PROCESS FOR REGIOSELECTIVE PREPARATION OF

GLYCEROL DERIVATIVE AND INTERMEDIATE THEREFOR Technical Field [1] This invention relates to a process for regioselective preparation of a glycerol derivative and an intermediate therefor, and more specifically to a process for re¬ gioselective preparation of l-palmitoyl-2-linoleoyl-3-acetylglycerol (octadeca-9,12-dienoic acid-l-acetoxymethyl-2-hexadecanoyloxy ethyl ester: PLA) of the following Formula 1 and an intermediate therefor. [2] [Formula 1] [3]

Background Art [4] PLA of Formula 1 is an ingredient of a deer antler, and is known as having activities for proliferation of hematopoietic stem cells and megakaryocytes. PLA can be separated from the chloroform extracts of the deer antler (Korean Patent No. 0283010), or alternatively can be prepared by the following 2 methods (Korean Patent Application No. 2000-0045168). The first method includes the steps of obtaining reaction products by the reaction of glycerol and palmitic acid, separating 1-palmitoylglycerol by using a column from the reaction products, and successively carrying esterification reactions for the separated 1-palmitoylglycerol to obtain PLA. However, this method is not a regioselecive process, and it has demerits in that it requires separation and purification steps using column-chromatography after each reaction step, and the yield is not desirable (about 3.21% from glycerol). Furthermore, the reaction should be carried out at a low temperature of about 0°C, and expensive 4-dimethylamino pyridine(DMAP) should be used in the same equivalents for the reaction of glycerol(starting material) and palmitic acid, and for the reaction of l-palmitoyl-3-acetyl glycerol(intermediate) and linoleic acid, which makes it difficult to produce PLA at low price and in large amounts. The second method for preparing PLA utilizes an acetolysis reaction of phosphatidyl choline. The yield of the second method is relatively desirable (about 74.5%), but expensive phosphatidyl choline should be used in large amounts for this method. Therefore, it is also difficult to produce PLA at low price and in large amounts. [5] Disclosure of Invention Technical Problem [6] Accordingly, it is an object of the present invention to provide a process for re- gioselective preparation of a glycerol derivative, which does not require separation or purification steps using column-chromatography and is capable of producing PLA in high yield and high purity. [7] It is other object of the present invention to provide a process for regioselective preparation of a glycerol derivative, which can produce PLA at room temperature without or with only small amount of expensive 4-dimethylaminopyridine, and prepare PLA at low price and in large amounts. [8] It is another object of the present invention to provide a process for regioselective preparation of a glycerol derivative which can use various glycerol derivatives as a starting material and prepare optically active PLA. [9] It is yet another object of the present invention to provide a novel intermediate for the regioselective preparation of a glycerol derivative. [10] Technical Solution [11] To achieve these objects, this invention provides a process for regioselective preparation of a glycerol derivative which includes the steps of obtaining (2-alkoxy-2-methyl-[l,3]-dioxolane-4-yl)methanol of the following Formula 2 by reacting glycerol and trialkylorthoacetate; obtaining 1 -acetyl glycerol of the following Formula 3 by hydrolyzing (2-alkoxy-2-methyl-[l,3]-dioxolane-4-yl)methanol; obtaining l-palmitoyl-3-acetyl glycerol of the following Formula 4 by reacting 1 -acetyl glycerol and palmitic acid derivative; and reacting l-palmitoyl-3-acetyl glycerol and linoleic acid derivative to produce l-palmitoyl-2-linoleoyl-3-acetylglycerol. [12] [Formula 2] [13] -OH -CX ^OR 1 LO^CH3 [14] [Formula 3] [15] -OH -OH

H3C [16] [Formula 4] [17]

[18] In Formula 2, R is a lower alkyl group having 1 to 5 carbon atoms. [19] [20] This invention also provides a process for regioselective preparation of PLA which includes the steps of obtaining l-palmitoyl-3-acetyl glycerol of Formula 4 by hy- drolyzing palmitic acid (2-alkoxy-2-methyl-[l,3]dioxolane-4-yl)methyl ester of the following Formula 5; and reacting l-palmitoyl-3-acetyl glycerol and linoleic acid derivative. [21] [Formula 5] [22]

E C XH, [23] In Formula 5, R is a lower alkyl group of 1 to 5 carbon atoms. [24] [25] The present invention further provides an intermediate of the following Formula 6 for preparation of a glycerol derivative. [26] [Formula 6] [27] pOR2 -0^ .OR 1 Q^CH3 [28] In Formula 6, R is a lower alkyl group of 1 to 5 carbon atoms and R is a hydrogen or a lower alkyl group of 1 to 20 carbon atoms. [29] Mode for the Invention [30] A more complete appreciation of the invention, and many of the attendant advantages thereof, will be better appreciated by reference to the following detailed de¬ scription. [31] A process for regioselective preparation of l-palmitoyl-2-linoleoyl-3-acetylglycerol (PLA) of Formula 1 according to the present invention uses glycerol as a starting material, and the exemplary process is shown in the following Reaction 1. [32] [Reaction 1] [33] -O H -O H I— OH I O H ~ ON Z O R 1 ^OH 1— O. L-O H L- OΛ CH, ">=o H3CT glycerol

-E-

