CA1132985A - Ketal and thioketal derivatives of mercaptoacyl prolines - Google Patents

Abstract

ABSTRACT

This invention is directed to compounds of the formula

Description

H~180a I~TAL AN~ M~IOKETAL DERI~ATIV~S OF MERCAPTOACYL
PROLINES
_ This invention relates to new derivatives of mercaptoacyl prolines and pipecolic acids of formula I and salts thereof (I) Il l2 ~C~
/ \, R4 R3 l (H21)p (I 2)q R5-S-(CH)m-C -C N - C*COOR

R and R6 are independently selected from hydrogen and lower alkyl pr~vided that R6 is lower alkyl only if R3 is also lcwer alkyl.
R3 and R4 are independently selected from hydrogen, lower alkyl, lower alkylthio, -(CH2)n-SH, and halo substituted lower alkyl.
Xl, X2 and X3 are independently selected from oxygen and sulfur.
Rl and R2 are independently selected from lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, halo substituted lower alkyl, hydroxy substituted lower alkyl, r~~~
( 2)n ~ (CH2?n~
R7 x3 ~L
. ;.

..

, , :; :

. .
~ ~ -ll~Z98~
HA180a

-2-and -(CH2) ~ J or Rl and R2 join in a polymethyl-ene chain to complete an unsubstituted or substituted S- or 6-membered ring.
When Rl and R2 are joined together in a polymethylene chain of 2 or 3 carbons, these cyclic ketal and thioketals can be represented as follows:

Rlo Rg 10~ 9 10 ~ Rg ~(C)t~\ ~(C)t~ /(C)t \~ . ~.~ \~
wherein t is 2 or 3 and Rg and Rlo are both hydrogen, both lower alkyl, or one is hydrogen and the other is lower alkyl, halo substituted lower alkyl, hydroxy substituted lower alkyl, ( 2 n ~ R7 ~(CH2)n~ ~ , or (CH2) `X3 N
Preferably, only one carbon of the polymethylene chain will be substituted.
R7 is hydrogen, lower alkyl of 1 to 4 carbons, espe-cially methyl, lower alkoxy of 1 to 4 carbons, especially methoxy, lcwer alkylthio of 1 to 4 car~ons, especially methylthio, chloro, bramo, fluoro, trifluor~methyl, or hydroxy.
R5 is hydrogen, a hydrolyzably removable protect-ing group, a chemically removable protecting group, or when R3 and R~ are other than -(CH2)n-SH a sulfide of the formula .13Z9t35 _3_ HAl80a Il l2 ~ C /

,~ \
S (H2C)p ~CH2)q l~ l3 ~
-~-(CH)m-C- C N C-COOR

m is zero, one, or two.
n is one, two or three.
p and q are each one or two provided that both are not two.
The asterisk in the above formula indicates a center of asymmetry in the ring. In the case of proline, i.e., p and q are both one, this center is in the L-configuration. In the case of pipecolic acid, i.e., one of p and q is two, this center is in the D, L or L-configuration.
Asymmetric centers can also be present in the mercaptoacyl sidechain depending upon the definition of R3, R4 and R6. Another assymmetric center may also be present in the ring when ~-Rl and X2-R2 are different. The products can accordingly exist in stereoisomeric forms or as racemic mLxtures thereof. All of these æe within the scope of the invention. The synthesis described below can utilize the racemate or one of the enantiomers as starting materials. ~hen the racemic starting material is used in the synthesis procedure, the stereoisomers obtained in final product can be separated by conventional chromatographic or fractional crystallization methods.

: . .
:. ' :: -~s _4_ HA180a Preferably, if there is an asymmetric center in the mercaptoacyl sidechain, it is in the D-confi~uration.

s This invention in its broadest aspect relates to the mercaptoacyl derivatives of proline and pipe-colic acid having formula I above and to salts thereof, to compositions containing such compounds and to the method for using such compounds as anti-hypertensive agents. This invention is also directed to certain novel intermediates useful in the preparation of compounds of formula I.
The term lower alkyl as used in defining the y , R1, R2, R3, R4, and R6 are straight or branched chain hydrocarbon radicals having up to seven carbons, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, iso-pentyl, etc. The preferred lower alkyl groups are up to four carbons with methyl and ethyl being most preferred. Similarly, the terms lower alkoxy and lower alkylthio refer to such lower alkyl groups attached to an oxygen or sulfur.
The term cycloalkyl refers to saturated rings of 3 to 7 carbon atoms with cyclohexyl being most preferred.
The term halo substituted lower alkyl refers to such lower alkyl groups described above in which one or more hydrogens have been replaced by chloro, bromo or fluoro groups such as trifluoromethyl, pentafluoroethyl, 2,2,2-trichloroethyl, chloromethyl, :. ~
. . . . . . .
, .......... ..

.: :
.
, .
,. .

\
1132~85 HA180a bromomethyl, etc.
The term hydroxy substituted lower alkyl refers to such lower alkyl groups described above in which one hydrogen has been replaced by a hydroxy group such as hydroxymethyl, 2-hydroxyethyl, etc.
The term lower alkenyl as used in defining the symbols Rl and R2 are mono-saturated straight or branched chain hydrocarbon groups of from 2 to 7 carbons such as ethenyl, propenyl, isopropenyl, butenyl, and the like. The lower alkynyl groups are straight or branched chain hydrocarbon groups of from 2 to 7 carbons having one triple bond, e.g., propargyl. The preferred lower alkenyl groups are from 2 to 5 carbons and the preferred lower alkynyl groups are from 2 to 4 carbon atoms.
The term hydrolyzably removable protecting group employed in defining R5 refers to a group that can be removed by conventional hydrolysis or o ammonolysis. Acyl groups of the formula are suitable for this purpose wherein R8 can be lower alkyl of 1 to 7 carbons, lower alkyl substituted with one or more chloro, bromo or fluoro groups, -(CH2)r-cycloalkyl, an aryl group such as ( 2)r ~ ; a hetero group such as -(C~2) ~ , or -(CH2)r ~ wherein r is zero, one, two or three, and R7 and X3 are as defined above.

:~
.,. --':. ' ' ~ . i ,, ~:, ", - ,, .".
. - ~. :. .

1~3Z985 ~ 80a Preferred groups are the lower alkanoyl groups hav-ng up to four carbons, especially acetyl, and benzoyl.
The term chemically removable protecting group employed in defining R5 refers to groups such as p-methoxybenzyl, p-methoxybenzyloxycarbonyl, trityl, t-butoxycarbonyl, etc. These groups can be removed after the completion of the acylation reaction by various means depending upon the definition of Xl-Rl and X2-R2 such as by treatment with trifluoro-acetic acid and anisole, sodium and liquidammonia, or mercuric trifluoroacetate. Preferred compounds of formula I are the L-proline containing derivative, i.e., p and q are both one, and R is hydrogen.
With respect to the mercaptoacyl sidechain, preferred as final products are those compounds wherein R5 is hydrogen; m is zero or one; R4 is hydro-gen; and R3 and R6 are both lower alkyl of 1 to 4 carbons, especially both methyl, or R6 is hydrogen and R3 is hydrogen, lower alkyl of 1 to 4 carbons, especially methyl, trifluoromethyl, methylthio, or mercaptomethyl. .~lso preferred as both intermediates and final products are the above sidechains wherein R5 is lower alkanoyl or 1 to 4 carbons, especially acetyl, or benzoyl.
Especially preferred as final products are the compounds of formula I having the mercaptoacyl sidechain wherein R5 is hydrogen; m is one; R4 and R6 are hydrogen; R3 is methyl; and the asymmetric carbon atom to which R3 is attached is in the D-configuration.

11 3Z985 HAl 8 0 a Preferred compounds with respect to the substi-tuents on the proline ring are those wherein Rl and R2 are independently selected from lower alkyl of 1 to 4 carbons, especially methyl or ethyl;
S
~ R7 ~5 ~ , -CH2 ~ 1l , or ~\
-CH2~ 0 ¦ ; and R7 is hydrogen, methyl, methoxy, ~N
methylthio, C1, Br, F, trifluoromethyl, or hydroxy; or Xl-Rl and X2-R2 join to form R~ Rg Rio I I Rlo O O

C C Rg /R9 ~ ~
R ~ I I ~ Rlo Rlo I ¦ Rlo lCH2 S~S o S ~

>~ ~<
25H2C CH2 ' or H21C ICH2 wherein Rg and 0 ~ 0 S, S
Rlo are both hydrogen or both lower alkyl of 1 to 4 carbons, especially both hydrogen or both methyl, or Rg is hydrogen and Rlo is lower alkyl of 1 to 4 carbons, .

. .
- . .

-8- HA180a especially methyi, hydroxy substituted lower alkyl of l to 4 carbons, especially hydroxymethyl, or halo substituted lower alkyl, especially trifluoromethyl.
Most preferred compounds with respect to the substituen~s on the proline ring are those wherein Xl and X2 are the same especially those wherein Xl-Rl and X2-R2 are both methoxy or both ethoxy or Xl-~l and X2-R2 join together to form H C CH H C CH , H2C - CH2 ,.

~_, S~ ~ S

/CH3 CH ~ /CH~

20 H2C - fH , H2C CH2 ~ H2l IH2 S~ S S S o, ~ o The compounds of formula I are obtained by coupling the substituted proline or pipecolic acid of the formula . , . ~ .

:

J ~3Z98S EiP.l ~ Oa _~_ (II) ,1 ,2 / C\2 (H2Cj p t ICH2) ~

HN C-COOR
H

with an acid or its chemical equivalent of the formula ~III) R4 ~3 R5-S-(CH)m C ~ COOH

wherein R5 is hydrogen, a hydrolyzably or chemically removable protecting Sroup to yield the product of the formula (IV) Rl lR2 ~ C ~ 2 R4 13 (H2C)p (CH2 q R5-S-(CH)m C - CO - N - C-COOR

.

1~32985 HA180a This reaction can be effected in the presence of a coupling agent such as dicyclohexylcarbodiimide or the like, or the acid can be activated by formation of its mixed anhydride, symmetrical anhydride, acid halide, active ester or use of Woodward reagent K, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline or the like. For a review of the methods of acylation, see Methoden der Organishchen Chemie (Houben-~eyl), ~ol. XV, part II, page 1 et seq. (1974). Preferably, the acid halide, especially the acid chloride, of formula III is reacted with the acid of formula II.
If the proline or pipecolic acid of formula II is reacted in the ester form the resulting ester product o formula IV, i.e., R is alkyl, can be converted to the free acid, i.e., R is hydrogen, by conventional means. For example, if R is ethyl this ester protecting group can be removed by saponification.
The product of formula IV is preferably isolated and purified by crystallization, e.g., by forming the dicyclohexylamine salt and then converting the salt to the free acid form by treatment with an aqueous solution of an acid, such as potassium acid sulfate.
The product of formula IV bearing the acyl group R8-CO- can be converted to the products of formula I wherein R5 is hydrogen by conventional hydrolysis or by ammonolysis.

:

.

~13Z98S HA18Q~

The products of formula I wherein R3 and R4 are other than -(CH2)n-SH and R5 is Il l2 X~ ~X2 t \
l4 l3 (H2l)p (1~2)q -S-(CH) - C - Co - N C*COOH
I H

are obtained by directly oxidizing with iodine a product of formula I wherein R5 is hydrogen.
The esters of formula I wherein R is lower alkyl can be obtained from the carboxylic acid compounds, i.e., wherein R is hydrogen, by conventional esterification procedures, e.g., by esterification with a diazoalkane such as diazomethane, a 1-alkyl-3-p-tolyltriazene, such as l-n-butyl-3-p-tolyltriazene, or the like.
The disubstituted prolines or pipecolic acids of formula II wherein Xl-Rl and X2-R2 are the same, can be obtained by reacting an N-protected keto compound of the formula (V) o C

(H2C) p (CH2 ) q Cbz - N C*COOR

,:
.
. :,~ . , - ;
,; , . ~, . , : ;

:- . . .. ,, . :. -"- -. . -~ . ,. . ... , - ~ :i ~13Z!W5 H~laOa wherein Cbz represents carbobenzyloxy with an alcohol or thiol having the formula (VI) Rl-Xl-H

in the presence of an orthoformate or thioformate of the formula HC(X -R ) and an acid such as concentrated sulfuric acid or p-toluenesulfonic acid. This reaction can be effected in an inert organic solvent such as benzene, acetic acid, ether, cyclohexane or the like, preferably with heating, e.g., at about reflux temperature.
See Buehler et al., Survey of Organic Syntheses tWiley & Sons, 1977) Vol. 1, pages 516-519. The lS product of this reaction is the N-protected intermediate of the formula (VII) R R

/~ C~\

(H2C)p (C 2)q Cbz - N C*COOR
H

The N-protected intermediate of formula VII
can be treated with a molar equivalent of an alcohol or thiol of the formula ~132985 ~A180a ~VIII) according to the conditions described above to yield 5 the intermediate (IX) 1 R2 ~ C / 2 . / \
tH2C) p , (CH2 ) q Cbz N - C-COOR
H s lS ~y employing a molar excess of the alcohol or thiol of formula VIII one obtains the intermediate (X) R2 l2 \2 ~ 2 /c ~\
(H2C)p (CH2)q Cbz N ~*COOR
H
The N-protecting group can then be remcved by conventional procedures, for example, when Xl and X2 are both oxygen by hydrogenolysis in the presence of palladium carbon catalyst or when either or both 30 Xl and X2 are sulfur by treatment with HBr and acetic acid to yield the disubstituted compounds of formula II.

1.', . , ",-~ ~ -.. . .

~3Z985 H.~180a Similarly, the spiro compounds of formula II
(i.e~, Rl and R2 are joined together in a polymethylene chain) can be obtained by reacting the keto compound of formula V with the alcohol or thiol of the formula (XI) /
~C,\

Xl X2 H H
wherein Rg, Rlor and t are are defined above in the presence of an acid such as p-toluenesulfonic acid, to yield the intermediate (XII) ~9 ,/ 10 ~(C)t~

Xl X2 ~ C
(H2C)p (CH2 Cbz-N C-COOR
H

Alternatively, the disubstituted compound .

. ~ .

li3Z~i HA180a of formula ~II can be treated directly with a molar excess of the alcohol or thiol of formula XI
to yield the intermediate of formula XII. This procedure is particularly useful when Rg and Rlo are either or both other than hydrogen.
As described above, the N-protecting group can then be removed to yield the spiro compounds of formula II.
As an alternative procedure, the introduction of the Xl-Rl and X2-R2 groups can be effected later in the sequence. According to this modification, the protected keto compound of formuia V is treated to remove the protecting group, e.g., with hydrogen bromide, resulting in an intermediate having the formula (XIII) o Il / C
(H2C) p (CH2 ) ~E

H - N - C~COOH
H

which is then acylated with the acid, preferably the acid halide, of formula III to yield the compound of the formula ' . ~ ' ` :` ~ ' .
.
. :

: .

~13Z985 HA180a (XIV) / C
l4 l3 o (H2C)p (CH2) R' - S -(CH) - C- C - N ~ C~-COOH

The groups Xl-Rl and X2-R2 or the spiro group ~ / 10 can then be introduced at this ~ (C)t\
Xl X2 \~, ./
point by the procedures described above to yield the product of formula IV.
Reference is also made to the following publications for additional illustrative methodology for producing starting materials and intermediates:
U.S. Patents 4,046,889, 4,105,776, 4,154,935 and 4,116,962; Can. J. Biochem. & Physiol. 37, 584 (1959); J.A.C.S. 79, 189 (1957); J. Med. Chem. 21, 445 (1978); Aus. J. Chem. 20, 1493-1509 (1967~;
Buehler et al., Survey of Organic Syntheses (Wiley & Sons, 1977), Vol. 1, pages 516-519, Vol. 2 pages 461-470; Chem. Pharm. Bull., Tokyo 26, 2209 and 2217 (1978); Can. J. Chem. 47, 860 (1969);

, .

