A novel domino approach for synthesis of indolyl tetrahydropyrano[4,3-c]pyrazole derivatives as anticancer agents

Tetrahedron 72 (2016) 5769e5775

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A novel domino approach for synthesis of indolyl tetrahydropyrano [4,3-c]pyrazole derivatives as anticancer agents Fu-Qiang Wang a, y, Hui Yang a, y, Bin He a, Yong-Kang Jia a, c, Shi-Yao Meng a, c, Chao Zhang a, Hong-Min Liu a, b, Feng-Wu Liu a, b, * a b c

School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, 450001, PR China Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou, 450001, PR China People’s Hospital of Zhengzhou, Zhengzhou 450053, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 June 2016 Received in revised form 19 July 2016 Accepted 30 July 2016 Available online 6 August 2016

A series of indolyl substituted 1,4,6,7-tetrahydropyrano[4,3-c]pyrazole derivatives were synthesized via a domino method based on the 1,4:3,6-Dianhydrofructose. The mechanism was proposed and proved by calculation with a density functional theory (DFT), which includes formation of phenylhydrazone, furanone ring opening, Fischer indole synthesis, pyrazole ring closing and a furan ring expansion. In vitro antitumor activity of all synthesized compounds against four human cancer cell lines (MCF-7, EC-109, HGC-27, and PC-3 cell lines) were then evaluated, using MTT assay. We found that most of the compounds were more potent than the positive control 5-fluorouracil. Particularly, compound 4i against HGC-27 and PC-3, 4j against EC-109 cell lines showed three fold greater activity than 5-fluorouracil, respectively. The activity of compounds 4c and 4d against MCF-7 cell line was close to that of doxorubicin. Ó 2016 Elsevier Ltd. All rights reserved.

Keywords: Domino reaction Indole 1,4:3,6-Dianhydrofructose 1,4,6,7-tetrahydropyrano[4,3-c]pyrazole In-vitro antitumor activity

1. Introduction The aza-heterocyclic rings exist widely in natural and synthetic drugs. Pyrazole and indole derivatives are two important class of nitrogen-containing heterocyclic compounds due to their extensive range of pharmacological activities such as anti-inflammatory,1 antimicrobial,2,3 antifungal,4 anticancer,5,6 and antiviral activities.7e9 Therefore, construction of pyrazole and indole rings has drawn much attention for decades.10,11 Furthermore, heterocycles condensed to pyrazole ring are an important source of bioactive molecules.10,12 Compounds containing both pyrazole and other important heterocyclic active structural units usually exhibit more outstanding biological activities. A series of condensed pyrazole derivatives were reported as antibacterial agents with fourfold of activities against Gram-positive as well as Gram-negative bacteria, compared with general pyrazole compounds.13 Pyranopyrazoles, referring to a fused five membered pyrazole ring to a six-membered pyran ring, are also a versatile source of biomolecules. Tremendous developments have been carried on the

* Corresponding author. Fax: þ86 371 6778 1739; e-mail address: [email protected]. cn (F.-W. Liu). y F.-Q. Wang and H. Yang contributed equally to this work. http://dx.doi.org/10.1016/j.tet.2016.07.078 0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.

synthesis of functionalized pyrano[2,3-c]pyrazoles and its application in medicinal chemistry,14 but only limited studies focused on the pyrano[4,3-c]pyrazoles.15,16 In the course of our application of 1,4:3,6-dianhydrofructose in the field of medicinal chemistry, we have synthesized a series of bioactive substituted a,b-unsaturated furofuran-3-ones (Scheme 1).17 Taking account of the skeleton of the molecule containing a free carbonyl and a latent carbonyl group (as shown in Scheme 1), we assumed that it may serve as a synthetic equivalent of dicarbonyl synthon and then attempted to utilize the synthon in synthesis of other bioactive molecules. As a first result, a series of novel indolyl substituted 1,4,6,7-tetrahydropyrano[4,3-c]pyrazole derivatives, which contain both pyranopyrazole and indole in one molecular frame, were obtained via a new domino method. Considering the previous reported anticancer activity of both pyrano [2,3-c]pyrazole derivatives and indole derivatives, it was thought worthwhile to explore the anticancer potency of the novel 1,4,6,7tetrahydropyrano[4,3-c]pyrazole derivatives. Herein we report the application of those substituted a,b-unsaturated furofuran-3-ones in construction of aza-heterocyclic compounds and investigation on antitumor activity of the obtained indolyl substituted 1,4,6,7tetrahydropyrano[4,3-c]pyrazole derivatives against four human cancer cell lines.

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F.-Q. Wang et al. / Tetrahedron 72 (2016) 5769e5775 Table 1 Optimization of reaction conditions

Scheme 1. a,b-unsaturated furofuran-3-ones.

2. Results and discussion 2.1. Chemistry The starting materials of a,b-unsaturated furofuran-3-ones (1 and 2) were synthesized by following the procedures described in our previous paper.17 1,4:3,6-Dianhydro-D-fructose was treated with various substituted benzaldehydes in the presence of basic catalyst KF/Al2O3 to afford a series of compounds 1 and 2 (Scheme 1). When 8R-isomer of unsaturated furofuran-3-one (1a, R¼3-F) was treated with 2 equiv of phenylhydrazine in the presence of an equivalent of aqueous HCl in methanol, a white solid (3a) and a light yellow syrup (4a) were obtained. Compounds 3a and 4a were elucidated by their HRMS, 1H NMR, 13C NMR, and 2D NMR spectra as a couple of isomers of indolyl pyranopyrazole derivatives. The absolute structure of 3a was further confirmed by Xray crystallographic analysis as an indolyl pyrano[4,3-c]pyrazole derivative with R-configuration at C-8 (shown as C-8 in crystal data in Fig. 1). Compound 4a was accordingly deduced as 8S-diastereoisomer of 3a. When 1a was substituted with 2a (8S-isomer) in the reaction, the same result was obtained, which indicated that the configuration of benzyl carbon (C-8) in the starting compound does not determine the C-8 configurations of products. The fact revealed an SN1 pathway at C-8 in the process of formation of pyran ring.

Fig. 1. X-ray crystallography for 3a.

The reaction conditions were optimized starting from utilization of different solvents and acids in the reaction (entry 1e8, Table 1). We found that isopropanol and 4-methylbenzenesulfonic acid (pTsOH) are favorable to the reaction. Then the influence of the amount of p-TsOH and reaction temperature on the yield of product was investigated (entry 9e15, Table 1). The total yield of both 3a and 4a was improved to 63% in the optimized conditions (entry 10).

Entry

Acids (eq)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

aq HCl aq HCl aq HCl AcOH p-TsOH PPAc BF3 Anh. AlCl3 p-TsOH p-TsOH p-TsOH p-TsOH p-TsOH p-TsOH p-TsOH

a b c

1 1 1 1 1 1 1 1 3 5 7 9 5 5 5

T ( C)

Solvents

Yieldsa

Reflux Reflux Reflux Reflux Reflux Reflux Reflux Reflux Reflux Reflux Reflux Reflux 70 60 50

MeOH EtOH iPrOH iPrOH iPrOH iPrOH iPrOH iPrOH iPrOH iPrOH iPrOH iPrOH iPrOH iPrOH iPrOH

21 22 29 NRb 35 12 22 9 50 63 59 55 57 49 36

Total isolated yields of product 3 and 4. No reaction. Polyphosphoric acid.

