Chiral 2-(2-benzyl-3-mercaptopropionylamino)-1-alkanol derivatives and chiral 2-(2-benzyl-3-mercaptopropionylamino)-4-methylthiobutyric acids are inhibitors of enkephalinase enzyme, reflecting their clinical utility as analgesics or anticonvulsant agents, or as therapy for disorders in which endogenous enkephalin levels are below normal.

BACKGROUND OF THE INVENTION 
The present invention is concerned with a class of chiral 
2-(2-benzyl-3-mercaptopropionyl)amino-1-alkanols, as well as chiral 
2-(2-benzyl-3-methylmercaptopropionylamino-4-methylthiobutyric acids, 
having beneficial CNS (central nervous system) effects including clinical 
utility as analgesics or anticonvulsants, or as therapy for disorders in 
which endogenous enkephalin levels are below normal. It is believed that 
these activities are mediated through the ability of these compounds to 
inhibit enkephalinase, a dipeptidyl carboxypeptidase which specifically 
cleaves the Gly.sup.3 -Phe.sup.4 bond of enkephalins. The enkephalins are 
compounds understood to be signal conveying compounds within the central 
nervous system. Inhibitors of enkephalins have useful CNS activity. 
Roques et al. [Nature 288, pp. 286-288 (1980)] have recently reported that 
N-(2-benzyl-3-mercaptopropionyl)glycine ("thiorphan") is a specific 
enkephalinase inhibitor. This compound is reported to elicit analgesic 
(antinociceptive) activity in mice in the so-called hot plate jump test 
but not in the so-called tail withdrawal test. 
Compounds of the very broad formula 
##STR1## 
wherein R.sup.a is H or R.sup.f CO; 
R.sup.b and R.sup.c are H, alkyl or phenylalkyl; 
R.sup.d is H, OH or alkyl; 
R.sup.e is OH, alkoxy or NH.sub.2 ; 
R.sup.f is alkyl, phenyl or phenylalkyl; 
p is 0, 1 or 2; and 
q is 1 to 3; 
have been reported by Ondetti et al. [U.S. Pat. No. 4,046,889 (July 6, 
1977)] as inhibitors of angiotensin converting enzyme, useful as 
hypotensive agents. Ondetti et al. define a subgenus of "broadly 
preferred" compounds as including R.sup.b and R.sup.c as other than 
phenylalkyl and "especially preferred" derivatives are further restricted 
to proline derivatives (i.e. q=2, R.sup.d =H). One compound of this class, 
having the formula 
##STR2## 
has been assigned the generic name captopril. 
SUMMARY OF THE INVENTION 
The present invention relates to chiral compounds of the formula 
##STR3## 
wherein X is hydrogen, (C.sub.1 -C.sub.3)alkyl, (C.sub.1 -C.sub.3)alkoxy, 
fluoro, chloro, bromo or trifluoromethyl; 
R is hydrogen or (C.sub.1 -C.sub.3)alkyl; 
n is 1 to 4; 
m is 0, 1 or 2; and 
R.sup.1 is (C.sub.1 -C.sub.3)alkyl. 
Of particular value are the compounds wherein n=2, m=0 and R.sup.1 =methyl. 
Within this subgenus, the preferred value of R is hydrogen and the most 
valued species further have X as p-chloro, p-methoxy or hydrogen. 
The present invention also relates to compounds of the formula 
##STR4## 
as well as the pharmaceutically acceptable cationic salts thereof. Such 
salts include the alkali metal salts, e.g., sodium and potassium; alkaline 
earth metal salts such as calcium and magnesium; aluminum salts; ammonium 
salts; and salts with organic bases, e.g., amines such as benzathine 
(N,N'-dibenzylethylenediamine), choline, diethanolamine, ethylenediamine, 
meglumine (N-methylglucamine), benethamine (N-benzylphenethylamine), 
diethylamine, piperazine and tromethamine 
(2-amino-2-hydroxymethyl-1,3-propanediol). Other salts, e.g. the 
dicyclohexylamine salt, not generally considered to be pharmaceutically 
acceptable, can also be used for purposes of isolation, purification or 
testing. 
The compounds of the present invention are enkephalinase enzyme inhibitors. 
It is by this mechanism that these compounds are believed to function as 
analgesic and anticonvulsant agents in mammals, including man. 
Also encompassed by the present invention are methods of alleviating pain 
(analgesia) or preventing convulsions in a mammal by administering to said 
mammal and analgesic or an anticonvulsant quantity of a compound of the 
present invention. 
DETAILED DESCRIPTION OF THE INVENTION 
The hydroxy compounds of the formulae (I) and (II) are readily prepared by 
coupling of the appropriate, protected mercapto acid with the appropriate 
amine, viz., 
##STR5## 
wherein R, X, n, m and R.sup.1 are as herein before defined and P is a 
sulfur protecting group such as benzoyl or acetyl which is selectively 
removed by a subsequent solvolysis step. 
The coupling of the acid with the amine is accomplished by a host of 
methods well known in the art of peptide chemistry employing essentially 
molar equivalents of acid and amine so as to maximize yields and minimize 
possible acylation of the alcohol. In the present instance, a particularly 
convenient method is to simply convert the acid to acid chloride with an 
excess of acid chloride forming reagent (e.g. oxalyl chloride, sulfonyl 
chloride) in an inert, low boiling solvent such as methylene chloride. 
Temperature is not critical, but temperatures in the range 
20.degree.-50.degree. C. are preferred (moderate pressure being required 
at the upper end of the range if methylene chloride is employed as 
solvent). The acid chloride is isolated free of solvent and excess reagent 
by simple evaporation in vacuo, redissolved in an inert solvent and added 
slowly to a solution of the amino alcohol and at least one molar 
equivalent of a tertiary amine such as triethylamine or 
N-methylmorpholine. Temperature again is not critical; for example 
0.degree.-50.degree. C. are well suited. Temperatures at the lower end of 
the range are preferred. If the acid employed in the synthesis is racemic, 
then it is generally possible to separate the resulting pair of 
diastereoisomers (epimers) by standard methods of fractional 
crystallization or chromatography. Alternatively, the diastereoisomers can 
be separated by the same such methods after removal of the protecting 
group. When one particular diastereoisomer is desired, it is preferable to 
use the appropriate chiral acid as the starting material. In any event, 
the protecting group P is removed selectively by standard solvolytic 
methods, which generally employ a strong base in water and/or an alcohol, 
optionally in the presence of a reaction inert, miscible organic solvent. 
A preferred method is to react the acylthio compound with at least one 
molar equivalent of sodium methoxide in anhydrous methanol. Generally, no 
more than a 20% molar excess of the methoxide is used in practice. 
Temperature is not critical (e.g. 0.degree.-50.degree. C. is well suited); 
conveniently, ambient temperatures are employed. 
The racemic acids required in the above synthesis are readily obtained by 
condensation of a thiocarboxylic acid with the appropriate 
3-phenyl-2-methylenepropionic acid: 
##STR6## 
The chiral forms of these acids, when desired, are obtained by forming 
salts with a chiral amine (e.g. d-(+)-alpha-methylbenzylamine), then 
employing fractional crystallization techniques well known in the art to 
separate the diastereomeric salts. 
