Vitamin a-acid esters of {60 -tocopherol and preparation thereof
United States Patent [191 Fukawa et al.  
 [451 Apr. 15, 1975 [73] Assignee: Nisshin Flour Milling Co., Ltd.,  
  Tokyo, Japan [22] Filed: May 17, 1972 [21] Appl. No.: 254,046  
 [52] US. Cl. 260/240 II; 260/345.5; 424/284 [51] Int. Cl C07d 7/22 [58] Field of Search 260/240 H, 345.5  
 [56] References Cited UNITED STATES PATENTS 2,231,125 2/1941 Karrer 260/345.5 3,151,127 9/1964 Spanel 260/345.5 X  
 Primary Examiner-John M. Ford Attorney, Agent, or Firm-Wenderoth, Lind and Ponack [5 7] ABSTRACT This invention relates to vitamin A-acid esters of an a-tocopherol prepared by subjecting an a-tocopherol to ester condensation with a vitamin A-acid or an active substituted compound thereof such as an acid chloride to form the ester bond.  
 &#39;3 Claims, No Drawings VITAMIN A-ACID ESTERS OF a-TOCOPIIEROL AND PREPARATION THEREOF BACKGROUND OF THE INVENTION It is already well known that a-tocopherol stabilizes vitamin A in a living body and enhances the strength of its action. Recently. it is emphasized that they have a cooperative action against the destruction of epithelial tissues, for example. in the lungs to death. Further. it is discovered that lesions due to excess vitamin A can be prevented by the coexistence of a-tocopherol.  
  On the other hand. vitamin A-acid which is an active intermediate in the pathway to vitamin A action is biosynthesized from vitamin A alcohol in a living body. Namely, it is clarified that the functions of vitamin A to the growth-stimulation, protein metabolism, stabilization of ephithelial tissues, etc., take place through vitamin A-acid. Although vitamin A-acid may be considered to therefore be an active form of vitamin A, it has such a defect that it is liable to give rise to hypervitaminosis.  
  From the above-mentioned standpoint, an attempt was made to synthesize the vitamin A-acid esters of a-tocopherol and the obtained vitamin A-acid esters of 7 a-tocopherol were tested by animal experiments to investigate their pharmacological effect. As a result, it was-discovered that the present substances have effects of a-tocopherol and vitamin A-acid, and moreover have a more excellent pharmacological effect, obviating the defects which will be developed when they are present independently.  
 SUMMARY OF THE INVENTION This invention relates to vitamin A-acid esters of an a-tocopherol and their preparation method comprising subjecting a tocopherol to the ester condensation with a vitamin A-acid or an active substituted compound thereof such as an acid chloride to form the ester bond.  
  The a-tocopherol mentioned here refers to DL-atocopherol, D-a-tocopherol and natural mixed tocopherol containing D-a-tocopherol, while the vitamin A- acid mentioned here refers to retinoic acid. particularly all-trans-retinoic acid. The tocopheryl retinoate of the present invention has the following general formula:  
 CH CH (Hi CH CIEB l i p %\o/i\ a-Tocopheryl retinoate PREFERRED EMBODIMENTS OF THE INVENTIVE METHOD As for the synthesis process, there are several known ester-bond formation processes, for example, the direct condensation of a-tocopherol with vitamin A-acid over a dehydration catalyst such as dicyclohexylcarbodiimide or trifluoroacetic acid anhydride. or the condensation with vitamin A-acid in the form of an acid chloride thereof or an active ester thereof such as the p-toluensulfonic acid ester. However. in order to retain the steric structure of the conjugated double bonds of vitamin A-acid and prevent the isomerization and cyclization reaction, it is desirable to employ as mild reaction conditions as possible. From this point of view. the esterification reaction over trifluoroacetic acid anhydride is most preferable. Further. in case an acid halide-process is employed. it is necessary that vitamin A-acid or an alkali metal salt thereof is allowed to react with oxalyl chloride to produce the acid chloride. which is then subjected to the esterification in the presence of a base such as pyridine.  
  If vitamin A-acid esters of a-tocopherol obtained in such processes are purified by adsorption chromatography or molecular distillation, it easily becomes so pure that it can be used as a medicine.  
