TWI398277B - Compound for inhibiting proliferation of smooth muscle cell and pharmaceutical composition thereof - Google Patents
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本發明是有關於一種化合物及其醫藥組合物，特別是有關於一種用於抑制平滑肌細胞增生之化合物及其醫藥組合物。The present invention relates to a compound and a pharmaceutical composition thereof, and more particularly to a compound for inhibiting proliferation of smooth muscle cells and a pharmaceutical composition thereof.
血管平滑肌細胞位於血管的中皮層(media)，於一般的狀況下，血管平滑肌細胞受到周遭的細胞外間質(extracellular matrix,ECM)之調控，處於靜止狀態(quiescence state)，其增生和移動之能力都受到抑制，此時的血管平滑肌細胞具有收縮之作用，負責血管收縮之功能。Vascular smooth muscle cells are located in the mesoderm of the blood vessels. Under normal conditions, vascular smooth muscle cells are regulated by the extracellular matrix (ECM), in a quiescence state, and their proliferation and movement. The ability is inhibited, and the vascular smooth muscle cells at this time have a contracting effect and are responsible for the function of vasoconstriction.
在動脈粥狀硬化(atherosclerosis)、靜脈移植後閉合(vein graft occlusion)與支架內再狹窄(in-stent restenosis)等疾病發生過程中，當內皮細胞受到刺激而受損時，血管平滑肌細胞會受到許多外來物質之刺激，例如血小板衍生性生長因子、細胞激素與血小板反應蛋白-1(thrombospondin-1)等，會使血管平滑肌細胞從靜止狀態轉為增生狀態，並自中皮層移動至內皮層(intima)，使內管內膜增厚，造成血管狹窄。In the process of atherosclerosis, vein graft occlusion, and in-stent restenosis, when endothelial cells are stimulated and damaged, vascular smooth muscle cells are affected. Many foreign substances, such as platelet-derived growth factor, cytokines and thrombospondin-1, cause vascular smooth muscle cells to change from a resting state to a proliferative state and move from the middle cortex to the endothelial layer ( Intima) thickens the inner membrane of the inner tube, causing the blood vessels to narrow.
經皮冠狀動脈介入治療干預(percutaneous coronary intervention,PCI)目前廣泛的用於冠狀動脈栓塞性疾病之治療方式。傳統的裸金屬支架(bare-metal stent)在使用上常有支架內再狹窄之不良副作用。為了減低此不良副作用，隨後即開發出塗藥支架(drug-eluting stent)，利用塗於支架上之化合物控制其內混和的藥物釋放，直接讓藥物作用於支架之周圍，抑制血管平滑肌細胞之增生與移動，降低支架內再狹窄之發生。Percutaneous coronary intervention (PCI) is currently widely used for the treatment of coronary embolism. Conventional bare-metal stents often have the undesirable side effects of in-stent restenosis in use. In order to reduce this adverse side effect, a drug-eluting stent was developed, and the drug-applied compound was used to control the drug release in the mixture, and the drug was directly applied around the stent to inhibit the proliferation of vascular smooth muscle cells. With movement, reduce the occurrence of restenosis in the stent.
現今上市之塗藥支架主要使用之藥物為細胞穩定型之mTOR抑制劑(例如西羅莫司(Sirolimus)、依維莫司(Everolimus)等)與細胞毒殺型之太平洋紫杉醇(Paclitaxel)，其主要的作用均為抑制血管平滑肌細胞之異常增生，以達到減低血管內皮異常增厚之作用。然而，臨床上所使用之塗藥支架仍有例如延緩血管修復與造成支架血栓等問題需要解決，且其減低支架內再狹窄之作用，仍未盡善盡美，因此新型藥物之開發仍有其必要。The drugs currently used in the coated stents are cell-stable mTOR inhibitors (such as sirolimus, Everolimus, etc.) and cytotoxic Paclitaxel, which are mainly The role is to inhibit the abnormal proliferation of vascular smooth muscle cells, in order to reduce the abnormal thickening of the vascular endothelium. However, the clinically used coated stents still have problems such as delaying vascular repair and causing stent thrombosis, and their effects of reducing in-stent restenosis are still not perfect, so the development of new drugs is still necessary.
有鑑於上述習知技藝之問題，本發明之目的就是在提供一種用於抑制平滑肌細胞增生之化合物及其醫藥組合物，使本發明之化合物及其醫藥組合物應用於塗藥支架時，可抑制使用者血管平滑肌增生，進而達到降低血管再狹窄率。In view of the above-mentioned problems of the prior art, it is an object of the present invention to provide a compound for inhibiting proliferation of smooth muscle cells and a pharmaceutical composition thereof, which can inhibit the compound of the present invention and a pharmaceutical composition thereof when applied to an applicator stent The user's vascular smooth muscle hyperplasia, thereby reducing the rate of vascular restenosis.
根據本發明之目的，提出一種用於抑制平滑肌細胞增生之化合物，其為下式(1)所示，其中，n為1至10之整數，且R1 、R2 、R3 及R4 係分別選自氫、烷氧基、羥基、羧基、烷基、鹵烷基及氧基之其中之一。According to the object of the present invention, there is provided a compound for inhibiting proliferation of smooth muscle cells, which is represented by the following formula (1), wherein n is an integer of 1 to 10, and R 1 , R 2 , R 3 and R 4 are They are each selected from the group consisting of hydrogen, alkoxy, hydroxy, carboxy, alkyl, haloalkyl and oxy.
其中，當n=2時，本發明之化合物的結構係為以下式(2)至式(4)所示。當n=3時，本發明之化合物的結構係為以下式(5)至式(7)所示。當n=4時，本發明之化合物的結構係為以下式(8)至式(10)所示。Here, when n = 2, the structure of the compound of the present invention is represented by the following formulas (2) to (4). When n = 3, the structure of the compound of the present invention is represented by the following formulas (5) to (7). When n = 4, the structure of the compound of the present invention is represented by the following formulas (8) to (10).
此外，本發明更提出一種用於抑制平滑肌細胞增生之醫藥組合物，其包含上述之式(1)化合物及一醫藥學上可接受之賦形劑。其中，本發明之式(1)化合物進一步係為式(2)~式(10)化合物(其各結構如上所示)。醫藥學上可接受之賦形劑係選自由調味劑、防腐劑、抗氧化劑、螯合劑、等滲透劑、錠劑佐劑、著色劑、結合劑以及醫藥可相容載劑所組成之群組中。Further, the present invention further provides a pharmaceutical composition for inhibiting smooth muscle cell proliferation comprising the compound of the above formula (1) and a pharmaceutically acceptable excipient. Among them, the compound of the formula (1) of the present invention is further a compound of the formula (2) to the formula (10) (each structure of which is as shown above). The pharmaceutically acceptable excipient is selected from the group consisting of a flavoring agent, a preservative, an antioxidant, a chelating agent, a penetrant, a lozenge adjuvant, a coloring agent, a binding agent, and a pharmaceutically compatible carrier. in.
