Note: Descriptions are shown in the official language in which they were submitted.
M--1076--Cl
,t
1~4~33
METHOD OF I2aHIBITING THE t;ROWTH
OF PROT020A
Technical Fie_d
This invention relates to certain ~-substituted
amines and ~-substituted-amino acids which are useful in
inhibiting the growth of protozoa in animals and
particularly in poultry.
Background Art
Polyamines have been implicated in many aspects of
cell division. Impairment of the biosynthesis of poly-
amines by means of enzyme inhibitors is believed to cause
a decrease in cell proliferation in mammals. Although the
physiological role of polyamines has not been clearly
delineated, there is evidence to suggest their
involvement with cell division and growth, H.G. Williams-
Ashman et al., The Italian J. Biochem. 25, 5-32 (1976),
!~
~ 3 M-1076-C1
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
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-
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,
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-
ation product of lysine, has been shown to stimulate theactivity of S-adenosylmethionine decarboxylase and is
known to be essential to the growth processes of many
M-1076-C1
117~
--3--
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 ornithinedecarboxylase as a source of putrescine further supports
our observations 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-
carboxylase are useful in inhibiting the growth of protozoa.
Moreover, this inhibition occurs throughout a wide spectrum
of protozoa such as with members of the subphylum
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 or a-substituted-a-amino acids
having the general formula
y
Z-C-R
NH2
(I)
wherein Rl is hydrogen or carboxy; Y is selected from the
group consisting of CH2F, CHF2, CF3 and C-CH; 2 is
,
- M-1076-Cl
~7~ ;3
--4--
selected from the group consisting of H2N-~CH2)3,
H2N-CH-(CH2)~ and H2N-CH2CH=CH; with the proviso that
CH3
when Rl is hydrogen, Y cannot be CF3 and Z must be
H2N-CH-(CH2)2; and the salts and individual optical
CH3
isomers thereof.
When administered 1n 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 (I) above the symbol Rl is
represented either by hydrogen or a carboxyl group. Where
the symbol Rl is hydrogen a class of a-substituted amines
is delineated. Where the symbol Rl is the carboxyl group,
a class of ~-substituted-~-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-
methyl, difluoromethyl or trifluoromethyl radicals.
The symbol Z represents either the 3-aminopropyl
group, the 3-amino-3-methylpropyl group or the 3-amino-1-
propylene group. The saturated groups, viz. the 3-amino-
propyl group and the 3-amino-3-methylpropyl group represent
the preferred side chains.
M-1076-Cl
~L7~6~3
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 ~-substituted diamines where-
in the symbol Z is the 3-aminopropyl group or the 3-amino-
l-propylene group, i.e., those compounds having the
general formulae
y
H2N- (CH2) 3-CH-NH2
(II)
and
y
H2N-CH2CH=cH-cH-NH2
(III)
wherein the symbol Y is as previously defined.
Specifically excluded from the remaining ~-substituted
diamines is the species wherein the symbol Y represents
the trifluoromethyl group. Thus, compound (IV), 4-methyl-
1-trifluoromethyl-1,4-butanediamine, is specifically
excluded from the scope of compounds which can be usefully
employed.
C,F3
H2N-C,H- (CH2) 2-CH-NH2
CH3
(IV)
Included within the scope of compounds that can be
employed are ~-substituted amino acids having the formula:
:, Y,
H2N-(CH2)3-C-COOH
NH2
(V)
M-1076-Cl
~17.~ 3
H2N--CH- (CH2) 2-C--COOH
CH3 ~H2
(VI)
and
y
H2N-CH2CH=CH-C-COOH
NH2
(VII)
In formulae (V), (VI) and (VII) above, the symbol Y is as
previously defined.
The ~-substituted amines included within the scope
of compounds that can be usefully employed in the
practice of this invention are defined by the general
formula
y
H2N-CH- (CH2) 2-CH--NH2
CH3
(VIII)
wherein the symbol Y represents the CH2F, CHF2 and C--CH
groups, but in the case of the diamines exludes the CF3
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.
~7~3 M-1076-cl
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 (I) 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 ~-carbon
atom possesses an asymmetric center and that individual
optical isomers of these compounds exist. Accordingly,
both the d- and l-optical isomers as well as the racemic
mixtures are contemplated as being within the scope of
this invention
Lactam formation can occur where the symbol Rl
represents the carboxyl group and the symbol Z represents
the 3-aminopropyl moiety or the 3-amino-3-methylpropyl
moiety as represented by the following general formula
NH2
I
Y - C -- C = O
(CH2)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.
~Lf.7a~3 M-1076-Cl
--8--
Illustrative examples of compounds useful in
accordance with the teachings of this invention include:
2,5-diamino-2-(fluoromethyl)pentanoic acid,
2,5-diamino-2-(difluoromethyl)pentanoic acid,
2,5-diamino-2-(trifluoromethyl)pentanoic acid,
2,5-diamino-2-(ethynyl)pentanoic acid,
2,5-diamino-2-fluoromethyl-5-methylpentanoic acid,
2,5-diamino-2-difluoromethyl-5-methylpentanoic acid,
2,5-diamino-2-trifluoromethyl-5-methylpentanoic acid,
2,5-diamino-2-ethynyl-5-methylpentanoic acid,
2,5-diamino-2-fluoromethyl-3-pentenoic acid,
2,5-diamino-2-difluoromethyl-3-pentenoic acid,
2,5-diamino-2-trifluoromethyl-3-pentenoic acid,
2,5-diamino-2-ethynyl-3-pentenoic acid,
1-fluoromethyl-4-methyl-1,4-butanediamine,
2-difluoromethyl-4-methyl-1,4-butanediamine, and
l-ethynyl-4-methyl-1,4-butanediamine.
The compounds of general formula (I) wherein Z is
H2N-(CH2)3; Y is CH2F, CHF2 and CF3, 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 F2CH-,
at a temperature of about -120C to 120C, preferably
about 25 to 50C, for about 1/2 hour to 48 hours
followed by acid or base hydrolysis. This can be
represented by the following reaction sequence.
.
