新的核苷类化合物β-L-D4A的化学合成及体外抗HBV作用

目的以D型谷氨酸为原料，通过一系列化学转化，合成了新的核苷类化合物β-L-D4A，并初步探索其体外抗HBV作用。方法合成β-L-D4A，用红外光谱、核磁共振氢谱和质谱确证目标化合物的结构，以2.2.15细胞(HepG2细胞进行HBV基因组转染后所得)培养为基础，Southern印迹法检测不同浓度化合物体外抑制HNV DNA复制作用，并求出50%抑制的药物浓度，即EC₅₀。以四噻唑蓝(MTT)比色分析法检测不同浓度药物的细胞毒性，求出IC₅₀。结果化合物β-L-D4A经红外光谱、核磁共振氢谱和质谱确证；2.2.15细胞培养上清液病毒DNA的Southern印迹、自显影结果显示病毒的抑制呈明显的浓度依赖性，计算出EC₅₀为0.2 μmol·L^-1，胞内DNA的Southern印迹、自显影显示类似的结果；细胞毒性实验显示IC₅₀为200 μmol·L^-1。结论体外实验显示β-L-D4A具有明显的抑制病毒DNA复制作用，且无明显的细胞毒性，TI值为1 000，高于临床用Lamivudine (750)，有望开发为临床抗HBV用药。     

含有芳基脲结构的4H-吡喃类化合物的设计、合成及抗肿瘤活性研究

目的 设计并合成一系列含有芳基脲结构的4H-吡喃类化合物，评价该类化合物的体外抗肿瘤活性。方法 以间硝基苯甲醛、丙二腈和丙酮二羧酸二甲酯为原料，通过“一锅法”合成含有硝基的吡喃中间体，该中间体的硝基经铁粉还原为氨基，再与取代异氰酸苯酯反应得到一系列目标化合物。以人大细胞肺癌细胞H460、人肺癌细胞A549和人结肠癌细胞HT-29 3种肿瘤细胞为测试细胞株，采用MTT法评价了目标化合物的抗肿瘤活性。结果 合成了11个含有芳基脲结构的4H-吡喃类化合物。体外抗肿瘤活性试验表明，11个化合物对3种肿瘤细胞株均具有很好的抑制活性。其中化合物7c活性突出，对H460和A549细胞的IC₅₀值分别为0.82，0.98 μmol·L^-1, 优于阳性对照药索拉非尼(IC₅₀=3.20， 2.83 μmol·L^-1)。结论 含有芳基脲结构的4H-吡喃类化合物具有很好的抗肿瘤活性，可作为抗肿瘤化合物的结构骨架进一步研究。     

R,S-1-(取代苯基)-4-[3-(萘-1-氧基)-2-羟基丙基]哌嗪类化合物的设计、合成及血管舒张活性

设计合成具有血管舒张活性的苯基哌嗪类系列化合物。以naftopidil活性代谢产物为先导化合物，根据已报道的苯基哌嗪类α₁-受体拮抗剂构效关系研究结论对先导化合物进行结构优化，设计并合成一系列新的苯基哌嗪类化合物，确定其结构，并通过测定其对苯肾上腺素引起家兔胸主动脉条收缩的抑制作用评价其血管舒张活性。发现其中5个化合物显示出不同程度的活性，其中化合物16的血管舒张活性较强，其在0.01和1 μmol·L^-1条件下血管收缩抑制率分别达到7.03%和22.72%，化合物16拟采用自发性高血压大鼠降压试验等进行进一步抗高血压活性研究。以上研究提示已报道苯基哌嗪类化合物的构效关系研究结论可适合于naftopidil的结构修饰。     

N-取代-O-对甲脒苯胺基羰甲基-L-酪氨酸甲酯类化合物的合成及抗血小板聚集活性

目的：根据RGD肽及目前活性较好的非肽类纤维。0蛋白原受体拮抗剂的结构特点,设计、合成一系列有抗血小板聚集活性的化合物。方法：以酪氨酸及对硝基苯甲酸等为主要原料经多步合成,对所得化合物用比浊法测定在1×10^-6 mol.L^-1时对体外血小板聚集的抑制率。结果：合成了18个N-取代-O-对甲脒苯胺基羰甲基-L-酪氨酸甲酯类化合物(Ia～r),均为新化合物。结论：其中10个化合物(Ia,f,g,i～m,q,r)显示一定的活性,活性最高的（Ig）抑制率达64%。     

