Dihydropyrazole derivatives as telomerase inhibitors: Structure-based design, synthesis, SAR and anticancer evaluation in vitro and in vivo

Accepted Manuscript Dihydropyrazole derivatives as telomerase inhibitors: structure-based design, synthesis, SAR and anticancer evaluation in vitro and in vivo Yang Wang, Fei Xiong Cheng, Xiao Long Yuan, Wen Jian Tang, Jing Bo Shi, Chen Zhong Liao, Xin Hua Liu PII:

S0223-5234(16)30079-4

DOI:

10.1016/j.ejmech.2016.02.009

Reference:

EJMECH 8360

To appear in:

European Journal of Medicinal Chemistry

Received Date: 24 November 2015 Revised Date:

3 February 2016

Accepted Date: 4 February 2016

Please cite this article as: Y. Wang, F.X. Cheng, X.L. Yuan, W.J. Tang, J.B. Shi, C.Z. Liao, X.H. Liu, Dihydropyrazole derivatives as telomerase inhibitors: structure-based design, synthesis, SAR and anticancer evaluation in vitro and in vivo, European Journal of Medicinal Chemistry (2016), doi: 10.1016/ j.ejmech.2016.02.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical abstract Dihydropyrazole derivatives as telomerase inhibitors: structure-based design, synthesis, SAR and anticancer evaluation in vitro and in vivo

Chen Zhong Liao a, Xin Hua Liua,*

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Yang Wang a,#, Fei Xiong Cheng b,#, Xiao Long Yuan c, Wen Jian Tang a, Jing Bo Shi a,

School of Pharmacy, Anhui Medical University, Hefei, 230032, P. R. China

b

Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China

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a

The First Affiliated Hospital of Anhui Medical University, Hefei, 230032, P. R. China

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c

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University of Technology, Shanghai 200237, P. R. China

Based on structure-based drug design, 78 compounds as potential human telomerase inhibitors were designed. In vivo studies showed that compound 13i displayed potent anticancer activity with inhibition tumor growth of S180 and HepG2 tumor-bearing mice. It also significantly enhanced the survival rate of EAC tumor-bearing mice.

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Dihydropyrazole derivatives as telomerase inhibitors: structure-based design, synthesis, SAR and anticancer evaluation in vitro and in vivo Yang Wang a,#, Fei Xiong Cheng b,#, Xiao Long Yuan c, Wen Jian Tang a, Jing Bo Shi a,

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Chen Zhong Liao a, Xin Hua Liua,* a

School of Pharmacy, Anhui Medical University, Hefei, 230032, P. R. China

b

Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China

c

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University of Technology, Shanghai 200237, P. R. China

The First Affiliated Hospital of Anhui Medical University, Hefei, 230032, P. R. China

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Abstract: It is of our interest to generate and identify novel compounds with regulation telomerase for cancer therapy. In order to carry out more rational design, based on structure-based drug design, several series of N-substituted-dihydropyrazole derivatives, totally 78 compounds as potential human telomerase inhibitors were designed and synthesized. The results demonstrated that some compounds had potent anticancer activity

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against four tumor cell lines, and showed good selectivity on tumor cells over somatic cells. By the modified TRAP assay, compound 13i exhibited the most potent inhibitory activity against telomerase with an IC50 value of 0.98 µM. In vivo evaluation results indicated that compound 13i could inhibit growth of S180 and HepG2 tumor-bearing mice, and it also

vivo

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significantly enhanced the survival rate of EAC tumor-bearing mice. The further results in confirmed

that

it

could

significantly

improve

pathological

changes

of

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N,N-diethylnitrosamine (DEN)-induced rat hepatic tumor. These data support further studies to assess rational design of more efficient telomerase inhibitors in the future.

Keywords: Dihydropyrazole; selective anticancer activity; telomerase; inhibitor _________________ #

These authors contributed equally to this work. *Corresponding author. Tel.: +86 551 65161115; fax: +86 551 65161115. E-mail: [email protected].

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1. Introduction Telomerase plays important roles in early stage of life-maintaining telomere and chromosomal integrity of frequently dividing cells. It turns dormant in most somatic cells during adulthood [1]. However, in cancer cells telomerase gets reactivated and works

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tirelessly to maintain the short length of telomeres, resulting in immortality [2]. About 80-90% of various cancer cells have detectable telomerase activity, telomerase is hence believed as a potential anticancer target [3-6]. Human telomerase consists of two parts: a template-encoding RNA named TER (telomerase RNA), and a primary protein component

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TERT (telomerase reverse transcriptase) which includes several functional domains: TEN (telomerase essential N-terminal domain), TRBD (telomerase RNA binding domain), RT

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(reverse transcriptase domain), and the C-terminal extension [7, 8]. In the past decade, several types of telomerase inhibitors were reported, including 2’-O-MeRNA oligonucleotides and peptide nucleic acids targeting the telomerase RNA template [9], ligands targeting telomeric DNA [10], and nucleosidic reverse transcriptase inhibitors [11]. Recently, a number of G-quadruplex stabilizing compounds were also designed and extensively studied as telomerase inhibitors [12-13].

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Among the whole structure of human telomerase, the expression level of human TERT (hTERT) is the rate-limiting factor for telomerase activity, and most human somatic cells do not show detectable telomerase activity due to lack of hTERT, hTERT is hence regarded as the key part of telomerase in discovery of anticancer drug [14]. Several hTERT

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inhibitors with good anticancer effects, including BIBR1532 [15], symmetrical bis-substituted derivatives of anthraquinone [16], rhodacyanine FJ5002 [17], MST-312 [18]

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and isothiazolone derivative TMPI [19] were reported. Among them, BIBR1532 was defined as a novel class of selective hTERT inhibitor with the mechanism of action similar to non-nucleosidic inhibitors of HIV-1 reverse transcriptase. However, most of the reported hTERT inhibitors also exhibited potential toxicities against somatic cells, and so far there are no inhibitors targeting hTERT have been approved by FDA. Therefore, design of potent hTERT inhibitors with high selectivity against tumor cells and low toxicity against somatic cells is a very urgent task. Dihydropyrazole [20-22] and coumarin [23-28] moieties are prominent structural

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motifs presented in numerous drugs. In our previous studies [29-30], several dihydropyrazole derivatives (LXH-1~LXH-5, Figure 1) showed potent telomerase inhibition activity, in which these compounds can be divided into two basic scaffolds phenyl-dihydropyrazole colored by blue, and courmarin colored by red. Based on

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structure-activity relationships (SAR), there are some motivations provided in the design process. For example, in order to compare the activity of 4,5-dihydropyrazole moiety with carbonyl or sulfone, some sulfonyl-4,5-dihydropyrazole derivatives were synthesized. Meanwhile, because coumarin derivatives showed potent anti-HIV-1 activity, some

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dihydropyrazole-coumarin derivatives were designed and synthesized, mainly due to the

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mechanistic and structural similarities between hTERT and HIV-1 reverse transcriptase.

Figure 1

Structure-based drug design has played very important roles for hits identification and optimization [31]. However, to date, for hTERT, there are no X-ray crystal structures available. Based on our above SAR, in order to carry out more rational drug design, in this

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report, we employed a three-dimension human telomerase model [7] to explore the binding mode of BIBR1532, and then designed drug-like hTERT inhibitor scaffolds which incorporate dihydropyrazole and courmarin, two motifs presented in many drugs to try to discover novel potent hTERT inhibitors.

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The antiproliferative activity of the title compounds against gastric cancer cell (MGC-803), breast cancer cell (Bcap-37), prostate cancer cell (PC3) and human hepatoma

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cell (HepG2) will be assayed. In order to determine the selective cancer cell toxicity of some compounds, proliferative inhibition assay against human normal cells will be conducted. Compounds with good antiproliferative activity will be further evaluated on telomerase to validate the antitumor. In order to further confirm anticancer activity in vivo, sarcoma cells S180, hepatoma cells HepG2 and ehrlich ascites cells EAC models will select to evaluate their antitumor pharmacodynamics, the effect on DEN-induced rat hepatic tumor will be explored to find pathological changes.

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2. Results and discussion 2.1 Modeling and scaffold design To date, there are no X-ray crystal structures available for hTERT. Recently, the X-ray structures of the full length T. castaneum telomerase alone (PDB IDs: 3DU5 and 3DU6)

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and in complex with a TNA: DNA hairpin (PDB ID: 3KYL) have been published [32]. Based on this information and the crystal structures for separate TRBD and TEN domains from T. castaneum, Steczkiewicz et al. has constructed a three-dimension human telomerase model by employing computational methods for distant homology detection,

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comparative modeling and molecular docking, guided by available experimental data [7]. In order to employ structure-based drug design methods for identifying novel hTERT

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inhibitors, we decided to use this model to explore the binding mode of BIBR1532 (Figure 2A), a promising and potent hTERT inhibitor with an IC50 value of 0.093 µM. Considering the model is an apoprotein, in silico induced fit docking (IFD) of Schrödinger was employed to dock BIBR1532 and BIBR1591 (Figure 2B, IC50 = 0.47 µM) into the active site. IFD can consider the flexibility both of the receptor and the ligand so that it may give us more reasonable docking poses. In our IFD study, a docking pose which can account for

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the limited SAR of BIBR1532 and BIBR1591 were chosen and submitted to do a 10 ns molecular dynamics (MD) simulation employing the program of Desmond, which was developed at D. E. Shaw Research to perform high-speed molecular dynamics simulations of biological systems on conventional commodity clusters.

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The MD simulation result shows that BIBR1532 binds to hTERT mainly with a hydrogen bond (the carboxylic acid group with Lys 710) and several hydrophobic

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interactions: the naphthalene ring with Phe 568 and Tyr 949; the methyl group with Pro 267 and the benzene ring with Lys 902 and Val 904 (Figure 2B). This model can explain why BIBR1591 has less potent inhibition against hTERT than BIBR1532 since the introduction of the morpholine in the meta-position of the benzoic acid may have confliction with the backbone of the protein.

Figure 2

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Based on the structure of BIBR1532, preliminary analysis of SAR about our earlier reported hTERT inhibitors [33-34] and the volume of the active site of hTERT, we designed dozens of synthesizable drug-like scaffolds which incorporate the moieties of dihydropyrazole and courmarin. The prinicipal is that the designed scaffolds should have

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an aromatic ring on which a hydrogen bond receptor is to mimic the benzoic acid of BIBR1532; additionaly, the other end of the designed scaffolds should be a hydrophobic moiety to mimic the naphthalene ring of BIBT1532. All these designed scaffolds were then docked into the active site of the hTERT-BIBR1532 complex. Only the scaffolds whose

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docking poses have similar interactions with hTERT as BIBR1532 were considered for further synthesis. At last, a small library of 78 novel dihydropyrazole derivatives were

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designed and synthesized as potential telomerase inhibitors in this study (Figure 3).

Figure 3

2.2 Chemistry

47 N-substituted-dihydropyrazoles and 31 dihydropyrazole-coumarin derivatives were

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designed and synthesized. The synthesis of compound 1 (Scheme 1) started from salicylaldehyde and acetone catalyzed by NaOH at 20 oC. The synthesis of α, β-unsaturated ketone 5 (Scheme 2) started from salicylaldehyde and acetoacetate catalyzed by piperidine at 50~60

o

C. Claisen-Schmidt condensation of 3-acetyl-2H-chromen-2-one with

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substituted-benzaldehyde was catalyzed by piperidine. Catalysts tosyl chloride and DMAP were proved to be efficient alternatives for the synthesis of compounds 7, 9 and 11.

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However, piperazine was a better catalyst for the synthesis of compound 13. In the process of synthesis of compounds 5, 6, 11, 12, 13 the pH value should be less than 8.2. Otherwise, the coumarin ring will open.

Schemes 1-2

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2.3 Structural Determination of Compounds 20 compounds (7d, 7f, 7j, 7l, 7m, 7n, 7o, 7t, 7u, 8d, 9d, 9g, 9l, 9n, 9r, 9s, 13f, 13x, 13y and 13z) were determined by X-ray diffraction analysis. Representative compounds (7j, 8d, 9d, 9n, 13x, 13y) crystallographic and experimental data were presented in Tables

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1~3. Another data were presented in Supporting Information (Tables S1~S8). Structures of the compounds 7j, 8d, 9d, 9n, 13x, 13y were shown in Figure 4, another structures of the compounds were shown in Supporting Information (Figures S1~S15). Crystallographic data (excluding structure factors) for the structures were deposited into the Cambridge Center

(Registered

No.

CCDC-813727,

CCDC-819490,

CCDC-819491,

CCDC-837389,

CCDC-836556,

CCDC-836555,

CCDC-837388,

CCDC-837390,

CCDC-814973,

CCDC-813726,

CCDC-824623,

CCDC-824625,

CCDC-834936,

CCDC-826456,

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Data

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Crystallographic

CCDC-861071,

CCDC-861072,

CCDC-834938,

CCDC-861070, CCDC-861075, CCDC-883316, respectively).

Tables 1~3

2.4 Anticancer Activity

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Figure 4

At first, compounds 7~13 were evaluated for their antiproliferative activity against

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PC3, Bcap-37, MGC-803 and HepG2 cell lines. The cells were allowed to proliferate in presence of tested material for 48 h, and the results were reported in terms of IC50 values

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(Table 4). From Table 4 it is obvious to see that compounds 10i, 10n, 11i and 13i exhibited high activity against MGC-803 with IC50 values at 3.51±0.21, 3.01±0.13, 2.02±0.23 and 2.96±0.15 µM, respectively. Compounds 9c, 10n, 11i and 13i possesseded potent activity against HepG2 with IC50 = 3.02±0.33, 2.52±0.09, 2.98±0.19 and 2.45±0.11 µM, respectively. Compounds 11i, 13c and 13f occupied high activity against PC3 with IC50 = 2.98±0.15, 1.87±0.11 and 1.92±0.13 µM, respectively. Compounds 13c, 13i and 13w showed high activity against Bcap-37 with IC50 values at 2.21±0.10, 3.37±0.30 and

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3.50±0.99 µM respectively, which is comparable with the positive control AMD. As given in Table 4, compounds 7~10 (phenyl-4,5-dihydropyrazole moiety) and 11~13 (phenyl-coumarin-4,5-dihydropyrazole moiety) exhibited similar antiproliferative activity against the above cancer cells. The introduction of N-sulfonyl group did not

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improve the activity (8d, 8e, 8f, 12k and 12l) obviously, neither did the coumarin unit. We then investigated the SAR profiles of the N-acetyl substituted group of dihydropyrazole (9, 10 and 13) to find a permissive region. Transformation of acetyl group could significantly improve the anticancer activity (10o, 11h, 11i, 13f, 13d, 13w and 13z). Further, the

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introduction of short-chain aliphatic amine group in the N-acetyl part played an important

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role (such as 13f, 13d, 13w and 13y).

Table 4

2.5 Inhibition assay of human normal cell

In order to determine wether the title compounds have selective activity on cancer

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cells. We subsequently conducted a proliferative inhibition assay against human normal gastric mucosa cell (GES-1) and liver cell (L-02). As shown in Table 5, all compounds manifested obvious un-toxic effect on GES-1 and L-02 with IC50 from 1.2 to 2.5 mM. The data indicated that the compounds have good selectivity against tumor cells over somatic

Table 5

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cells (Tables 4 and 5).

2.6 Anticancer activity evaluation in vivo Mice sarcoma cells S180 model, hepatoma cells HepG2 model and ehrlich ascites

cells EAC model are recognized as the reliable research models for study of antitumor pharmacodynamics. Pathological changes of N,N-diethylnitrosamine (DEN)-induced rat hepatic tumor is also a classic induced tumor model as for antitumor pharmacodynamics study. Based on the results of tables 4 and 5, the compounds 9c and 13i showed good

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selectivity on HepG2 cells over GES-1 cells in vitro. To further verify the inhibitory effect on the growth of tumor cells in vivo, sarcoma cells S180, hepatoma cells HepG2 and ehrlich ascites cells EAC were selected to evaluate effects of compounds 9c and 13i. 2.6.1 Antitumor effects of title compounds against tumor growth

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The inhibitory effects of title compounds against growth of transplanted S180 or HepG2 carcinoma were presented in Table 6 and Figure 5. The results revealed that title compounds significantly decreased the tumor weights of S180 and HepG2 tumor-bearing mice. The potent activity was showed by compound 13i with inhibitory rates 48.9% and

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Table 6

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46.1% for S180 and HepG2 tumor-bearing mice, respectively.

Figure 5

2.6.2 Effects of title compounds against the survival of EAC-bearing mice EAC tumor-bearing mice were observed for mean survival time. Based on the mortality, the effect of percentage increases in life span was calculated. Survival response

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of untreated EAC-bearing mice died within 16.6 days (Table 7). A similar phenomenon with above was observed that mice displayed longer survival times treated with compounds 9c and 13i. Among them, the compound 13i was observed to enhance the

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survival rate to about 44%.

Table 7

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2.6.3 Histopathology effect of title compound In order to further reveal the pathological effects of the title compounds in vivo, the

histopathology effect on DEN-induced rat hepatic tumor was explored. Microscopic features of the liver were examined. In the control group, as expected, integral liver cell structure, hepatic lobular architecture and hepatic nuclei were clearly observed (Figure 6A). In the DEN model group, normal liver lobular structure was completely destroyed. The tumor cells showed low differentiation and evident atypia (including multinuclear, karyopyknosis, nuclear vacuoles, etc.), with larger nuclei and nucleoli, chromatin rough. In

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addition, tumor giant cells and cancer nests infiltrating surrounding tissues were also observed (Figure 6B). The experimental group markedly abated pathological changes of hepatic lobules. The hepatic cells exhibited evidently reduced atypia, high differentiation and full cytoplasm. The cells were also observed similar multinuclear, karyopyknosis and

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nuclear vacuoles to those of normal cells. These results showed that title compound 13i could significantly improve pathological changes of DEN-induced rat hepatic tumor (Figure 6C).

