Synthesis, Crystal Structure, and Antitumor Activity of 1-(4-Methoxybenzylidene)-2-(1-phenyl-6-trifluoromethyl- 1H-pyrazolo[3,4-d]pyrimidin-4-yl)hydrazine Monohydrate①

YANG Ping GAO Hui SONG Xin-Jin

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Synthesis, Crystal Structure, and Antitumor Activity of 1-(4-Methoxybenzylidene)-2-(1-phenyl-6-trifluoromethyl- 1-pyrazolo[3,4-]pyrimidin-4-yl)hydrazine Monohydrate①

YANG Pinga, bGAO HuiaSONG Xin-Jiana, b②

a(445000)b(445000)

The novel title compound 1-(4-methoxybenzylidene)-2-(1-phenyl-6-trifluoromethyl- 1-pyrazolo[3,4-]pyrimidin-4-yl)hydrazine monohydrate (C20H15F3N6O×H2O,M= 430.40) has been synthesized by a four-step procedure including the cyclization, chlorination, hydrazinolysis and condensation reaction, and its crystal structure was determined by single-crystal X-ray diffraction. The crystal belongs to orthorhombic, space groupwith= 8.3779(13),= 17.607(3),= 26.774(4) ?,= 3949.2(11) ?3,= 8,D= 1.448 g/cm3,= 0.117 mm–1,(000) = 1776, the final= 0.0553 and= 0.1516 for 2354 observed reflections with> 2(). X-ray diffraction analysis reveals that the title compound is almost coplanar except for the tri?uoromethyl and phenyl moieties. In the crystal packing, the molecules are linked by intermolecular O(1W)–H(1WA)···N(2), O(1W)–H(1WA)···N(4) and N(5)–H(5A)···O(1W) hydrogen bondswater molecules and stacked through-stacking interactions. The preliminary bioassay suggested that the title compound exhibits relatively good antitumor activity against HepG2 and BCG-823.

pyrazolo[3,4-]pyrimidine, hydrazone, trifluoromethyl, antitumor activity

1 INTRODUCTION

Both pyrimidine and pyrazole are important he- terocycles exhibiting remarkable biological activities. From biological point of view, fused hetero-aromatic ring systems are often of much greater interest than their constituting monocyclic fragments. Pyrazolo- [3,4-]pyrimidines occupy a special position among them as they serve as structural analogues of biogenic purines and hence exhibit signi?cant biolo- gical activities, such as antimicrobial[1-3], antitu- mor[4-7], antiviral[8], anti-inflammatory[9, 10], and so on. As is well known, hydrazones are a kind of spe- cial Schiff bases bearing C=N–NH- functional groups. Many hydrazone derivatives are reported to possess a multitude of biological effects[11-14], such as fungicidal, insecticidal, anticancer and anti-inflam- matory activities. Moreover, ?uorinated com- pounds have become a hot topic in the agrochemical and medicinal fields. In general, incorporation of a fluoro or trifluoromethyl group provides compounds with increased biological activity due to the enhanced pharmacokinetic and physicochemical properties as compared to their non-fluorinated ana-logues[15-17].

In the light of mentioned observations, and based upon the principle of superposition, novel pyrazolo- [3,4-]pyrimidine compounds incorporating both hydrazone and trifluoromethyl groups have aroused our great interest. In this contribution, we report the synthesis of 1-(4-methoxybenzylidene)-2-(1-phenyl- 6-trifluoromethyl-1-pyrazolo[3,4-]pyrimidin-4-yl)-hydrazine, focusing on its crystal structure and anti- tumor activity.

2 EXPERIMENTAL

2.1 Materials and instruments

All chemicals used for the preparation were of analytical grade. Reaction solvents were dried by standard methods and distilled prior to use. IR spectra were recorded on a Nicolet NEXUS 470 FT-IR spectrophotometer in the range of 4000～400 cm-1, using KBr pellets. NMR spectra were obtained on a Varian Mercury Plus-400 MHz spectrometer with TMS as internal standard and DMSO-6as the solvent. MS spectra were performed by a Thermo DSQ II mass spectrometer using the electron ioni- zation (EI) method. Elemental analysis was carried out on a Vario EL III CHNSO analyzer. X-ray dif- fraction data were collected on a Bruker Smart AP- EX-II CCD diffractometer equipped with a graphite- monochromatized Mo(= 0.71073 ?) radiation. Melting points were measured with an X-4 digital melting-point apparatus and uncorrected.

