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Design, Synthesis, and Biological Evaluation of 1-Methyl-1,4dihydroindeno[1,2-c]pyrazole Analogues as Potential Anticancer Agents Targeting Tubulin Colchicine Binding Site Yan-Na Liu, Jing-Jing Wang, Ya-Ting Ji, Guo-dong Zhao, LongQian Tang, Cheng-Mei Zhang, Xiu-Li Guo, and Zhao-Peng Liu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00071 • Publication Date (Web): 12 May 2016 Downloaded from http://pubs.acs.org on May 12, 2016

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Journal of Medicinal Chemistry

Design, Synthesis, and Biological Evaluation of 1-Methyl-1,4-dihydroindeno[1,2-c]pyrazole Analogues as Potential Anticancer Agents Targeting Tubulin Colchicine Binding Site Yan-Na Liu,†,§ Jing-Jing Wang,‡,§ Ya-Ting Ji,† Guo-Dong Zhao,† Long-Qian Tang,† Cheng-Mei Zhang,† Xiu-Li Guo,*,‡ and Zhao-Peng Liu*,†

†

Institute of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of

Education), School of Pharmaceutical Sciences, Shandong University, Jinan 250012, P. R. China

‡

Department of Pharmacology, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, P. R. China

ABSTRACT: By targeting a new binding region at the interface between αβ-tubulin heterodimers at the colchicine binding site, we designed a series of 7-substituted 1methyl-1,4-dihydroindeno[1,2-c]pyrazoles inhibitors.

Among

the

as

compounds

potential

tubulin

synthesized,

polymerization 2-(6-ethoxy-3-(3-

ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2-c]pyrazol-7-yloxy)acetamide

ACS Paragon Plus Environment


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6a

and

2-(6-ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2-

c]pyrazol-7-yloxy)-N-hydroxyacetamide 6n showed noteworthy low nanomolar potency against HepG2, Hela, PC3, and MCF-7 cancer cell lines. In mechanism studies, 6a inhibited tubulin polymerization and disorganized microtubule in A549 cells by binding to tubulin colchicine binding site. 6a arrested A549 cell in G2/M phase that was related to the alterations in the expression of cyclin B1 and p-cdc2. 6a induced A549 cells apoptosis through the activation of caspase-3 and PARP. In addition, 6a inhibited capillary tube formation in a concentration-dependent manner. In non-small cell lung cancer (NSCLC) xenografts mouse model, 6a suppressed tumor growth by 59.1 % at a dose of 50 mg/kg (ip) without obvious toxicity, indicating its in vivo potential as anticancer agent.

INTRODUCTION Microtubules are cytoskeleton protein polymers comprised of α- and β-tubulin heterodimers that are vital components of all cells and play important roles in cell division, cell structure maintenance, cell signaling as well as transport of organelles inside the cell.1,2 Because the rapid dividing cancer cells are highly dependent on tubulin polymerization/depolymerization, interfering with tubulin dynamics becomes a well verified strategy for the development of antimitotic drugs.3−9 There are at least four known binding domains on tubulin. The taxane site (on the microtubule interior)


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and the laulimalide site (on the microtubule exterior) binding agents are microtubule stabilizers that prevent the depolymerization of microtubules to form stable, nonfunctional microtubules, represented by paclitaxel, epothilones, discodermolide, and laulimalide, while the colchicine site binding agents, such as colchicine (1),10 podophyllotoxin (2),11 and combretastatin A-4 (CA-4; 3),12 and the vinca site binding agents, such as vincristine and vinblastine,13−15 are microtubule disrupters that inhibit tubulin polymerization to interfere with the formation of the necessary mitotic assembly required for cell division.16−18 Currently, a number of antitubulin agents binding to the taxane or vinca alkaloid sites have been approved by the Food and Drug Administration (FDA) and achieved notable success in cancer chemotherapy, however, the emergence of multidrug resistance (MDR) due to the overexpression of the P-glycoprotein(P-gp) drug transporter and the class III β-tubulin limits their efficiency and long-term use.19−22 Unlike the taxane or vinca alkaloid sites binding drugs, the colchicine site binding agents are generally not P-gp substrate and are still active in cells overexpressing class III β-tubulin, and therefore, are hopeful to overcome resistant phenotypes of carcinoma.23,24 In addition, the colchicine site binding agents also exhibit vascular disrupting effects on tumor endothelial cells required for the growth of the cancer and thus act as vascular disrupting agents (VDAs).25−29 Extensive efforts have been made in the discovery of natural or



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synthetic small-molecule tubulin inhibitors targeting the colchicine binding site, and a number of drug candidates are under clinical investigation as potential new anticancer agents.27,30−34 Based on the X-ray crystal structures of tubulin in complex with colchicine site ligands, the colchicine domain can be divided into three zones and a bridge.35,36 Zone 1 is a mostly hydrophobic pocket boxed by α-tubulin Asn101, Thr179, Ala180, Val181, and β-tubulin Lys254, Leu255, Asn258, Met259, Thr314, Val315, Ala316, Asn350, Val351, Lys352. The 2-methoxytropolone moiety in 1 (PDB: 3UT5 and 3E22),37,38 or the benzodioxole ring in 2 (PDB: 1SA1)39 is accommodated in this pocket. Zone 2 is hydrophobic pocket formed by β-tubulin Tyr202, Val238, Thr240, Cys241, Leu242, Ala250, Asp251, Leu252, Lys254, Leu255, Asn258, Met259, Ala316, Lys352, Thr353, Ala354, Ile378. In zone 2, Asp251 and Leu255 can adopt two alternative dispositions, one of which forms a common pocket that can accommodate the larger trimethoxyphenyl ring in 1, 2 and other structurally related ligands, while the second one opens the groove leading to zone 3, and as a result, the space available for zone 2 is reduced to host only smaller moieties. The zone 3 is a deep hydrophobic pocket formed by β-tubulin Ile4, Tyr52, Gln136, Asn167, Phe169, Glu200, Val238, Thr239, Thr240, Leu242, and Leu252, connected with zone 2 by a polar groove formed by Glu200, Tyr202, Cys241, Leu248, Ala250, Leu255, Ala256,


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Met259, Phe268, Ala316, Val318, and Ile378. In general, the typical colchicine site binding inhibitor (1, 2, 3 and their related analogues) features with two aryl systems that occupy either the zone 1 or the zone 2 and a bridge with different length that determines their relative orientations.36,40 The pyrrolidindione TN16 (4; PDB: 3HKD)41 represents another kind of colchicine site binding inhibitor that only interacts with zone 2 and zone 3. The sulfonamide derivative ABT-751 (5),42 an orally active antimitotic agent discovered by Eisai Company, is in clinical trials for cancer treatment.43−45 5 is currently the only known ligand contacting all the three zones in its X-ray structure (Figure 1).41 O

OH O

HN

O

O

O

O

O

O O

O O

O

O O

α-TUB

O zone 1

O

NH

O

O 3 (CA-4)

O

1 (colchicine)

O

H N

4 (TN16)

2 (podophyllotoxin)

O2 S

bridge NH

N zone 2

O

α-TUB

H N

R1R2N OH

N N

O 7 O 6

N H

O

zone 3

β-TUB

6 (1-methyl-1,4-dihydroindeno[1,2-c]pyrazoles)

5 (ABT-751)

Figure 1. Colchicine site ligands (1, 2, 3, 4, 5 and the designed indenopyrazoles) with their binding modes with tubulin (zone 1 in red, zone 2 in blue, zone 3 in pink, the predicted new binding region in zone 1 in green, and the bridge in thin black).



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1,4-Dihydroindeno[1,2-c]pyrazole is a core scaffold found in a number of inhibitors, including cyclin dependent kinase (CDK),46,47 platelet-derived growth factor receptor (PDGFR) tyrosine kinase,48 checkpoint kinase 1 (Chk1),49,50 epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR)-2 tyrosine kinases,51 and hypoxia inducible factor 1 (HIF-1).52 To prevent the pyrazole tautomerisation, we placed a methyl group at the N1 position and used the 1-methyl-1,4-dihydroindeno[1,2-c]pyrazole framework for the design of novel tubulin polymerization inhibitors binding to the colchicine site. Based on the X-ray crystal structures of tubulin in complex with 5 (PDB: 3HKC)41, we made docking simulations of the designed indenopyrazoles and the preliminary results indicated that they may interact with tubulin in a similar way as that of 5. As shown in Figure 1, the m-ethoxyaniline moiety occupies the zone 3, the 1-methyl-1,4-dihydroindeno[1,2c]pyrazole portion is accommodated at the zone 2 pocket, the ethyl group in the ethoxy substituent at the 6-position is partially located in the zone 1. Moreover, the substituent at the 7-position may form additional interactions with tubulin in an open cavity up to the α-tubulin subunit in the interfacial region. As far as we know, this new binding region in zone 1 has not been explored in the design of tubulin inhibitors. In this report, we focused on the substituents at the 7-position to investigate their effects on antiproliferative activities and discovered two indenopyrazole analogues as


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potent tubulin polymerization inhibitors targeting colchicine binding site. It is of note that during the progress of our project, Nakamura’s group just recently identified a 1,4-dihydroindeno[1,2-c]pyrazole

analogue,

dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate

methyl (GN39482),

3-((6-methoxy-1,4as

a

tubulin

polymerization inhibitor without confirming its binding site.53 RESULTS AND DISCUSSION Chemistry.

The

synthesis

of

1-methyl-1,4-dihydroindeno[1,2-c]pyrazole

derivatives 6a−p was shown in Scheme 1. After selective ethyl ether formation by the treatment of the readily available 5,6-dihydroxy-1-indenone 7 with diethyl sulfate in DMF in the presence of K2CO3, the resulting 5-ethoxy-6-hydroxy-1-indenone 8 was reacted with TBSCl to protect its hydroxyl group. Deprotonation of α-proton of carbonyl in indanone 9 was carried out using lithium hexamethyldisilazide (LHMDS), the resulting enolate was reacted with 3-ethoxyphenylisothiocyanate to give a thioamide intermediate, which underwent condensation with 1-methylhydrazine to form the 1-methyl-1,4-dihydroindeno[1,2-c]pyrazole 10 in a moderate yield (54 %). Deprotection of the TBS group in 10 with TBAF in THF provided indenopyrazole 11, which was reacted with methyl chloroacetate and K2CO3 as a base in acetone under reflux conditions to generate the methyl ester 12 in 74 % yield. Finally, aminolysis of the ester 12 with a variety of amines, including ammonia (a), methylamine (b),



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dimethylamine (c), ethylamine (d), 2-hydroxyethylamine (e), 2-cyanoethylamine (f), 2-aminoethylamine (g), 2-dimethylaminoethylamine (h), 2-methoxyethylamine (i), cyclopropylamine (j), cyclopropylmethylamine (k), 2-propynylamine (l), allylamine (m), hydroxyamine (n), methoxyamine (o), hydrazine monohydrate (p), provided the corresponding amides 6a−p. All the newly synthesized compounds were identified by 1

H NMR, 13C NMR, MS spectra, and elemental analysis.

