Note: Descriptions are shown in the official language in which they were submitted.
,r~ ,~_ ,
- 1 - 15920Y
1 TITLE OF THE INVENTION
Novel Fluorinated Amino Acids
ABSTRACT OF THE DISCLOSURE
Novel substituted ~-fluoromethyl-a-
amino alkanoic acids and esters thereof are dis-
closed. The novel compounds have biological
activity including decarboxylase inhibition.
BACKGROUND OF THE INVENTION
The present invention is concerned with
novel substituted a-fluoromethyl-a-amino alkanoic -
acids.
An unsubstituted a~fluoromethyl-a-
amino alkanoic acid, namely 2-fluoromethylalanine,
having the formula:
Cl H 2 F
CH3-- C--COOH
2 0 NH 2
(A)
is known lKollonitsch et al, J. Org. Chem. 40,
.
; `
- 2 - 15920Y
3808-9 (1975)]. No specific biological acti-
vity for this compound is suggested. This com-
pound (A) is prepared by fluorodehydroxylation
of the corresponding 2-hydroxymethylalanine.
S a-Methyl amino acids, such as L-a-
methyl-3,4-dihydroxyphenylalanine (a-methyldopa,
an antihypertensive agent, are known to have
decarboxylase inhibiting activity (Goodman, et al.,
The Pharmacological Basis of Therapeutics, Mac
Millan Company, New York, New York 1970, p. 577;
Canadian Patent 737,907) .
Novel substituted a-fluoromethyl-a-
amino alkanoic acids have been discovered. These
novel acids have decarbo~ylase inhibiting acti-
vity significantly greater than that of a-methyl
amino acids~
SUMMARY OF THE INVENTION
~,
Novel substituted a-fluoromethyl-a-
amino alkanoic acids and esters thereof. ~ -
DESCRIPTION OF THE PREFERRED EMBODIMENTS -~
.. _ . _ . .. .
An embodiment of the present invention ~-
is compounds having the formula
IH2F
R IC--COOR
;~
wherein R is a substituted Cl-C4 alkyl group
and Rl is H or Cl-C18alkyl.
The pharmaceutically acceptable acid
.~
:
$~
- 3 - 15920
1 addition salts of the formula I compounds are
also included. In general, the salts are those
of the formula I base with a suitable organic
or inorganic acid. Preferred inorganic acid
S salts are the hydrohalides, e.g., hydrochlorides,
hydroiodides, hydrobromides; the sulfates, and
the phosphates. The hydrohalides, and especially
the hydrochlorides, are more preferred.
The formula I compounds have a chiral
center and may occur in optically active forms
i.e., as optical isomers. These isomers are
designated conventionally by the symbols L and D,
+ and -, 1 and d, S and R or combinations thereof.
Where the compound name or formula has no isomer
designation, the name or formula includes the
individual isomer mixtures thereof and racemates.
The compounds having the S-isomer con-
figuration are, in general, preferred.
R is a substituted alkyl group exempli-
fied by
R20 ~\
2O ~ CH2 where R2 is H or C2-C6
alkanoyl e.g., CH3-CO,
CH3(C~2)4-CO,(CH3)3~-CO,
and the like;
N- CH2-
- 4 - 15920 Y
0~
2- ~3CH2
; preferably
HO
CH- HO ~ CH2
CH2 HO ~ CH
H ; H
-
: 1 HOOC-CH-2; ~H2
HOOC-CH2-CH-2; HOOC-CH ~CH2-CH2CH2;
H2N-CH2-CH2-CH2; H2N-CH2CH2CH2CH2; HO-CH2;
NH -~
¦¦ H
H2N C ~ CH2 CH2 CH2; and
H3CS CH2 CH2
Rl is H or Cl-C18 alkyl- Examples
of suitable alkyl groups are methyl, octadecyl,
2-ethylhexyl, t-butyl, hexyl, isopropyl, ethyl,
undecyl aAd the like; Cl-C6 alkyl is preferred
and ethyl is especially preferred. H is a
most preferred definition of Rl.
Preferred compounds of formula I are
those where R is
3~L~
- 5 - 15920y
R2O HO
R2O_ ~ CH2 , ~ H2-
~ CE12 ~ CE~2
HO ~ CH 2
H
H2N-(CH2)2 HOOC-CH2-CH2
EIO ~ CEl2 ' Hooc-cEl-cE2-cEl2-cEl
H N ~C N CH CH CH and
.
H3CS-CH2-CH2-
1 especially where Rl is hydrogen.
Compounds which are particularly pre-
ferred have the formula
CH F
R2 ~ ~ 2
II NH2
.
~ , ' ~ ' , ' :
- 6 - 15920Y
l More preferred formula II compounds are those
werein R2 is hydrogen and Rl is hydrogen or
ethyl. Especially preferred formula II compounds
are those wherein Rl and R2 are hydrogen, with
the S-isomer configuration being most preferred.
Another particularly preferred com-
pound has the formula
lo 7 fH2F
~- ~ CH2-C COOR
~\ i NH2
III
especially where Rl is hydrogen.
The S-isomer of formula III is most preferred.
Especially preferred compounds are those
of the formulae
HO
CH2F
~ CH2 - C - COOH
~H2
HO ~ CH2 - l ~ COOH
~H2
fH2F ~,
~ CH2 - C ~ COOH
,
.r-
~V~
_ 7 - 15920Y
1 HO ICH2F
~ ~H 2
H CH2F
HOOC CH2 ~ CH2 - C - COOH, and
~H2
NH ~ IH2F
H2N - ~ - N-(CH2)~ - C - COOH
NH2
The compounds of the present invention
have physiological or chemotherapeutic uses. In
most cases, the biological activities of these
compounds are in large measure a consequence of
their potent decarboxylase inhibiting activities.
