Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
ilAl 3 Sb
~3J~
This invention relates to new proline derlvatives
and related compounds which have the general formula -
(I) :
IR4 IRl U2C -- (C*H)
R2- S - (~II)n- ~H - CO - N ~ COR
wherein R is hydroxy, NH2 or lower alkoxy;
Rl and R4 each is hydrogen, lower alkyl,
phenyl or phenyl-lower alkyl;
R2 is hydrogen, lower alkyl, phenyl,
substituted phenyl wherein the phenyl
substituent is halo, lower alkyl or
lower alkoxy, phenyl-lower alkyl,
diphenyl-lower alkyl, triphenyl-lower
alkyl, lower alkyl-thiomethyl, phenyl-
lower alkylthiom thyl, loMer alkanchyl-
amidomethyl, R5-C-, R5-M-C-,R5-Nil-C-,R6-S- or R7;
R3 is hydrogen,hyclroxy or lower alkyl;
R5 is lower alkyl, phenyl or phenyl-lower
~IA135b
alkyl;
R6 is lower alkyl, phenyl, substituted
phenyl, (wherein the phenyl substituent
is halo, lower alkyl or lower alkoYy),
hydroxy-lower alkyl or amino(carboxy)-
lower alkyl;
R3
m(HC) - CH2 Rl R4 ,!
R7 is R-OC-HC-- N - CO - CH -(CH)- S(O) ;
M is 0 or S;
m is 1 to 3;
n and p each is 0 to 2,
and to processes for making them.
The asterisks indicate asymmetric carbon atoms.
Each of the carbons bearing a substituent Rl, R3 and R4
is asymmetric when that substituent is other than hydrogen.
The invention in its broad aspects includes proline
and related derivatives having formula I above. Within this
broad group, because of their properties, certain subgroups
are preferred over others.
Broadly preferred are those compounds of formula I
wherein R is hydroxy or lower alkoxy; Rl is hydrogen or
lower alkyl; R;2 is hydrogen, R5-CO, R6-S-, or R7; R3 and
R4 each is hydrogen; R5 is lower alkyl, especially methyl
or phenyl; R6 is lower alkyl, especially methyl or ethyl;
m is 2, n is 0, 1 or 2, especially 1; and R7 wherein R, Rl,
R3, R4, m and n have the same preferences as above and p is 0.
_ . _ .. . . _ , . .. : . .~ . . . . ___
~ lr ~
Especially preferred are those compounds which have the
formula
(Il)
R2 * COR
wherein R is hydroxy or lower alkoxy;
Rl is hydrogen or lower alkyl;
R2 is hydrogen, R -CO-, R -S- or R ;
R5 is lower alkyl or phenyl, especially the first;
R6 is lower alkyl;
and n is 0, 1 or 2.
Within the group of compounds represented by formula
II, the following are still more preferred subgrouFs in
the order (a to r) of increasing preference to the com-
pounds which are especially preferred embodiments:
a) R is hydroxy
b) n is 1
c) R2 is hydrogen or lower alkanoyl
d) R2 is hydrogen
e) R2 is acetyl
f) Rl is hydrogen or lower alkyl
g) Rl is hydrogen or methyl
h) R is hydroxy, Rl is hydrogen or methyl
i) R is hydroxy, Rl is hydrogen or methyl, R2 is hydro- :
gen or acetyl and n is 0, 1 or 2
j) R is hydro~y, Rl and R2 each is hydrogen and n is 0
- 3 -
. . ~, -
.
k) R is hydroxy, Rl is hydrogen, R2 is acetyl and n is
1) R is hydroxy, Rl is methyl, R2 is acetyl and n is 1
m) R is hydroxy, Rl and R2 each is hydrogen and n is 1
n) R is hydroxy, Rl is methyl, R2 is hydrogen and n is
o) R is hydroxy, Rl is hydrogen, R2 is lower alkylthio
and n is 1
p) R2 is
~ 11
R-OC N - OC - CH - (CH2)n-S-;
each R is hydroxy; Rl is hydrogen or lower alkyl, es-
pecially hydrogen or methyl; and n is 0 to 2, es-
pecially 1
M
q) R2 is R5-M-C- wherein M is O or S
M
r) R2 is R5-NH-C- wherein M is O or S, preferably S.
It will be appreciated that combinations of the fore-
going, where applicable, are among the preferred groups.
The stereoisomers in which the proline is in the L-
form are especially preferred.
The lower alkyl groups represented by any of the vari-
ables include straight and branched chain hydrocarbon
radicals from methyl to heptyl, for example, methyl, ethyl,
propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, iso-
pentyl and the like. The lower alkoxy groups are of the
same kind having 1 to 7 carbons linked to oxygen, for ex-
ample, methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-
butoxy, t-butoxy and the like. The Cl-C4 members, espe-
cially Cl and C2 members, of both
, 4
I-IA135b
types are preferred. Phenylmethyl is the preferred phenyl-
lower alkyl group.
The lower alkanoyl groups are those having the acyl
radicals of the lower (C2-C7~ fatty acids, for example,
acetyl, propionyl, butyryl, isobutyryl and the like. Similarly,
those lower alkanoyl groups having up to four carbons, and
especially acetyl, are preferred.
The four common halogens are included by the term
"halo" but chlorine and bromine are preferred. The substi-
tuted phenyl groups preferably bear the substituent in the
4-position of the ring. The hydroxy-lower alkyl yroups
have a hydroxy group on an alkyl chain like those described
above, preferably on the terminal carbon, e.g., hydroxymethyl,
2-hydroxyethyl, etc. The amino(carboxy)lower alkyl groups
have one amino and one carboxy on a lower alkyl group such
as those described above, preferably both on one carbon,
e.g., on the ~erminal carbon as in the preferred 2-amino-2-
carboxyethyl group.
The products of formula I and the preferred subgroups
can be produced by various methods of synthesis.
In general, the products of this invention are
produced by acylating a compound of the formula
(III)
R3
H C (Cll)
21 I m
HN - C~l - COR
with an acid of the formula `
(IV) R Rl
R2 _ S ~ (C~l)n C~l - COO~I
_ 5
~ 55 HA135b
or its chemical equivalent.
Thus, the final product can be produced not only by direct
acylation with an acid of formula IV but also by intermediates
such as (a) ~-haloalkanoic acids of the formula
R4 Rl
(V)
X (CH)n CH - COOH
wherein X is bromo, chloro or iodo, or (b) a tosyloxyalkanoic
acid, i.e., X in formula V is tosyloxy (CH3 ~ SO2O-)
(c) a substituted acrylic acid of the formula
14 1l
(VI) CH - C COOH
The product of this acylation is then subjected to displacement
or addition with the anion of a thiol or thioacid of the
formula
(VII) R2 SH
Acylation can also be effected with a thiolactone of the
formula
14 IR
(VIII) tfH)n fH
s . =o
wherein n is 1 or 2, or a mercaptoalkanoic acid of the formula
IR4 IRl
(IX) Y S (CH) - CH COR
ilA13Sb
wherein Y is R2 or, in addition, if a product of formula I
wherein R2 is hydrogen is desired, then Y can also be a
protecting c3roup such as (a) Cil3O- ~ _C~l2-, (b) ~ ,
(c) C~3CON~C~l2, (d) R-O-C-CI-I-(C~I) -S- or other sulfur
protecting group. "Deprotection" can be effected by
conventional means such as treatment with hot trifluoroacetic
acid, cold trifluoromethanesulfonic acid, mercuric acetate,
sodium in liquid ammonia, zinc and hydrochloric acid or the
like. For a review of these methods see Methoden der
Organischen Chemie (Houben-Weylj, Vol. XV, part I, page
736 et seq. (1974).
When the acid of formula IV is used as the acylatinc3
agent, the acylation can be effecied in the presence of a
coupling agent like dicyclohexycarbodiimide or the li~e,
or the acid can be activated by formation of its mixed
anhydride, symmetrical anhydride, acid chloride, acid
ester or use of Woodward reagent ~, N-ethoxycarbonyl-2-
ethoxy-1,2-dihydroquinoline or the like. For a review of the
methods for acylation, see Methoden der Organischen Chemie
(Houben-Weyl), Vol. XV, ?art II, page 1 et seq. (1974).
Compounds of formula III include, for example,
proline, hydroxyproline, 4-methylproline, pipecolic acid,
5-hydroxypipecolic acid, azetidine-2-carboxylic acid, their
lower alkyl esters and the like. The acylation of such
compounds is described in greater de-tail below.
According to a preferred method for producing compounds
of formula I, especially wherein R2 is ~5-CO-, an acid or ` -
ester of formula III is coupled with a haloalkanoic acid of
the formula
--7--
__ .. .. _ . . _ . ... . _ . . _ . . _
~ IA135b
(V)
l4 ll
X-(CH) - CH- COOH
wherein X is a halogen, preferably ehlorine or bromine. This
ean be effeeted by one of the known procedures in which the acid
IV is aetivated, prior to reaction with the acid III, involving
formation of a mixed anhydride, symmetrical anhydride, acid
ehloride, active ester, or use of Woodward reagent K, EEDQ
(~-ethoxyearbonyl-2-ethoxy-1,2-dihydroxyquinoline) or the
like.
The product of this reaction is a eompound of the
formula
(X) R3
14 1l H2C (CH)m
X - (CH)n CH - CO - N - CH-COR
The produet of formula X is subjeeted to a dis~laeement
reaction with the anion of a thioaeid of -the formula
(VII)
R -SH
yielding a produet of the formula
(XI)
l4 1l ~l2C- (CH)m
R2- S - (CH)n- CH -CO - N - C~l-COR
When R2 is R5CO, this product can then be converted to
the product .~ ,
(XII) R3
R4 ll 1l2C (Cll)
~IS - (Cll) - Cll - CO ~ N CII-COR
}IA13Sb
by ammonolysis. ~hen R2 is a protecting ~roup, then the
compound of foL~ula XII can be obtained by"deprotection"
as described above. When R is an ester ~roup (i.e., R
is lower alkoxy), the ester cJroup can be removed, e.cJ.,
when R is tert. butoxy or tert. amyloxy, by treatment of
the ester of formula XI or XII with trifluoroacetic acid
and anisole to give the correspondin~ free acid. When other
alkoxy groups are presen-t, alkaline hydrolysis will yield
the correspondin~ acid.
A variation of this procedure involves the use of
an acrylic acid of the formula
(VI) l4 IRl
CH=C-COOH
as startin~ material. This acrylic acid is first converted to
the acid halide form then made to react with a compound of
formula III to obtain a compound of the formula
(XIII) 13
l4 IRl ~12C (CH)
CH=C - CO- N CH-COR
and this intermediate is subjected to the addition reaction
with the thiol or thioacid VII as described above.
A tosyloxyalkanoic acid of the formula
(XIV)
R R
3 ~ SO2O-(CH)n- CH- COOII
can also be usecl as the a~ent to acylatc thc acid of formula
III, then the acylation product is subjected to the displacc- ` `-
~ment reaction, etc., as descrihed above.
The acrylic acid of formula VI can alterna-tively be
first made to react with -tlle thioacid c~f formula VII to
_9_
IIA135b
obtain a product of the formula
( XV )
R~ R
R2-S-CH-CH-COOH
whieh is eonverted to its acid halide, e.g., with thionyl
ehloride, then eoupled to the eompound of formula III and
the same sequenee as above then followed.
The aeid or ester of formula III can also be aeylated
~ith a "protected" form of a ~-mercaptoalkanoic acid of the
formula
~XVI) R4 Rl
R8-s-(cH)n-cH-cooH
wherein R8 is the "proteeting" group. Such "protectincJ" groups
ean take the form described above.
Following the acylation, the product can be "deprotected"
by one fo the known methods referred to above.
Still another aeylating agent can take the form of a
thiolaetone, e.g., ~-propiothiolactone, a-methyl-~-propiothio-
lactone or the like.
Additional details of preferred modes of producing
eompounds of this invention can be found in the-followincJ
and in the specific examples.
Aceording to a particularly preferred modification,
the aeid or ester of formula III is acylated with a halo-
alkanoyl halide of the formula
(XVII) R4 Rl
. ,;~,
X-(CH)n- CH-COX
wherein eaeh X is independently a halogen, preferably ehlorine
or bromine, Rl is hydrogcn, lower alkyl or phenyl-lower alkyl
_ l ()--
3~
and n is 0, 1 or 2. This reaction is effected in an alka-
line medium, e.g., dilute alkali metal hydroxide solution,
alkali metal dicarbonate or alkali metal carbonate solution
at a reduced temperature, e.g., about 0 -to 15C. The re-
action product is subjec-ted to displacement with -the anion
of the thiol or thio acid of -the formula VII above, also
in alkaline medium, preferably alkali metal carbonate so-
lution, and then worked up in conventional manner. The
product of this reaction, wherein R2 of formula I is
R5-CO, is converted to the product wherein R2 is hydrogen
by ammonolysis, e.g., alcoholic ammonia or concentrated
ammonium hydroxide solution, or alkaline hydrolysis, e.g.,
with aqueous metal hydroxide. When an acid of formula
III is used as starting material, the final product ob-
tained as the free carboxylic acid can then be converted
to its ester, for example by esterification with a dia-
zoalkane, like diazomethane, l-alkyl-3-p-tolyl-triazene,
like l-n-butyl-3-p-tolyltriazene or the like. Treatment
of an ester, preferably the methyl ester, with an alco-
holic ammonia solution, converts the free acid to the ~ ;
amide, i.e., R is NH2.
According to another variation, an ester, prefer-
ably the t-butyl ester, of formula III, in an anhydrous
medium such as dichloromethane, tetrahydrofuran, dioxane -
or the like, is treated with a thioalkanoic acid of the
formula
(~YVIII) 1l
R2-S-(CH2)n - C~ - COOH
in the presence of dicyclohexylcarbodiimide, N,N'-car-
bonyl-bisimidazole, ethoxyacetylene, diphenylphosphoryl
azide or similar coupling agents at a temperature in the
range of about 0 to 10C. The ester group (R) can then
be removed, for
-11-
s~
~IAl 3 5 b
e~ample, by treatment wi~h trifluoroacetic acid and anisole
at about room temperature.
When an ester of formula I:[I (e.g., R is lower
alko~y, especially, -t-butoxy) is acylated with a thiolactone,
e.g., ~-propiothiolactone, a-methyl-~-propiothiolactone or the
like, the reaction can be effected in an anhydrous solvent
like tetrahydrofuran, dioxane, methylene chloride or the
like at about 0 C. to about room temperature. The ester
group ean be removed with anisole and trifluoroacetic acid
as deseribed above. M
In similar manner, when R2 is R5-M-C-, products of
formula I having this substituent are formed by reacting a
-eompound of formula XII with the halogenated compound
(XIX) M
R5-M-C-X
or alternatively reaeting a compound of formula X with an
alkali metal salt or alkaline earth metal sal-t of the
lormula
(XX) 1~1
R5-M-C-S-Me
wherein lMe represents the alkali metal or alkaline earth metal.
When R2 is R5-NH-C-, products of formula I having this
substituent are produced by reaeting a compound of formula XII
with the appropriately substituted isocyana-te or isothio-
eyanate of the formula
(XXI)
R5-N=~=M
Alternatively, the same products can be produeed by coupling
an aeid of the formula
-12-
HA135b
(XXII)
~1 IR4 11
R5-NH-C-S-(CH) - CH-COOH
with an amino acid of formula III.
Compounds of formula I, wherein R2 is lower alkyl, phenyl,
substituted phenyl, phenyl-lower alkyl, triphenyl-lower alkyl,
lower alkylthiomethyl or phenyl-lower alkylthiomethyl are
produced by reacting a compound of formula XII with the
corresponding halide R2X or by reacting a compound of formula X
with the corresponding thiol R2SH in the same manner as
described above.
~ hen R2 is lower alkanoylamidomethyl, the product of
formula I is produced by condensing a compound of formula XII
with the corresponding hydroxymethyl-lower alkanoylamide of
the formula
~XXIII) lower alkyl-CO-NHCH2OH
in the presence of an acid catalyst like tri~luoroacetic acid.
Products of formula I wherein R2 is R6-S can be ;
prepared by any of the known methods for the synthesis of
mixed disulfides, e.g., by the reaction of a compound of
formula(XII)with a thiosulfinate(xxIv), thiosulfonate (XXV), ~ -
sulfenyl halide (XXVI), thiosulfate (XXVII) or sulfenyl
thiocyanate (XXVIII) O
(XXIV) R6-S-S-R6 , (XXV) R6-S-S-R6 , (XXVI) R6S-X,
(XXVII) R6-S-SO3H, (XXVIII) R6-S-SCN
In the particular case wherein R7 is
l4 IRl fH2 - (CH)n
-S(O)-- (CH) -- CH - CO - N ~ CH-COR,
-13-
~IA135b
R, Rl, R3 and R4 are the same as the corresponding substituents
in formula I and p is O, the symmetrical disulfides can be
obtained by direct oxidation of a compound of formula XII F
with iodine. When p is 1 or 2, such products are obtained
by the stepwise oxidation of the corresponding compound wherein
p is O. Mixed disulfides are obtained by the modification
shown in the examples.
