TWI754474B - 具選擇性第二型環氧合酶抑制活性之1,2,4-三氮唑衍生物及其用途 - Google Patents
具選擇性第二型環氧合酶抑制活性之1,2,4-三氮唑衍生物及其用途 Download PDFInfo
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Abstract
本專利申請案揭示一種1,2,4-三氮唑環類衍生物為新穎之選擇性第二型環氧合酶抑制劑，在體外對第二型環氧合酶具有良好抑制效果，在小鼠體內比市售藥物吲哚美辛還更具有抗發炎活性，並且不會有胃部損傷之副作用。
Description
本發明係關於一種三氮唑類衍生物；詳言之，本發明係關於一種具新型選擇性第二型環氧合酶抑制活性之1,2,4-三氮唑環類衍生物之用途。
已知大多數臨床使用的非甾體抗炎藥(NSAID)抑制環氧合酶的兩種同功型：COX-1(組成型)和COX-2(誘導型)。非選擇性NSAID的使用會同時作用於兩種環氧合酶亞型，並增加胃腸道(GI)並發症的風險(C.Sostres,Best Pract.Res.Clin.Gastroenterol,2010,24,121-132)。選擇性第二型環氧合酶抑制劑顯示出有效的抗炎活性，而不會引起明顯的胃腸道損傷(P.McGettigan,J.Am.Med.Assoc.,2006,296,1633-1644)。除此之外，第二型環氧合酶抑制劑也被報導具有抗癌之活性(C.G.Crosby,Expert Opinion on Emerging Drugs,2003,8,1-7)。因此，對於修飾選擇性第二型環氧合酶抑制劑之研究已引起人們的關注，成為一個重大挑戰。
據文獻報導，1,2,4-三氮唑環類衍生物具有廣泛的生物活性(Q.Zhang,J Med Chem.2006,49,4044-4047)。1,2,4-三氮唑環類衍生物對大鼠使用卡拉膠(Carrageenans)誘導之發炎反應，具有顯著的抗發炎效果，
其抗發炎效果與市售藥物吲哚美辛(Indomethacin)相當(A.M.Megee,European Journal of Medicinal Chemistry.2009,44,117-123.)。
本實驗室合成1,2,4-三氮唑環類衍生物，作為抗發炎藥物(S.M.,Li,Bioorganic Chemistry,2020,104,104333)。並於本專利申請案揭示一種新穎之選擇性第二型環氧合酶抑制劑，在體外對第二型環氧合酶具有抑制效果，在小鼠體內比市售藥物Indomethacin還更具有抗發炎活性，並且不會有胃部損傷之副作用。
本發明揭示一種新穎之選擇性第二型環氧合酶抑制劑，其包含投與治療上有效量之式(I)化合物，其立體化學異構物或其醫藥上可接受鹽類至有需求之個體：
其中，R1為獨立地為芳基、雜芳基、雜環基。R2獨立地為亞甲基鹵素，該鹵素為氟、氯、溴或碘。
下列描述僅在說明本發明之各具體實施例。因此，本文所論及之特定具體實施例或改良不可解釋為侷限本發明之範疇。熟習本領域之技術人員可顯見的是，所進行的各種變更或等同物並未背離本發明之範疇。
為了使本發明更明確且易於理解，必須先定義特定術語。額
外的定義係列於實施方式全文中。除非另有定義，本文所使用的技術性及科學性術語具有本發明領域之技術人員所能常規理解的意義。
本文所使用的冠詞「一」與「一者」是指一或大於一者(亦即，至少一者)的該冠詞語法對象。舉例而言，「一元件」是指一元件或大於一者之元件。
1.材料及方法
1.1 材料
本發明之所用測試藥品純度皆為95%以上，根據文獻方法合成(S.M.,Li,Bioorganic Chemistry,2020,104,104333)，對照藥品吲哚美辛及塞來昔布(celecoxib)購自Sigma Chemical Co.。COX抑制篩選檢測試劑盒(catalog no.560131)購自Cayman Chemical。雄性ICR小鼠購自BioLASCO Taiwan Co.,Ltd。
1.2 體外COX-1和COX-2抑制試驗
按照製造商建議的程序操作，使用酶免疫測定(EIA)試劑盒(編號560131)檢測受試化合物抑制COX-2和COX-1的能力(IC50值，μM)。半數最大抑制劑濃度IC50(μM)由Excel forecast函數計算出，並將IC50(COX-1)除以IC50(COX-2)計算出選擇性指數(SI)值。
1.3 卡拉膠誘發小鼠腳水腫發炎試驗
