WO1988008256A1

WO1988008256A1 - Dihydroflavonol derivatives useful as sweeteners

Info

Publication number: WO1988008256A1
Application number: PCT/US1988/001202
Authority: WO
Inventors: A. Douglas Kinghorn; N. P. Dhammika Nanayakkara; Djaja D. Soejarto
Original assignee: University Patents Inc
Current assignee: University Patents Inc
Priority date: 1987-04-28
Filing date: 1988-04-18
Publication date: 1988-11-03
Anticipated expiration: 1989-10-28
Also published as: AU1708388A

Abstract

Certain dihydroflavonol derivatives are disclosed as being intensely sweet.

Description

Dihydroflavonol derivatives useful as sweeteners

Sucrose is unique among sweet compounds in that it produces a sweetness which is unmasked by any other taste sensation. However, relatively large amounts of sucrose have previously been used in sweetening of foodstuffs, and it is now generally accepted that this has been responsible as a major dietary source of dental caries. As a result of this, there has been a continued interest in discovering other sweetening agents besides sucrose that are characterized in being highly sweet, are not cariogenic, are not caloric, are not toxic, are not mutagenic, and exhibit acceptable taste qualities.

At the present time, the two intensely sweet sucrose substitutes used in the United States are saccharin and aspartame. Questions have, however, been raised about both these substances, especially in respect to their safety when used over long periods of time.

Researchers in both the United States and Japan have been turning to plant derived materials as an alternative resource pool for the isolation and development of new high-intensity sweetening agents, and now an ever growing number of compound classes of plant-derived "natural" sweeteners as known. In Japan, for example, the natural sweeteners, thaumatin, stevioside, phyllodulcin, and glycyrrhizin are all employed for sweetening foods, beverages, and medicines. In the United States, there is a great public demand for natural chemicals, and much effort is currently being expended in the quest for additional examples of acceptable naturally occurring sweeteners. Our research goal has also been to pursue research into the isolation of intensely sweet compounds from plants. One of the plants which we have examined as a potential source of intensely sweet compounds is Tessaria dodoneifolia (Hook and Arnott) Cabrera (family Compositae) which was obtained in Paraguay where it is sold as a plant under the name of "Hierba dulce" for sweetening foods and beverages. While attempts to isolate the sweet constituent of this plant were not successful from the plant material at hand, seeds of T. dodoneifolia were successfully cultivated at the University of Ilionois Pharmacognosy Field Station. By bioactivity-guided fractionation, we have now shown that the sweet compound present in shoots of "Hierba dulce" is dihydroquercetin-3-acetate. Realizing that we had discovered a new class of intensely sweet compounds, which are derivatives of dihydroflavonol, we decided to continue our research with this class of compounds which has led to the present invention.

Tessaria dodoneifolia, the plant used in our investigations, is known to be sweet, and is colloquially called, "chilca dulce" in Argentina (see E. Guerreiro, J. Kavka, and O . S. Giordano, Anales Assoc. Quim,. Argentina, 61:161-164[1973]). Only three previous chemical investigations have been conducted on this plant, and these have led to the isolation of two flavanones, additional flavonoids including dihydroquercetin-3-acetate (see J. Kavka, E. Guerreiro, and 0. S. Giordano, An. Quim. 73:305-306 [1978]), and a sesquiterpene. Dihydroquercetin-3-acetate, the compound we have identified as being the sweet constituent in the shoots of T. dodoneifolia, was not recognized as being sweet at the time of its isolation in 1978. The compound has since been isolated from two other plant sources (F. Bohlmann, C. Zdero, M. Grenz, A. K. Dhar, H. Robinson, and R. M. King, Phytochemistry, 20:281-286[1985], and M. Grande, F. Piera, A. Cuenca, P. Torres, and I. S. Bellido, Planta Med., 414-419 [1981]), where again its sweet properties were not recognized.

Recently, in 1984, Takahashi et al. published a procedure for the total synthesis of the 3-deacetyl derivative of dihydroquercetin-3-acetate (Heterocycles 22:1147-1153), which was utilized, in part, to synthesize the compounds of the present invention, and in 1986, Zdero et al. (Phytochemistry, 25:2841-2855) described (although he did not recognize its sweet nature), as a natural product, the positional isomer (3,5,7,4'-tetrahydroxy-3'-methoxyflavonone-3-0-acetate) of dihydroquercetin-3-acetate-4'-methyl ether, a compound belonging to this new class of highly sweet dihydroflavonol derivates.

Broadly stated, the dihydroflavonol derivatives of the present invention are compounds of the general formula

wherein R₁ may be H, OH, or OC_1-4 alkyl;

wherein R₂ may be H, OH, or OC_1-4 alkyl;

wherein R₃ may be OH, OCOC_1-10 alkyl, OC_1-10 alkyl, or

wherein R₄ may be H, OH, OC_1-10 alkyl; and

wherein R₅ may be H, OH, O(CH₂)_n CH(NH₂)COOH, O(CH_{_}

₂ ⁾ _n ^COO-_M+, O(CH₂)_nSO₃M⁺, O(CH₂)_nNH₃X-,

O(CH₂)_nNHSO₃-M+, wherein M⁺ is a physiologically acceptable cation such as Na ⁺, K⁺, or Ca⁺⁺, wherein X- is a physiologically acceptable anion such as Cl-, PO₃ ^-3, and wherein n is a number from 1 to 10, wherein

R₅ is a sugar, e.g. glucose, rhamnose, rutinose, or neohesperidose, or other acceptable nontoxic sugar.

More specifically, the preferred intensely sweet dihydroflavonols according to the present invention

(these have been chosen as being examplary of those compounds according to the broad general formula depicted above) are dihydroquercetin-3-acetate

(Compound I) which has the structure:

and its synthetic derivative, dihydroquercetin-3- acetate-4'-methyl ether (5,7,3'-trihydroxy-4'-methoxy- dihydroflavonol-3-acetate) (Compound II) which has the structure:

By the indication of the substitutions labeled

"C.....alkyl" is meant any alkyl radical having the number of carbon atoms in the subscript notation. For example, the radical when the label is defined as specifically being "C₄ alkyl" would be a butyl, isobutyl, or tertiary butyl radical. The carbon designation is meant to encompass any and all of the isomeric forms of the radical defined by carbon number. EXAMPLE I

1. Isolation Method for the Sweet Compound, Dihydroquercetin-3-acetate (I) from the Paraguayan Plant, Tessaria dodoneifolia

Dried, ground young growing tips of T. dodoneifolia (600 g) were extracted with methanol-water (4:1, 3 x 2 liters) by percolation. The combined extracts were evaporated to dryness at 50°C to afford a gum (240 g), which was dissolved in water (4 liters) and extracted with ethyl acetate (6 x 1.5 liters). The organic layer was washed with water (2 x 500 ml), and evaporated to dryness to afford 116 g of a residue. The sweetness of the methanol-water extract of T. dodoneifolia was found to concentrate in the ethyl acetate fraction, which was determined as nonmutagenic in forward mutation assays with S. typhimurium strain TM677, and was nontoxic for mice when a 2 g/kg dose level was administered by oral intubation.