The chiral amino alcohols required for the present syntheses are in many 
cases commercially available. Alternatively, they are obtained by 
esterification/hydride reduction of chiral acids/esters available 
commercially or by literature methods, i.e.: 
##STR7## 
wherein R, n, m and R.sup.1 are as defined above. Alternatively the chiral 
amino alcohol is obtained by standard methods of resolution using a chiral 
amine at the acid stage, or a chiral acid at the acid, ester or alcohol 
stage. 
The carboxylic acids of the formulae (III) and (IV) are similarly prepared 
by coupling a protected mercapto acid with L-methionine ester, viz.: 
##STR8## 
wherein P is as previously defined and R.sup.2 is an acid protecting group 
removable by hydrolysis. The simplest possible ester, i.e. R.sup.2 
=methyl, is perfectly well-suited for the purpose. The coupling is carried 
out as described above for the coupling of protected mercapto acid with 
amino alcohols. In this case however, an excess of the acid chloride (or 
other suitable activated form of the acid) can be used if desired, since 
no alcohol group is present in the amino ester. Comments presented earlier 
concerning separation of diastereoisomers, or use of chiral acids as 
starting materials, apply to this case as well. The protecting groups P 
and R.sup.1 are likewise removed by solvolysis, generally simultaneously 
by using at least two equivalents of a strong base. One method 
particularly well suited is to carry out hydrolysis using a small excess 
of aqueous sodium hydroxide in a water miscible reaction inert solvent, 
e.g. a lower alcohol, or 1,2-dimethoxyethane. 
The pharmaceutically-acceptable cationic salts of the compounds of the 
formula (III) and (IV) are readily prepared by reacting the acid forms 
with an appropriate base, usually one equivalent, in a cosolvent. Typical 
bases are sodium hydroxide, sodium methoxide, sodium ethoxide, sodium 
hydride, potassium methoxide, magnesium hydroxide, calcium hydroxide, 
ammonium hydroxide, benzathine, choline, diethanolamine, ethylenediamine, 
meglumine, benethamine, diethylamine, piperazine and tromethamine. The 
salt is isolated by concentration to dryness, or by addition of a 
non-solvent. In some cases, salts can be prepared by mixing a solution of 
the acid with a solution of a different salt of the cation (e.g. sodium 
ethylhexanoate, magnesium oleate), employing a solvent in which the 
desired cationic salt precipitates, or can be otherwise isolated by 
concentration and/or addition of a non-solvent. When a salt which is not 
generally considered to be pharmaceutically acceptable is employed for 
isolation or purification, it can be readily converted to the free acid 
form by acidification of an aqueous slurry or solution of the salt, 
extraction into a water immiscible organic solvent and evaporation. 
The compounds of the present invention are studied in vitro for inhibition 
of enkephalinase enzyme in the following manner. To prepare the enzyme, 
whole brain (minus cerebellum) is removed from Sprague-Dawley CD male rats 
(Charles River Breeding Laboratories, Inc., Wilmington, MA; 200-250 g.) 
following decapitation. The tissue is homogenized (Polytron, Brinkmann 
Instruments, Inc., Westbury, NY) in 30 volumes (w/v) of ice-cold 50 mM 
Tris-HCl tris(hydroxymethyl)methylamine hydrochloride, Fisher Scientific 
Co., Fair Lawn, NJ) pH 7.7 buffer. The homogenate in centrifuged at 
50,000.times.g. for 15 minutes. The pellet is resuspended in 50 mM 
Tris-HCl pH 7.7 buffer and centrifuged at 50,000.times.g. for 15 minutes. 
The resultant pellet is resuspended and centrifuged 3 additional times as 
described above. The membrane pellet is dispersed in 15 volumes of 50 mM 
Tris-HCl pH 7.7 buffer containing 1% Triton X-100 (Rohm and Haas, 
Philadelphia, PA) and incubated at 37.degree. C. for 45 minutes. After 
centrifugation at 100,000.times.g. for 60 minutes, the solubilized enzymes 
are frozen in 2 ml. aliquots (this preparation can be stored frozen for 3 
months). 
To assay inhibitors for inhibition of enkephalinase, triplicate mixtures 
comprised of 90 microl 50 mM Tris-HCl pH 7.7 buffer containing puromycin 
dihydrochloride (Sigma Chemical Co., St. Louis, MO), 3.2 mM; 200 microl 
solubilizing buffer (from final step of enzyme preparation); 5 microl 
inhibitor (various concentrations, dissolved in water); 200 microl enzyme; 
and 5 microl .sup.3 H-leucine-enkephalin (26.8 Ci/mmol, New England 
Nuclear, Boston, MA), 144 nM, are incubated for 1 hour at 37.degree. C. in 
1.8 ml. polyethylene tubes (Beckman Microfuge tubes, Beckman Instruments, 
Inc., Palo Alto, CA). One set of control tubes contains boiled enzyme 
(heated at 100.degree. C. for 10 minutes) for blank. The reaction is 
stopped by boiling tubes for 10 minutes, followed by centrifugation for 1 
minute in the Beckman Microfuge centrifuge. A ten microl aliquot of each 
supernatant is spotted on a thin layer chromatography (TLC) plate (Silica 
Gel 60, 20.times.20 cm, E. Merck, Darmstadt, Germany) using 1-8 samples 
per plate. Ten microl of a standard mixture consisting of 0.4 mg./ml. 
tyrosine (tyr, Mann Research Laboratories, Inc., New York, NY), 1 mg./ml. 
L-tyrosylglycine (tyr-gly Sigma), 1 mg./ml. L-tyrosylglycylglycine 
(tyr-gly-gly, Sigma) and 1 mg./ml. leucine-enkephalin (leu-E, 
Calbiochem-Behring Corp., La Jolla, CA) are spotted on top of each 
incubation mixture spot, as well as separately on each plate, to aid in 
zone indentification. 
Plates are dried and placed in covered glass tanks equilibrated with 100 
ml. of isopropanol:ethyl acetate:5% acetic acid (2:2:1). After development 
of the TLC plates, the separated products were visualized by spraying with 
a ninhydrin:acetone (0.5%, w/v) solution and heating for 10 minutes at 
100.degree. C. in a vacuum oven. R.sub.f values of standard markers are: 
tyr, 0.33; tyr-gly, 0.20: tyr-gly-gly, 0.13; leu-E, 0.47. The radioactive 
spots are identified by the added markers and scraped into scintillation 
vials to which 1 ml. ethanol is added, followed by 10 ml. Aquasol-2 (New 
England Nuclear). Vials are kept overnight and counted for radioactivity 
in a liquid scintillation counter. Tyr-gly-gly formation is based on the 
difference between total counts per minute for this TLC zone minus the 
corresponding value obtained by using boiled enzyme (blank). Activity is 
reported as the inhibiting molar concentration (IC.sub.50, M) which will 
inhibit the enzyme activity to 50% of its normal value. Typical results 
obtained with various compounds of the present invention are shown in 
Table I in comparison with thiorphan in the same test. 