Products of formula I have one or more asymmetric carbons.
When Rl, R3 or R4 is other than hydrogen the carbon to which
10 it is attached is asymmetric. These carbon atoms are
indicated by an asterisk in formula I. The compounds
accordingly exist in stereoisomeric forms or in racemic
mixtures thereof. All of these are within the scope of
the invention. The above described synthesis can utilize
the racemate or one of the enantiomers as starting material.
When the racemic starting material is used in the synthetic
procedure, the stereoisomers obtained in the product can be
separated by conventional chromatographic or fractional
crystallization methods. In general, the L-isomer with
20 respect to the carbon of the amino acid constitutes the
preferred isomeric form. Also the D-isomer with respect to
the ~-carbon in the acyl side chain (i.e., the carbon
bearing Rl) is preferred.
The compounds of this invention form basic salts
with various inorganic and organis bases which are also
within the scope of the invention. Such salts include
ammonium salts, alkali metal salts like sodium and potassium ~.
salts (which are preferred), alkaline earth metal salts like
the calcium and magnesium salts, salts with organic bases,
e.g., dicyclohe~ylamine salt, benzathine, N-methyl-D-glucamine,
--1~--
~L~$ ~
HA13Sb
hydrabamine salts, salts with amino acids like arginine,
lysine and the like. The non-toxic, physioloqically acceptable
salts are preferred, although other salts are also useful,
e.g., in isolating or purifying the product, dS illustrated
in the examples in the case of the dicyclohexylamine salt.
The salts are formed in conventional manner by reacting
the free acid form of the product with one or more equivalents
of the appropriate base providing the desired cation in a
solvent or medium in which the salt is insoluble, or in
water and removing the water by freeze drying. By neutralizing
the salt with an insoluble acid like a cation exchange resin
in the hydroge~ form (e.g., polystyrene sulfonic acid resin
like Dowex 50) or with an aqueous acid and extraction with an
organic solvent, e.g., ethyl acetate, dichloromethane or the
like, the free acid form can be obtained, and, if desired,
another salt formed.
-15-
... , . . . _ _
Additional experimental details are found in the
examples which are preferred embodimen-ts and also serve as
models for the preparation of other members of -the group.
The compounds of this invention inhibit -the conver-
sion of the decapeptide angiotensin I to angiotensin II
and therefore are useful in reducing or relieving angio-
tensin related hypertension. The action of the enzyme re-
nin on angiotensinogen, a pseudoglobulin in blood plasma,
produces angiotensin I. Angiotensin I is converted by
angiotensin converting enzyme (ACE) to angiotensin II.
The latter is an active pressor substance which has been
implicated as the causative agent in various forms of
hypertension in various mammalian species, e.g., rats and
dogs. The compounds of this invention intervene in the
angiotensin(renin)-~angiotensin I -~angiotensin II sequence
by inhibiting angiotensin converting enzyme and reducing
or eliminating the formation of the pressor substance
angiotensin II. Thus by the administration of a composi-
tion containing one or a combination of compounds of for-
mula I or physiologically acceptable salt thereof, angio-
tensin dependent hypertension in the species of mammal
suffering therefrom is alleviated. A single dose, or pre-
ferably two to four divided daily doses, provided on a
basis of about 0.1 to 100 mg. per kilogram per day, pre-
ferably about 1 to 50 mg. per kilogram per day is appro-
priate to reduced blood pressure as indicated in the ani-
mal model experiments described by S.L. Engel, T.R.
Schaeffer, M.H. ~augh and B. Rubin, Proc. Soc. Exp. Biol.
Med. 143, 483 (1973). The substance is preferably admin-
istered orally, but parenteral routes such as subcutaneous,intramuscular, intravenous or
-16-
. '`'~
intraperi-toneal can also be employed.
The ccmpounds of this invention can be utilized -to achieve the
reduction of blood pressure by Eormula-ting in CQmpositiOnS such as
tablets, capsules or elixirs for oral adminis-tration or in sterile
solutions or suspensions for parenteral ac~ministration. About 10 to
500 mg. of a compound or mixture of compolmds of formula I or physio-
logically acceptable salt is compounded with a physiologically accep-
table vehicle, carrier, excipient, binder, preservative, stabilizer,
flavor, etc., in a unit dosage form as called for by accepted phar-
~aceutical practice. The amount of active substance in these com-
positions or preparations is such that a suitable dosage in the range ~.
indicated is obtained.
Illustrative of the adjuvants which may be incorporated intablets, capsules and the like are the following: a binder such as
gum tragacanth, acacia, corn starch or gelatin; an excipient such
as dicalcium phosphate; a disintegrating agent such as corn starch,
potato starch, alginic acid and -the like; a lubricant such as
magnesium stearate; a sweetening agent such as sucrose, lactose or
saccharin; a flavoring agent such as peppermint, oil of winter-
green or cherry. When the dosage unit form is a capsule, it maycontain in addition to materials of the above type a liquid carrier
suc~ as a fatty oil. Various other materials may be present as
ceatings or to otherwise modify the physical form of the dosage
unit. For instance, tablets may be coated with shellac, sugar or
both~ A syrup or elixir may contain the active compound, sucrose
as a sweetening agent, methyl and propyl parabens as preservatives,
a dye and a flavoring such as cherry or orange flavor.
Sterile compositions for injec-tion can be formulated
~;~ ,!
according to conventional pharmaceutical practice by dis-
solving or suspending the active substance in a vehicle
such as water for injection, a na-turally occurring vege-
table oil like sesame oil, coconu-t oil, peanut oil, cotton-
seed oil, etc., or a synthetic fa-tty vehicle like ethyl
oleate or the like. Buffers, preservatives, antioxidants
and the like can be incorporated as required.
The following examples are illustrative of the in-
vention and constitute especially preferred embodiments.
All temperatures are in degrees celsius.
-18-
.`~ I ..
5~;
Example 1
1-(2-Benzoylthioacetyl)-L-Proline
L-Proline (5.75 g.) is dissolved in N sodium hydr-
oxide (50 ml.) and the solution is chilled in an ice-water
bath. Sodium hydroxide 2N (26 ml.) and chloroacetyl chlo-
ride (5.65 g.) are added and the mixture is stirred vigo-
rously at room temperature for three hours. A suspension
of thiobenzoic acid (7.5 g.) and potassium carbonate (4.8
g.) in water (50 ml.) is added. After 18 hours stirring
at room temperature, the reaction mixture is acidified and
extracted with ethyl aceta-te. The ethyl acetate layer is
washed with water, dried over magnesium sulfate and con-
centrated to dryness in vacuo. The residue (14.6 g.) is
dissolved in ethyl acetate (150 ml.) and dicyclohexylamine
(11 ml.) is added. The crystals are filtered and recry-
stallized from ethyl aeetate, yield 5.7 g. m.p. 151-152.
To convert the salt to the acid, the crystals are dissol-
ved in a mixture of 5~ aqueous potassium bisulfate (100
ml.) and ethyl acetate (300 ml.). The organie phase is
washed onee with water, dried over magnesium sulfate and
eoneentrated to dryness in vacuo, yield 3.45 g.
Example 2 `
1-(2-~ereaptoacetyl)-L-Proline
1-(2-Benzoylthioaeetyl)-L-proline (3.4 g.) is dis-
solved in a mixture of water (10.5 ml.) and eoneentrated
ammonia (6.4 ml.). After one hour, the reaetion mixture
is diluted with water and filtered~ The Eiltrate is ex-
traeted with ethyl aeetate and then aeidified with con-
eentrated hydroehlorie aeid, sa-turated with sodium ehlo-
ride and extraeted twiee with ethyl aeetate. The ethylaeetate extraets are washed with saturated sodium ehlo-
ride and eoneentrated to dryness, yield 1.5 g.
--19--
.,
~ , ` '
3 ~
The product, l~ mercaptoacetyl)-L-proline is crystallized
from ethyl acetate (m.p. 133-135).
Example 3
1-(2-Benzoylthioacetyl)-L-Proline Methyl Ester
1-(2-Benzoylthioacetyl)-L-proline obtained in Example
1, is dissolved in methanol and an ~ethereal solution of
diazomethane is added until there is a persistent yellow
color. After 15 minutes, a few drops of acetic acid are
added and the solvent is removed in vacuo to obtain 1-(2-
benzoylthioacetyl)-L-proline methyl ester.
Example 4
1-~2-Mercaptoacetyl)-L-Proline Amide
The product of Example 3 is dissolved in 10~ metha-
nolic ammonia and the solution is stored at room tempera-
ture in a pressure bottle. When thin layer chromatogra-
phic analysis indicates that the two ester functions have
been ammonolyzed, the reaction mixture is concentrated to
dryness to obtain l-t2-mercaptoacetyl)-L-proline amide.
Example 5
1-(2-Benzoylthioacetyl)-L-Hydroxyproline
By substituting L-hydroxyproline for the L-proline in
the procedure of Example 1, 1-(2-benzoylthioacetyl)-L-
hydroxyproline is obtained.
Example 6
1-(2-Mercaptoacetyl)-L-Hydroxyproline
By treating the product of Example 5 with ammonia as
in Example 2, 1-(2-mercaptoacetyl)-L-hydroxyproline is
obtained.
Example 7
1-(2-Benzoylthioacetyl)-L-Azetidine-2-Carboxylic Acid
By substituting L-azetidine-2-carboxylic acid for the
- 20 -
~r
L-proline in the procedure of Example 1, 1-(2-benzoylthio-
acetyl)-L-azetidine-2-carboxylic acid is obtained.
Example 8 ~.
1-(2-Mercaptoacetyl?-L-Azetidine-2-Carboxylic Acid
By treating the product of Lxample 7 with ammonia
as in Example 2, 1-(2-mercaptoacetyl)-L-azetidine-2-car-
boxylic acid is obtained.
Example 9
1-(2-Benzoylthioacetyl)-L-pipecolic Acid
By substituting L-pipecolic acid for the L-proline
in the procedure of Example 1, 1-(2-benzoylthioacetyl)-L-
pipecolic acid is obtained.
Example 10
1-(2~Mercaptoacetyl)-L-Pipecolic Acid
By treating the product of Example 9 with ammonia
as in Example 2, 1-(2-mercaptoacetyl)-L-pipecolic acid is
obtained.
Example 11
1-(2-Benzoylthiopropanoyl)-L-Proline
L-Proline (5.75 g.) is dissolved in aqueous N so-
dium hydroxide (50 ml.) and the solution is chilled in an
ice bath with stirring. 2N sodium hydroxide (25 ml.) and
2-bromopropionyl chloride (8.57 g.) are added in that
order and the mix-ture is removed from the ice bath and
stirred at room temperature for one hour. A mixture of
thiobenzoic acid (7.5 g.) and potassium carbonate (4.8 g.)
in water (50 ml.) is added and the mixture is stirred over-
night at room temperature. After acidification with con-
centrated hydroch:Loric acid, the aqueous solution is ex-
tracted with ethy:L acetate and the organic phase is washed
with water, dried and
~3~
concentrated to clryness. The residue (1~.7 g.) is chroma-
to~raphed on a column of 440 ~. of silica gel with a mix-
ture of benzene-acetic acid (7:1). The fractions contain-
ing the desired material are pooled, concen-trated to dry-
ness, and the residue is precipitated twice with e-ther-
hexane and converted to a dicyclohexylamine salt in e-ther-
hexane, yield 9.4 g. m.p., (142) 148-156. The dicyclo-
hexylamine salt is converted back to the acid as in Exam-
ple 1, yield 5.7 g.
Example 12
1-(2-Mercaptopropanoyl)-L-Proline
1-(2-Benzoylthiopropanoyl)-L-proline (5.7 g.) is
dissolved in a mixture of water (12 ml.) and concentrated
ammonium hydroxide (9 ml.) with stirring. After one hour,
the mixture is diluted with water (10 ml.) and filtered.
The ~iltrate is extracted twice with ethyl acetate, con-
centrated to one-third of the original volume, acidified
with concentrated hydrochloric acid and extracted with
ethyl acetate. The organic phase is washed with satura-
ted sodium chloride, dried and concentrated to dryness invacuo. The residue, 1-(2-mercaptopropanoyl)-L-proline, is
crystallized from ethyl aceta-te-hexane, yield 3 g., m.p.
(105) 116-120.
Example 13
1-(3-Benzoylthiopropanoyl)-L-Proline
L-Proline (5.75 g.) is dissolved in normal sodium
hydroxide (50 ml.) and the solution is chilled in an ice
bath. 3-Bromopropionyl chloride (8.5 g.) and 2N sodium
hydroxide (27 ml.) are added and the mixture is stirred
for 10 minutes in the ice bath and three hours at room
temperature. A suspension of thiobenzoic acid (7.5 g.)
and potassium carbonate (4.5 g.) in water (50 ml.) is
added and the mixture is stirred
~J~
for 18 hours at room temperature. AEter acidiEica-tion wi-th
concentrated hydrochloric acid, the aqueous phase is ex-
tracted twice Wit~l ethyl acetate. The organic layers are
dried over ma~nesium sulfate and concentrated -to dryness
_ vacuo to obtain 1-(3-benzoylthiopropanoyl)-L-proline,
yield 7.1 g., m.p. 101-102 (ethyl acetate-hexane).
Example 14
L-Proline tert.-butyl ester
L-Proline (230 g.) is dissolved in a mixture of
water (1 1.) and 5 N sodium hydroxide (400 ml.). The so-
lu~ion is chilled in an ice bath, and under vigorous stir-
ring, 5 N sodium hydroxide (460 ml.) and benzyloxycarbonyl
chloride (340 ml.) are added in five equal aliquots during
a half hour period. After one hour stirring at room tem-
perature, the mixture is extracted twice with ether and
acidified with concentrated hydrochloric acid. The preci-
pitate is filtered and dried. Yield 442 g., m.p. 78-80.
The benzyloxycarbonyl-L-proline thus obtained (180
g~) is dissolved in a mixture of dichloromethane (300 ml.),
liquid isobutylene (800 ml.) and concentrated sulfuric acid
(7.2 ml.). The solution is shaken in a pressure bottle
for 72 hours. The pressure is released, the isobutylene
is allowed to evaporate and the solution is washed with 5%
sodium carbonate, water, dried over magnesium sulfate and
concentrated to dryness in vacuo, -to obtain benzyloxycar-
bonyl-L-proline tert. butyl ester, yield 205 g.
Benzoyloxycarbonyl-L-proline tert. butyl ester `
(205 g.) is disso:Lved in absolute ethanol (1.2 1) and hy-
drogenated at normal pressure with 10% Pd on carbon (10
g.) until only a -trace of carbon dioxide is observed in
-the hydrogen exi-t gas
-23-
HA135b
(24 hours). ~ e catalyst is Eilter~d of r ancl ~hc filtrate
is concentrated in vacuo at 30 mm. ~kJ. The residue is
distilled in vacuo,.~o obtain L-proline tert.~utyl ester,
b.p. 50-51.
lmM
~ aml~lc 15
1-(3-~cctylthioproi>anoyl)-L-Prolinc tert-butyl Ester
~ L-Proline tert-butyl ester (5.13 cJ.) is dissolved in
dichloromethane (40 ml.) and the solution is chilled in an
ice-water bath. A solution of dicyclohexylcarbodiimide
(6.18 g.) in dichloromethane (20 ml.) is added followed
immediately by 3-acetylthiopropionic acid (q.45 y.). After
15 minutes stirring in the ice-water bath and 16 hours at
room temperature, the precipitate is filtered off and the
filtrate is concentrated to dryness in vacuo. The residue is
dissolved in ethyl acetate and washed neutral. The organic
layer is dried over magnesium sulfate and conccntrated to
dryness in vacuo to obtain 9.8 g. of 1-(3-acetylthiopropanoyl)-L-
proline tert-butyl ester.
Example lG
1-(3-~cetyltllio~ropanoyl)-L-Prolinc
1-(3-~cetylthiopropanoyl)-L-proline-t-butyl ester
(4.7 g.) is dissolved in a mixture of anisole (34 ml.) and
trifluoroacetic acid (68 ml.) and -the mix~ure is ke~t at
room temperature for one hour. The solven~s are removed
in vacuo and the residue is precipitated from ether-hexane
several times. The residue (3.5 g.) is dissolved in `
acetonitrile (25 ml.) and dicyclohexylamine (2.8 ml.) is
added. The crystalline salt is filtered and recrystallizcd
from isopropanol. Yield 3.8 g.. m.p. 176-177 . The salt
is reconverted to 1-(3-acetylthiopro~anoyl)-L-proline as in ',
-24-
HA135b
Example 1, yield 1.25 g., m.p. 89-90 (etllyl acetat~-hcxane).