1~32985 HA180a J~ Amer. Chem. Soc., 80, 6350 (1958); Harrison et al., Compendium o~ Organic Synthetic Methods, (Wiley-Interscience, New Yor~, 1971), pages 449-456;
J. Amer. Chem. Soc., 79, 192 (1956); ~ull. Soc.
Chem., 1965(8) pages 22~53-2259; J. Org. Chem. 25, p. 521-530 (1960).
The procedures illustrated therein can be utilized as general methods for the synthesis of compounds and separation of isomers which can be utilized in the invention described in this application. Additional illustrative details are found in the examples which serve as models for the preparation of other members of the group.
The compounds of this invention form basic salts with a variety of inorganic or organic bases. The salt forming ion derived from such bases can be metal ions, e.g. ! aluminum, alkali metal ions, such as sodium or potassium, alkaline earth metal ions such as calcium or magnesium, or an amine salt ion, of which a number are known for this purpose, for example aralkylamines like, dibenzylamine, N,N-dibenzyl-ethylenediamine, lower alkylamines like methylamine, t-butylamine, procaine, lower alkylpiperidines like N-ethylpiperidine, cycloalkylamines, like cyclo-hexylamine or dicyclohexylamine, l-adamantanamine, benzathine, or salts derived from amino acids like arginine, lysine or the like. The physio-logically acceptable salts like the sodium or potassium salts can be used medicinally as described .

1~32985 HA180a - below and are preferred. These and other salts which are not necessarily physiologically accepta~le are useful in isolating or purifying a product accepta-ble for the purposes described below, as illustrated S with the dicyclohexylamine salt in the examples.
The salts are produced by reacting the acid form of the compound with a equivalent of the base supplying the desired basic ion in a medium in which the sait precipitates or in aqueous medium and then lyophilizing.
The free acid form can be obtained from the salt by conventional neutralization techniques, e.g., with potassium bisulfate, hydrochloric acid, etc.
The compound of formula I wherein R5 is g ~ R8-C- ~ or the disulfide type substituent, especially wherein R5 is hydrogen, are useful as hypotensive agents. They inhibit the conversion of the decapeptide angiotensin I to angiotensin II and, therefore, are useful in relieving angiotensin related hypertension. The actiQn of the enzyme renin on angiotensinogen, a pseudo-globulin 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 angiotensinogen ~ (renin) ~ angiotensin I ~ (ACE) angiotensin II sequence by inhibiting angiotensin ' ' ' , , .

, ' . ~ ~

~132985 ~A180a converting enzyme and reducing or eliminating t.ie formation of the pressor substance angiotensin II.
Thus by the administration of a composition containing one, or a combination of compounds of formula I
S angiotensin dependent hypertension in the species of mammal suffering therefrom is alleviated.
single dose, or preferably two to four divided daily doses, provided on a basis of about 0.1 to 100 mg.
per kilogram of body weight per day, preferabiy about 1 to 15 mg. per kilogram of body weight per day is appropriate to reduce blood pressure. The substance is preferably administered orally, but parenteral routes such as the subcutaneous, intramuscular, intravenous or intraperitoneal routes can also be employed.
The compounds of this invention can also be formulated in combination with a diuretic for the treatment of hypertension. A combination product comprising a compound of this invention and a diuretic can be administered in an effective amount which comprises (for a 70 kg. mammal) a total daily dosage of about 30 to 600 mg., preferably about 30 to 300 mg., of a compound of this invention, and about 15 to 300 mg., preferably about 15 to 200 mg.
of the diuretic, to a mammalian species in need thereof. Exemplary of the diuretics contemplated for use in combination with a compound of this invention are the thiazide diuretics, e.g., chlor-thiazide, hydrochlorthiazide, flumethiazide, hydro-glumethiazide, benzdroflumethiazide, methchlothiazide, : ~ .
.:, -~ .
- :-.

~.Al~Oa --~u--trichlormethiazide, polythiazide or benzthiazide, as well as ethacrynic acid, ticrynafen, chlorthal-idone, furosemide, musolimine, bumetanide, triam-terene, amiloride and spironolactone, and salts of such compounds.
The compounds of formula I can be formulated for use in the reduction of blood pressure in compo-sitions such as tablets, capsules or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration. About 10 to 500 mg.
of a compound or mixture of compounds of formula I
is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form as called for by accepted pharmaceutical practice.
The amount of active substance in these compositions or preparations is such that a suitable dosage in the range indicated is obtained.
Illustrative process details are set forth in the following examples for the various reactions.
These examples are preferred embodiments and also serve as models for the prepar~tion of other compounds of this invention. The temperatures are given in degrees on the centrigrade scaie.

- . .

. . .

`- 1132985 HA180a Example 1 ~7(S),8S]-7-(Acetylthio)-2-methyl-1-oxopropyl]-1, 4-dioxo-7-azaspiro~4.4]nonane-8-carboxylic acid a) N-Carbobenzyloxy-4-hydroxy~L-proline 26.5 g. (0.20 mole) of 4-hydroxy-L-proline and 32.8 ml. (0.23 mole) of benzyl chloroformate are reacted in 200 ml. of water and 100 ml. of acetone in the presence of 20 g. (0.02 mole) of potassium bicarbonate and 69.2 g. (0.50 mole) of potassium carbonate and worked up with 90 ml. of concentrated hydrochloric acid as described in Can.
J. Biochem. & Physiol. 37, 584 (1959) to obtain N-carbobenzyloxy-4-hydroxy-L-proline. This product is reacted with cyclohexylamine to form the cyclohexylamine salt yield 69 g. , m.p. 193-195. The salt (34 g.) is neutralized with N-hydrochloric acid to obtain 27 g- of free acid as a colorless glass ~a]D ~70~ (c, 1~ in chloroform).
b) N-carbobenzyloxy-4-keto-L-proline 21.5 g. (0.81 mole) of N-carbobenzyloxy-4-hydroxy-L-proline is oxidized in 1.2 liters of acetone with 83 ml. of 8N chromic acid in sulfuric acid as described in J.A.C.S. 79, 189 (1957). In order to facilitate the subsequent filtration of chromium salts, 30 g. of Celite (diatomaceous earth) is added to the acetone solution before introduction of the oxidizing agent. An air stirrer is employed. The reaction mixture is filtered and the acetone filtrate is concentrated to approximately 300 ml. before diluting with 1 liter of chloroform. The solution is ; washed with 300 ml. of saturated sodium chloride (four times~, dried (Mg5O4), filtered and the solvent evaporated to give :
, , ' ':

~132985 H~180a N-carbobenzyloxy-4-keto-L-proline(22.8 g.) which is crystallized from ether (50 ml.)-hexane (150 ml.) to obtain 17.2 g. (81%) of product, m.p. 99-101, ~a]26 +17 (c, 1~ in chloroform).
c) N-Carbobenzylox~I-4,4-ethylenedioxy-L-proline A stirred mixture of 12.8 g. (0.049 mole) of N-carbobenzyloxy-4-keto-L-proline, 53 ml. (0.095 mole) of ethylene glycol, and 0.35 g. of p-toluene-sulfonic acid H2O in 1.31 1. of benzene is heated and the resulting solution is refluxed for 7 hours (water formed is collected in a Dean-Stark apparatus).
After standing overnight at room temperature, the lower glycol layer is separated and the benzene solution is washed with 150 ml. of saturated sodium chloride, dried (MgSO4), and the solvent evaporated to give 14.6 g. of N-carbobenzyloxy-4,4-ethylenedioxy-L-proline as a syrupy residue. The latter is dissolved in 60 ml. of ethanol, filtered, treated with 5 g.
of cyclohexylamine, and diluted with ether. On seeding and rubbing, the crystalline cyclohexylamine salt separates; weight after cooling overnight, 9.0 g., m.p. 179-180 (s. 173). The material is recrystallized from acetonitrile, m.p. 182-184 (s. 179 )~ [a]D -21 (c, 1% in EtOH).
The cyclohexylamine salt (8.4 g.) is suspended in 40 ml. of ethyl acetate, stirred, cooled, and treated with 40 ml. of lN hydrochloric acid. The layers are separated, the aqueous phase extracted with additional ethyl acetate ( 3 x 40 ml.), the combined organic layers are dried (MgSO4), and the solvent .
:, : .
~ , 113Z985 HA180a evaporated, finally at 0.2 mm. The syrupy residue which begins to Crfstallize is rubbed under ether and the ether evaporated to give 6.4 g. ~42~) of nearly colorless N-carbobenzyloxy-4,4-ethylene-S dioxy-L-proline, m.p. 101-103 (s. 98), la~26 _34 (c, 1~ in CHC13).
d) 4,4-Ethvlenedioxv-L-~roline A solution o~ 3.2 g. (o.0104 mole) of N-carbobenzyloxy-4,4-ethylenedioxy-L-proline in 100 ml.
of methanol-water (2:1) is treated with 1 g. of 5%
palladium-carbon and shaken on the Parr hydrogenator for 6 hours. The catalyst is filtered off under nitrogen, washed with methanol, and the combined filtrates evaporated, finally at 0.1 - 0.2 mm, to give 1.7 g. (94%) of colorless solid, 4,4-ethylene-dioxy-L-proline; m.p. 245-247 (dec.); [a]26 -32 (c,0.5~ in 1:1 MeOH-H2O).
e) [? (s), 8S]-7-[3-(Acetylthio)-2-methyl-1-o:~opropyl]-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid A stirred solution of 3.2 g. of 4,4-ethylene-dioxy-L-proline (0.0185 mole) in 50 ml. of water is cooled to 5 and treated portionwise with solid sodium carbonate to pH 8.5. Then while continuing stirring and cooling, a solution of 3.7 g. (0.020 mol.) of D-3-acetylthio-2-methylpropanoyl chloride in 5 ml.
of ether is added portionwise while maintaining the pH at 8.5 with 25% sodium carbonate solution (about 14 ml.). After 1 1~4 :~ours, the solution istreated with 50 ml. of ethyl acetate,stirred, cooled,acidified carefully with hydrochloric acid (1:1) to pH 2.0, :, .:, ' , . :
.

,, :

H~180a saturated with sodium chloride and the layers are separated. The aqueous phase is extracted with additional ethyl acet~te (3 x 50 ml.), the combined organic layers are dried (MgSO4) and the solvent evaporated finally at 0.2 mm. The solid residue is rubbed under ether and the evaporation repeated to obtain 5.9 g. (100%) of [7(S), 8S]-7-[3-(acetylthio)-2-methyl-1-oxopropyl]-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid; m.p. 108-111.
The product is converted to the dicyclohexyl-amine salt with 3.4 g. of dicyclohexylamine in 70 ml.
of ethyl acetate. On seeding and rubbing, the crystalline salt precipitates and is recrystallized from 95 ml. of acetonitrile; yield 6.7 g., m.p.
187-189 (s. 184), [al25 -59 (c, 1~ in EtOH).
The dicyclohexylamine salt is converted to the free acid by suspending it in ethyl acetate and treating wi~h 75 ml.of 10% potassium bisulfate and stirring until two layers are obtained. After separating, the aqueous phase is extracted with ethyl acetate ( 4 x 75 ml.), the organic layers are combined, dried (MgSO4) and the solvent is evaporated to give 4.1 g. of colorless [7(S),8S]-7-[3-(acetylthio)-2-methyl-1-oxopropyl]-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid, m.p. 120-122 (s. 117) [a]25 -118 (c, 1% in EtOH).
Example 2 [7(S)-8S]-7-(3-Mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid Argon is passed through a cold solution of . .

- -- . :

113Z985 HA180a 8.5 ml. of concentrated ammonium hydroxide in 20 ml.
of water. 4.0 g. (0.013 mole) of [7(S),85]-7-[3-tacetylthio)-2-methyl-1-oxopropyl]-1,4-diOxO-7-azaspiro-~4.4]nonane-8-carboxylic acid from Example le are then added and the mixture is stirred in an ice bath for a few minutes and then a room temperature under argon for two hours. The solution is treated with 30 ml. of ethyl acetate,cooled, stirred, and acidified with 16 ml. of of hydro-chloric acid (1:1). The layers are separated, the aqueous phase is extracted with additional 30 ml.
of ethyl acetate (twice),the ethyl acetate extracts are combined, dried (MgSO4) and the solvent evaporated to give 17(s),8s]-7-(3-mercapto-2-methyl-l-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid as a solid residue. The product is rubbed under ether and the evaporation is repeated.
The product is then triturated with 30 ml. of hexane, cooled for one hour, filtered under argon and dried in vacuo to give 2.7 g. of colorless solid ~7(S),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid, m.p. 131-133 (s. 125), ta]25 -66 (c, 1~ in Et~
Example 3 (S)-1-[(3-Acetylthio)-2-methv]-1-oxopropyll-4,4-dimethoxy-L-proline a) N-Carbobenzyloxy-4,4-dimethoxy-L-proline, methyl ester A stirred solution of 7.8 g. (0.03 mole) of N-carbobenzyloxy-4-keto-L-proline from Example 1 in , '" ' . :
, : .

~ .
.~ . . . . . . . .
, .; :

HA180a 60 ml. of methanol is treated with 96 ml. of tri-methyl orthofo~mate, followed by 0.6 ml. of concentrated sulfuric acid and allowed to stand overnight at room temperature.
The pale yellow solution is stirred, treated with 1.5 g. of potassium carbonate, followed by 30 ml. of water and the bul~ of the solvent is removed on a rotary evaporator to give a syrupy residue which is shaken with 30 ml. of water and 30 ml. of chloroform. After separating the layers the a~ueous phase is extracted with additional chloroform (3 x 30 ml.) and the combined organic layers are washed with 45 ml. of saturated sodium chloride solution and dried (MgSO4). Evaporation of the solvent yields 8.4 g. (88~) of N-carbobenzyl-oxy-4,4-dimethoxy-L-proline, methyl ester.
b) N-Carbobenzyloxy-4,4-dimethoxy-L-proline The ester (8.4 g., 0.026 mole) from part a is dissolved in 80 ml. of methanol, treated dropwise at -1 to 4 with 18 ml. (0.036 mole) of 2N sodium hydroxide kept at 0 for one hour, and at room temperature overnight. After removing about one half of the solvent on a rotary evaporator, the solution is diluted with 150 ml. of water, washed with 100 ml.
of ether (wash discarded), acidified while cooling with 63 ml. of 1:1 hydrochloric acid to pH 2, and extracted with ethyl acetate (4 x 750 ml.). The combined extracts are washed with 50 ml. of saturated sodium chloride solution, dried (MgS04), and the solvent evpaorated to give 8.0 g. of a pale yellow ~ "
.

., . , : . . :~

,.
.
: : "; . ~
:~ : ~ : . ~ . .

1~3Z985 HA18Oa viscous oil. The oil is dissolved in 35 ml. ethancl, treated with 3.0 g. of cyclohexylamine in 10 ml. of ethanol and diluted to 500 ml. with ether. On seeding and rubbing, the crystalline N-carbobenzyloxy-4,4-dimethoxy-L-proline cyclohexylamine salt separated;
weight after cooling overnight, 7.0 g., m.p. 157-159 (s, 151) [~]D ~34 (c, 1~ in EtOH). This material is recrystallized from 100 ml. of acetonitrile to give the salt as a colorless solid, m.p. 158-160 (s, 154) [a]26 _33 (c, 1% in EtOH).
The N-carbobenzyloxy-4,4-dimethoxy-L-proline cyclohexylamine salt is suspended in 40 ml. of ethyl acetate, stirred and treated with 25 ml. cf lN
hydrochloric acid. When two clear layers are obtained lS they are separated, the aqueous phase is extracted with additional ethyl acetate (3 x 40 ml.), the combined organic layers are dried tMgSO4), and the solvent evaporated, finally at 0.2 mm and 40 to yield 7.2 g. (70%) of N-carbobenzyloxy-4,4-dimethoxy-L-proline as a pale yellow viscous syrup.
c) 4,4-Dimethoxy-L-proline .

A solution of N-carbobenzyloxy-4,4-dimethoxy-L-proline (72 g., 0.022 mole) in 210 ml. of methanol-water (2:1) is treated with 2.3 g. of 5% palladium-carbon and shaken on a Parr hydrogenator for 6 hours.The catalyst is filtered off under nitrogen, washed with methanol, and the combined filtrates are evaporated, finally at 0.1 - 0.2 mm., to give a partly crystalline residue. This residue is taken up in 200 ml. of methanol and the evaporation repeated.