. M-1076-Cl
'16~3
g
Zl-,CH-COOR2 Zl-C( ) COOR2
R4 strong base~ ' N=C-R3
compounds 1
alkylating
reagent
~ ~0
Y, Y
z2-C-COOR2dilute aqueous Zl-C-COOR2
NH2~ acld/hydrazine_ N=,C-R3
R4
compounds 3 compounds 2
IH2O
I(acid/base)
H2N-tcH2)3-c-cooH
NH2
(Formula V)
In the above reaction sequence Y is FCH2-, F2CH-,
F3C-; R2 is a lower alkyl group, for example, methyl,
ethyl, isopropyl, n-propyl or n-butyl; R3 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 R3 and R4 taken together may form an alkylene
group of from 5 to 7 carbon atoms, that is,
-CH2-(CH2)m-CH2- wherein m is an integer of from 3 to 5.
Illustrative examples of straight or branched alkyl
groups of from 1 to 8 carbon atoms which R3 and R4 may
M-1076-Cl
~17~6~3
--10--
represent are, for example, methyl, ethyl, n-propyl,
isopropyl, n-butyl, tert-butyl, n-pentyl, neopentyl or
triethylmethyl groups. The symbol Zl is R3-C=N(CH2)3-,
O o R4
Rs-CN~(CH2)3- or R6-OCNH(CH2)3-; R3 and R4 are the same
and have the meanings defined above; and each of Rs and
R6 is phenyl, benzyl or a lower alkyl group of from 1 to
4 carbon atoms that is straight or branched, for example,
methyl, ethyl or isopropyl; Z2 is H2N(CH2)3-,
O O
Rs-CNH(CH2)3- or R6-OCNH(CH2)3- wherein Rs and R6 have
the above defined meanings.
Suitable strong bases which may be employed in the
above reaction se~uence 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, triethylamine, lithium
acetylide or dilithium acetylide. Lithium acetylide,
dilithium acetylide, sodium hydride, and lithium diiso-
propylamide are particularly preferred bases.
Suitable alkylating reagents which may be employed
in the above reaction sequence are illustratively chloro-
fluoromethane, bromofluoromethane, fluoroiodomethane,
chlorodifluoromethane, bromodifluoromethane, difluoro-
iodomethane, bromotrifluoromethane, chlorotrifluoromethane,
trifluoroiodomethane, bromochloromethane, dichloromethane,
chloroiodomethane, bromodichloromethane and dichloroiodo-
methane. These alkylating reagents ~re well known to
the art.
- M-1076-Cl
1~7'~ 3
--11--
Removal of the protecting groups of the amine and
carboxylic acid function may be achieved in one step by
treatment of compound 2 with aqueous acid, for example,
hydrochloric acid or toluenesulfonic acid, at a tempera-
ture of about 0 to 100C for about 4 to 24 hours to
give compounds of general Formula V. It is preferred
to remove first the protecting groups of the amine
function(s) of compounds 2 when said functions are pro-
tected as a Schiff's base by treating compounds 2 with
dilute aqueous acid, for example, hydrochloric acid or
with hydrazine or phenylhydrazine in solvents, such as,
lower alcohols, for example, methanol or ethanol, ethers,
chlorinated hydrocarbons, benzene and water. Remova
of the protecting groups of the carboxylic acid function
and the amine groups when the amine groups are protected
other than as a Schiff's base is achieved by treatment
of compounds 3 with concentrated aqueous acids, for
example, hydrobromic acid at a temperature of about 0 to
100C or in aqueous base, for example, ammonium hydroxide.
The amine protected ester derivatives, that is,
compounds 1, wherein R3 is other than methoxy or ethoxy,
are prepared by treating an appropriate amino acid ester
with a carbonyl bearing compound to form a Schiff's base
in a generally known manner, specifically: (a) when R3
is hydrogen, by treating the appropriate amino acid ester
with benzaldehyde or an alkanal having from 1 to 9
carbon atoms being straight or branched, for example, 1-
propanal, l-butanal, 2,2-dimethylpropan-1-al or 2,2-
diethylbutan-l-al; (b) when R3 is phenyl by treating
the appropriate amino acid ester with benzophenone or
phenyl alkyl ketone wherein the alkyl moiety has from 1
to 8 carbon atoms and is straight or branched, for
example, phenyl methyl ketone, phenyl ethyl ketone, phenyl
isopropyl ketone, phenyl n-butyl ketone or phenyl tert-
.
M-1076-C1
3L~746~3
-12-
butyl ketone; and (c) 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 alkyl
ketone as described above or with a di-alkyl ketone
wherein each alkyl moiety has from 1 to 8 carbon atoms
and is straight or b anched, for example, dimethyl ketone,
diethyl ketone, methyl isopropyl ketone, di-n-butyl
ketone or methyl tert-butyl ketone. The carbonyl bearing
compounds are known in the art or may be prepared by
procedures well known in the art.
When R3 is methoxy or ethoxy in compound 1, an
appropriate amino acid ester derivative is reacted with
benzoyl halide or an alkanoic acid halide wherein the
alkanoic acid has from 1 to 9 carbon atoms and may be
lS straight or branched, such as, acetyl chloride, propionyl
chloride, butyryl chloride, tert-butyrl chloride, 2,2-
diethylbutyric acid chloride or valeryl chloride. The
reaction is conducted at 0C in an organic solvent such
as ether, methylenechloride, dimethylformamide, dimethyl-
acetamide or chlorobenzene in the presence of an organic
base such as triethylamine or pyridine. ~ollowing the
reaction, the reaction mixture is allowed to warm to
about 25C for one hour. The resulting amide derivative
is combined with an alkylating reagent, such as methyl-
fluorosulfonate, dimethylsulfate, methyliodide, methyl
~-toluenesulfonate or trimethyloxonium hexafluorophosphate
(when R3 is methoxy) or triethyloxonium tetrafluoroborate
(when R3 is ethoxy) at about 25C 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 25C and an organic base
such as triethylamine or pyridine is added after which
the solution is extracted with brine and the product
isolated.
a~
3 M- l ~ 7 6 - C I
When R3 and R4 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.
o
When the symbol Zl in compounds 1 is RsCNH(CH2)3-
O O O
or R6OCNH(CH2)3-, the -CRs and -COR6 protecting groups
are added to the corresponding free amino acid, i.e.,
ornithine, 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
o
(when Zl is RsCNH(CH2)n-) or an appropriate alkyl or
aryl haloformate (when Zl is R6OCNH(CH2)n-), in a solvent
such as acetone in the presence of a base sush 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 Rl is carboxy are prepared from the corresponding
amino acid esters having the structure
y
H2N(CH2)3-C-cOR7
NH2
(X)
wherein Y 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,
M-1076-Cl
~a17466?3
-14-
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 hour to 24 hours at
a temperature of from about 0 to 120C, optionally under
a nitrogen atmosphere. The compounds of general Formula
(X) 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 HCl gas.
