FI79834C - D-amino acid amide compound useful as an intermediate in the preparation of L-aspartyl-D-amino acid dipeptide amides - Google Patents

Description

1,79834 D-amino acid amide compound useful as an intermediate in the preparation of L-aspartyl-D-amino acid di-peptide amides 5 Separated from patent application 810148 (patent 77044)

The present invention provides a D-amino acid amide compound useful as an intermediate in the preparation of L-as-10 partyl D-amino acid dipeptide amides.

FI patent 77044 relates to novel amides of L-aspartyl-D-alanine, L-aspartyl-D-2-aminobutyric acid and L-aspartyl-D-valine, which are particularly useful due to their strong sweetening properties and contain edible suitable composition.

In U.S. Patent 3,492,131, certain lower alkyl esters of L-aspartyl-L-phenylalanine have been found to be up to 200 times sweeter than sucrose and to be substantially free of the bitter flavors that have been disadvantages of previous artificial sweeteners such as saccharin. The stability of these compounds was later found to be limited in aqueous mixtures, due to the formation of diketopiperazine, especially under the neutral-acidic pH conditions prevailing in most food mixtures.

Mazur et al., J. Med. Chem. 16 (1973) 1284 have shown that the lower alkyl esters of L-aspartyl-D-alanine lower alkyl esters and some of their homologues, in particular L-aspartyl-D-alanine isopropyl ester, are up to 125 times that of sucrose.

Sukehiro et al., Seikatsu Kagaku 11 (1977) 9-16; Chem. Abstr. 87 (1977) 168407h disclosed certain amides of L-aspartyl-D-alanine of formula

NH O

35 X / NH 1 nhr1

COOH "I

O ch3 2 79834 wherein R 1 "is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, secondary butyl, cyclohexyl or a carbon residue of methyl esters of glycine, D-alanine or L-alanine. The most effective were compounds with 5 Since n-butylamide was found to be 125 times sweeter than sucrose and isobutyl and secondary butylamides are 100 times sweeter than sucrose, it was concluded that the potency of these amides is mainly affected by the presence of alkyl amylose and cyclohexyl, which are 100-125 and 100 times sweeter, respectively. R 1 is the number of carbon atoms, and that the structural isomerism of the alkyl group has little effect on the sweetening power.

It has now surprisingly been found that not only the size of the amide-15 substituent has a decisive effect on the high sweetness of the L-asparyl-D-alaninamides, but on the contrary, the precise spatial arrangement of the amide substituent R is crucial. It has been found that certain L-aspartyl-D-alanine amides having a carbon atom having a carbon atom having an amide nitrogen atom) also have a side chain and a side chain again at one or both of the β and β 'carbon atoms. , has significant benefits.

FI patent 77044 relates to novel side-chain amides of L-aspartyl-D-alanine and certain other L-aspartyl-25-C1-alkyl-uC-amino acid dipeptides which have a surprisingly high sweetening power and do not have undesirable and flavoring agents in conventional amounts, as well as compositions containing said amides which can be used to sweeten edible substances. Also, their stability has been found to be surprisingly good both in solid form and in aqueous mixtures in the characteristic pH range of most food mixtures, even at higher temperatures used in baking and conventional food preparation.

35 The novel compounds which are the subject of FI patent 77044

are L-aspartyl-D-amino acid dipeptide amides of formula I

3 79334

NH0 O

I 2 II

II NHR (I) 5 COOH ° Ra and their physiologically acceptable cationic and acid addition salts. These new compounds are characterized in that in formula I R is methyl, ethyl or isopropyl; and R is phenyl; di-isopropyylikarbinyyli; d-methyl-t-10 butylcarbinyl; d-ethyl-t-butyylikarbinyyli; di-t-butyl-tyylikarbinyyli; disyklopropyylikarbinyyli; dimetyylikar-binyyli; methyl-isobutyylikarbinyyli; isopropyl-t-butyl-tyylikarbinyyli; 2-methylthio-2,4-dimethyl-3-yl; a group of formula (a) or (b) 15 4 r3 \ / r3 * ^.

“~ Y ^ (CH2> m ^^ X

K (a) (b) wherein at least one of R 3, R 4, R 3 and R 2 is alkyl of 1 to 4 carbon atoms and the others are hydrogen or alkyl of 1 to 4 carbon atoms; X is O, S or SO_; m is 0, Z 3 4 1, 2 or 3; n and p are each 0 or 1; and the sum of the carbon atoms of R 1, R 2, R 2 and R 2 is not greater than 6, and when both R 3 and R 2 or R 3 and R 2 are alkyl, they are methyl; a group of formula (c) Ί „8 30 (o Η1ΰ ^ 1 - (CH-) 2 m 7 8.9 35 wherein R, R 1 and R each represent hydrogen or alkyl,

7 O Q

having 1 to 4 carbon atoms, and the sum of the carbon atoms of the groups R, R and R 4 79834 is not more than 6, R 3 to 4 is hydrogen or alkyl having 1 to 4 carbon atoms, and m is 0 to 2; a group of formula (d) 5 H 3 Cl 2 CH 3 - / (d)

O

10 or a group of formula (e), CH-.

H-.C —-_ y J 3 ——— OH

\ .H

15 H3C

Preferred sweeteners are dipeptide amides of formula (I) wherein the aspartyl amino acid dipeptide group is a derivative of L-aspartic acid and D-amino acid, RaCH (NH2) COOH.

Novel intermediate amines of formula (I) may be used in the preparation of compounds of formula (I)

R b NH 2 wherein R b is a) d-ethyl-t-butylcarbinyl, cyclopropyl-t-butylcarbinyl or cyclopentyl-t-butylcarbinyl; b) R 3 / R 4 -ÖCH 2, * i R 6 ^ 5 35 wherein m ^ is 1, 2 or 3 and when m ^ is 1: R 3 -R 6 each represent methyl, when m ^ is 2: R 3 is methyl, ethyl or isopropyl and ς 79834 R 1 -R 8 are each hydrogen and when 3 is: R 1 is t-butyl and R 4 -R 8 are each hydrogen; or c) CH 3 \ CH 3 CH 3 CH 3 10 wherein when n 1 is O and p 1 is 1, X 1 is O, S or SO 2 and when n 1 is each O, X 1 is S, SO 2 or C = O .

The valuable novel intermediate compounds of the present invention useful in the preparation of compounds of formula (I) are characterized in that the D-amino acid amide compounds have the formula

RaCHCONHRC

20 NH2

a C

wherein R is methyl, ethyl or isopropyl and R is diisopropylcarbinyl, di-t-butylcarbinyl, dicyclopropylcarbinyl or a group of formula 25 3 CH3 ^: * 2 30 x - ^ ^ si 2 P2

ch3 R

wherein when n2 and p2 are each O, R43, and R61 are each methyl and X- is S or SO9, when n2 is O and p2 is 1, R and R0 are each methyl 6 79334 and X9 is S or SO9, and when n2 is 1 and P2 is 1, R and R0 are each hydrogen and X2 is S.

As used herein, the suffix "carbinyl" means 5 groups -CH-. Thus, for example, diisopropylcarbinyl is a group (i-C 1 H 2) 2 -CH- and dicyclopropylcarbinylamine is (A) 2CHNH 2.

The dipeptide amides of formula (I) are conveniently prepared by methods suitable for amino acid coupling. The preferred process for the preparation of di-peptide amides of formula (I) is outlined below.

_NHQ Ra L "/ 7Λ + D-NHoCHC00R1; L (1) condensation COOR COOH (2) H 2 O '15 or carboxyl-activated derivative a

NHQ Ra NHQ R

COO ^ "cbNH ^ - 5 * COO ^^ ONH ^^ CONHR

(II) / 20 (A protection ^ 111 ^

Uj removal

In the above L-aspartic acid derivatives, Q has one of the well-known amino protecting groups that can be selectively removed, such as those described by Boissonnas, Advances in Organic Chem. 3 (1963) 159-190. Particularly preferred amino protecting groups are benzyloxycarbonyl and tert-butyloxycarbonyl. R 1 is mainly an alkyl group having one to four carbon atoms or benzyl. The D-alanine, D-2-aminobutyric acid or D-va-30 line used may be a free amino acid in which R 1 is hydrogen, but is essentially a carboxyl-protected derivative in which R 1 may be a residue of an ester group such as methyl or ethyl, but mainly it is a silyl group such as trialkylsilyl having 3 to 12 carbon atoms. For economic and te-35 efficiency reasons, a particularly preferred such group is trimethylsilyl.

, 79334

In the first step of the above reaction sequence, the di-protected L-aspartic acid is condensed with the appropriate D-amino acid or carboxy-protected derivative to give the di-protected dipeptide of formula (II). Although each of these steps can be performed using di-protected aspartic acid in the presence of condensing agents such as dicyclohexylcarbodiimide, it is more preferable to use an OC-carboxyl-activated derivative of di-protected aspartic acid. Preferred such carboxyl-activated derivatives are chloride, bromide, anhydride or mixed anhydride. For reasons of efficiency, the mixed anhydrides of the abovementioned di-protected L-aspartic acids with chlorocarboxylic acid esters, in particular alkyl esters having one to four carbon atoms in said alkyl, are particularly preferred. The most preferred mixed anhydrides are anhydrides prepared from methyl and ethyl esters of chlorocarboxylic acid, for economic reasons.

