Method of synthesising sulforaphane

Abstract

The present invention relates to a method of synthesizing sulforaphane by reacting a compound of formula (A) with an oxidizing agent in an aqueous solvent and in the presence of a catalyst. The invention further provides a method of synthesizing a stabilized complex of sulforaphane and cyclodextrin by mixing the sulforaphane prepared by the methodology defined herein with cyclodextrin in an aqueous solvent. ##STR00001##

Claims

1. A process for the preparation of a complex of sulforaphane and cyclodextrin, the process comprising: (i) reacting, in an aqueous solvent, a compound of formula A: ##STR00014## with an oxidizing agent and in the presence of a catalyst to form sulforaphane; and (ii) mixing the sulforaphane from step (i) with cyclodextrin in an aqueous solvent to form a precipitate of the sulforaphane-cyclodextrin complex, wherein in step (i) 1 to 2 molar equivalents of oxidizing agent, relative to the compound of formula A, is used.

2. A process according to claim 1, wherein the solvent in step (i) and step (ii) is water.

3. A process according to claim 1 or claim 2, wherein the oxidizing agent is hydrogen peroxide or a water soluble or miscible organic per-acid, or a mixture thereof.

4. A process according to claim 3, wherein the oxidizing agent is hydrogen peroxide.

5. A process according to claim 1, wherein the catalyst is selected from cyclodextrin and organic or inorganic acids, such as ascorbic acid, formic acid, acetic acid, and/or sulphuric acid.

6. A process according to claim 5, wherein the catalyst is a cyclodextrin.

7. A process according to claim 6, wherein the catalyst is alpha-cyclodextrin.

8. A process according to claim 1, wherein 0.0001 to 1.0 molar equivalents of catalyst are present, relative to the compound of formula A.

9. A process according to claim 1, wherein in step (i) the temperature of the reaction is maintained at a temperature of 25 C. or less or 15 C. or less when the oxidising agent is added to the reaction mixture.

10. A process according to claim 1, wherein the cyclodextrin used in step (ii) is selected from one or more of W6 alpha cyclodextrin, a six sugar ring molecule, W7 beta cyclodextrin, a seven sugar ring molecule, W8 gamma cyclodextrin, an eight sugar ring molecule, derivatives thereof, and mixtures thereof.

11. A process according to claim 10, wherein the cyclodextrin used in step (ii) is alpha-cyclodextrin.

12. A process according to claim 11, wherein the mixture is cooled to a temperature within the range of about 8 C. to about 10 C.

13. A process according to claim 11, wherein the mixture is cooled to a temperature within the range of about 5 C. to about 4 C.

14. A process according to claim 11, wherein the molar ratio of sulforaphane to cyclodextrin in the resultant complex is within the range of 0.4:1 to 1:1.

15. A process according to claim 1, wherein the process comprises a further step of recrystallizing the resulting complex.

16. A sulforaphane-cyclodextrin complex obtainable by a process as defined in claim 1, wherein the cyclodextrin used in step (ii) is alpha-cyclodextrin and wherein the sulforaphane and alpha-cyclodextrin are present in the complex in a molar ratio of 0.98:1 to 1:1, and wherein the complex has a purity by HPLC greater than 98%.

17. A method of treating and/or preventing microbial infections and/or cancer, the method comprising administering to an individual in need of such treatment a therapeutically effective amount of a sulforaphane-cyclodextrin complex according to claim 16.

18. A pharmaceutical composition comprising a sulforaphane-cyclodextrin complex according to claim 16 and one or more additional pharmaceutical excipients.

19. A process according to claim 1, wherein in step (i) 1 to 1.5 molar equivalents of oxidizing agent, relative to the compound of formula A, is used.

20. A process according to claim 1, wherein in step (i) 1 to 1.1 molar equivalents of oxidizing agent, relative to the compound of formula A, is used.

21. A sulforaphane-cyclodextrin complex according to claim 16, wherein the purity by HPLC is greater than 98.5%.

Description

EXAMPLES

(1) The invention will now be illustrated in the following Examples.

(2) General Materials and Methods

(3) .sup.1H and .sup.13C NMR spectra were recorded on a Oxford 400 MHz spectrometer using TMS as the internal standard and the chemical shifts are reported in ppm.

(4) Electrospray ionization mass spectrometry (ESI-MS) was performed on a Micromass Platform LCZ connected to Waters 2695 separations module and Water 996 photodiode array detector. GC-MS spectrometry was performed on a Agilent 7820A/5975 MSD series.

(5) HPLC was performed on a HP 1050 Module, Column: Phenomenex Gemini C18, 5 110A, 2504.6 mm. Total run time: 40 min. MeCN in H2O+0.1% TFA. Flow: 1.5 mL/min. Detector: 244 nm (VWD).