Scheme 1. Synthesis of 1-Methyl-1,4-dihydroindeno[1,2-c]pyrazoles 6a−pa

O HO

O (a)

HO

HO

O (b)

10 (54%)

O (f)

NH EtO

H3CO

OEt

N N

O

(g)

NH EtO

11 (91%) O R R N

NH

9 (91%)

N N

(e)

1 2

(c), (d)

EtO 8 (77%)

HO

N N

TBSO EtO

EtO 7

TBSO

O

OEt

12 (74%)

OEt

N N NH

EtO 6a-p (58-81%)

OEt

a: R1 = R2 = H; b: R1 = Me, R2 = H; c: R1 = R2 = Me; d: R1 = Et, R2 = H; e: R1 = 2-hydroxyethyl, R2 = H; f: R1 = 2-cyanoethyl, R2 = H; g: R1 = 2-aminoethyl, R2 = H; h: R1 = 2-dimethylaminoethyl, R2 = H; i: R1 = 2-methoxyethyl, R2 = H; j: R1 = cycloproyl, R2 = H; k:R1 = cycloproylmethyl, R2 = H; l: R1 = prop-2-ynyl, R2 = H; m: R1 = allyl, R2 = H; n: R1 = OH, R2 = H; o: R1 = MeO, R2 = H; p: R1 = NH2, R2 = H a

Reagents and conditions: (a) (EtO)2SO2, K2CO3, DMF, 50 °C, 9 h; (b) TBSCl, imidazole, DMF, 1 h; (c) LHMDS, 3-EtOPhNCS, THF, −78 °C ~ rt, 16 h; (d) CH3NHNH2, 1,4-dioxane/EtOH (1:1), 0 °C ~ rt, 84 h; (e) TBAF, THF, 1 h; (f) ClCH2CO2Me, K2CO3, acetone, reflux, 14 h; (g) R1R2NH, MeOH, reflux, 1 ~ 12 h.


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In Vitro Cell Growth Inhibitory Activity. The newly synthesized 1-methyl-1,4dihydroindeno[1,2-c]pyrazoles 6a−p were initially evaluated for their antiproliferative activity against four human cancer cell lines, HepG2 (human liver carcinoma), Hela (human cervical carcinoma), PC3 (human prostate cancinoma), and MCF-7 (human breast adenocarcinoma), by the conventional MTT assay, using 1, 3, 5, and paclitaxel as reference drugs. The results are shown in Table 1. The acetamide 6a exhibited excellent growth inhibitory activity against all the four cancer cell lines, with IC50 values ranging from 7.4 to 32.3 nM. It was 17- to 123-fold more potent than 5, and was just as potent as 1 and paclitaxel. In comparison with 6a, the amide N-methylation (6b) led to 5.5- to 27-fold decrease in potency, while the amide N-dimethylation (6c) further deteriorated its activity. N-Methylacetamide 6b was still more potent than 5 against cancer HepG2, Hela, and MCF-7 cell lines, but was slight less active against PC3 cells. Replacing the N-methyl by an ethyl (6d) group also reduced its potency. However, further introducing a hydroxyl (6e), an amino (6g), a dimethylamino (6h), or a methoxy (6i) group at the β-position of ethyl substituent improved the sensitivity of 6d towards some cancer cell lines in different degrees, but a cyano group at the same position was not tolerated. Compounds 6h and 6i exhibited similar level of potency as that of 5. The N-cyclopropylacetamide 6j maintained considerable potency (IC50 = 0.65–3.07 µM), while its counterpart 6k with



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one carbon elongation displayed much reduced activity. Introduction of an unsaturated 2-propnyl (6l) or allyl (6m) group was considerable tolerated, though the resulting analogues 6l and 6m were less potent than the unsubstituted acetamide 6a. The N-hydroxyacetamide 6n exhibited potency comparable to that of 6a with IC50 values ranging from 13.2 to 33.5 nM, but the methylation of 6n greatly reduced its activity. Though the hydrazide 6p was as potent as 5, it was much weaker than 6a and 6n. In addition, these indenopyrazoles were generally more sensitive towards human breast cancer MCF-7 cells than 5, except the N-dimethyacetamide 6c, the N-(2cyanoethyl)acetamide 6f, and the N-cyclopropylmethylacetamide 6k. It is of note that 6a and 6n were 17- to 122-fold more potent than 5 and just as potent as the drug paclitaxel and 1 against these four cancer cell lines, though they were less active than 3. In summary, we investigated the antiproliferative activities of a series of 1-methyl1,4-dihydroindeno[1,2-c]pyrazoles with 7-oxoacetamides of 4- to 8-atom lengths. The substituents at the acetamide nitrogen had great influences on their tumor cell growth inhibitory potency. The 7-oxoacetamides with a 4- or 5-atom length gave the best results, possible due to the limited distance and space at the interface between the αand β-subunit of tubulin heterodimers. In general, a hydrogen bond donor/aceptor


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atom (O, N) at or near the terminal of the substituents is preferred for the better antiproliferative activity. Table 1. Growth Inhibitory Activities of Compounds 6a−p against Human Cancer Cell Lines O 1 2

R R N

O

N N N H

EtO

OEt

IC50 mean ± SDa (µM) Compd R1

R2

HepG2

Hela

6a

H

H

0.026 ± 0.003

0.0074 ± 0.019 ± 0.0005 0.002

0.0323 ± 0.0032

6b

Me

H

0.142 ± 0.013

0.090 ± 0.005

0.640 ± 0.022

0.892 ± 0.041

6c

dimethyl

Me

0.920 ± 0.045

0.720 ± 0.015

1.78 ± 0.12

6.16 ± 0.50

6d

Et

H

1.31 ± 0.13

0.623 ± 0.033

3.74 ± 0.22

1.47 ± 0.15

6e

2-hydroxyethyl

H

1.80 ± 0.15

0.415 ± 0.017

3.68 ± 0.18

0.662 ± 0.020

6f

2-cyanoethyl

H

2.42 ± 0.44

1.45 ± 0.32

8.60 ± 0.70

4.61 ± 0.65

6g

2-aminoethyl

H

1.40 ± 0.15

0.865 ± 0.017

1.52 ± 0.09

1.85 ± 0.20

6h

2dimethylaminoethyl

H

0.340 ± 0.020

0.375 ± 0.016

0.460 ± 0.020

2.99 ± 0.16

6i

2-methoxyethyl

H

0.447 ± 0.017

0.490 ± 0.010

0.800 ± 0.020

1.53 ± 0.042

6j

cyclopropyl

H

0.653 ± 0.013

0.720 ± 0.020

1.40 ± 0.10

3.07 ± 0.30

6k

cyclopropylmethyl

H

1.70 ± 0.10

3.20 ± 0.32

4.60 ± 0.20

> 50.0


PC3

MCF-7


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6l

2-propynyl

H

0.587 ± 0.020

0.505 ± 0.012

1.78 ± 0.13

2.11 ± 0.05

6m

allyl

H

0.853 ± 0.014

0.690 ± 0.022

1.94 ± 0.024

0.765 ± 0.020

6n

OH

H

0.0164 ± 0.0132 ± 0.0235 ± 0.0335 ± 0.0011 0.0021 0.0010 0.0020

6o

MeO

H

0.853 ± 0.034

0.470 ± 0.010

2.90 ± 0.14

1.67 ± 0.15

6p

NH2

H

0.400 ± 0.010

0.350 ± 0.010

0.760 ± 0.012

0.579 ± 0.020

1

0.019 ± 0.002

0.0076 ± 0.015 ± 0.0007 0.001

0.047 ± 0.001

3

0.0021 ± 0.0093 ± 0.0034 ± 0.0125 ± 0.0005 0.0009 0.0002 0.0006

5

0.447 ± 0.018

0.355 ± 0.020

0.460 ± 0.010

3.79 ± 0.02

PTXb

0.0101 ± 0.018 ± 0.0050 0.001

0.021 ± 0.001

0.0124 ± 0.0050

a

SD: standard deviation. bPTX: paclitaxel. All experiments were independently performed at least three times. In growth inhibitory test against two representative non-small cell lung cancer (NSCLC) cell lines, A549 and NCI-H460, 6a displayed higher or comparable potency as 1 and 3 (Table 2). Table 2. Growth Inhibitory Effects of 6a on NSCLC Cell Lines. IC50 mean ± SD (nM) Compound

A549

NCI-H460

6a

41.46 ± 0.86

37.32 ± 2.40

1

59.06 ± 3.48

49.29 ± 2.79

3

49.05 ± 2.63

35.81 ± 1.14


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Colony formation assay were used to test the sensitivity of NSCLC cells to 6a over a relatively long exposure time (9 days). In the concentrations ranging from 2 to 10 nM, 6a significantly and dose-dependently reduced the colony formation in A549 (Figure 2A) and NCI-H460 cells (Figure 2B). At the concentration of 10 nM, 6a inhibited the colony formation by 91.05 % in A549 and by 97.34 % in NCI-H460. Trypan blue staining was further used to investigate the cytotoxicity of 6a. In the presence of 6a at concentrations ranging from 5 nM to 80 nM, the density of cells slightly decreased but the dead cells were negligible (Figure 2C). These results indicated that 6a had a strong antiproliferative activity on NSCLC cells in the absence of evident cytotoxicity.



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Figure 2. Growth inhibitory effects of 6a on NSCLC cell lines by colony formation assay and trypan blue staining. Representative colony formation images on A549 cells (A) and NCI-H460 cells (B) after exposed to 6a for 9 days. The results were denoted as a percentage of the vehicle-treated cells. (C) The cytotoxicity of 6a on A549 cells was estimated by trypan blue exclusion. After exposed to 6a for 24 h, A549 cells were harvested and stained with trypan blue solution. Arrows indicated the dead cells. The results were expressed as a percentage based on the ratio of the number of viable


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treated cells to that of vehicle control. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 vs. the vehicle control.

Inhibition of Tubulin Polymerization and Colchicine Binding. To investigate whether the activities of the synthetic 1-methyl-1,4-dihydroindeno[1,2-c]pyrazoles were related to the interactions with microtubule systems, the most cytotoxic compounds 6a and 6n, the medium potent 6b, the least active 6k, and reference compounds 1, 3, and 5 were evaluated as tubulin polymerization inhibitors. Both 6a and 6n produced a concentration-dependent inhibition of tubulin polymerization (Figure 3), with calculated IC50 values of 4.62 µM and 5.33 µM, respectively (Table 3). The medium cytotoxic 6b exhibited weak tubulin polymerization inhibition (IC50 = 22.0 µM), while 6k showed no inhibitory activity (Figure 1S, Table 1S; Supporting Information). In addition, 6a and 6n competed with [3H]colchicine in binding to tubulin. The binding potency of 6a and 6n to the tubulin colchicine binding site was as strong as that of 1 or 5 but less than that of 3 (Table 3).



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Figure 3. Effect of 6a and 6n on tubulin polymerization in vitro. Purified tubulin protein at 2 mg/mL in a reaction buffer was incubated at 37 °C in the presence of 1 % DMSO, test compounds (6a or 6n at 1.25 µM, 2.5 µM, 5 µM or 10 µM) or 1 (20 µM). Polymerizations were followed by an increase in fluorescence emission at 460 nm over a 60 min period at 37 °C. The experiments were performed three times.

Table 3. Inhibition of Tubulin Polymerizationa and Colchicine Binding to Tubulinb Inhibition of tubulin polymerization Compound

Inhibition of colchicine binding (%) inhibition ± SD

IC50 (µM)

1 µM

5 µM

6a

4.62

37 ± 0.7

67 ± 0.4

6n

5.33

31 ± 0.5

63 ± 0.6

1

1.65

40 ± 1.0

72 ± 0.2

3

2.48

83 ± 1.2

92 ± 1.5

5

2.79

39 ± 0.6

68 ± 0.9

a

The tubulin assembly assay measured the extent of assembly of 2 mg/mL tubulin after 60 min at 37 °C. Data are presented as mean from three independent experiments. bTubulin, 1 µM; [3H]colchicine, 5 µM; and inhibitors, 1 or 5 µM. The inhibitory effects of 6a on microtubules organization were then confirmed by immunofluorescence staining of α-tubulin in A549 cells. In the vehicle-treated cells, the microtubule networks exhibited a normal arrangement with slim and fibrous microtubules wrapped around the cell nucleus. However, when cells were treated with


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6a (40 nM) for 24 h, the microtubule networks became disorganized and shrunk to the cell border, similar to the disruption produced by 1 at 40 nM (Figure 4).

