阅读说明：本技术 柔茎香茶菜甲素o-14苯甲酸酯化衍生物及其制备方法和用途 (O-14 benzoic acid esterified derivative of isodon softescens, preparation method and application thereof ) 是由 可钰 王淙 刘宏民 徐霞 胡天星 郑姿君 王妮 于 2020-06-03 设计创作，主要内容包括：本发明涉及天然产物及药物化学领域,公开柔茎香茶菜甲素O-14苯甲酸酯化衍生物类化合物及其制备方法和应用。其制备方法：以柔茎香茶菜甲素为起始原料,在不破坏其活性中心的前提下,经过与苯甲酸或取代苯甲酸发生O-14位酯化反应,得到柔茎香茶菜甲素O-14苯甲酸酯化衍生物。该类化合物具有抗癌活性和良好的稳定性,可用于制备抗肿瘤药物,应用于临床治疗食道癌、胃癌、原发性肝癌、胰腺癌、乳腺癌以及急性髓系白血病等。其具有如下通式：<Image he="100" wi="203" file="212139DEST_PATH_IMAGE002.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>。(The invention relates to the field of natural products and pharmaceutical chemistry, and discloses isodon malabarica O-14 benzoate derivative compounds, and a preparation method and application thereof. The preparation method comprises the following steps: the O-14 site esterification reaction is carried out on rabdosia leptandra komarovii A serving as a starting material and benzoic acid or substituted benzoic acid on the premise of not damaging the active center of the rabdosia leptandra komarovii A to obtain the O-14 benzoic acid esterified derivative of rabdosia leptandra komarovii A. The compound has anticancer activity and good activityThe stability of the compound can be used for preparing anti-tumor drugs, and is applied to clinical treatment of esophagus cancer, gastric cancer, primary liver cancer, pancreatic cancer, breast cancer, acute myeloid leukemia and the like. It has the following general formula:)

柔茎香茶菜甲素O-14苯甲酸酯化衍生物及其制备方法和用途

技术领域

本发明涉及天然产物及药物化学领域，具体涉及新型柔茎香茶菜甲素类化合物柔茎香茶菜甲素O-14苯甲酸酯化衍生物类化合物及其制备方法和应用。

背景技术

冬凌草，学名碎米桠，为唇形科香茶菜属植物，其最早于1972年从河南林县民间草药发掘而出，以往历代草本均无记载。该植物药用部位为茎和叶，含有单帖，倍半萜，二萜，三萜，挥发油，生物碱，氨基酸，多糖，黄酮及甾体等化学成分。药理活性数据指出冬凌草中的抗肿瘤活性成分主要为二萜类化合物。

本申请发明人在文献调研和总结前人工作的基础上，通过一系列方法从济源冬凌草中提取纯化得到骨架为对映-贝壳杉烯型二萜的本发明先导化合物：柔茎香茶菜甲素(Flexicaulin A)。体外活性研究表明，该先导化合具有良好的生物活性和较广的抗肿瘤谱。

α-亚基环戊酮结构作为该类二萜化合物抗肿瘤活性中心已经得到证实，该类化合物具有优越的药理活性，但是其较差的稳定性和其相对温和的抗肿瘤活性制约了其在临床上的应用与发展。此外，有关柔茎香茶菜甲素的衍生物合成及构效关系研究也相对较为薄弱。因此，研究柔茎香茶菜甲素的衍生物合成及化学结构修饰并研究其构效关系，从而进一步从中寻找化学性质更优越，抗肿瘤活性更高的新的化学实体具有重要的理论意义和实际应用价值。

发明内容

本发明目的在于提供一系列柔茎香茶菜甲素衍生物及其光学活性体或消旋体，非对映异构体混合物或药学可接受的盐，并提高其抗肿瘤作用及稳定性，为其在临床上的应用提供可能。

本发明的另一目的在于提供其制备方法以及其在制备抗肿瘤药物的应用。

为实现本发明目的，本发明将柔茎香茶菜甲素与苯甲酸或取代苯甲酸酯化得到系列衍生物，提高其稳定性，同时保留或增强其抗肿瘤活性。

本发明提供的柔茎香茶菜甲素O-14苯甲酸酯化衍生物结构通式如下所示：

其中R在苯环上单取代或双取代。可以为氢，直链或含支链C1-C10烷基，被卤素取代的直链或含支链的C1-C5烷基，卤素，甲氧基，三氟甲基，硝基，羟基，氨基，叠氮基，磺酸基等。

R优选：氢，C1-C3烷基，被卤素取代的直链C1-C3烷基，卤素，甲氧基，三氟甲基。

R更优选：氢，；甲基、氟、溴、氯、甲氧基、三氟甲基、氯甲基或溴甲基。

优选的R取代后的苯环表述如下：

本发明所述的柔茎香茶菜甲素O-14苯甲酸酯化衍生物通过如下合成路线得到：

柔茎香茶菜甲素(1)为原料，二氯甲烷(DCM)为溶剂，在搅拌条件下分别加入苯甲酸或取代苯甲酸，然后加入催化剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)，4-二甲氨基吡啶(DMAP)，N,N-二异丙基乙胺(DIPEA)，柱色谱纯化得到产物2(通式I)。