 EXAMPLE 1 Ten grams of trifluoroacetic acid anhydride dissolved in absolute dioxane was added dropwise below 10C while stirring into 9g retinoic acid and 14g DL-atocopherol dissolved in ml absolute dioxane. After the dropwise addition, the mixture was allowed to stand for 3 hours at 40C and the reaction product was extracted with ether, and washed with water, dilute alkali and water successively. The other layer was dehydrated with anhydrous sodium sulfate, then the solvent was re- I moved by distillation under a reduced pressure to obtain 22g of a light yellow oily residue. The residue was adsorption-chromatographed over silica gel and the main elution band showing yellow in color which was eluted with 2 percent ethyl ether-containing hexane was collected and solvent of the eluent was removed to obtain 15g of light yellow oily DL-a-tocopheryl retinoate. The DL-a-tocopheryl retinoate thus obtained was a single substance in thin layer chromatography, and the assignments made from its infrared absorption spectrum, nuclear magnetic resonance spectrum and ultraviolet absorption spectrum are consistent with the molecular structure. The actual values found in elemental analysis agreed with the theoretical values.  
  Thin layer chromatograph on a silica gel plate Rf value 0.57 n-hexane diethyl ether methanol (65:30:5)  
 0.68 benzene chloroform (4:1)  
  Reversed phase paper chromatograph on 5 percent n-paraffin impregnated paper Rf value: 0.28 (paraffin-saturated dimethylformamide) Elemental Analysis Found Theoretical (CM-I o Continued Ultraviolet Absorption Spectrum A max 365nm E,,.,,,&#34;&#39; 642 (ethanol) A shoulder 298nm E...,,,&#34;&#39; I36 (ethanol) A min 255nm (ethanol) EXAMPLE 2 Eleven grams of retinoic acid was dissolved under cooling in 25g of a 2N potassium hydroxide aqueous solution, and freeze-drying was carried out to produce potassium retioatt which was then suspended retioate 200ml of benzene. A trace of pyridine was added into the suspension, then ml of oxalyl chloride was added dropwise with caution while stirring at 0C in a stream of nitrogen. The reaction was allowed to take place for min at 0C and further for I hour more at room temperature. The reaction mixture was concentrated below 5C under a reduced pressure. The residue was dissolved in ethylene dichloride and added dropwise in a period of about 5 min. into a solution of g DL-atocopherol and 20ml pyridine dissolved in 200ml ethylene dichloride. After that, the mixture was allowed to stand for a night at room temperature, then it was poured into ice-water. extracted with ether and washed with water, dilute hydrochloric acid, dilute alkali and water successively. The ether layer was dried with anhydrous sodium sulfate, then the solvent was removed by distillation under a reduced pressure. The residue was purified by chromatography in the same way as in Example Ito obtain 12g of thin layer chromatographically and reversed phase chromatographically single DL-a-tocopheryl retinoate/ EXAMPLE 3 Trifluoroacetic acid anhydride (2.3g) dissolved in l0ml dioxane was added dropwise in a period of 15 minutes at 0C while stirring in a stream of nitrogen into 3.5g retinoic acid and 4.3g D-a-tocopherol (obtained by subjecting natural mixed tocopherol to methylation, isolating it as D-a-tocopheryl hemisuccinate, then hydrolyzing it) dissolved in ml dioxane. The temperature was then increased to C at which the reaction was allowed to take place for 4 hours. and the reaction product was then poured into ice-water and washed with water, dilute alkali and water. The ether layer was dehydrated with anhydrous sodium sulfate and the solvent was removed under a reduced pressure to obtain a residue, which was then purified by chromatography using 80g of silica gel in the same way as in Example I to obtain 4.3g of thin layer chromatographically and reversed phase chromatographically single D-a-tocopheryl retinoate.  
  Its Rf values in chromatography and physicochemical properties were completely the same as those of DL-a-tocopheryl retinoate and the two compounds did not separate in the mixed chromatography.  
  Pharmacological properties of DL-a-tocopheryl retinoate shall be stated hereunder:  
 1. Acute Toxicity Studies on acute toxicity of DL-a-tocopheryl retinoate and related compounds were carried out. The trials were conducted with ICR mice, weighing 25-30g, by oral and intravenous administration. Each compound was given to one group of 6 animals. The observations were continued for 7 days. Lichfield and Wilcoxon method was used for the calculation of LD50. The compound was synthesized through the condensation of DL-atocopherol and retinoic acid.  