TABLE I 
______________________________________ 
Enkephalinase Inhibition 
Compound 
I/II/III/IV X R n m R.sup.1 
IC.sub.50, M 
______________________________________ 
I H H 2 0 CH.sub.3 
4.8 .times. 10.sup.-7 
II H H 2 0 CH.sub.3 
9.6 .times. 10.sup.-6 
III -- -- -- -- -- 9.1 .times. 10.sup.-9 
IV -- -- -- -- -- 7 .times. 10.sup.-8 
Thiorphan - 1.5 .times. 10.sup.-8 
______________________________________ 
To test for the analgesic activity of the compounds of the present 
invention, a chemical nociceptive stimulus is employed, viz., blockade of 
abdominal stretching after phenylbenzoquinone (PBQ). The Carworth CF-1 
mouse strain is used, based on the fact that this strain exhibits a 
particularly clear stretching response. The animals to be used are fasted 
for 15-16 hr. before the start of the experiment. Fifteen mice (groups of 
5) weighing 11-15 g. are employed per dose level. Compounds are given by 
the oral or subcutaneous route. Drug pre-treatment times are 1 hr. (p.o.) 
and 20 min. (s.c.). Pairs of mice are injected with 2 mg./kg. of PBQ i.p. 
and placed in a lucite box (11.times.7.times.9.5 in.) maintained at 
40.degree. by a thermostatically controlled water bath. Starting 5 min. 
later the animals are observed for 5 min. and the number of abdominal 
stretching responses per animal is recorded. A stretch is considered to 
represent an intermittent contraction of the abdomen, hind limb extension, 
pelvic rotation, or opisthotonos (the abdomen of the mouse touches the 
floor of the cage and is dragged the length of the cage). The degree of 
analgesic protection is calculated on the basis of the suppression of 
writhing relative to control animals run on the same day (% MPE). Typical 
results obtained with compounds of the present invention are shown in 
Table II. This test reflects the ultimate clinical utility of the 
compounds as analgesic agents. 
TABLE II 
______________________________________ 
Percent Blockade of Abdominal Stretching 
(% MPE) After Phenylbenzoquinone 
Compound % Dose (s.c., 
I/II/III/IV 
X R n m R.sup.1 
MPE mg./kg.) 
______________________________________ 
I H H 2 0 CH.sub.3 
100 320 
69 233 
23 170 
I 4-OCH.sub.3 
H 2 0 CH.sub.3 
62 320 
I 4-Cl H 2 0 CH.sub.3 
86 320 
II H H 2 0 CH.sub.3 
(a) 320 
II 4-OCH.sub.3 
H 2 0 CH.sub.3 
46 320 
II 4-Cl H 2 0 CH.sub.3 
75 320 
III -- -- -- -- -- 57 160 
IV -- -- -- -- -- 48 160 
______________________________________ 
(a) No significant activity noted at this dose level. 
The anticonvulsant activity of the compounds of the present invention is 
determined by testing in mice, viz., Charles River males, Swiss CD strain 
(17-21 g.), fasted for 18 hr. before testing. Groups of mice are treated 
with a range of levels of the test compounds, and 1 hr. later with 
supramaximal electroconvulsive shock (ECS), administered for 0.2 sec. at 
50 mA, 60 Hz, through transcorneal electrodes. After administration of the 
electrical stimulus, each mouse is observed for 10 sec. for the presence 
or absence of hind limb tonic extension. All control mice exhibit such 
convulsions. Data are summarized in Table III. Blockade of supramaximal 
ECS in mice is a common laboratory test for clinical utility as an 
anticonvulsant, and certain known anticonvulsant drugs show activity in 
this test. It will be further noted that thiorphan was inactive even at a 
much higher dose. 
TABLE III 
______________________________________ 
Anticonvulsant Effect of Enkephalinase 
Inhibitors vs. Supramaximal 
Electroconvulsive Shock (ECS) in Mice 
Compound Ed.sub.50 
I/II/III/IV 
X R n m R.sup.1 
(s.c., mg./kg.) 
______________________________________ 
I H H 2 0 CH.sub.3 
178 
III -- -- -- -- -- 74.1 
IV -- -- -- -- -- 140.8 
Thiorphan - &gt;320 
______________________________________ 
The hydroxy derivatives (formulae I and II), based on the enkephalinase 
inhibitory and analgesic activities described above, are useful clinically 
as analgesic agents, i.e., for use in alleviation of pain, while the acid 
derivatives based on their enkephalinase inhibitory and anticonvulsant 
activities are useful clinically in preventing convulsions. 
In either case, the compounds of the present invention can be formulated in 
a variety of pharmaceutical preparations which contain the derivative 
alone or in combination with pharmaceutical carriers such as inert solid 
diluents, aqueous solutions or various non-toxic, organic solvents and in 
dosage forms such as gelatin capsules, tablets, powders, lozenges, syrups, 
injectable solutions and the like. Such carriers include water, ethanol, 
gelatins, lactose, starches, vegetable oils, petroleum jelly, gums, 
glycols, talc, benzoyl alcohols, and other known carriers for medicaments. 
If desired, these pharmaceutical preparations can contain additional 
material such as preserving agents, wetting agents, stabilizing agents, 
lubricating agents, absorption agents, buffering agents and isotonic 
agents. 
The derivatives are administered to a patient in need of the particular 
treatment by a variety of conventional routes of administration such as 
oral, intravenous, intramuscular or subcutaneous. In general, small doses 
will be administered initially with a gradual increase in the dose until 
the optimum level is determined. However, as with any drug the particular 
dose, formulation and route of administration will vary with the age, 
weight and response of the particular patient and will depend upon the 
judgment of his attending physician. 
In the usual course of treatment, a total dose of a derivative of 
approximately 0.1 mg. per day to 100 mg. per day in single or divided 
doses, will provide effective treatment for the human patient. When the 
derivative has a prolonged effect, the dose can be administered less 
frequently, such as every other day or in 1 or 2 divided doses per week.

The present invention is illustrated by the following examples. However, it 
should be understood that the invention is not limited to the specific 
details of these examples. 