The experiment was performed on millimole scale: 1 mmol of compound 1, 2.1 mmol of phenylhydrazine, 15 mL of solvent. The reaction was monitored by TLC to the disappearance of compound 1 except the case of no reaction. The generality and the scope of the transformation was investigated under the optimum conditions by using unsaturated furofuran-3-one derivatives (1) from various substituted benzaldehydes (as shown in Table 2). From the results we noted that all the unsaturated furofuran-3-one with halo-substitutions (1ae1j) readily afforded corresponding indolyl pyranopyrazole derivatives 3ae3j and 4ae4j (entry 1e10, Table 2). However, the unsaturated furofuran-3-ones derivatives from other substituted benzaldehydes such as alkylbenzaldehyde, and alkyloxybenzaldehyde did not give target products in the reaction (entry 11e15, Table 2). Table 2 Summary for the synthesis of indolyl pyrano[4,3-c]pyrazole derivatives

Entry

1

R

3,4

Yields (3:4)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1a 1b 1c 1d 1e 1f 1g 1h 1i 1j 1k 1l 1m 1n 1o

3-F 2-F 4-F 3,4-diF H 3-Cl 4-Cl 3-Br 4-Br 2-F, 4-Br 3-OMe 4-OMe 2,3-diOMe 3,4,5-triOMe 4-isoPr

3a,4a 3b,4b 3c,4c 3d,4d 3e,4e 3f,4f 3g,4g 3h,4h 3i,4i 3j,4j NPa NP NP NP NP

63 65 67 70 52 57 62 54 59 67 d d d d d

a

(33:30) (34:31) (36:31) (36:34) (26:26) (29:28) (32:30) (28:26) (30:29) (35:32)

NP: no product obtained.

The proposed pathway for the transformation was illustrated in Scheme 2. Unsaturated furofuran-3-one was condensated firstly with amino group of a phenylhydrazine to give phenylhydrazone (A). Oxygen of a water molecule attacked C-4 of the

F.-Q. Wang et al. / Tetrahedron 72 (2016) 5769e5775

dianhydrofructose skeleton in the presence of strong acid to open furan ring, giving a 4-hydroxyl-1-oxo intermediate (B). The reversible transformation of A to B could be speeded up by the condensation of newly generated carbonyl group and another phenylhydrazine to furnish intermediate C, and also consequent formation of indole derivative (D) via a Fischer indole synthesis process. Subsequent acid-catalytic domino reactions including elimination of hydroxyl from benzyl, 1,2-shift of oxygen in furan ring to form a pyran ring, and formation of epoxy ring at C-3 and C-4 afforded an epoxy-pyran derivative E. Then the nitrogen in phenylhydrazone attacked C-4 to form a dihydropyrazole ring and open epoxy ring at same time, giving an indolyl dihydropyrazole (F). Finally the removal a H2O molecule from C-3 and C-4 at dihydropyrazole F generated more stable pyranopyrazole molecules containing C3eC4 double bond (3 and 4) with a greater conjugate system.

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Table 3 Inhibitory activity of pyrazole derivatives against four human cancer cell lines Compounds

R

8R/8S IC50 (mM)

3a 4a 3b 4b 3c 4c 3d 4d 3e 4e 3f 4f 3g 4g 3h 4h 3i 4i 3j 4j 5-fluorouracil doxorubicin

3-F 3-F 2-F 2-F 4-F 4-F 3,4-diF 3,4-diF H H 3-Cl 3-Cl 4-Cl 4-Cl 3-Br 3-Br 4-Br 4-Br 2-F, 4-Br 2-F, 4-Br

R S S R R S R S R S R S R S R S R S S R

MCF-7

EC-109

HGC-27

PC-3

22.621.35 30.211.32 >64 22.141.32 28.361.25 3.540.55 >64 7.740.89 12.541.10 24.521.42 25.961.41 20.391.31 23.561.37 15.571.19 23.101.36 19.541.29 33.631.53 >64 17.281.24 18.561.27 d 1.580.20

>64 >64 >64 >64 >64 >64 >64 23.071.36 24.791.39 >64 >64 25.071.40 >64 >64 >64 31.191.49 25.291.40 17.051.23 >64 7.740.89 24.641.39 d

>64 >64 25.331.40 24.951.40 17.011.23 12.281.09 >64 >64 >64 >64 >64 >64 >64 >64 22.551.35 17.391.24 11.371.13 7.700.89 14.221.53 14.501.34 25.891.41 d

>64 >64 >64 14.231.15 >64 15.531.19 22.221.35 >64 >64 12.421.09 >64 15.101.18 >64 10.701.03 >64 >64 >64 5.720.76 >64 21.701.34 16.651.22 d

potent compounds including compounds 4c, 4d, 4i and 4j, had the same structural features at C-8 of pyrano[4,3-c]pyrazole core, suggesting that the 8-phenyl and 5S-hydroxyl locating at different face of pyran ring might be favorable to anticancer activity. 3. Conclusion

Scheme 2. Proposed pathway for synthesis of indolyl pyranopyrazoles.

2.2. Anticancer activity The anticancer activity of all synthesized compounds were evaluated, using four human cancer cell lines, including EC-109 (human esophageal cancer cell line), MCF-7(human breast cancer cell line), HGC-27 (human gastric cancer cell line), and PC-3 (human prostate cancer cell line). The IC50 values (half maximal inhibitory concentration) were determined using MTT assay with the typical anticancer drug 5-fluorouracil or doxorubicin as positive control. The inhibition rates of all obtained compounds at 64 mM and 32 mM were firstly detected against the four human cancer cell lines, respectively. Some compounds with potent activity were selected and further evaluated. The IC50 values were listed in Table 3. As shown in Table 3, many tested compounds exhibited more potent anticancer activity than that of the positive control 5fluorouracil (5-FU) against HGC-27 and PC-3 cell lines. Among them, compound 4i containing a 4-Br substitution has the IC50 values of 7.700.89 and 5.720.76 mM against HGC-27 and PC-3 cell lines, respectively, being about three times more effective than that of positive control 5-FU. Compounds 4c and 4d showed inhibition of MCF-7 with IC50 values of 3.540.55 and 7.740.89 mM, respectively, close to that of doxorubicin (1.580.20 mM). Moreover, almost a third of the compounds had similar activities to 5-FU against EC-109 cell line. In particular, the compound 4j, derivative from 4-bromo-2-fluorobenzaldehydes, showed three-times greater inhibitory rate to EC-109 than 5fluorouracil (IC50: 7.740.89 mM vs 24.641.39 mM). All the most