其中R定义同上。

本发明创新点及优点：以柔茎香茶菜甲素为起始原料，在不破坏其活性中心的前提下，与苯甲酸或取代苯甲酸发生O-14位酯化反应，得到目标产物，收率高，达45％以上。该类化合物具有良好的抗癌活性和稳定性，可用于制备抗肿瘤药物，应用于临床作为治疗食道癌、胃癌、原发性肝癌、胰腺癌、乳腺癌以及急性髓系白血病等药物，具有很好的开发前景。

具体实施方式

通过以下具体实例进一步举例说明本发明，但应注意本发明的范围并不接受这些实施例的任何限制。

实施例1：

将100mg(0.25mmol)柔茎香茶菜甲素(1)溶解于8mL二氯甲烷(DCM)中，在搅拌条件下加入46mg(0.38mmol)的苯甲酸，然后加入催化剂EDCI 48mg(0.25mmol)，DMAP 3mg(0.025mmol)，DIPEA 32mg(0.25mmol)，TLC板检测反应完成后，加入二氯甲烷30mL稀释反应体系，并用饱和氯化铵溶液洗涤反应体系三次，合并水层并反萃一次，合并有机相，无水硫酸钠干燥，浓缩，柱色谱纯化得白色固体产物I-1。¹H NMR(400MHz,DMSO-d₆)δ7.84(d,J＝7.9Hz,2H),7.61(t,J＝7.3Hz,1H),7.49(t,J＝7.7Hz,2H),5.86(s,2H),5.42(s,1H),4.94(d,J＝12.3Hz,1H),4.53(d,J＝12.5Hz,1H),4.37(d,J＝4.3Hz,2H),3.14(d,J＝3.6Hz,1H),2.65(dt,J＝13.8,4.1Hz,1H),2.15(s,3H),2.03–1.93(m,2H),1.86–1.60(m,4H),1.49(q,J＝16.5,14.5Hz,4H),1.14(d,J＝11.7Hz,1H),0.99(d,J＝12.4Hz,1H),0.94(s,3H),0.93(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ204.15,170.28,165.96,148.33,132.50,130.94,129.24(×2),128.17(×2),112.80,76.94,72.07,66.09,62.80,62.01,59.16,52.93,42.99,41.14,40.67,38.98,33.74,32.93,32.51,27.93,22.20,20.77,17.74.HR-MS(ESI):Calculated for C₂₉H₃₆NaO₇[M+Na]⁺:519.2359,found 519.2390。收率89％。

实施例2：

用53mg(0.38mmol)2-氟苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-2。¹H NMR(400MHz,DMSO-d₆)δ7.70(td,J＝7.7,1.9Hz,1H),7.58(tdd,J＝7.0,4.9,1.9Hz,1H),7.28–7.21(m,2H),5.78(s,2H),5.34(s,1H),4.89(d,J＝12.5Hz,1H),4.49–4.42(m,2H),4.32(d,J＝1.9Hz,1H),3.11–3.06(m,1H),2.60(dt,J＝14.1,4.2Hz,1H),2.11(s,3H),2.01–1.88(m,2H),1.76(q,J＝12.5Hz,1H),1.62(d,J＝25.1Hz,2H),1.53–1.38(m,3H),1.34–1.14(m,2H),1.12–1.06(m,1H),0.99–0.92(m,1H),0.90(s,3H),0.88(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ204.05,170.31,163.17,159.59,148.15,134.38,132.05,124.04,119.36,116.42,112.87,77.23,72.03,66.11,62.77,61.93,59.12,52.88,42.84,41.13,40.65,38.94,33.74,32.85,32.50,27.90,22.21,20.79,17.71.HR-MS(ESI):Calculatedfor C₂₉H₃₅FNaO₇[M+Na]⁺:537.2265,found 537.2253。收率88％。

实施例3：

用60mg(0.38mmol)2-氯苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-3。¹H NMR(400MHz,DMSO-d₆)δ7.70(d,J＝7.8Hz,1H),7.51–7.47(m,2H),7.36(dt,J＝8.3,4.3Hz,1H),5.78(d,J＝14.6Hz,2H),5.32(s,1H),4.92(d,J＝12.4Hz,1H),4.57(dd,J＝4.3,1.7Hz,1H),4.45(d,J＝12.5Hz,1H),4.30(d,J＝2.0Hz,1H),3.08(d,J＝3.9Hz,1H),2.61(dt,J＝14.1,4.3Hz,1H),2.11(d,J＝1.8Hz,3H),1.98–1.86(m,2H),1.77(q,J＝12.4Hz,1H),1.68–1.58(m,2H),1.54–1.29(m,4H),1.24(dd,J＝11.1,5.2Hz,1H),1.10(d,J＝10.7Hz,1H),0.94(d,J＝8.8Hz,1H),0.91(s,3H),0.88(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ203.91,170.31,164.43,148.08,132.62,132.02,130.40,130.32,126.79,112.99,77.68,72.12,66.14,62.77,61.94,59.20,52.89,42.83,41.21,40.66,38.96,33.74,32.91,32.51,27.87,22.23,20.80,17.74.HR-MS(ESI):Calculated for C₂₉H₃₅ClNaO₇[M+Na]⁺:553.1969,found 553.2032。收率88％。