Decarboxylases are enzymes which act on a~amino
acid substrates, effecting decarboxylation to
produce the corresponding amine. This action is
illustrated by the following equation:
CO H DeCarboxylas-e-~ L-CH2 ~=Alkyl or
2 group
~-amino acid substrate amine
. .
- 8 - 15920Y
1 By inhibiting this decarboxylation,
the biosynthestic pathway to a number of biologi-
cally significant amines can be modulated or inhibited
with physiologically useful consequences. ~or ex-
ample, a-fluoromethyl dopa inhibits dopa
decarboxylase and can be used in combination
with dopa to potentiate the latter's usefulness
in the treatment of Parkinson's disease. a-
Fluoromethyl histidine inhibits biosynthesis of
histamine via decarboxylation of histidine (ED50
in mice ~ 0.4 mg/kg). Consequently, it and combin-
ations with histamine antagonists have utilities
in the prevention of gastric lesions and in
treating allergic conditions. a-Fluoromethyl
ornithine by virtue of its ornithine decarboxy-
lase inhibition interrupts polyamine biosynthesis
and is of utility in the treatment of some neo-
plasms. a-Fluoromethyl arginine is an effective
antibacterial. a-Fluoromethyl glutamic acid is a
CNS stimulant.
The present compounds also are substan-
tially specific in their decarboxylase inhibition
activity, that is an a-fluoromethyl-a-amino acid
generally inhibits the decarboxylation of the
25 corresponding non a-fluoromethyl acid. For ex- ~:
ample, a-fluoromethyl dopa inhibits the decar-~ -;
boxylation of dopa; a-fluoromethyl histidine ~ill
inhibit the decarboxylation of histidine, etc.
Because of this specificity and potency
as decarboxylase inhibitors, the present compounds
are also useful as diagnostic tools to determine
the presence and importance of the correspond-
; ing decarboxylase in relation to diseases or to
the functioning of biological systems. ~or ex-
ample, the importance of ~-amino-butyric
acid, in the central nervous system (CNS) may
~.
- 9 - 15920~
1 be studied by inhibiting its biosynthesis using
an a-fluoro-methyl glutamic acid, etc. This
diagnostic utility is aided by the potent and
in many instances irreversible decarboxylase
inhibiting activity of the present a-fluoro-
methyl amino acids.
Representative compounds have been
determined to have decarboxylase inhibiting
activity using conventional in-vitro assays.
a-Fluoromethyl-3/4-dihydroxyphenylala-
nine ~ a-fluoromethyl tyrosine, and a-fluoromethyl-
meta-tyrosine have also been found to have anti-
hypertensive activity. This activity is deter-
mined by observing the antihypertensive effect
(blood pressure reduction) on (peroral or parenteral)
administration of the compounds to a spontaneously
hypertensive (SH) rat. This observed effect indi-
cates that the compounds axe effective as anti-
hypertensive agents, when conventionally adminis-
tered in suitable amounts in an appropriate pharma-
ceutical dosage form to a hypertensive human. The
pharmaceutical dosage form is conventionally pre-
pared and generally includes conventional, pharma- -
ceutically acceptable diluents.
The compounds of the present invention
may be prepared using any convenient method.
One such useful process involves the
reaction of an a-hydroxymethyl-a-amino acid with
SF4 in liquid HF, as illustrated by the following
30 equation: - -
,
CH2H IH2F
R - ~ _ COOH S~ /HF ~ R C ~ COOH
NH2 NH2
:, . , :
~ 15920Y
l The reaction is general].y carried out at tempera-
tures ranging from about -80C to about 20C.
This general reaction is also referred to as
fluorodehydroxylation and is described in the
5 Journal of Organic Chemistry 40, 3809-lO (1975).
BF3 may be used to promote the reaction.
It has now been discovered that the fluoro-
dehydroxylation of certain aryl substituted a-
hydroxymethyl-a-amino acids is substantially im-
proved by utilizing BF3 or AlCl3 as a co-reactant
with SF4. Specifically, this is an improved process
for preparing a compound having the ~ormula
ICH2F
R' - CH2 ~ C- COOH
~H2
(IV)
wherein R' is an aryl group which comprises reacting
a compound l~aving the formula
CH2OH
R' - CH2 - C COOH
~H2 '
(V)
with a) SF4 and b) BF3 or AlCl3, in liquid HF at
temperatures ranging from about -80C to about 20C.
~. 2~3~4~3
- 11 - 15920Y
1 R' is an aryl group exemplified by ~
HO-
HO ~ preferablY H ~ HO ~
HO H
, ~ , ~ , and ~ ~ .
H HO H N
0 Preferred R' groups are H~ , HO ~ , and
H
This present process is preferably carried
out at atmospheric pressure although pressures above
atmospheric may be used. The reaction temperature
ranges from about -80C to about 20, -80C to 0C
being preferred~
The present process may conveniently be
carried out by introducing the 5F4 and BF3 or AlC13
into the Formula V/HF reaction system initially~
The process may also be carried out by first adding
the SF4 to the reaction system, allowing the reac-
tion to proceed for a period of time and then adding
the BF3 or AlC13 and allowing the reaction to go to
completion.
The use of BF3 or AlC13 in the SF4/HF reac-
tion system substantially improves the yield of Form-
ula IV product.