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Figure 6

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2.7 Telomerase Activity

To confirm if the synthesized compounds performed anticancer activities via telomerase inhibition as designed, some title compounds were evaluated by a modified TRAP assay using an extraction from MGC-803 cells. Modified TRAP is a powerful technique to determine small molecules inhibiting telomere elongation qualitatively and quantitatively [35]. The results were summarized in Table 8. Among them, compounds 7l,

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9c, 9o, 10e, 10n, 11i, 13a and 13f showed potent inhibitory activity against telomerase with IC50 values less than 5 µM, better than positive control staurosporine. In particular, compound 13i showed the most potent activity against telomerase with IC50 = 0.98 µM. In primary SAR analysis, the nine series compounds are divided into two types, one is skeleton

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5-aryl-dihydropyrazole

(compounds

7~10),

the

other

is

3-coumarin-5-aryl-dihydropyrazole skeleton (compounds 11~13). In general, the

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introduction of the coumarin moiety in the dihydropyrazole ring (compounds 11~13) significantly increased the inhibitory activity (compounds 11h, 11i, 13a, 13b, 13c, 13f, 13j, 13x, 13z). When piperidine ring was replaced by substituted-piperidine (compounds 13u, 13v, 13p, 13r), compound 13p lost activity against telomerase and the inhibitory activity of compound 13u decreased 41 times. Furthermore, compounds 13p and 13u did not show any antiproliferative activity against PC3, Bcap-37 and MGC-803 cells (Table 8). Further SAR analysis was focused on different substitutes of 5-aryl. For the 5-aryl-dihydropyrazole skeleton (compounds 7~10), the phenyl bromine substitution

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(compounds 7 and 9) showed a little increasing effect on the activity against telomerase (compounds 7l, 9c, 9d, 9o and 7q). The un-substitution at the 5-aryl resulted in considerable loss of potency (compound 7j). For 3-coumarin-5-aryl-dihydropyrazole skeleton (compounds 11~13), the introduction of an electron-withdrawing (trifluoromethyl)

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group in the phenyl ring increased the activity (compounds 13x and 13y) comparing to those with un-substituted phenyl ring. Therefore the presence of trifluoromethyl group in the phenyl ring played an important role for the telomerase activity. These SAR

2.8 Proposed Binding Model

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Table 8

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observations could offer the useful information for further structural optimiztion.

In this study, a three-dimension human telomerase model [7] and an advanced docking method–IFD were employed to explore the binding mode of BIBR1532, a potent hTERT inhibitor. The docked complex was then done a 10 ns MD simulation. Our designed scaffolds were docked into the active site of the modelled hTERT– BIBR1532

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complex to see if these designed scaffolds have common interactions with the hTERT as BIBR1532. Based on these criteria, a library compounds were synthesized, and some of them showed good inhibition against hTERT. Among them, compound 13i has an IC50 value of 0.98±0.07 µM against human telomerase.

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Compound 13i was docked into the complex of hTERT-BIBR1532, which was gotten by the induced fit docking method as discussed. Modeling showed that compound

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13i has similar active site as BIBR1532 shown in Figure 7: the moiety of courmarin of 13i can mimic benzoic acid of BIBR1532 and form a hydrogen bond with Lys 710 and have hydrophobic interactions with Lys 902, Val 904 and Pro 929; one fatty chain has hydrophobic interactions mainly with Phe 568; the dihydropyrazole ring and the benzene ring have hydrophobic interactions mainly with Pro 627.

Figure 7

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3. Conclusions In

this

study,

nine

series

of

N-substituted-dihydropyrazoles

and

dihydropyrazole-coumarin derivatives were designed, synthesized and evaluated as

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potential telomerase inhibitors. Among the 78 compounds, compounds 10i, 10n, 11i and 13i exhibited potent activity against MGC-803 cell with lower toxicity on normal cells in vitro. In vivo studies showed that compound 13i displayed potent anticancer activity with inhibition tumor growth of S180 and HepG2 tumor-bearing mice. It also significantly

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enhanced the survival rate of EAC tumor-bearing mice. Furthermore, compound 13i could significantly improve pathological changes of DEN-induced rat hepatic tumor. Moreover,

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compound 13i showed high inhibitory activity against telomerase with IC50 = 0.98±0.07 µM. The binding modes of several inhibitors on hTERT indicated that the conserved residue Lys710 is important for ligand binding via hydrogen bond interaction, which could explain the SAR of these compounds and match reported mutation data. These results are of help in rational design of more efficient telomerase TERT inhibitors for cancer therapy.

4.1 Chemistry

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4. Experimental section

All reagents were purchased from Aldrich (U.S.A) and Sinopharm Chemical Reagent Co., Ltd (China). Separation of the compounds by column chromatography was carried out

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with silica gel 60 (200~300 mesh ASTM, E. Merck). The quantity of silica gel used was 30~70 times the weight charged on the column. Then, the reactions were monitored using

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TLC. Melting points (uncorrected) were determined on a XT4 MP apparatus (Taike Corp., Beijing, China). 1H NMR spectra were recorded on a Bruker DPX400 or DPX300 spectrometer at 25 °C with TMS and solvent signals allotted as internal standards. Chemical shifts were reported in ppm (δ). Elemental analyses were performed on a CHN-O-Rapid instrument and were within ± 0.4 % of the theoretical values. 4.1.1 General procedure for preparation of compounds 7c~7u Carboxylic acid (12 mmol) was dissolved in 20 mL of chloroform, at room

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temperature was added tosyl chloride (12 mmol) and DMAP (12 mmol), then the substituted-2-(3-methyl-4,5-dihydro-1H-pyrazol-5-yl) phenol 2 (10 mmol) was added, the reaction mixture was refluxed for 3 h. The mixture was cooled, washed with water, and allowed to stand at 0~5 ˚C over night. The product was collected by filtration and the crude

compounds 7c~7u (Scheme 1) as colorless solids.

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residue was purified by chromatography on SiO2 (acetone/petroleum, v:v=2:1) to give title 7c: 1-[5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-3-phenylprop-2-en-1

-one, colorless crystals, yield, 66%; mp 265~267 oC; 1H NMR (CDCl3, 300 MHz): δ 2.25

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(3H, s, -Me), 3.13 (1H, dd, J= 18.0 and 3.0 Hz, pyrazole, 4-Ha), 3.40 (1H, dd, J= 12.0 and 18.0 Hz, pyrazole, 4-Hb), 5.80 (1H, dd, J= 12.0 and 3.0 Hz, pyrazole, 5-H), 6.88-7.75 (11H,

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m, -CH=CH- and ArH) , 9.69 (1H, brs, -OH); 13C NMR (CDCl3, 125 MHz): δ 16.3, 31.0, 43.5, 53.3, 117.3, 119.7, 121.3, 126.2, 128.3, 128.9, 130.0, 133.5, 134.4, 135.0, 143.2, 155.6; Anal. calcd for C19H18N2O2: C, 74.49; H, 5.92; N, 9.14%. Found: C, 74.78; H, 6.13; N, 9.00%.

7d: 1-[5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)(4-nitrophenyl)]methan-one, colorless crystals, yield, 80%; mp 224~225 oC; 1H NMR (CDCl3, 300 MHz): δ 2.21 (3H, s,

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-Me), 3.12 (1H, dd, J= 18.9 and 3.6 Hz, pyrazole, 4-Ha), 3.46 (1H, dd, J= 11.3 and 18.8 Hz, pyrazole, 4-Hb), 5.94 (1H, dd, J= 11.4 and 3.9 Hz, pyrazole, 5-H), 6.93-8.29 (8H, m, ArH), 8.76 (1H, brs, -OH); 13C NMR (CDCl3, 125 MHz): δ 16.3, 43.8, 54.7, 119.1, 121.4, 122.9, 126.0, 126.7, 130.1, 131.1, 139.4, 149.1, 155.0, 160.8, 164.8; Anal. calcd for

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C17H15N3O4: C, 62.76; H, 4.65; N, 12.92%. Found: C, 63.02; H, 4.80; N, 12.61%. 7f:1-[5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-3-phenylpropan-1-one,

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colorless crystals, yield, 81%; mp 202~203 oC; 1H NMR (CDCl3, 300 MHz): δ 2.08 (3H, s, -Me), 2.88 (4H, m, -2CH2-), 2.96 (1H, dd, J= 18.5 and 3.3 Hz, pyrazole, 4-Ha), 3.22 (1H, dd, J= 11.2 and 18.5 Hz, pyrazole, 4-Hb), 5.57 (1H, dd, J= 11.2 and 3.5 Hz, pyrazole, 5-H), 6.81-7.18 (9H, m, ArH), 9.00 (1H, brs, -OH); 13C NMR (CDCl3, 125 MHz): δ 16.2, 30.8, 35.6, 39.9, 43.5, 55.1, 115.7, 119.3, 125.7, 126.4, 128.3, 128.8, 128.9, 141.8, 154.3, 157.8, 168.9; Anal. calcd for C19H20N2O2: C, 74.00; H, 6.54; N, 9.08%. Found: C, 74.32; H, 6.80; N, 9.35%. 7g: 5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)(p-tolyl)methanone, colorless

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crystals, yield, 54%; mp 232~233 oC; 1H NMR (CDCl3, 300 MHz): δ 2.23 (3H, s, -Me), 2.46 (3H, s, -Me), 3.15 (1H, dd, J= 18.0 and 3.5 Hz, pyrazole, 4-Ha), 3.49 (1H, dd, J= 11.2 and 18.0 Hz, pyrazole, 4-Hb), 5.77 (1H, dd, J= 11.2 and 3.6 Hz, pyrazole, 5-H), 6.80-7.86 (8H, m, ArH), 9.20 (1H, brs, -OH); Anal. calcd for C18H18N2O2: C, 73.45; H, 6.16; N,

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9.52%. Found: C, 73.17; H, 6.32; N, 9.88%.

7i: 2-chlorophenyl)(5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)methan- one, colorless crystals, yield, 62%; mp 207~209 oC; 1H NMR (CDCl3, 300 MHz): δ 2.10 (3H, s, -Me), 3.08 (1H, dd, J= 18.6 and 3.3 Hz, pyrazole, 4-Ha), 3.45 (1H, dd, J= 11.1 and 18.6

SC

Hz, pyrazole, 4-Hb), 5.90 (1H, dd, J= 11.1 and 3.3 Hz, pyrazole, 5-H), 6.74-7.40 (8H, m, ArH), 8.97 (1H, brs, -OH); 13C NMR (CDCl3, 125 MHz): δ 16.2, 44.2, 55.9, 121.1, 125.9,

M AN U

126.6, 128.7, 129.6, 130.7, 155.0, 160.2, 165.9; Anal. calcd for C17H15ClN2O2: C, 64.87; H, 4.80; N, 8.90%. Found: C, 65.06; H, 4.92; N, 9.11%.

7j: 2-[(4-chlorophenoxy)-1-(5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)] ethanone, colorless crystals, yield, 80%; mp 198~199 oC; 1H NMR (CDCl3, 300 MHz): δ 2.11 (3H, s, -Me), 2.82 (1H, dd, J= 18.5 and 4.5 Hz, pyrazole, 4-Ha), 3.39 (1H, dd, J= 11.0 and 18.0 Hz, pyrazole, 4-Hb), 4.12 (2H, s, -CH2-), 5.68 (1H, dd, J= 11.5 and 4.5 Hz,

TE D

pyrazole, 5-H), 6.79-7.22 (8H, m, ArH); 13C NMR (CDCl3, 125 MHz): δ 16.2, 39.9, 45.1, 55.7, 66.0, 115.8, 116.8, 119.4, 124.9, 126.2, 127.9, 128.6, 129.6, 154.3, 157.5, 159.1, 164.3; Anal. calcd for C18H17ClN2O3: C, 62.70; H, 4.97; N, 8.12%. Found: C, 63.06; H, 5.13; N, 8.44%.

EP

7l: 1-[5-(5-bromo-2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-2-chloroethan-one, colorless crystals, yield, 88%; mp 160~161 oC; 1H NMR (CDCl3, 300 MHz): δ 2.21 (3H, s,

AC C

-Me), 3.00 (1H, dd, J= 18.9 and 3.6 Hz, pyrazole, 4-Ha), 3.42 (1H, dd, J= 11.7 and 19.2 Hz, pyrazole, 4-Hb), 4.49 (2H, m, -CH2-), 5.67 (1H, dd, J= 11.4 and 3.9 Hz, pyrazole, 5-H), 6.76-7.28 (3H, m, ArH), 8.65 (1H, brs, -OH); Anal. calcd for C12H12BrClN2O2: C, 43.47; H, 3.65; N, 8.45%. Found: C, 43.68; H, 4.02; N, 8.17%. 7m:

1-[5-(5-bromo-2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)]propan-1-one,

colorless crystals, yield, 75%; mp 180~181 oC; 1H NMR (CDCl3, 300 MHz): δ 1.14 (3H, t, J= 7.5 Hz, -CH3 ), 2.18 (3H, s, -Me), 2.67 (2H, m, -CH2-), 2.96 (1H, dd, J= 18.6 and 3.3 Hz, pyrazole, 4-Ha), 3.35 (1H, dd, J= 11.4 and 18.9 Hz, pyrazole, 4-Hb), 5.60 (1H, dd, J=

ACCEPTED MANUSCRIPT

11.1 and 3.6 Hz, pyrazole, 5-H), 6.74-7.26 (3H, m, ArH), 9.53 (1H, brs, -OH); Anal. calcd for C13H15BrN2O2: C, 50.18; H, 4.86; N, 9.00%. Found: C, 50.00; H, 5.07; N, 8.81%. 7n: 5-(5-bromo-2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl(p-tolyl)methan- one, colorless crystals, yield, 68%; mp 208~210 oC; 1H NMR (CDCl3, 300 MHz): δ 2.20 (3H,

RI PT

m, -CH3 ), 2.38 (3H, s, -Me), 3.04 (1H, dd, J= 18.9 and 3.6 Hz, pyrazole, 4-Ha), 3.40 (1H, dd, J= 11.1 and 18.9 Hz, pyrazole, 4-Hb), 5.83 (1H, dd, J= 11.1 and 3.9 Hz, pyrazole, 5-H), 6.83-7.82 (7H, m, ArH); Anal. calcd for C18H17BrN2O2: C, 57.92; H, 4.59; N, 7.51%. Found: C, 58.21; H, 4.52; N, 7.89%.

SC

7o: 1-[5-(5-bromo-2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-3-phenylpro- pan1-one, colorless crystals, yield, 82%; mp 179~180 oC; 1H NMR (CDCl3, 300 MHz): δ 2.06

M AN U

(3H, s, -Me), 2.86 (1H, dd, J= 16.8 and 3.6 Hz, pyrazole, 4-Ha), 2.88 (4H, m, -2CH2-), 3.22 (1H, dd, J= 10.8 and 18.6 Hz, pyrazole, 4-Hb), 5.51 (1H, dd, J= 11.4 and 3.3 Hz, pyrazole, 5-H), 6.67-7.19 (8H, m, ArH); Anal. calcd for C19H19BrN2O2: C, 58.93; H, 4.95; N, 7.23%. Found: C, 59.07; H, 4.60; N, 7.45%.

7p:1-[5-(5-bromo-2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-2-(4chlorophenoxy)ethanone, colorless crystals, yield, 77%; mp 211~212 oC; 1H NMR (CDCl3,

TE D

300 MHz): δ 2.14 (3H, s, -Me), 2.93 (1H, dd, J= 18.6 and 3.3 Hz, pyrazole, 4-Ha), 3.32 (1H, dd, J= 11.1 and 18.9 Hz, pyrazole, 4-Hb), 4.88 (2H, s, -CH2-), 5.59 (1H, dd, J= 11.4 and 3.9 Hz, pyrazole, 5-H), 6.64-7.34 (7H, m, ArH); Anal. calcd for C18H16BrClN2O3: C, 51.03; H, 3.81; N, 6.61%. Found: C, 51.00; H, 4.17; N, 6.21%. 2-chloro-1-[5-(3,5-dibromo-2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-

EP

7q:

ethanone, colorless crystals, yield, 89%; mp 136~137 oC; 1H NMR (CDCl3, 300 MHz): δ

AC C

2.07 (3H, s, -Me), 2.79 (1H, dd, J= 18.6 and 4.2 Hz, pyrazole, 4-Ha), 3.32 (1H, dd, J= 11.7 and 18.3 Hz, pyrazole, 4-Hb), 4.37 (2H, s, -CH2-), 5.54 (1H, dd, J= 11.4 and 4.2 Hz, pyrazole, 5-H), 6.96-7.50 (3H, m, ArH); Anal. calcd for C12H11Br2ClN2O2: C, 35.11; H, 2.70; N, 6.82%. Found: C, 35.44; H, 3.03; N, 7.10%. 7r: 1-[5-(3,5-dibromo-2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]propan-1-one, colorless crystals, yield, 68%; mp 124~125 oC; 1H NMR (CDCl3, 300 MHz): δ 1.07 (3H, t, J= 7.5 Hz -Me), 2.10 (3H, s, -Me), 2.59 (2H, m, -CH2-), 2.85 (1H, dd, J= 18.6 and 3.9 Hz, pyrazole, 4-Ha), 3.28 (1H, dd, J= 11.4 and 18.6 Hz, pyrazole, 4-Hb), 5.51 (1H, dd, J= 11.4

ACCEPTED MANUSCRIPT

and 3.9 Hz, pyrazole, 5-H), 6.91-7.52 (2H, m, ArH), 9.12 (1H, brs, -OH); Anal. calcd for C13H14Br2N2O2: C, 40.03; H, 3.62; N, 7.18%. Found: C, 39.91; H, 3.99; N, 6.87%. 7s: 1-[5-(3,5-dibromo-2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-3phenylpropan-1-one, colorless crystals, yield, 70%; mp 143~144 oC; 1H NMR (CDCl3, 300

RI PT

MHz): δ 2.12 (3H, s, -Me), 2.88 (1H, dd, J= 18.6 and 3.9 Hz, pyrazole, 4-Ha), 2.96 (4H, m, -2CH2-), 3.31 (1H, dd, J= 11.1 and 18.6 Hz, pyrazole, 4-Hb), 5.56 (1H, dd, J= 11.4 and 3.9 Hz, pyrazole, 5-H), 6.74-7.60 (7H, m, ArH), 9.05 (1H, brs, -OH); Anal. calcd for C19H18Br2N2O2: C, 48.95; H, 3.89; N, 6.01%. Found: C, 49.21; H, 4.04; N, 5.76%.

SC

7t: 2-(4-chlorophenoxy)-1-[5-(3,5-dibromo-2-hydroxyphenyl)-3-methyl-4,5

-dihydropyrazol-1-yl]ethanone, colorless crystals, yield, 93%; mp 208~210 oC; 1H NMR

M AN U

(CDCl3, 300 MHz): δ 2.16 (3H, s, -Me), 2.87 (1H, dd, J= 18.6 and 4.5 Hz, pyrazole, 4-Ha), 3.38 (1H, dd, J= 10.8 and 17.7 Hz, pyrazole, 4-Hb), 4.99 (2H, m, -CH2-), 5.63 (1H, dd, J= 11.7 and 4.5 Hz, pyrazole, 5-H), 6.85-7.57 (6H, m, ArH); Anal. calcd for C18H15Br2ClN2O3: C, 43.02; H, 3.01; N, 5.57%. Found: C, 43.00; H, 3.32; N, 6.00%. 7u:

5-(3,5-dibromo-2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl](p-tolyl)-

methanone, colorless crystals, yield, 81%; mp 164~165 oC; 1H NMR (CDCl3, 300 MHz): δ

TE D

2.17 (3H, s, -Me), 2.38 (3H, s, -Me), 2.94 (1H, dd, J= 18.6 and 4.2 Hz, pyrazole, 4-Ha), 3.40 (1H, dd, J= 11.1 and 18.3 Hz, pyrazole, 4-Hb), 5.82 (1H, dd, J= 11.4 and 4.2 Hz, pyrazole, 5-H), 7.09-7.84 (6H, m, ArH) , 8.92 (1H, brs, -OH); Anal. calcd for C18H16Br2N2O2: C, 47.82; H, 3.57; N, 6.20%. Found: C, 48.11; H, 3.85; N, 5.92%.