[34] As shown in Reaction 1, in order to prepare l-palmitoyl-2-linoleoyl-3-acetylglycerol regioselectively by using glycerol as a starting material, firstly, the reaction of glycerol and trialkylorthoacetate is carried out to prepare (2-alkoxy-2-methyl-[l,3]-dioxolane-4-yl)methanol of Formula 2. The step can be carried out by reacting 1 equivalent of glycerol with 1 to 1.5 equivalents of tri¬ alkylorthoacetate with stirring in the presence of 5 to 10ml of an organic solvent with respect to Ig of glycerol(5 to 10 ml/g) and, if desired, in the presence of 0.5 to lmol of an acid catalyst with respect to lOOmol of glycerol, at 0 to 30°C, preferably about 5°C for 1 to 3 hours, for example about 2 hours. The alkyl group of trialkylorthoacetate can be a lower alkyl group of 1 to 5 carbon atoms, and the preferable example of trialky¬ lorthoacetate is trimethylorthoacetate or triethylorthoacetate, and the more preferable example is trimethylorthoacetate. If the amount of trialkylorthoacetate is less than 1 equivalent, glycerol may not be reacted completely, and if the amount of trialky¬ lorthoacetate is more than 1.5 equivalents, it is economically undesirable without any special advantages and byproducts may be formed by a side reaction on the other primary hydroxyl group. As the organic solvent for the reaction, any conventional nonpolar aprotic organic solvent, preferably methylene chloride, can be used. If the amount of the organic solvent is less than 5ml with respect to Ig of glycerol, the reaction may not be carried out completely and the reaction rate may become slow due to alcohol formed during the reaction. If the amount of the organic solvent is more than 10ml with respect to Ig of glycerol, it is economically undesirable without any special advantages. The acid catalyst is used to increase the reaction rate. As the acid catalyst, any conventional acid catalyst, preferably pyridinium p-toluenesulfonate(PPTS) can be used. If the amount of the acid catalyst is less than 0.5mol with respect to lOOmol of glycerol, the reaction rate may become slow, and if the amount of the acid catalyst is more than lmol, it is economically undesirable without further increase in the reaction rate. Also, if the reaction temperature is less than 0°C, the reaction rate may become slow, and if the reaction temperature is more than 30°C, byproducts may be formed due to side reactions. If the reaction time is too short, the reaction may not be carried out completely, and if the reaction time is too long, the total reaction time is prolonged without any special advantages. [35] [36] Then, the produced (2-alkoxy-2-methyl-[l,3]-dioxolane-4-yl)methanol is hydrolyzed to prepare 1 -acetyl glycerol(AG) of Formula 3. This step can be carried out by reacting 1 equivalent of (2-alkoxy-2-methyl-[l,3]-dioxolane-4-yl)methanol with 1 to 3 equivalents of water, preferably 2 to 3 equivalents of water, at 0 to 30°C, preferably about 5°C, for 30 minutes to 2 hours, preferably about 1 hour. If the amount of water is less than 1 equivalent, the reaction may not be carried out completely, and if the amount of water is more than 3 equivalents, the reaction yield may decrease without any special advantages. If the reaction temperature is less than 0°C, the rate of hydrolysis may become slow, and if the reaction temperature is more than 30°C, byproducts may be formed due to a side reaction. If the reaction time is too short, the reaction may not be carried out completely, and if the reaction time is too long, the total reaction time is just prolonged without any special advantages. The prepared 1 -acetyl glycerol is an important intermediate for preparing PLA, and is obtained by regioselectively introducing an ester group on one of the two primary hydroxyl groups of glycerol. Glycerol has two primary hydroxyl groups having high reactivities and one secondary hydroxyl group having a low reactivity. Accordingly it is important to introduce two different ester groups regioselectively on the two primary hydroxyl groups in order to introduce three different ester groups regioselectively on the three hydroxyl groups. The 1 -acetyl glycerol regioselectively obtained according to this invention has a high purity so that it can be used in the step of preparing l-palmitoyl-3-acetyl glycerol without any additional purification. [37] [38] Then, the step of obtaining l-palmitoyl-3-acetyl glycerol of Formula 4 by reacting the produced 1 -acetyl glycerol with palmitic acid derivative is carried out. This step can be carried out by reacting 1 equivalent of 1 -acetyl glycerol and 1 to 1.5 equivalents of palmitic acid derivative with stirring in the presence of an organic base and, if desired, in the presence of an organic solvent at 0 to 20°C, preferably about 5°C, for 1 to 3 hours, preferably about 2 hours. Examples of palmitic acid derivative include palmitic acid, palmitoyl halide, and so on. When palmitic acid is used, the reaction efficiency can be improved by adding a water-removing agent such as dicyclohexyl- carbodiimide(DCC) in the presence of 4-N,N-dimethylaminopyridine(DMAP). However, it is preferable to use palmitoyl chloride because the amount of impurities and byproducts may decrease, the reaction rate may increase, and the reaction yield may also increase when using palmitoyl chloride. If the amount of palmitic acid derivative is less than 1 equivalent with respect to 1 equivalent of 1 -acetyl glycerol, 1 -acetyl glycerol may not be reacted completely, and if the amount thereof is more than 1.5 equivalents, it is economically undesirable without any special advantages and the amount of impurities may increase. As the organic base, any conventional organic base can be used without limitation. Preferably, pyridine or triethylamine, more preferably pyridine, which is a weak base, can be used as the organic base. The regios- electivity is improved and the amount of byproducts decreases when using pyridine as the organic base. When the organic base is used together with the organic solvent, the amount of the organic base is preferably 3 to 10 equivalents with respect to 1 equivalent of 1 -acetyl glycerol. When the organic base is used without the organic solvent, the preferable amount of the organic base is 6 to 30 equivalents considering that the solubilities of the cooled palmitic acid derivative and the produced salts, which are formed during the reaction, are small and the stirring of the reaction mixture is difficult. The amount of the organic base is 2 to 3 times as much as those in case