Examplc 1~ 1
1-(3-i~eL-captopropalloyl)-L-proline tert-butyl Ester
To a solution of L-proline tert-butyl ester (3.42 g.)
in dry tetrahydrofuran (10 ml.) chilled in an ice ~ath, L
propiothiolactone (1.76 g.) is addcd. ~fter 5 n!inutes storage in 1'
- the ice bath and three hours at room temperature, the reaction
mixture is diluted with ethyl acetate (~00 ml.) and washed
~ith 5% potassium bisulfate, and water. The organic layer L
is dried over magnesium sulfate and concentrated to dryness in
vacuo. The residue 1-(3-mercaptopropanoyl)-L-proline tert-
butyl ester is crystallized from ether-hexane, yield 3.7 g.,
m.p. 57-58.
Exam~le 18
1-(3-1~lercaptopropanoyl)-L-Proline - -
Proceclure
r
1-(3-Benzoylthiopropanoyl)-L-proline (4.9 g.) is r
dissolved in a mixture of water (8 ml.) and concentrated ammonium
hydroxide (5.6 ml.) and the solution is stored with stirring
under argon for one hour. The reaction mixture is diluted
with water, filtered, and the filtrate is extracted with
ethyl acetate. The aqueous phase is acidified with concentrated
hydrochloric acid, saturated with sodium chloride and extracted j;
with ethyl acetate. The organic layers are washed with
saturated sodium chloride, dried over magnesium sulfate, and
concentra-ted to dryness in vacuo. The residue,l-(3-mercapto-
propanoyl)-L-proline, is crystallized from ethyl acetate
hexane, yield 2.5 ~., M.p. 68-70 .
Procedure B
1-(3-Acetylthiopropanoyl)-L-proline (0.8 g.) is dissolved
-25-
i
~3~
HA135b
in 5.5 N methanolic ammollia (5 Inl.) ancl ~lle solu~ion ~e~
under aryon at room temperature. After 2 hours ~lle solvent is
removed in vacuo, the residue is dissolved in water and applied
to an ion e~change column on the 1l cycle[Dowex 50tAnalytical
c3rade)~and eluted with water. The frac~ions tl~at CJiVe thiol
positive reaction are pooled and concentrated to dryness,
yield 0.6 g. This product is crystallized from ethyl acetate-
hexane as in Procedure A to obtain 1-(3-merca~topropanoyl)-L-
proline. ~'
Procedure C
1-(3-Mercaptopropanoyl)-L-proline t-butyl ester ~2.3 g.)
is dissolved in a mixture of anisole (20 ml.~ and trifluoro-
acetic acid (45 ml.). After one hour storac~e at room temperature
under argon, the reaction mixture is concentra~ed ~o dryness
in vacuo and the residue precipitated from ethyl acetate-
hexane several times. 'l'he residue (1.9 g.) is dissolved in
ethyl acetate (30 ml.) and dicyclohexylamine (1.~5 ml.) is
added. Tlle crystalline salt is filtered and recrystallizcd
from isopropanol, yield 2 g. m.p. 187-188 .
The salt is converted to the acid as in ~xample 1,
yield 1.3 c3. The product is crystallized from ethyl acetate
hexane as in Procedure ~.
Salts
Sodium
1-(3-Meroaptopropanoyl)-L-proline (500 my.) is dissolved
in a mixture of water (2.5 ml.) and N sodium hydroxide ~2.5 ml.).
The solution is freeze dried to o~tain ~l~e sodium salt.
Ma~nesium
1-(3-Me~-captopropanoyl)-L-prolille (500 mc~.),
magnesium oxide (49.5 mg.), and ~a~er (10 ml.) are s~irred
. _ . . .................................... _ . . . , . ... _ . _
, .
with slight heating until complete solution is obtained.
Then the solvent is removed by Ereeze dryin~ to obtain the
magnesium salt.
Calcium
l-t3-Mercaptopropanoyl)-2-p:roline (500 mg.) is
dissolved in a mixture of calcium hydroxide (91 mg.) and
water (10 ml.), and the solution is freeze dried to obtain
the calcium salt.
Potassium
1-(3-Mercaptopropanoyl)-L-proline (500 mg.) is
dissolved in a mixture of potassium bicarbonate (246 mg.)
and water (10 ml.) and freeze dried to obtain the potassium
salt.
N-Methyl-D-Glucamine
1-(3-Mercaptopropanoyl)-L-proline(500 mg.) and N-
methyl-D-glucamine (480 mg.) are dissolved in water (10 ml.)
and freeze dried to obtain the N-methyl-D-glucamine salt.
Example 19
1-(3-Mercaptopropanoyl)-L-Hydroxyproline
By substituting L-hydroxyproline for the L-proline
in the procedure of Example 11 and then treating the pro-
duct by Procedure A of Example 18, 1-(3-benzoylthiopropan-
oyl)-L-hydroxyproline and 1-(3-mercaptopropanoyl)-L-hydr-
oxyproline, respectively, are obtained.
Example 20
1-(3-Mercaptopropanoyl)-L-Azetidine-2-Carboxylic Acid
By substituting L-azetidine-2-carboxylic acid
tert-butyl ester (prepared by substituting L-azetidine-2-
carboxylic acid for the proline in Example 14) for the L-
proline tert-butyl ester in the procedure of Example 15,
3~
HA135b
treating ~he product as in ~ample 16 and tl~e 1-(3-acetyl-
thiopropanoyl)-L-azetidine-2-carboxylic acid tllus obtained by
Procedure B of Example 18, 1-(3-acetylthiopropanoyl~-L-
azetidine-2-carboxylic acicl tert-butyl ester and 1-(3-mercapto-
propanoyl)-L-aze~idine-2-carboxylic acid, respectively,
are obtained.
Example 21
1-(3-Mercaptopropanoyl?-L-Pipccolic ~cid
By substituting L-pipecolic acid tert-butyl ester
(prepared by substituting L-pipecolic acid for tlle L-proline
in Example 14) for the L-proline tert-butyl cster in the
procedure of Example lS and ~reating the product by Procedure C
of Example 18, 1-(3-mercaptopropanoyl)-L-pipecolic acid tert-
butyl ester and l-(3-mercaptopropanoyl)-L-pipecolic acid,
respectively, are obtained.
Example 22
1-(3-l~iercaptopropanoyl)-4--'/letllyl-L-Prolinc
By substituting 4-methyl-L-proline for L-prolinc-
in tlle procedure of Example 13 and then treating the product
by Procedure A of Example 18, 1-(3-benzoyltlliopropanoyl)-
4-methyl-L-proline and 1-(3-mercaptopropanoyl)-4-mctllyl-L-
proline, are obtained.
ExalllL~lc 23
1-(3-~1ercaptoproL~anoyl)-5-llydroxy-L-Pipecolic ~cid
~ y substituting 5-hydroxy-L-pipecolic acid for L-
proline in the procedure of Example 13 and then treating the
product by the Procedure A of Example 18, 1-(3-ben~oyl-
thiopropanoyl)-5-hydroxy-L-pipecolic, and 1-(3-mercapto-
propanoyl)-5-hydroxy-L-pipccolic acid ar~ obtained.
-28-
, _ , _ _ . . . . . .
~3~
Example 24
1-(3-Mercaptopropanoyl)-D-Proline
By substituting D-proline for L-proline in the pro-
cedure of Example 13 and then treating the product by Pro-
cedure A of Example 18, 1-(3-benzoylthiopropanoyl)-D-proline
and l-(3-mercaptopropanoyl)-D-proline, m.p. 68-70, are ob-
tained.
Example 25
3-Acetylthio-2-Methylpropanoic Acid
A mixture of thioacetic acid (50 g.) and methacry- ,
lic acid (40.7 g.) is heated on the steam bath for one hour
and then stored at room temperature for 18 hours. After
confirming by nmr spectroscopy that complete reaction of
the methacrylic acid has been achieved, the reaction mix-
ture is distilled in vacuo and the desired 3-acetylthio-2-
methylpropanoic acid is separated in the fraction with
boiling point 128.5-131 (2.6 mmHg.), yield 64 g.
Example 26
3-Benzoylthio-2-Methylpropanoic Acid
By substituting thiobenzoic acid for the thioace-
tic acid in the procedure of Example 25, 3-benzoylthio-2- `;
methylpropanoic acid is obtained.
Example 27
3-Phenylacetylthio-2-Methylpropanoic Acid
By substituting thiophenylacetic acid for the thio-
acetic acid in the procedure of Example 25, 3-phenylacetyl
thio-2-methylpropanoic acid is obtained.
Example 28
1-(3-Acetylthio-2-methylpropanoyl)-L-Proline tert-butyl
Ester
L-Proline tert-butyl ester (5.1 g.) is dissolved
in dichloromethane (40 ml.) and the solution stirred and
chilled
-29-
,~
;5
in an ice bath. Dicyclohexylcarbocliimide (6.2 g.) dissolved
in dichloromethane (15 ml.) is added Eollowed immediately by
a solution of 3-acetylthio-2-me-thylpropanoic acid (4.9 g.)
in dichloromethane (5 ml.). After 15 minutes stirring in
the ice bath and 16 hours at room temperature, the precipi~
tate is filtered off and -the filtrate is concentrated to
dryness in vacuo. The residue is dissolved in e-thyl acet-
ate and washed neu-tral. The organic phase is dried over
magnesium sulfate and concentrated to dryness in vacuo.
The residue 1-(3-acetylthio-2-methylpropanoyl)-L-proline
tert-butyl ester is purified by column chromatography
(silica gel-chloroform), yield 7.9 g.
Example 29
1-(3-Acetylthio-2-methylpropanoyl)-L-Proline
Procedure A
The 1-(3-acetylthio-2-methylpropanoyl)-L-proline
tert-butyl es-ter of Example 28 (7.8 g.) is dissolved in a
mixture of anisole (55 ml.) and trifluoroacetic acid (110
ml.). After one hour storage at room temperature the sol-
vent is removed in vacuo and the residue is precipita-ted
several times from ether-hexane. The residue (6.8 g.) is
dissolved in acetonitrile (40 ml.) and dicyclohexylamine
(4.5 ml.) is added. The crystalline salt is boiled with
fresh acetonitrile (100 ml.), chilled to room temperature
and filtered, yield 3.8 g., m.p. (165) 187-188. This ma-
terial is recrystallized from isopropanol [~]D-67 (C 1.4,
EtOH). The crystalline dicyclohexylamine salt is suspen-
ded in a mixture of 5% aqueous potassium bisulfate and
ethyl acetate. The organic phase is washed with water
and concentrated to dryness. The residue is crystallized
from ethyl acetate-hexane to yield the l-(3-acetyl-
-30-
^ r~
thio-2-D-methylpropanoyl-B-proline, m.p. 83-85[a]D -162
(c,1.7,EtO~).
Procedure B
3-Acetylthio-2-methylpropanoic acid (8.1 g.) and thio-
nyl chloride (7 g.) are mixed and the suspension is stirred
for 16 hours at room temperature. The reaction mixture is
concentrated to dryness and distille~d in vacuo (b.p. ~0).
This 3-acetylthio-2-methylpropanoic acid chloride (5.4 g.)
and 2N sodium hydroxide (15 ml.) are added to a solution of
L-proline (3.45 g.) in normal sodium hydroxide (30 ml.)
chilled in an ice water bath. After 3 hours stirring at
room temperature, the mixture is extracted with ether, the
aqueous phase is acidified and extracted with ethyl acetate.
The organic phase is dried over magnesium sulfate and con-
centrated to dryness to obtain l-(3-acetylthio-2-DL-methyl-
propanoyl-L-proline).
Procedure C
Methaeryloyl ehloride (4.16 g.) is added to a solu-
tion of L-proline (3.45 g.) in a mixture of water (100 ml.)
and sodium bicarbonate (12 g.) chilled in an ice water
bath, ~ith vigorous stirring. When the addition is com-
pleted, the mixture is stirred at room temperature for two
hours, and then extracted with ether. The aqueous phase
is acidified with N hydrochloric acid and extracted with
ethyl acetate. The organic phase is eoncentrated to dry-
ness in vacuo, the residue is mixed with thiolacetic acid
(3.5 g.), a few crystals of azobisisobutyronitrile are
added and the mixture is heated on the steam bath for two
hours. The reaction mixture is dissolved in benzene-ace-
-tie aeid (75:25), and applied to a eolumn of siliea gel.
Elution with the same solvent mixture yields -the 1-(3-ace-
tylthio-2-DL-methylpropanoyl)-L-proline.
5~
Example 30
1-(3-Benzoylthio-2-methylpropanoyl)-L-proline tert-butyl
Ester
By substituting 3-benzoylthio-2-methylpropanoic acid
for the 3-ace-tylthio-2-methylpropanoic acid in the procedure
of Example 28, 1-(3-benzoylthio-2-me-thylpropanoyl)-L-proline
tert.butyl ester is obtained.
Example 31
1-(3-Phenylacetylthio-2-methylpropanoyl)-L-Proline tert-
butyl Ester
By substituting 3-phenylacetylthio-2-methylpropanoic
acid for the 3-acetylthio-2-methylpropanoic acid in the
procedure of Example 28, 1-(3-phenylacetylthio-2-methyl-
propanoyl)-L-proline tert butyl ester is obtained.
Example 32
1-(3-Benzoylthio-2-methylpropanoyl)-L-proline
By substituting 1-(3-benzoylthio-2-methylpropanoyl)-
L-proline tert-butyl ester for the 1-(3-acetylthio)-2-
methylpropanoyl-l-proline tert-butyl ester in Procedure A
of Example 29, 1-(3-benzoylthio-2-methylpropanoyl)-L-pro-
line is obtained.
Example 33
1-(3-Phenylacetylthio-2-methylpropanoyl)-L-proline
By substituting 1-(3-phenylacetylthio-2-methylpro-
panoyl)-L-proline tert-butyl ester for 1-(3-acetylthio-2-
methylpropanoyl)-L-proline tert-butyl es-ter in Procedure
A of hxample 29, 1-(3-phenylacetylthio-2-methylpropanoyl)-
L-proline is obtained.
Example 34
1-(3-Mercapto-2-D-methylpropanoyl)-L-Proline
1-(3-Mercapto-2-methylpropanoyl)-L-proline is ob-
tained by treating the product of each of Examples 29,
32 and 33 as follows:
-32-
~ 3~
The thioester (0.85 g.) is dissolved in 5.5 N metha-
nolic ammonia and the solu-tion is ~ept a-t room temperature
for 2 hours. The solvent is removed in vacuo and -the re-
sidue is dissolved in water, applied to a ion exchange co-
lumn on the H cycle (Dowex 50, analytical grade) and
eluted with water. The frac-tions that give positive -thiol
reaction are pooled and freeze dried. The residue is cry-
stallized Erom ethyl acetate-hexane, yield 0.3 g. The 1-
(3-mercapto-2-D-methylpropanoyl-L-proline has m.p. 103-104
[~]D -131 (C,2,EtOH).
Example 35
1-(3-Acetylthio-2-methylpropanoyl)-L-Proline Methyl Ester
1-(3-Acetylthio-2-methylpropanoyl)-L-proline is re-
acted with an ethereal solution of diazomethane according
to the procedure described in Example 3 to obtain 1-(3-
acetylthio-2-methylpropanoyl)-L-proline methyl ester.
Example 36
1-(3-Mercapto-2-methylpropanoyl)-L-Proline amide
By substitu-ting 1-(3-acetylthio-2-methylpropanoyl)-
L-proline methyl ester in the procedure of Example 4, 1-
(3-mercapto-2-methylpropanoyl)-L-proline amide is obtained
Example 37
3-Acetylthio-2-Benzylpropanoic Acid
By substituting 2-benzylacrylic acid Eor the metha-
crylic acid in the procedure of Example 25, 3-acetylthio-
2-benzylpropanoic acid is obtained.
Example 38
1-(3-Acetylthio-2-benzylpropanoyl)-L-Proline tert-butyl
Ester
. _ _
By substituting 3-acetylthio-2-benzylpropanoic acid
~or the 3-acetylthio-2-methylpropanoic acid in the proce-
dure of
-33-
.
~ 13Sb
E:;ample 22, 1-(3-acetylthio-2-benzylpropanoyl)-L-proline
tert-b~tyl ~ster is obtained.
Example 39
1-(3-Acetylthio-2-benzylpropano~ -L-Proline
_ _ _ ~ _ _ _ _ _ _ _ _ _ _ _
The product of Æxample 38 is subst:ituted for -the 1-(3-
acetylthio-2-methylpropanoyl-L-proline tert-butyl ester in
the Procedure A of E~ample 29 to obtain 1-(3-acetylthio-2-
benzylpropanoyl)-L-proline.
Example 40
.
1-(3-Mercapto-2-benzylpropanoyl)-L-Proline
.
1-(3-Acetylthio-2-benzylpropanoyl)-L-proline is treated
with metnanolic ammonia according to the procedure o~ Example
34 to obtain 1-(3-mercapto-2-benzylpropanoyl)-L-proline as an
oil, R~ = 0.47 (silica gel, benzene-acetic acid (75:25).