The compounds of general formula I wherein Z is
H2N-CH~CH2)2; Y is CH2F, CHF2 and CF3, and R1 is carboxy
CH3
are prepared via procedures that are analogous to those
just described.
Compounds of general Formula I wherein Z is
H2N-CH(CH2)2 or H2N-(CH2)3, Y is C CH and Rl is carboxy
CH3
are prepared by treating a suitably protected
propargylamine derivative, such as a silyl derivative,
with a strong base to form a protected propargylamine
carbanion intermediate. This carbanion intermediate is
reacted with an alkylating reagent of the formula RgX,
wherein X is a halogen such as chlorine or bromine, and
R8 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
~746~3
alkylated protected propargylamine carbonaion. The
second carbanion 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:
(Rg)3-Si-c-c-clH2 strong (Rg)3-Si-C--C-CH( )
N=C,-Rll base ~ N=C~-Rll
Rlo ,Rlo
compounds 4 ~Rgx
(R7)3-si-c~c-cq-) strong (Rg)3-Si-C=c-cH
N=C-Rll ~base N=C-Rll
Rll) , Rlo
acylating compounds 5
!reagent
,R8 z
(Rg)3-Si-C~C-C-R12 H2o HC~C-C-COOH
~ (acid/base)
Rlo
compounds 6 Formula XI
In the above reaction scheme R~ and X have the meanings
defined hereinabove, Ph represents phenyl, R1o is hydrogen,
methoxy or ethoxy, Rll is phenyl, tert-butyl, or triethyl-
methyl, Rg is a straight or branched lower alkyl group
having from 1 to 4 carbon atoms, R12 is a carboxy anion,
M-1076-Cl
~7~3
a carboxylic acid ester, a carboxamide, a nitrile or other
group capable of being hydrolyzed to a carboxylic acid
function, and Z is H2N-c-(cH2)2 or H2~(C~2)3-
CH3
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
alkylithium compounds that may be employed include butyl
lithium or phenyl lithium, lithium diisopropylamide,
lithium amide, tertiary potassium butylate and sodium amide.
The alkylating reagents, RgX, 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
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.
Solvents that can be employed include diethyl ether, tetra-
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,
N,N-dimethylcarbamoyl chloride, 2-methylthio-1,3-dithio-
linium iodide, ethylene carbonate or ethylene trithio-
carbonate. When 2-methylthio-1,3-dithiolinium iodide is
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.
M-1076-Cl
:~17~6~3
-17-
The reaction temperature varies from -120C to about 25C,
a preferred reaction temperature being about -70C, with
the reaction period ranging frvm about l/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
Rlo 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 propargylamine is accomplished by forming a Schiff's
base with a nonenolizable carbonyl bearing compound such
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 l
to 4 carbon atoms and is either straight or branched.
Trialkylsilyl chlorides that can be utilized include
trimethylsilylchloride or triethylsilylchloride.
The propargylamine derivatives of compounds 4, wherein
Rlo is methoxy or ethoxy are prepared by reacting propar-
gylamine wherein the acetylene function is protected by
a trialkylsilyl group with benzoyl chloride, pivalic acid
chloride, or 2,2-diethylbutyric acid chloride at 0C 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 25C for
.
M-1076-Cl
6~3
-18-
one hour. The resulting amide derivative is combined
with an alkylating reagent, such as, methylfluorosulfonate,
dimethylsulfate, methyliodide, methyl p-toluenesulfonate
or trimethyloxonium hexafluorophosphate when Rlo is
methoxy, or triethyloxonium tetrafluoroborate when Rlo is
ethoxy, at about 25C in a chlorinated hydrocarbon solvent
such as methylene chloride, chlorobenzene or chloroform.
The reaction mixture is refluxed for about 12 to 20 hours,
cooled to about 25C and an organic base such as triethyl-
amine or pyridine is added. The resulting solution isextracted with brine and the desired product isolated
therefrom.
The protected propargylamine starting material is
obtained by treating a 3-trialkylsilylprop-2-ynyl-1-imino-
benzyl derivative, that is compounds 4 wherein Rlo ishydrogen and Rll is phenyl, with hydrazine or phenylhydra-
zine at about 25C for about 1/2 hour after which the
mixture is diluted with, for example, petroleum ether,
benzene or toluene and the amine isolated. Alternatively,
the imine is hydrolyzed with 0.5 to 1 N HCl solution, an~
the aqueous phase evaporated to afford the amine as the
hydrochloride salt.
Compounds of formula I wherein Z is H2N-CH(CH2)2;
CH3
Y is CH2F or CHF2; and Rl is hydrogen are prepared by
reducing a ketone of the formula
o
Z '--C--Y
(XII)
,R13 R113
wherein Z' is phthaloyl-NCH(CH2)2-, benzoyl-NHCH(CH2)2-,
- M-1076-Cl
~17~3
--19--
R,13 R,13
alkanoyl-NHCH(CH2)2-, alkoxycarbonyl-NHCH(CH2)2- or
R,l~
benzyloxycarbonyl-NHCH(CH2)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 CHF2 and R13 is methyl.
The ketones are reduced to the corresponding alcohol
which is treated with one equivalent of an imide, such
as, phthalimide, succinimide 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
solvent. Suitable solvents include ethers such as diethyl
ether, tetrahydrofuran or ~-dioxane, benzene or dimethoxy-
ethane. The reaction is conducted at about 0 to 100C,
preferably about 25C, for a period of about one-half
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.
The compounds of general formula (XII) wherein Y is
FCH2- are prepared by treating a compound of the formula
R
z ' -C-CH2R14
(XIII)
wherein Z' is defined as above and R14 is a suitable
leaving group, such as chlorine, bromine or iodine,
mesylate, tosylate, triflate or trifluoroacetate with an
appropriate fluorinating reagent, such as, potassium
fluoride, silver fluoride, cesium fluoride, thallium
fluoride, tetrabutylammonium fluoride in a suitable
solvent. Suitable solvents include solvents such as
M-1076-Cl
3L174~;Q3
-20-
dimethoxyethane, dimethylsulfoxide, dimethylformamide,
ethylene glycol, acetonitrile, acetone, benzene or hydrogen
fluoride. The reaction is conducted at a temperature of
from about 0 to 200C for a period of about 2 to 48 hours.