实施例4：

用76mg(0.38mmol)2-溴苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-4。¹H NMR(400MHz,DMSO-d₆)δ7.69–7.65(m,2H),7.43–7.37(m,2H),5.81–5.78(m,1H),5.75(d,J＝5.3Hz,2H),5.32(s,1H),4.93(d,J＝12.5Hz,1H),4.61(d,J＝4.3Hz,1H),4.43(d,J＝12.4Hz,1H),4.32(d,J＝2.0Hz,1H),3.13–3.08(m,1H),2.61(dt,J＝13.8,4.0Hz,1H),2.12(s,3H),1.97–1.87(m,2H),1.83–1.57(m,4H),1.44(td,J＝18.6,16.6,10.8Hz,3H),1.10(dd,J＝12.8,2.3Hz,1H),0.94(d,J＝8.9Hz,1H),0.91(s,3H),0.88(s,4H).¹³CNMR(101MHz,DMSO-d₆)δ203.90,170.32,165.03,148.04,133.53,132.66,132.40,132.06,127.29,120.51,113.06,77.79,72.12,66.12,62.76,61.91,59.20,54.86,52.87,42.78,41.22,40.65,33.74,32.91,32.52,27.85,22.24,20.81,17.73.HR-MS(ESI):Calculatedfor C₂₉H₃₅BrNaO₇[M+Na]⁺:597.1464,found 597.1508。收率83％。

实施例5：

用52mg(0.38mmol)2-甲基苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-5。¹H NMR(400MHz,DMSO-d₆)δ7.72–7.67(m,1H),7.37(td,J＝7.5,1.6Hz,1H),7.21(d,J＝7.7Hz,2H),5.78(d,J＝10.2Hz,2H),5.33(s,1H),4.91(d,J＝12.4Hz,1H),4.48(d,J＝12.2Hz,1H),4.42(d,J＝4.4Hz,1H),4.29(d,J＝2.0Hz,1H),3.08(d,J＝3.9Hz,1H),2.60(dt,J＝14.2,4.2Hz,1H),2.44(s,3H),2.11(s,3H),1.98–1.87(m,2H),1.80(q,J＝12.7Hz,1H),1.66–1.56(m,2H),1.54–1.32(m,4H),1.31–1.21(m,1H),1.09(d,J＝10.8Hz,1H),0.97–0.92(m,1H),0.90(s,3H),0.89(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ204.15,170.30,166.93,148.50,138.97,131.42,131.10,130.67,130.40,125.38,112.65,76.99,72.09,66.12,62.80,62.00,59.25,52.95,43.01,41.20,40.67,33.76,32.91,32.51,27.99,22.25,21.06,20.79,17.74.HR-MS(ESI):Calculated for C₃₀H₃₈NaO₇[M+Na]⁺:533.2595,found 533.2515。收率83％。

实施例6：

用72mg(0.38mmol)2-三氟甲基苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-6。¹H NMR(400MHz,DMSO-d₆)δ7.78(t,J＝8.8Hz,2H),7.70(dd,J＝6.3,2.7Hz,2H),5.81(s,1H),5.73(s,1H),5.30(s,1H),4.93(d,J＝12.5Hz,1H),4.68(d,J＝4.0Hz,1H),4.39(d,J＝12.5Hz,1H),4.34(s,1H),3.05(s,1H),2.63–2.54(m,1H),2.11(s,3H),1.93(d,J＝14.4Hz,2H),1.81–1.65(m,2H),1.59(s,2H),1.43(t,J＝16.9Hz,3H),1.17(d,J＝11.4Hz,1H),1.11(d,J＝12.4Hz,1H),0.95(s,1H),0.91(s,3H),0.89(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ203.71,170.30,165.50,147.84,132.26,131.38,131.31,130.85,126.86,126.15,124.76,122.04,113.08,78.11,72.10,66.09,62.70,61.90,59.19,52.85,42.74,41.22,40.64,33.72,32.99,32.52,27.88,22.20,20.77,17.74.HR-MS(ESI):Calculated for C₃₀H₃₅F₃NaO₇[M+Na]⁺:587.2233,found 587.2208。收率85％。

实施例7：

用73mg(0.38mmol)2,3-二氯苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-7。¹H NMR(400MHz,DMSO-d₆)δ7.77(dd,J＝8.0,1.8Hz,1H),7.63(dd,J＝7.8,1.7Hz,1H),7.40(t,J＝8.0Hz,1H),5.78(d,J＝18.8Hz,2H),5.33(s,1H),4.92(d,J＝12.3Hz,1H),4.66(d,J＝4.1Hz,1H),4.43(d,J＝12.5Hz,1H),4.33(d,J＝1.9Hz,1H),3.10(d,J＝4.1Hz,1H),2.61(dt,J＝14.1,4.1Hz,1H),2.12(s,3H),1.97–1.88(m,2H),1.83–1.58(m,4H),1.54–1.36(m,4H),1.10(d,J＝10.7Hz,1H),0.95(t,J＝9.4Hz,1H),0.91(s,3H),0.88(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ203.78,170.31,164.06,147.92,133.13,133.08,132.90,132.66,130.20,129.80,127.98,113.14,78.19,72.10,66.14,62.73,61.89,59.17,52.84,42.73,41.19,40.64,33.72,32.89,32.50,27.82,22.21,20.80,17.72.HR-MS(ESI):Calculated for C₂₉H₃₄Cl₂NaO₇[M+Na]⁺:587.1579,found 587.1633。收率67％。