Example 41
1-(3-Mercapto-2-methylpropanoyl) L-Hydroxy Proline
By substituting L-hydroxy proline tert-butyl ester in
the procedure of Example 28, treating the product according
to Procedure A of Example 29 and then continuing as in Example
34, 1-(3-acetylthio-2-methylpropanoyl)-L-hydroxyproline
tert-butyl ester, l-(3-acetylthio-2-methylpropanoyl)-L-
hydroxyproline and l-(3-mercapto-2-methylpropanoyl)-L-
hydroxyproline, respectively, are obtained.
Example 42
1-(3-Mercapto-2-methylpropanoyl)-L-Azetidine-2-Carboxylic Acid
By substituting L-azetidine-2-carboxylic acid tert-
b-~tyl ester in the procedure of Example 28, treating the product
according to the Procedure A of Example 29 and then continuing as
-34-
-: :
5~
in Example 34, 1-(3-acetylthio-2-methylpropanoyl)-L-~zetidine-
2-carboxylic acid tert-butyl ester, 1,3-acetylthio-2-me-thyl-
propanoyl)-L-azetidine-2-carboxylic acid and 1-(3-mercapto-
2-methylpropanoyl-L-azetidine-2-carboxylic acid are obtained.
Example 43
1-(3-Mercapto-2-methylpropanoly)-L-Pipecolic Acid
sy substituting L-pipecolic acid in the procedure of
Example 28, treating the product according to Procedure A of
Example 29 and then continuing as in Example 34, 1-(3-acetyl-
thio-2-methylpropanoyl)-L-pipecolic acid tert-butyl ester,
1-~3-aCetylthio-2-methylpropanoyl)-L-pipecolic acid and 1-(3-
mercapto-2-methylpropanoyl)-L-pipecolic acid, respectively,
are obtained.
Example 44
1_(4-Penzoylthiobutanoyl)-L-Proline_
To a solution of L-proline (2.88 g.) in normal sodium
hydroxide ~25 ml.) chilled in an ice bath, 2N sodium hydroxide
~12.5 ml.) and 4-chlorobutyryl chloride ~3.5 g.) are added.
The reaction mixture is stirred at room temperature for 3.5
hours and a suspension of thiobenzoic acid ~3.75 g.) and
potassium carbonate ~2.4 g.) in water (25 ml.) is added. After
overnight stirring at room temperature, the reaction mixture is
acidified with concentrated hydrochloric acid and extracted with
ethyl acetate. The organic layer is dried over magnesium sul-
fate and concentrated to dryness in vacuo. The residue is
chromatographed on a column of silica gel with benzene-acetic
acid (7:1). The fractions containing the desired material are
pooled and concentrated to dryness, yield 1.35 g. A small
aliquot of this mat:erial is dissolved in ethyl acetate and
dicyclohexylamine is added until pH 8-10 (on a wet pH paper).
~ -35-
The dicyclohexylamine salt crystallizes out, immediately, m.p.
159-161.
Example 45
1-(4-Mercaptobutanoyl)-L-Proline
1-(4-Benzylthiobutanoyl)-L-proline (1.08 g.) is
dissolved in a mixture of water (4 ml.) and concentrated
ammonia (2.7 ml.). After one hour stirring at room tempera-
ture, the mixture is diluted with wc~ter, filtered, extracted with
ethyl acetate, and the aqueous phase was concentrated in vacuo.
This ammonium salt of 1-(4-mercaptobutanoyl)-L-proline is
purified by ion exchange chromatography on a column of diethyl-
aminoethyl-Sephadex (cross linked dextran) with a gradient of
ammonium bicarbonate, yield 0.7 g. The ammonium salt is
dissolved in water (2 ml.) and applied to a column of
Dowex 50 sulfonic acid resin analytical grade in the hydrogen
form, and the free acid is eluted with water. The fractions
containing the desired material tsulfhydryl reagent and
carboxyl reagent positive) are pooled and freeze dried to
obtain l-(4-mercaptobutanoyl)-L-proline. The dicyclohexyl
ammonium salt is produced by the procedure of Example 44,
m.p. 157-158~.
Example 46
4-Bromo-2-Methylbutanoic Acid
Ethyl-4-bromo-2-methylbutanoate [G. Jones and J. Wood,
Tetrahedron, 21, 2961 (1965)] (1.04 g.) is dissolved in
dichloromethane (50 ml.) and cooled to -10. A 1 M solution
of boron tribromide in dichloromethane (50 ml.) is added
dropwise with stirring and the stirring is continued for
1 hour at -10 and 2 hours at 25. The reaction is terminated
by the careful addition of water. The layers are separated
and the organic phase is washed with water, dried and concen-
trated to dryness to obtain 4-bromo-2-methylbutanoic acid.
Example 47
1-(4-Benzoylthio-2-methylbutanoyl)-I.-Proline
a) 4-Bromo-2-methylbutanoic acid (~ g.) and thionyl chloride
(7 g.) are mixed and the mixture is stirred for 16 hours at room
temperature. The reaction mixture is concentrated to dryness
and distilled in vacuo.
b) To a solution of L-proline (2.88 g.) in normal sodium
hydroxide (25 ml.) chilled in an ice bath, 2N sodium hydroxide
(12.5 ml.) and the 4-bromo-2-methylbutanoic acid chloride
obtained in part (a) (3.9 g.) are added. The reaction
mixture is stirred at room temperature for 3.5 hours and a
suspension of thiobenzoic acid (3.75 g.) and potassium
carbonate (2.4 g.) in water (25 ml.) is added. After overnight
stirring at room temperature, the reaction mixture is acidified
with concentrated hydrochloric acid and extracted with ethyl
acetate. The organic layer is dried over magnesium sulfate
and concentrated to dryness in vacuo. The residue is
chromatographed on a column of silica gel with benzene-acetic
acid (7:1). The fractions containing the desired product,
1-(4-benzoylthio-2-methylbutanoyl)-L-proline are pooled
and concentrated to dryness in vacuo.
Example 48
. _ _
1-(4-Mercapto-2-methylbutanoyl)-L-Proline
By substituting 1-(4-benzoylthio-2-methylbutanoyl)-
L-proline for the 1-(4-benzoylthiobutanoyl)-L-proline in
the procedure of Example 45, 1-(mercapto-2-methylbutanoyl)-
L-proline in obtained.
-37-
~ .
Example 49
4-Bromo-2-benzylbutanoic acid
By substituting ethyl-4-bromo-2-benzylbutanoate
[prepared by the procedure of G. Jones and J. Wood [Tetrahedron,
_, 2961 (1965) starting with diethylbenzylmalonate]] for
the ethyl-4-bromo-2-methylbutanoate in the procedure of
Example 46, 4-bromo-2-benzylhutanoic acid is obtained.
Example 50
1-(4-Benzoylthio-2-benzylbutanoyl)-L-Proline
By substituting 4-bromo-2-benzylbutanoic acid for
the 4-bromo-2-methylbutanoic acid in the procedure of
Example 47, 1-(4-benzoylthio-2-benzylbutanoyl)-L-proline
is obtained.
Example 51
1-(4-Mercapto-2-benzylbutanoyl)-L-Proline
By substituting 1-(4-benzoylthio-2-benzylbutanoyl)-L-
proline for the l-(4-benzoylthiobutanoyl)-L-proline in the
procedure of Example 45, 1-(mercapto-2-benzylbutanoyl)-
L-proline is obtained.
Example-52
1-(4-Mercaptobutanoyl)-L-Hydroxyproline
By substituting L-hydroxyproline for the L-proline
in the procedure of Example 44 and subjecting the product to
ammonolysis as in Example 45, 1-~4-benzoylthiobutanoyl)-L-
hydroxy-proline and l-(4-mercaptobutanoyl)-L-hydroxyproline,
respectively, are obtained.
Exampl'e 53
1-(4-Mercaptobutanoyl)-L-Azetidine-2-Carboxylic Acid
By substituting L-azetidine-2-carboxylic acid for the
L-proline in the procedure of Example 44 and subjecting the
-38-
product to ammonolysis as in Example 45, 1-(4-benzoylthio-
~utanoyl)-L-azetidine-2-carboxylic acid and l-(~-mercapto-
bu-tanoyl)-L-azetidine-2-carboxylic acid, respectively, are
obtained.
Example 54
.
1-(4-Mercaptobutanoyl)-L-Pipecolic Acid
By substituting L-pipecolic acid for the L-proline in
the procedure of Example 44 and subjecting the product to
ammonolysis as in Example 45, l-(~-benzoylthiobutanoyl)-L-
pipecolic acid and 1-~4-mercaptobutanoyl)-L-pipecolic acid,
respectively, are obtained~
Example 55
1-(3-Acetylthiobutanoyl)-L-Proline tert-butyl Ester
Dicyclohexylcarbodiimide (6.2 g.) and 3-acetylthio-
butyric acid (4.86 g.) are added to a solution of L-proline
tert-butyl ester (5.1 g.) in dichloromethane (60 ml.) stirred
in an ice bath. After 15 minutes the ice bath is removed
and the mixture is stirred at room temperature for 16 hours.
The precipitate is filtered, the filtrate is concentrated to
dryness and the residue is chromatographed on a column of
silica gel with chloroform to obtain l-(3-acetylthiobutanoyl)-
L-proline tert-butyl ester, yield 5.2 g.
Example 56
1-(3-Acetylthiobutanoyl)-L-Proline
The 1-(3-acetylthiobutanoyl)-L-proline tert-butyl ester
of Example 55 (5.2 g.) is dissolved in a mixture of trifluoro-
acetic acid (60 ml.) and anisole (30 ml.) and the solution
is kept at room ternperature for one hour. The solvents are removed
in vacuo and the residual l-(3-acetylthiobutanoyl)-L-proline
is reprecipitated i~rom ether-hexane several times, yield
-39-
5~;
4 g.. The dicyclohexylamine salt is made by the procedure
of Example 44, m.p. 175-176.
Example 57
1-(3-Mercaptobutanoyl)-L-Proline
_ .
The l-t3-acetylthiobutanoyl)--L-proline of
Example 56 (0.86 g.) is dissolved in 5.5 N. methanolic
ammonia (20 ml.) and the reaction m:ixture is stored at room
temperature for 2 hours. The solvent is removed in vacuo
and the residue chromatographed on an ion exchange column
(Dowex 50) with water. The fractions containing the desired
1-(3-mercaptobutanoyl)-L-proline are pooled and lyophilized,
yield 0.6 g. The dicyclohexylamine salt is produced by the
procedure of Example 44, m.p. 183-184.
Example 58
1-~3-[[(Ethoxy)carbonyl]thio]propanoyl]-L-proline
Ethyl chloroformate (1.2 g.) is added to a solution of
3-mercaptopropanoyl-L-proline (2.03 g.) in normal sodium bicar-
bonate (30 ml.) and the mixture is stirred vigorously at 5
for one hour, and for two hours at room temperature. After
acidification with concentrated hydrochloric acid, the mixture
is extracted with ethyl acetate. The organic phase is ~ -
washed with water, dried over magnesium sulfate, and concentra-
ted to dryness to yield l-[3-[[(ethoxy)carbonyl]thio]propanoyl]- .
L-proline.
-40-
~u ~5~
Example 59
1-[3-[[(Ethoxy?thiocarbonyl]thio]propanoyl]-L-proline
Aqueous 2N sodium hydroxide (25 ml) and 3-bromopropionyl
chloride (8.5 g) are added to a solution of L-proline (5.75 g)
in N sodium hydroxide (50 ml) chilled and stirred in an ice
bath. After five minutes the ice bath is removed and the
stirring is continued at room temperature. After three hours
ethyl xantogenic acid potassium salt: (9.6 g) is added and the
mixture is stirred overnight at room temperature. The solution
is acidified with concentrated hydrochloric acid and extracted
with ethyl acetate. The organic layer is concentrated to dry-
ness and the residue is chromatographed on a column of silica
gel with a mixture of benzene-acetic acid (7:1) as solvent, to
yield l-[3-[[(ethoxy)thiocarbonyl]thio]propanoyl]-L-proline,
m.p. 94-95.
Example 60
1-[3-[[(Benzylthio?carbonyl]thio]propanoyl]-L-proline
A solution of benzylthiocarbonyl chloride (11 ml) in
dioxane (20 ml) is added in rive portions to a solution of
1-(3-mercaptopropanoyl)-L-proline (1.6 g) in normal sodium
bicarbonate (24 ml) chilled in an ice bath, over a period of
30 minutes. The ice bath is removed and the stirring is
continued for 2.5 hours at room temperature. After acid-
ification with concentrated hydrochloric acid, the aqueous
phase is extracted with ethyl acetate. The organic phase
is dried over magnesium sulfate and concentrated to dryness
to yield l-[3-[[(benzylthio)carbonyl]thio]propanoyl]-L-proline.
-41-
~i
;5
Example 61
1-[3-[[(Ethylthio)thiocarbonyl]thio]propanoyl]-L-proline
Aqueous 2N sodium hydroxide (25 ml) and 3-bromopropionyl
chloride (8.5 g) are added to a solution of L-proline (5.75 g)
in N sodium hydroxide (50 ml) chilled and stirred in an ice
bath. After five minutes, the ice bath is removed and the
stirring is continued at room temperature. After three hours,
ethyl trithiocarbonate potassium salt (10.5 g) is added and
the mixture is stirred at room temperature overnight. After
acidification with concentrated hydrochloric acid, the mixture
is extracted with ethyl acetate. The organic layer is dried
over magnesium sulfate and concentrated to dryness to yield
1-~3-[[(ethylthio~hiocarbonyl]thio]propanoyl]-L-proline.
Example 6~
3-l[(Methylamino)thiocarbonyl]thio]propionic acid
Methylisothiocyanate (4 g) is added to a solution of
3-mercaptopropionic acid (5.3 g) in a mixture of pyridine
(250 ml) and 0.5 N sodium hydroxide (100 ml). The solution
is kept at 40 for two hours and concentrated to dryness
in vacuo. The residue is dissolved in water (100 ml.),
acidified with concentrated hydrochloric acid and extracted
with ether. The organic phase is concentrated to dryness
to yield 3-[[(methylamino)thiocarbonyl]thio]propionic acid,
m.p. 86-87.
-42-
Example 63
1-[3-[[(Methylamlno)thiocarbonyl]thio~propanoyl]-L-proline
tert-butyl ester
To a solution of L-proline tert-butyl ester (1.71 g) and
hydroxybenzotriazole (1.35 g) in dichloromethane (10 ml) chilled
and stirred in an ice bath, dicyclohexylcarbodiimide (2.06 g)
and 3-methylaminothiocarbonylthiopropionic acid (1.79 g) are
added. After 15 minutes, the bath is removed and the stirring
is continued overnight. The precipitate is filtered off and
the filtrate is diluted with ethyl acetate and washed neutral.
The organic phase is concentrated to dryness to yield
1-[3-r[(methylamino)thiocarbonyl]thio]propanoyl]-L-proline
tert-butyl ester, m.p. 129-130.
Example 64
1-~3-[[(Methylamino)thiocarbonyl]thio]propanoyl]-L-proline
A) l-(Methylaminothiocarbonylthiopropanoyl)-L-proline tert-
butyl ester (0.98 g) is dissolved in a mixture of anisole (3.6
ml) and trifluoroacetic acid (7~5 ml). After one hour at room
temperature the mixture is concentrated to dryness in vacuo and
the residue precipitated from ether-hexane three times. This
material is chromatographed on a column of silica gel with a
solvent mixture of benzene-acetic acid (75:25) to yield 1-[3-
[t(methylamino)thiocarbonyl]thio]propanoyl]-L-proline,
Rf = 0.4 rsilica gel-benzene:acetic acid (75:25)]. The
dicyclohexylammonium salt has m.p. 127-129.
-43-
-
B) Methylisothiocyanate (4 g) is added to a solution of 3-
mercaptopropanoyl-L-proline (10.1 g) in a mixture of pyridine
(250 ml) and 0.5 N sodium hydro~ide (100 ml). The solution is
kept at 40 for two hours and concentrated to dryness in vacuo.
The residue is dissolved in water (100 ml), acidified with con-
centrated hydrochloric acid and extracted with ethyl acetate.
The organic phase is concentrated to dryness to yield
1-[3-~[(methylamino)thiocarbonyl]thio]propanoyl]-L-proline.
Example 65
1-13-[_[(Ethylamino)carbonyl]thio]propanoyl]-L-_roline
Ethylisocyanate (0.45 ml) is added to a solution of
1-(3-mercaptopropanoyl)-L-proline (1 g) in a mixture of N
sodium hydroxide (5 ml) and pyridine (5 ml). The solution
is heated at 40 for four hours and concentrated in vacuo.
The residue is distributed between 0.1 N hydrochloric acid ~,
and ethyl acetate. The organic layer is washed with water,
dried over magnesium sulfate and concentrated to dryness to
yield l-13-[[(ethylamino)carbonyl]thio]propanoyl]-L-proline.