of using the organic solvent together with the organic solvent. As the organic solvent, any conventional nonpolar aprotic organic solvent, preferably methylene chloride, can be used without limitation. The amount of the organic solvent is preferably 5 to 10ml with respect to Ig of 1 -acetyl glycerol. If the amount thereof is less than 5ml, the reaction mixture cannot be stirred adequately due to the salts formed during the reaction, and if the amount thereof is more than 10ml, there is no special advantage. If the reaction temperature is less than 0°C, the reaction rate may become slow, and if the reaction temperature is more than 20°C, byproducts may be formed due to side reactions. If the reaction time is too short, the reaction may not be carried out completely, and if the reaction time is too long, the total reaction time is just prolonged without any special advantages. [39] [40] If necessary, the possible impurities exist in the reaction solution of the produced l-palmitoyl-3-acetyl glycerol, for example, palmitic acid, l-acetyl-2-palmitoyl glycerol and l-palmitoyl-2-pamitoyl-3-acetyl glycerol, can be removed by the following processes. Palmitic acid can be removed by adding 1 to 2 equivalents of calcium hydroxide(Ca(OH) ) with respect to 1 equivalent of l-palmitoyl-3-acetyl glycerol into the reaction solution at room temperature, stirring the reaction solution for 1 hour, and filtering out the insoluble calcium salts formed during stirring, l-acetyl-2-palmitoyl glycerol and l-palmitoyl-2-pamitoyl-3-acetyl glycerol can be removed by adding 90% methanol aqueous solution into the reaction solution at 15 to 25°C, stirring the reaction solution for about 1 hour, and filtering out the precipitates formed during stirring. This is based on the fact that the solubilities of the impurities with respect to 90% methanol aqueous solution are lower than that of l-palmitoyl-3-acetyl glycerol. The amount of 90% methanol (MeOH) aqueous solution is preferably 5 to 10ml with respect to Ig of l-palmitoyl-3-acetyl glycerol. If the dissolving temperature is less than 15°C, l-palmitoyl-3-acetyl glycerol can also be crystallized and precipitated, and the yield of the process can decrease. If the dissolving temperature is more than 25°C, the impurities may remain in the dissolved state in the reaction solution, and the purity of PLA may decrease. If the dissolving time is less than 1 hour, the impurities may not be completely precipitated, and if the dissolving time is more than 1 hour, the purification step is just prolonged without any special advantages. [41] [42] Then, as shown in Reaction 1, the step of obtaining PLA of Formula 1 by reacting l-palmitoyl-3-acetyl glycerol and linoleic acid derivative is carried out. The reaction can be carried out in the presence of an organic solvent and, if desired, in the presence of a catalyst. The reaction can be carried out by reacting 1 equivalent of l-palmitoyl-3-acetyl glycerol and 1 to 1.05 equivalents of linoleic acid derivative and, if desired, 1 to 1.1 equivalents of dicyclohexylcarbodiimide at 0 to 30°C, for example at room temperature, for 3 to 5 hours, preferably about 4 hours. Examples of linoleic acid derivative include linoleic acid, linoleic active ester, linoleic active amide, linoleoyl chloride and so on, and the preferable example is linoleic acid. If the amount of linoleic acid derivative is less than 1 equivalent, l-palmitoyl-3-acetyl glycerol may not be reacted completely, and if the amount thereof is more than 1.05 equivalents, it is economically undesirable without any special advantages and the unreacted linoleic acid derivative may remain as impurities. [43] [44] Dicyclohexylcarbodiimide improves the reaction yield, the reaction rate, and the purity and color of PLA by reacting with and removing the byproducts formed during the reaction of l-palmitoyl-3-acetyl glycerol and linoleic acid derivative. If the amount of dicyclohexylcarbodiimide is less than 1 equivalent, the reaction may not be carried out completely and the reaction yield may be lower, and if the amount thereof is more than 1.1 equivalents, it remains as impurities in the reaction solution and the purity of PLA may decrease. As the organic solvent, conventional organic solvents can be used. Preferable examples of the organic solvent include hexane, cyclohexane, ethyl acetate, methylene chloride, tetrahydrofuran(THF) and so on. When linoleic acid is used as the reactant, it is preferable to use hexane, such as n-hexane and cyclohexane, as the organic solvent. This is because that the solubility of dicyclohexylurea formed by the reaction of dicyclohexylcarbodiimide and water, which is formed during the reaction and the solubility of PLA are greatly different from each other in hexane or cy¬ clohexane solvent. The amount of the organic solvent can be varied within a range that the reaction solution can be stirred, and the preferable amount is 5 to 10ml with respect to Ig of l-palmitoyl-3-acetyl glycerol. The catalyst used in the reaction of l-palmitoyl-3-acetyl glycerol and linoleic acid is to improve the poor reactivity and the low reaction rate of the secondary hydroxyl group on which an ester group is introduced. The secondary hydroxyl group has poor reactivity and the low reaction rate due to a steric hindrance. A preferable catalyst is 4-dimethylaminopyridine. The preferable amount of 4-dimethylaminopyridine is 0.5 to lmol with respect to lOOmol of l-palmitoyl-3-acetyl glycerol. If the amount is less than 0.5mol, the improvement of the reaction rate is not satisfactory, and if the amount thereof is more than lmol, it is economically undesirable without further improving the reaction rate and the purity of PLA may decrease due to 4-dimethylaminopyridine remained after completion of reaction. If the reaction temperature is less than 0°C, the reaction rate may become slow, and if the reaction temperature is more