齊敦果酸衍生物的 3-O-葡萄醣醛酸化反應

在 2.1.2 中，我們準備了 5 種以 TCA 作為離去基的醣基給予者，還有 1 個以 TES 保護、STol 為離去基醣基給予者。以 STol 為離去基，一般會用 NIS/TfOH 活 化，但因為 benzyl oleanolate 在此條件下有重排的現象，所以我們改用 AgOTf、

p-NO2PhSCl 活化。（Scheme 19）

實驗方式為：將起始物 73、AgOTf 和 4Å 分子篩在氮氣環境、室溫下溶於 CH₂Cl₂ 中，冰浴下加入 p-NO2PhSCl，反應半小時後加入 benzyl oleanolate，攪拌 3 小時，

發現不反應，升溫後結果亦然，且起始物 73 的離去基已被活化，以矽膠管柱層析 純化後並未得到想要的產物且 benzyl oleanolate 也受到了影響，產生重排的現象。

Scheme 19. 以 triethylsilyl group 保護進行葡萄醣醛酸化反應

以 TCA 作為葡萄醣醛酸化反應中的離去基，一般會用 BF₃·OEt₂ 或 TMSOTf 等 路易士酸（Lewis acid）活化。參考相關研究發現，若使用活性高的 BF3·OEt2 會使 triterpene 更容易產生其它副反應，因此不使用 BF₃·OEt₂ 為活化劑⁶³，而是以 TCA 作為離去基進行反應（Scheme 20、表四、表五）。

實驗步驟為：先將 1.2 當量的化合物 25、31、32、79 或 90 和 1 當量的化合物

1. 比較 entry 1&2、entry 3&4、entry 8&9，發現不管二號保護基是 Ac、Bz 或 Piv，

B(PhF5)3較不易有副反應的產生，其可能原因為酸性較低，所以即使反應溫度

Scheme 20. Glucuronidation with 28-O-benzyl oleanolic acid 表 四、化合物 25、31、32 和 67 的反應結果

Entry Compound Promotor Solvent Temperature Product

1 25 TMSOTf CH2Cl2 0 ℃ 94：23%; orthoester: none 2 25 B(PhF5)3 CH2Cl2 rt 94：33%; orthoester: none 3 31 TMSOTf CH2Cl2 0 ℃ 91：12%; orthoester: 35%

4 31 B(PhF5)3 CH2Cl2 0 ℃ 91：52%; orthoester：10%

5 31 B(PhF5)3 CH2Cl2 rt 91：60%; orthoester: none 6 31 B(PhF₅)₃ CH₂Cl₂ 0 ℃ → rt 91：43%; orthoester: none

7 31 B(PhF₅)₃ THF rt no product

8 32 TMSOTf CH₂Cl₂ 0 ℃ 95：44%; orthoester ~ 5%

9 32 B(PhF₅)₃ CH₂Cl₂ rt 95：48%; orthoester: none 10^a 32 B(PhF₅)₃ CH₂Cl₂ rt 95：17%; orthoester: none

a Without 4Å MS.

雖然 Ac、Bz、Piv 為保護基可以利用鄰基效應增加 β selectivity，但是他們皆 為”disarmed”的保護基，反應性較低，所以我們設計了將 C-3、C-4 位置以 benzyl group 保護的化合物 90，增加反應性，另外又合成反應性高的 TBS group 為保護基

合成化合物 79，來看他們是否能更加優化葡萄醣醛酸化反應（Scheme 21、表五）。

Scheme 21. 以”Armed”保護基和 28-O-benzyl oleanolic acid 進行反應

反應的結果整理於表五，可以發現：

1. 以 TBS 為保護基（79），因為 TBS 為”Armed”的保護基，反應活性較大，但在 酸性下不穩定，以 TMSOTf 為活化劑時，因為酸性較高，所以有去掉 TBS 保

護基的副產物。而以 B(PhF5)3 為活化劑時，因為無鄰基效應，在室溫下反而

會有α 異構物的產生，透過溫度控制，在 0 ℃時具有很好的選擇性，產率可

到達 63%。

2. 溶劑仍以 CH₂Cl₂為佳，THF 為溶劑之反應差（entry 4）。

3. 將 C-3、C-4 位置以 benzyl group（Bn）保護和原本使用 Bz 保護比起來並較好 的結果，產率甚至較原本的低［表三（entry 5）v.s.表四（entry 6）］。

表 五、化合物 79、90 和 67 反應結果

Entry Compound Promotor Solvent Temperature Product

1 79 TMSOTf CH2Cl2 0 ℃ 96^a：25%

2 79 B(PhF5)3 CH2Cl2 0 ℃ 96：63%; orthoester：none 3 79 B(PhF5)3 CH2Cl2 rt 96：50%; α：13%

