抗肿瘤生物碱类化合物，其药物组合物及其制备方法和应用转让专利
申请号 : CN201110114123.4
文献号 : CN102225937B
文献日 : 2012-07-25
发明人 : 郝小江 , 张于 , 何红平 , 李艳 , 郭伶俐 , 邸迎彤
申请人 : 中国科学院昆明植物研究所
摘要 :
提供结构式（1-4）所示的具有抗肿瘤活性的vobasine-iboga类型二聚单萜吲哚生物碱ervachininesA-D（1-4），其制备方法，含有该类化合物的药物组合物，该类化合物及其药物组合物在制备抗肿瘤药物中的应用。
权利要求 :
1.如下结构式1-4所示的二聚单萜吲哚生物碱Ervachinines A-D或其药学上可接受的盐，其中化合物1、2、3、4分别对应代表化合物Ervachinines A、B、C、D，
2.抗肿瘤组合物，含有权利要求1所述的结构式1-4所示的Ervachinines A-D或其药学上可接受的盐，及可药用载体或赋形剂，其中化合物1、2、3、4分别对应代表化合物Ervachinines A、B、C、D。
3.权利要求1的所述的结构式1-4所示的化合物在制备抗肿瘤药物中的应用。
4.权利要求2所述的药物组合物在制备抗肿瘤药物中的应用。
说明书 :
抗肿瘤生物碱类化合物，其药物组合物及其制备方法和应
用
技术领域
[0001] 本发明属于药物技术领域，具体地，涉及具有抗肿瘤活性的式（1-4）的二聚单萜吲哚生物碱Ervachinines A-D,其为药物活性成分的药物组合物，其制备方法与其在制备抗肿瘤药物中的应用。
背景技术
[0002] 狗牙花属（Ervatamia）我国有15种，5变种，主要分布于我国西南到华南地区，目前，现有技术中未见二聚单萜吲哚生物碱Ervachinines A-D的报道，更未有其活性的报道。
发明内容
[0003] 本发明的目的在于提供式（1-4）的二聚单萜吲哚生物碱ervachinines A-D及其药学上可接受的盐（有机酸、或无机酸），该类化合物为抗肿瘤药物活性成分组成的药物组合物，其制备方法与其在制备抗肿瘤药物中的应用。
[0004] 本发明的上述目的是通过下面的技术方案得以实现的：
[0005] 如下结构式1-4所示的二聚单萜吲哚生物碱Ervachinines A-D或其药学上可接受的盐，
[0006] 。
[0007] 抗肿瘤组合物，含有上述的结构式1-4所示的药物有效成分Ervachinines A-D或其药学上可接受的盐，及可药用载体或赋形剂。
[0008] 式1-4所示的化合物Ervachinines A-D的制备方法，取中国狗牙花Ervatamia.chinensis，用95 %乙醇加热回流提取三次，每次4 小时，合并提取液，减压回收乙醇，将浓缩后的浸膏用3%的酒石酸水溶液捏溶并调节pH值2～3，分别用石油醚，乙酸乙酯各萃取三次，然后用饱和Na2CO3溶液调节pH值9～10，用CHCl3萃取得到总碱部分，总碱部分利用硅胶柱层析，石油醚-丙酮梯度洗脱得到四部分F1–F4，F3利用硅胶柱层析，石油醚/丙酮/二乙胺，10:1:0.2 ® 4:1:0.2和Sephadex LH-20甲醇分离得到化合物ervachinine A和ervachinine C；F4 利用反相柱层析甲醇/水得到三个部分P1–P3，P2 部分经过硅胶柱层析，石油醚/乙酸乙酯，10:1 ® 3:1和Sephadex LH-20甲醇得到化合物 ervachinine B和ervachinine D。
[0009] 结构式1-4所示的化合物在制备抗肿瘤药物中的应用。
[0010] 上述的药物组合物在制备抗肿瘤药物中的应用。
[0011] 本发明同时提供了式（1-4）化合物在制备抗肿瘤药物中的应用。
[0012] 进一步的，本发明还提供了式（1-4）化合物为药物有效成分的药物组合物在制备抗肿瘤药物中的应用。
[0013] 本发明化合物用作药物时，可以直接使用，或以药学上可接受的盐以及药物组合物的形式使用。该药物组合物含有0.1—99%，优选为0.5-90%的本发明化合物，其余为药物学上可接受的，对人和动物无毒和惰性的可药用载体和/或赋形剂。
[0014] 所述的药用载体或赋形剂是一种或多种固体、半固体和液体稀释剂、填料以及药物制品辅剂。将本发明的药物组合物以单位体重服用量的形式使用。苔黑酚-1-氧-β-D-葡萄糖吡喃苷、苔黑酚及其衍生物的组合物采用制药和食品领域公认的方法制备成各种剂型、如液体制剂（注射剂、混悬剂、乳剂、溶液剂、糖浆剂等）、固体制剂（片剂、胶囊剂、颗粒剂，冲剂等）、喷剂、气雾剂等。本发明的药物可经注射（静脉注射、静脉滴注、肌肉注射、腹腔注射、皮下注射）和口服、舌下给药、粘膜透析等给药途径进行抗艾滋病的治疗。
[0015] 具体实施方式：
[0016] 下面结合本发明实施例来进一步说明本发明的实质性内容，但不以此来限定本发明。
[0017] 实施例1：