than 30°C, the color of reaction mixture can be deteriorated. If the reaction time is too short, the reaction may not be carried out completely, and if the reaction time is too long, the reaction step is just prolonged without any special advantages. The step of reacting l-palmitoyl-3-acetyl glycerol with linoleic acid derivative according to this invention has the following merits compared with a conventional method. In the conventional method, the reaction was carried out at 0°C in the presence of a solvent such as dichloromethane, dicyclohexylcarbodiimide of about 6 equivalents with respect to l-palmitoyl-3-acetyl glycerol, and the same equivalents of expensive 4-dimethylaminopyridine. In contrast, the method of this invention is carried out at room temperature in the presence of a solvent such as n- hexane, dicyclohexylcarbodiimide of 1 to 1.1 equivalents, and 1/100 to 1/200 equivalents of 4-dimethylaminopyridine. The produced PLA is in the liquid state at room temperature, and can be separated and purified with a column chromatography, in which a stationary phase can be a silica gel and an eluent can be the mixture of hexane and ethylacetate (hexane : ethylacetate = 18:1 in volume) to obtain PLA in high yield and purity. [45] [46] An alternative method for regioselective preparation of PLA of Formula 1 according to the present invention is shown in the following Reaction 2. [47] [Reaction 2] [49] In Reaction 2, R is a lower alkyl group of 1 to 5 carbon atoms. [50] As shown in Reaction 2, in order to produce l-palmitoyl-2-linoleoyl-3-acetylglycerol, firstly, the step of obtaining l-palmitoyl-3-acetyl glycerol (PAG) by hydrolyzing palmitic acid (2-alkoxy-2-methyl-[l,3]dioxolane-4-yl)methyl ester of Formula 5 is carried out. The hydrolysis can be carried out under the same conditions as described for the hydrolysis in Reaction 1. However, the preferable amount of water is 1 to 5 equivalents, and more preferably 2 to 5 equivalents, with respect to 1 equivalent of the compound of Formula 5. Then, the prepared l-palmitoyl-3-acetyl glycerol is reacted with linoleic acid derivative to produce the target PLA. The reaction of l-palmitoyl-3-acetyl glycerol and linoleic acid derivative can also be carried out under the same conditions as described for the linoleic acid derivative addition reaction in Reaction 1 [51] [52] Palmitic acid (2-alkoxy-2-methyl-[l,3]dioxolane-4-yl)methyl ester of Formula 5 used in Reaction 2 can be prepared by various methods. [53] [54] The first method for preparing palmitic acid (2-alkoxy-2-methyl-[l,3] dioxolane- 4-yl)methyl ester includes the steps of reacting glycerol and trialkylorthoacetate to obtain (2-alkoxy-2-methyl-[l,3]-dioxolane-4-yl)methanol (Formula 2), and reacting the obtained (2-alkoxy-2-methyl-[l,3]-dioxolane-4-yl)methanol with palmitic acid derivative, as shown in the following Reaction 3. [55] [Reaction 3] [56] O. I O H O H O^ v — ' O H R , OyOR 1 I O H - E Ov / O O^ CH, - E

[57] In Reaction 3, R is a lower alkyl group of 1 to 5 carbon atoms. [58] The reaction of glycerol and trialkylorthoacetate and the reaction of introducing palmitic acid derivative can be carried out under the same conditions as described for the reaction of glycerol and trialkylorthoacetate and the reaction of introducing palmitic acid derivative in Reaction 1. Therefore, the species and the amount of the organic base and the solvent for the reactions are same as described for Reaction 1. However, in the step of preparing palmitic acid (2-alkoxy-2-methyl-[l,3] dioxolane- 4-yl)methyl ester of Formula 5, when the organic base is used together with the organic solvent, the amount of the organic base is preferably 3 to 5 equivalents with respect to 1 equivalent of (2-alkoxy-2-methyl-[l,3]-dioxolane-4-yl)methanol, and when the organic base is used without the organic solvent, the amount of the organic base is preferably 6 to 15 equivalents. The yield of preparing 1-palmi toyl- 2-linoleoyl-3-acetylglycerol according to the present invention depends on the regiose- lectivity of the two primary hydroxyl groups having same reactivity in the ester- ification reactions. Accordingly, a process which utilizes Reaction 3, in which palmitic acid derivative reacts with only one primary hydroxyl group of glycerol, in combination with Reaction 2, is more preferable in the reaction yield than a process which utilizes Reaction 1, in which palmitic acid derivative competitively reacts with one primary hydroxyl group and one secondary hydroxyl group. [59] [60] The second method for preparing the compound of Formula 5 uses glycidol (including derivatives thereof) as a starting material, as shown in Reaction 4. [61] [Reaction 4] [62]

C— k

[63] In reaction 4, R is a lower alkyl group of 1 to 5 carbon atoms. [64] As shown in Reaction 4, in order to obtain palmitic acid (2-alkoxy-2-methyl-[l,3] dioxolane-4-yl)methyl ester, firstly, the hydroxyl group of glycidol is reacted with palmitic acid derivative, such as palmitoyl chloride, to prepare palmitic acid oxiranyl methyl ester. The reaction of palmitic acid derivative and glycidol can be carried out in the presence of an organic base such as pyridine and a aprotic nonpolar organic solvent such as dichloromethane. In this reaction, the amount of palmitoyl chloride is preferably 1 to 1.5 equivalents with respect to 1 equivalent of glycidol, and the amount of the organic solvent is preferably 5 to 10ml with respect to Ig of glycidol(5 to 10 ml/ g), and the amount of the organic base is preferably 3 to 5 equivalents with respect to 1 equivalent of glycidol. If the amount of the organic solvent is too small, the reaction mixture may not be stirred adequately, and if the amount thereof is too large, it is eco¬ nomically undesirable without any special advantages. If the amounts of the organic base and palmitoyl derivative are too small, the reaction may not be carried out completely, and if the