承上所述，依本發明之用於抑制平滑肌細胞增生之化合物及其醫藥組合物，其可具有一或多個下述優點：According to the present invention, a compound for inhibiting smooth muscle cell proliferation and a pharmaceutical composition thereof according to the present invention may have one or more of the following advantages:
(1) 當本發明之化合物及其醫藥組合物應用於塗藥支架時，其可抑制血管平滑肌增生，進而達到降低血管再狹窄率。(1) When the compound of the present invention and its pharmaceutical composition are applied to an applicator stent, it inhibits vascular smooth muscle hyperplasia and thereby reduces the rate of vascular restenosis.
(2) 本發明之化合物係以天然物蛇床子素(osthole為起始物，經由化學方式導入活性基團，蛇床子素(osthole)係為傳統中藥蛇床(Cnidium monnieri )之主要活性成分之一，因蛇床子素本身就具有多種藥理活性，故利用此蛇床子素與肉桂酸醯胺結合成本發明之化合物，對於抑制平滑肌細胞增生具有加乘效果。(2) The compound of the present invention is a natural active product of osthole, which is chemically introduced into an active group, and osthole is the main active ingredient of a traditional Chinese medicine snake bed ( Cnidium monnieri ). First, since osthole itself has various pharmacological activities, the combination of the osthole and the cinnamate cinnamate to the compound of the invention has an additive effect on inhibiting smooth muscle cell proliferation.
實施例1：本發明之化合物之結構Example 1: Structure of the compound of the present invention
本發明之用於抑制平滑肌細胞增生之化合物係為下式(1)所示之化合物，其中，n為1至10之整數，且R1 、R2 、R3 及R4 係分別選自氫、烷氧基、羥基、羧基、烷基、鹵烷基及氧基之其中之一。The compound for inhibiting smooth muscle cell proliferation of the present invention is a compound represented by the following formula (1), wherein n is an integer of 1 to 10, and R 1 , R 2 , R 3 and R 4 are each selected from hydrogen. One of an alkoxy group, a hydroxyl group, a carboxyl group, an alkyl group, a haloalkyl group and an oxy group.
較佳地，當n=2時，本發明之化合物的結構係為以下式(2)至式(4)所示。當n=3時，本發明之化合物的結構係為以下式(5)至式(7)所示。當n=4時，本發明之化合物的結構係為以下式(8)至式(10)所示(不以此為限)。Preferably, when n = 2, the structure of the compound of the present invention is represented by the following formulas (2) to (4). When n = 3, the structure of the compound of the present invention is represented by the following formulas (5) to (7). When n = 4, the structure of the compound of the present invention is represented by the following formula (8) to formula (10) (not limited thereto).
此外，本發明之用於抑制平滑肌細胞增生之醫藥組合物係包含上述之式(1)化合物及一醫藥學上可接受之賦形劑。其中，本發明之式(1)化合物進一步係為式(2)~式(10)化合物(其各結構如上所示)。醫藥學上可接受之賦形劑係選自由調味劑、防腐劑、抗氧化劑、螯合劑、等滲透劑、錠劑佐劑、著色劑、結合劑以及醫藥可相容載劑所組成之群組中，可將本發明之醫藥組合物製作為粉劑、液劑或濃稠乳劑等形式，以供醫事人員使用。Further, the pharmaceutical composition for inhibiting smooth muscle cell proliferation of the present invention comprises the compound of the above formula (1) and a pharmaceutically acceptable excipient. Among them, the compound of the formula (1) of the present invention is further a compound of the formula (2) to the formula (10) (each structure of which is as shown above). The pharmaceutically acceptable excipient is selected from the group consisting of a flavoring agent, a preservative, an antioxidant, a chelating agent, a penetrant, a lozenge adjuvant, a coloring agent, a binding agent, and a pharmaceutically compatible carrier. The pharmaceutical composition of the present invention can be prepared into a powder, a liquid or a thick emulsion for use by medical personnel.
實施例2：本發明之化合物之合成Example 2: Synthesis of a compound of the invention
本實施例係以蛇床子素(osthole)為起始物，進行結構修飾以得到具有不同碳鏈長度的化合物(14)~(16)，其合成如流程步驟1所示。蛇床子素(11,osthole)以附著於活性碳上之鈀金屬(palladium on carbon,Pd/C)為催化劑進行常壓下氫化反應，得到化合物(12)，產率為85%，其氫-核磁共振光譜(1 H-NMR)分析顯示兩個雙鍵被氫化而使δ6.82(1H,d),6.22(1H,d),5.21(1H,t)的峰值(peak)消失，並利用質譜(MS)所測得之分子量為249.2[M+H]+ 。接著，利用強路易士酸(Lewis acid)三溴化硼(BBr3 )進行去甲基化反應，在冰浴下慢慢滴入反應溶液後再回至室溫，得到去甲基化產物(13)，產率約為99%，其1 H-NMR分析顯示甲氧基(-OCH3 )的峰值δ3.82(s,3H)消失，其分子量為235.2[M+H]+ 。此步驟在激發(work-up)的過程原先在冰浴下以甲醇破壞，進而使內酯(lactone)開環後進一步使肉桂酸甲酯化，得到肉桂酸甲酯產物(methyl cinnamic ester)。爾後，以碳酸鉀(K2 CO3 )當鹼，將合成的化合物(13)與2~4個碳鏈長度的烷基二鹵化物(alkyl dihalide)進行烷基化反應(alkylation)，得到不同碳鏈長度的化合物(14)~(16)，其產率約為85-93%。In this example, osthole was used as a starting material to carry out structural modification to obtain compounds (14) to (16) having different carbon chain lengths, and the synthesis was as shown in Scheme 1. The osthole is hydrogenated under normal pressure by using palladium on carbon (Pd/C) attached to activated carbon to obtain compound (12) in a yield of 85%. Nuclear magnetic resonance spectroscopy ( 1 H-NMR) analysis showed that the two double bonds were hydrogenated and the peaks of δ 6.82 (1H, d), 6.22 (1H, d), 5.21 (1H, t) disappeared and utilized. The molecular weight measured by mass spectrometry (MS) was 249.2 [M+H] + . Next, demethylation reaction is carried out using Lewis acid boron tribromide (BBr 3 ), and the reaction solution is slowly added dropwise to the room temperature in an ice bath, and then returned to room temperature to obtain a demethylated product ( 13), the yield was about 99%, and its 1 H-NMR analysis showed that the peak δ 3.82 (s, 3H) of methoxy (-OCH 3 ) disappeared, and its molecular weight was 235.2 [M+H] + . This step is initially destroyed by methanol in an ice bath under a work-up process, and the lactone is further methylated after ring opening of the lactone to obtain a methyl cinnamic ester. Thereafter, alkylation of the synthesized compound (13) with an alkyl dihalide of 2 to 4 carbon chain lengths is carried out with potassium carbonate (K 2 CO 3 ) as a base to obtain a difference. The compound having a carbon chain length (14) to (16) has a yield of about 85 to 93%.