The leaving group Rl4 may also be a diazo group in which
case the fluorinating reagent employed is hydrogen fluor-
ide/pyridine. Suitable solvents for the reaction wherein
R14 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 -20 to 65C. Illustratively, a com-
pound of the formula
o
Z '-C-R14
as defined above wherein Rl4 is a diazo group in a suitable
aprotic solvent is added to a solution of hydrogen fluor-
ide/pyridine and cooled to -10C. The reaction mixture
is stirred vigorously at -10C for l hour, warmed to about
25C for 2 hours and then poured over ice. The organic
phase is separated, washed with a base such as sodium
bicarbonate, dried over magnesium sulate and concentrated
under vacuum to afford the appropriate fluoromethyl ketone
derivative of formula (XII).
The diazo ketone derivatives, that is, the compounds
of formula (XIII) wherein R14 is a diazo group, can be
obtained via the corresponding acid halide represented by
a compound of the formula
o
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 -~0 to 20C, followed
M-1076-Cl
-21- 1174~3
by vigorous stirring at about 25C for about l to 24
hours. The diazo ketone derivative so obtained can be
isolated using standard procedures such as evaporation of
the solvent with subsequent purification by recrystallization
or chromatography. ~lternatively, 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 R14 is, for example, halogen,
mesylate, tosylate, triflate, or trifluoroacetate using
procedures generally known to the art. To obtain compounds
of general formula (XIII) wherein R14 is halogen, such
as, chlorine, bromine, or iodine the corresponding compound
of formula (XIII) in which Rl4 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 Rl4
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,
the 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
o
Z'-C-halide
as described above, are known compounds which can be pre-
pared from the corresponding acids. Thus, for example,
M-1076-Cl
~'7~ 33
-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 0C 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 0 to 40C for about 1 to 24 hours also results
in the preparation of the corresponding acid halide.
The compounds of general formula (XII) wherein Y
Rl
is FCH2- and Z' is other than benzoyl-NHC(CH2)2- or
,Rl
alkanoyl-NHC(CH2)2- may also be obtained by treating a
compound of the formula
z2-Rl5
(XIV)
R,13
wherein Z2 is phthaloyl-N-CH(CH2)2-, alkoxycarbonyl-
R,13 R,13NHC(CH2)2-, benzyloxycarbonyl-NHC(CH2)2-, ~-methylthioethyl
or ~-benzylthioethyl; R13 is methyl; and Rls is chlorine,
bromine or iodine, mesylate or tosylate. Thus, a compound
of formula (XIV) is reacted with triphenylphosphine or
tri-(lower)-alkylphosphine, for example, tri-n-butyl-
phosphine, in a solvent such as benzene, toluene, methanol,
ethanol, acetonitrile, tetrahydrofuran, diethyl ether or
dimethoxyethane. The reaction is conducted at a temperature
ranging from 25C to the reflux temperature of the solvent
M-1076-Cl
-23-
for ajperiod 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 trialkylphos-
phonium salt is added to an excess (up to 25%) of sodiumor lithium 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,
nitrogen or argon. An appropriate solvent, such as,
benzene, toluene, diethyl ether, tetrahydrofuran or di-
methoxyethane is added and the resulting substituted
methylidenephosphorane is collected. 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 0C
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
hydrochloric or hydrobromic acid or an organic acid such
as trifluoroacetic acid or ~-toluene sulfonic acid in the
presence of a cosolvent such as tetrahydrofuran, diethyl
ether, or benzene for a period of from about 30 minutes
to 24 hours at a temperature ranging from about 0C to
the reflux temperature of the solvent. The amount of
acid employed may vary from a catalytic amount to a
concentrated acid solution.
As used in general formula (XIV) the term
R13
phthaloyl-N-CH(CH2)2- is taken to mean the group
o
C Rl 3
~-~ M-1076-Cl
4~3
-24-
R113
the term alkoxycarbonyl-NHCH(CH2)2- is taken to mean
O R13
the group alkyl-O-C-NHCH(CH2)2-, the term benzyloxycar-
R13bonyl-NHCH(C~2)2- is taken to mean the group ~ -CH2-
O ,Rl 3
OCNHCH(CH2)2-, wherein R13 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
F2CH- are obtained by treating ~[(methylsulfinyl)methyll-
- thio]methane or [[(ethylsulfinyl)methyl]thio]ethane 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 R16 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
from chlorodifluoromethane, bromodifluoromethane, and
difluoriodomethane. The alkylation reaction is followed
by a hydrolysis using an aqueous acid solution.
Suitable strong bases which may be employed in
preparing the difluoromethyl substituted ketone derivatives
as described above are sodium hydride, dilithium acetylide,
lithium diisopropylamide, butyllithium, potassium tert-
butoxider sodium tert-butoxide, lithium tert-butoxide,
phenyllithium, methyllithium, sodium amide, lithium amide
or potassium hydride.
M-1076-C1
~7a~6~3
-25-
The alkylation reaction described in preparing the
difluoromethyl ketone derivatives are carried out in an
appropriate solvent, for example, tetrahydrofuran, di-
ethyl ether, hexamethylphosphortriamide, dimethylsul-
foxide, or benzene. The reaction is conducted at atemperature ranging from about -78 to 65C for a period
of from about 30 minutes to 24 hours. Preferably, a
temperature of about 40C is utilized for the difluoro-
methyl alkylation step. The alkylated sulfinyl inter-
mediates are isolated by quenching with a brine solutionfollowed by extraction utilizing diethyl ether, dichloro-
methane, or benzene. The alkylated sulfinyl intermediates
are recovered from the combined extracts.
Hydrolysis of the alkylated sulfinyl derivatives to
the ketone is achieved using an aqueous mineral acid
solution, such as, hydrochloric, hydrobromic, perchloric
or sulfuric acids in a solvent such as tetrahydrofuran,
acetonitrile, diethyl ether or benzene. The hydrolysis
is conducted at a temperature ranging from about -20 to
105C, preferably about 25C for a period of from about
30 minutes to 24 hours, preferably about 2 hours. Generally,
a solution of 0.3 equivalents of mineral acid in 1.5~
water is employed. The specific examples described below
further illustrate the preparation of the difluoromethyl
ketone derivatives of formula (XII).