We found a domino method for synthesis of novel indolyl substituted 1,4,6,7-tetrahydropyrano[4,3-c]pyrazole derivatives, starting from phenylhydrazine and unsaturated furofuran-3-one derived from 1,4:3,6-dianhydrofructose. A series of target compounds have been synthesized and also the pathway of transformation has been proposed. The bioactive evaluation of the obtained compounds revealed that most of them have potent antitumor activity against four human cancer cell lines. 4. Experimental section 4.1. General methods Reagents and solvents were purchased from commercial sources and were used without further purification. Melting points were determined on an X-5 micromelting apparatus and are uncorrected. 1H and 13C NMR spectra were acquired on a Bruker AVANCE III-400 spectrometer with chemical shifts (d) given in parts per million relative to tetramethylsilane as the internal standard (d¼0.00 ppm). All assignments were based upon the twodimensional NMR spectra. HRMS (high resolution mass spectra) were taken with a Q-Tof Micromass spectrometer. TLC was performed on silica gel GF254 plates and detected by placing under the UV lamp. Column chromatography was conducted using a column of silica gel (200e300 mesh). 4.2. X-ray diffraction data collection X-ray diffraction analysis was carried out on an Agilent Xcalibur Gemini, Eos CCD diffractometer with graphite-monochromated Cu Ka (l¼1.5418  A) radiation. A monoclinic crystal was selected and mounted on a glass fiber. All data were collected at a temperature of 291(2) K. X-ray diffraction intensities were collected, integrated

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F.-Q. Wang et al. / Tetrahedron 72 (2016) 5769e5775

and scaled with CrysAlisPro suite of programs. The structure was solved via direct methods and expanded using the Fourier technique. The non-hydrogen atoms were refined with anisotropic thermal parameters. Hydroxyl hydrogen atoms were refined with isotropic thermal parameters. Other hydrogen atoms were included but not refined. All calculations were performed using the SHELX97 crystallographic software package.18 4.3. General procedure for the synthesis of compounds 3aej and 4aej In a round-bottom flask equipped with a magnetic stirred bar, compound 1 or 2 (1 mmol), phenylhydrazine (4 mmol), TsOH (5 mmol), and isopropanol (15 mL) were added. The resulting mixture was stirred at refluxing temperature. The disappearance of compound 1 or 2 was monitored by TLC. Upon completion, the reaction mixture was concentrated to dryness under reduced pressure. The residue was dissolved in EtOAc (30 mL) and washed with water for three times (330 mL). The organic layer was dried over anhydrous Na2SO4. After removal of the solvent, the residue was purified by flash column chromatography with elution of ethyl acetate (EA)/petroleum ether (PE) to give the corresponding title compound 3 and 4. 4.4. Characterization of compounds 3aej and 4aej 4.4.1. (4R,7S)-4-(3-Fluorophenyl)-3-(3-(3-fluorophenyl)-1H-indol-2yl)-7-hydroxyl-1-phenyl-1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (3a). White solid, yield 33%, Rf¼0.30 (EA/PE 1:3); mp 150e153  C; 1 H NMR (400 MHz, CDCl3) d 8.67 (s, 1H), 8.02 (d, J¼7.7 Hz, 2H), 7.52 (td, J¼8.0, 5.0 Hz, 3H), 7.43 (t, J¼7.4 Hz, 1H), 7.35 (td, J¼8.0, 6.1 Hz, 1H), 7.28 (d, J¼8.1 Hz, 1H), 7.18 (t, J¼7.6 Hz, 1H), 7.05 (qd, J¼7.9, 6.5, 2.7 Hz, 2H), 7.00e6.90 (m, 2H), 6.85 (dt, J¼10.2, 2.0 Hz, 1H), 6.75 (td, J¼8.6, 2.7 Hz, 1H), 6.58e6.39 (m, 2H), 4.62 (s, 1H), 4.58 (s, 1H), 4.15 (dd, J¼11.8, 1.6 Hz, 1H), 3.77e3.67 (m, 1H)$13C NMR (101 MHz, CDCl3) d163.8 (d, J¼74 Hz), 161.3 (d, J¼74 Hz), 141.5, 140.3 (d, J¼7 Hz), 139.8, 138.9, 136.9 (d, J¼9 Hz), 135.6, 129.8 (d, J¼8 Hz), 129.6, 129.3(d, J¼8 Hz), 127.8, 126. (d, J¼46 Hz), 125.6 (d, J¼3 Hz), 124.0 (d, J¼3 Hz), 123.1, 122.8, 120.3, 119.2, 116.8, 116.5 (d, J¼22 Hz), 115.8 (d, J¼21 Hz), 115.5 (d, J¼2 Hz), 115.1 (d, J¼22 Hz), 113.2 (d, J¼21 Hz), 111.0, 76.64, 76.63, 71.6. HRMS (ESI) Calcd for C32H24F2N3O2: 520.1837, found: m/z: 520.1839 [MþH]þ. Anal. Calcd for C32H23F2N3O2: C, 73.98; H, 4.46; N, 8.09. Found: C, 74.10; H, 4.58; N, 8.05. 4.4.2. (4S,7S)-4-(3-Fluorophenyl)-3-(3-(3-fluorophenyl)-1H-indol-2yl)-7-hydroxyl-1-phenyl-1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (4a). Yellow syrup, yield 30%; Rf¼0.26 (EA/PE 1:3); 1H NMR (400 MHz, CDCl3) d 8.86 (s, 1H), 8.03 (d, J¼7.6 Hz, 2H), 7.60e7.53 (m, 5H), 7.46e7.37 (m, 4H), 7.22 (m, 3H), 7.08 (m, 3H), 6.65 (d, J¼8.4 Hz, 2H), 4.76 (s, 1H), 4.53 (d, J¼10.6 Hz, 1H), 3.72 (dd, J¼12.5, 1.7 Hz, 1H), 3.42 (dd, J¼12.4, 2.0 Hz, 1H), 2.76 (d, J¼10.8 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 163.2 (d, J¼71 Hz), 161.7(d, J¼71 Hz), 141.8, 139.4, 138.9, 136.5, 135.5, 133.5, 131.8, 131.7, 131.0, 130.1, 129.5, 127.8, 126.6, 123.3, 122.7, 122.4, 121.0, 120.2, 119.2, 115.5, 113.7 (d, J¼7 Hz), 112.9 (d, J¼9 Hz), 110.9, 73.1, 64.9, 60.6. HRMS (ESI) Calcd for C32H24F2N3O2: 520.1837, found: m/z: 520.1837 [MþH]þ. Anal. Calcd for C32H23F2N3O2: C, 73.98; H, 4.46; N, 8.09. Found: C, 74.22; H, 4.58; N, 8.12. 4.4.3. (4S,7S)-4-(2-Fluorophenyl)-3-(3-(2-fluorophenyl)-1H-indol-2yl)-7-hydroxyl-1-phenyl-1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (3b). Yellow syrup, yield 34%; Rf¼0.30 (EA/PE 1:3); 1H NMR (400 MHz, CDCl3) d 8.74 (s, 1H), 7.98 (d, J¼7.9 Hz, 2H), 7.49 (t, J¼7.7 Hz, 2H), 7.43e7.31 (m, 3H), 7.26 (t, J¼8.4 Hz, 1H), 7.21e6.98 (m, 6H), 6.88 (dd, J¼10.5, 8.2 Hz, 1H), 6.71e6.54 (m, 2H), 4.97 (s,