4 79 B(PhF₅)₃ THF 0 ℃ messy, 96 ~ 5%

5 90 TMSOTf CH₂Cl₂ 0 ℃ 97：35%; orthoester：20%

6 90 B(PhF₅)₃ CH₂Cl₂ rt 97：50%; orthoester：none

a 有去掉 1~2 個 TBS 保護基的副產物生成

2.2.2 皂皮酸衍生物（allyl quillaic ester）的 3-O-葡萄醣醛酸化反應

2. 比較 entry 1&2、3&4、6&7、9&10，以 TMSOTf 和 B(PhF5)3活化，無一定論，

保護基與活化劑需要搭配嘗試，才能獲得較高產率。

6. 這兩個反應的純化以 B(PhF5)3為活化劑較佳，因為較無 orthoester 和 α-form 產 物產生，所以產物和其他副產物在層析時比較容易分離，而且反應可以在室溫 中反應，有量化的潛力。

Scheme 22. Glucuronidation with Allyl quillaic ester 表 六、不同葡萄醣醛酸給予者和 Allyl quillaic ester 的反應結果

Entry Compound Promotor Temperature Product 1 25 TMSOTf 0 ℃ messy^a, 98 < 5%

2 25 B(PhF5)3 rt messy^a, 98：17%; orthoester：20%

3 31 TMSOTf 0 ℃ messy^a, 99：26%

4 31 B(PhF₅)₃ 0 ℃ 99：32%; orthoester：none 5 31 B(PhF5)3 rt 99：38%; orthoester：none 6 32 TMSOTf 0 ℃ 66：60%; orthoester：12%

7 32 B(PhF₅)₃ 0 ℃ 66：49%; orthoester：none 8 32 B(PhF₅)₃ rt 66：45%; orthoester：none 9 79 TMSOTf 0 ℃ messy^a, 100：55%; α：10%

10 79 B(PhF₅)₃ 0 ℃ 100：41%; α：12%

11 79 B(PhF₅)₃ rt 100：59%; α < 5%

12 90 B(PhF5)3 rt 101：50%; orthoester：none

a messy: side reactions of quillaic acid transacylation 產物：

2.3 玻尿酸分解酶抑制劑活性

我們以兩種方法檢測玻尿酸分解酶抑制劑的活性：混濁度試驗和 Morgan-Elson 反應法，分別以完整玻尿酸含量和玻尿酸降解產物含量兩個角度來評估玻尿酸分 解酶的活性。

混濁度試驗是利用玻尿酸會和具有長碳鏈的 CTAB（cetyltrimethylammonium bromide）反應產生不溶性複合物，因此，可依據混濁度檢測玻尿酸含量，若玻尿 酸分解酶活性高，玻尿酸被分解，反應後加入 CTAB 產生的不溶性複合物少，混

濁度低；反之，混濁度高，則表玻尿酸分解酶活性被抑制⁷⁰。

圖十顯示了不同濃度的 92 反應後的混濁度，可以發現即使將濃度提升至 1 mM，

其混濁度和完全不加抑制劑的混濁度無明顯差異，表 92 並無顯著抑制活性。

圖 十、不同濃度之化合物 92 抑制玻尿酸水解酶之能力

Morgan-Elson 反應法是利用玻尿酸被降解後會產生 free form 的 N-terminal，會

第三章 結論

我們針對三萜類進行葡萄醣醛酸化反應做優化研究，探討 6 種不同的葡萄醣 醛酸基給予者和兩種作為醣接受者的三萜類化合物在不同活化劑、溫度、溶劑中

的反應結果。（圖十二）

我們發現，當使用 B(PhF5)3為活化劑時，具有較好的β 選擇性，值得一提的 是，使用此活化劑時，反應溫度為一個重要的因素，冰浴下反應時容易有 orthoester 的副產物生成，但如果將反應升溫至室溫，orthoester 副產物消失。

醯類保護基方面，Ac 保護基因為立障較小，反應時容易有副反應的發生。Bz 和 Piv 保護基兩者沒有太大的差別，端看葡萄醣醛酸基接受者的不同而有不同的產 率，接受者為齊墩果酸時，Bz 保護產率較高；皂皮酸為接受者時，Piv 保護的產 率較高。但在選擇性方面，Piv 保護基因為立障效應，較不會原酯的副產物生成。