n-(4-芳酰胺基苯基)甲磺酰胺类化合物的合成及抗炎活性

目的研究n-(4-芳酰胺基苯基)甲磺酰胺类化合物的抗炎作用。方法以对硝基苯胺为起始原料经三步反应合成目标化合物，并以二甲苯致小鼠耳肿胀模型测试目标化合物的抗炎活性。结果共合成11个化合物，经IR，¹HNMR和MS光谱确证结构；初步药理试验结果显示大部分化合物对二甲苯致小鼠耳肿胀具有较强的抑制作用。结论n-(4-芳酰胺基苯基)甲磺酰胺类化合物具有较强的抗炎活性。     

α-胺基苄基膦酸酯的合成及其生物活性

目的寻找具有内皮依赖性舒张血管活性的新结构胺基膦酸酯类化合物。方法运用一种新型的类Mannich反应，以生物电子等排体原理设计合成了一系列α-胺基苄基膦酸酯类化合物，并运用离体大鼠血管环和离体豚鼠回肠收缩实验观察其舒张血管活性及其对M受体的激动作用。结果共合成了7个新的α-胺基苄基膦酸酯类目标化合物，其结构经IR，¹H NMR和元素分析确定。初步离体血管环实验和离体豚鼠回肠收缩实验结果显示，化合物2a，2b和2c有一定的舒张血管活性且不激活平滑肌M受体。结论在浓度为1×10^-5 mol·L^-1时，化合物2b和2c有较强的舒张血管活性，其舒张率分别为(67±21)%，(82±18)%，而且不激动M受体，其中，化合物2b有内皮依赖性的舒张血管反应。     

人血浆中O-去甲右美沙芬的测定及药代动力学研究

目的建立直接测定人血浆中O-去甲右美沙芬的方法,并应用于药代动力学研究。方法18名健康受试者单剂量po氢溴酸右美沙芬60 mg后,血浆样品经液-液萃取,通过液相色谱-质谱-质谱联用法测定其活性代谢物O-去甲右美沙芬的浓度,用非室模型计算药代动力学参数。结果O-去甲右美沙芬测定的线性范围为0.2～80 μg·L^-1;其主要药代动力学参数T_max为(2.1±0.7) h,C_max为(14±8) μg·L^-1,T_1/2为(3.8±1.8) h,用梯形法计算,AUC_0-t为(60±37) μg·h·L^-1。结论该法灵敏度高,操作简便,可直接测定活性代谢物,适用于右美沙芬的临床药代动力学研究及制剂的生物等效性评价。     

液相色谱-质谱-质谱联用法测定猕猴血浆中阿德福韦液相色谱-质谱-质谱联用法测定猕猴血浆中阿德福韦

目的 建立测定猕猴血浆中阿德福韦(adefovir)的液相色谱-质谱-质谱联用法。方法 取血浆样品0.25 mL经甲醇沉淀蛋白后,以甲醇-水-甲酸(20∶80∶1)为流动相,用Diamonsil C₁₈柱分离,通过电喷雾离子化四极杆串联质谱,以选择离子反应监测方式进行检测。用于定量分析的离子反应分别为m/z 274→m/z 162(阿德福韦)和m/z 288→m/z 176［内标,9-(3-膦酸甲氧基丙基)腺嘌呤］。结果阿德福韦线性范围为0.02～4.00 mg·L^-1,最低定量限为20 μg·L^-1,日内、日间精密度(RSD)小于5.8%,准确度(RE)在±4.5%范围内。在临床前药代动力学研究中,应用此法测试了3只猕猴po给予阿德福韦地匹福酯(adefovir dipivoxil)后血浆中阿德福韦的浓度。结论该法操作简便,准确,适用于临床前药代动力学研究。     

新型含二硫键和硫酯键的乙酰半胱氨酸衍生物的合成与抗肝损伤活性评价

目的 设计、合成N-乙酰半胱氨酸衍生物,并评价目标化合物对H₂O₂诱导的LO2细胞氧化损伤的保护作用。方法 以L-半胱氨酸和N-乙酰半胱氨酸为起始原料,采用酰氯酯化法合成具有全新结构的乙酰半胱氨酸衍生物;以H₂O₂损伤LO2人肝细胞建立体外氧化损伤模型,利用CCK-8法检测不同浓度H₂O₂对LO2细胞存活率的影响,并检测细胞上清中MDA含量和SOD活性。结果 共合成了6个全新结构的N-乙酰半胱氨酸衍生物,其结构经¹H-NMR、¹³C-NMR、ESI-MS确证,目标化合物能够抑制H₂O₂诱导的LO2氧化损伤,并能够降低MDA含量和提高SOD活性（P<0.01或P<0.05）。结论 本研究快速、高效地合成了N-乙酰半胱氨酸系列衍生物,目标化合物对体外肝细胞损伤具有保护作用。     

