CN114957379A - 一种二氢嘧啶硫酮类化合物及其制备方法与应用 - Google Patents
一种二氢嘧啶硫酮类化合物及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开一种二氢嘧啶硫酮类化合物及其制备方法与应用,所述化合物如下式所示(DHPM 1);本发明所述的化合物其具有较好的水溶性、生物相容性和UV辐射吸收能力,具有较强的抗UV‑B和UV‑C辐射损伤的作用。
Description
技术领域
本发明涉及药物化学领域,具体涉及一种化合物DHPM 1及其应用。
背景技术
紫外线(ultraviolet,UV)是波长在100-400nm电磁波的总称。自然界中绝大多数UV来自于太阳光,根据其波段可以分为UV-A(320-400nm)、UV-B(280-320nm)和UV-C(100-280nm,其中100-200nm又称真空UV)。通常情况下太阳光中UV-C段被大气中臭氧层吸收,只有极少部分到达地球表面,周围环境中暴露的UV主要是UV-A(97%)和UV-B(3%)。UV在日常生产生活中应用非常广泛,例如适量UV可促进人体维生素D合成、实验室和医用装备UV灭菌、诱杀害虫等等,同时过度UV暴露会带来极大的危害,眼睛和皮肤表现尤为明显。许多人知道UV会导致皮肤癌,但却不知眼睛才是对UV最为敏感的部位,比皮肤更为脆弱,因为眼睛不像皮肤一样存在较厚的上皮层、角质层和黑素细胞可以吸收UV辐射减轻损伤。UV波长越短、能量越强,例如UV在300nm波长下对眼组织的损伤作用要比325nm波长下强600倍。过量UV辐射可起严重眼病-翼状胬肉、电光性角膜炎、白内障、黄斑退化、视网膜灼伤、雪盲等。同时,臭氧层的破坏,高原、低纬度等地区的强UV辐射,雪地、海滨等场所的强UV反射,电焊等特殊行业,以及现代电子产品使用过程中产生的UV辐射等都增加了现代生活中眼睛UV暴露的风险。
UV的危害主要是通过损伤DNA实现的:UV-A辐射主要通过促使细胞产生活性氧自由基(ROS),损伤DNA;UV-B辐射可直接被细胞DNA碱基吸收,破坏DNA结构,进而通过增加p53表达或激活caspase-3、8、9引起细胞凋亡。UV波长决定其穿透能力,进而决定UV损伤的眼部组织:角膜主要吸收300nm以下的UV-B辐射;晶状体主要吸收370nm以下的UV-A辐射。角膜和晶状体是UV辐射影响的主要眼部组织,另有少量UV辐射可穿透至视网膜损害感光细胞和色素上皮功能。
目前文献报道的具有抗角膜UV-B损伤功能的化合物主要有烟酰胺腺嘌呤二核苷酸(NAD)及其前体烟酰胺(NAM)、烟酰胺单核苷酸(NMN)、垂体腺苷酸环化酶激活多肽(PACAP)、顺式-尿刊酸(cis-CUA)、维生素C、岩藻黄素(Fucoxanthin)、洋甘菊和小米草提取物滴眼液(DacriovisTM)、表没食子儿茶素没食子酸酯(EGCG)、瑞巴派特、卡替洛尔、杜氏盐藻、枸杞多糖、4-香豆酸、丹参素等。上述化合物的发挥抗角膜UV-B损伤的主要作用机制如下:(1)抗氧化:清除OH自由基,降低MDA水平,增强SOD、过氧化氢酶、GSH-Px、GSH-Rd、GSH活性,增加Nrf2的表达,抑制脂质过氧化、蛋白氧化、DNA氧化等;(2)抗炎:抑制炎症相关因子IL-1β、IL-6、IL-8、IL-18、COX-2、iNOS、LDH、MCP-1、TNF-α、VEGF等的表达或分泌(3)抗凋亡:促进抗凋亡基因Bcl-2的表达,抑制促凋亡基因Bax、cleaved caspase-3的表达,降低Bax/Bcl-2比例。
由于UV穿透能力有限,主要影响眼表组织(尤其是角膜),深入研究角膜UV损伤机制,并开发相关药物或防护技术,对于呵护眼健康具有重要意义。