使用 TBS 為保護基反應活性最好，和兩個三萜類化合物的反應產率都高，我 們認為其具有發展的可能性，未來要進行三萜類化合物的葡萄醣醛酸化反應時，

TBS 保護基是一個很好的選擇。

圖 十二、（A）醣給予者；（B）醣接受者

（A） （B）

在以本研究中所獲得的葡萄醣醛酸衍生物進行玻尿酸分解酶抑制劑之活性探 討實驗中，發現在 1 mM 下仍無明顯抑制活性。這些化合物的活性將再繼續進行探 討。

本研究提供了優化的葡萄醣醛酸化反應條件，未來有助於進一步合成更多葡 萄醣醛酸衍生物。

第四章 實驗部分

saccharic acid 1,4-lactone (β-glucuronidase inhibitor), hyaluronic acid (HA)

Sigma-Aldrich:

Hyaluronidase (HAse) from bovine testes, Type I-S, lyophilized powder: Sigma H3506, Lot. SLBV1921, 587 units/mg Solid

6. DMAB 溶液(10X)：100 mg DMAB 完全溶解於 125 μL 10 N HCl 後加入 875 μL 2.5% hexadecyltrimethylammonium bromide (CTAB)之 2% NaOH 終止反應，以 SpectraMax^® Paradigm^® Multi-Mode Microplate Reader (Molecular Devices, p-dimethylaminobenzaldehyde 溶於 125 L 10 N HCl 後加入 9.875 mL 冰醋酸），於 37 ℃下反應 20 分鐘後，以 13,200 r.p.m.於 4 ℃下離心 15 分鐘，取 200 mL 至 96 孔盤中 ，以 SpectraMax^® Paradigm^® Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA)偵測吸收值（λ = 585 nm），計算抑制率後，以 GraphPad Prism 7 軟體繪圖。

4.2 一般實驗方法

Acetic acid, acetonitrile (ACN), acetone, Amberlyst IR120 (H⁺), boron trifluoride diethyl etherate (BF3·OEt2), N-bromosuccinimide (NBS), 1,8-Diazabicyclo[5.4.

0]undec-7-ene (DBU), 4-dimethylamino pyridine (DMAP), dimethylformamide (DMF), N,N-Diisopropylethylamine (DIPEA), Na2S2O3, NH4Cl, pyridine, tetrahydrofuran (THF), p-thiocresol, trifluoromethanesulfonate (TMSOTf), triethylamine, D-glucose, Hydrogen bromide solution 33 wt. % in acetic acid (33% HBr/AcOH),

tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf), tetrabutylammonium

iodide (TBAI), benzoyl chloride (BzCl), tris(pentafluorophenyl)borane (B(PhF₅)₃), pivaloyl chloride (PivCl), dimethoxymethyl benzene, iodobenzene diacetate (TBAI), 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO)

Alfa Aesar:

Trichloroacetonitrile (Cl3CCN), p-toluenethiol (TolSH)

Fisher Scientific:

2,2-dimethoxypropane, NaHCO3, K2CO3,NaOH, KOH, Pd/C

RDH:

Molecular sieves 4 Å , Celite^®

Merck KGaA:

CDCl3, Kieselgrl 60 silica gel 40-63 μm (230-400 mesh)

Sigma-Aldrich:

Ac2O, CD3OD, borane tetrahydrofuran (BH3．THF)

友和貿易股份有限公司:

ACS-CHCl3, methanol, EtOAc, hexane

景明貿易股份有限公司:

Dichloromethane (CH2Cl2), dimethylformamide (DMF)

4.3.2 實驗儀器

4.3.2.1 核磁共振（NMR）

使用 Bruker AMX-400（400 MHz）、Bruker DPX-200（200 MHz）或 AV III-600

（600 MHz），以溶劑 CDCl3或 CD3OD 配置樣品，以 ppm 為化學位移之單位，校 正標準為 CDCl₃（1H: 7.26 ppm, 13C: 77.2 ppm）、CD₃OD（1H: 3.31 ppm, 13C: 49.0 ppm）。1H-NMR 分裂形式定義如下：s, 單峰（singlet）; d, 雙分裂（doublet）; t, 三 分裂（triplet）; q, 四分裂（quartet）; m, 多分裂（multiplet）。耦合常數（coupling constant）以 J 表示，單位為 Hz。

4.3.2.2 薄層層析（Thin layer chromatography, TLC）

使用 Merk Kieselgrl 60 F254 之矽膠（silica gel）薄層層析片，利用流動相展開 後，以紫外燈（254 nm）照射觀察，也可以顯色劑 anisaldehyde 或 cerium 染片後 加熱觀察。

4.3.2.3 管柱層析

Silicycle 60 矽膠 40-63 μm（230-400 mesh, Merck）

4.3.2.4 質譜

Bruker BioTOF II^TM ESI-TOF

4.3.2.5 高壓液相層析（HPLC）

SHIMADZU (system controller: CBM-20A, photodiode array detector:

SPD-M20A, pump: LC-20AT, autosampler: SIL-20AHT). 純 化 使 用 HPLC Ascentis-RP18（5 μm, 250 mm × 10 mm）管柱進行分離，化合物 92 流動相分離條 件如表三所示，流速為 2.0 mL/min，以 210 nm 波長偵測。化合物 92 純度分析使 用 HPLC Mightysil-RP18（5μm，250 mm × 4.6 mm），流動相為 isocratic H₂O + 0.1%

TFA：30%，CAN：70%，流速為 1.0 mL/min，以 210 nm 波長偵測。

4.4 合成步驟及數據

 General procedure for imidate formation (Method A) Step 1.

To a stirred solution of thio compound (1 equiv.) in acetone/H2O, 9/1 (0.1~0.2 M) was added NBS (4 or 6 equiv.) at rt. After being stirred for 2 h, the reaction was quenched by NaHCO3(aq.), Na2S2O3(aq.) and then removed acetone under reduced pressure. The mixture was diluted with CH2Cl2, washed by NaHCO3(aq.), Na2S2O3(aq.)

brine, dried over MgSO4 and then concentrated under reduced pressure. The residue was purified by column chromatography to give hemiacetal product.

Step 2.

To the hemiacetal product (1 equiv.) dissolved in anhydrous CH2Cl2 (0.05~0.2 M) was added Cl3CCN (6 equiv.), DBU (0.2 equiv.) at rt under N2 atmosphere. The reaction mixture was stirred for 2 h. The resulting mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography to give imidate glucuronide.

 General procedure for glucuronidation (Method B)

To the suspension of glucuronyl imidate (1.2 equiv.), benzyl oleanolate or allyl quillaic ester (1 equiv.) and active 4 Å molecular sieve powder in anhydrous CH2Cl2

(40~60 mM) was added B(PhF₅)₃ or TMSOTf (0.1 eq) at rt under N₂ atmosphere. After being stirred for 30 min, the reaction was quenched by Et3N and then filtered. The resulting mixture was concentrated under reduced pressure and purified by column chromatography.

Methyl

(trichloroacetimidoyl-2,3,4-tri-O-acetyl-α-D-glucopyranosid)uronate (25)⁷²

Compound 25 (150 mg, 95%) as white foam: R_f 0.28 (EtOAc/Hexanes, 1/3) was synthesized according to method A by using compound 70 (250 mg, 0.57 mmol) and purified by flash chromatography (silica gel, EtOAc/Hexanes, 1/3). 25: ¹H NMR (200 MHz, CDCl3) δ 8.72 (s, 1H, NH), 6.63 (d, J = 3.6 Hz, 1H, H-1), 5.62 (dd, J = 10.2, 9.4 Hz, 1H, H-4), 5.26 (dd, J = 10.1, 9.4 Hz, 1H, H-3), 5.14 (dd, J = 10.1, 3.6 Hz, 1H, H-2), 4.49 (d, J = 10.2 Hz, 1H, H-5), 3.74 (s, 3H, OCH3), 2.01 (s, 9H, OCOCH3*3) ppm.

Methyl

(trichloroacetimidoyl-2,3,4-tri-O-benzoyl-α-D-glucopyranosid)uronate (31)

Compound 31 (207 mg, 56%) as white foam: R_f 0.49 (EtOAc/Hexanes, 1/3) was synthesized according to method A by using 71 (348 mg, 0.55 mmol) and purified by flash chromatography (silica gel, EtOAc /Hexanes, 1/4). 31: ¹H NMR (200 MHz, CDCl3) δ 8.68 (s, 1H, NH), 7.99-7.86 (m, 6H, Ar-H), 7.55-7.31 (m, 9H, Ar-H), 6.91 (d, J = 3.5 Hz, 1H, H-1), 6.29 (dd, J = 10.2, 9.9 Hz, 1H, H-3), 5.75 (dd, J = 10.1, 9.9 Hz,

1H, H-4), 5.63 (dd, J = 10.2, 3.5 Hz, 1H, H-2), 4.76 (d, J = 10.1 Hz, 1H, H-5), 3.69 (s, 3H, OCH3) ppm; BBD ¹³C NMR (50 MHz, CDCl3) δ 167.4, 165.6, 165.4, 160.4, 133.8, 133.6, 130.0, 129.9, 128.6, 92.9 (C-1), 90.6, 71.1, 70.3, 69.7, 69.4, 53.2 ppm.