[0018] 中国狗牙花（Ervatamia.chinensis）全株7.5 kg，用95 %乙醇加热回流提取三次（3 ´ 4 h），合并提取液，减压回收乙醇，将浓缩后的浸膏用3%的酒石酸水溶液捏溶并调节pH值2～3，分别用石油醚，乙酸乙酯各萃取三次，然后用饱和Na2CO3溶液调节pH值9～10，用CHCl3萃取得到总碱部分40 g。总碱部分利用硅胶柱层析（石油醚-丙酮梯度洗脱）得到四部分（F1–F4），F3 （1.2 g）利用硅胶柱层析（石油醚/丙酮/二乙胺，10:1:0.2 ® 4:1:0.2）和Sephadex LH-20（甲醇）分离得到化合物ervachinine A（1，17.0 mg），ervachinine C（3，25.0 mg）；F4 （1.5 g）利用反相柱层析（甲醇/水）得到三个部分（P1–P3），P2 部分（600 mg）经过硅胶柱层析（石油醚/乙酸乙酯，10:1 ® 3:1）和Sephadex LH-20（甲醇）得到化合物 ervachinine B （2，20.0 mg）和ervachinine D （4， 17.5 mg）。
[0019] Ervachinine A (1): light yellow amorphous powder; -13.8 (c 0.39, MeOH); UV (MeOH) λmax (log ε) 293 (3.82), 288 (3.83), 227 (4.28) nm; IR (KBr) vmax 3426, 2927, 1724, 1630, 1463, and 1287 cm–1; 1H and 13C NMR , ESIMS 735 [M + H]+; HRESIMS m/z 735.4131 (calcd for C44H55N4O6 [M + H]+, 735.4121). 1H NMR (CD3OD,400 MHz) d: 5.17 (1H, br d, J = 13.0 Hz, H-3), ca. 4.08 (1H, H-5), 3.36 (1H, m, H-6a), 3.58 (1H, m, H-6b), 7.56 (1H, br d, J = 7.5 Hz, H-9), 7.11 (1H, m, H-10), 7.10 (1H, m, H-11),7.08 (1H, m, H-12), 2.05 (1H, m, H-14a), 2.59 (1H, m, H-14b), 3.53 (1H, m, H-15), 3.66 (1H, d, J = 11.0 Hz, H-17a), ca 3.88 (1H, H-17b), 1.68 (3H, d, J = 8.0 Hz, H-18), 5.42 (1H, q, J = 8.0 Hz, H-19), 2.74 (1H, d, J = 10.0 Hz, H-21a), 2.91 (1H, d, J = 10.0 Hz, H-21b), 7.72 (1H, br s, H-NH), 2.42 (3H, br s, H-CO2Me), 2.60 (3H, br s, H-NMe), 3.14 (1H, dd, J = 11.0,
2.5 Hz, H-3a′), 3.68 (1H, br d, J = 11.0 Hz, H-3b′), 3.17 (1H, m, H-5a′),
3.40 (1H, m, H-5b′), 3.00 (1H, m, H-6a′), 3.12 (1H, m, H-6b′), 6.95 (1H, s, H-9′), 6.69 (1H, s, H-12′), 1.82 (1H, m, H-14′), 1.12 (1H, m, H-15a′),
1.70 (1H, m, H-15b′), 1.76 (1H, br d, J = 16.8 Hz, H-17a′), 2.48 (1H, br d, J = 16.5 Hz, H-17b′), 0.90 (3H, t, J = 8.0 Hz, H-18′), 1.44 (1H, m, H-19a′),
1.56 (1H, m, H-19b′), 1.29 (1H, m, H-20′), 3.54 (1H, br s, H-21′), 4.01
13
(3H, s, H-10′-OMe), 3.64 (3H, s, H-CO2Me′), C NMR (CD3OD 100 MHz).d: 138.0 (C-2, s), 39.5 (C-3, d), 60.0 (C-5, d), 17.4 (C-6, t), 109.9 (C-7, s), 129.8 (C-8, s), 117.5 (C-9, d), 118.9 (C-10, d), 120.4 (C-11, d), 110.2 (C-12, d),
136.0 (C-13, s), 36.4 (C-14, t), 35.7 (C-15, d), 52.0 (C-16, s), 70.3 (C-17, t), 12.2 (C-18, q), 121.7 (C-19, d), 136.1.0 (C-20, s), 51.8 (C-21, t), 50.4 (C-CO2Me, q), 173.9 (C-CO2Me, s), 42.70 (C-NMe, q), 137.2 (C-2′, s), 51.9 (C-3′, t), 53.0 (C-5′, t), 22.1 (C-6′, t), 110.2 (C-7′, s), 127.3 (C-8′, s), 99.1 (C-9′, d), 150.1 (C-10′, s), 129.9 (C-11′, s), 110.4 (C-12′, d), 130.2 (C-13′, s), 27.3 (C-14′, d), 31.9 (C-15′, t), 54.8 (C-16′, s),