T. dodoneifolia flowering tip ethyl acetate extract (100 g) was adsorbed to silica gel (800 g) and transferred to a glass column (1.5 m x 15 cm) containing 2.5 kg of silica gel. Separation was achieved by elution of the column with mixtures of petroleum ether (bp 60-80°C), chloroform, and methanol, of increasing polarity. Elution of the column with chloroformmethanol (39:1) afforded a sweet fraction (6.8 g) which was crystallized from methanol-chloroform to give pale yellow needles of I (900 mg, 0.17% yield).

2. Physical and Spectroscopic Characteristics of Compound I

The naturally occurring sweet compound, dihydroquercetin-3-acetate (I) exhibited: mp 133-135°C; [α]_D +40.7° (c 4.87, MeOH), UV, λ_max (MeOH) 223 (log ε 4.09) , 291 (4.14) , 333 sh nm (3.50) ; IR, ν_max (KBr)

3450-3250, 1745, 1642, 1590 cm^-1; ¹H NMR [300 MHz, (CD₂)₃CO)] δ 1.98 (3H, s, -OAc), 5.37 (1H, d, J = 12.0 Hz, 2-H), 5.84 (1H, d, J = 12.0 Hz, 3-H), 5.99 (1H, d, J = 2.8 Hz, 6-H), 6.02 (1H, d, J = 2.8 Hz, 8-H), 6.88

(2H, m, 5'-,6'-H), 7.06 (1H, d, J = 2.3 Hz, 2' -H), 11.59 (1H, s, -OH); ¹³C NMR (90 MHz, C₅D₅N)

20.1 (-OCOCH₃), 72.6 (C-3), 81.6 (C-2), 96.2, 97.5 (C-6, -8), 101.4 (C-10), 115.7, 116.2 (C-2', -5'), 119.9 (C-6'), 127.2 (C-1'), 147.1, 148.1 (C-3', -4'), 163.1, 164.6 (C-5, -9), 168.9 (C-7), 169.4 (OCOCH₃), 192.1 (C-4), MS, (rel. intensity) m/z 346 (M⁺, 8%), 304 (8), 286 (73), 275 (10), 153 (100), 150 (36), 123 (15).

EXAMPLE II Absolute Stereochemistry of Dihydroquercetin-3-Acetate

The absolute stereochemistry of I was determined as follows: A sample of commercial (+)-dihydroquercetin (taxifolin) (Roth-Atomergic Chemicals Corp., Plainview, NY) (50 mg, II) in pyridine (2 ml) was acetylated with acetic anhydride (1 ml) at 60°C for 1 hr. The reaction mixture was then poured into dil. HCl (5%, 200 ml), and extracted with ether. The ether layer was washed with NaHCO₃ (5%, 100 ml) and water, and evaporated to dryness. The residue was chromatographed over Florisil (10 g), and elution of the column with ethyl acetate afforded pure (+)-pentaacetoxy-dihydroquercetin (III, 48 mg), which exhibited the following physical and spectroscopic data: mp, 128-130°C; [α]_D +67.5°; ¹H-NMR (360 MHz, CDCl₃) α 7.39 (1H, dd, J = 8,2 Hz, 6'H), 7.29 (1H, d, J = 2 Hz, 2'-H), 7.27 (1H, d, J = 8 Hz, 5'-H), 6.79, 6.61 (1H each, d, J = 4 Hz, 6-,8-H), 5.66 (1H, d, J = 12 Hz, 2-H), 5.43 (1H, d, J = 12 Hz, 3-H), 2.38 (3H, s, -COCH₃), 2.31 (9H, s, 3 x - COCH₃ ), 2.05 (3H, s, -COCH₃); MS, m/z 514 (M⁺, 1%), 472 (25), 411 (20), 388 (15), 370 (17), 346 (13), 328 (37), 285 (17), 194 (22), 153 (46), 43 (100).

Natural dihydroquercetin-3-acetate (I, 50 mg) was acetylated under the same conditions as II, to yield pentaacetoxydihydroquercetin (III, 42 mg). This compound exhibited mp, 126-128°C, [α]_D +65.3° (c 1.1, CHCl₃), as well as identical 1H-NMR and MS data to

(+)-pentaacetoxydihydroquercetin produced from II.

Therefore, it may be concluded that the absolute stereochemistry of the sweet derivative of T . dodoneifolia is (2R^*,3R^*)-dihydroquercetin-3-acetate [= (2R^*, 3R^*)-5,7,3',4'-tetrahydroxydihydroflavonol-3-acetate, I]:

I R = H; R' = Ac II R = R' = H III R = R' = Ac

EXAMPLE III Physical and Spectroscopic Characteristics of Dihydroquercetin-3-Acetate-4'-Methyl Ether

The synthetic sweet isolate, 5,7,3'-trihydroxy- 4'-methoxydihydroflavonol-3-acetate (II) exhibited the following data: mp, 182-184°C, [α] 25 0; UV, λ_max

(MeOH) 240 (log ε 2.67), 289 (3.03), 337 sh nm (2.29);

IR, (KBr) 3513, 3236, 2937, 1733, 1641, 1585, 1517,

1465, 1273, 1245, 1166, 1086, 1032 cm ^_1; ¹H-NMR

(CDCl₃, 300 MHz) δ 2.05 (3H, s, -OAc), 3.91 (3H, s,

-OMe), 5.25 (1H, d, J = 12 Hz, H-2), 5.77 (1H, d, J =

12 Hz, H-3), 5.98 and 6.03 (1H each, d, J = 2 Hz, H-6,

-8), 6.87 (1H, d, J = 8 Hz, H-5'), 6.96 (1H, dd, J =8, 2 Hz, H-6'), 7.03 (1H, d, J = 2 Hz, H-2'); ¹³C-NMR