Methyl

(trichloroacetimidoyl-2,3,4-tri-O-pivaloyl-α-D-glucopyranosid)uronate (32)

Compound 32 (0.7 g, 56%) as white foam: R_f 0.41 (EtOAc/Hexanes, 1/10) was synthesized according to method A by using compound 72 (1.5 g, 2.65 mmol) and purified by flash chromatography (silica gel, EtOAc /Hexanes, 1/15). 32: ¹H NMR (400 MHz, CDCl3) δ 8.71 (s, 1H, NH), 6.65 (d, J = 3.3 Hz, 1H, H-1), 5.69 (dd, J = 10.1, 9.8 Hz, 1H, H-3), 5.30 (dd, J = 10.2, 9.8 Hz, 1H, H-4), 5.19 (dd, J = 10.1, 3.3 Hz, 1H, H-2), 4.48 (d, J = 10.2 Hz, 1H, H-5), 3.71 (s, 3H, OCH3), 1.15-1.10 (m, 27H, OCOC(CH3)3*3) ppm; BBD ¹³C NMR (100 MHz, CDCl3) δ 177.2 (OCOC(CH3)3), 176.9 (OCOC(CH3)3), 176.8 (OCOC(CH3)3), 167.4 (C-6), 160.5 (C=NH), 92.6 (C-1), 90.7 (CCl3), 70.8 (C-5), 69.5 (C-3), 68.8 (C-2), 68.7 (C-4), 53.1 (OCH3), 38.9 (OCOC(CH3)3), 38.8 (OCOC(CH3)3), 27.2 (OCOC(CH3)3), 27.1 (OCOC(CH3)3) ppm.

28-O-allyl quillate (61)

To a solution of quillaic acid (803 mg, 1.65 mmol) in THF/H₂O (10/1 v/v, 8 mL) was treated with allyl bromide (286 μL, 3.30 mmol), potassium carbonate (455 mg, 3.30 mmol) and TBAI (30 mg, 0.08 mmol) under rt. The mixture was heated at reflux for 3.5 h. The reaction mixture was concentrated under reduced pressure. The residue was then diluted with CH2Cl2, washed by H2O, dried over MgSO4 and concentrated to afford a crude residue which was purified by column chromatography (silica gel;

EtOAc/Hexanes, 1/3) to give compound 61 (797 mg, 92%) as white foam: Rf = 0.43 (EtOAc/Hexanes, 1/2); ¹H NMR (400 MHz, CDCl3) δ 9.35 (s, 1H. H-23), 5.87-5.78 (m, 1H, OCH2CHCH2), 5.36 (t, J = 3.4 Hz, 1H, H-12), 5.27 (d, J = 17.1 Hz, 1H, OCH2CHCH2), 5.18 (d, J = 10.4 Hz, 1H, OCH2CHCH2), 4.57-4.39 (m, 3H, H-16, OCH2CHCH2), 3.75 (dd, J = 11.3, 4.1 Hz, 1H, H-3), 3.03 (dd, J = 14.2, 3.5 Hz, 1H, H-18), 2.14 (t, J = 13.6 Hz, 1H, H-19), 2.08-1.82 (m, 5H), 1.81-1.56 (m, 7H), 1.55-1.40 (m, 2H), 1.38-1.28 (m, 4H), 1.28-1.20 (m, 2H), 1.17 (d, J = 8.7 Hz, 1H), 1.13-0.98 (m, 5H), 0.94-0.93 (m, 7H), 0.88 (s, 3H), 0.70 (s, 3H); BBD ¹³C NMR (100 MHz, CDCl3) δ 207.3, 176.4, 142.8, 132.0, 122.3, 118.0, 74.7, 71.7, 65.1, 55.2, 48.6, 48.0, 46.5, 46.3, 41.3, 40.5, 39.7, 38.0, 35.8, 35.3, 35.3, 32.7, 32.2, 30.7, 30.3, 26.7, 25.9, 24.5, 23.2, 20.6, 16.9, 15.6, 8.8 ppm; HRMS⁺ (ESI-TOF) calcd for C33H51O5 [M+H]⁺ 527.3731, found 527.3733.

28-O-Allyl-3-O-(methyl 2,3,4-tri-O-pivaloyl-β-D-glucopyranosiduronate)quillate (66)