The compounds of formulas (XIV) and (XV) wherein R15
and R16 are halogen are known to the art or can be pre-
pared from an appropriate carboxylic acid derivative
having the formula
Z4-COO~
(XVI)
-- M-1076-Cl
-26-
R13 R13
wherein Z4 is phthaloyl-NCH(CH2)-, benzoyl-NHCH(CH2)-,
R,13 R113
alkanoyl-NHCH(CH2)-, alkoxycarbonyl-N~CH(CH2)-,
R13
benzyloxycarbonyl-NHCH(CH2)-, methylthiomethyl or
benzylthiomethyl. These acids are known to the art or
can be obtained by known procedures from the corresponding
unprotected amino acids~ The compounds of formulas (XIV)
and (XV) wherein Rls and R16 are mesylate or tosylate
may be prepared by treating the corresponding derivatives
in which Rls and R16 are halogen with a metal salt of
methanesulfonic acid or ~-toluenesulfonic acid. Illustra-
tively, the sodium salt of methanesulfonic acid or ~-
toluenesulfonic acid can be utilized.
Reduction of the ketones of formula (XII) to the
corresponding alcohols is achieved chemically using 1 to
10 equivalents of a metal hydride, such as lithium boro-
hydride, sodium borohydride, sodium cyanoborohydride, or
lithium aluminum hydride. In addition the ketones can be
reduced with borane or dimethylthioborane or catalytically
reduced using, for example, Raney nickel, rhodium, palladium
on charcoal, or platinum oxide. In general, the reaction
time varies from about 10 minutes to 24 hours and the
temperature at which the reduction is conducted can range
from about -40C to 100C depending upon the particular
reducing reagent employed. When hydride or borane re-
duction is employed the reaction is conducted in a suitablesolvent for a period of time from about 10 minutes to 24
hours with temperatures ranging from about -40C to 65C.
Suitable solvents that can be employed for reduction of
compounds of general formula (XII) include lower alcohols,
such as methanol or ethanol, or ethers, such a.s diethyl
ether or tetrahydrofuran. When catalytic reduction is
.~
~ M-1076-Cl
~:7~6~;~
-27-
employed the reaction time varies from about 1 hour to 24
hours, the reaction temperature ranges from about 25 to
100C and the hydrogen pressure can range from 1 to 120
atmospheres.
Hydrolysis to the amine and the removal of any distal
amine protecting group is achieved using a strong mineral
acid such as hydrochloric acid, hydrobromic acid or sulfuric
acid, or an organic acid such as toluenesulfonic acid or
trifluoroacetic acid. The hydrolysis is conducted in
water or an aqueous solvent at the reflux temperature for
a period of from about 4 to 48 hours. Alternatively, 1
to 3 equivalents of hydrazine, methylhydrazine or
methylamine can be utilized at a temperature of from
about 25C to the reflux temperature of the solution for
about 1 to 12 hours, followed by treatment with a strong
mineral acid or organic acid as described above.
Compounds of formula I wherein Z is H2N-CH(CH2)2;
CH3
Rl is hydroxy; and Y is C CH, or as hereinafter indicated
the alkynyl group, are prepared by the hydrolysis of the
alkylated compounds 5, described above. The desired
alkylating reagents RgX that are employed can be prepared
by methods known to the art. Thus, the reagent
PhHc=NcH2(cH2)2-
can be prepared by reacting 3-bromo-n-propylamine
hydrochloride with benzaldehyde and an organic trialkyl-
amine, such as triethylamine, in a solvent such as diethyl
ether, tetrahydrofuran, dioxane, chloroform or dichloro-
methane. The reactant
C,H3
PhHC=~CH(CH2)2-
is prepared by reacting 3-aminobutylbromide hydrobromide
with benzaldehyde and an organic amine such as triethyl-
amine. The compound 3-aminobutylbromide hydrobromide is
M-1076-Cl
~L17~6~3
-28-
a known compound that can be prepared from the corres-
ponding alkanol by treatment with concentrated HBr at
a temperature of from 25C to 110C for a period of
from 1 to 12 hours. The ~-aminoalkanol derivative is
obtained by treating an appropriate ~-keto-alkanoic acid
ester the formula
0, 0
CH3C-CH2C-OcH2cH3
The ~-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 25 to 50C
for a period ranging from 1 to 12 hours. Subsequent
hydrolysis of the ester moiety results in the formation
~f the y-aminoalkanol.
The alkylating reactic,n may be carried out in an
aprotic solvent, for example, benzene, toluene, ether,
tetrahydrofuran, dimethylsulfoxide or hexamethyl phos-
phortriamide. The reaction temperature varies from about
-100 to 25C preferably about -70C and the reaction
2~ time varies from about 1/2 hour to 24 hours.
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 aqueous
base, for example, sodium hydroxide or potassium or treat-
ment with phenylhydrazine, hydroxylamine or hydrazine
then with aqueous base.
The individual optical isomers of compounds of
formula I wherein Rl is carboxy or hydrogen are resolved
using a (+) or (-) binaphthylphosphoric acid salt in
accordance with the procedure of R. Viterbo et al.,
Tetrahedron Letters 48, 4617 (1971). Other resolving
agents such as ~+) camphor-10-sulfonic acid may also be
employed. Alternatively, when Z is H2~-rH-(CH2)2 or
CH3
M-1076-Cl
117~6~3
-29-
H2N(CH2)n, resolution is achieved via the lactam of said
compounds. The thus resolved acids and amines ~ay 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 or 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
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.
3~ 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
M-1~76-Cl
:11746~3
-30-
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.
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.
Inter alia, the compounds described herein are irreversi-
ble inhibitors of ornithine decarboxylase and S-
adenoxylmethionine decarboxylase. As irreversible
inhibitors of these enzymes, these compounds inhibit
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 the compounds of this invention are
able to effectively inhibit the growth of protozoa.
M-1076-Cl
:a~746~3
The effect of the compounds of general for~ula (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 birds are kept in batteries 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 lO0,000
sporylated oocysts via gavage. The test compound is
administered in the particular dosage 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
parasites twenty four hours prior to testing. Control
animals so infected generally die 5 days subsequent to
innoculation. The compound to be tested is ad~inistered
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 controlanimals as indicated by an examina~ion of blood smears.
The compounds described herein are employed in
M-1076-Cl
-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 Trypanosoma 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
15 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 nature of the protozoal infection to be treated
whether prophylactic or therapeutic, the severity of
the infection and the period of treatment.
Thus, for example the compound 2-difluoromethyl-2,5-
diaminopentanoic acid can be effectively administered to
chickens for the treatment of coccidiosis one day prior
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-
diaminopentanoic acid as low as 0.015~ in the drinking
water of chickens. Preferably, a prophylactic concentra-
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
M-1076-Cl
4~;~3
-33-
the epithelial cells of the caecum as a trophozoite
stage. 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 doses of currently available chemotherapeutic
agents, that are primarily cidal in nature. Consequently,
medicated feeds are now routinely employed in commercial
flocks, so that all commercial fowl 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,
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 permanently immunize the
host from future E. tenella infections.