1H), 4.56 (s, 1H), 4.14 (dd, J¼11.8, 1.7 Hz, 1H), 3.73 (dd, J¼11.7, 1.7 Hz, 1H), 3.41 (s, 1H)$13C NMR (101 MHz, CDCl3) d 162.1(d, J¼140 Hz), 159.6 (d, J¼140 Hz), 141.6, 139.7, 139.0, 135.5, 132.4 (d, J¼4 Hz), 130.7 (d, J¼9 Hz), 129.5, 129.1 (d, J¼3 Hz), 128.7 (d, J¼7 Hz), 127.64, 127.57, 127.4, 125.1 (d, J¼12 Hz), 124.0 (d, J¼4 Hz), 123.5 (d, J¼3 Hz), 122.8, 122.7, 122.0 (d, J¼15 Hz), 120.0, 116.12, 116.10 (d, J¼22 Hz), 115.5 (d, J¼22 Hz), 110.9, 110.5, 71.3 (d, J¼3 Hz), 71.2, 61.0. HRMS (ESI) Calcd for C32H24F2N3O2: 520.1837, found: m/z: 520.1848 [MþH]þ. Anal. Calcd for C32H23F2N3O2: C, 73.98; H, 4.46; N, 8.09. Found: C, 74.37; H, 4.68; N, 7.84. 4.4.4. (4R,7S)-4-(2-Fluorophenyl)-3-(3-(2-fluorophenyl)-1H-indol2-yl)-7-hydroxyl-1-phenyl-1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (4b). Yellow syrup, yield 31%; Rf¼0.25 (EA/PE 1:3); 1H NMR (400 MHz, CDCl3) d 8.97 (s, 1H), 8.07 (d, J¼7.8 Hz, 2H), 7.57 (t, J¼7.9 Hz, 2H), 7.43 (t, J¼7.3 Hz, 2H), 7.38 (d, J¼8.1 Hz, 1H), 7.33 (d, J¼7.9 Hz, 1H), 7.20 (m, 5H), 7.04 (t, J¼7.5 Hz, 1H), 6.96 (dd, J¼10.1, 8.4 Hz, 1H), 6.75 (t, J¼7.5 Hz, 1H), 6.54 (t, J¼7.3 Hz, 1H), 5.17 (s, 1H), 4.53 (d, J¼10.7 Hz, 1H), 3.76 (d, J¼12.4, 1H), 3.46 (d, J¼12.4 Hz, 1H), 2.87 (d, J¼11.2 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 162.2 (d, J¼120 Hz), 159.8 (d, J¼120 Hz), 141.9, 139.8, 139.0, 135.5, 132.6 (d, J¼3 Hz), 130.5 (d, J¼8 Hz), 129.5, 129.3 (d, J¼8 Hz), 129.1 (d, J¼3 Hz), 127.7, 124.3(d, J¼4 Hz), 123.1, 123.0 (d, J¼3 Hz), 122.7, 121.8(d, J¼15 Hz), 120.0, 119.9, 116.2(d, J¼21 Hz), 115.8 (d, J¼21 Hz), 114.8, 110.9, 68.0 (d, J¼3 Hz), 65.1, 60.7. HRMS (ESI) Calcd for C32H24F2N3O2: 520.1837, found: m/z: 520.1846 [MþH]þ. Anal. Calcd for C32H23F2N3O2: C, 73.98; H, 4.46; N, 8.09. Found: C, 74.22; H, 4.58; N, 8.12. 4.4.5. (4R,7S)-4-(4-Fluorophenyl)-3-(3-(4-fluorophenyl)-1H-indol2-yl)-7-hydroxyl-1-phenyl-1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (3c). White solid, yield 36%; Rf¼0.31 (EA/PE 1:3); mp 155e158  C; 1 H NMR (400 MHz, CDCl3) d 8.51 (s, 1H), 8.01 (d, J¼7.5 Hz, 2H), 7.56e7.41 (m, 5H), 7.28 (t, J¼7.5 Hz, 2H), 7.24e7.03 (m, 6H), 6.85e6.73 (m, 4H), 4.62 (d, J¼9.5 Hz, 1H), 4.60 (s, 1H), 4.14 (dd, J¼11.9, 1.5 Hz, 1H), 3.73 (d, J¼11.8 Hz, 1H), 2.99 (d, J¼11.1 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 163.5 (d, J¼80 Hz), 161.0 (d, J¼80 Hz), 141.7, 139.8, 139.0, 135.6, 134.0 (d, J¼3 Hz), 131.5 (d, J¼80 Hz), 130.6 (d, J¼3 Hz), 130.1 (d, J¼80 Hz), 129.6, 127.8, 127.3, 126.2, 123.1, 122.8, 120.2, 119.2, 117.0, 115.7, 115.5, 115.3, 115.0, 114.8, 111.0, 76.5, 71.4, 61.2. HRMS (ESI) Calcd for C32H24F2N3O2: 520.1837, found: m/z: 520.1848 [MþH]þ. Anal. Calcd for C32H23F2N3O2: C, 73.98; H, 4.46; N, 8.09. Found: C, 74.29; H, 4.63; N, 8.15. 4.4.6. (4S,7S)-4-(4-Fluorophenyl)-3-(3-(4-fluorophenyl)-1H-indol-2yl)-7-hydroxyl-1-phenyl-1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (4c). White solid, yield 31%; Rf¼0.25 (EA/PE 1:3); mp 157e160  C; 1 H NMR (400 MHz, CDCl3) d 8.85 (s, 1H), 8.03 (d, J¼7.9 Hz, 2H), 7.57 (t, J¼7.8 Hz, 2H), 7.41 (m, 3H), 7.14 (m, 6H), 6.76 (m, 4H), 4.69 (s, 1H), 4.54 (d, J¼8.7 Hz, 1H), 3.70 (d, J¼12.3 Hz, 1H), 3.44 (d, J¼12.3 Hz, 1H), 2.76 (d, J¼11.0 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 163.5 (d, J¼55 Hz), 161.1 (d, J¼55 Hz), 142.0, 139.4, 139.1, 135.5, 133.4, 133.36, 131.8 (d, J¼7 Hz), 130.5, 130.2 (d, J¼7 Hz), 129.5, 128.3, 127.8, 126.7, 123.2, 122.8, 120.1, 119.3, 115.7 (d, J¼32 Hz), 115.6, 114.8 (d, J¼21 Hz), 110.9, 73.0, 64.8, 60.6. HRMS (ESI) Calcd for C32H24F2N3O2: 520.1837, found: m/z: 520.1836 [MþH]þ. Anal. Calcd for C32H23F2N3O2: C, 73.98; H, 4.46; N, 8.09. Found: C, 74.20; H, 4.53; N, 8.03. 4.4.7. (4R,7S)-4-(3,4-Difluorophenyl)-3-(3-(3,4-difluorophenyl)-1Hindol-2-yl)-7-hydroxyl-1-phenyl-1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (3d). White solid, yield 36%, Rf¼0.32 (EA/PE 1:3); mp 143e146  C; 1H NMR (400 MHz, CDCl3) d 8.65 (s, 1H), 8.00 (d, J¼7.9 Hz, 2H), 7.61e7.39 (m, 4H), 7.31 (d, J¼8.0 Hz, 1H), 7.27e7.15 (m, 2H), 7.09 (t, J¼7.5 Hz, 1H), 7.01e6.87 (m, 2H), 6.81 (dd, J¼18.0, 8.4 Hz, 1H), 6.62 (ddd, J¼10.5, 7.6, 1.2 Hz, 1H), 6.51 (m, 1H), 4.61 (d,