(6) Karl Fischer (H.sub.2O content) analysis was performed on a KF coulometer 831 equipped with Ti stand 703.

(7) All reactions were run under an atmosphere of dry nitrogen and the reported yields are isolated yields. All chemical reagents were purchased from commercial sources and used as received.

Preparation of Starting Materials

Preparation of 1-isothiocyanato-4-methylthiobutane (Formula A)

(8) ##STR00010##

(9) A 50-L multi-neck round bottom flask equipped with an overhead stirrer, a temperature probe and a 1 L addition funnel and a positive flow of N.sub.2 was cooled to 10 C. in MeOH/ice bath and charged with THF (EMD, reagent grade, 15.0 L). 1-Amino-4-methylthiobutane (Formula B; 1.5 Kg, 12.6 moles, 1.0 equiv.) and triethylamine (1.75 L, 1.0 equiv.) were added, and the solution was further stirred until it had cooled below 10 C. Carbon disulfide (755 mL, 1.0 equiv.) was added dropwise over 2 hours while keeping the internal temperature below 3 C. (bath temperature was 20 C.), after which the yellow-green solution had been warmed to 11 C. Hydrogen peroxide (35% aq, 1224 mL, 1.0 equiv.) was added slowly over 2.5 hours while keeping the internal temperature between 11 to 18 C. (bath temperature was 0 C.), which produced a dark orange-red suspension with swirling yellow particulates.

(10) Workup:

(11) After stirring overnight, an aliquot was checked by GC (75 C..fwdarw.200 at 15/min, then 40/min to 300, 2 min hold: 7.73 mins) and then the mixture was transferred into a 50-L workup station using a hose equipped with a filter head. The mixture was diluted with 4.5 L of ethyl acetate, and then washed with 10% HCl (6 L), water (6 L), and brine (7.5 L). The collected organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum to yield 2 kg of dark red oil.

(12) Distillation:

(13) The red oil was transferred into a 2 L (3 batches) round bottom flask and connected to the Kugelrohr. The apparatus was placed under high vacuum (0.3-0.5 torr), and the air bath heated to 85 C. The forerun (mostly ethyl acetate and trace unknown by-product) was discarded. After changing the receiver, the bath temperature was increased to 115 C. Pale yellow material distilled over at 100-110 C., and immediately froze upon contact with the dry-ice/acetone bath. After distillations (three batches) yielded 1.7 Kg (84% yield) material at 98% pure by HPLC and >99% pure by GC.

(14) .sup.1HNMR (CDCl.sub.3, 400 MHz); 1.7-1.85 (m, 4H), 2.2 (s, 3H), 2.55 (t, 2H), 3.56 (t, 2H)

Example 1Preparation of Sulforaphane (1-isothiocyanato-4-methylsulfinylbutane)

(15) ##STR00011##

(16) A 5-L multi-neck round bottom flask equipped with an overhead stirrer, a temperature probe and a 500 mL addition funnel was set-up with a positive flow of N.sub.2. -Cyclodextrin (30 g, 0.03 moles, 0.01 equivalents) was dissolved in 1 L of distilled water and degassed over 30 minutes by purging with nitrogen. To the above solution was added 501 g (3.1 moles, 1 equivalent) of 1-isothiocyanato-4-methylthiobutane (Formula A) and degassed again at 0 C. over 30 minutes. To this biphasic reaction mixture was added 305 mL of H.sub.2O.sub.2 (3.1 moles, 1 equivalent, 35% aq.) slowly while maintaining temperature between 0-2 C. [NOTE: Peroxide was dropped in at a rate sufficiently low so that the temperature did not increase above 10 C.]. Once the addition complete, the reaction mixture was stirred at ice bath temperature for about 8 hours and then slowly allowed to come to room temperature overnight. Reaction mixture was filtered to remove the light yellow insoluble solids and then the filtrate was kept in the refrigerator for 1 h. Based on the analytical HPLC the crude sulforaphane was 95% pure.

(17) This material was used for the complexation step (Example 2) without any further workup/purification.

(18) Summary of Three Repetitions

(19) TABLE-US-00001 Lot Reaction size Purity by HPLC (crude) Observations 1 501 g 95% H.sub.2O.sub.2 was added at <2 C. 2 500 g 95.6%. H.sub.2O.sub.2 was added at <4 C. 3 500 g 95.4% H.sub.2O.sub.2 was added at <2 C.

(20) All three hatches were conducted at the same reaction scale and the reactions proceeded in similar way in terms of reaction time and product purity.

(21) .sup.1HNMR (CDCl.sub.3, 400 MHz); 1.90 (m, 4H), 2.58 (s, 3H), 2.75 (m, 2H), 3.60 (t, 2H).