In a particularly preferred process for the preparation of the compounds of formula (I), β-benzyl-N-benzyloxycarbonyl-L-aspartic acid is reacted with ethyl chlorocarbonate by methods known in the art to form the corresponding mixed anhydride. D-amino acid,

RaCH (NH2) COOH obtained from commercial sources or by resolution of a racemic amino acid by known methods. e.g., Yamada et al., J. Org. Chem. , 38, 4408 (1973) / 7, is converted in a separate vessel to trimethylsilyl ester by contacting the amino acid with an equimolar amount of trimethylsilyl chloride in the presence of a reaction-neutral organic solvent. Suitable solvents for this purpose are, for example, pyridine, dimethylformamide or dimethylacetamide; particularly preferred is dimethylformamide.

In a typical reaction according to this method, the addition of 35 D-amino acids, e.g. D-alanine dissolved in dimethylformamide and an equimolar amount of trimethylchlorosilane takes place at room temperature. Β-Benzyl-β 79334 N-benzyloxycarbonyl-L-aspartic acid and an excess of molar acid scavenger, mainly triethylamine, are dissolved in a separate flask in a mixture of dimethylformamide and tetrahydrofuran and at room temperature 5 or below, mainly at about -25 ° C. At + 25 ° C, and especially at about -10 to 0 ° C, an equimolar amount of ethyl chlorocarbonate is added to form mixed anhydride. To this is added, for example, a solution of D-alanine trimethylsilyl ester, the temperature being essentially within the same range. The reaction is usually completed within 1-2 hours, after which the reaction mixture is poured into water or an aqueous acid, for example 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 general methods. . The di-protected dipeptide (II) is usually sufficiently purified for use in the next step, but may be further purified if desired, for example by column chromatography.

In a second step of this process, the di-protected dipeptide (II) is reacted with an equimolar amount of a primary amine of formula RN1 · ^ to give the corresponding di-protected dipeptide-amide intermediate of formula (III) wherein Ra, R, R1 ^ and Q are as previously defined.

As in the first step, the carboxylic acid form of the reagent (II) can be used successfully when using condensing agents, for example dicyclohexylcarbodiimide, to give intermediates of formula (II). However, it is preferred that the compound of formula (II) be converted into a carboxyl-activated derivative, for example a chloride, bromide or mixed anhydride, the latter being preferred. Thus, the mixed anhydride is prepared using in particular a compound of formula (II), wherein R 10 is benzyl and Q is benzyloxycarbonyl. As before, the preferred anhydrides are the anhydrides obtained from .9,7334 esters of chlorocarboxylic acid, and their methyl or ethyl esters are particularly preferred. The mixed anhydrides of compound (II) are prepared using the reagents and conditions described above for the first step of this series of reactions. In a typical reaction, a compound of formula (II) and triethylamine are combined in approximately equimolar amounts in a reaction-neutral organic solvent, for example tetrahydrofuran, the mixture is cooled to about -10 ° C and ethyl chlorocarbonate is added to give mixed anhydride. To this is then added an equimolar amount of an amine of formula RNH 2 or a solution thereof, for example in the same reaction-neutral solvent and at a temperature in the range of about -50 to + 25 ° C and most preferably in the range of -35 to -15 ° C. Upon completion of the amine addition, the reaction mixture is allowed to warm to approximately room temperature and maintained at this temperature until the reaction is substantially complete, usually about 1 to 20 hours. The desired intermediate of formula (II) is then isolated and purified, as desired, by the same methods as described above for compound (II).

In the final step of this process, the carboxyl protecting group is removed, and the amino protecting group, Q, is obtained to give the desired sweeteners of formula (I).

The method chosen to remove the protecting groups from the dipeptide amide of formula (III) depends on a number of factors which will be apparent to those skilled in the art.

Two important factors in making such a choice are the nature of the protecting groups R 1 and Q and the nature of the amide substituent R. For example, when R 1 and Q are, respectively, particularly preferred groups, benzyl and benzyloxycarbonyl and R is free of sulfur, the preferred method for removing said protecting groups is generally hydrogenolysis. However, when R 1 is benzyl or alkyl as defined above and Q is tert-butyloxycarbonyl and R has any of the above meanings, it is generally preferred to deprotect by hydrolysis. A combination of hydrolysis and hydrogenolysis is desirable in cases where I <is alkyl, Q is benzyloxycarbonyl and R is sulfur-free.

When hydrogenolysis is selected to remove protecting groups from an intermediate of formula (III), it is recommended that the reaction be carried out in the presence of a catalytic amount of noble metal catalyst, with palladium being particularly preferred and in the presence of a reaction-neutral solvent. Examples of such solvents include 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. Particularly preferred such solvents are methanol and ethanol for their economy and efficiency. Although hydrogenolysis can be performed successfully at higher pressures and temperatures, for reasons of economy and comfort, atmospheric pressures of about 1 to 10 and room temperature are preferred. When temperature and pressure are preferred, the reaction is generally complete within about 30 minutes to about six hours, after which the catalyst is removed, typically by filtration, the solvent is evaporated and the resulting product is purified, if desired, by standard methods such as recrystallization or chromatography. la column.

When hydrolysis is chosen to remove one or both of the protecting groups R1® and Q, any of the well-known methods for carrying out alkaline hydrolysis or acid hydrolysis of esters or the like can be used with some success. However, when it is necessary to remove the protecting groups R 1 by hydrolysis, alkaline hydrolysis is preferred and the use of at least an equivalent strong base base, for example sodium hydroxide or potassium hydroxide in the presence of water and a lower alkanol, especially methanol or ethanol, is to be considered as particularly preferred. at room temperature or at about room temperature. Under these primary conditions, the hydrolytic removal of the R 1 group is usually completed in a few hours or less.

When the amino protecting group Q is tert-butyloxycarbonyl, it is recommended to use acidic hydrolysis to remove it. It is particularly desirable to use an aqueous solution of dilute hydrochloric acid in the presence of methanol or ethanol and to heat the mixture to boiling temperature. Under these conditions, the hydrolysis is usually complete in a few hours or less.

To isolate the products of formula (I) after deprotection by any of the hydrolysis methods described above, general methods known in the art are used. For example, after acid hydrolysis, the reaction mixture is evaporated to dryness to remove the solvent, the aqueous residue is washed with a water-immiscible non-polar solvent such as ethyl ether or chloroform, then the aqueous layer is made alkaline and the product is extracted with a water-immiscible solvent. such as ethyl acetate and the product is recovered by evaporating the solvent. If desired, the product can be further purified, for example by recrystallization or column chromatography. When alkaline hydrolysis to remove the protecting group is followed by hydrogenolysis to remove the amino protecting group Q, it is desirable that the reaction mixture obtained by alkaline hydrolysis be neutralized by adding an acid, for example hydrochloric acid, and the neutralized reaction mixture is hydrogenolyzed as described above.

Another preferred method for preparing compounds of formula (I) is set forth below.

12 79834

Ra Ra

D-QNHCHCOOH + RNH QNHCHCONHR

-—> or carboxyl-activated derivative 5

NHQ

protection Ra L- / T ~

removal, COOR COOH

-> nh2chconhr -> (III) -> (I) 10 (V) a 10 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 the amine RNH2 using the methods and conditions described above for the preparation of intermediates (II) and (III) to give the amino-protected compound of formula (IV). D-amino acid amide. The protecting group Q is removed by hydrogenolysis or hydrolysis as described above, and the resulting free aminoamide (V) is condensed with a di-protected L-aspartic acid derivative or a carboxyl-activated derivative thereof as described above for the preparation of intermediates of formula (II). the di-protected dipeptide amide of formula (III) is obtained to give the desired sweetener of formula (I) as described above.

In a variation of this process, an intermediate of formula (IV) having a cyclic or acyclic sulfide group (-S-) in R may be oxidized to the corresponding sulfoxide or sulfone prior to its conversion to intermediate (V) and thereafter as described above. reactions to give compounds of formula (I) wherein R is a sulfoxide or sulfone.

