1 Introduction

Cancer is the predominant cause of death worldwide(Chen et al., 2016; Siegel et al., 2011). An estimated 4 million new cancer cases and almost 3 million cancerrelated deaths were reported in 2015 in China (Chen et al.,2016). Based on the GLOBOCAN 2012 data, leukemia is the most common cancer in the world. Currently, many drugs available to treat leukemias have been characterized with low selectivity, high toxicity, and acquired resistance.Thus, it is important to discover new drugs with high efficacy and safety to treat leukemia.

喘振控制主要包括控制最小流量、控制最大压力、控制压缩比与实际流量的关系。喘振控制模型通常采用压缩比与实际流量的关系。这个模型的优点在于能够提供准确的喘振预测，同时气体组分的变换对喘振线的形状和位置几乎没有什么影响，这样就能维持喘振控制点和实际喘振点之间的安全余量在一个比较稳定的范围内，避免操作过程中因气体组分发生变化使操作越过喘振安全余量区而导致喘振发生。

Cytarabine (1-β-D-arabinofuranosylcytosine, Ara-C) is the first marine antitumor agent approved by the US Food and Drug Administration in 1980. It is an antimetabolic anticarcinomic agent of pyrimidine derived from spongothymidine and mainly isolated from the Caribbean sponge species Tethyacrypta (Sagar et al., 2010). Cytarabine is metabolized by deoxycytidine kinase, NMP kinase, and NDP kinase to form the bioactive product triphosphate of cytarabine (Ara-CTp) (Braess et al., 1996; Cottam and Carson, 2007) (Fig.1); this metabolite is the key component to treat different types of leukemias (Gandhi et al.,1996), including acute myelocytic leukemia (Tamamyan et al., 2017; Woelich et al., 2017), lymphocytic leukemia,meningeal leukemia, and the blast crisis phase of chronic myelogenous leukemia (Montaser and Luesch, 2011;Pigneux et al., 2007). Many cytarabine derivatives and prodrugs have been reported to enhance absorption and bioavailability (Adema et al., 2010; Liu et al., 2017; Sun et al., 2008) (Fig.2). Among the prodrugs, cytarabine ocfosfate (YNK01) is a stearyl phosphate diester of cytarabine that was approved in 1992 and elacytarabine (CP-4055) is a fatty acid derivative and is still under phase III clinical trials (Chhikara and Parang, 2010; DiNardo et al.,2013).

Encouraged by the success of structurally modifying cytarabine, we designed and synthesized a lipophilic amide compound and a phosphate derivative by N4/O5’modification of cytarabine, and their antitumor activities were evaluated against the leukemia cell line HL-60 by using the MTT assay.

2 Materials and Methods

2.1 Chemistry

Fig.1 Metabolic pathway of cytarabine.

Fig.2 Structures of cytarabine derivativesand prodrugs.

All reagents and solvents were purchased from commercial sources and used without further purification.1H-nuclear magnetic resonance (NMR; 500 MHz) spectra was recorded on a Bruker Avance 400 spectrometer in CDCl3 or DMSO-d6, using tetramethylsilane as the internal standard. The abbreviations used for the NMR signals are as follows: s, singlet; d, doublet; and m, multiplet.Mass spectra (MS) were obtained from Agilent 1100 LC/MS Spectrometry Services (Agilent Technologies, Palo Alto, CA, USA). Electrospray ionization MS were recorded by using a Waters Quattro Micro mass instrument(Bedford, MA, USA). Lipophilicity of cytarabine and the new derivatives was parameterized by cLogP and predicted using MOE2015.10 (Chemical Computing Group Inc.).

The effects of the compounds on cellular proliferation and viability were determined by using the MTT assay.The cell lines were cultured in DMEM medium supplemented (Gibco, Grand Island, NY, USA) with 10% heatinactivated fetal bovine serum at 37℃ in a humid atmosphere (5% CO2 and 95% air). After a 24-h culture, the cell lines were concentrated and adjusted to 2 × 105 mL−1(cells). The cells were then seeded in 96-well plates. Each compound in the experimental group was set with nine concentration gradients, and each concentration had three replicates. The final concentrations of the compounds were 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.016, 0.008,0.004 and 0.002 mmol L−1. Then, 5 μL of the compounds were added at different concentrations into each well. As a blank control, 5 μL of normal saline was added to nine wells. After a 72-h incubation, 10 μL MTT solution was added to each well, and the cells were incubated for another 4 h. Each well was lysed with 100 mL of 10% sodium dodecyl sulfate (containing 0.01 mol L−1 HCl). After 24 h, the absorbance of each well was determined by Bio-Rad 680 ELISA reader (Hercules, CA, USA). Optical density was measured at a wavelength of 570 nm, and IC50 values were calculated by linear regression analysis.

