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Synthesis and biological evaluation of anthra[1,9-cd]pyrazol-6(2H)-one scaffold derivatives as potential anticancer agents
⁎Corresponding authors at: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan. Tel.: +886 2 87923100x19390; fax: +886 2 8792 7282 (D.-S. Yu). Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan. Tel.: +886 2 2736 1661x7525; fax: +886 2 87923169 (H.-S. Huang). yuds@ms21.hinet.net (Dah-Shyong Yu), huanghs99@tmu.edu.tw (Hsu-Shan Huang)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Abstract
Several anthrapyrazolone derivatives derived from 7-chloroanthra[1,9-cd]pyrazol-6(2H)-one and 7-chloro-2-(2-hydroxyethyl)anthra[1,9-cd] pyrazol-6(2H)-one have been prepared by the addition or substitution nucleophilic reactions and further transformed into extended tetracyclic systems and fused to different nitrogenheterocyclic rings into the pharmacophore structure moiety. The compounds synthesized were evaluated for their cytotoxic activity and telomerase activity in prostate cancer cell line by SRB assay and in human non-small cell lung carcinoma cell line by TRAP assay, respectively. Compounds 1–6, 13, 14, 16, 17, 19, 21, 23, 28 and 31 were selected by the NCI and only 1, 4, and 16 represent the GI50, TGI and LC50, respectively. Among them, 1 and 16 exhibited distinctive selectivity of GI50 of 10.498 μM and 4.542 μM over 60 cell lines which is better than the average GI50 (20.3 μM) for SP600125 (NSC75890). Overall, the test compounds exhibited different telomerase and cytotoxic activities and only few compounds displayed antitumor activity in the low range.
Keywords
Anthrapyrazolone
SRB assay
TRAP assay
Cytotoxicity
NCI 60-cell panel assay
Antiproliferation
1 Introduction
Many clinically successful anticancer drugs are either natural products or have been developed from naturally occurring lead compounds, such as taxol, camptothecin (topotecan and irinotecan), vinblastine, and anthracyclines (doxorubicin and mitoxantrone). The mechanism of anthracyclines is inhibiting topoisomerase II through binding a part of the catalytic cycle of the enzyme to stabilization of the cleavable complex (Suzuki et al., 1995), and intercalating into the minor groove of double-strand DNA base pairs via the core planar pharmacophore group (Wang et al., 1994). It is an amusive class of pyrazole and pyrazole-fused heterocyclic derivatives because of their synthetic gifted and efficacious biological activities. Besides, anthrapyrazole, a kind of anthrathenes having a pyrazole ring, is a series of structures which has found anticancer activities. They classified as alkaloids are rare in nature, but they still are the important components and have pharmacological effects on humans. There are several studies found that pyrazole derivatives had potent inhibitory activity against inflammatory (Tewari et al., 2010), bacterium (Genin et al., 2000a; Kucukguzel et al., 2000), virus (Genin et al., 2000b), adenosine receptors (Baraldi et al., 2002, 2003), and leukemia cell lines (Kostakis et al., 2002; Mansour et al., 2003).
Anthrapyrazole represents another successful bioisosterism lead structure as an alternative approach for antitumor application (Krapcho et al., 1995), which structurally related to mitoxantrone (MX) and its mechanism of action is mainly poisoning of the DNA Topoisomerase II enzyme (Faulds et al., 1991). To overcome the drawback of cardiotoxic effects, a pyrazole ring is fused to the anthraquinone moiety, giving a tetracyclic system in an attempt to reduce toxicity or modify the activity of the lead compound, and may alter the metabolism of the lead (Krapcho et al., 1985, 1994; Gandolfi et al., 1995). Losoxantrone (DUP-941), is an anthrapyrazole derivative and found that sharing similar pharmacokinetics (Graham et al., 1992), behaving almost similarly in the in vitro National Cancer Institute (NCI) cell screen and exhibiting the same anticancer spectrum of activity as MX with reduced cardiotoxicity (Leteurtre et al., 1994). There were at least two phase II studies of losoxantrone reported both included untreated and treated patients with metastatic breast cancer (Talbot et al., 1991) and prostate cancer (Huan et al., 2000). Simultaneously, one of DUP 941 analogues, DUP-937 (teloxantrone), is an intercalator and inhibits DNA synthesis which was conducted for phase I and phase II clinical trials (Renner et al., 1995). SP600125 (NSC75890), prevented cell injury induced by N-Acetyl-p-Aminophenol (APAP) exposure, is also a famous c-Jun N-terminal kinase (JNK) inhibitor having middle cytotoxicity (Fig. 1). To understand the molecular basis for a dramatically different response by otherwise very similar compounds, we performed a detailed investigation on the biological activities of several anthrapyrazoles belonging to the two families, on their cytotoxicity and specificity as well as on their telomerase activity.
Structural hybridization of some preclinical and clinical anticancer drugs.
As stated above, pyrazole systems are involved in a wide range of biological activities, and for that reason, in addition to the antineoplastic evaluation, several representative compounds have been prospectively evaluated for their antitumor potential. The search for additional enzyme inhibitor is confirmed by the extraordinary anticancer activity of pyrazole-fused and anthraquinones and by the feasibility that different enzyme poisons would exhibit different activities in cancer chemotherapy (Huang et al., 2009). According to our previous studies, a series of disubstituted anthraquinones was showed the inherent capacity against telomerase inhibition activity (Huang et al., 2005b, 2005a). In recent years, we hybridized the anthraquinone and some potent structures and found that imidazole-fused anthraquinones have potent inhibitory activities against telomerase (Huang et al., 2008, 2009; T.C. Chen et al., 2013; C.L. Chen et al., 2013) and poly (ADP-ribose) polymerase (PARP) (Lee et al., 2013). Thus, we try to design and synthesize a series of new derivatives which is substituted at position 7. To find such new heterocycle-fused small molecular or pharmacophore targets for anticancer drugs the results of their biological evaluation are reported and discussed here.
2 Chemistry
2.1 Material and instruments
The methods and physical data of synthesizing compounds 1–32 were described and all the synthetic compounds were compared to their growth inhibition, cytotoxicity and telomerase activity. In this investigation, we were dedicated on the role of our systematic synthesized tetracyclic and heterocyclic pharmacophore and introduced a series of side chains linked to the anthra[1,9-cd]pyrazol-6(2H)-one moiety (Scheme 1).
Synthesis of anthrapyrazolones. Reagents and conditions: (i) Hydrazine monohydrate, DMSO, DIPEA, reflux, 120 °C, 2 h. (ii) 2-Hydroxyethylhydrazine, pyridine, reflux, 120 °C, 2 h. (iii) Primary amines, DMSO, DMAP, mini-reactor, 120–140 °C, 4 h.
Each reaction was monitored by TLC (silica gel 60 F254) under standardized UV (254 nm). 1H NMR and 13C NMR were measured on Varian GEMINI-300 (300 MHz); δ values are in ppm relative to tetramethylsilane (TMS) as an internal standard. Multiplicities are recorded as s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), m (multiplet), and br (broadened). High resolution electron impact ionization (HREI) was measured on Finnigan MAT MAT-95XL (Instrumentation Center, National Tsing Hua University, Hsinchu, Taiwan). Melting points were obtained with a Büchi B-545 melting point apparatus. Typical experiments illustrating the synthetic procedures for the preparation of the anthrapyrazolones are described below.
2.2 Synthetic methods
Thirty-two compounds were synthesized, starting from 1,5-dichloroanthraquinone. All the organic solvents and chemical reactants required for synthesis were purchased from either Merck Chemical Company or Sigma–Aldrich Chemical Company without further purification.
2.2.1 Synthetic procedure I: Preparation of compound 1
1,5-Dichloroanthraquinone (2.00 g, 7.2 mmol) was dissolved in DMSO (20 mL) and hydrazine monohydrate (1.5 mL, 30 mmol) and diisopropylethylamine (DIPEA) (2 mL) was added. The reaction mixture was continued under refluxing condition for 2 h. The reaction mixture was cooled to room temperature, treated with crushed ice, neutralized and collected the ethyl acetate (EA) layer by partition. The organic layer was added anhydrous magnesium sulfate (MgSO4) to remove the water, dried and washed with dichloromethane to afford desired compound as a brown solid.