In a third preferred process for the preparation of compounds of formula (I), the D-amino acid amide of formula (V) described above is reacted with 13,79834 L-aspartic acid-N-thiocarboxyanhydride to give directly compounds of formula (I). In carrying out this process, intermediate (V) is contacted in a suitable solvent with an equimolar amount of L-aspartic-N-thiocarboxyanhydride at mildly temporal pH conditions at a temperature between about -25 and + 10 ° C to give a compound of formula (I). The alkaline pH required for this reaction is provided by a strong base such as sodium hydroxide or potassium carbonate. Suitable solvents for use in this reaction include solvents which dissolve at least a portion under the reaction conditions used in the reagents without reacting in substantial amounts with either reagent and allow the products formed in the reaction to be isolated relatively easily. Examples of such solvents used in this reaction include water, tetrahydrofuran, 1,2-dimethoxyethane, diethylene glycol dimethyl ether, dimethyl sulfoxide, dimethylformamide, and combinations thereof; the preferred solvents are water and mixtures thereof with tet-20 rahydrofuran. The preferred alkaline pH range to be used in this reaction is between about 8-10 and especially its preferred pH is about 9. The particularly preferred temperature is between about -10 and 0 ° C.

Under the above preferred conditions, the reaction is usually complete within 1-2 hours. The product of formula (I) is then isolated by general methods, for example the pH of the reaction mixture is adjusted to the isoelectric pH of the product, usually between about pH 5.0 and 5.6, whereupon the product of formula (I) precipitates, the solvent is removed by evaporation or by filtration and the crude product is slurried in an organic solvent, for example methanol, ethanol, ethyl ether, ethyl acetate or a mixture thereof. The product of formula (I) is then isolated, for example by filtration. It can be further purified if desired, e.g. by recrystallization or column chromatography.

14 79 834

The required amines of formula RNI, wherein R is as previously defined, are either commercially available or can be obtained from readily available starting compounds. For example, 2-alkylcyclohexylamines and 2,6-dialkyl-5-cyclohexylamines can be obtained by catalytic hydrogenation of the corresponding alkyl-substituted anilines. Many amines are obtained using reductive amination from the corresponding ketones using a variety of conditions known in the art. For example, reduction amination by a well-known Leuckhart reaction using formic acid and formamide as reducing agents may be useful; see. for example, Organic Reactions, Wiley and Sons, New York, Vol. 5, p. 301, 1949. Alternatively, the appropriate ketone can be aminated by reduction using sodium cyanoborohydride and ammonium acetate, cf. for example, J. Amer. Chem. Soc., 93, 2897 (1971) or with ammonia in ethanol in the presence of a hydrogenation catalyst such as Raney nickel, platinum or palladium; see. for example, Organic Reactions, 4, 174 (1948). Many of the amines of formula RNI are obtained from the corresponding ketones by formation of an oxime 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 or sodium hydrogenation in the presence of a low molecular weight alkanol at a higher than normal temperature.

In a particularly preferred process, which is particularly useful in the reduction of oximes of sulfur-containing ketones, the oxime is reduced in ethanol and using 30 moles of excess sodium at the boiling point of the mixture.

The required RN1 ketone starting materials for amines are either commercially available, known in the art, or are prepared by known methods. For example, ketones of formula (VII) 35 (VII) 79334 wherein, R 4, R 5, R 1, X, n and p are as defined above can be obtained by alkylation of the corresponding compounds wherein R, R, R 0 and R is each hydrogen to give compounds of the above formula wherein one to each of R 4, R, R, R 0 is alkyl as defined above. The alkylation is carried out, for example, using alkylating agents, such as a suitable alkyl halide or alkyl sulphate, under net or time conditions provided by strong bases, for example sodium hydride or sodium amide. Using the same procedure, compounds of formulas (VII) in which only the substituent in the -> L-position relative to 1, 2 or 3 keto groups is alkyl can be converted to compounds of the same formula in which two to four of the equivalents R 1, R 4 , R 1, R 6 are alkyls. According to formula (VII) ... 3.4. gem dialkyl compounds in which either R and R or 5 6

R and R are said alkyls, can be obtained from the appropriate monoalkyl compound by protecting the unsubstituted Cu position before alkylation and then removing the protecting group. For example, 2,2-dimethylcyclohexanone can be obtained by condensing 2-methylcyclohexanone with ethyl formate in the presence of sodium methylate and alkylating the resulting intermediate as outlined below. o O

X ^^ x ^ ONa CH3I

+ HCO C H -],

NaOCH3 NaNf, 2

30 O

m / z Na 35 16 79334

Certain ketones of formula (VII) are also obtained from acyclic starting compounds which are derivatives of ketones of formula (VIII), 5 VV r3 I 5 I 4 (VIII)

R R

wherein R 1, R 2, R 2 and R 2 are as previously defined. For example, four-membered ketones of formula (VII) wherein X is S are obtained by bromination of compound (VIII) with two moles of bromine and the resulting c, U * dibromo compound is cyclized with e.g. hydrogen sulphide to give thioethanone. The corresponding five-membered ring ketones (VII) are obtained by first reacting compound (VIII) with formaldehyde to give the ol-hydroxymethyl intermediate, the β-position of which is then brominated and then cyclized with hydrogen sulfide to give the formula (VII) wherein X is S.

Certain tetrahydrothiapyran-4-ones of formula (VII) are obtained by adding hydrogen sulphide to an appropriately substituted divinyl ketone.

The D-amino acid amide intermediates of formula 25 of the present invention

Ra CHCONHRC

nh2 wherein R is a group of formula 30 41

CH., R

1CHo> n \ where X2 is SO2 is obtained from the corresponding compounds where X 17 79834 X is S by oxidation using reagents and conditions known for the formation of sulfoxides and sulfones from sulfides. Alternatively, a suitable ketone of formula (VII) wherein X is S or an amine obtained from said ketone 5 as described above may be oxidized to a sulphoxide or sulphone before carrying out the coupling reaction to give an amide of formula (I). Preferred reagents and conditions for such oxidation of sulfides include the use of hydrogen peroxide in a solvent such as acetic acid or acetone. When reagents are used in equimolar amounts, the product is a sulfoxide that is readily converted to the corresponding sulfone by the addition of another mole of peroxide. Other preferred oxidants are potassium permanganate 15 or chromic acid, in the preparation of sulfones, and m-chloroperbenzoic acid. The latter reagent is particularly useful in converting the aforementioned thioketones (VII) to the corresponding sulfoxide using one mole of this reagent or to the sulfone using two moles of peracid.

The compounds of the formula (I) and their physiologically tolerable salts are preferred as sweeteners because of their high efficacy, physical form and stability.

The invention is further illustrated by the following examples, of which Examples 1F, 3B, 5B, 6B, 7C, 9B, 10B, 11A, 12B, 13 and 14 illustrate the preparation of the D-amino acid amides of the invention.

Example 1 L-aspartyl-D-alanine-N- (3,5-dimethyl-tetrahydro-thiopyran-4-yl) -amide; ie 79834 I, R = - / ® 'R * = CH3 CH3 mixture of cis / trans and trans / trans isomers 10 A. 3,5-dimethyltetrahydrothiopyran-4-one

A mixture of 2 g of sodium methoxide acetate and 25 ml of ethanol was saturated with hydrogen sulfide gas. To this was added 7.0 g (0.063 mol) of diisopropenyl ketone in an ice bath under cooling until the reaction was no longer exothermic.

The mixture was stirred at room temperature as hydrogen sulfide was passed through the mixture for four hours and then allowed to stand overnight. The ethanol and excess H 2 S were evaporated off in vacuo and the residue was dissolved in ethyl ether, washed successively with water, potassium carbonate solution, dilute hydrochloric acid and again with water. The ether extracts were dried (Na 2 O 2) and evaporated to dryness to give 6.8 g of an 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, which was used in the next step without further purification.

B. 4-Oxyimino-3,5-dimethyltetrahydrothiopyran A mixture of 1.67 g (0.011 mol) of the cyclic ketone obtained in Part 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 to reflux for three hours, cooled and the precipitate collected by filtration. Recrystallization from methanol-water (1: 1) gave 1.5 g of oxime as a white solid, m.p. 60-85 ° C, which is a mixture of isomers suitable for use in the next step.

C. trans / trans and cis / trans-4-amino-3,5-dimethyl-tetrahydrothiopyran

5 To a solution of 1.45 g (0.009 mol) in Part B.

obtained oxime in 15 ml of ethanol, 5 g of sodium spheres were added portionwise, followed by a further 25 ml of ethanol, and the resulting mixture was heated to reflux for about 30 minutes. The reaction mixture was diluted with water, extracted with ethyl ether, and the extracts were washed with water. The ether layer was extracted with dilute hydrochloric acid and the aqueous layer was washed with fresh ether. The aqueous layer was made alkaline by adding sodium hydroxide solution and re-extracted with ether. The organic layer was dried (MgSO 4) and the ether evaporated to give 1.1 g of a colorless oil as a residue. Gas-liquid chromatographic analysis (OV-1 column, with a temperature-program of 80-100 ° C) showed the product to contain two main components in a ratio of 60/40. 1 H-NMR (CDCl 3) showed the product to be a mixture of 4-amino-3-trans-5-trans-dimethyltetrahydrothiopyran and the corresponding 3-cis-5-trans isomer.