F.-Q. Wang et al. / Tetrahedron 72 (2016) 5769e5775

J¼4.0 Hz, 1H), 4.60 (s, 1H), 4.15 (d, J¼11.8 Hz, 1H), 3.73 (d, J¼11.8 Hz, 1H), 3.21 (d, J¼10.9 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 151.6 (d, J¼12 Hz), 150.6(dd, J¼60, 12 Hz), 149.1 (d, J¼12 Hz), 148.3 (dd, J¼60, 12 Hz), 141.2, 139.9, 138.8, 135.6, 135.0 (dd, J¼6, 4 Hz), 131.6 (dd, J¼6, 4 Hz), 129.6, 128.0, 126.8, 126.2, 125.8 (dd, J¼5.7, 3.6 Hz), 124.4 (dd, J¼5.7, 3.6 Hz), 123.4, 122.8, 120.6, 118.9, 118.4 (d, J¼17 Hz), 117.3 (d, J¼13 Hz), 117.2 (d, J¼13 Hz), 116.7 (d, J¼17 Hz), 116.4, 114.6 (d, J¼1 Hz), 111.1, 76.1, 71.5, 61.0. HRMS (ESI) Calcd for C32H22F4N3O2: 556.1648, found: m/z: 556.1653 [MþH]þ. Anal. Calcd for C32H21F4N3O2: C, 69.19; H, 3.81; N, 7.56. Found: C, 69.33; H, 3.93; N, 7.59. 4.4.8. (4S,7S)-4-(3,4-Difluorophenyl)-3-(3-(3,4-difluorophenyl)-1Hindol-2-yl)-7-hydroxyl-1-phenyl-1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (4d). White solid, yield 34%; Rf¼0.26 (EA/PE 1:3); mp 140e143  C; 1H NMR (400 MHz, CDCl3) d 9.01 (s, 1H), 8.01 (d, J¼7.9 Hz, 2H), 7.56 (t, J¼7.7 Hz, 2H), 7.48e7.32 (m, 3H), 7.26e7.16 (m, 2H), 7.08 (t, J¼7.5 Hz, 1H), 6.99 (m, 2H), 6.88 (dd, J¼18.0, 8.5 Hz, 1H), 6.64 (dt, J¼8.4, 1.6 Hz, 1H), 6.54 (m, 1H), 4.75 (s, 1H), 4.54 (d, J¼8.8 Hz, 1H), 3.73 (d, J¼12.1 Hz, 1H), 3.43 (dd, J¼12.3, 1.4 Hz, 1H), 2.91 (d, J¼9.8 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 151.6e150.7(m), 149.0e148.2 (m), 141.6, 139.6, 138.9, 135.6, 134.7(m), 131.6(dd, J¼6, 4 Hz), 129.6, 128.0, 127.9, 126.7, 126.3 (dd, J¼6, 3 Hz), 124.5 (dd, J¼6, 3 Hz), 123.5, 122.8, 120.5, 119.0, 118.9 (d, J¼17 Hz), 117.5 (d, J¼17 Hz), 117.3 (d, J¼17 Hz), 116.8 (d, J¼17 Hz), 115.4, 114.6, 111.1, 72.6, 65.1, 60.5. HRMS (ESI) Calcd for C32H22F4N3O2: 556.1648, found: m/z: 556.1651 [MþH]þ. Anal. Calcd for C32H21F4N3O2: C, 69.19; H, 3.81; N, 7.56. Found: C, 69.25; H, 3.96; N, 7.49. 4.4.9. (4R,7S)-1,4-Diphenyl-7-hydroxyl-3-(3-phenyl-1H-indol-2-yl)1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (3e). White solid, yield 26%; Rf¼0.30 (EA/PE 1:3); mp 151e153  C; 1H NMR (400 MHz, CDCl3) d 8.49 (s, 1H), 8.01 (d, J¼7.9 Hz, 2H), 7.50 (q, J¼8.1 Hz, 3H), 7.36 (td, J¼14.7, 13.4, 7.4 Hz, 4H), 7.25e7.09 (m, 4H), 7.01 (dq, J¼14.2, 7.0 Hz, 4H), 6.74 (d, J¼7.4 Hz, 2H), 4.57 (m, 2H), 4.11 (d, J¼11.8 Hz, 1H), 3.68 (d, J¼11.7 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 142.0, 139.6, 139.1, 138.2, 135.7, 134.7, 130.1, 129.5, 128.7, 128.4, 128.3, 127.8, 127.5, 127.2, 126.4, 126.2, 122.7, 122.6, 119.9, 119.4, 117.6, 116.8, 110.9, 77.2, 71.4, 61.2. HRMS (ESI) Calcd for C32H26N3O2: 484.2025, found: m/z: 484.2041 [MþH]þ. Anal. Calcd for C32H25N3O2: C, 79.48; H, 5.21; N, 8.69. Found: C, 79.55; H, 5.30; N, 8.60. 4.4.10. (4S,7S)-1,4-Diphenyl-7-hydroxyl-3-(3-phenyl-1H-indol-2yl)-1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (4e). Yellow syrup, yield 26%; Rf¼0.25 (EA/PE 1:3); 1H NMR (400 MHz, CDCl3) d 8.93 (s, 1H), 8.03 (d, J¼7.6 Hz, 2H), 7.65e7.53 (m, 4H), 7.46e7.40 (m, 2H), 7.39e7.24 (m, 6H), 7.20 (ddt, J¼9.0, 7.7, 1.3 Hz, 2H), 7.07 (ddd, J¼8.1, 7.0, 1.1 Hz, 1H), 6.96 (dd, J¼8.9, 7.0 Hz, 2H), 6.71 (d, J¼7.7 Hz, 1H), 4.69 (s, 1H), 4.55 (d, J¼10.5 Hz, 1H), 3.72 (dd, J¼12.4, 1.8 Hz, 1H), 3.47 (dd, J¼12.4, 2.1 Hz, 1H), 2.85 (d, J¼10.8 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 141.7, 139.8, 139.5, 139.0, 136.8, 135.6, 133.1, 131.6, 131.5, 130.1, 130.0, 129.6, 129.5, 129.0, 127.9, 127.8, 127.0, 126.9, 123.4, 122.8, 122.7, 122.3, 120.3, 119.2, 115.5, 115.2, 111.0, 73.2, 65.2, 60.6. HRMS (ESI) Calcd for C32H26N3O2: 484.2025, found: m/z: 484.2033 [MþH]þ. Anal. Calcd for C32H25N3O2: C, 79.48; H, 5.21; N, 8.69. Found: C, 79.79; H, 5.43; N, 8.52. 4.4.11. (4R,7S)-4-(3-chlorophenyl)-3-(3-(3-chlorophenyl)-1H-indol2-yl)-7-hydroxyl-1-phenyl-1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (3f). White solid, yield 28%; Rf¼0.30 (EA/PE 1:3); mp 134e136  C; 1 H NMR (400 MHz, CDCl3) d 8.60 (s, 1H), 8.02 (d, J¼7.9 Hz, 2H), 7.56e7.52 (m, 3H), 7.43 (t, J¼7.4 Hz, 1H), 7.37e7.32 (m, 2H), 7.29 (d, J¼8.1 Hz, 1H), 7.22e7.14 (m, 2H), 7.11e7.03 (m, 3H), 6.93 (t, J¼7.8 Hz, 1H), 6.79 (s, 1H), 6.65 (d, J¼7.7 Hz, 1H), 4.62 (m, 2H), 4.16 (d,