2.2.2 Synthetic procedure II: Preparation of compound 17
1,5-Dichloroanthraquinone (2.00 g, 7.2 mmol) and 2-hydroxyethylhydrazine (2 mL, 29.5 mmol) were dissolved in pyridine (20 mL) and stirred at 120 °C for 2 h. The reaction mixture was cooled to room temperature, treated with crushed ice and neutralized with 5% hydrochloric acid (HCl). And then, the mixture was partition with ethyl acetate. The organic layer was dried by anhydrous MgSO4, removed the solvent and washed with dichloromethane to afford desired compound as a yellow solid.
2.2.3 General procedure III: Preparation of compounds 2–16 and 18–32
Compound 1 or 17 (2 mmol), primary amine (10 mmol) and N,N-dimethylaminopyridine (DMAP) (2 mmol) were dissolved in DMSO (10 mL) stirred at room temperature for 4 h. The reaction was added water (100 mL), neutralized by 5% HCl and extracted with EA. The resulting precipitate was collected by filtration, washed with water and purified by crystallization from EA/hexane to afford desired compounds.
2.3 Physical data
2.3.1 7-Chloroanthra[1,9-cd]pyrazol-6(2H)-one (1)
The pure compound was obtained as a brown solid (yield 42%). (Rf = 0.43 at ethyl acetate:n-hexane = 1:1). Mp 310–311 °C (EtOH) (Kim et al., 2003). FT-IR (KBr; ν cm−1): 3234 (NH), 1637 (CO). 1H NMR (300 MHz, DMSO-d6): δ (ppm) 7.59 (dd, J = 7.8, 1.2 Hz, 1H, Ar–H10), 7.71 (t, J = 7.5 Hz, 1H, Ar–H4), 7.72 (t, J = 7.8 Hz, 1H, Ar–H9), 7.87 (d, J = 7.2 Hz, 1H, Ar–H3), 7.97 (d, J = 8.1 Hz, 1H, Ar–H8), 8.21 (dd, J = 7.5, 1.2 Hz, 1H, Ar–H5), 13.82 (br, 1H, –NH–). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 117.12, 120.66, 121.83, 122.20, 126.31, 128.86, 129.15, 132.67, 134.22, 135.21, 136.23, 139.59, 182.29 (C⚌O). HRMS (EI) m/z calcd for C14H7ClN2O+ [M]+ 254.0247, found, 254.0246 (100), 256.0214 (33).
2.3.2 7-[2-(Methyl)propylamino]anthra[1,9-cd]pyrazol-6(2H)-one (2)
Product 2 was prepared from 1 with isobutylamine. The red solid material was isolated in 19% yield. (Rf = 0.60 at ethyl acetate:n-hexane = 1:1). Mp 237–238 °C (EtOH). FT-IR (KBr; ν cm−1): 3249 (NH), 1622 (C⚌O), 1600 (C⚌N). 1H NMR (300 MHz, DMSO-d6): δ (ppm) 1.03 (d, J = 6.6 Hz, 6H, –CH3), 1.99 (m, 1H, –CH–), 3.13 (t, J = 6.0 Hz, 2H, –NCH2–), 6.85 (d, J = 8.7 Hz, 1H, Ar–H10), 7.36 (d, J = 7.2 Hz, 1H, Ar–H3), 7.51 (t, J = 7.8 Hz, 1H, Ar–H9), 7.64 (t, J = 7.8 Hz, 1H, Ar–H4), 7.79 (d, J = 7.2 Hz, 1H, Ar–H8), 7.86 (d, J = 8.1 Hz, 1H, Ar–H5), 10.17 (t, J = 5.1 Hz, 1H, –NH–), 13.55 (br, 1H, pyrazole-NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 20.56, 27.88, 50.22, 109.20, 112.41, 114.10, 116.31, 119.57, 122.09, 127.15, 128.94, 133.57, 135.67, 139.42, 139.96, 154.08, 187.12 (C⚌O). HRMS (EI) m/z calcd for C18H17N3O+, [M]+: 291.1372, found, 248.0815 (100), 291.1367 (15).
2.3.3 7-(Propylamino)anthra[1,9-cd]pyrazol-6(2H)-one (3)
Product 3 was prepared from 1 with propylamine. The red solid material was isolated in 34% yield (Rf = 0.58 at ethyl acetate:n-hexane = 1:1). Mp 247–248 °C (EtOH). FT-IR (KBr; ν cm−1): 3147 (NH), 1618 (C⚌O), 1599 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 1.10 (t, J = 7.5 Hz, 3H, –CH3), 1.74–1.86 (m, 2H, –CH2–), 3.30–3.37 (m, 2H, –NCH2–), 6.88 (dd, J = 8.1, 1.5 Hz, 1H, Ar–H10), 7.46 (dd, J = 7.5, 1.5 Hz, 1H, Ar–H3), 7.52 (t, J = 7.8 Hz, 1H, Ar–H9), 7.66 (t, J = 7.8 Hz, 1H, Ar–H4), 7.85 (d, J = 7.8 Hz, 1H, Ar–H8), 7.85 (d, J = 7.8 Hz, 1H, Ar–H5), 10.17 (br, 1H, –NH–), 12.60 (br, 1H, pyrazole-NH). 13C NMR (75 MHz, Acetone-d6): δ (ppm) 11.75, 22.15, 44.20, 109.16, 112.26, 114.06, 116.16, 119.44, 122.06, 127.12, 128.84, 133.53, 135.56, 139.33, 139.97, 153.86, 186.99 (C⚌O). HRMS (EI) m/z calcd for C17H15N3O+ [M]+: 277.1215, found, 248.0832 (100), 277.1218 (25).
2.3.4 7-[5-(Hydroxyl)pentylamino]anthra[1,9-cd]pyrazol-6(2H)-one (4)
Product 4 was prepared from 1 with 5-amino-1-pentanol. The red solid material was isolated in 44% yield (Rf = 0.18 at ethyl acetate:n-hexane = 1:1). Mp 206–207 °C (EtOH) (Pang et al., 2010). FT-IR (KBr; ν cm−1): 3159 (NH), 3078 (OH), 1602 (C⚌O), 1569 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 1.59–1.63 (m, 2H, –CH2–), 1.59–1.63 (m, 2H, –CH2–), 1.77–1.86 (m, 2H, –CH2–), 3.34–3.41 (m, 2H, –NCH2–), 3.47 (s, 1H, –OH), 3.60 (t, J = 4.8 Hz, 2H, –OCH2–), 6.88 (dd, J = 8.1, 1.8 Hz, 1H, Ar–H10), 7.45 (dd, J = 7.2, 1.5 Hz, 1H, Ar–H3), 7.52 (t, J = 7.5 Hz, 1H, Ar–H9), 7.66 (t, J = 7.7 Hz, 1H, Ar–H4), 7.85 (d, J = 7.5 Hz, 1H, Ar–H8), 7.85 (d, J = 7.5 Hz, 1H, Ar–H5), 10.16 (br, 1H, –NH–), 12.61 (br, 1H, pyrazole-NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 23.57, 28.84, 32.54, 42.49, 61.04, 109.14, 112.26, 114.04, 116.18, 119.45, 122.05, 127.12, 128.84, 133.52, 135.58, 139.34, 139.96, 153.83, 186.96 (C⚌O). HRMS (EI) m/z calcd for C19H19N3O2+ [M]+: 321.1477, found, 248.0818 (100), 321.1475 (20).