化合物(14)之1 H-NMR分析顯示乙烷基(ethyl group)之峰值出現在δ4.22(2 H,t,J=5.7 Hz),3.82(t,J=5.8 Hz,2H)，其分子量為296.1[M]+ 。化合物(15)之丙基(propyl group)之峰值出現在δ4.10(t,J=5.7 Hz,2H),3.76(t,J=6.2 Hz,2 H),2.25(m,2H)，其分子量為311.2[M+H]+ 。化合物(16)之丁基(butyl group)之峰值出現在δ3.99(t,J=5.7 Hz,2H)、3.62(t,J=6.0 Hz,2 H)及1.99(m,4H)，其分子量為324.2[M]+ 。 1 H-NMR analysis of the compound (14) showed that the peak of the ethyl group appeared at δ 4.22 (2H, t, J = 5.7 Hz), 3.82 (t, J = 5.8 Hz, 2H), The molecular weight was 296.1 [M] + . The peak of the propyl group of the compound (15) appears at δ 4.10 (t, J = 5.7 Hz, 2H), 3.76 (t, J = 6.2 Hz, 2 H), 2.25 (m, 2H), The molecular weight was 311.2 [M+H] + . The peak of the butyl group of the compound (16) appears at δ3.99 (t, J = 5.7 Hz, 2H), 3.62 (t, J = 6.0 Hz, 2 H) and 1.99 (m, 4H), which The molecular weight is 324.2 [M] + .
流程步驟1：Process step 1:
接續，以鄰位(ortho)、間位(mata)與對位(para)羥基(hydroxy)取代之肉桂酸(cinnamic acid)，在酸性條件下與甲醇分別進行甲酯化反應，得到甲酯化產物(17)~(19)，其產率為92-99%。化合物(20)、化合物(21)及化合物(22)之甲氧基(-OCH3 )在1 H-NMR分析顯示其峰值分別為δ3.82 ppm(s,3H),3.81 ppm(s,3H),3.80 ppm(s,3H)，其分子量為178.1[M]+ 。然後將化合物(20)~化合物(22)分別與不同碳鏈長度之化合物(14)~化合物(16)，在二-甲基甲醯胺(DMF)與碳酸鉀條件下，進行偶合反應(coupling reaction)，得到化合物(23)至化合物(31)。經1 H-NMR分析顯示其峰值確實為化合物(14)~化合物(16)與化合物(20)~化合物(22)互相偶合而成，其分子量為453.07[M+H]+ 。In the continuation, cinnamic acid substituted with ortho, mata and para hydroxy groups is subjected to methyl esterification under acidic conditions to obtain methyl esterification. The products (17) to (19) have a yield of 92-99%. The methoxy group (-OCH 3 ) of the compound (20), the compound (21) and the compound (22) showed peaks of δ 3.82 ppm (s, 3H) and 3.81 ppm (s, 3H) by 1 H-NMR analysis, respectively. ), 3.80 ppm (s, 3H), having a molecular weight of 178.1 [M] + . Then, the compound (20) to the compound (22) are separately coupled with the compound (14) to the compound (16) having different carbon chain lengths under the conditions of dimethylformamide (DMF) and potassium carbonate (coupling). Reaction), the compound (23) to the compound (31) are obtained. The 1 H-NMR analysis showed that the peak was obtained by coupling the compound (14) to the compound (16) and the compound (20) to the compound (22), and the molecular weight thereof was 453.07 [M+H] + .
化合物(23)~化合物(31)以氫氧化鋰(LiOH)產生皂化反應(saponification)，分別得到水解產物(23)~(31)，其甲氧基(-OCH3 )的訊號在1 H-NMR分析顯示，於δ3.80 ppm(s,3H)左右會消失，並利用高解析質譜(HR-MS)測得其分子量為452.2153。最後將肉桂酸衍生物(32)~(40)與氯甲酸乙酯(chloroethyl formate)反應，得到混合酸酐(mixed anhydride)中間體，以TLC偵測反應物耗盡後，即加入新鮮製備之羥胺游離鹼(hydroxylamine free base)溶於甲醇之溶液，進而得到本發明之化合物(41)~(49)。Compound (23) to compound (31) are saponification by lithium hydroxide (LiOH) to obtain hydrolyzate (23) to (31), respectively, and the signal of methoxy (-OCH 3 ) is at 1 H- NMR analysis showed disappearance at around δ 3.80 ppm (s, 3H) and its molecular weight was 452.2153 as determined by high-resolution mass spectrometry (HR-MS). Finally, the cinnamic acid derivatives (32)~(40) are reacted with chloroethyl formate to obtain a mixed anhydride intermediate. After the reactants are depleted by TLC, freshly prepared hydroxylamine is added. The hydroxyline free base is dissolved in a methanol solution to obtain the compounds (41) to (49) of the present invention.
再者，本實施例利用氫-核磁共振光譜(1 H-NMR)、碳13核磁共振光譜(13 C-NMR)及紅外線光譜儀(IR)，鑑定本發明之式(41)至式(49)之化合物的結構，其結果如下：Furthermore, this embodiment uses the hydrogen-nuclear magnetic resonance spectroscopy ( 1 H-NMR), carbon 13 nuclear magnetic resonance spectroscopy ( 13 C-NMR), and infrared spectrometer (IR) to identify the formula (41) to formula (49) of the present invention. The structure of the compound, the results are as follows:
本發明之式(41)化合物：1 H NMR(500 MHz,[D6 ]DMSO): δ=10.65(s,1H),10.44(s,1 H),7.72(d,J=15.9 Hz,1H),7.50(d,J=7.6 Hz,1H),7.33(t,J=7.5 Hz,1H),7.11(d,J=8.3 Hz,1H),6.97(t,J=7.5 Hz,1H),6.83(d,J=8.4 Hz,1H),6.47(d,J=15.8 Hz,1H),6.46(d,J=8.6 Hz,1H),4.35(t,J=6.3 Hz,2H),4.28(t,J=6.0 Hz,2H),2.71(t,J=7.0 Hz,2H),2.57(m,2H),2.23(t,J=7.1 Hz,2H),1.40(m,1H),1.20(m,2H),0.74(s,3H),0.73 ppm(s,3H)；13 C NMR(125 MHz,[D6 ]DMSO): δ=169.9,163.6,157.3,156.0,153.5,133.8,131.2,128.1,127.2,124.0,121.6,121.3,119.8,119.1,113.0,104.1,67.7,67.0,38.6,33.4,28.2,25.4,22.9,22.7,21.5 ppm；IR(KBr): νmax cm-1 =3647,1651,1488,752 cm-1 。The compound of the formula (41) of the present invention: 1 H NMR (500 MHz, [D 6 ] DMSO): δ = 10.65 (s, 1H), 10.44 (s, 1 H), 7.72 (d, J = 15.9 Hz, 1H) ), 7.50 (d, J = 7.6 Hz, 1H), 7.33 (t, J = 7.5 Hz, 1H), 7.11 (d, J = 8.3 Hz, 1H), 6.97 (t, J = 7.5 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.47 (d, J = 15.8 Hz, 1H), 6.46 (d, J = 8.6 Hz, 1H), 4.35 (t, J = 6.3 Hz, 2H), 4.28 ( t, J = 6.0 Hz, 2H), 2.71 (t, J = 7.0 Hz, 2H), 2.57 (m, 2H), 2.23 (t, J = 7.1 Hz, 2H), 1.40 (m, 1H), 1.20 ( m, 2H), 0.74 (s, 3H), 0.73 ppm (s, 3H); 13 C NMR (125 MHz, [D 6 ] DMSO): δ = 169.9, 163.6, 157.3, 156.0, 153.5, 133.8, 131.2, 128.1, 127.2, 124.0, 121.6, 121.3, 119.8, 119.1, 113.0, 104.1, 67.7, 67.0, 38.6, 33.4, 28.2, 25.4, 22.9, 22.7, 21.5 ppm; IR (KBr): ν max cm -1 = 3647, 1651, 1488, 752 cm -1 .