(90.8 MHz, CDCl₃) δ 20.3 (OCOCH₃), 55.7 (OCH₃), 72.3

(C-3), 80.7 (C-2), 95.9 (C-6), 97.2 (C-8), 101.3

(C-10), 110.5 (C-5'), 113.5 (C-2'), 119.3 (C-6'),

127.8 (C-1), 145.2 (C-3'), 147.3 (C-4'), 162.3 (C-9),

163.7 (C-5), 166.1 (C-7), 170.1 (OCOCH₃), 191.5 (C-4);

MS, (rel. intensity) m/z 360 (M⁺, 12), 318 (5), 300

(85), 289 (10), 166 (100), 164 (67), 153 (88), 151

(21), 137 (25), 133 (12); mass measurement, found

360.0845, cald. for C₁₈H₁₆O₇, 360,0853.

The synthetic route for the intensely sweet compound II from 2,4-dibenzyloxy-6-methoxymethoxy-acetophenone and 3-benzyloxy-4-methoxybenzaldehyde is depicted below:

2,4-Dibenzyloxy-6- 3-Genzyloxy-4- metlioxymetfioxy- metlioxybenz- 3,4',6'-Tribenzyloxy-2'- acetophenone aldehyde methoxymethoxy-4-methoxychalcone

α,β-Epoxy-3,4',6'-tribenzyloxy-2'-methoxy- 5,7,3'-Tribenzyloxy-4'- methoxy-4-methoxy- methoxydihydroflavonol chalcone

5,7,3'-Tribenzyloxy-4'-

R = Bz methoxydihydroflavonol- 5,7,3'-Trihydroxy-4^,-meth 3-acetate

R₁-CH₂OMe oxydihydroflavonol- 3-acetate (II) EXAMPLE IV

Synthetic Methods for the Production of the Sweet

Compound, 5,7,3'-Trihydroxy-4'-methoxydihydroflavonol- 3-acetate (II)

Synthesis from the. Commercially Available 2,4,6- Trihydroxyacetophenone and 3-Hydroxy-4-methoxybenz- aldehyde (Isovanillin)

i. Preparation of Benzyloxy Derivatives of the Starting Materials:

The first starting material (2,4-dibenzyloxy-6-methoxymethoxyacetophenone) was prepared from 2,4,6-trihydroxyacetophenone (Aldrich Chemical Co., Milwaukee, Wisconsin) according to literature procedures (M. L. Sehti, S. C. Taneja, K. L. Dhar, and C. K. Atal, Ind. J. Chem., 20B, 770-772, 1981; F. R. van Heerden, J. J. Van Zyl, G. J. H. Rail, E. V. Brandt, and D. G. Roux, Tetrahedron Lett., 661-662, 1978). 3-Benzyloxy-4-methoxybenzaldehyde, the other starting compound, was prepared from isovanillin (Aldrich) by the method of R. Robinson and S. Sugasawa (J. Chem. Soc., 134, 3163-3172, 1931).

ii. Synthesis of 3,4',6'-Tribenzyloxy-2'-methoxymethoxy-4-methoxychalcone:

A mixture of 2,4-dibenzyloxy-6-methoxymethoxyacetophenone (3.92 g), 3-benzyloxy-4-methoxybenzaldehyde (2.42 g), and sodium hydroxide (14 g) in ethanol (100 ml) was stirred at room temperature for 3 days. Solvent was removed under reduced pressure, and the residue was triturated with water, and filtered. The precipitate was washed with water and dried, to afford a yellow solid, which was crystallized from methanol to afford 3,4',6'-tribenzyloxy-2'-methoxymethoxy-4- methoxychalcone (4.8 g, 78%), as yellow needles, which exhibited the following data: mp 126-128 °C; IR, ν_max 3033, 2931, 1662, 1645, 1603, 1592, 1514, 1257, 1156, 1072 cm^-1; ¹H NMR (CDCl₃, 360 MHz) δ 7.5-7.2 (15H, m, aromatic), 7.38 (1H, d, J = 16 Hz, β-H), 7.69 (1H, d, J = 2 Hz, 2-H), 7.08 (1H, dd, J = 8,2 Hz, 6'-H), 6.87 (1H, d, J = 8 Hz, 5-H), 6.81 (1H, d, J = 16 Hz, -H), 6.49 and 6.31 (1H each, d, J = 2 Hz, 3'-,5',-H), 5.13 (2H, s, -OCH₂O), 5.10 (2H, s, -OCH₂Ph), 5.03 (4H, s, 2 x -OCH₂Ph), 3.90 (3H, s, -OCH₃), 3.37 (3H, s, CH₂ OCH₃); MS, m/z 616 (M⁺, 10%), 571 (19), 543 (7), 525 (10), 492 (7), 329 (12), 271 (16), 181 (14), 91 (100).

iii. Synthesis of -Epoxy-3,4',6'-tribenzyloxy-2'-methoxymethoxy-4-methoxychalcone:

A mixture of 3,4',6'-tribenzyloxy-2'methoxy-methoxy-4-methoxychalcone (4.3 g) in acetone (80 ml), 30% H₂O₂ (10 ml), 2N NaOH (10 ml), and methanol (30 ml) was shaken for 2 hours, and occasionally brought to boiling point. Solvent was evaporated in vacuo and the residue partitioned between water and ethyl acetate. The organic phase was washed with KI (10%, 200 ml), Na₂S₂O₃ (10%, 200 ml) , and water, and then evaporated to dryness. The residue was crystallized from methanol to give , -epoxy-3,4',6'-tribenzyloxy-2-methoxymethoxy-4-methoxychalcone as pale yellow needles (3.4 g, 77%). This compound exhibited the following physical and spectral, data: mp 125-127°C; IR, ν_max 3034, 2936, 1691, 1603, 1518, 1264, 1154,

1118, 1059, 1012 cm^-1; ¹H NMR (CDCl₃, 300 MHz)