2.3.5 7-(Phenethylamino)anthra[1,9-cd]pyrazol-6(2H)-one (5)
Product 5 was prepared from 1 with phenethylamine. The red solid material was isolated in 57% yield (Rf = 0.64 at ethyl acetate:n-hexane = 1:1). Mp 221–222 °C (EtOH). FT-IR (KBr; ν cm−1): 3178 (NH), 3078 (OH), 1620 (C⚌O), 1599 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 3.09 (t, J = 7.2 Hz, 2H, –CH2–), 3.60–3.67 (m, 2H, –NCH2–), 6.97 (d, J = 9.0 Hz, 1H, Ar–H10), 7.23 (t, J = 6.9 Hz, 1H, Ar′–H4), 7.34 (t, J = 7.8 Hz, 2H, Ar′–H3, H5), 7.41 (d, J = 7.8 Hz, 2H, Ar′–H2,6), 7.49 (t, J = 7.2 Hz, 1H, Ar–H3), 7.50 (t, J = 5.1 Hz, 1H, Ar–H9), 7.66 (t, J = 7.5 Hz, 1H, Ar–H4), 7.84 (d, J = 3.0 Hz, 1H, Ar–H8), 7.86 (d, J = 3.9 Hz, 1H, Ar–H5), 10.22 (br, 1H, –NH–), 12.62 (br, 1H, pyrazole-NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 35.09, 44.02, 109.38, 112.28, 114.20, 116.22, 119.49, 122.05, 126.68, 127.06, 128.83, 129.02, 129.16, 133.53, 135.56, 139.37, 139.72, 139.88, 153.56, 186.91 (C⚌O). HRMS (EI) m/z calcd for C22H17N3O+ [M]+: 339.1372, found, 248.0815 (100), 339.1366 (5).
2.3.6 7-(Cyclohexylamino)anthra[1,9-cd]pyrazol-6(2H)-one (6)
Product 6 was prepared from 1 with cyclohexylamine. The red solid material was isolated in 22% yield (Rf = 0.69 at ethyl acetate:n-hexane = 1:1). Mp 246–247 °C (EtOH). FT-IR (KBr; ν cm−1): 3145 (NH), 3078 (OH), 1620 (C⚌O), 1599 (C⚌N). 1H NMR (300 MHz, DMSO-d6): δ (ppm) 1.34–2.01 (m, 10H, –(CH2)5), 3.60 (s, 1H, –NCH–), 6.89 (d, J = 8.1 Hz, 1H, Ar–H10), 7.34 (dd, J = 7.2, 0.9 Hz, 1H, Ar–H3), 7.49 (t, J = 7.8 Hz, 1H, Ar–H9), 7.63 (t, J = 7.8 Hz, 1H, Ar–H4), 7.77 (d, J = 6.9 Hz, 1H, Ar–H8), 7.85 (d, J = 8.1 Hz, 1H, Ar–H5), 10.21 (d, J = 7.2 Hz, 1H, –NH–), 13.58 (br, 1H, pyrazole-NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 24.15, 25.66, 32.60, 49.83, 108.98, 112.74, 113.96, 116.17, 119.43, 122.07, 127.19, 128.87, 133.68, 135.50, 139.38, 139.97, 152.93, 186.97 (C⚌O). HRMS (EI) m/z calcd for C20H19N3O+ [M]+: 317.1528, found, 274.0982 (100), 317.1522 (60).
2.3.7 7-(Cyclopentylamino)anthra[1,9-cd]pyrazol-6(2H)-one (7)
Product 7 was prepared from 1 with cyclopentylamine. The red solid material was isolated in 21% yield (Rf = 0.58 at ethyl acetate:n-hexane = 1:1). Mp 212–213 °C (EtOH). FT-IR (KBr; ν cm−1): 3149 (NH), 1622 (C⚌O), 1602 (C⚌N). 1H NMR (300 MHz, DMSO-d6): δ (ppm) 1.54–2.08 (m, 8H, –(CH2)4–), 3.99–4.01 (m, 1H, –NCH–), 6.87 (d, J = 8.4 Hz, 1H, Ar–H10), 7.36 (d, J = 7.2 Hz, 1H, Ar–H3), 7.51 (t, J = 7.8 Hz, 1H, Ar–H9), 7.64 (t, J = 7.8 Hz, 1H, Ar–H4), 7.77 (d, J = 6.9 Hz, 1H, Ar–H8), 7.85 (d, J = 8.1 Hz, 1H, Ar–H5), 10.16 (d, J = 6.3 Hz, 1H, –NH–), 13.57 (br, 1H, pyrazole-NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 23.98, 33.42, 53.47, 109.18, 113.13, 114.09, 116.30, 119.53, 122.07, 127.16, 128.94, 133.60, 135.55, 139.42, 139.94, 153.33, 187.00 (C⚌O). HRMS (EI) m/z calcd for C19H17N3O+ [M]+: 303.1372, found, 274.0969 (90), 303.1371 (100).
2.3.8 7-(Methylcyclohexylamino)anthra[1,9-cd]pyrazol-6(2H)-one (8)
Product 8 was prepared from 1 with aminomethylcyclohexane. The brown solid material was isolated in 47% yield (Rf = 0.63 at ethyl acetate:n-hexane = 1:1). Mp 245–246 °C (EtOH). FT-IR (KBr; ν cm−1): 3177 (NH), 1620 (C⚌O), 1600 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 1.10–1.97 (m, 10H, –(CH2)5–), 1.10–1.97 (m, 1H, –CH–), 3.20 (t, J = 6.0 Hz, 2H, –NCH2–), 6.87 (dd, J = 8.4, 1.5 Hz, 1H, Ar–H10), 7.46 (dd, J = 7.5, 1.5 Hz, 1H, Ar–H3), 7.51 (t, J = 7.5 Hz, 1H, Ar–H9), 7.66 (t, J = 7.8 Hz, 1H, Ar–H4), 7.84 (dd, J = 7.8, 2.0 Hz, 2H, Ar–H8, Ar–H5), 10.28 (br, 1H, –NH–), 12.60 (br, 1H, pyrazole-NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 25.73, 26.33, 30.93, 37.33, 48.90, 109.07, 112.32, 114.05, 116.23, 119.48, 122.04, 127.12, 128.85, 133.52, 135.58, 139.35, 139.89, 154.01, 187.01 (C⚌O). HRMS (EI) m/z calcd for C21H21N3O+ [M]+: 331.1685, found, 248.0815 (100), 331.1676 (25).
2.3.9 7-[3-(Hydroxyl)propylamino]anthra[1,9-cd]pyrazol-6(2H)-one (9)
Product 9 was prepared from 1 with 3-amino-1-propanol. The red solid material was isolated in 24% yield (Rf = 0.18 at ethyl acetate:n-hexane = 1:1). Mp 147–149 °C (EtOH). FT-IR (KBr; ν cm−1): 3163 (NH), 3116 (OH), 1620 (C⚌O), 1598 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 1.94–2.02 (m, 2H, –CH2–), 3.45–3.51 (m, 2H, –CH2–), 3.78 (t, J = 5.7 Hz, 2H, –NCH2–), 6.92 (d, J = 8.7 Hz, 1H, Ar–H10), 7.47 (d, J = 8.1 Hz, 1H, Ar–H3), 7.52 (t, J = 7.5 Hz, 1H, Ar–H9), 7.66 (t, J = 7.5 Hz, 1H, Ar–H4), 7.85 (d, J = 7.8 Hz, 2H, Ar–H5,8), 10.18 (br, 1H, –NH–), 12.60 (br, 1H, pyrazole-NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 32.26, 39.52, 58.77, 109.15, 110.39, 112.27, 113.16, 116.18, 119.43, 125.20, 127.13, 128.86, 129.04, 139.34, 140.41, 153.88, 186.94 (C⚌O). HRMS (EI) m/z calcd for C17H15N3O2+ [M]+: 293.1164, found, 248.0809 (100), 293.1156 (30).