(22) .sup.13CNMR (CDCl.sub.3, 100 MHz); 130.2, 53.4, 44.5, 38.5, 29.5, 20.1

Example 2Preparation of Sulforaphane-Cyclodextrin Complex

(23) ##STR00012##

(24) -Cyclodextrin (Wacker CAVAMAX W6 Food Grade, 3015 g, 3.1 moles, 1 equivalent) was dissolved in distilled water (8 L) by heating up to 55 C. under nitrogen atmosphere. The homogeneous solution was cooled down to 25 C. using an ice-water bath and then degassed for 20 min by purging nitrogen. After degassing, it turned into a foggy solution. Aqueous solution of sulforaphane was removed from the refrigerator (see previous step) and then added to the above foggy -cyclodextrin solution at once. At this stage reaction temperature was 18 C., and continued stirring at room temperature overnight (16 h). The heterogeneous reaction mixture was cooled down to 1-2 C. using ice-methanol bath and stirred for 3 hr at that temperature. The precipitated white solid was filtered and dried overnight under high vacuum at room temperature by covering the filter funnel with a latex sheet. The white filter cake was transferred into a 10-L rotovap flask and dried further at room temperature under a high vacuum to afford 2,802 g of complex (98.7% pure by HPLC, 78.5% yield).

(25) Summary of Three Repetitions

(26) TABLE-US-00002 Lot Reaction size** Purity by HPLC Yield* 1 550.8 g 98.5% 78.5% 2 549.7 g 98.6% 76.9% 3 549.7 g 98.7% 73.2% *overall yield in last two steps, **based on the 100% conversion in the previous step.

(27) All three batches were conducted at almost same scale and the reactions proceeded in similar way in terms of reaction time, yield, product purity and percentage loading of sulforaphane on -cyclodextrin.

(28) .sup.1HNMR (D.sub.2O, 400 MHz); 1.99 (br, 4H), 2.73 (s, 3H), 2.98 (br, 2H), 3.60 (m, 12H), 3.70 (br, 2H), 3.92 (m, 24H), 5.11 (d, 6H).

(29) .sup.13CNMR (D.sub.2O, 100 MHz); 130.05, 101.82, 81.40, 74.05, 71.98, 71.84, 60.34, 52.02, 44.94, 37.03, 29.29, 20.08.

Example 3Preparation of Sulforaphane (1-isothiocyanato-4-methylsulfinylbutane) with Different Catalysts and Reaction Conditions

(30) General Procedure:

(31) ##STR00013##

(32) A multi-neck round bottom flask equipped with an overhead stirrer, a temperature probe and an addition funnel was set-up with a positive flow of N.sub.2. Acid catalyst (0.001 to 0.01 equivalents) was dissolved in solvent (water, acetonitrile, acetone etc.) and degassed over 30 minutes by purging with nitrogen. To the above solution was added 1 equivalent of thioether starting material (2) and degassed again at 0-5 C. over 30 minutes. To this biphasic reaction mixture was added 1 equivalent of oxidizing agent (H.sub.2O.sub.2, m-CBPA etc.) slowly while maintaining temperature between 0-10 C. Once the addition complete, the reaction mixture was stirred at ice bath temperature for about 8-24 hours and then slowly allowed to come to room temperature overnight. Reaction mixture was filtered to remove the insoluble solids and then the filtrate was kept in the refrigerator or used immediately in the following step. Based on the analytical HPLC the crude sulforaphane was 95% pure. This material was used for the complexation step without any further purification.

(33) The oxidation of compound 2 into sulforaphane in various solvents and/or in the presence of different catalysts/acids are shown in the Table below, along with the reaction conditions.

(34) TABLE-US-00003 H.sub.2O.sub.2/H.sub.2O, H.sub.2O.sub.2/H.sub.2O, H.sub.2O.sub.2/H.sub.2O, H.sub.2O.sub.2/H.sub.2O, H.sub.2O.sub.2/H.sub.2O, H.sub.2O.sub.2/H.sub.2O, H.sub.2O.sub.2/H.sub.2O, 0.01 eq 1.0 eq 0.05 eq 0.1% Reaction Acetone, Acetonitrile 0.1 eq -CD -CD, 0 C. -CD, 0 C. AcOH Fuller's Conditions 0 C. to RT 0 C. to RT 0 C. to RT to RT to RT 0 C. to RT Earth Purity by 95.6% 96.8% 96% 98% 77% 98.8% 98% HPLC (3.1% SM) (0.7% SM) (1% SM) (0.5% SM) (21% SM) (0.06% SM) (0.06% SM) Yield Not Not isolated Not isolated 82%* Not isolated Not isolated Not isolated isolated Final 98% purity *Isolated yield.

(35) Based on the above results, all of the listed catalysts produced similar results, but cyclodextrin is preferred since it is used in the next complexation step to stabilize the sulforaphane.

(36) Only trace amount of sulfonyl impurity (Erysolin) was detected by HPLC.