Compound 66 (16.5 mg, 60%) as a white solid: R_f 0.40 (EtOAc/Hexanes, 1/4) was synthesized according to method B by using 32 (20 mg, 0.033 mmol), 61 (15 mg, 0.028 mmol) and TMSOTf (0.5 μL, 0.0028 mmol); ¹H NMR (600 MHz, CDCl3) δ 9.40 (s, 1H, H-23), 5.88-5.82 (m, 1H, OCH2CHCH2), 5.38 (t, J = 3.4 Hz, 1H, H-12), 5.31-5.16 (m, 4H, OCH2CHCH2, H-3′, H-4′), 5.10 (t, J = 8.8, 8.2 Hz, 1H, H-2′), 4.52-4.47 (m, 4H, H-1′, OCH2CHCH2, H-16), 3.99 (d, J = 10.0, 1H, H-5′), 3.84 (dd, J = 11.7, 4.62 Hz, 1H, H-3), 3.72 (s, 3H, OCH3), 3.06 (dd, J = 14.3, 4.1 Hz, 1H, H-18), 2.14 (t, J = 13.6 Hz, 1H, H-19), 1.89-1.87 (m, 4H), 1.79-1.73 (m, 5H), 1.67-1.62 (m, 4H), 1.48-1.39 (m, 2H), 1.33-1.29 (m, 4H), 1.16-1.08 (m, 30H), 1.04-0.99 (m, 3H), 0.96 (s, 3H), 0.94 (s, 3H), 0.90 (s, 3H), 0.86 (m, 1H), 0.70 (s, 3H) ppm; BBD ¹³C NMR (150 MHz, CDCl3) δ 207.5 (C-23), 177.1, 176.6, 176.5, 167.5 (C-6′), 142.9, 132.3 (OCH2CHCH2), 122.7 (C-12), 118.3 (OCH2CHCH2), 100.8 (C-1′), 81.3 (C-3), 75.0 (C-16), 72.5 (C-5′), 72.1 (C-3′), 71.4 (C-2′), 69.7 (C-4′), 65.3 (allylic CH2), 54.6, 52.9, 49.2, 48.9, 46.7, 46.5, 41.5, 40.8, 39.9, 38.9, 38.8, 38.2, 36.1, 35.6, 35.6, 32.9, 32.4, 30.9, 30.5, 29.8, 27.3, 27.3, 27.2, 27.1, 24.9, 24.7, 23.4, 20.2, 17.2, 15.8, 10.4 ppm; HRMS⁺ (ESI-TOF) calcd for C55H85O14 [M+H]⁺ 969.5934, found 969.5904.

28-O-benzyl oleanolate(67)⁷³

To a stirred solution of oleanolic acid (10.0 g, 21.9 mmol), K₂CO₃ (4.55 g, 32.9 mmol) and TBAI (81.0 mg, 0.219 mmol) in aqueous THF (100 mL) was added benzyl chloride (3.8 mL, 32.9 mmol) at rt. The reaction was heated at reflux and stirred 12 h.

The resulting mixture was concentrated under reduced pressure, the residue was diluted with CH2Cl2, washed by water and brine, dried over MgSO4 and then concentrated. The residue was recrystallized for methanol to obtain 67 (10.16 g, 84%) as a white crystal;

1H NMR (200 MHz, CDCl3) δ 7.40-7.29 (m, 5H, Ar-H), 5.28 (t, J = 3.4 Hz, 1H, H-12), 5.08-5.05 (m, 2H, CO2CH2Ph), 3.28-3.12 (m, 1H, H-3), 2.88 (dd, 1H, H-18), 2.02-1.80 (m, 3H), 1.75-1.57 (m, 6H), 1.54-1.45 (m, 3H), 1.45-1.15 (m, 8H), 1.12 (s, 3H), 1.05-0.98 (m, 2H), 0.97 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.87 (s, 3H), 0.77 (s, 3H), 0.73 (s, 1H, H-5), 0.60 (s, 3H) ppm; BBD ¹³C NMR (50 MHz, CDCl3) δ 177.6, 143.8, 136.5, 128.5, 128.1, 122.6, 79.16 (C-3), 66.1, 55.3, 47.7, 46.8, 46.0, 41.8, 41.5, 39.4, 38.8, 38.6, 34.0, 33.3, 32.9, 32.5, 30.8, 27.7, 27.3, 26.0, 23.7, 23.5, 23.2, 18.5, 17.0, 15.7, 15.4 ppm; HRMS⁺ (ESI-TOF) calcd for C37H55O3 [M+H]⁺ 547.4146, found 547.4140.