2.3.10 7-(Butylamino)anthra[1,9-cd]pyrazol-6(2H)-one (10)
Product 10 was prepared from 1 with butylamine. The red solid material was isolated in 19% yield (Rf = 0.63 at ethyl acetate:n-hexane = 1:1). Mp 219–220 °C (EtOH) (Sokolyuk et al., 2002). FT-IR (KBr; ν cm−1): 3153 (NH), 1622 (C⚌O), 1604 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 1.02 (t, J = 7.5 Hz, 3H, –CH3), 1.50–1.63 (m, 2H, –CH2–), 1.73–1.82 (m, 2H, –CH2–), 3.34–3.40 (m, 2H, –NCH2–), 6.89 (d, J = 6.6 Hz, 1H, Ar–H10), 7.47 (d, J = 6.9 Hz, 1H, Ar–H3), 7.52 (t, J = 7.8 Hz, 1H, Ar–H9), 7.66 (t, J = 7.8 Hz, 1H, Ar–H4), 7.85 (d, J = 7.5 Hz, 2H, Ar–H5,8), 10.15 (br, 1H, –NH–), 12.62 (br, 1H, pyrazole-NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 13.88, 20.06, 30.99, 42.08, 109.13, 112.26, 114.06, 116.17, 119.43, 122.05, 127.10, 128.84, 133.54, 135.58, 139.32, 139.60, 153.85, 186.97 (C⚌O). HRMS (EI) m/z calcd for C18H17N3O+ [M]+: 291.1372, found, 248.0816 (100), 291.1365 (25).
2.3.11 7-(Methylamino)anthra[1,9-cd]pyrazol-6(2H)-one (11)
Product 11 was prepared from 1 with methylamine. The red solid material was isolated in 14% yield (Rf = 0.60 at ethyl acetate:n-hexane = 3:2). Mp 254–255 °C (EtOH). FT-IR (KBr; ν cm−1): 3147 (NH), 1622 (C⚌O), 1602 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 3.04 (d, J = 5.1 Hz, 3H, –NCH3), 6.85 (d, J = 8.4 Hz, 1H, Ar–H10), 7.48 (dd, J = 7.4, 1.5 Hz, 1H, Ar–H3), 7.55 (t, J = 7.5 Hz, 1H, Ar–H9), 7.66 (t, J = 8.1 Hz, 1H, Ar–H4), 7.84 (d, J = 2.4 Hz, 1H, Ar–H8), 7.87 (d, J = 3.3 Hz, 1H, Ar–H5), 10.01 (br, 1H, –NH–), 12.62 (br, 1H, pyrazole-NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 29.49, 109.23, 111.93, 116.25, 119.40, 127.13, 128.87, 129.06, 129.22, 132.73, 133.47, 134.29, 135.61, 154.61, 186.88 (C⚌O). HRMS (EI) m/z calcd for C15H11N3O+ [M]+ 249.0902, found, 232.0877 (50), 249.0897 (100).
2.3.12 7-(Benzylamino)anthra[1,9-cd]pyrazol-6(2H)-one (12)
Product 12 was prepared from 1 with benzylamine. The red solid material was isolated in 26% yield (Rf = 0.62 at ethyl acetate:n-hexane = 3:2). Mp 219–220 °C (EtOH) (Bennett et al., 2004). FT-IR (KBr; ν cm−1): 3153 (NH), 1622 (C⚌O), 1600 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 4.64 (d, J = 4.5 Hz, 2H, –NCH2–), 6.86 (dd, J = 6.9, 2.4 Hz, 1H, Ar–H10), 7.30 (t, J = 7.2 Hz, 1H, Ar′–H4), 7.39 (t, J = 7.5 Hz, 2H, Ar′–H3,5), 7.48 (d, J = 6.3 Hz, 2H, –Ar′–H2, H6), 7.48 (d, J = 6.3 Hz, 1H, Ar–H3), 7.49 (t, J = 7.5 Hz, 1H, Ar–H9), 7.66 (t, J = 7.8 Hz, 1H, Ar–H4), 7.85 (d, J = 2.1 Hz, 1H, Ar–H8), 7.88 (d, J = 3.0 Hz, 1H, Ar–H5), 10.54 (br, 1H, –NH–), 12.65 (br, 1H, pyrazole-NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 46.43, 109.70, 112.69, 114.54, 116.41, 119.63, 122.06, 127.03, 127.58, 127.74, 128.90, 129.09, 133.59, 135.54, 139.33, 139.40, 139.83, 153.55, 187.16 (C⚌O). HRMS (EI) m/z calcd for C21H15N3O+ [M]+ 325.1215, found, 248.0815 (25), 325.1209 (100).
2.3.13 7-[2-(1-Cyclohexenyl)ethylamino]anthra[1,9-cd]pyrazol-6(2H)-one (13)
Product 13 was prepared from 1 with 2-(1-cyclohexenyl)ethylamine. The red solid material was isolated in 31% yield (Rf = 0.69 at ethyl acetate:n-hexane = 3:2). Mp 213–214 °C (EtOH). FT-IR (KBr; ν cm−1): 3211 (NH), 1622 (C⚌O), 1600 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 1.59–1.66 (m, 8H, cyclohexene-(CH2)4), 2.40 (t, J = 6.6 Hz, 2H, –CH2–), 3.41–3.47 (m, 2H, –NCH2–), 5.67 (s, 1H, cyclohexene-CH–), 6.89 (dd, J = 8.1, 1.5 Hz, 1H, Ar–H10), 7.46 (dd, J = 7.5, 1.5 Hz, 1H, Ar–H3), 7.52 (t, J = 7.2 Hz, 1H, Ar–H9), 7.65 (t, J = 8.1 Hz, 1H, Ar–H4), 7.85 (d, J = 7.5 Hz, 1H, Ar–H8), 7.85 (d, J = 7.5 Hz, 1H, Ar–H5), 10.07 (br, 1H, –NH–), 12.60 (br, 1H, pyrazole-NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 22.22, 22.73, 25.05, 27.84, 37.15, 41.01, 109.18, 112.35, 114.18, 116.14, 119.40, 122.05, 123.41, 127.12, 128.80, 133.49, 135.01, 135.47, 139.38, 139.91, 153.63, 186.82 (C⚌O). HRMS (EI) m/z calcd for C22H21N3O+ [M]+: 343.1685, found 248.0820 (100), 343.1682 (30).
2.3.14 7-[2-(4-Methoxyphenyl)ethylamino]anthra[1,9-cd]pyrazol-6(2H)-one (14)
Product 14 was prepared from 1 with 2-(4-methoxyphenyl)ethylamine. The red solid material was isolated in 42% yield (Rf = 0.57 at ethyl acetate:n-hexane = 3:2). Mp 219–210 °C (EtOH). FT-IR (KBr; ν cm−1): 3167 (NH), 1620 (C⚌O), 1600 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 3.02 (t, J = 7.5 Hz, 2H, –CH2–), 3.55–3.61 (m, 2H, –NCH2–), 3.77 (s, 3H, –OCH3), 6.90 (d, J = 8.7 Hz, 4H, Ar′–H), 7.32 (d, J = 8.7 Hz, 1H, Ar–H10), 7.47 (dd, J = 7.2, 1.5 Hz, 1H, Ar–H3), 7.53 (t, J = 8.1 Hz, 1H, Ar–H9), 7.65 (t, J = 7.2 Hz, 1H, Ar–H4), 7.84 (d, J = 3.0 Hz, 1H, Ar–H8), 7.86 (d, J = 4.2 Hz, 1H, Ar–H5), 10.20 (br, 1H, –NH–), 12.61 (br, 1H, pyrazole-NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 34.21, 44.28, 55.38, 109.34, 112.30, 114.16, 114.36, 116.23, 119.50, 122.05, 127.08, 128.84, 130.14, 131.57, 133.53, 135.59, 139.37, 139.89, 153.59, 158.42, 186.91 (C⚌O). HRMS (EI) m/z calcd for C23H19N3O2+ [M]+: 369.1477, found 248.0821 (100), 369.1469 (5).