,~.

. " .` -:
:. . .

113Z98S HA18Oa When the solid is rubbed under ether (evaporation again repeated) there is obtained 3.6 g. (95%) of nearly colorless 4,4-dimethoxy-L-proline, m.p. 192-19~
(dec.) ; t~]D -47 (c, 1~ in MeOH).
A sample crystallized from methanol-ether is colorless and melts at i97-198 (dec.);[]D -49 (c, 1~ in MeOH).
d) (S)-1-[3-(Acetylthio)-2-methyl-1-oxoprop~1j-4,4-dimethoxy-L-proline A stirred solution of 3.3 g. (0.019 mole) of 4,4-dimethoxy-L-proline in 50 ml. of water is cooled to 5 and brought to pH ~.5 by the addition of 25~
sodium carbonate solution (w/v). Then while continuing stirring and cooling, a solution of 3.8 g. (0.021 mole) of D-3-acetylthio-2-methylpropanoyl chloride in 5 ml.
of ether is added portionwise while maintaining the pH at 7.5 - 8.5 by dropwise addition of 25~ sodium carbonate solution. When the pH has stabilized at 8.2 - 8.4 (after about 15 minutes), stirring and cooling is continued for a total of one hour. The solution is then washed with 50 ml. of ethyl acetate twash discarded, layered over with 50 ml. of ethyl acetate, cooled, stirred, acidified carefully with 1:1 hydrochloric acid to pH 2.0, saturated with sodium chloride, and the layers separated. The aqueous phase is extracted with additional ethyl acetate ( 3 x 50 ml.), the combined organic layers are dried (MgSO4), and the solvent evaporated, finally at 0.2 mm, to give 6.7 g. of syrupy product. This syrup is treated in 70 ml. of ethyl acetate with 3.9 g. of dicyclohexylamine to give 6.5 g.

.
, , .-~329~5 ~180a of colorless (S)-1-[3-(acetylthio)-2-methyl-1-oxo-propyl]-4,4-dimethoxy-L-proline dicyclohexylamine salt in crops (3.1 g. and 3.4 g.), m.p. 158-160 (s, 145 ). [a]D -71 (c, 1% in EtOH).
Following recrystallization from 20 ml. of hot ethyl acetate-60 ml. of hexane, the colorless solid salt weighs 6.0 g., m.p. 158-166 (s, 155), []D -69 (c, 1% in EtOH~.
The dicyclohexylamine salt is c~nverted to the free acid by suspending 5.0 g. in 50 ml. of ethyl acetate, cooling and treating with 60 ml. of 10%
potassium bisulfate solution to give 2 clear layers.
After separating, the aqueous phase is extracted with ethyl acetate (3 x 50 ml.), the combined organic layers are dried (MgSO4), and the solvent evaporated, finally at 0.1 - 0.2 mm. and 45 to give 4.1 g.
(69%) of (S)-1-[3-(acetylthio)-2-methyl-1-oxopropyl]-4,4-dimethoxy-L-proline as a viscous, almost glass-like material la]D -112 (c, 1% in EtOH).
Example 4 (S)-1-(3-Mercapto-2-methyl-1-oxopropyl)-4,4-dimethoxy-L-proline Argon is passed through a cold solution of 8.5 ml. of concentrated ammonium hydroxide in 20 ml. of water for 0.25 hour. The latter is then added while cooling and under a blanket of argon to 4.1 g.
(0.013 mole) of (S)-1-[3-(acetylthio)-2-methyl-1-oxopropyl]-4,4-dimethoxy-L-proline and the mixture is swirled in an icebath until a pale yellow solution is obtained (about 15 minutes). Stirring under J~ -, ,, .
,,: ,: .

113Z985 HA18Oa argon is continued at room temperature for an additional 2 hours, then the solution is extracted with 30 ml. of ethyl acetate (this and subsequent operations are carried out as much as possible under an argon atmosphere~. The aqueous layer is cooled, stirred, layered over with 30 ml. of ethyl acetate, and acidified portionwi~e with approximat~ly 16 ml.
of 1:1 hydrochloric acid. The layers are separated, the aqueous phase is extracted with additional ethyl acetate (3 x 30 ml.), the combined ethyl acetate layers are dried (~gSO4), and the solvent evaporated to gi~e 3.5 g. (100%) of (S)-1-(3-mercapto-2-methyl-l-oxopropyl)-4,4-dimethoxy-L-proline as a colorless, viscous syrup, []D -72 (c, 1% in EtOH).
The latter (3.4 g.) is triturated with 20 ml.
of ethyl acetate, rubbed, diluted with 30 ml. of hexane, and cooled to give a colorless solid, weight 2.6 g., m.p. 108-110 , ~a]D ~77 (c,l~ in EtOH).
Example 5 (S)-1-[3-(~cetylthio)-2-methyl-1-oxopro~yll-4,4-diethoxy-L-proline a) N-Carbobenzyloxy-4,4-diethoxy-L-proline, ethYl ester Following the procedure of Example 3 (a) but substituting triethyl orthoformate for the tri-methyl orthoformate and ethanol for the methanol one obtains 10.8 g. of N-carbobenzyloxy-4,4-diethoxy-L-proline, ethyl ester as a yellow oil.

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113Z985 HA180a b) N Carbobenz lox -4 4-diethox -L- roline ~ Y Y , ~ P
The crude ester from part (a) (10.8 g., 0.03 mole) is saponified with 70 ml. of lN sodium hydroxide according to the procedure of Example 4 (b), 30 ml.
of ethanol is added in 10 ml. portions to obtain a solution, to give 10.5 g. of a yellow viscous oil.
This oil is dissolved in 100 ml. of ether and treated with cyclohexylamine (3.0 g.). On seeding and rubbing, 8.3 g. of the crystalline N-carbobenzyloxy-4,4-diethoxy-L-proline cyclohexylamine salt separates; m.p.
123-125 (s. 114 ) ~]D -32 (c, 1% in ethanol).
This material is recrystallized from 20 ml. of acetonitrile to give 7.0 g. of the salt as a colorless solid: m.p. 125-128 (s. 115) [al26 -31 (c, 1% in ethanol).
The N-carbobenzyloxy-4,4-diethoxy-L-proline cyclohexylamine salt is suspended in 40 ml. of ethyl acetate, stirred and treated with 20 ml. of 1 N
hydrochloric acid. The layers are separated and the aqueous phase is extracted with additional ethyl acetate (3 x 40 ml.), the organic layers are combined, dried (MgSO4), and the solvent evaporated to give 5.6 g. (56~) of N-carbobenzyloxy-4,4-diethoxy-L-proline as a light yellow oil.
c) 4,4-Diethoxy-L-proline A solution of the N-carbobenzyloxy-4,4-diethoxy-L-proline (5.6 g., 0.017 mole) in 180 ml. of 2:1 ethanol-water is treated with 2 g. of a 5~ palladium carbon catalyst and shaken under 3 atmospheres of hydrogen for six hours. The crude partly solid : .
" ' . . .
- :
.

1~3Z985 HA18Oa product is rubbed first under ethanol, then ether, and the evaporation repeated each time to give 3 g.
(91%) of nearly colorless solid 4,4-diethoxy-~-proline; m.p. 172-174 (dec.); preceded by gradual darkening and sintering ~a]D6 -40 (c, 1~ in methanol).
Anal. Calc'd. for CgH17N04 : 0.25 H20:
C, 52.03; H, 8.49; N, 6.74 Found: C, 52.22; H, 8.59; N, 6.69.
d) (S)-1-[3-(acetylthio)-2-methyl-1-oxopropyl]-4,4-diethoxy-L-proline The 4,4-diethoxy-L-proline (2.9 g., 0.014 mole) from part (c) and 3 g. ~0.017 mole) of D-3-acetylthio-2-methylpropionyl chloride dissolved in 3.5 ml. of ether are reacted in 35 ml. of water in the presence of sodium carbonate according to the procedure of Example 3 (d) to yield 5.4 5. of pale yellow viscous oil. This oily product is treated in 40 ml. of ethyl acetate with 2.6 g. of dicyclo-hexylamine and diluted with 60 ml. of hexane toyield in two crops 4.9 g. of (S)-1-~(3-acetylthio)-2-methyl-l-oxopropyl]-4,4-diethoxy-L-proline dicyclo-hexylamine salt; m.p. 135-138 (s. 132). Following recrystallization from 15 ml. of hot ethyl acetate-45 ml. of hexane, the colorless solid salt weighs4.2 g.: m.p. 138-140 (s. 135), []26 -63 (c, 1~ in ethanol).
Anal. Calc'd. for C15H25N06S C12 23 C, 61.33; H, 9.15; N, 5.30; S, 6.06 30 Pound: C, 61.48; H, 9.55; N, 5.25; S, 5.91.

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-~13Z985 HA18Oa The dicyclohexylamine salt is converted to the free acid by suspendin~ 4.2 g. in 40 ml. of ethyl acetate, cooling and treating with 40 ml. of 10 potassium bisulfate solution to give two layers.
After separa~ing, the aqueous phase is extracted with ethyl acetate (3 x 50 ml.), the combined organic layers are dried (MgS04), and the solvent evaporated to give 3.0 g. (61%) of (S)-1-[3-(acetylthio)-2-methyl-l-oxopropyll-4~4-diethoxy-L-proline as a pale yellow viscous syrup.
Example 6 (S)-4,4-Diethoxy-1-(3-Mercapto-2-methyl-1-oxopropyl)-L-proline Argon is passed through a cold solution of 5.5 ml. of concentrated ammonium hydroxide in 13 ml.
of water for 0.25 hour. The latter is then added while cooling under a blank~t of argon to 3.0 g.
~0.0086 mole) of (S)-1-[3-~acetylthio)-2-methyl-l-oxopropyl]-4,4-diethoxy-L-proline and the mixture is worked up as described in Example 4 to yield 2.4 g. (92~) of (S)-4,4- diethoxy-1-(3-mercapto-2-methyl-1-oxopropyl)-L-proline as a nearly colorless viscous syrup ~al26 -64, (c, 1% in ethanol).
Anal. Calc d. for C13 23 5 2 C, 50.38; H, 7.64; N, 4.52; S, 10.35 Found: C, 50.68; H, 7.96; N, 4.78; S, 10.07.
Example 7 [2(S),3S]-2-[3-(Acetylthio)-2-methyl-1-oxopropyl]-6,10-dioxo-2-azaspiro[4.5]decane-3-carboxylic acid ~
a) N-Carbobenzy oxy-4,4-trimethylenedioxy-L-proline ~; ~

, , , - : :

: .

~A18Oa _3a_ Interaction of 8.2 g. (0.031 mole) of N-carbobenzyloxy-4-keto-L-proline and 45 ml. (0.62 mole) of 1,3-propanediol in 450 ml. of benzene in the presence of 500 mg. of p-toluenesulfonic acid gi~es 12.3 g. of crude viscous ester nroduct.
This product is saponified with 70 ml. of lN
sodium hydroxide to give 10.6 g. of crude N-carbo-benzyloxy-4,4-trimethylenedioxy-L-proline as a yellow oil. The latter is dissolved in 40 ml.
of ethanol - 400 ml. ether and treated with

3.2 g. of cyclohexylamine to yield 10.1 g. ofW-carbo-benzyloxy-4,4-trimethylenedioxy-L-proline, cyclohexyl-amine salt; m.p. 163-165 (s. 160), [a]26 -27 (c, 1% in ethanol). Crystallization of 9.8 g. of the salt from 300 ml. of acetonitrile yields 9.5 g. of colorless solid cyclohexylamine salt;
m.p. 165-167 (s, 162) [al25 -27 (c, 1~ in ethanol).
The cyclohexylamine salt (9.0 g.) is suspended in 40 ml. of ethyl acetate, stirred, cooled, and treated with 45 ml. of 1 N hydrochloric acid.
The layers are separated, the aqueous phase extracted with additional ethyl acetate (3 x 40 ml.), the combined organic layers are dried (MgS04), and the solvent evaporated to give 7.1 g. (75%) of glass-like N-carbobenzyloxy-4,4-trimethylenedioxy-L-proline.
b) 4,4-Trimethylenedioxy-L-proline A solution of 7.1 g. (0.022 mole) of N-carbobenzyloxy-4,4-trimethylenedioxy-L-proline in 200 ml. of 2:1 methanol-water is hydrogenated in ..

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~ ., -1~32985 HAlaOa-35-the presence of 2 g. of 5~ palladium- carbon catalyst to give 3.8 g. (93~) of nearly colorless 4,4-tri-methylenediaxy-L-proline; m.p. 234-236 (dec.); preceded by gradual darkening and sintering; [a]25 -36 (c, 0.5~ in 1:1 methanol-water).
Anal. Calc'd. for C8~13NO4:
C, 51.33; H, 7.00; N, 7.48 Found: C, 51.42; H, 7.11; N, 7.40.
c) ~2(S),3S~-2-[3-(Acetylthio)-2-methyl-1-oxopropyl]-6,10-dioxo-2-azaspiro[4,5]decane-3-carboxylic acid

4,4-Trimethylenedioxy-L-proline (3.7 g., 0.02 mole) is acylated with 4.0 g. (0.022 mole) of D-3-acetylthio-2-methylpropionyl chloride in 50 ml.
of water in the presence of sodium carbonate according to the procedure of Example 1 (e) to give 7.3 g.
of glass-like crude product.
The product is converted to its dicyclohexyl-amine salt with 3.6 g. of dicyclohexylamine in 70 ml.
of ethyl acetate. On seeding and rubbing, the crystalline salt precipitates to yield 7.5 g. of dicyclohexylamine salt; m.p. 168-170 (s. 166), [a]D ~59 (c, 1% in ethanol). Recrystallization from 30 ml. of acetonitrile gives 6.5 g. of colorless solid salt; m.p. 169-171, [a]25 -63, (c, 1~ in ethanol).
The dicyclohexylamine salt is converted to the free acid by suspending 6.4 g. in 75 ml.
of ethyl acetate and treating with 75 ml. of 10 potassium bisulfate and stirring until two layers are obtained. After separating, the aqueous 1~3Z985 HA18Oa phase is extracted with ethyl acetate (4 x 75 ml.), the organic layers are combined, dried ~MgS04), and the solvent e~aporated to give 4.3 g. ~67%) of glass-like ~2(S),3S]-2-[3-(acetylthio)-2-methyl-1-oxopropyl]-6,10-dioxo-2-azaspiro~4,5]decane-3-carboxylic acid.
Example 8 [2(S),3S~-2-(3-~1ercapto-2-methyl-1-oxopropyl)-h,10-dioxo-2-azas~iro14,5]decane-3-carboxylic acid [2(S),3S]-2-[3-(Acetylthio)-2-methyl-1-oxopropyl]-6~lo-dioxo-2-azaspiro[4~5]decane-3-carboxylic acid (4.3 g., 0.013 mole) is hydrolyzed with 8.5 ml.
of concentrated ammonia in 20 ml. according to the procedure of Example 2 to yield 0.9 g. of colorless solid product; ~]25 -64 (c, 0.5% in ethanol). An additional 0.8 g. of product is obtained by extracting the aqueous phase uith chloroform; [a]D5 -66 . The two crops are dissolved in chloroform, evaporated, rubbed under ether, and the evaporating repeated to yield 1.7 g. (46~) of t2(S),3S1-2-(3-mercapto-2-methyl-1-oxopropyl)-6,10-dioxo-2-azaspiro[4,5]decane-3-carboxylic acid; m.p. 169-171 (s. 167 ), [a]25 -71 (c, 1% in methanol).
Anal. Calc'd. for C12HlgN05s:
C, 49.81; H, 6.62; N, 4.84; S, 11.08 Found: C, 49.67; H, 6.67; N, 4.93; S, 11.10.
Example 9 [7(S),8S]-7-[3-(AcetYlthio)-2-methyl-1-oxopro?vl]-7-aza-1,4-dithiaspiro[4.4]nonane-8-carboxylic acid a) N-Carbobenzyloxy-4,4-ethylenedithio-L-proline, methyl ester A stirred solution of 3.9 g. (0.014 mole) of HA180a N-carbobenzyloxy-4-keto-L-proline, methyl ester in 6~ ml. of methylene chloride is treated with 3 ml.
(0.036 mole) of ethanedithiol, cooled to 8, and treated under an argon blanket with 3 ml. (0.024 mole) of boron trifluoride etherate. After removing the cooling bath, the pale yellow solution is stirred for an additional hour and kept overnight at room temperature. The solution is stirred, treated with several pieces of crushed ice, followed by 20 ml.
of water. After 30 minutes the layers are separated and the aqueous phase (50 ml.) is extracted with additional methylene chloride (3 x 30 ml.). The combined organic layers are washed with 50 ml. of saturated sodium chloride solution, dried (MgSO4), and the solvent removed on a rotary evaporator to give 6 g. (100%) of a pale yellow oil N-carbo-benzyloxy-4,4-ethylenedithio-L-proline, methyl ester.
b) N-Carbobenzyloxy-4,4-ethylenedithio-L-proline The methyl ester product from part (a) (7.4 g., approximately 0.018 mole) is dissolved in 65 ml. of methanol, treated dropwise at -1 to 4 with 14.5 ml. (0.029 mole) of 2N sodium hydroxide, kept at 0 for one hour, and at room temperature overnight. After removing about half the solvent on a rotary evaporator, the solution is diluted with 125 ml. of water, washed with ether (wash discarded), acidified while cooling with 5 ml. of 1:1 hydro-chloric acid to a pH of 2, and extracted with ethyl acetate (4 x 50 ml.). The combined extracts are washed with S0 ml. of saturated sodium chloride, `
`
.

