carboxyl group. Where the symbol R 1 is hydrogen a class of a-substituted amines is delineated. Where the symbol R 1 is the carboxyl group, a class of a-subst:tuted-a-amino acids is delineated. The symbol Y represents either an acetylenic group or a fluoro-substituted methyl group. The fluoro-substi- tuted methyl groups are illustrated by the monofluoro- o c methyl, difluoromethyl or trifluoromethyl r-dicals. U4: U The symbol Z represents either the 3-aminopropyl r group, the 3-amino-3-methylpropyl group or the 3-amino-l- propylene group. The saturated groups, viz. the 3-amino- propyl group and the 3-amino-3-methylpropyl group represent the preferred side chains.

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The proviso limitation is intended to exclude certain classes of diamines from the scope of compounds encom- passing this invention. Excluded from the invention via this proviso limitation are a-substituted diamines where- in the symbol Z is the 3-aminopropyl group or the 3-amino- 1-propylene group, those compounds having the general formulae Y H 2 N-(CH2) 3 -CH-NH 2

(II)

and

Y H2N-CH 2 CH=CH-CH-NH2

(III)

wherein the symbol Y is as previously defined. Specifically excluded from the remaining a-substituted diamines is the species wherein the symbol Y represents a- the trifluoromethyl group. Thus, compound 4-methyl- l-trifluoromethyl-l,4-butanediamine, is specifically e excluded from the scope of compounds which can be usefully employed.

CF3 H2 N-CH-(CH2 2 -CH-NH 2 CH 3

(IV)

Included within the scope of compounds that can be employed are a-substituted amino acids having the formula: Y H2 N-(CH2 3 -C-COOH 4H 2

9k

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Y H2 N-CH-(CH 2 )2-C-COOH CH 3 lH2

(VI)

and Y H2N-CH 2 CH=CH-C-COOH NH2

(VII)

In formulae (VI) and (VII) above, the symbol Y is as previously defined. The a-substituted amines included within the scope 4t of compounds that can be usefully employed in the bP practice of this invention are defined by the general formula 'O

Y H 2 N-CH-(CH2) T 2 -C -NH 2 61(* CH3

(VIII) a

wherein the symbol Y represents the CH 2 F, CHF 2 and CECH groups, but in the case of the diamines exludes the CF 3 group. Illustrative examples of the salts of the compounds of this invention include non-toxic acid addition salts formed with inorganic acids, such as, hydrochloric, hydrobromic, sulfuric and phosphoric acid, and organic acids, such as, methanesulfonic, salicyclic, maleic, malonic, tartaric, citric, cyclamic and ascorbic acids.

1r-.

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-7- A preferred class of compounds of this invention are those compounds in which the symbol Y represents the difluoromethyl group. Another preferred class of com- pounds is delineated where the symbol Z represents the 3-aminopropyl moiety or the 3-amino-3-methylpropyl moiety. In addition to the salts indicated above, the term salts is taken to include those internal salts or zwitter- ions of those compounds of formula above that are amphoteric in nature. Moreover, whereas the optical configuration for the compounds described herein is not specifically designated, it is recognized that the a-carbon atom possesses an asymmetric center and that individual optical isomers of these compounds exist. Accordingly, Sboth the d- and 1-optical isomers as well as the rac;emic mixtures are contemplated as being within the scope of this invention. Lactam formation can occur where the symbol R 1 repr sents the carboxyl group and the symbol Z represents the 3-aminopropyl moiety or the 3-amino-3-methylpropyl 20 moiety as represented by the following general formula

NH 2

Y C C=O I I a tB (CH 2 3 NH

(IX)

In the above general formula the symbol Y is as originally described. Where the symbol Z represents the 3-amino- 3-methylpropyl moiety, the (CH2 3 group in formula (IX) above can be additionally substituted with a 3-methyl group.

won.

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Illustrative examples of compounds useful in accordance with the teachings of thiE invention include: 2,5-diamino-2-(fluoromethyl)pentanoic acid, amino- 2- (difluoromethyl)pentanoic acid, 2,5-diamino-'2-(trifluoromethyl)pentanoic acid, 2,5-dianino-2-(ethynyl)pentanoic acid, 2.5-diamino-2-fluoromethyl-5-methylpentanoic acid, 2,5Zdiamino-2-difluoromethyl-5-methylpentanoi( acid, 2,5-diamino-2-trifluoromethyl-5-methvlpentanoic acid, 2,5-diamino-2-ethynyl-5-methylpentanoic acid, 2,5-diamino-2-fluoromethyl-3-pentenoic acid, 2,5-diamino-2-difluoromnethyl-3-pentenoic acid, 2,5--diainino-2-trifluoromethyl-3-pentenoic acid, 2,5-diamino-2-ethynyl-3-pentenoic acid, .l-fluoromethyl-4-nethyl-1,4-butanediamine, 1-difl uoromethyl-4-methyl-1 ,4-butanediamine, and 1-ethynyl-4-methyl-1,4-butanediemine.

The compounds of general formula wherein Z is

o W4 H 2 N-(CH 2 Y is CH 2 F, CHF 2 and CF 3 3 and Rl is carboxy are prepared by treating respectively an ester derivative of ornithine, wherein the amino groups are suitably pro- tected, with a strong base to form the carbanion inter- mediate. This is reacted with a suitable halomethyl-halo alkylating reagent in an aprotic solvent, such as di- methylsulfoxide, dimethylformamide, dimethylacetamide, benzene, toluene, ethers, such as, tetrahydrofuran, diethyl ether or dioxane, and in the presence of a hexamethylphosphortriamide when Y is other than F 2 CH-, at a temperature of about -120 0 C to 1200C, preferably about 250 to 500 C, for about 1/2 hour to 48 hours followed by acid or base hydrolysis. This can be represented by the 'ollowing reaction sequence.

<AL~

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SZI-CH-COOR 2 Zl-C COOR2 N=C-R3 strong base NC-R3 COOR 2 R4 R4

compounds 1 alkylating reagent

Y Y Z 2 -C-COOR 2 dilute aqueous Zl-C-COOR 2 NH Sacid/hydrazine 82 2 N=C-R 3 R4

compounds 3 compounds 2

NN O 112 0 I (acid/base)

Y (rd u, H2N-(CH 2 3 -C-COOH NH2

(Formula V)

In the above reaction sequence Y is FCH 2 F2CH-, 4, F3 R 2 is a lower alkyl group, for example, methyl,

I~ C~ I" ethyl, isopropyl, n-propyl or n-butyl; R 3 is hydrogen, phenyl, a straight or branched alkyl group having from 1 to 8 carbon atoms, methoxy or ethoxy; R4 is phenyl or a straight or branched alkyl group of from 1 to 8 carbon atoms; or R 3 and R 4 taken together may form an alkylene group of from 5 to 7 carbon atoms, that is, -CH2-(CH 2 )m-CH2- wherein m is an integer of from 3 to Illustrative examples of straight or branched alkyl groups of from 1 to 8 carbon atoms which R 3 and R4 may

ii

M-1 (n7C-r

M-1076-C1

represent are, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, neopentyl or triethylmethyl groups. The symbol Zl is R 3 -C=N(CH 2 3 -r

O O R4 R_-CNH(CH 2 3 or R6 -OCNH(CHi 2 3 R 3 and R4 are the same and have the meanings defined above; and each of R 5 and R 6 is phenyl, benzyl or a lower alkyl group of from 1 to 4 carKon atoms that is straight or branched, for example, methyl, ethyl or isopropyl; Z 2 is H 2 N(CH 2 3

O 0 R 5 -CNH(CH 2 3 or R 6 -OCNH(CH2) 3 wherein R5 and R6 have the above defined meanings. Suitable stronq bases which may be employed in the above reaction sequence to form the carbanion intermediate are those which will abstract a proton from the carbon atom alpha to the carboxy group, such as, alkyl lithium, for example, butyl lithium or phenyl lithium, lithium di-alkylamide, for example, lithium diisopropylamide, lithium amide, tertiary potassium butylate, sodium amide, metal hydrides, for example, sodium hydride or potassium hydride, tertiary amines, such as, triefhylamine, lithium acetylide or dilithium acetylide. Lithium acetylide, dilithium acetylide, sodium hydride, and lithium diiso- propylamide are particularly preferred es. Suitable alkylating reagents whici -y be employed in the above reaction sequence are il..ustratively chloro- 25 fluoromethane, bromofluoromethane, fluoroiodomethane, chlorodifluoromethane, bromodifluorc ,thane, difluoro- iodomethane, bromotrifluoromethane, chlorctrifluoromethane, trifluoroiodomethane, bromochloromethane, dichloromethane, chlcroiodomethane, bromodichloromethane and dichloroiodo- methane. These alkylating reagents are well known to the art.