36.4 (C-17′, t), 11.6 (C-18′, q), 26.7 (C-19′, t), 38.9 (C-20′, d), 57.0 (C-21′, d), 52.4 (C- CO2Me′, q), 175.2 (C-CO2Me′, s), 56.0(C- 10′-OMe, q).[0020] Ervachinine B (2): light yellow amorphous powder; -29.9 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 295 (3.56), 287 (3.56), 226 (4.05) nm; IR (KBr) vmax 3420, 2925, 1726, 1628, 1464, and 1287 cm–1; ESIMS 677 [M + H]+; HRESIMS m/z 677.4058 (calcd for C42H53N4O4 [M + H]+, 677.4066).1H NMR (CD3OD , 500 MHz) d:
5.11 (1H, br d, J = 13.0 Hz, H-3), 3.77 (1H, t, J = 6.0 Hz, H-5), 3.26 (1H, m, H-6a), 3.55 (1H, m, H-6b), 7.50 (1H, br d, J=5.6 Hz, H-9), 6.98 (1H, m, H-10),
6.98 (1H, m, H-11), 7.08 (1H,br d, J = 5.6 Hz, H-12), 2.04 (1H, m, H-14a), 2.49 (1H, m, H-14b), 3.64 (1H, dd, J =7.6, 4.8 Hz, H-15), 3.59 (1H, d, J =7.2 Hz, H-17a), 3.80 (1H, d, J = 7.2 Hz, H-17b), 1.68 (3H, d, J = 4.0 Hz, H-18), 5.36 (1H, q, J = 4.0 Hz, H-19), 2.87 (1H, d, J = 9.2 Hz, H-21a), 3.56 (1H, d, J =
9.2 Hz, H-21b), 2.30 (3H, s, H-CO2Me), 2.49 (3H, s, H-NMe ), 2.84 (1H, br d, J = 7.2 Hz H-3a′), 2.99 (1H, br d, J = 7.2 Hz, H-3b′), 3.03 (1H, m, H-5a′),
3.29 (1H, m, H-5b′), 2.62 (1H, m, H-6a′), 3.25 (1H, m, H-6b′), 6.91 (1H, s, H-9′), 6.52 (1H, s, H-12′), 1.73 (1H, m, H-14′), 1.73 (1H, br d, J = 8.0 Hz, H-15a′), 1.77 (1H, m, H-15b′), 2.81 (1H, m, H-16′), 1.45 (1H, m, H-17a′),
1.94 (1H, brt, J = 8.0 Hz, H-17b′), 0.88 (3H, t, J = 4.8 Hz, H-18′), 1.42 (1H, m, H-19a′), 1.50 (1H, m, H-19b′), 1.50 (1H, m, H-20′), 2.74 (1H,
13
s, H-21′ ), 3.95 (3H, s, H- OMe′), C NMR (CD3OD 125 MHz) d: 139.9 (C-2, s), 39.5 (C-3, d), 62.3 (C-5, d), 18.7 (C-6, t), 111.4 (C-7, s), 131.5 (C-8, s), 118.3 (C-9, d), 119.3 (C-10, d), 122.2 (C-11, d), 111.1 (C-12, d), 138.3 (C-13, s), 38.6 (C-14, t), 36.6 (C-15, d), 54.2 (C-16, s), 70.4 (C-17, t), 12.5 (C-18, q), 120.4 (C-19, d), 139.0 (C-20, s), 53.1 (C-21, t), 50.6 (C-CO2Me, q),
174.9 (C- CO2Me, s), 42.7 (C-NMe, q ),143.4 (C-2′, s), 50.9 (C-3′, t), 55.8 (C-5, t), 21.7 (C-6′, t), 108.8 (C-7′, s), 129.4 (C-8′, s), 99.7 (C-9′, d), 152.1 (C-10′, s), 130.6 (C-11′, s), 111.5 (C-12′, d), 131.6 (C-13′, s), 27.8 (C-14′, d), 33.0 (C-15′, t), 42.0 (C-16′, d), 35.2 (C-17′, t),
12.5 (C-18′, q), 28.8 (C-19′, t), 43.4 (C-20′, d), 59.5 (C-21′, d), 56.6 (C-10′-OMe, q).