113~85 HA180a dried (MgSO4), and the solvent evaporated to give fi g.
of a pale yellow viscous oil. This oil is dissolved in 25 ml. of ethanol, treated with 1.8 g. of cyclohexylamine in 5 ml. of ethanol, and diluted to 300 ml. with ether. On seeding and rubbing, the crystalline cyclohexylamine salt separates to yield after overnight cooling 5.7 g. of N-carbo-benzyloxy-4,4-ethylenedithio-L-proline cyclohexyl-amine salt; m.p. 205-207 (s. 201). Recrystallization from 50 ml. of ethanol-400 mi. ether yields 4.9 g. of colorless solid salt; m.p. 207-209 (s. 201), [a]25 -15 (c, 1% in chloroform).
The cyclohexylamine salt (4.8 g.) is suspended in 25 ml. of ethyl acetate, stirred, and treated with 25 ml. of lN hydrochloric acid. When two clear layers are obtained, they are separated, the aqueous phase is extracted with additional ethyl acetate (3 x 25 ml.), the combined organic layers are dried (MgSO4), and the solvent evaporated to give 3.8 g.
(62%) of N-carbobenzyloxy-4,4-ethylenedithio-L-proline as a pale yellow viscous syrup.
c) 4,4-Ethylenedithio-L-proline, hydrobromide N-Carbobenzyloxy-4,4-ethylenedithio-L-proline ~3.7 g., 0.011 mole) is treated with 20 ml. of hydrogen bromide in acetic acid (30 - 32~), stoppered loosely, and stirred magnetical~y. ~ixing is difficult due to the viscosity of the starting material and the latter is broken up as much as possible with a spatula. In the meantime, the crystalline product begins to separate. Further .
::

;., . , ~ .

ll~Z98~
H~180a quantities of hydrogen bromide in acetic acid are added after 15 minu'es (10 ml.) and after 2; minutes (5 ml.) and stirrin~ is continued for a total of 35 minutes. Ether (250 ml.) is added to complete precipitation of the product and after cooling for 15 minutes the cream colored material is filtered under nitrogen, washed with ether, and dried in vacuo to give 2.7 g. of 4,4-ethylenedithio-L-proline, hydrobromide; m.p. 240-242 (dec.); sintering and darkening from approximately 200; [a]D6 _40o (c, 0.5% in 1:1 chlo~oform-methanol).
d) [7(5),8S]-7-[3-(Acetylthio)-2-methYl-l-oxoproPyl]-7-aza-1,4-dithias~iro[4 4]nonane-8-carboxylic acid A stirred solution of 2.6 g. (0.~091 mole) of 4,4-ethylenedithio-L-proline, hydrobromide in 25 ml. of water is cooled to 5 and brought to pH 8.2 by the addition of 25% sodium carbonate (wt./vol.). While continuing stirring and cooling, a solution of 1.9 g. (0.01 mole) of D-3-acetylthio-2-methylpropionyl chloride in 2.5 ml. of ether isadded portionwise whiIe maintaining the pH at J.5 - 8.2 by the dropwise addition of 25% sodium caxbonate. When the pH is stabilized at 8.2 - a . s (after about 15 minutes), stirring and cooling are continued for a total of one hour. The solution is then washed with 25 ml. of ethyl acetate ~wash discarded), layered over with 25 ml. of ethyl acetate, cooled, stirred, acidified carefully with 1:1 hydrochloric acid to pH 2.0, saturated with sodium chloride, and the layers separated. The HA180a _4~_ aqueous phase is extracted with additional ethyl acetate (3 x 25 ml.), the combined organic layers dried (MgSO4), and the solvent evaporated, finally at 0.2 mm., to give 2.6 g. of syrupy product which

5 begins to crystallize. The latter is treated in 30 ml.
of ethyl acetate with l.S g. of dicyclohexylamine to give 3.0 g. of colorless dicyclohexylamine salt;
m.p. 176-178 (s, 170 ); [a]D ~55 (C~ 1~ in ethanol). This material is ground in a mortar under lS ml. of acetonitrile, cooled for one hour, filtered, washed with 5 ml. of cold acetonitrile and with ether, and dried to give 2.9 g. of dicyclohexylamine salt; m.p. 177-179 (s, 172); 1~26 56 in ethanol).
Anal. Calc d 13 19 4 12 23 C, 56.56; H, 7.98; N, 5.28; S, 18.12 Found: C, 56.21; H, 8.18; ~I, 5.05; S, 18.00.
The above dicyclohexylamine salt is converted to the free acid by suspending 2.8 g. in 30 ml. of ethyl acetate, cooling, and treating with 30 ml. of 10~
potassium bisulfate to give two clea~r layers. After separating, the aqueous phase is extracted with ethyl acetate (3 x 30 ml.), the combined organic layers dried (MgSO4), and the solvent evaporated, finally at 0.1 -0.2 mm and 45, to give 2.0 g.
(63%) of colorless solid f7(S),8S]-7-[3-(acetylthio)-2-methyl-1-oxopropyll-7-aza-1,4-dithiaspiro f 4.4] -nonane-8-carboxylic acid, m.p., 125-126 (S, 122);
- 1]D6 -101 (C, 1~ in ethanol).

, . :' ,.~` -` 1132985 H~180a Example 10 ~7(S),8S]-7-(3-Mercapto-2-methyl-1-oxo~ropyl)-7-aza-1,4-dithiaspiro~4.4~nonane-8-carbo~ylic acid Argon is passed through a cold solution of 3.5 ml. of concentrated ammonia in 8.5 ml. of water for lS minutes. The latter is then added while cooling and under a blanket of argon to 1.9 g.
(0.0054 mole) of [7(S),8S~-7-[3-(acetylthio)-2-methyl-l-oxopropyl]-7-aza-1,4-dithiaspiro[4.4]nonane-8-carboxylic acid and ~he mixture is swirled in an ice-bath until a solution is obtained. Stirring under argon is continued at room temperature for an additional two hours, then the solution is extracted with 15 ml. of ethyl acetate under an argon atmosphere. The aqueous layer is cooled, stirred, layered over with 15 ml. of ethyl acetate, and acidified portionwise with approximately 6.5 ml.
of 1:1 hydrochloric acid. The layers are separated, the aqueous phase extracted with additional ethyl acetate (3 x 15 ml.), the combined acetate layers dried (MgSO ), and the solvent evaporated to give a glass-like residue which solidifies when rubbed under ether. The evaporation is repeated and the colorless product is suspended in 30 ml. of hexane, filtered and dried in vacuo to give 1.4 g. (84~) of [7~5),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-7-aza-1,4-dithiaspirot4.4]nonane-8-carboxylic acid;
m.p. 116-118 (s, 105); [a]26 -44 (c, 1% in ethanol).
Anal. Calc'd. for C11~17NO3S3:

:

~-HA180a C, 42.97; ~, 5.5~; N, 4.56; S, 31.29; SH, 100~
Found: C, 42.70, H, 5.71; N, 4.54; S, 31.16; SH, 100%.
Example 11 [7(S)r8S3-7-(3-Mercapto-2-methyl-1-oxopropYl)-1,4-dioxo-7-azaspiro[4.5]decane-8-carboxylic acid a) [7(S),8S~-7-[3-(~cetylthio)-2-methyl-1-oxopropyl~-1,4-dioxo-7-azaspiro[4.5]decane-8-carboxylic acid Following the procedure of Example 1 but sub-stituting an equivalent amount of N-carbobenzyloxy-5-keto-L-pipecolic acid for the N-carbobenzyloxy-4-keto-L-proline in part (c) and then following the procedure of parts (d) and (e) one obtains, [7(S),8 ~-7-~3-(acetylthio)-2-methyl-1-oxopropyl]-1,4-dioxo-7-azaspiro[4.5]decane-8-carboxylic acid.
b) ~7(S)!8S]-7-(3-Mercapto-2-methyl-l-oxopropyl)-1,4-dioxo-7-azaspiro[4.5]decane-8-carboxylic acid The product from part (a) is hydrolyzed with concentrated ammonia according to the procedure of Example 2 to yield [7(S),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.5]decane-8-carboxylic acid.
Example 12 [l(S),2s]-1-(3-Mercapto-2-methyl-1-oxopropyl)-5,5-dimethoxy-2-piperidinecarboxylic ~cid a) [l(S),2S]-1-[3-(Acetylthio-2-methyl-l-oxopropyl]-5,5-dimethoxy-2-piperidinecarboxylic acid Following the procedure of Example 3 but substituting an equivalent amount of N-carbobenzyloxy-5-keto-Lopipecolic acid for the N-carbobenzyloxy-4-keto-L-proline in part (a) one obtains [l~S),25l-1-~, ~

~13Z985 HAl~Oa t3-(acetylthio)-2-methyl-1-oxopropyl3-5,s-dimethoxy-2-piperidinecarboxylic acid.
b) ll(S),2S]-1-(3-Mercapto-2-methyl-1-oxopropyl)-5,5-dimethoxy-2-piperidinecarboxylic acid The product from part ~a) is hydrolyzed with concentrated ammonia according to the procedure of Example 4 to yield [l(S),2S]-1-(3-mercapto-2-methyl-l-oxopropyl)-5,5-dimethoxy-2-piperidinecarboxylic acid.
Example 13 [l(S),2-1-1-(3-Mercapto-2-methyl-1-oxopropyl)-4,4-dimethoxy-2-piperidinecarboxylic acid a) [l(S),2-]-1-[3-(Acetylthio)-2-methyl-1-oxopropyl]-4,4-dimethoxy-2-piperidinecarboxylic acid Following the procedure of Example 3 but substituting an equivalent amount of N-carbobenzyloxy-4-keto-2-pipecolic acid for the N-carbobenzyloxy-4-keto-L-proline in part (a) one obtains [l(S),2-]-1-13-(acetylthio)-2-methyl-1-oxopropyll-4,4-diethoxy-2-piperidinecarboxylic acid.
b) [l(S),2-]-1-[3-Mercapto-2-methyl-1-oxopropyl)-4, 4-dimethoxy-2-piperidinecarboxylic acid - The product from part (a) is hydrolyzed with concentrated ammonia according to the procedure of Example 4 to yield [l(S),2-]-1-(3-mercapto-2-methyl-1-oxopropyl)-4,4-dimethoxy-2-piperidinecarboxylic acid.

, .

113298~ HAl 8 0 a Example 14 [2(S),3S]-2-(3-Mercapto-2-methyl-1-oxopropyl)-6,10-di-thia-2-azaspiro~4.5]decane-3-carboxvlic acid a) [2(S),3S]-2-~3-(Acetylthio)-2-methyl-l-oxopropyl)-6,10-dithia-2-azaspiro[4.5~decane-3-carboxy-lic acid Following the procedure of Example 9 but substituting 1,3-propanedithiol for the ethanedithiol in part (a), one obtains [2(S),3S]-2-[3-(acetylthio)-2-methyl-1-oxopropyl~-6,10-dithio-2-azaspiro[4.5~decane-3-carboxylic acid.
b) [2(S),3S~-2-(3-Mercapto-2-methyl-1-oxoProPvl)-6,10-dithia-2-azaspiro~4.5~decane-3-carboxylic acid The product from part (a) is hydrolyzed lS with concentrated ammonia according to the proced~re of Example 10 to yield [2(S),3S~-2-(3-mercapto-2-methyl-l-oxopropyl)-6,10-dithia-2-azaspiro[4.5~decane-3-carboxylic acid.
Example 15 [7~S),8S]-7-(3-Mercapto-2-methyl-1-oxopropyl)-1-oxo-4-thia-7-azaspiro[4.4~nonane-8-carboxylic acid a) [7(S),8S]-7-[3-(Acetylthio)-2-methyl-1-oxoproPyl]-l-oxo-4-thia-7-azaspiro[4.4]nonane-8-carboxylic acid Following the procedure of Example 1 but substituting 2-mercaptoethanol for the ethylene glycol in part (c), one obtains [7(S).8S]-7-[3-(acetylthio)-2-methyl-1-oxopropyl]-1-oxo-4-thia-7-azaspiro[4.4]nonane-8-carboxylic acid.

- , , ` ~ ' - ~`

~i32985 H~180a b) [7~S),8S]-7-(3-Mercapto-2-methYl-l-oxopropYl)-l-oxo-4-thia-7-azaspiro~4.4]nonane-8-c~rbo~ylic acid The product from part (a) is hydrolyzed with concentrated ammonia according to the procedure S of Example 2 to yield [7(S),85]-7-(3-mercapto-2-methyl-1-oxopropyl)-1-oxo-~-thia-7-azaspiro[4.4]nonane-8-carboxylic acid.
Example 16 (8S)-7-[3-(Acetylthio)-2-trifluoromethyl-1-oxopropyl]-1,4-dioxo-7-azaspiro[4.4~nonane-8-carboxylic acid (Isomers A and ~) a) D,L-3-(Acetylthio)-2-trifluoromethylpropionic acid a-Trifluoromethyl acrylic acid (10 g., 0.071 mole) ~prepared according to the procedure set forth J. Chem. Soc., 1954, p. 371] is cooled in a salt-ice-water bath, stirred and treated portionwise with 5.7 ml. (0.075 mole) of 97% thiolacetic acid.
After the addition, the yellow liquid is stirred in the cold for one hour, allowed to warm to room temperature, and distilled to yield 14 g. (91%) of D,L-3-(acetylthio)-2-trifluoromethylpropionic acid as a light yellow oil, b.p. 149-153/13 mm. The material solidifies on storing in the cola.
b) D,~-3-(Acetylthio)-2-trifluoromethylpropionyl chloride The D,L-3-(acetylthio)-2-trifluoromethyl-propionyl acid (7 g., 0.032 mole) is treated with 18 ml. (0.25) of redistilled thionyl chloride and the mixture is refluxed for three hours. After ,:

. --H.~l8oa removing the excess thionyl chloride on a rotary evaporator, the residue is distilled to give 6.8 g.
of D,L-3-(acetylthio)-2-trifluoromethylpropionyl chloride as a pale yellow oil; b.p. 80-82/16 mm.
c) (8S)-7-[3-(Acetylthio)-2-trifluoromethyl-1-oxopropy~ 4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid (Isomers A and B) .
4,4-Ethylenedioxy-L-proline (2.4 g., 0.014 mole) is reacted with 3.4 g. (0.014 mole) of D.L-3-(acetylthio)-2-trifluoromethylpropionyl chloride in 40 ml. of water in the presence of sodium carbonate according to the procedure of Example 1 (e) to yield 4.5 g. of nearly colorless solid product; m.p.
126-145 (s. 115 ), [a~D -34 (c, 1~ in ethanol).
The mixture of diastereoisomers (4.2 g.) is suspended in 45 ml. of ether, stirred for two hours, cooled for 20 minutes, and the undissolved solid is filtered, washed with cold ether, and air dried to yield 2.7 g. of product; m.p. 166-172 (s. 140 ), ~al25 -62 (c, 13 in ethanol). The material is then ground in a mortar under 25 ml. of ether, filtered after 15 minutes, washed with some ether, and again air-dried to yield 2.1 g. of product; m.p. 172-177 (s. 143). Following crystallization from 11 ml. of boiling isopropanol and cooling overnight, -1.55 g. of colorless (8S)-7-[3-(acetylthio)-2-trifluoromethyl-l-oxopropyl]-1,4-dioxo-7-azaspir~-~4.4]-nonane-8-carboxylic acid, isomer A is obtained; m.p.
192-194 (s, 183), [~25 -32 tc, 1% in ethanol).
I 3~ A sample is recrystaLlized from isopropanol~ m.p.