实施例8：

用73mg(0.38mmol)2,4-二氯苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-8。¹H NMR(400MHz,DMSO-d₆)δ7.69(d,J＝8.5Hz,2H),7.50(dd,J＝8.5,2.0Hz,1H),5.78(d,J＝6.5Hz,2H),5.33(s,1H),4.91(d,J＝12.4Hz,1H),4.63(d,J＝4.1Hz,1H),4.43(d,J＝12.3Hz,1H),4.34–4.32(m,1H),3.13–3.06(m,1H),2.61(dt,J＝14.3,4.3Hz,1H),2.11(s,3H),2.03–1.85(m,2H),1.80–1.55(m,3H),1.52–1.27(m,4H),1.24–1.07(m,2H),0.95(d,J＝6.0Hz,1H),0.90(s,3H),0.88(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ203.87,170.32,163.66,147.97,136.73,133.40,132.36,129.94,129.08,127.20,113.11,77.89,72.07,66.11,62.73,61.88,59.14,52.83,42.72,41.16,40.63,38.92,33.73,32.86,32.50,27.80,22.22,20.80,17.71.HR-MS(ESI):Calculated for C₂₉H₃₄Cl₂NaO₇[M+Na]⁺:587.1579,found 587.1563。收率67％。

实施例9：

用71mg(0.38mmol)5-氯-2-甲氧基苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-9。¹H NMR(400MHz,DMSO-d₆)δ7.54–7.48(m,2H),7.11(d,J＝8.9Hz,1H),5.77(t,J＝1.2Hz,1H),5.74(d,J＝2.5Hz,2H),5.32(s,1H),4.89(d,J＝12.4Hz,1H),4.63(d,J＝4.4Hz,1H),4.43(d,J＝12.3Hz,1H),4.30(d,J＝2.0Hz,1H),3.78(s,3H),3.04(d,J＝4.3Hz,1H),2.59(dt,J＝13.8,4.3Hz,1H),2.10(s,3H),1.97–1.86(m,2H),1.78–1.69(m,1H),1.66–1.57(m,2H),1.53–1.33(m,4H),1.12–1.06(m,1H),0.94(d,J＝9.7Hz,1H),0.90(s,3H),0.87(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ204.07,170.29,163.49,156.94,148.10,132.37,130.53,123.44,122.24,114.19,112.96,77.31,72.17,66.17,62.79,61.96,59.15,56.04,54.85,52.91,42.87,41.18,40.65,33.73,32.91,32.50,27.85,22.20,20.78,17.73.HR-MS(ESI):Calculated for C₃₀H₃₇ClNaO₈[M+Na]⁺:583.2075,found583.2157。收率67％。

实施例10：

用53mg(0.38mmol)3-氟苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-10。¹H NMR(400MHz,DMSO-d₆)δ7.64(dt,J＝7.7,1.4Hz,1H),7.54–7.47(m,2H),7.46–7.40(m,1H),5.87–5.78(m,2H),5.39(s,1H),4.88(d,J＝12.4Hz,1H),4.48–4.42(m,2H),4.34(d,J＝2.0Hz,1H),3.12–3.08(m,1H),2.60(dt,J＝13.9,4.1Hz,1H),2.11(s,3H),1.99–1.87(m,2H),1.74(q,J＝12.7Hz,1H),1.65–1.30(m,6H),1.25–1.14(m,1H),1.09(dd,J＝12.7,2.2Hz,1H),0.98–0.92(m,1H),0.90(s,3H),0.88(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ204.06,170.28,164.88,162.84,160.41,148.20,133.30,130.50,125.38,119.64,115.62,112.95,77.34,72.00,66.06,62.77,61.96,59.11,52.88,42.87,41.10,40.65,33.72,32.92,32.49,27.86,22.17,20.77,17.72.HR-MS(ESI):Calculated for C₂₉H₃₅FNaO₇[M+Na]⁺:537.2265,found 537.2253。收率67％。

实施例11：

用60mg(0.38mmol)3-氯苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-11。¹H NMR(400MHz,DMSO-d₆)δ7.76–7.72(m,2H),7.67–7.63(m,1H),7.50(t,J＝7.9Hz,1H),5.84–5.79(m,2H),5.39(s,1H),4.88(d,J＝12.4Hz,1H),4.48–4.42(m,2H),4.34(d,J＝1.6Hz,1H),3.11(d,J＝3.9Hz,1H),2.59(dt,J＝14.2,4.3Hz,1H),2.11(s,3H),1.98–1.87(m,2H),1.73(q,J＝12.7Hz,1H),1.61(d,J＝13.7Hz,3H),1.53–1.37(m,4H),1.09(dd,J＝12.6,2.2Hz,1H),0.97–0.92(m,1H),0.89(s,3H),0.87(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ204.08,170.29,164.81,148.18,132.94,132.41,130.33,128.78,127.87,113.00,77.41,71.98,66.07,62.76,61.94,59.10,52.86,42.82,41.10,40.64,38.94,33.72,32.90,32.50,27.85,22.18,20.79,17.72.HR-MS(ESI):Calculated for C₂₉H₃₅ClNaO₇[M+Na]⁺:553.1969,found 553.1976。收率79％。

