DK154652B - L-ASPARTYL-D-AMINO ACID DIPEPTIDAMIDES AND D-AMINO ACIDAMIDS FOR USE IN THEIR PREPARATION - Google Patents

Description

DK 154652 B

The invention relates to novel L-aspartyl-D-amino acid dipeptide amides, with the general formula I as defined in claim 1, as well as physiologically acceptable cationic salts and acid addition salts thereof.

These compounds are distinguished by their powerful sweetening properties. Furthermore, the invention relates to novel compounds for use as starting compounds in the preparation of the subject amides.

In U.S. Patent No. 3,492,131, certain lower-10 alkyl esters of L-aspartyl-L-phenylalanine are stated to be up to 200 times as sweet as sucrose and substantially free of bitter odor which impaired prior artificial sweeteners such as saccharin. These compounds were then found to have only limited stability in aqueous systems due to diketopiperazine formation, especially at the neutral to acidic pH conditions prevalent in most food systems.

Mazur et al., J. Med. Chem., 16, page 1284 (1973), has described that lower alkyl esters of L-aspariyl-D-20 alanine and certain homologues thereof, especially L-aspartyl-D-alanine isopropyl ester, have sweetening properties up to 125 times sucrose.

Sukehiro et al., Seikatsu Kagaku, 11, pp. 9-16 (1977); Chem. Abstr., 87, 168407h (1977), disclosed 25 certain amides of L-aspartyl-D-alanine of the formula NH 2 0 nhr 1 COOH 0 CH 3 wherein R 1 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl , secondary butyl, cyclohexyl or the carbon residue of the methyl esters of glycine, d-alanine or 1-alanine. The most potent compounds were those in which R 1 is one of the above-mentioned butyl groups or cyclohexyl with 100-125 and 100 times the sweetness of sucrose respectively. Since the n-butyl amide was found to have 125 times the sweetness and iso-

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butyl and secondary butyl amides 100 times sucrose, it was concluded that the sweetness of these amides is affected substantially by the number of carbon atoms in the alkyl group r and that structural isomerism in the alkyl group has little effect on the sweetness.

Esters of L-aspartyl-D-serine and L-aspartyl-D-threonine have Ariyoshi et al., Bull. Chem. Soc. Japan, 47, page 326 (1974), found to be sweeter than the corresponding esters of L-aspartyl. -D-alanine and L-aspartyl-D-2-aminobutyric acid, respectively. The most powerful of these 10 esters, L-aspartyl-D-serine-n-propyl ester, was 320 times as sweet as a 5% sucrose s tandard, U.S. Patent No. 3,971,822 describes esters of L-aspartyl-D -alaninol with carboxylic acids, including 2-methyl-butter, cyclopropane carboxylic, cyclobutane carboxylic and 2-15 methylcyclobutane carboxylic acids. The esters of cyclopropane and cyclobutane carboxylic acid were 200 and 220 x sucrose, respectively. The ester with 2-methylcyclobutane carboxylic acid was only 160 times sucrose. Also described are similar L - asparty 1 -D serinol esters, of which the sweetest, the ester with 20 propionic acid, is 160 times sucrose.

U.S. Patents 3,959,245 and 3,907,766 disclose L-aspartylaminomalonic acid methyl-2-methyl-cyclohexyl diester and the corresponding alkylphenyl diester, respectively.

The first is stated to be 6600 times sucrose, the last 25 4200-33000 times sucrose. In a related publication by the inventors, chem. Pharm. Bull., 24, page 2112 (1976), discloses a series of Ti-aspartylaminomalonic acid diesters, one of the ester groups being methyl or ethyl, the other being one of a number of branched alkyl and cycloalkyl groups.

U.S. Patent No. 4,111,925 found that not only is the size of the amide substituent critical to a high degree of sweetness in L-aspartyl-D-alanine amides, but on the contrary, it is the exact spatial arrangement of the amide substituent R, that is critical. Certain L-aspartyl-D-alanine amides branched at ·. x, the carbon network (the carbon carrying the amide nitrogen atom), which in turn is 3

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branched by one or both (Jtf 'and', 3} carbon atoms, were found to have significant advantages.

The present invention provides certain novel L-aspartyl, -D-amino acid di-peptidamides, which have an unexpectedly high sweetness, are free of undesirable odor properties at conventional levels of use and surprisingly have high stability in both solid and aqueous systems. above the pH range found in most food systems, even at the elevated temperatures, 10 used in baking and conventional food processing.

The novel compounds of the invention are L-aspartyl-D-amino acid dipeptide amides of formula Ia - (I)

COOH R

20 as well as physiologically acceptable cationic salts and acid addition salts thereof, wherein formula R is Cl 2 OH or CH 2 OCH 2 and R is a branched group selected from fenchyl, diisopropylcarbinyl, d-methyl-t-butylcarbinyl, d-ethyl-t butylcarbinyl, di-t-butylcarbinyl, 2-methylthio-2,4-dimethylpentane

3_y1 '3 R1 R3 X

RS <7ScH2> n _

RT rs R V

30 R

wherein at least one of R 1, R 1, R 3 and R 4 is alkyl having from one to four carbon atoms and the balance is hydrogen or alkyl having from one to four carbon atoms; the sum of the carbon atoms 1 R, R, R and 6 3 35 R is not greater than 6 and if both R and 5 6 R or R and R are alkyl, they are methyl or ethyl; X is O, S, SO, SO2, C = O or CHOH; m is zero, 1, 2, 3 or 4 / and n and p are each zero, 1, 2

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4

"V

5 η 7 8 9 where m is as defined above, one of R, R and R and 10 alkyl having from one to four carbon atoms and the remainder being hydrogen or alkyl having from one to four carbon atoms, and 7 8 9 the sum of the carbon atoms in R, R and R are not greater than six; 15 j-fCH2> m - <k> q 20 * where m has the meaning given above and q is 0, 1, 2, 3 or 4; R12 R13 25 y- <C | 2> t

ISLAND

12 13 12

wherein each of R and R is methyl or ethyl, or R

13 is hydrogen and R is alkyl having from one to four carbon atoms, Z is O or NH and t is 1 or 2;

R16 ^ 5T

35

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5 wherein w is 0, 1, 2, 3 or 4, R 1 and R 16 are each alkyl 15

with from one to four carbon atoms, R is hydrogen, OH

or alkyl having from one to two carbon atoms where the sum of 14 15 16 of the carbon atoms in R, R and R is not greater than 14 15 six and if both R and R are alkyl, they are methyl or ethyl, and R 17 Wherein R and R are alkyl having from one to four carbon atoms, R and R are hydrogen or alkyl having from one to two carbon atoms, and when taken individually, A is OH, and B is hydrogen, OH or methyl and, if taken together, A and B are -CHOOC-, -CH9NHC-, -OCCH9-, -NHCCH--, -OC-, -ώ ti ~ ιι II ^ il * ti 0 0 0 0 0 17 -NHC 'or -OCO- where the sum of the carbon atoms in R

II II

o o 29 R 18, R 19 and R 29 are not greater than six, and if both R 1 and R 2 or R 9 and R 29 are alkyl, they are methyl or ethyl.

A particularly preferred group of L-aspartyl-D-amino acid amides of formula (I) are those wherein R is an acyclic group selected from the group consisting of diisopropyl-carbinyl, d-methyl-t-butylcarbinyl and di t-butylcarbinyl.

Another particularly preferred group of L-aspartyl-D-amino acid amides of formula (I) are those wherein R is a group selected from the group consisting of

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6 R1 R2 R1 -R2

~ \ \ __X

R6 ^ \ 5 A- (CH2> e 5 R R R3 4 5

RV6 D

, o O <"<CH2) m '- (CR) - (CH0) q 2'm 15 r1 ^, R13 ε14 \ / ε15

- <T2> t -O-A

- R 2 -O

Wherein R-R, R-R, A, B, X, Z, m, n, p, g, t and w are defined as above, and are particularly preferred 30 linkages of formula (I) wherein R has one of the first 3 four values in the group immediately above.

4 Particularly preferred amides of formula (I) are the L-5 in 6 aspartoyl-D-serinamides, i. those wherein R is CR 2 OR.

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7

By physiologically acceptable cationic salts of the compounds of the invention is meant those salts formed by neutralizing the free carboxylic acid group of the compounds of formula I with bases of physiologically acceptable metals, ammonia and amines. Examples of 5 such metals are sodium, potassium, calcium and magnesium. Examples of such amines are N-methylglucamine and ethanolamine.

By the term physiologically acceptable acid addition salts is meant the salts formed between the free amino group of the compound of formula I and a physiologically acceptable acid. Examples of such acids are acetic, benzoate, hydrogen bromide, salt, lemon, fumar, gluconr, lactic, maleic, apple, nitric, phosphorus, saccharin, succinic and tartaric acids.

The invention further provides novel D-amino acid amide compounds for use as starting compounds in the preparation of compounds of formula I and peculiar in that they have the general formula 20

Ra-CHCONHR ° NH₂ wherein Ra is C ^OH or CE ^OCH ^ and Rc is a group selected from phenyl, diisopropylcarbinyl, d-methyl-t-butylcarbinyl, d-ethyl-t-butylcarbinyl, di-t-butyl -carbinyl, cyclopropyl-t-butylcarbinyl, cyclopentyl-t-butylcarbinyl, dicyclopropylcarbinyl, 30 V ° P40

K

γ> Η2, ™ ι r6 ° Ar50 which is 1, 2 or 3 and if m ^ is 1: R R, R4 ^, R ^^ og and R ^ are each methyl,

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8 if m is 2: R is methyl, ethyl or isopropyl and R 40, R 30 and R 60 are each hydrogen, or R and R are each methyl and R and 60 are each hydrogen and if m 1 is 3: 30 (A) R is isopropyl or t-butyl and R, R3, 1 and R3® are each hydrogen, (b) R30 is ethyl, R33 is methyl, and R40 and R are each hydrogen, or (c) R33 and R 43 each is methyl and R 33 and cr \ 10 R each are hydrogen or methyl, and V (CH 2> pr 2 CH 2 1 1) 1 in 61 wherein n 2 and p 2 are each 0, R and R each are methyl, and X ~ is S, SO0, C = 0 or CHOH, or n2 is 0, and p2 is 1 R4 and R ° are each methyl, and X- is 0, S or SO ~, or ^^ 4 n1 is 1, and p 2 is 1, R 1 and R 2 are each hydrogen and X 2 is S or SO 2

The suffix "carbinyl" is used here to denote the part -CH-. Thus, e.g. the diisopropylcarbinyl group (i-C-jH-) O-CH- and dicyclopropylcarbinylamine is 25 J Δ (A) 2chnh2.

Said dipeptide amides are conveniently prepared by methods suitable for coupling amino acids.

A preferred method for preparing the dipeptidamides of formula I is outlined below.

30 35

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9 / NH ^ 5______11 (1) condensation v L- / -ζ + D-NH₂iHCOOR (2) -H₂O- *

COOR10 COOH

or carboxyl-activated derivative 5 NHQ a _ mQ Ra / \ Λ RNH2 v L '

COOR10 COHN COOH - “~ ^ COOR10 COHN CONHR

10 (II) γ / from (III) γ / Cleavage (I) In the above L-aspartic acid derivative, Q is one of the 15 well-known amino protecting groups which can be selectively removed, such as those described by Boissonnas, Advances in Organic Chem , Vol. 3, pages 159-190 (1963). Particularly preferred amino protecting groups are benzyloxy-carbonyl and tert-butyloxycarbonyl. R 10 is preferably an alkyl group having from one to four carbon atoms or benzyl. The D-serine or DO-methylserine used may be in the form of the free amino acid in which R 11 is hydrogen, but it is preferably a carboxyl protected derivative, 11 wherein R may be the residue of an ester group such as methyl 25 or ethyl, but preferably a silyl group such as trialkylsilyl having from three to twelve carbon atoms. One such preferred group due to economy and efficiency is trimethylsilyl.

In the first step of the above reaction sequence, the double-protected L-aspartic acid is condensed with the appropriate D-amino acid or a carboxy-protected derivative to give the double-protected dipeptide of formula II. Although this step can be carried out with the double-protected aspartic acid in the presence of condensing agents such as e.g. dicyclohexylcarbodiimide, it is preferred to use an (oC) carboxyl activated derivative of the double protected aspartic acid. such

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10 preferred carboxyl activated derivatives are the chloride, bromide, anhydride or mixed anhydride. Particularly preferred because of the efficiency are the mixed anhydrides of the above double-protected L-aspartic acid with estereaf chlorocarbon acid, especially those alkyl esters wherein said alkyl moiety has from one to four carbon atoms. The most preferred mixed anhydrides are because of costs made from the methyl and ethyl esters of chlorocarbon acid.

In a preferred process for the preparation of the compounds of Formula I, (benzyl) -N-benzyloxycarbonyl-L-aspartic acid is reacted with ethyl chlorocarbon to give the corresponding mixed anhydride by methods known in the art. In a separate vessel, the D-amino acid / RaCH (NH 2) COOH obtained from 15 commercial sources or by isolation from the racemic amino acid is converted by known methods [see e.g. Yamada et al., J. Org. Chem., 38, 4408 (1973)], to the trimethylsilyl ester by contacting the amino acid with an equimolar amount of trimethylsilyl chloride in the presence of a reaction-inorganic solvent; in the case where Ra is CE 2 OH, two molar equivalents of silylating agent are commonly used. Suitable solvents for this purpose are e.g. pyridine, dimethylformamide or dimethylacetamide, particularly preferred is dimethylformamide.

In a typical reaction following this method, the D-amino acid, e.g. D-O-methylserine, dissolved in dimethylformamide and an equimolar amount of trimethylchlorosilane at room temperature. In a separate flask, β-benzyl-N-benzyloxycarbonyl-L-aspartic acid and a molar excess of an acid-binding agent, preferably triethylamine, are dissolved in a mixture of dimethylformamide and tetrahydrofuran, and an equimolar amount of ethyl chloro carbonate is added. room temperature or below, preferably at approx. -25 to 25 ° C and especially at approx. -10 to 0 ° C to form the mixed anhydride. To this is added the solution of e.g. D-O-methylserine trimethylsilyl ester, 11

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preferably at a temperature within the same range. The reaction is generally completed within one to two hours, after which the reaction mixture is poured into water or aqueous acid, e.g. hydrochloric acid and the product of formula II is extracted with a water-immiscible solvent, typically chloroform, methylene chloride or ethyl ether, and isolated by standard methods. The double-blocked dipeptide (II) generally has a sufficient purity to be used in the next step, but can, if desired, be further purified e.g.

10 by column chromatography.

In the second step of this process, the double-blocked dipeptide II is reacted with an equimolar amount of primary amine of formula RNH2 to obtain the corresponding double-blocked dipeptidamide intermediate α 10 of formula III wherein R, R, R and Q are as previously defined. As in the first step, the carboxylic acid form of reactant II can be used successfully using condensing agents, e.g. dicyclohexylcarbodiimide to give intermediates of formula III. However, it is preferred to convert the compound of formula II into a carboxyl activated derivative e.g. the chloride, bromide or mixed anhydride, the latter being preferred. Thus, using the particularly preferred compound of formula II wherein R R is benzyl and Q is benzyloxycarbonyl, the mixed anhydride is prepared. As above, the preferred anhydrides are those obtained from esters of chlorocarbon acid and The 30 mixed anhydrides of Compound II are prepared using reactants and conditions as described above for the first step of this scheme. In a typical reaction, the compound of Formula II and triethylamine are combined for approx. equimolar amounts in a reaction inert organic solvent, for example tetrahydrofuran, the mixture is cooled to about -10 ° C and ethyl chlorocarbonate is added to give the mixed anhydride, then an equimolar amount of amine is added.

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Or a solution thereof, for example, in the same reaction-inert solvent and at a temperature in the range of ca. -50 to 25 ° C, and preferably at -35 to -5 ° C. After completion of the addition of the amine, the reaction mixture is allowed to warm to ca.

5 to room temperature and maintained at this temperature until the reaction is substantially complete, generally from ca. 1 to .20 hours. The desired intermediate of formula IH is then isolated and purified, if desired, by the same methods described above for Compound II.

In the final step of this process, the carboxyl protecting group R10 and the amino protecting group Q are removed to obtain the desired dipeptide amides of formula I.

The method chosen to remove the protecting groups from the dipeptide amide of formula III will vary according to many different factors which will be apparent to those skilled in the art. Two major factors for such selection are the nature of the protecting groups R and Q and the nature of the amide substituent R. For example. if R and Q, respectively, are the particularly preferred groups benzyl and benzyloxycarbonyl and R does not contain sulfur, a preferred method of removing said protecting groups is

generally by hydrogenolysis. However, if 23 R R is benzyl or alkyl as defined above and Q

is tert-butyloxycarbonyl and R has one of the above values, it is generally preferred to remove the protecting groups by hydrolysis. A combination of hydrolysis and hydrogenolysis is preferred in cases where 30 R 3 · 0 is alkyl, Q is benzyloxycarbonyl and R does not contain sulfur.