Figure 4. Effects of 6a on the organizations of cellular microtubule network. A549 cells were treated with (a) 0.1 % DMSO, (b) 6a (40 nM), (c) 1 (40 nM) for 24 h. Microtubules were visualized with an anti-α-tubulin antibody (red), and the cell nucleus was visualized with Hoechst 33342 (blue).

Molecular Modeling. The molecular docking study was carried out to elucidate the binding features of the 1-methyl-1,4-dihydroindeno[1,2-c]pyrazoles with tubulin. Since the αβ-tubulin heterodimers complex with different ligands in certain flexibility at the colchicine site, we performed the docking studies of 6a with five representative crystal structures in which the ligands interacted with tubulin in the typical zones 1 and 2 (PDB code: 1SA0, 1SA1, 3HKE)39,41, zones 2 and 3 (PDB code: 3HKD)41, and all the three zones (PDB code: 3HKC)41. The 3D-structure of each ligand was built using the Sybyl sketch module followed by energy minimization using the Tripos force field. The Surflex docking program was used to automatically dock the selected



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compounds into the binding pocket of tubulin.56 The docking of 6a with 3HKC generated a higher Surflex docking score of 9.09, while that for 3HKE, 1SA0, 1SA1 and 3HKD were 7.99, 7.81, 5.64 and 5.49, respectively (Supporting Information), indicating that 6a tended to bind most favorably to the 3HKC model. With the full consideration of the hydrophobic interactions with the three zones in the tubulin binding pocket, the hydrogen bindings, and the Surflex docking scores, we concluded that 6a could bind to tubulin in the similar way as that of 5. As shown in Figure 5, in comparison with 1 that only occupied zones 1 and 2, 6a formed hydrophobic interaction with all the three zones. The m-ethoxyaniline group of 6a superimposed with the phenol substituent of 5, protruded into zone 3 more deeply, formed the same hydrogen bonding with Tyrβ202. Like the o-aminopyridine core in 5, the 1-methyl-1,4-dihydroindeno[1,2-c]pyrazole portion was accommodated at the zone 2 pocket. The amino group in the m-ethoxyaniline moiety formed hydrogen bonding with Valβ238 in zone 2. Though the ethoxy substituent at the 6position in 6a occupied only partial hydrophobic cavity in zone 1, the acetamide substituent at the 7-position interacted additionally with tubulin in an open cavity up to the α-tubulin subunit in the interfacial region, its amide nitrogen formed a critical hydrogen bonding with Serα178 at the interfacial surface.


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The medium potent 6b and the least active 6k showed similar binding modes as that of 6a (Supporting Information, Figures 8S and 9S). However, in comparison with 6a, the amino group in the m-ethoxyaniline moiety of 6b did not have hydrogen bonding with Valβ238. For compound 6k, the amide nitrogenat the 7-position could not form hydrogen bonding with Serα178. Moreover, the cyclopropylmethyl group in 6k protruded out of the interfacial surface that might account for its loss of tubulin polymerization inhibitory activity. It is apparent that the substituents at the 7-position of 1-methyl-1,4dihydroindeno[1,2-c]pyrazoles have great influences on their conformations and bindings with the tubulin. Besides the hydrophobic interactions, this modeling investigation stressed the hydrogen bondings with Valβ238, Tyrβ202, and Serα178 that seemed to be critical for the anticancer potency of these indenopyrazole derivatives. However, due to the limited resolution of the known crystal structures (3.58–4.20 Å), this docking modeling was just speculative. Further high resolution crystal structure studies of 6a or related indenopyrazole derivatives will provide true interactions of these inhibitors with tubulin. Other computational methods in combination of HINT scores were succefully used for the rational prediction of the binding mode of colchicine site ligands and for the SAR analysis.57 By isolating the HINT score for hydrogen bonding interactions involving Cysβ241 as the parameter,



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Kellogg’s group proposed a molecular modeling for a series of novel C-4 analogues of pyrrole-based antitubulin agents that could rationalize the observed SAR with a linear correlation between the antiproliferative activity and the microtubule inhibitory activity. Besides the hydrophobic interactions, a weak hydrogen bond with Cysβ241 was found to be critical for the microtubule depolymerizing activity.57

Figure 5. Docking of 6a (gray) and 1 (green) into the tubulin colchicine binding site and overlapping with 5 (red). In comparison with 1 that only occupied the zones 1 and 2, 6a formed hydrophobic interaction with all the three zones. In addition, the 7oxoacetamide substituent occupied an additional region in zone 1 and its amide nitrogen formed critical hydrogen bonding with Serα178 at the interfacial surface.

A549 Cell Cycle Arrest and G2/M-related Proteins Regulation. Both microtubule stabilizers and destabilizers alter the tubulin-microtubule equilibrium causing mitotic arrest at G2/M phase and ultimately apoptotic cell death. The most active compound 6a was therefore examined for its effect on cell cycle progression of A549 cells by flow cytometry. Treatment of 6a resulted in a gradual accumulation of


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cells in the G2/M phase of the cell cycle in a concentration-dependent manner, whereas the control cells were mainly in the G1 phase (Figure 6A). At a concentration of 40 nM, the percentage of cells in the G2/M phase increased to 68.1 %. This result compares favorably to 37.0 % in the G2/M phase for cells treated with 1 at the same concentration. We next studied the association between 6a-induced G2/M arrest and alterations in expression of proteins that regulate cell division. The mitosis promoting factor, a complex formed between cyclin B1 and cdc2, plays an important role in the transition from interphase to mitotic phase and governs cell cycle progression by enhancing cell cycle distribution in the G2/M fraction.54 As shown in Figure 6B, 6a caused concentration-dependent increases in cyclin B1. In contrast, p-cdc2, the inactive form of cdc2, was down-regulated in a concentration-dependent manner. These alterations were consistent with G2/M phase arrest induced by 6a.



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Figure 6. (A) Effects of 6a on A549 cell cycle progress. Cells were treated with different concentrations of 6a or 1 (40 nM) for 24 h and then analyzed by flow cytometry. (B) Effects of 6a on G2/M regulatory protein. A549 cells were treated for 24 h with the indicated concentration of 6a. The cells were harvested and lysed for the detection of cyclin B1, p-cdc2. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 vs. the vehicle control.

A549 and NCI-H460 Cell Apoptosis and Caspase-3 Activation. Mitotic arrest of tumor cells by tubulin-directed agents is generally associated with cellular apoptosis. The effects of 6a on A549 and NCI-H460 cell apoptosis was evaluated by observation


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of cellular morphology and Annexin V-FITC cell surface binding. As shown in Figure 7A, the vehicle-treated cells were polygon and attached to the plate bottom, while the 6a or 1 treated cells were round with cytoplasmic membrane blebbing and cell shrinkage. In comparison with the compact rounded nucleus of the vehicle-treated cells, 6a or 1 treated cells exhibited evident apoptosis features with nucleus fragmentation and chromatin condensation (Figure 7B). In addition to the morphological evaluation, the effect of 6a on apoptosis was confirmed by flow cytometric analysis. In normal live cells, plasma membrane is integrated, with phosphatidylserine (PS) located on the cytoplasmic surface. At an early stage of apoptosis, PS is translocated to the extrocytoplasmic surface. Annexin V-FITC is used to detect the externalization of PS. PI is impermeable to live and apoptotic cells due to plasma membrane integrity, but can bind tightly to the nucleic acids in necrotic cells. The cell populations can be differentiated using the Annexin V and the PI double-staining system. Exposure to 6a at 10, 20 and 40 nM for 24 h, the number of apoptotic cells were increased by 3.4 %, 8.0 % and 13.7 %, respectively (Figure 7C). 6a and 1 demonstrated no significant difference in inducing cell apoptosis at the same concentration of 40 nM. Caspase activation is a critical step in the final execution of the apoptotic cellular death process and can be induced by a variety of cellular stimuli including growth



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factor withdrawal, growth factor receptor inhibition or through disruption of the microtubule apparatus involved in cell division. The expressions of cleaved caspase-3 and cleaved PARP in A549 cells were both enhanced significantly by exposure to 6a for 24 h in a concentration-dependent manner (Figure 7D). These effects on the cleavage of caspase-3 and PARP with 6a are consistent with concentrations at which it induces the G2/M cell cycle arrest in A549 cell lines.


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Figure 7. 6a induces cell apoptosis. Exposed to 6a or 1 at 40 nM for 24 h, A549 and NCI-H460 cellular (A) and nuclear (B) morphological changes were imaged. Arrows indicated characteristics of apoptotic cells. (C) Detection of apoptotic cells after Annexin-V/PI staining by flow cytometry analysis. Exposed to 6a or 1 for 24 h, A549



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cells were harvested and stained with Annexin-V/PI for analysis. The different cell stages were assigned as live (Q1-LL), early apoptotic (Q1-LR), late apoptotic (Q1-UR) and necrotic cells (Q1-UL). (D) Western blotting analysis of the expression of cleaved caspase-3 and cleaved PARP. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 vs. the vehicle control.

Inhibition of Capillary Tube Formation of HUVECs. Angiogenesis, the growth of the new blood vessels, is essential for human cancer progression, development, and metastasis. Colchicine site binding agents could act as VDAs that cause vascular shutdown within solid tumors and a number of them have been in clinical investigations.28 The HUVEC tube formation assays is a dynamic in vitro assay representative of the key steps in angiogenesis, including proliferation, adhesion, and the formation of tube-like vascular structures. In an in vitro endothelial cell tube formation assay on 3-D Matrigel, 6a inhibited HUVEC cord formation in a concentration-dependent manner at concentrations that had minimal effects on HUVEC proliferation over the 8-hour time-course of the tube formation assay (Figure 8). Tube formation was inhibited by nearly 39, 58 and 69 % at concentrations of 10, 20 and 40 nM, respectively.


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Figure 8. 6a inhibits capillary tube formation of HUVECs. HUVECs were plated on Matrigel and allowed to form capillary tubes followed by exposure to different concentrations of compound 6a. HUVECs were seeded into 96-well plate which had been pre-coated with Matrigel and incubated with 6a for 8 h. Cultures were photographed, and the number of capillary tube networks was counted under a microscope (original magnification of 100×). Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 vs. the vehicle control.

Growth Inhibition of Human Non-small Cell Lung Cancer (NSCLC) Xenografts in Vivo. To validate further antitumor activity in vivo, nude mouse NSCLC xenograft models were established. Once a tumor was approximately 100 to 200 mm3 in size and was palpable, the mice were randomized into blank, vehicle control and treatment groups (6 mice per group). Control mice received the vehicle [PEG 400:ethanol:5 % dextrose in water (D5W) = 4:1:5]. In two treatment groups, 6a was administrated by intraperitoneal injection (ip) at a dose of 12.5 mg/kg or 50 mg/kg every day for 21 consecutive days. As shown in Figure 9, no significant differences between blank group and vehicle group were observed in the body weight,



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tumor volumes and weight, so the vehicle had neither toxicity nor inhibitory effect. Compared with the vehicle group, administration of 6a at 12.5 and 50 mg/kg by ip resulted in 33.8 % and 59.1 % reduction of tumor growth respectively (Figure 9B). The antitumor effect of 6a was also observed by a delayed increase in the volume of xenografts (Figure 9C). No significant differences in body weight or other adverse effects were observed upon treatment with compound 6a (Figure 9D). Hence, compound 6a showed strong antitumor activity on a well-tolerated dose schedule.