2.2 Synthesis of the title compound

The general procedure for synthesis of the title compound 5 is shown in Scheme 1. The starting material,5-amino-1-phenyl-1-pyrazole-4-carbonitri-le (1), was synthesized as described in the litera- ture[18].

Scheme 1. Procedure for synthesis of the title compound 5

Preparation of 4-chloro-1-phenyl-6-trifluorome- thyl-1-pyrazolo[3,4-]pyrimidine(3): A mixture of compound 1 (1.84 g, 10 mmol), trifluoroacetic acid (TFA, 30 mL) and phosphoryl trichloride (2 mL) was refluxed for 2.5 h in a round-bottomed flask. The remaining TFA was removed by vacuum dis- tillation. Then ice water was added and neutralized with potassium carbonate till no bubble occurs. The resulting white precipitate was collected by filtration, washed with cold water, and dried to deliver1- phenyl-6-trifluoromethyl-1-pyrazolo[3,4-]pyrimidin-4(5)-one (2), which was then added directly to a round-bottomed flask without further purification and refluxed with phosphoryl trichloride (15 mL) for 5 h. The excess of phosphoryl trichloride was removed under reduced pressure. The residue was poured over crushed ice, followed by neutralization with sodium bicarbonate solution and then filtrated. The collected solid precipitate was recrystallized from n-hexane to obtain 4-chloro-1- phenyl-6-(tri- fluoromethyl)-1-pyrazolo[3,4-]pyrimidine(3) in 68% yield as white solid. m.p.: 187～188 ℃.EI-MS:/298 (M+).

Preparation of 1-(1-phenyl-6-trifluoromethyl-1-pyrazolo[3,4-]pyrimidin-4-yl)hydrazine (4): To a solution of 3 (1.49 g, 5 mmol) in acetonitrile (10 mL) was added hydrazine monohydrate (3 mL). The mixture was stirred under reflux for 2 h and cooled to room temperature. The precipitate was collected by filtration, and washed with-hexane to afford 1-(1-phenyl-6-trifluoromethyl-1-pyrazolo[3,4-]py-rimidin-4-yl)hydrazine (4) in 90% yield as whitesolid. m.p. 233～234 ℃. EI-MS:/294 (M+).

Preparation of the title compound 5: A mixture of compound 4 (0.59 g, 2 mmol) and 4-methoxy- benzaldehyde (0.29 g, 2.1 mmol) in EtOH (10 mL) was stirred under reflux for 1 h and cooled to room temperature. The reaction mixture was concentrated under reduced pressure. The residue was recrys- tallized from ethanol to give 1-(4-methoxybenzyli- dene)-2-(1-phenyl-6-trifluoromethyl-1-pyrazolo[3,4-]pyrimidin-4-yl)hydrazine (5)in 83% yield as white solid. m.p. 238～239 ℃;1H NMR (DMSO-6, 400 MHz):12.78 (s, 1H, NH), 8.73 (s, 1H, pyrazolylN=CH), 8.28 (s, 1H, N=CH-Ar), 8.16～7.06 (m, 9H, Ar-H), 3.83 (s, 3H, OCH3);13C NMR (DMSO-6, 100 MHz):160.97, 156.20, 153.07, 152.55, 147.55, 138.13, 136.74, 129.03, 128.72, 126.63, 126.09, 121.00, 118.27, 114.37, 100.91, 55.13;19F NMR (DMSO-6, 376 MHz):?69.14; FT-IR (KBr, cm-1): 3435 (N–H), 1601, 1506 (C=N, C=C), 1300, 1137 (CF3); EI-MS:/412 (M+). Anal. Calcd. forC20H15F3N6O:C,58.25; H, 3.67; N, 20.38%. Found: C, 58.09; H, 3.85; N, 20.26%.

2.3 Crystal data and structure determination

Block-shaped colorless crystals of compound 5 for X-ray analysis were obtained by vapour diffusion from ethanol at room temperature. A single crystal with dimensions of 0.22mm′0.20mm′0.18mm was mounted on a Bruker Smart APEX-II CCD dif- fractometer equipped with a graphite-monochro- mated Mo(= 0.71073 ?) radiation. The inten- sity data were collected by using a-scan mode in the range of 2.31≤≤26.00° at 298(2) K. A total of 20341 reflections were collected and 3872 were independent withint= 0.0500, of which 2354 were observed with> 2() and used in the succeeding refinements. Absorption correction was not applied. The structure was solved by direct methods with SHELXS-97[19]and expanded using Fourier dif- ference techniques. The non-hydrogen atoms were refined anisotropically, and the hydrogen atoms were added according to theoretical models. Struc- tural refinement was carried out by full-matrix least-squares techniques on2with SHELXL-97[19]. The final refinement gave= 0.0553 and= 0.1516 (= 1/[2(F2) + (0.1000)2+ 0.0358], where= (F2+ 2F2)/3).= 1.078, (Δ/)max= 0.000, (Δ)max= 0.441 and (Δ)min= ?0.319 e/?3.