查耳酮类化合物合成及抗乳腺癌活性研究

目的 设计、合成并筛选具有抗乳腺癌活性的查耳酮类化合物。方法 利用Claisen-Schmidt 羟醛缩合反应,合成了一系列的查耳酮类化合物,并通过MTT法测试化合物抗乳腺癌活性。结果 合成了36个查耳酮类化合物,其结构均通过¹H-NMR和¹³C-NMR进行了表征。初步生物活性结果表明大部分目标分子查耳酮对MCF-7有较强的抑制活性,对MDA-MB-231也展现出了中等的抑制活性,其中化合物27(IC₅₀=11.3 μmol·L^-1)对MCF-7的抑制活性是他莫昔芬的1.5倍,同时这些查尔酮类化合物对正常细胞均没有毒性。结论 本研究为开发具有抗乳腺癌活性的查耳酮类化合物提供了参考和依据。     

Inhibition of the in vitro growth of Plasmodium falciparum by acyclic nucleoside phosphonates.

Forty-eight acyclic nucleoside phosphonates (putative prodrugs of acyclic nucleoside triphosphate inhibitors of DNA replication) have been evaluated for in vitro antiplasmodial activity. Only certain purine derivatives with a hydroxyl group attached to the acyclic sugar moiety displayed antiplasmodial activity. The two most active analogs were (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine ((S)-HPMPA, IC50=0.18+/-0.07 microM) and (S)-3-deaza-HPMPA (IC50=0.29+/-0.08 microM). Their cyclic derivatives, containing an ester bond between the phosphonate and the hydroxyl group, were slightly less active. All tested compounds that lacked the hydroxyl group, including potent antiretrovirus analogs such as 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and the (S)-HPMPA derivatives (R)-PMPA and (S)-FPMPA, did not show any activity, even at very high concentrations ( >250 microM). Similarly, pyrimidine analogs of (S)-HPMPA, such as (S)-HPMPT, (S)-HPMPU and the anti-herpesvirus analog (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine ((S)-HPMPC), were devoid of any antiplasmodial activity. In addition, 11 acyclic nucleoside (non-phosphorylated) analogs--which in contrast to the acyclic nucleoside phosphonates require the presence of a monophosphorylating enzyme for the first activation step--were tested. None of them inhibited the growth of the parasite. In short three chemical entities seem to be imperative for antiplasmodial activity: a purine base, a hydroxyl group in the acyclic side chain and a phosphonate group terminating this chain.     

灯盏乙素苷元4'-L-氨基酸衍生物的设计、合成与抗氧化活性

为了设计合成具有较强神经细胞氧化损伤保护作用及较好理化性质的灯盏乙素苷元4'-L-氨基酸衍生物,以灯盏乙素苷元为先导化合物,根据主动转运原理在改善口服药物生物利用度应用上取得的成功经验,采用拼合设计原理在先导化合物的4'-羟基上引入L-氨基酸酯、醚结构,设计、合成灯盏乙素苷元4'-L-氨基酸衍生物。采用H2O2诱导PC12细胞氧化损伤模型对设计化合物进行了体外抗氧化活性评价,同时进行了目标化合物理化性质研究。结果发现设计的化合物均具有抗氧化活性,5个化合物抗氧化活性优于VE,灯盏乙素苷元L-氨基酸醚类化合物在缓冲液中稳定性(t1/2 9～92 h)优于酯类衍生物(t1/2 0.5 h),灯盏乙素苷元L-氨基酸酯类衍生物18、19与醚类衍生物22、24～27的水溶解度分别为1 796～4 100μg·mL-1和27.7～81.1μg·mL-1,两者水溶性分别达到灯盏乙素的120～280倍和2～6倍。以上研究提示L-氨基酸前药设计策略可适用于灯盏乙素苷元的结构修饰,以获得具有较好抗氧化活性及理化性质的灯盏乙素苷元前药。     