Figure 9. 6a inhibits the growth of human NSCLC xenografts in athymic mice. The images (A) and weights (B) of excised tumors from each group. Inhibition rates were defined as a percentage of vehicle-treated tumor weight. The tumor volumes (C) and


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body weights (D) were detected every three days. Data are presented as mean ± SD of 6 mice. *P < 0.05 vs. the vehicle control.

CONCLUSIONS By targeting an unexplored new binding region at the interface between both the αand β-subunit of tubulin heterodimers in zone 1, a series of 7-substituted 1-methyl1,4-dihydroindeno[1,2-c]pyrazoles (6a−p) were designed, efficiently synthesized, and evaluated in cellular and tubulin inhibition assays, resulting in the discovery of a new class of antitumor agents as tubulin polymerization inhibitors. The most potent compounds, 6a and 6n, exhibited noteworthy low nanomolar potency against a set of cancer cell lines, significant inhibition of tubulin polymerization, and competed with 1 at the tubulin colchicine binding site. 6a arrested most cells in the G2/M phase of the cell cycle and disrupted cellular microtubules, and induced A549 cell apoptosis, thus providing evidence that these active compounds are a new kind of tubulin polymerization inhibitors acting at the colchicine site. Mechanism considerations suggested that the blockade in G2/M phase of cell cycle was associated with alterations in the expression of cyclin B1 and p-cdc2, the induction of apoptosis was related to activation of caspase-3 and PARP. Furthermore, 6a inhibited capillary tube formation in a concentration-dependent manner and has the potential as a vascular disrupting agent. In NSCLC xenografts mouse model, 6a displayed strong in vivo



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antitumor activity, suppressing tumor growth by 59.1 % at a dose of 50 mg/kg (ip) with no obvious signs of toxicity. Molecular modeling results suggested the acetamide substituent at the 7-position might be responsible for the high potency of 6a by occupying an additional open cavity up to the α-tubulin subunit in the interfacial region and forming critical hydrogen bonding with Serα178 at the interfacial surface. In summary, these newly developed compounds with a novel 7-substituted 1-methyl1,4-dihydroindeno[1,2-c]pyrazole skeleton showed marked biological activity both in vitro and in vivo and have potential for further development as a novel class of anticancer agents.

EXPERIMENTAL SECTION Chemistry. Melting points were determined on an X-6 micro-melting point apparatus (Beijing Tech. Co., Ltd). 1H and

13

C NMR spectra were recorded on

Bruker-400 NMR or Bruker-600 NMR spectrometers, using tetramethylsilane (TMS) as the internal standard. The solvent was CDCl3 unless otherwise indicated. 1H spectra were recorded relative to DMSO-d6 (2.50 ppm) or TMS (0.00 ppm) as internal standard, and 13C NMR spectra were recorded relative to DMSO-d6 (39.52 ppm). All spectra were recorded at room temperature for DMSO or CDCl3 solutions. Electrospray-ionization mass spectrometry (ESI-MS) was performed on an API 4000


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instrument. Thin layer chromatography (TLC) was performed on silica gel GF254 plates. Silica gel GF254 and silica gel (200−300 mesh) from Qingdao Haiyang Chemical Company were used for TLC and column chromatography, respectively. All reagents are commercially available and were used as purchased without further purification. All reactions involving oxygen- or moisture-sensitive compounds were carried out under a dry N2 atmosphere. Unless otherwise noted, reagents were added by syringe. THF was distilled from sodium/benzophenone immediately prior to use. Elemental analyses were recorded with CHN model on FLASH 2000 organic elemental analyzer, and the observed values were considered acceptable within ± 0.3 % of calculated values, supporting > 95 % purity. 5-Ethoxy-6-hydroxyl-1-indanone (8). To a mixture of 5,6-dihydroxy-1-indenone 7 (125 g, 0.76 mol) and K2CO3 (105 g, 0.76 mol) in DMF (1000 mL), diethyl sulfate (88 mL, 0.76 mol) was added. The mixture was heated to 50 °C and stirred vigorously for 9 h. The mixture was cooled to room temperature and poured slowly into 2 M HCl (800 mL). The resulting precipitate was filtered, washed with a small amount of petroleum ether, dried, to give 112 g (77 %) of 8 as a yellow solid, mp 157–159 °C. 1

H NMR (400 MHz, DMSO-d6) δ 1.37 (t, J = 10.2 Hz, 3H), 2.5 (s, 2H), 2.94 (s, 2H),

4.13 (q, J = 10.2 Hz, 2H), 6.94 (s, 1H), 7.04 (s, 1H), 9.33 (s, 1H); MS (ESI) m/z 193.4 (M + H) +, 215.4 (M + Na)+.



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5-Ethoxy-6-tert-butyldimethylsiloxy-1-indanone (9). To a solution of 5-ethoxy-6hydroxyl-1-indanone (8, 3.20 g, 16.6 mmol) in DMF (25 mL) was added TBSCl (3.76 g, 24.9 mmol) and imidazole (1.70 g, 24.9 mmol). The mixture was stirred at room temperature for 1 h. Aqueous 10 % citric acid solution (100 mL) was added. The resulting precipitate was filtered, washed with a small amount of petroleum ether, dried, to give 4.65 g (91 %) of 9 as a yellow solid, mp 142–143 °C. 1H NMR (400 MHz, CDCl3) δ 0.16 (s, 6H), 1.00 (s, 9H), 1.49 (t, J = 10.2 Hz, 3H), 2.65 (t, J = 8.4 Hz, 2H), 3.02 (t, J = 8.4 Hz, 2H), 4.11 (q, J = 10.2 Hz, 2H), 6.83 (s, 1H), 7.18 (s, 1H); 13

C NMR (100 MHz, DMSO-d6) δ 204.9, 156.7, 151.6, 144.7, 129.7, 120.1, 113.3,

109.9, 64.5, 36.7, 26.0, 25.6, 18.6, 15.0, -4.33; MS (ESI) m/z 307.5 (M + H) +, 329.5 (M + Na)+; Anal. Calcd for C17H26O3Si: C, 66.62; H, 8.55. Found: C, 66.32; H, 8.23. 7-(tert-Butyldimethylsilyloxy)-6-ethoxy-N-(3-ethoxyphenyl)-1-methyl-1,4dihydroindeno[1,2-c]pyrazol-3-amine

(10).

A

solution

of

5-ethoxy-6-tert-

butyldimethylsiloxy-1-indanone (9, 4.65 g, 15.2 mmol) in anhydrous THF (60 mL) was cooled to –78 °C under nitrogen atmosphere. LHMDS (18.2 mL, 1.0 M THF solution) was added dropwise. The mixture was stirred at –78 °C for 2 h and then warmed to –45 °C in 45 min. A solution of 1-ethoxy-3-isothiocyanatobenzene (3.17 g, 17.7 mmol) in anhydrous THF (200 mL) was added, the resulting mixture was stirred at room temperature overnight. Acetic acid (2 mL) was added and the mixture was


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stirred at room temperature for 10 min then evaporated via rotary evaporation. The resulting residue was dissolved in dichloromethane (500 mL), washed with 1 M HCl, distilled water and brine, dried over Na2SO4. After filtration and evaporation, the resulting crude brown oil was dissolved in 1,4-dioxane (100 mL) and ethanol (100 mL), cooled to 0 °C. Methylhydrazine (4 mL,74.1 mmol) was added dropwise. After stirring at 0 °C for 2 h, the mixture was moved to room temperature and stirred for 82 h. After the solvent was evaporated, the residue was extracted with dichloromethane (100 mL), washed with brine, dried over Na2SO4. After filtration and evaporation, the residue was purified by column chromatography on silica gel (50–100 % EtOAc in hexane) to afford 3.91 g of 10 (54 %) as a light yellow solid, mp 110–112 °C. 1H NMR (600 MHz, DMSO-d6) δ 0.17 (s, 6H), 1.00 (s, 9H), 1.32 (t, J = 7.2 Hz, 3H), 1.37 (t, J = 7.2 Hz, 3H), 3.37 (s, 2H), 3.91 (s, 3H), 3.97 (q, J = 7.2 Hz, 2H), 4.06 (q, J = 7.2 Hz, 2H), 6.29 (dd, J = 7.8,1.8 Hz, 1H), 6.82 (dd, J = 7.8,1.8 Hz, 1H), 6.96 (t, J = 1.8 Hz, 1H), 7.06 (t, J = 7.8 Hz, 1H), 7.12 (s, 1H), 7.22 (s, 1H), 8.36 (s, 1H);

13

C

NMR (100 MHz, CDCl3) δ 160.1, 150.0, 149.5, 144.7, 144.1, 142.7, 129.8, 124.7, 113.3, 111.5, 111.3, 108.8, 105.9, 102.6, 64.2, 63.3, 37.1, 30.0, 25.8, 18.5, 15.0, 14.9, -4.57; MS (ESI) m/z 480.5 (M + H) +, 502.5 (M + Na)+; Anal. Calcd for C27H37N3O3Si: C, 67.60; H, 7.77; N, 8.76. Found: C, 67.52; H, 7.66; N, 8.60.



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6-Ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2-c]pyrazol7-ol (11). To a solution of 10 (3.91 g, 8.39 mmol) in THF (70 mL) was added TBAF (2.63 g, 10.1 mmol). The mixture was stirred at room temperature for 1 h, and the reaction was completed monitored by TLC. EtOAc (150 mL) was added, and the organic solution was washed with saturated aqueous NH4Cl solution and brine, dried over Na2SO4. After filtration and evaporation, the residue was purified by column chromatography on silica gel (hexane/AcOEt = 5:1–3:1) to give 2.68 g of 11 (91 %) as a light yellow solid, mp 153–155 °C. 1H NMR (600 MHz, DMSO-d6) δ 1.32 (t, J = 7.2 Hz, 3H), 1.35 (t, J = 7.2 Hz, 3H), 3.32 (s, 2H), 3.89 (s, 3H), 3.97 (q, J = 7.2 Hz, 2H), 4.06 (q, J = 7.2 Hz, 2H), 6.27 (dd, J = 7.8,1.2 Hz, 1H), 6.83 (dd, J = 7.8,1.2 Hz, 1H), 6.96 (t, J = 1.2 Hz, 1H), 7.05 (t, J = 7.8 Hz, 1H), 7.11 (s, 1H), 7.15 (s, 1H), 8.34 (s, 1H), 8.83 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 159.7, 148.9, 146.3, 146.0, 145.4, 144.8, 140.1, 129.8, 125.1, 112.7, 112.6, 108.3, 106.7, 104.3, 101.8, 64.7, 63.0, 37.3, 29.3, 15.3, 15.2. MS (ESI) m/z 366.4 (M + H) +, 388.4 (M + Na)+; Anal. Calcd for C21H23N3O3: C, 69.02; H, 6.34; N, 11.50. Found: C, 69.28; H, 6.31; N, 11.45. Methyl 2-(6-ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2c]pyrazol-7-yloxy)acetate (12). To a solution of 11 (1.10 g, 3.13 mmol) in acetone (35 mL) was added methyl chloroacetate (0.55 mL, 6.26 mmol) and K2CO3 (0.87 g, 6.26 mmol). The mixture was refluxed for 14 h. Distilled water (35 mL) was added.