3 RESULTS AND DISCUSSION

The structure of the title compound 5 was con- firmed by IR,1H NMR,13C NMR,19F NMR, EI-MS and elemental analysis. These spectroscopic data are in accordance with the assumed structure. Its structure was further determined by X-ray single- crystal diffraction method. Its important bond leng- ths and bond angles can be seen in Table 1, and hydrogen bond details and-stacking interactions are listed in Tables 2 and 3, respectively. Its molecu- lar structure is given in Fig. 1, and its packing dia- gram showing hydrogen bond details and-stacking interactions is revealed in Fig. 2.

Fig. 1. Molecular structure of compound 5 showing intramolecular hydrogen bonds

Table 1. Selected Bond Lengths (?) and Bond Angles (°) for Compound 5

BondDist.BondDist.BondDist. N(1)–N(2)1.372(3)C(1)–C(2)1.380(4)C(16)–C(15)1.387(4) C(8)–C(7)1.390(3)C(5)–C(4)1.385(4)C(14)–C(13)1.451(3) C(12)–C(8)1.412(3)C(7)–N(1)1.363(3)C(6)–C(5)1.385(3) C(10)–N(4)1.333(3)C(11)–F(3)1.289(3)C(17)–O(1)1.364(3) C(12)–N(5)1.336(3)C(11)–F(2)1.317(4)C(17)–C(18)1.395(4) C(14)–C(15)1.387(3)O(1)–C(20)1.431(3)C(10)–C(11)1.506(4) C(14)–C(19)1.389(3)C(9)–N(2)1.313(3)C(2)–C(3)1.383(4) C(6)–C(1)1.382(4)C(8)–C(9)1.406(3)C(7)–N(3)1.350(3) C(6)–N(1)1.422(3)C(10)–N(3)1.328(3)C(4)–C(3)1.367(4) C(17)–C(16)1.377(4)C(13)–N(6)1.279(3)C(11)–F(1)1.301(3) C(18)–C(19)1.368(4)C(12)–N(4)1.351(3)N(6)–N(5)1.374(3) Angle(°)Angle(°)Angle(°) N(5)–C(12)–N(4)116.0(2)O(1)–C(17)–C(16)124.2(2)C(7)–C(8)–C(9)104.9(2) N(5)–C(12)–C(8)124.8(2)O(1)–C(17)–C(18)116.2(2)C(10)–N(3)–C(7)110.3(2) N(4)–C(12)–C(8)119.2(2)N(3)–C(10)–N(4)130.6(2)C(7)–C(8)–C(12)115.6(2) N(3)–C(7)–N(1)125.9(2)N(3)–C(10)–C(11)115.5(2)C(13)–N(6)–N(5)115.4(2) N(3)–C(7)–C(8)127.0(2)N(4)–C(10)–C(11)114.0(2)C(9)–C(8)–C(12)139.4(2) N(1)–C(7)–C(8)107.0(2)N(6)–C(13)–C(14)122.4(2)C(9)–N(2)–N(1)106.4(2) N(2)–C(9)–C(8)111.4(2)C(7)–N(1)–N(2)110.30(19)C(10)–N(4)–C(12)117.2(2) C(1)–C(6)–N(1)120.5(2)C(7)–N(1)–C(6)129.4(2)C(12)–N(5)–N(6)119.6(2) C(5)–C(6)–N(1)119.3(2)N(2)–N(1)–C(6)119.9(2)C(17)–O(1)–C(20)117.6(2)

D–H···Ad(D–H)d(H···A)d(D···A)∠D–H···ASymmetry code C(1)–H(1)···N(3)0.932.553.054(3)114 C(9)–H(9)···N(6)0.932.593.000(3)107 O(1W)–H(1WA)···N(2)#10.852.182.984(3)158#1: –x+1/2, y+1/2, z O(1W)–H(1WA)···N(4)#20.852.383.150(3)151#2: x+1/2, –y+1/2, –z+1 N(5)–H(5A)···O(1W)#30.862.172.879(4)139#3: x, y–1, z