本發明之式(42)化合物：1 H NMR(500 MHz,CD3 OD): δ=7.91(d,J=15.9 Hz,1H),7.51(d,J=7.4 Hz,1H),7.30(t,J=7.6 Hz,1H),7.00(d,J=8.3 Hz,1H),6.93(t,J=7.4 Hz,1H),6.83(d,J=8.3 Hz,1H),6.50(d,J=15.9 Hz,1H),6.44(d,J=8.3 Hz,1H),4.26(t,J=6.1 Hz,2H),4.13(t,J=5.7 Hz,2H),2.82(t,J=7.2 Hz,2H),2.61(t,J=8.2 Hz,2H),2.36(t,J=7.1 Hz,2H),2.29(t,J=5.9 Hz,2H),1.52(m,1H),1.30(m,2H),0.88(s,3H),0.87 ppm(s,3H)；13 C NMR(125 MHz,[D6 ] DMSO): δ=169.8,159.3,156.0,153.4,138.7,136.7,130.5,130.4,127.2,121.4,120.3,119.9,118.7,116.1,113.6,103.7,64.8,64.5,38.8,33.4,29.3,28.4,25.4,23.0,21.6 ppm；IR(KBr): νmax cm-1 =3218,2951,1650,1490,1455,1246,1120,1072,751 cm-1 。The compound of the formula (42) of the present invention: 1 H NMR (500 MHz, CD 3 OD): δ = 7.91 (d, J = 15.9 Hz, 1H), 7.51 (d, J = 7.4 Hz, 1H), 7.30 (t) , J=7.6 Hz, 1H), 7.00 (d, J=8.3 Hz, 1H), 6.93 (t, J=7.4 Hz, 1H), 6.83 (d, J=8.3 Hz, 1H), 6.50 (d, J = 15.9 Hz, 1H), 6.44 (d, J = 8.3 Hz, 1H), 4.26 (t, J = 6.1 Hz, 2H), 4.13 (t, J = 5.7 Hz, 2H), 2.82 (t, J = 7.2) Hz, 2H), 2.61 (t, J = 8.2 Hz, 2H), 2.36 (t, J = 7.1 Hz, 2H), 2.29 (t, J = 5.9 Hz, 2H), 1.52 (m, 1H), 1.30 ( m, 2H), 0.88 (s, 3H), 0.87 ppm (s, 3H); 13 C NMR (125 MHz, [D 6 ] DMSO): δ = 169.8, 159.3, 156.0, 153.4, 138.7, 136.7, 130.5, 130.4, 127.2, 121.4, 120.3, 119.9, 118.7, 116.1, 113.6, 103.7, 64.8, 64.5, 38.8, 33.4, 29.3, 28.4, 25.4, 23.0, 21.6 ppm; IR (KBr): ν max cm -1 =3218, 2951, 1650, 1490, 1455, 1246, 1120, 1072, 751 cm -1 .
本發明之式(43)化合物：1 H NMR(500 MHz,CD3 OD): δ=7.92(d,J=16.0 Hz,1H),7.51(d,J=7.5 Hz,1H),7.31(t,J=7.6 Hz,1H),7.00(d,J=8.4 Hz,1H),6.94(t,J=7.4 Hz,1H),6.83(d,J=8.4 Hz,1H),6.51(d,J=15.9 Hz,1H),6.41(d,J=8.3 Hz,1H),4.14(t,J=6.1 Hz,2H),3.99(t,J=6.0 Hz,2H),2.82(t,J=7.2 Hz,2H),2.62(m,2H),2.36(t,J=7.1 Hz,2H),2.07(m,2H),1.98(m,2H),1.54(m. 1H),1.32(dd,J=7.2,15.6 Hz,2H),0.92(s,3H),0.89 ppm(s,3H)；13 C NMR(125 MHz,[D6 ]DMSO): δ=169.9,163.6,157.3,156.2,153.4,134.0,131.2,128.5,127.2,123.8,121.2,119.9,118.7,113.0,103.8,68.1,67.5,38.8,33.4,28.3,26.2,26.0,25.4,23.1,22.9,21.9,21.6 ppm；IR(KBr): νmax cm-1 =2953,1652,1489,1246,1118,752 cm-1 。The compound of the formula (43) of the present invention: 1 H NMR (500 MHz, CD 3 OD): δ = 7.92 (d, J = 16.0 Hz, 1H), 7.51 (d, J = 7.5 Hz, 1H), 7.31 (t) , J=7.6 Hz, 1H), 7.00 (d, J=8.4 Hz, 1H), 6.94 (t, J=7.4 Hz, 1H), 6.83 (d, J=8.4 Hz, 1H), 6.51 (d, J =15.9 Hz, 1H), 6.41 (d, J = 8.3 Hz, 1H), 4.14 (t, J = 6.1 Hz, 2H), 3.99 (t, J = 6.0 Hz, 2H), 2.82 (t, J = 7.2) Hz, 2H), 2.62 (m, 2H), 2.36 (t, J = 7.1 Hz, 2H), 2.07 (m, 2H), 1.98 (m, 2H), 1.54 (m. 1H), 1.32 (dd, J = 7.2, 15.6 Hz, 2H), 0.92 (s, 3H), 0.89 ppm (s, 3H); 13 C NMR (125 MHz, [D 6 ] DMSO): δ = 169.9, 163.6, 157.3, 156.2, 153.4, 134.0, 131.2, 128.5, 127.2, 123.8, 121.2, 119.9, 118.7, 113.0, 103.8, 68.1, 67.5, 38.8, 33.4, 28.3, 26.2, 26.0, 25.4, 23.1, 22.9, 21.9, 21.6 ppm; IR (KBr): ν max cm -1 = 2953, 1652, 1489, 1246, 1118, 752 cm -1 .