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The reaction mixture was extracted with EtOAc (200 mL), washed with 1 M HCl (6 mL), distilled water (80 mL) and brine (80 mL), dried over Na2SO4. After filtration and evaporation, the residue was purified by column chromatography on silica gel (hexane/AcOEt = 2:1–1:1) to give 1.02 g of 12 (74 %) as a white solid, mp 139–141 °C. 1H NMR (400 MHz, DMSO-d6) δ 1.32 (t, J = 10.2 Hz, 3H), 1.35(t, J = 10.2 Hz, 3H), 3.38 (s, 2H), 3.72 (s, 3H), 3.93 (s, 3H), 3.98 (q, J = 10.2 Hz, 2H), 4.09 (q, J = 10.2 Hz, 2H), 4.88 (s, 2H), 6.29 (d, J = 6.8 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.98 (s, 1H), 7.06 (t, J = 8.0 Hz, 1H), 7.24 (s, 1H), 7.27 (s, 1H), 8.38 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 169.9, 159.7, 148.6, 147.6, 146.9, 145.4, 144.9, 142.9, 129.8, 124.9, 113.1, 112.7, 108.3, 106.2, 104.4, 101.9, 66.4, 64.7, 63.1, 52.2, 37.5, 29.4, 15.3, 15.2; MS (ESI) m/z 438.5 (M + H) +, 460.5 (M + Na)+; Anal. Calcd for C24H27N3O5: C, 65.89; H, 6.22; N, 9.60. Found: C, 65.72; H, 6.31; N, 9.45. 2-(6-Ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2c]pyrazol-7-yloxy)acetamide (6a). To a solution of 12 (15.0 g, 34.0 mmol) in MeOH (50 mL) was added aqueous ammonia (25 %, 52 mL). The reaction mixture was heated to 65°C and stirred for 12 h. A white solid was precipitated out. It was filtered, washed with distilled water and a small amount of MeOH, dried, to give 11.3 g of 6a (80 %) as a white solid, mp 186–188 °C. 1H NMR (600 MHz, DMSO-d6) δ 1.32 (t, J = 7.2 Hz, 3H), 1.37 (t, J = 7.2 Hz, 3H), 3.39 (s, 2H), 3.94 (s, 3H), 3.98 (q, J = 7.2 Hz,



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2H), 4.10 (q, J = 7.2 Hz, 2H), 4.53 (s, 2H), 6.30 (dd, J = 8.4,1.8 Hz, 1H), 6.83 (dd, J = 8.4,1.8 Hz, 1H), 6.98 (t, J = 1.8 Hz, 1H) , 7.06 (t, J = 7.8 Hz, 1H), 7.27 (s, 1H), 7.37 (s, 1H), 7.39 (s, 1H), 7.50 (s, 1H), 8.38 (s, 1H);

13

C NMR (100 MHz, DMSO-d6) δ

170.9, 159.7, 148.6, 147.9, 147.2, 145.4, 144.9, 143.4, 129.8, 124.9, 112.8, 112.7, 108.3, 107.6, 104.4, 101.9, 69.8, 64.7, 63.1, 37.5, 29.5, 15.3; MS (ESI) m/z 423.4 (M + H) +, 445.5 (M + Na)+; Anal. Calcd for C23H26N4O4: C, 65.39; H, 6.20; N, 13.26. Found: C, 65.43; H, 6.19; N, 13.18. 2-(6-Ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2c]pyrazol-7-yloxy)-N-methylacetamide (6b). To a solution of 12 (111.5 mg, 0.25 mmol) in MeOH (2.5 mL) was added aqueous methylamine solution (40 %, 4.0 mL). The mixture was heated to 65 °C and stirred for 2 h. After the completion of the reaction indicated by TLC, the solvent was evaporated. The residue was purified by column chromatography on silica gel (hexane/AcOEt = 1:1) to give 88.5 mg of 6b (81 %) as a light yellow solid, mp 163–165 °C. 1H NMR (600 MHz, DMSO-d6) δ 1.30 (t, J = 7.2 Hz, 3H), 1.35 (t, J = 7.2 Hz, 3H), 2.68 (d, J = 4.8 Hz, 3H), 3.36 (s, 2H), 3.91 (s, 3H), 3.94 (q, J = 7.2 Hz, 2H), 4.08 (q, J = 7.2 Hz, 2H), 4.52 (s, 2H), 6.26 (dd, J = 7.8,1.8 Hz, 1H), 6.80 (dd, J = 7.8,1.8 Hz, 1H), 6.95 (t, J = 1.8 Hz, 1H), 7.03 (t, J = 8.4 Hz, 1H), 7.24 (s, 1H), 7.36 (s, 1H), 7.88 (s, 1H), 8.36 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 169.3, 160.1, 149.4, 148.3, 146.8, 144.5, 144.3, 144.2, 128.8, 125.1, 113.5,


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111.8, 108.8, 107.7, 106.0, 102.7, 70.9, 64.7, 63.3, 37.2, 30.1, 25.7, 14.93, 14.90; MS (ESI) m/z 437.6 (M + H) +, 459.6 (M + Na)+; Anal. Calcd for C24H28N4O4: C, 66.04; H, 6.47; N, 12.84. Found: C, 66.15; H, 6.26; N, 12.70. 2-(6-Ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2c]pyrazol-7-yloxy)-N,N-dimethylacetamide (6c). To a solution of 12 (95 mg, 0.22 mmol) in MeOH (2 mL) was added aqueous dimethylamine solution (30 %, 2 mL). The mixture was heated to 65°C and stirred for 4 h. After the solvent was evaporated, the residue was purified by column chromatography on silica gel (hexane/AcOEt = 1:1–1:4) to give 65.2 mg of 6c (67 %) as a light yellow solid, mp 137–139 °C. 1H NMR (600 MHz, DMSO-d6) δ 1.32 (t, J = 7.2 Hz, 3H), 1.35 (t, J = 7.2 Hz, 3H), 2.87 (s, 3H), 3.06 (s, 3H), 3.37 (s, 2H), 3.93 (s, 3H), 3.97 (q, J = 7.2 Hz, 2H), 4.09 (q, J = 7.2 Hz, 2H), 4.86 (s, 2H), 6.29 (dd, J = 7.8,1.8 Hz, 1H), 6.84 (dd, J = 7.8,1.8 Hz, 1H), 6.99 (t, J = 1.8 Hz, 1H), 7.06 (t, J = 7.8 Hz, 1H), 7.23 (s, 1H), 7.25 (s, 1H), 8.38 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 168.2, 160.1, 149.7, 148.5, 147.0, 144.7, 144.2, 143.7, 129.8, 125.0, 113.4, 112.1, 108.7, 107.5, 105.9, 102.5, 70.2, 64.8, 63.3, 37.2, 36.8, 35.8, 30.0, 14.94, 14.90; MS (ESI) m/z 451.6 (M + H) +, 473.4 (M + Na)+; Anal. Calcd for C25H30N4O4: C, 66.65; H, 6.71; N, 12.44. Found: C, 66.85; H, 6.66; N, 12.30.



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2-(6-Ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2c]pyrazol-7-yloxy)-N-ethylacetamide (6d). To a solution of 12 (70 mg, 0.16 mmol) in MeOH (2 mL) was added aqueous ethylamine (70 %, 2 mL). The mixture was heated to 65°C and stirred for 2 h. After the solvent was evaporated, the resulting residue was purified by column chromatography on silica gel (hexane/AcOEt = 2:1– 1:1) to give 49 mg of 6d (67 %) as a white solid, mp 146–148 °C. 1H NMR (600 MHz, DMSO-d6) δ 1.07 (t, J = 7.2 Hz, 3H), 1.32 (t, J = 7.2 Hz, 3H), 1.38 (t, J = 7.2 Hz, 3H), 3.20 (q, J = 7.2 Hz, 2H), 3.39 (s, 2H), 3.93 (s, 3H), 3.97 (q, J = 7.2 Hz, 2H), 4.11 (q, J = 7.2 Hz, 2H), 4.55 (s, 2H), 6.29 (d, J = 8.4 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.98 (s, 1H), 7.06 (t, J = 8.4 Hz, 1H), 7.27 (s, 1H), 7.38 (s, 1H), 7.93 (s, 1H), 8.38 (s, 1H);

13

C NMR (100 MHz, CDCl3) ) δ 168.4, 160.1, 149.4, 148.3, 146.8,

144.5, 144.21, 129.8, 125.1, 113.6, 111.8, 108.8, 107.4, 105.9, 102.7, 70.7, 64.7, 63.3, 37.2, 33.9, 30.1, 14.94, 14.90, 14.8; MS (ESI) m/z 451.6 (M + H) +, 473.4 (M + Na)+; Anal. Calcd for C25H30N4O4: C, 66.65; H, 6.71; N, 12.44. Found: C, 66.75; H, 6.76; N, 12.30. 2-(6-Ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2c]pyrazol-7-yloxy)-N-(2-hydroxyethyl)acetamide (6e). To a solution of 12 (100 mg, 0.23 mmol) in MeOH (3 mL) was added 2-aminoethanol (0.21 mL, 3.5 mmol). The mixture was heated to 65°C and stirred for 1 h. A white solid was precipitated out. It


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was filtered and washed with distilled water and a small amount of MeOH, dried, to give 93 mg of 6e (86 %), mp 195–197 °C. 1H NMR (600 MHz, DMSO-d6) δ 1.32 (t, J = 7.2 Hz, 3H), 1.38 (t, J = 7.2 Hz, 3H), 3.26 (t, J = 5.4 Hz, 2H), 3.39 (s, 2H), 3.46 (q, J = 5.4 Hz, 2H), 3.94 (s, 3H), 3.97 (q, J = 7.2 Hz, 2H), 4.10 (q, J = 7.2 Hz, 2H), 4.58 (s, 2H), 4.77 (t, J = 5.4 Hz, 1H), 6.29 (dd, J = 7.8,1.8 Hz, 1H), 6.83 (dd, J = 7.8,1.8 Hz, 1H), 7.00 (t, J = 1.8 Hz, 1H), 7.06 (t, J = 7.8 Hz, 1H), 7.27 (s, 1H), 7.38 (s, 1H), 7.90 (t, J = 5.4 Hz, 1H), 8.38 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 168.5, 159.7, 148.6, 148.0, 147.1, 145.4, 144.9, 143.4, 129.8, 124.9, 112.7, 108.3, 107.6, 104.4, 101.9, 70.0, 64.7, 63.0, 60.2, 41.6, 37.5, 29.5, 15.3; MS (ESI) m/z 467.5 (M + H) +, 489.6 (M + Na)+; Anal. Calcd for C25H30N4O5: C, 64.36; H, 6.48; N, 12.01. Found: C, 64.66; H, 6.62; N, 11.96. N-(2-Cyanoethyl)-2-(6-ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4dihydroindeno[1,2-c]pyrazol-7-yloxy)acetamide (6f). To a solution of 12 (100 mg, 0.23 mmol) in MeOH (2.5 mL) was added 3-aminopropanenitril (0.25 mL, 0.35 mmol). The mixture was heated to 65°C and stirred for 10 h. After the solvent was evaporated, the resulting residue was purified by column chromatography on silica gel (hexane/AcOEt = 1:1–1:2) to give 70 mg of 6f (64 %) as a white solid, mp 175–177 °C. 1H NMR (600 MHz, DMSO-d6) δ 1.32 (t, J = 7.2 Hz, 3H), 1.38 (t, J = 7.2 Hz, 3H), 2.73 (t, J = 7.2 Hz, 2H), 3.39 (s, 2H), 3.43 (q, J = 7.2 Hz, 2H), 3.93 (s, 3H), 3.96