If hydrogenolysis is selected to remove protecting groups from the intermediate of formula III, it is preferred to carry out the reaction in the presence of an o: catalytic amount of noble metal catalyst, palladium being particularly preferred and in the presence of a reaction inert solvent. Examples of such solvents

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13 include the lower alkanols such as methanol, ethanol, isopropanol and n-butanol, ethers such as tetrahydrofuran, ethyl ether, 1,2 dimethoxyethane and diethylene glycol dimethyl ether, esters such as ethyl acetate, methyl propionate and dimethyl succinate, and dimethylformamide. From economy 5 and efficiency, particularly preferred solvents are methanol and ethanol. Although hydrogenolysis can be successfully carried out at higher pressures and temperatures, it is preferable to use pressures from 1 to 10 atmospheres and room temperature based on economy and convenience. At the preferred temperature and pressure, the reaction is generally completed at from ca. 30 min. to approx. Typically, the catalyst is removed by filtration, the solvent is evaporated and the resulting product is purified if desired by standard methods, e.g. by recrystallization or column chromatography.

When hydrolysis is selected to remove one or both of the protecting groups R ** and Q, any of the well-known methods of alkaline or acidic hydrolysis of esters and the like can be used with some success. blocking groups by hydrolysis, however, alkaline hydrolysis is preferred, and particularly preferred conditions are the use of at least one equivalent amount of strong base, for example, sodium hydroxide or potassium hydroxide in the presence of water and a lower one.

2 S

alkanol, especially methanol or ethanol, at or about room temperature. Under these preferred conditions, hydrolytic removal of the R10 group is generally completed in a few hours or less.

If the amino protecting group Q is tert-butyloxycarbonyl, it is preferred to use acid hydrolysis for its removal. Particularly preferred is dilute aqueous hydrochloric acid in the presence of methanol or ethanol and heating the mixture at reflux.

Under these conditions, hydrolysis is generally completed in a few hours or less.

Isolation of the products of formula I after removal of protecting groups by any of the 35

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The above hydrolysis methods employ standard procedures well known in the art. For example, the reaction mixture is evaporated after acid hydrolysis to remove solvent, the aqueous residue is washed with a nonpolar solvent which is immiscible with water, e.g. ethyl ether or chloroform, after which the aqueous layer is made alkaline and the product is extracted with a water-immiscible solvent such as e.g. ethyl acetate and the product is obtained by evaporation of the solvent. If desired, the product can be further purified e.g. by recrystallization or column chromatography. If alkaline hydrolysis to remove a protecting group is followed by hydrogenolysis to remove the amino protecting group Q, the reaction mixture from the alkaline hydrolysis is preferably neutralized by the addition of acid e.g. hydrochloric acid, and the neutralized reaction mixture is subjected to hydrogenolysis as described above.

Another preferred process for preparing the subject compounds of formula I is shown below.

25 30 35

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and 15 a

Ra Ra »l

D-QNHCHCOOH + RNH0 QNHCHCONHR

or a carboxyl-activated derivative 5

NHQ

a ίΓ ^

cleavage? C00R C00H

-> NH 2 CHCONHR -> (III) -> (I) (V) a 10 wherein R, R, R and Q are as defined above.

The amino-protected D-amino acid or its carboxyl-activated derivative is reacted with an equimolar amount of amine RNI RN using methods and conditions as described above for the preparation of intermediates II and III to obtain an amino-protected D-amino acid amide of formula IV. The protecting group 20 Q is removed by hydrogenolysis or hydrolysis as described above, and the resulting free aminoamide V is condensed with a double-blocked L-aspartic acid derivative or a carboxyl-activated derivative thereof, as described above for the preparation of Formula II intermediates, to obtain the diblocked dipeptidamide of formula III, from which the desired dipeptidamide of formula I is obtained as described previously.

By modifying this process, an intermediate of formula IV wherein R contains a cyclic or acyclic sulfide (-S -) can be oxidized to the corresponding sulfoxide or sulfone prior to its conversion to intermediate V and subsequent reactions as described above. , to give compounds of formula I wherein R is a sulfoxide or sulfone.

In a third preferred process for preparing the subject compounds, the D-amino acid amide of formula V described above is reacted with L-aspartic acid N-thiocarboxyanhydride to directly obtain

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In the course of this process, the intermediate V in a suitable solvent is contacted with an equimolar amount of L-aspartic acid-N-thiocarboxyanhydride at a mild alkaline pH at temperatures of from ca. -25 to 10 ° C to give the compound of formula I. The alkaline pH of this reaction is obtained by a strong base e.g. sodium hydroxide or potassium carbonate. Suitable solvents for this reaction are those which dissolve at least a portion of the reactants under the reaction conditions used without appreciably reacting with any of the reactants, and which allow the products formed by the reaction to be isolated with relative ease. Examples of such solvents for this reaction are water, tetrahydrofuran, 1,2-dimethoxyethane, diethylene glycol-dimethyl ether, dimethyl sulfide oxide, dimethylformamide and combinations thereof; preferred solvents are water and its mixtures with tetrahydrofuran. A preferred alkaline pH range for this reaction is from ca. 8 to 10, and in particular a pH of approx. 9. A particularly preferred temperature is in the range of approx. -10 to 0 ° C.

Under the above preferred conditions, the reaction is generally completed in 1 to 2 hours.

The product of formula I is then isolated by standard methods e.g. the pH of the reaction mixture is adjusted to the isoelectric pH of the product generally approx. pH 5.0 to 5.6 to precipitate the product of formula I, most of the solvent is removed by evaporation or filtration, and the feedstock is slurried with an organic solvent e.g. methanol, ethanol, ethyl ether, ethyl acetate or mixtures thereof. The product of formula I is then isolated by filtration. If desired, it can be further purified by e.g. recrystallization or column-3 chromato gra f i.

The sweetness of the compounds of the invention was determined by comparing their taste sweetness with sucrose. Aqueous solutions of the compound of formula I diluted to

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17 a suitable concentration range was compared to a sucrose standard by an expert taste panel. Comparisons were generally made with aqueous sucrose solutions of 7-9%, i.e. 7-9 g per 100 ml. Higher sucrose-5 concentrations have a particular mouthfeel that may affect the results, and lower sucrose concentrations are not representative of normal use situations. For example, a 0.014% solution of the compound of formula I is judged to be as sweet as a 7% sucrose solution, the compound's sweetness is 7 / 0.014 = 500 x sucrose. All the sweetness values listed herein for the compounds of the invention were determined by this method. At threshold concentrations (i.e., the lowest concentration at which sweetness is first noticed, which for sucrose is usually at concentrations in the range of 2-3%), the sweetness of a sweetener such as the compounds of the invention generally is twice that observed at comparing its taste sweetness with 7-9% solutions of sucrose.

The required amines of the formula RN 1, wherein R is defined as before, are either commercially available or can be obtained from readily available precursors. Eg. For example, the 2-alkylcyclohexylamines and 2,6-dialkylcyclohexylamines can be obtained by catalytic hydrogenation of the corresponding alkyl-substituted anilines. Many of the amines are obtained by reductive amination of the corresponding ketone, using a number of conditions known in the art. Eg. For example, reductive amination may be used with the well-known Leuckhart reaction using formic acid and formamide as reducing agents, see e.g. The overview in "Organic Reactions", Wiley and Sons, New York, Vol. 5, pages 301, 1949. Alternatively, the ketone in question can be reductively aminated using sodium cyanoborohydride. and ammonium acetate, see e.g. J. Amer. Chem. Soc., 93, 2897 (1971), or by ethanolic ammonia in the presence of a hydrogenation catalyst such as Raneynickel, platinum or palladium, see e.g. "Organic Reactions", Volume 4,

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18, page 174 (1948). Many of the amines of formula RN3 are obtained from the corresponding ketones by forming an oxime as an intermediate formed by reacting the ketone with hydroxylamine or its salts under conditions well known in the art. The oxime intermediate is then reduced by catalytic hydrogenation or by sodium in the presence of a lower alkanol at elevated temperatures. A particularly preferred process, particularly useful for reducing sulfur-containing ketone oxides, uses reduction of the oxime in ethanol and a molar excess of sodium at the reflux temperature of the mixture.

The necessary ketone precursors for the amines RNE 1 are either commercially available, known in the art, or prepared by known methods. Eg. can ketones of formulas VI and VII

4

20 = V'CHA KV <ch2 ^ X

R6 R5 R6 - ^ \ 5 x \

VI VII

2 Λ S 6 wherein R, R, R, R °, X, m, n and p are as defined above except those of formula VII, wherein X is C = 0, is obtained by alkylating the corresponding compounds wherein R 1, R 4, R 5 and R 6 are each hydrogen to give compounds of the above formulas, wherein from one to three of all 6 of R, R, R, R are alkyl as defined above. The alkylation is carried out e.g. using alkylating agents such as the appropriate alkyl halide or alkyl sulfate under neutral or alkaline conditions obtained with strong bases e.g. sodium hydride or sodium amide. Using the same method 35, compounds of formulas VI and VII wherein only 1, 2 or 3 of the substituents lying (a) to the keto group are alkyl can be converted to compounds of 34 the same formula wherein from two to four among R, R, 19 ·

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R 1 is alkyl. Geminal dialkyl compounds of Forms -A 4 5 6, layers VI and VII, wherein either R and R or R and R are alkyl, can be obtained from the appropriate monoalkyl compounds by blocking the unsubstituted α-position 5 before alkylation and then removing it. blocking group. Eg. For example, 2,2-dimethylcyclohexanone can be obtained by condensation of 2-methylcyclohexanone with ethyl formate in the presence of sodium methoxide, and the resulting intermediate can be alkylated as outlined below.

10

Cj + HCO2C2H5.

NaNH2 '15 o jj> Q] ^ V'0Ma H20' 0H-? (\ '·

Ketones of formulas VI or VII, wherein one or both of R and R are propyl or butyl, can be obtained by condensing the corresponding (a) unsubstituted compound with the aldehyde or ketone under alkaline conditions to an a or a, α'-alkylidene ketone intermediate which can then be hydrogenated to give the desired ketone.

The required cyclobutanones are obtained by methods described by Conia et al., Bull. Soc. chim. France, 726 (1963) and Conia, Ind. chim. Beige, 31, 981 (1966).

An alternative method for preparing the ketones of formulas VI and VII involves cyclizing an acyclic precursor. Eg. by means of the well-known Dieckmann cyclization of dicarboxylate esters and subsequent hydrolysis and decarboxylation, see e.g.

35 "Modern Synthetic Reactions", W.A. Benjamin, Menlo Park, California, 1972, page 740. The α-keto esters produced, especially those where no other α-substituent, can also be alkylated before hydrolysis and decarboxylation, if desired.

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20 ring. This reaction can also be used to give ketones VI and VII which are unsubstituted by the carbon atoms adjacent to the carbonyl group which can be alkylated as described above.

For the preparation of diketones of formula VII wherein X is C = 0, the keto group in an acyclic keto-dicarboxylate ester precursor is converted to a ketal or thioketal, e.g. dime thylke number ·, diethylthioketal, ethylenedioxyke number or ethylene dithioketal prior to Dieckmann cycling. Ester group hydrolysis and decarboxylation give a ketoketal which can be converted to the corresponding amino ketal by methods described above, followed by hydrolysis of the ketal group by methods well known in the art. The resulting amino ketone may be hydrogenated to the corresponding hydroxyamine (X = CHOH) if desired, by known methods, for example, by reduction with sodium borohydride.

2,2,4,4-Tetraalkyl-3-hydroxycyclobutylamines are prepared from the corresponding 1,3-dione by the procedure described in U.S. Patent No. 3,125,569.

The amines of the formula R 3 - / R 4 - R 5 R 5 o 6 wherein X is CHOH and R-R, n and p are as defined above, or N-protected derivatives thereof, e.g. N-benzyl oxycarbonyl derivatives may be oxidized e.g. with chromium trioxide for the corresponding compounds wherein X is C = 0. Alternatively, the hydroxyamine may first be reacted with e.g. a carboxyl activated derivative of an N-protected D-O-methylserine, and the resulting intermediate of formula IV wherein R is the said hydroxy-containing group is oxidized e.g. with chromium trioxide to give the corresponding ketone. The resulting ketone of formula IV is then converted to the desired product of formula I,

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Wherein R is a keto-containing group as desired above.

Some of the ketones of formula VII are also obtained from acyclic precursors derived from ketones of formula VIII

wherein R8, R4, R8 and R4 are as previously defined. Eg. four-membered ketones of formula VII are obtained, where X is O or S by bromination of VIII with two moles of bromine and the resulting a, a'-dibromo compound is cyclized with f.

15 sodium hydroxide to give an oxethanone or hydrogen sulfide to give a thiethanone. The corresponding five-membered ketones VII are obtained if VIII is first reacted with formaldehyde to give an α-hydroxymethyl intermediate, which is then brominated at the 20 α VII, wherein X is O or S, respectively.

Certain of the tetrahydropyran-4-ones and tetrahydro-thiapyran-4-ones of formula VII are obtained by adding 25 water or hydrogen sulfide to the appropriately substituted divinyl ketone.

Ketone intermediates of formula IX which can be converted to amines via the oxime are obtained by methods outlined below, wherein R, R, R and R are as defined above.

r2VÅ / R20 X R17 ΛΛ R17 —2% - => r19Xj r18 COOC-, Η ,. CO-C-H ^ OH O 0 ώ j 2 (X) (XI) (IX)

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22

The appropriately substituted acetic acetic ester X is condensed with formaldehyde e.g. under alkaline conditions, and the resulting hydroxymethylated intermediate XI is cyclized e.g. with heating in the presence of a mild acid or base, removing ethanol during its formation.

Bromination of acetic acid esters of formula X and subsequent treatment of the product with e.g. sodium hydroxide gives ketones of formula XII which are converted to the corresponding amine as described above.

R20 in R17 r20? r17 r191Y18 -? r19> 0v8 co2c2h5 0 * ~ °

15 XII

Alternatively, the ketolactones XII can be prepared by the procedure described in Zeit. Chemistry, 13, 11 (1973); Chem. Abstr., 78, 135596e (1973) by reacting the appropriate cyclobutane-1,3-dione with hydrogen peroxide.

The dibromo derivative of VIII described above can also be treated with alkali hydroxides e.g. sodium hydroxide under mild conditions to give the corresponding 1,3-dihydroxy ketone which is converted to the corresponding 1,3-dioxane-2,5-dione of formula XIII by reaction with phosgene. ~ n n r20 r17 r20 fl R17 R V ^ <Rl7 VI11> ijif ic i ^ iYr18 .c-ocl2 ^ Ri9i r r18

30 J »1® ^« 19L OH V

The Br Br $ (XIII) 5-oxime amino intermediate of XIII, upon treatment with sodium in ethanol, gives the corresponding 5-amino compound.

Treatment of a ketone monobromo derivative of formula VIII with e.g. ethyl malonate and subsequently t 23

DK 154652 B

hydrolysis, decarboxylation and ester formation of the resulting product gives the intermediates of formula XIV, which serve as precursors for the ketones XV, e.g. as shown below.

5 71) S R17 R20 X r17 r20 I. R17 * 19> Λ ^ 18 -> R1 ^^ 8 \ c02C2h5 C02C2H5 g

10 XIV XV

The ketolactones XV are then converted to the corresponding 4-amino compound, e.g. by reducing the oxime as described above.

The 1,3-dibromo ketone derivatives of VIII described above can also be converted to the corresponding 1,3-dimercaptoketones by reaction with at least two moles of sodium hydrogen sulfide. Treatment of the dimercaptoketone with reagents such as iodine, hydrogen peroxide or hypochlorous acid under disulfide forming conditions well known in the art yields ketones of formula XVI which are converted to amines by reduction of the oxime using e.g. sodium in ethanol.

R6 11 R3 25 5> wv

R S— S

(XVI)

Amines of formula XVII are obtained directly e.g. by the method of Nagase et al., Chem. Pharm. Bull. 17, 30 398 (1969) as shown below.

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24 12v R12. NaCN R1 ^ * _ / * NH0 R V-CHO CH2 ° _v. 13 ^> pCHO NH cl> 1? ^ \ CN2

^ r13 CH, OH 4 Rl3CH2OHCN

5 K 2 nh2 h + 10 (XVII)

Use of ethylene oxide in place of formaldehyde in the first step of the above Scheme 15 gives the corresponding 3-amino-2-pyrones.