Another method for preparation of the sub-
stituted a-fluoromethyl a-amino alkanoic acids in-
volves the application of photofluorination. For
a description of this method, see Journal of the
American Chemical Society, 92, 7494 (1970)
...
f~
- 12 - 159~0Y
1 and ibid., 98, 5591 (1976). For example, ~-fluoromethyl-
glutamic acid is prepared:
CIH3 CIH2F
1 2 H2COOE~ fluorination> HOOC-f-CH2CH2cooH
NH2 NH2
Both optical isomers of a-methylglutamic acid are
known; thus this method is useful for preparation of
both optical isomers of a-fluoromethylglutamic acid.
Similarly, a-fluoromethyl-ornithine is pre-
pared by photofluorination of a-methyl-ornithine:
15 CIH3 CH2F
2 2 H2NH2 fluorination~Hooc-c-cH2cH2cH2NH2
~H 2 NH 2
Since both optical isomers of a-methylornithine are
available, this method of synthesis can deliver both
of the two optical isomers of a-fluoromethyl~ornithine.
a-Fluoromethyl-ornithine is a suitable start-
ing material for synthesis of a-fluoromethyl-arginine
by reaction with S-methylisothiourea:
- 13 - 15920Y
CH2F CH2F
HC-C-CH2CH2CH2NH2 ~ HC-f-CH2CH2CH2-NH-C-NH2
~H2 NH2 NH
An acid addition salt of a compound
of the present invention may be prepared by
conventional treatment of the free a-amino acid
with a useful acid generally in a suitable sol-
vent.
A single enantiomer of the presentcompounds may also be obtained by (1) resolving the
fluorinated amino acid racemate using conventional
resolution techniques or (2) resolving the pre-
cursor a-hydroxymethyl-a-amino acid using con-
ventional resolution techniques and then fluoro-
dehydroxylating the precursor enantiomer. A
conventional resolution technique involves form-
ing a salt of the a-amino acid with an optically
active base and subsequently recovering the
specific enantiomer from the salt.
Compounds of the formula
CH2F
R20 ~ ~ - CH2 - C - C00
where R2 is C2-C6 alkanoyl are prepared by acy-
lating the corresponding compound where R2 is
hydrogen. Conventional acylating agents and
conditions are employed.
Compounds of the formula
CH2F
~ 35 R - - IC ~ COORl
NH2
- 14 - 15920Y
1 where Rl is Cl-C18 alkyl are prepared by ester-
ifying the corresponding compound where Rl is
hydrogen. Again, conventional esterification
reagents and conditions are employed.
The following examples illustrate pre-
paration of representa~ive compounds of the pre-
sent invention. All temperatures are in C.
The fluorodehydro~ylation reactions described
in the examples were performed in reactors made
of KEL-~. Melting points are determined in
open capillary and are uncorrected.
EXAMPLE l
Preparation of R,S-Alpha-(Fluoromethyl)-3-
15 HYdroxy-Tyrosine
CH2F
HO ~ CH2-C - COOH
HO ~ ~H2
One and S/10 g of R,S, a-(hydroxymethyl)-3-
hydroxytyrosine hydrochloride (a-hydroxymethyl-
DOPA HCl) was dissolved in 50 ml of anhydrous
hydrogen fluoride, while being cooled in a dry-
25 ice-acetone bath. The HF solvent was then evap-
orated after removal of the cooling bath with
a stream of nitrogen gas. This operation trans- ~-
forms the HCl salt into the HF salt of the start- -
ing material. (Alternatively 1.3 g of the free
30 amino acid may be used as starting material, thus
eliminating the need for the above operation.)
The HF salt thus obtained is redissolved by pass-
., .
- 15 - 15920Y
1 ing into the reactor a stream of HF gas after
cooling it in a dry-ice-acetone bath, until a
30 ml liquid HF was collected in the reactor.
SulEur tetrafluoride gas (1.2 ml, measured in
liquid state at -78C) was then passed in, the
dry-ice-acetone cooling bath was then removed
and replaced by a cooling bath kept at -12C.
After 15 hours of aging, the solvent was evap-
orated with a stream of N2, the residue was
dissolved in 50 ml of 2.5M aqueous HCl, evapor-
ated to dryness in vacuo and subjected to amino
acid analysis on Spinco-Beckman amino acid analy-
zer. This analysis indicated the formation of
a-fluoromethyl-3-hydroxy~tyrosine. The product
R,S-alpha-fluoromethyl-3-hydroxy-tyrosine is iso-
lated by ion-exchange chromtography in the same
manner as it is described in Example 2 for S-
alpha-fluoromethyl-3-hydroxy-tyrosine.
EXAMPLE 2
Preparation of S-alpha-Fluoromethyl-3-Hydroxy-
Tyrosine
A.) Preparation of R-a-hydroxymethyl-3-
hydroxy-tyrosine
50 g of 3[3',4'-diacetoxyphenyl]-2-
acetamino-2-acetoxymethyl-propionic acid is
added into 204 ml of 4M aqueous KOH with stir-
ring. After 1 hour of stirring (under nitrogen),
the solution contains potassium salt of 3(3',4'-
dihydroxyphenyl)-2-acetamino-2-hydroxymethyl-
propionic acid, formed in essentially quantita-
tive yield. Without isolation, by methylation
with dimethyl sulfate, this compound is trans-
- 16 - 15920Y
1 formed into 3(3',4'-dimethoxyphenyl)-2-acetamino-
2-hydroxymethyl-propionic acid. This operation
is performed at room temperature under N2 gas
by dropwise addition with vigorous stirring of
dimethyl sulfate (about 64 ml) and 4M aqueous
KOH solution (about 148 mlJ over a period of
about 1 hour.