amounts thereof are too large, it is undesirable because it works as impurities. Alternatively, palmitic acid oxiranyl methyl ester can be obtained by reacting palmitic acid and glycidol in the presence of a water-removing agent such as dicyclohexylcarbodiimide (DCC), or can also be obtained by reacting palmitic acid and epichlorohydrin, which is one of glycerol derivative, in the presence a base such as tributylamine. However, it is preferable to obtain palmitic acid oxiranyl methyl ester by using palmitoyl chloride as a reactant because the reaction can be carried out readily, and the reaction yield and the purity of the product can be improved. [65] [66] Then, the obtained palmitic acid oxiranyl methyl ester is converted into a 1,2-diol compound by the ring opening reaction of an epoxy group to produce 1 -palmitoyl glycerol. The ring opening reaction of epoxy group can be carried out by reacting palmitic acid oxiranyl methyl ester and water in the presence of an organic acid, an organic acid and a catalyst, an organic acid and a base, an oxidizing agent such as CAN(ceric ammonium nitrate), or an inorganic acid such as HClO 4. In the ring opening reaction, the amount of the acid can be used in a catalytic amount. For example, the acid of 1 to 5mol with respect to lOOmol of palmitic acid oxiranyl methyl ester can be used. If the amount of the acid is too small, the reaction rate may become slow, and if the amount thereof is too large, it is economically undesirable without any special advantages. If the organic acid is used as an acid, the regioselectivity of the organic acid ester formed during the reaction is not preferable, and an additional process for hydrolyzing the organic acid may be necessary. Also, if the oxidizing agent such as CAN is used, water should be used as a solvent. In this case, it takes much time for the ring opening reaction of epoxy group due to the low solubility of palmitic acid oxiranyl methyl ester in water. Therefore, it is preferable that the ring opening reaction is carried out by using the inorganic acid such as HClO 4 in the presence of a mixed solvent of water and a water-miscible organic solvent such as tetrahydrofuranCTHF). In this case, a volume ratio of THF : H O is preferably about 2:1 to 5:1, and the amount of the mixed solution is preferably 5 to 10ml with respect to Ig of palmitic acid oxiranyl methyl ester. If the amount of the organic solvent in the mixed solvent is less than the above range, the reaction rate may become slow due to the low solubility of palmitic acid oxiranyl methyl ester, and if the amount thereof is more than the above range, the reaction rate may become slow due to the insufficiency of water. [67] [68] Then, the step of obtaining palmitic acid (2-alkoxy-2-methyl-[l,3] dioxolane- 4-yl)methyl ester of Formula 5 by reacting the obtained 1 -palmitoyl glycerol with tri- alkylorthoacetate. This step can be carried out by reacting 1 equivalent of 1 -palmitoyl glycerol and 1 to 1.5 equivalents of trialkylorthoacetate in the presence of 5 to 10ml of an organic solvent with respect to Ig of 1-palmitoyl glycerol, and, if desired, 0.5 to lmol of an acid catalyst with respect to lOOmol of 1-palmitoyl glycerol. If the amount of trialkylorthoacetate is less than 1 equivalent, glycerol cannot be reacted completely, and if the amount thereof is more than 1.5 equivalents, it is economically undesirable without any special advantages. As the organic solvent, a conventional nonpolar aprotic organic solvent, preferably dichloromethane, can be used. If the amount of the organic solvent is less than 5ml with respect to Ig of glycerol, the reaction may not be carried out completely and the reaction rate may become slow due to alcohol formed during the reaction. If the amount thereof is more than 10ml, it is economically un¬ desirable without any special advantages. The acid catalyst increases the reaction rate. As the acid catalyst, a conventional acid catalyst, preferably pyridinium p- toluenesulfonate(PPTS) can be used. If the amount of the acid catalyst is less than 0.5mol with respect to lOOmol of glycerol, the reaction rate may become slow, and if the amount thereof is more than lmol, it is economically undesirable without any special advantages. [69] [70] The third method for preparing the compound of Formula 5 uses 1,2-isopropylidene glycerol (including derivatives thereof) as a starting material, as shown in Reaction 5. [71] [Reaction 5] [72]

pOH 5 x

[73] In Reaction 5, R is a lower alkyl group of 1 to 5 carbon atoms. [74] As shown in Reaction 5, in order to obtain palmitic acid (2-alkoxy-2-methyl-[l,3] dioxolane-4-yl)methyl ester, firstly, the step of obtaining palmitic acid (2,2-dimethyl-[l,3]dioxolane-4-yl)methyl ester by reacting the hydroxyl group of 1,2-isopropylidene glycerol and palmitic acid derivative such as palmitoyl chloride is carried out. The reaction can be carried out in the presence of an organic base such as pyridine and an aprotic nonpolar organic solvent such as dichloromethane. In the above reaction, the amount of palmitoyl chloride is preferably 1 to 1.5 equivalents with respect to 1 equivalent of 1,2-isopropylidene glycerol, and the preferable amount of the organic solvent is 5 to 10ml with respect to Ig of 1,2-isopropylidene glycerol (5 to 10 ml/g), and the amount of the organic base is preferably 3 to 5 equivalents with respect to 1 equivalent of 1,2-isopropylidene glycerol. If the amount of the organic solvent is too small, the reaction mixture may not be stirred adequately, and if the amount thereof is too large, it is economically undesirable without any special advantages. If the amount of the organic base or the amount of palmitic acid derivative are too small, the reaction may not be carried out completely, and if the amounts thereof are too large, it is undesirable because the organic base and palmitic acid derivative work as impurities. The