NH2 12 t * · 20

Lactams corresponding to the above-mentioned lactone intermediates or those of formulas IX, XII, XV or XVII are obtained by reacting the lactone in question with ammonia; eg. the above-mentioned lactone is contacted with an excess of anhydrous ammonia in ethanol and the mixture is left stirring overnight at room temperature to give compounds of formula Γ 2 R12 30 ° T Y R13

Alternatively, certain lactam intermediates are obtained following Scheme 35

DK 154652 B

25 r2i? JL / R17 CH2 ° X T ^ R18 ir (X) -: - \ "19 Jo C H -

r (C.H.CH.) 0NH ^ k 2c2n5 Pd / C

5 6 5 2 2> (ch2c6h5) 2 r2 ° Xr17 "isY ^ * 18 ->" 19> '* 18 10 l CO2C2H5 ^ N / ^ 0 1 «H2

The resulting ketones are then converted to the necessary amines by methods described above.

The isomeric ketolactams are obtained by the following reaction scheme: Br2 ^ K2 ^ * 17. R2lA ^ 17 20 R19T R18 2 · (C6H5CH2) 2NH V9> f Tr ^ R19 ^ jjl8-

The corresponding 5-membered lactams are also obtained by methods described in U.S. Patent 3,125,569: NOH PCI, - 20 ft 17 30? 9 ^ S <^ e or H2 SO4, *

0 polyphosphoric acid O

Cyclic or open-chain α-hydroxy ketones or 35 α, α-dihydroxy ketones of the formulas

DK 154652 B

26 14 H15 R17 r18

R rA

° = (, (CH2) m or O 5 R16 OH R2C ^ R ^ 9 where R 1 -R 2 ^, m, A and B are defined as before, are prepared by bromination with one or two moles of bromine and treatment of the bromine or dibromo intermediate having a hydroxy-containing base, for example, sodium hydroxide or potassium hydroxide, as described above.

ISLAND ISLAND

1. Br2 CH3 \ ^ N ^ CH3

1 \ Ύ. NaOH, ^ L J OH

ethanol

20 O

In 2 Br2. CHhA ^ CH3 2 · Xioi ho- ^ Joh 25

Dicycloalkylketones XVIII and alkylcycloalkylketones XIX are prepared by reacting the appropriate acid halide and Grignard reagent using conditions and reagents well known in the art, e.g. as shown below 35

DK 154652B

27 COC1 MgCl - (CH2'm ri * rf -VVi 5 I- (CH2) L_ (CH2) m l_ ^ 2 q (XVIII) 8 / R7

coci RV

-MgCl R \ tr8 10 L (CH2, m ^ 0 ΓΊ

- (CH.J

2 m (xix) Amines of the formula RNH2, wherein R is defined as before, are also obtained by the well-known Hofmann reaction during conversion of the appropriate carboxamide with alkali metal hypohalogenite. This process is particularly useful for the preparation of cyclopropylamines. The corresponding cyclopropylamides are obtained and converted to amines, e.g. as shown below.

63 R6 R3 R \ / R + n2chcc2c2h5 --Τ 'R5 "" V ~ T ~ R4 25 4' i r6 r3 C02C2H5 1- ", R5A \ 7 ^ R4

2. NaOBr ^ Y

30 NH2

The first step of the above scheme for forming the cyclopropylcarboxylic acid ester is known in the art, see e.g. Mescheryakov et al., Chem.

Abstr., 54, 24436 (1960).

The compounds of formula I or intermediate amides thereof wherein R is

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28 X r4 R V - (CH2> n- 5 Rff R ^ where X is SO or SC ^ is obtained from the corresponding compounds wherein X is S through oxidation using reagents and conditions known to form Alternatively, the appropriate ketone of formula VII wherein X is S or the amine derived from the ketone as described above can be oxidized to the sulfoxide or sulfone prior to coupling to form the dipeptide amide of the formula I. Preferred reagents and conditions for such oxidation of sulfides include the use of hydrogen peroxide in a solvent, for example, acetic acid or acetone.

If equimolar amounts of reactants are used, the product is sulfoxide which is readily converted to the corresponding.

20 sulfur with an additional mole of peroxide. Other preferred oxidizing agents are potassium permanganate, sodium metaperiodate or chromic acid for the preparation of the sulfones and m-chloroperbenzoic acid. two moles of peracid are used.

The compounds of formula I and their physiologically acceptable salts provide advantages as sweeteners in view of their high sweetness, their physical form and stability. They are generally crystalline, not hygroscopic, water-soluble solids. They are uniquely characterized in that they have a sweet taste without undesirable rancid or bitter taste properties at common levels of use. They can be used to give edible materials sweetness. The term "edible materials" as used herein refers to all non-toxic substances which can be consumed by humans or other animals

DK 154652 B

29 in solid or liquid form. Examples of such substances are: foodstuffs, including foods, prepared foods, chewing gum and soft drinks, food additives, including flavoring and coloring agents, and flavor enhancers and pharmaceuticals.

The present compounds can be prepared in a number of forms suitable for use as sweeteners. Typical forms that can be used are solid forms such as powders, tablets, granules and dragees and liquid forms such as solutions, suspensions, syrups, emulsions and other commonly used forms which are particularly suitable for combination with edible materials.

These forms may consist of the compounds of formula I or their physiologically acceptable salts either alone or together with non-toxic sweetening carriers, i.e. non-toxic substances commonly used with sweeteners. Such suitable carriers include liquids such as water, ethanol, sorbitol, glycerol, citric acid, corn oil, peanut oil, soybean oil, seed oil, propylene glycol, corn syrup, maple syrup and liquid paraffin, and solids such as lactose, cellulose, starch, dextrins, dextrins, dextrins, , polysaccharides such as polydextrose (see, e.g., U.S. Pat.

Similarly useful and compatible are compositions containing a compound of the invention combined with a known sweetening agent such as e.g. sucrose, saccharin, cyclamate and L-aspartyl-L-phenylalanine methyl esters useful for sweetening edible materials. Particularly useful are mixtures of compounds of formula I and saccharin or a physiologically acceptable salt thereof, e.g. sodium, potassium, calcium or ammonium salts of saccharin. In the aforementioned mixtures with saccharin, the compounds of formula I completely reduce or mask the well-known undesired bitter aftertaste of the saccharin.

Particularly useful sweetening compositions are those

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Containing saccharin in admixture with compounds of formula I which are at least 400 times as sweet as sucrose, especially those in which Ra is CH 2 OH and R is dicyclopropylcarbinyl, 2,2,4,4-tetramethylthietan-3-yl or 2 , 2,4,4-tetramethylpiperidine-thyl-l, l-dioxothietan-3-yl. Particularly preferred are such mixtures of saccharin and L-aspartyl-D-serine-N- (2,2,4,4-tetramethylthietan-3-yl) amide, especially such mixtures containing the last compound of formula I and saccharin in a weight ratio ranging from 1: 1 to 1: 8. Not only do these blends have a pleasant sweet taste and are noticeably without bitter aftertaste, they are surprisingly considerably sweeter than intended by summing up the sweetness of the individual components of the mixture. Ie they have a synergistic effect, being up to 50% sweeter than expected. In mixtures of saccharin or its salts and L-aspartyl-D-serin-N- (2,2,4,4-tetramethylthietan-3-yl) amide outside the above range, it is reduced. synergistic effect significantly.

The present L-aspartyl-D-amino acid dipeptide-2Q mites and the corresponding dipeptide amides of U.S. Patent No. 4,411.925 of the formula

HOOCCH 'CHCONHCHCONHR

^ I 1 and 1

NH 2 R

α * wherein R is as previously defined and R is methyl, ethyl, n-propyl or isopropyl are also applicable to the applications described in the art of L-aspartyl-L-phenylalanine methyl ester and analogs thereof.

Eg. are useful in the same functions described in the following patents and patent applications for L-aspartyl-L-phenylalanine methyl ester. In these applications, they have both the advantages described for the dipeptide ester and their previously mentioned advantages in stability and sweetness:

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31 US Patent No. 3,642,491 3,971,857 3,761,288 3,982,023 3,800,046 4,001,456 5 3,818,077 4,004,039 3,829,588 4,007,288 3,865,957 4,031,258 3,875,311 4,036,992 3,875,312 4,051,268 10 3,886,295 4,059,706 3,922,369 4,122,195 3,934,048 4,139,636 3,947,600 4,143,170 3,955,000 4,153,737 15 3,956,507

Canadian Patent No. 1,026,987 1,027,113 1,028,197 1,043,158 1,046,840

Dutch patent application no .; 73-04.314 73-11.307 25 75-14.921 76-05.390 76-08.963

West German of publication_script No: 2,438,317 30 2,456,926 2,509,257 2,518,302 2,609,999 2,646,224 35 2,713,951

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+ 3g

Belgian Patent No. 830,020 838,938 863,138 5,882,672

British patent riding .; 1464571

Japanese Kokai no .: 77-04.176

French Patent Nos. 2,338,651 and

Swiss Patent No. 590,615.

10

The invention is further illustrated in the following examples.

Example 1 L-Aspartyl-D-Serine-N- (dicyclopropylcarbinyl) amide

A

I, Ra = CH 2 OH, R =

V

A. D-HOCH and CHC00H 11 _NHCbz

A solution of 4.41 g (0.042 mol) of D-serine in 21 ml of 2N sodium hydroxide was cooled to 5-10 ° C, adjusted with concentrated hydrochloric acid to pH 10.0 - 11.5 and 6.9 ml ( 0.048 mol) benzyl chloroformate in 1.5 hour portions with simultaneous addition of 2N sodium hydroxide to keep the mixture within the above pH range.

The mixture was stirred overnight at room temperature, washed with ethyl ether and the aqueous phase acidified (pH 2.5-3.0) with 6N hydrochloric acid. Extraction with ethyl acetate, washing the extracts with brine and drying (MgSO 4) gave 3.14 g of product as a colorless solid which was recrystallized in 20 ml of ethyl acetate to give 2.64 g of product, R [thin layer chromatograph (TLC) ethyl acetate / hexane / acetic acid, 9: 9: 2, by volume].

DK 154652 B

33

A

B. D-HQCH2CHC0NEK

NHCbz V-y

5 'V

To a slurry of 2.4 g (0.01 mole) of N-Cbz-D-serine obtained under A in 75 ml of chloroform was added 1.1 ml (0.01 mole) of N-methylmorpholine. A solution was cooled to -12 ° C. To this was added 0.96 ml (0.01mol) of ethyl chloroformate, the mixture was stirred at -10 ° C for five minutes, a solution of 1.11 g (0.01mol) of dicyclopropylamine in 5 ml of chloroform was added and stirring was continued for 5 minutes. minutes at -15 ° C. The reaction mixture was allowed to warm to room temperature, washed successively with 0.5 N hydrochloric acid, 5% sodium hydrogencarbonate solution, water and the chloroform evaporated in vacuo. The aqueous washings were collected and extracted with ethyl acetate. The ethyl acetate extracts were combined with the residue obtained by evaporation of chloroform and the ethyl acetate 20 dried (MgSO 4) and evaporated in vacuo to give a white solid which was dried in a vacuum oven overnight to give 3.2 g of the desired product. 0.54 used in the next step.

25 A

C. D-HOCH-CHCONH- <X v

The 3.2 g (9.6 mmol) N-Cbz amide obtained in Step B was dissolved in 70 ml of methanol, 1.0 g of 5% Pd / C catalyst was added and the mixture was hydrogenated at 60 psi ( 4.2 kg / cm) for 30 minutes. The catalyst was removed by filtration and the filtrate was evaporated in vacuo to give 1.93 g of product as a soap-like solid.

35 Λ D. C6H5CH2OCOCH2CHCONHCH (CH2OH) CONHCH (/ Λ) 2 ____ NHCbz _

A mixture of 3.4 g (9.5 mmol) of β-benzyl-N-benzyl

DK 154652 B

34 oxycarbonyl-L-aspartate, 1.0 ml (9.5 mmol) of N-methylmorpholine and 0.9 ml (9.5 mmol) of ethyl chloroformate were stirred at -15 to -10 ° C for five minutes and added a solution of 1.9 g (9.5 mmol) of D-serine-N-dicyclopropyl-5-carbinylamide, obtained in step C, in 10 ml of chloroform at -15 ° C. The resulting mixture was stirred at -10 ° C for five minutes, allowed to warm to room temperature, and stirred for 1 hour. The reaction mixture was evaporated in vacuo to remove solvent, the residue was taken up in ethyl acetate (250 ml), washed alternately with 1N-hydrochloric acid, 5% sodium hydrogencarbonate solution, brine and dried over anhydrous magnesium sulfate. The solvent was evaporated in vacuo to give a gelatinous solid. This was taken up in 75 ml of hot ethyl acetate. On cooling, crystalline solid was dried which was dried in vacuo at 40 ° C to give 2.75 g of the desired anti-dipeptide amide as a fine white solid, Rf 0.30.

E. A mixture of 2.75 g of the diprotide dipeptide amide obtained in Step D, 200 ml of methanol and 1.0 g of 5% Pd / C catalyst was hydrogenated at 60 psi (4.2 o kg / cm) for 1 hour during which the product precipitated. The catalyst / product mixture was filtered, the filter cake was heated in 100 ml of warm water and filtered again. The collected filtrates were evaporated to dryness, triturated with water, filtered and dried in vacuo to give 260 mg of product as a fine white, soft solid,

Mp. 252-254 ° C, R f 0.58 (TLC, n-butanol / water / acetic acid, 4: 1: 1, ninhydrin injection).

The filter cake from the hydrogenation was slurried in 50 ml of 0.1N hydrochloric acid, the mixture was filtered through diatomaceous earth (Supercel), the filtrate (pH 1.6) adjusted with sodium hydroxide solution to pH 5.9, and the precipitated product was collected by filtration and dried in vacuo obtaining an additional 800 mg of product. Total step yield 68%.

Mass Spectrum (m / e) 313 (M).

Sweetness: 700 x sucrose.

DK 154652 B

35

Example 2 L-Aspartyl-D-serin-N- (2,2,4,4-tetramethylthietan-3-yl) amide.

CH3 ch3 5 I, Ra = CH2OH, R =. CH3. ch3 A. D-HOCH-CHCOOH 21 NHt-Boc 10 The procedure is that described by Moroder et al., Z. Physiol.Chem. 357, 1651 (1976) to prepare t-Boc amino acids. To 10 ml of both dioxane and water was added 2.18 g (10 mmol) of di-t-butyl dicarbonate, 1.6 ml (11.5 mmol) of triethylamine. and 1.05 g (10 mmol) of D-serine.

The mixture was stirred for 30 minutes at room temperature and the dioxane was evaporated in vacuo. The aqueous residue was cooled in ice which was added ethyl acetate and the mixture was stirred while diluted potassium hydrogen sulfate solution was added to pH 2-3. The aqueous layer was separated, extracted twice with ethyl acetate and the combined extracts washed with water, dried (Na 2 SO 4) and the solvent evaporated in vacuo to give 1.7 g of product as a viscous paste.

ch3 ch3 25 B. D-HOCH2CHCONH - <^ 7lS ^ s nh2 ch3 ch3

A mixed anhydride was prepared from 2.85 g (14 mmol) of Nt-butoxycarbonyl-D-serine, 1.55 ml of N-methyl-morpholine, 1.34 ml of ethyl chloroformate in 75 ml of methylene chloride at -12 to - To this mixture was added 2.01 g (14 mmol) of 3-amino-2,2,4,4-tetramethylthietane and stirring was continued for 5 minutes at -12 ° C. C. The product was isolated as described in Example 1, Step B, to give 4 g of viscous liquid residue. The residue was dissolved in 40 ml of methylene chloride, 12 ml of trifluoroacetic acid (d = 1,480) was added and the mixture was stirred at room temperature for 3 hours. reaction

DK 154652 B

The mixture was made alkaline with 40% sodium hydroxide solution, the organic layer removed, the aqueous layer saturated with sodium chloride and extracted with methylene chloride. The combined extracts were dried (MgSO4) and concentrated in vacuo to give 2.21 g of amorphous off-white solid. Crystallization in ethyl ether / hexane gave 1.92 g of product as a fine white solid.

(013) 2

C. t-ButylOCOCH2CHCONHCHCONH - <^^ S

in CH 2 OH I

I (cHo) 2 10 ........... NH-t-BOC ............