The dicyclohexylammonium salt is prepared by adding dicyclo-
hexylamine to a solution of the free acid in ethyl acetate,
m.p. 150-152.
-Example 66
.
1-[3- r 1 (Ethoxy)carbonyl]thio]-2-methylpropanoyl]-L-proline
By substituting 1-(3-mercapto-2-methylpropanoyl]-L-proline
for the 3-mercaptopropanoyl-L-proline in the procedure of
Example 58, 1-[3-[~(ethoxy)carbonyl]thio]-2-methylpropanoyl]-L-
proline is obtained.
-44-
Example 67
1-[3-[[(Ethoxy)carbonyl]thio]butanoyl]-L-proline
By substituting 1-(3-mercaptohutanoyl)-L-proline for
the 3-mercaptopropanoyl-L-proline in the procedure of
Example 58, 1-[3-[[(ethoxy)carbonyl]thio]butanoyl]-L-pro-
line is obtained.
Example 68
1-[3-[[(Ethoxy)thiocarbonyl]thio]propanoyl]-L-azetidine-2-
carboxylic acid
By substituting L-az~tidine-2-carboxylic acid for
L-proline in the procedure of Example 59, 1-[3-[[(ethoxy)-
thiocarbonyl]thio]propanoyl]-L-azetidine-2-carboxylic acid
is obtained.
Example 69
1-[3-[[(Ethoxy)thiocarbonyl]thio]propanoyl]-L-pipecolic
acid
By substituting L-pipecolic acid for L-proline in
the procedure of Example 59, 1-[3-[[(ethoxy)thiocarbonyl]-
thio]propanoyl]-L-pipecolic acid is obtained.
Example 70
-
1-[4-[[(Benzylthio)carbonyl]thio]butanoyl]-L-proline
By substituting 4-mercaptobutanoyl-L-proline for the
3-mercaptopropanoyl-L-proline in the procedure of Example
60, 1-[4-[[(benzylthio)carbonyl]thio]butanoyl]-L-proline
is obtained.
Example 71
1-[2-[[(Benzylthio)carbonyl]thio]propanoyl]-L-proline
By substituting 2-mercap-topropanoyl-L-proline for
the 3-mercaptopropanoyl-L-proline in the procedure of
Example 60, 1-[2-[~(benzylthio)carbonyl]thio]propanoyl]-
L-proline is obtained.
-45-
Example 72
1-[3-[[(Ethylthio)thiocarbonyl]thio]propanoyl]-L-proline methyl
ester
A solution of 1-[3-[[(ethylthio)thiocarbonyl]thio]-
propanoyl]-L-proline in ethyl acetate is treated with an ethereal
solution of diazomethane until persistent yellow color. After
discharging the yellow color with a few drops of acetic acid,
the solvents are removed ln vacuo to yield l-[3-[[(ethylthio~-
thiocarbonyl]thio~propanoyl]-L-proline methyl ester.
Example 73
l-t3-[[(methylamino)thiocarbonyl]thio]propanoyl]-5-hydroxy-L-
pipecolic acid
By substituting 1-(3-mercaptopropanoyl)-S-hydroxy-L-
pipecolic acid for the 3-mercaptopropanoyl-L-proline in the
Procedure B of Example 64, 1-~3-[[(methylamino)thiocarbonyl]thio]-
propanoyl]-5-hydroxy-L-pipecolic acid is obtained.
Example 74
1-13-[[(Methylamino)thiocarbonyl]thio]-2-methylpropanoyl]-L-
proline amide
By substituting 1-(3-mercapto-2-methylpropanoyl)-L-
proline amide for the 3-mercaptopropanoyl-L-proline in the
Procedure B of Example 64, 1-[3-[[(methylamino)thiocarbonyl]thio]-
2-methyl-propanoyl]-L-proline amide is obtained.
Example 75
_
1-[3-[l~Phenoxy)carbonyl]thio]propanoyl]-L-proline
By substituting phenylchloroformate for ethyl chloroformate
in the procedure of Example 58, 1-[3-[[(phenoxy)carbonyl]thio]-
propanoyl]-L-proline is obtained.
-46-
i5
Example 76
1-~3-~[(Phenoxy)carbonyl]thio]butanoyl]-L-proline
By substituting phenylchloroformate for the ethyl
chloroformate and 4-mercaptobutanoyl-L-proline for the
3-mercaptopropanoyl-L-proline in the procedure of Example 58,
- [ 3- [ E (phenOxy)Carbonyl]thio]butanoyl]-L-proline is obtained.
Example 77
- [3- [ [ (Phenylamino)carbonyl]thio]propanoyl]-L-proline
_
By substituting phenylisocyanate for the ethylisocyanate
in the procedure of Example 65, 1-[3-[[(phenylamino)carbonyl]-
thio]propanoyl]-L-proline is obtained.
Example 78
1-13-[[(Phenethylamino?carbonyl]thio]propanoyl]-L-proline
By substituting phenethylisocyanate for the ethylisocyanate
in the procedure of Example 65, 1-[3-[[(phenethylamino)carbonyl]-
thio]propanoyl]-L-proline is obtained.
Example 79
1-[3- r t(Ethylamino)carbonyl]thio]-2-benzylpropanoyl]-L-proline
By substituting 1-~3-mercapto-2-benzylpropanoyl)-L-
proline for the l-(3-mercaptopropanoyl)-L-proline in the
procedure of Example 65, 1-[3-[[(ethylamino)carbonyl]thio]-
2-benzylpropanoyl]-L-proline is obtained.
-47-
~3~5~
Example 80
1-(3-Methylthiopropanoyl)-L-proline
A) Methyl 3-methylthiopropionate t51 g) is saponified with
a 10~ sodium hydroxide solution (150 ml, 30 minutes at 100).
The cooled solution is extracted with ether and then acidified.
The crude acid thus obtained is distilled and converted to
the acid chloride with thionyl chloride.
A solution of L-proline (11.5 g) in N sodium hydroxide (100 cc)
is chilled in an ice bath and the 3-methylthiopropanoic acid
chloride (6.9 g) is added dropwise with vigorous stirring over
a ten minutes period. After five hours the reaction mixture
is acidified and extracted with ethyl ether to yield 1-(3-
methylthiopropanoyl)-L-proline. The dicyclohexylammonium salt
is prepared by adding dicyclohexylamine to a solution of the
free acid in ethyl acetate, m.p. 169-171.
B) Methyl iodide (71 g) is added to a solution of 1-(3-
mercaptopropanoyl)-L-proline ethyl ester (115 g) and sodium
(11.5 g) in ethanol (400 ml). The reaction is allowed to
proceed overnight, the ethanol is removed in vacuo and the
residue is dissolved in a mixture of ethyl acetate and water.
The organic layer is dried and concentrated to dryness in vacuo.
The resulting 1-(3-methylthiopropanoyl)-L-proline ethyl ester
(98 g) is suspended in a mixture of methanol (200 ml) and 5 N
sodium hydroxide t200 ml) and stirred at room temperature for
five hours. The methanol is removed in vacuo, and the aqueous
phase is extracted with ethyl acetate, acidified and reextracted
with ethyl acetate. This last organic phase is washed with
waler, dried and concentrated to dryness to yield l-t3-methylthio-
propanoyl)-L-proline.
~ -48-
.
~3~
Example 81
-[3-(4-Chlorophenylthio)propanoyl]-L-proline
Aqueous 2 N sodium hydroxide (25 ml) and 3-bromopropionyl
chloride (8.5 g) are added to a sol~tion of L-proline (5~75 g)
in N-sodium hydroxide (50 ml) chilled and stirred in an ice
bath. After five minutes, the ice bath is removed and the
stirring is continued for three hours at room temperature. The
reaction mixture is acidified with concentrated hydrochloric acid
~nd extracted with ethyl acetate. The organic layer is washed
with ~Jater, dried and concentrated to dryness in vacuo. The
residue is dissolved in a mixture of 4-chlorobenzenethiol (8 g),
sodium hydroxide (4.2 g) and ethanol (300 ml). The solution is
refluxed for 6 hours. The solvent is removed in vacuo and the
residue is dissolved in water, acidified with concentrated hydro-
chloric acid and extracted with ethyl acetate. The organic layer
is washed with water, dried, and concentrated to dryness in
vacuo to yield l-[3-(4-chlorophenylthio)propanoyl]-L-proline.
Example 82
1,~[(3-Benzylthiomethyl)thio]propanoyl]-L-proline
1-(3-Mercaptopropanoyl)-L-proline (8.1 g) is dissolved
in boiling liquid ammonia (100 ml) and small pieces of sodium
are added until permanent blue color is obtained which is then
discharged with a small piece of ammonium chloride. Benzyl-
thiomethyl chloride (6.9 g) is added and the ammonia is allowed
to evaporate. The final traces of ammonia are removed in
vacuo, the residue is dissolved in water and extracted with
ethyl acetate. The aqueous phase is acidified with concentrated
hydrochloric acid and extracted with ethyl acetate. The organic
layer is washed with water, dried and concentrated to dryness
-49-
25~i;
to yield l-[[(3-benzylthiomethyl)thio]propanoyl]-L-proline.
Example 83
1-[[(3-Acetamidomethyl)thio]propanoyl]-L-proline
1-(3-Mercaptopropanoyl)-L-proline (2 g) and N-hydroxy-
methylacetamide (0.89 g) are dissolved in trifluoroacetic acid
(10 ml) and the solution is stored at room temperature for one
hour. The excess tirfluoroacetic acid is removed in vacuo and
the residue is precipitated several times from ether-hexane.
Finally, the residue is distributed between dilute hydrochloric
acid and ethyl acetate. The organic layer is washed with water,
dried and concentrated to dryness to yield l-[[3-acetamido-
methyl)thio]propanoyl]-L-proline.
Example 84
l-(Methylthioacetyl)-L-proline
By substituting methyl methylthioacetate for the methyl
3-methylthiopropionate in the Procedure A of Example 80, 1- ;
(methylthioacetyl)-L-proline, m.p. 123-124, is obtained.
Example 85
l-(Benzylthioacetyl)-L-proline
By substituting benzylthioacetyl chloride for the 3-
methylthiopropanoyl chloride in the Procedure A of Example 80,
l-(benzylthioacetyl)-L-proline, m.p. 86-88, is obtained.
i,
-50-
Example 86
1-[3-[(2-Phenylethyl)thio]propanoyl]-L-proline
By substituting phene-thylbromide for the methyl io-
dide in the Procedure B of Example 80, 1-[3-[(2-phenyl-
ethyl)thio]propanoyl]-L-proline is obtained.
Example 87
1-[3-[(Triphenylmethyl)thio]propanoyl]-L-proline
By substituting triphenylmethyl chloride for the
methyl iodide in the Procedure B of Example 80, 1-[3-
~(triphenylmethyl)thio]propanoyl]-L-proline is obtained.
Example 88
1-(3-Methylthio-2-methylpropanoyl)-L-proline amide
By substituting 1-(3-mercapto-2-methylpropanoyl)-L-
proline amide for the 1~(3-mercaptopropanoyl)-L-proline
ethyl ester in the Procedure B of Example 80 and elimina-
ting the saponification step, l-(3-methylthio-2-methylpro-
panoyl)-L-proline amide is obtained.
Example 89
1-(3-Methylthiopropanoyl)-L-azetidine-2-carboxylic acid
By substituting L-azetidine-2-carboxylic acid for
the L-proline in the Procedure A of Example 80, 1-(3-
methylthiopropanoyl)-L-azetidine-2-carboxylic acid is
obtained.
-51-
~`3~5S
Example 90
l-[3-(4-~lethoxyphenylthio)propanoyl~-L-proline
By substituting 4-methoxyben~:enethiol for the 4-chloro-
benzenethiol in the procedure of Example 81, l-[3-(4-methoxy-
phenylthio)propanoyl]-L-proline is obtained.
Example 91
l-(3-~ethylthiopropanoyl)-L-pipecolic acid
By substituting L-pipecolic acid for the L-proline in
the Procedure A of Example 80, 1-(3-methylthiopropanoyl)-L-
pipecolic acid is obtained.
Example 92l-[2-(4-Chlorophenylthio propanoyl]-L-proline
By substituting 2-bromopropionyl chloride for the
3-bromopropionyl chloride in the procedure of Example 81,
1-[2-(4-chlorophenylthio-propanoyl]-L-proline is obtained.
Example 93
l-E3-[(Diphenylmethyl)thio]-2-benzylpropanoyl)]-L-proline
Diphenylmethanol (0.92 g) and 1-(3-mercapto-2-benzyl-
propanoyl)-L-proline (1.5 g) are dissolved in trifluoroacetic
acid (lO ml) and the solution is kept at room temperature for
30 minutes. The excess trifluoroacetic acid is removed in vacuo
to yield l-[3-[(diphenylmethyl)thio]-2-benzylpropanoyl]-L-proline.
-52-
~7
Example ~4
1-[4~ Chlorophenylthio)butanoyl]-L-proline
By substituting 4-bromopropionyl chloride for the 3-
bromopropionyl chloride in the procedure of Example 81, 1-[4-(4-
chlorophenylthio)butanoyl]-L~proline is obtained.
Example 95
_ _
1-13-~(Benzylthiomethyl)thio]butano~rl]-L-proline
By substituting 3-mercaptobutanoyl-L-proline for the
3-mercaptopropanoyl-L-proline in the procedure of Example 82,
1-~3-[(ben~ylthiomethyl)thio]butanoyl]-L-proline is obtained.
Example 96
1-[[4-[(Acetamidomethyl)thio]-2-methylbutanoyl]-L-proline
By substituting 1-~4-mercapto-2-methylbutanoyl)-
L-proline for the 1-(3-mercaptopropanoyl)-L-proline in the
procedure of Example 83, 1-[[4-(acetamidomethyl)thio]-2-
methylbutanoyl]-L-proline is obtained.
Example 97
1-[3-(Ethyldithio)propanoyl-]-L-proline
A) 3-Mercaptopropanoyl-L-proline (10 g) is added to a
solution of ethylthiosulfinate (8.4 g) in methanol (100 ml)
and the reaction mixture is stirred vigorously at room
temperature for four hours. The methanol is removed in
vacuo to yield l-(3-ethyldithiopropanoyl)-L-proline.
B) A solution of ~ethylthiosulfinate (8.4 g) in
ethanol (50 ml) is added to an aqueous solution of 3-mercapto-
propanoyl-L-proline (10 g) maintained at pH 6-7 by careful
addition of sodium hydroxide. The mixture is stirred vigorously
-53-
~3;~5~;
at room temperature until negative thiol reaction. The mix-
ture is diluted with water, adjusted to pH 8 and extracted
with ethyl acetate, the aqueous phase is acidified to pH 3
and extracted again with ethyl acetate. This latter extract
is washed with water, dried and concentrated to dryness to
yield l-13-(ethyldithio)propanoyl]-L-proline.
Example 98
1-[3-[(4-Methylphenyl)dithio]propanoyl]-L-proline
A solution of 4-methylphenylsulfenyl chloride (1.76 g.)
in ether (20 ml) is added to a solution of 3-mercaptopropanoyl-
L-proline (2 g) in 0.5 N sodium hydroxide (20 ml) chilled in
an ice bath. The mixture is stirred vigorously for one hour,
and the aqueous phase is separated, acidified with concentrated
hydrochloric acid and extracted with ethyl acetate. The organic
phase is washed with water, dried and concentrated to dryness
to yield l-[[3-(4-methylphenyl)dithio]propanoyl]-L-proline.
Example 99
1-13-(Phenyldithi'o-propanoyl]-L-proline
By substituting phenylthiosulfinate [prepared from phenyl-
disulfide according to U. Weber and P. Hartter, Z. Physiol. Chem.,351, 1384 (1970)] for the ethylthiosulfinate in the procedure of
Example 97, 1-[3-(phenyldithio-propanoyl]-L-proline is obtained.
Example 100
1-13-[(2-Phenylethyl)dithi'o]propanoyl]-L-proline
By substituting 2-phenylethylthiosulfinate (prepared from
phenethyldisulfide) for the ethylthiosuflinate in the procedure
of Example 97, 1-[3-1(2-phenylethyl)dithio]propanoyl]-L-proline
is obtained.
-54-
~3~
Example 101
1-[3-[(2-Hydroxyethyl)dithio]propanoyl]-L-proline
. .
To a solution of 1,1'-[(sulfinylthio)-bis-(3-propanoyl)]-
bis-L-proline (21 g) in methanol (100 ml), mercaptoethanol(4.2 g)
is added and the reaction mixture is stirred vigorously at room
temperature for four hours. The methanol is removed in vacuo
and the residue is pruified by chromatography on a silica gel
column to yield l-13-[(2-hydroxyethyl)dithio]propanoyl]-L-proline.
Example 102
1-12-(Ethyldithio)propanoyl]-L-proline
. _
By substituting 2-mercaptopropanoyl-L-proline for 3-
mercaptopropanoyl-L-proline in the procedure of Example 97,
1-12-(ethyldithio)propanoyl]-L-proline is obtained.