2.1.1 General procedure

Organic acids a or d (5.8 mmol) with DMF (2 drops)were added to SOCl2 (15 mL). The mixture was heated under reflux for 4 hours and dried in vacuo to obtain intermediate b or e. Lauryl alcohol (1.01 g) and Et3N (3 mL)were added to a solution of b or e in dioxane (30 mL), and the mixture was placed in an ice bath for 4 h. After concentrating the solution, the solid residue was dissolved in water (100 mL). Then, the solution was titrated to pH 2 with HCl (1 mol L−1). The aqueous layer was extracted using diethyl ether (100 mL). Then, the organic layer was separated and dried with Na2SO4, filtered, and evaporated in vacuo to obtain intermediate c or f.

Cytarabine was purchased from SunRayPharma (Shanghai, China) and was used as a positive control drug. The anticancer activities of the prepared derivatives were evaluated against leukemia cell line HL-60 by using the MTT assay. HL-60 was purchased from American Type Cell Culture Collection (ATCC, Manassas, VA, USA).

2.1.2 Procedure for synthesis of compound 1

Compound 1 was obtained after purification using the general procedure with thiophene-2,5-dicarboxylic acid a1 (1.0 g) as the starting material.

2.1.3 Procedure for synthesis of compound 2

Compound 2 was obtained after purification using the general procedure with 1H-pyrazole-3,5-dicarboxylic acid a2 (0.9 g) as the starting material.

2.1.4 Procedure for synthesis of compound 3

Compound 3 was obtained after purification using the general procedure with pyrazine-2,3-dicarboxylic acid d3(1.0 g) as the starting material.

Table 1 Analytical data of the prepared compounds

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2.1.5 Procedure for synthesis of compound 4

POCl3 (4.75 mmol) and DIPEA (6.48 mmol) were dissolved in anhydrous THF (5 mL). Compound 3 (1.21 g,475 mmol) was dissolved in anhydrous THF and added to the mixture. The mixture was reacted at 0℃ until compound 3 was consumed completely (determined by thinlayer chromatography). Then, DIPEA (10 mL) and lauryl alcohol (10.0 g, 53.8 mmol) were added to the mixture.After reacting for 12 h at room temperature, the reaction liquid was filtered and the filtrate was concentrated in vacuo to obtain the solid residue. The solid residue was dissolved in water (50 mL) and titrated to pH7 with HCl(1 mol L−1). The aqueous layer was extracted two times with diethyl ether (100 mL), and the combined organic layers were dried over Na2SO4, filtered, and evaporated in vacuo. Then, the residue was purified by silica gel column chromatography (dichloromethane: methyl alcohol= 20:1) to obtain compound 4.

2.1.8 Procedure for synthesis of compound 7

式中：F1是O1点的静摩擦力(N)； F2是O2点的静摩擦力(N)；α是光伏面板与水平面的夹角(°)；N1是O1点的反支力(N)；N2是O2点的反支力(N)；P1是O1点的等效吸附力(N)；P2是O2点的等效吸附力(N)。

Compound 5 was obtained after separation using the general procedure with pyridine-2,5-dicarboxylic acid d5(1.0 g) as the starting material.

2.1.7 Procedure for synthesis of compound 6

Compound 6 was obtained after purification using the general procedure with pyridine-2,4-dicarboxylic acid d6(1.0 g) as the starting material.

2.1.6 Procedure for synthesis of compound 5

Compound 7 was obtained after separation using the general procedure with pyridine-3,4-dicarboxylic acid d7(1.0 g) as the starting material.

2.1.9 Procedure for synthesis of compound 8

（二）从材料包含的知识深度上看，要做到“三个思考”，即是什么，为什么，怎么样。深层次解读信息，是什么（什么现象、问题的实质）、为什么（原因、作用、危害等）、怎么样（措施、建议、态度等）。当然，不一定每题都同时回答三个层次，通常用在认识、评价类试题，要根据问题并结合材料做到具体问题具体分析。

2.1.10 Procedure for synthesis of compound 9

1）喘证急性加重期；2）排除间质性肺病，肺癌等其他需要治疗的慢性肺部疾病；合并严重的呼吸、循环、泌尿、血液、神经和内分泌系统等疾病、精神患者、恶性肿瘤、传染病患者；妊娠期或哺乳期妇女；近半年内已接受过中药贴敷治疗、刮痧、穴位拔罐的患者；3）贴敷穴位、刮痧部位有皮损或瘢痕体质者，及对药物或敷料特别敏感的患者；4）研究者认为不适合参加本研究者。

The general procedure used pyridine-2,5-dicarboxylic acid d9 (1.0 g) as the starting material. In the second step,pyridine-2,5-dicarbonyl dichloride e reacted with decyl alcohol h (0.9 g) and converted to 6-((decyloxy)carbonyl)nicotinic acid f9. After separation, f9 reacted with cytarabine. Compound 9 was obtained after purification.