. .
, .

-' ~ ' ,--- ' ~

9 ~ HA18Oa 193-195 ~s, 184), ta]D -134 (c, 1% in ethanol).
Isomer ~ is obtained by combining the above ether and isopropanol filtrates and removing the solvents under reduced pressure to give S 2.2 g. of a pale yellow solid; m.p. 108-109 ~5, 95 ); [a]25 + 30 (c, l~ in ethanol). This material is purified by crystallization from 6 ml.
of isopropanol to give 1.2 g. of nearly colorless solid; m.p. 153-155 (s, 130?, [a]25 +40 (c, 1% in ethanol). After crystallization from 4 ml. of ethyl acetate -6 ml. of hexane, 1.1 g. of (8S)-7-[3-(acetylthio)-2-trifluoromethyl-l-oxopropyl]-1,4-dioxo-7-aza-spiro[4.4lnonane-8-carboxylic acid, isomer B; m.p.
lS 153-lSS (s, 141), ~a]25 +41 (c, 1% in ethanol) is obtained.
Example 17 (8S)-7-(3-Mercapto-2-trifluoromethyl-1-oxopropyl)-1,4-dioxo-7-azaspiro~4.4]nonane-8-carboxylic acid, Isomer A
Isomer A product from Example 16 (1.45 g., 0.0039 mole) is hydrolyzed with 2.5 ml. of concentrated ammonia in 6 ml. of water over a period of one hour as described in Example 2 to yield 1.25 g. ~97%) of colorless ~8S)-7-(3-mercapto-2-trifluoromethyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid, isomer A, as a glass-like product, [a]D -61 (c, 1% in ethanol). TLC:Rf 0.40 (95:5:5 methylene chloride -methanol - acetic acid;
vis. SH reagent, PMA and heat).

.
- . .
:, . . ;
. .

. ~

1~3Z985 HA180a -4~-Anal. Calc'd. for CllHl~F3NOss:
C, 40.12; H, 4.28; N, 4.25; 5, 9.74; F, 17.31 Found: C, 40.10, H, 4.43; N, 4.51; S, 9.63; F, 17.10.
The above acid is dissolved in ethyl acetate and treated with l-adamantanamine to yield ~he 1-adamantanamine salt; m.p. 213-215 (dec.), [al25 -47 c, 1% in methanol).
Example 18 (8S)-7-(2-Mercapto-2-trifluoromethyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-c2rboxylic acid, Isomer B
Isomer B product from Example 16 (1.05 g., 0.0028 mole) is hydrolyzed with 2 ml. of concentrated ammonia in S ml. of water according to the procedure described in Example 2 to yield 0.9 g. (97~) of (8S)-7-(3-mercapto-2-trifluoromethyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid, Isomer B as a pale yellow viscous syrup,; [a]D5 -16 (c, 1~ in ethanol).
The material sets to a waxy solid;m.p.61-64 (s .SS ) Anal. Calc d- for: 11 14 3 S 2 C, 39.5,8; H, 4.38; N, 4.20; S, 9.61; F, 17.01 Found: C, 39.60; H, 4.28; ~, 4.26; S, 9.62; F, 16.89.
Example 19 1-~3-(Acet~lthio)-2-trifluoromethyl-1-oxopropyl]-4,4-dimethoxy-L-proline, Isomers A and B
Following the procedure of Example 16 but substituting 4,4-dimethoxy-L-proline for the 4,4-ethylenedioxy-L-proline in part (c), one obtains 1-[3-(acetylthio)-2-trifluoromethyl-1-oxopropyl]-4,4-dimethoxy-L-proline as a racemic mixture. The ~13Z98S
HAl~Oa _aa_ individual isoners can be separated as taught in Example 16.
Example 2~
1-(3-Mercapto-2-trifluoromsthyl-1-oxopropyl)-4~4-di-methoxy-L-proline, Isomer .i and Isomer B
Each individual isomer product from Example 19 is hYdrolyzed with concentrated ammonia according to the procedure of Example 4 to yield 1-(3-mercapto-2-trifluoromethyl-l-oxopropyl)-4,4-dimethoxy-L-proline,isomer A and 1-(3-mercapto-2-trifluoro-methyl-l-oxopropyl)-4,4-dimethoxy-L-proline, isomer B.
Example 21 [7(S),85]-7-(3-Mercapto-2-mercaptomethyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid a) [7(S),8S]-7-~3-(Acetylthio)-2-(acetylthiomethyl)-l-oxopropyl]-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid Following the procedure of Example 1 but substituting D-2-acetylthiomethyl-3-acetylthiopro-pionyl chloride for the D-3-acetylthio-2-methylpropio~l chloride in part (e), one obtains ~7(S),8S]-7-~3-(acetylthio)-2-(acetylthiomethyl)-1-oxopropyl]-1,4-dioxo-7-azaspirol4.4]nonane-8-carboxylic acid.
b) [7(5),8(S)]-7-(3-Mercapto-2-mercaptomethyl-1-_~propy~ 4-dioxo-7-aza~ro[4~4inonan-e-8 carhoxvlic acid l'he product fro~:~ part (-~ is hydroLyzed with concentrated an~onia accordin~ to the pro~edure of Example 2 to yield [7(S),8S]-7-(3-mercapto-2-mercaptomethyl-1-oxopropyl)-1,4-dioxo-7-azaspiro-. ~ ~ .

: ' ' ' , ~32985 HA18Oa ~4.4]nonane-8-carboxylic acid.
E~ample 2~
(S)-1-(3-Mercapto-2-merca~tomethyl-1-oxopropyl)-4,4-dimetho~y-L-proline a) (S)-1-~3-(.~cet~lthio)-2-(acetylthiomethyl)-1-oxopro-pyl~-4,4-dimethoxy-L-proline Following the procedure of Example 3 but substituting D-2-acetylthiomethyl-3-acetylthiopro-pionyl chloride for the D-3-acetylthio-2-methylpropionyl chloride in part (d), one obtains (S)-1-~3-(acetylthio~-2-(acetylthiomethyl)-1-oxopropyl]-4,4-dimethoxy-L-proline.
b) (S)-1-(3-Mercapto-2-mercaptomethyl-1-oxopropyl)-4,4-dimethoxy-L-proline The product from part (a) is hydrolyzed with concentrated ammonia according to the procedure of Example 4 to yield (S)-1-(3-mercapto-2-mercapto-methyl-l-oxopropyl)-4,4-dimethoxy-L-proline.
Example 23 ~8S)-7-(3-MercaPto-2-methvlthio-l-oxopropyl)-l~4 dioxo-7-azaspiro~4.4]nonane-8-carboxylic acid a) 3-(Acetylthio)-2-(meth~Ilthio)propionic acid 12.5 g. (0.094 mole) of methyl-2-~methylthio)-[prepared from methyl 2-chloroacrylate according to the procedure of Gundesmann et al., Chemische Berichte 94, 3254 (1916)] is stirred with lN
aqueous sodium hydroxide (94 ml.) with ice cooling.
The mixture is allowed to warm to ambient temperature, then stirred for five hours. The resulting solution is washed with ether, then acidified to p~ 2 with .

., : .
, .
.: - . . -.
., . : . .

HA180a concentrated hydrochloric acid. The solid precipitate i 5 extracted into methylene chloride, and the solution is washed with saturated sodium chloride and the solvent evaporated. The solid residue, 2-tmethylthio) acrylic acid; m.p. 70-75, is used immediateiy in the following reaction.
Equimolar amounts of 2-(methylthio)acrylic acid and thiolacetic acid are mixed under argon and stirred at 80 for several hours to yield 3-tacetylthio)-2-(methylthio~propionic acid.
b) 3-(Acetylthio)-2-~methylthio)propionic acid chloride The 3-(acetylthio)-2-(methylthio)propionic acid is refluxed in thionyl chloride for two hours. The reaction mixture is distilled to remove excess thionyl chloride and the product is distilled in vacuo to yield 3-(acetylthio)-2-(methylthio)propionic acid chloride.
c? (8S)-7-[3-(Acetylthio)-2-methylthio-1-oxopropyl]-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid 4,4-Ethylenedioxy-L-proline is reacted with 3-(acetylthio)-2-(methylthio)propionic acid chloride according to the procedure of Example 1 (e) to yield (8S)-7-13-(acetylthio)-2-methylthio-1-oxopropyll-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid.
d) (8S)-7-(3-Mercapto-2-methylthio-1-oxopropyl)-1, 4-dioxo-7-azaspiro~4.4]nonane-8-carboxylic acid The product from part (c) is treated with concentrated ammonia according to the procedure of Example 2 to yield (8S)-7-(3-mercapto-2-methylthio-1-oxopropyl)-1,4-dioxo-7-azaspiro~4.4]nonane-8-,~

: , ~

: : ~ .:
. , . .:: ':

, . : ~-::
. . :
~ . ~ - - ~ : . ' : ., ~13Z985 HAl~Oa carboxylic acid.
Example 24 1-~3-Mercapto-2-methylthio-1-oxopropyl)-4,4-dimethoxy-L-proline S a) 1-~3-~Acetylthio)-2-methylthio-1-oxoDropyl]-4,4-dimethoxv-L-proline 4,4-Dimethoxy-L-proline is reacted with 3-(acetylthio)-2-(methylthio)propionic acid chloride according to the procedure or Example 3(d) to yield 10 1-[3-[aCetYlthi)-2-methylthio-l-oxopropyl]-4~4 dimethoxy-L-proline.
b) l-(3-Mercapto-2-methylthio-1-oxopropyl)-4,4-dimethoxy-L-proline The product from part (a) is hydrolyzed with concentrated ammonia according to the procedure of Example 4 to yield 1-(3-mercapto-2-methylthio-1-oxo-propyl)-4,4-dimethoxy-L-proline.
Example 25 (85)-7-(3-~ercapto-1-oxopropyl)-1,4-dioxo-7-azaspiro-[4.4~nonane-8-carboxylic acid a) (8S)-7-[3-(Acetylthio)-l-oxopropyl]-1,4-dioxo-7-azas~iro r4 . 4]nonane-8-carboxylic acid 4,4-Ethylenedioxy-L-proline is reacted with 3-acetylthiopropionyl chloride according to the procedure of Example 1 (e) to yield (8S)-7-[3-(acetyl-thio)-1-oxopropyl]-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid.

", .. . . . .. .
. ' .~, , ' '' ' ' , ; : . ' .

:' `. " ' ,' ' ~ ~

H~l 8 0 a b) (8S)-7-(3-Mercapto-l-oxopropyl)-1,4-dioxo-7-azaspiro[4.41nonane-8-carboxylic acid The product ~rom part (a) is hydrolyzed with concentrated ammonia according to the procedure of Example 2 to yield 18S)-7-(3-mercapto-1-oxopropyl)-1,4-dioxo-7-azaspirot4.4]nonane-8-carboxylic acid.
Example 26 1-(3-Mercapto-l-oxopropyl)-4,4-dimethoxy-L-proline a) l-[3-(Acetylthio)-l-oxopropyl]-4,4-dimethoxy-L-proline 4,4-Dimethoxy-L-proline is reacted with 3-acetylthiopropionyl chloride according to the procedure of Example 3 (d) to yield 1-[3-(acetylthio)-l-oxopropyl]-4,4-dimethoxy-L-proline.
b) 1-(3-Mercapto-l-oxopropyl)-4,4-dimethoxy-L-~roline The product from part (a) is hydrolyzed with concentrated ammonia according to the procedure of Example 4 to yield 1-(3-mercapto-1-oxopropyl)-4,4-dimethoxy-L-proline.
Example 27 (85)-?-(4-MercaPto-l-oxobutyl)-1,4-dioxo-7-azas~iro~4.4]-nonane-8-carboxylic acid a) (8S)-7-[4-(Acetylthio)-l-oxobutyl]-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid 4,4-Ethylenedioxy-L-proline is reacted with 4-acetylthiobutyroyl chloride according to the procedure of Example 1 (e) to yield 8(S)-7-[4-(acetylthio)-l-oxobutyl]-1,4-dioxo-7-azaspiro[4.4]-nonane-8-carboxylic acid.

.
. , : .
-, `,'.: ;
, . .. ., , . . -. .. ~
.

113Z985 HA180a b) (8S)-7-~4-Mercapto-l-oxobutyl)-1,4-dioxo-7-aza-spiro~4,4~nonane-8-carboxylic acid The product from part (a) is hydrolyzed with concentrated ammonia according to the pro-cedure of Example 2 to yield (8S)-7-(4-mercapto-1-oxobutyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid.
Example 28 1-(4-Mercapto-l-oxobutyl)-4,4-dimethoxy-L-proline a) 1-[4-(Acetylthio)-l-oxobutyl]-4,4-dimethoxy-L-proline 4,4-Dimethoxy-L-proline is reacted with 4-acetylthiobutyroyl chloride according to the procedure of Example 3 (d) to yield 1-[4-(acetylthio)-1-oxobutyl]-4,4-dimethoxy-L-proline.
b) l-(4-Mercapto-l-oxobutyl)-4,4-dimethoxy-L-proline The product from part (a) is hydrolyzed with concentrated ammonia according to the procedure of Example 4 to yield 1-(4-mercapto-1-oxobutyl)-4,4-dimethoxy-L-proline.
Example 29 (851-7-(2-Mercapto-l-oxoethyl)-1,4-dioxo-7-azaspiro-[4.4]nonane-8-carboxylic acid a) (8S)-7-[2-(Acetylthio)-l-oxoethyl]-1,4-dioxo-7-azaspiro[4.41nonane-8-carboxylic acid 4,4-Ethylenedioxy-L-proline is reacted with acetylthioacetyl chloride according to the procedure of Example 1 (e) to yield (8S)-7-~2-(acetylthio)-1-oxoethylJ-1,4-dioxo-7-azaspiro[4.41nonane-8-~, .,;

1132~5 H~180a carboxylic acid.
b) (8S)-7-(2-Mercapto-l-o~oethyl)-1,4-dioxo-7-azaspirot4.41nonane-~-carboxylic acid The product from part (a~ is hydrolyzed with concentrated ammonia according to the procedure of Example 2 to yield (8S)-7-(2-mercapto-1-oxoethyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid.
Example 30 1-(2-Mercapto-l-oxoethyl)-4,4-dimethoxy-L-proline a) 1-[2-(Acetylthio)-l-oxoethyl]-4,4-dimethoxy-L-proline 4,4-Dimethoxy-L-proline i5 reacted with acetylthioacetyl chloride according to the procedure of Example 3 (d) to yield 1-[2-(acetylthio)-1-oxoethyl]-4,4-dimethoxy-L-proline.
b) l-(2-Mercapto-l-oxoethyl)-4,4-dimethoxy-L-proline The product from part (a) is hydrolyzed with concentrated ammonia according to the procedure of Example 4 to yield 1-(2-mercapto-1-oxoethyl)-4,4-dimethoxy-L-proline.
Example 31 ( a s ) - 7-(3-Mercapto-2,2-dimethyl-1-oxopropyl)-1,4-dioxo-7-azaspirol4.4~nonane-8-carboxylic acid a) (8S)-7-[3-(Acetylthio)-2~2-dimethyl-l-oxopropYl]
1,4-dioxo-7-azaspirot4.4]nonane-8-carboxylic acid 4,4-Ethylenedioxy-L-proline is reacted with 3-acetylthio-2,2-dimethylpropionyl chloride according : to the procedure of Example 1 (e) to yield ~8S)-7-13-(acetylthio)-2,2-dimethyl-1-oxopropy11-1,4-.
;

.