本發明之式(44)化合物：1 H NMR(500 MHz,[D6 ]DMSO): δ=7.34(d,J=15.8 Hz,1H),7.30(t,J=7.8 Hz,1H),7.12(m,2H),6.95(d,J=8.2 Hz,1H),6.82(t,J=8.4 Hz,1H),6.44(d,J=15.8 Hz,1H),6.43(d,J=7.8 Hz,1H),4.28(t,J=6.1 Hz,2H),4.21(t,J=6.0 Hz,2H),2.69(t,J=7.2 Hz,2H),2.51(m,2H),2.22(t,J=7.2 Hz,2H),1.54(m,1H),1.24(dd,J=7.2,15.6 Hz,2H),0.79(s,3H),0.77 ppm(s,3H)；13 C NMR(125 MHz,[D6 ]DMSO): δ=169.8,163.2,159.3,156.0,153.5,138.7,136.7,130.5,127.2,121.6,120.3,119.9,119.1,116.2,113.8,104.2,67.6,67.1,38.6,33.4,28.2,25.4,22.9,22.8,21.6 ppm；IR(KBr): νmax cm-1 =3224,1652,1489,1257,1121,1058,784 cm-1 。The compound of the formula (44) of the present invention: 1 H NMR (500 MHz, [D 6 ] DMSO): δ = 7.34 (d, J = 15.8 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 7.12 (m, 2H), 6.95 (d, J = 8.2 Hz, 1H), 6.82 (t, J = 8.4 Hz, 1H), 6.44 (d, J = 15.8 Hz, 1H), 6.43 (d, J = 7.8 Hz) , 1H), 4.28 (t, J = 6.1 Hz, 2H), 4.21 (t, J = 6.0 Hz, 2H), 2.69 (t, J = 7.2 Hz, 2H), 2.51 (m, 2H), 2.22 (t , J = 7.2 Hz, 2H), 1.54 (m, 1H), 1.24 (dd, J = 7.2, 15.6 Hz, 2H), 0.79 (s, 3H), 0.77 ppm (s, 3H); 13 C NMR (125 MHz, [D 6 ] DMSO): δ = 169.8, 163.2, 159.3, 156.0, 153.5, 138.7, 136.7, 130.5, 127.2, 121.6, 120.3, 119.9, 119.1, 116.2, 113.8, 104.2, 67.6, 67.1, 38.6, 33.4 , 28.2, 25.4, 22.9, 22.8, 21.6 ppm; IR (KBr): ν max cm -1 = 3224, 1652, 1489, 1257, 1121, 1058, 784 cm -1 .
本發明之式(45)化合物：1 H NMR(500 MHz,CD3 OD): δ=7.51(d,J=15.8 Hz,1H),7.28(t,J=8.0 Hz,1H),7.11(d,J=7.6 Hz,1H),7.09(s,1H),6.95(d,J=8.1 Hz,1H),6.84(d,J=8.4 Hz,1H),6.43(d,J=15.8 Hz,1H),6.42(s,1H),4.21(t,J=6.1 Hz,2H),4.10(t,J=5.9 Hz,2H),2.82(t,J=7.2 Hz,2H),2.62(t,J=8.3 Hz,2H),2.36(t,J=7.2 Hz,2H),2.23(t,J=6.0 Hz,2H),1.52(m,2H),1.30(m,2H),0.88(s,3H),0.87 ppm(s,3H)；13 C NMR(125 MHz,[D6 ]DMSO): δ=169.9,163.6,157.3,156.0,153.4,133.9,131.2,128.8,127.2,123.7,121.3,120.1,118.7,112.8,103.7,65.3,64.5,38.8,33.4,29.3,28.4,25.4,23.1,22.9,21.6 ppm；IR(KBr): νmax cm-1 =3209,2951,1627,1491,1266,1159,1119,1078,1005,776 cm-1 。The compound of the formula (45) of the present invention: 1 H NMR (500 MHz, CD 3 OD): δ = 7.51 (d, J = 15.8 Hz, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.11 (d) , J = 7.6 Hz, 1H), 7.09 (s, 1H), 6.95 (d, J = 8.1 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H), 6.43 (d, J = 15.8 Hz, 1H ), 6.42 (s, 1H), 4.21 (t, J = 6.1 Hz, 2H), 4.10 (t, J = 5.9 Hz, 2H), 2.82 (t, J = 7.2 Hz, 2H), 2.62 (t, J =8.3 Hz, 2H), 2.36 (t, J = 7.2 Hz, 2H), 2.23 (t, J = 6.0 Hz, 2H), 1.52 (m, 2H), 1.30 (m, 2H), 0.88 (s, 3H) ), 0.87 ppm (s, 3H); 13 C NMR (125 MHz, [D 6 ] DMSO): δ = 169.9, 163.6, 157.3, 156.0, 153.4, 133.9, 131.2, 128.8, 127.2, 123.7, 121.3, 120.1, 118.7, 112.8, 103.7, 65.3, 64.5, 38.8, 33.4, 29.3, 28.4, 25.4, 23.1, 22.9, 21.6 ppm; IR (KBr): ν max cm -1 = 3209, 2951, 1627, 1491, 1266, 1159, 1119, 1078, 1005, 776 cm -1 .
本發明之式(46)化合物：1 H NMR(500 MHz,CD3 OD): δ=7.26(d,J=15.7 Hz,1H),7.30(t,J=7.8 Hz,1H),7.12(m,2H),6.95(d,J=8.2 Hz,1H),6.82(t,J=8.4 Hz,1H),6.44(d,J=15.8 Hz,1H),6.43(d,J=7.8 Hz,1H),4.28(t,J=6.1 Hz,2H),4.21(t,J=6.0 Hz,2H),2.69(t,J=7.2 Hz,2H),2.51(m,2H),2.22(t,J=7.2 Hz,2H),1.54(m,1H),1.24(dd,J=7.2,15.6 Hz,2H),0.79(s,3H),0.77 ppm(s,3H)；13 C NMR(125 MHz,[D6 ]DMSO): δ=169.9,163.6,157.5,156.3,153.4,144.4,133.9,131.2,128.8,126.9,121.2,120.1,118.7,117.3,112.8,103.7,67.3,67.5,38.8,34.8,28.4,26.1,25.7,23.0,22.9,21.6 ppm；IR(KBr): νmax cm-1 =3627,3564,1651,1540,1507,1455 cm-1 。The compound of the formula (46) of the present invention: 1 H NMR (500 MHz, CD 3 OD): δ = 7.26 (d, J = 15.7 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 7.12 (m) , 2H), 6.95 (d, J = 8.2 Hz, 1H), 6.82 (t, J = 8.4 Hz, 1H), 6.44 (d, J = 15.8 Hz, 1H), 6.43 (d, J = 7.8 Hz, 1H) ), 4.28 (t, J = 6.1 Hz, 2H), 4.21 (t, J = 6.0 Hz, 2H), 2.69 (t, J = 7.2 Hz, 2H), 2.51 (m, 2H), 2.22 (t, J) = 7.2 Hz, 2H), 1.54 (m, 1H), 1.24 (dd, J = 7.2, 15.6 Hz, 2H), 0.79 (s, 3H), 0.77 ppm (s, 3H); 13 C NMR (125 MHz, [D 6 ] DMSO): δ = 169.9, 163.6, 157.5, 156.3, 153.4, 144.4, 133.9, 131.2, 128.8, 126.9, 121.2, 120.1, 118.7, 117.3, 112.8, 103.7, 67.3, 67.5, 38.8, 34.8, 28.4 , 26.1, 25.7, 23.0, 22.9, 21.6 ppm; IR (KBr): ν max cm -1 = 3627, 3564, 1651, 1540, 1507, 1455 cm -1 .