A mixture of 2.3 g (8.0 mmol) of β-t-butyl-Nt-butoxycarbonyl-L-aspartate, 0.88 ml (8.0 mmol) of N-methyl-morpholine and 0.77 ml (8, 0 mmol) ethyl chloroformed in 40 ml of methylene chloride was stirred at -12 ° C for 5 minutes. A solution of 1.85 g (8.0 mmol) of D-serine * N- (2,2,4,4-tetramethyl-thietan-3-yl) amide in 5 ml of the same solvent was added and stirring continued at -12 to -10 ° C for 10 minutes. The mixture was allowed to warm to room temperature, stirred for 1 hour at this temperature and the solvent evaporated. The residue was taken up in ethyl acetate, washed with dilute hydrochloric acid, sodium hydrogen carbonate solution, brine, dried (MgSO 4) and the ethyl acetate evaporated to give 3.34 g of amorphous solid. Crystallization in ethyl ether / hexane gave 2.91 g of colorless solid, R f 0.70 (ethyl acetate / hexane, 7: 3).

D. A solution of 2.4 g (4.78 mmol) of the product of step C above in 60 ml of chloroform was provided with a gas feed tube and anhydrous hydrogen chloride was bubbled through the solution. After 5 minutes, solid precipitation was observed. The hydrogen chloride addition was continued for 10 minutes, then the mixture was stirred at room temperature for 1 hour and evaporated to dryness in vacuo. The residue was taken up in water, washed with chloroform, the pH adjusted to 35 to 5.6, washed again with chloroform and the aqueous phase evaporated in vacuo. Ethanol was added to the residue and the mixture was evaporated to dryness in vacuo. The residue was dissolved in 37

DK 15 4 6 5 2 B

25 ml of warm water and allow to cool. The precipitated product was collected by filtration and dried in vacuo to give 1.12 g (67%) of product, m.p. 193-196 ° C.

0.32 (n-butanol / water / acetic acid, 4: 1: 1).

Analysis calculated for ΐ4Ν255305δ: 5 C: 48.39, H: 7.25, N: 12.09, S: 9.23

Found: C: 46.77, H: 7.48, N: 11.91, S: 8.82.

Sweetness: 1200 x sucrose.

Example 3 L-Aspartyl-D-serine-N- (2,2,4,4-tetramethyl-1,1-dioxo-thietan-3-yl) amide CH3 CH3 I, Ra = CH2OH, R = - < 3 CH3 CH3 A. 3-Amino-2,2,4,4-tetramethylthietane-1,1-dioxide.

A solution of 14.53 g (0.1 mole) of 3-amino-2,2,4,4-tetramethylthietane and 64.17 g (0.3 mole) of sodium m-periodate in 500 ml of water was stirred overnight. over at room temperature. The reaction mixture was adjusted to pH 13 with sodium hydroxide solution and the precipitated sodium iodate removed by filtration. The filtrate was washed with 100 ml of ethyl ether, the aqueous phase was continuously extracted with 2 µ of methylene chloride over 18 hours, the extract dried (Mg-SO 2) and the solvent evaporated in vacuo. The residual solid was recrystallized from ethyl acetate to give 8.5 g of product, m.p. 104 to 106.5 ° C. A second portion of crystals, 2.7 g, m.p.103-lo6 ° C was obtained. Total 2Q yield: 63%.

B. D-Serine-N- (2,2,4,4-tetramethyl-1,1-dioxo-thietan-3-yl) amide.

In the procedure of Example 1, Part B, gave 1.33 g (6.5 mmol) of N-benzyloxycarbonyl-D-serine, 0.715 ml of N-meth-22 methylmorpholine, 0.62 ml of ethyl chloroformate and 1.15 g (6, 5 mmol) 3-amino-2,2,4,4-tetramethyl-thietane-1,1-dioxide, 2.3 g of N-benzyloxycarbonyl-D-serine-N- (2,2,4,4-tetramethyl 38

DK 1546S2B

1,1-dioxo-thietan-3-yl) amide as a viscous liquid, 0.37 (ethyl acetate / hexane, 7: 3). This liquid was dissolved in methanol, 750 mg of 5% Pd / C catalyst was added and the mixture was hydrogenated according to the procedure of Example 1, Part C.

After filtration to remove the catalyst, the methanol was evaporated in vacuo, the residue was taken up in 1N hydrochloric acid and extracted with chloroform. The aqueous layer was made alkaline with sodium hydroxide saturated with sodium chloride and continuously extracted with chloroform overnight. Evaporation of the solvent gave 1.54 g of product as a 10-viscous solid which solidified on standing, 0.32 (m-butanol / water / acetic acid, 4: 1: 1).

CH, CH.J

J J

C. C6H5CH20C0CH2CHC0NHCHC0NH - <^ _ ^ SO2

NHCbz CH2OH

15 3 3

The diblocked dipeptidamide of the above formula was prepared on a 4.7 millimolar scale using the procedure of Example 1, Step D with 1.7 g of β-benzyl-N-benzyloxycarbonyl-L-aspartate, 0.51 ml of N-methyl mother -20 pholine, 0.45 ml of ethyl chloroformate and 1.24 g of D-serine-N- (2,2,4,4-tetramethyl-1,1-dioxothietan-3-yl) amide. The product, 2.45 g, was obtained as a colorless amorphous solid.

Two grams of this material were purified by chromatography on 60 g of silica gel eluting with ethyl acetate to give 1.2 g of amorphous solid product, R f 0.30 (ethyl acetate / hexane, 7: 3).

D. A mixture of 1.2 g of purified product from Part C above, 75 ml of methanol and 0.6 g of 5% Pd / C was hydrogenated at 80 psi 2 (5.6 kg / cm). When hydrogen uptake ceased, the catalyst was removed by filtration and the filtrate was evaporated to give a colorless solid residue. The residue was taken up in water, washed with chloroform and the aqueous layer was evaporated in vacuo. The residual solid was crystallized in ethanol to give 255 mg of the desired dipeptide amide as a fine white solid, m.p. 170-173 ° C, Rf 0.20. An additional 180 mg of product was obtained by working up the filter cake from the hydrogenation.

DK 154652 B

39 Sweetness: 850 x sucrose.

Example 4 D-O-Methylserine.

A. N-Choracetyl-dl-O-methylserine.

125 ml of water was added 59.55 g (0.5 mole) of dl-O-methyl serine, the mixture was stirred and 20 g (0.5 mole) of sodium hydroxide was added. The resulting solution was cooled in ice, and at the same time, a solution of 20 g of sodium hydroxide in 125 ml of water and 47.8 ml (0.6 mole) of chloroacetyl chloride was added from two drip funnels over 1 hour. keep the reaction mixture at pH 9.0-9.5. After the addition was complete, the resulting mixture was stirred for 1 hour at pH 9.0-9.5. The mixture was washed twice with methylene chloride, the aqueous phase acidified to pH 1.5 with concentrated hydrochloric acid under cooling in ice, saturated with sodium chloride and extracted several times with chloroform.

The combined extracts were dried (MgSO4) and the solvent was evaporated in vacuo. The residue was stirred with methyl ether 2g to precipitate a yellow solid, which was collected by filtration and dried, 69.38 g (71%). Mp. 104-107 ° C,

R 0.23 (ethyl acetate / hexane / acetic acid, 9: 9: 2, phosphorus-molybdate spray). A second portion of the aqueous phase was obtained by adding more sodium chloride and extracting with chloroform. Evaporation of chloroform gave 3.65 g of product. Total yield: 75%.

B. N-Chloroacetyl-D-O-methylserine.

To 3000 ml of water at 35-37 ° C was added 73.03 g (0.37 mol) of N-chloroacetyl-dl-O-methylserine and the mixture was adjusted to 2 g to pH 7.18 by the addition of concentrated ammonium hydroxide. Water was added to give a total volume of 3700 ml. To this was added 17 mg of commercial pig kidney aminoacylase, N-acylamino acid amidohydrolase; EC

3.5.1.14 (Acylase I) at 1845 units / mg. (1 unit is defined as the amount required to hydrolyze 1 micromole of N-acetyl-L-methionine per hour at pH 7.0 and 25 ° C.

The amount of enzyme added was that calculated to hydrolyze the susceptible isomer in 6 hours. The result-

DK 154652 B

40

they were kept at 37-38 ° C for 28 hours, occasionally adding ammonium hydroxide to maintain the pH of 7.1 to 7.2. An additional 5 mg of enzyme was added after 24 hours. The hydrolysis mixture was acidified to pH 4.5 with glacial acetic acid, filtered through a 0.6 µm millipore filter 5 (type BD) and the filtrate evaporated in vacuo below 35 ° C.

to reduce the total volume to 100-150 ml. The residue was acidified to pH 2.00 with hydrochloric acid and extracted several times with ethyl acetate and further extracted with chloroform. The individual organic extracts were each washed with water, dried (MgSO 4), and solvent was evaporated in vauum to give a yellow liquid residue. Addition of hexane and evaporation in vacuo induced crystal formation. The ethyl acetate extracts gave 16.67 g (46%) of N-chloroacetyl-D-O-methylserine, m.p. 95-96 ° C, 15 µl] D - 15.5 (c = 1, 1N NaOH). The chloroform extracts gave 4.61 g (13%) of the same product, m.p. 90-94 ° C (odor of chloroacetic acid). Both product portions gave a single spot by thin layer chromatography on silica gel plates, Rf 0.35, 9: 9: 2, ethyl acetate / hexane / acetic acid, phosphorolybdate spraying.

C. To 16.67 g (0.085 mole) of N-chloroacetyl-D-O-methylserine was added 25 ml of 2N hydrochloric acid and the mixture was heated under reflux for 3 hours. The mixture was concentrated in vacuo, removing the remaining chloroacetic acid with additional water. The solid residue was washed with ethyl ether and collected by filtration to give 12.31 g (93%) of D-O-methylserine hydrochloride, m.p. 188-190 ° C; [α] D - 16.7 ° (c = 0.7, CH 3 OH).

Example 5 L-Aspartyl-D-O-methylserine-N- (dicyclopropylcarbinyl) amide.

35

DK 154652 B

. "A

..................... ...... V

_ A. D-CH 0 AND nCHCOOH

5 3 21 NHCbz

A solution of 12.31 g (0.079 mol) of DO-methylserine containing 6.32 g (0.158 mol) of sodium hydroxide was cooled to 5-10 ° C and 11.74 ml (0.0806 mol) of benzyl chloroformate and 1Q 4M sodium hydroxide were added simultaneously at pH 8-9. The resulting mixture was stirred until the pH remained at 8 without further addition of base. After washing with methylene chloride, the aqueous phase was acidified with concentrated hydrochloric acid, extracted four times with methylene chloride, the extracts dried (MgSO 4) and the solvent evaporated in vacuo. The viscous liquid residue was stirred with hexane and the precipitated solid was collected by filtration to give 18.6 g of product (93%), Γ ct] β - 2.7 °

(c = 1, IN NaOH), R ~ 0.43.A

2o Δ b. D-ch, och9chconh - / NHCbz \ y

To a solution of 3.80 g (0.015 mol) of N-benzyloxycarbonyl-D-O-methylserine in 75 ml of methylene chloride was added 1.68 ml (0.015 mol) of N-methylmorpholine and the mixture was cooled to -15 ° C. To this was added 1.43 ml (0.015 mol) of ethyl chloro format, the mixture was stirred at -20 to -15 ° C for 10 minutes and 1.68 g (0.015 mol) of dicyclopropylcarbinylamine was added. The mixture was allowed to warm to. room temperature and stirred for 2 hours. The resulting mixture was washed twice with 1N sodium hydroxide, twice with 1N hydrochloric acid and dried over magnesium sulfate. Evaporation of solvent in vacuo gave 5.1 g (98%). of the desired product, R f 0.59 on silica gel TLC in 1: 1 ethyl acetate / hexane, phosphorus molybdate spray.

The structure of the product was confirmed by 1 H-NMR spectroscope! .

\

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42

A

C. D-CH3OCH2CH (NH2) CONH- ^ _V_

The 5.1 g of N-benzyloxycarbonyl-DO-methylserine N- (di-cyclopropylcarbinyl) amide obtained in Part B above was hydrogenated by the procedure of Example 1, Part C to give 2.96 g (95% ) DO-methylserine-N- (dicyclopropyl-carbinyl) amide as a liquid, Rf 0.39; [α] D - 23.2 (c = 0.7, 1N HCl). The structure was confirmed by 1 H-NMR spectroscopy.

D. CgH5CH2.OCOCH2CH (NHCbz) CONHCH (CH2OCH3) CONHCH (/ \) 2

A mixture of 4.97 g (13.9 mmol) of β-benzyl-N-benzyloxycarbonyl-L-aspartate, 1.55 ml (13.9 mmol) of N-methyl-morphine and 1.33 ml ( Ethyl chloroformate (13.9 mmol) was reacted as described in Example 1, Part D to give 7.43 g (97%) of the diblocked dipeptide amide which was recrystallized twice in ethyl acetate to give 4.25 g of colorless product, R 0.45 (7: 3 ethyl acetate / hexane). The structure was confirmed by 1 H NMR.

E. Hydrogenation of 4.25 g of purified diblocked dipeptidamide obtained in Part D above, following the procedure of Example 1, Part E, gave 2.4 g (95%) of the desired dipeptide amide, 25 m.p. 215-217 ° C (dec.); [α 3D + 37.6 ° (c = 0.8, 1.2N HCl); Rf 0.41.

Sweetness: 85 x sucrose.

Example 6 L-Aspartyl-D-O-methylseriesHSK 2,2,4,4-tetramethylthietan-3-yl) amide.

CH-. CH, a

I, R = CH 2 OCH 3, R = S

ch3 35 A. D-CHoOCHOCHCOOH 3 2 j NHt-Boc

To a solution of 2.89 g (18.6 mmol) of D-O-methyl-43

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Serine hydrochloride in 11 ml of water was added 6.48 ml (46.5 mmol) of triethylamine, 5.10 g (20.7 mmol) of 2- (t-butoxycarbonyloxy-imino) -2-phenylacetonitrile ("BOC-ON") and 11 ml of tetrahydrofuran. The mixture was stirred at room temperature overnight, diluted with 25 ml and washed with ethyl acetate. The aqueous layer was acidified to pH 1.8 with 1M hydrochloric acid, extracted with ethyl acetate (3 x 75 mL), dried (MgSO4) and evaporated in vacuo to give 4.2 g of product as a viscous liquid, , 65 (9: 9: 2 ethyl acetate / hexane / acetic acid, phosphomolybdate spray).

B. N-t-Boc-D-O-Methylserin-N- (2,2,4,4-tetramethylthietan-3-yl) amide.

To a solution of 4.2 g (18.6 mmol) of Nt-Boc-DQ-methylserine in 90 ml of methylene chloride was added 2.08 ml of N-methylmorpholine, the mixture was cooled to -15 ° C and added to -15 ° C. 1.78 ml of ethyl chloroformate was added. After stirring for 8 minutes at -20 to -15 ° C, 2.70 g (18.6 mol) of 3-amino-2,2,4,4-tetramethyl-thietane dissolved in 10 ml of methylene chloride were added at the same temperature and the mixture allowed to warm to room temperature. After stirring for 2 hours, the mixture was washed with dilute sodium hydroxide, dilute hydrochloric acid, dried over anhydrous magnesium sulfate and the solvent was evaporated in vacuo to give 6.04 g (94%) of colorless solid, 0.35 (3: 7 ethyl acetate). (hexane, phosphomolybdate spray). The structure was confirmed by 1 H NMR.

C. D-O-Methylserine N- (2,2,4,4-tetramethylthietan-3-yl) -amide.

To a solution of 6.04 g (17.4 mmol) of Nt-Boc-DO-methylserin-N- (2,2,4,4-tetramethylthietan-3-yl) amide in 13.4 ml of methylene chloride was added 6, 7 ml (87 mmol) of trifluoroacetic acid (specific gravity 1,480) and the mixture was stirred at room temperature for 3 hours. An additional 1.0 ml was added in 2 ml of methylene chloride and stirring was continued for 1 hour. The reaction mixture was made alkaline with 35% (w / w) sodium hydroxide solution, the organic layer was removed and the aqueous layer extracted several times with fresh methylene chloride. The total extracts

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44 was dried (MgSO 4) and the solvent was evaporated in vacuo to give 4.29 g of liquid crude product. This was taken up in 20 ml of 1N hydrochloric acid, washed with ethyl ether, the aqueous layer made alkaline with sodium hydroxide (40% w / w), saturated with sodium chloride and extracted with methylene chloride. Evaporation of the extracts gave 3.21 g (75%) of the desired product, 0.41; [α] D - 19.8 ° (c = 0.8, 1N HCl). The structure of this product was confirmed by its 1 H-NMR spectrum.

CH7 CH,

D. t-Butyl- OCOCH2CH CONH CH CONH - ^^^ S

NH-t-BocCH2OCH3

Using 3.76 g of the product obtained by the process in Part C above, the procedure of Example 2, step C was repeated on a 13 millimolar scale using 50 ml of methylene chloride as solvent to give 5.0 g (74% ) of the desired diblocked dipeptide amide as a brittle foam, R ^ 0.40 20 I * !? phosphorus molybdate spraying).