发明内容
本发明的目的是提供一种抗角膜UV-B损伤的化合物。
本发明的目的通过以下措施达到:
一种化合物,结构如下:
本发明还提供本发明所述的化合物的制备方法:
本发明还提供一种药物组合物,包括本发明所述的化合物和药学上可接受的载体。
本发明还提供所述化合物或药物组合物在用于制备抗紫外线损伤或者防紫外线产品中的应用。
本发明还提供所述化合物或药物组合物在用于制备抗角膜紫外线损伤或抗皮肤紫外损伤产品中的应用。
本发明所述的产品可以包括但不限于药品、医疗器械、保健品、化学涂层或护肤品等。
本发明所述的化合物或组合物可制备为药学上允许的任何剂型,例如为适于眼内、外用、口服、肠胃外、腹膜内、静脉内、动脉内、透皮、舌下、肌内、直肠、透颊、鼻内、吸入、阴道、局部、皮下、脂肪内、关节内、腹膜内或鞘内任意给药方式的制剂。
在一种优选的实施方式中,本发明所述的化合物或组合物的剂型为滴剂、膏剂、搽剂、喷雾剂、凝胶剂、贴剂、水剂、片剂、冲剂、口服液剂、胶囊剂、滴丸剂、灌肠剂、膜剂或注射剂。
本发明所述的化合物可以单独使用也可以联合其他产品使用,为以上疾病的治疗提供了新的产品。
本发明的有益效果:本发明所述的化合物其具有较好的水溶性,吸收UV辐射能力强,同时具有较强的抗UV-B和UV-C辐射的作用。
附图说明
图1是DHPM 1的1H-NMR;
图2是DHPM 1的13C-NMR;
图3是DHPM 1在PBS溶液中(20μg/mL)的紫外吸收光谱;
图4是DHPM 1与HCE-2细胞共同孵育24h后的细胞毒作用;
图5是DHPM 1抗UV-B诱导的HCE-2细胞损伤作用(Control组不进行UV-B辐射,其余各组接受不同剂量UV-B辐射;UV-B:302nm);
图6是DHPM 1抗UV-C诱导的HCE-2细胞损伤作用(Control组不进行UV-C辐射,其余各组接受不同剂量UV-C辐射;UV-C:254nm);
图7是Calcein AM/PI活死HCE-2细胞双染:(A)无UV-B辐射对照组;(B)UV-B辐射组(0.1J/cm2);(C)UV-B辐射(0.1J/cm2)+0.5mg/mL DHPM 1组;
图8是DHCF-DA法测定HCE-2细胞内ROS水平:(A)无UV-B辐射对照组;(B)UV-B辐射组(0.1J/cm2);(C)UV-B辐射(0.1J/cm2)+0.5mg/mL DHPM 1组(荧光强度与细胞内ROS水平成正比);
图9流式检测HCE-2细胞的DNA损伤(A)无UV-B辐射对照组;(B)UV-B辐射组(0.1J/cm2);(C)UV-B辐射(0.1J/cm2)+0.1mg/mL DHPM 1组;(D)UV-B辐射(0.1J/cm2)+0.2mg/mLDHPM 1组;(E)UV-B辐射(0.1J/cm2)+0.5mg/mL DHPM 1组(荧光强度与DNA损伤程度成正比);
图10DHPM 1抗UV-B诱导的HCE-2细胞损伤作用(Control组不进行UV-B辐射,其余各组接受不同剂量UV-B辐射;UV-B:302nm)。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。
实施例1化合物DHPM 1的制备:
其中:(a)20mol%MgCl2,AcOH,100℃;(b)NaOH;(c)(1)DIEA,DMF;(2)20%piperidine in DMF;(d)(1)Glycine,HBTU,DIEA,DMF;(2)20%piperidine in DMF;(e)compound 5,HBTU,DIEA,DMF;(f)95%TFA in CH2Cl2.