2.5 Hz, H-3a′), 3.68 (1H, br d, J = 11.0 Hz, H-3b′), 3.17 (1H, m, H-5a′),
3.40 (1H, m, H-5b′), 3.00 (1H, m, H-6a′), 3.12 (1H, m, H-6b′), 6.95 (1H, s, H-9′), 6.69 (1H, s, H-12′), 1.82 (1H, m, H-14′), 1.12 (1H, m, H-15a′),
1.70 (1H, m, H-15b′), 1.76 (1H, br d, J = 16.8 Hz, H-17a′), 2.48 (1H, br d, J = 16.5 Hz, H-17b′), 0.90 (3H, t, J = 8.0 Hz, H-18′), 1.44 (1H, m, H-19a′),
1.56 (1H, m, H-19b′), 1.29 (1H, m, H-20′), 3.54 (1H, br s, H-21′), 4.01
13
(3H, s, H-10′-OMe), 3.64 (3H, s, H-CO2Me′), C NMR (CD3OD 100 MHz).d: 138.0 (C-2, s), 39.5 (C-3, d), 60.0 (C-5, d), 17.4 (C-6, t), 109.9 (C-7, s), 129.8 (C-8, s), 117.5 (C-9, d), 118.9 (C-10, d), 120.4 (C-11, d), 110.2 (C-12, d),
136.0 (C-13, s), 36.4 (C-14, t), 35.7 (C-15, d), 52.0 (C-16, s), 70.3 (C-17, t), 12.2 (C-18, q), 121.7 (C-19, d), 136.1.0 (C-20, s), 51.8 (C-21, t), 50.4 (C-CO2Me, q), 173.9 (C-CO2Me, s), 42.70 (C-NMe, q), 137.2 (C-2′, s), 51.9 (C-3′, t), 53.0 (C-5′, t), 22.1 (C-6′, t), 110.2 (C-7′, s), 127.3 (C-8′, s), 99.1 (C-9′, d), 150.1 (C-10′, s), 129.9 (C-11′, s), 110.4 (C-12′, d), 130.2 (C-13′, s), 27.3 (C-14′, d), 31.9 (C-15′, t), 54.8 (C-16′, s),
36.4 (C-17′, t), 11.6 (C-18′, q), 26.7 (C-19′, t), 38.9 (C-20′, d), 57.0 (C-21′, d), 52.4 (C- CO2Me′, q), 175.2 (C-CO2Me′, s), 56.0(C- 10′-OMe, q).[0020] Ervachinine B (2): light yellow amorphous powder; -29.9 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 295 (3.56), 287 (3.56), 226 (4.05) nm; IR (KBr) vmax 3420, 2925, 1726, 1628, 1464, and 1287 cm–1; ESIMS 677 [M + H]+; HRESIMS m/z 677.4058 (calcd for C42H53N4O4 [M + H]+, 677.4066).1H NMR (CD3OD , 500 MHz) d:
5.11 (1H, br d, J = 13.0 Hz, H-3), 3.77 (1H, t, J = 6.0 Hz, H-5), 3.26 (1H, m, H-6a), 3.55 (1H, m, H-6b), 7.50 (1H, br d, J=5.6 Hz, H-9), 6.98 (1H, m, H-10),
6.98 (1H, m, H-11), 7.08 (1H,br d, J = 5.6 Hz, H-12), 2.04 (1H, m, H-14a), 2.49 (1H, m, H-14b), 3.64 (1H, dd, J =7.6, 4.8 Hz, H-15), 3.59 (1H, d, J =7.2 Hz, H-17a), 3.80 (1H, d, J = 7.2 Hz, H-17b), 1.68 (3H, d, J = 4.0 Hz, H-18), 5.36 (1H, q, J = 4.0 Hz, H-19), 2.87 (1H, d, J = 9.2 Hz, H-21a), 3.56 (1H, d, J =
9.2 Hz, H-21b), 2.30 (3H, s, H-CO2Me), 2.49 (3H, s, H-NMe ), 2.84 (1H, br d, J = 7.2 Hz H-3a′), 2.99 (1H, br d, J = 7.2 Hz, H-3b′), 3.03 (1H, m, H-5a′),
3.29 (1H, m, H-5b′), 2.62 (1H, m, H-6a′), 3.25 (1H, m, H-6b′), 6.91 (1H, s, H-9′), 6.52 (1H, s, H-12′), 1.73 (1H, m, H-14′), 1.73 (1H, br d, J = 8.0 Hz, H-15a′), 1.77 (1H, m, H-15b′), 2.81 (1H, m, H-16′), 1.45 (1H, m, H-17a′),
1.94 (1H, brt, J = 8.0 Hz, H-17b′), 0.88 (3H, t, J = 4.8 Hz, H-18′), 1.42 (1H, m, H-19a′), 1.50 (1H, m, H-19b′), 1.50 (1H, m, H-20′), 2.74 (1H,
13
s, H-21′ ), 3.95 (3H, s, H- OMe′), C NMR (CD3OD 125 MHz) d: 139.9 (C-2, s), 39.5 (C-3, d), 62.3 (C-5, d), 18.7 (C-6, t), 111.4 (C-7, s), 131.5 (C-8, s), 118.3 (C-9, d), 119.3 (C-10, d), 122.2 (C-11, d), 111.1 (C-12, d), 138.3 (C-13, s), 38.6 (C-14, t), 36.6 (C-15, d), 54.2 (C-16, s), 70.4 (C-17, t), 12.5 (C-18, q), 120.4 (C-19, d), 139.0 (C-20, s), 53.1 (C-21, t), 50.6 (C-CO2Me, q),
174.9 (C- CO2Me, s), 42.7 (C-NMe, q ),143.4 (C-2′, s), 50.9 (C-3′, t), 55.8 (C-5, t), 21.7 (C-6′, t), 108.8 (C-7′, s), 129.4 (C-8′, s), 99.7 (C-9′, d), 152.1 (C-10′, s), 130.6 (C-11′, s), 111.5 (C-12′, d), 131.6 (C-13′, s), 27.8 (C-14′, d), 33.0 (C-15′, t), 42.0 (C-16′, d), 35.2 (C-17′, t),
12.5 (C-18′, q), 28.8 (C-19′, t), 43.4 (C-20′, d), 59.5 (C-21′, d), 56.6 (C-10′-OMe, q).