本發明之式(47)化合物：1 H NMR(500 MHz,CD3 OD): δ=7.52(d,J=16.9 Hz,1H),7.49(d,J=8.9 Hz,2H),6.98(d,J=8.6 Hz,2H),6.86(d,J=8.4 Hz,1H),6.46(t,J=8.4 Hz,1H),6.32(d,J=15.8 Hz,1H),4.33(t,J=4.7 Hz,2H),4.25(t,J=4.2 Hz,2H),2.84(t,J=7.2 Hz,2H),2.61(m,2H),2.37(t,J=7.2 Hz,2H),1.50(m,1H),1.32(dd,J=7.1,15.6 Hz,2H),0.85(s,3H),0.84 ppm(s,3H)；13 C NMR(125 MHz,[D6 ]DMSO): δ=169.8,160.1,156.0,153.5,138.5,129.5,128.0,127.2,121.6,119.1,117.0,115.3,104.2,67.2,67.1,38.6,33.4,28.2,25.4,22.9,21.5 ppm；IR(KBr): νmax cm-1 =2951,1601,1511,1256,1176,1127,1069,835 cm-1 。The compound of the formula (47) of the present invention: 1 H NMR (500 MHz, CD 3 OD): δ = 7.52 (d, J = 16.9 Hz, 1H), 7.49 (d, J = 8.9 Hz, 2H), 6.98 (d) , J=8.6 Hz, 2H), 6.86 (d, J=8.4 Hz, 1H), 6.46 (t, J=8.4 Hz, 1H), 6.32 (d, J = 15.8 Hz, 1H), 4.33 (t, J =4.7 Hz, 2H), 4.25 (t, J = 4.2 Hz, 2H), 2.84 (t, J = 7.2 Hz, 2H), 2.61 (m, 2H), 2.37 (t, J = 7.2 Hz, 2H), 1.50 (m, 1H), 1.32 (dd, J = 7.1, 15.6 Hz, 2H), 0.85 (s, 3H), 0.84 ppm (s, 3H); 13 C NMR (125 MHz, [D 6 ] DMSO): δ=169.8,160.1,156.0,153.5,138.5,129.5,128.0,127.2,121.6,119.1,117.0,115.3,104.2,67.2,67.1,38.6,33.4,28.2,25.4,22.9,21.5 ppm;IR(KBr): ν max cm -1 = 2951, 1601, 1511, 1256, 1176, 1127, 1069, 835 cm -1 .
本發明之式(48)化合物：1 H NMR(500 MHz,[D6 ]DMSO): δ=7.48(d,J=8.3 Hz,2H),7.39(d,J=15.7 Hz,1H,),7.03(d,J=8.4 Hz,1H),6.96(d,J=8.5 Hz,2H),6.74(d,J=8.4 Hz,1H),6.30(d,J=15.8 Hz,1H),4.18(t,J=6.0 Hz,2H),4.11(t,J=5.9 Hz,2H),2.86(t,J=7.4 Hz,2H),2.70(t,J=7.5 Hz,2H),2.57(t,J=7.8 Hz,2H),2.18(m,2H),1.45(m,1H),1.27(dd,J=7.1,15.6 Hz,2H),0.86(s,3H),0.84 ppm(s,3H)；13 C NMR(125 MHz,[D6 ]DMSO):δ=169.8,163.6,160.1,156.0,153.4,138.5,130.4,129.5,127.9,127.2,121.4,118.7,117.0,115.3,103.7,64.9,64.4,38.8,33.4,29.3,28.4,25.4,23.0.21.6 ppm；IR(KBr): νmax cm-1 =3205,2948,1644,1604,1513,1492,1467,1258,1176,1119,1062,821 cm-1 。The compound of the formula (48) of the present invention: 1 H NMR (500 MHz, [D 6 ] DMSO): δ = 7.48 (d, J = 8.3 Hz, 2H), 7.39 (d, J = 15.7 Hz, 1H,), 7.03 (d, J = 8.4 Hz, 1H), 6.96 (d, J = 8.5 Hz, 2H), 6.74 (d, J = 8.4 Hz, 1H), 6.30 (d, J = 15.8 Hz, 1H), 4.18 ( t, J = 6.0 Hz, 2H), 4.11 (t, J = 5.9 Hz, 2H), 2.86 (t, J = 7.4 Hz, 2H), 2.70 (t, J = 7.5 Hz, 2H), 2.57 (t, J = 7.8 Hz, 2H), 2.18 (m, 2H), 1.45 (m, 1H), 1.27 (dd, J = 7.1, 15.6 Hz, 2H), 0.86 (s, 3H), 0.84 ppm (s, 3H) 13 C NMR (125 MHz, [D 6 ] DMSO): δ = 169.8, 163.6, 160.1, 156.0, 153.4, 138.5, 130.4, 129.5, 127.9, 127.2, 121.4, 118.7, 117.0, 115.3, 103.7, 64.9, 64.4 , 38.8, 33.4, 29.3, 28.4, 25.4, 23.0.21.6 ppm; IR (KBr): ν max cm -1 = 3205, 2948, 1644, 1604, 1513, 1492, 1467, 1258, 1176, 1119, 1062, 821 Cm -1 .
本發明之式(49)化合物：1 H NMR(500 MHz,[D6 ]DMSO): δ=7.51(d,J=15.8 Hz,1H),7.47(d,J=8.4 Hz,2H),6.92(d,J=8.5 Hz,2H),6.83(d,J=8.3 Hz,1H),6.40(d,J=8.4 Hz,1H),6.31(d,J=15.7 Hz,1H),4.08(t,J=5.8 Hz,2H),3.97(t,J=5.8 Hz,2H),2.83(t,J=7.2 Hz,2H),2.63(t,J=7.9 Hz,2H),2.37(t,J=7.2 Hz,2H),1.98(m,2H),1.94(m,2H),1.55(m,1H),1.34(dd,J=7.2,9.8 Hz,2H),0.92(s,3H),0.91 ppm(s,3H)；13C NMR(125 MHz,[D6 ]DMSO):δ=169.9,160.2,156.2,153.4,138.6,129.5,127.8,127.2,121.2,118.7,116.9,115.3,103.8,67.8,67.5,38.8,33.4,28.4,26.1,26.0,25.4,23.0,21.6 ppm；IR(KBr): νmax cm-1 =3218,1603,1511,1256,1173,1117,1056,823 cm-1 。The compound of the formula (49) of the present invention: 1 H NMR (500 MHz, [D 6 ] DMSO): δ = 7.51 (d, J = 15.8 Hz, 1H), 7.47 (d, J = 8.4 Hz, 2H), 6.92 (d, J = 8.5 Hz, 2H), 6.83 (d, J = 8.3 Hz, 1H), 6.40 (d, J = 8.4 Hz, 1H), 6.31 (d, J = 15.7 Hz, 1H), 4.08 (t , J = 5.8 Hz, 2H), 3.97 (t, J = 5.8 Hz, 2H), 2.83 (t, J = 7.2 Hz, 2H), 2.63 (t, J = 7.9 Hz, 2H), 2.37 (t, J = 7.2 Hz, 2H), 1.98 (m, 2H), 1.94 (m, 2H), 1.55 (m, 1H), 1.34 (dd, J = 7.2, 9.8 Hz, 2H), 0.92 (s, 3H), 0.91 Pd (s, 3H); 13C NMR (125 MHz, [D 6 ] DMSO): δ = 169.9, 160.2, 156.2, 153.4, 138.6, 129.5, 127.8, 127.2, 121.2, 118.7, 116.9, 115.3, 103.8, 67.8, 67.5, 38.8, 33.4, 28.4, 26.1, 26.0, 25.4, 23.0, 21.6 ppm; IR (KBr): ν max cm -1 = 3218, 1603, 1511, 1256, 1173, 1117, 1056, 823 cm -1 .