EXAMPLE 1 
Methyl 2S-(3-Acetylthio-2R-benzylpropionylamino)-4-methylthiobutyrate 
and 
Methyl 2S-(3-Acetylthio-2S-benzylpropionylamino)-4-methylthiobutyrate 
Thiolacetic acid (10.65 gm., 140.0 mmoles) was added to 2-benzylacrylic 
acid (10.0 gm., 62.0 mmoles) and the resulting solution was heated at 
90.degree. C. under a nitrogen atmosphere for one hour at which time 
silica gel TLC (ether eluant) indicated that the reaction was essentially 
complete (R.sub.f 0.3--product; R.sub.f 0.6--starting material). The 
reaction mixture was cooled and the excess thiolacetic acid was removed 
under vacuum. The residue was azeotroped once with benzene, triturated 
three times with hexane and decanted to remove the last traces of 
thiolacetic acid. The residual red oil was promptly dissolved in methylene 
chloride (25.0 ml.) and treated with oxalyl chloride (21.8 gm., 170.0 
mmoles). The solution was heated at 40.degree. C. in a nitrogen atmosphere 
for one hour, by which time gas evolution had ceased. The reaction mixture 
was cooled, evaporated under vacuum and azeotroped once with benzene. The 
residue was dissolved in methylene chloride (25.0 ml.) and added dropwise 
over a 0.5 hr. period to a cold (0.degree. C.) stirred mixture of 
L-methionine methyl ester hydrochloride (12.3 gm., 61.6 mmoles), methylene 
chloride (75.0 ml.) and triethylamine (14.51 gm., 140.0 mmoles). After the 
addition was complete, the reaction was stirred for one hour at room 
temperature under a nitrogen atmosphere, by which time TLC monitoring 
(ether) indicated the reaction was complete (R.sub.f 0.48, 0.51--products, 
R.sub.f 0.3--starting material). The reaction was washed with 2 N 
hydrochloric acid (2.times.25.0 ml.), water (1.times.25.0 ml.) and 
saturated sodium bicarbonate (1.times.25.0 ml.). The organic layer was 
dried over anhydrous magnesium sulfate, filtered and concentrated under 
vacuum to give 25.0 gm. of a mixture of the title products as an oil. The 
product mixture was separated into two isomers via medium pressure liquid 
chromatography, using a 7.5 gm. sample mixture and eluting at 35 psi on a 
25 mm.times.1000 mm column with 20% ether/hexane, 25% ether/hexane and 30% 
ether/hexane. This process was repeated until all of the mixture was 
separated. There resulted methyl 
2S-(3-acetylthio-2S-benzylpropionylamino)-4-methylthiobutyrate: 4.1 gm. 
(17.3% yield) colorless crystals m.p. 68.degree.-70.degree. C. from 
ether/hexane (1:1); 
Analysis Calcd. for C.sub.18 H.sub.25 O.sub.4 NS.sub.2 : C, 56.37; H, 
6.57%; N, 3.65. Found: C, 56.19; H, 6.26; N, 3.64. 
and methyl 2S-(3-acetylthio-2R-benzylpropionylamino)-4-methylthiobutyrate: 
5.4 gm. (22.8% yield) colorless crystals m.p. 65.degree.-7.degree. C. from 
ether/hexane (1:1). 
Analysis Calcd. for C.sub.18 H.sub.25 O.sub.4 NS.sub.2 : C, 56.37; H, 6.57; 
N, 3.65. Found: C, 56.48; H, 6.35; N, 3.68. 
By the same procedure 3-acetyl-2R-benzylpropionic acid and 
3-acetyl-2R-benzylpropionic acid are reacted with oxalyl chloride and then 
L-methionine methyl ester to form, respectively, methyl 
2S-(3-acetylthio-2R-benzylpropionylamino)-4-methylthiobutyrate and 
2S-(3-acetylthio-2S-benzylpropionylamino)-4-methylthiobutyrate, avoiding 
the need for chromatographic separation of these isomers. 
EXAMPLE 2 
2S-(2R-Benzyl-3-mercaptopropionylamino)-4-methylthiobutyric Acid 
and 
2S-(2S-Benzyl-3-mercaptopropionylamino)-4-methylthiobutyric Acid 
Product mixture of esters of the preceding example (18.82 g., 49.0 mmoles) 
in 1,2-dimethoxyethane (72.0 ml.) was stirred at room temperature under a 
nitrogen atmosphere. 2 N Sodium hydroxide (55.0 ml., 110 mmoles) was added 
and the resulting solution was stirred for one hour, at which time silica 
gel TLC (9:1 chloroform:ethanol) indicated that the reaction was complete 
(R.sub.f 0.2--product; R.sub.f 0.75--starting material). 
1,2-Dimethoxyethane was removed under vacuum and the residue acidified 
with 2 N hydrochloric acid to pH 2. The residue was extracted with ethyl 
acetate (3.times.100 ml.) and the combined extracts were dried over 
anhydrous magnesium sulfate. The dried solution was concentrated to give 
16.4 gm. of a crude oil product. The oil was chromatographed on 350 gm. 
silica gel (230-400 mesh) eluting with chloroform then 1% 
ethanol/chloroform and finally 2% ethanol/chloroform. The product was 
isolated as a mixture of the two title isomers: 3.4 gm. (21% yield) of a 
crystalline product, m.p. 130.degree.-2.degree. C. One recrystallization 
from ethyl acetate gave 2.3 gm. pure product, m.p. 137.degree.-8.degree. 
C. 
Analysis Calcd. for C.sub.15 H.sub.21 O.sub.3 NS.sub.2 : C, 55.02; H, 6.46; 
N, 4.28. Found: C, 54.66; H, 6.18; N, 4.25. 
EXAMPLE 3 
Dicyclohexylammonium 
2S-(2R-Benzyl-3-mercaptopropionylamino)-4-methylthiobutyrate 
A solution of methyl 2S-(3-acetylthio-2R-benzylpropionylamino)-4-methylthio 
butyrate (0.5 gm., 1.3 mmoles) in 1,2-dimethoxyethane (1.9 ml.) was 
stirred at room temperature under a nitrogen atmosphere. 2 N Sodium 
hydroxide (1.3 ml., 2.6 mmoles) was added and the resulting solution was 
stirred for one hour, with TLC monitoring as in the preceding example. 
1,2-Dimethoxyethane was removed under vacuum and the residue acidified 
with 2 N hydrochloric acid to pH 2. The acidified solution was extracted 
with ethyl acetate (3.times.15 ml.) and the combined extracts were dried 
over anhydrous magnesium sulfate. The dried solution was concentrated to 
give 484 mg. of a crude oil product. The oil product was converted to the 
dicyclohexylamine salt in ether to give 479 mg. (72% yield) product, m.p. 
152.degree.-5.degree. C. One recrystallization with isopropanol yielded 
171 mg. pure product, m.p. 164.degree.-7.degree. C. 
Analysis Calcd. for C.sub.15 H.sub.21 O.sub.3 N.sub.2 S.C.sub.12 H.sub.23 
N: C, 63.74; H, 8.72; N, 5.51. Found: C, 63.51; H, 8.35; N, 5.30. 
EXAMPLE 4 
2S-(2S-Benzyl-3-mercaptopropionylamino)-4-methylthiobutyric Acid 
and 
Dicyclohexylamine Salt 
By the procedure of the preceding example, methyl 
2S-(3-acetylthio-2S-benzylpropionylamino)-4-methylthiobutyrate (1 g.) was 
converted to 939 mg. of the title product as an oil. The oil was 
chromatographed on 20 gm. of silica gel (230-400 mesh), eluting with 
ether, to give 716 mg. (84% yield) of pure title product as a clear oil. A 
565 mg. sample of the oil was converted to the dicyclohexylamine salt in 
ether to give 565 mg. product, m.p. 136.degree.-41.degree. C. One 
recrystallization with benzene yielded 428 mg. product, m.p. 
140.degree.-3.degree. C. 
Analysis Calcd. for C.sub.15 H.sub.21 O.sub.3 N.sub.2 S.C.sub.12 H.sub.23 
N: C, 63.74; H, 8.72; N, 5.51. Found: C, 63.96; H, 8.58; N, 5.29. 