The structure was confirmed by the 1 H NMR spectrum of the product.

E. Anhydrous hydrogen bromide was bubbled through a solution of 5.0 g (9.7 mmol) of the diblocked dipeptide amide obtained in step D above, with stirring at room temperature for 1 hour. The resulting mixture was evaporated to dryness in vacuo and the resulting yellow solid residue dissolved in water. The solution was washed twice with ethyl ether, once with methylene chloride, the aqueous phase was adjusted to pH 5.8 with sodium hydroxide solution and evaporated to dryness in vacuo. The residue was dissolved in 10 ml of 95% ethanol and ethyl ether was added to precipitate the title compound in two portions: 1.66 g [a] D + 13.4 ° (c = 0.9, 1.2N HCl ), m.p. 85-90 ° C; 1.20 g, [α] D + 13.9 ° (c = 0.8, 1.2N HCl). TLC of each portion showed a product stain at R ^ 0.51 with small amounts of material with R ^ 0.44.

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45 F. Purification via p-toluenesulfonate salt.

To 10 ml of water was added 1.39 g (3.85 mmol) of the total portions of product obtained above and 0.66 g (3.83 mmol) of p-toluenesulfonic acid. The resulting solution was stirred at room temperature for 2 hours. The precipitated solid was collected by filtration and washed with a small amount of water to give 0.94 g of p-toluenesulfone salt. The salt was combined with 3 ml of liquid anion exchange resin (Amberlite LA-F ^ f, 6 ml of hexane, 2 ml of water, and ^ Registered trademark of Rohm and Haas Co.

The mixture was stirred for 2 hours. The aqueous phase was separated, washed with hexane and evaporated to dryness in vacuo to give 0.72 g of off-white solid, [α] D + 22.43 ° (c = 0.8, 1.2N HCl); Rf 0.48.

Sweetness: 320 x sucrose. The sweet taste was judged to be exceptionally clean, free of flavor and with a rapid lake degradation.

Example 7 L-Aspartyl 1- D-O-methylserin-N- (2,2,4,4-tetramethyl-1,1-20 dioxothietan-3-yl) amide.

ch3ch3a. d -ch3och2chconh - <3S NHCbz <3 CH3 3.8 g (15 mmol) of N-benzyloxycarbonyl-D-O-methylserine was dissolved in 75 ml of methylene chloride. N-methyl-morpholine (1.68 ml) was added, the solution cooled to -15 ° C and 1.43 ml of ethyl chloroformate added. The resulting mixture was stirred for 8 minutes at -15 ° C, then 2.18 g (15 mmol) of 3-amino-2,2,4,4-tetramethylthietane was added and the mixture was allowed to warm to room temperature. Stirring was continued for 2 hours at room temperature, the reaction mixture was washed with dilute sodium hydroxide, dilute hydrochloric acid, dried (MgSO 4) and the solvent was evaporated in vacuo to give 6.11 g of liquid product, R f 0.58 (ethyl acetate / hexane , 1: 1 ·, phosphorus molybdate spray).

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46 B. Oxidation to 1,1-dioxide.

6.11 g of the product from Part A above was dissolved in 75 ml of chloroform and cooled in ice with the addition of 7.12 g of m-chloroperbenzoic acid in portions. The reaction mixture is allowed to warm to room temperature and stirred for 3 hours. Additional chloroform (75 πύ) was added and the solution was washed twice each time with 5% (w / v) sodium carbonate solution, 0.5N sodium thiosulfate and 1N hydrochloric acid. After drying the organic phase (MgSO 4) and evaporation of the solvent, 6.24 g of product was obtained as a viscous liquid, Rf 0.22 (ethyl acetate / hexane, 1: 1? Phosphor molybdate spray) with traces of the starting material and sulfoxide. The 1 H NMR spectrum was consistent with the structure of the desired sulfone with a small amount of chloroform.

CH-CHO C. D-CH3OCH2CHCONH - <^ SO2 * H2 C <^ 3.

A mixture of 6.11 g of N-benzyloxycarbonyl-DO-methylserin-N- (2,2,4,4-tetramethyl-1,1-dioxothietan-3-yl) -amide, 250 ml of methanol and 3, 0 g of 5% palladium-on-carbon catalyst was hydrogenated at 50 psi (3.52 kg / cm) for 2 hours. The catalyst was removed by filtration, the filtrate was evaporated in vacuo and the residue was taken up in 35 ml of 1N hydrochloric acid. The acid solution was washed three times with chloroform, made alkaline with solid sodium hydroxide, saturated with sodium chloride and extracted with 3 x 50 ml of chloroform.

The combined extracts were dried (MgSO 4) and the solvent was evaporated in vacuo to give 3.37 g (80%) of the α-aminoamide product as a colorless liquid, R [α] n - 15.7 ° (c = 0.8, 1N HCl). The structure was confirmed ± by H-NMR spectroscopy.

35

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47 CH, CHq D. C6H5CH2OCOCH2CH (NHCbz) CONHCH (CH2OCH3) CONH-C2 "'" "SO2 .................... chJ> <ch3

Using 3.37 g (12 mmol) of the product obtained in step c above as the starting material of the procedure of Example 1, step D, the desired diblocked dipeptidamide as a clear glass was obtained, 6.73 g (91%) 0.28 (ethyl acetate / hexane, 70:30). The 1 H NMR spectrum was consistent with the structure of this compound.

E. A mixture of 6.73 g of β-benzyl-N-Cbz-L-aspartoyl-DO-methylserin-N- (2,2,4,4-tetramethyl-1,1-dioxothietan-3-yl) amide, 250 ml of methanol and 2.0 g of 5% Pd / C catalyst were hydrogenated according to the procedure of step C above.

15

The residue remaining after evaporation of the solvent was stirred overnight in ethyl ether and the solid product was collected by filtration and dried in a vacuum oven to give 3.3 g (77%) of the desired dipeptidamide, R, 0.23; mp. 140-150 ° C (dec.); [α] D 20 + 20.3 ° (c = 1; 1.2N 20 D - HCl).

Sweetness: 200 x sucrose.

Example 8 L-Aspartyl -D-ser in-N- (dl-cis, trans-2,6-dimethylcyclohexyl) amide.

ch3 I, Ra = CH2OH, - R = _ CJ13 A. dl-cis, trans-2,6-Dimethylcyclohexylamine.

A solution of 2.1 g of trans-2,6-dimethylcyclohexanone oxime in 30 ml of dry ethanol was heated under reflux. To this was added 3.1 g of metallic sodium in portions.

When the addition was complete, the mixture was refluxed for 30 minutes and allowed to cool to room temperature. The resulting gel was dissolved in water, adjusted to pH 2.0 with hydrochloric acid and washed with ethyl acetate.

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48 ether. The aqueous phase was made alkaline with sodium hydroxide, extracted with ether, the extracts dried (MgSO4) and evaporated to give the desired amine as a colorless liquid.

B. D-Serine N- (dl-cis, trans-2,6-dimethylcyclohexyl) amide 5 Using 1.47 g (6.15 mmol) of N-Cbz-D-serine, 775 mg (6 , 15 mmol) dl-cis, trans-2,6-dimethylcyclohexylamine and equimolar amounts of N-methylmorpholine and ethyl chloroformate, and subsequent removal of amino protecting group by catalytic hydrogenation following the procedures of Example 1, Step B and C, 0.70 g of the desired D-serinamide was obtained as a white solid, 0.64 (ethyl acetate / hexane, 7: 3).

C. Protected Dipeptidamide.

Using 600 mg (2.8 mmol) of D-serine-N-15 (dl-cis, trans-2,6-dimethylcyclohexyl) amide in the procedure of Example 1, Step D, the corresponding β-benzyl-N- benzyloxycarbonyl-L-aspartoyl-D-serinamide, 1.2 g, as a colorless solid. Recrystallization in isopropyl ether gave 1.0 g, 0.35 (ethyl acetate / hexane, 7: 3).

D. Catalytic hydrogenation of the diprotid dipeptidamide from step C above (1.0 g) in methanol in the presence of 0.6 g of 5% palladium / carbon catalyst according to the procedure of Example 1, Part E gave 435 mg of the title compound as an off-white crystalline solid.

Sweetness: 200 x sucrose.

Example 9 β-Benzyl-N-benzyloxycarbonyl-L-aspartyl-D-O-methylserine.

30 C cCH oOCCHoCHC0NHCHC00H

JU 6 5 2 „2; , O NH CH2OCH3

Cbz D-O-Methylserine (6.65 g, 56.1 mmol) was dissolved in 100 ml of Ν, Ν-dimethylformamide (DMF) and the solution was added dropwise to 6.74 g (62.4 mmol) of trimethylchlorosilane at room temperature. In a separate flask was placed β-benzyl-N-benzene.

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49 zyloxycarbonyl-L-aspartate (18.0 g, 50.4 mmol), triethylamine (12.35 g, 122 mmol) and 110 ml of both DMF and tetrahydrofuran and the resulting solution cooled to -15 ° C. To the solution was added ethyl chloroformate (5.95 g, 55.1 mmol) and the resulting mixture was stirred for 10 minutes at -10 ° C. To this was added dropwise the DMF solution of silylated D-O-methylserine, as prepared above, while maintaining the mixture at -5 to -10 ° C. The mixture was stirred at -5 ° C for 1 hour, 0.2N hydrochloric acid was added until the mixture was acidic and the resulting mixture extracted with chloroform. The chloroform extracts were collected 10 and washed several times with dilute hydrochloric acid to remove residual DMF. The solvent was evaporated in vacuo to give the title compound.

By repeating the procedure, using D-serine instead of D-O-methylserine, and twice the above-mentioned amount of trimethylchlorosilane, β-benzyl-N-Cbz-L-aspartoyl-D-serine was obtained in a similar manner.

Example 10 2Q β-Methyl-N-benzyloxycarbonyl-L-aspartyl-D-serine.

A suspension of 80.7 g (0.78 mole) of D-serine in 200 ml of DMF was cooled to 10 ° C, 184 g (1.70 mole) of trimethyl chlorosilane was added in portions, and the resulting mixture was stirred at 25 ° C for 1 hour. hour.

2. In a separate flask, a solution of 158 g (0.86 mol) of β-methyl-L-aspartic acid hydrochloride was placed in 1 liter of water. 34.5 g (0.86 mole) of sodium hydroxide were added followed by 80 g of sodium bicarbonate and the resulting mixture was stirred vigorously. After cooling to 5-10 ° C, 161 g (0.94 mol) of benzyloxycarbonyl chloride were added in portions and stirring was continued for 2 hours at this temperature. The reaction mixture was washed with 100 ml of ethyl acetate, acidified by the addition of 80 ml of concentrated hydrochloric acid and extracted with ethyl acetate (2 x 450 ml).

The extract (900 ml) was found to contain 218 g (0.78 mol, 90% yield) of β-methyl-N-benzyloxycarbonyl-L-aspartate.

It was used in the next step without further purification.

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50

The ethyl acetate extract was cooled to -20 ° C, 165 g (1.63 mole) of triethylamine and 84 g (0.78 mole) of ethyl chloroformate were added. The solution was incubated at -15 ° to -20 ° C for 30 minutes, then quickly treated with the DMF solution of silylated D-serine prepared above and the resulting mixture allowed to warm to room temperature for 1 hour with stirring. The reaction mixture was washed with water (3 x 500 ml), the organic layer dried over sodium sulfate and evaporated in vacuo to give the title compound.

By repeating the procedure, but using D-O-methylserine instead of D-serine and only half of the above trimethylchlorosilane, β-methyl-N-Cbz-L-aspartyl-D-O-methylserine was obtained.

Example 11 1 g of t-Butylcyclopropylcarbinylamine.

To 0.5 mole of both cyclopropanecarbonyl chloride and cuprous chloride in 500 ml of dry ethyl ether was added dropwise under a nitrogen atmosphere 238 ml (0.5 mole) of 2.1 M t-butyl-magnesium chloride in the same solvent at -10 ° C. The reaction mixture was poured into a mixture of 250 ml of 3M hydrochloric acid and 700 g of ice, the organic layer was isolated, washed with water, sodium hydrogen carbonate solution, brine and dried over anhydrous magnesium sulfate. The ether was evaporated at reduced pressure and the residue distilled at atmospheric pressure to give 45 g (72%) of t-butylcyclopropyl ketone, b.p. 145-153 ° C.

The 45 g (0.36 mol) of ketone was reacted with hydroxylamine hydrochloride and sodium acetate in 1: 1 ethanol / water according to process N (see below). After heating under reflux for 3 h overnight, the reaction mixture was cooled and the precipitated oxime was collected and washed with cold ethanol to give 23.5 g of t-butylcyclopropyl ketoxime.

An additional 7.7 g of the mother liquor was obtained. The combined yields were recrystallized in 1: 1 ethanol / water to give 25.2 g (50%) of oxime, m.p. 113.5 to 114 ° C.

To a solution of 5.0 g (0.035 mole) of oxime in 80 ml of ethanol was added 8.04 g (0.35 mole) of sodium and the reaction was carried out and the product isolated as described in

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51 procedure Q, to obtain 3.31 g of crude dl-t-butylcyclopropylcarbinylamine. This was distilled at atmospheric pressure to give 2.01 g (45%) of product, boiling at 153-155 ° C.

Example 12 5: 4-Oximino-3,5-dimethyltetrahydrothiopyran.

A. 3,5-Dimethyltetrahydrothiopyran-4-one.

A mixture of 2 g of sodium acetate and 25 ml of ethanol was saturated with hydrogen sulfide gas. To this, 7.0 g (0.063 mole) of diisopropenyl ketone was added under cooling in an ice bath until the reaction was no longer exothermic. The mixture was stirred at room temperature while hydrogen sulfide was passed through the mixture for 4 hours, then the mixture was allowed to stand overnight. The ethanol and the excess were evaporated in vacuo and the residue was taken up in ethyl ether, washed in turn with water, potassium carbonate solution, dilute hydrochloric acid and again water. The ether extracts were dried (Na 2 SO 4) and evaporated to give 6.8 g of oil. This was distilled in vacuo through a 10 cm Vigreaux column to give 1.67 g of product, b.p. 83-86 ° C / 9 mm used in the next step without further purification.

B. 4-Oximino-3,5-dimethyltetrahydrothiopyran.

A mixture of 1.67 g (0.011 mol) of the cyclic ketone obtained in step A, 1.6 g (0.023 mol) of hydroxylamine hydrochloride and 1.9 g (0.023 mol) of sodium acetate in 30 ml of water and 10 ml of ethanol heated under reflux for 3 hours, cooled and the precipitate collected by filtration. After recrystallization in 1: 1 methanol / water, 1.5 g of oxime was obtained as a white solid, m.p. 60-85 ° C, which is a mixture of the isomers of appropriate purity for use in the next step.

35

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52

Example 13 2-Methylthio-2,4-dimethyl-3-aminopentane.

A. 2-Methylthio-2,4-dimethylpentan-3-one

A solution of 200 ml of methanol containing 9.2 g of 5 (0.40 mol) of metallic sodium was cooled in an ice bath and saturated with gaseous methyl mercaptan. To this was added 77.2 g (0.40 mole) of 2-bromo-2,4-dimethylpentan-3-one at room temperature and the resulting mixture was stirred for two hours. The reaction mixture was diluted with water, extracted with ethyl ether, the extracts washed with water, brine and dried over anhydrous sodium sulfate.

The ether was evaporated and the residue distilled in vacuo to give 50.4 g of product, (bp 76 ° C (20 mm)).

B. 2-Methylthio-2,4-dimethyl-3-aminopentane A solution of 6.0 g (0.038 mol) of 2-methylthio-2,4-dimethylpentan-3-one, 9.9 g of formamide and 2.1 g of 100% formic acid was heated at reflux while water formed during the reaction was removed by a fractionation head. After 12 hours, an additional 20 g of hydrochloric acid was added and reflux continued for another 24 hours in the same manner, at which time the reaction mixture reached a temperature of 190 ° C. The mixture was cooled, diluted with water and extracted with ethyl acetate. The extracts were washed with water and evaporated to dryness under reduced pressure to give 5.3 residual oil. The oil was refluxed with 40 ml of 6N hydrochloric acid for 6 hours, diluted with water, washed with ether and the aqueous phase made strongly alkaline with sodium hydroxide. After extraction with ethyl ether and evaporation of the extract, 3.3 g (56%) of colorless amine was obtained, giving a single peak by gas-liquid chromatography on a 6-foot OV-1 column at 110 ° C, retention time 412 seconds.