將小鼠(約29-39g)隨機分為6組(n=6隻小鼠/組)，並以低劑量(1.25mg/kg)，中劑量(2.5mg/kg)和高劑量(5mg/kg)的待測化合物加入1%CMC為測試組。陽性對照組和賦形劑對照組分別口服相同體積之1%CMC及相同體積含吲哚美辛10mg/kg之1%CMC。給藥
後三十分鐘，通過右足底注射50μL1%的卡拉膠於0.9%生理鹽水中誘發急性足水腫。使用Plethysmometer在注射前和注射後1、2、3、4和5小時的間隔測量腳掌的體積。
1.4 測量小鼠血清中一氧化氮(NO)濃度
在卡拉膠誘發小鼠腳水腫發炎試驗後抽取小鼠血液，經由格里斯反應(Griess reaction)的比色法測定，評估誘導發炎物質NO的產生。
1.5 酶聯免疫吸附法測定血清細胞激素
根據製造商的說明書，使用市售的ELISA試劑盒(Biosource International Inc.)測定小鼠的血清TNF-α和IL-6濃度。TNF-α和IL-6的濃度表示為pg/mL。
1.6 組織切片
小鼠抽完血後，在室溫下，將足部組織切片在1.85%甲醛及1%乙酸中固定1週，用乙醇脫水並包埋在石蠟中。組織切片用二甲苯脫蠟，並用蘇木精和曙紅染色以進行細胞計數。觀察所有樣品並用BH-2奧林巴斯顯微鏡拍照。
1.7 胃潰瘍保護活性
犧牲小鼠後，取出胃，延曲線切開，用蒸餾水洗滌，並浸入鹽水中輕輕清洗，進行組織病理學檢查以確認在治療的小鼠胃的胃粘膜層中的炎症反應程度。
2.下面所列的例子為1,2,4-三氮唑環類衍生物對COX-2選擇性抑制及小鼠體內的抗發炎相關實例，用以更詳細地解釋本發明；但本發明不為這些實例所限。
3.實施案例一至十一
本發明之實施實例一至十一(表1，列1-11)，將1,2,4-三氮唑環類衍生物1a-1k進行體外COX抑制測定，探索COX-1/COX-2選擇性之研究。COX-2抑制效果在苯環上取代位置的趨勢是間位>對位>鄰位(表1，列1-6)。化合物1b，1e，1g和1h在一號氮上苯環上具有間位取代基，包括氟、三氟甲基、氯和甲基，具有顯著COX-2抑制能力(0.00712、1.58、0.773和2.69μM，表1，列2、5、7和8)。結果表示與間位給電子基團相比，間位鹵代芳基的化合物對抑制COX-2的活性更有利(表8，列8)。氟原子更顯著有COX-2抑制活性。帶有間位和對位氟的化合物1b(0.00712μM)和1c(0.0179μM)是上市藥品塞來昔布的COX-2抑制活性0.05至7倍。在一號氮苯環上取代對於COX-2抑制的趨勢為：2,4-二氯<溴<氰<甲基<三氟甲基<氯<氟。
1,2,4-三氮唑環類衍生物普遍顯示出對COX-2的抑制作用比對COX-1的抑制作用強(表1)，顯示胃腸道毒性的發生率較低(S.Kawai,Inflammation Research,1998,47,102-106)。根據選擇性指數結果，1,2,4-三氮唑環類衍生物1c具有出色的選擇性(COX-1/COX-2=1080，表1，列3)和對COX-2的抑制活性。因此，1,2,4-三氮唑環類衍生物1c在體外試驗為安全且有效的選擇性COX-2抑制劑。
基於體外COX-2篩選結果，選擇本案實例三進行體內抗炎活性測試。使用卡拉膠誘導的小鼠足部水腫作為急性炎症模型，卡拉膠刺激3小時後，所有組均達到足部水腫的高峰(圖1)。未注射化合物的陰性對照組通過注射卡拉膠而增加了0.18毫升的足部體積。在陽性對照組中，吲哚美辛可在卡拉膠刺激3小時後有效減輕足部水腫。1,2,4-三氮唑環類衍生物1c顯示出劑量依賴性地減少小鼠足部水腫(圖1)。濃度為5毫克每公斤的1,2,4-三氮唑環類衍生物1c比對照組吲哚美辛10毫克每公斤表現出更多的抗炎活性(圖1)。
實例三中1,2,4-三氮唑環類衍生物1c在小鼠足部水腫實驗後，犧牲小鼠，抽取小鼠血液並離心除去上清液。透過Griess試劑測量一氧化氮釋放量。在圖2A中，卡拉膠注射後5小時，血清中的一氧化氮濃度顯著增加，代表發炎現象，然而1,2,4-三氮唑環類衍生物1c可以顯著逆轉一氧化氮濃度，並且有劑量依賴性。以5毫克每公斤劑量的1,2,4-三氮唑環類衍生物1c處理可將一氧化氮的血漿濃度顯著降低至86.4%，這類似於吲哚美辛組別(10毫克每公斤，圖2A)。同樣，在卡拉膠注射後5小時，水血液中TNF-α和IL-6濃度均顯著增加(p<0.001)，給予1,2,4-三氮唑環類衍生物1c組別，呈現劑量依賴性降低了TNF-α和IL-6濃度(圖2B和2C)。
小鼠足部水腫和炎症趨化因子實驗均顯示，一半劑量5毫克每公斤的1,2,4-三氮唑環類衍生物1c與正常劑量10毫克每公斤的吲哚美辛表現出相似抗發炎的結果。因此，使用較低劑量的1,2,4-三氮唑環類衍生物1c有著與市售藥相同的抗發炎活性，並且因為低劑量，副作用的風險更低。
