Hu Xiangzheng ¹ , Liu Anjun², Wang Lixia², Liu Yanhong¹, Ni Liqin¹
(1.College of Science, Tianjin University of Science and Technology, Tianjin, 300457, China; 2. College of Food Science and Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, China)

Received Nov. 21, 2006.

Abstract Acylating activity of hydroxyl groups in 2' - hydroxyethyl 3a , 7a , 12a - trihydroxy - 5b - cholan - 24 - ate has been determined by the use of methacryloyl chloride, methacryloyl anhydride and methacrylic acid as the acylating agents. The results show: that the primary hydroxyl group has the highest reactivity in all four hydroxyl groups. The relative reactivity of secondary hydroxyl groups on steroids skeleton is various with three kinds of acylating agents.
Ketword 2'- hydroxyethyl 3a , 7a , 12a - trihydroxy - 5b-cholan- 24- ate; acylating; reactivity

1 INTRODUCTION
Bile acids are natural compounds that are synthesized from cholesterol in the liver, as sodium salts of N-acyl derivatives of glycine and taurine and play important physiological roles in the body. They are stored in the gallbladder and released during meals for the digestion of fats and lipids in food and reabsorbed in the intestine to complete the enterohepatic circulation with very little loss. All bile acids possess a steroid skeleton, a carboxylic acid group and different hydroxyl groups, chemical modifications can be made easily on these groups. They are all amphiphilicity molecules and can form micelles and other supramolecular structures in stepwise manner ^{[1, 2, 3]}. Compounds derived from bile acids are expected to be safe and nontoxic when used in biomedical and pharmaceutical fields. In recent years, bile acids have been used in the development of new polymeric materials used in drug delivery system^[4,5], asymmetric synthesis^[6,7], molecular recognition ^[8,9] and polymeric biomaterials^[10-12].
    3a , 7a , 12a - trihydroxy - 5b - cholan - 24 - oic acid, or cholic acid , one of the most commonly occurring bile acids, consists of a steroid skeleton, one carboxylic group and three hydroxyl groups, which exhibit very different reactivity depending on their location on the steroid skeleton ^[13]. Literature work reported that the reactivity of the three hydroxyl groups on steroid skeleton follows the order C3 OH >> C7 OH > C12 OH for esterification^[1,2,14]. In the previous work, the authors used methacryloyl chloride as acylating agent to modify cholic acid derivatives and found the reactivity of secondary hydroxyl groups on steroids backone follows the order C3 OH > C12 OH > C7 OH ^[15]. In this paper, the authors used methacryloyl chloride, methacryloyl anhydride and methacrylic acid as the acylating agents to study acylation activity of hydroxyl groups in 2' - hydroxyethyl 3a , 7a , 12a - trihydroxy - 5b - cholan - 24- ate (1) (Figure 1).

2 EXPERIMENTAL
2.1 Materials and instruments                                             
All the chemicals were of reagent grade and used without further purification. All the anhydrous solvents were freshly purified by standard techniques. IR spectra were record on FTS135-FTIR spectrophotometer (Bio-Rad Instruments) with KBr pellets. NMR spectra were recorded at 23°C on a Bruker AMX- 400 operating at 400.4 MHz for ¹H in CDCl₃ and TMS were used as the solvent and internal standard. Elemental analyses were carried out on Foss Heraeus.
    The chemical structures of 1 and its methacrylate derivatives (2 ~ 6) are shown in Figure 1.

2.2  2'-Hydroxyethyl 3a ,7a ,12a -trihydroxy-5b -cholan-24-ate (1)
The product 1 was synthesized by using a procedure of the one published in the literature ^[15]. m.p. 135-136°C; IR /cm^-1: 3351.7, 2935.8, 2866.5, 1733.7, 1464.0, 1375.5, 1176.5, 1078.6, 980.9, 913.4; ¹H NMR (400MHz, CDCl₃), d : 0.69 (s, 3H, 18-H₃), 0.89 (s, 3H, 19-H₃), 1.00 (d, J = 6.0Hz, 3H, 21-H₃), 3.43 (m, 1H, 3a -H), 3.82 (m, 3H, 7a -H, COOCH₂CH₂OH), 3.97 (s, 12a -H), 4.21 (t, 2H, COOCH₂CH₂OH); Anal. Calcd for C₂₆H₄₄O₆ (452.63): C 68.99 %, H 9.80 %; found: C 68.84 %, H 9.87 %.