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(q, J = 7.2 Hz, 2H), 4.11 (q, J = 7.2 Hz, 2H), 4.60 (s, 2H), 6.29 (dd, J = 7.8,1.8 Hz, 1H), 6.83 (dd, J = 7.8,1.8 Hz, 1H), 6.97 (t, J = 1.8 Hz, 1H), 7.07 (t, J = 7.8 Hz, 1H), 7.28 (s, 1H), 7.37 (s, 1H), 8.33 (t, J = 6.0 Hz, 1H), 8.39 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 169.1, 159.7, 148.5, 148.1, 147.1, 145.4, 144.8, 143.6, 129.8, 124.9, 119.6, 112. 9, 112.73, 108.3, 107.9, 104.4, 101.9, 70.0, 64.7, 63.0, 37.5, 35.0, 29.5, 18.1 15.3; MS (ESI) m/z 476.4 (M + H) +, 498.6 (M + Na)+; Anal. Calcd for C26H29N5O4: C, 65.67; H, 6.15; N, 14.73. Found: C, 65.55; H, 6.27; N, 14.88. N-(2-Aminoethyl)-2-(6-ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4dihydroindeno[1,2-c]pyrazol-7-yloxy)acetamide (6g). To a solution of 12 (63 mg, 0.14 mmol) in MeOH (2.0 mL) was added ethane-1,2-diamine (0.15 mL, 2.2 mmol). The mixture was heated to 65°C and stirred for 5 h. After the solvent was evaporated, the residue was crystalized from MeOH/H2O (20:1) to give 53 mg of 6g (77 %) as a light yellow solid, mp 175–177 °C. 1H NMR (600 MHz, DMSO-d6) δ 1.32 (t, J = 7.2 Hz, 3H), 1.38 (t, J = 7.2 Hz, 3H), 1.47 (br s, 2H), 2.62 (t, J = 6.0 Hz, 2H), 3.17 (q, J = 6.0 Hz, 2H), 3.39 (s, 2H), 3.94 (s, 3H), 3.97 (q, J = 7.2 Hz, 2H), 4.10 (q, J = 7.2 Hz, 2H), 4.57 (s, 2H), 6.29 (d, J = 7.8 Hz, 1H), 6.83 (d, J = 7.8 Hz, 1H), 6.98 (s, 1H), 7.06 (t, J = 7.8 Hz, 1H), 7.27 (s, 1H), 7.37 (s, 1H), 7.93 (br s, 1H), 8.39 (s, 1H); 13C NMR (150 MHz, DMSO-d6) δ 168.8, 159.7, 157.9, 148.6, 148.1, 147.1, 145.3, 144.9, 143.5, 129.8, 124.9, 112.8, 112.7, 108.3, 107.9, 104.4, 101.8, 70.1, 64.7, 63.0, 39.1,


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37.4, 29.5, 15.3; MS (ESI) m/z 466.5 (M + H)+, 488.5 [M + Na]+; Anal. Calcd for C25H31N5O4: C, 64.50; H, 6.71; N, 15.04. Found: C, 64.65; H, 6.76; N, 15.20. N-(2-(Dimethylamino)ethyl)-2-(6-ethoxy-3-(3-ethoxyphenylamino)-1-methyl1,4-dihydroindeno[1,2-c]pyrazol-7-yloxy)acetamide (6h). To a solution of 12 (85 mg, 0.19 mmol) in MeOH (3.0 mL) was added N,N-dimethylethane-1,2-diamine (0.26 mL, 2.9 mmol). The mixture was heated to 65°C and stirred for 4 h. After the solvent was evaporated, the residue was crystalized from MeOH/H2O (10:1) to give 65 mg of 6h (72 %) as a light yellow solid, mp 192–194 °C. 1H NMR (600 MHz, DMSO-d6) δ 1.32 (t, J = 7.2 Hz, 3H), 1.38 (t, J = 7.2 Hz, 3H), 2.15 (s, 6H), 2.34 (t, J = 6.6 Hz, 2H), 3.27 (q, J = 6.6 Hz, 2H), 3.39 (s, 2H), 3.95 (s, 3H), 3.97 (q, J = 7.2 Hz, 2H), 4.10 (q, J = 7.2 Hz, 2H), 4.58 (s, 2H), 6.29 (d, J = 7.8 Hz, 1H), 6.83 (d, J = 7.8 Hz, 1H), 6.98 (s, 1H), 7.06 (t, J = 7.8 Hz, 1H), 7.27 (s, 1H), 7.37 (s, 1H), 7.77 (t, J = 6.6 Hz, 1H), 8.39 (s, 1H);

13

C NMR (100 MHz, CDCl3) δ 168.6, 160.1, 149.4, 148.1, 146.6, 144.7,

144.2, 143.8, 129.8, 125.1, 113.7, 112.0, 108.7, 106.6, 105.78, 102.5, 69.9, 64.8, 63.3, 58.0, 45.3, 37.3, 36.6, 30.0, 14.9; MS (ESI) m/z 494.6 (M + H)+, 516.7 (M + Na)+; Anal. Calcd for C27H35N5O4: C, 65.70; H, 7.15; N, 14.19. Found: C, 65.93; H, 7.22; N, 13.95. 2-(6-Ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2c]pyrazol-7-yloxy)-N-(2-methoxyethyl)acetamide (6i). To a solution of 12 (85 mg,



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0.20 mmol) in MeOH (3.0 mL) was added 2-methoxyethylamine (0.25 mL, 2.9 mmol). The mixture was heated to 65°C and stirred for 6 h. After the solvent was evaporated, the residue was crystalized from MeOH/H2O (20:1) to give 74 mg of 6i (79 %) as a light yellow solid, mp 197–199 °C. 1H NMR (600 MHz, DMSO-d6) δ 1.32 (t, J = 7.2 Hz, 3H), 1.38 (t, J = 7.2 Hz, 3H), 3.25 (s, 3H), 3.35 (t, J = 5.4 Hz, 2H), 3.39 (s, 2H), 3.40 (t, J = 5.4 Hz, 2H), 3.94 (s, 3H), 3.97 (q, J = 7.2 Hz, 2H), 4.10 (q, J = 7.2 Hz, 2H), 4.59 (s, 2H), 6.29 (d, J = 7.8 Hz, 1H), 6.83 (d, J = 7.8 Hz, 1H), 6.98 (s, 1H), 7.06 (t, J = 7.8 Hz, 1H), 7.27 (s, 1H), 7.38 (s, 1H), 7.91 (t, J = 4.8 Hz, 1H), 8.39 (s, 1H);

13

C NMR (100 MHz, CDCl3) δ 168.7, 160.1, 149.6, 148.5, 146.7, 144.3,

144.2, 129.9, 124.8, 113.2, 111.8, 109.0, 107.2, 106.2, 102.9, 71.2, 70.3, 64.7, 63.4, 58.8, 38.8, 37.2, 30.2, 14.9, 14.8; MS (ESI) m/z 481.5 (M + H)+, 503.4 (M + Na)+; Anal. Calcd for C26H32N4O5: C, 64.98; H, 6.71; N, 11.66. Found: C, 64.76; H, 6.71; N, 11.43. N-Cyclopropyl-2-(6-ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4dihydroindeno[1,2-c]pyrazol-7-yloxy)acetamide (6j). To a solution of 12 (90 mg, 0.21 mmol) in MeOH (3.0 mL) was added cyclopropylamine (0.21 mL, 3.1 mmol). The mixture was heated to 65°C and stirred for 12 h. After the solvent was evaporated, the residue was recrystallized from MeOH/H2O (20:1) to give 60 mg of 6j (63 %) as a light yellow solid, mp 175–177 °C. 1H NMR (600 MHz, DMSO-d6) δ 0.47–0.49 (m,


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2H), 0.66–0.68 (m, 2H), 1.32 (t, J = 7.2 Hz, 3H), 1.37 (t, J = 7.2 Hz, 3H), 2.70–2.73 (m, 1H), 3.38 (s, 2H), 3.93 (s, 3H), 3.97 (q, J = 7.2 Hz, 2H), 4.10 (q, J = 7.2 Hz, 2H), 4.53 (s, 2H), 6.29 (dd, J = 7.8,1.8 Hz, 1H), 6.83 (d, J = 7.8 Hz, 1H), 6.98 (s, 1H), 7.06 (t, J = 7.8 Hz, 1H), 7.26 (s, 1H), 7.34 (s, 1H), 8.01 (d, J = 3.6 Hz, 1H), 8.39 (s, 1H); 13

C NMR (100 MHz, CDCl3) δ 170.1, 160.1, 149.3, 148.2, 146.7, 144.6, 144.3, 144.2,

129.0, 125.2, 113.6, 111.9, 108.8, 107.5, 105.9, 102.7, 70.8, 64.8, 63.3, 37.2, 30.1, 22.1, 14.94, 14.91, 6.4; MS (ESI) m/z 463.5 (M + H)+, 485.6 (M + Na)+; Anal. Calcd for C26H30N4O4: C, 67.51; H, 6.54; N, 12.11. Found: C, 67.28; H, 6.64; N, 11.91. N-(Cyclopropylmethyl)-2-(6-ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4dihydroindeno[1,2-c]pyrazol-7-yloxy)acetamide (6k). To a solution of 12 (90 mg, 0.21 mmol) in MeOH (3 mL) was added cyclopropanemethylamine (0.27 mL, 3.1 mmol). The mixture was heated to 65°C and stirred for 10 h. After the solvent was evaporated, the residue was recrystallized from MeOH/H2O (20:1) to give 71 mg of 6k (77 %) as a light yellow solid, mp 192–194 °C. 1H NMR (600 MHz, DMSO-d6) δ 0.18–0.21 (m, 2H), 0.40–0.43 (m, 2H), 0.94–0.97 (m, 1H), 1.32 (t, J = 7.2 Hz, 3H),1.39 (t, J = 7.2 Hz, 3H), 3.06 (t, J = 6.0 Hz, 2H), 3.39 (s, 2H), 3.94 (s, 3H), 3.97 (q, J = 7.2 Hz, 2H), 4.11 (q, J = 7.2 Hz, 2H), 4.58 (s, 2H), 6.29 (dd, J = 7.8,1.8 Hz, 1H), 6.83 (d, J = 7.8 Hz, 1H), 6.98 (s, 1H), 7.06 (t, J = 7.8 Hz, 1H), 7.28 (s, 1H), 7.38 (s, 1H), 7.95 (t, J = 6.0 Hz, 1H), 8.39 (s, 1H);

13

C NMR (100 MHz, DMSO-d6) δ



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168.3, 159.7, 148.6, 147.9, 147.1, 145.3, 144.9, 143.4, 129.8, 124.9, 112.8, 112.7, 108.30, 107.5, 104.4, 101.9, 69.9, 64.7, 63.0, 43.0, 37.5, 29.5, 15.30, 15.26, 11.3, 3.59; MS (ESI) m/z 477.5 (M + H)+, 499.6 (M + Na)+; Anal. Calcd for C27H32N4O4: C, 68.05; H, 6.77; N, 11.76. Found: C, 67.95; H, 6.76; N, 11.50. 2-(6-Ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2c]pyrazol-7-yloxy)-N-(prop-2-ynyl)acetamide (6l). To a solution of 12 (102 mg, 0.23 mmol) in MeOH (3.5 mL) was added prop-2-yn-1-amine (0.24 mL, 3.5 mmol). The mixture was heated to 65°C and stirred for 12 h. After the solvent was evaporated, the resulting residue was recrystallized from MeOH/H2O (20:1) to give 70 mg of 6l (69 %) as a light yellow solid, mp 195–197 °C. 1H NMR (600 MHz, DMSO-d6) δ 1.32 (t, J = 7.2 Hz, 3H), 1.38 (t, J = 7.2 Hz, 3H), 3.16 (t, J = 2.4 Hz, 1H), 3.39 (s, 2H), 3.94 (s, 3H), 3.97 (q, J = 7.2 Hz, 4H), 4.11 (q, J = 7.2 Hz, 2H), 4.61 (s, 2H), 6.29 (dd, J = 7.8,1.8 Hz, 1H), 6.83 (d, J = 7.8 Hz, 1H), 6.98 (s, 1H), 7.06 (t, J = 7.8 Hz, 1H), 7.28 (s, 1H), 7.35 (s, 1H), 8.39 (s, 1H), 8.42 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 168.6, 160.1, 149.3, 148.3, 146.7, 144.6, 144.6 144.3, 129.9, 125.2, 113.7, 111.8, 108.8, 108.0, 105. 9, 102.7, 79.1, 71.6, 71.0, 64.7, 63.3, 37.2, 30.1, 28.7, 14.93, 14.91; MS (ESI) m/z 461.5 (M + H)+, 483.5 (M + Na)+; Anal. Calcd for C26H28N4O4: C, 67.81; H, 6.13; N, 12.17. Found: C, 67.54; H, 6.14; N, 11.99.