依據上述各種實驗結果整理如下表1所示，進而判定本發明之式(41)至式(49)之化合物的結構分別為氮-氫氧-2[2-(3,4-二氫-8-異戊-2-羰基-2氫-吡喃-7-氧基)乙氧基]肉桂醯胺(N -Hydroxy-2-[2-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)ethoxy]cinnamide)、氮-氫氧-2-[3-(3,4-二氫-8-異戊-2-羰基-2氫-吡喃-7-氧基)丙氧基]肉桂醯胺(N -Hydroxy-2-[3-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)propoxy]cinnamide)、氮-氫氧-2-[4-(3,4-二氫-8-異戊-2-羰基-2氫-吡喃-7-氧基)丁氧基]肉桂醯胺(N -Hydroxy-2-[4-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)butoxy]cinnamide)、氮-氫氧-3-[2-(3,4-二氫-8-異戊-2-羰基-2氫-吡喃-7-氧基)乙氧基]肉桂醯胺(N -Hydroxy-3-[2-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)ethoxy]cinnamide)、氮-氫氧-3-[3-(3,4-二氫-8-異戊-2-羰基-2氫-吡喃-7-氧基)丙氧基]肉桂醯胺(N -Hydroxy-3-[3-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)propoxy]cinnamide)、氮-氫氧-3-[4-(3,4-二氫-8-異戊-2-羰基-2氫-吡喃-7-氧基)丁氧基]肉桂醯胺(N -Hydroxy-3-[4-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)butoxy]cinnamide)、氮-氫氧-4-[2-(3,4-二氫-8-異戊-2-羰基-2氫-吡喃-7-氧基)乙氧基]肉桂醯胺(N -Hydroxy-4-[2-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)ethoxy]cinnamide)、氮-氫氧-4-[3-(3,4-二氫-8-異戊-2-羰基-2氫-吡喃-7-氧基)丙氧基]肉桂醯胺(N -Hydroxy-4-[3-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)propoxy]cinnamide)、以及氮-氫氧-4-[4-(3,4-二氫-8-異戊-2-羰基-2氫-吡喃-7-氧基)丁氧基]肉桂醯胺(N -Hydroxy-4-[4-(3,4-Dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)butoxy]cinnamide)。According to the above various experimental results, as shown in the following Table 1, it was further determined that the structures of the compounds of the formula (41) to (49) of the present invention are respectively nitrogen-hydrogen-oxy-2[2-(3,4-dihydro-8). -isopent-2-carbonyl-2hydro-pyran-7-oxy)ethoxy]cinnamoamine ( N- Hydroxy-2-[2-(3,4-dihydro-8-isopentyl-2-oxo) -2H-chromen-7-yloxy)ethoxy]cinnamide), nitrogen-hydrogenoxy-2-[3-(3,4-dihydro-8-isopen-2-carbonyl-2-hydropyran-7-oxygen N- Hydroxy-2-[3-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)propoxy]cinnamide), nitrogen-hydrogen Oxy-2-[4-(3,4-dihydro-8-isopen-2-carbonyl-2-hydro-pyran-7-oxy)butoxy]cinnamoylamine ( N- Hydroxy-2-[ 4-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)butoxy]cinnamide), nitrogen-hydrogen oxy-3-[2-(3,4-dihydro-8-) Isovaler-2-carbonyl-2hydro-pyran-7-oxy)ethoxy]cinnamoamine ( N- Hydroxy-3-[2-(3,4-dihydro-8-isopentyl-2-oxo-) 2H-chromen-7-yloxy)ethoxy]cinnamide), nitrogen-hydrogenoxy-3-[3-(3,4-dihydro-8-isopen-2-carbonyl-2hydro-pyran-7-oxyl N- Hydroxy-3-[3-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)propoxy]c Innamide), nitrogen-hydrogen oxy-3-[4-(3,4-dihydro-8-isopen-2-carbonyl-2-hydropyran-7-oxy)butoxy]cinnamoylamine ( N -Hydroxy-3-[4-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)butoxy]cinnamide), nitrogen-hydrogen oxy-4-[2-(3,4 -Dihydro-8-isopent-2-carbonyl-2hydro-pyran-7-oxy)ethoxy]cinnamoamine ( N- Hydroxy-4-[2-(3,4-dihydro-8-) Isopentyl-2-oxo-2H-chromen-7-yloxy)ethoxy]cinnamide), nitrogen-hydrogenoxy-4-[3-(3,4-dihydro-8-isopen-2-carbonyl-2-hydro-pyridyl) N- Hydroxy-4-[3-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)propoxy]cinnamide And nitrogen-hydrogenoxy-4-[4-(3,4-dihydro-8-isopent-2-carbonyl-2hydro-pyran-7-oxy)butoxy]cinnamoylamine ( N -Hydroxy-4-[4-(3,4-Dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)butoxy]cinnamide).
於本實施例中，本發明之化合物(41)~(49)係為化合物(2)~(9)的R1 、R2 、R3 及R4 皆表示為氫，且n之數目為2~4之整數，不以此結構為限，亦可將氫修飾取代為烷氧基、羥基、羧基、烷基、鹵烷基及氧基之其中之一，更或者可減低n為1之整數、或增加n為5~10之整數。綜合以上，本發明之化合物(41)~(49)之結構如下所示：In the present embodiment, the compounds (41) to (49) of the present invention are the compounds (2) to (9), and R 1 , R 2 , R 3 and R 4 are all represented as hydrogen, and the number of n is 2 An integer of ~4, not limited to this structure, may also be substituted with a hydrogen modification to one of an alkoxy group, a hydroxyl group, a carboxyl group, an alkyl group, a haloalkyl group, and an oxy group, or may reduce the integer of n to 1. , or increase n to an integer from 5 to 10. In summary, the structures of the compounds (41) to (49) of the present invention are as follows:
實施例3：本發明之醫藥組合物用於抑制平滑肌細胞增生Example 3: The pharmaceutical composition of the invention is used for inhibiting smooth muscle cell proliferation
為了證明本發明之化合物(41)~(49)可用於抑制平滑肌細胞增生，因此檢測本發明之醫藥組合物對人類冠狀動脈平滑肌細胞增生的影響。In order to demonstrate that the compounds (41) to (49) of the present invention can be used for inhibiting smooth muscle cell proliferation, the effect of the pharmaceutical composition of the present invention on proliferation of human coronary artery smooth muscle cells was examined.
(1) 人類冠狀動脈平滑肌細胞之培養(1) Culture of human coronary artery smooth muscle cells
人類冠狀動脈平滑肌細胞(HCASMC)購自於PromoCell，取自於59歲高加索男性捐贈者之冠狀動脈。細胞培養採用Smooth Muscle Cell Growth Medium 2(PromoCell)培養基，內含5%培養基補充物，每2至3天更換培養基。待細胞貼附率達90-95%時繼代培養，除去培養基後，以PBS沖洗細胞乙次，吸去PBS後加入細胞分離溶液(Accutase)，於顯微鏡下觀察細胞去貼附後，加入新的培養基將細胞懸浮，稀釋至所需比例移至新的培養皿中進行培養。Human coronary artery smooth muscle cells (HCASMC) were purchased from PromoCell and taken from the coronary artery of a 59-year-old Caucasian male donor. The cell culture was performed using Smooth Muscle Cell Growth Medium 2 (PromoCell) medium containing 5% medium supplement, and the medium was changed every 2 to 3 days. When the cell attachment rate is 90-95%, the cells are subcultured. After removing the medium, the cells are washed twice with PBS, the PBS is aspirated, and the cell separation solution (Accutase) is added. After the microscope is observed, the cells are attached and added. The medium is suspended, diluted to the desired ratio and transferred to a new culture dish for culture.