i

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Removal of the protecting groups of the amine and pyl carboxylic acid function may be achieved in one step by tyl or treatment of compound 2 with aqueous acid, for example, CH2) 3-, hydrochloric acid or toluenesulfonic acid, at a tempera- 0 ture of about 00 to 100 C for about 4 to 24 hours to he same give compounds of general Formula V. It is preferred R 5 and to remove first the protecting groups of the amine rom 1 to function(s) of compounds 2 when said functions are pro- example, tected as a Schiff's base by treating compcunds 2 'ith 6ilute aqueous acid, for example, hydrochloric acid or with hydrazine or phenylhydrazine in solvents, such as, 6 have lower alcohols, for example, methanol or ethanol, ether-s chlorinated hydrocarbons, benzene and water. Removal in the of the protecting groups of the carboxylic acid function termediate 15 and the amine groups when the amine groups are protected carbon other than as a Schiff's base is achieved by treatment lithium, of compounds 3 with concentrated aqueous acids, for thium example, hydrobrcmic.acid at a temperatura of about 00 to mide, 1000 C or in aqueous base, for example, ammonium hydroxid,. um amide, a a 20 The amine protected ester derivatives, that is, Otassiim compounds 1, wherein R 3 is other than methoxy or ethoxv, lithium as a, uasas are prepared by treating an appropriate amino acid ester lide, with a carbonyl bearing compound to form a Schiff's base diiso- in a generally known manner, specifically: when R3 is hydrogen, by treating the appropriate amino acid ester nployed a with benzaldehyde or an alkanal having from 1 to.9 chloro- as a s J carbon atoms being straight or branched, for example, 1- 4 I 4P( lane, propanal, 1-butanal, 2,2-dimethylpropan-l-al or 2,2- Luoro- diethylbutan-l-al; when P.3 is phenyl by treating )romethane, 30 the appropriate amino acid ester with benrophenone or -omethane, phenyi alkyl ketone wherein the alkyl moiety has from I .oroiodo- to 8 carbon atoms and is straight or branched, for in to example, phenyl methyl ketone, phenyl ethyl ketone, phenyl isopropyl ketone, phenyl n-butyl ketone or phenyl tert-

M-l'J76-C1 1076-Cl .1 nY I14U, al, 409 Z

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butyl ketone; and when R3 is a straight or branched alkyl group having from 1 to 8 carbon atoms, treating the appropriate amino acid ester with a phenyl U"yl ketone as described anove or with a di-alkyl ketone wherein each alkyl moiety has from 1 to 8 carbon atoms ind is straight or branched, for example, dimethyl ketone, liethyl ketone, methyl isopropyl kecone, di-n-butyl ketone or methyl tert-butyl ketone. The carbonvl bearingc compounds are known in the art cr may be prepared by procedures well known in the art. When R3 is methoxy or ethoxy in compouno an appropriate amino acid cter derivat-ve is reacted with benzoyl halide or an alkanoic acid halide wherein thte alkanoic acid has from 1 to 9 carbon atoms and may be straight or branched, such as, ac tyl chloride, propionyl chloride, butyryl chloride, tert-butyrl cnloride, 2,2- diethylbutyric acid chloride or valeryl chloride. The reaction is conducted at 00 C in an organic scivent such as ether, methylenechloride, dimethylformamide, dimethyl- acetamide or chlorobenzene in the presence of an organic base such as triethylamine or pyridine. Following the reaction, the reaction mixture is allowed to warm to 0 about 25 C for one hour. The resulting arride derivative is combined with an alkylating reagent, such as methyl- fluorosulfonate, dimethylsulfate, methyliodide, methyl p-toluenesulfonate or trimethyloxonium hexafluorophosphate (when R 3 is methoxy) or triethyloxonium tetrafluoroborate (when R3 is ethoxy) at about 250C in a chlorinated hydro- carbon solvent such as methylene chloride, chlorobenzene or chloroform. The reaction mixture is refluxed for about 12 to 20 hours, cooled to about 250C and an organic base such as triethylamine or pyridine is added after which the solution is extracted with brine and the product isolated.

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When R3 and R 4 together form an alkylene group in compounds 1 having from 5 to 7 carbon atoms, the corresponding amino acid ester derivatives are obtained by treating the amino acid ester with a cyclic alkanone to form a Schiff's base by procedures generally known in the art. Cyclic alkanones that can be employed include cyclopentanone, cyclohexanone and cycloheptanone. 0 When the symbol Z1 in compounds 1 is R 5 CNH(CH2) 3 O O 0 or R 6 OCNH(CH 2 3 the -CR 5 and -COR 6 protecting groups are added to the corresponding free amino acid, i.e., ornit.iine, by treatment of said amino acid with an excess of a copper salt, such as copper carbonate, in boiling water for about 1 to 6 hours. Upon cooling to room temperature the insoluble materials are filtered and the filtrate is treated with an appropriate acid halide

0 (when ZI is R5CNH(CH2)n-) or an appropriate alkyl or 0 aryl haloformate (when Z1 is R60CNH',CH 2 in a solvent such as acetone in the presence of a base such as sodium bicarbonate or sodium hydroxide. This treatment is followed by treatment with hydrogen sulfide. Illustra- tive acid halides which may be employed include acetyl chloride, propionyl chloride, benzoyl chloride or 2- phenylacetyl chloride. Illustrative haloformates which may be employed include benzyl chloroformate, phenyl chloroformate, methyl chloroformate or ethyl chloroformate. The lactams of the compounds of general Formula I wherein R 1 is carboxy are prepared from the corresponding amino acid esters having the structure Y H 2 N CH 2 3 -C-COR 7 NH 2 (X) wherein V has the meaning defined in Formula I and R7 is a straight or branched alkoxy group of from 1 to 8 carbon atoms, illustratively methoxy, ethoxy, isopropoxy,

_1 applicant(s)

P 19/11/7

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butoxy or hexyloxy. The lactams are prepared by treating said amino acid esters with an appropriate base, such as sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium methoxide, potassium methoxide, potassium tert-butoxide, sodium amide, or an organic amine such as a trialkylamine, for example, triethylamine in a solvent such as a lower alcohol, for example, methanol, ethanol, isopropyl alco- hol, n-butanol, water, dimethylformamide, dimethylsulfoxide, hexamethylphosphortriamide or mixtures thereof. The reaction is conducted for a period of from 1/2 hoir to 24 hours at a temperature of from about 00 to 1200C, optionally under a nitrogen atmosphere. The compounds of general Formula are obtained by procedures well known to the art, for example, by utilizing the corresponding amino acid and treating said amino acid with an appropriate alcohol such as methanol, ethanol, isopropyl alcohol, n-butanol or n- heptanol saturated with HC1 gas. The compounds of general formula I wherein Z is H2N-CH(CH 2 2 Y is CH 2 F, CHF 2 and CF3, and R 1 is carboxy CH 3 o are prepared via procedures that are analogous to those just described. O ,o Compounds of general Formula I wherein Z is H2N-CH(CH2)2 or H 2 N-(CH 2 3 Y is C CH and Ri is carboxy CH3

25 are prepared by treating a suitably protected propargylamine derivative, such as a silyl derivative, S° with a strong base to form a protected propargylamine carbanion intermediate. This carbanion intermediate is reacted with an alkylating reagent of the formula R 8 X, wherein X is a halogen such as chlorine or bromine, and Rg is PhHC=N(CH2)n in which n is the integer 3. The thus formed alkylated protected propargylamine derivative is then treated with a strong base to form an

M-1076-Cl

alkylated protected propargylamine carbonaion. The second carba-nion is reacted with an acylating reagent -and the protecting groups are subsequently removed by acid or base hydrolysis as indicated in the following reaction scheme:

(R9 3 -Si-C-;C-CH 2 strong N=C-Rll base N=C-Rll

compounds 4 SR8 X

R8 strong (R9 3 -Si-C-C-CH N=C-Rlj <base N=C-Rll R1 o C

Iacylating reagent compounds

a~ z *gt* (Rq) 3 -Si-C!C-C-Rl2 HCEC -C-GOH 11i20 -4 04 N=CRl NH-2 (acid/base)

compounds 6 Formula XI

4

C

In the above reaction scheme R3 and X have the meanings defined hereinabove! Ph represents phenyl, R1O is hydrogen, methoxy or ethoxy, Rll is phenyl, tert-butyl, or triethyl- mncthyl, R9 is a straight or branched lower alkyl group Ihaving from 1 to 4 carbon atoms, R1 2 is a carboxy anion,

I

M-1076-Cl

-16-

a carboxylic acid ester, a carboxamide, a nitrile or other group capable of beine hydrolyzed to a carboxylic acid function, and Z is H2N-C-(CH 2 2 or H 2 N(CH 2 3 CH 3 Suitable strong bases which may be employed in the above reaction to form each carbanion are those which will abstract a proton from the carbon atom adjacent to the acetylene moiety, such as an alkyl lithium. Suitable alkylithfilm compounds that may be employed include butyl lithium or phenyl lithium, lithium diisopropylamide, lithium amide, tertiary potassium butylate and sodium amide. The alkylating reagents, R 8 X, employed in the above reaction are known to the art and can be prepared by standard procedures known in the art. Thus, the reactant PhHC=N(CH2)n can be prepared, for example, by reacting 15 3-bromo-n-propylamine hydrochloride or 4-bromo-n-butylamine hydrochloride with benzaldehyde in the presence of an organic amine, such as, triethylamine in a suitable solvent. ,D Solvents that can be employed include diethyl ether, tetra- C so hydrofuran, dioxane, chloroform or dichloromethane. Suitable acylating reagents which may be employed in the above reaction are halo-formates, such as chloro methylformate or chloro ethylformate, azido tert-butyl- formate, cyanogen bromide, carbon dioxide, diethylcarbonate, phenylisocyanate, triethoxymethylium tetrafluoroborate, 00 N,N-dimethylcarbamoyl chloride, 2-methylthio-1,3-dithio- linium iodide, ethylene carbonate or ethylene trithio- carbonate. When 2-methylthio-l,3-dithiolinium iodide is O 0r employed the additional step of alcoholysis by means of a lower alcohol, such as ethanol or isopropyl alcohol is required prior to hydrolysis of the protecting groups. The alkylating reaction is readily conducted in the presence of an aprotic solvent such as benzene, toluene, ether, tetrahydrofuran, dimethylsulfoxide, dimethylform- amide, dimethyl acetamide, hexamethyl phosphortriamide.