35 53

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Example 14 3-Amino-2-hydroxy-2,4-dimethylpentane A. 2-Hydroxy-2,4-dimethyl-3-pentanone

To a stirred solution of 28.4 ml (0.2 mole) of 2,4-dimethyl-3-pentanone in 100 ml of chloroform was added dropwise 10.3 ml (0.2 mole) of bromine in 30 ml of the same solvent. The resulting mixture was stirred for a few minutes, the solvent was evaporated in vacuo, the residue was taken up in 100 ml of water. Water, 50 ml, and 10 M sodium hydroxide, 50 ml, were added to the solution. The result. The running mixture was stirred at reflux for 1 hour, diluted with 200 ml of water and extracted with 3 x 50 ml of ethyl ether. The extracts were dried (MgSO4), evaporated to dryness, and the residue distilled to give 15.95 g (61%) of hydroxy ketone, k p. 60-62 ° C / 18 mm.

B. 3-Amino-2-hydroxy-2,4-dimethylpentane The hydroxy ketone of Step A, 15 g (0.115 mol) was reduced in a refluxed formamide and formic acid mixture to give 4.5 g (30%) of hydroxyamine, bp 80-81 ° C / 17 mm.

Example, DL-2-Amino-3,3-dimethyl-4-hydroxybutyric acid lactone hydrochloride

Manufactured according to the procedure of Wieland, Chein.

25 Ber., Vol. 81, page 323 (1948): 2-Keto-3,3-dimethyl-4-hydroxybutyric acid lactone, 3.5 g was neutralized with dilute sodium hydroxide and the aqueous solution was evaporated to dryness in vacuo. The residue was taken up in 100 ml of hot ethanol, filtered hot, 30 and a solution of 700 mg of metallic sodium in 10 ml of ethanol containing 2 g of hydroxylamine hydrochloride was added. The sodium salt of 3,3-dimethyl-4-hydroxy-2-oximino butyric acid lactone, 5 g, was precipitated and recrystallized from methanol. The oxime was formed by decomposition of the sodium salt in 2N hydrochloric acid, from which it slowly crystallized. After recrystallization in benzene hexane, m.p. 160 ° C.

A solution of 25 g of oxime in 100 ml of ethanol

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54 were added in portions to 5 g of platinum oxide suspended in 150 ml of 2N hydrochloric acid and the mixture was hydrogenated at atmospheric pressure for 2 days. The catalyst was filtered off, the filtrate was evaporated and the residue was taken up in 150 ml of ethanol. Treatment with 500 ml of ethyl ether precipitated DL-2-amino-3,3-dimethyl-4-hydroxybutyric acid lactone hydrochloride, 22 g, which was recrystallized from ethanol / ether, srap. 208-212 C.

Example 16

Carbonated cola soft drink.

A carbonated cola soft drink was prepared according to the composition set forth below. The resulting soft drink was judged to have a sweetness intensity comparable to a control soft drink containing 11% sucrose.

15 Ingredients Weight%

Caffeine (1% aqueous solution) 0.700 L-Aspartyl-D-serine-N- (cis, trans-2,6-dimethylcyclohexyl) amide (10% aqueous) 0.540

Cola flavor concentrate 0.080 Phosphoric acid (50% aqueous) 0.040

Citric acid (50% aqueous) 0.066

Sodium citrate (25% aqueous) 0.210

Caramel color (25% aqueous) 0.370

Lemon Oil Extract 0.012 Lime Oil Extract 0.021

Carbonated water (3.5 volumes of carbon dioxide) g.s.

100000

Replacement of the L-aspartyl-D-serine-N- (cis, trans-2,6-dimethylcyclohexyl) amide in the above recipe with 0.090% 10% aqueous L-aspariyl-D-seric acid N- (dicy-clopropylcarbinyl) amide or 1.35% 10% aqueous L-aspartyl · -DO-methylserine-N- (dicyclopropylcarbinyl) amide gives carbonated cola soft drinks of similar quality.

Example 17

Dietetic hard candy.

35

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55

Hard buns were prepared according to the following recipe and procedure:

Ingredients Gram L-Aspartyl-D-Serine-N- (dicyclopropylcarbyl) -amide 0.59 5 Water 4.00 FD and C red £ 40 (10% aqueous) 0.30

Cherry flavor 0.60

Citric acid 6.00

Polydextrose * 420.00 10 Water 180.00 * U.S. Patent No. 3,766,165 In a small beaker, the sweetener was dissolved in water, color, flavor and citric acid added and mixed well to dissolve. In a separate beaker, polydextrose and water were collected. It was heated to 140 ° C with stirring, then allowed to cool to 120-125 ° C. The other small cup ingredients were added and mixed or thoroughly blended. The pulp was transferred to an oil-covered marble slab and allowed to cool to 75-80 ° C.

The mass was extracted through an oil covered pressure roller.

Use of 0.49 g of L-aspartyl-D-serine-N- (2,2,4,4-tetramethyl-1,1-dioxothitan-3-yl) amide or 2.33 g of L-aspartyl-D- serine-N- (2,2,4,4-tetramethyl-3-pentyl) amide as a sweetener instead of L-aspartyl-D-serine-N- (dicyclopropylcarbinyl) amide gave similar results.

Example 18 A gelatin dessert was prepared according to the following composition and procedure.

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56

Ingredients Gram

Gelatin 225 Bloom 7.522

Citric Acid 1.848

Sodium citrate 1,296 Strawberry flavor 0.298 L-Aspartyl-D-serin-N- (2,2,4,4-tetramethyl-thietan-3-yl) amide 0.036

Boiling water 240,000

Cold water '240,000 10 491,000

The first five ingredients were pre-mixed, added to boiling water and stirred to complete dissolution. Cold water was added and stirred rapidly. The mixture was transferred to plates and refrigerated until solidified.

Example 19

Low-calorie sweeteners were prepared for use at the table according to the following formulations: A. A powdered sweetener was prepared by mixing the following ingredients.

Ingredients Weight% L-Aspartic acid -D-serine-N- (2,2,4,4-tetramethyl-1,1-dioxothietan-3-yl) amide 0.42

Crystalline sorbitol 49.52

Dextrin (dextrose equivalent 10) 50.00

Monosodium glutamate 0.02

Glucon-delta-lactone 0.02 Sodium citrate 0.02 100.00

One gram of the resulting mixture corresponds to approx. three grams of sucrose.

B. A liquid sweetener for use at the table was prepared as follows.

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57

Ingredients Weight% L-Aspartyl, -D-Serine-N- (dicyclopropylcarbinyl) -amide 0.17

Water 99.73 5 Sodium Benzoate 0.10 100.00

One gram of the resulting solution corresponds to approx. 1.2 g of crystalline sucrose.

If the sweetener of formula I used in step 10 above is 0.83 g of a 1: 4 mixture of L-aspartyl-D-serine-N- (2,2,4,4-tetramethylthietane) 3-yl) amide and sodium saccharin, comparable results are obtained. Similarly, if L-aspartyl-D-serine-N- (di-cyclopropylcarbinyl) amide used in step B above is obtained, 0.34 g of a 1: 6 mixture by weight of the same compound is obtained. sodium saccharin, a comparable liquid table sweetener.

Example 20 20 Frozen dessert.

A sugarless frozen vanilla dessert was prepared according to the following formulation according to usual practice.

Ingredients Weight% 25 Heavy cream (35% butterfat) 23.00

Fat-free dry milk 10.50

Mono- and diglyceride emulsifier 0.25

Polydextrose *, 11.20

Water 54.49 L-Aspartyl-D-O-methylserine-N- (2,2,4,4-tetramethyl-1,1-dioxothietan-3-yl) amide 0.06

Gelatin (225 Bloom) 0.50 100.00 * US Patent No. 3,766,165 35

. . DK 154652 B

S8

Example 21

Pickled pears.

Fresh pears were washed, peeled, cut, cut and immersed in an aqueous solution containing 0.05% by weight of ascorbic acid. The cut fruit was placed in screw-cap jars, and the jars were filled with a syrup containing the following ingredients.

Ingredients Weight%

Sorbitol 25,000 L-Aspartyl-D-Serin-N- (2,2,4,4-tetramethyl-thietan-3-yl) amide 0.025

Citric Acid 0.125

Water, e.g.

100,000 The glasses were loosely closed and placed in an autoclave containing hot water and treated at 100 ° C for 45 minutes. The glasses were removed, immediately sealed by tightening the lids, and allowed to cool.

Example 22 Soft drink powder concentrate.

Ingredients Weight%

Citric acid 31.78

Sodium citrate 5.08 25 Strawberry flavor 57.72

Strawberry FD and C color 0.54 L-Aspartyl-D-O-methylserin-N- (2,2,4,4-tetramethylthietan-3-yl) amide 2.44

Carboxymethyl cellulose 2.44 30.00

All the ingredients were combined in a blender and mixed until the mixture was homogeneous. For use, 1.73 g of soft drink powder concentrate was used in 4 fluid ounces (118 ml) of water.

35

Example 23

Cake.

An extremely acceptable vanilla ice cream was prepared.

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59 ge for the following recipe:

Ingredients Gram

Emulsified cleared 16.09

Water 20.83 Eggs 23.00 5 Sodium Hydrogen Carbonate 1.10

Vanilla extract, single fold 0.28

Glucon-delta-lactone 1.75

Polydextrose *, 70% aqueous solution 80.00

Fat-free thaw milk 2.50 10 Baking flour 56.20

Pulse milk 0.80

Wheat Starch 1.40 L-Aspartyl-D-Serin-N ~ (2,2,4,4-Tetramethylthietan-3-yl) amide 0.05 15 204.00 * U.S. Patent No. 3,766,165

Fat-free thaw milk, powdered milk, polydextrose solution and emulsified claret were combined. This was mixed at low speed until soft and smooth (about 3 minutes), 20 eggs were added and whipped until a homogeneous soft mixture was obtained. The sweetener was dissolved in water, added to the soft mixture and mixed for 2-3 minutes. The remaining ingredients were added and mixed until the mixture was soft and smooth (3-5 minutes). 120 g of dough 25 were placed in a small greased form and baked at 350 ° F (176 ° C) for 30 minutes.

30 35

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60

Example 24

Sodium salt of L-aspartyl '-D-serine-N- (dicyclopropylcarbinyl) amide

To a solution of 3.12 g (0.01 mole) of L-aspartyl-D-serine-N- (dicyclopropylcarbinyl) amide in 100 ml of ethanol 5 was added 2 ml of 5N sodium hydroxide. The resulting mixture was stirred for 10 minutes at room temperature, then evaporated to dryness in vacuo. The residue was triturated with anhydrous ethyl alcohol, filtered, and air dried.

If sodium hydroxide used above was substituted with an equivalent amount of potassium hydroxide, calcium hydroxide, magnesium hydroxide or ammonium hydroxide, the corresponding potassium, calcium, magnesium and ammonium salts were formed in a similar manner.

The remaining L-aspartyl amino acid dipeptide amides of formula I are also converted to carboxylate salts as described above.

Example 25 Acid addition salts.

The L-Aspartyl-D-amino acid dipeptide amide of formula I is slurried in a small amount of water and an equivalent amount of acid such as salt, phosphorus, sulfur, vinegar, maleic, fumar, milk, wine, is added. citric, gluconic or saccharinic acid. The resulting mixture is stirred for 15-30 minutes, then evaporated to dryness or precipitated by the addition of a cosolvent such as methanol or ethanol.

30 35

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61

Process A

Alkylcycloalkylcarbinylamines and dicycloalkylcarbinylamines

These are prepared as illustrated below with t-butylcyclopentylcarbinylamine.

To a mixture of 1.0 mole of cyclopentylcarbonyl chloride and 99 g (1.0 mole) of cuprous chloride in 1000 ml of dry ether under a nitrogen atmosphere is added dropwise 478 ml (1.0 mole) of 2M t-butyl magnesium chloride in the same solvent. . The addition is carried out at -5 to -15 ° C.

10 ° a resulting mixture is poured into 500 ml of 3M hydrochloric acid and 700 g of ice, the organic layer is removed and washed successively with water, sodium bicarbonate solution, brine and dried (MgSO4). The dried ether extract is evaporated under reduced pressure and the residue distilled off to obtain t-butylcyclopentyl ketone.

ii. The ketone (0.75 mole) is mixed with hydroxylamine hydrochloride 38.3 g (1.16 mole) and sodium acetate, 123 g (1.50 mole) in sufficient water to effect solution, heated on a steam bath for one hour, cooled and the mixture is adjusted to pH 7.5 with sodium hydroxide solution. After extraction of the mixture with ether, the extracts (MgSO 4) are dried and evaporated to dryness to give the oxime. The oxime is dissolved in anhydrous ethanol (ca.

2 liters per mole oxime) and the solution is heated under reflux. Metallic sodium (about 10 moles per mole of oxime) is added in portions at a rate sufficient to maintain reflux temperature. When all sodium is added, the resulting mixture is cooled and 200 ml of ethanol is added followed by 300 ml.

30 water. The mixture is acidified with hydrochloric acid, evaporated to remove ethanol and the residue made alkaline (pH 12-13) with 10 M sodium hydroxide. The alkaline mixture is extracted several times with ether and the combined extracts are dried (MgSO 4). Dry hydrogen chloride is passed through the dried extracts until precipitation is complete. That

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62 precipitated hydrochloride salt is collected by filtration, washed with ether and air dried. The salt is converted to the free base by aqueous sodium hydroxide, extraction with ethyl ether and evaporation of the extracts. The product t-butylcyclopentylcarbinylamine, 5 kp. 80-90 ° C (21 mm), has a purity sufficient for use in preparing the subject amides, but may, if desired, be further purified e.g. by distillation or column chromatography.

10 The following amines are also prepared by this process: 2,2-dimethyl-3-aminopentane, b.p. 123-126 ° C, atmospheric pressure; 2,2,4-trimethyl-3-aminopentane, b.p. 149-150 ° C, at-

1 R

moss ferry pressure.

Method B

2.2- Dimethylcyclohexylamine. i. 2,2-Dimethylcyclohexanone.

on

To a suspension of 13.5 g (0.25 mole) of sodium methoxide in 500 ml of ethyl ether was added 30.8 g (0.28 mole) of 2-methylcyclohexanone and 20.3 g (0.28 mole) of ethyl formate. The mixture was stirred at room temperature for 12 hours, filtered under a nitrogen atmosphere, the solids were washed with ethyl ether and dried in a vacuum oven at 75 ° C.

The dried cake was crushed in a mortar with a pestle to a fine powder to give 17.5 g (43%) of sodium 2-formyl-6-methylcyclohexanone used in the next step.

30

The above product, 17.5 g (0.11 mol) was added to a mixture of 2.88 g (0.13 mol) of sodium, 500 ml of anhydrous ammonia and ca. 0.1 g of ferric chloride. The resulting gray suspension was cooled to -45 ° C and stirred for 1 hour at reflux temperature of the system. To this was added 20.86 g (0.15 mol) of methyl iodide, the mixture was stirred at reflux for three hours and allowed to cool.

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63 vapors under heating to room temperature overnight.

The residue was suspended in 300 ml of ethyl ether, refluxed to disperse ammonia traces and water was added to dissolve the solids, the ether was extracted with water (3 x 100 ml), the combined aqueous layers treated with 6 g of 5 sodium hydroxide and heated to steam distillate. ketone. The steam distillate was extracted with ethyl ether, the extracts washed with brine, dried and the ether evaporated to give 2,2-dimethylcyclohex anone as a colorless liquid, 2.0 g.

Ii. The ketone obtained above is converted to the oxime and the latter reduced with sodium in ethanol as described in Method A, step ii, to give 3.1 g of 2,2-dimethylcyclohexylamine.

Process C

2,2,6,6-Tetramethylcyclohexylamine.

i. 2,2,6,6-Tetramethylcyclohexanone.

A 50% suspension of sodium hydroxide in mineral oil, 14.3 g (0.30 mol), was suspended in tetrahydrofuran, the liquid decanted, and the solid resuspended and decanted again to remove the oil. Then, 15 g (0.12 mole) of 2,2-dimethylcyclohexanone was added, after dropwise addition of a mixture of 11 g of t-butanol and 20 ml of tetrahydrofuran (vigorous hydrogen evolution), and the resulting mixture was refluxed, until the hydrogen evolution was complete. To this mixture was added dropwise 37.8 g (0.30 mole) of methyl sulfate and heated under reflux for 24 hours. After dilution with water, extraction with ethyl ether, washing the extracts with water, drying and evaporation of the solvent below 40 ° C, 17 g of tetramethyl ketone were obtained.

This was distilled to give 14.6 g of product, b.p.