[0021] Ervachinine C(3): light yellow amorphous powder; -42.7 (c 0.31, MeOH); UV (MeOH) λmax (log ε) 295 (4.05), 227 (4.64) nm; IR (KBr) vmax 3426,2927, 1723, 1629, 1464, and 1287 cm–1; ESIMS 735 [M + H]+; HRESIMS m/z 735.4135 (calcd for C44H55N4O6 [M + H]+, 735.4121). 1H NMR (CD3OD, 400 MHz) d: 5.11(1H, br d, J = 12.8 Hz, H-3), 3.74 (1H, t, J = 10.4 Hz, H-5), 3.23 (1H, dd, J = 15.2,
8.4 Hz, H-6a), 3.51 (1H, m, H-6b), 7.51 (1H, dd, J = 5.6, 2.4 Hz, H-9), 6.98 (1H, m, H-10), 6.98 (1H, m, H-11), 7.10 (1H, dd, J = 6.4, 3.2 Hz, H-12), 2.00 (1H, m, H-14a), 2.48 (1H, m, H-14b), 3.62 (1H, dd, J = 12.0, 5.6 Hz, H-15),
3.56 (1H, d, J = 10.8 Hz, H-17a), 3.78 (1H, d, J = 10.8 Hz H-17b), 1.65 (3H, d, J = 6.0 Hz, H-18), 5.29 (1H, q, J = 6.0 Hz, H-19), 2.75 (1H, d, J = 10.0Hz, H-21a), 3.48 (1H, d, J = 10.0Hz, H-21b), 2.28 (3H, br s, H-CO2Me), 2.39 (3H, brs, H-NMe), 2.81 (1H, dd, J = 13.2, 6.4Hz, H-3a′), 3.10 (1H, m, H-3b′),
2.56 (1H, m, H-5a′), 2.66 (1H, m, H-5b′), 2.59 (1H, m, H-6a′), 2.71 (1H, m, H-6b′), 6.97 (1H, s, H-9′), 6.83 (1H, s, H-12′), 1.55 (1H, m, H-14′), 0.96 (1H, m, H-15a′), 1.59 (1H, m, H-15b′), 1.71 (1H, br d, J = 14.0 Hz, H-17a′),
2.55 (1H, m, H-17b′), 0.81 (3H, t, J = 7.2 Hz, H-18′), 1.32 (1H, m, H-19a′),
1.42 (1H, m, H-19b′), 1.24 (1H, m, H-20′), 3.40 (1H, brs, H-21′), 3.92 (3H, s, H-11′-OMe), 3.62(3H, s, H-CO2Me′). 13C NMR (CD3OD 100 MHz) d: 139.7 (C-2, s), 39.1 (C-3, d), 62.1 (C-5, d), 18.7 (C-6, t), 111.3 (C-7, s), 131.3 (C-8, s), 118.4 (C-9, d), 119.2 (C-10, d), 122.0 (C-11, d), 110.0 (C-12, d), 138.0 (C-13, s), 38.6 (C-14, t), 36.4 (C-15, d), 54.0 (C-16, s), 70.3 (C-17, t), 12.4 (C-18, q), 120.3 (C-19, d), 138.7 (C-20, s), 52.9 (C-21, t), 50.3 (C-CO2Me, q),
174.6 (C-CO2Me, s), 42.5 (C-NMe, q), 136.7 (C-2′, s), 54.5 (C-3′, t), 53.7 (C-5′, t), 22.7 (C-6′, t), 110.4 (C-7′, s), 123.5 (C-8′, s), 118.3 (C-9′, d), 128.5 (C-10′, s), 154.5 (C-11′, s), 93.9 (C-12′, d), 136.6 (C-13′, s), 28.6 (C-14′, d), 33.0 (C-15′, t), 56.1 (C-16′, s), 36.9 (C-17′, t),
12.1 (C-18′, q), 27.8 (C-19′, t), 39.9 (C-20′, d), 58.0 (C-21′, d), 56.2 (C-11′-OMe′, q), 52.8 (C-CO2Me′, q), 176.4 (C-CO2Me′,s).