'4 M-1076-Cl

Pr~n~mt',~nv 1

M-1076-C1

-17-

0 The reaction temperature varies from -1200C to about 25 C, a preferred reaction temperature being about -700C, with the reaction period ranging from about 1/2 hour to 24 hours. Removal of the protecting groups, as represented in the foregoing reaction scheme in the step relating to the conversion of compounds 6 to the compounds of Formula XI, is achieved by treatment of compounds 6 with an aqueous acid such as hydrochloric acid or toluene sulfonic acid. Alternatively, an aqueous base such as sodium hydroxide or potassium hydroxide, or hydrazine or phenylhydrazine may be employed. The propargylamine derivatives of compounds 4 wherein is hydrogen are prepared by the addition of protecting groups on the acetylene function and the nitrogen function of propargylamine. Protection of the nitrogen function of the ?ropargylamine is accomplished by forming a Schiff's base with a nonenolizable carbonyl bearing compound such o as benzaldehyde, 2,2-dimethylpropanal or 2,2-diethylbutanal. Protection of the acetylenic function is accomplished by reacting the above-described Schiff's base with a tri- alkylsilyl chloride wherein the alkyl moiety has from 1 to 4 carbon atoms and is either straight or branched. Trialkylsilyl chlorides that can be utilized include trimethylsilylchloride or tr-.ethylsilylchloride. The propargylamine derivatives of compounds 4, wherein R 1 0 is methoxy or ethoxy are prepared by reacting propar- gylamine wherein the acetylene function is protected by Sa trialkylsilyl group with benzoyl chloride, pivalic acid t0 chloride, or 2,2-diethylbutyric acid chloride at 0°C in a suitable solvent. Suitable solvents include diethyl ether, dioxane, tetrahydrofuran, chloroform, methylenechloride, dimethylformamide, dimethylacetamide, or chlorobenzene. The reaction is conducted in the presence of an organic base such as triethylamine or pyridine after which the reaction mixture is allowed to warm to about 250C for

M-076-C

M-1076-C1

B M-1076-C1

f 'j.1 -18-

I1. one hour. The resulting amide derivative is combined with an alkylating reagent, such as, methylfluorosulfonate, dimethylsulfate, methyliodide, methyl e-toluenesulfonate or trimethyloxonium hexafluorophosphate when R 1 0 is methoxy, or triethyloxonium tetrafluoroborate when R1 0 is ethoxy, at about 250 C in a chlorinated hydrocarbon solvent such as methylene chloride, chlorobenzene or chloroform. The reaction mixture is refluxed for about L2 to 20 hours, cooled to about 250C and an organic base such as triethyl- amine or pyridine is added. The resulting solution is extracted with brine and the desired product isolated therefrom. The protected propargylamine zcarting material is obtained by treating a 3-trialkylsilylprop-2-ynyl-l-imino- 15 benzyl derivative, that is compounds 4 wherein R 1 0 is 04 (A hydrogen and R 1 1 is phenyl, with hydrazine or phenylhydra- 4EI 0.4* zine at about 25 0 C for about 1/2 hour after which the mixture is diluted with, for example, petroleum ether, 04 B 4 benzene or toluene and the amine isolated. Alternatively, 44( 20 the imine is hydrolyzed with 0.5 to 1 N HC1 solution, and the aqueous phase evaporated to afford the amine as the hydrochloride salt. Compounds of formula I wherein Z is H 2 N-CH(CH 2 2 CH 3 Y is CH 2 F or CHF2; and R 1 is hydrogen are prepared by reducing a ketone of the formula 0 4 0 -0-0.r V 0 Z'-C-Y 4s (XII)

R13 13 wherein Z' is phthaloyl-NCH(CH2) 2 benzoyl-NHCH(CH2) 2

W. C Ri-

M-1076-C1

-19-

R13 R13 alkanoyl-NHCH(CH 2 2 alkoxycarbonyl-NHCH(CH 2 or

R13 benzyloxycarbonyl-NHCH(CH 2 2 wherein the alkanoyl moiety has from 2 to 5 carbon atoms and is straight or branched, the alkoxy moiety has from 1 to 4 carbon atoms and is straight or branched, Y is CH2F and CHF 2 and R13 is methyl. The ketones are reduced to the corresponding alcohol which is treated with one equivalent of an imide, such as, phthalimide, succirimide or maleimide, 1.1 equivalents of a phosphine, for example, triphenylphosphine or a trialkylphosphine, such as, tri-n-butylphosphine and 1.1 equivalents of diethyl azodicarboxylate in a suitable asolvent. Suitable solvents include ethers such as diethyl ether, tetrahydrofuran or p-dioxane, benzene or dimeihoxy- 0 :ooa ethane. The reaction is conducted at about 00 to 100 C, u

14 tu 15 preferably about 250C, for a period of about one-half rr hour to 24 hours under an inert atmosphere such as nitrogen or argon. The thus obtained imido derivative is then hydrolyzed to the free amine. o The compounds of general formula (XII) wherein Y is f FCH 2 are prepared by treating a compound of the formula 20 *i. I0 0 I 9 Z '-C-CH Rj 4 i ii teE

(XIII)

wherein Z' is defined as above and R 14 is a suitable leaving group, such as chlorine, bromine or iodine, i esylate, tosylate, triflate or trifluoroacetate with an appropriate fluorinating reagent, such as, potassium fluoride, silver fluoride, cesium fluoride, thallium fluoride, tetrabutylammonium fluoride in a suitable Ssolvent. Suitable solvents include solvents such as

-i

M-1076-Cl

dimethoxyethane, dimethylsulfoxide, dimethylformamide, ethylene glycol, acetonitrile, acetone, benzene or hydrogen fluoride. The reaction is conducted at a temperature of from about 00 to 2000C for a period of about 2 to 48 hours. The leaving group RI4 may also be a diazo group in which case the fluorinuting reagent employed is hydrogen fluor- ide/pyridine. Suitable solvents for the reaction wherein R 1 4 is a diazo group are aprotic solvents such as diethyl ether, tetrahydrofuran and pentane. The reaction time varies from about 30 minutes to 24 hours at a temperature ranging from about -200 to 650C. Illustratively, a com- pound of the formula 0 Z'-C-R 1 4

as defined above wherein R14 is a diazo group in a suitable 15 aprotic solvent is added to a solution of hydrogen fluor- ide/pyridine and cooled to -100 C. The reaction mixture o" is stirred vigorously at -10 0 C for 1 hour, warmed to about 0 C for 2 hours and then poured over ice. The organic phase is separated, washed with a base such as sodium bicarbonate, dried over magnesium sulfete and concentrated under vacuum to afford the appropriate fluoromethyl ketone derivative of formula (XII). The diazo ketone derivatives, that is, the compounds AV of formula (XIII) wherein RI4 is a diazo group, can be obtained via the corresponding acid halide represented by a compound of the formula.

Z'-C-halide

wherein halide may be, for example, chloride and Z' has the meaning defined in formula (XII). The acid halide contained in an aprotic solvent, as for example diethyl ether, tetrahydrofuran, pentane, hexane, benzene, dimeth- oxyethane or dioxane, is added to a solution of diazo- methane in ether cooled to about -400 to 20 0 C, followed

M-1076-C1

-34-

with pharmaceutically accPnt-hi .h1

SM-1076-Cl

-21-

by vigorous stirring at about 25 0 C for about 1 to 24 hours. The diazo ketone derivative so obtained can be isolated using standard procedures such as evaporation ot the solvent with subsequent purification by recrystallization or chromatography. Alternatively, the reaction mixture can be treated with an appropriate fluorinating reagent as described above without isolation. The appropriately substituted diazo ketone derivative described above can be used to prepare compounds of formula (XIII) wherein R 1 4 is, for example, halogen, mesylate, tosylate, triflate, or trifluoroacetate using procedures generally known 'o the art. To obtain compounds of general formula (XIII) wherein RI4 is halogen, such as, chlorine, bromine, or iodine the corresponding comround of formula (XIII) in which R14 is a diazo group is treated with either aqueous hydrogen chloride, hydrogen bromide or hydrogen iodide in a suitable aprotic solvent. In order to obtain compounds of formula (XIII) wherein R 1 4 S. is mesylate, tosylate, triflate or trifluoroacetate, the corresponding diazo ketone derivative, wherein R14 is a diazo group is dissolved in a suitable aprotic solvent and treated with dilute sulfuric acid to yield the "corresponding benzyl methanol ketone derivative. Lastly, Sthe benzyl methanol ketone is esterified with an appropriate acid chloride or acid anhydride utlizing methane sulfonic acid, p-toluene sulfonic acid, tri- fluoromethyl sulfonic acid or trifluoroacetic acid. The acid halides, that is, compounds of the formula 0 Z '-C-halide

as described above, are known compounds which ccn be pre- pared from the corresponding acids. Thus, for exampi,

Y~i iA

k '7 S

;I 1

M-1076-C1

-22-

treatment of the appropriate acid with thionyl chloride in an aprotic solvent, such as, diethyl ether, tetra- hydrofuran, benzene or dichloromethane at a temperature ranging from about 00 C to the reflux temperature of the solvent for'about 1 to 24 hours results in the foundation of the corresponding acid halide. Alternatively, treat- ment of the appropriate acid with oxalyl chloride in one of the aprotic solvents described above at a temperature of about 00 to 40 0 C for about 1 to 24 hours also results in the preparation of the corresponding acid halide. The compounds of general formula (XII) wherein Y