(1)乙酰乙酸甲酯1(580mg,5.0mmol)、苯甲醛2(530mg,5.0mmol)、N-甲基硫脲3(675mg,7.5mmol)、无水硫酸镁(95mg,1.0mmol)和冰醋酸(5.0mL)于100℃封管反应2小时,反应毕冷却,倒入碎冰中剧烈搅拌并过滤,粗产物经水洗、乙醚洗得白色固体1.2g,即化合物4。HRMS calcd for C14H17N2O2S[M+H]+277.1005,found 277.1011;1H NMR(400MHz,DMSO-d6):δ9.87(d,J=4.8Hz,1H),7.36-7.18(m,5H),5.21(d,J=4.8Hz,1H),3.64(s,3H),3.48(s,3H),2.53(s,3H);13C NMR(100MHz,DMSO-d6):δ150.4,140.5,126.5,121.5,110.8(2C),110.0,108.7(2C),92.0,49.6,49.1,36.8,20.9.
(2)化合物4(552mg,2.0mmol)加入到甲醇(5mL)和1N氢氧化钠水溶液(10mL)的混合溶液中回流1小时,冷却至室温倾入碎冰中,1N盐酸水溶液酸化,过滤并干燥得白色固体430mg,即化合物5。HRMS calcd for C14H17N2O2S[M+H]+263.0849,found 263.0850;1H NMR(400MHz,DMSO-d6):δ12.56(s,1H),9.78(d,J=3.6Hz,1H),7.36-7.31(m,2H),7.28-7.20(m,3H),5.21(d,J=3.6Hz,1H),3.47(s,3H),2.52(s,3H);13C NMR(100MHz,DMSO-d6):δ178.1,167.1,147.4,142.2,128.6(2C),127.6,126.0(2C),106.2,52.3,36.1,16.2.
(3)通过固相合成的方法制备:2-氯三苯甲基氯树脂(2-Chlorotrityl chlorideresin,1.0g,1mmol)在无水二氯甲烷中溶胀20分钟,Fmoc-L-Tyr(H2PO3)-OH(1.45g,3mmol)、DIEA(825μL,5mmol)溶于无水DMF与树脂反应2小时,DCM/MeOH/DIEA(80:15:5)封闭、20%哌啶脱Fmoc保护基。Fmoc-Gly-OH(890mg,3mmol)经HBTU/DIEA活化后,与上述树脂继续缩合反应2小时,20%哌啶脱Fmoc保护基。然后,化合物5(780mg,3mmol)经HBTU/DIEA活化后,继续与上述树脂进行缩合反应2小时,利用三氟乙酸将产物从树脂上切下、氮气吹干溶剂、乙醚超声、过滤得粗产品,然后经HPLC制备得纯品DHPM 1 420mg。HRMS for C24H28N4O8PS[M+H]+:563.1366;found 563.1357;1H NMR(图1,400MHz,DMSO-d6):δ9.48(d,J=4.8Hz,1H),8.23(dt,J=5.6,2.4Hz,1H),8.19(t,J=7.2Hz,1H),7.35-7.02(m,9H),5.13(d,J=4.0Hz,1H),4.45-4.37(m,1H),3.92-3.75(m,1H),3.73-3.56(m,1H),3.41(s,3H),2.22(s,3H),3.05-2.95(m,1H),2.90-2.78(m,1H);13C NMR(图2,100MHz,DMSO-d6):δ177.8,172.7,168.8,166.8,150.1,142.1,137.3,132.8,130.1(2C),128.5(2C),127.4,126.0(2C),119.8,119.7,112.1,53.6,53.1,41.8,36.0,35.8,16.6.