EP

4.1.2 General procedure for preparation of compounds 8d~8g Substituted-2-(3-methyl-4,5-dihydro- 1H-pyrazol-5-yl) phenol 2 (10 mmol) was

AC C

dissolved in 20 mL of chloroform, at room temperature was added tosyl chloride (10 mmol) or nitrophenyl chloride (10 mmol), the reaction mixture was refluxed for 1 h. The mixture was cooled, washed with water, and allowed to stand at 0~5 ˚C over night. The product was collected by filtration and the crude residue was purified by chromatography on SiO2 (ethyl acetate/petroleum, v:v=3:1) to give title compounds 8d~8g (Scheme 1) as colorless solids. 8d: 4-bromo-2-(3-methyl-1-tosyl-4,5-dihydro-1H-pyrazol-5-yl)phenol, colorless crystals, yield, 89%; mp 184~186 oC; 1H NMR (CDCl3, 300 MHz): δ 1.93 (3H, s, -Me), 2.38 (3H, s,

ACCEPTED MANUSCRIPT

-Me), 2.70 (1H, dd, J= 9.6 and 17.7 Hz, pyrazole, 4-Ha), 2.99 (1H, dd, J= 11.1 and 17.7 Hz, pyrazole, 4-Hb), 4.78 (1H, dd, J= 9.6 and 10.8 Hz, pyrazole, 5-H), 6.66-7.71 (7H, m, ArH); Anal. calcd for C17H17BrN2O3S: C, 49.89; H, 4.19; N, 6.84%. Found: C, 50.12; H, 4.31; N, 7.15%.

RI PT

8e: 2,4-dibromo-6-[3-methyl-1-(4-nitrophenylsulfonyl)-4,5-dihydro-1H-pyrazol-

5-yl]phenol, colorless crystals, yield, 85%; mp 230~231 oC; 1H NMR (CDCl3, 300 MHz): δ 1.93 (3H, s, -Me), 2.60 (1H, dd, J= 8.6 and 18.0 Hz, pyrazole, 4-Ha), 3.12 (1H, dd, J= 11.7 and 18.3 Hz, pyrazole, 4-Hb), 5.09 (1H, dd, J=8.7 and 11.4 Hz, pyrazole, 5-H),

SC

7.18-8.29 (6H, m, ArH); Anal. calcd for C16H13Br2N3O5S: C, 37.02; H, 2.52; N, 8.09%. Found: C, 36.89; H, 2.80; N, 8.17%.

8f: 2,4-dibromo-6-(3-methyl-1-tosyl-4,5-dihydro-1H-pyrazol-5-yl)phenol, colorless crystals,

M AN U

yield, 82%; mp 217~219 oC; 1H NMR (CDCl3, 300 MHz): δ 1.94 (3H, s, -Me), 2.17 (3H, s, -Me), 2.75 (1H, dd, J= 8.6 and 18.0 Hz, pyrazole, 4-Ha), 3.17 (1H, dd, J= 11.4 and 18.3 Hz, pyrazole, 4-Hb), 4.89 (1H, dd, J=8.6 and 11.4 Hz, pyrazole, 5-H), 7.05-7.60 (6H, m, ArH); Anal. calcd for C17H16Br2N2O3S: C, 41.82; H, 3.30; N, 5.74%. Found: C, 42.03; H, 3.51; N, 5.52%.

TE D

8g: 4-chloro-2-[3-methyl-1-(4-nitrophenylsulfonyl)-4,5-dihydro-1H-pyrazol-5-yl] phenol, colorless crystals, yield, 80%; mp 223~224 oC; 1H NMR (CDCl3, 300 MHz): δ 2.02 (3H, s, -Me), 2.78 (1H, dd, J= 9.0 and 18.3 Hz, pyrazole, 4-Ha), 3.17 (1H, dd, J= 11.1 and 18.3 Hz, pyrazole, 4-Hb), 5.08 (1H, dd, J=9.0 and 11.1 Hz, pyrazole, 5-H), 6.74-8.36 (7H, m,

EP

ArH); Anal. calcd for C16H14ClN3O5S: C, 48.55; H, 3.57; N, 10.62%. Found: C, 48.70; H, 3.21; N, 10.99%.

AC C

4.1.3 General procedure for preparation of compounds 9a~9u 2-chloro-1-(5-(2-substituted-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)

ethanone 3 (10 mmol) was dissolved in 20 mL of acetone, at room temperature was added amine (12 mmol), DMAP (12 mmol) and catalytic KI, the reaction mixture was allowed to stand at 40 ˚C for 2 h. The mixture was cooled, washed with water. The product was collected by filtration and the crude residue was purified by chromatography on SiO2 (dichloromethane/methanol, v:v= 68:1) to give compounds 9a~9u (Scheme 1) as colorless solids. Acetylation of the hydroxyl of compound 3, then according to the general procedure

ACCEPTED MANUSCRIPT

for preparation of compounds 9a~9u, compounds 10b~10p were synthesized. 9a: 2-(diethylamino)-1-[5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]- ethanone, colorless crystals, yield, 60%; mp 203~204 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.02 (t, 6H, J = 7.2 Hz), 2.18 (s, 3H), 2.65 (q, 4H, J = 7.2 Hz), 3.02 (dd, 1H, J1 = 18.4 Hz, J2 =

RI PT

2.8 Hz, pyrazole, 4-Ha), 3.32 (dd, 1H, J1 = 18.4 Hz, J2 = 11.6 Hz, pyrazole, 4-Hb), 3.61 (m, 2H), 5.62 (dd, 1H, J1 = 11.6 Hz, J2 = 2.8 Hz, pyrazole, 5-H), 6.85-6.93 (m, 3H), 7.14-7.18 (m, 1H), 9.29 (brs, 1H). 13C NMR (CDCl3, 125 MHz): δ 12.0, 16.2, 44.0, 47.9, 53.8, 53.9, 118.4, 120.3, 125.3, 127.1, 129.3, 154.9, 158.7, 169.4; Anal. calcd for C16H23N3O2: C,

SC

66.41; H, 8.01; N, 14.52%. Found: C, 66.06; H, 7.89; N, 14.70%.

9b: 2-(diisopropylamino)-1-[5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-

M AN U

yl]- ethanone, colorless crystals, yield, 75%; mp 211~212 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 0.97-1.00 (m, 12H), 2.20 (s, 3H), 3.01-3.10 (m, 3H, containing pyrazole 4-Ha), 3.32 (dd, 1H, J1 = 18.4 Hz, J2 = 11.2 Hz, pyrazole, 4-Hb), 3.58 (s, 2H), 5.65 (dd, 1H, pyrazole, 4-Hb), 6.85-6.97 (m, 3H), 7.15-7.19 (m, 1H), 9.34 (brs, 1H, -OH). Anal. calcd for C18H27N3O2: C, 68.11; H, 8.57; N, 13.24%. Found: C, 67.85; H, 8.80; N, 13.02%. 9c: l-[5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-2-(piperidin-1-yl)

TE D

ethan one, colorless crystals, yield, 80%; mp 225~227 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.41-1.42 (m, 2H), 1.57-1.63 (m, 4H), 2.17 (s, 3H), 2.50-2.51 (m, 4H), 3.01 (dd, 1H, J1 = 18.4 Hz, J2 = 2.8 Hz, pyrazole, 4-Ha), 3.31 (dd, 1H, J1 = 18.4 Hz, J2 = 11.2 Hz, pyrazole, 4-Hb), 3.49 (m, 2H), 5.66 (dd, 1H, J1 = 11.6 Hz, J2 = 2.8 Hz, pyrazole, 5-H),

EP

6.84-6.93 (m, 3H), 7.14-7.18 (m, 1H), 9.25 (brs, 1H). 13C NMR (CDCl3, 125 MHz): δ 16.2, 24.1, 25.8, 43.8, 53.7, 55.0, 59.8, 118.9, 120.7, 125.7, 127.2, 129.6, 155.1, 158.7, 168.4;

AC C

Anal. calcd for C17H23N3O2: C, 67.75; H, 7.69; N, 13.94%. Found: C, 68.06; H, 7.44; N, 14.22%.

9d:1-[5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-2-(pyrrolidin-1-yl) ethanone, colorless crystals, yield, 65%; mp 200~201 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.73-1.78 (m, 4H), 2.17 (s, 3H), 2.61-2.65 (m, 4H), 3.02 (dd, 1H, J1 = 18.4 Hz, J2 = 2.8 Hz, pyrazole, 4-Ha), 3.29 (dd, 1H, J1 = 18.4 Hz, J2 = 11.2 Hz, pyrazole, 4-Hb), 3.65 (m, 2H), 5.67 (dd, 1H, J1 = 11.2 Hz, J2 = 2.8 Hz, pyrazole, 5-H), 6.84-6.92 (m, 3H), 7.14-7.18 (m, 1H). 13C NMR (CDCl3, 125 MHz): δ 16.2, 23.7, 43.6, 53.5, 54.6, 56.9, 119.4, 120.9, 125.8,

ACCEPTED MANUSCRIPT

127.3, 129.7, 155.3, 158.7, 168.8; Anal. calcd for C16H21N3O2: C, 66.88; H, 7.37; N, 14.62%. Found: C, 67.05; H, 7.33; N, 14.99%. 9g: 1-[5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-2-(4methoxyphenyl amino)ethanone, colorless crystals, yield, 82%; mp 225~226 oC; 1H NMR

RI PT

(400 MHz, CDCl3) δ (ppm): 2.20 (s, 3H), 3.01 (dd, 1H, J1 = 18.4 Hz, J2 = 2.8 Hz, pyrazole, 4-Ha), 3.36 (dd, 1H, J1 = 18.4 Hz, J2 = 11.2 Hz, pyrazole, 4-Hb), 3.74 (s, 3H), 4.17 (m, 2H, -CH2- ), 5.71 (dd, 1H, J1 = 11.2 Hz, J2 = 2.8 Hz, pyrazole, 5-H), 6.62-6.64 (m, 2H), 6.76-6.78 (m, 3H), 6.84-7.13 (m, 4H), 8.79 (brs, 1H, -NH). 13C NMR (CDCl3, 125 MHz):

SC

δ 16.2, 43.9, 46.9, 53.8, 55.9, 114.6, 114.9, 118.8, 121.0, 125.6, 126.7, 129.8, 141.7, 152.5, 154.8, 159.6, 168.4; Anal. calcd for C19H21N3O3: C, 67.24; H, 6.24; N, 12.38%. Found: C,

M AN U

67.00; H, 6.45; N, 12.05%.

9j: 1-[5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-2-(4-phenylpiperidin-1 -yl)ethanone, colorless crystals, yield, 59%; mp 198~200 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.34-1.49 (m, 2H), 1.50-1.60 (m, 2H), 2.04-2.10 (m, 4H), 2.17 (s, 3H), 2.92-3.05 (m, 2H, containing pyrazole, 4-Ha), 3.36 (dd, 1H, J1 = 18.4 Hz, J2 = 11.2 Hz, pyrazole, 4-Hb), 3.49 (m, 2H), 5.65 (dd, 1H, J1 = 11.2 Hz, J2 = 3.2 Hz, pyrazole, 5-H), 6.85-6.95 (m,

TE D

3H), 7.11-7.18 (m, 4H), 7.24-7.28 (m, 2H), 9.21 (brs, 1H). Anal. calcd for C23H27N3O2: C, 73.18; H, 7.21; N, 11.13%. Found: C, 73.57; H, 7.44; N, 11.01%. 9l: 2-(4-chloropiperidin-1-yl)-1-[5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1yl]ethanone, colorless crystals, yield, 80%; mp 220~222 oC; 1H NMR (400 MHz, CDCl3) δ

EP

(ppm): 1.89-1.97 (m, 2H), 2.08-2.14 (m, 2H), 2.16 (s, 3H), 2.43-2.48 (m, 2H), 2.82-2.84 (m, 2H, containing pyrazole, 4-Ha), 3.00 (dd, 1H, J1 = 18.4 Hz, J2 = 3.6 Hz, pyrazole,

AC C

4-Hb), 3.28-3.36 (m, 1H), 3.54 (m, 2H), 4.05 (brs, 1H, - piperidine-Cl), 5.66 (dd, 1H, J1 = 11.2 Hz, J2 = 3.6 Hz, pyrazole, 5-H), 6.84-6.92 (m, 3H), 7.13-7.17 (m, 1H), 9.09 (brs, 1H, -OH). 13C NMR (CDCl3, 125 MHz): δ 16.2, 35.3, 43.5, 51.4, 53.3, 57.2, 58.9, 119.6, 121.2, 126.0, 127.2, 129.9, 155.3, 159.0, 168.3; Anal. calcd for C17H22ClN3O2: C, 60.80; H, 6.60; N, 12.51%. Found: C, 61.17; H, 6.94; N, 12.50%. 9m:

2-(3,5-dimethylpiperidin-1-yl)-1-[5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol

-1-yl]ethanone, colorless crystals, yield, 83%; mp 217~218 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 0.87-0.92 (m, 6H), 2.08-2.14 (m, 2H), 2.13 (s, 3H), 2.17-2.22 (m, 2H),

ACCEPTED MANUSCRIPT

2.36-2.48 (m, 2H), 2.91 (dd, 1H, J1 = 18.8 Hz, J2 = 4.4 Hz pyrazole, 4-Ha), 3.23-3.27 (m, 2H), 3.40 (dd, 1H, J1 = 18.4 Hz, J2 = 11.6 Hz pyrazole, 4-Hb), 3.98 (m, 2H), 5.67 (dd, 1H, J1 = 11.6 Hz, J2 = 4.4 Hz pyrazole, 5-H), 6.87-6.95 (m, 2H), 7.05-7.07 (m, 1H), 7.14-7.18 (m, 1H). Anal. calcd for C19H27N3O2: C, 69.27; H, 8.26; N, 12.76%. Found: C, 69.05; H,

RI PT

8.34; N, 13.02%.

9n:Tert-butyl4-{2-[5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-2-oxo

ethyl}piperazine-1-carboxylate, colorless crystals, yield, 80%; mp 189~190 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.44 (s, 9H), 2.17 (s, 3H), 2.52-2.52 (m, 4H), 3.02 (dd, 1H, J1

SC

= 18.8 Hz, J2 = 3.2 Hz pyrazole, 4-Ha), 3.33 (dd, 1H, J1 = 18.8 Hz, J2 = 11.6 Hz pyrazole, 4-Hb ), 3.44-3.47 (m, 4H), 3.53 (m, 2H), 5.66 (dd, 1H, J1 = 11.2 Hz, J2 = 3.6 Hz pyrazole,

M AN U

5-H ), 6.86-6.92 (m, 3H), 7.14-7.19 (m, 1H), 9.04 (brs, 1H). 13C NMR (CDCl3, 125 MHz): δ 16.2, 28.5, 43.5, 53.3, 53.4, 59.0, 79.7, 119.6, 121.3, 126.0, 127.2, 130.0, 154.8, 155.2, 159.0, 168.0; Anal. calcd for C21H30N4O4: C, 62.67; H, 7.51; N, 13.92%. Found: C, 63.01; H, 7.22; N, 13.61%.

9o: 1-[5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-2-(4-methylpiperazin-1yl)ethanone, colorless crystals, yield, 69%; mp 184~185 oC; 1H NMR (400 MHz, CDCl3) δ

TE D

(ppm): 2.13 (s, 3H), 2.56 (s, 3H), 2.87-2.88 (m, 4H), 2.92-2.96 (m, 5H, containing pyrazole, 4-Ha), 3.33 (dd, 1H, J1 = 18.4 Hz, J2 = 11.6 Hz pyrazole, 4-Hb), 3.61 (m, 2H), 5.61 (dd, 1H, J1 = 11.6 Hz, J2 = 4.0 Hz pyrazole, 5-H), 6.82-6.86 (m, 1H), 6.88-6.92 (m, 2H), 7.10-7.14 (m, 1H). Anal. calcd for C17H24N4O2: C, 64.53; H, 7.65; N, 17.71%. Found: C, 64.81; H,

EP

7.94; N, 18.10%.

9q:1-[5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-2-(3-methylpiperidin-

AC C

1-yl)ethanone, colorless crystals, yield, 65%; mp 200~201 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 0.80-0.83 (m, 3H), 1.56-1.66 (m, 2H), 1.69-1.73 (m, 4H), 1.94-2.03 (m, 1H), 2.16 (s, 3H), 2.86-2.95 (m, 2H), 3.00 (dd, 1H, J1 = 18.4 Hz, J2 = 3.2 Hz pyrazole, 4-Ha ), 3.31 (dd, 1H, J1 = 18.8 Hz, J2 = 11.2 Hz pyrazole, 4-Hb), 3.49 (m, 2H), 5.66 (dd, 1H, J1 = 11.2 Hz, J2 = 18.4 Hz pyrazole, 5-H), 6.84-6.92 (m, 3H), 7.13-7.17 (m, 1H). 13C NMR (CDCl3, 125 MHz): δ 16.2, 19.7, 25.5, 31.0, 32.8, 43.5, 53.3, 54.4, 59.5, 62.4, 119.5, 121.1, 126.0, 127.4, 129.8, 155.3, 158.6, 168.6; Anal. calcd for C18H25N3O2: C, 68.54; H, 7.99; N, 13.32%. Found: C, 68.17; H, 8.22; N, 13.07%.

ACCEPTED MANUSCRIPT

9r:1-[5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-2-thiomorpholino- ethanone, colorless crystals, yield, 86%; mp 177~178 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 2.20 (s, 3H), 2.71 (4H, d, J= 4.0 Hz), 2.83 (4H, d, J= 4.0 Hz), 3.07 (1H, dd, J= 3.2 and 18.6 Hz, pyrazole, 4-Ha), 3.40 (1H, dd, J= 11.2 and 18.8 Hz, pyrazole, 4-Hb), 3.55 (2H, m), 5.65

RI PT

(1H, dd, J= 12.0 and 4.0 Hz, pyrazole, 5-H), 6.90-7.23 (4H, m), 9.09 (1H, brs, -OH). Anal. calcd for C16H21N3O2S: C, 60.16; H, 6.63; N, 13.16%. Found: C, 60.01; H, 6.87; N, 13.40%.