The reaction mixture was stirred for
another hour, then left standing overnight.
Acidification (at 5-10C with 55 ml of conc.
aqueous HCl), extraction with ethyl acetate
(12 X 300 ml), drying over Na2SO4 and evapora-
tion in vacuo gave R,S-3(3',4'-dimethoxyphenyl-
2-acetamino-2-hydroxymethyl-propionic acid. It
was purified by recrystallization from 1325 ml
of acetonitrile, m.p. 154-6C (dec).
Twenty-nine and 1/10 g of strychnine
was suspended in 1.12 1 of ethanol 2BA, heated
to reflux, then 26.1 g of R,S-3(3',4'-dimethoxy-
phenyl)-2-acetamino-2-hydroxymethyl-propionic
acid was added. The solution thus obtained was -
allowed to cool down and left standing overnight
at room temperature. Crystals of the strychnine
salt of antimer, "A" separate; m.p. 193-194C
("HM").
The mother-liquor of the above named
precipitation was evaporated in vacuo to dryness
and recrystallized from 270 ml of ethanol 2BA
the hot solution is allowed to cool to room
temperature and left standing at room temperature
for ~ 3 hours, then kept in the refrigerator
for ~ 4 hours. The crystals formed were collect-
ed on a filter and after drying, recrystallized
- 17 - 15920Y
1 from acetonitrile to give strychine salt of antimer
"B" of 3(3'4'-dimethoxy-phenyl)-2-acetamino-2-
hydroxymethyl-propionic acid m.p. 130-132C
(dec.). Yield 17.5 g.
Seventeen g of this strychnine salt
was decomposed by dissolving it first in 160
ml of water; 31 ml of lM aq. NaOH solution was
added. The strychnine separated was removed
by filtration and the solution evaporated to
small volume in vacuo and applied onto a small
ion exchange resin co~ mn ~150 ml of AG-X2
cation exchange Dowexl 50 resin, 200/400 mesh).
Elution with water, followed by evaporation in
vacuo of the fractions which showed absorption,
as indicated by an LKB UV absorption monitor
(UVICORD II - 8300). This compound, antimer
"B" of 3(3',4'-dimethoxyphenyl)-2-acetamino-
2-hydroxymethyl-propionic-acid showed [a] D:
78.3 + 0.5 (C, 1.425 in 0.1M aq. NaOH).
Transformation of the above compound
into the corresponding stereo-isomer of a-hydroxy-
methyl-3-hydroxytyrosine: Four and 43/100 g
of antimer 'IB'' of 3(3',4'-dimethoxyphenyl)-2-
acetamino-2-hydroxymethylpropionic acid is di-
ssolved in 100 ml conc. ~Cl and sealed and
heated for 90 minutes in a Fisher-porter tube
immersed into an oil bath of 130C. The sol-
vent was evaporated in vacuo and the above HCl
treatment repeated. The residue thus obtained
30 represents R-a-hydroxymethyl-3-hydroxy tyrosine
hydrochloride.
B.) Fluorodehydroxylation
8 g of R-a-hydroxymethyl-3-hydroxy-
: '
.
.
r ~
- 18 - 15920Y
1 tyrosine.HCl is charged to a 1 1. reactor.
The reactor is immersed into a dry-ice acetone
bath and 80 ml of liquid HF is condensed on top
of the substrate. To remove the HCl present,
the cooling-bath is removed and the HF solvent
removed by passing in a stream of N2 gas. The
reactor is immersed into the cooling bath again
and a stream of HF gas is passed in until a
liquid volume of ~ 250 ml collects. 6.2 ml of
SF4 (17.6 mmol/ml: ~ 109 mmol) is then bubbled
in, the solution aged for f_~ hour, the cooling
bath exchanged for an ethylene-glycol bath kept
at -16C and the solution aged for ~ 22 hours.
Boron trifluoride gas is passed in until satura-
tion and the solution aged again at -16C for 46
hours. The cooling bath is removed and the sol-
vent evaporated by passing through it a vigorous
steam of N2 gas. The residue is quenched in
~ 100 ml of ice-cold aqueous HCl (2.5M), evapor-
ated in vacuo, the residue dissolved in waterand added onto a column of cation-exchange resin.
2.2 1 of AG-50-X-8 resin (200/400 mesh) was
employed. Elution with 0.25M aq. HCl, containing
5% methanol; in ~ 8.5 hours, 7.2 1 of this sol-
vent is pumped through the column. This is follow-
ed by 7.2 1 of 0.4M aq. HC1 with 7.5% methanol
in 8.5 hours, then concluding with 0.6M aq.
HCl wi~h 10% methanol. 22 ml fractions are
collected, 10 tubes per rack. Tubes in racks
No 45-66 contained the desired compound. Evap-
oration in vacuo gave HCl salt of S isomer of
a-fluoromethyl-3-hydroxy-tyrosine.
For liberation of the free amino acid,
4.826 g of this compound was dissolved in 90 ml
- 19 - 15920Y
1 of isopropanol, filtered through Celitel. 6.2
ml of propylene oxide was added to the filtrate
and the suspension kept at room temperature for
3.5 hours, then at ~ 5C for another 2.5 hours.
The S a-fluoromethyl-3-hydroxy-tyrosine thus
formed was collected by filtration, washed with
isopropanol and dried overnight in vacuo at 76.