R! is FCH 2 and Z' is other than benzoyl-NHC(CH2)2- or R 1 alkanoyl-NHC(CH 2 2 may also oe obtained by treating a compound of the formula

4I Q 15 Z 2 -RI1 0 il

(XIV)

R13 '4 wherein Z2 is pnthaloyl-N-CH(CH2)2-, alko.-ycarbonyl- R R13 13 NHC(CH2)2- benzyloxycarbonyl-NHC(CH2)2-, 8-methylthioethyl S 40 04 a or 8-benzylthioethyl; R 1 3 is methyl; and R15 is chlorine, '4400,

bromine or iodine, mesylate or tosylate. Thus, a compound 20 of formula (XIV) is reacted with triphenylphosphine or S NC'44

tri-(lower)-alkylphosphine, for example, tri-n-butyl- 0. u

I0, C phosphine, in a solvent such as benzene, toluene, methanol, ethanol, acetonitrile, tet-ahydrofuran, diethyl ether or dimethoxyethane. The reaction is conducted at a temperature ranging from 25 0 C to the reflux temperature of the solvent

-C. '-A

.M-1076-C1

-23- for a period ranging from about 10 minutes to 48 hours. On cooling the precipitate which forms is washed with solvent and recrystallized to give the appropriate phos- phonium salt. The triphenylphosphonium or trialkylpnos- phonium salt is added to an excess (up to 25%) of sodium or l:thium metal dissolved in liquid ammonia to which a catalytic amount of ferric nitrate is added. Stirring is continued for about 10 minutes to 3 hours after which the ammonia is evaporated under an inert atmosphere, such as, nitrogpn or argon. An appropriate solvent, such as, benzene, toluene, diethyl ether, tetrahydrofuran or di- methoxyethane is added and the resulting substituted methylidenephosphorane is :ollected. The methylidene- phosphorane is treated with a lower alkyl ester of mono- fluoroacetic acid in a solvent such as benzene, toluene, ,diethyl ether, tetrahydrofuran or dimethoxyethane. The reaction is conducted under an inert atmosphere such as nitrogen or argon at a temperature ranging from about 00 C o to the reflux temperature of the solvent for a period of from about 30 minutes to 24 hours. The reaction mixture is concentrated by distillation to yield an olefin. The olefin is treated with an aqueous mineral acid, such as o hydrochloric or hydrobromic acid or an organic acid b'ich as trifluoroacetic acid or p-toluene sulfonic acid in the presence of a cosolvent such as tetrahydrofuran, diethyl ether, or benzene for a period of from about 30 minutes .Duo to 24 hours at a temperature ranging from about 0 0 C to

the reflux temperature of the solvent. The amount of acid employed may vary from a catalytic amount to a concentrated acid solution. 4. As used in general formula (XIV) the term

R13 phthaloyl-N-CH(CH 2 2 is taken to mean the group

R13 i J^C"N-CH(CH2

M-1076-Cl

-24-

R13 the term alkoxycarbcnyl-NHCH(CH2) 2 is taken to mean

0 R13 the group alkyl-O-C-NHCH(CH 2 2 the term benzyloxycar-

R13 bonyl-NHCH(CH 2 2 is taken to mean the group (7j-CH 2 9 R13 OCNHCH(CH 2 2 wherein Ri1 3 has the meanings defined in

formula (XIV) and alkyl is a straight or branched group having from 1 to 4 carbon atoms. Compounds of general formula (XII) wherein Y is F 2 CH- are obtained by treating [[(methylsulfinyl)methyll- thiojmethane or [[(ethylsulfinyl)methyl]thiolethane with a suitable strong base followed by alkylation with an appropriate derivative of the formula

Z'-R16

(XV)

wherein in formula (XV) the symbol Z' has the meaning previously defined in formula (XII) and R 1 6 is chlorine, bromine, iodine, mesylate or tosylate. The thus formed Z' substituted sulfinyl derivative is treated with a suitable strong base followed by alkylation using an appropriate halomethylhalo alkylating reagent selected 0 from chlorodifluoromethane, bromodifluorcmethane, and difluoriodomethane. The alkylation reaction is followed by .a hydrolysis using an aqueous acid solution. 0 Suitable strong bases which may be employed in preparing the difluoromethyl substituted ketone derivatives as described above are sodium hydride, dilithium acetylide, lithium diisopropylamide, butyllitnium, potassium tert- butoxide, sodium tert-butoxide, lithium tert-butoxide, phenyllithium, methyllithium, sodium amide, lithium amide or potassium hydride.

Iu U

M-lJ 76--Cl

The alkylation reaction described in preparing the difluoromethyl ketone derivatives are carried out in an appropriate solvent, for example, tetrahydrofuran, di- ethyl ether, hexamethylphosphortriamnide, dimethylsul- foxide, or benzene. The reaction is conducted at a temperature ranging from about -780 to 650 C for a period of from about 30 minutes to 24 hours. Preferabiv. temperature of about 400 C is utilized for the if'c~ methyl alkylation step. Th e ci1 Ky 1a ted, 7 Z: int er mediates are isolated by quenching wvith a olution followed by extraction utilizing diethy. etl-.er, zrro metnane, or benzene. The alkvJ.Iated sinI:e'ed3tes are recovered from the combined extracts. Hydrolysis of the alkylated sulfinvl der:;v±tive e- S 15 the ketone is achieved using an aqueous mineral acid solution, such as, hydrochloric, hydzrbr=_ic, per-hioric or sulfuric acids in a solvent such as tetr~jLydro-f,_ran, acetonitrile, diethyl ether or benzene. The hydrol',Sis is conducted at a temperature ranainc from abcurt -200 tc 105 0 C, preferably about 25 0 C- for a period of fr.-_m abcour minutes to 24 hourS, preferably abot >c1%.2n-r~y a solution of 0.3 equivalents of mineral acd I water is employed. The specific examples aescribeac ecw further illustrate the preparation ot the difl :rorethy ketone derivatives of formula (XII). The compounds of formulas 'XIV/' anc.- ',ihereion P, and R1 6 are halogen are known to thfe art ran be pre- pared from an appropriate carboxylic ard~'~~ having the formula

Z4 -COC:- (XV2N

I M- 1076-Cl 4

-26-

wherein ZA is phthaloyl-NCH(CH2 benzovYl-NHCH0-CH-; 143 13 alkanoyl-NHCH(CHn)-, alkoxvcarbonvl-NH FC"C§-,

I 113 benzv'Io;7ycarbony1--NHCH (CH2) methylthiomethyl or benzyltniometh,.I. These acids are known to "he art- or can be obtainEd bv known procedures from the corr,2spond n, unprotected anaino acids-. The cononds rm:'Xas and :XV wnercsin T-7 and R1 6 are mesylIate tosylate may Oe prepared -reating the re: deiave ~rWhich 3 and R- 6 are "alogen zh a meta -salt of methja nesul fonic aciLd or p-tcluenssu I7_7nic lra- tve v1 the sod.,um sa-'t of_' r, an, -lf -,ic c toluenesu)lfonic :icid can be ~:izc Reduction of" the ketones of forrral_-tXMI' to the corresponding alcohols is ach~eved chemicallyt using I 15 10 equivalents of a neta- hydride, such aslibm boro- hjdride, sodium bcrohydride, Sodium cyanobcrch.'drid:_, OL lithium aluminum hydride. In addition the ke,,.r'es can, be reduced with borane or dmtvtibrreor r-aIaytical>' reduced using, for example, Rane- nickel_, frhodlum, palladiu- cC C 3 C 3 20 on charcoal, or platinum oxl.de. In ctaoa. te reaction time varies from about 10 pinutes to> tc r an,, tme temperature at which the reduction is condced can rancne from about -400 C to l00OC Lepenriing upon t-ne particular31 ""3

reducing reagent empl.oyed. When hydride oran-: re- duction employed the react--on is odtr naut.C solvent for a period of time -from abcu t mir tez IT hours with temperatures rangjing fro-m a;Do;ut -0Ct Suitable solvents that can be emicloved for rdcir~ compounds of general formula ce iwr lch~s such as methanol or etnanc-, r u ado. ether or tetrah-'drofoiran. W.-en aarrm

M- 1076-Cl

of 40 ml of RF/pyrid-ne precooled to CoC. The resultinq

76-Cl

-27-

employed the reaction time varies from about 1 hour to 24 bours, the reaction temperature ranges tram about 250 to 1000C and the hydrogen pressure carn range from 1 to 120 atmospheres. Hydrolysis to the amine and the removal at any distal amine protecting group is achieved using a strong mineral acid such as 1ydrochloric acid, hydrobromic acid or sulfuric acid, or an organic acid such as tcluenesulfcnic acid or trifluoroacetic acid. The hydrolysis is conducted in water or an aarueous solvent at the reflujx temperature for a period of from about 4 to hours. Alternatively, I to 3 equivalents of hyvdraz~ne, rethvihvldrazine or meth-lamine can be utilized at a temperature offrom about 25 0 C to the -ef lux tempe.-ature of thle so",-ut ion f or about 1 to hours, fcilowed by treatment wi::h a strona mineral acid or organic acid as described above. Comipounds of formula i wtnereirn Z is HiN-CHfCH2,2; CH 3 .Rl is hydroxy; ana Y iz: C CF, or as hereinarcer indicated the alkynyl group, are prepared by the hydrolysis of h-e a! k~lated compounds 3, described acoDve. The desired alkylating rea-gentz R8 X tnat are empioved can bIe prepared by methois known to the art. Thius, he reaagent