9s: 2-(diisobutylamino)-1-[5-(2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]

SC

ethanone, colorless crystals, yield, 81%; mp 172~173 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 0.81 (m, 12H), 1.64 (m, 2H), 2.18 (s, 3H), 2.30 (4H, d, J= 8.0 Hz), 3.04 (1H, dd,

M AN U

J= 4.0 and 20.0 Hz, pyrazole, 4-Ha), 3.30 (1H, dd, J= 12.0 and 20.0 Hz, pyrazole, 4-Hb), 3.59 (2H, m), 5.65 (1H, dd, J= 12.0 and 4.0 Hz, pyrazole, 5-H), 6.87-7.20 (4H, m), 9.48 (1H, brs, -OH). Anal. calcd for C20H31N3O2: C, 69.53; H, 9.04; N, 12.16%. Found: C, 69.17; H, 8.85; N, 12.44%.

9t: 1-[5-(5-bromo-2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-2-(dipropylamino) ethanone, colorless crystals, yield, 74%; mp 180~181 oC; 1H NMR (CDCl3, 400 MHz): δ

TE D

0.85 (6H, t, J= 8.0 Hz), 1.46 (4H, m), 2.16 (3H, s, -Me), 2.55 (4H, m), 2.89 (1H, dd, J= 20.0 and 4.0 Hz, pyrazole, 4-Ha), 3.32 (1H, dd, J= 12.0 and 20.0 Hz, pyrazole, 4-Hb), 3.67 (2H, m, -CH2-), 5.62 (1H, dd, J= 12.0 and 4.0 Hz, pyrazole, 5-H), 6.67-7.18 (3H, m, ArH); 10.91%.

EP

Anal. calcd for C18H26BrN3O2: C, 54.55; H, 6.61; N, 10.60%. Found: C, 54.19; H, 6.35; N, 9u: 1-[5-(5-bromo-2-hydroxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-2-(diallylamino)

AC C

ethanone, colorless crystals, yield, 80%; mp 167~168 oC; 1H NMR (CDCl3, 400 MHz): δ 2.00 (3H, s, -Me), 2.59 (1H, dd, J= 20.0 and 4.0 Hz, pyrazole, 4-Ha), 3.26 (5H, m, containing pyrazole, 4-Hb), 3.74 (2H, m, -CH2-), 5.12 (4H, m), 5.60 (1H, dd, J= 12.0 and 4.0 Hz, pyrazole, 5-H), 5.84 (2H, m), 6.28-6.94 (3H, m, ArH); Anal. calcd for C18H22BrN3O2: C, 55.11; H, 5.65; N, 10.71%. Found: C, 55.34; H, 5.89; N, 10.42%. 10b: 2-(1-(2-(diethylamino)acetyl)-3-methyl-4,5-dihydro-1H-pyrazol-5-yl)phenyl acetate, colorless crystals, yield, 81%; mp 228~230 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.17 (t, 6H, J = 7.2 Hz), 2.06 (s, 3H), 2.29 (s, 3H), 2.67-2.73 (m, 1H), 2.91 (m, 4H, containing

ACCEPTED MANUSCRIPT

pyrazole, 4-Ha ), 3.27 (dd, 1H, J1 = 18.4 Hz, J2 = 12.8 Hz pyrazole, 4-Hb ), 3.85 (s, 2H), 5.46 (dd, 1H, J1 = 12.0 Hz, J2 = 5.2 Hz pyrazole, 5-H), 7.02-7.04 (m, 1H), 7.18-7.20 (m, 2H), 7.27-7.31 (m, 1H). Anal. calcd for C18H25N3O3: C, 65.23; H, 7.60; N, 12.68%. Found: C, 65.01; H, 7.25; N, 13.04%.

RI PT

10c:2-[1-(2-(diisopropylamino)acetyl)-3-methyl-4,5-dihydro-1H-pyrazol-5-yl]phenyl

acetate, colorless crystals, yield, 80%; mp 218~219 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.39-1.43 (m, 12H), 2.17 (s, 3H), 2.34 (s, 3H), 2.84 (dd, 1H, J1 = 18.4 Hz, J2 = 4.4 Hz pyrazole, 4-Ha ), 3.47 (dd, 1H, J1 = 18.4 Hz, J2 = 12.4 Hz pyrazole, 4-Hb ),

3.69-3.75

SC

(m, 3H), 4.33-4.70 (m, 1H), 5.42-5.54 (m, 1H, pyrazole, 5-H), 7.03-7.15 (m, 1H), 7.23-7.27 (m, 2H), 7.33-7.36 (m, 1H). Anal. calcd for C20H29N3O3: C, 66.83; H, 8.13; N,

M AN U

11.69%. Found: C, 66.45; H, 8.52; N, 12.02%.

10d: 2-(diisopropylamino)-1-[5-(2-methoxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl] ethanone, colorless crystals, yield, 64%; mp 210~212 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.43-1.62 (m, 12H), 2.11 (d, 3H, J = 6.8 Hz), 2.58-2.79 (m, 1H), 3.28 (s, 1H), 3.37-3.47 (m, 1H), 3.82 (s, 3H), 4.14 (s, 1H),

4.29-4.62 (m, 2H), 5.51-5.64 (m, 1H),

6.87-6.92 (m, 2H), 6.98-7.08 (m, 1H), 7.25-7.26 (m, 1H). Anal. calcd for C19H29N3O2: C,

TE D

68.85; H, 8.82; N, 12.68%. Found: C, 69.17; H, 9.07; N, 12.99%. 10e: 2-[3-methyl-1-(2-(piperidin-1-yl)acetyl)-4,5-dihydro-1H-pyrazol-5-yl]phenyl acetate, colorless crystals, yield, 85%; mp 225~227 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.40-1.42 (m, 2H), 1.57-1.62 (m, 4H), 2.03 (s, 3H), 2.28 (s, 3H), 2.59-2.61 (m, 4H), 2.65

EP

(dd, 1H, J1 = 19.2 Hz, J2 = 5.6 Hz), 3.18-3.26 (m, 1H), 3.56 (s, 2H), 5.46 (dd, 1H, J1 = 12.0 Hz, J2 = 5.6 Hz), 7.01-7.03 (m, 1H), 7.13-7.19 (m, 2H), 7.24-7.27 (m, 1H). Anal. calcd for

AC C

C19H25N3O3: C, 66.45; H, 7.34; N, 12.24%. Found: C, 66.31; H, 7.22; N, 12.01%. 10g: 2-[3-methyl-1-(2-(pyrrolidin-1-yl)acetyl)-4,5-dihydro-1H-pyrazol-5-yl]phenyl acetate, colorless crystals, yield, 76%; mp 220~221 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.59-1.78 (m, 4H), 2.10-2.12 (m, 3H), 2.29 (s, 3H), 2.67-2.81 (m, 1H), 3.21-3.40 (m, 3H), 3.48-3.53 (m, 2H), 4.11-4.72 (m, 2H), 5.42-5.46 (m, 1H), 7.00-7.04 (m, 1H), 7.18-7.25 (m, 2H), 7.27-7.33 (m, 1H). Anal. calcd for C18H23N3O3: C, 65.63; H, 7.04; N, 12.76%. Found: C, 66.02; H, 7.41; N, 12.51%. 10i: 2-{1-[2-(4-hydroxypiperidin-1-yl)acetyl]-3-methyl-4,5-dihydro-1H-pyrazol-5-yl}

ACCEPTED MANUSCRIPT

phenyl acetate, colorless crystals, yield, 67%; mp 220~221 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.63-1.69 (m, 2H), 1.92-1.94 (m, 2H), 2.07 (s, 3H), 2.31 (s, 3H), 2.42-2.47 (m, 2H), 2.69 (dd, 1H, J1 = 18.8 Hz, J2 = 5.2 Hz),

2.92-2.94 (m, 2H), 3.26 (dd, 1H, J1 = 18.8

Hz, J2 = 12.4 Hz), 3.61 (s, 2H), 3.70-3.72 (m, 1H), 5.49 (dd, 1H, J1 = 12.0 Hz, J2 = 5.2 Hz),

RI PT

7.04-7.06 (m, 1H), 7.16-7.22 (m, 2H), 7.27-7.30 (m, 1H). Anal. calcd for C19H25N3O4: C, 63.49; H, 7.01; N, 11.69%. Found: C, 63.11; H, 7.39; N, 12.38%.

10j: 2-{3-methyl-1-[2-(4-acetylpiperidin-1-yl)acetyl]-4,5-dihydro-1H-pyrazol-5-yl}-

phenyl acetate, colorless crystals, yield, 81%; mp 208~209 oC; 1H NMR (400 MHz,

SC

CDCl3) δ (ppm): 1.70-1.78 (m, 2H), 1.89-1.934 (m, 2H), 2.02 (s, 3H), 2.03 (s, 3H), 2.28 (s, 3H), 2.53-2.58 (m, 2H), 2.66 (dd, 1H, J1 = 18.8 Hz, J2 = 5.2 Hz), 2.92-2.95 (m, 2H), 3.23

M AN U

(dd, 1H, J1 = 18.4 Hz, J2 = 12.0 Hz), 3.63 (s, 2H), 4.75-4.80 (m, 1H), 5.45 (dd, 1H, J1 = 12.0 Hz, J2 = 5.2 Hz), 7.01-7.03 (m, 1H), 7.13-7.20 (m, 2H), 7.24-7.28 (m, 1H). Anal. calcd for C21H27N3O4: C, 65.44; H, 7.06; N, 10.90%. Found: C, 65.31; H, 7.01; N, 10.67%. 10k: 1-(5-(2-methoxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl)-2-(4-methoxypiperidin-1-yl)ethanone, colorless crystals, yield, 80%; mp 203~204 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 2.10 (s, 3H), 2.14-2.18 (m, 4H), 2.69 (dd, 1H, J1 = 18.4 Hz, J2 = 4.0 Hz),

TE D

3.41 (dd, 1H, J1 = 18.4 Hz, J2 = 11.6 Hz), 3.51 (s, 3H), 3.85 (s, 3H), 3.89-4.04 (m, 4H), 4.28-4.29 (m, 1H), 4.73-4.80 (m, 2H), 5.61 (dd, 1H, J1 = 11.6 Hz, J2 = 4.4 Hz), 6.90-6.97 (m, 2H), 7.02-7.04 (m, 1H), 7.26-7.30 (m, 1H). Anal. calcd for C19H27N3O3: C, 66.06; H, 7.88; N, 12.16%. Found: C, 66.00; H, 8.17; N, 12.45%.

EP

10l: 2-{3-methyl-1-[2-(2-methylpiperidin-1-yl)acetyl]-4,5-dihydro-1H-pyrazol-5- yl}phenyl acetate, colorless crystals, yield, 62%; mp 211~212 oC; 1H NMR (400 MHz, CDCl3) δ

AC C

(ppm): 1.24-1.28 (m, 3H), 1.38-1.42 (m, 2H), 1.69-1.75 (m, 4H), 1.90-1.96 (m, 1H), 2.07 (s, 3H), 2.28 (s, 3H), 2.69-2.78 (m, 1H), 3.09-3.10 (m, 2H), 3.24-3.31 (m, 1H), 3.88 (dd, 1H, J1 = 16.8 Hz, J2 = 8.0 Hz), 4.10-4.13 (m, 1H), 5.40-5.43 (m, 1H), 7.01-7.04 (m, 1H), 7.18-7.20 (m, 2H), 7.28-7.31 (m, 1H). Anal. calcd for C20H27N3O3: C, 67.20; H, 7.61; N, 11.76%. Found: C, 67.38; H, 8.00; N, 11.43%. 10n: 2-[1-(2-chloroacetyl)-3-methyl-4,5-dihydro-1H-pyrazol-5-yl]phenyl acetate, colorless crystals, yield, 70%; mp 205~206 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 2.09 (s, 3H), 2.32 (s, 3H), 2.75 (dd, 1H, J1 = 18.0 Hz, J2 = 5.6 Hz), 3.31 (dd, 1H, J1 = 18.0 Hz, J2 = 12.0

ACCEPTED MANUSCRIPT

Hz), 4.43 (s, 2H), 5.51 (dd, 1H, J1 = 12.4 Hz, J2 = 5.6 Hz), 7.07-7.09 (m, 1H), 7.17-7.24 (m, 2H), 7.28-7.32 (m, 1H). Anal. calcd for C14H15ClN2O3: C, 57.05; H, 5.13; N, 9.50%. Found: C, 56.87; H, 5.44; N, 9.22%. 10o: N,N-diethyl-2-[5-(2-methoxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-N-

RI PT

methyl-2-Oxoethanaminium, colorless crystals, yield, 80%; mp 227~229 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.38 (6H, t, J = 7.2 Hz), 2.08 (s, 3H), 2.68 (1H, dd, J= 4.4 and 18.4 Hz, pyrazole, 4-Ha), 3.40 (1H, dd, J= 11.6 and 18.4 Hz, pyrazole, 4-Hb), 3.46 (3H, s), 3.79-3.94 (7H, m), 4.64 (2H, m), 5.58 (1H, dd, J= 11.6 and 4.8 Hz, pyrazole, 5-H),

SC

6.88-6.94 (2H, m), 6.98-7.01 (1H, m), 7.24-7.29 (1H, m). Anal. calcd for C18H28N3O2+: C, 67.89; H, 8.86; N, 13.20%. Found: C, 68.14; H, 9.13; N, 12.98%.

10p: 1-[5-(2-methoxyphenyl)-3-methyl-4,5-dihydropyrazol-1-yl]-2-(pyrrolidin-1-

M AN U

yl) ethanaminium, colorless crystals, yield, 70%; mp 219~221 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 2.06 (s, 3H), 2.21-2.24 (m, 2H), 2.34-2.37 (m, 2H), 2.66 (1H, dd, J = 18.0 and 4.8 Hz, pyrazole, 4-Ha), 3.38 (1H, dd, J = 18.8 and 11.6 Hz, pyrazole, 4-Hb), 3.44 (3H, s), 3.85 (3H, s), 4.03-4.13 (4H, m), 4.98 (2H, m), 5.60 (1H, dd, J = 11.6 and 4.8 Hz, pyrazole, 5-H), 6.88-6.95 (2H, m), 7.01-7.29 (2H, m). Anal. calcd for C18H26N3O2+: C,

TE D

68.33; H, 8.28; N, 13.28%. Found: C, 68.60; H, 8.41; N, 13.31%. 4.1.4 General procedure for preparation of compounds 11f~11j A solution of carboxylic acid (12 mmol) and nitropheny chloride (12 mmol) in chloroform (20 mL) was stirred, 3-(5-substituted-phenyl-4,5-dihydro-1H-pyrazol- 3-yl)-

EP

2H-chromen-2-one (10 mmol) and DMAP (14 mmol) were added, then the reaction mixture was refluxed for 5 h. The mixture was cooled, washed with water, and allowed to

AC C

stand at 0 ˚C over night. The product was collected by filtration and the crude residue was purified by chromatography on SiO2 (acetone/petroleum, v:v=4:1) to give title compounds 11f~11j (Scheme 2) as colorless solids. 11f:

3-(1-benzoyl-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-2-one,

colorless

crystals, yield, 62%; mp 220~222 oC; 1H NMR (CDCl3, 300 MHz): δ 3.49 (1H, dd, J= 18.9 and 5.1 Hz , pyrazole, 4-Ha), 4.03 (1H, dd, J= 12.0 and 18.9 Hz, pyrazole, 4-Hb), 5.83 (1H, dd, J= 12.0 and 5.4 Hz , pyrazole, 5-H), 7.25-8.03 (14H, m, C5-8-H and ArH ), 8.35 (1H, s, C4-H); 13C NMR (CDCl3, 125 MHz): δ 43.8, 61.8, 116.7, 118.8, 119.7, 125.0, 125.7,

ACCEPTED MANUSCRIPT

127.9, 128.9, 129.0, 130.1, 131.2, 133.0, 134.3, 141.3, 141.5, 151.7, 154.2, 159.3, 166.7; Anal. calcd for: C25H18N2O3: C, 76.13; H, 4.60; N, 7.10%. Found: C, 76.42; H, 4.91; N, 7.00%. 11g: 3-[1-(4-methylbenzoyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl]-2H-chromen

RI PT

-2- one, colorless crystals, yield, 69%; mp 197~198 oC; 1H NMR (CDCl3, 300 MHz): δ 2.42 (3H, s, -Me), 3.45 (1H, dd, J= 18.9 and 5.1 Hz , pyrazole, 4-Ha), 4.00 (1H, dd, J= 12.0 and 18.9 Hz, pyrazole, 4-Hb), 5.81 (1H, dd, J= 12.0 and 5.4 Hz , pyrazole, 5-H), 7.24-7.92 (13H, m, C5-8-H and ArH ), 8.34 (1H, s, C4-H); 13C NMR (CDCl3, 125 MHz): δ

SC

21.7, 43.7, 61.8, 116.7, 117.9, 118.9, 119.8, 125.0, 125.4, 125.7, 127.8, 128.6, 128.9, 129.0, 130.2, 131.3, 132.9, 141.2, 141.6, 151.4, 154.2, 159.3, 166.6; Anal. calcd for: C26H20N2O3:

M AN U

C, 76.45; H, 4.94; N, 6.86%. Found: C, 76.71; H, 5.21; N, 7.09%.

11h: 3-(5-phenyl-1-propionyl-4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-2-one, colorless crystals, yield, 78%; mp 217~218 oC; 1H NMR (CDCl3, 300 MHz): δ 1.22 (3H, t, J= 7.5 Hz, -Me), 2.84 (2H, q, J= 7.5 Hz, -CH2-), 3.41 (1H, dd, J= 18.9 and 5.1 Hz , pyrazole, 4-Ha), 3.96 (1H, dd, J= 12.0 and 18.9 Hz, pyrazole, 4-Hb), 5.59 (1H, dd, J= 12.0 and 4.8 Hz , pyrazole, 5-H), 7.21-7.64 (9H, m, C5-8-H and ArH ), 8.44 (1H, s, C4-H);

13

C NMR

TE D

(CDCl3, 125 MHz): δ 9.0, 27.6, 44.2, 60.6, 116.7, 118.9, 119.9, 125.0, 125.6, 127.7, 128.9, 129.0, 132.9, 140.9, 141.8, 150.5, 154.2, 159.3, 172.4; Anal. calcd for: C21H18N2O3: C, 72.82; H, 5.24; N, 8.09%. Found: C, 73.04; H, 5.02; N, 8.43%. 11i: 3-[1-(2-chloroacetyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl]-2H-chromen

EP

-2-one, colorless crystals, yield, 93%; mp 210~212 oC; 1H NMR (CDCl3, 300 MHz): δ 3.49 (1H, dd, J= 18.9 and 4.8 Hz , pyrazole, 4-Ha), 3.99 (1H, dd, J= 12.0 and 19.2 Hz,

AC C

pyrazole, 4-Hb), 4.55 (2H, s, -CH2-), 5.59 (1H, dd, J= 11.7 and 4.8 Hz , pyrazole, 5-H), 7.21-7.65 (9H, m, C5-8-H and ArH ), 8.46 (1H, s, C4-H); 13C NMR (DMSO-d6, 125 MHz): δ 42.9, 44.4, 60.4, 116.6, 119.1, 125.5, 126.0, 128.0, 129.3, 129.9, 133.8, 141.9, 143.2, 152.8, 154.0, 158.4, 164.0; Anal. calcd for: C20H15ClN2O3: C, 65.49; H, 4.12; N, 7.64%. Found: C, 65.70; H, 3.95; N, 8.00%. 11j: 3-[1-(2-chlorobenzoyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl]-2H-chromen -2- one, colorless crystals, yield, 63%; mp 194~195 oC; 1H NMR (CDCl3, 300 MHz): δ 3.50 (1H, dd, J= 19.2 and 5.1 Hz , pyrazole, 4-Ha), 4.08 (1H, dd, J= 12.0 and 18.9 Hz,

ACCEPTED MANUSCRIPT

pyrazole, 4-Hb), 5.78 (1H, dd, J= 12.0 and 5.1 Hz , pyrazole, 5-H), 7.26-7.55 (13H, m, C5-8-H and ArH ), 8.22 (1H, s, C4-H); 13C NMR (DMSO-d6, 125 MHz): δ 44.6, 60.6, 116.6, 118.9, 119.5, 125.4, 126.2, 127.6, 128.0, 129.3, 129.5, 130.0, 130.5, 131.4, 133.7, 136.2,

N, 6.53%. Found: C, 70.39; H, 3.90; N, 6.47%.