D. N-tertiary-butoxycarbonyl-D-alanine

To a mixture of 7.0 mL of tetrahydrofuran and 7 mL of water was added 2.71 g (11 mmol) of N- (t-butoxycarbon-; 2-yloxyimino) -2-phenylacetonitrile (BOC-ON, Aldrich

Chemical Co.), 0.89 g (10 mmol) of D-alanine and 1.5 g (15 mmol) of triethylamine and the resulting biphasic mixture were stirred at room temperature. After about two hours, the mixture became homogeneous and stirring was continued for 30 nights. The mixture was diluted with water, washed with ethyl acetate and acidified to pH 1.5 with dilute hydrochloric acid. The acidified solution was extracted with ethyl acetate, the extracts were washed with water, saturated brine and dried over sodium sulfate. The solvent was evaporated in vacuo to give 1.9 g of product as a colorless oil suitable for use in the next step.

20 79834 E. N- (3,5-dimethyltetrahydrothiopyran-4-yl) -t-butoxycarbonyl-D-alaninamide 5 CH-.

} ~ \

t-Boc-NH-CHCONH- ^ S

ch3 CH3 10

To a mixture of 1.7 g (8.9 mmol) of Nt-Boc-D-alanine from Part D, 1.98 g (19 mmol) of triethylamine and 40 ml of tetrahydrofuran, under dry conditions, cooled to -10 ° C to, was added dropwise 0.96 g (8.9 mmol) of ethyl chloroformate, and the resulting mixture was stirred at this temperature for 20 minutes. To this was added 1.1 g (7.5 mmol) of a mixture of isomers of 4-amino-3,5-dimethyltetrahydro-rothiopyran obtained in Part C, and the resulting mixture was stirred at -10 ° C for 10 minutes, then allowed to warm to room temperature. temperature. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with sodium bicarbonate solution, dilute hydrochloric acid, water, brine, then dried (Na 2 SO 4) and the solvent evaporated in vacuo to give 2.4 g of product as a foam. Thin layer chromatography (TLC) on silica gel plates, eluting with ethyl acetate / hexane 1: 1, showed a large main spot of product at 0.5 and minor impurities at 30 at 0.6 and 0.9.

F. N- (3,5-Dimethyltetrahydrothiopyran-4-yl) -D-alaninamide CH3

NHoCHCONH- (S

2V in CH3 35 2i 79334

The t-Boc-amide obtained in Part E was dissolved in 15 ml of ethanol and a mixture of 5 ml of concentrated hydrochloric acid and 10 ml of water was added. The resulting mixture was heated at reflux for 30 minutes, cooled and the ethanol removed by evaporation in vacuo. The aqueous residue was washed with ethyl ether, basified with sodium hydroxide solution, extracted with ether and the extracts dried (Na 2 SO 4). Evaporation of the solvent gave 1.1 g (67%) of a colorless oil which crystallized on standing.

10 G. Coupling of D-alaninamide with L-azoartic acid N-thiocarboxyanhydride

D-alaninamide from Part F, 1.1 g (5.1 mmol); was dissolved in 5 ml of tetrahydrofuran and 5 ml of water were added. The clear solution was cooled on ice and 0.89 g (5.1 mmol) of L-aspartic acid N-thiocarboxyanic anhydride was added in one portion. To this was added the required amount of 0.5 M sodium hydroxide to maintain the pH of the mixture at 9. After stirring for 30 minutes, the reaction mixture was washed with ethyl ether and then ethyl acetate, and the washings were discarded. The aqueous phase was acidified with dilute hydrochloric acid to pH 5.6 and evaporated to dryness in vacuo. The residue was dissolved in hot methanol (100 ml), filtered and the methanol evaporated. The residue was redissolved in hot methanol, filtered and the filtrate was decolorized with charcoal, filtered through diatomaceous earth and the filtrate evaporated to dryness to give 1.67 g of crude product as a powder. The crude product was dissolved in water (11 ml) and filtered, evaporated to 5 ml under a stream of nitrogen and cooled on ice to precipitate the product as a gel solid. The product was collected by filtration, sucked dry, washed with 2 ml of cold water, 2 ml of cold methanol and finally with a mixture of 1 ml of methanol and 4 ml of ethyl ether to give a granular product which was dried in a vacuum oven at 50 ° C. to give 0.3 g of the desired product, m.p. 225-227 ° C (dec.); silica gel TLC: Rf 0.57, butanol / acetic acid / water 4: 1: 1.

Sweetness, 200 x sweetness of sucrose.

22 79334

Example 2 3-Amino-2,2,4,4-tetramethyltetrahydrothiophene A. 1-Hydroxy-2,2,4-trimethylpentan-3-one

To sodium methylate prepared from 7.5 g of sodium metal and 250 ml of methanol was added 72.5 g (2.4 mol) of paraformaldehyde, followed by 250 g (2.2 mol) of diisopropyl ketone and the resulting the mixture was heated at reflux for three hours. The reaction was quenched with water, neutralized with hydrochloric acid, extracted with ethyl ether, washed with water, brine and the solvent evaporated. The residual oil (90 g) was distilled in vacuo to give 28 g of the desired product, which boiled at 92-98 ° C under 16-20 mm vacuum. GLC assay on OV-1 column at 107 ° C, retention time 314 seconds, purity 96%. When the above procedure was repeated on the same scale, but the reaction mixture was boiled for 16 hours, 31 g of product with a purity of 96% by GLC were obtained.

B. 4-Bromo-1-hydroxy-2,2,4-trimethylpentan-3-one To a stirred, boiling solution of 69 g (0.48 mol) of 1-hydroxy-2,2,4-trimethylpentan-3-one -one in 500 ml of chloroform, a solution of 77 g (0.48 mol) of bromine in 100 ml of chloroform was added dropwise. At the end of the addition, the mixture was stirred at reflux for 25 hours, allowed to cool and to stand at room temperature overnight. Evaporation of the solvent in vacuo gave 127 g of product which was used in the next step without purification.

C. 2,2,4,4-Tetramethyltetrahydrothiophen-3-one The product obtained in Part B, 79 g (0.3 mol) was dissolved in 300% dry pyridine, cooled to 0 ° C and 114 ° C was added portionwise. g (0.6 mol) of p-toluenesulfonyl chloride. The resulting mixture was stirred at this temperature for three hours 15 minutes, poured into ice / water and extracted with ethyl ether. Extracts 23 79334 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 mol), was dissolved in a liter of pyridine, 180 g (0.75 mol) of sodium sulfide monohydrate was added and the mixture was heated to 75 ° C and kept at this temperature for one hour and allowed to stand at room temperature overnight. . Water was added and the mixture was 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 in 89% yield. The product gave only one spot on silica gel TLC analysis eluting with ethyl acetate / hex-15 (v / v 1: d, Rf 0.5). The 1 H-NMR spectrum was consistent with the structure of the title compound.

D. Lueckart Reduction of Ketone To a 100 mL round bottom three neck flask equipped with a stirrer, thermometer, and condenser with a fractionating head was added 10.0 g (0.063 mol) of 2,2,4,4-tetramethyltetrahydrothiophen-3-one. 15.2 ml (0.38 mol) of formamide and 3.5 ml (0.092 mol) of formic acid and the mixture were heated to reflux (163 ° C) to remove water. The reaction mixture was kept at 160-180 ° C for 20 hours with the occasional addition of formic acid (10 ml). During this time, the temperature of the mixture rose to 200 ° G. The reaction mixture was cooled, water was added, and the mixture was extracted with ethyl acetate. The extracts were evaporated to dryness in vacuo. The residue was boiled with 20 ml of 6-norm.

30 hydrochloric acid for two hours, cooled, the resulting mixture was washed with ethyl ether, the pH of the aqueous phase was adjusted to 11 with sodium hydroxide solution and extracted with ethyl ether. The extracts were dried and evaporated to dryness to give 2 g of 3-amino-2,2,4,4-tetramethyltetrahydro-35 thiophene, which was identified by 1 H-NMR and showed 24 79334 ti to be homogeneous by silica gel TLC analysis. in two systems: Rf 0.63, ethyl acetate / hexane 7: 4 and Rf 0.53, butanol / acetic acid / water 4: 1: 1, nin-hydrine positive The product was a yellow oil.