Figure 1 The chemical structures of 2¢ - hydroxyethyl 3a , 7a , 12a - trihydroxy - 5b - 24 - ate and its methacrylate derivatives

2.3 2'-Methacryloyloxyethyl 3a ,7a ,12a -trihydroxy-5b -cholan-24-ate(2)
The product 2 was synthesized by the use of a procedure of the one published in the literature^[15]. IR /cm^-1: 3492.1, 2939.8, 2868.6, 1717.9, 1637.4, 1452.8, 1379.6, 1268.7, 1168.8, 940.2; ¹H NMR (400MHz, CDCl₃), d : 0.67 (s, 3H, 18-H), 0.88 (s, 3H, 19-H), 0.97 (d, J = 6.0Hz, 3H, 21-H), 1.94 (s, 3H, C(CH₃)=CH₂), 3.43 (m, 1H, 3-H), 3.84 (s, 1H, 7-H), 3.96 (s, 1H, 12-H), 4.33 (s, 4H, COOCH₂CH₂COO)，5.60 (s ,1H, =CH), 6.13 (s ,1H, =CH); Anal. Calcd for C₃₀H₄₈O₇ (520.71): C69.20 %, H9.29 %; found: 69.02 %, H 9.39 %.
2.4 2'-Methacryloyloxyethyl 3a -methacryloyloxy-7a ,12a -dihydroxy-5b -cholan-24-ate(3)
The product 3 was synthesized by the use of a procedure of the one published in the literature^[15]. IR /cm^-1: 3508.0, 2942.8, 2871.2, 1717.4, 1635.9, 1452.3, 1377.3, 1296.7, 1072.9, 940.1; ¹H NMR (400MHz, CDCl₃), d : 0.69 (s, 3H, 18-H₃), 0. 91 (s, 3H, 19-H₃), 0.98 (d, J = 6.4Hz, 3H, 21-H₃), 1.91 (s, 3H, C(CH₃)=CH₂), 1.94 (s, 3H, C(CH₃)=CH₂), 3.86 (s, 1H, 7a -H), 3.99 (s, 1H，2a -H), 4.33 (s, 4H, COOCH₂CH₂OCO), 4.63 (m, 1H, 3a -H), 5.50 (s, 1H，=CH), 5.60 (s, 1H， =CH), 6.07 (s, 1H, =CH), 6.13 (s, 1H, =CH); Anal. Calcd for C₃₄H₅₂O₈ (588.78): C 69.36 %, H 8.90 %; found: C 69.15 %, H 9.06 %.
2.5  2'-Methacryloyloxyethyl 3a ,7a -dimethacryloyloxy-12a -hydroxy-5b -cholan-24-ate(4)
The product 4 was synthesized by the use of a modified procedure of the one published in the literature^[14]. 1（2.00 g,4.42 mmol）, DCC (4.56g, 22.1mmol) and DMAP (0.27 g ,2.21 mmol) were dissolved in DCM (40 mL).Thereafter, methacrylic acid(1.90g,22.1 mmol，dissolved in10 mL DCM) was added dropwise and the reaction allowed to proceed overnight with stirring. The solid was filtered off and the solution washed successively with brine, 5% acetic acid, 5 % aqueous Na₂CO₃, and brine. After drying the organic layer over anhydrous Na₂SO₄, the solvent was evaporated under reduced pressure, and the crude product was purified by column chromatography (silica gel, ethyl acetate/petroleum = 3/7 as the eluent). Yield:1.79 g (62 %). IR /cm^-1: 2943.5, 1711.4, 1637.2, 1450.8, 1295.3, 1159.1 cm-1 ; ¹H NMR (400MHz, CDCl₃), d : 0.75 (s, 3H, 18-H₃), 0.83 (s, 3H, 19-H₃), 0.89 (d, J = 6.4Hz, 3H, 21-H₃), 1.89 (s, 3H, C(CH₃)=CH₂), 1.93 (s, 3H, C(CH₃)=CH₂), 1.98 (s, 3H, C(CH₃)=CH₂), 3.98 (s, 1H，12a -H), 4.31 (s, 4H, COOCH₂CH₂OCO), 4.60 (m, 1H, 3a -H), 5.02 (s, 1H, 7a -H), 5.50 (s, 1H, =CH), 5.56 (s, 1H, =CH), 5.58 (s, 1H, =CH), 6.03 (s, 1H,=CH), 6.11 (s, 1H, =CH), 6.13 (s,1H, =CH)；Anal. Calcd for C₃₄H₅₂O₈ (656.86): C 69.49%, H 8.59%; found: C 69.73%, H 8.42%。
2.6  2'-Methacryloyloxyethyl 3a ,12a -dimethacryloyloxy-7a -hydroxy-5b -cholan-24-ate(5)
Starting with 1 (1.00 g, 2.21 mmol), DMAP (0.08 g, 0.65mmol) , Et₃N (0.9 g, 8.9 mmol) and methacryloyl chloride (0.93 g, 8.9 mmol), the synthesis took place as described for 3. Yield: 0.62 g (43 %); IR /cm^-1: 2943.2, 2871.5, 1711.9, 1636.4, 1450.1, 1320.3, 1294.9, 1158.6, 938.6; ¹H NMR (400MHz, CDCl₃), d : 0.75 (s, 3H, 18-H₃), 0.81 (d, J = 6.4Hz, 3H, 21-H₃), 0.89 (s, 3H, 19-H₃), 1.89 (s, 3H, C(CH₃)=CH₂), 1.93 (s, 3H, C(CH₃)=CH₂), 1.98 (s, 3H, C(CH₃)=CH₂), 3.87 (s, 1H，7a -H), 4.31 (s, 4H, COOCH₂CH₂OCO), 4.59 (m, 1H, 3a -H), 5.16 (s, 1H, 12a -H), 5.50 (s, 1H, =CH), 5.56 (s, 1H, =CH), 5.58 (s, 1H, =CH), 6.03 (s, 1H,=CH), 6.11 (s, 1H, =CH), 6.13 (s,1H, =CH); Anal. Calcd for C₃₄H₅₂O₈ (656.86): C 69.49 %, H 8.59 %; found: C 69.77 %, H 8.51 %.
2.7  2'-Methacryloyloxyethyl 3a ,7a ,12a -trimethacryloyloxy -5b -cholan-24-ate(6)
Starting with 1 (1.00 g, 2.21 mmol), Et₃N (2.24 g, 22.1 mmol) and methacryloyl chloride (1.84 g, 17.68 mmol), The synthesis was carried out as described for 3. The crude product was purified by column chromatography (silica gel, ethyl acetate/petroleum = 1/5 as the eluent). Yield: 1.27 g (79 %); IR /cm^-1: 2954.2, 2871.5, 1711.7, 1637.2, 1450.3, 1317.6, 1294.0, 1149.4, 936.5, 812.8; ¹H NMR (400MHz, CDCl₃), d : 0.75 (s, 3H, 18-H₃), 0.81 (d, J = 6.0Hz, 3H, 21-H₃), 0.94 (s, 3H, 19-H₃), 1.88 (s, 3H, C(CH₃)=CH₂), 1.92 (s, 3H, C(CH₃)=CH₂), 1.95 (s, 3H, C(CH₃)=CH₂), 1.98 (s, 3H, C(CH₃)=CH₂), 4.30 (s, 4H, COOCH₂CH₂OCO), 4.63 (m, 1H, 3a -H), 5.01 (s, 1H, 7a -H), 5.20 (s, 1H， 12a -H), 5.49 (s, 1H, =CH), 5.54 (s, 1H, =CH), 5.56 (s, 1H, =CH), 5.57 (s, 1H, =CH), 5.99 (s, 1H, =CH), 6.10 (s, 2H, =CH×2), 6.12 (s, 1H, =CH); Anal. Calcd for C₄₂H₆₀O₁₀ (724.93): C 69.59 %, H 8.34 %; found: C 69.68 %, H 8.52 %.