Ph!HC=NCH 2 (Ch2'§,

can be prepared by reacting 3-bromc-n- propy amine hydrochloride wlth benzaldeh-'de and an oraanic trialkvl7- amine, such. as triethylamine, in a so'_-:ent s ich. as diethyl ether, tetrahydrofuran, dioxane, chroroforo rc dlchlor3- methane. Thne rea :tan t CH. PhF.C=NCH (F

is prepared by reacting 3-aminobt.-vlrbromide hydrabromide with benzaldehyde and an organic amine such as triethyl- amine. The comcound 3- ou~rm.ehyd!:oiroillidp i_:

M- 1076-Cl

A

IIJLILU~e oEr rom Q to IIU'-t. nor a period or from 1 to 12 hours. The Y-aminoalkanol derivative is obtained by treating an appropriate $-keto-alkanoic acid ester the formula 0 0 CH 3 C-CHi-C-OCH 2CH3 The B-keto-alkanoic acid ester is treated with hydroxylamine to form the corresponding oxime, which is reduced with lithium aluminum hydride in ether or tetrahydrofuran at. a temperature: of from 2150 to 500C for a period ranging from I to 12 hours. Subsequent hydrolysis of 'the~ester moiety resultS in the formation of the y-aminoalkanol. The alkylating reaction may be carried out in an aprotic solvent, for example, benzene, toluene, ether, tetrahydrofuran, dimethy'lsulfoxide or hexamethyl phos- phortriamide. The reaction temperature vari4es from about -1000 to 250 C preferably about -C and the reaction time varies from abou 1:L ort 4hus Removal of the protecting groups, as represented in the reaction scheme in the step going from compounds 5 to the desired amines, is achieved by treatment with aqueous acid, for exaple, hydrochloric acid followed by aq~ueous base, for example, sodium hydroxide cr ootassium or treat- ment with phenylhydrazine, hydroxylamine or hydrazine then with aciueous base.

The individual optical isomers of compounds of formula I wherein Rl is carboxy or hydrogen are resolved

acodacqith tepoduefR.Viterbo et al., TerhdonLtes_8_ 67 17) Other resolvinc agents such as campbor-lC-.sAUtonic acid may also be Ateratiely whn ZisH2N--CH-(CH2)) or empoye. CH 3

M- 1076-Cl

-42-

The 2 -difluoromethyl-2,5-diaminopentanoic acid is mixed with aPproximattzv A

S M-1076-C1

-29-

H 2 N(CH 2 resolution is achieved via the lactam of said compounds. The thus resolved acids and amines may be employed in the same manner as described hereinabove for the racemic mixtures. The compounds described herein are useful in in- hibiting the growth of protozoa in animals. The term "animals" is intended to include inter alia mammals, such as mice, rats, guinea pigs, rabbits, ferrets, dogs, cats, cows, horses and primates including man. Also encom- passed within the term animals are both fish and fowl. The term "fowl" is intended to include male cr female birds of any kind including parrots and canaries, but is primarily intended to encompass poultry which are commercially raised for eggs or meat. Accordingly, the term "fowl" is particularly intended to encompass hens, cocks and drakes of chickens, turkeys and ducks. The term "protozoa" is intended to include those members of the subphyla Sarcomastigophora and Sprozoa of the phylum Protozoa. More particularly, the term "protozoa" as used herein is intended to include those genera of parasitic protozoa which are important to man because they either cause disease in man or his domestic animals. These genera are for the most part found o. classified in the superclass of Mastigophora of the subphylum Sarcomastigophora and the class of Telosporea of the subphylum Sporozoa in the classification according to Baker (1969). Illustrative genera of these parasitic protozoa include Histomonas, Trypanosoma, Giardia, Trichomonas, Eimeria, Isopora, Toxoplasma and Plasmodium. I .o 30 Excluded from the superclass of Mastigophora is the genus Leishmania, certain species of which cause the tropical disease of Leishmaniasis in man. Also, specifi- cally excluded from the genus Trypanosoma, as used in this invention, are the species Trypanosoma cruzi, which can cause Chagas' disease in man, and the species

-I

M1076-Cl

Trypanosoma lewisi. The compounds described herein have been found not to be particularly effective against these species. On the other hand, the compounds of Formula I are particularly useful in inhibiting the growth of Trypanosoma brucei, the causative agent for nagana, or the tsetse-fly disease of horses and cattle in central Africa. The compounds described herein are also re- markably'effective in inhibiting the growth of Eimeria tenella, a species of protozoa causing coccidiosis in fowl. Indeed, a preferred embodiment of the present invention is the use of these compounds to inhibit the growth of intestinal coccidia in commercial poultry. P 15 The economic importance of intestinal coccidia is highly significant. Thus in 1972, the estimated loss to the poultry industry in the United States due to coccidial infections was approximately 47 million dollars. Due to the rapid development of drug resistance by coccidia, and due to the relatively high toxicity of some of the drugs used in the treatment of coccidiosis, there is a need for effective coccidiostats that are non-toxic and to which intestinal coccidia do not develop rapid drug resistance. It is not exactly understood how the compounds of this invention are able to inhibit the growth of protozoa. s" Inter alia, the compounds described herein are irreversi- ble inhibitors of ornithine decarboxylase and S- adenoxylmethionine decarboxylase. As irreversible i inhibitors of these enzymes, these compounds inhibit o 30 polyamine formation which may be required for protozoal cell division. In any event, the practice of this invention is not limited to any particular mode or theory of action whereby tne compounds of this invention are able to effectively inhibit the growth of protozoa.

M-1076-C]

-44-

TABLE I

M-1076-C1

-31-

The effect of the compounds of general formula (I) upon the growth of protozoa, and more particularly upon the growth of coccidia, can be demonstrated using Eimeria tenella and two week old male white leghorn chicks as the test animals. The bir6s are kept in batt ries and both the infected and non-infected birds are housed in separate rooms to assure the maintenance of coccidia-free birds. Each experimentally infected bird receives 100,000 sporylated oocysts via gavayc. The test compound is administered in the particular dosaye desired through the drinking water and drug-free mash is provided ad libitum. To evaluate the effect of the active ingredient on E. tenella infections, the chicks are sacrificed with carbon dioxide, necropsied, generally at day five of the study, and cecal lesions evaluated. The inhibition of protozoal growth can also be determined using Trypanosoma brucei brucei, which is the causative agent of bovine trypanosomiasis (nagana) in Africa. The related species Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense are the causative agents for African sleeping sickness in humans. In general, drug activity is tested against established infections of a pleomorphic EATRO 110 isolate of T. b. brucei in mice. Test animals are infected with 5 x 105 Soe 25 parasites twenty four hours prior to testing. Control animals so infected generally die 5 days subsequent to innoculation. The compound to be tested is administered o. to the test animals via their drinking water in varying dosages. Animals cured of the infection remain parasite- free more than 30 days after the deaths of the control animals as indicated by an examination of blood smears. The compounds descr.bed herein are employed in

r

M-1076-C1

-32-

amounts that are effective in inhibiting protozoal growth. These amounts will depend, of course, upon various factors, such as the type and nature of the protozoal infection, the activity of the specific compound, the age, sex and species of animal treated and whether the treatment is prophylactic or therapeutic. In general, the compounds described herein can be orally or parenterally administered at a daily dose ranging from 5 mg/kg to 7 g/kg. Preferably, in the case of Tr-ypanosoma infections the dosage range is from about 600 mg/kg to about 2 g/kg. In the case of Eimeria in- fections the dose can be lowered, ranging from about 13 mg/kg to about 1 g/kg. Due to the low toxicity of the compounds described herein, the compounds can be safely administered ad libitum via the drinking water of the test animals in the treatment of coccidiosis in fowl. Generally speaking, concentrations of the active ingredient ranging from about 0.01% to about 2% are suitable, depending primarily upon the na-ure of the protozoal infection to be treated whether prophylactic or therapeutic, the severity of the infection and the period of treatment. co Thus, for example the compound 2--difluoromethyl-2,5- .o diaminopentanoic acid can be effectively administered to chickens for the treatment of coccidiosis one day prior 25 to infection as a 2% solution. Alternatively, a prophylactic course of treatment can be utilized 8'days prior to in- fection utilizing concentrations of 2-difluoromethyl-2,5- ,aoT diaminopentanoic acid as low as 0.015% in the drinking o water of chickens. Preferably, a prophylactic concentra- S 30 tion of from 0.06% to about 1.0% is preferred. This prophylactic treatment for the inhibition of protozoal growth provides one of the principle advantages to the use of the decarboxylase inhibitors described herein. Thus, in the case of coccidia infections in chickens, for example, Eimeria tenella grows intracellularly in

i I

S M-1076-C1

-33-

the epithelial cells of the caecum as a trophozoite Sstage. Subsequently, these cells undergo a form of multiple mitosis to form a large number of merozoites. These merozoites are released as the host cell lyses and serve to extensively infect fresh cells. The result is that the wall of the caecum is badly damaged, leading to severe blood and fluid loss and finally death. More- over, during the life cycle of E. tenella, resistant oocysts are produced which are voided in the faeces of chickens. ChicKens being coprophagous in nature, the disease is rapidly spread by contamination of their food supply. Accordingly, coccidial infections in commercial flocks, when they occur, are epidemically treated with massive dcles of currently available chemotherapeutic agents, that are primarily cidal in nature. Consequently, medicated feeds are now routinely employed ir commercial flocks, so t'at all commercial Zowl now receive almost constant medication to prevent outbreaks of coccidiosis from occurring. The fact that the decarboxylase inhibitors herein described can be prophylactically administered, enables the host to overcome either a subsequent natural or artificial induced infection enzymatically via an inhi- bitory mechanism rather than via a cidal action. Thus, S o 25 in the case of an E. tenella infection, the infection is curtailed in a manner that enables the host to avail itself of its own body defense mechanisms. The resulting antibodies which are produced via such a controlled infection, serve to further permarnently immunize the host from future E. tenella infections. The pharmaceutical compositions that are particularly suited for the prophylaxis or treatment of protozoal infec- Stions in fowl comprise the heretofore described a-substituted- amines or the a-substituted-a-amino acids in combinatio-.