7.42-7.28 (15H, m, aromatic), 6.81 (2H, m, 5-,6-H), 6.74 (1H, br s, 2-H), 6.42, 6.27 (1H each, d, J = 2 Hz, 2'-,5'-H), 5.09, 5.04, (1H each, d, J = 12Hz, -OCH₂O) , 5.04, 5.00 (1H each, d, J = 12 Hz, -OCH₂Ph) , 5.01 (4H, S, 2 x -OCH₂Ph), 3.89, 3.85 (1H each, d, J = 2 Hz,α- and/β-H), 3.87 (3H, s, -OCH₃), 3.32 (2H, s, -OCH₃); MS, m/z 632 (M⁺ 12%), 587 (2), 575 (3), 377 (24), 303 (5), 282 (16), 91 (100).

iv. Synthesis of 5,7,3'-Tribenzyloxy-4'- methoxydihydroflavonol:

A mixture of α , β-epoxy-3,4',6'-tribenzyloxy-2'- methoxymethoxy-4-methoxychalcone (3.2 g), conc. HCl (10 ml), methanol (100 ml), and tetrahydrofuran (10 ml) was refluxed for 1 hour. The reaction mixture was evaporated to dryness in vacuo, and the residue was crystallized from methanol to give a yellow powder, and was further recrystallized from the same solvent as yellow needles of 5,7,3'-tribenzyloxy-4'-methoxydihydroflavonol (600 mg, 20%), with mp 196-198° C; IR, ν_max 3464, 3031, 2905, 1668, 1606, 1574, 1516, 1260

1158, 1143, 1118, 741 cm^-1; ¹H NMR (CDCl₃, 300 MHz) 7.57-7.30 (15 H, m, aromatic), 7.14 (1H, dd, J = 9,2 Hz , 6 ' -H) , 7 . 13 ( 1H , d , J = 2 Hz , 2 ' -H) , 6 . 96 ( 1H , d , J = 9Hz, 5'-H), 6.27, 6.20 (1H each, d, J = 2 Hz, 6-, 8-H), 5.19, 5.15 (1H each, d, J = 8 Hz, -OCH₂Ph), 5.18 (2H, s, -OCH₂Ph), 5.05 (2H, s, -OCH₂Ph), 4.90 (1H, d, J = 11 Hz, 2-H), 4.42 (1H, d, J = 11 Hz, 3-H), 3.90 (3H, s, -OCH₃); MS, m/z 588 (M⁺, 8%) 559 (34), 497 (10), 467 (5), 361 (5), 333 (48), 256 (16), 227 (83), 181 (28), 167 (10), 137 (26), 91 (100).

v. Synthesis of 5,7,3'-Tribenzyloxy-4'- methoxydihydroflavonol-3-acetate

A total of 550 mg of 5,7,3'-tribenzyloxy-4'-methoxydihydroflavonol was heated on a steambath with acetic anhydride (2 ml) and pyridine (1 ml) for 30 min. The reaction mixture was cooled, poured into HCl (5%, 200 ml), and extracted with diethyl ether. The organic layer was washed with NaHCO₃ (5%, 200 ml), and water, and evaporated to dryness. The residue was crystallized from methanol to give 5,7,3'-tribenzyloxy-4'-methoxydihydroflavonol-3-acetate (white plates, 500 mg, 85%) that exhibited the following data: mp 130-131° C; IR (KBr) v_max 3031, 2932, 1751, 1685, 1608, 1570, 1516, 1436, 1261, 1223, 1166, 1155, 1111, 1017, 739 cm^-1; ¹H NMR (CDCl₃, 300 MHz) δ 7.54-7.30 (15H, m, aromatic), 7.05 (1H, dd, J = 8,2 Hz, 6'-H), 7.04 (1H, d, J = 2 Hz, 2'-H), 6.92 (1H, d, J = 8 Hz, 5'-H), 6.24, 6.18 (1H each, d, J = 2.5 Hz, 6-, 8-H), 5.79 (1H, d, J = 13 Hz, 3-H), 5.25 (1H, d, J = 13 Hz, 2-H), 5.16 (4H, s, 2 x -OCH₂Ph), 5.03 (2H, s, -OCH₂Ph), 3.91 (3H, s, -OCH₃), 1.98 (3H, s, COCH₃); MS, m/z, 630 (M, 8%), 570 (41), 479 (8), 298 (38), 256 (77), 181 (15), 165 (10), 135 (6), 91 (100).

vi. Removal of the Benzyl Protecting Groups:

5,7,3'-Tribenzyloxy-4-methoxhydihydroflavonol-3-acetate (40.0 mg) in dimethylformamide (40 ml), containing 10% Pd-C (0.25 g) and cone. HCl (15 drops) was stirred under hydrogen for 2 hours. The catalyst was removed by filtration, and the solvent evaporated in vacuo, with the residue being chromatographed over silica gel (50 g). Elution with chloroform afforded pure 5,7,3'-trihydroxy-4'-dihydroflavonol-3-acetate (170 mg, 70%), which exhibited identical physical and spectral data to those indicated for this sweet compound (II) in Example III.

The synthetic route for the intensely sweet compound II from Hesperetin is depicted below:

3,2',4',6'-Tetrabenzyloxy-4- Hesperetin methoxychalcone

4'-Methoxytaxifolin α,β-Epoxy-3,2',4',6'-Tetrabenzyloxy-4-methoxychalcone

3-Acetoxy-5,7,3'-tri-t-

3'-5,7-Tri-t-butyldimethyl- butyldimethylsilylsilyloxy-4'-methoxyoxy-4'-methoxytaxitaxifolin folin

R = Bz

R = Sit-BuMe₂

5,7,8'-Trihydroxy-4 -methoxy- dihydroflavonol-3-acetate (II) EXAMPLE V Synthetic Methods for the Production of the Sweet Compound 5,7,3'-Trihydroxy-4'-methoxydihydro- flavonol-3-Acetate

Synthesis from Hesperitin

i. Synthesis of 3,2',4',6'-Tetrabenzyloxy-4-methoxychalcone:

A mixture of hesperitin (30 g), anhydrous K₂CO₃ (150 g), benzyl chloride (75 ml), in anhydrous DMF (500 ml) was heated at 120°C for 5 hr. The reaction mixture was filtered, and the solvent was evaporated to dryness in vacuo to give a yellow oil. This was poured into water and extracted with chloroform. The organic 1.c. Phase was washed with water and evaporated to dryness. The residue was crystallized from ethyl acetate to give 3,2',4',6'-tetrabenzyl- oxy-4-methoxychalcone (54 g, 82%). This compound exhibited, mp, 158-160°C; IR, (KBr) ν_max 3031, 2876, 1644, 1600, 1513, 1432, 1260, 1157, 1116, 699 cm^-1, ¹H-NMR (300 MHz, CDCl₃) δ 7.5-7.2 (21H, m, 4 x CH₂C₆H₅ and -H), 7.08 (1H, d, J = 2 Hz, 2-H), 7.06 (1H, dd, J = 8, 2 Hz, 6-H) 6.87 (1H, d, J = 8 Hz, 5H), 6.83 (1H, d, J = 16 Hz, α-H), 6.25 (2H, s, 3'-5'-H), 5.11 (2H, s, CH₂C₆C₅), 5.03 (4H, s, 2 x CH₂C₆H₅), 4.90 (2H, s, CH₂C₆H₅), 3.92 (3H, s, OMe); MS, m/z 662 (M⁺, 2%), 571 (5), 543 (3), 329 (28), 239 (5) 181 (7), 91 (100).

ii. Synthesis of , -Epoxy-3,2',4',6'-Tetrabenzyloxy 4-methoxychalcone:

A mixture of 3, 2',4',6'-tetrabenzyloxy-4-methoxychalcone (21 g), sodium hydroxide (6 g in 30 ml of water), hydrogen peroxide (30%, 20 ml), chloroform (525 ml), and methanol (300 ml) was stirred at room temperature for 18 hr. Solvents were evaporated in vacuo, and the products were partitioned between chloroform and water. The organic phase was washed with KI (10%, 100 ml), Na₂S₂O₃ (10%, 200 ml), and water, and evaporated to dryness. The residue was crystallized from chloroform-methanol to give α, β -epoxy-3,2',4',6'-tetrabenzyloxy-4-methoxychalcone (19.2 g, 89.3%). This compound exhibited, mp, 124- 126°C; IR, (KBr) ν_max 3032, 1696, 1605, 1517, 1436,

1262, 1160, 1131, 1123, 746, 696 cm^-1; ¹H-NMR (360

MHz, CDCl₃) δ 7.5-7.2 (20H, m, 4 x OCH₂C₆H₅), 6.77

(1H, d, J = 8 Hz, 5-H) 6.70 (1H, dd, J = 8,2 HZ,6-H),

6.68 (1H, d, J = 2 Hz, 2-H), 6.21 (2H, s, 3'-, 5'-H),

5.01 (4H, s, 2 x CH₂C₆H₅), 5.00 (2H, AB_q, J = 12,.6

Hz, CH₂C₆H₅), 4.98 (2H, s, CH₂C₆H₅), 3.89, 3.86 (1H each, d, J = 2 Hz, - and -H) , 3.85 (3H, s, OMe); MS, m/z 678 (M⁺, 0.5%), 423 (13), 396 (5), 282 (3), 181 (5), 91 (100).

iii. Synthesis of 4'-Methoxytaxifolin:

A mixture of α,β-epoxy-3,2',4',6'-tetrabenzyloxy-4-methoxychalcone (15 g) Pd-C (10%, 6 g) , conc. HCl (4.5 ml) in DMF (600 ml) was stirred under hydrogen for 4 hr. The reaction mixture was filtered, poured into water and extracted with ethyl acetate. The organic layer was washed with water and evaporated to dryness. The residue was chromatographed over silica gel, and elution of the column with petroleum ether:acetone (85:15) afforded pure 4'-methoxytaxifolin (6.2 g, 88%). mp, 190-192°C, IR (KBr) \) max.3412,

3077, 1637, 1590, 1519, 1474, 1270, 1155, 1131, 1083, 985, 798 cm^-1; ¹H-NMR (360 MHz, DMSO)δ6.96 (1H, d, J = 8 Hz, 5'-H), 6.94 (1H, br s, 2-H), 6.91 (1H, d, J = 8 Hz, 6'-H), 5.95, 5.89 (1H each, d, J = 2 Hz, 6, 8-H), 5.04 (1H, d, J = 11 Hz, 2-H) 4.83 (1H, d, J = 11 Hz, 3-H), 3.79 (3H, s, OMe); MS, m/z 318 (M⁺, 30%), 289 (49), 166 (47), 165 (49), 164 (38), 153 (100), 151 (38), 137 (30), 85 (40); mass measurement, observed, 318,0739, calcd. for C₁₆H₁₄O₇, 318,0733.

iv. Partial Silylation of 4'-Methoxytaxifolin:

A mixture of 4'-methoxytaxifolin (3.18 g) in imidazole (4.5 g), and t-butyldimethylsilyl chloride (4.8 g) in dry dimethylformamide (100 ml), was stirred at 0° C for 12 hr. The reaction mixture was poured into water, and extracted with ether, and the organic layer washed with water and evaporated to dryness to give a white crystalline solid. This residue was purified by chromatography over silica gel, by elution with petroleum ether-acetone (98:2) to afford pure 3',5,7-tri-t-butyldimethylsilyloxy-4'-methoxytaxifolin (4.8 g, 72%), which exhibited the following characteristics: mp. 122-124°C, IR (KBr) ^νmax 3453, 2930,

2860, 1680, 1603, 1558, 1514, 1427, 1254, 1168, 1097, 840, 782 cm^-1; ¹H-NMR (300 MHz, CDCl₃) δ7.09 (1H, dd, J = 9, 2Hz, 6'-H), 7.04 (1H, d, J = 2 Hz, 6-,2'-H), 6.90 (1H, d, J = 8 Hz, S'-n), 6.09 and 5.97 (1H each, d, J = 2 Hz, 6'-, 8'-H), 4.87 (1H, d, J = 12 Hz, 2-H), 4.38 (1H, d, J = 12 Hz, 3H), 3.82 (3H, s, -OMe), 1.06, 0.99, 0.97 (9H each, s, -Si-C- (CH₃)₃), 0.30 and 0.25 (3H each, s, Si(CH₃)₂), 0.23 (6H, s, -Si(CH₃)₂), 0.18 and 0.17 (3H each, s, -Si(CH₃)₂); MS, m/z 660 (M⁺, 0.7%), 645 (3), 631 (3), 605 (23), 603 (100), 381 (9), 231 (20), 251 (16), 179 (24), 73 (71).

v. Acetylation of 5,7,3'-Tri-t-butyldimethylsilyloxy-4'-methoxytaxifolin:

