High-Potency Sweeteners Derived from -Amino Acids - American

Chapter 9

High-Potency Sweeteners Derived from β-Amino Acids

Downloaded by UNIV OF MISSOURI COLUMBIA on April 12, 2017 | http://pubs.acs.org Publication Date: December 31, 1991 | doi: 10.1021/bk-1991-0450.ch009

George W. Muller, Darold L. Madigan, J. Chris Culberson, D. Eric Walters, Jeffery S. Carter, Carrie A. Made, Grant E. DuBois, and Michael S. Kellogg The NutraSweet Company, Mt Prospect, IL 60056

Over the last 20-25 years, intense effort has been focused on the discovery of novel sweeteners. Suosan, N-(4-nitrophenyl)-N'-(2-carboxyethyl)urea is a known synthetic sweetener which contains an aryl substituent and an acidic moiety. Aspartyl amide sweeteners contain a hydrophobic component and an acidic moiety. We designed analogues in which a hydrophobic unit was incorporated in suosan type sweeteners providing increased potency. The commercialization of aspartame has revealed the large market for a safe, low calorie synthetic sweetener with a taste profile accurately reproducing that of sucrose. For a high potency sweetener to be commercially successful, it must meet the following criteria. It must: 1) be safe for human consumption under the conditions of approved use, 2) have a taste profile very close to that of sucrose, 3) have the required solubilities for commercial applications, 4) be stable to the application conditions, and 5) be economically competitive with sucrose and the high fructose corn syrups. Herein, we wish to report a new series of high potency sweeteners related to suosan, 1 (I). Examination of the literature on sweeteners reveals several other high potency sweeteners containing electron deficient aryl moieties which may function as π-stacking recognition units(2-4) in a sweet taste perception mechanism. Ο

1

OO97-^156/91/045O-O113$O6.0O/0 © 1991 American Chemical Society Walters et al.; Sweeteners ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

114

SWEETENERS:

DISCOVERY, M O L E C U L A R DESIGN, A N D CHEMORECEPTION

Suosan, N-(4-nitrophenyl)-N'-carboxyethylurea (1), contains an aryl moiety and a short chain carboxylic acid. In 1972, Budesinsky and Vavrina reported 5-bromo-4-cyanopyrimidine (2) to be sweet (5). Compound 2 has a sweetness potency (5) of 700 times sucrose (on a weight basis) relative to a 7% sucrose solution [abbreviation: P (7) = 700]. The cyanopyrimidine 2 is an electron deficient aryl group. Saccharin (3) is a well known commercial sweetener exhibiting a P (10) = 300 which contains both aryl and sulfonamide moieties (6). w


w

Ο

2

3

Examination of the aspartyl amide sweetener class reveals a great diversity of structure with sweetness potencies exceeding 30,000 times sucrose (7). The dipeptide ester known generically as aspartame (4) is the only aspartyl amide sweetener approved by regulatory agencies to date (8, 9). It has a sweetness potency ranging from 400 times sucrose versus a 0.34% sucrose solution to 100 times sucrose versus a 15% sucrose solution (8). The amino malonic acid derived sweetener 5 prepared by Fujino and coworkers has a reported sweetness potency of 22,000-33,000 times sucrose (7). Alitame (6), derived from L-aspartic acid, Dalanine, and a tetramethyl thietanylamine, exhibits a P U0) = 2,000 (10). These three aspartyl amide sweeteners have in common a free carboxylic acid and a large hydrophobic group. w

Ο

NH

2

H

Ο

5

4

6

Walters et al.; Sweeteners ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

9. MULLER ET AL.

High-Potency Sweeteners front β-Amino Acids

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Combination of Aryl and Aspartyl Amide Sweeteners


Several examples of high potency sweeteners may be viewed as combining the attributes of both the aryl and aspartyl amide sweeteners. In 1973, Lapidus and Sweeney (I J) reported that a-Laspartyl anilide derivatives were sweet. N-Trifluoroacetyl- and Ntrichloroacetyl- a-L-aspartyl-p-cyanoanilide (7 and 8, respectively) were reported to have sweetness potencies of 3,000 times sucrose (J J). Inspection of structures 7 and 8 shows an electron deficient aryl moiety, a carboxylic acid and a hydrophobic substituent (trihaloacetyl).