[a]D: ~9.3 + 0.5, c, 1.82 in 1:1 mixture of tri-
fluoracetic acid and water.
EXAMPLE 3
Preparation of R-a-Fluoromethyl-3-Hydroxy-Tyrosine
For preparation of the above named com-
pound, the strychnine salt of antimer A of
3(3',4'-dimethoxyphenyl)-2-acetamino-2-hydroxy-
; 15 methyl-propionic acid (Example 2 "HM") was carried
through steps analogous to those in Example 2.
The final product of the sequential steps was R-
~-fluoromethyl-3-hydroxy-tyrosine, with ~a]D: ~9
(c, 2.5 in a 1:1 mixture of H2O-trifluoroacetic
acid).
EXAMPLE 4
R,S-a-Fluoromethyl-Tyrosine
one and 5/100 g (0.005 mol) of R,S-
25 a-hydroxymethyl-tyrosine is charged into a reac- ~ -
tor. The reactor is immersed into a dry-ice-
acetone bath and ~ 50 ml of liquid HF is collected
by passing in a stream of HF gas. Under contin-
uing cooling, SF4 gas (4 ml, measured in liquid
state at -78C) is passed in, then BF3 gas until
saturation at -78C. (Stirring with magnetic
stirrer). The deep-red solution thus obtained
is aged overnight at -78C, the cooling bath is
removed then, and the solvent evaporated by blow-
ing a dry stream of nitrogen gas through it.
r~
- 20 - 15920Y
1 The residue is dissolved in 20 ml of 2.5M aq.
HCl and evaporated to dryness in vacuo. The resi-
due is dissolved in water and applied to a strong
acid cation-exchange resin column, prepared with
100 ml of AG50-X-8 resin (200/400 mesh). The
column is first washed with water (1.8 1),
followed by 0.5M aq. HCl. 20 ml fractions of the
effluent are collected and the course of the elution
is followed by W monitor of LKB, Model WICORD II.
The fractions corresponding to the main peak in
the UV curve are combined and evaporated to dry-
ness in vacuo, to yield hydrochloride salt of
R,S-fluoromethyl-tyrosine. 400 mg of this salt
is dissolved in 6 ml of water; after a few minutes,
crystallization of R,S-fluoromethyl-tyrosine begins.
After standing overnight at 5C, the product is
filtered, washed with water, ethanol and diethyl-
ether and driPd in vacuo at 76C, to give R,S-
a-fluoromethyl tyrosine.
EXAMPLE 5
R,S-~-Fluoromethyl-Histidine (FM HIST)
H CH2OH
CH2-C--~O~H CH2--C-COH
N ~ ~ 1) Racemization ~ ~ l
2 2) ~ ~N~ NH2
CH2C6H5 3) H2C= CH2C6H5
(L) SD,L)
I II
`
~` f ~
-
9~ ~ ~
~ 21 - 15920Y
1 fH2OH CH2
&H2-C-C~2 CH -C-COOH
N /~H3 ~ ~ NF/SF4BF3 ~ 2 ~ 2
(D,L) FM-HIST
III IV
~'
.:
A) Racemic Ntim)Benzyl-HistidinP
Thirty g of N(im)Benzyl-L-histidine
is dissolved in 600 ml H2O and the soltuion
heated in a high-pressure autoclave at 200C
fox 8 hours with shaking. The autoclave is
cooled to room temperature, the clear super-
natant solution evaporated in vacuo to dryness
to give the R,S-a-fluoromethyl-histidine as a ~-
colorless crystal.
B) R,S-a-Hydroxymethyl-N(im)Benzyl-Histi-
dine (II)
Twenty g of rac. N(im)benzyl-histi-
dine is dissolved in 1 1 of hot water, then 40
g of basic cupric carbonate is added in portions
and the mixture refluxed with stirring for 1 hour.
The mixture is filtered while hot and the filtrate - -
,~' .,. ~.
` ~ r~
.,~.~
~:~2~
- 22 - 15920Y
1 is evaporated in vacuo to give Cu chelate of
racemic N(im)benzyl-histidine as a blue solid.
A mixture of 31 ml of formalin (38%
H2CO), 3.1 ml of pyridine and 2.13 g of Na2C03 is
5 heated with stirring to 70C then 20 g of the
above named Cu-chelate is added and the system
heated and stirred at 75 for 90 minutes. Evapora-
tion in vacuo gives a blue solid residue. ThiS
is dissolved in a mixture of 50 ml of H20 with 50
1~ ml of conc. NH40H and char~ed onto a cation-ex-
B change resin column (Dowex1 50-X-8, 300 ml resin
in the NH4-form) and eluted with 2M aq. NH40H
solution. The effluent is monitored with LKB
UVICORD II UV absorption monitor and the 1.1 1.