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N-Allyl-2-(6-ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2c]pyrazol-7-yloxy)acetamide (6m). To a solution of 12 (95 mg, 0.22 mmol) in MeOH (3.0 mL) was added allylamine (0.25 mL, 3.3 mmol). The mixture was heated to 65°C and stirred for 12 h. The solvent was evaporated. The residue was recrystallized from MeOH/H2O (20:1) to give 69 mg of 6m (69 %) as a light yellow solid, mp 172–174 °C. 1H NMR (600 MHz, DMSO-d6) δ 1.32 (t, J = 7.2 Hz, 3H), 1.37 (t, J = 7.2 Hz, 3H), 3.39 (s, 2H), 3.81 (br s, 2H), 3.93 (s, 3H), 3.97 (q, J = 7.2 Hz, 2H), 4.10 (q, J = 7.2 Hz, 2H), 4.62 (s, 2H), 5.07 (d, J = 10.2 Hz, 1H), 5.16 (d, J = 17.4 Hz, 1H), 5.82–5.86 (m, 1H), 6.28 (d, J = 7.8 Hz, 1H), 6.83 (d, J = 7.8 Hz, 1H), 6.98 (s, 1H), 7.06 (t, J = 7.8 Hz, 1H), 7.28 (s, 1H), 7.39 (s, 1H), 8.01 (br s, 1H), 8.39 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 168.6, 160.1, 149.4, 148.3, 146.7, 144.5, 144.3, 144.2, 133.9, 129.8, 125.1, 116.5, 113.6, 111.7, 108.8, 107.6, 106.0, 102.7, 70.8, 64.7, 63.3, 41.3, 37.2, 30.1, 14.9; MS (ESI) m/z 463.5 (M + H)+, 485.6 (M + Na)+; Anal. Calcd for C26H30N4O4: C, 67.51; H, 6.54; N, 12.11. Found: C, 67.54; H, 6.57; N, 11.98. 2-(6-Ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2c]pyrazol-7-yloxy)-N-hydroxyacetamide (6n). To a solution of hydroxylamine hydrochloride (1.6 g, 24 mmol) in MeOH (60 mL) was added 28 % MeONa/MeOH solution (5.0 mL). After stirring for 30 min, the resulting sodium chloride was



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filtered. The filtrate was added to the solution of 12 (1.0 g, 2 mmol) in MeOH (15 mL). The mixture was heated to 65°C and stirred for 6 h. A white solid was precipitated out. It was filtered, washed with distilled water and a small amount of MeOH, dried, to give 0.78 g of 6n (77 %) as a white solid, mp 183–185 °C. 1H NMR (600 MHz, DMSO-d6) δ 1.32 (t, J = 7.2 Hz, 3H), 1.37 (t, J = 7.2 Hz, 3H), 3.38 (s, 2H), 3.94 (s, 3H), 3.97 (q, J = 7.2 Hz, 2H), 4.09 (q, J = 7.2 Hz, 2H), 4.54 (s, 2H), 6.29 (dd, J = 7.8,1.8 Hz, 1H), 6.83 (dd, J = 7.8,1.8 Hz, 1H), 6.98 (s, 1H), 7.06 (t, J = 7.8 Hz, 1H), 7.25 (s, 1H), 7.35 (s, 1H), 8.39 (s, 1H), 9.04 (s, 1H), 10.74 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 165.1, 159.7, 148.6, 147.9, 147.3, 145.4, 144.9, 143.2, 129.8, 124.9, 112.9, 112.7, 108.3, 107.2, 104.4, 101.9, 68.5, 64.7, 63.1, 37.5, 29.5, 15.29, 15.27; MS (ESI) m/z 439.5 (M + H)+, 461.5 (M + Na)+; Anal. Calcd for C23H26N4O5: C, 63.00; H, 5.98; N, 12.78. Found: C, 62.86; H, 6.07; N, 12.62. 2-(6-Ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2c]pyrazol-7-yloxy)-N-methoxyacetamide (6o). To a solution of methoxylamine hydrochloride (2.5 g, 30 mmol) in MeOH (20 mL) was added 28 % MeONa/MeOH solution (7.0 mL). After stirring for 30 min, the resulting sodium chloride was filtered. The filtrate was added to the solution of 12 (100 mg, 0.23 mmol) in MeOH (15 mL). The mixture was heated to 65°C and stirred for 12 h. The solvent was evaporated. The residue was purified by column chromatography on silica gel


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(hexane/AcOEt = 1:4) to give 60 mg of 6o (58 %) as a white solid, mp 146–148 °C. 1

H NMR (600 MHz, DMSO-d6) δ 1.28 (t, J = 7.2 Hz, 3H), 1.33 (t, J = 7.2 Hz, 3H),

3.39 (s, 2H), 3.64 (s, 3H), 3.90 (s, 3H), 3.94 (q, J = 7.2 Hz, 2H), 4.11 (q, J = 7.2 Hz, 2H), 4.54 (s, 2H), 6.26 (dd, J = 7.8,1.8 Hz, 1H), 6.83 (dd, J = 7.8,1.8 Hz, 1H), 6.95 (t, J = 1.8 Hz, 1H), 7.06 (t 1H, J = 7.8 Hz), 7.24 (s, 1H), 7.31 (s, 1H), 8.36 (s, 1H), 11.36 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 165.2, 159.7, 148.6, 148.0, 147.1, 145.4, 144.9, 143.5, 129.8, 124.9, 113.0, 112.7, 108.3, 107.5, 104.4, 101.9, 68.6, 64.7, 63.8, 63.1, 37.4, 29.5, 15.3; MS (ESI) m/z 453.5 (M + H)+, 475.4 (M + Na)+; Anal. Calcd for C24H28N4O5: C, 63.70; H, 6.24; N, 12.38. Found: C, 63.67; H, 6.28; N, 12.40. 2-(6-Ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2c]pyrazol-7-yloxy)acetohydrazide (6p). To a solution of 12 (90 mg, 0.21 mmol) in MeOH (3.0 mL) was added hydrazine hydrate (1 mL). The mixture was heated to 65°C and stirred for 1 h. The solvent was evaporated. The residue was recrystallized from MeOH/H2O (20:1) to give 76 mg of 6p (84 %) as light yellow solid, mp 178– 180 °C. 1H NMR (600 MHz, DMSO-d6) δ 1.29 (t, J = 7.2 Hz, 3H),1.35 (t, J = 7.2 Hz, 3H), 3.35 (s, 2H), 3.91 (s, 3H), 3.94 (q, J = 7.2 Hz, 2H), 4.07 (q, J = 7.2 Hz, 2H), 4.34 (s, 2H), 4.56 (s, 2H), 6.26 (dd, J = 7.8,1.8 Hz, 1H), 6.80 (d, J = 7.8,1.8 Hz, 1H), 6.95 (s, 1H), 7.03 (t, J = 7.8 Hz, 1H), 7.23 (s, 1H), 7.35 (s, 1H), 8.36 (s, 1H), 9.12 (s, 1H); 13

C NMR (100 MHz, DMSO-d6) δ 167.6, 159.7, 148.6, 147.9, 147.3, 145.4, 144.9,



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143.3, 129.8, 124.9, 112.8, 112.7, 108.3, 107.5, 104.4, 101.9, 69.3, 64.7, 63.1, 37.5, 29.5, 15.3; MS (ESI) m/z 438.5 (M + H)+, 460.6 (M + Na)+; Anal. Calcd for C25H27N5O4: C, 63.14; H, 6.22; N, 16.01. Found: C, 63.34; H, 6.19; N, 16.23. Cell Growth Inhibitory Assay. Target compounds were assayed by conventional MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) method. In brief, the exponentially growing cells were seeded into 96-well cell plates at density of 1−6 × 103 cells per well and allowed to adhere overnight. Cells were incubated with various concentrations of the test compounds for 48 h (Table 2) or 72 h (Table 1). Then 20 µL of MTT (5 mg/mL) was added, the cells were incubated at 37 °C for another 4 h. The reduced MTT crystals were dissolved in DMSO and the absorbance was measured at 570 nm by a microplate spectrophotometer. The growth inhibitory effects of each compound were expressed as IC50 values, which represent the molar drug concentrations required to cause 50 % tumor cell growth inhibition. In colony formation assay, A549 and NCI-H460 cells (500 per well) grown in 6well plates were exposed to different concentrations of 6a at 37 °C for 9 days. The colonies (greater than 50 cells) were fixed, stained with 0.5 % crystal violet, and then counted. In trypan blue staining assay, A549 cells were seeded in 6-well plates at density of 2 × 105 cells per well and after overnight adherence they were exposed to increasing


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concentrations of 6a for 24 h. Afterwards, cell suspension was mixed with 0.4 % trypan blue solution at a 1:9 ratio. The number of viable cells (clear) vs. dead cells (blue) was determined by a hemocytometer. Cell morphology. A549 and NCI-H460 cells were seeded in 24-well plates at density of 5 × 104 cells per well. After overnight adherence, they were exposed to increasing concentrations of 6a for 24 h. Cells were imaged using fluorescence microscope. In order to visualized nuclear morphological changes, cells were stained with Hoechst 33342 (Sigma, USA). In vitro tubulin polymerization assay. The fluorescence-based in vitro tubulin polymerization assay was performed using Tubulin Polymerization Assay Kit (BK011P, Cytoskeleton, USA) according to the manual. The tubulin reaction mix contained 2 mg/mL porcine brain tubulin (> 99 % pure), 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP and 15 % glycerol. Firstly, 96-well plate was incubated with 5 µL of inhibitors in various concentrations at 37 °C for 1 min. Then 50 µL of the tubulin reaction mix was added. Immediately, the increase in fluorescence was monitored by excitation at 355 nm and emission at 460 nm in a multimode reader. Competitive inhibition assays. The competitive binding activity of inhibitors was evaluated using a radiolabeled [3H]colchicine competition scintillation proximity (SPA) assay.55 In brief, 0.08 µM [3H]colchicine was mixed with the test compound