RI PT

142.1, 142.5, 153.6, 154.0, 158.5, 164.8; Anal. calcd for: C25H17ClN2O3: C, 70.01; H, 4.00; 4.1.5 General procedure for preparation of compounds 12k~12l 3-(5-substituted-phenyl-4,5-dihydro-1H-

pyrazol-3-yl)-2H-chromen-2-one

6

(10

mmol) was dissolved in 20 mL of chloroform, at room temperature was added tosyl

SC

chloride (10 mmol) or nitrophenyl chloride (10 mmol) and DMAP (10 mmol), the reaction mixture was refluxed for 2 h. The mixture was cooled, washed with water, and allowed to

M AN U

stand at 0~5 ˚C over night. The product was collected by filtration and the crude residue was purified by chromatography on SiO2 (acetone/petroleum, v:v=2:1) to give title compounds 12k~12l (Scheme 2) as colorless solids.

12k: 3-[1-(4-nitrophenylsulfonyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl]-2H-chromen-2 -one, colorless crystals, yield, 89%; mp 217~219 oC; 1H NMR (CDCl3, 300 MHz): δ 3.46 (1H, dd, J= 18.6 and 7.5 Hz , pyrazole, 4-Ha), 3.94 (1H, dd, J= 11.7 and 18.9 Hz, pyrazole,

TE D

4-Hb), 5.19 (1H, dd, J= 11.4 and 7.5 Hz , pyrazole, 5-H), 7.23-8.26 (13H, m, C5-8-H and ArH ), 8.49 (1H, s, C4-H); Anal. calcd for: C24H17N3O6S: C, 60.63; H, 3.60; N, 8.84%. Found: C, 61.02; H, 3.41; N, 9.12%.

12l:3-(5-phenyl-1-tosyl-4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-2-one,colorless crystals,

EP

yield, 91%; mp 208~210 oC; 1H NMR (CDCl3, 300 MHz): δ 2.42 (3H, s, -Me), 3.27 (1H, dd, J= 18.6 and 9.3 Hz , pyrazole, 4-Ha), 3.85 (1H, dd, J= 11.7 and 18.6 Hz, pyrazole,

AC C

4-Hb), 4.95 (1H, dd, J= 11.7 and 9.3 Hz , pyrazole, 5-H), 7.26-7.74 (13H, m, C5-8-H and ArH ), 8.53 (1H, s, C4-H); 13C NMR (CDCl3, 125 MHz): δ 21.7, 45.7, 65.9, 116.7, 118.8, 118.9, 125.2, 126.8, 128.2, 128.5, 128.9, 129.1, 129.7, 133.2, 140.6, 141.9, 142.3, 144.5, 153.7, 154.3, 159.3; Anal. calcd for: C25H20N2O4S: C, 67.55; H, 4.54; N, 6.30%. Found: C, 67.19; H, 4.50; N, 6.02%. 4.1.6 General procedure for preparation of compounds 13a~13z 3-(1-(2-chloroacetyl)-5-substituted-phenyl-4,5-Dihydro-1H-pyrazol-3-yl)-2H-chrome n-2-one 6’ (10 mmol) was dissolved in 20 mL of acetone, at room temperature was added

ACCEPTED MANUSCRIPT

amine (12 mmol), piperazine (10 mmol) and catalytic KI, the reaction mixture was allowed to stand at 50 ˚C for 5 h. The mixture was cooled, washed with water. The product was collected by filtration and the crude residue was purified by chromatography on SiO2 (dichloromethane /methanol, v:v= 70:1) to give title compounds 13a~13z (Scheme 2) as

RI PT

colorless solids.

13a:3-{1-[2-(methylamino)acetyl]-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl}-2H-

chromen-2-one, colorless crystals, yield, 68%; mp 208~209 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 2.46 (s, 3H), 3.43 (dd, 1H, J1 = 19.2 Hz, J2 = 4.8 Hz pyrazole, 4-Ha),

SC

3.87-4.00 (m, 3H, containing pyrazole, 4-Hb), 5.57 (dd, 1H, J1 = 11.6 Hz, J2 = 5.2 Hz pyrazole, 5-H), 7.19-7.24 (m, 3H), 7.26-7.36 (m, 4H), 7.57-7.68 (m, 2H), 8.35 (1H, s,

M AN U

C4-H), 9.28 (1H, brs, -NH); Anal. calcd for: C21H19N3O3: C, 69.79; H, 5.30; N, 11.63%. Found: C, 70.05; H, 4.99; N, 12.02%.

13b: 3-{1-[2-(ethylamino)acetyl]-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl}-2H-chro- men2-one, colorless crystals, yield, 62%; mp 206~207 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 2.46 (t, 3H, J = 7.2 Hz), 2.64-2.73 (m, 2H), 3.43 (dd, 1H, J1 = 19.2 Hz, J2 = 4.8 Hz pyrazole, 4-Ha), 3.90-4.00 (m, 3H, containing pyrazole, 4-Hb), 5.58 (dd, 1H, J1 = 12.0 Hz,

TE D

J2 = 4.8 Hz pyrazole, 5-H ), 7.19-7.27 (m, 3H), 7.30-7.37 (m, 4H), 7.57-7.65 (m, 2H), 8.45 (1H, s, C4-H); Anal. calcd for: C22H21N3O3: C, 70.38; H, 5.64; N, 11.19%. Found: C, 70.65; H, 6.01; N, 11.54%.

13c: 3-{1-[2-(isopropylamino)acetyl]-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl}-2H-

EP

chromen-2-one, colorless crystals, yield, 60%; mp 202~204 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.07-1.11 (m, 6H), 2.81 (m, 1H), 3.43 (dd, 1H, J1 = 18.8 Hz, J2 = 4.8 Hz

AC C

pyrazole, 4-Ha ), 3.90-4.01 (m, 3H, -CH2 and containing pyrazole, 4-Hb), 5.58 (dd, 1H, J1 = 12.0 Hz, J2 = 5.2 Hz pyrazole, 5-H), 7.20-7.25 (m, 3H), 7.30-7.37 (m, 4H), 7.58-7.66 (m, 2H), 8.45 (1H, s, C4-H); Anal. calcd for: C23H23N3O3: C, 70.93; H, 5.95; N, 10.79%. Found: C, 71.21; H, 6.24; N, 11.10%. 13d: 3-{3-phenyl-1-[2-(propylamino)acetyl]-4,5-dihydro-1H-pyrazol-5-yl}2H-chromen-2-one, colorless crystals, yield, 67%; mp 212~214 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 0.92 (t, 3H, J = 7.2 Hz), 1.54 (q, 2H), 2.57-2.64 (m, 2H), 3.44 (dd, 1H, J1 = 19.2 Hz, J2 = 4.8 Hz pyrazole, 4-Ha), 3.89-4.01 (m, 3H, containing pyrazole, 4-Hb ), 5.58

ACCEPTED MANUSCRIPT

(dd, 1H, J1 = 11.6 Hz, J2 = 4.8 Hz pyrazole, 5-H), 7.20-7.24 (m, 3H), 7.31-7.37 (m, 4H), 7.58-7.65 (m, 2H), 8.45 (1H, s, C4-H); Anal. calcd for: C23H23N3O3: C, 70.93; H, 5.95; N, 10.79%. Found: C, 71.05; H, 5.68; N, 10.59%. 13e: 3-{1-[2-(cyclopropylamino)acetyl]-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl}-

RI PT

2H-chromen-2-one, colorless crystals, yield, 55%; mp 208~209 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 0.44-0.52(m, 4H), 2.26 (t, 1H, J2 = 5.2 Hz ), 3.44 (dd, 1H, J1 = 18.8 Hz, J2 = 4.8 Hz pyrazole, 4-Ha), 3.92-4.02 (m, 3H, containing pyrazole, 4-Hb), 5.59 (dd, 1H, J1 = 12.0 Hz, J2 = 4.8 Hz pyrazole, 5-H), 7.19-7.25 (m, 3H), 7.27-7.37 (m, 4H), 7.58-7.66 (m, 13

C NMR (CDCl3, 125 MHz): δ 6.3, 6.5, 30.5, 44.1, 51.1, 60.9,

SC

2H), 8.45 (1H, s, C4-H);

116.8, 118.8, 119.5, 125.1, 125.7, 127.9, 129.0, 133.1, 141.2, 141.4, 151.7, 154.3, 159.3,

M AN U

169.6; Anal. calcd for: C23H21N3O3: C, 71.30; H, 5.46; N, 10.85%. Found: C, 71.66; H, 5.19; N, 10.48%.

13f: 3-{1-[2-(isobutylamino)acetyl]-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl}-2Hchromen-2-one, colorless crystals, yield, 62%; mp 214~216 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 0.92-0.94 (m, 6H), 1.74-1.81 (m, 1H), 2.45-2.48 (m, 2H), 3.44 (dd, 1H, J1 = 19.2 Hz, J2 = 4.8 Hz pyrazole, 4-Ha), 3.89-4.01 (m, 3H, containing pyrazole, 4-Hb), 5.59

TE D

(dd, 1H, J1 = 12.0 Hz, J2 = 4.8 Hz pyrazole, 5-H), 7.21-7.24 (m, 3H), 7.27-7.37 (m, 4H), 7.58-7.64 (m, 2H), 8.45 (1H, s, C4-H); Anal. calcd for: C24H25N3O3: C, 71.44; H, 6.25; N, 10.41%. Found: C, 71.20; H, 5.97; N, 10.75%. 13g: 3-{1-[2-(tert-butylamino)acetyl]-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl}-2H-

EP

chromen-2-one, colorless crystals, yield, 69%; mp 202~203 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.15 (s, 9H), 3.43 (dd, 1H, J1 = 19.2 Hz, J2 = 5.2 Hz pyrazole, 4-Ha),

AC C

3.89-4.01 (m, 3H, containing pyrazole, 4-Hb), 5.57 (dd, 1H, J1 = 12.0 Hz, J2 = 4.8 Hz pyrazole, 5-H), 7.20-7.24 (m, 3H), 7.29-7.37 (m, 4H), 7.58-7.67 (m, 2H), 8.45 (1H, s, C4-H); Anal. calcd for: C24H25N3O3: C, 71.44; H, 6.25; N, 10.41%. Found: C, 71.69; H, 6.38; N, 10.70%.

13h: 3-{1-[2-(cyclohexylamino)acetyl]-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl}2H-chromen-2-one, colorless crystals, yield, 60%; mp 212~214 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.09-1.25 (m, 4H), 1.66-1.71 (m, 4H), 1.89-1.91 (m, 2H), 2.40-2.46 (m, 1H), 3.44 (dd, 1H, J1 = 18.2 Hz, J2 = 4.8 Hz pyrazole, 4-Ha), 3.91-3.98 (m, 3H, containing

ACCEPTED MANUSCRIPT

pyrazole, 4-Hb), 5.56-5.59 (m, 1H, pyrazole, 5-H), 7.20-7.25 (m, 3H), 7.30-7.37 (m, 4H), 7.58-7.65 (m, 2H), 8.45 (1H, s, C4-H); Anal. calcd for: C26H27N3O3: C, 72.71; H, 6.34; N, 9.78%. Found: C, 73.08; H, 6.30; N, 9.46%. 13i: 3-{1-[2-(dibutylamino)acetyl]-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl}-2H-chromen-2

RI PT

-one, colorless crystals, yield, 69%; mp 207~208 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 0.90 (t, 6H, J1 = 7.2 Hz,), 1.25-1.34 (m, 4H), 1.58-1.64 (m, 4H), 2.92-2.96 (m, 4H), 3.45 (dd, 1H, J1 = 19.6 Hz, J2 = 4.8 Hz pyrazole, 4-Ha), 3.94-4.23 (m, 3H, containing pyrazole, 4-Hb), 5.56 (dd, 1H, J1 = 12.0 Hz, J2 = 4.8 Hz pyrazole, 5-H), 7.18-7.24 (m, 2H), 7.26-7.37

SC

(m, 5H), 7.58-7.62 (m, 1H), 7.69-7.72 (m, 1H), 8.59 (1H, s, C4-H); Anal. calcd for: C28H33N3O3: C, 73.18; H, 7.24; N, 9.14%. Found: C, 73.51; H, 7.00; N, 9.41%. 13j: 3-{3-phenyl-1-[2-(pyrrolidin-1-yl)acetyl]-4,5-dihydro-1H-pyrazol-5-yl}-2H-

M AN U

chromen-2-one, colorless crystals, yield, 59%; mp 211~212 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.84-1.84 (m, 4H), 2.78-2.84 (m, 4H), 3.42 (dd, 1H, J1 = 19.2 Hz, J2 = 4.8 Hz pyrazole, 4-Ha), 3.85-4.01 (m, 3H, containing pyrazole, 4-Hb), 5.59 (dd, 1H, J1 = 12.0 Hz, J2 = 4.8 Hz pyrazole, 5-H), 7.19-7.24 (m, 3H), 7.26-7.37 (m, 4H), 7.57-7.66 (m, 2H), 8.46 (1H, s, C4-H); Anal. calcd for: C24H23N3O3: C, 71.80; H, 5.77; N, 10.47%. Found: C,

TE D

72.17; H, 6.09; N, 10.65%.

13l: 3-{1-[2-(2-methylpiperidin-1-yl)acetyl]-3-phenyl-4,5-dihydro-1H-pyrazol5-yl}- 2H-chromen-2-one, colorless crystals, yield, 57%; mp 212~213 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.22-1.25 (m, 3H), 1.37-1.40 (m, 1H), 1.57-1.76 (m, 4H), 2.70-3.20

EP

(m, 4H), 3.41-3.47 (m, 1H, pyrazole, 4-Ha), 3.67-4.27 (m, 3H, containing pyrazole, 4-Hb), 5.57 (dd, 1H, J1 = 12.0 Hz, J2 = 4.8 Hz pyrazole, 5-H), 7.19-7.22 (m, 2H), 7.25-7.37 (m,

AC C

5H), 7.57-7.69 (m, 2H), 8.52 (1H, s, C4-H); Anal. calcd for: C26H27N3O3: C, 72.71; H, 6.34; N, 9.78%. Found: C, 72.40; H, 6.61; N, 10.11%. 13m: 3-{1-[2-(3-methylpiperidin-1-yl)acetyl]-3-phenyl-4,5-dihydro-1H-pyrazol5-yl}-2H-chromen-2-one, colorless crystals, yield, 50%; mp 218~219 oC;

13

C NMR

(CDCl3, 125 MHz): δ 19.5, 24.8, 24.9, 30.6, 32.0, 44.2, 53.7, 58.8, 60.9, 61.2, 116.7, 118.8, 125.2, 125.7, 128.0, 129.0, 129.2, 133.2, 141.1, 141.9, 152.1, 154.3, 159.3, 166.1; Anal. calcd for: C26H27N3O3: C, 72.71; H, 6.34; N, 9.78%. Found: C, 72.89; H, 6.20; N, 9.54%. 13n: 3-{1-[2-(3,5-dimethylpiperidin-1-yl)acetyl]-3-phenyl-4,5-dihydro-1H-

ACCEPTED MANUSCRIPT

pyrazol-5- yl}-2H-chromen-2-one, colorless crystals, yield, 55%; mp 210~211 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 0.83 (d, 6H, J = 6.0 Hz), 1.67-1.93 (m, 6H), 2.92-3.00 (m, 2H), 3.40 (dd, 1H, J1 = 19.2 Hz, J2 = 4.8 Hz pyrazole, 4-Ha), 3.71 (dd, 2H, J1 = 32.0 Hz, J2 = 16.8 Hz pyrazole, 4-Hb), 3.93 (dd, 1H, J1 = 18.8 Hz, J2 = 12.0 Hz pyrazole, 5-H),

RI PT

5.57 (dd, 1H, J1 = 12.0 Hz, J2 = 4.8 Hz), 7.19-7.25 (m, 3H), 7.26-7.36 (m, 4H), 7.56-7.62 (m, 2H), 8.41 (1H, s, C4-H); Anal. calcd for: C27H29N3O3: C, 73.11; H, 6.59; N, 9.47%. Found: C, 73.50; H, 6.85; N, 9.22%.

13o: 3-{1-[2-(4-(diethylamino)piperidin-1-yl)acetyl]-3-phenyl-4,5-dihydro-1H-

SC

pyrazol-5-yl}-2H-chromen-2-one, colorless crystals, yield, 62%; mp 198~199 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.54 (d, 6H, J = 7.2 Hz), 1.97-2.07 (m, 2H), 2.20-2.20 (m,

M AN U

2H), 2.41-2.48 (m, 2H), 3.15-3.23 (m, 6H), 3.39-3.45 (m, 2H, containing pyrazole, 4-Ha), 3.78 (m, 2H), 3.96 (dd, 1H, J1 = 18.8 Hz, J2 = 12.4 Hz pyrazole, 4-Hb), 5.55 (dd, 1H, J1 = 12.0 Hz, J2 = 4.8 Hz pyrazole, 5-H), 7.18-7.24 (m, 3H), 7.29-7.37 (m, 4H), 7.58-7.62 (m, 1H), 7.70-7.71 (m, 1H), 8.49 (1H, s, C4-H); Anal. calcd for: C29H34N4O3: C, 71.58; H, 7.04; N, 11.51%. Found: C, 71.29; H, 6.99; N, 11.57%.