15 portion of the ef~luent with W absorption is
combined, evaporated in vacuo to a solid. The
residue is dissolved in a mixture of 60 ml of H20
with 5 ml of conc. aq.NH40H and charged onto~an
anion exchange resin column (300 ml of Dowex~ l-X-2
20 resin in the OH form). The column is washed with
water (2 1.) and eluted with 2M aq. HCl, moni-
tored with a UVICORD II for UV absorption. The
effluent fractions with ultraviolet absorption
were combined and evaporated to dryness, to give
25 substantially pure HCl salt of N~im)benzyl-~-hydroxy-
methyl-histidine (II) (new compound). This compound
is transformed into ~-hydroxymethyl-histidine (III)
in the following way: 12.5 g of II is dissolved in
200 ml of liquid NH3(3-neck flask, equipped with
30 "cold-finger" condenser filled with dry-ice-ace-
tone), then sodium is added (5.5 g, cut in small
pieces) until the blue color persists for 10
minutes. NH4C1 is added then to consume the
excess Na (indicated by decolorization) and the
35 NH3 solvent is allowed to evaporate under a stream
- 23 - 15920Y
of N2. The product III thus obtained is purified
by chromato~raphy on a cation-exchange resin column
B (2.2 1. of Dowel-50-X-8, 200/400 mesh). Crude
III is dissolved in 100 ml of H2O and applied
5 onto the resin column. The column is washed
first with water ( 4 1.) then developed with
aq. HCl (1.5 M, then 2 M). 20 ml fractions are
collected, flow rate 600 ml/hr.
Fraction No. Pauly Reaction
1-400 1.5M HCl
401-670 2M HCl
671 & later +
Fractions 671-760 are combined and evaporated in
vacuo to dryness, to give III: R,S-a-hydroxymethyl-
histidine-2HCl (new compound).
C) R,S-a-Fluoromethyl-Histidine (IV
Two and 73/100 g of R,S-a-hydroxymethyl-
histidine-2 HCl(III) is dissolved in 70 ml of
liq. HF, then evaporated to dryness by passing
in a stream of N2. The residue thus o~tained
represents the hydrofluoride salt of a-hydroxy-
methyl-histidine. It is redissolved in 200 ml
of liq. HF (dry-ice-acetone cooling bath), then
25 9 ml SF4 is passed in (measured as liquid at
-78C). The solution is stored overnight, while
being kept in a cooling bath of -12C. The solu-
tion is saturated then with BF3 gas, left standing
for 5 hours, saturated again at -12C and left
aging at the same temperature for 66 hours. The
cooling-bath is then removed and the solvent
evaporated by passing in a stream of N2. The
residue represents mainly HBF4 salt of a-fluoro-
methyl-histidine. This is dissolved in 100 ml
:
., .
~2a;~
- 24 ~ 15920Y
1 of 2.5M aq. HCl, evaporated to dryness and
transformed into the HCl salt as follows: It
is redissolved in H2O and applied onto a cation-
exchange resin column (100 ml of AG50-X-2, 200/
400 mesh), eluted with H2O until effluent is
neutral and free of F . The product is released
then from the column by 3M aq. HCl, evaporated
to dryness in vacuo, to result in a residue,
consisting mainly of dihydrochloride of IV.
For final purification, this is rechromatographed
on another AG-50-X-2 column (900 ml resin).
Elution with: 0.5M aq. HCl - 1 1.
1.0M aq. ~Cl - 1.5 1.
1.5M aq. HCl - 3.3 1 (collection
begins here,
20-ml frac-
tions)
2.0M aq. HCl - 8.00 1.
The desired product IV was located by ~auly test.
Fractions 390-470 are combined, evaporated to
dryness in vacuo, to give pure dihydrochloride
of _ . Recrystallization from water-isopropanol
(1:9 v/v) gives the crystalline monohydrochloride
salt of a-fluoromethyl-histidine, m.p. 226 7
(dec.).
EXAMPLE 6
Synthesis of R,S-a-Fluoromethyl-Ornithine
- - .
A) R,S a-Hydroxymethyl-S-N-Benzoyl-Ornithine
Copper chelate of R,S-~N-benzoyl-orni-
thine (7.995 g) is added in small portions onto
a mixture made of formalin (38~ H2CO; 12.45 ml),
pyridine (1.25 ml), and sodium carbonate (0.81 g)
at 70C, under mechanical stirring. After
4~3
- 25 - 15920Y
1 further 90 minutes stirring at 75C, it is evap-
orated to dryness in vacuo, the dark blue residue
dissolved in a mixture of 30 ml of H2O and 30
ml of conc. aq. NH3 solution and charged to a
cation-exchange resin column (130 ml of Dowex
50-X-8 in the NH4 form) to remove Cu +. The
column is eluted with 250 ml of 2M aq. NH3 and
the effluent evaporated to dryness in vacuo.
The residue is redissolved in H20 and~applied onto
an anion exchange resin column (Dowex11-X-2,
OH form, 130 ml resin). The column is washed
with H2O (250 ml) and eluted with 3M aq. HCl.
The HCl effluent is concentrated in vacuo to give
R,S-a-Hydroxymethyl-~-N-Benzoyl-Ornithine.
B) R,S-a-Hydroxymethyl-Ornithine Dihy-
drochloride
Three and 5/10 g of the product ob-
tained in a) is dissolved in 40 ml of 6M aq. HCl
and refluxed ~or 21 hours. The solution is ex-
tracted with toluene (2 x 40 ml) and the aqueousphase evaporated in vacuo to dryness, to give
R,S-a-hydroxymethyl-ornithine dihydrochloride
(new compound).
C) R,S-a-Fluoromethyl Ornithine
One and 1/10 g of the product obtained
under b) is placed into a reactor, the reactor
immersed into a dry-ice-acetone bath and HF qas
passed in until HF solution of 25 ml volume is
formed in the reactor. The cooling bath is re-
moved and the solvent evaporated by passing in
a stream of N2. The residue thus obtained repre-
sents the HF salt of R,S-a-hydroxymethyl-orni-
thine. This residue is redissolved in HF, by
cooling the reactor in the dry-ice-acetone bath
- ~6 - 15920Y
1 and passing in HF gas until 50 ml volume is
reached. SF4 gas is passed in (4 ml as measured
in liquid state at -78C), the dry-ice-acetone
cooling bath removed and replaced by a bath
5 kept at -15C. After aging for 16 hours at
-15C, BF3 gas is passed in for saturation.