实施例12：

用76mg(0.38mmol)3-溴苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-12。¹H NMR(400MHz,DMSO-d₆)δ7.92(t,J＝1.9Hz,1H),7.85–7.80(m,2H),7.48(t,J＝7.9Hz,1H),5.86(d,J＝7.3Hz,2H),5.44(s,1H),4.93(d,J＝12.4Hz,1H),4.53–4.47(m,2H),4.39–4.36(m,1H),3.18–3.13(m,1H),2.64(dt,J＝14.1,4.3Hz,1H),2.15(s,3H),2.03–1.93(m,2H),1.83–1.62(m,4H),1.59–1.41(m,4H),1.13(dd,J＝12.8,2.0Hz,1H),1.02–0.96(m,1H),0.94(s,3H),0.92(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ204.06,170.27,164.71,148.19,135.28,133.12,131.67,130.56,128.23,121.33,112.98,77.43,71.99,66.08,62.76,61.95,59.10,52.87,42.82,41.10,40.65,38.96,33.72,32.91,32.50,27.85,22.18,20.78,17.72.HR-MS(ESI):Calculated for C₂₉H₃₅BrNaO₇[M+Na]⁺:597.1464,found 597.1476。收率84％。

实施例13：

用58mg(0.38mmol)3-甲氧基苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-13。¹H NMR(400MHz,DMSO-d₆)δ7.36(d,J＝6.8Hz,2H),7.33–7.31(m,1H),7.13(dt,J＝6.7,2.7Hz,1H),5.80(d,J＝8.7Hz,2H),5.37(s,1H),4.87(d,J＝12.4Hz,1H),4.47(d,J＝12.5Hz,1H),4.36(d,J＝4.4Hz,1H),4.33(d,J＝2.4Hz,1H),3.76(s,3H),3.08(d,J＝3.8Hz,1H),2.59(dt,J＝13.9,4.3Hz,1H),2.10(s,3H),1.98–1.87(m,2H),1.75(q,J＝12.7Hz,1H),1.61(d,J＝16.5Hz,3H),1.54–1.35(m,4H),1.09(d,J＝12.9Hz,1H),0.97–0.92(m,1H),0.89(s,3H),0.88(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ204.15,170.28,165.74,158.87,148.36,132.24,129.35,121.50,118.03,114.58,112.76,77.03,72.04,66.06,62.79,62.00,59.13,55.17,52.92,42.98,41.11,40.66,38.93,33.74,32.92,32.50,27.94,22.19,20.77,17.73.HR-MS(ESI):Calculated for C₃₀H₃₈NaO₈[M+Na]⁺:549.2464,found 549.2485。收率80％。

实施例14：

用52mg(0.38mmol)间甲基苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-14。¹H NMR(400MHz,DMSO-d₆)δ7.63–7.56(m,2H),7.37(d,J＝7.8Hz,1H),7.31(t,J＝7.6Hz,1H),5.80(d,J＝4.9Hz,2H),5.37(s,1H),4.88(d,J＝12.4Hz,1H),4.48(d,J＝12.4Hz,1H),4.29(dd,J＝7.3,3.0Hz,2H),3.08(d,J＝3.6Hz,1H),2.59(dt,J＝14.2,4.2Hz,1H),2.31(s,3H),2.10(s,3H),1.97–1.87(m,2H),1.76(q,J＝12.5Hz,1H),1.60(d,J＝16.1Hz,2H),1.54–1.33(m,4H),1.31–1.20(m,1H),1.08(d,J＝11.0Hz,1H),0.98–0.91(m,1H),0.89(s,3H),0.88(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ204.19,170.26,166.05,148.34,137.38,133.10,130.90,129.61,128.07,126.50,112.80,76.93,72.07,66.12,62.81,62.02,59.18,52.95,42.99,41.16,40.68,39.04,33.74,32.95,32.51,27.92,22.19,20.81,20.77,17.75.HR-MS(ESI):Calculated for C₃₀H₃₈NaO₇[M+Na]⁺:533.2515,found 533.2556。收率53％。