8.4 Hz, H-6a), 3.51 (1H, m, H-6b), 7.51 (1H, dd, J = 5.6, 2.4 Hz, H-9), 6.98 (1H, m, H-10), 6.98 (1H, m, H-11), 7.10 (1H, dd, J = 6.4, 3.2 Hz, H-12), 2.00 (1H, m, H-14a), 2.48 (1H, m, H-14b), 3.62 (1H, dd, J = 12.0, 5.6 Hz, H-15),
3.56 (1H, d, J = 10.8 Hz, H-17a), 3.78 (1H, d, J = 10.8 Hz H-17b), 1.65 (3H, d, J = 6.0 Hz, H-18), 5.29 (1H, q, J = 6.0 Hz, H-19), 2.75 (1H, d, J = 10.0Hz, H-21a), 3.48 (1H, d, J = 10.0Hz, H-21b), 2.28 (3H, br s, H-CO2Me), 2.39 (3H, brs, H-NMe), 2.81 (1H, dd, J = 13.2, 6.4Hz, H-3a′), 3.10 (1H, m, H-3b′),
2.56 (1H, m, H-5a′), 2.66 (1H, m, H-5b′), 2.59 (1H, m, H-6a′), 2.71 (1H, m, H-6b′), 6.97 (1H, s, H-9′), 6.83 (1H, s, H-12′), 1.55 (1H, m, H-14′), 0.96 (1H, m, H-15a′), 1.59 (1H, m, H-15b′), 1.71 (1H, br d, J = 14.0 Hz, H-17a′),
2.55 (1H, m, H-17b′), 0.81 (3H, t, J = 7.2 Hz, H-18′), 1.32 (1H, m, H-19a′),
1.42 (1H, m, H-19b′), 1.24 (1H, m, H-20′), 3.40 (1H, brs, H-21′), 3.92 (3H, s, H-11′-OMe), 3.62(3H, s, H-CO2Me′). 13C NMR (CD3OD 100 MHz) d: 139.7 (C-2, s), 39.1 (C-3, d), 62.1 (C-5, d), 18.7 (C-6, t), 111.3 (C-7, s), 131.3 (C-8, s), 118.4 (C-9, d), 119.2 (C-10, d), 122.0 (C-11, d), 110.0 (C-12, d), 138.0 (C-13, s), 38.6 (C-14, t), 36.4 (C-15, d), 54.0 (C-16, s), 70.3 (C-17, t), 12.4 (C-18, q), 120.3 (C-19, d), 138.7 (C-20, s), 52.9 (C-21, t), 50.3 (C-CO2Me, q),
174.6 (C-CO2Me, s), 42.5 (C-NMe, q), 136.7 (C-2′, s), 54.5 (C-3′, t), 53.7 (C-5′, t), 22.7 (C-6′, t), 110.4 (C-7′, s), 123.5 (C-8′, s), 118.3 (C-9′, d), 128.5 (C-10′, s), 154.5 (C-11′, s), 93.9 (C-12′, d), 136.6 (C-13′, s), 28.6 (C-14′, d), 33.0 (C-15′, t), 56.1 (C-16′, s), 36.9 (C-17′, t),
12.1 (C-18′, q), 27.8 (C-19′, t), 39.9 (C-20′, d), 58.0 (C-21′, d), 56.2 (C-11′-OMe′, q), 52.8 (C-CO2Me′, q), 176.4 (C-CO2Me′,s).
[0022] Ervachinine D (4):light yellow amorphous powder; -43.4 (c 0.34, MeOH); UV (MeOH) λmax (log ε) 295 (4.12), 226 (4.72) nm; IR (KBr) vmax 3415,2927, 1722, 1629, 1464, and 1288 cm–1; ESIMS 677 [M + H]+; HRESIMS m/z 677.4080 (calcd for C42H53N4O4 [M + H]+, 677.4066). 1H NMR (CD3OD, 400 MHz).d: 5.13 (1H, br d, J =
13.0Hz, H-3), 3.78 (1H, t, J = 11.2 Hz, H-5), 3.26 (1H, dd, J = 14.8, 8.0 Hz, H-6a), 3.56 (1H, m, H-6b), 7.53 (1H, br d, J = 5.6 Hz, H-9), 7.00 (1H, m, H-10), 7.00 (1H, m, H-11), 7.10 (1H, dd, J = 5.6, 2.4 Hz, H-12), 2.01 (1H, m, H-14a), 2.51 (1H, m, H-14b), 3.63 (1H, m, H-15), 3.60 (1H, d, J = 12.0 Hz, H-17a), 3.81 (1H, d, J = 12.0 Hz, H-17b), 1.69 (3H, d, J = 6.4 Hz, H-18), 5.36 (1H, q, J = 6.4 Hz, H-19), 2.87 (1H, d, J = 13.2 Hz, H-21a), 3.57 (1H, d, J =
13.2 Hz, H-21b), 2.31 (3H, s, H-CO2Me), 2.49 (3H, s, H-NMe), 2.75 (1H, brd, J = 8.4 Hz, H-3a′), 2.90 (1H, brd, J = 8.4 Hz, H-3b′), 2.82 (1H, m, H-5a′),