EXAMPLE 5 
2S-(3-Benzoylthio-2R-benzylpropionylamino)-4-methylthio-1-butanol 
and 
2S-(3-Benzoylthio-2S-benzylpropionylamino)-4-methylthio-1-butanol 
A solution of 2-benzyl-3-benzoylthiopropionic acid (4.44 gm., 14.8 mmoles) 
in methylene chloride (25.0 ml.) was stirred at room temperature under a 
nitrogen atmosphere. Oxalyl chloride (9.3 gm., 73 mmoles) was added and 
the resulting solution heated at 40.degree. C. for one hour by which time 
the evolution of gas ceased. The reaction was cooled, evaporated under 
vacuum and azeotroped once with benzene to yield the acid chloride. The 
latter was dissolved in methylene chloride (25.0 ml.) and added dropwise 
over a 0.5 hr. period to a cold (0.degree. C.) stirred solution of 
2S-amino-4-methylthio-1-butanol (2.0 gm., 14.8 mmoles) in methylene 
chloride (50.0 ml.) and triethylamine (1.65 gm., 16.0 mmoles). After the 
addition was complete, the reaction was stirred for one hour at room 
temperature under a nitrogen atmosphere. The reaction was monitored via 
tlc (silica gel) using 9:1 chloroform:ethanol and after one hour, the 
reaction was complete (R.sub.f 0.55--products; R.sub.4 0.40--starting 
material). The reaction was washed with 2 N hydrochloric acid (2.times.25 
ml.), water (1.times.25.0 ml.) and saturated sodium bicarbonate 
(1.times.25.0 ml.). The organic layer was dried over anhydrous magnesium 
sulfate and concentrated under vacuum to give 5.4 gm. (87% yield) of 
product, m.p. 95.degree.-7.degree. C. One recrystallization with ethyl 
acetate gave an analytical sample of the mixed title products melting at 
104.degree.-7.degree. C. 
Analysis Calcd. for C.sub.22 H.sub.27 O.sub.3 NS.sub.2 : C, 63.28; H, 6.52; 
N, 3.35. Found: C, 63.24; H, 6.32; N, 3.25. 
By the same method 2R-benzyl-3-benzoylthiopropionic acid and 
2S-benzyl-3-benzoylthiopropionic acid are reacted with oxalyl chloride and 
then 2S-amino-4-methylthio-1-butanol to yield, respectively, the 
individual isomeric title products. 
By the same method 2-benzyl-3-benzoylthiopropionic acid is reacted with 
oxalyl chloride and then with 2R-amino-3-methylthio-1-propanol, 
2S-amino-5-methylthio-1-pentanol, 2S-amino-6-methylthio-1-hexanol, 
2S-amino-4-propylthio-1-butanol, 2S-amino-4-methanesulfinyl-1-butanol and 
2S-amino-4-methanesulfonyl-1-butanol to yield respectively: 
2R-(3-benzoylthio-2R-benzylpropionylamino)-3-methylthio-1-propanol and 
2R-(3-benzoylthio-2S-benzylpropionylamino)-3-methylthio-1-propanol; 
2S-(3-benzoylthio-2R-benzylpropionylamino)-5-methylthio-1-pentanol and 
2S-(3-benzoylthio-2S-benzylpropionylamino)-5-methylthio-1-pentanol; 
2S-(3-benzoylthio-2R-benzylpropionylamino)-6-methylthio-1-hexanol and 
2S-(3-benzoylthio-2S-benzylpropionylamino)-6-methylthio-1-hexanol; 
2S-(3-benzoylthio-2R-benzylpropionylamino)-4-propylthio-1-butanol and 
2S-(3-benzoylthio-2S-benzylpropionylamino)-4-propylthio-1-butanol; 
2S-(3-benzoylthio-2R-benzylpropionylamino)-4-methanesulfinyl-1-butanol and 
2S-(3-benzylthio-2S-benzylpropionylamino)-4-methanesulfinyl-1-butanol; and 
2S-(3-benzoylthio-2R-benzylpropionylamino)-4-methanesulfonyl-1-butanol and 
2S-(3-benzylthio-2S-benzylpropionylamino)-4-methanesulfonyl-1-butanol. 
EXAMPLE 6 
2S-(2R-benzyl-3-mercaptopropionylamino)-4-methylthio-1-butanol 
and 
2S-(2S-benzyl-3-mercaptopropionylamino)-4-methylthio-1-butanol 
A solution of the mixed title products of the preceding example (4.5 gm., 
10.8 mmoles) in anhydrous methanol (50.0 ml.) was stirred at room 
temperature under a nitrogen atmosphere. Solid sodium methoxide (0.7 gm., 
12.96 mmoles) was added portionwise over a ten minute period. The 
resulting solution was stirred and the reaction monitored via tlc (silica 
gel) using 9:1 chloroform:ethanol; after one hour the reaction was 
complete (R.sub.f 0.35, 0.30--products, R.sub.f 0.40--starting material). 
The reaction mixture was then evaporated under vacuum. The residue was 
acidified with 2 N hydrochloric acid to pH 2 and extracted with ethyl 
acetate (3.times.25.0 ml.). The combined extracts were dried over 
anhydrous magnesium sulfate and concentrated under vacuum to give 4.98 gm. 
of a crude oil product mixture. The product mixture was chromatographed on 
150 gm. silica gel (230-400 mesh) with chloroform as eluant to give two 
isomers: 2S-(2R-benzyl-3-mercaptopropionylamino)-4-methylthio-1-butanol, 
0.564 gm. (16% yield) colorless crystals m.p. 118.degree.-120.degree. C. 
from ether/pet. ether; 
Analysis Calcd. for C.sub.15 H.sub.23 O.sub.2 NS.sub.2 : C, 57.47; H, 7.40; 
N, 4.47. Found: C, 57.48; H, 7.42; N, 4.52. 
and 2S-(2S-benzyl-3-mercaptopropionylamino)-4-methylthio-1-butanol, 0.6 gm. 
(17% yield) colorless crystals, m.p. 67.degree.-9.degree. C. from 
ether/hexane. 
Analysis Calcd. for C.sub.15 H.sub.23 O.sub.2 NS.sub.2 : C, 57.47; H, 7.40; 
N, 4.47. Found: C, 57.43; H, 7.20; N, 4.42. 
Alternatively, the individual title isomers of the preceding example are 
solvolyzed to yield the individual title isomers of the present example, 
avoiding the chromatographic separation of isomers. 