(4)研究证实:DHPM 1作为单一的有机小分子化合物,其在PBS中的溶解度>2.5mg/mL,具有较好的水溶性;DHPM 1在PBS溶液中(20μg/mL)的紫外吸收光谱(图3)显示:DHPM 1可以有效地吸收UV-B(280-320nm)和UV-C(200-280nm)波段的紫外辐射。
实施例2生物相容性测定
为了评价DHPM 1的生物相容性,采用CCK-8法考察其在无UV辐射下的细胞毒作用。选取对数生长期的人角膜上皮细胞(HCE-2,1×104cells/well),接种于96孔板,置于37℃、5%CO2的培养箱孵育24h,使其贴壁生长。移去培养液,加入含有不同浓度DHPM-1(0、0.1、0.2、0.5、1.0、2.0mg/mL)的新鲜培养液,继续培养24h。采用CCK-8法测定450nm下的吸光度,按如下公式计算细胞存活率:
细胞存活率=(A实验组-A空白组)/(A对照组-A空白组)×100%
结果如图4所示:在无UV辐射条件下,DHPM 1在2mg/mL浓度下对HCE-2细胞没有明显细胞毒作用,提示其生物相容性好。
实施例3抗UV-B损伤测定
为了评价DHPM 1的抗UV-B损伤性能,采用CCK-8法考察其分别在UV-B辐射后的细胞活力。选取对数生长期的HCE-2细胞(1×104cells/well),接种于96孔板,置于37℃、5%CO2的培养箱孵育24h,使其贴壁生长。移去培养液,加入含有不同浓度DHPM 1(0、0.1、0.2、0.5或1.0mg/mL)的新鲜培养液,然后经不同剂量UV-B照射(25、50或100mJ/cm2)诱导HCE-2细胞凋亡,辐射完毕后继续培养24h。采用CCK-8法测定450nm下的吸光度,按如下公式计算细胞存活率:
细胞存活率=(A实验组-A空白组)/(A对照组-A空白组)×100%
结果如图5所示:UV-B(25mJ/cm2)可诱导42%HCE-2细胞凋亡,DHPM 1在0.1mg/mL浓度时已经表现出明显的保护作用,细胞活力提高28%(cell viability=87%);高剂量UV-B辐射(0.1J/cm2)可诱导79%HCE-2细胞凋亡,0.1mg/mL DHPM 1依然具有显著的细胞保护作用(cell viability=44%),尤其是DHPM 1达到0.5mg/mL时可保护HCE-2细胞免受UV-B辐射损伤(cell viability=100%)。同时实施例2的的结果表明,DHPM 1在至少2mg/mL浓度下对HCE-2细胞没有明显细胞毒作用,生物相容性好,可见本申请所述化合物具有抗高剂量的UV-B辐射损伤的效果。
实施例4抗UV-C损伤测定
为了评价DHPM 1的抗UV-C损伤性能,采用CCK-8法考察其分别在UV-C辐射后的细胞活力。选取对数生长期的HCE-2细胞(1×104cells/well),接种于96孔板,置于37℃、5%CO2的培养箱孵育24h,使其贴壁生长。移去培养液,加入含有不同浓度DHPM-1(0、0.1、0.2、0.5或1.0mg/mL)的新鲜培养液,然后经不同剂量UV-C照射(25、50或100mJ/cm2)诱导HCE-2细胞凋亡,辐射完毕后继续培养24h。采用CCK-8法测定450nm下的吸光度,按如下公式计算细胞存活率:
细胞存活率=(A实验组-A空白组)/(A对照组-A空白组)×100%