2.2 Pharmacology

Cytarabine (406 mg, 1.67 mmol), intermediate c or f(2.01 mmol), PyBOP (955 mg, 1.84 mmol), and DMAP(244 mg, 2.0 mmol) were dissolved in DMF (10 mL). The solution was stirred for 12 h at room temperature. Then,the mixture was evaporated in vacuo and purified by silica gel column chromatography (dichloromethane:methyl alcohol =10:1) to obtain the products. The analytical data are summarized in Table 1.

结合工程建设进展情况，积极开展质量监督巡查工作，每年年初制定工作计划，邀请有关领导和专家组成巡查组，对监督的在建大中型工程，开展1至2次全面的质量监督巡查工作，对巡查发现的问题及时提出质量监督巡查通报，分年度有侧重地对混凝土、金属结构以及土方等专项施工重要节点开展巡查。

(2)从时间演变来看，成都平原城市群县域经济实力存在排名变化，整体变化相对较小.上升型县域主要分布在成都市区周围，下降型县域主要分布于经济实力相对薄弱的研究区外围，实力相对稳定的县(区)占到研究区的51.7%，空间分布比较分散.

3 Results and Discussion

3.1 General

Cytarabine is a polar nucleoside antileukemia drug with limited plasma stability and cell permeability. It can be metabolized by the deaminating action of enzymes (FADL et al., 1995; VandeVoorde et al., 2015). The N4-amino groups were protected by acylation to minimize the enzymatic deamination of Ara-C. Long fatty acyl chains have been used to enhance the drug’s lipophilicity (Nam et al., 2004). On the basis of these concepts, we designed and synthesized a series of compounds, including compounds 1–3 and 5–7, to modify the N4 of cytarabine, block the metabolic site, and enhance lipophilicity.

左小龙一把搭住店主的肩膀，道：好了，我也不跟你瞎说了，我不出差，但我得等月底有了钱以后才能取，现在我取不了，你就先帮我装好弄好，然后就锁起来，我也不开走，隔天来看看就成。

黑鹰山铁矿床磷灰石的引人注目之点是其稀土元素含量颇高。根据两个磷灰石精样品的原子吸收光谱法测定结果，其稀土氧化物总量w(TR2O3)为2.5407%～2.6948%，可称为含稀土磷灰石[2]。

The antitumor activities of the prepared compounds were evaluated against human leukemia cancer cell line HL-60 by using the MTT assay. Cytarabine was used as the reference drug. The IC50 and predicted cLogP values are summarized in Table 2.

3.2 Synthesis

The synthetic route is illustrated in Scheme 1. Using organic acid a or d as the starting material, compounds 1–3 and 5–9 were prepared by means of chloroformylation, esterification, and amination with cytarabine, as shown in Route 1 of Scheme 1. Compound 4 was prepared as shown in Route 2. The intermediate g was obtained by reacting POCl3 and DIPEA using compound 3 as the starting material. Then, g was hydroxylated with lauryl alcohol after an overnight reaction at room tem-perature to obtain compound 4. The structures of the pre- pared compounds are summarized in Table 1.

The general procedure was followed using pyrazine-2,3-dicarboxylic acid d8 (1.0 g) as the starting material. In the second step, pyrazine-2,3-dicarbonyl dichloride e reacted with decyl alcohol h (0.9 g) and converted to 3-((decyloxy)carbonyl)pyrazine-2-carboxylic acid f. Compound 8 was obtained after purification.

(2)通入Cl2是为了将Fe2+(或FeCl2)氧化为Fe3+(或FeCl3),工业上不用HNO3是因为会产生污染性气体NOx,也会引入杂质离子。

Scheme 1 Synthesis of cytarabine derivatives.

3.3 Bioactivity Evaluation

Cytarabine was intracellularly converted into the mono-,di-, and triphosphate derivatives (cytarabine triphosphate,Ara-CTp) (Braess et al., 1996; Cottam and Carson, 2007).Monophosphorylation was generally the rate-determining step, and delivery of monophosphorylated nucleosides that bypass this step is highly desired (Chhikara and Parang,2010). Thus, we synthesized and evaluated a monophosphorylated prodrug of cytarabine, compound 4, to enhance its bioavailability. Phosphate ester prodrugs have excellent chemical stability and adequate bioconversion to the parent drugs by alkaline phosphatases in the intestine, liver,and blood (Flores-Ramos et al., 2017; Hanson et al., 2003;Parker et al., 2016). Moreover, we prepared compounds 8 and 9 with shorter fatty acyl chains to investigate the effects of lipophilicity on antitumor activity.