By the same method the other benzoyl compounds of the preceding example are 
converted to: 
2R-(2R-benzyl-3-mercaptopropionylamino)-3-methylthio-1-propanol and 
2R-(2S-benzyl-3-mercaptopropionylamino)-3-methylthio-1-propanol; 
2S-(2R-benzyl-3-mercaptopropionylamino)-5-methylthio-1-pentanol and 
2S-(2S-benzyl-3-mercaptopropionylamino)-5-methylthio-1-pentanol; 
2S-(2R-benzyl-3-mercaptopropionylamino)-6-methylthio-1-hexanol and 
2R-(2S-benzyl-3-mercaptopropionylamino)-6-methylthio-1-hexanol; 
2S-(2R-benzyl-3-mercaptopropionylamino)-4-propylthio-1-butanol and 
2S-(2S-benzyl-3-mercaptopropionylamino)-4-propylthio-1-butanol; 
2S-(2R-benzyl-3-mercaptopropionylamino)-4-methanesulfinyl-1-butanol and 
2S-(2S-benzyl-3-mercaptopropionylamino)-4-methanesulfinyl-1-butanol; and 
2S-(2R-benzyl-3-mercaptopropionylamino)-4-methanesulfonyl-1-butanol and 
2S-(2S-benzyl-3-mercaptopropionylamino)-4-methanesulfonyl-1-butanol. 
EXAMPLE 7 
2S-[3-Benzoylthio-2R-(p-chlorobenzyl)propionylamino]-4-methylthio-1-butanol 
and 
2S-[3-Benzoylthio-2S-(p-chlorobenzyl)propionylamino]-4-methylthio-1-butanol 
3-Benzoyl-2-(p-chlorobenzyl)propionic acid (6.34 gm., 18.9 mmoles) and 
oxalyl chloride (6.5 gm., 4.5 ml., 51.6 mmoles) in 40 ml. of methylene 
chloride were heated at 40.degree. C. for 1 hour. The reaction mixture was 
cooled and evaporated to dryness and azeotroped with benzene to yield the 
corresponding acid chloride. 2S-Amino-4-mercapto-1-butanol (2.56 gm., 18.9 
mmoles) and triethylamine (2.76 ml., 19.8 mmoles) were combined in 50 ml. 
of methylene chloride and cooled to 0.degree. C. The above acid chloride 
was dissolved in 10 ml. of methylene chloride and added dropwise to the 
chilled solution. The reaction mixture was warmed to room temperature, 
stirred for 16 hours, then sequentially washed with two 25 ml. portions of 
2 N hydrochloric acid, 25 ml. of water and 25 ml. of saturated sodium 
bicarbonate, dried over anhydrous magnesium sulfate, filtered, evaporated 
to dryness, and the residue crystallized with ether to yield 6.69 gm. of 
the title isomers as a mixture; m.p. 116.degree.-121.degree. C.; ir (KBr) 
1645, 1659 cm.sup.-1. Evaporation of the mother liquor gave an additional 
1.6 g. of less pure product. 
By the same method 3-benzoyl-2-(m-chlorobenzyl)propionic acid, 
3-benzoyl-2-(o-chlorobenzyl)propionic acid, 
3-benzoyl-2-(p-fluorobenzyl)propionic acid and 
3-benzoyl-2-(p-bromobenzyl)propionic acid are converted, respectively, to: 
2S-[3-benzoylthio-2R-(m-chorobenzyl)propionylamino]-4-methylthio-1-butanol 
and 
2S-[3-benzoylthio-2S-(m-chlorobenzyl)propionylamino]-4-methylthio-1-butano 
l; 
2S-[3-benzylthio-2R-(o-chlorobenzyl)propionylamino]-4-methylthio-1-butanol 
and 
2S-[3-benzoylthio-2S-(o-chlorobenzyl)propionylamino]-4-methylthio-1-butano 
l; 
2S-[3-benzylthio-2R-(o-fluorobenzyl)propionylamino]-4-methylthio-1-butanol 
and 
2S-[3-benzoylthio-2S-(o-fluorobenzyl)propionylamino]-4-methylthio-1-butano 
l; and 
2S-[3-benzylthio-2R-(p-bromobenzyl)propionylamino]-4-methylthio-1-butanol 
and 
2S-[3-benzoylthio-2S-(p-bromobenzyl)propionylamino]-4-methylthio-1-butanol 
EXAMPLE 8 
2S-[2R-(p-Chlorobenzyl)-3-mercaptopropionylamino]-4-methylthio-1-butanol 
and 
2S-[2S-(p-Chlorobenzyl)-3-mercaptopropionylamino]-4-methylthio-1-butanol 
The mixed title compounds of the preceding example (6.67 gm., 14.76 mmole) 
were solvolyzed according to Example 6, yielding 6.27 g. of a mixture of 
the title isomeric compounds. The first named of the title isomers (2.0 
g.) crystallized directly when the mixture was taken up in ether; m.p. 
152.degree.-154.degree. C. Recrystallization from ethyl acetate gave 1.2 
g. (m.p. 154.degree.-155.degree. C.) and a second crop of 0.37 g. 
Analysis Calcd. for C.sub.22 H.sub.26 O.sub.3 ClNS.sub.2 : C, 51.78; H, 
6.37; N, 4.03. Found: C, 52.34; H, 6.13; N, 4.01. 
The ether mother liquor from the initial crystallization of the first 
isomer was evaporated to dryness (4.2 g.) and chromatographed on MPLC at 
70 psi on a Brinkmann 25.times.1000 mm column with chloroform as eluant. 
The second isomeric product fractions were collected and evaporated to 
dryness. Crystallization from ether gave 0.5 g. of the second named isomer 
of the title products, m.p. 89.degree.-91.degree. C. Recrystallization 
from ethyl acetate/hexane gave a 354 mg. first crop (m.p. 
90.degree.-92.degree. C.) and 145 mg. second crop. 
Analysis Calcd. for C.sub.22 H.sub.26 O.sub.3 ClNS.sub.2 : C, 51.78; H, 
6.37; N, 4.03. Found: C, 52.06; H, 6.25; N, 4.05. 
By the same method the other halo compounds of the preceding example are 
converted to: 
2S-[2R-(m-chlorobenzyl)-3-mercaptopropionylamino]-4-methylthio-1-butanol 
and 2S-[2S-(m-chlorobenzyl)-3-mercaptopropionylamino]-4-methylthio-1-butan 
ol; 
2S-[2R-(o-chlorobenzyl)-3-mercaptopropionylamino]-4-methylthio-1-butanol 
and 2S-[2S-(o-chlorobenzyl)-3-mercaptopropionylamino]-4-methylthio-1-butan 
ol; 
2S-[2R-(p-fluorobenzyl)-3-mercaptopropionylamino]-4-methylthio-1-butanol 
and 2S-[2S-(p-fluorobenzyl)-3-mercaptopropionylamino]-4-methylthio-1-butan 
ol; and 
2S-[2R-(p-bromobenzyl)-3-mercaptopropionylamino]-4-methylthio-1-butanol and 
2S-[2S-(p-bromobenzyl)-3-mercaptopropionylamino]-4-methylthio-1-butanol. 