13p: 3-{1-[2-(4-hydroxypiperidin-1-yl)acetyl]-3-phenyl-4,5-dihydro-1H-pyrazol

TE D

-5- yl}-2H-chromen-2-one, colorless crystals, yield, 60%; mp 220~221 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.63-1.69 (m, 2H), 1.90-1.93 (m, 2H), 2.38-2.49 (m, 2H), 2.89-2.96 (m, 2H), 3.39 (dd, 1H, J1 = 19.2 Hz, J2 = 4.8 Hz pyrazole, 4-Ha), 3.69-3.79 (m, 3H), 3.94 (dd, 1H, J1 = 19.2 Hz, J2 = 12.0 Hz pyrazole, 4-Hb ), 5.57 (dd, 1H, J1 = 12.0 Hz, J2 = 4.8

EP

Hz pyrazole, 5-H), 7.19-7.25 (m, 3H), 7.28-7.37 (m, 4H), 7.57-7.63 (m, 2H), 8.40 (1H, s, C4-H); Anal. calcd for: C25H25N3O4: C, 69.59; H, 5.84; N, 9.74%. Found: C, 69.77; H, 6.05;

AC C

N, 10.02%.

13r:3-{1-[2-(4-chloropiperidin-1-yl)acetyl]-3-phenyl-4,5-dihydro-1H-pyrazol-5yl}-2H-chromen-2-one, colorless crystals, yield, 51%; mp 219~221 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.91-1.99 (m, 2H), 2.10-2.15 (m, 2H), 2.51-2.61 (m, 2H), 2.88-2.91 (m, 2H), 3.40 (dd, 1H, J1 = 18.8 Hz, J2 = 4.8 Hz pyrazole, 4-Ha), 3.74 (m, 2H), 3.95 (dd, 1H, J1 = 18.8 Hz, J2 = 12.0 Hz pyrazole, 4-Hb), 4.07-4.07 (s, 1H), 5.57 (dd, 1H, J1 = 12.0 Hz, J2 = 4.8 Hz pyrazole, 5-H), 7.19-7.25 (m, 3H), 7.29-7.37 (m, 4H), 7.58-7.63 (m, 2H), 8.41 (1H, s, C4-H); 13C NMR (CDCl3, 125 MHz): δ 35.5, 44.0, 50.9, 51.3, 57.3, 59.2, 60.8,

ACCEPTED MANUSCRIPT

116.8, 118.8, 119.7, 125.1, 125.6, 127.8, 128.9, 129.0, 133.1, 141.2, 141.4, 151.2, 154.2, 159.2, 167.7; Anal. calcd for: C25H24ClN3O3: C, 66.74; H, 5.38; N, 9.34%. Found: C, 67.03; H, 5.06; N, 9.74%. 13s: Tert-butyl4-{2-oxo-2-[5-(2-oxo-2H-chromen-3-yl)-3-phenyl-4,5-

RI PT

dihydropyrazol- 1-yl]ethyl}piperazine-1-carboxylate, colorless crystals, yield, 64%; mp 203~204 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.45 (s, 9H), 2.58-2.63 (m, 4H), 3.40 (dd, 1H, J1 = 18.8 Hz, J2 = 4.8 Hz pyrazole, 4-Hb ), 3.46-3.47 (m, 4H), 3.73 (m, 2H), 3.95 (dd, 1H, J1 = 18.8 Hz, J2 = 12.4 Hz pyrazole, 4-Hb), 5.57 (dd, 1H, J1 = 12.0 Hz, J2 = 4.8 Hz

SC

pyrazole, 5-H), 7.19-7.24 (m, 3H), 7.29-7.37 (m, 4H), 7.58-7.64 (m, 2H), 8.40 (1H, s, C4-H); Anal. calcd for: C29H32N4O5: C, 67.43; H, 6.24; N, 10.85%. Found: C, 67.21; H,

M AN U

6.42; N, 11.07%.

13t: 3-{1-[2-(4-methylpiperazin-1-yl)acetyl]-3-phenyl-4,5-dihydro-1H-pyrazol5-yl}-2H-chromen-2-one, colorless crystals, yield, 60%; mp 200~201 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 2.73 (s, 3H), 3.11-3.26 (m, 8H), 3.43 (dd, 1H, J1 = 19.6 Hz, J2 = 4.8 Hz pyrazole, 4-Ha), 3.85 (m, 2H), 3.97 (dd, 1H, J1 = 19.6 Hz, J2 = 11.6 Hz pyrazole, 4-Hb), 5.54 (dd, 1H, J1 = 11.6 Hz, J2 = 4.8 Hz pyrazole, 5-H), 7.16-7.26 (m, 3H), 7.29-7.38

TE D

(m, 4H), 7.58-7.63 (m, 1H), 7.70-7.72 (m, 1H), 8.51 (1H, s, C4-H); 13C NMR (CDCl3, 125 MHz): δ 44.0, 44.2, 50.4, 54.0, 58.7, 61.0, 116.8, 118.8, 119.2, 125.2, 128.0, 129.1, 129.2, 133.3, 141.1, 141.7, 152.2, 154.3, 159.2, 166.7; Anal. calcd for: C25H26N4O3: C, 69.75; H, 6.09; N, 13.01%. Found: C, 69.47; H, 6.38; N, 13.25%.

EP

13u:3-{3-phenyl-1-[2-(4-phenylpiperidin-1-yl)acetyl]-4,5-dihydro-1H-pyrazol-5yl}-2H-chromen-2-one, colorless crystals, yield, 68%; mp 220~221 oC; 1H NMR (400

AC C

MHz, CDCl3) δ (ppm): 1.55-1.72 (m, 2H), 2.53-2.54 (m, 2H), 2.77-2.88 (m, 2H), 3.24-3.27 (m, 2H), 3.41 (dd, 1H, J1 = 18.8 Hz, J2 = 4.8 Hz pyrazole, 4-Ha), 3.53-3.53 (m, 1H), 3.96 (dd, 1H, J1 = 18.8 Hz, J2 = 12.0 Hz pyrazole, 4-Hb), 4.07-4.18 (m, 2H), 5.53 (dd, 1H, J1 = 12.0 Hz, J2 = 4.8 Hz pyrazole, 5-H), 7.09-7.16 (m, 2H), 7.17-7.26 (m, 5H), 7.29-7.36 (m, 5H), 7.57-7.59 (m, 1H), 7.74-7.76 (m, 1H), 8.65 (1H, s, C4-H);

13

C NMR (CDCl3, 125

MHz): δ 30.5, 30.6, 36.4, 42.5, 44.4, 57.9, 61.0, 117.7, 118.7, 118.8, 125.2, 125.6, 126.2, 128.1, 128.4, 129.1, 129.5, 133.4, 139.8, 140.8, 142.6, 153.0, 154.3, 159.3, 164.5; Anal. calcd for: C31H29N3O3: C, 75.74; H, 5.95; N, 8.55%. Found: C, 76.02; H, 6.11; N, 8.91%.

ACCEPTED MANUSCRIPT

13v:1-{2-oxo-2-[5-(2-oxo-2H-chromen-3-yl)-3-phenyl-4,5-dihydropyrazol-1-yl]ethyl}piperidine-4-carboxamide, colorless crystals, yield, 51%; mp 202~203 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 1.76-1.90 (m, 4H), 2.13-2.20 (m, 1H), 2.28-2.39 (m, 2H), 3.04-3.12 (m, 2H), 3.40 (dd, 1H, J1 = 18.8 Hz, J2 = 4.8 Hz pyrazole, 4-Ha), 3.74 (m, 2H),

RI PT

3.95 (dd, 1H, J1 = 18.8 Hz, J2 = 12.4 Hz pyrazole, 4-Hb ), 5.37-5.53 (m, 2H), 5.58 (dd, 1H, J1 = 12.0 Hz, J2 = 4.8 Hz pyrazole, 5-H), 7.19-7.24 (m, 3H), 7.29-7.37 (m, 4H), 7.58-7.64 (m, 2H), 8.41 (1H, s, C4-H); Anal. calcd for: C26H26N4O4: C, 68.11; H, 5.72; N, 12.22%. Found: C, 68.43; H, 5.38; N, 12.45%.

SC

13w: 3-{1-[2-(diethylamino)acetyl]-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl}-2H

-chromen-2-one, colorless crystals, yield, 59%; mp 200~201 oC; 13C NMR (CDCl3, 125

M AN U

MHz): δ 10.4, 25.8, 44.1, 44.9, 52.0, 58.3, 60.3, 60.9, 116.7, 118.8, 125.2, 125.6, 127.9, 129.1, 129.3, 133.2, 141.3, 141.8, 151.9, 154.3, 159.2, 167.3; Anal. calcd for: C24H25N3O3: C, 71.44; H, 6.25; N, 10.41%. Found: C, 71.69; H, 5.98; N, 10.20%. 13x: 3-[5-phenyl-1-(2-thiomorpholinoacetyl)-4,5-dihydro-1H-pyrazol-3-yl]-2H -chromen-2-one, colorless crystals, yield, 75%; mp 215~216 oC; 1H NMR (CDCl3, 400 MHz): δ 2.74 (4H, brs), 2.94 (4H, brs), 3.41 (1H, dd, J= 19.2 and 4.8 Hz, pyrazole, 4-Ha),

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3.74 (2H, m), 3.96 (1H, dd, J= 12.0 and 19.2 Hz, pyrazole, 4-Hb), 5.57 (1H, dd, J= 12.0 and 4.8 Hz , pyrazole, 5-H), 7.19-7.62 (9H, m, C5-8-H and ArH), 8.41 (1H, s, C4-H); Anal. calcd for: C24H23N3O3S: C, 66.49; H, 5.35; N, 9.69%. Found: C, 66.71; H, 5.07; N, 9.88%. 13y: 3-{1-[2-(dihexylamino)acetyl]-5-[2-(trifluoromethyl)phenyl]-4,5-dihydro

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-1H-pyrazol-3-yl}-2H-chromen-2-one, colorless crystals, yield, 77%; mp 219~220 oC; 1H NMR (CDCl3, 500 MHz): δ 0.85 (6H, t, J= 6.8 Hz), 1.27-1.50 (16H, m), 2.64 (4H, t, J=7.4

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Hz), 3.29 (1H, dd, J= 19.2 and 5.0 Hz , pyrazole, 4-Ha), 3.78 (2H, m), 3.97 (1H, dd, J= 11.9 and 19.2 Hz, pyrazole, 4-Hb), 5.93 (1H, dd, J= 11.9 and 5.0 Hz , pyrazole, 5-H), 7.25-7.64 (8H, m, C5-8-H and ArH), 8.43 (1H, s, C4-H);

13

C NMR (CDCl3, 125 MHz): δ

14.2, 22.8, 27.2, 27.9, 32.0, 44.4, 54.6, 57.2, 116.8, 118.8, 119.6, 125.0, 126.7, 127.6, 128.9, 129.5, 132.7, 133.0, 140.4, 141.1, 150.3, 154.2, 159.2, 169.3; Anal. calcd for: C33H40F3N3O3: C, 67.91; H, 6.91; N, 7.20%. Found: C, 67.77; H, 7.20; N, 7.05%. 13z: 3-{1-(2-morpholinoacetyl)-5-[2-(trifluoromethyl)phenyl]-4,5-dihydro1H-pyrazol-3-yl}-2H-chromen-2-one, colorless crystals, yield, 81%; mp 232~234 oC; 1H

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NMR (CDCl3, 400 MHz): δ 2.73 (4H, t, J= 4.0 Hz), 2.93 (4H, t, J=4.0 Hz), 3.30 (1H, dd, J= 20.0 and 8.0 Hz , pyrazole, 4-Ha), 3.82 (2H, m), 3.96 (1H, dd, J= 12.0 and 20.0 Hz, pyrazole, 4-Hb), 5.93 (1H, dd, J= 12.0 and 8.0 Hz , pyrazole, 5-H), 7.14-7.69 (8H, m, 8.66%. Found: C, 62.01; H, 4.41; N, 8.82%.

4.2 Crystallographic Studies

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C5-8-H and ArH ), 8.40 (1H, s, C4-H); Anal. calcd for: C25H22F3N3O4: C, 61.85; H, 4.57; N,

X-ray single-crystal diffraction data of compounds 7d, 7f, 7j, 7l, 7m, 7n, 7o, 7t, 7u,

SC

8d, 9d, 9g, 9l, 9n, 9r, 9s, 13f, 13x, 13y and 13z were collected on a Bruker SMART APEX CCD diffractometer at 296(2) K using MoKα radiation (λ = 0.71073 Å) by the ω scan

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mode. The program SAINT was used for integration of the diffraction profiles. The structures were solved by direct methods using the SHELXS program of the SHELXTL package and refined by full-matrix least-squares methods with SHELXL [36]. All non-hydrogen atoms of compounds 7d, 7f, 7j, 7l, 7m, 7n, 7o, 7t, 7u, 8d, 9d, 9g, 9l, 9n, 9r, 9s, 13f, 13x, 13y and 13z were refined with anisotropic thermal parameters. All hydrogen atoms were generated theoretically onto the parent atoms and refined isotropically with

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fixed thermal factors.

4.3 Cell Proliferation Assays

The antiproliferative activities evaluation was conducted by using a modified

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procedure as described in the literature [37]. Briefly, target tumor cells were grown to log phase in RPMI 1640 medium supplemented with 10% fetal bovine serum. After diluting to

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3×104 cells mL-1 with the complete medium, 100 µL of the obtained cell suspension was added to each well of 96-well culture plates. The subsequent incubation was performed at 37 ˚C, 5% CO2 atmosphere for 24 h before subjecting to antiproliferation assessment. Tested samples at pre-set concentrations were added to 6 wells with Doxorubicin (AMD) co-assayed as a positive reference. After 48 h exposure period, 25 µL of PBS containing 2.5 mg·mL-1 of MTT was added to each well. After 4 h, the medium was replaced by 150 µL DMSO to dissolve the purple formazan crystals produced. The absorbance at 570 nm of

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each well was measured on an ELISA plate reader. The data represented the mean of three experiments in triplicate and were expressed as means ±SD using Student’s test. The IC50 value was defined as the concentration at which 50% of the cells could survive. The following human tumor cell lines were used in the assay: human gastric cancer cell

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MGC-803, human breast cancer cell Bcap-37 and Prostate cancer cell PC3.

4.4 Telomerase Activity Assays

Compounds 7~13 were tested in a search for small molecule inhibitors of telomerase

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activity by using the TRAP-PCR-ELISA assay. In detail, the MGC-803 cells were firstly maintained in RPMI 1640 buffer (Hyclone, Miami, FL, USA), supplemented with 10%

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fetal bovineserum (GIBCO, New York, USA), streptomycin (0.1 mg/mL) and penicillin (100 IU/mL) at 37°C in a humidified atmosphere containing 5% CO2. After trypsinization, 5×104 cultured cells in logarithmic growth were seeded into T25 flasks (Corning, New York, USA) and cultured to allow to adherence. The cells were then incubated with Staurosporine (Santa Cruz, Santa Cruz, USA) and the drugs with a series of concentration as 60, 20, 6.67, 2.22, 0.75, 0.25 and 0.082l g/mL, respectively. After 24 h treatment, the

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cells were harvested by cell scraper orderly following by washed once with PBS. The cells were lysed in 150 µL RIPA cell lysis buffer (Santa Cruz, Santa Cruz, USA), and incubated on ice for 30 min. The cellular supernatants were obtained via centrifugation at 12,000 g for 20 min at 4 °C and stored at -80 °C. The TRAP-PCR-ELISA assay was performed

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using a telomerase detection kit (Roche, Basel, Switzerland) according to the manufacturer’s protocol. In brief, 2 µL of cell extracts were mixed with 48 µL TRAP

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reaction mixtures. TRAP primers and Taq polymerase and incubated at 25oC for 30 min. PCR was then initiated at 94 °C, 120 s for predenaturation and performed using 35 cycles each consisting of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 90 s. Then 20 µL of PCR products were hybridized to a digoxigenin (DIG)-labeled telomeric repeat specific detection probe. And the PCR products were immobilized via the biotin-labeled primer to a streptavidin-coated microtiter plate subsequently. The immobilized DNA fragments were detected with a peroxidase-conjugated anti-DIG antibody and visualized following addtion of the stop regent. The microtitre plate was assessed on TECAN Infinite M200 microplate

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reader (Mannedorf, Switzerland) at a wavelength of 490 nm, and the final value were presented as mean±SD. 4.5 Molecular Modeling In this study, a three-dimension human telomerase model [7] and in silico

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Schrödinger’s IFD were used to model the binding poses of our designed compounds with Mcl-1. Before doing the IFD, the structural errors of the model was first corrected manually and then the complex was treated by Protein Prepared Wizard of Schrödinger. IFD is allowing incorporation of the protein and ligand flexibility in the docking

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protocol, which is consisted of the following steps: (i) constrained minimization of the protein with an RMSD cutoff of 0.18 Å; (ii) initial Glide docking of the ligand using a

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softened potential (Van der Waals radii scaling); (iii) one round of Prime side-chain prediction for each protein/ligand complex, on residues within defined distance of any ligand pose; (iv) prime minimization of the same set of residues and the ligand for each protein/ligand complex pose; (v) Glide re-docking of each protein/ligand complex structure within a specified energy of the lowest energy structure; (vi) estimation of the binding energy (IFDScore) for each output pose. All docking calculations were run in the extra

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precision (XP) mode of Glide. The center of the grid box of the hTERT was defined by two residues in the active site: Lys 710 and Lys 902. The size of the grid box was set to 15 Å. Default values were used for all other parameters. A further 10 ns MD simulation was submitted using a docking pose which can

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account for the limited SAR of BIBR1532 and BIBR1591 by employing the program of Desmond, which was developed at D. E. Shaw Research to perform high-speed molecular

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dynamics simulations of biological systems on conventional commodity clusters. All suggested values of Desmond were employed to run this 10 ns MD simulation. The MD simulation include the following steps: (i) a solvated system for simulation was generated; (ii) positive or negative counter ions were distributed to neutralize the system and additional ions (Na+ and Cl-) to were introduced to set the desired ionic strength; (iii) OPLS-AA force field parameters were assigned to the entire molecular system; (iv) the system was relaxed or minimized; (v) the Desmond simulation was run.

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4.6 Evaluation of the antitumor activities in vivo 4.6.1 Animals and Cell Lines Kunming mice (SPF, male or female, 20 ± 2 g, 60 rats were divided randomly into two groups containing 10 normal rats and 50 administrated rats.) were purchased from the

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experimental animal center of China Pharmaceutical University. Animals were housed in a temperature (22 ± 2 °C) and relatively humidity (50%)-controlled room on a 12 h light/dark cycle, given free access to food and water, and acclimatized for at least one week prior to use. All the animal experiments were performed in accordance with the

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Regulations of the Experimental Animal Administration issued by the State Committee of Science and Technology of China. Efforts were made to minimize the number of animals

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used and their suffering. Animals were maintained in accordance with the Guides of Center for Developmental Biology, Anhui Medical University for the Care and Use of Laboratory Animals and all experiments used protocols approved by the institutions’ subcommittees on animal care.