结果如图6所示:UV-C(25mJ/cm2)可诱导HCE-2细胞完全凋亡,DHPM 1在0.1mg/mL浓度时细胞活力接近10%,0.2mg/mL浓度时已经表现出明显的保护作用(cell viability=27%),0.5mg/mL时保护作用达到100%;即使在高剂量UV-C辐射(0.1J/cm2)下,1.0mg/mLDHPM 1仍然可实现接近100%的HCE-2细胞保护作用,使其免受UV-C辐射损伤。与文献(Tengfei Mao,et al.J.Am.Chem.Soc.2018,140,6865-6872;陶磊,毛腾飞.CN108219084A)报道的共聚物相比,DHPM 1为水溶性有机小分子,更容易进行质量控制,成药性更有优势;同时实现100%细胞保护作用,文献中报道共聚物浓度达到5mg/mL,比DHPM 1(0.5mg/mL)高10倍,可见本申请的化合物显著优于文献报道的共聚物。
实施例5 Calcein-AM/PI双染
选取对数生长期的HCE-2细胞(5×104cells/well),接种于35mm激光共聚焦培养皿,置于37℃、5%CO2的培养箱孵育24h,使其贴壁生长。移去培养液,加入含有不同浓度DHPM 1(0或0.5mg/mL)的新鲜培养液,然后经UV-B照射(0.1J/cm2)诱导HCE-2细胞凋亡,辐射完毕后继续培养24h。弃去培养液,PBS洗3次。加入含有Calcein-AM(2μM)和PI(4.5μM)的培养液,置于培育箱中继续培养7min后,用PBS缓冲液洗1次,加入live cell imagingsolution。激光共聚焦显微镜进行显微观察(活细胞Calcein-AMλex:488nm,λem:500-530nm;死细胞PI:λex:561nm,λem:600-700nm),以无UV辐射组为对照。
结果如图7所示:(A)无UV-B辐射对照组细胞被Calcein-AM染色,为正常活细胞;(B)UV-B辐射(0.1J/cm2)组,大部分细胞被PI染色,仅少部分可被Calcein-AM染色,表明UV-B诱导大部分细胞凋亡;(C)UV-B辐射(0.1J/cm2)+DHPM 1(0.5mg/mL)组,细胞都被Calcein-AM染色,且不能被PI染色,表明0.5mg/mL DHPM 1能够有效抵抗UV-B诱导的HCE-2细胞损伤。
实施例6细胞内ROS水平测定
选取对数生长期的HCE-2细胞(5×104cells/well),接种于35mm激光共聚焦培养皿,置于37℃、5%CO2的培养箱孵育24h,使其贴壁生长。移去培养液,加入含有不同浓度DHPM 1(0或0.5mg/mL)的新鲜培养液,然后经UV-B照射(0.1J/cm2)诱导HCE-2细胞凋亡,辐射完毕后继续培养一定时间(7h)。弃去培养液,PBS洗3次。加入含有DCFH-DA(10μM)的培养液,置于培育箱中继续培养15min后,用PBS缓冲液洗3次,加入live cell imagingsolution。利用激光共聚焦显微镜观察探针的荧光强度(激发波长λex:488nm;发射波长λem:500-650nm),细胞内的荧光强度反映细胞内的ROS水平。
结果如图8所示:(A)无UV-B辐射对照组细胞内荧光较弱;(B)UV-B辐射(0.1J/cm2)对照组部分细胞内的荧光强度较强;(C)UV-B辐射(0.1J/cm2)+DHPM 1(0.5mg/mL)组,细胞内的荧光强度较A组强,但明显比B组细胞内荧光强度弱,表明0.5mg/mL DHPM1能够有效降低UV-B诱导的HCE-2细胞内ROS的产生,从而发挥抗UV-B损伤作用。
实施例7抗DNA损伤
选取对数生长期的HCE-2细胞(5×104cells/well),接种于6孔板,置于37℃、5%CO2的培养箱孵育24h,使其贴壁生长。移去培养液,加入含有不同浓度DHPM 1(0、0.1、0.2或0.5mg/mL)的新鲜培养液,然后经UV-B照射(0.1J/cm2)诱导HCE-2细胞凋亡,辐射完毕后继续培养24h。弃去培养液,PBS洗3次,消化收集细胞,4%多聚甲醛溶液固定15min,PBS洗2次,90%预冷甲醇通透15min,PBS洗2次,Alexa488conjugated phospho-histoneH2A.X(Ser139)(20E3)rabbit mAb(1:50,Cell Signaling Technology)避光孵育1h,PBS洗2次,加入0.6mL PBS进行流式测定(激发波长λex:488nm;发射波长λem:515-560nm),荧光强度反应DNA损伤程度成正比。