In 1987, Nofre and Tinti at the Université Claude Bernard reported aryl ureido and thioureido derivatives of aspartame with P (2) as high as 40,000 (12; potencies were recalculated from molar to weight ratios.). Ureido derivative 9 was reported to have a P (2) = 7,800. These derivatives contain an electron deficient aryl moiety, a carboxylic acid and a hydrophobic substituent. More recently, Nofre and Tinti reported that N-aryl-N'-alkylN"-carboxymethylguanidines are high potency sweeteners with potencies up to 170,000 times that of sucrose (13). Compound 10 was reported to have a P (2) = 170,000 (13). Again, the same three recognition units are present. w

w

w

^_ff

H

V^N^CC^CHa έ

Ν

H

C0 H 2

9

NH

> C0 H 2

10

Thus, combination of the recognition units present in the prototype compounds yielded sweeteners with increased potency relative to members of either class. Suosan as a Template Nofre and co-workers had previously reported that a cyano moiety was a functional replacement group for the nitro group in suosan with only a small drop in sweetness potency (14; potencies were


116

SWEETENERS:


recalculated from molar to weight ratios.). These workers reported a P (2) = 870 for suosan and a P (2) = 600 for the cyanophenyl substituted analogue. A report by Goodman and Rodriguez, along with the initial report by Lapidus and Sweeney, demonstrated that 4-nitrophenyl and 4-cyanophenyl were optimal groups for sweetness potency in the aspartyl anilide sweetener series (II, 15). We chose to use N-(4-cyanophenyl)-N'-carboxyethylurea (11, A = Β = H) as our template instead of suosan. w

w


Ο

Β

11 Using examples from the above mentioned classes of sweeteners, a composite model of sweet taste agonists was developed at The NutraSweet Company (16). Suosan contains the requisite carboxylic acid moiety and an electron déficient aryl ring but lacks the large hydrophobic recognition unit common to a large number of high potency sweeteners. Examination of suosan in The NutraSweet Company sweet taste agonist model revealed the possibility of increasing the potency by incorporation of this hydrophobic recognition unit. Our examination of suosan suggested substitution at either of two positions, A or Β of analogue 11, might lead to higher potency sweeteners. N-Substitution. The N-substituted compounds were prepared as shown in Scheme I. Treatment of β-propiolactone with amines yielded the desired N-substituted β-alanines (17). Treatment of 4cyanophenyl isocyanate with the N-substituted β-alanines yielded the the desired analogues.

12 R= cyclohexyl 13 R= benzyl Scheme I


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The analogues in which cyclohexyl and benzyl were substituted on the nitrogen were not sweet at concentrations up to 1 mg/mL in water. Both the cyclohexyl analogue 12 and benzyl analogue 13 were found to be bitter. These results confirmed that the p-NH moiety played a role in the recognition of an agonist for sweet taste perception.


β-Substitution. β-Substitution on the β-alanine portion of compound 11 incorporates a hydrophobic group while retaining the β-ΝΗ moiety. We first prepared analogues that were substituted with cycloalkyl groups via the route illustrated in Scheme II.

Among the analogues prepared were cyclohexyl, cyclooctyl, and spirocyclohexyl analogues 14, 15, and 16 respectively. Cycloalkyl substituted β-alanines were prepared by treatment of an aldehyde or ketone with malonic acid and ammonium acetate in refluxing 95% ethanol (J8, 19). Treatment of 4-cyanophenyl isocyanate with the β-substituted β-alanine yielded the desired analogues (This reaction was run with either the neutral or sodium salt of the β-amino acid.). Analogues 14-16 were found to be very bitter, yet, interestingly all of these compounds exhibited a low level of sweetness in their taste profiles.


118

SWEETENERS:


NC


Ο

Ο

NC

By an analogous procedure, the β-phenyl analogue 17 was prepared (3-amino-3-phenylpropionic acid is commercially available from Aldrich Chemical Company). Compound 17 was sweet with a P (5) = 5,000. The observed potency was over an order of magnitude greater than the parent compound 11 (14). The flavor profile of compound 17 was found to be very similar to sucrose with a clean sweet taste. The large increase in potency of 17 over 11 spurred exploration of the structure activity relationships (SAR) of this series. To expand the SAR, we prepared homologues in which the phenyl ring was extended by methylene units. Thus, the benzyl and phenethyl analogues 18 and 19 were prepared. The synthetic route to 18 and 19 is illustrated in Scheme III. The desired β-substituted acrylates were prepared by treatment of the requisite aldehyde with a stabilized Wittig reagent (20). Amination was accomplished by Michael addition of benzyl amine {21-23) followed by catalytic hydrogenolysis (24). The resultant amino esters were then condensed with 4-cyanophenyl isocyanate followed by cleavage of the methyl ester with sodium hydroxide to yield the desired analogues. Benzyl analogue 18 was sweet but less potent than the parent compound 17, having a P (3) = 3,000. The phenethyl analogue 19 was empirically equivalent to compound 18 with a P (2.5) = 2,500. Extension of the aromatic group from the β position provided no advantage. We also examined the effects on taste activity of various substitutions on the β aryl substituent of 17. Electron withdrawing w

w

w


MULLER ET AL.