E. Using the appropriate ketone as a starting material instead of diisopropyl ketone in the above process, the following amine is obtained in the same manner: nh2 10

H3C JL

H 3 S- 15

When the above tetrahydrothiophenes are contacted with an equimolar amount of hydrogen peroxide or m-chloroperbenzoic acid, the corresponding sulfoxide (X = SO) is formed in both cases. Treatment of the same starting material or sulfoxide using an excess of the same reagents or potassium permanganate in moles gives the corresponding sulfone (X = SC> 2) ·

Example 3 L-aspartyl-D-alanine-N- (2,2,4,4-tetramethyl-tetrahydrothiophen-3-yl) amide: ·: CHO CH., 3v 3 1 'R = - (la , R6 = CH3 30 CH3 / XCH3 CH., CH, t-Boc-NHCH-CONH _ / (35 A · CH- dk3 ch3 25 79834

To a solution of 2.09 g (11 mmol) of N-t-butoxycarbonyl-D-alanine in 75 mL of tetrahydrofuran was added 1.47 mL (10 mmol) of triethylamine and the mixture was cooled to -10 ° C. At this temperature, 0.96 ml of 5 (10 mmol) of ethyl chloroformate was added and stirring was continued for 15 minutes. After cooling to -20 ° C, 1.6 g (10 mmol) of dl-3-amino-2,2,4,4-tetramethyltetrahydrothiophene were added and the resulting mixture was allowed to warm to room temperature. Ethyl acetate was added and the mixture was washed twice with 50 mL portions of 5% (w / w) aqueous citric acid, aqueous sodium bicarbonate (1 x 50 mL) and brine (1 x 50 mL). The organic layer was dried (Na 2 SO 4) and evaporated to dryness in vacuo to give 3 g of N- (2,2,4,14-tetramethyltetrahydrothiophen-3-yl) -t-butoxycarbonyl-1-D-alaninamide as an oil. The structure of the product was confirmed by its 1 H-NMR spectrum. It was substantially homogeneous by silica gel TLC analysis. This product was used without further purification in the next 20 steps.

ch3 ch3

b. ch3ch (nh2) conh- / J

25 3 CH3

To the product of Part A were added 5 ml of methanol and 40 ml of 1 mol. hydrochloric acid and the mixture was heated on a water bath for 30 minutes. The methanol was removed by evaporation and the residue was extracted with ether. The ether was discarded and the pH of the aqueous phase was adjusted to 11.0 with sodium hydroxide solution, extracted with ethyl acetate, the extracts dried (Na 2 SO 4) and evaporated to dryness to give 1.0 g of N- (2,2,4,4-tetramethyl-tetrahydrothiophen-3-yl) -D-alaninamide, identified by its nuclear magnetic resonance spectrum. It was homogeneous by silica gel TLC.

26 79834 C. Coupling to form dipeptide amide

The D-alaninamide from Part B, 0.97 g (4.25 mmol), was mixed with 10 mL of water, cooled on ice, and the pH of the mixture was adjusted to 0.5 norm. sodium hydroxide solution to 9.2. To this was added portionwise and with stirring 0.8 g (4.25 mmol) of L-apartaric acid N-thiocarboxyanhydride, maintaining the pH of the mixture at 9 with sodium hydroxide solution (0.5-norm). At the end of the addition, the resulting mixture was stirred at 0 ° C for 45 minutes, the pH was adjusted to 5.2 with hydrochloric acid and evaporated to dryness in vacuo. The residue was slurried with methanol, filtered to remove precipitated solids and the methanol was removed from the filtrate by evaporation in vacuo. Ethyl ether was added to the residue to give a solid. This was collected by filtration, reslurried in ethyl ether, filtered again and the solid recrystallized from 8 ml of water. After drying in a vacuum oven, about 1 g of product was obtained, m.p. 124-126 ° C. A second batch was obtained by further treating the uu-20 mother liquors.

Sweetness: 500 x sweetness of sucrose.

When left-turning 3-amino-2,2,4,4-tetramethyltetrahydrothiophene, isolated by standard methods, was used instead of the racemic amine in the above method, the resulting L-aspartyl-D-alaninamide was found to be 800 times sweeter than sucrose.

Example 4 3-Amino-2,2,4,4-tetramethylthioethane 30 A. 2,4-Dibromo-2,4-dimethylpentan-3-one To 136 g (1.2 mol) of diisopropyl ketone was added 2 ml of phosphorus tribromide. and the mixture was cooled to 10 ° C. To this was added dropwise 384 g (2.4 mol) of bromine, and the mixture was allowed to warm to room temperature.

After two hours at this temperature, the mixture was heated at 55-60 ° C for 1 hour, after which the mixture was cooled and partitioned between chloroform and water-soluble portions. The water was discarded and the organic layer was washed with sodium carbonate solution until neutral. The organic layer was dried (MgSO 4) and the solvent was evaporated to give 316 g (97%) of the desired product.

5 B. 2,2,4,4-tetramethyl-3-oxothioethane

Sodium metal, 23 g (1.0 mol) was dissolved in 500 ml of dry methanol and the resulting mixture was cooled to 10 ° C. Hydrogen sulfide was passed through the mixture until the mixture was saturated. 10 136 g (0.5 mol) of the dibromoketone obtained in Part A were then added dropwise and hydrogen sulfide was continued to pass through the reaction mixture. At the end of the addition, the mixture was stirred for two hours at 10 ° C, allowed to warm to room temperature and stirred overnight. After the reaction mixture was poured into water, it was extracted with ethyl ether, and the extracts were washed with dilute hydrochloric acid and brine. After drying over magnesium sulfate, the ether was evaporated off, the residue was slurried in methanol, cooled and filtered to give 46 g (64%) of a solid product which was used without purification in the next step.

C. Reduction Amination of Ketone To 75 mL of dry methanol was added 4.5 g (0.031 mol) of 2,2,4,4-tetramethyl-3-oxothioethane, 23.9 g (0.31 mol) of ammonium acetate, and 1.36 g (0, 0217 mol) of sodium 25 cyanoborohydride and the resulting mixture was heated at reflux for four hours. Additional sodium cyanoborohydride (1.36 g) was added and boiling was continued for three days with the addition of a third portion of the same reagent at the beginning of the third day. The resulting mixture was acidified to pH 30 with 2N hydrochloric acid and evaporated to dryness on a rotary evaporator in vacuo. The residue was dissolved in water, washed with ethyl ether, the pH of the aqueous phase was adjusted to 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 of 28 79334 (42%) of the desired amine as a crystalline solid. The structure of the product was confirmed by its 1 H-NMR spectrum, m.p. about 45 ° C, b.p. 90 ° C (20 mm).

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

The amine obtained in Part C above, 29 g (0.2 mol) was dissolved in 50 ml of acetonitrile and 250 ml of water were added. Maintaining the pH of the mixture with sodium hydroxide at 10 over 30 minutes, 35.8 g (0.21 mol) of carbo-10-benzoxychloride were added, the mixture was stirred for 1 hour, filtered, the precipitate was washed with water and dried in vacuo at 50 ° C to give NCbz- amine, Rf 0.7 (hexane / ethyl acetate, 4: 1 v / v, phosphomolybdic acid spray), 52.1 g (93.4%). This was dissolved in 70 ml of methylene chloride, added slowly, keeping the temperature below 45 ° C (20-42 ° C), 77 g (0.372 mol) of m-chloroperbenzoic acid. The precipitated solid was collected by filtration, the filtrate was washed with 1-norm. with hydrochloric acid, aqueous sodium hydrogen carbonate solution, dried (MgSO 4) and the solvent evaporated. The residue was crystallized from acetone-water to give 42 g (73%) of Cbz-protected amine 1,1-dioxide, Rf 0.7 (hexane / ethyl acetate v / v 1: 1, phosphomolybdic acid spray).

The protecting group was removed by hydrogenation of 5 g of Cbz-amine in 250 ml of methanol, using 5 ml of concentrated hydrochloric acid and 2 g of 5% Pd / C catalyst (humidity 50%). The product was isolated as usual. Yield: 2.4 g (85%), Rf 0.6. The retention time for gas-liquid chromatography on a 1 m OV-1 column at 180 ° C was 1.3 minutes. For the three steps, the overall yield was 65% from the 3-amino-2,2,4,4-tetramethylthioethane starting material.

Using an equivalent amount of amine and m-chloro-35 riperbenzoic acid in the above procedure, the corresponding sulfoxide is obtained in the same manner as 29,79834 rows.

E. Using the ketone of formula RJR uHCOCHR ^ R * 5, wherein R3 and R5 are methyl and R4 and R5 are hydrogen, in place of the diisopropyl 3 4 5 6 liketone in the processes of Parts A-C, the corresponding amine of the following formula is obtained.

nh2 H-, C rjj

B

The corresponding sulfoxides and sulfones are prepared by the method of Part D above.

Example 5 L-aspartyl-D-alanine-N- (2,2,4,4-tetramethylthio-ethan-3-yl) amide; 20 CH, CH- 1r R = -, Ra = ch3 CH3 25 30 CH3 CH3 A. t-Boc-NHCH (CH -) CONOH-- ^ Xch3 CH3 3 35 30 79 834

To a solution of 2.09 g (11 mmol) of N-t-butoxycarbinyl-D-alanine in 75 mL of tetrahydrofuran was added 1.47 mL (10 mmol) of triethylamine and the mixture was cooled to -10 ° C. 0.96 ml (10 mmol) of ethyl chloro-5 formate were added, the mixture was stirred for 10 minutes, then cooled to -20 ° C and 1.70 g (11 mmol) of 3-amino-2,2,4,4-tetramethylthioethane were added. dissolved in 5 ml of tetrahydrofuran. The reaction mixture was allowed to warm to room temperature, ethyl acetate was added and the mixture was washed with 5% w / w citric acid solution (2 x 50 mL), sodium bicarbonate solution until neutral, then brine. The organic layer was dried (280280%) and the solvent removed by evaporation to give 2.7 g (78%) of the desired solid product. The 1 H-NMR spectrum of the product was as expected. Based on silica gel TLC, eluting with 1: 1 ethyl acetate / hexane, the product appeared homogeneous, Rf 0.77.