Synthesis and antiviral evaluation of alkoxyalkyl esters of acyclic purine and pyrimidine nucleoside phosphonates against HIV-1 in vitro

Alkoxyalkyl esters of cidofovir, an acyclic nucleoside phosphonate, have been shown to have antiviral activities several orders of magnitude greater than unmodified cidofovir against cytomegalovirus, herpes simplex virus, vaccinia, cowpox, ectromelia and adenoviruses in vitro. Hexadecyloxypropyl-cidofovir is orally bioavailable and active in lethal animal models of vaccinia, cowpox, ectromelia and cytomegalovirus. To see if this strategy is also applicable to other acyclic nucleoside phosphonates, we have converted several phosophonomethoxyethyl purines and pyrimidines to their hexadecyloxypropyl, octadecyloxyethyl and oleyloxyethyl esters and compared their activity against HIV-1 with the activity of the respective unmodified acyclic nucleoside phosphonates. The hexadecyloxypropyl esters of phosphonomethoxyethyl-adenine, phosphonomethoxyethyl-2,6-diaminopurine and phosphonomethoxyethyl-N(6)-cyclopropyl-diaminopurine were 3-5 orders of magnitude more active against HIV-1 in vitro than the parent nucleotides. The EC(50) values for these compounds were in the 10-20 pM range with selective indexes of 1,250 to >4,000. The acyclic pyrimidine phosphonates were generally inactive against HIV-1 in vitro. Phosphonomethoxyethyl-cytosine and phosphonomethoxyethyl-5-fluorocytosine were inactive against HIV-1. Surprisingly, hexadecyloxypropyl-phosphonomethoxyethyl-5-fluorocytosine was active against HIV-1 with a submicromolar EC(50) and a selective index of 174. Esterification of acyclic nucleoside phosphonates with alkoxyalkyl moieties may represent a general approach for increasing antiviral activity and selectivity of this class of antivirals.     

新型核苷膦酸酯类化合物的合成及其抗乙肝病毒活性研究

目的设计合成新型核苷膦酸酯类化合物,并进行体外抗乙肝病毒活性评价。方法以不同取代的硫酚与2-氨基-9-[2-[二(2,2,2-三氟乙氧基)膦酰甲氧基]乙基]-6-氯嘌呤进行烃化反应合成目标化合物,化合物结构经1H-NMR和FAB-MS谱确证。采用HepG2.2215细胞进行体外抗乙肝病毒活性评价。结果与结论设计合成了9个核苷膦酸酯类新化合物,这些化合物均有一定的抗乙肝病毒活性。化合物4a、4b的活性强于拉米夫定、阿德福韦酯。苯环上取代基的类型显著影响核苷膦酸酯类化合物抗乙肝病毒活性。     

2-(8-氮杂-6-取代苯硫基-9H-嘌呤-9-基)乙氧基甲基磷酸类化合物的设计、合成及抗病毒活性

目的设计、合成新型抗乙型肝炎病毒的开环核苷膦酸类似物。方法根据阿德福韦（PMEA）的结构特点,采用生物电子等排体理论,引入8．氮杂-6-取代苯硫基嘌呤,设计、合成系列目标化合物。化合物结构经IR,1H-NMR谱进行确证。对1a-1e化合物进行了体外抗HBsAg、HBeAg和抗HBV—DNA活性筛选试验。结果设计合成了10个目标化合物;所测化合物抗HBsAg、HBeAg活性均好于PMEA,化合物1e抗HBV—DNA活性与PMEA相当,值得继续研究。结论PMEA引入8-氮杂-6-取代苯硫基嘌呤抗HBV—DNA活性保持,抗HBsAg、HBeAg活性提高。     

Adenine: an important drug scaffold for the design of antiviral agents

Adenine derivatives, in particular the scaffold bearing the acyclic nucleoside phosphonates (ANPS), possess significant antiviral and cytostatic activity. Till now, several effective adenine derivatives have been marketed for the treatment of HIV, HBV, CMV and other virus-infected diseases. These compounds are represented by tenofovir (PMPA), a medicine for both HIV and HBV, and adefovir as an anti-HBV agent. More than this, other analogs, such as GS9148, GS9131, and GS7340, are also well-known anti-viral agents that have been progressed to the clinical studies for their excellent activity. In general, the structures of these compounds include an adenine nucleobase linked to a phosphonate side chain. Considerable structural modifications on the scaffold itself and the peripheral sections were made. The structure-activity relationships (SARs) of this skeleton will provide valuable clues to identify more effective adenine derivatives as antiviral drugs. Here, we systematically summarized the SARs of the adenine derivatives, and gave important information for further optimizing this template.KEY WORDS: Antiviral, Structure–activity relationship, Adenine, Acyclic nucleoside phosphonates, Scaffold     