62-64 ° C (15 mm).

ii. The 2,2,6,6-tetramethylcyclohexane (8 g) obtained above was converted to the oxime and the latter compound was reduced according to the procedure of Method A, step ii, to give 1.4 g of the desired amine as a colorless liquid which had an appropriate purity for use

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64

Method D

2.2.5.5- Tetramethylcyclopentanone.

To a slurry of 2.0 moles sodium hydride (washed to remove oil) in tetrahydrofuran was added 5 190 ml (2.0 moles) of methyl sulfate. At the same time, 35.7 g (0.425 mol) of cyclopentanone in 50 ml of the same solvent was added slowly. The reaction mixture spontaneously warmed to a gentle reflux and hydrogen evolution was vigorous. At the end of the addition, the mixture is left stirring overnight at room temperature. After refluxing for two additional hours, a mixture of t-butanol in tetrahydrofuran was added and reflux continued for three hours. The reaction mixture was diluted with water, extracted with ethyl ether, the extracts washed with water, brine, dried over anhydrous MgSO4 and the solvent evaporated to give 48.2 g of crude product.

This was distilled to give 24.2 g of tetramethyl ketone, b.p. 63-68 ° C, 40 mm.

Method E

2.2.5.5- Tetramethylcyclopentylamine.

A flask was filled with 35 g (0.61 mol) of 40% sodium dispersion in mineral oil. The oil was removed by washing with ethyl ether and decanting. The sodium was then mixed with 400 ml of ether and a mixture of 32.8 g (0.20 mol) of 2,2,5,5-tetramethyladiponitrile prepared by the method of Coffman et al., J. Am. Chem. Soc., 80, 2868 (1957), and 400 ml of tetrahydrofuran were added slowly. The resulting mixture was stirred at room temperature for 4 hours, excess sodium was degraded by dropwise addition of saturated aqueous ammonium chloride, the organic layer was washed with water, dried (Na 2 SO 4) and evaporated to give 25.1 g of crude 2.2. 5,5-tetra-methylcyclopentylimin. The imine was dissolved in 75 ml of ethanol 35 and added dropwise to a flask containing 23.3 g (1 mole) of sodium. An additional 75 ml of ethanol was added and the mixture was heated at reflux until the remaining sodium metal was consumed. Reaktionsbian-

DK 154652 B

The mixture was diluted with water, acidified to pH 1 with concentrated hydrochloric acid, the aqueous phase washed with ether, then made strongly basic by the addition of sodium hydroxide. The organic layer was extracted with ether, washed with brine, dried (Na 2 SO 4) and evaporated to dryness. The residue was distilled in vacuo to give 6.6 g (23%) of the desired amine, bp.

60-61 ° C (20 mm).

Method F

2-Alkyl and 2,6-dialkylcyclohexylamines.

To a solution of 25 g of 2,6-diisopropylaniline in 250 ml of both ethanol and water was added 10 g of dry 5% ruthenium-on-carbon catalyst. The mixture was hydrogenated in an autoclave at 100 ° C, 1000 psi (70.4 kg / cm 2) until hydrogen uptake ceased. The catalyst was removed by filtration and the filtrate was evaporated to remove solvent. The residue was distilled in vacuo to give 11.2 g of 2,6-diisopropylcyclohexylamine in the form of a mixture of cis, trans and trans, trans isomers, Kp. 122-2Q 124 ° C at 22 mm.

Using the appropriate 2-alkylaniline or 2,6-dialkylaniline as starting material and by hydrogenation according to the above method, the following cyclohexylamines are also obtained.

2-methyl-6-ethylcyclohexylamine, Kp. 82-87 ° C at 19 mm (50% yield); 2-methyl-6-isopropylcyclohexylamine, Kp. 86 ° C at 14 mm (45% yield); trans-2-ethylcyclohexylamine, bp.77-78 ° C (Z3 mm); 2,6-diethylcyclohexylamine, bp 96 ° C (17 mm).

Method G

2-t-butylcyclohexylamine. i. 2-t-Butylcyclohexanone.

A solution of 31.25 g (0.20 mol) of t-butylcycloro hexanol in 80 ml of ethyl ether was cooled to 10 ° C. To this was added dropwise with stirring a solution of 21.0 g (0.07 mole) of sodium dichromate dihydrate and 15.75 ml (0.30 mole) of concentrated sulfuric acid in 100 ml of water while stirring.

. · DK 154652 B

66 then stirred to room temperature, stirred for 2 hours, poured into ice water, the ether layer removed, the aqueous phase extracted again with ether and the combined extracts washed with water, sodium hydrogen carbonate and dried (MgSO4). Evaporation of the ether gave 30.6 g (99%) of the desired ketone.

ii. Leuckart reduction of ketone.

A mixture of 2-t-butylcyclohexanone 30.6 g (0.20 mol) of formamide 50 ml (1.2 mol) and formic acid (10 ml) was heated at reflux while water was removed during its reaction with the reaction, while the ketone returned to the reaction vessel. Formic acid (10 ml) was added as needed to control the deposition of ammonium carbonate in the condenser. After 4 hours, the reaction temperature reached 197 ° C and distillation ceased. The mixture was cooled, diluted with water (50 ml) and extracted with ethyl acetate (75 ml). The organic layer was evaporated, concentrated hydrochloric acid was added (50 ml per 100 ml of residue), the mixture was boiled overnight, cooled and washed with 50 ml of ethyl ether. The aqueous phase was adjusted to pH 11 with 9 Ω sodium hydroxide, cooled, extracted with ether (2 x 40 ml) and the extracts dried over sodium hydroxide beads. The solvent was evaporated and the residue distilled through a 10 cm column to give 21.9 g of the title amine (71%) Kp. 86-88 ° C (21 mm) as a mixture of cis and trans isomers.

iii. dl-Eenchon and 1-fenchon are reduced to the corresponding fenchylamines according to the Leuckart reduction method in step ii above. (-) Fenchylamine is obtained as a water-clear liquid Kp. 55-60 ° C (6 mm), [α] n -21.9 ° in 30% yield.

30

Method H

2,4-dimethyl-3-aminopentane.

Place 0.2 g of platinum dioxide and 10 ml of water in a shake flask. The slurry was hydrogenated at 50 psi (3.5 2 kg / cm) for 15 minutes. To the resulting slurry of platinum black was added 34.26 g (0.30 mol) of 2,4-dimethyl-3-pentanone, 20.0 g (0.37 mol) of ammonium chloride, 225 ml of ammonium chloride.

DK 154652 B

67 mono-saturated methanol and 25 ml of concentrated ammonium hydroxide. The resulting slurry was hydrogenated at 60 psi (4.2 kg / cm) and room temperature for 20 hours, filtered, refluxed for 1 hour and cooled. The mixture was adjusted to pH 2.0 with concentrated hydrochloric acid, and the volume was reduced by evaporation under reduced pressure. After washing with 75 ml of ethyl ether, the aqueous solution was brought to pH 13 with 10 M sodium hydroxide solution and extracted with three 100 ml portions of ether. The extracts were pooled, dried over anhydrous MgSO 4 and saturated with gaseous hydrogen chloride. The precipitated amine hydrochloride was collected by filtration, air dried and decomposed with 75 ml of 10M sodium hydroxide solution.

The oily amine layer was removed and distilled at atmospheric pressure, Kp. 129-132 ° C, with 17.6 g of yield.

15

Process I trans-2-E thylcyclopentylamine i. 2-Ethylcyclopentanone.

In a three-neck flask, 5.0 g of metallic 2 ° sodium was dissolved in 250 ml of dry ethanol and 31.24 g (0.20 mole) of 2-carboethoxycyclopentanone were added. To the resulting yellow solution was added dropwise 18.4 ml (0 Ethyl iodide (23 moles) and the mixture heated under reflux for 2 hours. After cooling, 250 ml of brine 25 and 50 ml of water were added and the mixture extracted with ethyl ether (2 x 100 ml). After drying (MgSO4) and solvent evaporation, 36.5 g (99%) of 2-ethyl-2-carboethoxycyclopentanone was obtained.

This was decarboxylated by heating under reflux with a mixture of 200 ml of concentrated hydrochloric acid and 100 ml of water. After 4 hours at reflux, carbon dioxide evolution was completed. The mixture was cooled, saturated with sodium chloride, extracted with ethyl ether, the extracts dried (MgSO 4) and ether evaporated. The residue was distilled to give 12.62 g (56%) of 2-ethylcyclopentanone, Kp. 97-98 ° C (100 mm).

DK 154652 B

68 ii. The product obtained above was converted to trans-2-ethylcyclopentylamine according to the procedure of Method A, step ii, Kp. 150-151 ° C in 35% yield. The identity of the product was confirmed by its 1 H-NMR spectrum.

Method J

5 trans-2-Isopropylcyclopentylamine.

i. 2-Isopropylcyclopentanone.

To a solution of 10 g of sodium metal in 670 ml of ethanol was added dropwise a mixture of 100 g (1.19 mol) of cyclopentanone and 60 g (1.03 mol) of acetone, and the resulting mixture was refluxed for 1.5 hours. The solvent was evaporated in vacuo, the residue taken up in ether, the solution washed with 3M hydrochloric acid (5 x 200 ml), 5% sodium hydrogen carbonate (3 x 200 ml), brine (1 x 200 ml) and dried (MgSO4). The ether was evaporated under gentle heating to give 97 g of dark liquid, distilled in vacuo to give 55 g of 2-isopropylidene cyclopentanone, Kp. 96-100 ° C (2.7 mm).

To 12.75 g of the above product in 250 ml of ethanol was added 2.0 g of 5% palladium-on-carbon catalyst 2 tor, and the mixture was hydrogenated at 50 psi (3.5 kg / cm). After 1 hour, hydrogen uptake was complete. The catalyst was removed and the solvent was evaporated in vacuo to give 12.75 g of colorless liquid. This was distilled to give 9.64 g of 2-isopropylcyclopentanone, 25 Kp. 74-76 ° C (20 mm).

Reduction of 2-isopropylcyclopentanone by the method of method A, step ii, gave the corresponding amine, Kp. 167 ° C (atmospheric pressure) in 31% yield.

Method K

2,2-Dimethyl-3-aminobutane.

Into a 500 ml flask were placed 10.0 g (0.10 mole) of 2,2-dimethyl-3-butanone, 250 ml of methanol, 76.94 g (1.0 mole) of ammonium acetate and 4.37 g (0.07 mole) sodium cyanoborohydride,

o C

and the mixture was allowed to stir at room temperature for 24 hours. The pH was adjusted to 2.0 with concentrated hydrochloric acid and the methanol removed under reduced 69

DK 1S4652 B

pressure. The residual solid was dissolved in 500 ml of water and washed with three 100 ml portions of ether. The pH of the aqueous solution was adjusted to 13 with 10M sodium hydroxide and the mixture extracted with three 100 ml portions of ether. The extracts were collected, dried over anhydrous 5 MgSO 4, filtered and distilled. The amine (2.4 g) distilled at 102-103 ° C at atmospheric pressure.

The racemic amine is divided by the polarimetric control method described by Bruck et al., J.

Chem. Soc., 921 (1956), using the amine hydrogen-10 tartrates and crystallization in 70:30 methanol / water (by volume) to obtain right-turn amine with 93 in 4% purity and left-turn amine with 80 ± 4% purity.

Method L

L- ^ sparaginsyre-N-thiocarboxyanhydride.

A. L-Aspartic acid (582 g, 4.29 mol) was gradually added with stirring to 350.9 g (8.58 mol) of 50% sodium hydroxide solution at 0 ° C. Subsequently, methylreethylxanate (550 g, 4.51 mol) in 405 ml of methanol was added as quickly as possible. The mixture was heated at 45 ° C for 1.5 hours, cooled to room temperature and washed with two portions of methylene chloride. The methylene chloride washings were discarded and the aqueous phase acidified with concentrated hydrochloric acid at 0 ° C. The solution was extracted with three portions of ethyl acetate and the combined extracts were washed with brine and dried over anhydrous magnesium sulfate. The solvent was evaporated in vacuo to give a yellow oil which crystallized by the addition of ethylene dichloride and n-hexane. N-Methoxy-thiocarbonyl-L-aspartic acid was collected by filtration, washed with fresh n-hexane and dried (420 g, 47%).

Mp. 128-130 ° C; 1 H-NMR (DMSO-d 6), (6) 2.73 (d, 2H, J = 6 Hz), 3.63 (s, 3H), 4.43 (dt, 1H, J = 6 Hz, 8 Hz) ), 6.63 (d, 1H, J = 8 Hz); IR spectrum (KBr) 1715.1515 cm · 1 ·.

B. N-Methoxythiocarbonyl-L-aspartic acid (207.0 g, 1.00 mol) was dissolved in 1200 ml of ethyl acetate at 0 ° C and phosphorus tribromide (47 ml, 0.50 mol) was added in one pore.

DK 154652 B

? o tion. The cooling bath was removed and the temperature allowed to rise spontaneously to 35 ° C. The solution was stirred for 10 minutes, after which a granular white precipitate formed. The reaction mixture was cooled to 0-5 ° C, the product collected by filtration, washed with a small volume of ether and dried. The yield of analytically pure L-aspartic acid N-thiocarboxyanhydride was 157.4 g (90%).

Mp. 200-225 ° C (roof composition); [α] 25 = -109.5 ° (c = 1, THF); IR spectrum (KBr) 3225, 1739, 1724, 1653, 1399 10 cm -1; 1 H-NMR (DMSO-d 6) ppm (6) 2.83 (d, 2H, J = 5.0 Hz), 4.70 (t, 1H, J = 5.0 Hz), 9.23 (bs, 2H, ex); mass spectrum (m / e) 175 (M +), 87, 60.

Method M

2,2,3,3-Tetramethylcyclopropylamine.

i. Ethyl 2,2,3,3-tetramethylcyclopropane carboxylate.

The method of Mesheheryakov, Chem.

Abstr., 54, 24436d (1960). To a mixture of 19 g (0.226 mole) of 2,3-dimethyl-2-butene and 2 g of cupric sulfate is refluxed with a mixture of 51 g (0.447 mole) of ethyl diazoacetate and 19 g of 2,3-dimethyl-2 butene. The resulting mixture is refluxed for 3 hours, cooled, filtered and distilled to give 19.8 g (26%) of the desired cyclic ester, bp. 76-25 77 ° C (15 mm).

ii. To 300 ml of methanol containing 40 g of ammonia is added 17 g (0.10 mol) of the ester obtained above and the resulting mixture is left to stand overnight. After refluxing for 1 hour, the ethanol was evaporated in vacuo to give 2,2,3,3-tetramethylcyclopropane carboxamide.

A solution of 2.82 g (0.02 mol) of the amide in 8 ml of tetrahydrofuran and 4 ml of water is cooled to 5 ° C and 10 ml of 2M sodium hypochlorite is added dropwise followed by 35 of 8 ml of 20% (w / v) sodium hydroxide. . The two-phase mixture is stirred at 5 ° C for 30 minutes then at 20 ° C for 1 hour. The organic layer is extracted with ether, the ether layer is extracted with 2M hydrochloric acid (3 x 20 ml),

DK 154652 B

71 acidic layers are made strongly alkaline with sodium hydroxide and extracted with ether. The extracts are dried (Na 2 SC> 4) and the ether is evaporated at 25 ° C (50 mm) to give 0.67 g (25%) of 2,2,3,3-tetramethylcyclopropylamine. 1 H-NMR (CDCl 3) ppm (δ): δ 0.95 (6H, singlet); 1.00 (6H, singlet); 1.83 (1H, multiplet); 1.7 (2H, multiplet).

Process N Dicyclopropylcarbinylamine.

Into a 500 ml round bottom flask was placed 41.7 g of 10 (0.60 mol) of hydroxylamine hydrochloride and 80 ml of water. With stirring, 44 ml of 10 M sodium hydroxide solution and 44.4 g (0.40 mol) of dicyclopropyl ketone were added. The mixture was stirred at reflux for 3 hours. After cooling, 60 ml of methylene chloride was added and the mixture was stirred until all of the oxime had dissolved. The methylene chloride layer was removed and dried over anhydrous magnesium sulfate. The solvent was removed by evaporation under reduced pressure and the residue was recrystallized in 55 ml of hexane to give 40.0 g of dicyclopropyl ketoxime, m.p. 69-72 ° C.