(2) BrdU嵌入試驗(BrdU Incorporation Assay)與細胞毒性試驗(2) BrdU Incorporation Assay and Cytotoxicity Test
於本實施例中，利用BrdU嵌入試驗觀察細胞增生情形。將細胞懸浮於含有0.1%培養基補充物之培養基中，於96孔細胞培養盤之每個孔洞中加入1×104 個細胞，移至二氧化碳培養箱中，於37℃、5%二氧化碳與高濕度下培養24個小時，移除培養基並更換為含5%培養基補充物之培養基，同時加入本發明之化合物(分別為式(41)~(49)之化合物)，於培養箱中培養72個小時。達最終培養時間前12小時，加入BrdU試劑於各培養孔中繼續培養。待試驗完成後，收集上清培養基，進行乳酸去氫酶(lactosedehydrogenase,LDH)之活性偵測。隨後移除剩餘培養基，加入固定液進行固定，完成固定後，以沖洗液進行沖洗，加入BrdU偵測抗體，靜置於4℃冰箱中一夜。隔日，移除BrdU偵測抗體，以沖洗液沖洗後，加入呈色用二次抗體，室溫反應60分鐘後，移除二次抗體。接著，完成清洗後，加入呈色受質，避光反應30分鐘，加入停止液停止呈色反應，以多孔讀盤機進行偵測(偵測波長為450 nm，參考波長為550 nm)。In this example, the cell proliferation was observed using the BrdU insertion assay. The cells were suspended in a medium containing 0.1% medium supplement, and 1 × 10 4 cells were added to each well of a 96-well cell culture plate, and transferred to a carbon dioxide incubator at 37 ° C, 5% carbon dioxide and high humidity. The culture was carried out for 24 hours, the medium was removed and replaced with a medium containing 5% medium supplement, and the compound of the present invention (the compound of the formula (41) to (49), respectively) was added, and cultured in an incubator for 72 hours. . Twelve hours before the final incubation time, BrdU reagent was added to continue culture in each well. After the test is completed, the supernatant medium is collected for activity detection of lactose dehydrogenase (LDH). Subsequently, the remaining medium was removed, and the fixing solution was added for fixation. After the fixation was completed, the solution was washed with a rinse solution, BrdU was added to detect the antibody, and it was left to stand in a refrigerator at 4 ° C overnight. On the next day, the BrdU detection antibody was removed, washed with a rinse solution, and a secondary antibody for coloration was added thereto, and after reacting for 60 minutes at room temperature, the secondary antibody was removed. Then, after the cleaning was completed, the coloring substrate was added, and the reaction was protected from light for 30 minutes. The stop solution was added to stop the color reaction, and the detection was performed by a multi-layer disk reader (detection wavelength was 450 nm, reference wavelength was 550 nm).
細胞毒性以上清培養基中LDH之含量作為評斷，當LDH含量越高，表示待測樣品之細胞毒性越強。收集100 μl上清培養基，加入100 μl細胞毒性檢測套組(Cytotoxicity Detection Kit)試劑，室溫避光反應30分鐘後，以多孔讀盤機進行偵測(偵測波長為492 nm，參考波長為690 nm)，高控制組為加入細胞裂解緩衝液(lysis buffer)之控制組，且其細胞毒性訂為100%。The content of LDH in the cytotoxic supernatant medium was judged. The higher the LDH content, the stronger the cytotoxicity of the sample to be tested. Collect 100 μl of the supernatant medium, add 100 μl of Cytotoxicity Detection Kit reagent, and react at room temperature for 30 minutes in the dark. The detection is performed with a multi-well reader (detection wavelength is 492 nm, reference wavelength is 690 nm), the high control group was added to the control group of cell lysis buffer, and its cytotoxicity was set at 100%.
請參閱第1圖及第2圖，其係為本發明之化合物對抑制BrdU嵌入作用及細胞毒性之影響。圖中，靜止之細胞組係指其細胞處於靜止期，而非增生期。由圖中結果可知，本發明之化合物(42)、(43)、(44)、(45)、(47)、(48)與(49)在10 M的濃度下，可明顯看到具有明顯抑制BrdU嵌入之作用，化合物(46)之化合物亦有相同效果(無顯示於圖式中)。式(41)之化合物亦可看出有些微抑制之作用，但其強度不如其他化合物。本結果顯示，其對人類冠狀動脈平滑肌細胞之增生具有明顯抑制之作用。另外，所有化合物之細胞毒性，均與控制組相當，顯示本發明之各化合物於此濃度下，對細胞並無毒性。Please refer to Fig. 1 and Fig. 2, which are the effects of the compounds of the present invention on inhibiting BrdU insertion and cytotoxicity. In the figure, a resting cell group means that its cells are in a stationary phase, not a proliferative phase. As can be seen from the results in the figures, the compounds (42), (43), (44), (45), (47), (48) and (49) of the present invention are clearly visible at a concentration of 10 M. The compound of compound (46) also has the same effect (not shown in the figure) by inhibiting the effect of BrdU intercalation. The compound of formula (41) can also be seen to have some effect of microinhibition, but its strength is inferior to other compounds. The present results show that it has a significant inhibitory effect on the proliferation of human coronary artery smooth muscle cells. In addition, the cytotoxicity of all the compounds was comparable to that of the control group, indicating that each compound of the present invention is not toxic to cells at this concentration.
據上所述，本發明之化合物及其醫藥組合物對於細胞而言，並無明顯地毒性產生。因此，本發明之化合物及其醫藥組合物應用於塗藥支架時，可抑制使用者血管平滑肌增生，進而達到降低血管再狹窄率。According to the above, the compounds of the present invention and pharmaceutical compositions thereof are not significantly toxic to cells. Therefore, when the compound of the present invention and the pharmaceutical composition thereof are applied to an applicator stent, the vascular smooth muscle hyperplasia of the user can be suppressed, thereby reducing the rate of restenosis of the blood vessel.
以上所述僅為舉例性，而非為限制性者。任何未脫離本發明之精神與範疇，而對其進行之等效修改或變更，均應包含於後附之申請專利範圍中。The above is intended to be illustrative only and not limiting. Any equivalent modifications or alterations to the spirit and scope of the invention are intended to be included in the scope of the appended claims.
第1圖 係為本發明之化合物(42)、(45)、(47)、(48)及(49)對抑制BrdU嵌入作用及細胞毒性之影響。Fig. 1 is a graph showing the effects of the compounds (42), (45), (47), (48) and (49) of the present invention on inhibition of BrdU insertion and cytotoxicity.
第2圖 係為本發明之化合物(41)、(43)及(44)對抑制BrdU嵌入作用及細胞毒性之影響。Fig. 2 is a graph showing the effects of the compounds (41), (43) and (44) of the present invention on inhibition of BrdU insertion and cytotoxicity.
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