. .

.. . .
.. . . . - , .

HA18Oa -~6-dioxo-7-azaspiro[4.4~nonane-8-carboxylic acid.
b)( S)-7-(3-Mercapt^-2 2-dimethvl-1-oxo~ropyl)-1,4-dioxo-7-azaspiro~4.4~nonane-8-carboxylic acid The product from part (a) is hydrolyzed with S concentrated ammonia to yield (8S)-7-~3-mercapto-2,2-dimethyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]-nonane-8-carboxylic acid.
Example 32 1-(3-Mercapto-2~2-dimethyl-l-oxopropyl)-4~4-dimeth L-proline a) l-[3-(Acetylthio~-2,2-dimethyl-1-oxopropyl~-4,4-dimethoxy-L-proline 4,4-Dimethoxy-L-proline is reacted with 3-acetylthio-2,2-dimethylpropionyl chloride according to the procedure of Example 3 (d) to give 1-[3-(acetylthio)-2,2-dimethyl-1-oxopropyl]-4,4-dimethoxy-L-proline.
b) 1-(3-Mercapto-2,2-dimethyl-1-oxopropyl)-4,4-. .
dimethoxy-L-proline The product from part (a) is hydrolyzed with concentrated ammonia according to the procedure of Example 4 to give 1-(3-mercapto-2,2-dimethyl-1-oxopropyl)-4,4-dimethoxy-L-proline.
Example 33 17(s)~8s]-7-(3-Mercapto-2-ethyl-l-oxopropyl]-l~4 dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid a) [7(S),8S]-7-13-(Acetylthio)-2-ethyl-1-oxopropyl]-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid 4,4-Ethylenedioxy-L-proline is reacted with D-3-acetylthio-2-ethylpropionyl chloride according , .
-: .-... .

, ' ' HA180a to the procedure of Example 1 (e) to give ~1(5),8S~-7-[3-(acetylthio)-2-ethyl-1-oxopropyl]-1,4-dioxo-7-azaspiro~4.4]nonane-8-carboxylic acid.
b) 17(S),8S]-7-(3-Mercapto-2-ethyl-1-oxopropyl)-1,4-dioxo-7-azaspiro~4.4]nonane-8-carboxylic acid The product from part (a) is hydrolyzed with concentrated ammonia according to the procedure of Example 2 to give [7(S),8S]-7-(3-mercapto-2-ethyl-1-oxopropyl)-1,4-dioxo-7-azaspiro~4.4]nonane-8-carboxylic acid.
Example 34(S?-1-(3-Mercapto-2-ethyl-1-oxopropyl-4,4-dimethoxy-L-proline a) (S)-1-13-(Acetylthio)-2-ethyl-1-oxopropyl]-4,4-dimethoxy-L-proline 4,4-Dimethoxy-L-proline is reacted with D-3-acetylthio-2-ethylpropionyl chloride according to the procedure of Example 3 (d) to give (S)-1-[3-(acetyl-thio)-2-ethyl-1-oxopropyl]-4,4-dimethoxy-L-proline.
b) (S)-1-(3-Mercapto-2-ethyl-1-oxopropyl)-4,4-dimethoxy-L-proline ~ he product from part (a) is hydrolyzed with concentrated ammonia according to the procedure of Example 4 to give (5)-1-(3-mercap_o-2-ethyl-1-oxopropyl)-4,4-dimethoxy-L-proline.

~,. , . . , ~
~ , -. , 1~3Z985 HA180a Example 35 ~8S] -7-(3-Mercapto-2-trifluo_ometnYL-l-oxo-propyl)-7-aza-1,4-dithiaspiro[4.4lnonane-8-carboxylic acid a) 3-~[(4-Methoxy)phenylmethyl]thio]-2-trifluoro-meth 1 ro ionvl chloride Y P p A neat mixture of l-trifluoromethylacrylic acid (3.9 g.) and 4-methoxybenzylthiol (4.3 g.) is stirred at 100-110 for one hour. The mixture is allowed to cool to room temperature and the solid is recrystallized from cyclohexane to yield 3-~[(4-methoxy)phenylmethyllthio]-2-trifluoromethylpro-pionic acid; m.p. 72-74.
Treatment of this acid with thionyl chloride lS yields 3-[1(4-methoxy)phenylmethyl]thio]-2-trifluoro-methylpropionyl chloride.
b) [8S]-7-[3-~t(4-Methoxv)PhenvlmethYllthiol-2-trifluoromethyl-l-oxopropyl]-7-aza-1,4-dithiaspiro-[4.4]nonane-8-carboxylic acid The 3-[1(4-methoxy)phenylmethyl]thio]-2-trifluoromethyl propionyl chloride from part (a) is reacted with 4,4-ethylenedithio-L-proline according to the procedure of Example 9 (d) to yield [8S]-7-[3-[[(4-methoxy)phenylmethyllthio]-2-trifluoromethyl-1-oxopropyl]-7-aza-1,4-dithiaspiro[4.4]nonane-8-carboxylic acid.
c) [8S]-7-(3-Mercapto-2-trifluoromethyl-1--oxopropyl)-7-aza-1,4-dithiaspiro[4.4]nonane-8-carbox~lic acid The product from part (b) is mixed with tri-1132985 HA180a fluoroacetic acid and anisole under nitrogen. The solvents are removed under vacuum to yield as a ~esidue [8S]-7-(3-mercapto-2-trifluoromethyl-1-oxopropyl)-7-aza-1,4-dithiaspiro[4.4]nonane-8-carboxylic acid.
Example 36 [8S]-7-(3-Mercapto-2-methylthio-1-o~opropyl)-7-aza-1,4-dithiaspiro[4.4]nonane-8-carboxylic acid a) 3-[~(4-Methoxy)phenylmethyl]thio]-2-methylthio-propionyl chloride 3-1[(4-Methoxy)phenylmethyl~thio]-2-methyl-thiopropanoic acid prepared according to the procedure of Example 10 in U.S. Patent 4,116,962 is treated with thionyl chloride to yield 3-[[(4-methoxy)phenylmethyl]-thio]-2-methylthiopropionyl chloride.
b) 18S]-7-~3-[[(4-Methoxy)phenylmethyl]thio]-2-methylthio-l-oxopropyl]-7-aza-1,4-dithiaspiro~4.4]-nonane-8-carboxylic acid The 3-[~(4-methoxy)phenylmethyl]thio]-2-methylthiopropionyl chloride from part (a) is reacted with 4,4-ethylenedithio-L-proline according to the procedure of Example 9 (d) to yield [8S]-7-[3-l~(4-methoxy)phenylmethyl]thio]-2-methylthi oxopropyl]-7-aza-1,4-dithiaspiro~4.4]nonane-8-carboxylic acid.
c) [8Sl-7-(3-Mercapto-2-methylthio-1-oxo-propyl)-7-aza-1,4-dithiaspiro[4.4]nonane-8-carboxylic acid The product from part (b) is mixed with tri-fluoroacetic acid and anisole under nitrogen. The , ,, , ;~ - .

~, ' ;' '. -: - ': ~ -.:. ::

Y.A180a solvents are removed under vacuum to yield as a residue ~8S]-7-(3-mercapto-2-methylthio-1-oxo-propyl)-7-aza-1,4-dithiaspiro[4.4]nonane-8-carboxylic acid.
Example 37 [7(S),8S]-7-(3-Mercapto-3-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid a) ~7(S),8S~-7-~3-(Acetylthio)-3-methyi-1-oxopropyl]-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid 4,4-Ethylenedioxy-L-proline is reacted with D-3-acetylthio-3-methylpropionyl chloride according to the procedure of Example 1 (e) to yield ~7(S~,8S~-7-[3-(acetylthio)-3-methyl-1-oxopropyl]-1,4-dioxo-7-azaspiro~4.4]nonane-8-carboxylic acid.
b) [7(S),8S]-7-(3-Mercapto-3-methyl-1-oxopropyl)-1, 4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid The product from part (a) is h~drolyzed with concentrated ammonia according to the procedure of Example 2 to gi~e [7(S),8S]-7-(3-mercapto-3-methyl-l-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid.
Example 38 (S)-1-(3-Mercapto-3-methyl-1-oxopropyl)-4,4-dimethoxy-L-proline a) (S)-1-[3-(Acetylthio)-3-methyl-1-oxopropyl]-4,4-dimethoxy-L-proline 4,4-Dimethoxy-L-proline is reacted with D-3-acetylthio-3-methylpropionyl chloride according to the procedure of Example 3 (d) to yield (S)-1-[3-(acetyl-.:

. .

.
' ~ ;' .:

1~3Z985 HA180a thio)-3-methyl-1-oxopropyl]-4,4-dimethoxy-L-proline.
b) (S?-1-(3-Mercapto-3-methyl-1-oxopropyl-4,4-dimethoxy-L-proline The product from part (a) is hydrolyzed with concentrated ammonia according to the procedure of Example 4 to yield (S)-1-(3-mercapto-3-methyl-1-oxopropyl)-4,4-dimethoxy-L-proline.
Example 39 1-(3-Mercapto-l-oxopropyl)-4,4-dimethylthio-L-proline a) 4-Keto-L-proline, hydrobromide To 4.0 g. (0.015 mole) of N-carbobenzyloxy-4-keto-L-proline are added 20 ml. of hydrogen bromide in acetic acid (30-32%). The mixture is frequently swirled over a period of eight minutes. At the end of this period (effervescence has stopped), the yellow-orange solution is layered over with 25 of ether, triturating the gummy product. The ether is discarded and the resulting tacky solid is triturated with fresh ether and finally with 50 ml. of acetoni-trile to give 4-keto-L-proline, hydrobromide as a crystalline solid weighing 2.7 g. (85%), m.p.
153-155 (dec.,), ta]26 -49 (c, 1% in water).
b) l-t3-(Acetylthio-l-oxopropyl]-4-oxo-L-proline A stirred solution of 4.1 g. (0.0195 mole) of 4-keto-L-proline, hydrobromide in 50 ml. of water is cooled to 5 and treated portionwise with solid sodium carbonate (foaming is controlled by adding a few drops of ether) to pH 8.0 (approx. 2 g. required).
Then while continuing stirring and cooling, a solution of 3.5 g. (0.012 mole) of 3-acetylthiopropanoyl chloride ., ~ .

" ` 113Z~S
HA180a in 5 ml. of ethyl acetate is added portionwise by means of a pipette while maintaining the p~ at 7.0 -8.0 by dropwise addition of 25% (w/v) sodium carbonate solution (about 10 ml.~. After about 10 minutes the pH stabilizes at 8.0 - 8.4. After continued stirring and cooling for a total of 1 hour, the solution is washed with ethyl acetate (2 x 50 ml.), layered over with 50 ml. of ethyl acetate, stirred, cooled, acidified carefully with concentrated hydrochloric acid to pH 2.0, saturated with sodium chloride, and the layers are separated. The a~ueous phase is extracted with additional ethyl acetate (3 x 50 ml.), the combined organic layers dried (MgSO4) and the solvent evaporated, finally at 0.2 mm. to give 4.8 g.
of a yellow-orange glass-like residue. This residue is dissolved in 35 ml. of ethyl acetate and treated with a solution of 3.5 g. of dicyclohexylamine in 5 ml. of ethyl acetate. On seeding and rubbing, - crystalline 1-[3-(acetylthio-1-oxopropyl)]-4-oxo-L-proline dicyclohexylamine salt separated, weight after cooling overnight, 2.7 g. (nearly colorless), m.p.
191-193 (dec.), Ea]26 -24 (c, 1~ in CHC13).
This dicyclohexylamine salt is converted to the free acid using potassium bisulfate as described in Example 1 (e) to give 3.7 g. of 1-~3-(acetylthio)-1-oxopropyl~-4-oxo-L-proline as a pale yellow glass-like solid.
c) 1-[3-(Acetvlthio)-l-oxopropyl]-4,4-dimethylthio-L-.
proline The 1-[3-(acetylthio)-1-oxopropyl]-4-oxo-L-113Z9~ ~18Oa

-6,-proline is reacted with methylthiol according to the procedure of Example 9 (a) to yield 1-[3-(acetylthio)-1-oxopropyl]-4,4-dimethylthio-L-proline.
d) 1-(3-Mercapto-l-oxopropyl)-4,4-dimethylthio-L-proline The product from part (a) is hydrolyzed with concentrated ammonia according to the procedure of Example 2 to yield 1-(3-mercapto-1-oxopropyl)-4,4-dimethylthio-L-proline.
Example 40 [2(S),3S]-2-(3-Mercapto-2-methyl-1-oxopropyl)-8,8-dimethyl-6,10-dioxo-2-azaspiro[4.51decane-3-carboxylic acid a) ~2(S),3S]-2-[3-(Acetylthio)-2-methyl-1-oxoPropvl]-8,8-dimethyl-6,10-dioxo-2-azaspiro[4.5]decane-3-carboxylic acid Utilizing the procedure of Example 1 but substituting 2,2-dimethyl-1,3-propanediol for the ethylene glycol in part (c), one obtains [2(S),3S]-2-[3-(acetylthio)-2-methyl-1-oxopropyll-8,8-dimethyl-6,10-dioxo-2-azaspiro[4.5~decane-3-carboxylic acid.
b) [2(S),3S]-2-(3-Mercapto-2-methyl-1-oxopropyl)-8,8-dimethyl-6,10-dioxo-2-azaspiro[4.5]decane-3-car-boxylic acid The product from part (a) is hydrolyzed with concentrated ammonia according to the procedure of Example 2 to yield [2(S),3S]-2-(3-mercapto-2-methyl-l-oxopropyl)-8,8-dimethyl-6,10-dioxo-2-azaspiro-~4.51decane-8-carboxylic acid.

~,, .
.
, . . . .
:, , -- :
" ' '; , : ' ~ ' 113Z98S HA180a Example 41 ~7(S),8S~-7-~3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-2-methyl-8-carboxylic acid a) N-Carbobenzyloxy-4,4-(1-methylethylenedioxy)-L-proline, methyl ester A stirred mixture of 8 g. (0.025 mole) of N-carbobenzyloxy-4,4-dimethoxy-L-proline, methyl ester, from Example 3 a, 2.4 g. (0.032 mole) of 1,2-propanediol, 0.4 g. of p-toluenesulfonic acid monohydrate, and 400 ml. of toluene is heated to reflux (110-112). The rate of reflux is regulated so that solvent slowly distills by means of a Dean-Stark tube into a graduated cylinder. When 80 ml. of solvent are collected an equal volume of fresh solvent is added to the reaction flask through an addition funnel. This procedure of removing and replacing 80 ml. of solvent is repeated four times during a total reflux period of 1.25 hours.
After standing overnight, the mixture is washed with water. (2 x 100 ml.), the combined washes are back extracted with 100 ml. of toluene, the combined organic layers are dried (MgSO4), and the solvent is removed on a rotary evaporator, finally at 0.2 mm., to give 8.2 g. (99~) of N-carbobenzyloxy-4,4~ methylethylenedioxy)-L-proline, methyl ester as a yellow viscous oil.
b) N-Carbobenzyloxy-4,4-(1-methylethylenedioxy)-L-proline The crude methyl ester product from part (a) (8.2 g., 0.025 mole) is dissolved in 80 ml. of methanol, .