After 5 hours further aging, the cooling bath
is removed and the solvent evaporated by passing
in a stream of N2. The residue is dissolved in
10 6M aq. HCl, evaporated to dryness in vacuo and
redissolved in H2O (~0 ml). This solution is
B applied onto a Dowex150-X-8 cation-exchange resin
column (400 ml resin, 200/400 mesh, H form).
The column is first washed with H2O (800 ml);
15 elution with 2M aq. HCl, 15 ml fractions are
collected. Flow rate 600 ml/h. Every 5th frac-
tion is spotted on TLC plate and developed with
ninhydrin spray. Fractions No. 171-220 are com-
bined and evaporated to dryness in vacuo, to de-
20 liver a mixture of amino acids, the main component
being R,S-~-fluoromethyl-ornithine-2HCl. For
further purification, this product is rechA~oma-
tographed on another column, made of Dowe ~50-X-8
cation exchange resin (200/400 mesh). For devel-
25 opment, the column is first washed with water,
then eluted with 1.5 aq. HC1, flow rate 0.6 l./h.
20-ml fractions are collected. The residue ob-
tained on evaporation of fractions No. 521-540
represents pure R,S-~-fluoromethyl-ornithine
30 dihydrochloride.
EXAMPLE 7
Synthesis of S-~-Fluoromethyl-Tyrosine
A.) Preparation of The Copper Chelate of
Tyrosine Methyl ether
Twentyfive g of R,S-tyrosine methyl ether
- 27 - 15920Y
1 (128 mmol) was dissolved in 646 ml of 0.2N NaOH at
80C and this solution was added to 16.1 g of copper
sulfate pentahydrate dissolved in 1600 ml of water
at 80C. An immediate precipitate was formed and
the solution was allowed to cool overnight after
which it was filtered affording 28.9 g of the copper
chelate of R,S-tyrosine methyl ether.
B.) R,S-a-Hydroxymethyl tyrosine methyl ether
Twentynine g of the copper (Cu++) chelate
of tyrosine methyl ether (0.064 mole) was added at
70C under stirring a solution o~ 3.9 g sodium car-
bonate, 52 ml of 37% aqueous formaldehyde and 5.2 ml
of pyridine (nitrogen blanket). After completion of
addition, there was added another 18 ml of formalde-
hyde solution and 1.6 ml of pyridine. After heatingat 70~C for 3.5 h and allowing the solution to cool
to room temperature in an additional 1.5 h, the solu-
tion remained at room temperature overnight. In the
morning, there appeared copious blue crystals which
were filtered and the filtrate concentrated to dryness
in vacuo. After the residue was dissolved in water and
reconcentrated to dryness, it was dissolved in 90 ml
of 4N HCl. After filtration the solution was used to
dissolve the above blue crystals. This required an
additional 300 ml of 4N HCl. The solution was then
treated with hydrogen sulfide, filtered through a
diatomaceous earth filter aid and concentrated to about
40 g of crude product. This was applied ~o a strong
acid cation exchange resin (0.5% of Dowe~Y50 X 8),
eluted with 4 1 of water and then 2N aqueous ammonia.
The effluent was monitored with UVICORD II (recording
ultraviolet spectrophotometer) and the UV absorbing
fraction was concentrated in vacuo to 22.16 g of
pure R,S-a-hydroxymethyl-tyrosine methyl ether.
,, , .:
r~ ~
~ ..~
- 28 - 15920Y
1 C.) R,S-N-Acetyl-~-hydroxymethyl-Tyrosine
Methyl Ether
Nineteen and 7/10 g of R,S-~-hydroxy-
methyl-tyrosine methyl ether (87.5 mmol) was sus-
pended in 2 00 ml o dry pyridine, then 68 ml of acetic
anhydride was added. After aging overnlght at room
temperature, the solution was concentrated in vacuo
to dryness and azeotroped with 2 X 50 ml toluene.
The residue was dissolved in 118 ml of methanol and
130 ml of aqueous 2.5N NaOH solution and stirred at
room temperature for 3.5 h. Acidification with 30
ml of conc. HCl followed by extraction (with 4 X 200
ml of ethyl acetate, and then drying and concentration
afforded 21 g of crude product. This was recry-
stallized from 75 ml of acetonitrile yielding 9.35
g of R,S-N-acetyl-~-hydroxymethyl-tyrosine methyl
ether, mp 151-152C dec.
D.) Optical Resolution of R,S-N-Acetyl~a-hydroxy-
methyl-tyrosine methyl ether
Ten g of R,S-N-acetyl-~-hydroxymethyl-tyrosine
methyl ether and 6.18 g of d-ephedrine were dissolved
in 50 ml of methanol. The solution was concentrated
to dryness in vacuo and then redissolved in 50 ml of
warm acetonitrile. Crystallization afforded 7.34 g
25 of the d-ephedrine salt of R-N-acetyl-a hydroxymethyl- ~ ;
tyrosine methyl ether, mp 125-131C (Crop A). Crop A
was recrystallized from 40 ml of acetonitrile af~ording
4.78 g of Crop B, mp 130-134C. The mother liquors
from A and B were combined, concentrated, the residues
dissolved in 22.4 ml of 2.5N NaOH and 50 ml of H2O.