palmitic acid (2,2-dimethyl-[l,3]dioxolane-4-yl)methyl ester can be obtained by reacting palmitic acid and 1,2-isopropylidene glycerol in the presence of dicyclohexylcarbodiimide(DCC), or can also be obtained by reacting palmitic acid and (2,2-dimethyl-[l,3]dioxolane-4-yl)methyl chloride, which is one of 1,2-isopropylidene glycerol derivatives, in the presence of a base such as K CO and an aprotic and polar solvent such as acetonitrile. However, it is preferable to obtain palmitic acid (2,2-dimethyl-[l,3]dioxolane-4-yl)methyl ester by reacting 1,2-isopropylidene glycerol and palmitoyl chloride considering the reaction process, the reaction yield and the purity of a product. [75] [76] Then, the obtained palmitic acid (2,2-dimethyl-[l,3]dioxolane-4-yl)methyl ester can be converted into 1 -palmitoyl glycerol by a conventional deprotection reaction of acetonide. However, considering the compound is insoluble in water, it is preferable to carry out the deprotection reaction by heating under reflux in the presence of 5 to 10ml of a polar protic organic solvent such as methanol with respect to Ig of (2,2-dimethyl-[l,3]dioxolane-4-yl)methyl ester(5 to 10ml/g), and 0.5 to 5mol of an acid catalyst such as pyridinium p-toluenesulfonate(PPTS) with respect to lOOmol of (2,2-dimethyl-[l,3]dioxolane-4-yl)methyl ester. The 1-palmitoyl glycerol obtained by the deprotection reaction is reacted with trialkylorthoacetate to prepare palmitic acid (2-alkoxy-2-methyl-[l,3]dioxolane-4-yl)methyl ester of Formula 5. [77] [78] The above mentioned reaction can be carried out by using optically active 1,2-isopropylidene glycerol as a starting material to obtain optically active l-palmitoyl-2-linoleoyl-3-acetylglycerol. In order to prepare optically active l-palmitoyl-2-linoleoyl-3-acetylglycerol, the deprotection reaction of palmitic acid (2,2-dimethyl-[l,3]dioxolane-4-yl)methyl ester can be carried out in the presence of 5 to 10 times (volume/weight) of a aprotic nonpolar organic solvent such as dichloromethane with respect to palmitic acid (2,2-dimethyl-[l,3] dioxolane- 4-yl)methyl ester and 3 to 5 equivalents of dimethylboronbromide at the low temperature of -30 to 50°C to prevent a chiral center from being racemized. If the reaction temperature is less than the range, the reaction rate may become slow, and if the reaction temperature is more than the range, a side reaction may occur. If the amount of dimethylboronbromide is less than the range, the reaction may not be carried out completely, and if the amount thereof is more than the range, it is eco¬ nomically undesirable without any special advantages. [79] [80] Yet another method for preparing the compound of Formula 5 uses allyl alcohol (including derivatives thereof) as a starting material. In the process, allyl alcohol or its derivatives is reacted with palmitic acid derivative to prepare palmitic acid allyl ester under the process conditions similar to those described above, and then palmitic acid allyl ester is oxidized to prepare 1-palmitoyl glycerol. 1-palmitoyl glycerol is reacted with trialkylorthoacetate to prepare palmitic acid (2-alkoxy-2-methyl-[l,3] dioxolane- 4-yl)methyl ester of Formula 5. Then l-palmitoyl-3-acetyl glycerol having re- gioselective acetyl group can be prepared according to the process of Reaction 2. [81] [82] The present invention also provides an intermediate of the following Formula 6 for preparing the target glycerol derivative. [83] [Formula 6] [84] I— OR ^ ,2 -CX ^OR 1 L0^CH3 [85] In Formula 6, R is a lower alkyl group of 1 to 5 carbon atoms, and preferably methyl or ethyl group, and R is a hydrogen or an alkyl group of 1 to 20 carbon atoms, and preferably a hydrogen or a palmitoyl group. The intermediate of Formula 6 can be obtained as described above by the reaction of glycerol and trialkylorthoacetate. The intermediate can also be obtained by the hydrolysis, the deprotection reaction, or the oxidization of starting materials such as glycidol, 1,2-isopropylidene glycerol, allyl alcohol and derivatives thereof. Besides, the intermediate of Formula 6 can also be obtained by the reaction of the compound of the Formula 6, wherein R is a hydrogen, and various carboxylic compounds or acyl halide compounds. [86] [87] As described above, the process for regioselective preparation of glycerol derivative according to this invention can prepare PLA by regioselectively introducing palmitoyl group, linoleoyl group, and acetyl group to glycerol or its derivatives, and does not require an additional separation step or an purification step using column chro¬ matography, and can prepare highly pure PLA in high yield. Also, the present invention can prepare PLA regioselectively by using various glycerol derivatives as starting materials and prepare optically active PLA by using optically active glycerol derivatives. Also, according to the present invention, PLA can be prepared at low price and in large amounts because PLA can be prepared without a process using a silica gel column, or the process using the silica gel column can be limited to only one process. [88] [89] Hereinafter, the preferable examples are provided for better understanding of the present invention. However, the present invention is not limited by the following examples. [90] [91] [Example] [92] A. Preparation of 1 -acetyl glycerol [93] 92.09g of glycerol and 153.4ml of trimethylorthoacetate were added into 500ml of methylene chloride(MC). The reaction mixture was cooled to 5°C, 2.52g of pyridinium-p-toluenesulfonate(PPTS) was added thereto, and the reaction was carried out for 2 hours with stirring. After completion of the reaction, 36ml of water was added to the reaction mixture, the reaction mixture was further stirred for 1 hour, and the solvent was removed by distillation under reduced pressure. 