20 ch3 ch3

B. d-ch3ch (nh2) conh- / S

CHftH3 Using the procedure of Example 1, Part F, the t-Boc-D-alaninamide obtained in Part A of this example was hydrolyzed to give the free O 1 -aminoamide in 43% yield. The product was homogeneous by silica gel TLC and gave a 1 H-NMR spectrum consistent with its structure.

C. Coupling to form dipeptide amide

A solution of 0.7 g (3.2 mmol) in Part B above

prepared from Of aminoamide, mixed with 10 ml of 35 water, adjusted to pH 10.1, cooled in an ice bath and added portionwise 0.567 g (3.2 mmol) of L-aspartic acid N-thiocarboxyanhydride, maintaining the pH of the mixture. 3i 79834 9 by adding 0.5-norm. sodium hydroxide solution. The mixture was then stirred at 0 ° C for 45 minutes, the pH adjusted to 5.2 and evaporated to dryness in vacuo. The residue was slurried in methanol, the solids removed by filtration and the filtrate evaporated to dryness. To the residue was added 150 ml of ethyl ether, the precipitated product was collected by filtration and dried in a vacuum oven overnight to give 1.5 g of crude material. After resuspension in ethyl ether twice more, 0.9 g of product (85%) were obtained, m.p. 135 ° C (dec.). Sweetness, sweetness of 2,000 x sucrose.

Example 6 L-aspartyl-D-alanine-N- (2,2,4,4-tetramethyl-1,1-dioxothioethan-3-yl) amide CH-CH_ I, Ra = CH3, R = ch3 ^ ^ ch3 20 ch3 ch3 A. t-Boc-NHCH (CH3) CONH - <^ v'SO2 CH3 3 30

A solution of 31.6 g (0.10 mol) of Nt-butoxycarbonyl-D-alanine-N- (2,2,4,4-tetramethylthioethan-3-yl) amide obtained in Part 16 of Example 16 A, in 500 mL of chloroform, was cooled to 10-20 ° C and 41.3 g (0.24 mol) of m-chloroperbenzoic acid was added again, keeping the temperature of the reaction mixture below 45 ° C. The resulting mixture 32 79334 was stirred for 1 hour, diluted with chloroform and washed successively with 5% aqueous sodium hydrogen carbonate solution, 0.1 NM. hydrochloric acid, 5% sodium thiosulfate and saturated brine. The washed organic layer was dried (MgSO 4) and evaporated to dryness to give 33.7 g of a residual oil (97%) which was suitably pure for use in the next step.

10>% / CH3 B. D-CH, CH (NH2) CONH-CH2O2SO2 CH3 ch3 15

The t-Boc amide obtained in Part A above, 34.8 g (0.10 mol), dissolved in a mixture of 50 ml of concentrated hydrochloric acid and 100 ml of water and 100 ml of ethanol, was heated at reflux for 20 hours. The mixture was cooled, washed with ethyl ether, the pH of the aqueous phase was adjusted to 12 with 50% aqueous sodium hydroxide solution and extracted with ethyl acetate. The extracts were dried (MgSO 4) and evaporated to dryness in vacuo to give 18.7 g (75%) of a crystalline solid.

This was recrystallized from carbon tetrachloride to give 80% recovery of the product.

C. Di-protected dipeptide amide

To a solution of 14.28 g (0.04 mol) of γ-β-benzyl-N-benzyloxycarbonyl-L-aspartate in 700 ml of tetra-30 hydrofuran was added 4.4 ml (0.04 mol) of N-methylmorpho. line and the mixture was cooled to -10 ° C. Ethyl chloromorphate, 3.9 ml (0.04 mol), was added, the mixture was stirred at -10 ° C for 15 minutes and cooled to -20 ° C. A solution of 9.92 g (0.04 mol) of D-alanine-N- (2,2,4,4-tetramethyl-1,1-dioxothioetan-3-yl) amide obtained in Part B 35 above was added in 50 ml. in tetrahydrofuran and the resulting mixture was stirred at ambient temperature for one hour. Reaction mixture 33 79834

The reaction mixture was poured into 700 ml of ethyl acetate, washed successively with 1-norm. with hydrochloric acid, sodium bicarbonate solution, 0.1-norm. with sodium hydroxide and brine. The washed extracts were dried (MgSO 4) and evaporated to dryness in vacuo. The residue was slurried in ethyl ether and the solid was collected by filtration to give 19.8 g (84%) of the di-protected dipeptide diamide, which was recrystallized from ethylene glycol dimethyl ether / isopropyl ether to give a yield of 77%.

D. To a solution of 10 g of the product from Part C above in 250 mL of methanol was added 3 g of 5% Pd / C catalyst (50% moisture) and 1 mL of concentrated hydrochloric acid and the mixture was hydrogenated at 50 psi for 1 hour: at a pressure of 2 15 (3.52 kg / cm). The catalyst was removed by filtration, the pH of the filtrate was adjusted to 5.2 with sodium hydroxide solution and evaporated to near dryness in vacuo. The residue was stirred with ethyl ether and the solid was collected by filtration and dried in a vacuum oven at 45 ° C to give 5.5 g, 89%, of the desired dipeptide diamide, m.p. 135 ° C (dec.). Sweetness: 1,000 x sucrose sweetness.

Example 7 L-aspartyl-D-alanine-N- (2-methylthio-2,4-di-25-methylpentan-3-yl) amide A. 2-methylthio-2,4-dimethylpentan-3-one

A solution of 200 ml of methanol and 9.2 g (0.40 mol) of sodium metal was cooled in an ice bath and saturated with gaseous methyl mercaptan. To this was added 77.2 g (0.40 mol) 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 were washed with water, brine and dried over anhydrous sodium sulfate. The ether was evaporated off and the residue 34,79834 was distilled in vacuo to give 50.4 g of product, b.p. 76 ° (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 to reflux while removing the reaction mixture. water by fractional droplet. After 12 hours, an additional 2.5 g of formic acid was added and boiling was continued for another 24 hours in the same manner during which time the reaction mixture reached 190 ° C. The mixture was cooled, diluted with water and extracted with ethyl acetate. The extracts were washed with water and evaporated to dryness in vacuo to give 5.3 g of an oil, the oil was boiled in 40 ml of 6-norm 6-cassette. hydrochloric acid for six 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 to dryness, 3.3 g (56%) of a colorless amine were obtained, which gave a single peak when gas-liquid chromatographed on a six-foot OV-1 column at 110 ° C; the reaction time was 412 seconds.

C. D-Alanine-N- (2-methylthio-2,4-dimethylpentan-3-yl) amide To a solution of 3.2 g (0.017 mol) of Nt-butoxycarbonyl-D-alanine and 2.5 g of g (0.017 mol) of triethylamine in 100 ml of tetrahydrofuran at -15 ° C, 1.64 ml of ethyl chloroformate were added. After stirring for 15 minutes, 2.49 g (0.017 mol) of 2-methylthio-2,4-dimethyl-3-aminopentane were added and the mixture was stirred for 1 hour. The reaction mixture was diluted with ethyl acetate, washed with water, 5% aqueous citric acid (w / v), sodium hydrogen carbonate solution and brine. The organic phase 35 79834 was evaporated to dryness to give 6.0 g of residue. This was dissolved in 100 ml of methanol, 60 ml of concentrated hydrochloric acid was added and the mixture was boiled for one hour. After evaporation of the methanol, the residue was dissolved in water, washed with ether, the pH of the aqueous phase was adjusted to 12 with sodium hydroxide and extracted with ethyl ether. Evaporation of the extracts to dryness gave 3.2 g (87%) of the product as a colorless oil, 0.56 (4: 1: 1 by volume ratio of butanol / water / acetic acid).

D. A solution of 3.1 g (0.013 mol) of D-alaninamide obtained in Part C in 30 ml of cetone and 17 ml of water was adjusted to pH 9.9 with sodium hydroxide solution and cooled to -2 ° C. To this was added in small portions 2.78 g (0.013 mol) of L-aspartic acid N-thiocarboxy-15 anhydride over 20 minutes while maintaining the pH at 9.9 at 1-norm. sodium hydroxide. At the end of the addition, the resulting mixture was stirred for 30 minutes at -2 ° C, washed with ethyl acetate, acidified to pH 2 with hydrochloric acid and washed again with ethyl acetate. The pH of the aqueous phase was then adjusted to 5.2 and evaporated to dryness. The crude dipeptide amide was obtained by slurrying the residue in methanol, filtering, treating the filtrate with ether and filtering to give a second crop of product. The total yield of crude product was 4.7 g.