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and biotinylated porcine tubulin (0.5 µg) in a buffer of 100 µL containing 80 mM PIPES (pH 6.8), 1 mM EGTA, 10 % glycerol, 1 mM MgCl2, and 1 mM GTP) for 2 h at 37 °C. Then streptavidin-labeled SPA beads (80 µg) were added to each mixture. The radioactive counts were measured directly with a scintillation counter. Immunofluorescent staining. A549 cells were seeded in 24-well plate (with cover slips plated) at density of 5 × 104 cells. After overnight adherence, they were exposed to 6a and 1 at 40 nM respectively for 24 h. The cover slips were fixed in ice-cold methanol/acetic acid (3:1) for 10 min and blocked with 3 % bovine serum albumin for 20 min at room temperature. Then they were incubated with mouse anti-α-tubulin antibody (T5168, sigma, USA) overnight at 4 °C and incubated with goat anti-mouse IgG-TRITC antibody (ZF-0313, ZSGB-BIO, China) at 37 °C for 2 h. Hoechst 33342 was used to counterstain the nuclear. The cover slips were visualized under fluorescence microscope. Molecular modeling. The crystal structure of tubulin in complex with different ligands (PDB: 1SA0, 1SA1, 3HKC, 3HKD, 3HKE) was used as the template in the docking study. The protein structure was prepared using the SYBYL-X suite (version 1.3, Tripos). After extracting the ligand, hydrogen atoms were added to the crystal. Charges were added to biopolymer by AMBER7 FF99 force field and ligand by


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Gasteiger-Huckel force field. The extracted ligand was used to generate the protomol with threshold of 0.99 and bloat of 0 Å. The 3D-structure of each ligand was constructed by the Sketch module of Sybyl, and energy minimization was performed using the Tripos force field. A non-bonded cutoff distance of 8 Å was adopted to consider the intramolecular interaction. Surflexdock module was used for the docking studies and the related parameters implied in the program were kept at default. Cell cycle analysis. A549 cells were seeded in 6-well plates at density of 2 × 105 cells per well and after overnight adherence they were exposed to increasing concentrations of 6a and positive control (1) for 24 h. Then cells were harvested, fixed in cold 70 % ethanol overnight, treated with RNase A at 37 °C for 30 min and incubated with propidium iodide (PI) solution (Solarbio, China) at 4 °C for 15 min. Cell cycle distribution was analyzed using a FACS flow cytometer. Annexin V-FITC/PI staining assay. A549 cells were treated as cell cycle analysis mentioned above. The percentage of apoptotic cells was quantitatively estimated by using Annexin V-FITC kit (ydjmbio, China) according to the manufacturer's instructions. The analysis was performed on a FACS flow cytometer (Beckman coulter, USA).



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Western blotting analysis. A549 cells were treated as cell cycle analysis mentioned above. Then cells were homogenised in cell lysis buffer for Western and IP (Beyotime, China) and the protein concentrations were determined by a BCA Protein Assay Kit (Thermo Scientific, USA). The protein extracts were reconstituted in loading buffer (Beyotime, China) and inactivated at 100 °C for 5 min. 30 µg of proteins were fractionated by 10 % or 12 % SDS-PAGE and were transferred to PVDF membranes (Millipore, USA) and then the protein levels were estimated by using the primary antibodies with appropriate dilution. The primary antibodies included those to cyclin B1 (1495-S, Eptomics, USA), p-cdc2 (9111), cleaved caspase-3 (9661), cleaved PARP (9541s, Cell Signaling Technology, USA) and βactin (ZF-0313, ZSGB-BIO, China). The primary antibodies were washed and then incubated

with

HRP-conjugated

secondary

antibody

(ZSGB-BIO,

China).

Immunoreactive bands were visualized using an enhanced chemiluminescence reagent (Millipore, USA) and quantified by densitometry using a ChemiDoc XRS + molecular imager. The intensities of the blots were quantified by AlphaEaseFC 4.0. Capillary tube formation assay. Matrigel (50 µL, Corning, USA) at 4 °C was used to coat each well of a 96-well plate and allowed to polymerize for 1 h at 37 °C. 50 µL of HUVECs (4 × 105 cells/mL) were seeded on Matrigel-coated 96-well plate, together with increasing concentrations of 6a. HUVECs were incubated for 8 h to


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allow formation of tube-like structures at 37 °C. The formed tubes were counted in three random observations under fluorescence microscope. Antitumor activity in vivo. The experimental procedures conformed to the animal experiment guidelines of the Animal Care & Welfare Committee of Shandong University. Five-week-old pathogen-free female Balb/c-nu mice were purchased from the Animal Centre of China Academy of Medical Sciences (Beijing, China). The nude mice were housed under pathogen-free conditions. Human NSCLC xenografts were established by inoculating human NSCLC tissue pieces which provided by the Second Hospital of Shandong University into the armpits of mice by trocar. When the tumor proliferating exuberantly, they were extracted and cut into 1 mm3 fragments (about 20 mg/fragment), and the fragments were subcutaneously inoculated into the armpits of each nude mouse. When the tumor volume reached approximately 100 mm3 to 200 mm3, the mice were randomly divided into four groups (n = 6): blank group, vehicle group [PEG 400:ethanol:dextrose 5 % in water (D5W)] = 4:1:5), 6a (12.5 mg/kg) group and 6a (50 mg/kg) group. Both drugs and vehicle were administrated intraperitoneal injection (ip) every day for 21 consecutive days. Tumor volume and body weight were measured every three days. Tumor volumes were calculated with a formula of W2 × L / 2, where W is width (short axis) and L is length (long axis). At the end of the experiment, the mice were sacrificed and the tumors were removed and



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weighed. Therapeutic effect of the 6a on xenografts growth was expressed as a percentage of the vehicle group.

ASSOCIATED CONTENT

Supporting Information

In vitro tubulin polymerization assay, molecular modeling, NMR spectra for all screening compounds and synthetic intermediates. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors

*For Z.-P.L.: phone +86 (0)531 8838006; E-mail: [email protected].

*For X.-L.G.: phone +86 (0)531 88382490; E-mail: [email protected].

Author Contributions

§

These two authors contributed equally to this paper.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGEMENTS


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This work was supported by the National Natural Science Foundation of China (NSFC, Grant No. 81573275).

ABBREVIATIONS LHMDS, lithium hexamethyldisilazide; SD, standard deviation; NSCLC, non-small cell lung cancer; D5W, 5 % dextrose in water; ip, intraperitoneal injection; TMS, tetramethylsilane; ESI-MS, electrospray-ionization mass spectrometry; TLC, thin layer

chromatography;

MTT,

(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-

tetrazolium bromide); PI, propidium iodide

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Table of Contents Graphic

KEYWORDS:

Colchicine

binding

site,

tubulin

polymerization

antiproliferative activity, docking, G2/M phase arrest, apoptosis.


inhibitor,

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Figure 1. Colchicine site ligands (colchicine, podophyllotoxin, CA-4, TN16, ABT-751 and the designed indenopyrazoles) with their binding modes with tubulin (zone 1 in red, zone 2 in blue, zone 3 in pink, the predicted new binding region in zone 1 in green, and the bridge in thin black). 173x98mm (300 x 300 DPI)



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Figure 2. Growth inhibitory effects of 6a on NSCLC cell lines by colony formation assay and trypan blue staining. Representative colony formation images on A549 cells (A) and NCI-H460 cells (B) after exposed to 6a for 9 days. The results were denoted as a percentage of the vehicle-treated cells. (C) The cytotoxicity of 6a on A549 cells was estimated by trypan blue exclusion. After exposed to 6a for 24 h, A549 cells were harvested and stained with trypan blue solution. Arrows indicated the dead cells. The results were expressed as a percentage based on the ratio of the number of viable treated cells to that of vehicle control. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 vs. the vehicle control. 167x186mm (300 x 300 DPI)


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Figure 3. Effect of 6a and 6n on tubulin polymerization in vitro. Purified tubulin protein at 2 mg/mL in a reaction buffer was incubated at 37 °C in the presence of 1 % DMSO, test compounds (6a or 6n at 1.25 µM, 2.5 µM, 5 µM or 10 µM) or colchicine (20 µM). Polymerizations were followed by an increase in fluorescence emission at 460 nm over a 60 min period at 37 °C. The experiments were performed three times. 157x44mm (300 x 300 DPI)



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Figure 4. Effects of 6a on the organizations of cellular microtubule network. A549 cells were treated with (a) 0.1 % DMSO, (b) 6a (40 nM), (c) colchicine (40 nM) for 24 h. Microtubules were visualized with an anti-αtubulin antibody (red), and the cell nucleus was visualized with Hoechst 33342 (blue). 155x51mm (300 x 300 DPI)


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Figure 5. Docking of 6a (gray) and 1 (green) into the tubulin colchicine binding site and overlapping with 5 (red). In comparison with 1 that only occupied the zones 1 and 2, 6a formed hydrophobic interaction with all the three zones. In addition, the 7-oxoacetamide substituent occupied an additional region in zone 1 and its amide nitrogen formed critical hydrogen bonding with Serα178 at the interfacial surface. 83x46mm (300 x 300 DPI)



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Figure 6. (A) Effects of 6a on A549 cell cycle progress. Cells were treated with different concentrations of 6a or colchicine (40 nM) for 24 h and then analyzed by flow cytometry. (B) Effects of 6a on G2/M regulatory protein. A549 cells were treated for 24 h with the indicated concentration of 6a. The cells were harvested and lysed for the detection of cyclin B1, p-cdc2. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 vs. the vehicle control. 170x146mm (300 x 300 DPI)


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Figure 7. 6a induces cell apoptosis. Exposed to 6a or 1 at 40 nM for 24 h, A549 and NCI-H460 cellular (A) and nuclear (B) morphological changes were imaged. Arrows indicated characteristics of apoptotic cells. (C) Detection of apoptotic cells after Annexin-V/PI staining by flow cytometry analysis. Exposed to 6a or colchicine for 24 h, A549 cells were harvested and stained with Annexin-V/PI for analysis. The different cell stages were assigned as live (Q1-LL), early apoptotic (Q1-LR), late apoptotic (Q1-UR) and necrotic cells (Q1-UL). (D) Western blotting analysis of the expression of cleaved caspase-3 and cleaved PARP. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 vs. the vehicle control. 171x221mm (300 x 300 DPI)



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Figure 8. 6a inhibits capillary tube formation of HUVECs. HUVECs were plated on Matrigel and allowed to form capillary tubes followed by exposure to different concentrations of compound 6a. HUVECs were seeded into 96-well plate which had been pre-coated with Matrigel and incubated with 6a for 8 h. Cultures were photographed, and the number of capillary tube networks was counted under a microscope (original magnification of 100×). Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 vs. the vehicle control. 166x38mm (300 x 300 DPI)


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Figure 9. 6a inhibits the growth of human NSCLC xenografts in athymic mice. The images (A) and weights (B) of excised tumors from each group. Inhibition rates were defined as a percentage of vehicle-treated tumor weight. The tumor volumes (C) and body weights (D) were detected every three days. Data are presented as mean ± SD of 6 mice. *P < 0.05 vs. the vehicle control. 166x113mm (300 x 300 DPI)



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Scheme 1. Synthesis of 1-Methyl-1,4-dihydroindeno[1,2-c]pyrazoles 6a−pa 154x113mm (300 x 300 DPI)


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Table of Contents Graphic 96x54mm (300 x 300 DPI)


Design, Synthesis, and Biological Evaluation of 1 ... - ACS Publications

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