3 RESULTS AND DISCUSSION
3.1  Synthesis of 2' - hydroxyethyl 3a ,7a ,12a - trihydroxy - 5b - cholan - 24 - ate derivatives
1 ~ 6 were synthesized from cholic acid. Methacryloyl chloride, methacryloyl anhydride and methacrylic acid were used as the acylating agents in order to determine the hydroxyl groups reactivity of 1. The results show: When methacrylic acid was used with DCC (dehydrating agent) and DMAP (catalyst), the products obtained were 2, 3, 4 and 6 respectively, indicating that the order of reactivity for four hydroxyl groups in 1 is 1° OH > C3 OH > C7 OH> C12 OH. When methacryloyl chloride was used, compounds 2, 3, 5 and 6 obtained. Thin-lay chromatography was used to follow the tracks of the reaction, which shown 1° OH 、C3 OH、C12 OH and C7 OH reacted with methacryloyl chloride on this order in the course of reaction with the increase of methacryloyl chloride. Therefore, the reactivity of the OH groups follow the order of 1° > C3 > C12 > C7 for methacryloyl chloride. To this reaction, we found it is difficult to make it ceasing on 1° OH by controlling the addition of methacryloyl chloride. When the input of methacryloyl chloride is enough, C3 OH reacted with it before the reaction on 1° OH finished.
    All five compounds 2, 3, 4, 5 and 6 can be obtained by the use of methacryloyl anhydride as acylating agent. For the reaction, 2, 3 and 6 can be obtained as the only product by controlling the adding of methacryloyl anhydride, while 4 and 5 are always obtained at the same time. The addition of DMAP accelerates the reaction. The higher temperature also shortens the reaction time and seems to favor the formation of 5. This indicating the reactivity for four hydroxyl groups in 1 is 1° > C3 > C7 » C12 when methacryloyl anhydride was used.
3.2 Reactivity of hydroxyl groups in 2' - hydroxyethyl 3a , 7a , 12a - trihydroxy - 5b - cholan - 24- ate
Cholic acid belong to coprostane steroids, all the flat rings of skeleton are on the same plane, where the cis connection ring A and ring B imparts a curvature to the steroids skeleton, making possible the formation of a cavity. C18 - CH₃, C19 - CH₃ and C21 - CH₃ are on completely hydrophobic b face, C3 OH, C7 OH and C12 OH are directed to a face, forming the hydrophilic part together with the carboxylic side chain. The three secondary hydroxyl groups of 1 are on the same face of the steroid skeleton, C3 OH is at an equatorial position therefore less sterically hindered and has a higher reactivity than C7 OH and C12 OH ^[16]. C12 OH should have a lower reactivity than that of C7 OH because the distance between this group and 21-CH₃ is shorter and thus more sterically hindered.
    The acylation easily occurs on the hydroxyl groups of 1 when methacryloyl chloride is used as the acylating agent, Et₃N as absorption agent of HCl. In the solution, methacryloyl cation can be released smoothly from methacryloyl chloride and it attacks the hydroxyl groups of 1, the reaction occurred. Compared to the secondary hydroxyl groups on skeleton, the primary hydroxyl has less sterically hindered, so a higher reactivity is. C3 OH has less sterically hindered than C7 OH and C12 OH, when methacryloyl chloride is used, the reaction occurs on it before C7 OH and C12 OH. Once C3 OH is acylated, the reactivity of C12 OH is enhanced since the methacryloyl group is electron-withdrawing ^[17], while steric hindrance of the same group for C7-OH is more significant. Therefore, the reactivity of the hydroxy groups of 1 follows the order of 1° > C3 > C12 > C7.
    When methacrylic acid is used as the acylating reagent, the methacryloyl cation can not be created in the solution directly, so the acylating reaction do not occur directly. The complex between methacrylic acid and DMAP is formed when the DMAP presented, the acylating reactivity of methacrylic acid is improved, so the reaction can occur. To four hydroxyl groups of 1, the primary hydroxyl group has less sterically hindrance and the highest reactivity. For the three second hydroxyl groups, the sterically hindered follows the order C3 < C7 < C12, therefore the reactivity follows the order C3 OH > C7 OH > C12 OH.
    Methacryloyl cation can be created from methacryloyl anhydride in the solution and react with the hydroxyl groups of 1. The primary hydroxyl group has the least sterically hinderence, so 2 can be produced at first. The producing rate of methacryloyl cation from methacryloyl anhydride is not as quick as from methacryloyl chloride, so the reaction can take place only on primary hydroxy group by controlling the adding of methacryloyl anhydride. For the three second hydroxyl groups, the steric hindrance follows the order C3 > C7 > C12, therefore, after primary hydroxyl is acylated, the reaction goes to on C3 OH and 3 presented. Continue to increase the input of methacryloyl anhydride, C7 OH is acylated and 4 is created. After the methacryloyl group is bound on C3 OH, the reactivity of C12 OH is enhanced, while steric hindrance of the same group for C7-OH is more significant, which leads the reactivity difference between C7 OH and C12 OH little. So 4 and 5 can be produced at the same time in this system.

ACKNOWLEDGEMENTS  The authors thank Tianjin University of Science and Technology for the financial support of this work.

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2'-羟基胆酸乙酯羟基酰化反应活性研究
胡祥正， 刘安军²，王丽霞²， 刘雁红¹，倪丽琴^1
(1.天津科技大学理学院， 天津 300457； 2.天津科技大学食品科学与生物技术学院， 天津 300457)
摘要 应用甲基丙烯酰氯、甲基丙烯酸酐和甲基丙烯酸作酰化试剂考察2'- 羟基胆酸乙酯分子中羟基的酰化反应活性。结果显示：与三种酰化试剂反应，2'- 羟基胆酸乙酯分子四个羟基中的伯羟基反应活性都最高，甾体骨架上三个仲羟基的相对反应活性顺序因酰化剂活性差别而不同。
关键词 2'- 羟基胆酸乙酯；酰化；反应活性

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