The pharmaceutical compositions that are particularly
suited for the prophylaxis or treatment of protozoal infec-
tions in fowl comprise the heretofore described ~-substituted-
amines or the ~-substituted-~-amino acids in combination
M-1076-Cl
~1'74~ 3
-34-
with a 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 aqueous solution of the
active ingredient is employed in the management and treat-
ment of coccidiosis 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 (I) 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
aceturate; Pentamidine isethionate; Primaquine; Tryparsamide;
Amicarbalide; Amprolium; Amphotericin B; quinine; ~onensin;
Minocycline, 7-dimethylamino-6-demethyl-6-deoxytetracycline;
Clindamycin, 7-deoxy-7(S)-chlorolincomycin; Buquinolate;
Robenidine; and Nicarbazin. In some instances the compounds
of formula (I) 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
M-1076-Cl
:~17~6~3
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 (I) can be
used in combination with other known cytotoxic agents for
the chemotherapy and chemoprophylaxis of parasitic
diseases, particularly trypanosomiasis. Such cytotoxic
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 mustard, 1,3-bis(2-
chloroethyl)-l-nitrosourea (BCNU), 1-(2-chloroethyl)-3-
cyclohexyl-l-nitrosourea (CCNU), busulfan or adriamycin.
Of particular interest 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 Bleomycin. 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-Cl
~7~6~3
-36-
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 dose of the drug used 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 moreparticularly illustrated in conjunction with the following
Examples specifically describing how the compounds of
this invention can be prepared and utilized.
EXAMPLE 1
2-Difluoromethyl-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 l.S liters of tetrahydrofuran
at -78C 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
temperature is raised to 40C and maintained between 40
and 50C for 3 hours during which time chlorodifluoro-
methane gas is bubbled through the mixture with stirring.
The reaction mixture is then treated with a saturated
solution of sodium chloride. The organic material is
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 lN HCl (1.5 1) for 3 hours, the
mixture extracted several times with chloroform and the
aqueous solution evapo.ated to dryness. The oily residue
is refluxed with 12N hydrochloric acid (1.5 1)
M-1076-Cl
~7a~6!r~3
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 i5 adjusted to 3.5 by the 2ddition
S of triethylamine, the ~olution treated again with char-
coal before concentration to about 5û0 ml and dilution
with 7-8 liters of acetone. The precipitated product is
filtered off and washed with ethanol. The crude product
is recrystallized by dissolving in about 150 ml hot
10 water and treatment of the solution with hot ethanol
(450 ml). On cooling crystals of 2-difluoromethyl-2,5-
diaminopentanoic acid hydrochloride monohydrate separaLe
71 g (37%), m.p. 183C.
EXAMPLE 2
lS ~-Ethynyl-~,~-diaminovaleric acid
11.~ 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 (6.78 ml,
0.048 M) of diisopropylamide in 60 ml of tetrahydrofuran
20 and 23.6 ml of 2.05 M solution of n-butyllithium at -70C
after which 9.5 g (0.042 M) of N-(3-bromopropyl)benzylimine
is added, and the mixture is stirred at -70C for 5-1/2
hours. To the reaction mixture is added 23.6 ml of a
2.05 M solution of n-butyllithium followed by the addition
25 of 4 5 g (3.67 ml, 0.048 M) of methyl chloroformate.
After 30 minutes at -78C the mixture is treated with
brine, and the reaction product ls isolated by ether
extraction. The ether extract is e~aporated and 300 ml
of 3 N HCl is added to the resulting residue and the
30 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 dryne ,s. The residue is triturated with
ethanol, filtered and the ethanol ~vaporated. The residue
` 11746~3 M-1076-Cl
-38-
is dissolved in water, the pH adjusted to 6, and the
solution is applied to a column of Amberlite 120 H+,
eluting with 1 M NH40H which affords, upon recrystalliza-
tion from ethanol-water, ~-ethynyl-~, ~-diaminovaleric
acid, M.P. 168-169 (dec.).
In the above procedure N-(3-bromopropyl)benzylimine
is prepared from 3-bromopropylamine and benzaldehyde by
procedures generally known in the art.
EXAMPLE 3
1-Fluorometh 1-1 4-butanediamine dihvdrochloride
Y
To a solution of 40 mmole of diazomethane in 110 ml
of ether cooled to 0C and magnetically stirred is added
under nitrogen dropwise over a period of 1 hour a solution
of 20 ml of 4-phthalimidobutyryl chloride in 75 ml of
ether. Stirring is continued for 1 hour at 25C after
which the reaction mixture is added to a solution of
40 ml of HF/pyridine precooled to 0C. The resulting
heterogeneous mixture is stirred at 25C for 1-1/2 hours
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. Concentra-
tion of the solvent under reduced pressure affords a
solid which is recrystallized from diethylether/pentane
to give fluoromethyl 3-phthalimidopropyl ketone, m.p.
92C.
To a solution of 550 mg (2.2 mmole) of fluoromethyl
3-phthalimidopropyl ketone in a mixture of 5 ml of
tetrahydrofuran and 5 ml of methanol cooled to -20C is
added a solution of 0.8 mmole of sodium borohydride in
a mixture of 5 ml of tetrahydrofuran and 5 ml of methanol
precooled to -20C. The reaction mixture is stirred for
15 minutes at -20C and then neutralized with 2 M ~Cl to
a pH of 1. The solvents are evaporated under reduced
pressure and the residue is partitioned between water
* Trade Mark
~ ~746~3 M-1076-Cl
-39-
and chloroform. The organic phase is washed with brine,
dried over magnesium sulfate and concentrated to give a
residue which is recrystallized from tetrahydrofuran-
diethylether to afford l-fluoro-5-phtllalimido-2-pentanol,
m.p. 85C. A mixture of 264 mg (1.05 mmole) of l-fluoro-
5-phthalimido-2-pentanol, 170 mg (1.05 mmole) of the
phthalimide, 302 mg (1.05 mmole) of triphenylphosphine and
201 mg (1.15 mmole) of diethylazodicarboxylate in 8 ml of
tetrahydrofuran is stirred under nitrogen for 2 hours at
25C. The solvent is evaporated under reduced pressure
and the residue taken up in benzene. The insoluble
material is discarded and the residue obtained after con-
centration of the filtrate is recrystallized from tetra-
hydrofuran-diethylether to give l-fluoromethyl-1,4-butane-
diyl-bis-phthalimide, m.p. 112C. A suspension of 3.1 g
of l-fluoromethyl-1,4-butanediyl-bis-phthalimide in 140 ml
of concentrated HC1 is heated at a reflux temperature for
3 days. The phthalic acid which precipitates on cooling
to 4C is filtered off. The filtrate is concentrated to
about 20 ml and cooled to 4C. 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
and then recrystallized from absolute ethanol to give
1-fluoromethyl-1,4-butanediamine dihydrochloride, m.p.