' :,, ' ; :

~ \
~13~ HAl80a treated dropwise at -1 to 4 with 18 ml: (0.036 mole) of 2N sodium hydroxide, ~ept at 0 for one hour, and at room temperature overnight. After removing about half of the solvent on a rotary evaporator, the S solution is diluted with lS0 ml. of water, washed with lO0 ml. of ether (wash discarded), acidified while cooling with 6.3 ml. of 1:1 hydrochloric acid to pH2, and extracted with ethyl acetate (4 x 75 ml.). The combined extracts are washed with 50 ml. of saturated sodium chloride, dried (MgSO4), and the solvent evap-orated to give 8 g. of a red-orange viscous oil. This oil is dissolved in 50 ml. of acetonitrile, warmed, stirred, and treated with 3.8 g. of l-adamantanamine.
The solid salt rapidly separates. After coolin~
overnight, the material is filtered, washed with cold acetonitrile and with ether, and dried in vacuo to yield 10.3 g. of crude adamantanamine salt; m.p.
202-204 (dec.), ta]26 -13 (c, 1% in methanol).
Following trituration with 50 ml. of ~oiling acetoni-trile and cooling, the pale tan solid salt weighed9.4 g.; m.p. 202-204 (dec.), [a]26 -13 (c 1% in methanol).
The above adamatanamine salt is suspended in 40 ml. of ethyl acetate, stirred, and treated with lN
hydrochloric acid. When two clear layers arè obtained they are separated, the aqueous phase is extracted with additional ethyl acetate (3 x 40 ml.), the combined organic layers are dried tMgSO4), and the solvent evaporated, finally at 0.2 mm. and 40, to yield 5.8 g. (72%) of N-carbobenzyloxy-4,4-(l-methylethyl-, ,~''..
. ~, . ., - - , ~ ~

.. . .
-:...... . .
,. .

~132985 HA180a enedioxy)-L-proline as a yellow-orange viscous syrup.
c) 4,4~ ethylethylenedioxy)-L-proline A solution of the above N-carbobenzyloxy-4,4-(l-methylethylenedioxy)-L-proline (5.6 g., 0.017 mole) in 150 ml. of 2:1 methanol-water is treated with 1.6 g. of 5~ palladium-carbon catalyst and shaken under 3 atmospheres of hydrogen for five hours.
The catalyst is filtered off under nitrogen, washed with methanol, and the combined filtrates evaporated, finally at 0.1 - 0.2 mm. to give a crystalline residue.
The latter is suspended in 200 ml. of methanol and the evaporation repeated. The solid residue is rubbed under ether (evaporation again repeated) to yield 3.0 g. (94%) of pale tan 4,4-(1-methylethylenedioxy)-L-proline; m.p. 219-221 (dec.); preceded by gradual darkening and sintering; [~125 -22 (c, 1~ in 1:1 ethanol-water).
d) [7(S)~8S]-7-[3-(Acetylthio)-2-methyl-l-oxopropYl]
1,4-dioxo-7-azaspiro[4.4]nonane-2-methyl-8-carboxylic acid The 4,4-(1-methylethylenedioxy)-L-proline (2.8 g., 0.015 mole) is reacted with 3.0 g. (0.017 mole3 of D-3-acetylthio-2-methylpropionyl chloride in 40 ml. of water according to the procedure of Example 1 (e) to give 5.0 g. of a viscous yellow product. The latter is treated with 2.8 g. of dicyclo-hexylamine in 45 ml. of ethyl acetate to yield 4.2 g. of nearly colorless [7(S),8S]-7-[3-(acetylthio)-2-methyl-1-oxopropyll-1,4-dioxo-7-azaspiro[4.4]nonane-2-methyl-8-carboxylic acid, dicyclohexylamine salt;

' , :~ .
. . .

.
' HA180a m.p. 170-172 (s, 168); ~a]DS -58; (c, 1~ in ethanol). Following crystallization from 12 ml. of acetonitrile, the colorless solid salt weighs 3.85 g. (51~); m.p. 170 - 172 (s. 168);
[]D5 ~57~ (c, 1% in ethanol).

C, 60.90; H, 8.65; r~, 5.46: S, 6.26 Found: C, 60.93; H, 8.72; ~, 5.43; S, 6.35.
The dicyclohexylamine salt is converted to the free acid by suspending 3.8 g. in ethyl acetate and treating with 45 ml. of 10% potassium bisulfate and stirring until two layers are obtained. After separa-ting, the aqueous phase is extracted with ethyl acetate (4 x 40 ml.), the organic layers are combined, dried (MgSO4) and the solvent evaporated to give 2.5 g.
(51~) of colorless solid [7(S),8S~-7-[3-(acetylthio)-2-met~yl-l-oxopropyl]-1,4-dioxo-7-azaspiro~4.4]nonane-2-methyl-8-carboxylic acid, m.p. 65 - 68 (s. 48);
[a]D5 -100, (c, 1% in ethanol).
_~ [7(S),8S~-7-(3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-2-methyl-8-carboxylic acid The product from part (d) is hydrolyzed with concentrated ammonia according to the procedure of E~ple 2 to give 2.05 g. of [7(S),85]-7-(3-mercapto-2-methyl-1-oxcpr~pyl)-l~4-dioxor7-azaspiror4.4]nonane-2-methyl-8-carboxylic acid as a vi~us colorless oil, [a]D -57 (c, 1% in ethanol).
Examples 42 - 75 Following the procedure of Example 41 the dimethoxy substituted compound of Column I (or its alkyl ester) is treated with the diol or dithiol ,"

1~3ZgaS
HA180a of Column II to yield the spiro in~ermediate of column III. Removal of the N-protecting group (and the alkyl ester group) and acylation with the acyl chloride or Column IV yields the product of Column V which can then be hydrolyzed to the product of Column VI.
Col. I Col. II

9~ ~ 10 C \ (C)t (H ~C)p (CH2)q ~~ Cbz-N- C-COOH Hl H

Col. III Col. IV

Rg Rl o 1l 1 4 1 3 11 '~ / R8-C-S- (CH)m-C-- C-Cl ~ (C),t~ R6 1~ ,/
C
(H2C) p ( ~CH2 q Cb z -N ----C- COOH
H

~1 32985 HA18 0 a Col. V

~, Xf (C)~

1\ /' 2 / C, il lR4 IR3 O (H2CI )p ( ICH2)q R -C-S- (CH)-- C-- C N - C-COOH

Col. VI
\ /

~ (C),~

1\ / 2 / C~

IR4 R3 ( 2 1 ) P ( ICH2 ) q H5--~CH)-- C--C N - C-COOH
m l I

,~ ' . ` " ' ` .i `

, , :

- ~i3Z98S
~A180a s S a ~ S

S O ~

C~ ~ O X
~ X 5: ~ O ~ 0~ ~ U~ O ~

C.) x .~ ~

:
.

- `` 113Z3H5 HAlaOa u ((~ J?

3 ~ :~

.'~ ~ ~.
U U

In t~ =
O ~ U ~ U--O
~-U-- ~ ~/ \ I I
-- -- --O = C~

- ' ~

HAl 80a S U

~ N Cl 2 5: ~--O 3~ ~ O 5:
k'-v U~--O :~ U~ O ~ O ~ O r _ _ cn ~ "~ u~

'~

, , . :
, :

~ '- ' ' ~:: ' : ~

li3298~ HA180a O~ .
C~)N ~

.

u~l u7 u~ n ~. ~

~329B5 HA180a 2~ ~

N _i N

~ :C

V

V V--O ~ q v_oO~ O~aU, I

:C

.

. . . . .
, . . ; .

, . . . I
.
. . ~ ~ - .
, . . . . .
- . .
.

1132~5 HA180a ~,~

,, ,~ ,, . _, ~ s :::
u u u u u U ~ ~ U _ O
~ u ---o u -- u~

= ~ o ~ = u,~ o o .
'~ ' : . ~

HA180a U ~ oN U (~)~

I ~ . N

~' U S~

~ ,_ ~
U--~ ~ S ~

S ~ ~ -- ~
U~--~n U~--O I U--U
u~--o t, - 8 ~ , , , , ~ ~

"

. ~ , - .
"
. . .. , . . . .~ . ~ . .. ~ .
.. ~ :, . .. ...
. ~, ; ; .. .. .
, ... ., , : , , . -,,`~ ~3Z9~
~!Al 80a 2: S -- ~

~ Ul ~ 7 2 0 ~ r~
O ~ ~

_N N ~ -- O

I ~I N

_I N N ~

r r 1~ ~

~ : , , , , .. - .: ~

`'' ~13~5 HAl~Oa Examples 75 - 124 Following the procedure of Example 41 the disubstituted compound of Column I ~or its alkyl ester) is treated with a molar equivalent of the alcohol or thiol of Column II to yield the compound shown in Column III. Alternatively, by treating the dimethoxy substituted compound of formula I (or its alkyl ester) with a molar excess of the alcohol or thiol of Column II one obtains the disubstituted compound of Column IV. Removal of the N-protecting group (and the alkyl.ester group) from the inter-mediate of either Column III or IV followed by acylation with the acid chloride of Column v yields the product of Columns VI and VII, respectively.
These compounds can then be hydrolyzed to the products of Columns VIII and IX, respectively, Col. I Col. II

Rl 1 1 X~ Xl R -X-H
/

~ C~
(H~C) (IC 2 q Cbz-N - C-COOH

.' , .. ' , .:
:

, '`: ' '' ~:
~- . . : ~ : . :
.. ..

HA180a Col. III Col. IV

Rl lR2 1 2 lR2 2 ~C / 2 \~ / \
(H2iC)p (ClH2)q (H2C) (CH
Cbz-N~ C-COOH Cbz-N-- - C-COOH
H H

Col. V
O R4 R3 e R8-C-S- (CH) m~C--C-Cl Col. VI

IRl IR2 X~ /X2 ,/ \
O R4 R3 ( 21 )p (1CH2)q R8-c-s-(cH)m-c--C --N -- C-COOH

.~
.

11'~
HA180a Col. VII

X;~, X2 ~ C~

O R4 R O 2 1 ) p (Cl H2 ) q R -C-S- ~CH) m~C-- C N _ C-COOH

R6 ` H

Col. VIII

1 l 1 2 Xl X2 , / C

1 4 1 3 l ~H2C) p ( ICH2) q HS- (CH)m-C--C--N C-COOH
H

~ .
, ..-,, .
., , . ~ ~ . ' ' ~ . ' ' '................... . . '.`. ' . i ~ ' ', . . ' ' . ' . _ ~13Z985 K~18 0a Col. IX

i 2 lR2 / C ,~
R4 R3 O~H2 I p ( ICH2) q HS- (CH~ m-- C--- C - . N --- C-COOH

~' ' "', ' ~ .

. ' '- , HA180a --~2--~jJ
u C~n u ~ ~

N ~ 1 N _I

~1 = = = = ~ , ,, ~1 3; ~ 1 '' 2 ~

y y ~r~

O

~I ~ Y Y 2 X y y ., vl y y CJ
X ~ ~ 0~ O ~0 0 ~1 Y -Y -Y Y Y Y Y
X-~l , , , _I
~ r ` ~ o ? ~ , ; ~ i ``

~13Z985 HA180a !~ ~ _N
S~ u~

~ X `C 3 = ~ ...

". U U U _~ S

3~ ~ ~ o o o o o o o o o , . . . . . ..

N '9 0 IO

.

:~3;~85 HAl~Oa 1) 0 Nt--l ~i _I _I .--1 '.'`1 _I N --I

t,~
~ 3 ~ _ ~ 3 ~

,.

U ~ T

! J I ~ ~ U~
SC,) U ~ ~ o o o U ~ U 1~
t~ t-l _I t~ ~

a~ o ~ Ln ~D '.~0 ~' ' .

: ~ :
: ' ` ` ' ' ~ , .

~180a r' ~ 5~ ur~

I N _I N

3~ 2 = ~:

=

~ u~ ~ r-5~

U O U ' U C~ U

a~ o O O O O O O O

. .

HA180a IN
I N U
S ~S 2 N ~) ~
_~ u t~ u c u c~
o N _I ~ N _I _I N ~

~: 2 2 1 3: ~ -- S S

2 2 ~ ~ 5:

t~ U t.) S

N ~) N (~ O
(~) N N C~
o~ ~ ~ o o o o o u~ . tn N c~ J 2 ~ I N N ~

O O ~

, . . ~ .
. ~ . .

}i~180a --~7--u c~

' X

t~ U X C~ U
3: 1 5: ~ I I I I

~ (~(,)J ~ ) o $
~`J ~ 3 ~ ~ N ~ ~
U
u~ ~n o o o o u~

X
O O O O O O O O

1~a~ ~ o _, t~l ~er .. . .
.

,, ' ' ' ~13;~i HA180a --8~--Example 125 (S,S,S,S)-7,7'-~Dithiobis(2-methyl-1-oxo-3,1-propane-diyl)]bis~1,4-dioxa-7-azaspiro[4.4~nonane-8-carboxylic acid]
~7(S),8Sl-7-(3-Mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro~4.4]nonane-8-carboxylic acid t3.0 g., 0.0109 mole) from Example 2 is dissolved in 80 ml. of water and the pH is adjusted to 6.5 with lN sodium hydroxide. To this stirred solution is added dropwise a total of 11 ml. of 0.5 M iodine solution in 95% ethanol (6.34 g. iodine/50 ml. solution) while maintaining the pH at 5.5 to 6.5 with lN sodium hydrox-ide. After 15 minutes, a trace of excess iodine is removed with dilute sodium thiosulfate and ~e solution is concentrated to approx~tely 50 ml., ccoled and acidified with 1:1 hydrochloric acid. Methylene chloride (30 ml.) is added and the mixture is saturated with sodium chloride, stirred, and the layers separated. The aqueous phase is extracted with additional methylene chloride (3 x 20 ml.), the combined organic layers dried (MgSO4), and the solvent evaporated, finally at 0.2 mm. The brittle residue is rubbed under ether and the evaporation repeated to give 2.8 g. of a pale yellow solid residue. The material is redisso ved in 50 ml. of methylene chloride, washed with water (3 x 10 ml.), the combined a~ueous layers back-extracted with 20 ml. of methylene chloride, and the combined layers dried (MgSO4). Evaporation and tri-turation with ether as above yields 2.2 g. (73%) of cream-colored amorphous solid (S,S,S,S)-7,7'-, -. . . :~

1~32~ H~18Oa -8~-Edithiobis(2-methyl-1-oxo-3,1-propanediyl)lbis[1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid];
m.p. 61-63 (foaming), (s. 50), [a]26 -92 (c, 1~ in ethanol~.
Anal. Calc d- for C22~32 2 10 2 C, 46.63; H. 6.05; N, 4.94; S, 11.31 Found: C, 46.52; H, 6.29; N, 4.63; S, 10.96.
Example 126 (S,S,S,S)-7,7'-[Dithiobis(2-methyl-oxo-3,1-propane-diyl)]bis[7-aza-1,4-dithiaspiro[4.4]nonane-8-carboxylic acid]
[7~S),8S]-7-(3-Mercapto-2-methyl-1-oxopropyl)-

7-aza-1,4-dithiaspiro[4.4]nonane-8-carboxylic acid from Example 10 is reacted with iodine according to the procedure of Example125 to yield (S,S,S,S)-7,7'-~dithiobis(2-methyl-1-oxo-3,1-propanediyl)]bis-[7-aza-1,4-dithiaspiro[4.4]nonane-8-carboxylic acid].
Example 127 (S,S,S,S)-l,l'-[Dithiobis(2-methyl-1-oxo-3,1-pro-panediyl)]bis[4,4-dimethoxy-L-proline]
(5)-1-(3-Mercapto-2-methyl-1-oxopropyl)-4,4-dimethoxy-L-proline from Example 4 is reacted with iodine according to the procedure of Example 125 ~o yield (S,S,S,S)-l,l'-[dithiobis(2-methyl-1-oxo-3,1-propanediyl)]bis[4,4-dimethoxy-L-proline].
Example 128 ~7(S),8(S)]-7-[3-(Acetylthio)-2-methyl-1-oxopropyl]-1,4-dioxo-7-azaspiro~4.4]nonane-8-carboxylic acid, methyl ester A solution of the product of Example 1 in .~

-` 113298S
HAlaOa --so--ether is treated with a slight excess of diazo-methane. After standing at room temperature for two hours, the solvent is evaporated to give t7~S),8S]-7-13-(acetylthio)-2-methyl- 1-oxopropyl]-1,4-dioxo-7-azaspiro~4.4]nonane-8-carboxylic acid, methyl ester.
Example 129 ~7(S),8S]-7-(3-Mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspirot4.4]nonane-8-carboxylic acid, methyl ester The product from Example 128 is hydrolyzed with concentrated ammonia according to the procedure of Example 2 to yield ~7(S),8S]-7-(3-mercapto-2-methyl-l-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-

8-carboxylic acid, methyl ester.
Example 130 (S)-1-~3-(Acetylthio)-2-methyl-1-oxopropyl]-4,4-dimethoxy-L-proline, methyl ester A solution of the product from Example 3 in ether is treated with a slight excess of diazo-methane. After standing at room temperature, the solvent is evaporated to give (S)-1-[3-(acetylthio)-2-methyl-1-oxopropyl]-4,4-dimethoxy-L-proline, methyl ester.
Example 131 (S)-1-(3-Mercapto-2-methyl-1-oxopropyl)-4,4-dimethoxy-L-proline, methyl ester The product from Example 130 is hydrolyzed with concentrated ammonia according to the procedure of Example 4 to yield (S)-1-(3-mercapto-2-methyl-1-. . . ; ~, .