2.3.15 7-(2-Methoxybenzylamino)anthra[1,9-cd]pyrazol-6(2H)-one (15)
Product 15 was prepared from 1 with 2-methoxybenzylamine. The red solid material was isolated in 39% yield (Rf = 0.60 at ethyl acetate:hexane = 3:2). Mp 217–218 °C (EtOH). FT-IR (KBr; ν cm−1): 3159 (NH), 1622 (C⚌O), 1601 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 3.93 (s, 3H, –OCH3), 4.59 (d, J = 5.7 Hz, 2H, –NCH2–), 6.87 (t, J = 5.1 Hz, 1H, Ar–H10), 6.94 (t, J = 7.8 Hz, 1H, Ar′–H5), 7.06 (d, J = 8.1 Hz, 1H, Ar–H3), 7.29 (t, J = 7.8 Hz, 1H, Ar–H9), 7.38 (d, J = 7.8 Hz, 1H, Ar′–H3), 7.47 (d, J = 4.5 Hz, 1H, Ar′–H6), 7.47 (t, J = 4.5 Hz, 1H, Ar′–H4), 7.66 (t, J = 7.5 Hz, 1H, Ar–H4), 7.87 (d, J = 7.8 Hz, 1H, Ar–H8), 7.87 (d, J = 7.81 Hz, 1H, Ar–H5), 10.50 (br, 1H, –NH–), 12.63 (br, 1H, pyrazole-NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 41.55, 55.87, 109.50, 111.52, 112.47, 114.50, 116.25, 119.55, 120.88, 122.10, 126.73, 127.11, 128.83, 129.02, 129.13, 133.60, 135.50, 139.39, 153.65, 157.69, 187.04 (C⚌O). HRMS (EI) m/z calcd for C22H17N3O2+ [M]+: 355.1321, found 355.1319.
2.3.16 7-(Piperonylamino)anthra[1,9-cd]pyrazol-6(2H)-one (16)
Product 16 was prepared from 1 with piperonylamine. The red solid material was isolated in 22% yield (Rf = 0.58 at ethyl acetate:n-hexane = 3:2). Mp 217–218 °C (EtOH). FT-IR (KBr; ν cm−1): 3173 (NH), 1622 (C⚌O), 1600 (C⚌N). 1H NMR (300 MHz, DMSO-d6): δ (ppm) 4.45 (d, J = 5.7 Hz, 2H, –NCH2–), 6.00 (s, 2H, –OCH2O–), 6.83 (d, J = 8.4 Hz, 1H, Ar–H10), 6.90 (s, 2H, Ar′–H5,6), 6.98 (s, 1H, Ar′–H2), 7.39 (d, J = 6.9 Hz, 1H, Ar–H3), 7.49 (t, J = 7.8 Hz, 1H, Ar–H9), 7.64 (t, J = 7.8 Hz, 1H, Ar–H4), 7.79 (d, J = 7.2 Hz, 1H, Ar–H8), 7.87 (d, J = 8.1 Hz, 1H, Ar–H5), 10.31 (t, J = 5.4 Hz, 1H, –NH–), 13.60 (br, 1H, pyrazole-NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 46.32, 101.44, 108.36, 108.82, 109.81, 112.81, 114.60, 116.47, 119.74, 121.09, 122.14, 127.12, 129.03, 129.19, 133.21, 133.63, 135.64, 146.99, 148.15, 153.53, 187.23 (C⚌O). HRMS (EI) m/z calcd for C22H15N3O3+ [M]+: 369.1113, found, 369.1107.
2.3.17 7-Chloro-2-(2-hydroxyethyl)anthra[1,9-cd]pyrazol-6(2H)-one (17)
The pure compound was obtained as a yellow solid (yield 37%). (Rf = 0.26 at ethyl acetate:n-hexane = 6:1). Mp 185–186 °C (EtOH) (Zhang et al., 2010). FT-IR (KBr; ν cm−1): 3481 (OH), 1622 (C⚌O), 1600 (C⚌N). 1H NMR (300 MHz, DMSO-d6): δ (ppm) 3.87–3.93 (m, 2H, –NCH2–), 4.63 (t, J = 5.1 Hz, 2H, –OCH2–), 4.95 (t, J = 5.7 Hz, 1H, –OH), 7.60 (dd, J = 7.8, 1.2 Hz, 1H, Ar–H10), 7.72 (t, J = 8.1 Hz, 1H, Ar–H4), 7.73 (t, J = 7.2 Hz, 1H, Ar–H9), 7.89 (d, J = 6.9 Hz, 1H, Ar–H3), 8.09 (d, J = 7.8 Hz, 1H, Ar–H8), 8.17 (dd, J = 7.8, 1.5 Hz, 1H, Ar–H5). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 53.00, 60.75, 117.34, 120.92, 122.23, 126.29, 128.77, 129.03, 132.69, 134.30, 134.92, 136.29, 137.33, 140.21, 182.10 (C⚌O). HRMS (EI) m/z calcd for C16H11ClN2O2+ [M]+: 298.0509, found, 267.0325 (100), 298.0513 (35), 300.0498 (10).
2.3.18 (7-[2-(Methyl)propylamino]-2-(2-hydroxyethyl)anthra[1,9-cd]pyrazol-6(2H)-one (18)
Product 18 was prepared from 17 with isobutylamine. The red solid material was isolated in 46% yield (Rf = 0.41 at ethyl acetate:n-hexane = 6:1). Mp 134–135 °C (EtOH). FT-IR (KBr; ν cm−1): 3423 (OH), 1618 (C⚌O), 1596 (C⚌N). 1H NMR (300 MHz, DMSO-d6): δ (ppm) 1.03 (t, J = 6.6 Hz, 6H, –CH3), 1.93–2.02 (m, 1H, –CH–), 3.12 (t, J = 5.4 Hz, 2H, –NCH2–), 3.88 (m, 2H, –NCH2–), 4.57 (t, J = 5.4 Hz, 2H, –OCH2–), 4.94 (t, J = 5.4 Hz, 1H, –OH), 6.82 (d, J = 8.1 Hz, 1H, Ar–H10), 7.30 (d, J = 7.2 Hz, 1H, Ar–H3), 7.49 (t, J = 8.0 Hz, 1H, Ar–H9), 7.63 (t, J = 7.5 Hz, 1H, Ar–H4), 7.78 (d, J = 6.9 Hz, 1H, Ar–H8), 7.93 (d, J = 8.1 Hz, 1H, Ar–H5), 10.17 (t, J = 5.1 Hz, 1H, –NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 20.40, 27.79, 50.13, 52.71, 60.66, 109.10, 112.24, 113.93, 116.21, 119.53, 122.46, 127.05, 128.55, 133.18, 135.46, 139.01, 139.85, 153.99, 186.71 (C⚌O). HRMS (EI) m/z calcd for C20H21N3O2+ [M]+: 335.1643, found, 292.1081 (100), 335.1628 (30).
2.3.19 7-Propylamino-2-(2-hydroxyethyl)anthra[1,9-cd]pyrazol-6(2H)-one (19)
Product 19 was prepared from 17 with propylamine. The red solid material was isolated in 71% yield (Rf = 0.41 at ethyl acetate:n-hexane = 6:1). Mp 181–182 °C (EtOH). FT-IR (KBr; ν cm−1): 3282 (OH), 1616 (C⚌O), 1595 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 1.10 (t, J = 7.5 Hz, 3H, –CH3), 1.79 (p, J = 7.2 Hz, 2H, –CH2–), 3.32 (q, J = 6.9 Hz, 2H, –NCH2–), 4.09 (t, J = 6.9 Hz, 2H, –NCH2–), 4.65 (t, J = 5.1 Hz, 2H, –OCH2–), 6.86 (d, J = 8.4 Hz, 1H, Ar–H10), 7.40 (dd, J = 7.5, 1.2 Hz, 1H, Ar–H3), 7.51 (t, J = 7.8 Hz, 1H, Ar–H9), 7.64 (t, J = 7.5 Hz, 1H, Ar–H4), 7.83 (d, J = 7.2 Hz, 1H, Ar–H8), 7.90 (d, J = 8.1 Hz, 1H, Ar–H5), 10.16 (br, 1H, –NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 11.78, 22.16, 44.20, 52.77, 60.78, 109.19, 112.25, 113.91, 116.31, 119.58, 122.49, 127.09, 128.64, 133.23, 135.56, 139.06, 139.91, 153.86, 186.71 (C⚌O). HRMS (EI) m/z calcd for C19H19N3O2+ [M]+: 321.1477, found, 292.1096 (100), 321.1475 (30).