犧牲後的小鼠進行足部切片確認對照組及控制組的發炎狀態。正常的足部結締組織並沒有細胞浸潤現象(圖3A)。相比之下，卡拉膠誘導的小鼠組別，足部結締組織中明顯的細胞浸潤，並且浸潤在膠原纖維和細胞間隙積累(圖3B)。上市藥物10毫克每公斤的吲哚美辛明顯的預防組織浸潤物蓄積(圖3C)。經1,2,4-三氮唑環類衍生物1c處理的小鼠的足部活檢顯示卡拉膠誘導的炎症反應呈劑量依賴性降低(圖3D-F)。在5毫克每公斤的化合物1c組中，炎症細胞顯著減少，並且幾乎未觀察到細胞浸潤在細胞間隙中，只侷限於血管區域(圖3F)。此外，在足部組織切片的棕色部分，觀察到許多COX-2免疫反應性細胞(圖3B-F)。然而，
給予2.5和5毫克每公斤的1,2,4-三氮唑環類衍生物1c及10毫克每公斤吲哚美辛皆顯著降低了足部中COX-2免疫反應細胞的增加(圖3C和3E-F)。
為了進一步證明三氮唑環類衍生物1c是否有造成胃潰瘍的副作用，口服給予1.25、2.5和5毫克每公斤的1,2,4-三氮唑環類衍生物1c以及10毫克每公斤吲哚美辛之組別，觀察小鼠胃粘膜的組織病理學觀察有無損傷，用於測定化合物的致潰瘍能力。正常胃黏膜並未觀察到上皮脫落和細菌脫落(圖4A)。使用卡拉膠誘導發炎的組別發現有幾個上皮脫落和細菌粘附(圖4B)。吲哚美辛組的切片與卡拉膠誘導的組別雖然相似，但具有輕度至中度的胃粘膜糜爛(圖4C)。小鼠胃的炎症反應程度，表明三氮唑環類衍生物1c組別沒有潰瘍、胃粘膜浸軟、壞死或淋巴細胞浸潤，這表明三氮唑環類衍生物1c的胃黏膜副作用遠低於上市藥吲哚美辛(圖4D-F)。
第(一)圖為卡拉膠(Carr)誘導發炎時間及小鼠腳掌體積變化(Change of edema volume)圖。
第(二)圖為化合物(Comp.)1c和吲哚美辛(Indo)對小鼠血清中(A)一氧化氮(Nitrite)，(B)TNF-α和(C)IL-6產生的影響。*，**和***分別表示與對照組相比p<0.05，p<0.01和p<0.001。
第(三)圖為卡拉膠(Carr)誘導發炎的足部水腫組織切片病理檢查。(A)對照組(僅生理食鹽水；Control)，(B)誘導組(Carr)，(C)吲哚美辛組(Indo)，(D)1.25毫克每公斤的1c組(E)2.5毫克每公斤的1c組(F)5毫克每公斤的1c組。
第(四)圖為卡拉膠(Carr)誘導發炎的胃黏膜組織切片病理檢查(A)對照組(僅生理食鹽水；Control)，(B)誘導組(Carr)，(C)吲哚美辛組(Indo)，(D)1.25毫克每公斤的1c組(E)2.5毫克每公斤的1c組(F)5毫克每公斤的1c組。
Claims (4)
- 一種使用如式(I)的1,2,4-三氮唑-3-羧酸甲酯類雜環化合物或其在藥學上可接受的鹽用於作為選擇性第二型環氧合酶抑制劑之用途：
- 一種使用含有如請求項1所述式(I)的化合物或其在藥學上可接受的鹽用於製造具第二型環氧合酶選擇性抑制的藥物之用途。
- 一種使用含有如請求項1所述式(I)的化合物或其在藥學上可接受的鹽用於製造可有效治療與第二型環氧合酶介導相關之疾病的藥物有效成分之用途。
- 一種使用含有如請求項1所述式(I)的化合物或其在藥學上可接受的鹽用於製造治療第二型環氧合酶介導之發炎、關節炎或發燒等作用的醫療組成物之用途。
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| EP1099695A1 (en) * | 1999-11-09 | 2001-05-16 | Laboratoire Theramex S.A. | 5-aryl-1H-1,2,4-triazole compounds as inhibitors of cyclooxygenase-2 and pharmaceutical compositions containing them |
| TWI729406B (zh) * | 2019-06-04 | 2021-06-01 | 翁豐富 | 具抗發炎活性之1,2,4-三氮唑衍生物之製備及其用途 |
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| TWI729406B (zh) * | 2019-06-04 | 2021-06-01 | 翁豐富 | 具抗發炎活性之1,2,4-三氮唑衍生物之製備及其用途 |
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