Cg(I)···Cg(J)Symmetry codeDist. centroidsDihedral angle CgI_Perp dist. CgJ_Perp dist. Cg(1)→Cg(3)–x+1, –y, –z+13.5340(16)2.42(13)3.3479(10)3.3803(10) Cg(2)→Cg(3)–x+1, –y, –z+13.5275(16)1.25(12)3.3566(10)3.3686(10)

Ring Cg(1): N(1)→N(2)→C(9)→C(8)→C(7)→

Ring Cg(2): N(3)→C(7)→C(8)→C(12)→N(4)→C(10)→

Ring Cg(3): C(14)→C(15)→C(16)→C(17)→C(18)→C(19)→

According to the structural analysis, the crystal structure of compound 5 consists of one 1-(4-me- thoxybenzylidene)-2-(1-phenyl-6-trifluoromethyl-1-pyrazolo[3,4-]pyrimidin-4-yl)hydrazine (5)mole- cule and one isolated water, among which hydrogen bonds can be observed for structure stabilization. In detail, in compound 5, the pyrazolo[3,4-]pyri- midine ring (ring 1)exhibits good coplanar nature with the deviation of about 0.0021 ?. In this plane, the C–C, C–N and N–N bond lengths are normal in the range of 1.313(3)～1.406(3) ?, suggesting its2hybrid nature and delocalization on the con- jugated system. But the phenyl ring (ring 2) is not coplanar with ring 1, and a clear dihedral angle of 33.50(11) ? can be found. In addition, the C(13)– N(6) bond distance of 1.279(3) ? indicates its double bond nature, and the C(14)–C(13)–N(6) bond angle of 122.36° also illustrates its2hybrid nature (close to 120°). Obviously, the hydrazone moiety assumes anconfiguration with the 4-methoxy- phenyl ring(ring 3) and ring 1 located on opposite sides of the C(13)=N(6) bond. Interestingly, ring 3 generally is coplanar with ring 1 with a dihedral angle of only 1.21°. Specially, intramolecular hydro- gen bonds (C(1)–H(1)···N(3) and C(9)–H(9)···N(6)) and intermolecular hydrogen bonds (O(1W)– H(1WA)···N(2), O(1W)–H(1WA)···N(4) and N(5)– H(5A)···O(1W), Table 2) between the title hydra- zone molecule 5 and water molecule can be found for structural stabilization. Also present are inter- molecular-stacking interactions (Table 3). In sum, the molecules form by staking a three-dimen- sional framework which results from intermolecular hydrogen bondswater molecules together with-stacking interactions (Fig. 2).

4 ANTITUMOR ACTIVITY

Theantitumor activity of the title com- pound against HepG2 (human hepatocellular liver carcinoma cell line) and BGC-823 (human gastric cancer cell line) was evaluated by the standard MTT assay[20]. 5-Fluorouracil (5-FU) was used as a posi- tive control. As described in Table 4, the result of preliminary bioassay shows that compound 5 ex- hibits higher antitumor activity against HepG2 and BGC-823 than 5-FU.

Table 4. In Vitro Antitumor Activity for Compound 5

Fig. 2. Packing diagram of compound 5 showing hydrogen bonds

(1) Khobragade, C. N.; Bodade, R. G.; Konda, S. G.; Dawane, B. S.; Manwar, A. V. Synthesis and antimicrobial activity of novel >pyrazolo[3,4-]pyrimidine derivatives.2010, 45, 1635–1638.

(2) Shamroukh, A. H.; Rashad, A. E.; Ali, H. S.; Abdel-Megeid, F. M. E. Some new pyrazole and pyrazolopyrimidines: synthesis and antimicrobial evaluation.2013, 50, 758–765.

(3) Chauhan, M.; Kumar, R. Medicinal attributes of pyrazolo[3,4-]pyrimidines: a review.2013, 21, 5657–5668.

(4) Cozzi, M.; Giorgi, F.; Marcelli, E.; Pentimalli, F.; Forte, I. M.; Schenone, S.; D'Urso, V.; De Falco, G.; Botta, M.; Giordano, A.; Indovina, P. Antitumor activity of new pyrazolo[3,4-]pyrimidine SRC kinase inhibitors in Burkitt lymphoma cell lines and its enhancement by WEE1 inhibition.2012, 11, 1029–1039.

(5) Abd El Hamid, M. K.; Mihovilovic, M. D.; El-Nassan, H. B. Synthesis of novel pyrazolo[3,4-]pyrimidine derivatives as potential anti-breast cancer agents.2012, 57, 323–328.