实施例15：

用72mg(0.38mmol)3-三氟甲基苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-15。¹H NMR(400MHz,DMSO-d₆)δ8.08–8.02(m,2H),7.95(d,J＝8.3Hz,1H),7.72(t,J＝7.8Hz,1H),5.84(d,J＝5.1Hz,2H),5.40(s,1H),4.89(d,J＝12.5Hz,1H),4.50–4.43(m,2H),4.34(s,1H),3.13(s,1H),2.64–2.56(m,1H),2.11(s,3H),1.98–1.88(m,2H),1.75(q,J＝12.5Hz,1H),1.62(d,J＝12.7Hz,2H),1.55–1.35(m,4H),1.21–1.15(m,1H),1.09(d,J＝12.5Hz,1H),0.95(d,J＝14.2Hz,1H),0.89(s,3H),0.88(s,4H).¹³C NMR(101MHz,DMSO)δ204.00,170.28,164.81,148.21,133.15,131.95,129.76,129.13,125.49,125.07,122.37,112.94,77.56,71.99,66.08,62.76,61.96,59.09,52.86,42.80,41.10,40.65,38.93,33.70,32.92,32.50,27.86,22.16,20.77,17.72.Calculated for C₃₀H₃₅F₃NaO₇[M+Na]⁺:587.2233,found 587.2211。收率88％。

实施例16：

用69mg(0.38mmol)3，4-二甲氧基苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-16。¹H NMR(400MHz,DMSO-d₆)δ7.41–7.36(m,2H),6.99(d,J＝8.4Hz,1H),5.79(d,J＝13.9Hz,2H),5.36(s,1H),4.86(d,J＝12.3Hz,1H),4.48(d,J＝12.7Hz,1H),4.30(dd,J＝6.5,3.1Hz,2H),3.79(s,3H),3.75(s,3H),3.08(d,J＝3.5Hz,1H),2.59(dt,J＝13.8,4.0Hz,1H),2.11(s,3H),1.98–1.88(m,2H),1.81–1.57(m,4H),1.55–1.36(m,4H),1.10(d,J＝12.1Hz,1H),0.94(d,J＝12.5Hz,1H),0.90(s,3H),0.88(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ204.24,170.26,165.62,152.43,148.49,148.02,123.23,123.16,112.62,112.20,110.82,76.75,72.10,66.07,62.81,62.08,59.71,59.15,55.62,55.41,52.98,43.09,41.10,40.67,33.75,32.98,32.51,27.98,22.18,20.76,17.75.HR-MS(ESI):Calculated for C₃₁H₄₀NaO₉[M+Na]⁺:579.2570,found 579.2644。收率75％。

实施例17：

用53mg(0.38mmol)4-氟苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-17。¹H NMR(400MHz,DMSO-d₆)δ7.86–7.81(m,2H),7.30–7.25(m,2H),5.82–5.78(m,2H),5.37(s,1H),4.88(d,J＝12.4Hz,1H),4.46(d,J＝12.7Hz,1H),4.36(d,J＝4.1Hz,1H),4.32(d,J＝2.0Hz,1H),4.07(d,J＝4.6Hz,1H),3.11–3.06(m,1H),2.59(dt,J＝13.8,4.1Hz,1H),2.10(s,3H),1.99–1.85(m,2H),1.73(q,J＝12.7Hz,1H),1.60(d,J＝15.8Hz,2H),1.44(q,J＝10.5Hz,3H),1.24–1.15(m,1H),1.08(dd,J＝12.7,1.9Hz,1H),0.97–0.91(m,1H),0.89(s,3H),0.87(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ204.11,170.28,165.05,163.41,148.25,132.06,131.96,127.53,115.40,115.18,112.88,77.06,72.02,66.06,62.77,61.97,59.11,52.89,42.92,41.11,40.65,38.96,33.73,32.90,32.50,27.89,22.19,20.78,17.72.HR-MS(ESI):Calculated for C₂₉H₃₅FNaO₇[M+Na]⁺:537.2265,found537.2250。收率75％。

实施例18：

用60mg(0.38mmol)4-氯苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-18。¹H NMR(400MHz,DMSO-d₆)δ7.78(d,J＝2.1Hz,1H),7.77(d,J＝2.0Hz,1H),7.53(d,J＝2.0Hz,1H),7.51(d,J＝2.0Hz,1H),5.81(d,J＝4.5Hz,2H),5.37(s,1H),4.88(d,J＝12.4Hz,1H),4.46(d,J＝12.5Hz,1H),4.36(d,J＝4.3Hz,1H),4.32(d,J＝2.1Hz,1H),3.11–3.07(m,1H),2.59(dt,J＝14.2,4.1Hz,1H),2.10(s,3H),1.98–1.88(m,2H),1.79–1.56(m,4H),1.54–1.36(m,4H),1.11–1.05(m,1H),0.97–0.91(m,1H),0.89(s,3H),0.87(s,4H).¹³CNMR(101MHz,DMSO-d₆)δ204.06,170.27,165.18,148.19,137.48,131.06(×2),129.77,128.41(×2),112.94,77.21,72.02,66.09,62.78,61.97,59.12,52.89,42.88,41.12,40.66,38.98,33.72,32.91,32.50,27.89,22.19,20.77,17.73.HR-MS(ESI):Calculatedfor C₂₉H₃₅ClNaO₇[M+Na]⁺:553.1969,found 553.2048。收率78％。