5,7,3'-Tri-t-butylidmethylsilyloxy-4'-methoxytaxifolin (3 g) in triethylamine (15 ml), was treated with acetic anhydride (2 ml) in the presence of 4-(dimethylamino) pyridine (250 mg) at 0°C for 15 min. The reaction mixture was poured onto ice, and extracted with ether. After the usual workup, the ether layer was purified over silica gel, by elution with petroleum ether-acetone (98:2), to yield pure 3- acetoxy-5,7,3'-tri-t-butyldimethysilyloxy-4'-methoxytaxifolin (2.8 g, 87%), which was crystallized from methanol-water to give white needles, and exhibited: mp, 80-82°C, IR (KBr) ν_max 2931, 2860, 2680, 1680,

1603, 1558, 1514, 1427, 1254, 1168, 1097, 840, 782 cm^-1; ¹H-NMR (300 MHz, CDCl₃) δ 7.04 (1H, dd, J = 8, 2, Hz, 6'-H), 6.93 (1H, d, J = 2 Hz, 2'-H), 6.86 (1H, d, J = 8 Hz, 5'-H), 6.10 and 5.98 (1H each, d, J = 3Hz, 6-, 8-H), 5.56 (1H, d, J = 12 Hz, 2 H), 5.19 (1H, d, J = 12 Hz, 3-H), 3.82 (3H, s, -OMe), 1.99 (3H, s, -OAc), 1.03, 0.99 and 0.96 (9H each, s, -Si-C- (CH₃)₃), 0.26 and 0.23 (3H each, s, -Si(CH₃)₂), 0.23 and 0.15 (6H each, s, -Si(CH₃)₂); MS, m/z M⁺ missing, 687 (M⁺ - CH₃, 3%), 648 (18), 645 (100), 603 (4), 585 (15), 267 (20), 73 (60).

vi. Removal of Silyloxy Groups:

3-Acetoxy-5,7,3'-tri-t-butyldimethylsilyloxy-4'-methoxytaxifolin (1.0 g) in tetrahydrofuran (15 ml), was treated with tetrabutylammonium fluoride (1.8 g) at 0°C for 15 min. The reaction mixture was poured into water, and extracted with ether. On workup, and purification by chromatography over silica gel by elution with petroleum ether-acetone (85:15), pure 3-acetoxy-4'-methoxytaxifolin (5,7,3'-trihydroxy-4'-methoxydihydroflavonol-3-acetate, II) (0.4 g, 78%) was found to exhibit identical data to those described in Example III.

Two methods have been shown for the synthesis of sweet compound II. In the first of these methods, compound II was produced from the commercially available 2,4 ,6-trihydroxyacetophenone and 3-hydroxy-4- methoxybenzaldehyde as precursors. This scheme allows the variation of the functional groups at positions 3, 5, 7, 3', and 4' of sweet compound II. The second method uses the widely available compound, hesperetin, as starting material, and accomplishes the synthesis of II in six stages. Hesperetin is the aglycone of hesperidin, a glycoside that is a by-product of the citrus industry. The remaining compounds, according to the broad general formula can easily be made conventional modifications in the starting materials well known, in the synthesis art. Furthermore the solubility of these compounds can be improved by the insertion of a polar group at position 7 in the general formula; such polar groups appear in the definition of R₅.

The natural compound, I and the synthetic derivative, II, were nonmutagenic when tested in a forward mutation assay utilizing Salmonella typhimurium strain TM677, both in the presence and absence of a metabolic activator (S-9) from the livers of Aroclor-1254 pretreated rates. Mutagenicity was assessed for compound I over the concentration range 0.039-0.625 mg/ml and for compound I over the concentration range 0.039-0.625 mg/ml and for compound II over the range, 0.17-1.38 mg/ml. Details of the protocols used are given in Tables 1 and 2, respectively. Preliminary Safety Test Data for the Sweet Natural Product, I, and the Sweet Synthetic Derivative, II

Table I. Results of Forward Mutation Assays on

Dihydroquercetin-3-acetate (I) Utilizing

Salmonella typhimurium TM 677^a

Compound Concentration Solvent Activating

Added (mg/ml) System Fraction

( x10⁵) none - - DMSO none 7.7 (100)

I 0.039 DMSO none 3.9 (100)

0.078 DMSO none 5.0 (100)

0.156 DMSO none 5.2 (89)

0.312 DMSO none 5.3 (85)

0.625 DMSO none 5.2 (56) none - - DMSO Aroclor S-9 3.9 (100)

I 0.039 DMSO Aroclor S-9 3.9 (100)

0.078 DMSO Aroclor S-9 4.1 (100)

0-156 DMSO Aroclor S-9 3.5 (100)

0.312 DMSO Aroclor S-9 3.7 (100)

0.625 DMSO Aroclor S-9 3.5 (87)

Each compound was assayed in duplicate at the indicated concentration. Each duplicate was placed in triplicate, both in the presence and absence of 8-azaguanine (8-AG), and the data were averaged. The spontaneous mutant fraction (i.e., average number of clones on 8-AG plates/average number of clones on plates without 8-AG) for this assay is 7.4± 5x10 ^-5 (n=145). A compound is said to cause significant mutation if the induced mutant fraction is greater than or equal to two times the spontaneous mutant fraction.

Percentage of bacteria surviving the treatment. Table 2. Results of Forward Mutation Assays on 5,7,3'-Trihydroxy-4'-methoxydihydroflavonol-3-acetate (II) Utilizing Salmonella typhimurium TM677^a

Compound Concentration Solvent Activating Mutant

Added (mg/ml) System Fraction

( x10⁵) none - - DMSO none 13.9(100)

II 0.17 DMSO none 7.8(100)

0.34 DMSO none 8.6(97)

0.69(ppt) DMSO none 8.6(69)

1.38 (ppt) DMSO none 5.2(53) none - - DMSO Aroclor S-9 4.0(100)

II 0.17 DMSO Aroclor S-9 5.5(100)

0.34 DMSO Aroclor S-9 6.7(100)

0.69 (ppt) DMSO Aroclor S-9 4.0(100)

0.38 (ppt) DMSO Aroclor S-9 8.2(87)

a Each compound was assayed in duplicate at the indicated concentration. Each duplicate was plated in triplicate, both in the presence and absence of 8-azaguanine (8-AG), and the data were averaged. The spontaneous mutant fraction (i.e., average number of clones on 8-AG plates/average number of clones on plates without 8-AG) for this assay is 7.4± 5x10 ^-5 (n=145). A compound is said to cause significant mutation if the induced mutant fraction is greater than or equal to two times the spontaneous mutant fraction.