20.8 g (0.15 mol) of dicyclopropyl ketoxime and 150 ml of anhydrous ethanol were placed in a 500 ml three-necked round bottom flask. With effective stirring, 19.2 g (0.83 mol) of sodium spheres was added in portions as quickly as possible while maintaining reflux during the addition. After dissolving the sodium, the reaction was cooled to 60 ° C and 60 ml of water was added. After cooling, 78 ml of concentrated hydrochloric acid was added dropwise with stirring. Ethanol was distilled under reduced pressure and 50 ml of water was added to dissolve salts. The mixture was adjusted to pH 13 with 10 M sodium hydroxide solution and extracted with three 40 ml portions of methylene chloride. The extracts were collected, dried over anhydrous magnesium sulfate, filtered and evaporated under reduced pressure. The remaining amine was distilled at 88-90 ° C / 355 mm Hg to give 11.0 g of the desired product.

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72

Method O

2-Amino-3,3-dimethyl-γ-butyrolactone hydrochloride. The method is by Nagase et al., Chem. Pharm.

Bull., 17, 398 (1969).

For a stirred solution of 2,2-dimethylhydroacryl-5aldehyde [prepared from sec-butylaldehyde and formaldehyde according to the method of Stiller et al., J.Am.Chem.

Soc., 62, 1785 (1940)] 5.11 g in methanol (25 ml) is added dropwise a solution of ammonium chloride (2.94 g) and sodium cyanide (2.9 g) in water (40 ml). After stirring for 3 hours, the mixture is saturated with ammonia gas and left at room temperature overnight. The resulting mixture is concentrated in vacuo to a small volume and 40 ml of concentrated hydrochloric acid is added. After refluxing for 3 hours, the mixture is evaporated in vacuo and the residue is crystallized in ethanol-ethyl ether and then in ethanol to give 2.2 g of the title compound, m.p. 214-215 ° C (decomposition).

Process of 2q 3-Amino-2,2,4,4-tetramethylthietane and its 1,1-dioxide.

A. 2,4-D.ibromo-2,4-dimethylpentan-3-one.

To 136 g (1.2 mole) of diisopropyl ketone was added 2 ml of phosphorus tribromide and the mixture was cooled to 10 ° C. To this was added 384 g (2.4 moles) of bromine dropwise, allowing the mixture to warm to room temperature. After 2 hours at this temperature, the mixture was heated at 55-60 ° C for 1 hour, then cooled and partitioned between chloroform and water. The water was discarded and the organic layer was washed with sodium carbonate solution until neutral. The organic layer was dried (MgSO 4) and solvent evaporated to give 316 g (97%) of the desired product.

B. 2,2,4,4-Tetramethyl-3-oxothietane.

Sodium metal, 23 g (1.0 mole), was dissolved in 500 ml of dry methanol and the resulting mixture was cooled to 10 ° C. Hydrogen sulfide gas was passed through the mixture,

DK 154652B

73 until it was saturated. Then, 136 g (0.5 mol) of dibromo ketone obtained in Step A was added dropwise while hydrogen sulfide continued to pass through the reaction mixture. After the addition was complete, the mixture was stirred for 2 hours at 10 ° C, allowed to warm to room temperature 5 and stirred overnight. After the reaction mixture was poured into water, it was extracted with ethyl ether and the extracts washed with dilute hydrochloric acid and brine. After drying over magnesium sulfate, the ether was evaporated, the residue was slurried with methanol, cooled and filtered to give 46 g (64%) of solid product used in the next step without purification.

C. Reductive amination of ketone.

To 75 ml of dry methanol were added 4.5 g (0.031 mol) of 2,2,4,4-tetramethyl-3-oxothietane, 23.9 g (0.31 mol) of ammonium acetate and 1.36 g (0 Sodium cyanoborohydride, and the resulting mixture was heated under reflux for 4 hours. Additional sodium cyanoborohydride (1.36 g) was added and reflux was continued for 3 days with a third increment of the same reagent added 20 after the start of the third day. The resulting mixture was acidified to pH 2 with hydrochloric acid and evaporated to dryness on a rotary evaporator under reduced pressure. The residue was dissolved in water, washed with ethyl ether, the aqueous phase adjusted to pH 11 with sodium hydroxide solution and extracted with ethyl ether. The extracts were washed with brine, dried (MgSO 4) and evaporated to dryness to give 1.9 g (42%) of the desired amine as a crystalline solid. The structure of the product was confirmed by its 1 H-NMR spectrum.

D. 3-Amino-2,2,4,4-tetramethylthietane-1,1-dioxide.

29 g (0.2 mole) of the amine obtained in step C above was dissolved in 50 ml of acetonitrile and 250 ml of water was added. While keeping the mixture at pH 10 with sodium hydroxide, 35.8 g (0.21 mole) of carhobenzoxy chloride was added over 30 minutes, the mixture was stirred for 1 hour, filtered, the precipitate was washed with water and dried in vacuo at 50 ° C. to obtain the NCfaz amine, 74

DK 154652 B

Rf 0.7 (hexane / ethyl acetate, 4: 1, volume / volume phosphorus-molybdenum acid spray), 52.1 g (93.4%). This was dissolved in 700 ml of methylene chloride, 77 g (0.372 mol) of m-chloroperbenzoic acid was added slowly while keeping the temperature below 45 ° C (20-42 ° C). The precipitated solid was collected by filtration, the filtrate washed with 1N hydrochloric acid, aqueous sodium hydrogen carbonate solution, dried (MgSO4) and the solvent evaporated. The residue was crystallized in acetone water to give 42 g (73%) of the Cbz-protected amine-1,1-dioxide, R f 0.7 (hexane / ethyl acetate, 1: 1, 10 volume / volume phosphorolybdic acid spray). .

The protecting group was removed by hydrogenolysis of 5 g of Cbz-amine in 250 ml of methanol, 5 ml of concentrated hydrochloric acid and 2 g of 5% Pd / C (50% wet). The product is isolated in the usual manner. Yield: 2.4 g (85%), Rf 0.6. The retention time by gas-liquid chromatography on a 1-meter OV-1 column at 180 ° C was 1.3 minutes. The total yield for the three steps starting from 3-amino-2,2,4,4-tetramethylthietane was 65%.

By using equivalent amounts of amine and m-20-chloroperbenzoic acid in the above procedure, the corresponding sulfoxide is similarly obtained.

Process Q

3-Amino-2,2,4,4-tetramethyltetrahydrothiophen.

A. 1-Hydroxy-2,2,4-trimethylpentan-3-one.

To sodium methoxide prepared from 7.5 g of sodium metal and 250 ml of methanol was added 72.5 g (2.4 moles) of paraformaldehyde, followed by 250 g (2.2 moles) of diisopropyl ketone and the resulting mixture was heated under reflux for 30 minutes. 3 hours. The reaction was quenched with water, neutralized with hydrochloric acid, extracted with ethyl ether, washed with water, brine, and the solvent evaporated. The remaining oil (90 g) was distilled in vacuo to give 28 g of the desired product boiling at 92-98 ° C at 16-20 mm. GLC on OV-1 column at 107 ° C, retention time 314 seconds, 96% pure.

DK 154652 B

75 If the above procedure was repeated on the same scale but the reaction mixture was refluxed for 16 hours, 31 g of product with 96% purity after GLC were obtained.

B. 4-Bromo-1-hydroxy-2,2,4-trimethylpentan-3-one.

To a stirred solution of 69 g (0.48 mol) of 1-hydroxy-2,2,4-trimethylpentan-3-one in 500 ml of refluxing chloroform was added dropwise a solution of 77 g (0.48 mol) bromine in 100 ml of chloroform. When the addition was complete, the mixture was stirred under reflux for 1 hour, allowed to cool and allowed to stand overnight at room temperature. Evaporation of the solvent under reduced pressure gave 127 g of product which was used in the next step without purification.

C. 2,2,4,4-Tetramethyltetrahydrothiophen-3-an.

The product obtained in step B, 79 g (0, 3 mol), dissolved in 300 ml of dry pyridine, cooled to 0 ° C and 114 g (0.6 mol) of p-toluenesulfonyl chloride added in portions at 0 ° C. . The resulting mixture was stirred at this temperature for 3 hours and 15 minutes, poured into ice / water and extracted with ethyl ether. The extracts 20 were washed with dilute hydrochloric acid, water and brine, then dried over anhydrous magnesium sulfate. The solvent was evaporated to give 111 g (98%) of crystalline tosylate.

The tosylate, 94 g (0.25 mole), was dissolved in 1 liter of pyridine, 180 g (0.75 mole) of sodium sulfide monohydrate was added, and the mixture was heated to 75 ° C and kept at this temperature for 1 hour and allowed to stand at room temperature overnight. over. Water was added and the mixture extracted with ether. The extracts were washed with hydrochloric acid, brine, dried (MgSO 4) and the solvent evaporated to give 35 g of the title compound, 89% yield. The product showed only one spot by silica gel TLC, eluted with ethyl acetate / hexane (1: 4 by volume, 35

DK 154652 B

76 R ^ 0/5. The 1 H NMR spectrum was consistent with the structure of the title compound.

D. Leuckart reduction of ketone.

To a 100 ml round-bottomed, three-necked flask equipped with a stirrer, thermometer and condenser with fractionation head was added 10.0 g (0.063 mole) of 2,2,4,4-tetra-methyltetrahydrothiophen-3-one, , 2 ml (0.38 mol) of formamide and 3.5 ml (0.092 mol) formic acid, and the mixture was heated under reflux (163 ° C) while water was removed. The reaction mixture was maintained at 160-180 ° C for 20 hours with the addition of formic acid (10 ml) at intervals. The mixing temperature rose to 200 ° C during this time. The reaction mixture was cooled, water was added and the mixture extracted with ethyl acetate. The extracts were evaporated in vacuo. The residue was refluxed with 20 ml of 6N hydrochloric acid for 2 hours, cooled, the resulting mixture washed with ethyl ether, the aqueous phase adjusted to pH 11 with sodium hydroxide solution and extracted with ethyl ether. The extracts were dried and evaporated to give 2 g of 3-amino-2,2,4,4-tetramethyl tetrahydrothiophene, identified by 1 H-NMR and found homogeneous by silica gel TLC.

Process R

3- Amino-2,2,4,4-tetramethyltetrahydrofuran.

A. 2,2,4,4-Tetramethyltetrahydrofuran-3-one.

4- Bromo-1-hydroxy-2,2,4-trimethylpentan-3-one (prepared as described in process Q, steps A and B), dissolve 25 g (0.1 mole) in 160 ml of ethanol and add a solution of 8 g (0.2 mole) of sodium hydroxide in 80 ml of water. The resulting mixture was stirred at room temperature for 30 minutes, diluted with water, extracted with ethyl ether, the extracts washed with water,

DK 154652 B

77 brine and dried on anhydrous magnesium sulfate. The solvent was evaporated to give 17/7 g of 2,2,4-trimethylpentane-1 * 4-diol as a colorless liquid identified by 1 H-NMR. The diol was dissolved in 5 ml of chloroform, 1.5 ml of concentrated sulfuric acid was added dropwise. The mixture was heated at reflux for 3 hours while a water / chloroform azeotrope was distilled from the mixture. After standing overnight at room temperature, the reaction mixture was washed with water, the organic layer was dried (MgSO 4) and solvent was evaporated in vacuo to give 13.9 g of colorless liquid. Distillation gave 8.3 g of the desired product, b.p. 70-72 ° C (50 mm), utilizing a total of 58%.

B. The ketone obtained in step A, 8.0 g (0.056 mol), hydroxylamine hydrochloride, 8.0 g (0.113 mol), and sodium acetate, 2.3 g (0.113 mol), was combined with 85 ml of ethanol, and the mixture was heated at reflux for 48 hours.

The resulting mixture was diluted with water, extracted with ethyl ether and the extracts washed with water, dried and evaporated to give 9.0 g of the mixture of syn and antioximes identified by its ³ ^ spectrum.

The oxime obtained above, 1.3 g (8.28 mmol), was dissolved in 70 ml of dry ethanol, filtered with 1.9 g of sodium metal, and the mixture was heated under reflux and kept at this temperature for 15 minutes. Heating was continued for 2 hours, with the addition of two additional increments (each 1.9 g) of sodium. The reaction mixture was then gently diluted with water, extracted with ethyl ether. The ether layer was extracted with dilute hydrochloric acid, the aqueous phase was made alkaline with sodium hydroxide and re-extracted with ether. The extracts were dried (MgSO4) and evaporated to dryness and the residue distilled to give the desired amine, bp. 68-35 69 ° C (15 mm). After further purification by precipitation of the hydrochloride salt in ethyl ether-methanol, the salt was basified and extracted again with ether to give 0.87 g of amine with 93% purity by gas chromatography (OV-1 column).

Claims (2)

1. L-Aspartyl-D-amino acid dipeptidamide characterized in that it has the general formula I 5 f 2 HH I r'V \ Vnhr COOH J Ra 10 as well as physiologically acceptable cationic salts and acid addition salts thereof, wherein formula R is CH 2 OH or CH 2 OCH 3, and R is a branched group selected from fenchyl, diisopropylcarbinyl, d-methyl-t-butyl-carbinyl, d-ethyl-t-butylcarbinyl, di-t-butylcarbinyl, 2-methylthio-2,4-dimethylpentane. 3-yl, 20 \ \ J08! 1? 31 R ^ r * 6V p where at least one of R ^, R ^, R ~ and R ^ is alkyl having from one to four carbon atoms and the remainder is hydrogen or alkyl having from one to four carbon atoms, and the sum of carbon atoms in R, R, R and 6 3 R is not greater than 6 and if both R and Ry or R and R are alkyl, they are methyl or ethyl, 30. is 0, S, SO, SO 2, C = 0 or CHOH, i.e. 0, 1, 2, 3 or 4, and n and p are each 0, 1, 2 or 3, the sum of n + p is not greater than 3, V * 2 m DK where one of R, R and R is alkyl having from one to four car carbon atoms, and the balance is hydrogen or alkyl having from one to four carbon atoms, and the sum is 7 8 9. of carbon atoms in R ', R and R are not greater than 6 and m has the meaning given above, J- (CH 2 ». UcH 2) g 10 where m has the meaning given above and q is 0, 1, 2 , 3 or 4, R 12? 13 -z / 12 13 where each of R and R is methyl or ethyl, or 12 13 R is hydrogen and R is alkyl having from one to Ure carbon atoms, Z is O or NH, and t is 1 or 2, r »£ 1 - \ ^> H2> w 25. OH where w is 1, 2, 3 or 4, R 14 and R 16 are each alkyl having 15 from one to four carbon atoms, R is hydrogen, OH, or alkyl having from one to two carbon atoms, the sum of car-14 being 16 30 the atoms of R, R and R are not greater than 6, and 14 15 if both R and R are alkyl, they are methyl or ethyl, and 35 ~ \ 20 \ -— BR ii ^ 19 80- DK 154652 B 17 19 wherein R and R are alkyl of from one to four carbon atoms, R and R are hydrogen or alkyl of from one to two carbon atoms, and taken separately A is OH and B is hydrogen, OH or methyl, and if taken together A and B are CH, OC-, CHONHC-, -O-OCHO-, -NHCCH--, -OC-, ^ II II ^ II ^ II 5 OO 00 17 -NHC- or -0C0- where the sum of carbon atoms in R, »f II 0 O R 3, R 9 and R 29 are not greater than 6, and if both R 1 and R 3 or R 19 and R 23 are alkyl, they are methyl or ethyl, D-amino acid amide compound for use as a starting compound in preparing a compound of formula I according to claim 1, characterized in that it has the general formula Ra-CHCOONHRC nh2 wherein Ra is C1 OH or CH2 CH2 and Rc is a group selected from fenchyl, diisopropylcarbinyl, d-methyl-t-butylcarbinyl, d-ethyl-t-butylcarbinyl, di-t-butylcarbinyl, cyclopropyl-t-butylcarbinyl, cyclopentyl-t-butylcarbinyl, dicyclopropylcarbinyl, V ° Wherein m ^ is 1, 2 or 3 and if m is 1: R33, R43, R33 and R33 are each methyl, if m is 2: R is methyl, ethyl or isopropyl, and R43, R 0 and R 33 are each hydrogen or R 30 and R 53 are each methyl and R 43 and SO R are each hydrogen and if m 2 is 3: (a) R 33 is isopropyl or t-butyl, and R 43, R 33 and R 33 each is hydrogen, (b) R 3 ^ is ethyl, R 3 ^ is methyl, and R 4® and: R 1 is each hydrogen, or (c) R 1 and R 40 are each methyl and R 50 and R 60 are each hydrogen or methyl, and 5 cH3> xR41 _ / ^ {CH2) n> ^ Λ. (cH 2) pr 2 X 2 CH 3 R 31 10 wherein n 2 and p 2 are each 0, R 41 and R 61 are each methyl and X 9 is S, SO 9, C = 0 or CHOH, or and p2 is 1, R and R are each methyl and X2 is O, S or SO2, or n2 is 1, and p2 is 1, R41 and R61 are each hydrogen, and X2 is S or SO2 · 20 25 35