实施例19：

用76mg(0.38mmol)4-溴苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-19。¹H NMR(400MHz,DMSO-d₆)δ7.72–7.65(m,4H),5.80(d,J＝6.5Hz,2H),5.38(s,1H),4.88(d,J＝12.4Hz,1H),4.46(d,J＝12.4Hz,1H),4.37(d,J＝4.1Hz,1H),4.33(d,J＝2.1Hz,1H),3.10(d,J＝3.8Hz,1H),2.59(dt,J＝13.9,4.0Hz,1H),2.10(s,3H),1.98–1.86(m,2H),1.73(q,J＝12.6Hz,1H),1.60(d,J＝15.6Hz,3H),1.53–1.35(m,4H),1.08(dd,J＝12.7,2.2Hz,1H),0.94(dd,J＝13.0,4.3Hz,1H),0.89(s,3H),0.87(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ204.08,170.28,165.35,148.17,131.38(×2),131.20(×2),130.11,126.56,112.97,77.22,72.00,66.08,62.77,61.94,59.10,52.87,42.85,41.10,40.65,38.96,33.73,32.88,32.50,27.88,22.19,20.78,17.72.HR-MS(ESI):Calculated forC₂₉H₃₅BrNaO₇[M+Na]⁺:597.1464,found 597.1459。收率73％。

实施例20：

用58mg(0.38mmol)对甲氧基苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-20。¹H NMR(400MHz,DMSO-d₆)δ7.77–7.71(m,2H),6.99–6.94(m,2H),5.78(d,J＝10.5Hz,2H),5.36(s,1H),4.88(d,J＝12.4Hz,1H),4.47(d,J＝12.2Hz,1H),4.31(d,J＝2.0Hz,1H),4.27(d,J＝4.3Hz,1H),3.79(s,3H),3.06(d,J＝3.6Hz,1H),2.59(dt,J＝14.2,4.2Hz,1H),2.10(s,3H),2.00–1.87(m,2H),1.74(q,J＝12.4Hz,1H),1.60(d,J＝17.4Hz,3H),1.53–1.35(m,4H),1.08(dd,J＝12.7,2.1Hz,1H),0.96–0.91(m,1H),0.89(s,3H),0.87(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ203.16,169.16,164.46,161.47,147.30,130.16(×2),122.10,112.35(×2),111.59,75.46,70.96,64.95,61.69,60.88,58.03,54.25,51.82,41.92,40.01,39.55,37.86,32.63,31.80,31.39,26.82,21.09,19.65,16.62.HR-MS(ESI):Calculated for C₃₀H₃₈NaO₈[M+Na]⁺:549.2464,found 549.2465。收率66％。

实施例21：

用52mg(0.38mmol)对甲基苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-21。¹H NMR(400MHz,DMSO-d₆)δ7.70–7.66(m,2H),7.23(d,J＝8.3Hz,2H),5.79(d,J＝6.7Hz,2H),5.37(s,1H),4.88(d,J＝12.5Hz,1H),4.47(d,J＝12.7Hz,1H),4.31(d,J＝2.0Hz,1H),4.28(d,J＝4.4Hz,1H),3.07(d,J＝3.6Hz,1H),2.59(dt,J＝13.8,4.1Hz,1H),2.33(s,3H),2.10(s,3H),1.98–1.85(m,2H),1.75(q,J＝12.6Hz,1H),1.60(d,J＝15.8Hz,3H),1.54–1.34(m,4H),1.08(dd,J＝12.8,2.0Hz,1H),0.96–0.91(m,1H),0.89(s,3H),0.87(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ204.24,170.28,165.93,148.35,142.67,129.30(×2),128.71(×2),128.22,112.79,76.75,72.06,66.08,62.80,61.99,59.15,52.93,42.99,41.13,40.66,38.99,33.74,32.91,32.50,27.93,22.20,21.09,20.77,17.73.HR-MS(ESI):Calculated for C₃₀H₃₈NaO₇[M+Na]⁺:533.2515,found 533.2516。收率45％。

实施例22：

用72mg(0.38mmol)4-三氟甲基苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-22。¹H NMR(400MHz,DMSO-d₆)δ7.97(d,J＝8.2Hz,2H),7.84(d,J＝8.4Hz,2H),5.84(d,J＝4.8Hz,2H),5.39(s,1H),4.89(d,J＝12.3Hz,1H),4.48(d,J＝12.4Hz,1H),4.42(d,J＝4.4Hz,1H),4.34(s,1H),3.12(s,1H),2.61(dd,J＝14.0,4.9Hz,1H),2.11(s,3H),1.99–1.87(m,2H),1.75(q,J＝12.4Hz,1H),1.62(d,J＝14.2Hz,2H),1.44(q,J＝16.2,14.1Hz,4H),1.20(dt,J＝14.3,7.2Hz,1H),1.09(d,J＝12.3Hz,1H),0.95(d,J＝11.0Hz,1H),0.89(s,3H),0.88(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ203.99,170.28,165.01,148.11,134.65,130.01(×2),125.33,122.39,113.04,77.53,72.00,66.10,62.77,61.94,59.10,52.86,42.82,41.11,40.65,38.96,33.71,32.89,32.49,27.87,22.17,20.77,17.71.Calculatedfor C₃₀H₃₅F₃NaO₇[M+Na]⁺:587.2233,found 587.2239。收率89％。