Antiviral prodrugs - the development of successful prodrug strategies for antiviral chemotherapy

Following the discovery of the first effective antiviral compound (idoxuridine) in 1959, nucleoside analogues, especially acyclovir (ACV) for the treatment of herpesvirus infections, have dominated antiviral therapy for several decades. However, ACV and similar acyclic nucleosides suffer from low aqueous solubility and low bioavailability following oral administration. Derivatives of acyclic nucleosides, typically esters, were developed to overcome this problem and valaciclovir, the valine ester of ACV, was among the first of a new series of compounds that were readily metabolized upon oral administration to produce the antiviral nucleoside in vivo, thus increasing the bioavailility by several fold. Concurrently, famciclovir was developed as an oral formulation of penciclovir. These antiviral 'prodrugs' thus established a principle that has led to many successful drugs including both nucleoside and nucleotide analogues for the control of several virus infections, notably those caused by herpes-, retro- and hepatitisviruses. This review will chart the origins and development of the most important of the antiviral prodrugs to date.     

The clinical potential of the acyclic (and cyclic) nucleoside phosphonates: the magic of the phosphonate bond

The use of the acyclic nucleoside phosphonates, starting with (S)-HPMPA as the prototype, yielded three clinically approved antiviral drugs, cidofovir for the treatment of CMV retinitis in AIDS patients, adefovir dipivoxil for the treatment of chronic hepatitis B and tenofovir disoproxil fumarate for the treatment of HIV infections (AIDS) and HBV infections. This era has now grown to many more acyclic (and cyclic) nucleoside phosphonates (such as the “open ring” DAPy and Fd4A phosphonates) and alkoxyalkyl and phosphonoamidate prodrugs thereof, as well as new clinical applications, including new drug combination regimens for the treatment of AIDS, the chemoprophylaxis of HIV infections, and the anticancer potential against some malignant disorders.     

5′-Amino Acid Esters of Antiviral Nucleosides, Acyclovir, and AZT Are Absorbed by the Intestinal PEPT1 Peptide Transporter

Purpose. General use of nucleoside analogues in the treatment of viral infections and cancer is often limited by poor oral absorption. Valacyclovir, a water soluble amino acid ester prodrug of acyclovir has been reported to increase the oral bioavailability of acyclovir but its absorption mechanism is unknown. This study characterized the intestinal absorption mechanism of 5-amino acid ester prodrugs of the antiviral drugs and examined the potential of amino acid esters as an effective strategy for improving oral drug absorption. Methods. Acyclovir (ACV) and Zidovudine (AZT) were selected as the different sugar-modified nucleo-side antiviral agents and synthesized to L-valyl esters of ACV and AZT (L-Val-ACV and L-Val-AZT), D-valyl ester of ACV (D-Val-ACV) and glycyl ester of ACV (Gly-ACV). The intestinal absorption mechanism of these 5-amino acid ester prodrugs was characterized in three different experimental systems; in siturat perfusion model, CHO/hPEPTl cells and Caco-2 cells. Results. Testing 5-amino acid ester prodrugs of acyclovir and AZT, we found that the prodrugs increased the intestinal permeability of the parent nucleoside analogue 3- to 10-fold. The dose- dependent permeation enhancement was selective for the L-amino acid esters. Competitive inhibition studies in rats and in CHO cells transfected with the human peptide transporter, hPEPTl, demonstrated that membrane transport of the prodrugs was mediated predominantly by the PEPT1 H⁺/dipeptide cotransporter even though these prodrugs did not possess a peptide bond. Finally, transport studies in Caco-2 cells confirmed that the 5-amino acid ester prodrugs enhanced the transcellular transport of the parent drug. Conclusions. This study demonstrates that L-amino acid-nucleoside chimeras can serve as prodrugs to enhance intestinal absorption via the PEPT1 transporter, providing a novel strategy for improving oral therapy of nucleoside drugs.