5773

J¼11.8 Hz, 1H), 3.74 (d, J¼11.8 Hz, 1H), 3.09 (d, J¼10.9 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 141.4, 139.9, 139.8, 138.9, 136.5, 135.6, 134.3, 133.8, 129.7, 129.7, 129.6, 1292, 129.0, 128.9, 128.1, 127.8, 126.9, 126.6, 126.5, 126.2, 123.2, 122.8, 120.3, 119.2, 116.7, 115.3, 111.0, 76.8, 71.6, 61.1. HRMS (ESI) Calcd for C32H24Cl2N3O2: 552.1246, found: m/z: 552.1262 [MþH]þ. Anal. Calcd for C32H23Cl2N3O2: C, 69.57; H, 4.20; N, 7.61. Found: C, 69.76; H, 4.39; N, 7.44. 4.4.12. (4S,7S)-4-(3-Chlorophenyl)-3-(3-(3-chlorophenyl)-1H-indol2-yl)-7-hydroxyl-1-phenyl-1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (4f). Yellow syrup, yield 29%; Rf¼0.26 (EA/PE 1:3); 1H NMR (400 MHz, CDCl3) d 8.96 (s, 1H), 8.03 (d, J¼7.3 Hz, 1H), 7.56 (t, 2H), 7.45e7.30 (m, 5H), 7.24 (t, J¼7.6 Hz, 1H), 7.18 (m, 3H), 7.10 (t, J¼7.5 Hz, 1H), 7.04 (t, J¼7.7 Hz, 1H), 6.78 (s, 1H), 6.64 (d, J¼7.7 Hz, 1H), 4.70 (s, 1H), 4.55 (d, J¼10.8 Hz, 1H), 3.71 (dd, J¼12.6, 1.7 Hz, 1H), 3.47 (dd, J¼12.6, 2.1 Hz, 1H), 2.87 (d, J¼10.8 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 141.8, 139.5, 139.0, 136.5, 135.6, 134.5, 134.0, 130.3, 129.9, 129.6, 129.2, 128.7, 128.6, 128.5, 128.0, 127.8, 127.1, 126.9, 126.6, 123.3, 122.8, 120.3, 119.2, 115.5, 115.3, 111.0, 73.2, 65.2, 60.6. HRMS (ESI) Calcd for C32H24Cl2N3O2: 552.1246, found: m/z: 552.1259 [MþH]þ. Anal. Calcd for C32H23Cl2N3O2: C, 69.57; H, 4.20; N, 7.61. Found: C, 69.70; H, 4.48; N, 7.37. 4.4.13. (4R,7S)-4-(4-Chlorophenyl)-3-(3-(4-chlorophenyl)-1H-indol2-yl)-7-hydroxyl-1-phenyl-1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (3g). White solid, yield 32%; Rf¼0.30 (EA/PE 1:3); mp 152e154  C; 1 H NMR (400 MHz, CDCl3) d 8.60 (s, 1H), 8.02 (d, J¼7.8 Hz, 2H), 7.53 (t, J¼7.7 Hz, 2H), 7.51 (d, J¼8.1 Hz, 1H), 7.45 (t, J¼7.3 Hz, 1H), 7.36 (d, J¼8.4 Hz, 2H), 7.30 (d, J¼8.1 Hz, 2H), 7.23 (t, J¼7.5 Hz, 1H), 7.10 (d, J¼7.5 Hz, 2H), 7.06 (t, J¼8.1 Hz, 1H), 6.99 (d, J¼8.4 Hz, 2H), 6.69 (d, J¼8.4 Hz, 2H), 4.63 (s, 1H), 4.61 (m, 1H), 4.15 (d, J¼11.8 Hz, 1H), 3.73 (d, J¼11.8 Hz, 1H), 3.20 (d, J¼10.1 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 141.5, 139.8, 138.9, 136.5, 1357, 134.5, 133.1, 132.3, 131.2, 129.64, 129.61, 128.1, 127.8, 127.0, 126.2, 123.2, 122.6, 120.3, 119.1, 116.7, 115.5, 111.0, 76.5, 71.4, 61.1. HRMS (ESI) Calcd for C32H24Cl2N3O2: 552.1246, found: m/z: 552.1251 [MþH]þ. Anal. Calcd for C32H23Cl2N3O2: C, 69.57; H, 4.20; N, 7.61. Found: C, 69.83; H, 4.49; N, 7.42. 4.4.14. (4S,7S)-4-(4-Chlorophenyl)-3-(3-(4-chlorophenyl)-1H-indol2-yl)-7-hydroxyl-1-phenyl-1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (4g). White solid, yield 30%; Rf¼0.25 (EA/PE 1:3); mp 152e154  C; 1 H NMR (400 MHz, CDCl3) d 8.95 (s, 1H), 8.02 (d, J¼7.7 Hz, 2H), 7.56 (t, J¼7.8 Hz, 2H), 7.46e7.35 (m, 5H), 7.22 (t, J¼7.6 Hz, 1H), 7.14 (d, J¼8.0 Hz, 2H), 7.09 (t, J¼8.4 Hz, 1H), 7.08 (d, J¼8.4 Hz, 2H), 6.71 (d, J¼8.4 Hz, 2H), 4.75 (s, 1H), 4.53 (d, J¼9.4 Hz, 1H), 3.71 (dd, J¼12.4 Hz, 1H), 3.42 (dd, J¼12.4, 1.5 Hz, 1H), 2.87 (d, J¼10.6 Hz, 1H). 13 C NMR (101 MHz, CDCl3) d 141.9, 139.5, 139.0, 136.0, 135.6, 134.2, 133.1, 132.9, 131.5, 129.8, 129.6, 128.8, 128.1, 128.0, 127.8, 126.7, 123.3, 122.8, 120.3, 119.2, 115.7, 115.4, 111.0, 73.0, 64.9, 60.6. HRMS (ESI) Calcd for C32H24Cl2N3O2: 552.1246, found: m/z: 552.1255 [MþH]þ. Anal. Calcd for C32H23Cl2N3O2: C, 69.57; H, 4.20; N, 7.61. Found: C, 69.88; H, 4.48; N, 7.39. 4.4.15. (4R,7S)-4-(3-Bromophenyl)-3-(3-(3-bromophenyl)-1H-indol2-yl)-7-hydroxyl-1-phenyl-1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (3h). White solid, yield 28%, Rf¼0.31 (EA/PE 1:3); mp 153e156  C; 1 H NMR (400 MHz, CDCl3) d 8.60 (s, 1H), 8.03 (d, J¼7.7, 1.3 Hz, 2H), 7.55 (m, 3H), 7.52e7.41 (m, 3H), 7.33e7.27 (m, 2H), 7.25e7.14 (m, 4H), 7.08 (dt, J¼7.8, 0.6 Hz, 1H), 6.94 (m, 1H), 6.90 (t, J¼7.8 Hz, 1H), 6.73 (d, J¼7.7 Hz, 1H), 4.63 (d, J¼11.8 Hz, 2H), 4.17 (dd, J¼11.9, 1.6 Hz, 1H), 3.76 (dd, J¼12.0, 1.7 Hz, 1H), 2.99 (d, J¼10.9 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 141.4, 140.0, 139.9, 138.9, 136.8, 135.6, 132.5, 131.9, 131.8, 130.0, 129.6, 129.5, 129.4, 128.5, 127.9, 126.9, 126.64,