实施例23：

用65mg(0.38mmol)对氯甲基苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-23。¹H NMR(400MHz,DMSO-d₆)δ7.71(d,J＝8.0Hz,2H),7.43(d,J＝8.3Hz,2H),5.76–5.72(m,2H),5.30(s,1H),4.82(d,J＝12.5Hz,1H),4.71(s,2H),4.40(d,J＝12.4Hz,1H),4.29–4.23(m,2H),3.02(d,J＝3.8Hz,1H),2.53(dt,J＝14.1,4.1Hz,1H),2.03(s,3H),1.95–1.81(m,3H),1.54(d,J＝14.7Hz,3H),1.47–1.30(m,4H),1.04–0.99(m,1H),0.87(dd,J＝13.1,4.6Hz,1H),0.82(s,3H),0.81(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ204.15,170.28,165.61,148.25,142.04,130.77(×2),129.59(×2),128.59,112.89,77.04,72.04,66.09,62.79,61.98,59.14,52.90,45.30,42.93,41.12,40.66,38.98,33.73,32.91,32.50,27.91,22.20,20.78,17.73.HR-MS(ESI):Calculated for C₃₀H₃₇ClNaO₇[M+Na]⁺:567.2126,found 567.2149。收率87％。

实施例24：

用82mg(0.38mmol)对溴甲基苯甲酸代替苯甲酸，其他操作同实施例1，得白色固体产物I-24。¹H NMR(400MHz,DMSO-d₆)δ7.78(d,J＝8.2Hz,2H),7.49(d,J＝8.3Hz,2H),5.81(s,2H),5.37(s,1H),4.88(d,J＝12.4Hz,1H),4.78(s,2H),4.47(d,J＝12.4Hz,1H),4.33(d,J＝7.0Hz,2H),3.08(d,J＝3.9Hz,1H),2.60(dt,J＝14.1,4.1Hz,1H),2.10(s,3H),1.98–1.86(m,2H),1.80–1.54(m,4H),1.53–1.30(m,4H),1.08(d,J＝11.9Hz,1H),0.94(dd,J＝13.4,3.8Hz,1H),0.89(s,3H),0.87(s,4H).¹³C NMR(101MHz,DMSO-d₆)δ204.14,170.28,165.61,148.26,142.04,130.77,129.59(×2),128.58(×2),112.89,77.04,72.04,66.09,62.79,61.99,59.14,52.91,45.30,42.94,41.13,40.67,38.99,33.73,32.92,32.50,27.91,22.20,20.78,17.73.HR-MS(ESI):Calculated for C₃₀H₃₇BrNaO₇[M+Na]⁺:611.1620,found 611.1664。收率66％。

实施例25：本发明合成的柔茎香茶菜甲素衍生物对人食管癌细胞EC109，食管癌细胞TE-1，胃癌细胞MGC-803，乳腺癌细胞MCF-7的抗肿瘤活性。

实验方法

选用以上四种癌细胞测所合成化合物的72h抗增殖活性；将对数生长期细胞用胰酶消化后配制成浓度为1-10×10⁴个/ml的细胞悬液，按1000-10000个细胞/孔接种于96孔板，每孔加100μl。将平板置37℃、体积百分比5％CO₂湿度培养箱中24h后，加入含有不同受试化合物浓度的培养液100μl，每个化合物按一定浓度梯度设平行的三个孔，对照组加空白的培养液100μl，再放入37℃、体积百分比5％CO₂湿度培养箱中孵育72h。用快速翻板法倒掉培养液，每孔加入用PBS缓冲液按5mg/ml新鲜配制的MTT溶液20μl，置37℃、体积百分比5％CO₂湿度培养箱温育4h，使MTT还原为Formazan，再次倒掉上清液，每孔加DMSO(二甲基亚枫)150μl，用平板摇床摇15min，摇匀后，使用酶标仪测定光密度值(OD)(检测波长570nm)，以溶剂对照处理细胞为对照组，按下述公式计算化合物对细胞的抑制率，并按中效方程计算半数抑制浓度IC₅₀：抑制率(％)＝(对照组OD均值-给药组OD均值)/对照组OD均值*100％

柔茎香茶菜甲素O-14苯甲酸酯化衍生物抗肿瘤活性

Oridonin为冬凌草甲素

FA为先导化合物柔茎香茶菜甲素(Flexicaulin A)

上述实验结果表明：本发明所述化合物具有较好的体外抗肿瘤活性和稳定性，可用于制备抗肿瘤药物，应用于临床治疗食道癌、胃癌、原发性肝癌、胰腺癌、乳腺癌以及急性髓系白血病等。以本发明化合物作为活性成分用于制备新的抗癌药物，具有潜在的应用价值。

稳定性实验评价

根据《中国药典》2015版稳定性评价实验原则，对本系列柔茎香茶菜甲素衍生物中活性最好的化合物I-23进行稳定性试验评价。稳定性试验包括影响因素试验，加速试验，长期试验。影响因素试验用一批原料药进行；加速试验与长期试验要求用三批供试品进行。实验名称，条件，仪器及结果如下表所示。

I-23稳定性评价实验(加速实验与长期实验)

I-23稳定性评价实验(影响因素实验)

其中光照实验在光照箱中进行。