200ml of toluene was added to the residue, and the solvent was removed again by distillation under reduced pressure. Then, 1,340ml of methylene chloride and 33.5g of anhydrous magnesium sulfate were added to the residue. The reaction mixture was stirred for 30 minutes, and then filtered. The filtered reaction mixture was used as a reactant of the following step without any additional purification. [94] [95] B-I. Preparation of 1-palmitoyl glycerol using glycidol [96] 74.08g of glycidol was dissolved with 350ml of dichloromethane. 237g of pyridine was added thereto, and the reaction mixture was cooled to 0°C. Then 274.9g of palmitoyl chloride and 275ml of dichloromethane were dropwisely added to the reaction mixture. After completion of the reaction, 1000ml of water was added for an extraction. Then, dichloromethane was removed by distillation under reduced pressure. 1250ml of tetrahydrofuran(THF) and 312.5ml of water were added thereto, and 5ml of 70 weight% HClO 4 diluted with 50ml of water was dropwisely added thereto. The reaction mixture was stirred for 2 hours at room temperature. Then small amount of NaCl was added and an extraction step was carried out by using dichloromethane. The reaction mixture was dehydrated with anhydrous MgSO 4 , and filtered. The remaining solvent was removed by distillation under reduced pressure to obtain 241.6g of the target material (yield: 73.1%). [97] [98] B-2. Preparation of 1-palmitoyl glycerol using racemic 1.2-isopropylidene glycerol [99] 132.16g of racemic 1,2-isopropylidene glycerol was dissolved with 660ml of dichloromethane. 237g of pyridine was added thereto and the reaction mixture was cooled to 0°C. Then 274.9g of palmitoyl chloride and 275ml of dichloromethane were dropwisely added thereto, and the reaction was carried out. After completion of the reaction, 1000ml of water was added for an extraction. Then dichloromethane was removed by distillation under reduced pressure. 1850ml of methanol and 12.5g of pyridinium-p-toluenesulfonate(PPTS) were added thereto, and the reaction mixture was refluxed for 2 hours. After completion of the reaction, the reaction solvent was removed by distillation under reduced pressure, 1000ml of water and small amount of NaCl were added thereto, and an extraction step was carried out with dichloromethane. The reaction mixture was dehydrated with anhydrous MgSO 4 , and filtered. The solvent was removed by distillation under reduced pressure to obtain 281.9g of the target material (yield: 85.3%). [100] [101] B -3. Preparation of 1 -palmitoyl glycerol using optically activefD or L-forrn) 1.2-isopropylidene glycerol [102] 132.16g of optically active 1,2-isopropylidene glycerol was dissolved with 660ml of dichloromethane. 237g of pyridine was added thereto, and the reaction mixture was cooled to 0°C. Then 274.9g of palmitoyl chloride was dropwisely added thereto and the reaction was carried out. After completion of the reaction, 1000ml of water was added for an extraction. The organic layer of the reaction mixture was dehydrated with anhydrous MgSO 4 , and filtered, and the solvent was removed by distillation under reduced pressure. Then 1850ml of dichloromethane was added thereto, the reaction mixture was cooled to -50°C, and 2L of dichloromethane solution including dimethyl boron bromide of 2.5M concentration was added thereto. After completion of the reaction, 2.4L of saturated NaHCO solution was slowly added with stirring. Then, the organic layer was separated, and was dehydrated with anhydrous sodium sulfate, and filtered. The solvent was removed under reduced pressure to obtain 277.9g of the target material (yield: 84.1%). The obtained 1-palmitoyl glycerol was analyzed with H- NMR, and the results and other property are as follows. [103] (1) (R)-enantiomer: [α] = -8.92(c=0.58, EtOH), 1H NMR (250MHz, CDCy:δ 0.88(t, J=7.5Hz, 3H), 1.20-1.35(m,24H), 1.62(m,2H), 2.17(brs,lH), 2.35(t, J=7.5Hz,2H), 2.60(brs,lH), 3.59(dd, J=3.75,11.5Hz,lH), 3.72(dd, J=5.7,ll .3Hz5IH), 3.92(m,lH), 4.18(m,2H) [104] (2) (S)-enantiomer: [α]D=8.90(c=0.58, EtOH), 1K NMR (250MHz, CDCl3): δ 0.88(t, J=7.5Hz, 3H), 1.22-1.35(m,24H), 1.61(m,2H), 2.18(brs,lH), 2.35(t, J=7.5Hz,2H), 2.60(brs,lH), 3.60(dd, J=3.75,11.5Hz,lH), 3.73(dd, J=5.7,ll. 3Hz5IH), 3.94(m,lH), 4.19(m,2H) [105] [106] C-I. Preparation of l-palmitoyl-3-acetyl glyceroKPAG) using 1 -acetyl glycerol [107] 1 -acetyl glycerol obtained from the above process A was dissolved with 670ml of dichloromethane. 407.7ml of pyridine was added thereto, and the reaction mixture was cooled to 0°C. Then, 303.7ml of palmitoyl chloride dissolved with 303.7ml of methylene chloride was dropwisely added to the reaction mixture, and the reaction mixture was stirred for 2 hours at the same temperature. Then 2000ml of water was added for an extraction. 22.2g of Ca(OH) was added into the organic layer of the reaction mixture, and the reaction mixture was stirred for additional 1 hour, and filtered. The organic layer was separated from the filtrate, and the solvent was removed by distillation under reduced pressure. 1,862ml of 90% MeOH aqueous solution was added to the residue. The reaction mixture was cooled to 15°C, stirred for 1 hour, then heated to 25°C, and then filtered. The filtrate was distilled under reduced pressure, and 1,500ml of hexane was added thereto. The reaction mixture was extracted three times with 300ml of 60% MeOH aqueous solution in each time. The hexane layer was dehydrated with MgSO 4 and distilled under reduced pressure to obtain 232.4g of the target material (theoretical amounts: 372.55g, yield: 62.38%). [108] [109] C-2. Preparation of l-palmitoyl-3-acetyl glycerolfPAG*) using glycerol [110] 92.09g of glycerol and 153.4ml of trimethylorthoacetate were added into 500ml of methylene chloride(MC), and the reaction mixture was cooled to 5°C. 2.52g of pyridinium-p-toluenesulfonate(PPTS) was added thereto, and the reaction was carried out for 2 hours with stirring. After completion of the reaction, the solvent was removed by distillation under reduced pressure. 