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F.-Q. Wang et al. / Tetrahedron 72 (2016) 5769e5775

126.59, 123.2, 122.9, 122.5, 121.9, 120.4, 119.1, 116.6, 115.1, 111.1, 76.8, 71.7, 61.1. HRMS (ESI) Calcd for C32H24Br2N3O2: 640.0235, found: m/ z: 640.0240 [MþH]þ. Anal. Calcd for C32H23Br2N3O2: C, 59.93; H, 3.61; N, 6.55. Found: C, 60.25; H, 3.90; N, 6.73. 4.4.16. (4S,7S)-4-(3-Bromophenyl)-3-(3-(3-bromophenyl)-1H-indol2-yl)-7-hydroxyl-1-phenyl-1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (4h). Yellow syrup, yield 26%; Rf¼0.25 (EA/PE 1:3); 1H NMR (400 MHz, CDCl3) d 8.96 (s, 1H), 8.05 (d, J¼7.8 Hz, 2H), 7.59e7.60 (m, 3H), 7.46e7.40 (m, 2H), 7.39e7.32 (m, 2H), 7.31e7.24 (m, 3H), 7.20 (t, J¼7.7 Hz, 2H), 7.07 (t, J¼7.5 Hz, 1H), 6.99 (m, 2H), 6.68 (d, J¼7.8 Hz, 1H), 4.70 (s, 1H), 4.54 (s, 1H), 3.71 (dd, J¼12.5, 1.0 Hz, 1H), 3.46 (dd, J¼12.5, 1.4 Hz, 1H), 2.91 (s, 1H). 13C NMR (101 MHz, CDCl3) d 141.7, 139.8, 139.5, 139.0, 136.8, 135.6, 133.1, 131.6, 131.5, 130.1, 130.0, 129.6, 129.5, 129.0, 127.9, 127.8, 127.0, 126.9, 123.4, 122.8, 122.7, 122.3, 120.3, 119.2, 115.6, 115.3, 111.0, 73.2, 65.2, 60.6. HRMS (ESI) Calcd for C32H24Br2N3O2: 640.0235, found: m/z: 640.0241 [MþH]þ. Anal. Calcd for C32H23Br2N3O2: C, 59.93; H, 3.61; N, 6.55. Found: C, 60.22; H, 3.86; N, 6.69. 4.4.17. (4R,7S)-4-(4-bromophenyl)-3-(3-(4-bromophenyl)-1H-indol2-yl)-7-hydroxyl-1-phenyl-1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (3i). White solid, yield 30%; Rf¼0.30 (EA/PE 1:3); mp 143e146  C; 1 H NMR (400 MHz, CDCl3) d 8.55 (s, 1H), 8.01 (d, J¼7.6 Hz, 2H), 7.55 (m, 6H), 7.47 (t, J¼7.5 Hz, 1H), 7.33 (d, J¼8.1 Hz, 1H), 7.25e7.18 (m, 3H), 7.12e7.07 (m, 3H), 6.70 (d, J¼8.0 Hz, 2H), 4.69 (s, 1H), 4.65 (d, J¼10.8 Hz, 1H), 4.19 (dd, J¼11.9, 1.1 Hz, 1H), 3.78 (dd, J¼11.9, 1.0 Hz, 1H), 3.06 (d, J¼11.0 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 141.5, 139.8, 138.9, 137.1, 135.6, 133.6, 131.5, 131.1, 129.9, 129.6, 127.8, 127.0, 126.2, 123.2, 122.9, 122.7, 120.4, 120.3, 119.2, 116.7, 115.5, 111.0, 76.6, 71.4, 61.1. HRMS (ESI) Calcd for C32H24Br2N3O2: 640.0235, found: m/ z: 640.0239 [MþH]þ. Anal. Calcd for C32H23Br2N3O2: C, 59.93; H, 3.61; N, 6.55. Found: C, 60.06; H, 3.78; N, 6.63. 4.4.18. (4S,7S)-4-(4-Bromophenyl)-3-(3-(4-bromophenyl)-1H-indol2-yl)-7-hydroxyl-1-phenyl-1,4,6,7-tetrahydropyrano[4,3-c]pyrazole (4i). Yellow syrup, yield 29%; Rf¼0.25 (EA/PE 1:3); 1H NMR (400 MHz, CDCl3) d 8.94 (s, 1H), 8.02 (d, J¼7.4 Hz, 2H), 7.58e7.52 (m, 4H), 7.45e7.36 (m, 4H), 7.22e7.19 (m, 3H), 7.09e7.06 (m, 3H), 6.68 (d, J¼8.2 Hz, 2H), 4.76 (s, 1H), 4.53 (s, 1H), 3.71 (dd, J¼12.4, 1.4 Hz, 1H), 3.41 (dd, J¼12.4, 1.8 Hz, 1H), 2.88 (s, 1H). 13C NMR (101 MHz, CDCl3) d 141.9, 139.5, 139.0, 136.5, 135.6, 133.6, 131.9, 131.8, 131.1, 130.2, 129.6, 128.0, 127.8, 126.6, 123.4, 122.8, 122.5, 121.0, 120.3, 119.3, 115.6, 115.4, 111.0, 73.1, 64.9, 60.6. HRMS (ESI) Calcd for C32H24Br2N3O2: 640.0235, found: m/z: 640.0243 [MþH]þ. Anal. Calcd for C32H23Br2N3O2: C, 59.93; H, 3.61; N, 6.55. Found: C, 60.11; H, 3.89; N, 6.58. 4.4.19. (4S,7S)-4-(4-Bromo-2-fluorophenyl)-3-(3-(4-bromo-2fluorophenyl)-1H-indol-2-yl)-7-hydroxyl-1-phenyl-1,4,6,7tetrahydropyrano[4,3-c]pyrazole (3j). White solid, yield 35%; Rf¼0.30 (EA/PE 1:3); mp 141e143  C; 1H NMR (400 MHz, CDCl3) d 9.00 (s, 1H), 8.04 (d, J¼7.6 Hz, 2H), 7.55e7.45 (m, 3H), 7.37 (d, J¼8.9 Hz, 3H), 7.31 (dd, J¼8.2, 1.8 Hz, 1H), 7.25 (dt, J¼8.1, 0.7 Hz, 1H), 7.14e7.09 (m, 2H), 6.97 (t, J¼7.9 Hz, 1H), 6.81 (dd, J¼8.2, 1.5 Hz, 1H), 6.41 (t, J¼8.0 Hz, 1H), 5.05 (s, 1H), 4.59 (d, J¼11.2 Hz, 1H), 4.20 (d, J¼11.2 Hz, 1H), 3.80 (dd, J¼11.2 Hz, 1H), 3.65 (d, J¼11.3 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 161.8 (d, J¼146 Hz), 159.3 (d, J¼146 Hz), 141.2, 140.1, 138.8, 135.6, 133.2 (d, J¼4 Hz), 129.8 (d, J¼4 Hz), 129.7, 127.8, 127.4 (d, J¼3.4 Hz), 127.3, 127.2, 126.8 (d, J¼3.4 Hz), 124.1, 123.28 (d, J¼10 Hz), 123.27, 122.6, 121.2 (d, J¼11.6 Hz), 121.0 (d, J¼5 Hz), 120.3, 119.8 (dd, J¼13.9, 11.6 Hz), 119.1 (d, J¼25 Hz), 115.1, 111.0, 109.3, 71.3, 70.6 (d, J¼3 Hz), 60.8. HRMS (ESI) Calcd for C32H22Br2F2N3O2: 676.0047, found: m/z: 676.0049 [MþH]þ. Anal.