Methyl 1,2,3,4-tetra-O-acetyl-α-D-glucopyranosiduronate (69)⁷⁴

Glucuronolactone (11 g, 62.4 mmol) in methanol (70 mL) was added NaOH (0.25 g, 6.24 mmol) and the reaction mixture was stirred for 5 h. The resulting mixture was concentrated by rotavapor to give a residue which was added pyridine (80 mL) and then slowly added AcOH (50 mL) at 0 ℃. The reaction mixture was stirred for 12 h, and then was concentrated, purified by column chromatography (silica gel, EtOAc/Hexanes, 1/2 to 1/1) to give compound 69 (19 g, 81%) as yellow foam: R_f 0.53 (EtOAc/Hexanes, 1/1); α-anomer: ¹H NMR (200 MHz, CDCl3) δ 6.39 (d, J = 3.6 Hz, 1H, H-1), 5.50 (dd, J

= 10.2, 9.4 Hz, 1H, H-4), 5.21 (dd, J = 10.1, 9.4 Hz, 1H, H-3), 5.11 (dd, J = 10.1, 3.6 Hz, 1H, H-2), 4.41 (d, J = 10.2 Hz, 1H, H-5), 3.74 (s, 3H, OCH3), 2.18 (s, 3H, OCOCH3), 2.03 (s, 6H, OCOCH3), 2.01 (s, 3H, OCOCH3*2) ppm.

Methyl p-tolyl 2,3,4-tri-O-acetyl-1-thio-β-D-glucopyranosiduronate (70)⁷⁵

To a stirred solution of compound 69 (13.8 g, 36.6 mmol) in anhydrous CH2Cl2 (40 mL) was added 33% HBr/HOAc (40 mL) in ice bath under N2 atmosphere. Upon completion of the reaction after 6 h, the reaction was quenched by ice water and then it was diluted with CH2Cl2, washed by ice water, H2O, brine, dried over MgSO4 and then concentrated to afford crude glucuronyl bromide. To a stirred solution of the glucuronyl bromide (14.6 g, 36.8 mmol) in 1.0 M Na2CO3(aq.) (40 mL)/EA (40 mL) was added HSTol (9.1 g, 73.5 mmol) then TBAI (2.72 g, 7.35 mmol) at rt. After being stirred for 12 h, the reaction was quenched by saturated NaHCO3 and then was diluted with EA, washed by saturated NaHCO3, brine, dried over MgSO4 and then concentrated. The residue was purified by column chromatography (silica gel, EtOAc/Hexanes, 1/5 to 1/2) to give compound 70 (12.1 g, 75%) as a white solid: Rf 0.38 (EtOAc/Hexanes, 1/3); ¹H NMR (200 MHz, CDCl3) δ 7.37 (d, J = 8.0 Hz, 2H, Ar-H), 7.11 (d, J = 8.0 Hz, 2H, Ar-H), 5.23 (dd, J = 9.5, 8.6 Hz, 1H, H-3), 5.11 (dd, J = 9.6, 9.5 Hz, 1H, H-4), 4.89 (dd, J = 8.6, 10.0 Hz, 1H, H-2), 4.64 (d, J = 10.0 Hz, 1H, H-1), 3.99 (d, J = 9.6 Hz, 1H, H-5), 3.73 (s, 3H, OCH3), 2.32 (s, 3H, SPhCH3), 2.06 (s, 3H, OCOCH3), 1.97 (s, 6H, OCOCH₃*2) ppm.

Methyl (p-tolyl 2,3,4-tri-O-benzoyl-1-thio-β-D-glucopyranosid)uronate (71)

To a stirred suspension of compound 70 (177 mg, 0.404 mmol) in MeOH (5 mL) was added Na (1 mg, 0.0404 mmol) at rt. Upon completion of the reaction after 3 h, the reaction was quenched by amberlite H+. The reaction mixture was filtered and concentrated. The residue was added anhydrous CH2Cl2 and then added BzCl (0.281 mL, 2.42 mmol), TEA (0.563 mL, 4.04 mmol) and DMAP (4.94 mg, 0.0404 mmol) in ice bath under N2 atmosphere. After being stirred for 6 h, the reaction was quenched by saturated NH4Cl and then it was diluted with CH2Cl2, washed by H2O, saturated brine, dried over MgSO4 and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel; EtOAc/Hexanes, 1/5) to afford compound 71 (220 mg, 87%) as white solid : R_f 0.33 (EtOAc/Hexanes, 1/4); ¹H NMR (400 MHz, CDCl3) δ 7.97-7.80 (m, 6H, Ar-H), 7.55-7.13 (m, 13H, Ar-H), 5.89 (dd, J = 9.6, 9.3 Hz, 1H, H-3), 5.60 (dd, J = 9.7, 9.6 Hz, 1H, H-4), 5.45 (dd, J = 9.6, 9.3 Hz, 1H, H-2), 4.98 (d, J = 9.8 Hz, 1H, H-1), 4.33 (d, J = 9.7 Hz, 1H, H-5), 3.71 (s, 3H, OCH3), 2.36 (s, 3H, SPhCH3) ppm; BBD ¹³C NMR (100 MHz, CDCl3) δ 167.2, 165.3, 139.2, 134.3, 133.5, 130.0, 129.9, 128.6, 128.4, 86.7 (C-1), 76.6, 73.6, 70.2, 53.1, 21.4 ppm;

HRMS⁺ (ESI-TOF) calcd for C₃₅H₃₁O₉S [M+H]⁺ 627.1683, found 627.1688.