 TABLE 1 Acute Toxicity ol- Retinyl Acetate. Retinoic Acid. and DL-u-toeopheryl retinoate in mice Treatment Route of LD50 Administration (mg/Kg) Retinyl Acetate Intravenous 432 Retinoic Acid Intravenous 92 Retinoic Acid Oral 780 Toco. Retinoate Intravenous above I.000 Toco. Retinoate ()ral ahove 2.000  
  As is shown in Table I. DL-a-tocopheryl retinoate shows very low acute toxicity in mice by oral and intravenous administration. It is very interesting that DL-atocopherol plays a very important role for the diminution of the toxicity of retinoic acid.  
 2. Wound Healing K. H. Lee*&#34; reported that retinoic acid and its related compounds promote skin wound healing in rats. K. H. Lee. J. Pharm. Sci.. 57. I042 I968) K. H. Lee and T. (3. Tong. ihid. 58. 773 (1909 The experiment was carried out to find the efficacy of DL-oz-tocopheryl retinoate by the revised method of Lees.  
  Sprague-Dawley male rats were anesthetized with ethyl ether. The hair on the back was depilated with an electric clipper. One incision, 6cm in length, was made through the skin and cutaneous muscles, at a distance about l.5cm from the midline on each side. The incisions were closed with metal clips. The wounds were left undressed. Retinyl acetate, DL-a-tocopheryl retinoate. suspended in non-ionic surfactant and retinoic acid, dissolved in a minimum amount of NaOH solution, were injected into rats daily for 5 days. The dosage level for each compound was 5.16mg per kg per day.  
 . HCO-60. polyoxyethylene-hydrogenated caster oil. made by Nikko Chemical Co.. Tokyo. was used.  
 TABLE II Effect of Retinyl Acetate. Retinoic Acid. and DL-a-Tocopheryl Retinoate on Skin Wound Healing in Rats. No. of Mean Tensile Percent Group Ani- Drugs Applied Strength (g/cm) Control mals I 8 Saline I807 l8.0 I00.0 ll 7 HCO-60 164.2 l3.8 90.) III 8 Retinyl Acetate I60.I I 9.0 88.6 IV 8 Retinoic Acid I803 9.l 99.8 V 8 Toco. Retinoate 204.2 l5.6 l I3.0  
 3. Granuloma Formation For the evaluation of granuloma formation, we modi- CH3 CH3 CH3 fied slightly the method of cotton pellet by Winter, et al.  
  Two acetate fiber disks were implanted symmetrically at subcutaneous of abdominal site on Wister male rats, weighing ISO-180g under ethyl ether anesthesia. Drugs. prepared in the same way as for the wound healing tests. were injected intraperitoneally during 5 days after operation. On the 6th day, granulomas were carefully separated and dried at 56C for 6 hours in an electric dryer. After drying the disks were weighed.  
 The results obtained are summarized in Table 1H.  
 TABLE III 1 CO&#39;O W 6 What is claimed is: l. A compound of the formula Effect of R.A., T.R. and V. E acetate on Granuloma Formation in Rats.  
 Drugs V.E acetate R.A. T.R.  
 Granuloma Treated 22.6 i 2.3mg 31.8 i 3.0mg 30.1 4.1mg Weight Control 21.5 i 2.l 26.9 i 5.3 23.1 3.7 Difference 1.0 i 1.6 5.5 i 3.0 6.9 t 2.9 Control Percent 105.1% l2037r- 130.3% lmpregnated Dose 2.7 mg 2.0 mg 4.7 mg No. of Animals 8 8 8 R.A.: Rctinoic Acid. T.R.: DL-a-Tocopheryl Retinoatc Granuloma: Induced by Acetate fiber disk Retinoic acid and DL-a-tocopheryl retinoate appear 2: to strengthen granuloma formation. 40  
  UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,878,202 Dated April 15, 1975 Inventor(5) Hideaki Fukawa and Kenichi &#39;Ianaka It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:  
 In the heading of the Patent, in the space provided for priority information, insert the following:  
 - Foreign Application Priority Data May 17, 1971 Japan a 46-32450 Signed and Scaled this Twenty-seventh D a y of November 1979 [SEAL] finest:  
 LUTRELLE F. PARKER RUTH C. MASON Arresting 0 Acting Commissioner ofPatellsud Trademarks