1 M-1076-C1

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for an additional 5 days or a total of 6 days. The

M-1076-Cl

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wit- pharmaceutically acceptable carrier. Advantageously, the antiprotozoal compositions are prepared by admixing the active compound with an inert carrier material. Typical carriers include talc, clay, pumice, silica, chalk, diatomaceous earth, walnut shell flour and equivalents thereof. Alternatively, the active ingredient can be admixed with a commercial feedstuff or vitamin and mineral pre-mix particularly adapted for fowl. In most cases a concentrated aquecus solution of the active ingredient is employed in the management and treat- ment of coccidioeis in fowl. The compounds described, for the most part, are highly soluble, particularly in the form of their salts. Such solutions may advantageously contain preservatives, such as parabens, benzyl alcohol, phenol or thimerosal. In addition, isotonic agents, sugars, stabilizing or buffering agents can be usefully employed. The compounds of formula can be used in conjunction with other known drugs currently in use for the chemotherapy and chemoprophylaxis of disease caused by parasitic protozoa. Generally, this has the effect of decreasing the amount of enzyme inhibitors administered. Such drugs include, among others: Antrycide, quinapyramine; Berenil, Diminazene 'O aceturate; Pentamidine isethionate; Primaquine; Tryparsamide; Amicarbalide; Amprolium; Amphotericin B; quinine; Monensin; o 0 Minocycline, 7-dimethylamino-6-deme'thyl-6-deoxytetracycline; Clindamycin, 7-deoxy-7(S)-chlorolincomycin; Buquinolate; Robenidine; and Nicarbazin. In some instances the compounds Soo- of formula actually enhance or potentiate the effects of these drugs. Of particular interest in this regard is the compound 2,5-diamino-2-difluoromethylpentanoic acid which has been shown to act synergistically with the antiprotozoal agents Antrycide, quinapyramine, Pentamidine isethionate and

I EM-1076-Cl

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EX'.MPLE 14

M-1076-C1

Amicarbalide. Thus, the 2,5--diamino-2-difluoromethyl- pentanoic acid concentration can be reduced by about four-fold when used in combination with subcurative doses (less than 1.0 mg/kg) of these drugs. Additionally, the compounds of formula can be used in combination with other known cytotoxic agents for the chemotherapy and chemoprophylaxis of parasitic diseases, particularly trypanosomiasis. Such cytotcxic agents include the antineoplastic antibiotic Bleomycin as well as other well-Known cytotoxic agents, as for example, cyclophosphamide, methotrexate, prednisone, 6-mercaptopurine, procarbozine, daunorubicin, vincristine, vindesine, vinblastine, chlorambucil, cytosine arabinoside, 6-thioguanine, thio TEPA, 5-fluorouracil, 5-fluoro-2- deoxyuridine, 5-azacytidine, nitrogen nustard, 1,3-bis(2- chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3- cyclohexyl-1-nitrosourea (CCNU), busulfan or adriamycin. Of particular i.terest in the treatment of trypanoso- miasis in general, and more particularly in the treatment of nagana in cattle, is the use of the enzyme inhibitor 2,5-diamino-2-difluoromethylpentanoic acid in combination with the antitumor antibiotic Blecmycin. This particular enzyme inhibitor appears to act synergistically with Bleomycin. Thus, mice infected with Trypanosoma brucei are cured after three days upon daily i.p. administration of Bleomycin at a dosage of 7 mg/kg. Similarly, trypanosome infections in mice are cured by the adminis- tration of a 1% solution of 2,5-diamino-2-difluoromethyl- pentanoic acid in the drinking water for 3 days. The results of several combination experiments indicate that cures are consistently effected with 0.5 mg/kg of Bleomycin in combination with 0.5% of 2,5-diamino-2- difluoromethylpentanoic acid administered via drinking water. Alternatively, cures are effected with concentrations

M-1076-C1

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of 0.25 mg/kg of Bleomycin in combination with only 0.25% of 2,5-diamino-2-difluoromethylpentanoic acid in the drinking water. A combination of 0.1 mg/kg of Bleomycin and 0.1% of 2,5-diamino-2-difluoromethylpentanoic acid has no effect. Thus. the curative dosage combinations reflect a reduction in Bleomycin drug dosage of from 1/2 to 1/28 of the curative dcse of the drug us.d singly, when used in combination with a subcurative dose of from 1/2 to 1/4 of the curative dose of 2,5-diamino-2- difluoromethylpentanoic acid. The invention described and claimed herein is more particularly illustrated in conjunction with the following Examples specifically describing how the compounds of this invention can be prepared and utilized.

EXAMPLE 1 2-Difluoroethyl-2,5-diaminopentanoic acid Under nitrogen a solution (500 ml) of 2M butyl- lithium in hexane is added to a stirred solution of 143.1 ml of diisopropylamine in 1.5 liters of tetrahydrofuran at -780 C after which 261 g (0.81 mole) of ornithine di- benzaldimine methyl ester in 1.5 liters of tetrahydrofuran is added. Upon completion of the addition the reaction I temperature is raised to 400C and maintained between 400 I and 500 C for 3 hours during which time chlorodifluoro- S"0 25 methane ga' is bubbled through the mixture with stirr-ing. The reaction mixture is then treated with a saturated solution of sodium chloride. The organic material is i extracted with ether, and the ether extract washed several times with sodium chloride solution, dried over magnesium sulfate and evaporated to give a viscous oil. The oil is stirred with IN HC1 (1.5 1) for 3 hours, the mixture extracted several times with chloroform and the aqueous solution evaporated to dryness. The oily residue is refluxed with 12N hydrochloric acid (1.5 1)

i I

S" M-1076-C1

-37-

for 16 hours, the cooled solution clarified by chloro- form extraction before concentration, decolorization (charcoal), and further concentration to about 750 ml. The pH of the solution is adjusted to 3.5 by the addition of triethy]amine, the solution treated again with char- coal before concentration to about 500 ml and dilution with 7-8 liters of acetone. The precipitated product i: filtered off and washel with ethanol. The crude product is recelftallized by dissolving in about 150 ml hot water and treatment of the solution with hot ethanol (450 ml). On cooling crystals of 2-difluoromethyl-2,5- diaminopentanoic acid hydrochloride monohydrate separate 71 g m.p. 1830C.

EXAMPLE 2 a-Ethynyl-a, 6-diaminovaleric acid 11.8 g (0.048 M) of N-(3-trimethylsilylprop-2-ynyl)- benzenecarboximidate in 20 ml of tetrahydrofuran is added to lithium diisopropylamide, prepared from 4.9 g (6.73 rl, 0.048 M) of diisopropylamide in 60 ml of tetrahydrofuran and 23.6 ml of 2.05 M solution of n-butyllithirm at after which 9.5 g (0.042 M) of N-(3-bromopropyl)benzylim:ne is added, and the mixture is stirred at -70°C for 5-1/2 .o hours. To the reaction mixture is added 23.6 ml of a S, 2.05 M solution of n-butyllithium followed by the additicn 25 of 4.5 g (3.67 ml, 0.048 M) of methyl chloroformate. After 30 minutes at -780 C the mixture is treated with brine, and the reaction product is isolated by etner Sextraction. The ether extract is evaporated and 300 m! of 3 N HCI is added to the resulting residue and the mixture is refluxed for 7 hours. On cooling the mixture is washed well with methylene chloride, made alkaline and washed again. The aqueous solution is acidified and concentrated to dryness. The residue is triturated with ethanol, filtered and tne ethanol evaporated. The residue

76-C1

-38-

is dissolved in water, the pH adjusted to and the soluti,)n is applied to a column of Amberlite 120 H+, eluting with I M NH 4 0H which affords, upon recrystalliza- tion from 3thanol-water, c-ethynyl-a, 6-diamino.'aleric acid, M.P. 168-169 (dec.). In the above procedure N-(2--bromopropyi)benzylimi~;e is prepared from 3-bromopropylamine and benzaldehyde bq procedures cenerally known In the art.