R-CHO

High-Potency Sweeteners front β-Amirw

Ph,PCHCO,CH, CH CN reflux

Acids

CO2CH3


3

RNH

2

CH3OH

18 R = CH Ph 19 R = CH CH Ph 2

2

2

Scheme ΠΙ


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SWEETENERS:



substituent effects were explored by preparing ortho-, meta-, and para-nitrophenyl analogues 20, 21, and 22.

C0 H 2

22 The meta analogue 21 had a sweetness potency [P (5.5) = 5,500] approximately equivalent to the parent compound 17 with para analogue 22 being slightly less potent [P (4) = 4,000]. However, the ortho analogue 20 retained only minimal activity with a P (2.5) = 25. To examine the effects on taste activity of electron donating groups on the aryl ring, the ortho-, meta-, and para-methoxyphenyl substituted analogues 23, 24, and 25 were prepared. w

w

w

C0 H 2

25 The meta analogue 24 had a P (4) = 4,000 and the para analogue 25 had a P (5.5) = 5,500. Again, as was observed for the nitro analogues 21 and 22» there were only moderate effects on activity by meta and para substitution relative to 17. Ortho-methoxy substitution resulted in complete elimination of sweet taste of the w

w


9.

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High-Potency Sweeteners from β-Amino

Acids

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compound such that no sweetness was perceptible at 1.0 mg/mL in water. Meta- and para-substitution with electron donating and electron withdrawing substituents on the aryl ring of 17 has minor effects on sweetness potency. However, ortho substitution obliterates the sweetness activity of these compounds. The effects of substitution of fused ring systems for the βphenyl moiety of 17 were probed by evaluation of analogues 26-28. Ο Downloaded by UNIV OF MISSOURI COLUMBIA on April 12, 2017 | http://pubs.acs.org Publication Date: December 31, 1991 | doi: 10.1021/bk-1991-0450.ch009

X

H

The 2-naphthalene analogue 26 exhibited a potency approximately equivalent to the parent compound 17. The 5-indanyl analogue 27 was found to be less potent with a P (2) = 4,000. However, the piperonyl analogue 28 was found to be more potent than the indanyl analogue with a P (2.5) = 25,000. This finding is consistent with the enhancing effect of oxygen (and sulfur) which has been observed on sweetness potency in several other sweetener classes (25). To explore this effect further, the 4-ethylphenyl isosteric analogue 29 of the 4-methoxyphenyl analogue 25 was prepared. Consistent with the heteroatom potency enhancement theory, 29 was found to exhibit a P (3) = 1,200 while 25 exhibits a P (5.5) = 5,500. w

w

w

w

NC

29

C0 H 2

Also explored were the effects on taste activity of heteroaromatic substitution for the phenyl moiety of 17. Specifically, the 4-pyridyl, 3-pyridyl, and 3-quinolyl analogues 30, 31, and 32 were prepared and evaluated. The 4-pyridyl analogue


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30 was found to exhibit a P (2) = 2,000. The 3-pyridyl analogue 31 and the 3-quinolyl 32 exhibited P (2) = 20,000. Heteroaromatic systems show potent activity in this series. w

w


Ο

Ο

32

C0 H 2

Isosteres The concept of isosteric replacement of functional groups is widely used in the design of pharmaceutical products (26). Thiourea, guanidine, and cyanoguanidine may function as isosteric equivalents of the urea functionality. In the initial report on the suosan series, the isosteric replacement of the urea functionality by a thiourea moiety was reported to yield an increase in potency. Nofre and Tinti in 1985 reported that cyanoguanidine and guanidine isosteric analogues of aryl urea sweeteners were active (27). With these precedents, we prepared the thiourea, guanidine, and cyanoguanidine analogues of compound 17. The thiourea 33 was sweet but empirically equivalent to 17, with a P (4) = 4,000. The guanidine analogue 34 [P (l) = 100] and the cyanoguanidine analogue 35 [P (3.5) = 350] were weakly sweet. Disappointingly, these isosteric replacements did not lead to increases in activity in the parent compound 17. w

w

w

S

33

NH

C0 H 2

C0 H 2

34


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NCN

35

C0 H 2


Chiiality All of the ureas previously discussed were prepared as racemates. In the aspartyl amide sweeteners, such as aspartame, only the enantiomer derived from L-aspartic acid is sweet (9). In the aspartyl anilides, initially reported by Sweeney and Lapidus, only the enantiomer derived from L-aspartic acid was found to have any sweetness activity (J J). The method of Fischer was employed for the resolution of 3-amino-3-phenylpropionic acid (28, 29). Both R and S enantiomers 36 and 37 of urea 17 were prepared. Optical purity was determined by HPLC analysis on a chiral stationary phase column. Analysis of the R enantiomer by HPLC showed a small amount (

High-Potency Sweeteners Derived from -Amino Acids - American

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