Example 103
1-13-1(4-Methylphenyl)dithio]butanoyl]-L-proline
By substituting 3-mercaptobutanoyl-L-proline for the 3-
mercaptopropanoyl-L-proline in the procedure of Example 98,
1-~3-(4-methylphenyl)dithio]butanoyl]-L-proline is obtained.
Example 104
1-13-(Ethyldithio?-2-methylpropanoyl]-L-proline methyl ester
By substituting 1-(3-mercapto-2-methylpropanoyl)-L-proline
for the 3-mercaptopropanoyl-L-proline in the procedure of Example
~7 and then treating the product with ethereal diazomethane as
in the procedure of Example 72, 1-13-(ethyldithio)-2-methylpro-
panoyl]-L-proline methyl ester is obtained.
-55-
. . ~ .
3~5
Example 105
_
1-[3-(Ethyldithio)propanoyl]-L-azetidine-2-carboxylic acid
By substituting 3-mercaptopropanoyl-L-azetidine-2-
carboxylic acid for the 3-mercaptopropanoyl-L-proline in the
procedure of Example 97, 1-[3-(ethyldithio)propanoyl]-L-aze-
tidine-2-carboxylic acid is obtained.
Example 106
1-[3-[(4-Methylphenyl)dithio]-2-methylpropanoyl]-L-hydroxyproline
By substituting 1-(3-mercapto-2-methylpropanoyl)-L-
hyd~oxy proline for the 3-mercaptopropanoyl-L-proline in the
procedure of Example 98, 1-[(3-14-methylphenyl)dithio]-2-
methylpropanoyl]-L-hydroxyproline is obtained.
Example-107
1-[4-~Ethyldithio)butanoyl]-L-pipecolic acid
By substituting 4-mercaptobutanoyl-~-pipecolic acid for
the 3-mercaptopropanoyl-L-proline in the procedure of Example 97,
1-[4-(ethyldithio-butanoyl]-L-pipecolic acid is obtained.
Example 108
1-~3-(Ethyldithio)propanoyl]-5-hydroxy-L-pipecolic acid
By substituting 1-(3-mercaptopropanoyl)-5-hydroxy-L-
pipecolic acid for the 3-mercaptopropanoyl-L-proline in the
procedure of Example 97, 1-[3-(ethyldithio)propanoyl]-5-hydroxy-
L-pipecolic acid is obtained.
-56-
3~
Example 10~
1-[3-[(2-Amino-2-carboxyethyl)dithio]propanoyl]-L-proline
A 0.5 M solution of thiocyanogen in glacial acetic acid
is prepared by shaking for ten minutes in a sealed flask 600 mg
of dry lead thiocyanate with a solution of 75 ~1 of bromine in
3 ml of acetic acid. After removal of lead bromide and excess
lead thiocyanate by centrifugation, 2.5 ml of this solution is
mixed with 2.5 ml of a 0.41 M solution of cysteine hydrochloride
previously neutralized with dilute sodium hydroxide. This
mixture is immediately added to 0.75 ml of a 1.9 M solution of
3-mercaptopropanoyl-L-proline previously neutralized with dilute
sodium hydroxide. After twenty minutes the mixture is titrated
to incipient brown color with alcoholic iodine, and adjusted to
pH 3. The precipitate is removed by filtration and the filtrate
is applied to a column of cation exchange resin (Dowex 50). The
column is washed with water until no more acidic material is
removed and then eluted with pyridine-acetate buffer pH 6Ø
The fractions containing the disulfide of cysteine and 3-
mercaptopropanoyl-L-proline are pooled and concentrated to
dryness.
Example llO
1,li-[Dithiobis)4-propanoyl)]-bis-L-proline
. .
3-Mercaptopropanoyl-L-proline (O.9S g) is dissolved in
water (20 ml) and the pH is adjusted to 6.5 with N-sodium
hydroxide. An ethanolic solution of iodine is added drop-
wise while mainta;.ning the pH at 6.5 with careful addition
of N sodium hydroxide. When a permanent yellow color is
obtained the addit:ion of iodine is stopped and the color
is discharged with a small amount of sodium thiosulEate.
The reaction mixture is acidified with concentrated hydro-
chloric acid and extracted with ethyl acetate. The organic
phase is washed with water, dried and concentrated to dryness
to yield 1,1'-[dithiobis(3-propanoyl)]-bis-L-proline. The di-
cyclohexylammonium salt is prepared by addition of dicyclo-
hexylamine to a solution of the free acid in acetonitrile,
m.p. 179-180D.
Example 111
0 ~ [Dithiobis(2~D-methyl-3-propanoyl)]-bis-L-proline
By substituting 3-mercapto-2-D-methylpropanoyl-L-
proline for the 3-mercaptopropanoyl-L-proline in the procedure
of Example 110, 1,1'-[dithiobis(2-D-methyl-3-propanoyl)]-bis- -
L-proline is obtained, m.p. 236-237.
Exampl-e 112
l,l'-tDithiobis(2-propanoyl)-bis-L-proline
.
By substituting 2-mercaptopropanoyl-L-proline for the
3-mercaptopropanoyl-L-proline in the procedure of Example 110,
1,1'-[dithiobis)2-propanoyl)]-bis-L-proline is obtained.
Example-113
l,l'-(Dithiobisacetyl)-bis-L-hydroxy proline
By substituting 1-(2-mercaptoacetyl)-L-hydroxyproline
for the 3-mercaptopropionyl-L-proline in the procedure of
Example 110, l,l'-(dithiobisacetyl)-bis-L-hydroxyproline is
obtained.
Example 114
1 ! 11- (Dithiobisacetyl)-bis-L-azetidine-2-carboxylic acid
By substituting 1-(2-mercaptoacetyl)-L-azetidine-2-
carboxylic acid for the 3-mercaptopropanoyl-L-proline in the
procedure of Example 110, l,l'-(dithiobisacetyl)-bis-L-
azetidine-2-carboxylic acid is obtained.
-58-
) ?~` -
. . -
33~
Example 115
1,1'-[Dithiobis(3-propanoyl)]-bis-L-pipecolic acid
By substituting 3-mercaptopropanoyl-L-pipecolic acid
for the 3-mercaptopropanoyl-L-proline in the procedure of
Example 110, 1,1'-[dithiobis~3-propanoyl)]-bis-L-pipecolic
acid is obtained.
Example 116
1,1'-[Dithiobis(3-propanoyl)]-bis-4-methyl-L-proline
By substituting 1-(3-mercaptopropanoyl)-4-methyl-L-proline
for the 3-mercaptopropanoyl-L-proline in the procedure of Example
110, 1,1'-[dithiobis(3-propanoyl)]-bis-4-methyl-L-proline is
obtained.
Example 117
1,1'-[Dithiobis(3-propanoyl)]-bis-5-hydroxy-L-pipecolic acid
By substituting 1-(3-mercaptopropanoyl)-5-hydroxy-L-
pipecolic acid for the 3-mercaptopropanoyl-L-proline in the pro-
cedure of Example 110, 1,1'-[dithiobis~3-propanoyl)]-bis-5-hydroxy-
L-pipecolic acid is obtained.
Example 118
1,1'-[Dithiobis(2-benzyl-3-propanoyl)]-bis-L-proline
By substituting 1-(3-mercapto-2-benzylpropanoyl)-L-proline
for the 3-mercaptopropanoyl-L-proline in the procedure of Example
110, 1,1'-[dithiobis(2-benzyl-3-propanoyl)]-bis-L-proline is
obtained.
_59_
3~
Example 119
1,1'-[Dithiobis)2-methyl-3-propanoyl)]-bis-L-pipecolic acid
By substituting 1-(3-mercapto-2-methylpropanoyl)-L-
pipecolic acid for the 3-mercaptopropanoyl-L-proline in the
procedure of Example 110, 1,1'-[dithiobis(2-methyl-2-propanoyl)]-
bis-L-pipecolic acid is obtained.
Example 120
_
1,1'-[Dithiobis)4-butanoyl)]-bis-L-proline
By substituting 4-mercaptobutanoyl-L-proline for the
lQ 3-mercaptopropanoyl-L-proline in the procedure of Example
110, 1,1'-[dithiobis(4-butanoyl)]-bis-L-proline is obtained.
Example 121
1,1'-~Dithiobis(2-benzyl-4-butanoyl)]-bis-L-pr_line
By substituting 1-(4-mercapto-2-benzylbutanoyl)-L-proline
for the 3-mercaptopropanoyl-L-proline in the procedure of
Example 110, 1,1'-[dithiobis(2-benzyl-4-butanoyl)]-bis-L-proline
is obtained.
Exampl-e 122
1,1'-[Dithiobis(3-butanoyl)]-bis-L-proline
By substituting 3-mercaptobutanoyl-L-proline for the
3-mercaptopropanoyl-L-proline in the procedure of ~xample
110, 1,1'-[dithiobist3-butanoyl)]-bis-L-proline is obtained.
-60-
5~.
Example 123
1,1'-[Dithiobis(3-propanoyl)]-bis-L-pxoline methyl ester
A solution of l,l'-[dithiobis(3-propanoyl)]-bis-L-
proline in methanol is treated with ethereal diazomethane
until persistent yellow color. After fifteen minutes a
few drops of acetic acid are added and the solvents are
removed in vacuo to yield, l,l'-[dithiobis(3-propanoyl)]-
bis-L-proline methyl ester.
Example 124
l,l'-[Dithiobis(3-propanoyl)]-bis-L-proline amide
A solution of l,l'-[dithiobis(3-propanoyl)]-bis-
L-proline methyl ester in methanol is saturated with am-
monia while cooling in an ice-water bath. The reaction
mixture is stored for 16 hours at room temperature in a
pressure bottle, and then the solvents are removed in
vacuo to yield l,l'-[dithiobis(3-propanoyl)]-bis-L-proline
amide.
Example 125
l,l'-[Dithiobis(2-phenyl-3-propanoyl)]-bis-L-proline
By substituting l-(3-mercapto-2-phenylpropanoyl~-
L-proline for the 3-mercaptopropanoyl-L-proline in the
procedure of Example llO, l,l'-Edithiobis(2-phenyl-3-pro-
panoyl)]-bis-L-proline is obtained.
-61-
._. . .
.5~
Example 126
1,1'-[(Sulfinylthio)-bis-(3-propanoyl)]-bis-L-proline
While cooling in an ice bath 0.12 mole of peracetic
acid is added to a stirred solution of l,l'-[dithiobis(3-
propanoyl)]-bis-L-proline (40 g) in glacial acetic acid
(500 ml). The reaction mixture is allowed to stand over-
night at room temperature and the solvent is then removed
_ vacuo to yield 1,1'-[(sulfinylthio)-bis-(3-propanoyl)]-
bis-L-proline.
Example 127
1,1'-[(Sulfonylthio)-bis-(3-propanoyl)]-bis-L-proline
A 30~ solution of hydrogen peroxide (2.0 ml) is
added to a solution of l,l'-[dithiobis(3-propanoyl)]-bis-
L-proline (4 g) in glacial acetic acid (80 ml) and the
solution is stored for thirty hours at room temperature.
The solvent is removed in vacuo to yield l,l'-[(sulfonyl-
thio)-bis-(3-propanoyl)]-bis-L-proline.
Example 128
1,1'-[(Sulfinylthio)-bis-(2-propanoyl)]-bis-L-proline
By substituting 1,1'-[dithiobis(2-propanoyl)]-
bis-L-proline for the 1,1'-[dithiobis(3-propanoyl)]-bis-
L-proline in the procedure of Example 126, l,l'-[(sul-
finylthio)-bis-(2--propanoyl)]-bis-L-proline is obtained.
-62-
Example 1~9
1,1'-[(SulEinylthio)-bis-acetyl]-bis-L-azetidine-2-carboxy-
lic acid
sy substituting l,l'-(dithiobisacetyl)-bis-L-azeti-
dine carboxylic acid for the l,l'-[dithiobis(3-propanoyl)]-
bis-L-proline in the procedure of Example 126, l,1'-[(sul-
finylthio)-bis-acetyl]-bis-L-azetidine-2-carboxylic acid
is obtained.
Example 130
1,1'-[~Sulfinylthio)-bis-(3-propanoyl)]-bis-4-methyl-L-
proline
By substituting 1,1'-[dithiobis(3-propanoyl)]-bis-
4-methyl-L-proline for the 1,1'-[dithiobis(3-propanoyl)]-
bis-L-proline in the procedure of Example 126, l,l'-~(sul-
finylthio)-bis-(3-propanoyl)]-bis-4-methyl-L-proline is
obtained.
Example 131
l~l'-[(Sulfinylthio)-bis-(2-ben~yl-3-propanoyl)]-bis-L
proline
By substituting 1,1'-[dithiobis(2-benzyl-3-propa-
noyl)]-bis-L-proline for the 1,1'- E dithiobis(3-propanoyl)]-
bis-L-proline in the procedure of Example 126, l,l'-[(sul-
finylthio)-bis-(2-benzyl-3-propanoyl)]-bis-L-proline is
obtained.
Example 132
1,1'-[(Sulfinylthio)-bis-(4-butanoyl)]-bis-L-proline
By substituting 1,1'-[dithiobis(4-butanoyl)]-bis-
L-proline for the 1,1'-[dithiobis(3-propanoyl)]-bis-L-
proline in the procedure of Example 126, l,l'-[(sulfinyl-
thio)-bis-(4-butanoyl)]-bis-L-proline is obtained.
~ -63-
-
Example 133
1,1'-[(Sulfinylthio)-bis-(3-butanoyl)~-bis~L-proline
By substituting 1,1'-[dithiobis(3-butanoyl)]-bis-L-
proline for the l,l'-[dithiobis(3-propanoyl)]-bis-L-proline
in the procedure of Example 126, l,l'-[(sulfinylthio)-bis-
(3-butanoyl)]-bis-L-proline is obtained.
Example 134
1,1'-[(Sulfinylthio)-bis-(2-methyl--3-propanoyl)-bis-L-pro-
line
By substituting 1,1'-[dithiobis(2-methyl-3-propan-
oyl)]-bis-L-proline for the 1,1'-[dithiobis(3-propanoyl)]-
bis-L-proline in the procedure of Example 126, l,l'-[(sul-
finylthio)-bis-(2-methyl-3-propanoyl)]-bis-L-proline is
obtained.
Example 135
1,1'-[(Sulfinylthio)-bis-(2-phenyl-3-propanoyl)]-bis-L-
proline
By substituting 1,1'-[dithiobis(2-phenyl-3-propa-
noyl)]-bis-L-proline for the 1,1'-[dithiobis(3-propanoyl)]-
bis-L-proline in the procedure of Example 126, l,l'-[(sul-
finylthio)-bis-(2-phenyl-3-propanoyl)]-bis-L-proline is
obtained.
Example 136
1-[3-[[3-(2-Carboxy-l-pyrrolidinyl?-3-oxopropyl]-dithio]-
2-methylpropanoyl]-L-proline
By substituting 1,1'-[(sulfinylthio)-bis-(2-methyl-
3-propanoyl)]-bis-L-proline for the ethylthiosulfinate in
the procedure of Example 97, 1-[3-[[3-(2-carboxy-1-pyrro-
lidinyl)-3-oxopropyl]dithio]-2-methylpropanoyl]-L-proline
is obtained.
-64-
: "~
Example 137
[(Sulfonylthio)-bis-acetyl)-bis-I,-hydroxyproline
By substituting l,l'-(dithiobisacetyl)-bis-L-hy-
droxy proline for the l,l'-~dithiobis(3-propanoyl)-bis-L-
proline in the procedure of Example 127, l,ll-[(sulfonyl-
thio)-bis-acetyl)-bis-L-hydroxyproline is obtained.
Example 138
1,1'-[~Sulfonylthio)-bis-(3-propanoyl)]-bis-L-pipecolic
aeid
By substituting 1,1'-[dithiobis(3-propanoyl)]-
bis-L-pipecolic acid for the 1,1'-[dithiobis(3-propanoyl)]-
bis-L-proline in the procedure of Example 127, l,l'-[(sul-
fonylthio)-bis-(3-propanoyl)]-bis-L-pipeeolic acid is ob-
tained.
Example 139
1,1'-[(Sulfonylthio)-bis-(3-propanoyl)]-bis-5-hydroxy-L-
pipeeolie aeid
By substituting 1,1'-[dithiobis(3-propanoyl)]-bis-
5-hydroxy-L-pipeeolie aeid for the 1,1'-[dithiobis-(3-
propanoyl)]-bis-L-proline in the procedure of Example 127,
1,1'-[(sulfonylthio)-bis-(3-propanoyl)]-bis-5-hydroxy-L-
pipeeolic aeid is obtained.