Cell lines used for evaluation of the in vivo antitumor activity in this study included three tumor cell lines, namely S180 (sarcoma tumer cell line), HepG2 (liver carcinoma cell

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line), EAC (Ehrlich ascites carcinoma cell line). All of cell lines were purchased by the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and the cells were cultured in RPMI-1640 medium, which was supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin and cultured in an

growth phase.

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atmosphere of 5% CO2 at 37 °C. Cells were collected for the experiments in the logarithmic

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To establish the tumor-bearing mouse model, the cell lines were harvested and inoculated subcutaneously into the right armpit region of the mice. On the 7th day, the tumor ascrites were obtained and washed with sterile PBS. Under sterile condition, the tumor ascrites were diluted with sterile nomal saline to 1 × 1010 /L cell suspension. Tumor ascites were maintained in vivo in mice by transplantation of 0.2 mL of ascites (2 × 106 cells) from the infected mice to the non-infected mice. 4.6.2. Tumor Xenograft model in vivo Each Kunming mouse (male or female, weight 20 ± 2 g, 60 rats were divided

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randomly into two groups containing 10 normal rats and 50 administrated rats.) were inoculated with seven-day-old ascrite (0.2 ml, 2 × 106 cells) subcutaneously into the right front armpit. 24 h after implantation of tumor cells, the mice were randomly divided into four test groups with 10 mice per group. Each mouse was weighed immediately after

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inoculation. The mice were treated by oral gavage with test samples (60 mg/kg/day) or normal saline (NS, 10 mL/kg) for ten days once daily. On day 11, the mice were sacrificed via cervical dislocation, and the mouse and tumor were excised and weighed for evaluating the tumor growth inhibition. The tumor inhibitory rate was calculated by the following

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formula:

  × 100% 

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W − Wtreated Tumor inhibitory rate rate (%) =  control Wcontrol 

(1)

Wcontrol and Wtreated were the average tumor weights of the control and treated mice, respectively. EAC tumor-bearing mice were observed for mean survival time. The effect of title compounds on percentage increases in life span was calculated on the basis of mortality of the experimental mice:

Σ Survival time (days) of each mouse in a group Total number of mice

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Mean survival time =

%ILS =

MST of treated group × 100 MST of control group

(2) (3)

4.6.3. In vivo tumor model induced by DEN

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SD rats, male, 120~160 g. 120 rats were divided randomly into two groups containing 12 normal rats and 108 DEN model rats. Rats were gavaged daily with DEN 8

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mg/kg, once a day, 6 days a week. Normal saline rats were given the same volume, molding to 10 weeks. 60 model rats were randomly divided into five groups, 12 rats in each group, continued to give DEN; normal rats were given normal saline, molding to 16 week, stopped giving DEN. Compound 13i treatment group from the tenth week, 50 mg·kg-1 was orally given, once a day, for 10 consecutive weeks. Similar parts of left lobe of liver were taken and fixed by 4% polyformaldehyde solution, embedded in paraffin, sliced and HE stained, for histopathological observation.

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4.7 Statistical Analysis All results are expressed as Mean ± SE. Statistical significance was determined by either the Student’s t-test for comparison between means or one-way analysis of variance

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with a post hoc Dunnett’s test. In all cases, p<0.05 was considered statistically significant.

5 Acknowledgments

The authors wish to thank the National Natural Science Foundation of China (No. 21272008, 21572003), Science and Technological Fund of Anhui Province for Outstanding

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Youth (1408085J04). We thank Prof. Robert L. Jernigan (Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, USA) for sharing the

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initial full length telomerase three-dimensional model with us. We thank Dr Marc C. Nicklaus at NCI, NIH of the United States to allow us to access the software, mainly Schrödinger.

6 Supporting information

CCDC-814973, CCDC-813726, CCDC-813727, CCDC-819490, CCDC-819491, CCDC-824625,

CCDC-834936,

CCDC-826456,

CCDC-837389,

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CCDC-824623,

CCDC-837388,

CCDC-836556,

CCDC-836555,

CCDC-837390,

CCDC-861071,

CCDC-861072, CCDC-834938, CCDC-861070, CCDC-861075, CCDC-883316 contain the supplementary crystallographic data for this paper. These data can be obtained free of

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charge via the URL http:// www.ccdc.cam.ac.uk/conts/retrieving. (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK;

fax: (+44) 1223 336033; e-mail:

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[email protected]). Tables S1~S11 and Figures S1~S15 are available free of charge.

References and notes

[1] W. E. Wright, J. W. Shay, Curr Opin Genet Dev 11 (2001), 98-103. [2] A. G. Bodnar, M. Ouellette, M. Frolkis, S. E. Holt, C. P. Chiu, G. B. Morin, C. B. Harley, J. W. Shay, S. Lichtsteiner, W. E. Wright, Science 279 (1998), 349-352. [3] S. A. Stewart, A. A. Bertuch, Cancer Res 70 (2010), 7365-7371. [4] J. W. Shay, W. E. Wright, Cancer Cell 2 (2002), 257-265.

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[5] J. W. Shay, W. E. Wright, Nat Rev Drug Discov 5 (2006), 577-584. [6] D. R. Corey, Chem Biol 16 (2009), 1219-1223. [7] K. Steczkiewicz, M. T. Zimmermann, M. Kurcinski, B. A. Lewis, D. Dobbs, A.

(2011), 9443-9448.

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Kloczkowski, R. L. Jernigan, A. Kolinski, K. Ginalski, Proc Natl Acad Sci USA 108

[8] C. Autexier, N. F. Lue, Annu Rev Biochem 75 (2006), 493-517.

[9] B. Herbert, A. E. Pitts, S. I. Baker, S. E. Hamilton, W. E. Wright, J. W. Shay, D. R. Corey, Proc Natl Acad Sci USA 96 (1999), 14276-14281.

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[10] D. Sun, B.Thompson, B. E. Cathers, M. Salazar, S. M. Kerwin, J. O. Trent, T. C. Jenkins, S. Neidle, L. H. Hurley, J Med Chem 40 (1997), 2113-2116.

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[11] C. Strahl, E. H. Blackburn, Mol Cell Biol 16 (1996), 53-65.

[12] S. Bhattacharya, P. Chaudhuri, A. K. Jain, A. Paul, Bioconjug Chem 21 (2010), 1148-1159.

[13] A. Paul, B. Maji, S. K. Misra, A. K. Jain, J Med Chem 55 (2012), 7460-7741. [14] W. C. Hahn, S. A. Stewart, M. W. Brooks, S. G.York, E. Eaton, A. Kurachi, R. L. Beijersbergen, J. H. Knoll, M. Meyerson, R. A. Weinberg, Nat Med 5 (1999),

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1164-1170.

[15] D. K. Barma, A. Elayadi, J. R. Falck, D. R. Corey, Bioorg Med Chem Lett 13 (2003), 1333-1336.

[16] H. S. Huang, J. F. Chiou, Y. Fong, C. C. Hou, Y. C. Lu, J. Y. Wang, J. W. Shih, Y. R.

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Pan, J. J. Lin, J Med Chem 46 (2003), 3300-3307. [17] I. Naasani, H. Seimiya, T. Yamori, T. Tsuruo, Cancer Res 59 (1999), 4004-4011.

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[18] E. P. Cohn, K. L. Wu, T. R. Pettus, N. O. Reich, J Med Chem 55 (2011), 3678-3686. [19] N. Hayakawa, K. Nozawa, A. Ogawa, N. Kato, K. Yoshida, K. Akamatsu, M. Tsuchiya, A. Nagasaka, S. Yoshida, Biochemistry 38 (1999), 11501-11507.

[20] K. M. Amin, A. A. M. Eissa, S. M. Abou-Seri, F. M. Awadallah, G. S. Hassan, Eur J Med Chem 60 (2013), 187-198.

[21] Dinesha, S. Viveka, P. Naik, G. K. Nagaraja, Med Chem Res 23 (2014), 4189-4197. [22] A. P. Kechea, G. D. Hatnapurea, R. H. Tale, A. H. Rodge, V. M. Kamble, Bioorg Med Chem Lett 22 (2012), 6611-6615.

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[23] K. M. Amin, N. M. A. Gawad, D. E. A. Rahman, M. K. M. El Ashry, Bioorg Chem 52 (2014), 31-43. [24] K. M. Amin, F. M. Awadallah, A. A. M. Eissa, S. M. Abou-Seri, G. S. Hassan, Bioorg Med Chem 19 (2011), 6087-6097.

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[25] A. Thakur, R. Singla, V. Jaitak, Eur J Med Chem 101 (2015), 476-495.

[26] K. M. Amin, S. M. Abou-Seri, F. M. Awadallah, A. A. M. Eissa, G. S. Hassan, M. M. Abdulla, Eur J Med Chem 90 (2015), 221-231.

[27] K. M. Chang, H. H. Chen, T. C. Wang, I. L. Chen, Y. T. Chen, S. C. Yang, Y. L. Chen,

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H. H. Chang, C. H. Huang, J. Y. Chang, C. Shih, C. C. Kuo, C. C. Tzeng, Eur J Med Chem 106 (2015), 60-74.

[28] P. R. Kamath, D. Sunil, A. A. Ajees, K. S. R. Pai, S. Das, Bioorg Chem 63 (2015),

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101-109.

[29] X. H. Liu, H. F. Liu, J. Chen, Y. Yang, B. A. Song, L. S. Bai, J. X. Liu, H. L. Zhu, X. B. Qi, Bioorg Med Chem Lett 20 (2010), 5705-5708.

[30] X. H. Liu, J. Li, J. B. Shi, B. A. Song, X. B. Qi, Eur J Med Chem 51 (2012), 294-2999.

1057-1085.

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[31] C. Liao, M. Sitzmann, A. Pugliese, M. C. Nicklaus, Future Med Chem 3 (2011),

[32] M. Mitchell, A. Gillis, M. Futahashi, H. Fujiwara, E. Skordalakes, Nat Struct Mol Biol 17 (2010), 513-518.

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[33] X. H. Liu, B. F. Ruan, J. X. Liu, B. A. Song, L. H. Jing, J. Li, Y. Yang, H. L. Zhu, X. B. Qi, Bioorg Med Chem Lett 21 (2011), 2916-2920.

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[34] X. Q. Wu, C. Huang, Y. M. Jia, B. A. Song, J. Li, X. H. Liu, Eur J Med Chem, 74 (2014), 717-725.

[35] Y. Y. Wu, A. M. Hruszkewycz, R. M. Delgado, A. Yang, A. O. Vortmeyer, Y. W. Moon, R. J. Weild, Z. P. Zhuang, A. T. Remaley, Clinica Chimica Acta 293 (2000) 199-212.

[36] G. M. Sheldrick, SHELXTL-97, Program for Crystal Structure Solution and Refinement; University of Göttingen: Göttingen, Germany, 1997. [37] X. Chen, C. Plasencia, Y. Hou, N. Neamati, J Med Chem 48 (2005), 1098-1106.

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Schemes HO

8d: R1=5-Br, R4=

CH3

8e: R1=3,5-2Br, R4=

N N O S

NO2

8f: R1=3,5-2Br, R4=

R

CH3

8g: R1=5-Cl, R4=

R1 8 O

4

NO2

(G)

(A)

R1

R1

OH

1

7

O

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(F)

HO

R1

N N H

2

(C)

HO

(D)

HO (D)

N N

R1

O

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N N

N N

Br

R5

Cl

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7d: R1=H, R2=

NO2

7f: R1=H, R2=

7g: R1=H, R2=

H2C Cl

7m: R1=5-Br, R2=

H 2C

7p: R1=5-Br, R2=

Cl Cl

10 O

R6 7r: R1=3,5-2Br, R2=

CH3

7t: R1=3,5-2Br, R2= 7u: R1=3,5-2Br, R2= Cl

O

7q: R1=3,5-2Br, R2= H2C

Cl

H2C

CH3

7s: R1=3,5-2Br, R2=

CH3

7o: R1=5-Br, R2=

7i: R =H, R =

O

C

7l: R1=5-Br, R2=

2

7j: R1=H, R2=

N N

7n: R1=5-Br, R2=

CH3

R7O

(E)

3

AC C

7c: R1=H, R2=

9a~9s

R5

O

9t~9u

1

R2

(B)

CH3

OH

HO

O

N N

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O CHO

R1

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HO

Cl

O

CH3

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CH3 N

9g: R5=

H N

9j: R5=

N

OMe

9n: R5=

N

N

9o: R5=

N

N CH3

CH3 H3C N CH3 H3C

9c: R5=

N

9d: R5=

N

9l: R5=

N

9r:

N

N

R7 =

CH3

O R 7=

6

10k: R6=

CH3

10g: R =

N

R 7=

R 6=

N

OH R7=

CH3

CH3

10i:

CH3

10n: R6=

CH3

O

H 3C CH3

CH3

N

CH2

OCH3 R7=

N

10l: R6=

O

H3C

N

R 5=

9u:

S

O

N

N

R=

H 3C

H3C

10d: R6=

R5 =

O

10e: R6=

CH3

CH3

10c: R6=

N

O R7 =

5

CH2

CH3 CH3

9t:

CH3 CH3 CH3

N

N

CH3

9m: R5=

10b: R6=

9q: R5=

R5 =

CH3

CH3

Cl

N

9s:

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9b: R =

CH3

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5

CH3

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9a: R5=

CH3 O CH3 CH3 O

6

10j: R = R7 =

CH3

CH3 H3C

N

Ac

7

R=

C H

3

O

R7 =

N

CH3

O Cl

R 7=

H3C

N

10o: R6=

CH3

R 7=

CH

3

CH3 CH3

CH3

10p: R6=

H3C

N

R 7=

CH3

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Scheme 1. Synthesis of title compounds 7~10

Reagent and conditions: (A) CH3COCH3, NaOH, C2H5OH, 20~25 oC, 10 h. (B) N2H4.H2O, 98% CH3CH2OH, reflux, 2 h. (C) CH2ClCOCl, reflux, 4 h. (D) amine, KI,

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DMAP, CH3COCH3, 40 oC. (E) H2SO4, CH3COOH, C2H5OH; amine, KI, DMAP, CH3COCH3, 40~50 oC. (F) Carboxylic acid, Tosyl chloride, DMAP, reflux, 3 h. (G)

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Substituted-benzenesulfonyl chloride, CHCl3, reflux.

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O

O O

O

(H)

O

H3C

OH

O

(I)

CH3

H

R

CH3

O

O

O

R8 (J)

O

HN N

R

(K) O

N N

R

O

O

R9 (M)

O S O N N

(L)

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R

O

Cl O

O

12

R10

O

N N R O

AC C

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O

N N

R

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(N) 6'

11

O

SC

6

O

5

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4

O

O 13

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11f:

R 8=

11g:

R= H;

R8 =

H2 C

11h: R8=

R= H; CH3

11i:

H2 C

R8=

Cl R= H; 11j:

R8 =

R= H;

CH3

R= H; Cl CH3

R =

12l:

R= H;

CH3

H N CH3 R= H;

13a: R 10 =

NH 10

13b:

H N

R10=

13g: R = H C 3

R= H;

R10= NH

H N

13e: R10= NH

CH3 R= H;

13i: R =

R= H;

R10=

13j:

R= H;

N

N

13l: R10=

13u: R10= N

NH2

R= H;

N

13x: R10=

N

13y: R10=

N

R= H;

N

R= H;

S

13r: R10=

N

13s: R10=

N

EP

CH3

R= H;

R= H;

OH

N

R= H;

Cl O CH3

N

10 13t: R = N

CH3

O

R= H;

CH3 N CH3

R= H;

R= 2-CF3; CH3

R= 2-CF3;

O

N

13p: R10=

R= H;

CH3

R= H;

TE D

H3C

13w: R10=

N

N

H3C

CH3 R= H;

13f: R =

13o: R10=

CH3

HN 10

CH3

R= H; CH3

H N

13d: R10=

R= H;

CH3

H3C

CH3 10

13v: R10=

N

M AN U

H3C

13h: R10=

R= H;

R= H;

N

13n: R10=

R= H;

CH3 CH3

CH3 13c:

13m: R 10=

R= H

R9=

NO2

RI PT

9

SC

12k:

CH3 13z: R10=

N

O

R= 2-CF3;

AC C

CH3

Scheme 2. Synthesis of title compounds 11~13

Reagent and conditions: (H) piperazine, 20~30 oC, 1 h. (I) benzaldehyde, piperidine, ethanol, reflux, 6 h. (J) NH2-NH2.H2O, C2H5OH, reflux, 3 h. (K) carboxylic acid, tosyl chloride, DMAP, reflux, 5 h. (L) CH2ClCOCl, reflux, 6 h. (M) substituted-benzenesulfonyl chloride, CHCl3, reflux 2 h. (N) amine, KI, piperazine, CH3COCH3, 50 oC.

ACCEPTED MANUSCRIPT

Tables Table 1. Crystallographical and experimental data for compounds 7j and 8d 7j

8d

Chemical formula

C18H16ClN2O3

C17 H17 Br N2 O3 S

Formula weight Crystal shape / color Crystal size (mm) Crystal system Space group a (Å) b (Å) c (Å) α (o) β (o) γ (o) V (Å3) Dcalc (mg⋅m-3) Z λ (MoKα) (Å) T (K) F(000) θ range (o) Index ranges(h, k, l) µ (mm-1)

343.78 Colorless 0.23 × 0.11 × 0.09 Triclinic P-1 8.060(6) 9.590(7) 11.308(8) 91.756(7) 107.241(6) 90.843(7) 834.1(10) 1.369 2 0.71073 296(2) 358 2.13 to 26.00

AC C

SC

Max. and min. trans. Data/restraints/parameters Goodness of fit on F2 R1/wR2 [I ≥ 2σ(I)]a R1/wR2 (all data)a ∆ρmax/∆ρmin (e⋅A-3)

R1 = ∑||Fo|-|Fc||/∑|Fo|

409.30 Colorless 0.34 x 0.29 x 0.21 Monoclinic P21/c 5.683(6) 26.86(3) 11.688(12) 90 102.509(15) 90 1741(3) 1.561 4 0.71073 296(2) 832 2.89 to 26.00 -6/6, -33/28, -13/14 2.498 8646 / 3357 0.6220 and 0.4839 3357 / 0 / 217 1.024 0.0684 / 0.1502 0.1826 / 0.1977

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-9/9, -11/11, -13/13

Reflections collected/unique

a

RI PT

Properties

0.247 6252 / 3208 0.9781 and 0.9453 3208 / 0 / 217 1.028 0.0533 / 0.1151 0.0909 / 0.1311 0.384 and -0.217

wR2 = [∑w(Fo2-Fc2)2/∑w(Fo2)2]1/2.