The bioassay results revealed that the IC50 values of compounds 5, 8 and 9 were 0.080, 0.090 and 0.057 μmol L−1, respectively. These three compounds were almost as potent as cytarabine (IC50 = 0.056 μmol L−1). Their cLogP values were in the range of 1–3, which is much more lipophilic than cytarabine at −2.27. Compounds 1–3, 6 and 7 showed a two- to four-fold reduced potency, while compound 4 was inactive, with an IC50 value of ＞5 μmol L−1.

Table 2 IC50 and cLogP values of the cytarabine derivatives

1 0.25 3.34 2 0.25 2.00 3 0.16 2.08 4 ＞5 11.01 5 0.08 2.68 6 0.25 2.68 7 0.25 2.68 8 0.090 1.29 9 0.057 1.90 CpID HL-60 IC50 (μmol L−1) cLogP Cytarabine 0.056 −2.27

3.3.1 Different linkers between N4 of cytarabine and long fatty acyl chains

现行水利工程供水价格水平较低，以2008年百家水利工程供水管理单位（以下简称水管单位）统计数据为例，供农业用水2.6分/m3，供工业用水26分/m3，供自来水用水39.81分/m3，供水力发电用水2.1分/m3。与成本水价相比，农业供水水价达不到成本的50%，非农业水价仅达到成本的80%左右。

Compounds 1–3 and 5–7 were designed and prepared to block the metabolic site and to further improve metabolic stability by introducing long fatty acyl chains with different 5- or 6-membered heterocyclic rings as linkers. The IC50 values of compounds 1–3 and 6, 7 were in the range of 0.16–0.31 μmol L−1, which was a decrease in potency compared to cytarabine (IC50 = 0.056 μmol L−1). Compound 5,with an IC50 value of 0.080 μmol L−1, was almost as potent as cytarabine, and it was more lipophilic than cytarabine.Compound 5 should have higher metabolic stability and bioavailability and could be developed as a promising anticancer agent in the future.

3.3.2 Monophosphorylated derivative of cytarabine

Phosphate compound 4 was prepared based on the successful phosphate prodrug strategies (Chhikara and Parang, 2010; Maloisel et al., 2002; Parker et al., 2016). A higher cLogP value was calculated at 11.01. The bioassay results revealed that it was inactive, which was in agreement with prodrug strategies.

3.3.3 Different lengths of long fatty acyl chains

As the length of long fatty acyl chains is related to molecular lipophilicity and further affects the cell permeability and bioavailability of a compound, we synthesized compounds 8 and 9 by replacing a decyl group with a dodecyl group in compounds 3 and 5. The bioassay result revealed that the IC50 values of compounds 8 and 9 improved to 0.090 and 0.057 μmol L−1, respectively, which is as potent as cytarabine. These results suggest that these two compounds exhibit their bioactivities directly, and they have advantages of stability and bioavailability over cytarabine.

4 Conclusion

Cytarabine is used to treat different types of leukemia.However, the high hydrophilicity of cytarabine is detrimental to cell permeability and bioavailability. To overcome this disadvantage, a series of novel lipophilic cytarabine derivatives were designed and synthesized by minimizing deamination to enhance metabolic stability and introducing long fatty acyl chains to increase cell permeability. All the newly synthesized compounds had higher lipophilicity compared with cytarabine. Compounds 5, 8 and 9 had comparable IC50 values with cytarabine against the HL-60 cell line. Thus, increasing the lipophilicity was a successful strategy to discover more cytarabine-derived antitumor drugs. By blocking the cytarabine metabolic site and improving lipophilicity in a suitable range,the compounds will likely have improved stability and bioavailability, which will further contribute to the drug efficacy. Compound 4 with a phosphate group is a promising compound to be developed as a prodrug.

在过去的几十年里，我国的旅游教育事业已经走过了漫长的道路，职业教育导游专业教育也掀起了一场大发展，大，各大中等职业学校都开设了导游专业。而到现在，各大中职院校每年培养数百导游专业学生，但也仍不能保证旅游市场的需求，尤其是在全域旅游时代概念引进的当下，更大的扩大了职业教育导游专业人才的缺口。

Acknowledgement

This study was supported by the ‘Zhufeng Scholar Program’ of Ocean University of China (No. 841412016),the ‘Outstanding Talents Plan’ of Qingdao (No. 15-10-3-15-(34)-zch) to Dr. Wenbao Li and youth special fund for PhD of Qingdao (No. 16-5-1-61-jch).

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