2.3.20 7-[5-(Hydroxyl)pentylamino]-2-(2-hydroxyethyl)anthra[1,9-cd]pyrazol-6(2H)-one (20)
Product 20 was prepared from 17 with 5-amino-1-pentanol. The red solid material was isolated in 48% yield (Rf = 0.18 at ethyl acetate:n-hexane = 6:1). Mp 183–184 °C (EtOH). FT-IR (KBr; ν cm−1): 3361 (OH), 1614 (C⚌O), 1595 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 1.61–1.63 (m, 4H, –CH2–), 1.77–1.84 (m, 2H, –CH2–), 3.34–3.40 (m, 2H, –NCH2–), 3.46 (t, J = 5.1 Hz, 1H, –OH), 3.60 (t, J = 5.4 Hz, 2H, –NCH2–), 4.09 (t, J = 4.8 Hz, 2H, –OCH2–), 4.65 (t, J = 5.1 Hz, 2H, –OCH2–), 6.86 (d, J = 8.4 Hz, 1H, Ar–H10), 7.40 (dd, J = 6.3, 1.2 Hz, 1H, Ar–H3), 7.51 (t, J = 8.1 Hz, 1H, Ar–H9), 7.64 (t, J = 7.8 Hz, 1H, Ar–H4), 7.83 (d, J = 6.9 Hz, 1H, Ar–H8), 7.90 (d, J = 8.4 Hz, 1H, Ar–H5), 10.16 (br, 1H, –NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 23.57, 28.85, 32.52, 42.49, 52.76, 60.75, 61.03, 109.16, 112.23, 113.90, 116.29, 119.57, 122.49, 127.08, 128.63, 133.21, 135.55, 139.05, 139.90, 153.83, 186.68 (C⚌O). HRMS (EI) m/z calcd for C21H23N3O3+ [M]+: 365.1739, found, 292.1078 (100), 365.1739 (20).
2.3.21 7-Phenethylamino-2-(2-hydroxyethyl)anthra[1,9-cd]pyrazol-6(2H)-one (21)
Product 21 was prepared from 17 with phenethylamine. The red solid material was isolated in 71% yield (Rf = 0.40 at ethyl acetate:n-hexane = 6:1). Mp 171–172 °C (EtOH). FT-IR (KBr; ν cm−1): 3298 (OH), 1618 (C⚌O), 1595 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 3.09 (t, J = 7.2 Hz, 2H, –CH2–), 3.59–3.66 (m, 2H, –NCH2–), 4.09 (t, J = 4.2 Hz, 2H, –NCH2–), 4.08–4.11 (br, 1H, –OH), 4.64 (t, J = 4.5 Hz, 2H, –OCH2–), 6.92 (d, J = 8.4 Hz, 1H, Ar–H10), 7.23 (t, J = 8.7 Hz, 2H, Ar′–H2,6), 7.33 (t, J = 6.9 Hz, 3H, Ar′–H3,4,5), 7.41 (d, J = 7.8 Hz, 1H, Ar–H3), 7.51 (t, J = 7.8 Hz, 1H, Ar–H9), 7.63 (t, J = 7.5 Hz, 1H, Ar–H4), 7.81 (d, J = 7.2 Hz, 1H, Ar–H8), 7.89 (d, J = 8.1 Hz, 1H, Ar–H5), 10.21 (br, 1H, –NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 35.08, 44.01, 52.77, 60.75, 109.39, 112.29, 114.04, 116.34, 119.62, 122.46, 126.68, 127.00, 128.63, 128.81, 129.16, 133.22, 135.57, 139.01, 139.69, 139.90, 153.56, 186.62 (C⚌O). HRMS (EI) m/z calcd for C24H21N3O2+ [M]+: 383.1634, found, 292.1084 (100), 383.1629 (7).
2.3.22 7-Cyclohexylamino-2-(2-hydroxyethyl)anthra[1,9-cd]pyrazol-6(2H)-one (22)
Product 22 was prepared from 17 with cyclohexylamine. The red solid material was isolated in 52% yield (Rf = 0.43 at ethyl acetate:n-hexane = 6:1). Mp 217–218 °C (EtOH). FT-IR (KBr; ν cm−1): 3419 (OH), 1652 (C⚌O), 1614 (C⚌N). 1H NMR (300 MHz, DMSO-d6): δ (ppm) 1.35–2.01 (m, 10H, cyclohexyl-CH2), 3.59–3.60 (m, 1H, –NCH–), 3.85–3.91 (m, 2H, –NCH2–), 4.58 (t, J = 5.4 Hz, 2H, –OCH2–), 4.94 (t, J = 5.4 Hz, 1H, –OH), 6.89 (d, J = 8.4 Hz, 1H, Ar–H10), 7.29 (d, J = 7.2 Hz, 1H, Ar–H3), 7.49 (t, J = 8.1 Hz, 1H, Ar–H9), 7.64 (t, J = 7.2 Hz, 1H, Ar–H4), 7.77 (d, J = 7.2 Hz, 1H, Ar–H8), 7.94 (d, J = 8.1 Hz, 1H, Ar–H5), 10.21 (d, J = 4.8 Hz, 1H, –NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 24.15, 25.65, 32.58, 49.84, 52.79, 60.78, 109.01, 112.76, 113.81, 116.29, 119.57, 122.49, 127.16, 128.69, 133.38, 135.53, 139.10, 139.91, 152.95, 186.71 (C⚌O). HRMS (EI) m/z calcd for C22H23N3O2+ [M]+: 361.1790, found, 318.1241 (100), 361.1794 (75).
2.3.23 7-Cyclopentylamino-2-(2-hydroxyethyl)anthra[1,9-cd]pyrazol-6(2H)-one (23)
Product 23 was prepared 17 with cyclopentylamine. The red solid material was isolated in 42% yield (Rf = 0.38 at ethyl acetate:n-hexane = 6:1). Mp 198–199 °C (EtOH). FT-IR (KBr; ν cm−1): 3392 (OH), 1649 (C⚌O), 1614 (C⚌N). 1H NMR (300 MHz, DMSO-d6): δ (ppm) 1.52–2.11 (m, 8H, cyclopentane-CH2), 3.85–3.91 (m, 2H, –NCH2–), 3.98–4.03 (m, 1H, cyclopentane-CH), 4.58 (t, J = 5.4 Hz, 2H, –OCH2–), 4.94 (t, J = 5.4 Hz, 1H, –OH), 6.87 (d, J = 8.4 Hz, 1H, Ar–H10), 7.30 (d, J = 7.2 Hz, 1H, Ar–H3), 7.51 (t, J = 8.1 Hz, 1H, Ar–H9), 7.64 (t, J = 7.5 Hz, 1H, Ar–H4), 7.77 (d, J = 7.2 Hz, 1H, Ar–H8), 7.95 (d, J = 8.4 Hz, 1H, Ar–H5), 10.17 (d, J = 6.0 Hz, 1H, –NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 24.02, 33.48, 52.86, 53.54, 60.85, 109.27, 113.19, 113.99, 116.46, 119.73, 122.55, 127.18, 128.83, 133.34, 135.63, 139.14, 140.01, 153.40, 186.79 (C⚌O). HRMS (EI) m/z calcd for C21H21N3O2+ [M]+: 347.1634, found, 318.1250 (80), 347.1624 (100).