Methyl (p-tolyl 2,3,4-tri-O-pivaloyl-1-thio-β-D-glucopyranosid)uronate (72)

To a stirred suspension of 70 (4.57 g, 10.3 mmol) in MeOH (15 mL) was added Na (23.6 mg, 1.03 mmol) at rt. Upon completion of the reaction after 3 h, the reaction was quenched by Amberlyst IR120 (H⁺). The reaction mixture was filtered and concentrated.

The residue (1.0 g, 3.2 mmol) was added anhydrous CH2Cl2 (10 mL) and then added PivCl (2.4 mL, 19.1 mmol), TEA (4.4 mL, 32 mmol) and DMAP (39.1 mg, 0.32 mmol) in ice bath under N2 atmosphere. After being stirred for 6 h, the reaction was quenched by NH4Cl and then it was diluted with CH2Cl2, washed by H2O, brine, dried over MgSO4 and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel; EtOAc/Hexanes, 1/8) to afford 72 (1.5 g, 83%) as white solid: Rf 0.72 (EtOAc/Hexanes, 1/2); ¹H NMR (200 MHz, CDCl3) δ 7.37 (d, 2H, Ar-H), 7.13 (d, 2H, Ar-H), 5.36 (dd, J = 9.3, 9.1 Hz, 1H, H-3), 5.19 (dd, J = 9.8, 9.3 Hz, 1H, H-4), 5.02 (dd, J = 10.1, 9.1 Hz, 1H, H-2), 4.67 (d, J = 10.1 Hz, 1H, H-1), 4.05 (d, J

= 9.8 Hz, 1H, H-5), 3.74 (s, 3H, OCH3), 2.35 (s, 3H, SPhCH3), 1.03-1.00 (m, 27H, OCOC(CH3)3) ppm; BBD ¹³C NMR (50 MHz, CDCl3) δ 176.8 (OCOC(CH3)3), 176.3 (OCOC(CH3)3), 176.1 (OCOC(CH3)3), 166.9 (C-6), 138.7 (Ar-C), 133.6 (Ar-C), 129.7 (Ar-C), 127.6 (Ar-C), 86.7 (C-1), 76.2 (C-5), 72.6 (C-3), 69.0 (C-2), 68.9 (C-4), 52.6 (OCH3), 38.6 (OCOC(CH3)3), 27.0 (OCOC(CH3)3), 21.1 (SPhCH3) ppm; HRMS⁺ (ESI-TOF) calcd for C₂₉H₄₃O₉S [M+H]⁺ 567.2622, found 567.2609.

Methyl

(p-tolyl 2,3,4-tri-O-tert-butyldimethylsilyl-1-thio-β-D-glucopyranosid)uronate (74) To a stirred suspension of 70 (4.57 g, 10.3 mmol) in MeOH (15 mL) was added Na (23.6 mg, 1.03 mmol) at rt. Upon completion of the reaction after 3 h, the reaction was quenched by amberlite H+. The reaction mixture was filtered and concentrated. The residue (1 g, 3.18 mmol) was added anhydrous DMF (5 mL) and then added TBSCl (2.88 g, 19.1 mmol), imidazole (0.78 g, 11.5 mmol) at rt under N2 atmosphere. The mixture was heated to 80℃. After being stirred for 12 h, the reaction was quenched by H2O, concentrated and then it was diluted with CH2Cl2, washed by H2O, brine, dried over MgSO4 and then concentrated under reduced pressure. The residue was purified by

(p-tolyl 2,3,4-tri-O-tert-butyldimethylsilyl-1-thio-β-D-glucopyranosid)uronate (74) To a stirred suspension of 70 (4.57 g, 10.3 mmol) in MeOH (15 mL) was added Na (23.6 mg, 1.03 mmol) at rt. Upon completion of the reaction after 3 h, the reaction was quenched by amberlite H+. The reaction mixture was filtered and concentrated. The residue (1 g, 3.18 mmol) was added anhydrous DMF (5 mL) and then added TBSCl (2.88 g, 19.1 mmol), imidazole (0.78 g, 11.5 mmol) at rt under N2 atmosphere. The mixture was heated to 80℃. After being stirred for 12 h, the reaction was quenched by H2O, concentrated and then it was diluted with CH2Cl2, washed by H2O, brine, dried over MgSO4 and then concentrated under reduced pressure. The residue was purified by

在文檔中 藉由保護基策略探討三萜之 3-O-葡萄糖醛酸化反應的 優化研究 (頁 44-0)