结果如图9所示:(A)无UV-B辐射对照组,荧光强度较弱;(B)UV-B辐射(0.1J/cm2)对照组荧光强度明显增强;(C-E)分别为UV-B辐射(0.1J/cm2)条件下,加入0.1、0.2、0.5mg/mL DHPM 1各组荧光强度变化,结果显示随着DHPM 1浓度的增高,荧光强度显著降低,表明DHPM 1可通过减轻UV-B引起的DNA损伤,发挥抗UV-B损伤作用。
实施例8DHPM 1抗UV-B损伤能力
为进一步评价DHPM 1的抗UV-B损伤性能,采用CCK-8法考察DHPM 1的抗UV-B辐射能力。选取对数生长期的HCE-2细胞(1×104cells/well),接种于96孔板,置于37℃、5%CO2的培养箱孵育24h,使其贴壁生长。移去培养液,加入含有不同浓度DHPM 1(0.2、或0.5mg/mL)的新鲜培养液,然后经不同剂量UV-B照射(25、50、100、150或200mJ/cm2)诱导HCE-2细胞凋亡,辐射完毕后继续培养24h。采用CCK-8法测定450nm下的吸光度,按如下公式计算细胞存活率:
细胞存活率=(A实验组-A空白组)/(A对照组-A空白组)×100%
结果如图10所示:在低剂量UV-B辐射(25或50mJ/cm2)情况下,0.2mg/mL DHPM1对HCE-2细胞就具有显著的保护作用;虽然随着UV-B辐射剂量增大,0.2mg/mL DHPM1细胞保护作用出现降低(cell viability=63%at 0.2J/cm2),但随着浓度升高,例如DHPM1在0.5mg/mL浓度下,依然可以表现出显著的保护作用(cell viability~100%at 0.2J/cm2),而实施例2的的结果表明,DHPM 1在至少2mg/mL浓度下对HCE-2细胞没有明显细胞毒作用,生物相容性好,可见本申请所述化合物具有抗高剂量的UV-B辐射损伤的效果。
Claims (10)
2.根据权利要求1所述的化合物,其特征在于,所述化合物制备为适于眼内、外用、口服、肠胃外、腹膜内、静脉内、动脉内、透皮、舌下、肌内、直肠、透颊、鼻内、吸入、阴道、局部、皮下、脂肪内、关节内、腹膜内或鞘内任意给药方式的制剂。
3.根据权利要求2所述的化合物,其特征在于,所述的制剂为滴剂、膏剂、搽剂、喷雾剂、凝胶剂、贴剂、水剂、片剂、冲剂、口服液剂、胶囊剂、滴丸剂、灌肠剂、膜剂或注射剂。
5.一种药物组合物,包括权利要求1所述的化合物和药学上可接受的载体。
6.根据权利要求5所述的组合物,其特征在于,所述组合物为适于眼内、外用、口服、肠胃外、腹膜内、静脉内、动脉内、透皮、舌下、肌内、直肠、透颊、鼻内、吸入、阴道、局部、皮下、脂肪内、关节内、腹膜内或鞘内任意给药方式的制剂。
7.根据权利要求6所述的组合物,其特征在于,所述的制剂为滴剂、膏剂、搽剂、喷雾剂、凝胶剂、贴剂、水剂、片剂、冲剂、口服液剂、胶囊剂、滴丸剂、灌肠剂、膜剂或注射剂。
8.权利要求1~3任一项所述的化合物或权利要求5~7任一项所述的药物组合物在用于制备抗紫外线损伤或者防紫外线产品中的应用。
9.权利要求1~3任一项所述的化合物或权利要求5~7任一项所述的药物组合物在用于制备抗角膜紫外线损伤或抗皮肤紫外损伤产品中的应用。
10.根据权利要求8或9所述的应用,其特征在于,所述的产品为药品、医疗器械、保健品、化学涂层或护肤品。
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