Percentage of bacteria surviving the treatment. Compounds I and II were administered to male Swiss Webster mice by oral intubation at 1 g/kg of body weight. Neither compound produced any lethalityfor up to 14 days after compound administration. No significant differences were observed for I and II between each test group of 10 mice and 10 controls, in terms of body weights, when assessed 1, 3, 7, and 14 days after compound administration. Details of the protocols used and test data for compounds I and II are shown in Tables 3 and 4, respectively.

Table 3. Results of Acute Toxicity Assays on Dihydroquercetin-3-acetate (I) Utilizing Swiss-Webster Male

Mice^a

Group Animal Animal Weight (g)^b

Number Day 0 Day 1 Day 3 Day 7 Day 14

test 1 18.8 18.6 20.5 21.1 22.3

2 20.7 21.9 24.1 25.3 27.1

3 20.3 21.6 22.1 23.5 24.9 4 21.2 23.3 23.2 24.3 25.0

5 20.3 21.0 23.1 23.0 23.8

6 21.3 22.2 23.8 25.7 26.3

7 20.6 20.2 22.1 24.3 25.4

8 19.4 18.7 22.6 23.9 21.7

9 21.9 21.0 22.8 25.2 26.9

10 20.0 20.8 22.8 25.2 23.4 control 1 20.5 21.2 23.4 24.7 26.4

2 19.8 21.1 22.8 25.0 26.5

3 19.7 20.5 21.0 22.3 23.2

4 20.2 23.2 23.9 25.4 26.9

5 19.4 20.0 22.3 23.2 24.8

6 22.7 24.7 25.3 25.7 27.4

7 19.2 19.0 ,22.0 23.4 .24.5

8 18.9 20.7 22.0 22.7 23.6

9 20.0 21.5 23.5 25.7 27.6

10 20.6 22.1 22.6 24.7 26.1 ^a Compound I was dispersed in 1% sodium (carboxymethyl)-cellulose (CMC) and mice in the test group were dosed by oral intubation at a level of 1 g/kg of body weight. Animals in the control group were treated with 1% CMC only. ^bt Test method was used to analyze these results for any significant variation; t for day 0, 0.789, day 1, 0.695, day 3, 0342, day 7, 0.167, day 14, 1.327. Therefore, there is no significant difference between the test and control groups (0.95 confidence). Table 4. Results of Acute Toxicity Assays on 5,7,3'-Trihydroxy-4'-methoxydihydroflavonol-3-acetate (II) Utilizing Swiss-Webster Male Mice^a

Group Animal Animal Weight (g)^b

Number Day 0 Day 1 Day 3 Day 7 Day 14

test 1 23.9 23.5 24.5 20.6 27.5

2 23.8 22.7 23.7 19.7 25.1

3 23.5 22.8 23.6 20.2 26.8

4 24.9 24.1 24.9 20.6 25.3

5 23.4 22.1 23.4 19.6 28.1

6 23.5 22.8 23.5 23.9 24.9

7 24.1 23.1 24.7 26.0 29.7

8 25.4 24.2 25.6 26.1 29.9

9 25.5 25.4 26.7 27.8 29.1

10 25.8 24.8 25.8 26.5 28.4 control 1 23.5 23.4 24.1 22.6 27.3

2 24.5 23.2 24.0 22.5 26.9

3 25.6 25.2 25.6 24.1 28.0

4 23.5 23.6 25.0 23.6 26.9

5 22.9 23.2 23.7 21.6 25.5

6 24.8 24.3 25.7 26.2 28.6.

7 22.5 22.4 23.6 23.1 28.4

8 23.5 22.5 23.3 24.8 26.4

9 22.4 21.8 22.8 20.4 24.5

10 23.8 23.5 24.7 23.5 27.8 ^aCompound II was dispersed in 1% sodium (carboxymethyl) cellulose (CMC) and mice in the test group were dosed by oral intubation at a level of 1 g/kg of body weight. Animals in the control group were treated with 1% CMC only. ^b t Test method was used to analyze these results for any significant variation; t for day 0, -1.560, day 1, -0.532, day 3, -0.829, day 7, 0.104, day 14, -.621. Therefore, there is no significant difference between the test and control groups (0.95 confidence).

In a preliminary taste testing of the sweet natural product I by an independent trained screening group, the product was rated 40-50 times sweeter than sucrose. More recently, a more extensive test was conducted on the sweet synthetic compound II by an independent trained screening group. A 3% ethanol- water, solution of compound II was prepared, and a 0.005% solution in this solvent was found to match the sweetness of 2% sucrose solution. Therefore, at this concentration, compound II was about 400 times sweeter than sucrose, and is thus about 10 times sweeter than natural compound I. The quality of the sweetness elicited by compound II was described as "slow onset -low sweet builds", and as "good within the context of natural sweeteners". No bitterness was reported, and the compound was found to have a quicker cutoff than sweeteners in the dihydrochalcone series.

Synthetic compound II has been subjected to preliminary stability studies, by storage for up to two weeks in solutions at the pH levels 3, 5, and 7, and at both room temperature and at 60°C. The compound was constituted at the appropriate pH in Teorell and Stenhagen's citrate-phosphate-borate buffer at a concentration of 1 mg/ml, and stability was assessed by the number of zones apparent by thin-layer chromatographic analysis at each time interval. Compound II was stable at all three pH levels at ambient temperature, throughout the course of the study. At the elevated temperature, II was more stable at acid rather than neutral conditions. While no breakdown was apparent at 60°C at pH 3 after two weeks, about 5-10% of the compound decomposed at pH 7 at this temperature and time intervals storage. It may be anticipated that most of the potential uses of compound II would involve acid pH levels.

Before a sweetener can be considered as being commercially feasible for use as a sucrose substitute, however, it must be examined in depth from several vantage points. A compound with the structure of didydroquercetin-3-acetate-4'-methyl ether (II) is unlikely to be caloric, and, since it does not possess a glycosidic residue that could be hydrolyzed to one or more sugars, II most probably will be noncariogenic. Although preliminary safety testing on this compound is encouraging, much more involved toxicological testing and metabolism studies would be needed before this substance could be marketed in the United States.