Example 140
1,1'-[(Sulfonylthio)-bis-(2-methyl-3-propanoyl)]-bis-L-
pipeeolie aeid
By substituting 1,1'-[dithiobis(2-methyl-3-propa-
noyl)]-bis-L-pipeeolie aeid for the 1,1'-[dithiobis~3-
propanoyl)]-bis-L-proline in the proeedure of Example 127,
1,1'-[(sulfonylthio)-bis-(2-methyl-3-propanoyl)]-bis-L-
pipeeolie aeid is obtained.
J -65-
Example 141
1,1'-[(Sulfonylthio)-bis-(2-benzyl-4-buta_oyl)]-bis-L-
proline
By substituting 1,1!-[dithiobis(2-benzyl-4-butan-
onyl)]-bis-L-proline for the 1,1'-[dithiobis(3-propanoyl)]-
bis-L-proline in the procedure of Example 127, l,l'-[(sul-
Eonylthio)-bis-(2-benzyl-4-butanoyl)]-bis-L-proline is ob-
tained.
Example 142
3-Acetylthio-2-phenylpropanoic acid
By substituting 2-phenylacrylic acid for the meth-
acrylic acid in the procedure of Example 25, 3-acetylthio-
2-phenylpropanoic acid is obtained.
1-~3-Acetylthio-2-phenylpropanoyl)-L-proline tert-butyl
ester
By substituting 3-acetylthio-2-phenylpropanoic acid
for the 3-acetylthio-2-methylpropanoic acid in the proce-
dure of Example 28, 1-(3-acetylthio-2-phenylpropanoyl)-L-
proline tert-butyl ester is obtained.
Example 143
1-(3-Mercapto-2-phenylpropanoyl)-L-proline
By substituting 1-(3-acetylthio-2-phenylpropanoyl)-
L-proline tert-butyl ester for the 1-(3-acetylthio-2-
methyl-propanoyl-L-proline tert-butyl ester in the pro-
cedure of Example 29, and subjecting the product to am-
monolysis as in Example 34, 1-(3-acetylthio-2-phenylpro-
panoyl)-L-proline and 1-(3-mercapto-2-phenylpropanoyl)-L-
proline are obtained.
-66-
z~
Example 14~
1-[3-(Acetylthio)-DL-propanoyl]pipecollc acid
Pipecolic acid (6.5 g.) is suspended in 200 ml. of
dimethylacetamide. 3-acetylthiopropanoyl chloride (~.3 g.)
is added dropwise at 23 to the suspension. A clear solu-
tion forms and -the temperature rises to 28. To this clear
solution is added N-methylmorpholine (10.1 g.). An imme-
diate precipitate forms and the temperature rises to 34.
The mixture is heated on a steam bath for 1 hour when a
clear solution forms. On cooling, the precipitated solid
is filtered to yield 5.1 g. of 1-[3-(aeetylthio)-DL-pro-
panoyl]pipecolic acid, m.p. 190-200. The solvent is re-
moved and the viscous residue is triturated with isopropyl
ether to yield 7.8 g. of product, m.p. 98-101. Recrystal-
lization from acetone-hexane yields a constant melting
solid, m.p. 102-104; Rf 0.72 [siliea gel, benzene, acetic
aeid (7:2)].
Example_145
DL-1-(3-Mereaptopropanoyl)pipeeolie acid
12 ml. of eoneentrated ammonium hydroxide is stirred
under nitrogen at 10 for about 15 minutes, then solid 1-
[3-laeetylthio)-DL-propanoyl]-pipeeolic acid (6.6 g.) is
added at 5 to 10. A clear solution forms after 2-3 min-
utes. The iee bath is removed and the solution is stirred
at room temperature under nitrogen for 45 minutes. The
solution is made strongly acid with 20% HC1 (cooling) and
the preeipitated oil is extraeted with 3 x 150 ml. of ethyl !
aeetate. The ethyl aeetate extraets are dried over magne- -
sium sulfate and the solvent is removed to yield 6.0 g. of
DL-1-(3-mereaptopropanoyl)pipeeolie aeid, Rf 0.77 [siliea
gel, benzene, aeetie aeid (7:1)].
-67-
,' '- . .. .
~r~ ~r~
I-IA135b
Example_1~6
1-(3-~1ercaptopropanoyl)-L-pipecolic acid
By substitutin~ L-pipecolic acid for the DL-
pipecolic acid in the procedure of Example 144 and then
submitting the product to the procedure of Example 145,
1-~3-(acetylthio)propanoyl]-L-pipecolic acid and 1-(3-
mercaptopropanoyl)-L-pipecolic acid Rf 0.80 [silica gel,
benzene, acetic acid (7:1)], [~] -51.5 (c, 1.0 abs.
ethanol), are obtained.
Example 147
1-[3-(Acetylthio)-2-methylpropanoyl=DL-pipecolic acid
6.5 g. (0.05 m.) of pipecolic acid are suspended
in dimethylacetamide (200 mg.), 9.0 g. (0.05 m.) of
3-acetylthio-2-methylpropanoyl chloride is added dropwise.
The temperature rises to 29 and a clear solution forms.
Then 10.1 g. of N-methylmorpholine is added all at once
and the temperature rises to 3~ . The mixture is heated
on a steam ~ath for 1 hour when a clear solution forms.
This is allowed to stand at room temperature overnight
and the solid which precipitates is filtered to yield
6.1 g., m.p. 203-204 . The solvent is removed and the
viscous residue is triturated with water and 20o HCl. The
yellow oil is extracted with 3 x 150 ml. of ethyl acetate.
The ethyl acetate extracts are dried over magnesium sulfate
and removed -to yield 14 t3, of 1-[3-(acetylthio)-2-methyl-
propanoyl-DL-pipecolic acid as a viscous oil.
Example 1~8
_ _
1-(3-Mercapto-2-methylpropanoyl)-DL-pipecolic acid
Aqueous NH4011 (30 ml. water and 20 ml. conc. NH40H)
is stirred under nitrogen at 10 for 15 minutes. This is
-6~-
. . .. .
J ~ ~
~IA135b
added to 13.0 g. (0.05 m) of 1-[3-(acetylthio)-2-
methylpropanoyl]-DL-pipecolic acid and the resulting solution
is stirred for 10 minutes under nitrogen; then at room
temperature for 50 minutes. It is then treated with water
and 20~ HCl and the yellow oil extracted with 3 x 150 ml.
of ethyl acetate. The ethyl acetate extract is dried over
magnesium sulfate and removed to yield 11.1 g. 1-(3-
mercapto-2-methylpropanoyl)-DL--pipecolic acid as a viscous
oil. Rf 0.62 [silica gel, ben~ene, acetic acid (7:2)~.
Example 149
3-[(4-Methoxyph~yl)methylthio]-2-methyl-propanoic acid
p-Methoxy-~-toluene thiol (li.4 g., 0.1 mol.) is
added to a solution of methacrylic acid (8.6 g., 0.1 mol.)
in 50 ml. 2N sodium hydroxide. The mixture is heated on
the steam bath for three hours, then refluxed for two
hours and cooled. The mixture is extracted with ether, then
the aqueous layer is acidified with concentrated HCl and
extracted with dichloromethane. The acidic extracts are
washed with brine, dried (MgSO4) and evaporated in vacuo.
The resulting semi-solid is taken up in 50 ml. of dichloro-
methane, diluted with 50 ml. hexane, and chilled. 3-[(4-
methoxyphenyl)methylthio]-2-methylpropanoic acid is
collected as a white crystalline solid, m.y. 74-8~
~5.5 g.).
Example 150
1-[3-(4-Metho~yphenyl)me;thylthio]-2-methylpropanoyl-L-
proline tert-butyl ester
I
3-~(4--methoxyphenyl)me-thylthio]-2-methylpropanoic
acid (3.6 g., 0.015 mol.), L-proline tert-butyl ester
(2.6 g., 0.015 mol.), and dicyclohexylcarbodiimide (3.1 g.,
-G~-
~ 5~ ~IA135b
0.015 mol.) are dissolved in 50 ml. of dichloromethane
and stirred thirty minutes at 0 . The cooling bath is
removed and the mixture stirred overnight (sixteen hours).
The resulting suspension is filtered and the filtrate
washed with 5~ potassium bisulfate, saturated sodium bicarbonate
and brine, then dried (Mc3SO~) ~nd evaporated in vacuo.
The resulting clear oil is applied to a 250 ml. silica
gel column and chromatographed using 20% ethyl acetate/
hexane as eluant. The main fraction (Rf = 0.70, silica
gel, ethyl acetate) is evaporated to 5.5 g. (93~ of 1-
[3-(4-methoxyphenyl)methylthio]-2-methylpropanoyl-L-proline
tert-butyl ester as a clear oil. Rf = 0.70 (silica gel,
ethyl acetate); Rf = 0.60 (silica gel, ether).
Example 151
-(3-Mercapto-2-methylpropanoyl)-L-prolinc
The ester from Example 150 (1.2 g., 0.003 mol.),
anisole (5 ml.) and trifluoromethanesulfonic acid (0.5 ml.)
are dissolved in 20 ml. of trifluoroacetic acid under
nitrogen, and the resulting red solution let stand one hour
at room temperature. The solution is evaporated in vacuo
to a red residue which is taken up in ethyl acetate and
washed with water, brine, then dried (MgSO4) and evaporated.
The residue is repeatedly triturated with hexane and the
residual hexane evaporated; the oil residue amounts to
0.4 g. A portion (180 mg.) of this material is subjected
to preparative thin-layer chromatography on 2 mm siLica gel
plates using benzene~acetic acid 75:25 as eluant. The .
main nitroprusside-positive band (Rf = 0.40) is recovered,
affording 135 mg. of 1-(3-mercapto-2-methylpropanoyl)-L-
proline as an oil. TLC usincJ benzene/acetic acid 75:25
-70-
. . _ . . _ . , . . _ .
3 S
HA135b
(~f = 0.40) and chloroform/methanol/acetic acid 50:~0:10
(Rf = 0.62).
Example 152
i
1-(3-Mercapto-2-D-me-thyl~ropanoyl)-L-proline
... . .
IJnder a blanket of argon 1-[3-(acetylthio)-2-
~methylpropanoyl]-L-proline (10.0 g.) is slurried in
water (lS0 ml.) at 10 . To this mixture is added 5~
sodium hydroxide and the pH of the solution maintained
at 13 for 1.5 hours. After this time, when the uptake
of sodium hydroxide had ceased, the solution is
acidified to a pH = 2.0 with concentrated sulfuric acid.
The aqueous solution is then extracted three
times with methylene chloride (3 x 150 ml.) and the
combined methylene chloride fractions concentrated to
an oil. The concentrate is taken up in ethyl acetate,
filtered and the filtrate diluted with hexane (30 ml.).
An additional amount of hexane is added after 1/2 hour
and then the mixture cooled to 10 for 1 hour.
The crystals are filtered and washed with hexane -`~
(2 x 25 ml.) and dried to constant weight to give ~
1-(3-mercapto-2-D-methylpropanoyl)-L-proline as white ~ ~-
crystals, 6.26 g., m.p. 100-102.
Example 153
1_[3-Tosyl xy-2-methylpropanoyll-L-proline
By substituting 3-tosyloxy-2-methylpropanoic acid
chloride for the 3-acetylthio-2-methylpropanoic
acid chloride in the procedure of ~xample 29b 1-[3-
... ., ~
tosyloxy-2-methylpropanoyl]-L-proline is obtained.
-71-
.. ..--
~ A135b
Example 15~
1-[3-~cetylthio-2-metllylpropanoyl~-L-proline
1-[3-Tosyloxy-2-methylpropanoyl]-L-proline
(3.5 g.) is added to a solution of thiolacetic acid
(1.14 g.), and triethylamine (3.5 ml.) in ethyl acetate
(20 ml.). The solution is maintained at 50 for three
hours, cooled, diluted with ethyl acetate (100 ml.), and
washed with dilute hydrochloric acid. The organic layer
is dried and concentrated to dryness in vacuo. The
residue is dissolved in acetonitrile and dicyclohexylamine
is added. The crystalline precipitate is recrystallized
from isopropanol to yield 1-[3-acetylthio-2-D-methyl-
propanoyl)-L-proline, dicyclohexylamine salt, m.p.
187-188 , [a]D -67 (c 1,4, EtOH). This salt is
converted to the free acid, m.p. 83-85 (an isomorphic
form of m.p. 104-105 is obtained if the crystallizing
solution is seeded with high melting material).
Example 155
1-(3-~ercaptopropanoyl)-L-proline, t-butyl ester
To a stirred solution of 1.71 g. (10 mmoles) of
proline t-butyl ester and 1.35 g. (10 mmoles) o~
hydroxybenzotriazole hydrate in 20 ml. of N,N-dimethyl-
formamide at 0-5 are added 2.06 g. (10 mmole) of N,N'-
dicyclohexylcarbodiimide. The mixture is stirred for
10 minutes, followed by the addition of 1.06 g. (10 mmole)
of 3-mercaptopropanoic acid in 2 ml. of N,N-dimethyl-
formamide- The mixture is then stirred at 0-5 for
1 hour, and at room temperature overnight. `-
The precipitated N,N'-dicyclohcxylurea is
filtered of~ and the filtrate concentrated in vacuo.
-72-
i l A l 3 S b
The residue is taken up in ethyl acetate, washed
thoroughly with saturated aqueous sodium bicarbonate, f
dried, and coneentrated in vacuo to 2.5 g. of oil.
The oil is taken up in 1:1 ethyl acetate-hexane
and applied to a silica gel column (100 g.). Elution
with 1:1 ethyl acetate-hexane affords 1.40 g. (54~) of
1-(3-mercaptopropanoyl)-L-proline, t-butyl ester as an
oil, which crystallizes on standing. Recrystallization
`from ether-hexane yields 0.9 g. of colorless crystalline
solid, m.p. 55-60, identical to the compound of
Example 17.
Example 156
1-(3-Mercaptopropanoyl)-L-proline
A solution of 75 mg. (0.27 mmole) of 1-[3-~(ethyl- `
amino)carbonyl~thio]propanoyl]-L-proline in 1 ml~ each of
eoneentrated ammonium hydroxide and water is allowed to
stand at room temperature for 18 hours under argon. The
solution is diluted with a small amount of water and
extràeted with ether. The aqueous layer is acidified with
cold eoneentrated hydroehloric acid and extracted with
ethyl aeetate. The eombined extraets are dried and
eoneentrated in vaeuo to give a eompound identical with
the product of Example 18. TLC (siliea gel; benzene:acetic
acid 7;3) Rf 0.4.
Example 157
Methacryloyl-L-Proline
L-proline (23.0 g., 0.2 mol.) is dissolved in
100 ml. water and stirred in an ice bath. Methacryloyl
ehloride (19.6 ml., 0.2 mol.) in 25 ml. of methyl isobutyl
ketone is added dropwise over three hours. Sodium hydroxide
.. , _ .. . .. _ . . _ _ ... ... .
3~Z55
solution (2N~ is added simult~neously, maintaining the pH
of the reaction mixture at 7Ø Addition of base is con-
tinued for four hours after addition of acid chloride has
been completed. The reaction mixture is adjusted to pH 5
with concentrated HCl and extracted with ethyl acetate.
The aqueous layer is then acidifie~ to pH 2.5 and extracted
thoroughly with ethyl acetate. The acidic extracts are
washed with brine and dried (MgSO4). The ethyl acetate
solution is treated with dicyclohexylamine (40 ml.) and
chilled overnight. The resulting white precipitate is
filtered and dried, yielding 29 g. (39%) of white solid,
m.p. 202-210. The solid is crystallized from 1.5 liters
3:1 acetonitrile/isopropanol to yield 19.7 g. of metha-
cryloyl-L-proline, dicyclohexylamine salt as fine white
needles, m.p. 202-210.
The salt is dissolved in water/ethyl acetate and
the mixture acidified with concentrated HCl. The result-
ing suspension is filtered to remove a fine white preci-
pitate which is washed well with ethyl acetate. The fil-
trate is saturated with sodium chloride and extractedthoroughly with ethyl acetate. The extracts are washed
with brine, dried (MgSO4) and evaporated to a clear oil
which solidifies. Crystallization from ethyl acetate/hex-
ane yields 7.5 g. (83~) of methacryloyl-L-proline as a
white crystalline solid, m.p. 89-93. An analytical
sample is obtained by recrystallization, m.p. 95-98.
Example 158
1-(3-Acetylthio-2--D-methylpropanoyl)-L-proline
Methacryloyl--L-proline (183 mg., 0.0001 mol.) is
dissolved in thio:Lacetic acid (0.5 ml.) and allowed
-74-
-
HA135b
to stand at room temperature for sixteen hours. The
solution is evaporated in vacuo to a yellow resiclue.
Preparative thin layer chromatography (silica ~el, di-
chloromethane/methanol/acetic acid ~0:5:5) allows isolation
of a clear oil (240 mg.) as the main fraction. TLC (diehloro-
methane~methanol/acetic acid go 5 :~r;) shows this material
to be l-(3-acetylthio-2-DL-methylpropanoyl)-L-proline
eorresponding to the product of Example 29B. Rf = 0.35;
(benzene/acetic acid 75:25) Rf = 0.38.