2.3.24 7-Methylcyclohexylamino-2-(2-hydroxyethyl)anthra[1,9-cd]pyrazol-6(2H)-one (24)
Product 24 was prepared from 17 with aminomethylcyclohexane. The red solid material was isolated in 54% yield (Rf = 0.41 at ethyl acetate:n-hexane = 6:1). Mp 163–164 °C (EtOH). FT-IR (KBr; ν cm−1): 3444 (OH), 1652 (C⚌O), 1616 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 1.08–1.93 (m, 10H, cyclohexane-CH2), 1.99 (br, 1H, cyclohexane-CH), 3.20 (t, J = 5.7 Hz, 2H, –NCH2–), 4.06–4.16 (m, 2H, –NCH2–), 4.06–4.16 (m, 1H, –OH), 4.64 (t, J = 5.1 Hz, 2H, –OCH2–), 6.85 (d, J = 8.7 Hz, 1H, Ar–H10), 7.39 (dd, J = 7.2, 1.2 Hz, 1H, Ar–H3), 7.48 (t, J = 8.1 Hz, 1H, Ar–H9), 7.63 (t, J = 7.5 Hz, 1H, Ar–H4), 7.82 (d, J = 7.2 Hz, 1H, Ar–H8), 7.88 (d, J = 8.4 Hz, 1H, Ar–H5), 10.21 (br, 1H, –NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 25.72, 26.31, 30.92, 37.33, 48.90, 52.77, 60.74, 109.10, 112.31, 113.91, 116.34, 119.61, 122.49, 127.09, 128.65, 133.23, 135.57, 139.04, 139.90, 154.02, 186.74 (C⚌O). HRMS (EI) m/z calcd for C23H25N3O2+ [M]+: 375.1947, found, 292.1080 (100), 375.1937 (25).
2.3.25 7-[3-(Hydroxyl)propylamino]-2-(2-hydroxyethyl)anthra[1,9-cd]pyrazol-6(2H)-one (25)
Product 25 was prepared from 17 with 3-amino-1-propanol. The red solid material was isolated in 75% yield (Rf = 0.18 at ethyl acetate:n-hexane = 6:1). Mp 208–209 °C (EtOH). FT-IR (KBr; ν cm−1): 3354 (OH), 1652 (C⚌O), 1614 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 1.95–2.00 (m, 2H, –CH2–), 3.44–3.51 (m, 2H, –CH2–), 3.77 (t, J = 5.1 Hz, 2H, –NCH2–), 4.09 (t, J = 4.8 Hz, 2H, –NCH2–), 4.08–4.11 (br, 1H, –OH), 4.65 (t, J = 4.8 Hz, 2H, –OCH2–), 6.90 (d, J = 7.8 Hz, 1H, Ar–H10), 7.41 (d, J = 7.2, 1.2 Hz, 1H, Ar–H3), 7.52 (t, J = 8.7 Hz, 1H, Ar–H9), 7.65 (t, J = 8.1 Hz, 1H, Ar–H4), 7.83 (d, J = 7.2 Hz, 1H, Ar–H8), 7.91 (d, J = 8.1 Hz, 1H, Ar–H5), 10.19 (br, 1H, –NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 32.28, 39.54, 52.77, 58.81, 60.80, 109.18, 112.19, 113.97, 116.22, 119.53, 122.49, 127.10, 128.61, 133.21, 135.50, 139.08, 139.88, 153.85, 186.62 (C⚌O). HRMS (EI) m/z calcd for C19H19N3O3+ [M]+: 337.1426, found, 292.1097 (100), 337.1432 (25).
2.3.26 7-Butylamino-2-(2-hydroxyethyl)anthra[1,9-cd]pyrazol-6(2H)-one (26)
Compound 26 was prepared from 17 with butylamine. The pure product was obtained as red powder (yield 58%) (Rf = 0.45 at ethyl acetate:hexane = 6:1). Mp 279–281 °C (EtOH). FT-IR (KBr; ν cm−1): 3319 (OH), 1649 (C⚌O), 1618 (C⚌N). 1H NMR (300 MHz, DMSO-d6): δ (ppm) 1.02 (t, J = 7.2 Hz, 3H, –CH3), 1.52–1.62 (m, 2H, –CH2–), 1.72–1.82 (m, 2H, –CH2–), 3.32–3.81 (m, 2H, –CH2–), 4.06–4.16 (br, 3H, –NCH2– & –OH), 4.64 (t, J = 5.1 Hz, 2H, –OCH2–), 6.85 (d, J = 8.7 Hz, 1H, Ar–H10), 7.39 (dd, J = 7.2, 1.2 Hz, 1H, Ar–H3), 7.49 (t, J = 8.1 Hz, 1H, Ar–H9), 7.63 (t, J = 7.5 Hz, 1H, Ar–H4), 7.81 (d, J = 6.9 Hz, 1H, Ar–H8), 7.88 (d, J = 8.1 Hz, 1H, Ar–H5), 10.13 (br, 1H, pyrazole-NH). 13C NMR (75 MHz, Acetone-d6): δ (ppm) 13.89, 20.09, 31.01, 42.11, 52.77, 60.77, 109.17, 112.22, 113.91, 116.28, 119.56, 122.49, 127.09, 128.62, 133.22, 135.54, 139.06, 139.89, 153.83, 186.69 (C⚌O). HRMS (EI) m/z calcd for C20H21N3O2+ [M]+: 335.1643, found 292.1085 (100), 335.1642 (20).
2.3.27 7-Methylamino-2-(2-hydroxyethyl)anthra[1,9-cd]pyrazol-6(2H)-one (27)
Compound 27 was prepared from 17 with methylamine. The pure product was obtained as red powder (yield 16%) (Rf = 0.24 at ethyl acetate:n-hexane = 3:2). Mp 213–214 °C (EtOH). FT-IR (KBr; ν cm−1): 3265 (OH), 1649 (C⚌O), 1620 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 3.04 (d, J = 4.8 Hz, 3H, –CH3), 4.11 (m, 3H, –NCH2– & –OH), 4.66 (t, J = 4.5 Hz, 2H, –OCH2–), 6.84 (d, J = 8.7 Hz, 1H, Ar–H10), 7.42 (d, J = 7.5 Hz, 1H, Ar–H3), 7.54 (t, J = 7.5 Hz, 1H, Ar–H9), 7.65 (t, J = 7.2 Hz, 1H, Ar–H4), 7.83 (d, J = 7.2 Hz, 1H, Ar–H8), 7.92 (d, J = 8.1 Hz, 1H, Ar–H5), 10.00 (br, 1H, –NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 29.47, 52.78, 60.76, 109.22, 111.93, 114.12, 116.37, 119.57, 122.51, 127.06, 128.67, 133.17, 135.61, 139.07, 139.94, 154.59, 186.59 (C⚌O). HRMS (EI) m/z calcd for C17H15N3O2+ [M]+: 293.1164, found 233.0775 (20), 244.0928 (35), 262.1017 (30), 276.1142 (20), 293.1160 (100).
2.3.28 7-Benzylamino-2-(2-hydroxyethyl)anthra[1,9-cd]pyrazol-6(2H)-one (28)
Compound 28 was prepared from 17 with benzylamine. The pure product was obtained as red powder (yield 54%) (Rf = 0.24 at ethyl acetate:n-hexane = 3:2). Mp 163–165 °C (EtOH). FT-IR (KBr; ν cm−1): 3309 (OH), 1652 (C⚌O), 1620 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 4.08–4.11 (m, 2H, –NCH2–), 4.08–4.11 (m, 1H, –OH), 4.65 (m, 4H, –CH2–), 6.85 (dd, J = 7.8, 2.1 Hz, 1H, Ar–H10), 7.30 (t, J = 7.2 Hz, 1H, Ar′–H4), 7.39 (t, J = 7.2 Hz, 2H, Ar′–H3,5), 7.48 (d, J = 6.3 Hz, 2H, Ar′–H2,6), 7.48 (d, J = 6.3 Hz, 1H, Ar–H3), 7.49 (t, J = 7.5 Hz, 1H, Ar–H9), 7.66 (t, J = 7.8 Hz, 1H, Ar–H4), 7.85 (d, J = 2.1 Hz, 1H, Ar–H8), 7.88 (d, J = 3.0 Hz, 1H, Ar–H5), 10.54 (br, 1H, –NH). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 46.42, 52.80, 60.75, 109.70, 112.65, 114.38, 116.49, 119.73, 122.49, 126.97, 127.56, 127.73, 128.67, 129.06, 133.28, 135.52, 138.96, 139.29, 139.93, 153.53, 186.86 (C⚌O). HRMS (EI) m/z calcd for C17H15N3O2+ [M]+: 369.1477, found, 292.1095 (20), 352.1239 (15), 369.1484 (100).