154C.
EXAMPLE 4
l-Fluoromethyl-4-methyl-1,4-butanediamine dihydrochloride
To a solution of 40 mmole of diazomethane in 110 ml
of ether cooled to 0C and magnetically stirred is added
under nitrogen dropwise over a period of 1 hour a solution
of 20 ml of 4-phthalimido-4-metnylbutyryl chloride in 7S
ml of ether. Stirring is continued for 1 hour at 25C
after which the reaction mixture is added to a solution
M-1076-Cl
11746~3
-40-
of 40 ml of HF/pyridine precooled to oC. The resulting
heterogeneous mixture is stirred at 25C for 1-1/2 hours
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. Concentration of the solvent
under reduced pressure affords a solid which is recrystal-
lized from diethylether/pentane to give fluoromethyl 3-
phthalimido-3-methylpropyl ketone.
To a solution of 550 mg (2.2 mmole) of fluoromethyl
3-phthalimido-3-methylpropyl ketone in a mixture o 5 ml
of tetrahydrofuran and 5 ml of methanol cooled to -20C is
added a solution of 0.8 mmole of sodium borohydride in a
mixture of 5 ml of tetrahydrofuran and 5 ml of methanol
precooled to -20C. The reaction mixture is stirred for
15 minutes at -20C and then neutralized with 2 M HCl to a
pH of 1. The solvents are evaporated under reduced
pressure and the residue is partitioned between water and
chloroform. The organic phase is washed with brine, dried
over magnesium sulfate and concentrated to give a residue
which is recrystallized from tetrahydrofuran-diethylether
to afford l-fluoro-5-phthalimido-5-methyl-2-pentanol. A
mixture of 264 mg (1.05 mmole) of 1-fluoro-5-phthalimido-
5-methylpentanol, 170 mg (1.05 mmole) of the phthalimide,
302 mg (1.05 mmole) of triphenylphosphine and 201 mg (1.15
mmole) of diethylazodicarboxylate in 8 ml of tetrahydro-
furan is stirred under nitrogen for 2 hours at 25C. the
solvent is evaporated under reduced pressure and the
residue taken up in benzene. The insoluble material is
discarded and the residue obtained after concentration of
the filtrate is recrystallized from tetrahydrofuran-
diethylether to give l-fluoromethyl-4-methyl-1,4-butane-
diyl-bis-phthalimide. A suspension of 3.1 g of l-fluoro-
methyl-4-methyl-1,4-butanediyl-bis-phthalimide in 140 ml
of concentrated HCl is heated at reflux temperature for 3
days. The phthalic acid which precipitates on cooling
M-1076-Cl
:~174~3
-41-
to 4C is filtered off. The filtrate is concentrated to
about 20 ml and cooled to 4C. 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 bolling isopropyl alcohol 3 times
and then recrystallized from absolute ethanol to give
l-fluoromethyl-4-methyl-1,4-butanediamine dihydrochloride.
EXAMPLE 5
l-Ethynyl-4-methyl-1,4-butanediamine
To 10.8 g (0.05 M) of 3-trimethylsilylprop-2-ynyl-1-
iminobenzyl in 500 ml of tetrahydrofuran under nitrogen
atmosphere at -78C is added n-butyllithium (0.05 M).
After 10 minutes the dark red carbanion is treated with
11.3 g (0.05 M) of 4-bromo-2-iminobenzylbutane in 20 ml
of tetrahydrofuran. After 3 hours at -78C, fifty ml of
water is added and the tetrahydrofuran is evaporated
leaving a residue which is heated at reflux under nitrogen
atmosphere with 100 ml of 6 N hydrochloric acid for 48
hours. Upon cooling the aqueous solution is washed with
methylene chloride, made alkaline with aqueous sodium
hydroxide and reextracted with methylene chloride. The
methylene chloride extract is dried over magnesium sul-
fate, filtered, concentrated and distilled to afford 1-
ethynyl-4-methyl-1,4-butanediamine.
EXAMPLE 6
Granules suitable for addition to the drinking water
of poultry are prepared as follows:
Grams
2-Difluoromethyl-2,5-diaminopentanoic acid 33.0
Corn starch 18.5
Lactose 48.2
Zinc stearate 0.3
100 .0
- M-1076-Cl
117~ 3
-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-
methyl-2,5-diaminopentanoic 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 38C to a moisture
content of approximately 3%, 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-
difluoromethyl-2,5-diaminopentanoic 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 useful for the prevention of coccidiosis
in poultry.
EXAMPLE 8
A medicated animal feed suitable for poultry is
prepared utilizing the following ingredients. The birds
are fed the medicated feed ad libitum.
'.
.
.:
M-1076-Cl
~1746~33
-43-
Percent by weight
Ground yellow corn......................... 60.3
Soy bean oil meal.......................... 33.0
Alfalfa leaf meal.......................... 1.0
Dicalcium phosphate........................ 3.0
Calcium carbonate.......................... 1.0
Iodized salt............................... 0.2
2-difluoromethyl-2,5-diaminopentanoic acid. .33
Vitamin-mineral-amino acid antibiotic mix
to furnish the following per 100 pounds
of feed:
Oxytetracycline....................... 0.5 gm
Penicillin ~as procaine salt)......... 0.25 gm
Manganese sulfate..................... 8 gm
DL-methionine......................... 22.7 gm
Riboflavin............................ 130 mg
DL-calcium pantothenate............... 930 mg
Niacin................................ 1400 mg
Pyridoxine............................ 130 mg
Vitamin B12........................... 1 mg
Choline chloride...................... 22.7 gm
Vitamin A............................. 300,000 units
Vitamin D3............................ 25,000 units
EXAMPLE 9
The following illustrates the effect of 2-difluoro-
methyl-2,5-diaminopentanoic acid on Trypanosoma brucei
brucei infections in mice.