The crude product was purified by preparative thin layer chromatography on silica gel plates (20 x 20 x 2 mm) eluting with butanol / water / acetic acid, 4: 1: 1 v / v. The band at Rf 0.42 was excised and eluted with methanol to give 30 pure L-aspartyl-D-alaninamide.

Sweetness: 150 x sweetness of sucrose.

Example 8 N-Benzyloxycarbonyl-D-2-aminobutyric acid To a 500 ml reaction vessel fitted with a mechanical stirrer, a thermometer and two additional funnels was added 20.0 g (0.194 mol) of commercially available D - (-) - 2-Amino acid and a solution of 7.76 g (0.194 mol) of sodium hydroxide in 97 ml of water. The mixture was stirred until complete dissolution and cooled to 5 ° C. From the addition funnels, 40.66 g (0.205 mol) of 86% benzyl chloroformate and a solution of 7.76 g of sodium hydroxide in 58 ml of water were added simultaneously. The temperature was maintained at 4-6 ° C during the addition. The reaction temperature was then slowly raised; 10 to 14 ° C. Stirring was continued for 15 minutes after the temperature was set, after which it was allowed to warm to room temperature. The reaction mixture was washed with 3 x 75 ml of ethyl ether, the pH of the aqueous phase was adjusted to 2 with concentrated hydrochloric acid (16 ml), causing the product to separate as an oil, the oil was extracted with 3 x 100 ml of ether, dried (MgSO 4) and the solvent evaporated. a clear, colorless liquid residue was obtained which was dried overnight under a vacuum of 0.3 mmHg, during which time the product crystallized to give 41.24 g (89.6%) of the title compound. A 1.0 g portion was recrystallized from 5 mL of xylene and the precipitate was washed with xylene and pentane. After drying overnight, 0.77 g of colorless crystals were obtained; Rf 0.57 in TLC on silica gel (solvent: ethyl acetate / hexane / acetic acid, vol.

25 ratio 4: 14: 2; plates were sprayed with 3% vanillin), m.p. 76-78 ° C, +10 (c = 2.8, ethanol).

Analysis for c 12 H 15 NO 4: Calculated: C 60.75 H 6.37 N 5.90 Found: C 60.61 H 6.41 N 5.97.

The remaining crude crystals (40 g) were recrystallized from xylene (150 ml), washed with xylene (3 x 25 ml) and pentane (2 x 25 ml) and dried in vacuo at 45 ° C to give 28.59 ( 71.7%) of colorless crystals, m.p. 78-79 ° C.

37 79834

Example 9 L-Aspartyl-D-2-aminobutyric acid N- (discyclopropylcarbinyl) amide

5 a / O

I, R = C «Hr / R = CH 2 2 A. D-CH 3 CH 2 CHCONHCH (R) N 10 Benzyloxycarbonyl-D-2-aminobutyric acid, 4.75 g (0.020 mol) was dissolved in 200 any tetrahydrofuran and cooled under a nitrogen atmosphere. protected to -15 ° C. Li-15 was adjusted with N-methylmorpholine, 2.02 g (0.020 mol) and ethyl chloroformate, 2.17 g (0.020 mol), and the resulting mixture was stirred at -15 to -10 ° C for 30 minutes. The resulting mixed anhydride solution was cooled to -20 ° C and 2.22 g (0.020 mol) of dicyclopropylcarbinylamine dissolved in any tetrahydrofuran was added. Stirring was continued for 15 minutes at -20 to -15 ° C, after which the mixture was allowed to warm to room temperature, 150 ml of ethyl acetate were added and the mixture was extracted with 2 x 100 ml of 1-norm. hydrochloric acid, 2 x 100 ml of 5% aqueous sodium hydrogen carbonate solution and 100 ml of saturated brine. The organic layer was dried (MgSO 4) and the solvent evaporated in vacuo to give 6.62 g (100%) of a colorless solid, 0.68 using silica gel TLC (solvent: ethyl acetate / hexane, 1: v / v). 1; the plate was sprayed with 3% vanillin).

b. d-ch3ch2chconhch (nh2 35 Cbz-amino-protected amide from Part A above, 6.62 g (0.020 mol) was dissolved in 250 ml of reagent grade methanol. 5% palladium on carbon catalyst 2 38 79834 1.5 g and the mixture was hydrogenated at 50 psi (3.52 kg / cm) for 45 minutes, the catalyst was removed by filtration and the filtrate was evaporated to dryness in vacuo to give a liquid residue, 1N hydrochloric acid (25 ml, 5 ml) was added. after which the mixture was stirred with 25 ml of ethyl ether, the aqueous phase was made strongly alkaline (pH 14) with 10 molar sodium hydroxide and the mixture was extracted with 3 x 25 ml of ether, the extracts were combined, dried (MgSO 4) and the ether evaporated, to give 3.29 g (83.9%) of a colorless solid, mp 63-64 DEG C. 0.60 (butanol / acetic acid / water, v / v; ninhydrin spray); (a) -46.7 °

• 1 U

(c = 2,1-norm. HCl). The 1 H NMR spectrum was consistent with the structure of D-2-aminobutyric acid N- (dicyclopropylcarbinyl) amide.

C. Di-protected dipeptide amide To a solution of N-benzyl-N-benzyloxycarbonyl-L-aspartate dissolved in 5.61 g (0.0157 mol) dissolved in 50 ml of tetrahydrofuran cooled to -15 ° C was added 1.59 g (0.0157 mol) of N-methylmorpholine. To this was added dropwise 1.70 g (0.0157 mol) of ethyl chloro-20 formate, the resulting mixture was stirred for 30 minutes at -15 ° C, and a solution of D-2-aminobutyric acid N- (dicyclopropylcarbinyl) amide was added dropwise. In 15 ml of tetrahydrofuran. Stirring was continued for 15 minutes at -15 ° C, after which the reaction mixture was allowed to warm to 25 room temperature. The solvent was evaporated in vacuo to give a solid residue. This was mixed with 150 ml of ethyl acetate and the resulting suspension was washed with 2 x 75 ml of 1-norm. hydrochloric acid. The clear organic layer was washed with 2 x 75 mL of 5% aqueous sodium bicarbonate solution, 75 mL of brine, dried (MgSO 4) and the solvent evaporated in vacuo to give 8.36 g (99.5%) of a colorless solid. solid, Rf 0.36 using TLC (ethyl acetate / hexane 1: 1, vanillin spray). Recrystallization from ethyl acetate gave 6.12 g (73%) of crystals, m.p. 167-168 ° C.

D. To a solution of the di-protected dipeptide diamide obtained in Part C in 250 mL of methanol was added 1.5 g of 5% Pd / C catalyst (50% moisture) and The 2 mixtures were hydrogenated at 50 psi (3.52 kg / cm) for 45 minutes, during which time the theoretical amount of hydrogen was consumed. The catalyst was removed by filtration, the filtrate was evaporated in vacuo to a volume of about 75 ml. The concentrate was filtered to remove the precipitated product, which was washed with methanol and dried in vacuo to give 2.85 g of a colorless powder, m.p. 225-227 ° C (dec.). An additional 0.76 g of product was obtained from the mother liquor. Total yield: 100%.

0.56 (butanol / acetic acid / water, 4: 1: 1, ninhydrin spray).

Sweetness: 500 x sweetness of sucrose.

Example 10 L-aspartyl-D-2-aminobutyric acid N- (2,2,4,4-tetrameth-1-5-yl-1,1-dioxothioethan-3-yl) amide: I, Ra = C2H5, R = - <^ SO2 20 a. D-ch3ch2chconh - <^ ^ so2 NHCbz Using 3-amino-2,2,4,4-tetramethylthioethane-11-dioxide instead of dicyclopropylcarbinylamine in the process of Example 9, Part A, N -benzyloxycarbonyl-D-2-aminobutyric acid N- (2,2,4,4-tetramethyl-1,1-dioxothioethan-3-yl) amide in 99% yield, Rf 0.50.

B. Hydrogenolysis of the product obtained above by the method of Example 30 9, Part B, afforded 18% of D-2-aminobutyric acid N- (2,2,4,4-tetramethyl-1,1-dioxothioethan-3-yl) amide: in step yields, Rf 0.41.

C. By the procedure of Example 9, Part C, the corresponding di-protected dipeptide amide, C6H5CH2OCOCH2CHCONHCH (C2) CONH - <^ ^ SO2, Rf 0.68 NHCOOCH_CcHc 2 6 5 35 40 79834 D was obtained in 99% step yield. C (1.16 g), by the method of Example 9, Part D, using 0.4 g of 5% Pd / C catalyst (50% moisture), gave the title compound in 92% step yields, m.p. 165-167 ° C (ha-5 so), Rf 0.4.

Sweetness: 200 x sweetness of sucrose.