EXAMPLE 9 
2S-[3-Benzoylthio-2R-(p-methoxybenzyl)propionylamino]-4-methylthio-1-butano 
l 
and 
2S-[3-Benzoylthio-2S-(p-methoxybenzyl)propionylamino]-4-methylthio-1-butano 
l 
3-Benzoylthio-2-(p-methoxybenzyl)propionic acid (6.6 g., 0.02 mole) was 
dissolved in 20 ml. of methylene chloride. Oxalyl chloride (4.8 ml., 0.055 
mole) was added and the mixture heated at 40.degree. C. for 1 hour. The 
resulting acid chloride was isolated by evaporation to an oil and 
azeotroping once with benzene. The acid chloride was redissolved in 10 ml. 
of methylene chloride and added dropwise to a cold (10.degree. C.) 
solution of 2S-amino-4-methylthio-1-butanol (2.7 g., 0.02 mole) and 
triethylamine (3.0 ml., 0.0215 mole) in 30 ml. of methylene chloride. 
After stirring for 16 hours at room temperature the reaction mixture was 
sequentially washed with 25 ml. portions of 1 N hydrochloric acid, water 
and saturated sodium bicarbonate, dried over anhydrous magnesium sulfate, 
filtered and evaporated to yield 7.1 g. of crude product. The crude was 
chromatographed by MPLC (silica gel, 25.times.1000 mm. column, 60 psi) 
with 10% ethanol in chloroform as eluant to yield a mixture of the title 
products [4.3 g.; R.sub.f 0.16 (ether); m.p. 88.degree.-92.degree. C.]. 
The reaction procedure was repeated, except that the crude was triturated 
with hexane, providing a lower yield (1.88 g., m.p. 84.degree.-87.degree. 
C.). 
By the same method 3-benzoylthio-(p-ethoxybenzyl)propionic acid, 
3-benzoylthio-2-(m-isopropylbenzyl)propionic acid, 
3-benzoylthio-2-(o-methylbenzyl)propionic acid and 
3-benzoylthio-2-(p-trifluoromethylbenzyl)propionic acid are converted, 
respectively, to: 
2S-[3-benzoylthio-2R-(p-ethoxybenzyl)propionylamino]-4-methylthio-1-butanol 
and 
2S-[3-benzoylthio-2S-(p-ethoxybenzyl)propionylamino]-4-methylthio-1-butano 
l; 
2S-[3-benzoylthio-2R-(m-isopropylbenzyl)propionylamino]-4-methylthio-1-buta 
nol and 
2S-[3-benzoylthio-2S-(m-isopropylbenzyl)propionylamino]-4-methylthio-1-but 
anol; 
2S-[3-benzoylthio-2R-(o-methylbenzyl)propionylamino]-4-methylthio-1-butanol 
and 
2S-[3-benzoylthio-2S-(o-methylbenzyl)propionylamino]-4-methylthio-1-butano 
l; and 
2S-[3-benzoylthio-2R-(p-trifluoromethylbenzyl)propionylamino]-4-methylthio- 
1-butanol and 
2S-[3-benzoylthio-2S-(p-trifluoromethylbenzyl)propionylamino]-4-methylthio 
-1-butanol. 
EXAMPLE 10 
2S-[3-Mercapto-2S-(p-methoxybenzyl)propionylamino]-4-methylthio-1-butanol 
and 
2S-[3-Mercapto-2R-(p-methoxybenzyl)propionylamino]-4-methylthio-1-butanol 
The mixed title isomers of the preceding example (5.69 g., 12.7 mmole) were 
solvolyzed by the procedure of Example 6, yielding 5.9 g. of crude product 
after evaporation. MPLC on Brinkmann silica gel (230-400 mesh, 
25.times.1000 mm. column) with chloroform as eluant gave the separated 
title isomers. There resulted pure fractions of the less polar, first 
named title isomer [1.2 g.; wax; m/e 342; R.sub.f 0.42 (19:1 
chloroform:ethanol)], mixed fractions (0.4 g.) and the more polar, second 
named title isomer [1.2 g.; R.sub.f 0.25 (19:1 chloroform:ethanol); m.p. 
130.degree.-134.degree. C.]. The second isomer was recrystallized from 
ethyl acetate (162 mg., m.p. 138.degree.-140.degree. C.). 
Analysis Calcd. for C.sub.16 H.sub.25 NO.sub.3 S.sub.2 : C, 55.95; H, 7.34; 
N, 4.08; m/e 343. Found: C, 56.16; H, 6.89; N, 4.08; m/e 343. 
By the same method the other benzoyl compounds of the preceding example are 
converted to: 
2S-[2R-(p-ethoxybenzyl)-3-mercaptopropionylamino]-4-methylthio-1-butanol 
and 2S-[2S-(p-ethoxybenzyl)-3-mercaptopropionylamino]-4-methylthio-1-butan 
ol; 
2S-[2R-(m-isopropylbenzyl)-3-mercaptopropionylamino]-4-methylthio-1-butanol 
and 
2S-[2S-(m-isopropylbenzyl)-3-mercaptopropionylamino]-4-methylthio-1-butano 
l; 
2S-[3-mercapto-2R-(o-methylbenzyl)propionylamino]-4-methylthio-1-butanol 
and 2S-[3-mercapto-2S-(o-methylbenzyl)propionylamino]-4-methylthio-1-butan 
ol; and 
2S-[3-mercapto-2R-(p-trifluoromethylbenzyl)propionylamino]-4-methylthio-1-b 
utanol and 
2S-[3-mercapto-2S-(p-trifluoromethylbenzyl)propionylamino]-4-methylthio-1- 
butanol. 
PREATION 1 
p-Chlorophenylmethylenemalonic Acid 
Under nitrogen, malonic acid (powdered, 20 g., 0.192 mole) was mixed with 
acetyl chloride (15 g., 0.192 mole) and heated to 65.degree. C. in an oil 
bath. p-Chlorobenzaldehyde (21.6 g., 0.153 mole) was added and the mixture 
further heated to 80.degree. C. A yellow, slowly refluxing solution 
resulted, followed by copious precipitation of solids. The mixture was 
cooled and product recovered by filtration, resuspended in water, stirred 
for 5 minutes and refiltered to yield title product [21.1 g.; 61% of 
theory; m.p. 215.degree. C. (dec.)]. 
By the same method m-chlorobenzaldehyde, o-chlorobenzaldehyde, 
p-fluorobenzaldehyde and p-bromobenzaldehyde are converted, respectively 
to: 
m-chlorophenylmethylenemalonic acid; 
o-chlorophenylmethylenemalonic acid; 
p-fluorophenylmethylenemalonic acid; and 
p-bromophenylmethylenemalonic acid. 
PREATION 2 
p-Methoxyphenylmethylenemalonic Acid 
By the procedure of the preceding preparation, p-methoxybenzaldehyde (21 
g., 0.154 mole) was converted to the title product (10.0 g; 58% of 
theory). 
By the same method, p-ethoxybenzaldehyde, m-isopropylbenzaldehyde, 
o-methylbenzaldehyde and p-trifluoromethylbenzaldehyde are converted to: 
p-ethoxyphenylmethylenemalonic acid; 
m-isopropylphenylmethylenemalonic acid; 
o-methylphenylmethylenemalonic acid; and 
p-trifluoromethylphenylmethylenemalonic acid. 