Calcd for C32H21Br2F2N3O2: C, 56.74; H, 3.13; N, 6.20. Found: C, 56.81; H, 3.26; N, 6.34. 4.4.20. (4R,7S)-4-(4-Bromo-2-fluorophenyl)-3-(3-(4-bromo-2fluorophenyl)-1H-indol-2-yl)-7-hydroxyl-1-phenyl-1,4,6,7tetrahydropyrano[4,3-c]pyrazole (4j). Yellow syrup, yield 32%; Rf¼0.26 (EA/PE 1:3); 1H NMR (400 MHz, CDCl3) d 9.20 (s, 1H), 8.05 (d, J¼8.0 Hz, 2H), 7.59 (t, J¼7.8 Hz, 2H), 7.46 (t, J¼7.4 Hz, 1H), 7.43e7.34 (m, 3H), 7.31 (d, J¼8.0 Hz, 1H), 7.27e7.19 (m, 2H), 7.09 (m, 2H), 6.90 (dd, J¼8.1, 1.8 Hz, 1H), 6.42 (t, J¼8.0 Hz, 1H), 5.35 (s, 1H), 4.58 (d, J¼8.9 Hz, 1H), 3.83 (dd, J¼12.6, 1.5 Hz, 1H), 3.46 (dd, J¼12.3, 1.9 Hz, 1H), 3.14 (d, J¼10.6 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 161.8 (d, J¼128 Hz), 159.4(d, J¼124 Hz), 141.5, 139.9, 138.8, 135.6, 133.5 (d, J¼4 Hz), 130.1 (d, J¼4 Hz), 129.6, 127.9, 127.8 (d, J¼34 Hz), 127.6 (d, J¼16 Hz), 126.5 (d, J¼3.7 Hz), 123.6 (d, J¼13.6 Hz), 123.4, 123.3 (d, J¼9 Hz), 122.8, 121.7 (d, J¼9 Hz), 121.2 (d, J¼16 Hz), 120.4, 119.8 (d, J¼25 Hz), 119.7, 119.5 (d, J¼25 Hz), 114.0, 111.0, 67.6 (d, J¼3 Hz), 65.2, 60.5. HRMS (ESI) Calcd for C32H22Br2F2N3O2: 676.0047, found: m/z: 676.0057 [MþH]þ. Anal. Calcd for C32H21Br2F2N3O2: C, 56.74; H, 3.13; N, 6.20. Found: C, 56.90; H, 3.21; N, 6.29. 4.5. MTT assays for anticancer activity The cells in the exponential growth phase were seeded into 96well microculture plates at a concentration of 5e6103 cells per well. The fresh medium containing the candidate compounds with different concentration were replaced for previous medium after 24 h incubation at 37  C. After incubated for 48 h, 20 mL of MTT solution (5 mg/mL) was applied to all wells and incubated for another 4 h. Then, all the plates were taken out from the 37  C incubation chamber, afterward, discarded the supernatant and added 150 mL of dimethyl sulfoxide (DMSO) to each well. Shook the plates for 10 min so that the violet blue insoluble substance (formazan) could be dissolved. Subsequently, the absorbance of the solution in each well was measured, using a microplate reader at a wavelength of 460 nm. The experiment was repeated twice and each concentration was analyzed in sextuplicates. The IC50 values were obtained by analysis of the obtained data using the IBM SPSS software. Acknowledgements This work was supported by National Natural Science Foundation of China (21372207), and Foundation of Educational Committee of Henan Province (13A350629), China. Supplementary data Supplementary data (Crystallographic data (excluding structure factors) for the structures in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication nos: CCDC 1481985. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: þ44(0) 1223 336033 or e-mail: [email protected]]) associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.tet.2016.07.078. References and notes 1. Raffa, D.; Migliara, O.; Maggio, B.; Plescia, F.; Cascioferro, S.; Cusimano, M. G.; Tringale, G.; Cannizzaro, C.; Plescia, F. Arch. Pharm. Chem. Life Sci. 2010, 10, 631e638. 2. Sridhar, R.; Perumal, P. T.; Etti, S.; Shanmugam, G.; Ponnuswamy, M. N.; Prabavathy, V. R.; Mathivanan, N. Bioorg. Med. Chem. Lett. 2004, 4, 6035e6040. 3. Gurkok, G.; Altanlar, N.; Suzen, S. Chemotherapy 2009, 55, 15e19.

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