44

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9d

9n

Chemical formula

C16 H21 N3 O2

C21 H29 N4 O4

Formula weight Crystal shape / color Crystal size (mm) Crystal system Space group a (Å) b (Å) c (Å) α (o) β (o) γ (o) V (Å3) Dcalc (mg⋅m-3) Z λ (MoKα) (Å) T (K) F(000) θ range (o) Index ranges(h, k, l) µ (mm-1)

287.36 Colorless 0.35 x 0.29 x 0.21 Triclinic P-1 10.034(4) 10.880(4) 15.438(6) 109.603(4) 96.330(4) 93.664(4) 1568.8(11) 1.217 4 0.71073 296(2) 616 2.05 to 25.99

401.48 Colorless 0.31× 0.23 × 0.21 Monoclinic P21/n 14.640(7) 11.068(5) 15.141(5) 90.00 119.70(3) 90.00 2131.1(16) 1.251 4 0.71073 296(2) 860 2.41 to 28.48

-12/11, -13/13, -19/19

-14/19, -14/14, -19/16

0.082 11270 / 6020 0.9830 and 0.9719 6020 / 0 / 379 1.029 0.0517 / 0.1285 0.1028 / 0.1563 0.223 and -0.212

0.088 12047 / 5126 0.9818 and 0.9733 5126 / 0 / 266 1.025 0.0630 / 0.1100 0.1459 / 0.1324 0.418 and -0.256

EP

TE D

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SC

Properties

RI PT

Table 2. Crystallographical and experimental data for compounds 9d and 9n

Reflections collected/unique

AC C

Max. and min. trans. Data/restraints/parameters Goodness of fit on F2 R1/wR2 [I ≥ 2σ(I)]a R1/wR2 (all data)a ∆ρmax/∆ρmin (e⋅A-3)

a

R1 = ∑||Fo|-|Fc||/∑|Fo|

wR2 = [∑w(Fo2-Fc2)2/∑w(Fo2)2]1/2.

45

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Table 3. Crystallographical and experimental data for compounds 13x and 13y 13x

13y

Chemical formula

C24 H23 N3 O3 S

C33 H40 F3 N3 O3

Formula weight Crystal shape / color Crystal size (mm) Crystal system Space group a (Å) b (Å) c (Å) α (o) β (o) γ (o) V (Å3) Dcalc (mg⋅m-3) Z λ (MoKα) (Å) T (K) F(000) θ range (o) Index ranges(h, k, l) µ (mm-1) Reflections Max. and min. trans. Data/restraints/parameters Goodness of fit on F2 R1/wR2 [I ≥ 2σ(I)]a R1/wR2 (all data)a ∆ρmax/∆ρmin (e⋅A-3)

433.51 Colorless 0.23 x 0.21 x 0.16 Orthorhombic Pbca 9.823(3) 19.365(5) 22.469(6) 90 90 90 4274.0(19) 1.347 8 0.71073 296(2) 1824 2.29 to 25.50 -11/11, -23/14, 0.183 22110 / 3972 0.9713 and 0.9591 3972 / 0 / 280 1.039 0.0542 / 0.1206 0.1007 / 0.1507 0.255 and -0.330

583.68 Colorless 0.15 x 0.13 x 0.08 Triclinic P-1 11.473(5) 13.612(5) 21.594(9) 74.192(4) 88.993(4) 76.119(4) 3146(2) 1.232 4 0.71073 296(2) 1240 2.10 to 24.79 -11/13, -16/16, 0.091 21328 / 10575 0.9928 and 0.9865 10575 / 204 / 761 1.006 0.1276 / 0.2004 0.3316 / 0.2724 0.338 and -0.285

SC

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EP

AC C

a

RI PT

Properties

R1 = ∑||Fo|-|Fc||/∑|Fo|, wR2 = [∑w(Fo2-Fc2)2/∑w(Fo2)2]1/2.

46

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Table 4. Antiproliferative activities of compounds 7~13 against PC3, Bcap-37 MGC-803 and HepG2 cell lines b HepG2 29.01±1.47 30.11±3.21 20.62±2.09 11.27±0.96 25.21±2.59 — 16.33±1.19 — 23.21±1.55 17.85±2.09 — 3.35±0.61 41.42±2.97 9.17±1.05 — — 20.77±1.31 — — — 51.57±2.81 15.11±1.71 3.02±0.33 5.54±1.00 39.06±1.88 23.32±2.45 42.10±2.80 51.91±2.94 5.00±0.44 — 17.91±2.91 45.33±3.01 49.17±2.89 9.81±0.99 7.87±0.35 33.21±1.50 34.09±1.30 8.40±0.52 19.01±0.97 5.09±0.22 12.31±0.65 1.34±0.15

SC

RI PT

MGC-803 39.57±2.25 50.00±4.10 24.12±2.00 26.33±0.98 18.34±3.01 — 13.47±1.26 50.33±4.07 21.00±1.24 21.20±2.38 23.55±0.98 4.91±0.77 38.57±2.41 17.11±1.31 50.01±3.01 56.51±2.00 30.71±1.00 40.10±1.62 — — 39.80±1.85 31.20±1.64 4.31±0.78 17.89±1.09 9.87±1.14 12.30±2.00 30.31±2.21 30.66±1.94 17.58±2.62 7.85±0.71 42.88±2.55 31.51±2.41 30.02±2.74 20.22±2.00 10.19±0.44 29.54±1.21 20.07±0.55 21.47±0.17 4.89±0.61 6.51±0.40 3.51±0.21 1.01±0.17

M AN U

IC50/µM Bcap-37 —a 45.20±3.87 25.66±2.10 9.97±1.51 30.17±2.41 — 32.32±2.29 20.84±1.91 32.58±1.54 41.29±3.07 36.80±3.01 51.10±2.29 40.11±2.20 27.21±1.20 46.31±2.20 — 36.80±2.00 — — 52.31±3.10 45.67±2.24 4.96±0.99 33.14±2.17 5.35±0.55 45.45±1.95 33.71±1.49 28.57±1.35 20.89±1.28 6.74±1.94 6.21±1.00 35.59±1.87 20.57±2.01 40.50±2.33 15.28±1.70 4.95±0.45 44.22±0.19 19.42±0.64 34.21±1.33 12.78±0.27 5.21±0.19 9.80±1.12 1.12±0.09

TE D

AC C

7c 7d 7f 7g 7i 7j 7l 7m 7n 7o 7p 7q 7r 7s 7t 7u 8d 8e 8f 8g 9a 9b 9c 9d 9g 9j 9l 9m 9n 9o 9q 9r 9s 9t 9u 10b 10c 10d 10e 10g 10i AMD

PC3 42.01±2.21 36.55±2.87 9.38±2.10 40.58±1.98 42.51±3.30 38.79±4.12 8.89±3.45 37.81±2.00 41.16±2.08 16.25±1.44 48.98±2.23 5.82±1.00 28.60±1.87 11.30±2.01 40.20±1.89 41.21±2.55 33.21±1.65 50.03±2.13 51.20±3.00 44.20±2.21 25.87±1.11 32.31±2.55 5.89±0.55 6.67±0.84 51.58±2.87 20.42±2.01 34.80±2.11 36.44±2.17 3.44±0.46 11.80±2.01 35.47±2.33 29.25±2.00 32.20±1.88 19.00±1.45 3.09±0.69 51.21±1.04 24.51±0.45 5.20±0.11 4.07±0.35 6.21±0.74 25.64±0.15 0.87±0.21

EP

Comp

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Table 4 (continue) IC50/µM

a b

HepG2 7.80±0.77 21.98±1.11 — 2.52±0.09 3.67±1.18 32.19±2.41 36.60±1.98 52.28±3.11 9.61±0.85 2.98±0.19 5.44±0.61 — — 24.01±1.64 30.59±1.28 6.00±0.42 10.75±0.52 — 9.97±0.95 31.40±1.56 34.40±0.67 2.45±0.11 44.30±2.78 2.19±0.10 — — — 19.28±0.99 — — 28.39±1.12 33.08±1.20 — 19.33±1.19 11.37±1.10 8.96±1.36 12.01±0.87 21.01±1.69 1.34±0.15

RI PT

MGC-803 4.12±0.11b 9.55±0.12 5.14±0.21 3.01±0.13 3.32±1.03 12.20±1.68 12.65±1.22 31.33±2.10 5.30±0.59 2.02±0.23 6.64±0.50 41.21±1.33 50.17±3.50 12.77±0.35 26.55±0.75 10.30±0.57 4.88±0.28 — 29.72±0.98 15.44±1.20 26.43±0.59 2.96±0.15 — 1.41±0.17 — — — 32.35±0.85 — — — 46.07±0.95 — 21.49±1.06 8.52±1.17 10.35±1.28 6.87±0.48 10.00±1.01 1.01±0.17

M AN U

Bcap-37 20.10±0.57 18.75±0.14 17.89±0.13 6.81±0.25 5.15±0.87 9.98±1.02 32.17±1.45 26.07±1.85 46.31±2.50 26.69±0.95 9.30±0.25 36.99±1.60 30.30±2.11 4.22±0.20 6.49±0.21 2.21±0.10 25.22±1.22 — 6.30±1.08 36.25±0.89 38.30±0.95 3.37±0.30 — 5.30±0.35 — — — 15.47±0.36 — 24.21±1.05 29.67±1.62 16.92±1.31 — 18.50±1.46 3.50±0.99 8.20±1.03 12.19±1.04 14.22±1.08 1.12±0.09

TE D

PC3 6.80±0.15 27.21±0.71 11.10±0.17 3.58±0.11 4.17±1.08 11.21±1.60 5.87±0.33 13.21±1.77 4.37±0.32 2.98±0.15 6.37±0.55 46.57±2.50 48.55±5.21 2.30±0.17 5.58±0.33 1.87±0.11 7.27±0.19 28.46±1.20 1.92±0.13 16.41±0.41 48.50±1.37 4.87±0.95 50.17±2.59 4.12±0.10 36.33±1.25 28.30±1.29 — 26.11±0.74 — — 36.35±0.59 25.77±0.58 — 8.31±1.30 6.41±1.01 4.00±0.77 6.03±0.45 12.07±0.62 0.87±0.21

EP

AC C

10j 10k 10l 10n 10o 10p 11f 11g 11h 11i 11j 12k 12l 13a 13b 13c 13d 13e 13f 13g 13h 13i 13j 13k 13l 13m 13n 13o 13p 13r 13s 13t 13u 13v 13w 13x 13y 13z AMD

SC

Comp.

not observed in the tested concentration range. The standard deviation (SD) of three time independent tests.

48

ACCEPTED MANUSCRIPT

Table 5. IC50 values of selected compounds against human normal cells L-02 and HK-2 proliferation a L-02 (IC50, mM)

9c

1.29±0.11

1.80±0.10

10i

2.51±0.17

2.23±0.20

10n

1.84±0.14

2.53±0.10

11i

2.11±0.20

1.28±0.22

13i

1.64±0.17

1.62±0.24

RI PT

GES-1 (IC50, mM)

SC

a

Compound

MTT assays were used for evaluation, and values were expressed as mean IC50 of the

AC C

EP

TE D

M AN U

triplicate experiment.

49

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Table 6. Antitumor effects of title compounds against tumor growth on the S180 and HepG2 xenograft mice a

(End, n)

Beginning

End

10

19.87 ± 1.25

23.84 ± 2.11

Control S180

HepG2

10

19.55 ± 1.00

24.89 ± 1.67

10

19.95 ± 1.31

24.64 ± 1.80

Control

10

19.50 ± 1.28

25.00 ± 1.65

9c

10

19.68 ± 1.55

26.00 ± 2.01

13i

10

19.61 ± 0.88

27.11 ± 1.50

(g)

Inhibition rate (%)

1.98 ± 0.22

1.10 ± 0.33 *

44.4

1.01 ± 0.11 **

48.9

1.93 ± 0.20

1.17 ± 0.40 **

39.4

1.04 ± 0.35 *

46.1

Mice were inoculated with S180 or HepG2 subcutaneously into the right front armpit and

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a

9c 13i

Tumor weight

RI PT

Groups

Body weight (g)

SC

Models

Animal number

randomly divided into four test groups. The mice were daily treated by compounds 9c and

13i (60 mg/kg/day), or normal saline (NS, 10 mL/kg) by oral gavage for ten consecutive days. Data were analyzed using SPSS11.0. Significant difference between each treatment

AC C

EP

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and the control are shown as P < 0.05 (*) and P < 0.01 (**).

50

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Table 7. Effects of title compounds against the survival of EAC-bearing mice a Groups Animal number (n) Body weight (g) Survival time a (d) Survival rate (%)

RI PT

16.60 ± 2.02 23.41 ± 3.21* 23.89 ± 4.65*

41.0 43.9

AC C

EP

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SC

Control 10 19.2± 1.17 9c 10 19.3 ± 1.07 13i 10 19.6 ± 1.15 a Time denoted by number of days. P < 0.05 (*)

51

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Table 8. Inhibitory activities of some titled compounds against telomerase b IC50 (µM)

Compound

IC50 (µM)

7l

3.17±0.10

10o

3.44±0.43

7f

10.97±1.12

10p

11.03±0.98

7q

3.68±0.14

11f

8.74±0.89

7r

18.15±1.43

11h

7j

54.36±1.01

11i

b

RI PT

Compound

3.63±0.06 2.27±0.16

8f

No

8g

41.18±0.94

12k

9c

4.63±0.18

13a

3.15±0.20

9d

5.44±0.21

13b

6.68±1.01

9g

22.25±1.09

13c

3.52±0.19

9n

6.77±0.38

13d

9.32±0.29

9o

2.10±0.26

13f

3.17±0.12

9u

11.85±0.17

13i

0.98±0.07

9t

9.94±0.19

13r

57.79±1.04

13p

nob

M AN U b

13.10±1.24

31.27±0.40

SC

11j

no

10d

14.25±2.20

13u

35.72±0.99

10e

2.12±0.07

13v

31.21±0.44

6.30±0.55

13w

5.57±0.70

5.69±0.13

13x

3.30±0.31

3.54±0.28

13y

4.77±0.73

8.40±0.74

13z

3.03±1.12

Staurosporine a

8.97 ±0.22

10g 10i 10j

EP

10k

TE D

10b

10l

AC C

10n

Staurosporine

a

6.20±0.28 1.27±0.11 8.97 ±0.22

a

Staurosporine is reported as a control.

b

The standard deviation (SD) of three time independent tests. no, not observed in the tested

concentration range 0–60 µM.

52

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Figure Legends

RI PT

Figure 1. The workflow of the general design strategy in this study Figure 2. (A) Chemical structures of BIBR1532 and BIBR 1591; (B) Binding mode of BIBR1532 to hTERT; (C) The modelled active site of hTERT

Figure 3. The workflow of the general design strategy in this study

Figure 5.

SC

Figure 4. ORTEP drawing of compounds 7j, 8d, 9d, 9n, 13x and 13y

Antitumor effect of compounds 9c and 13i against tumor growth of S180 and

M AN U

HepG2 xenograft mice. Mice were inoculated with S180 or HepG2 subcutaneously into the right front armpit and randomly divided into four test groups. The mice were daily treated by compounds 9c and 13i (60 mg/kg/day), or normal saline (NS, 10 mL/kg) by oral gavage for ten consecutive days. Data were analyzed using SPSS11.0 and the figures were made by GraphPad Prism5. Significant difference between each treatment and the control are

TE D

shown as P < 0.05 (*) and P < 0.01 (**).

Figure 6. Effect of compound 13i on pathological changes of DEN-induced rat hepatic tumor (HE staining ×200) (A) Control, (B) Model, (C) Compound 13i a Animals were randomly divided into 3 groups (n = 12 per group): control group, DEN

EP

a

model group and compound 13i group.

AC C

Figure 7. (A) Binding mode of compound 13i to hTERT; (B) Superimposing of BIBR1532 (green) and compound 13i (gold) in the active site of hTERT

53

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Figure 1. The workflow of the general design strategy in this study

O

CH3

2011

H3C OH

N N

N N

O

N N

LXH-3 hTERT: 4.0 µM

SC

Br

O hTERT: 1.8 µM

HO

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O LXH-2

O

O

LXH-1 hTERT: 2.0µM

CH3

O

H3C OH N N

H3C N

O

R

N N H

Focus

2012

RI PT

2010

HN N

R

O

O

Synthesis lab-based database 78 new entities

O

H3C

N N

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N

Optimization

LXH-4 hTERT: 5.9 µM

Lead compounds O O

HO

O

hTERT: 2.0 µM

N

N N

N N

O

N O

9c

13i

AC C

EP

LXH-5

O

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Figure 2. (A) Chemical structures of BIBR1532 and BIBR 1591; (B) Binding mode of

AC C

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BIBR1532 to hTERT; (C) The modelled active site of hTERT

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HO

HO N N O S R4

R1

R1

N N

Comp7

Comp 8 O

R2

Series 2

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Figure 3. The workflow of the general design strategy in this study

O

Series 1

R7 O

N N

HO N N C

Comp10

N N H

Series 3

O

R8

Comp11

O

CHO

Series 6

N N

R

R1

R5

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R6

SC

HO

R

R

O

Comp9a~s

Series 4

HO

N N Br

5

O

1

Comp9t~u

Series 5

OH

R9

O

Comp 12

O S O Series 7 N N R O

R

R10 O

HN N

N N

Comp 13 Series 8

R O

O

O

O

AC C

EP

O

TE D

O

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ACCEPTED MANUSCRIPT

M AN U

SC

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Figure 4. ORTEP drawing of compounds 7j, 8d, 9d, 9n, 13x and 13y

AC C

EP

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Compound 7j

Compound 8d

57

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AC C

EP

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Compound 9d

Compound 9n

58

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AC C

EP

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Compound 13x

Compound 13y

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Figure 5.

Antitumor effects of compounds 9c and 13i against tumor growth of S180 and

AC C

EP

TE D

M AN U

SC

RI PT

HepG2 xenograft mice

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Figure 6. Effect of compound 13i on pathological changes of DEN-induced rat hepatic tumor (HE staining ×200) (A) Control, (B) Model, (C) Compound 13i a Animals were randomly divided into 3 groups (n = 12 per group): control group, DEN

AC C

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model group and compound 13i group.

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a

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Figure 7. (A) Binding mode of compound 13i to hTERT; (B) Superimposing of BIBR1532

AC C

EP

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(green) and compound 13i (gold) in the active site of hTERT

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Highlights

► Structure-based design used as the new coumarin telomerase inhibitors.

RI PT

► Based on 78 new compounds, the rational SAR was analyzed.

AC C

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► In vivo studies showed that compound 13i displayed potent anticancer activity.