The oil is dissolved in 3 ml. acetonitrile,
treated with dicyclohexylamine until the solution is
basie, and chilled. ~ white crystalline solid (106 mg.)
m.p. 175-181 , is eolleeted. Crystallization from
isopropanol gives l-(3-aeetylthio-2-D-methylpropanoyl)-
L-proline, dieyclohexylamine salt, m.p. 187-188 ,
identieal with this product in Example 29A.
Example 159
! .
l-[Dithiobis-(2-methyl-3-propanoyl)]-bis-L-proline
By substituting 3,3'-dithiobis-2-methylpropanoic aeid
for the 3-acetylthio-2-methylpropanoie acid in the pro-
eedure of Example 29B, l-[dithiobis-(2-methyl-3-propanoyl)]-
bis-L-proline is obtained. r
Example 160
1-(3-Mercapto-2 - methylpropanoyl)-L-proline
Zinc dust (10.0 g.) is added to a slurry of the
product of Example 159 (5.0 g.) in 100 ml. of l.O.N
sulfuric aeid and the mixture is stirred at 18 for four -~
hours under a hlanket of nitrogen. The solution is then
fil-tered, the zine washed with wat~r (2n ml.) ancl the
eombined filtrates are extracted with methylene ehloride
-75-
. _ .. _ . ..... . _ _ _ ..
HA135b
(3 x 75 ml.). The methyl~ne chloride washes are back
extrac-ted with water (25 ml.) and then the organic
solution concentrated to an oil. This oil is taken up
in ethyl acetate (20 ml.) and filtered. Hexane (15 ml.)
is added to the filtrate and the mixture is stirred for k
15 minutes. Af~er this time, an additional volume of
hexane (30 ml.) is added and the solution cooled to 5
for 1 hour. The mixture is then filtered, and the product
is washed with hexane (2 x 10 ml.) and dried to give 4.17 g.
of white crystals of the product, 1-(3-mercapto-2-
methylpropanoyl)-L-proline. TLC, Rf = 0.60
(Solvent system: benzene/acetic acid 75:25).
Example 161
3-Benzylthio-2-methylpropanoic acid L
By substituting a-toluenethiol for p-methoxy-a- t
toluenethiol in the procedure of Example 149, 3-benzylthio-2-
methylpropanoic acid is obtained.
Example 162
1-[3-(Benzylthio)-2-methylpropanoyl]-L-proline tert. ~ -
butyl ester
By sub~tituting 3-benzylthio-2-methylpropanoic
acid for the 3-[(4-methoxyphenyl)methylthio]-2-methyl-
propanoic acid in the procedure of Example 150, 1-[3-
tbenzylthio)-2-methylpropanoyl~-L-proline tert. butyl ester
is obtained.
Example 163
1-[3-(Benzylthio)-2-methylpropanoyl]-L~proline
1-[3-(benzylthio)-2-methylpropanoyl]-L-proline
tert. butyl ester (7.8 g.) is dissolved in a mixture of
anisole (55 ml.) and trifluoroacetic acid (110 ml.)~ After
-76-
~LG ~
}IA135
one hour storage at room temperature, the solvent is
removed in vacuo and the residue is dissolved in ether,
~ashed several times with saturated sodium chloride, dried
over magnesium sulfate and evaporated to dryness in vacuo
to yield 1-[3-(benzylthio)-2-methylpropanoyl]-L-proline.
Rf 0.5 (Silica gel, ~enzene/acetic acid 3:1) Rf 0.5.
(Silica gel, Methyl-ethylketone/acetic acid/pyridine/water
14:1:2:1).
E mple 164
1-(3-Mercapto-2-methylpropanoyl)-L-proline
1-[3-(benzylthio)-2-methylpropanoyl]-L-proline
(0.1 g.) is suspended in boiling liquid ammonia (10 ml.)
and small pieces of sodium are added with stirring until
persistent blue color. The color is discharged with a
few crystals of ammonium sulfate and the ammonia is allowed
to evaporate under a current of nitrogen. The residue is
dissolved in a mixture of dilute hydrochloric acid and ethyl
acetate. The organic layer is dried and concentrated to
dryness in vacuo to yield l-(3-mercapto-2-methylpropanoyl)-
L-proline. Rf: 0.35 (Silica gel; ~enzene/acetic acid
3:1), R~ 0.5 (Silica gel; Methyl-ethylketone/acetic acid~ t
pyridine/water 14:1:2:1) identical to the compound of
Example 34.
Example 165
3-Triphenylmethylthio-2-methylpropanoic acid
A solution of 3-mercapto-2-methylpropanoic acid
(1.2 g.) and tritylchloride (2.9 g.) in methylene ` `~
chloride (50 ml.) is kept at room temperature for 2 hours.
The mixture is warmed in a steam bath for 20 minutes and then
evaporated to dryness in VACUO and the residue is dissolved
- :~'; ~ ' ,, . '
HA135b
in saturated aqueous sodi~un bicarbonate and the solution
is washed with ethyl ace-tate. The aqueous phase is
acidified to pH 3 and extracted with ethyl acetate. The
organic layer is dried and concentrated to dryness to give
3-triphenylmethylthio-2-methylpropanoic acid. Rf 0.8
(Silica gel, Benzene/acetic acid 3
Example 166
1-[3-(Triphenylmethylthio)-2-methylpropanoyll-L-proline
tert.butyl ester
By substituting 3-triphenylmethylthio-2-methyl-
propanoic acid for the 3-[(4-methoxyphenyl)methylthio]-
2-methylpropanoic acid in the procedure of Example 150,
1-[3-(triphenylmethylthio)-2-methylpropanoyl]-L-proline
tert.butyl ester is obtained. L
Example 167
1-[3-(Triphenylmethylthio)-2-methylpropanoyl]-L-proline i~
3-Triphenylmethylthio-2-methylpropanoic acid
(1.8 g.) and N,N'-carbonyldiimidazole (0.8 g.) are
dissolved in tetrahydrofuran (10 ml.) with stirring at
room temperature. After twenty minutes, the solution is
added to a mixture of L-proline (0.6 g.) and N-methyl-
morpholine (1 g.) in dimethylacetamide (20 ml.). The
resulting mix-ture is stirred overnight at room temperature,
çoncentrated to dryness and the residue dissolved in a
mixture of ethyl acetate and 10~ aqueous potassium bisulfate.
The organic layer is separated and dried and concentrated
to dryness in vacuo to obtain l-[3-(triphenylmethylthio)- ~ ,
2-methylproFIanoyl]-L-proline. Rf: - 0.4 (Silica gel, L
Benzene/acetic acid 3:1), Rf 1.0 (Silica gel, Methyl-ethyl-
ketone/acetic acid/pyridine/water 19:1:2:1).
-78-
, _ . . . ... _ _ _ _ .. .... _ _ . .
.._
IIA135b
Example 16~
1-[3-Mercapto-2-methylpropan_~)-L-prollne
1-[3-(triphenylmethylthio)-2-methylpropanoyl]-L-
proline tert.butyl ester (5 g.) is dissolved in a mixture
of anisole (55 ml.) and trifluoroacetic acid (110 ml.).
After one hour storage at room temperature, the solvents
are removed in vacuo and the r~esidue is applied to a
eolumn of silica gel equilibrated with benzene:acetic
aeid (75:25) and eluted with the same solvent. The
fraetions eorresponding to the component with Rf 0.40
(TLC silica gel with same system) are pooled and concentrated
-to dryness to yield 1-[3-mercapto-2-methylpxopanoyl)-L-
proline. Rf 0.62 (silica gel, chloroform/methanol:
aeetie aeid:water 50:40:10), identieal to the eompound
of Example 34.
Example 169
3-(Tetrahydropyran-2-ylthio)-2-methylpropanoic acid
To a solution of 3-mereapto-2-methylpropanoie
aeid (2.4 g.) and freshly distillled 2,3-dihydro-4H-
pyrane (1.9 g.) in benzene (60 ml.), boron trifluoride
etherate (2.8 g.) is added. After two hours, potassium
earbonate (4 g.) is added, the mixture is stirred and
filtered. The filtrate is concentrated to dryness to
yield 3-(tetrahydropyran-2-ylthio)-2-methylpropanoie aeid.
Example 170
1-[3-(Tetrahydropyran-2-ylthio)-2-methylpropanoyl]-L-proline
By substituting 3-(tetrahydropyran-2-ylthio)-2-
methylpropanoie aeid for the 3-triphenylmethylthio-2-
methylpropanoie aeid in the proeedure of Example 167,
1-[3-(tetrahydropyran-2-ylthio)-2-methylpropanoyl]-L-proline
-79-
.~r~ 5 ~1A135b
is obtained. Rf: 0.8 (Silica gel, senzene/acetic acid
3:1; Rf: 0.75 (Silica gel, I~lethyl-ethylketone/Acetic acid/
pyridine/water; 14:1:2:1).
Example 171
1-(3-Merc~E~o-2-methylpropanoyl)-L-proline
A solution of 1-[3-(tetrahydropyran-2-ylthio)-2-
methylpropanoyl)-L-proline (1 g.) in a mixture of methanol
(25 ml.) and concentrated hydrochloric acid (25 ml.) is
stored at room temperature for 30 minutes. The solvents
are removed in vacuo to yield 1-(3-mercapto-2-methyl-
propanoyl)-L-proline. Rf: 0.35 (silica gel, Benzene/ P
acetic acid, 3:1), Rf 0.5 (silica gel, Methyl-ethylketone/
acetic acid/pyridine/water 14:1:2:1) identical to the
compound of Example 34.
Example 172
3-Acetamidomethylthio-2-methylpropanoic acid
3-Mercapto-2-methylpropanoic acid (2.4 g.) and
N-hydroxymethylacetamide (1.8 g.) are dissolved in
trifluoroacetic acid and the solution is stored at room
temperature for one hour. The trifluoroacetic acid is
removed in vacuo and the residue is dried in vacuo over
potassium hydroxide to yield 3-acetamidomethylthio-2-
methylpropanoic acid.
Example 173
1-[3-(Acetamidomethylthio)-2-methylpropanoyl]-L-proline
By substituting 3-acetamidomethylthio-2-methyl-
propanoic acid for the 3-(tetrahydropyran-2-yl thio)-2-
methylpropanoic acid in the procedure of Example 170
1-[3-(acetamidomethylthio)-2-
!
-80-
`:
~ .2~ ~IA135b
methylpropanoyl]-L-proline is obtained. Rf 0.2 (Silica
gel, senzene/acetic acid 3:1) Rf 0.3 (Silica gel, Methyl-
ethylketone/ace-tic acid/pyridine/water 14:1:2:1).
Example 174
1-(3-~Iercapto-2-methylpropanoyl)-L-proline
1-[3-(acetamidomethylthio)-2-methylpropanoyl]-L-
proline (1.4 g.) and mercuric acetate (1.93 g.) are dissolved
in a mixture of acetic acid (;25 ml.) and water (25 ml.).
After one hour stirring on the steam bath, hydrogen sulfide
is bubbled through until no more precipitation of mercuric
sulfide is observed. The mixture is filterd, the precipitate
is washed with ethanol, and the filtrate is concentrated
to dryness in vacuo to yield l-(3-mercap-to-2-methylpropanoyl)-
L-proline. Rf: 0.35 (Silica gel, Benzene/Acetic acid 3:1);
Rf : 0.5 (Silica gel, Methyl-ethylketone/Acetic acid/
pyridine/water 14:1:2:1) identical to the compound of
Example 34.
Exam~le 175
1-(3-Mercapto-2-methylpropanoyl)-L-proline tert. butyl ester
To the cold (5 ) solution of 1.2 g. (10 mMol.) of 3_
mercapto-2-methylpropanoic acid and 1.7 g. (10 mMol.) of
L-proline tert. butyl ester in 25 ml. dichloromethane
2.26 g. of dicyclohexylcarbodiimide in 5 ml. dichloromethane
is added in portions. After 2 hours at room temperature,
5 drops of acetic acid are added, the mixture is filtered
and the filtrate evaporated to an oily residue. This
residue is taken up in 20 ml. of petroleum ether-ethyl
acetate (3:1) and applied to a 150 ml. silica gel column
prepared in petroleum ether. The fraction eluted with
petroleum ether-ethyl acetate (1:1) contains the product,
.
,
_ _ ,,,
3,~
HA135b
1-(3-mercapto-2-methylpropanoyl)-L-proline tert.butyl ester.
This fraction (0.6 g.) is dried over P205 in vacuo for
12 hours. R~ 0.6 (Silica gel, Ben~ene/Acetic acid 3:1),
Rf 0.8 (Silica gel, Methyl-ethylketone/acetic acid/pyridine/
water 14:1:2~
Example 176
1-(3-Mercapto-2-methylpropanoy:L)-L-proline
By substituting 1-(3-mercapto-2-methylpropanoyl)-L-
proline tert. butyl ester for the l-(3-mercaptopropanoyl-L-
proline tert.butyl ester in the procedure of Example 18C,
1-(3-mercapto-2-methylpropanoyl)-L-proline is obtained.
Rf 0.35 (Silica gel, Ben~ene/acetic acid 3:1), Rf 0.5
(Silica gel, ~lethyl-ethylketone/Acetic acid/Pyridine/Water
14:1:2:1), identical to the c~mpound of Example 34
! .
. ~ ~ .,
.'
(
--82--
..... _ _
. . . _._. ~. .
~ 5 HA135b
The raccmic form of the ~inal prod~lct in any of 3
the foregoing examples is produced by utilizing the DL-form
of the starting amino acid instead of the L-form.
Similarly, the D-form oE the final proclucts in ,any
of the foregoing examples is produced by utilizing the D-form
of the starting amino acid instead of the L-form.
Example 177
1000 tablets each containing 100 mg. of 1-(2- ,
mercaptopropanoyl)-L-proline are procluced from the following
10 ingredients:
1-(2-Mercaptopropanoyl)-L-proline 100 g.
Corn starch 50 ~J.
Gelatin 7.5 g.
Avicel (microcrystalline cellulose) 25 g-
Magnesiwn stearate 2.5 g.
The 1-(2-mercaptopropanoyl)-L-proline and corn I ,
starch are admixed with an aqueous solution,of the gelatin.
The mixture is dried and ground to a fine powder. The
Avicel and then the magnesium stearate are admixed with the
20 granulation. This is then compressed in a tablet to form t
1000 tablets each containing 100 mg. of active ingredient.
Example 178
By substituting 100 g. of 1-(3-mercapto-2-D-
methylpropanoyl)-L-proline for the 1-(2-mercaptopropanoyl)-
L-proline in Example 177, 1000 tablets each containing 100 mg.
of the l-~3-mercapto-2-D-methylpropanoyl-L-proline are
produced.
-~3-
S HA135b
xam~ 179
_ .
1000 tablets each containing 200 mg. of 1-(2-
mercaptoacetyl)-L-proline are produced from ~he following
ingredients:
1-(2-Mercaptoacetyl)-L-proline200 g.
Lactose 100 g.
Avicel 150 g.
Corn starch 50 g.
Magnesium stearate 5 y.
The 1-(2-mercaptoacetyl)-L-proline, lactose and
Avicel are admixed, then blended with the corn starch. I
Magnesium stearate is added. The dry mixture is compressed I ;
in a tablet press to form 1000 505 mg. tablets each containing
200 mg. of active ingredient. The tablets are coated ~/ith a
solution of Metho~el E 15 (methyl cellulose) includincJ as a
color a lake containing yellow ~6.
E~ample 180
Two piece ~1 gelatin capsules each containing 250 mg.
of l-(2-mercaptopropanoyl)-L-proline are filled with a mixture
of the following ingredients:
1-(2-Mercaptopropanoyl)-L-proline250 mg.
Magnesium stearate 7 mg.
USP lactose 193 my.
Example 181
An injectable solution is produced as follows: ,'
1-(2-Mercaptopropanoyl)-L-proline500 mg.
Methyl paraben 5 g.
Propyl paraben 1 g~
Sodium chloride 25 g. I
Watcr Eor illjCCtlOtl ~j!i. S 1. .¦
-84-
11~3 5:~5 ~IA135b
The active substance, preservatives and sodium
chloride are dissolved in 3 liters of water for injection and
then the volume is brought up to 5 liters. The solution is
filtered through a sterile filter and aseptically filled
into presterilized vials whicIl are then closed with pre-
sterilized rubber closures. Each vial contains 5 ml. of
solution in a concentration of 100 mg. of active ingredient
per ml. of solution for injection.
Example 182
By substituting 100 g. of 1,1'-[dithiobis(2-D-methyl-
3-propanoyl)]-bis-L-proline for the 1-(2-mercaptopropanoyl)-
L-proline in Example 177, 1000 tablets each containing 100 mg.
of the l,l'-[dithiobis[2-D-methyl-3-propanoyl)]-bis-L-
proline are produced.
Each of the products of the examples can be
similarly formulated by substituting it for the active
ingredient in Examples 177, 179, 180 or 181.
-85-
I