EXAMPLE 3 1-Fluoromethyl-1,4-b:utanediamine dihydrochioric-e- To a solution of 40 mmole of diazomethane in I>IC mi of ether cooled to 00C and magnetically stirred is add;-ed under nitrogen dropwise ovra period of I hrour a soluition of 20 ml of 4-ph-halimidobutyryl chloride in 75 ml of ether. Stirring is continued for 1 hour at 2300- after which the reaction mixture is added to a solution of ml of HF/pyridine precooled to 00C. The resulting 0 heteroigeneous mixture is stirred at 25 C for 1-1,,2 hour and then poured on ice water. The ether phase iS separated, washed with a solution of bicarbonate, then with brine and dried over magnesium sulfate. Concen:.fa- tion of the solvent under reduced oressur-e afford: a

solid which is recrystallized from aiethylether'pentaic

920 C. To a solution of 5 50 -ri~i 2.2 mmol e of fic-orometh% i

tetrahydrofuran and -5m! of methano] cooled to -'L00C U added a solutLio.n of C.3 mriolc of sodium borohydride In a mixture of 5 m-1 of !ietrahydrofuran and 5 ml1 of met-hanoj precooled to -200 C. The reaction mixture is stirred for minutes at -200 C and then neutralized with 2 M HCI to a pH of 1. The solvents are evaporated under reduosd-G pressure and the residue is Dartitioned between water

M- 1076-Cl

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arnd chloroform. The organic phase is washed with brine, dried over magnesium sulfate and concentrata d to gjive a residue which is recrystallized from tetranydrofuran- diethylether to ifford l-fluoro-5- -thalimido-'--pentaol., m.p. 850 C. A mixture of 264 mg (1.05 mmcle) of 1-fluoro- 5-phthalimido-2-pentanol, 170 mg (1.05 mmole) of the phthalimide, 302 mg '1.05 mmole) of triphenyiphosphine and 201 mn (1.15 m~mole) of diethylazodicarboxyviare in 6 il tetr-ahydr.,furan is stirred under nitroaien for n- ou~s it- 250 C. The solvent is evaporated lind r r::duced pre,:-sure and the residue taken up in benzene, The insoluble material is discarded and the residue obtained afterc- centration of the filtrate is recrys-al-ized from tet-a- hydrofuran-diethviethler to give i- f Iuorcne 1i-, a re- diYl-bis--hthal im~de, m.p. 1120C. A ucension of -2.1 9 of 1-fluoromethvl- I ,4-rutanedivl-bir--phlt.hal,,mide In 140 mi of concentrated RdIis neated at a reflux temperarrt Cl days. The phthalic acid whic'; Preciilares on 7aciinc to 40 C Is filtered off. The filtrate i3 concentr ar ed out 20 ml and cooled to 41)C. The.C re-maininc :h-!naiir acid which separates is filtered off a tde filca- concentrated under reduced Dressure. 1Z~cu treated with 4C ml of boiiina 'Sp c'I and then recryStallized fromr "-ooi:rE *:;-hanc-l 2- 1-f'Luoromethyl-1-,4-butane5i:amine dhdor'ra. 1-54 0 C.

EXAMPLE I-F2uoropmethv*l-4-methyI-_4 -b,..tard L ~ne Toc a s olujt ion o f 4 0 mmcl o c 31a zo- ie h ar ;n of ether cooled to GOC arid iracnet:,7, v ztirr~ad i: added under nitrogen dropwise o-.'er a PEri-a) >1 or On of 20 ml of 41-phthalilmido-4-mer, nvbur' idce T.l of ether. Stirring is corrudfcr I Y- Pr t LOC after which thI-e reactr is K1 rv.-

M- 1076-Cl

of 40 ml of RF/pyrit'fne precooled to OOC. The resultinq 0 C for heterogeneous mixture is st rred at '25 1-1,,2 hours and then poured on ice water. The ether phase is separated, washed with a solution of bicarbonate, then with briae andi dried over magnesium sulfate. Concentration of- the solvent under reduced pressure affords a solid which recrvl.sta>- lized from diethv-1ther/oentane to give furmeh 1 3-V phtha 'imidlo-3i-methylpropyl ketone. To a sol.-o,--on of 556l mg immoiLe' oI-f ]Luorow'e-',-" 3ohta~iidoS-mzo; prpylketo--ne in a mixture of tetrahvdrofuran arod 5 ml of methanol cooled to 00 added a solution of 0.S mmie or sodium borohycliide n a mixture of 5 ml or retrahvdrofulra, ard 35 ml of methanci precooled to -2COC The reaction 'rixture iS tre rr 1 15 m inu te--S Z0- -3n,2 -hen n eu t raiz ed wi-Lth 2 M HClto a 1 pH of I The ent-s are evaporated under reduced 1 pressure and the residue is partitioned between 'water and chloroform. The or-anic ohase is washed with brine, dried over magnesium s-ilfate and concentrated to ive a residuje which is recrystallized from tetralhvororuiran-diethvl'.ethpr to afford- 1-fl ujoro-5-phtual imido,,-E-methvl'-2- penranol. A\ mixture of 264 niPg (l.C05 mmcle' of lfutorptaa~ 72QMC 37 %zole -f rr~e phtha ii Je 302 mg (1.05 mmcle :of and 201 c I .rd7 1l rmm olIe of d ie th yIaz c- c a rbc xv Ia refnior 1 r 'd c- furan is str.red -underr n tr oen fr r sa solvent is evaporated u -nder rrc c- ornse l-2 residue taken in -enzere. Ihe i11 o u7 discarded and th-e r e s .iecra i ned af t T' f C'.o t 3C the f_4Iltr ate r allI Ized from trrt r r r, diethyl ether to give 1---luoromethyl--4 re 1-ltyIe diyl-bls-phthalimide. A su spc-n s ion ,r methyl-4-meth,, buane I-i, of concen-rated is nteated atrefo days. The ptial-'ic ac Id wnich+ prc-p i -r«

M- 1076-Cl

to 4 0 C is filtered off. The filtrate is concentrated to about 20 ml and cooled to 40C. The remaining phthalic acid which separates is filtered off and the filtrate is concentrated under reduced pressure. The residue is treated with 40 ml' of boiling isopropyl alcohol 3 times ar3 then recrvstailized from absolute ethanol to. give 1- fluioromethyl-4 -methvi-1,4-utianedami.~t dilh~drochloride.

EXALMPLE c I -Ethynyi1-4- metv -14-butan ediamine 10 8 q ~C MNY of1 3- tr imethyls I Iv-1Lro--vrll- Iminobenzyl In r53'0 m- of" tetrahvdrof'ura under nitroqen atmosphere at -70C C is added n-butvl lt-him. 705 M) After 11 minu-tes :he d: red cartanion iF treated withl 11.3 c .sM f4boo--mnonvbtn in 20 ml, of tetrahvNdrofuran. After '3hours atL Ififty' ml' of water is added and :he tetrabhydrofuran Is e'raporat,:d leaving a residue which is heated, at refl-'ux undfr nitrogen atmosphere with 100 ml 6 N hydrochloric acid for 48 hours. Upon cco-Ing the aqueous solu..tion is washed with methyl:ene chlor~de, made alkalirne wit-h aaeous sodium hydroxide and reextracted with methvlene chloride. The methv"lene chl-loride extract is drl-,ed over magnesium sul- fate, filtered, concentrated and diSt-,17-d to- afforo i-

et y y -e n d-

EXAMPLE ~ranue~ sutaciefor additionc rn~%wae orpoutryv art rprdasfoI-

2-Di*fluoromethv,-l-- ,14-2 n(,:nentanr-,,.1 idiam 3j-- 3 Cor-n starch Lactose 4~. Zinc stearate

SM-1076-Cl

-42-

The 2-difluoromethyl-2,5-diaminopentanoic acid is mixed with approximately 6 to 9 grams of lactose and passed through a fluid energy mill or micronizer to give a particle size of 1-25 microns. Water, 35 ml, is added to approximately 2.0 grams of the corn starch and blended to prepare a 5% starch paste. The micronized 2-difluoro- acid lactose powder, the remaining lactose and the remaining corn starch are well blended. The starch paste is added and blended, and the resulting mixture is passed through a No. 12 mesh screen. The resulting granules are dried at ?80C to a moisture content of approximately ground through a U.S. Standard No. 12 screen and lubricated by mixing with 0.3 grams of zinc stearate.

EXAMPLE 7 A 10% stock solution for use in the treatment of coccidiosis is prepared by dissolving 37.5 grams of 2- acid in one gallon of water at room temperature. One part of this stock solution diluted with nine parts of water results in the preparation of a 1% medicated drinking water solution for poultry which is usef:- c r the prevention of coccidiosis in poultry.

EXAMPLE 8

25 A medicated animal feed suitable for poultry is

prepared utilizing the fcllowing ingredients. The birds are fed the medicated feed ad libitum.

M-IU Ib-Cl WF

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Percent by weioht Ground yellow co 60.3 Soy bean oil 33.0 Alfalfa leaf Dicalcium Calcium 1 Iodized 0.2 2-difluorometiyl-2 acid. ,5-diaminopentanoic 3 Vitamin-minerali-amino acid antibiotic mix to furnish the following per 100 pounds of feed: 0. Penicillin (as procaine Manganese gm

130 DL-calcium pantotrtienate q3G m~q

Pyr gR Vitamin Choline C C A Vitamin 300 1000 Vitamin D3 251,0 0 .n~-ts

EXAMPLE 9 The following illustrates the effect: of 2-difl-icro- acid on T"rypanoscna brucei brucei infections n mice. Groups of fiv,,e mice weighing 20-21- g are inncculaited with T. b. brucei (EATRO 110 isolate; 5 x 105organisms/'- mouse) The compound is administered via drinking water, ad l~ibitumn, 24 hours following infection. Results are expressed as average survival (in days) beyond the death of the control animals, based upon an averag~e survival c control animals of five days. Berenil (dirninazene aceturate) is included as a control tr Ypanocide. The: results are indicated in Table I below.