3.04 (1H, m, H-5b′), 3.20 (1H, m, H-6a′), 3.00 (1H, m, H-6b′), 6.97 (1H, s, H-9′), 6.83 (1H, s, H-12′), 1.69 (1H, m, H-14′), 1.12 (1H, m, H-15a′),
1.79 (1H, m, H-15b′), 1.48 (1H, m, H-17a′), 2.01 (1H, m, H-17b′), 0.88 (3H, t, J = 6.8 Hz, H-18′), 1.41 (1H, m, H-19a′), 1.47 (1H, m, H-19b′),
1.53 (1H, m, H-20′), 2.70 (1H, brs, H-21′), 3.95 (3H, s, H-11′-OMe), 13C NMR (CD3OD 100MHz) d: 139.9 (C-2, s), 39.1 (C-3, d), 62.1 (C-5, d), 18.7 (C-6, t),
111.2 (C-7, s), 131.4 (C-8, s), 118.2 (C-9, d), 119.2 (C-10, d), 122.0 (C-11, d), 110.0 (C-12, d), 138.1 (C-13, s), 38.7 (C-14, t), 36.4 (C-15, d), 54.1 (C-16, s), 70.3 (C-17, t), 12.3 (C-18, q), 120.2 (C-19, d), 138.9 (C-20, s),
53.1 (C-21, t), 50.4 (C-CO2Me, q), 174.5 (C-CO2Me, s), 42.6 (C-NMe, q), 141.5 (C-2′, s), 50.6 (C-3′, t), 55.4 (C-5′, t), 21.3 (C-6′, t), 108.6 (C-7′, s), 124.7 (C-8′, s), 118.1 (C-9′, d), 128.2 (C-10′, s), 154.0 (C-11′, s), 93.7 (C-12′, d), 135.6 (C-13′, s), 27.7 (C-14′, d), 33.0 (C-15′, t),
41.9 (C-16′, d), 35.3 (C-17′, t), 12.3 (C-18′, q), 28.5 (C-19′, t), 43.3 (C-20′, d), 59.4 (C-21′, d), 56.3 (C-11′-OMe, q).
13.0Hz, H-3), 3.78 (1H, t, J = 11.2 Hz, H-5), 3.26 (1H, dd, J = 14.8, 8.0 Hz, H-6a), 3.56 (1H, m, H-6b), 7.53 (1H, br d, J = 5.6 Hz, H-9), 7.00 (1H, m, H-10), 7.00 (1H, m, H-11), 7.10 (1H, dd, J = 5.6, 2.4 Hz, H-12), 2.01 (1H, m, H-14a), 2.51 (1H, m, H-14b), 3.63 (1H, m, H-15), 3.60 (1H, d, J = 12.0 Hz, H-17a), 3.81 (1H, d, J = 12.0 Hz, H-17b), 1.69 (3H, d, J = 6.4 Hz, H-18), 5.36 (1H, q, J = 6.4 Hz, H-19), 2.87 (1H, d, J = 13.2 Hz, H-21a), 3.57 (1H, d, J =
13.2 Hz, H-21b), 2.31 (3H, s, H-CO2Me), 2.49 (3H, s, H-NMe), 2.75 (1H, brd, J = 8.4 Hz, H-3a′), 2.90 (1H, brd, J = 8.4 Hz, H-3b′), 2.82 (1H, m, H-5a′),
3.04 (1H, m, H-5b′), 3.20 (1H, m, H-6a′), 3.00 (1H, m, H-6b′), 6.97 (1H, s, H-9′), 6.83 (1H, s, H-12′), 1.69 (1H, m, H-14′), 1.12 (1H, m, H-15a′),
1.79 (1H, m, H-15b′), 1.48 (1H, m, H-17a′), 2.01 (1H, m, H-17b′), 0.88 (3H, t, J = 6.8 Hz, H-18′), 1.41 (1H, m, H-19a′), 1.47 (1H, m, H-19b′),
1.53 (1H, m, H-20′), 2.70 (1H, brs, H-21′), 3.95 (3H, s, H-11′-OMe), 13C NMR (CD3OD 100MHz) d: 139.9 (C-2, s), 39.1 (C-3, d), 62.1 (C-5, d), 18.7 (C-6, t),
111.2 (C-7, s), 131.4 (C-8, s), 118.2 (C-9, d), 119.2 (C-10, d), 122.0 (C-11, d), 110.0 (C-12, d), 138.1 (C-13, s), 38.7 (C-14, t), 36.4 (C-15, d), 54.1 (C-16, s), 70.3 (C-17, t), 12.3 (C-18, q), 120.2 (C-19, d), 138.9 (C-20, s),
53.1 (C-21, t), 50.4 (C-CO2Me, q), 174.5 (C-CO2Me, s), 42.6 (C-NMe, q), 141.5 (C-2′, s), 50.6 (C-3′, t), 55.4 (C-5′, t), 21.3 (C-6′, t), 108.6 (C-7′, s), 124.7 (C-8′, s), 118.1 (C-9′, d), 128.2 (C-10′, s), 154.0 (C-11′, s), 93.7 (C-12′, d), 135.6 (C-13′, s), 27.7 (C-14′, d), 33.0 (C-15′, t),
41.9 (C-16′, d), 35.3 (C-17′, t), 12.3 (C-18′, q), 28.5 (C-19′, t), 43.3 (C-20′, d), 59.4 (C-21′, d), 56.3 (C-11′-OMe, q).