Example 11 L-aspartyl-D-2-aminobutyric acid N- (2,4-dimethyl-3-pentyl) amide: 10 L, Ra = C2H5, R =

15 A. D-CH3CH2CHCONhY

Using the appropriate amine in Part A of Example 9 and hydrogenolyzing the crude product by the method of Part B of Example 9, diisopropylcarbinylamide of D-2-aminobutyric acid was obtained in 85% yield as an oil, Rf 0.56.

B. The procedure of Example 9, Part C afforded, in 76% yield, the corresponding di-protected amide, C6H5CH2OCOCH2CHCONHCH (C2H5) CONH-CH / CH (CH3) 2 and this hyd-25 NHCOOCH2C6H5 was prepared by the method of Example 9, Part D to give the title compound. 89% step yield (total yield 68%), m.p. 213-215 ° C (dec.), Rf 0.54.

Sweetness: 100 x sweetness of sucrose.

Example 12 L-aspartyl-D-valine-N- (discyclopropylcarboxylic acid) amide α / 0 35 I, R = CH (CHO), R = CHf 3 2] 79834 A. (CH3) 2CHCHCONHCH (s) 2 NHCbz Using commercially available D-valine instead of D-2-aminobutyric acid in the method of Example 8, N-benzyloxycarbonyl-D-valine was obtained in 82% yield as a white powder, m.p. 55-57 ° C, 0.45. This was reacted with dicyclopropylcarbinylamine according to the procedure of Example 9, Part A to give the desired protected amide in 100% yield, Rf 0.35.

B. Hydrogenolysis of the product obtained above by the method of Example 9, Part B, gave D-valine-N- (dicyclopropylcarbinyl) amide in 83.5% yield, Rf 0.53, as a colorless solid.

C. The procedure of Example 9, Part C, afforded the corresponding di-protected dipeptide amide, C6H5CH2OCOCH2CHCONHCH / CH (CH3) ^ CONHCHCA) 2 'in 92% yield, -, Ο, -Η ,.

2 6 5 ca., 0.55, as an off-white solid.

D. Hydrogenolysis of the product of Part C obtained above by the method of Part D of Example 9 gave the title compound in 85% yield as a colorless solid, Rf 0.54.

Sweetness: 110 x sweetness of sucrose.

Example 13 L-aspartyl-D-valine-N-isopropylamide i, Ra = ch (ch3) 2, r = ch (ch3) 2

Reaction of N-benzyloxycarbonyl-D-valine with isopropylamine by the method of Example 9, Part A gave, in quantitative yields, N-benzyloxycarbonyl-D-valine-isopropylamide having an Rf of 0.61 (TLC, ethyl acetate / hexane 1: 1, vanillin spray).

Hydrogenolysis with Pd / C on the method of Example 9, Part B, afforded D-valine isopropylamide in 71% yield, Rf 0.50. Coupling of this with β-benzyl N-Cbz-L-aspartate gave the di-protected dipeptide 42,79834 amide (by the method of Example 9, Part C), Rf 0.40 (TLC, ethyl acetate / hexane, 1: 1, vanillin spray), Yield 62% as an off-white solid. This was converted to the desired title compound by the method of Example 9, Part D, in 90.6% yield, m.p. 226-227 ° C (dec.), 0.44.

Sweetness: 1-2 x sweetness of sucrose.

Example 14 L-aspartyl-D-alanine-N- (2,2,4,4-tetramethyl-3-10 pentyl) amide / C (CH 3) 3 I, Ra = CH 3, R =

Xc (ch3) 3 A. 2,2,4,4-tetramethyl-3-aminopentane This was prepared by the method of J. Chem. Soc.,

Perkin I, 2087 (1976); ibid, 1797 (1974). Pivalonitrile (33.2 g) and t-butyl chloride (44.4 g) were added, under nitrogen protection, to a well-stirred suspension of sodium sand (18.4 g) in hexane (80 ml), tetrahydrofuran (20 ml) and methanol. (1 ml) in the mixture, within one hour at 15-20 ° C. The mixture was stirred for three hours, a solution of chlorobenzene (2 g) in tetrahydrofuran (5 ml) was added dropwise over 10 minutes and stirring was continued for 1 hour. Within 0.5 h, methanol (20 mL) was carefully added followed by water until the phases separated. The aqueous phase was extracted with ether and the combined organic phases were dried and concentrated in vacuo. The residue was distilled to give di-t-butyl ketone-imine, b.p. 62-63 ° C / 19 mm.

The imine (14 g) in dry ether (50 ml) was added to a suspension of lithium aluminum hydride (1.7 g) in dry ether (50 ml). The mixture was stirred at room temperature for 24 hours, boiled for two hours and cooled. Water (1.7 ml), 15% sodium hydroxide solution (5 ml) and water (5 ml) were carefully added successively, the mixture was filtered, concentrated and the residue was distilled to give the desired amine, b.p. 79-81 ° C / 40 mm.

B. N-Benzyloxycarbonyl-D-alanine, 7.59 g (0.034 mol) was added to 170 ml of methylene chloride containing 4.88 ml (0.035 mol) of triethylamine and the resulting solution was cooled to -10 ° C. Ethyl chloroformate, 3.35 ml (0.035 mol) was added, the mixture was stirred at -10 to -5 ° C for 25 minutes, after which 5.0 g (0.034 mol) of 2,2,4,4-tetramethyl-3-aminopentane were added. and stirring was continued overnight after allowing the mixture to warm to room temperature. The reaction mixture was washed with 2 x 100 mL of 5% aqueous sodium bicarbonate, 2 x 100 mL of 3 mol. hydrochloric acid, dried (MgSO 4) and the solvent evaporated in vacuo to leave 9.99 g (82%) of N-Cbz-D-alanine-N- (2,2,4,4-tetramethyl-3-pentyl). amide identified by 1 H-NMR spectrum, R f 0.72 (TLC, ethyl acetate / hexane, 1: 1, phosphomolybdate spray).

C. Cbz-D-alaninamide obtained above, 9.99 g Pd / C-20 catalyst (moisture 50%) and the mixture was hydrogenated at 45-75 psi 2 (12.8-21.3 kg / cm) for 30 minutes . The catalyst was filtered off, the filtrate was evaporated to dryness in vacuo to leave a white solid. To this was added 100 ml of 1-norm. hydrochloric acid, the mixture was stirred for two 25 hours, washed with ethyl ether, then basified with 40% by weight sodium hydroxide solution. The basic solution was extracted with ethyl ether, the ether layer dried over magnesium sulphate and evaporated to dryness to give 1.1 g of a colorless product which was identified as D-alanine-N- (2,2,4,4-tetramethyl-3-pentyl) amide, Rf .59 (butanol / water / acetic acid, 4: 1: 1, ninhydrin and then phosphomolybdate sprays).

D. By the method of Example 9, Part C, a di-protected dipeptide amide was prepared on a 0.005 mole scale from the above amide and N-benzyl-N-Cbz-L-aspartate using triethylamine and ethyl chloroformate in acetone (50 mL) in 80% yield, R ^ 0.4 44 79834 (TLC, ethyl acetate / hexane, 1: 1, phosphomolybdate spray). Purification was performed by chromatography on 25 g of silica gel, eluting with ethyl acetate / hexane, 2: 3 v / v to give 1.65 g of purified liquid product.

E. Di-protected dipeptide amide, 1.15 g (0.021 mol) was hydrogenolyzed in 150 mL of methanol with 0.2 g of 5% Pd / C catalyst by the method of Example 20, Part D to give 0 , 64 g (93%) of the title compound, 10 m.p. 145-149 ° C (dec.), 0.50.

Sweetness: 450 x sweetness of sucrose.

Claims (2)

A D-amino acid amide compound useful as an intermediate in the preparation of L-aspartyl-D-amino acid-5 dipeptide amides, characterized in that the D-amino acid amide compound has the formula Ra-CHCONHRC NH- 10 ^ ä c wherein R is methyl, ethyl or isopropyl and R is diisopropylcarbinyl, di-t-butylcarbinyl, dicyclopropylcarbinyl or a group of formula R41 CH3 ^ / _ /, CH2, "^ x2 Y- (CH2> p2 ^ wherein when n2 and P2 are each 0, R42 and R2 are both methyl and X2 is S or SC2 when n2 is 0 and p2 is 1, R41 and R6 are each methyl and X ~ is S or SO ~, and when n2 is 1 and P2 is 1, R4 and Rb are each hydrogen and X2 is S. 46 79834 D-aminosyramides which are the same as those used in the preparation of L-aspartyl D-amino-5 syradipeptide amide, which can be converted to D-aminosyramides in the form of Ra-CHCONHRC NH 10 color CH3'V 20 3r <CH2, p ^ CHrv color, d n 2 and P2 vardera is 0, is R4 "* - and R ^^ vardera 25 methyl or X2 is S or SC ^, then 1 * 2 is 0 and P2 is 1, is R4 "*" and is R® ^ vardera methyl or X2 is S or SC1, and is n2 is 1 or p2 is 1, is R4 ^ and is R2 is a value and X2®rs *