M-1076-C]

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TABLE I

Total Average Drug Treatment -regi.men Dose Survival (mg) (Days)

None 0 0 52-difluorc-methyl-2,5-diaminopentanoic acid 2% in drinkino water, 6 days 60O- 2% in drinking water, 3 days 3UC_313 1% in drinkin(g water, 6 days 300k_ 1% in drinkinc water, 3 days i50a 3C 0. 5% in drinking water, 3 days 0.1% in drinking water, 3 days 5a

300 mq/'kg p.c. dailyv 3 days 2 0 mg/kg p dailIy 3 d ay z 11.3 mg,,k g d aiIy 3 dza, s 50 mg, kg p.o. daily 3 d 3.6

2-methyi-2,5-diaminopentanoic acid 2% in drinkinq water 3 day.s 3J 7

diminazene acet'irate mg,/k" i.rL. dalv a's

Based upon a da-;il intake 5 ml water/25 g, mo,;se Considered c'jrative. Animal: survived montn nev;cnd controls; blood smears were necati ;e 'or parasites after 1 month. Attempts at subinoculation of' brain suspensions into uninfected animals remained neaticve after >30 days.

.4

M-1076-Cl

EXAMPLE The following Example illustrates the effect of a 2% solution of 2-difluoromethyl-2,5-diami ncp~ntanoic acid in the drinkinq water of chickens infected with cocysts of Eimer-ia tenella. Twanty chickens are infected pe as at day 1 with 100,000 oocysts of E. tenella. Ten of the animals are givyen drinking water containirg a 2% solotion of 2-diflucro- acid. The remaining animals 13 serve as controls. dav 3 alI cof the control animals demonstrate clirnical signs of the disease. On day 7 all ofl the animals are sacrificed, cecal lesions are macro- scoQ4icallv examined and quantified as follows. =No detectable macrosc,=cic lesions. I: =Few scattered petechiae in the cecal wall; no thinkening of the wall and normal taecal contents3 present. L~esions= are n-umerous w::n noticeable loss :.7the cecalI contents; cecai wall slighzly thickened. -I =Larae amoints o" blood and tissue iebris present, cec'ui cores; cecal. wail -re=rI,; thickened, little i.f an%, norma. cecal contents pres3ent. z Cecal wal1 rea t-'jy ds tended wi h much blo_od or cecal cores rresent. Iclde bri iacir'ao oK incliedin cores. ird also scores a!s c~a -4.

Averaqe L~esicn S'core For

-2 a. ia z r rou.

p-

I

M- 1

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EXAMPLE 11 Following essentially the same procedure as in the preceding Example, six chickens are administered a 2% solution of 2 -difluloromethyl-2,5-diaminopentanoic acid (DFMO) in their drinking water for a period of 3 days. At day 1 this group of six chickens, in addition to two groups of ten chickens each, are all infected per os with 100,100 oocysts of E. tenella per chicken. One group of ten chickens serves as the control group, -he other group of ten chickens receives a standard dose of AMprolium in their drinking water for the next 5 day,s. At present Amprolium is the coccidiostat of chcice. On day 5 all of the animals are sacrificed and examined t~c evidence of disease using the lesion scoring index described in the is preceeding Example. The following rsi are cotained.

TABLE !!I Mean Lrs4c'n Treatment No. Chickens D:ays cf Tr--atmen-: Sccre Control 3.7-- 2% solution of DFMO 0 S0.120% solut-ion of -Amproliam

EXAIMPLE 1_ The following Exampla _11 tat_-S -rne etfect Of varying dose-= of ~-L~~imioetn~ acid on Fimeria tenelia in~t:sIn chic~enz. Following essentia.-J: same !Drccedure as in Example 1C, the dosage of 2df~oehl',d~nio propionic acid (DFMO) iS var.'c:d as shown in Table 1V below. Treatment with DF.MO4 is Starta--i at ay Teoik are infected per os at day 0, ar- s contLr-ied

1 r M-1076-Cl

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for an additional 5 days or a tctal of 6 days. The birds are sacrificed at day 5 and examined for evidence of disease using the lesion scoring index described in Example

TABLE IV

Mean Lesion Treatment No. Chickens Davs of Treatment Score Control 3.29 DFIMO 6 0 1.0% DFMO 6 DFMO 6 1.

EXAMPLE 13 The fcllowing Example illustrates the effeutienes, of a low prophylactic dose upon Eimeria tenelia lesLcns in chickens. Following essentially the same procedure set forth in Example 10, but varying the icse of 2-difluorometnl-,> diaminopentanoic acid 'DFMO) administered and the pericd of administration, he fo11ow2ng results are ontainEd.

TABLE V '4

Mean Lesncn Treatment No. Chickenz Davs of Trmatment Score .4 .2 Control 9 2% DFMO -1 thru 1% DFMO 9 -I thru 9 DFMO -I thru 0 9 0.25% DFMO -8 thru +5 0.44 0.125% DFMO 9 -8 thru +5 2 0.0625% DFMO 9 -6 rhru -K.

U '4 V 76-Cl

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EX.'.MPLE 14 The following Example illuztrates the acquisition of a permanent immunity towards Eimeria tenella infections in chickens. S:irds that have previously been treated wtih 2-ditluoro- methyl-2,S-diaminopentanoic acid at concentrations as low as U.5% on days -8 through +i5 relative to infection are challenged one week following completion ot therapy as indicated in Table V! below. These results indicate that Prophylactic therapy at low doses permits an adequate development of parasites in the absence of a disease state, thereby enabling the development of an immunity to slil- sequent E. tenella infections.

TABLE VI

No. of Days of Mean Les'-on Mean Lesion Treatment Chickens Treatment Score-Initial, Score-Fina. Control 9 1 .000 DFNIO 9 -~thri -5 0.3 0.503 DFMO 9

-1076-Cl

3if uc'ro- 3 low ar e

2 .3 state, 4

6 aLesio- 7 ce-Firnal 8 50 9

11

12 13

M-1076-Cl

4. where

whe re 6. where tx- suL -49-- C~CE

TheC c'i3I flsJe f7 nl n( thci nvention are 1 5 Icx-7 7.

I I,1 A metho~d of in.hibitinq the qrowt- p~tzc 2 in. animals which-. compries administerinq a pocozoali ratic 3 inhibitina amount cf an a-su:stitute-O amino acie or an S. 4 c,--suLsti tuted amine having the formlula Y amincl '4 M9 2 6 wherein 7 R1 is hydrogen or carboxy; 8 Y is selected from the go up conF'.Ftinq of 1-HF', 9 CHF2, CF3 a. 7:CH; where z is selected from the group consisucing H 2 N-.ICH>) t~et R iS, 11 1 2 N-CH-(CH-42 and HnN-CH?CH=CH; with the proviso Y is 6H 3 CH 1 that when R i.s hydrcen, Y canrnot bec- andZ z is 13 must b H2 N-CH--(CH2,)2 anno H2 CH- 14 the salIts and inc:'':ou al optic-a-i cm r hereof.

1 2 method according to c1inm 1 wherziln RI is an hydrocgen. t- h 2 combi 13. Amthod :0dvto jclaim Iwherxein 1 RI is 2 car boxy. 9. *stitu acid. 1o. are f

4. A method according to any one of the precedirn71-airis wherein Z is H 2 N-CH--(CH 2 or H 2 2 N-(CCH2 3

CH 3

A method accordinq to any one of the p-recedirin- c~ where Y is CHP 2 6. t' method accordina, to iny one of the rc.:clin wherein the animals are fowl and thezx-st,,.stitite& ar'iro acoi d c subs t itute6 amine is ad9ministered tr, tf-ei r- n vii wter at J ce norcenrat±o o :rox C. L t 7. A mthdaccords rc, to ci al :v 6 woei.he -b;tiuo amino acid or nK-sulbstj toted ar'ine is amrs-2a1i ±cnot

ration of from 0.06i: to I> S. AP. rotczoal infecti(n .nO Lrophvlla-tic tic it-rnet tioi, for -the treatment of an' mrl sorrsiz a) a Tprotozoal irnbiing aro'auc.t ci an suist t' e, amino acidl or an o- subs t ituted dav'1i nc laav ii-c; r 111

I

wherein R is hydrogen or carboxy; Q W Y is selected from the cro-up,cnnrisstjir, c rf 't CHF 2 CF 3 and C=CHf; Z is selected from thle roup cnit~ fI'-N 2NCH-(CF 2 )2 and HIN-CH 2C~h'7 wit1 11 i S:.th ;he *0000CH 0 3 Ris hydrogen, Y cannot beC., I~ctu

and the salts and individual o-)ptica-' ISuiTorsfe,,n combination with b) a suitablec -arrier. 9. A composition accorcii.a u a nhrin~ni&s4 stituted amino acid is -d iur'~t:vi~ p erio rj

acid. A composition accordir.,cTt(,alir; l iereirn tlie ani-nals are fowl, the carrier is water and the sibstitute-' aniita acidr

I BL ~NT A is 2 ,S-diamino-2-difluoromethylpentanoic- acid present at a concentration of from 0.01% to 2'2. 11. A composition according to claimr 8 wherein the o-substituted amino acied is 2,5-diamino-2-difluoromretb ylpenitanc, acid in combination with a synergistically effective amount ef an antiprotozoa'l agent selected from the group consisting o' Antrycide, Pentamtidine, Amnicarbalide and Bleornycin. 12. A i-iethod, as claimed in claimn I, substantially i hereinbcf-ore described with reference tc any one xamrples. cfth ii A Compositionr CKaime., iin Clail' 3, subs n a v s hereinbe fore describec' with- i-efel-ence to any11 Oo ftecarl.

DATED. 29 19 q~

TI'LLTPS ORMONF;{ AND ;ZP7 -J C? A-trornevs -Ior:

MERRELL DOW PHARMACET1 CAL.- TN-'

-51- BL

I..

11 Cd C),