An item of great importance in the evaluation of a sweetener is its sensory profile. While compound I, the natural sweetener obtained from the Paraguayan plant, may not have sufficient sweetness intensity for potential commercialization, its synthetic derivative II does, in being about 400 times sweeter than sucrose. In addition, compound II, a dihydroflavonol, may be compared in terms of the quality of its test with three other classes of sweeteners, with which itis somewhat structurally related, namely, the dihydrochalcones (one of which has recently been approved for use as a sweetener in Belgium), and the flavans and flavanones. The dihydrochalcones are limited by the long time taken for the sweetness response to become manifest, and by a lingering aftertaste. The flavan and flavonone classes of sweeteners were designed to mimic structural characteristics of the sweet compound, phyllodulcin, and all compounds in these classes that were intensely sweet had unpleasant or unacceptable aftertastes. Compound II appears to be superior to the known members of the dihydrochalcone, flavan, and flavanone classes of sweeteners in terms of its taste qualities.

As mentioned previously the compounds according to the present invention have utility as sweetening agents for a variety of foodstuffs when added thereto in amounts sufficient to obtain the desired sweetness levels. Although such levels are arbitrary according to the perceived tastes of the user, some guidance as to the amount of compound to add in order to achieve the desired level of sweetness may be taken from comparing the sweetness of these compounds with the conventional agent, sucrose.

By foodstuffs is meant any material ingested for nutrition, including beverages such as sodas and coffee, powdered beverage mixes, bakery goods such as cakes, pies, and breads and miscellaneous items such as toothpastes, mouthwashes, candy, breath mints, and the like. The compounds according to the present invention may be added to such foodstuffs either before or after preparation for ingestion, much as the commonly accepted sucrose preparations, depending upon the wishes of the individual.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and without departing from the spirit and scope thereof can make various changes and/or modifications to the invention for adapting it to various usages and conditions. Thus, while we have illustrated and described the preferred embodiment of our invention, it is to be understood that this is capable of variation and modification, and we therefore do not wish to be limited to the precise terms set forth, but desire to avail ourselves of such changes and alterations which fall within the purview of the following claims:

Claims

WE CLAIM :

A compound according to the general formula

wherein R₁ is selected from the group

-H,

-OH, and

-OC_1-4 alkyl; wherein R₂ is selected from the group

-H

-OH, and

-OC_1-4 alkyl; wherein R₃ is selected from the group

-OH

-OC_1-10 alkyl ,

-OCOC_1-10 alkyl, and (CH₂)_n wherein n is from 1-10;

wherein R₄ is selected from the group

-H,

-OH, and

-OC_1-10 alkyl, and wherein R₅ is selected from the group

-H,

-OH,

-O (CH_{2) n}CH (NH₂ ) COOH,

-O(CH₂)_nCOO - M⁺ -O(CH₂)_nSO-3 M⁺,

-O(CH₂)_nNH⁺3 X-,

-O(CH₂)_nNHSO₃ M⁺, and

-sugar, wherein n is from 1 to 10, M is a physiological acceptable cation, and x is a physiologically acceptable anion; and with the proviso that R₁, R₂, R₄ and R₅ are not OH when R₃ is -OOCCH₃.

2. A compound according to Claim 1 wherein R₂, R₄, and R₅ are -OH.

3. A compound according to Claim 2 wherein R₁ is

-OC_1-4 alkyl .

4. A compound according to Claim 2 wherein R₃ is -OOCCH₃.

5. A compound according to Claim 2 wherein R₁ is -OC_1-4 alkyl and R₃ is -OOCCH₃.

6. The compound according to Claim 5 wherein R₂ is -OCH₃.

7. A method of imparting sweetness to a foodstuff which comprises adding a compound of the general formula wherein R₁ is selected from the group

-H,

-OH, and -OC_1-4 alkyl; wherein R₂ is selected from the group

-H -OH, and -OC_{1 -4} alkyl ; wherein R₃ is selected from the group

-OH

- OC _1-10 alkyl,

-OCOC_1-10 alkyl, and wherein n is

from 1-10; wherein R₄ is selected from the group

-H,

-OH, and

-OC_1-10 alkyl; and wherein R₅ is selected from the group

-H, -OH,

-O(CH_2)nCH(NH₂)COOH,

-O(CH₂)_nCOO- M⁺,

-O(CH₂)_nSO-3 M⁺,

-O(CH₂)_nNH⁺3 X-,

-O(CH₂)_nNHSO₃ M⁺, and

- sugar, wherein n is from 1 to 10, M⁺H is a physiological acceptable cation, and x- is a physiologically acceptable anion in the amount sufficient to impart the desired degree of sweetness to the foodstuff.

8 A method according to Claim 7 wherein R₂ R₄ and

R₅ are -OH .

9. The method according to Claim 8 wherein the compound is 5,7,3'-trihydro-4'-methoxydihydroflavonol-3-acetate.

10. The method according to Claim 8 wherein the compound is dihydroquercetin-3-acetate.

11. A foodstuff having added thereto a compound of the general formula wherein R₁ is selected from the group

-H ,

-OH, and

-OC_1-4 alkyl; wherein R₂ is selected from the group

-H

-OH, and - -OC_1-4 alkyl; wherein R₃ is selected from the group

-OH

-OC_1-10 alkyl

-OCOC_1-10 alkyl, and wherein n is

from 1-10; wherein R₄ is selected from the group

-H,

-OH, and

-OC_{1 -10} alkyl; and wherein R₅ is selected from the group

-H,

-OH,

-O(CH_{2 )n}CH(NH₂)COOH,

-O(CH₂)_nCOO- M⁺, -O(CH₂)_nSO-3 M⁺,

-O(CH₂)_n NH⁺3 X-,

-O(CH₂)_nNHSO₃ M⁺, and

- sugar, wherein n is from 1 to 10, M⁺H is a physiological acceptable cation, and x is a physiologically acceptable anion in the amount sufficient to impart the desired degree of sweetness to the foodstuff.

12. A foodstuff according to Claim 11 wherein the compound is dihydroquercetin-3-acetate.

13. A foodstuff according to Claim 11 wherein the compound is 5,7,3'-trihydro-4'-methoxydihydroflavonol-3-acetate.