The aqueous solutions were extracted with 2 X 75 ml
ethyl acetate. The aqueous solutions were cooled
and acidified with 5 ml of conc. HCl and the result-
ant solution e~tracted with 3 X 70 ml ethyl acetate.
The dried organic solution was concentrated to 7.73 g
(Crop C). Crop C and 4 . 7 g of l-ephedrine were di-
ssolved in 50 ml of methanol and concentrated to
- 29 - 15920Y
1 12.39 g (Crop D). This was recrystallized from 50
ml acetonitrile to yield 5.06 g of the l-ephedrine
were dissolved in 50 ml of methanol and concentrated
to 12.39 g (Crop D). This was recrystallized from
50 ml acetonitrile to yield 5.06 g of the l-ephedrine
salt of S-N-acetyl-~-hydroxymethyl-tyrosine methyl
ether (Crop E), mp 131.5-133.5C dec. Crop E was recrys-
tallized from 27 ml of acetonitrile to give Crop ~,
4.72 g, mp 130.5-134.5C dec. Combined the mother
liquors from Crop F and Crop E, and concentrated to
7.31 g of Crop G. Crop G was converted back to the
free acid usin~ the method which was used to obtain
Crop C and there was obtained 3.0 g of Crop H. This
was treated as was the initial R,S-material with 1.9
g of d-ephedrine. Recrystallization of the salt from
17 ml of acetonitrile afforded 2.4 g Crop J, mp 127-
130C. Crop J was recrystallized to 2.06 g of Crop K,
mp 130-134C dec.
Combined Crops ~ and K (6.52 g) were recrys-
tallized from 40 ml of acetonitrile affording 6.06 g
of the d-ephedrine salt of R-N-acetyl-a-hydroxymethyl-
tyrosine methyl ether (75.8% overall).
~ he ~ree acid was regenerated in the same
manner that the combined mother liquors of Crops A
and B were converted to Crop C and there was obtained
3.50 g of R-N-acetyl-a-hydroxymethyl-tyrosine methyl
ether : [a]D= ~92 (C, 1.35, 0.27N NaOH).
E.) R-~-Hydroxymethyl-Tyrosine
Three and 3/10 g of R-N-acetyl-a-hydroxy-
methyl-tyrosine methyl ether was dissolved in 100 ml
of conc. HCl and heated in a pressure tube at 130C
for 2 h. The solution was concentrated to dryness, the
residue dissolved in 35 ml of H2O, filtered and treated
with 1 ml of pyridine. 2.11 g of pure R-~-hydroxymethyl-
; tyrosine (81%) crystallized out: [~]D= 0.86 (C, 1.15,
50% aqueous trifluoroacetic acid). The circular
-
- 30 - 15920Y
1 dichroism (CD) spectrum has the same sense as the
CD of S-~-methyl-tyrosine.
F. ) S-a-Fluoromethyl-Tyrosine
Following the procedure of example 4,
S- a-fluoromethyl-tyrosine was prepared from R-a-
hydroxymethyl-tyrosine.
E~AMæLE 8
(+)-a-Fluoromethylglutamic Acid
6.56 g of a-methylglutamic acid hemi-
hydrate is photofluorinated in liquid HF solution,
employing the general technique described in Jouxnal
of the American Chemical Society, 92, 7494 (1970)
and 98, 5591 (1976). The substrate was dissolved in
120 ml of liquid HF and irradiated with a 2500W
ultraviolet light source under stirring while fluoroxy-
trifluoro-methane (CF30F) gas (3.0 ml as measured in
liquid form at -78C) was passed in the course of 80
min, under cooling in a dry-ice-acetone bath. After
another 80-minute period with irradiation under similar
conditions, an additional similar dose of CF30F was
added in 3 hours, continuing with the stirring, cool-
ing and irradiation. The mixture was kept overnight -~
in the dry-ice-acetone bath, then it was further
fluorinated (with 3 ml of CF30F, added in 5 hours
with irradiation). Nitrogen gas was blown through
the solution for removal of the solvent and the resi-
due was evaporated with 2.5N aq. HCl (2X) in vacuo.
The resiaue was dissolved in 40 ml of water. To 10
30 ml of this solution 10 ml of conc. HCl was added and
the mixture was refluxed for about 68 hours. After
treatment with DARCO G-60, the filtrate was evapor-
ated in vacuo and the residue refluxed with 3 0 ml
of conc. HCl for another 68-hour period. After
'h~ 3~
- 31 - 15920Y
1 treatment with DARCO, the solution was evaporated
to dryness, dissolved in 10 ml of conc. HCl and
heated in a sealed glass tube for 24 hours in an
oil-bath kept at 130-135. Evaporation in vacuo to
dryness gave a residue which was dissolved in H2O
and subjected to elution chromatography on a cation-
exchange resin column, made of 360 ml of AG50-X12
(mesh 200/400). Eluants: 2.6 1 of H2O, followed
by O.lN aq. HCl (1.5 1) then by 0.15N a~. HCl. UV
absorption of the effluent was monitored by a re-
cording UV at 206 nm. 15-ml fractions of effluent
were collected and 20 fractions, corresponding to ~ ~-
the first ultraviolet absorbing peak, were combined
and evaporatea in vacuo to dryness, to give a-fluoro-
methyl-glutamic acid hydrochloride. For liberation
of the amino acid, this was dissolved in isopropanol,
filtered, then propylenoxide added. a-Fluoromethyl-
glutamic acid 0.7 H2O crystallizes out of the solution.
This compound is a time-dependent inhibitor of glu-
tamic acid decarboxylase.