Groups of five mice weighing 20-25 g are innoculated
with _ b. brucei (EATRO 110 isolate; 5 x 105 organisms/-
mouse). The compound is administered via drinking water,
_ libitum, 24 hours following infection. Results are
expressed as average survival (in days) beyond the death
of the control animals, based upon an average survival of
control animals of five days. Berenil (diminazene
aceturate) is included as a control trypanocide. The
results are indicated in Table I below.
-- M-1076-Cl
1174t~3
-44-
TABLE I
TotalAverage
DrugTreatment regimen DoseSurvival
(mg)(Days)
None
52-difluoromethyl-2,5-diaminopentanoic acid
2% in drinkina water, 6 days 600a ~30b
2% in drinking water, 3 days 300a >30
1% in drinking water, 6 days 300a ~30
1% in drinking water, 3 days I50a >30
100.5% in drinking water, 3 days 75a 28.6
0.1% in drinking water, 3 days l5a 2
300 mg/kg p.o., daily 3 days 22.5 26.3
150 mg/kg p.o., daily 3 days 11.3 22.8
75 mg/kg p.o., daily 3 days 5.6 19.2
1550 mg/kg p.o., daily 3 days 3.8 0
2-methyl-2,5-diaminopentanoic acid
2~ in drinking water 3 days 3ooa o
diminazene aceturate
2.5 mg/kg i.p. daily, 3 days 0.2 >30
a Based upon a daily intake of 5 ml water/25 g mouse/day
b Considered curative. Animals survived ~1 month beyond
controls; blood smears were negative for parasites
after 1 month. Attempts at subinoculation of brain
suspensions into uninfected animals remained negative
25after >30 days.
.
M-1076-Cl
117~6~3
-45-
EXAMPLE 10
The following Example illustrates the effect of a 2~
solution of 2-difluoromethyl-2,5-diaminopentanoic acid in
the drinking water of chickens infected with oocysts of
Eimeria tenella.
Twenty chickens are infected F~_ S at day 1 with
100,000 oocysts of E. tenella. Ten of the animals are
given drinking water containing a 2~ solution of 2-difluoro-
methyl-2,5-diaminopentanoic acid. The remaining animals
serve as controls. By day 3 all of the control animals
demonstrate clinical signs of the disease. On day 7 all
of the animals are sacrificed, cecal lesions are macro-
scopically examined and quantified as follows.
0 = No detectable macroscopic lesions.
+1 = Few scattered petechiae in the cecal wall; no
thinkening of the wall and normal cecal contents
present.
+2 = Lesions are numerous with noticeable loss in the
cecal contents; cecal wall slightly thickened.
+3 = Large amounts of blood and tissue debris present,
i.e., cecal cores; cecal wall greatly thickened,
little if any normal cecal contents present.
+4 = Cecal wall greatly distended with much blood or
cecal cores present. Cecal debris lacking or
included in cores. (Dead bird also scores as +4.)
TABLE II
Average Lesion
Score For
Lesion Scores In Individual Animals Group
Controls +4 +3 +4 +4 +4 +4 +2 +4 +4 +4 3.6
~N = 10)
2-difluorometh 1-2 5-diamino entanoic acid treated
Y , P
(~ = 10) +1 +1 +0 +1 +1 +3 +4 +4 +1 +1 1.7
M-1076-Cl
~17~
-46-
EXAMPLE 11
Following essentially the same procedure as in the
preceding Example, six chickens are administered a 2~
solution of 2-difluoromethyl-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 ~er os with
100,000 oocysts of _ tenella per chicken. One group of
ten chickens serves as the control group, the other group
of ten chickens receives a standard dose of Amprolium in
their drinking water for the next 5 days. At present
Amprolium is the coccidiostat of choice. On day 5 all of
the animals are sacrificed and examined for evidence of
disease using the lesion scoring index described in the
preceeding Example. The following results are obtained.
TABLE III
Mean Lesion
~reatment No. Chickens Days of Treatment Score
Control 10 --- 3.70
20 2% solution
of DFMO 6 3 0
0.120% solution
of Amprolium 10 5 0.3
EXAMPLE 12
The following Example illustrates the effect of
varying doses of 2-difluoromethyl-2,5-diaminopentanoic
acid on Eimeria tenella infections in chickens.
Following essentially the same procedure as in
Example 10, the dosage of 2-difluoromethyl-2,5-diamino-
propionic acid (DFMO) is varied as shown in Table IV below.
Treatment with DFMO is started at day -1. The chickens
are infected per os at day 0, and treatment is continued
M-1076-C1
1~4f~3
-47-
for an additional 5 days or a total of 6 days. The
birds are sacrificed at day 5 and examined for evidence
of disease using the lesion scoring index described in
Example 10.
TABLE IV
Mean Eesion
Treatment No. Chickens Days of Treatment Score
Control 7 --- 3.29
2% DF~O 6 6 0
101.0% DFMO 6 6 0.5
0.5~ DFMO 6 6 1.00
EXAMPLE 13
The following Example illustrates the effectiveness
of a low prophylactic dose upon E1meria tenella lesions
in chickens.
Following essentially the same procedure set forth in
Example 10, but varying the dose of 2-difluoromethyl-2,5-
diaminopentanoic acid (DFMO) administered and the period
of administration, the following results are obtained.
TABLE V
Mean ~esion
Treatment No. Chickens Days of Treatment Score
Control 9 --- 3.33
2% DFMO 9 -1 thru +5 0
25 1% DFMO 9 -1 thru +S 0
0.5% DFMO 9 -1 thru +5 0
0.25% DFMO 9 -8 thru +5 0.44
0.125% DFMO 9 -8 thru +5 0
0.0625~ DFMO 9 -8 thru +5 0.66
- ~-1076-Cl
~174ti~3
-48-
. EXAMPLE 14
The following Example illustrates the acquisition of
a permanent immunity towards Eimeria tenella infections in
chickens.
Birds that have previously been treated wtih 2-difluoro-
methyl-2,5-diaminopentanoic acid at concentrations as low
as 0.5% on days -8 through +5 relative to infection are
challenged one week following completion of therapy as
indicated in Table VI 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 sub-
sequent E. tenella infections.
TABLE VI
No. of Days of Mean Lesion Mean Lesion
Treatment Chickens Treatment Score-Initial Score-Final
Control 9 --- 0 2.50
1.0% DFMO 9 -8 thru +5 0.33 0
0.5% DFMO g -8 thru +5 0 0