200ml of toluene was added to the residue, and the solvent was removed again by distillation under reduced pressure. Then, 1,340ml of methylene chloride(MC) and 407.7ml of pyridine were added to the residue, and the reaction mixture was cooled to 0°C. Then, 303.7ml of palmitoyl chloride dissolved with 303.7ml of methylene chloride(MC) was slowly added thereto. The reaction mixture was stirred for 2 hours at the same temperature. 74.09g of Ca(OH) and 500ml of H 2 O were added thereto, and the reaction mixture was stirred for 1 hour and filtered. The organic layer was separated from the filtrate, and the solvent was removed by dis¬ tillation under reduced pressure. 1,862ml of 90% MeOH aqueous solution was added to the residue, and the reaction mixture was cooled to 15°C. The reaction mixture was stirred for 1 hour, then heated to 25°C, and filtered. The filtrate was distilled under reduced pressure, and 1,500ml of hexane was added thereto, and the reaction mixture was extracted three times with 300ml of 60% MeOH aqueous solution in each time. Then the hexane layer was dehydrated with MgSO 4 , and distilled under reduced pressure to obtain 325.2g of the target material (theoretical amounts: 372.55g, yield: 87.30%). [111] [112] C-3. Preparation of l-palmitoyl-3-acetyl glyceroKPAG) using 1-palmitoyl glycerol [113] 330.51g of 1-palmitoyl glycerol obtained from the above process B and 153.4ml of trimethylorthoacetate were added into 1650ml of methylene chloride(MC), and the reaction mixture was cooled to 5°C. 2.52g of pyridinium p-toluenesulfonate(PPTS) was added thereto, and the reaction mixture was stirred for 2 hours. After completion of the reaction, 36ml of water was added, the reaction mixture was stirred for 1 hour, and the solvent was removed by distillation under reduced pressure. Then 200ml of toluene was added to the residue and the solvent was removed again by distillation under reduced pressure. Then 1,340ml of methylene chloride(MC) and 33.5g of anhydrous magnesium sulfate were added to the residue, and the reaction mixture was stirred for 30 minutes, and filtered. The solvent was removed by distillation under reduced pressure to obtain 340.5g of the target material (yield: 91.4%). [114] [115] C-4. Preparation of l-palmitoyl-3-acetyl glycerolfPAG*) using optically active 1-palmitoyl glycerol [116] 330.51g of optically active 1-palmitoylglycerol obtained from the above process B- 3, 102.09g of anhydrous acetic acid, 71. Ig of pyridine and 1.22g of 4-dimethylaminopyridine(DMAP) were successively added into 1650ml of ethylacetate. The reaction mixture was stirred for 2 hours at room temperature, and 1000ml of water and small amount of NaCl were added for an extraction. The solvent was removed under reduced pressure, and the remaining mixture was purified with a column chromatography to obtain 218.7g of the target material (yield: 58.7%). The obtained l-palmitoyl-3-acetyl glycerol was analyzed by H-NMR, and the results and other property are as follows. [117] (1) (R)-enantiomer: [α]D=-0.84(c=0.65, EtOH), 1H NMR(250MHz, CDCl3): δ 0.88(t, J=7.5Hz, 3H), 1.17-1.25(m,24H), 1.62(m,2H), 2.07(s,3H), 2.37(t, J= 7.5Hz, 2H), 2.43(d, J=4.25Hz, IH), 4.03-4.22(m,5H) [118] (2) (S)-enantiomer: [α]D=-0.85(c=0.65, EtOH), 1K NMR(250MHz, CDCl3): δ 0.87(t, J=7.5Hz, 3H), 1.18-1.25(m,24H), 1.61(m,2H), 2.08(s,3H), 2.36(t, J=7.5Hz,2H), 2.43(d, J=4.25Hz,lH), 4.01 - 4.22(m,5H) [119] [120] D-I. Preparation of l-palrmtoyl-2-linoleoyl-3-acetylglycerol(PLA*) [121] 232.4g of l-palmitoyl-3-acetyl glycerol obtained from the above process C was added into 1,162ml of hexane, and 183.7g of linoleic acid and 141.57g of dicyclo- hexylcarbodiimide(DCC) were added thereto at room temperature. 0.76g of dimethylamino pyridine(DMAP) was added to the reaction mixture, and the reaction mixture was stirred for 4 hours at the same temperature. Then 5.6 Ig of H O was added thereto, and the reaction mixture was stirred for additional 1 hour and filtered. The filtrate was separated and purified with a column chromatography in which the stationary phase was silica gel Si-60(230 to 400 mesh) and the eluent was the mixture of hexane and ethylacetate(the volume ratio of hexane and ethylacetate is 18: 1) to obtain 345.8g of the target material (theoretical amounts: 395.4g, yield: 87.4%). [122] [123] D-2. Preparation of optically active l-palmitoyl-2-linoleoyl-3-acetylglycerol(PLA) [124] 232.4g of optically active l-palmitoyl-3-acetyl glycerol obtained from the above process C-4 was added into 1,162ml of hexane, and 183.7g of linoleic acid and 141.57g of dicyclohexylcarbodiimide(DCC) were added thereto at room temperature. 0.76g of dimethylamino pyridine(DMAP) was added to the reaction mixture, and the reaction mixture was stirred for 4 hours at the same temperature. Then 5.6 Ig of H O was added thereto, and the reaction mixture was stirred for additional 1 hour and filtered. The filtrate was separated and purified with a column chromatography in which the stationary phase was silica gel Si-60(230 to 400 mesh) and the eluent was the mixture of hexane with ethylacetate(the volume ratio of hexane and ethylacetate is 18:1) to obtain 322.2g of the target material (theoretical amounts: 395.4g, yield: 81.5%). The obtained optically active l-palmitoyl-2-linoleoyl-3-acetylglycerol was analyzed by H-NMR, and the results and other property are as follows. [125] (1) (R)-enantiomer: 1K NMR (250MHz, CDCl3): δ 0.85 - 0.9(m, 6H), 1.20-1.3 l(m,38H), 1.61(m,4H), 2.03(m,4H), 2.07(s,3H), 2.37(t,J=7.5Hz,2H), 2.33(t,J=7.5Hz,2H), 2.77(m,lH), 4.14(dd, J=5.95,11.8Hz,2H), 4.29(ddd, J=4.14, 11.8, 12.8Hz, 2H), 5.26(m,lH) [126] (2) (S)-enantiomer: 1K NMR (250MHz, CDCl3): δ 0.84 - 0.93(m, 6H), 1.21 - 1.31 (m,38H), 1.60(m,4H), 2.02(m,4H), 2.07(s,3H), 2.38(t,J=7.5Hz,2H), 2.33(t,J=7.5Hz,2H), 2.76(m,lH), 4.15(dd, J=5.95,11.8Hz,2H), 4.30(ddd, J=4.14,11.8,12.8Hz,2H), 5.25(m,lH) [127]