PREATION 3 
p-Chlorobenzylmalonic Acid 
p-Chloromethylenemalonic acid (71.1 g.) in 600 ml. of ethyl acetate was 
hydrogenated over 10% Pd/C (3 g.) on a Paar shaker at 50 psig until 
substantially 1 equivalent of hydrogen was consumed. The catalyst was 
recovered by filtration and the mother liquor evaporated to dryness in 
vacuo. The residue was triturated with hexane to yield 69 g. (90%) of the 
title product. 
By the same method the other methylenemalonic acids of Preparation 1 are 
converted to: 
m-chlorobenzylmalonic acid; 
o-chlorobenzylmalonic acid; 
p-fluorobenzylmalonic acid; and 
p-bromobenzylmalonic acid. 
PREATION 4 
p-Methoxybenzylmalonic Acid 
The title product of Preparation 2 (15 g.) in 325 ml. of ethyl acetate was 
hydrogenated over 1 g. of 50% Pd/C according to the preceding example. 
There resulted 9.8 g. of title product. 
By the same method the other methylenemalonic acids of Preparation 2 are 
converted to: 
p-ethoxybenzylmalonic acid; 
m-isopropylbenzylmalonic acid; 
o-methylbenzylmalonic acid; and 
p-trifluoromethylbenzylmalonic acid. 
PREATION 5 
2-(p-Chlorobenzyl)acrylic Acid 
The title product of Preparation 3 (35 g., 0.153 mole) was combined with 20 
ml. of water, stirred in an ice bath and 25% aqueous dimethylamine was 
added dropwise to a pH of 7.5. A second portion (35 g.) of the malonic 
acid was added and sufficient additional water to achieve solution. 
Aqueous formaldehyde (35 ml. of 36%) was added and the stirred solution 
allowed to warm gradually to room temperature and stirred for 17 hours. 
The intermediate dimethylaminomethylated product was recovered by 
filtration and partially dried by suction (108 g. wet). The wet 
intermediate was combined with 500 ml. of water and heated on a steam bath 
for 2 hours, during which the evolution of carbon dioxide was evident. The 
reaction mixture was cooled, the pH adjusted to 2.0 with 6 N hydrochloric 
acid and the precipitated title product recovered by filtration (35.3 g., 
m.p. 95.degree.-96.degree. C.). 
By the same method, the other benzylmalonic acids of Preparation 3 are 
converted to: 
2-(m-chlorobenzyl)acrylic acid; 
2-(o-chlorobenzyl)acrylic acid; 
2-(p-fluorobenzyl)acrylic acid; and 
2-(p-bromobenzyl)acrylic acid. 
PREATION 6 
2-(p-Methoxybenzyl)acrylic Acid 
The title compound of Preparation 4 (25 g., 0.111 mole) was combined with 
25 ml. of water and the pH adjusted to 7.3 with 25% aqueous dimethylamine. 
The solution was cooled in an ice-acetone bath, additional malonic acid 
(25 g.) was added and the mixture stirred until solution resulted. 
Formaldehyde (35 ml. of 36% in water) was added. The mixture was stirred 
briefly in the ice-acetone bath and then warmed to room temperature. The 
precipitated title product was recovered by filtration (21 g., 49% of 
theory). 
By the same method, the other benzylmalonic acids of Preparation 4 are 
converted to: 
2-(p-ethoxybenzyl)acrylic acid; 
2-(m-isopropylbenzyl)acrylic acid; 
2-(o-methylbenzyl)acrylic acid; and 
2-(p-trifluoromethylbenzyl)acrylic acid. 
PREATION 7 
3-Benzoylthio-2-(p-chlorobenzyl)propionic Acid 
A solution of 2-(p-chlorobenzyl)acrylic acid (3.93 gm., 22 mmoles) and 
thiobenzoic acid (2.6 ml., 20 mmoles) in 40 ml. of methylene chloride was 
heated at reflux for 16 hours. The reaction mixture was evaporated to 
dryness and product crystallized from hexane (4.5 g.). Additional product 
was obtained by evaporation of the hexane mother liquor and chromatography 
of the residue (7.1 g.) on 200 g. of silica gel (230-400 mesh) with 
chloroform as eluant; 1.8 g., m.p. 111.degree.-114.degree. C. 
By the same method the other benzylacrylic acids of Preparation 5 are 
converted to: 
3-benzoylthio-2-(m-chlorobenzyl)propionic acid; 
3-benzoylthio-2-(o-chlorobenzyl)propionic acid; 
3-benzoylthio-2-(p-fluorobenzyl)propionic acid; and 
3-benzoylthio-2-(p-trifluoromethylbenzyl)propionic acid. 
PREATION 8 
3-Benzoylthio-2-(p-methoxybenzyl)propionic Acid 
By the method of Preparation 7, 2-(p-methoxybenzyl)acrylic acid (3.84 gm., 
20 mmoles) was reacted with thiobenzoic acid to yield the title product in 
essentially quantitative yield [6.61 gm.; R.sub.f 0.48 (ether)]. 
By the same method, the other benzylacrylic acids of Preparation 6 are 
converted to: 
3-benzoylthio-2-(p-ethoxybenzyl)propionic acid; 
3-benzoylthio-2-(m-isopropylbenzyl)propionic acid; 
3-benzoylthio-2-(o-methylbenzyl)propionic acid; and 
3-benzoylthio-2-(p-trifluoromethyl)propionic acid. 
PREATION 9 
Optical Resolution of 3-benzoylthio-2-benzylpropionic Acid 
Racemic 3-benzoylthio-2-benzylpropionic acid (10 g.) was dissolved in 150 
ml. of ether and slowly added to 4.24 gm. (35 mmole) of 
d-(+)-alpha-methylbenzylamine to obtain 14 g. of salt [alpha.sub.589.sup.D 
(C=1, CHCl.sub.3)+3.46.degree.]. Two recrystallizations from methylene 
chloride-hexane furnished 3 gm. of salt containing mostly 2(S) isomer 
[alpha.sub.589.sup.D (C=1, CHCl.sub.3)-21.2.degree.]. Further 
crystallization from isopropyl alcohol gave 1.56 g. of salt of 2(S) isomer 
[approx. 90% 2(S) isomer], [alpha.sub.589.sup.D (C=1, 
CHCl.sub.3)-25.degree.], m.p. 131.degree.-133.degree.. The free acid 
[approx. 90% 2(S) isomer]was liberated by dissolving in water, adding 2 
NHCl, and extracting the product into ethyl acetate. Drying over 
MgSO.sub.4 and concentrating furnished the free 2(S) acid, colorless 
crystals, m.p. 64.degree.-67.degree., alpha.sub.589.sup.D (C=1, 
CHCl.sub.3)-36.39.degree.. 
Coupling with L-methioninol and deprotection afforded predominantly 
2S-(2S-benzyl-3-mercaptopropionylamino)-4-methylthio-1-butanol of Example 
6. 
The pure 2(S)-acid is obtained by further recrystallizations from methylene 
chloride-hexane or isopropyl alcohol. The corresponding 2(R)-acid can be 
isolated from mother liquors or by use of 1-(-)-alpha-methylbenzyl amine 
as the resolving agent. 
In like manner other 3-acylthio-2-(substituted-benzyl)propionic acids of 
preceding examples are resolved into their enantiomers.