(6) Song, X. J.; Shao, Y.; Dong, X. G. Microwave-assisted synthesis of some novel fluorinated pyrazolo[3,4-]pyrimidine derivatives containing 1,3,4-thiadiazole as potential antitumor agents.2011, 22, 1036–1038.

(7) Yuan, L.; Song, C. W.; Li, C. H.; Li, Y.; Dong, L.; Yin, S. F. Synthesis and biological evaluation of pyrazolo[4,3-]pyrimidine analogues.2013, 67, 152–157.

(8) Rashad, A. E.; Hegab, M. I.; Abdel-Megeid, R. E.; Micky, J. A.; Abdel-Megeid, F. M. E. Synthesis and antiviral evaluation of some new pyrazole and fused pyrazolopyrimidine derivatives.2008, 16, 7102–7106.

(9) Yewale, S. B.; Ganorkar, S. B.; Baheti, K. G.; Shelke, R. U. Novel 3-substituted-1-aryl-5-phenyl-6-anilinopyrazolo[3,4-]pyrimidin-4-ones: docking, synthesis and pharmacological evaluation as a potential anti-inflammatory agents.2012, 22, 6616–6620.

(10) Karoui, A.; Allouche, F.; Deghrigue, M.; Agrebi, A.; Bouraoui, A.; Chabchoub, F. Synthesis and pharmacological evaluation of pyrazolopyrimido- pyrimidine derivatives: anti-inflammatory agents with gastroprotective effect in rats.2014, 23, 1591–1598.

(11) Altintop, M. D.; Ozdemir, A.; Turan-Zitouni, G.; Ilgin, S.; Atli, O.; Iscan, G.; Kaplancikli, Z. A. Synthesis and biological evaluation of some hydrazone derivatives as new anticandidal and anticancer agents.2012, 58, 299–307.

(12) Song, X. J.; Yang, P.; Wang, D. F.; Tan, Z. D. Synthesis and biological activity of new fluorinated thieno[2,3-]pyrimidinyl hydrazone derivatives.2014, 11, 380–385.

(13) Pan, X. Q.; Huang, S. S.; Wang, F.; Fan, H.; Leng, H.; Zhang, H. L.; Diao, Y. P. Synthesis, crystal structure and antibacterial activity of two hydrazones derived from 2-fluorobenzhydrazide.2013, 32, 142–148.

(14) Yang, P.; Wang, Y,; Duan, Z. C.; Shao, Y.; Nie, G. H.; Song, X. J.; Tian, D. T. Synthesis, crystal structure, and biological activity of 4-chloro- benzaldehyde (2-trifluoromethyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-]pyrimidin-4-yl)hydrazone monohydrate.2013, 32, 1023–1030.

(15) Hagmann, W. K. The many roles for fluorine in medicinal chemistry.2008, 51, 4359–4369.

(16) Song, X. J.; Duan, Z. C.; Shao, Y.; Dong, X. G. Facile synthesis of novel fluorinated thieno[2,3-]pyrimidine derivatives containing 1,3,4-thiadiazole.2012, 23, 549–552.

(17) Song, X. J.; Yang, P.; Gao, H.; Wang, Y.; Dong, X. G.; Tan, X. H. Facile synthesis and antitumor activity of novel 2-trifluoromethylthieno [2,3-]pyrimidine derivatives.2014, 25, 1006–1010.

(18) Das, J.; Moquin, R. V.; Pitt, S.; Zhang, R.; Shen, D. R.; McIntyre, K. W.; Gillooly, K.; Doweyko, A. M.; Sack, J. S.; Zhang, H.; Kiefer, S. E.; Kish, K.; McKinnon, M.; Barrish, J. C.; Dodd, J. H.; Schieven, G. L.; Leftheris, K. Pyrazolo-pyrimidines: a novel heterocyclic scaffold for potent and selective p38α inhibitors.2008, 18, 2652–2657.

(19) Sheldrick, G. M. A short history of SHELX.2008, A64, 112–122.

(20) Denizot, F.; Long, R. Rapid colorimetric assay for cell growth and survival.1986, 89, 271–277.

20 March 2014;

15 May 2014 (CCDC 956233)

① Supported by the National Natural Science Foundation of China (No. 21262012), the Open Fund of Key Laboratory of Biologic Resources Protection and Utilization of Hubei Province (No. PKLHB1314), the Project for Cultivating Excellent Postgraduate's Dissertation of Hubei Minzu University (PY201402), and the First-class Discipline of Forestry in Hubei Minzu University

. Song Xin-Jian, born in 1975, master, professor. E-mail: whxjsong@163.com