`` li3~5 HA180a oxopropyl)-4,4-dimethoxy-L-proline, methyl ester.
Example 132 [7(S),8S] _-t3-Mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro~4.4]nonane-8-carboxylic acid, sodium salt A solution of 1.0 g. of the product of Example 2 is dissolved in 10 ml. of water and treated with one equivalent of sodium bicarbonate. The solution is freeze-dried to give[7(S),8S]-7-(3-mercapto-2-methyl-1-oxopropyI)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid, sodium salt.
In a similar manner, by employing potassium bicarbonate the corresponding potassium salt is obtained.
Example 133 (S)-1-(3-Mercapto-2-methyl-1-oxopropyl)-4,4-dimethoxy-L-proline, sodium salt A solution of 1.0 g. of the product of Example 4 is dissolved in 10 ml.of water and treated with one equivalent of sodium bicarbonate. The solution is freeze-dried to give (S)-1-(3-mercapto-2-methyl-1-oxopropyl)-4,4-dimethoxy-L-proline, sodium salt.
In a similar manner, by employing potassium bicarbonate the corresponding potassium salt is obtained.
Example 134 1000 tablets each containing the following ingredients:
[7(S),8S]-7-(3-Mercapto-.

~132~
HA18Oa 2-methyl-1-oxopropyl)-1, 4-dioxo-7-azaspiro~4.4]nonane-8-carboxylic acid 100 mg.
Corn starch 50 mg.
Gelatin 7.5 mg.
~vicel (microcrystalline cellulose) 25 mg.
Magnesium stearate 2.5 mg.
185 mg.
10 are prepared (from sufficient bulk quantities) by mixing the [7(S),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid and corn starch with an aqueous solution of the gelatin. The mixture is dried and lS ground to a fine powder. The Avicel and then the magnesium stearate are admixed with granulation.
This mixture is then compressed in a tablet press to form 1000 tablets each containing 100 mg. of active ingredient.
Example 135 Tablets each containing 100 mg. of (S)-1-(3-mercapto-2-methyl-1-oxopropyl)-4,4-dimethoxy-L-proline are produced as described in Example 134.
Example 136 1000 tablets each containing 50 mg. of [7(S), 8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid are produced from the following ingredients , ` '' ~
~.

'' ~132~5 HA180a _93_ [7(S),8Sl-7-(3-mercapto-2-methyl-l-oxopropyl)-1,4-dioxo-?-azaspiro[4.4]nonane-8-carboxylic acid 50 g.
Lactose 100 g.
Avicel 150 g.
Corn starch S0 g.
Magnesium stearate S g.
The 17(S),8S]-7-[3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid, lactose, and Avicel are admixed, then blended with the corn starch. Magnesium stearate is added. The dry mixture is compressed in a ta~blet press to form 1000 355 mg. tablets each containing 50 mg. of active ingredient. The tablets are coated with a solution of Methocel E 15 (methyl cellulose) including as a color a lake containing yellow #6.
Example 137 Tablets each containing 50 mg. of (S)-l-(3-mercapto-2-methyl-l-oxopropyl)-4~4-dimethoxy-L
proline are produced as described in Example 136.
Example 138 Two piece #l gelatin capsules each containing 100 mg. of [7(S),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid, sodiun salt, are filled with a mixture of the following ingredients:

.

"
, : . -.
" , . , .. ,. ~
.

:

1~3Z~ HA18Oa ~7(S),8S]-7-(3-mercapto-2-methyl-l-oxopropyl)-1,4-dioxo-7-azaspirot4.4]nonane-8-carboxylic acid, sodium salt 100 mg.
Magnesium stearate 7 mg.
Lactose 193 mg.
Example 139 Gelatin capsules containing 100 mg. of (S)-1-(3-mercapto-2-methyl-1-oxopropyl)-4,4-dimethoxy-L-proline, sodium salt are produced as described in Example 138.
Example 140 An injectable solution is produced as follows:
[7(5),8S]-7-(3-Mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid 500 g.
Methyl paraben 5 g.
Propyl paraben 1 g.
Sodium chloride 25 g.
Water for injection qs. 5 The active substance, preservatives and sodium chloride are dissolved in 3 liters of water and then the v~lume is brought up to 5 liters. The solution is filtered through a sterile filter and aseptically filled into pre-sterilized vials which are then closed with pre-sterilized rubber closures. Each vial contains 5 ml. of solution in a concentration of 100 mg of , ';............ ' , ~:,.

. '-'~ ' . ~

... .

HA180a _95_ active ingredient per ml. of solution for injection.
Example 141 An injectable solution containing (S)-l-(3-mercapto-2-methyl-1-oxopropyl)-4,4-dimethoxy-L-proline is prepared as described in Example 140.
Example 142 6000 tablets each containing the following ingredients:
[7(S),8S3-7-(3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro-[4.4]nonane-8-carboxylic acid100 mg.
Avicel ~microcrystalline 100 mg.
cellulose) Hydrochlorothiazide - 12.5 mg.
Lactose U.S.P. 113 mg.
Corn starch U.S.P. 17.5 mg.
Stearic acid U.S.P. 7 mg.
350 mg.
are produced from sufficient bulk quantities by slugging the 17tS),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid, Avicel and a portion of the stearic acid. The slugs are ground and passed through a #2 screen, then mixed with the hydrochlorothiazide, lactose, corn starch and remainder of the stearic acid. The mixture is compressed into 350 mg. capsule shaped tablets in a tablet press. The tablets are scored for dividing in half.

:`

~ zg85 - HA180a Example 143 Tablets each containing (S)-1-(3-mercapto-2-methyl-l-oxopropyl)-4,4-dimethoxy-L-proline and hydrochlorothiazide can be prepared as described in Example 142.
The product of Examples 1, 3 and 6 to L33 can also be formulated according to the procedures of Examples 134-143.

- ~ :
. : - ~. :

Claims (28)

HA180a The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A process for preparing a compound of the formula or a pharmaceutically acceptable salt thereof, wherein R is hydrogen or lower alkyl;
R1 and R2 are independently selected from the group consisting of lower alkyl, or R1 and R2 join together in a polymethylene chain of the formula X1 and X2 are independently selected from the group consisting of oxygen and sulfur;
R3 and R4 are independently selected from the group consisting of hydrogen, lower alkyl, lower alkylthio, -(CH2)n-SH, and halo substituted lower alkyl;
R6 is hydrogen or lower alkyl provided that R6 is lower alkyl only when R3 is lower alkyl;
m is zero, one or two;
n is one, two or three;
p and q are each one or two provided that both are not two;
t is two or three;
R9 and R10 are both hydrogen, both lower alkyl, or one is hydrogen and the other is lower alkyl;
R5 is hydrogen, a hydrolyzably removable protecting, a chemically removable protecting group, or provided that neither R3 or R4 is -(CH2)n-SH

HA180a characterized by:
(1) coupling a substituted proline or pipecolic acid of the formula with an acid of the formula wherein R5 is hydrogen or a hydrolyzably or chemically removable protecting group, or (2) treating a compound of the formula wherein R5 is hereinabove defined with alcohols or thiols selected from the group consisting of R1X1H
R2X2H and to form a product wherein R5 is hydrogen or a hydrolyzably or chemically removable protecting group, and removing said protecting group to form a product wherein R5 is hydrogen, and oxidizing said products wherein R5 is hydrogen to form the product wherein R5 is

2. The process of claim 1 wherein p and q are both one; R1 and R2 each is lower alkyl of 1 to 4 carbons or form two joined methylene groups; R3 and R4 each is hydrogen or lower alkyl of 1 to 4 carbons; R5 is hydrogen, lower alkanoyl of 1 to 4 carbons, or benzoyl; R6 is hydrogen, and m is zero or one.

3. The process of claim 1 wherein X1 and X2 each is oxygen; R, R4, R5 and R6 each is hydrogen; R1 and R2 each is lower alkyl of 1 to 4 carbons or R1 and R2 each is methylene and join to complete the ethylenedioxy ring;
R3 is methyl; and m is one.

4. The process of claim l wherein X1-R1 and X2-R2 complete an ethylenedioxy ring.

5. The process of claim 1 wherein R is hydrogen;
R4 is hydrogen; R3 and R6 are both lower alkyl of 1 to 4 carbons or R6 is hydrogen and R3 is hydrogen, lower alkyl of 1 to 4 carbons, trifluoromethyl, methylthio, or mercaptomethyl; R5 is hydrogen, lower alkanoyl of 1 to 4 carbons, or benzoyl; X1 and X2 are the same; and R1 and R2 are lower alkyl of 1 to 4 carbons.

HA180a

6. The process of claim 1 wherein R, R4, R5 and R6 are hydrogen; R3 is methyl; m is one; and R
and R2 are both methyl or ethyl.

7. The process of claim 1 wherein 3-mercapto-2-methyl-propionic acid is coupled with 4,4-dimethoxy-L-proline to form (S)-1-(3-mercapto-2-methyl-1-oxo-propyl)-4,4-dimethoxy-L-proline.

8. The process of claim 1 wherein 3-mercapto-2-methyl-propionic acid is coupled with 4,4-diethoxy-L-proline to form (S)-1-(3-mercapto-2-methyl-1-oxopropyl)-4,4-diethoxy-L-proline.

9. The process of claim 1 wherein 3-mercapto-2-methyl-propionic acid is coupled with 4,4-ethylenedioxy-L-proline to form [7(S),8(S)]-7-(3-mercapto-2-methyl-l-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid.

10. The process of claim 1 wherein 3-mercapto-2-methyl-propionic acid is coupled with 4,4-trimethylene-dioxy-L-proline to form [2(S),3S]-2-(3-mercapto-2-methyl-l-oxopropyl),-6,10-dioxo-2-azaspirol4.5]deccane-3-carboxylic acid.

11. The process of claim 1 wherein 3-mercapto-2-methyl-propionic acid is coupled with 4,4-ethylenedithio-L-proline to form [7(S),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-7-aza-1,4-dithiaspirol4.4]nonane-8-carboxylic acid.

12. The process of claim 1 wherein 3-mercapto-2-methyl-propionic acid is coupled with 4,4-(1-methylene-dioxy)-L-proline to form [7(S),8S]-7-(3-mercapto-2-methyl-l-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-2-methyl-8-carboxylic acid.

13. The process of claim 1 wherein 3-mercapto-2-trifluoromethyl-propionic acid is coupled with 4,4-ethylenedioxy-L-proline to form (8S)-7-(3-mercapto-2-trifluoromethyl-l-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]-nonane-8-carboxylic acid.

14. The process of claim 1 wherein 3-mercapto-2-methyl-propionic acid is coupled with 4,4-ethylenedioxy-L-proline to form 17(S),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid which is then oxidized to form (S,S,S,S)-7,7l--[dithiobist2-methyl-1-oxo-3,1-propanediyl)]bis(1,4-dioxa-7-azaspiro[4.4]nonane-8-carboxylic acid. 100 HA180a

15. A compound of the formula or a pharmaceutically acceptable salt thereof, wherein R is hydrogen or lower alkyl;
Rl and R2 are independently selected from the group consisting of lower alkyl, or R1 and R2 join together in a polymethylene chain of the formula Xl and X2 are independently selected from the group consisting of oxygen and sulfur;
R3 and R4 are independently selected from the group consisting of hydrogen, lower alkyl, lower alkylthio, -(CH2)n-SH, and halo substituted lower alkyl;
R6 is hydrogen or lower alkyl provided that R6 is lower alkyl only when R3 is lower alkyl;
m is zero, one or two;
n is one, two or three;
p and q are each one or two provided that both are not two;
t is two or three;
Rg and R1o are both hydrogen, both lower alkyl, or one is hydrogen and the other is lower alkyl;
R5 is hydrogen, a hydrolyzably removable protecting, a chemically removable protecting group, or provided that neither R3 or R4 is -(CH2)n-SH

HA180a whenever prepared by the process of claim 1.

16. A compound according to claim 15 wherein p and q are both one; R1 and R2 each is lower alkyl of 1 to 4 carbons or form two joined methylene groups; R3 and R4 each is hydrogen or lower alkyl of 1 to 4 carbons; R5 is hydrogen, lower alkanoyl of 1 to 4 carbons, or benzoyl; R6 is hydrogen, and m is zero or one, whenever prepared by the process of claim 2.

17. A compound according to claim 15 wherein X1 and X2 each is oxygen; R, R4, R5 and R6 each is hydrogen; R1 and R2 each is lower alkyl of 1 to 4 carbons or R1 and R2 each is methylene and join to complete the ethylenedioxy ring; R3 is methyl; and m is one, whenever prepared by the process of claim 3.

18. A compound according to claim 15 wherein X1-R1 and X2-R2 complete an ethylenedioxy ring, whenever prepared by the process of claim 4.

19. A compound according to claim 15 wherein R is hydrogen;
R4 is hydrogen; R3 and R6 are both lower alkyl of 1 to 4 carbons or R6 is hydrogen and R3 is hydrogen, lower alkyl of 1 to 4 carbons, trifluoromethyl, methylthio, or mercaptomethyl; R5 is hydrogen, lower alkanoyl of 1 to 4 carbons or benzoyl; X
and X2 are the same; and R1 and R2 are lower alkyl of 1 to 4 carbons, whenever prepared by the process of claim 5.

20. A compound according to claim 15 wherein R, R4, R5, and R6 are hydrogen; R3 is methyl; m is one; and R1 and R2 are both methyl or ethyl, whenever prepared by the process of claim 6.

21. A compound according to claim 15 having the name (S)-1-(3 -mercapto- 2 -methyl-1-oxopropyl)-4,4-dimethoxy-L-proline, whenever prepared by the process of claim 7.

HA180a

22. The compound according to claim 15 having the name (S)-1-(3-mercapto-2-methyl-1-oxopropyl)-4,4-diethoxy-L-proline, whenever prepared by the process of claim 8.

23. A compound according to claim 15 having the name [7(S),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid, whenever prepared by the process of claim 9.

24. A compound according to claim 15 having the name [2(S),3S]-2-(3-mercapto-2-methyl-1-oxopropyl)-6,10-dioxo-2-azaspirol4.5]decane-3-carboxylic acid, whenever prepared by the process of claim 10.

25. A compound according to claim 15 having the name [7(S),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-7-aza-1,4-dithiaspiro[4.4]nonane-8-carboxylic acid, whenever prepared by the process of claim 11.

26. A compound according to claim 15 having the name [7(S),8S]-7-(3-mercapto-2-methyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-2-methyl-8-carboxylic acid, whenever prepared by the process of claim 12.

27. A compound according to claim 15 having the name (8S)-7-(3-mercapto-2-trifluoromethyl-1-oxopropyl)-1,4-dioxo-7-azaspiro[4.4]nonane-8-carboxylic acid, whenever prepared by the process of claim 13.

28. A compound according to claim 15 having the name (S,S,S,S)-7,7'-[dithiobis(2-methyl-1-oxo-3,1-propanediyl)]-bis(l,4-dioxa-7-azaspiro[4.4]nonane-8-carboxylic acid, whenever prepared by the process of claim 14.