[0023] 实施例2：
[0024] Ervachinines A-D（1-4）抗肿瘤药理作用实验结果：
[0025] 一、测定药物和化合物
[0026] 待测样品ervachinines A-D（1-4）由本发明实施例1制得。待测样品溶解于DMSO中，4℃保存，贮存浓度为40 μM；
[0027] 二、测试方法
[0028] 1. 接种细胞：用含10％胎牛血清的培养液（DMEM或者RMPI1640）配成单个细胞悬液，以每孔5000-10000个细胞接种到96孔板，每孔体积100μl，贴壁细胞提前12小时接种培养。
[0029] 2. 加入待测化合物溶液（固定浓度40μM初筛，在该浓度对肿瘤细胞生长抑制在50%附近的化合物设5个浓度进入梯度复筛），每孔终体积200μl，每种处理均设3个复孔。
[0030] 3. 显色：37摄氏度培养48小时后，每孔加MTT溶液20μl。继续孵育4小时，终止培养，吸弃孔内培养上清液，每孔加200μl的SDS溶液（10%），过夜孵育（温度37℃），使结晶物充分融解。
[0031] 4. 比色：选择595nm波长，酶联免疫检测仪（Bio-Rad 680）读取各孔光吸收值，记录结果，以浓度为横坐标，细胞存活率为纵坐标绘制细胞生长曲线，应用两点法（Reed and Muench法）计算化合物的IC50值。
[0032] 三、 实验结果
[0033] 表1. 化合物二聚吲哚生物碱ervachinines A-D及顺铂
[0034] 的抗肿瘤活性测试结果 (IC50, μM)
[0035]化合物 HL-60 SMMC-7721 A-549 MCF-7 SW480
1 3.58 3.35 2.86 3.20 2.39
2 11.77 11.40 15.98 15.17 15.33
3 0.84 3.46 3.25 3.25 3.66
4 3.41 4.63 3.10 11.76 3.63
顺铂（Cisplatin） 1.00 17.05 26.75 16.97 18.32
1 3.58 3.35 2.86 3.20 2.39
2 11.77 11.40 15.98 15.17 15.33
3 0.84 3.46 3.25 3.25 3.66
4 3.41 4.63 3.10 11.76 3.63
顺铂（Cisplatin） 1.00 17.05 26.75 16.97 18.32
[0036] 上述化合物对以上5个细胞株生长的半数抑制浓度（IC50）在0.66-23.41 mM之间，依据中国医药工业杂志1993，24：455-457，周建军等提出的评价抗肿瘤物质活性的改良MTT法得出结论：上述化合物（1，3，4）的活性数据显示了明显的抑制癌症的作用。本发明从产自云南西双版纳的中国狗牙花 (Ervatamia.chinensis )中分离鉴定的4个化学结构新颖的二聚单萜吲哚生物碱（1-4）经抗肿瘤活性筛选，发现该类化合物具有显著的体外肿瘤生长抑制活性，是一类有发展潜力的抗肿瘤候选药物。
[0037] 实施例3：
[0038] 按实施例1的方法先制得二聚吲哚生物碱ervachinines A-D（结构1-4所示），按常规分别加注射用水，精滤，灌封灭菌制成注射液。
[0039] 实施例4：
[0040] 按实施例1的方法先制得二聚吲哚生物碱ervachinines A-D（结构1-4所示），加入4%的有机酸或无机酸溶液，过滤，干燥，制成医学上可接受的盐。
[0041] 实施例5：
[0042] 按实施例1的方法先制得二聚吲哚生物碱ervachinines A-D（结构1-4所示），将其分别溶于无菌注射用水中，搅拌使溶，用无菌抽滤漏斗过滤，再无菌精滤，分装于2安瓿中，低温冷冻干燥后无菌熔封得粉针剂。
[0043] 实施例6：
[0044] 将所分离得到的二聚吲哚生物碱ervachinines A-D（结构1-4所示）分别与赋形剂重量比为9:1的比例加入赋形剂，制成粉剂。
[0045] 实施例7：
[0046] 按实施例1的方法先制得二聚吲哚生物碱ervachinines A-D（结构1-4所示），分别按其与赋形剂重量比为1:5-1:10的比例加入赋形剂，制粒压片。
[0047] 实施例8：