2.3.29 7-[2-(1-Cyclohexenyl)ethylamino]-2-(2-hydroxyethyl)anthra[1,9-cd]pyrazol-6(2H)-one (29)
Compound 29 was prepared from 17 with 2-(1-cyclohexenyl)ethylamine. The pure product was obtained as red powder (yield 62%) (Rf = 0.48 at ethyl acetate:hexane = 6:1). Mp 151–152 °C (EtOH). FT-IR (KBr; ν cm−1): 3446 (OH), 1652 (C⚌O), 1614 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 1.54–1.69 (m, 8H, cyclohexene-CH2), 2.40 (t, J = 6.6 Hz, 2H, –CH2–), 3.42 (dd, J = 13.8, 5.1 Hz, 2H, –CH2–), 4.08 (t, J = 5.1 Hz, 2H, –NCH2–), 4.04–4.10 (br, 1H, –OH), 4.64 (t, J = 5.4 Hz, 2H, –OCH2–), 5.66 (br, 1H, cyclohexene-CH), 6.84 (dd, J = 8.7, 0.9 Hz, 1H, Ar–H10), 7.38 (dd, J = 7.5, 1.5 Hz, 1H, Ar–H3), 7.49 (t, J = 8.1 Hz, 1H, Ar–H9), 7.61 (t, J = 7.8 Hz, 1H, Ar–H4), 7.79 (d, J = 6.9 Hz, 1H, Ar–H8), 7.87 (d, J = 8.1 Hz, 1H, Ar–H5), 10.13 (br, 1H, –NH–). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 22.26, 22.76, 25.10, 27.88, 37.19, 41.04, 52.76, 60.80, 109.24, 112.35, 114.06, 116.22, 119.55, 122.51, 123.46, 127.10, 128.61, 133.21, 135.04, 135.47, 139.10, 139.90, 153.65, 186.57 (C⚌O). HRMS (EI) m/z calcd for C24H25N3O2+ [M]+: 387.1947, found, 292.1094 (100), 387.1945 (5).
2.3.30 7-[2-(4-Methoxyphenyl)ethylamino]-2-(2-hydroxyethyl)anthra[1,9-cd]pyrazol-6(2H)-one (30)
Compound 30 was prepared from 17 with 2-(4-methoxyphenyl)ethylamine. The pure product was recrystallized from ethanol to get the red powder (yield 48%) (Rf = 0.35 at ethyl acetate:n-hexane = 6:1). Mp 151–152 °C (EtOH). FT-IR (KBr; ν cm−1): 3502 (OH), 1650 (C⚌O), 1616 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 3.02 (t, J = 7.2 Hz, 2H, –CH2–), 3.54–3.61 (m, 2H, –NCH2–), 3.71 (s, 3H, –OCH3), 4.07–4.90 (m, 2H, –NCH2– & –OH), 4.65 (t, J = 5.1 Hz, 2H, –OCH2–), 6.90 (d, J = 6.9 Hz, 4H, Ar′–H), 7.32 (d, J = 8.4 Hz, 1H, Ar–H10), 7.38 (dd, J = 7.2, 1.2 Hz, 1H, Ar–H3), 7.51 (t, J = 7.5 Hz, 1H, Ar–H9), 7.63 (t, J = 7.5 Hz, 1H, Ar–H4), 7.81 (d, J = 6.9 Hz, 1H, Ar–H8), 7.89 (d, J = 8.1 Hz, 1H, Ar–H5), 10.18 (br, 1H, –NH–). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 34.21, 44.27, 52.77, 55.39, 60.77, 109.36, 112.29, 114.03, 114.37, 116.31, 119.62, 122.48, 127.04, 128.63, 130.13, 131.57, 133.22, 135.56, 139.04, 139.91, 153.57, 158.42, 186.62 (C⚌O). HRMS (EI) m/z calcd for C25H23N3O3+ [M]+: 413.1739, found 292.1093 (100), 413.1736 (8).
2.3.31 7-(2-Methoxybenzylamino)-2-(2-hydroxyethyl)anthra[1,9-cd]pyrazol-6(2H)-one (31)
Compound 31 was prepared from 17 with 2-methoxybenzylamine. The pure product was recrystallized from dichloromethane and hexane to get the red powder (yield 54%) (Rf = 0.35 at ethyl acetate:hexane = 6:1). Mp 181–182 °C. FT-IR (KBr; ν cm−1): 3419 (OH), 1652 (C⚌O), 1618 (C⚌N). 1H NMR (300 MHz, Acetone-d6): δ (ppm) 3.93 (s, 3H, –OCH3), 4.08–4.11 (m, 2H, –NCH2– & –OH), 4.59 (d, J = 6.0 Hz, 2H, –NCH2–), 4.65 (t, J = 5.1 Hz, 2H, –OCH2–), 6.85 (dd, J = 8.1, 1.5 Hz, 1H, Ar–H10), 6.94 (t, J = 7.2 Hz, 1H, Ar′–H5), 7.06 (d, J = 7.8 Hz, 1H, Ar–H3), 7.29 (t, J = 7.2 Hz, 1H, Ar–H9), 7.37 (d, J = 7.2 Hz, 1H, Ar′–H3), 7.41 (dd, J = 7.5, 1.5 Hz, 1H, Ar′–H6), 7.47 (t, J = 7.8 Hz, 1H, Ar′–H4), 7.64 (t, J = 7.8 Hz, 1H, Ar–H4), 7.83 (d, J = 7.2 Hz, 1H, Ar–H8), 7.90 (d, J = 8.1 Hz, 1H, Ar–H5), 10.48 (br, 1H, –NH–). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 41.51, 52.77, 55.87, 60.75, 109.47, 111.53, 112.46, 114.30, 116.39, 119.66, 120.85, 122.49, 126.68, 127.02, 128.64, 128.80, 129.01, 133.27, 135.51, 139.00, 139.91, 153.63, 157.66, 186.72 (C⚌O). HRMS (EI) m/z calcd for C24H21N3O3+ [M]+: 399.1583, found, 292.1063 (15), 382.1311 (15), 399.1587 (100).
2.3.32 7-Piperonylamino-2-(2-hydroxyethyl)anthra[1,9-cd]pyrazol-6(2H)-one (32)
Compound 32 was prepared from 17 with piperonylamine. The pure product was obtained as red powder (yield 43%) (Rf = 0.35 at ethyl acetate:hexane = 6:1). Mp 219–220 °C. FT-IR (KBr; ν cm−1): 3273 (OH), 1616 (C⚌O), 1593 (C⚌N). 1H NMR (300 MHz, DMSO-d6): δ (ppm) 3.88 (q, J = 5.1 Hz, 2H, –CH2–), 4.45 (d, J = 5.4 Hz, 2H, –CH2–), 4.58 (t, J = 5.7 Hz, 2H, –CH2–), 4.94 (t, J = 5.4 Hz, 1H, –OH), 6.00 (s, 2H, –OCH2O–), 6.83 (d, J = 8.1 Hz, 1H, Ar–H10), 6.90 (d, J = 0.9 Hz, 2H, Ar′–H5,6), 6.98 (s, 1H, Ar′–H2), 7.33 (dd, J = 7.2, 1.2 Hz, 1H, Ar–H3), 7.49 (t, J = 8.7 Hz, 1H, Ar–H9), 7.65 (t, J = 7.5 Hz, 1H, Ar–H4), 7.78 (d, J = 6.9 Hz, 1H, Ar–H8), 7.96 (d, J = 8.1 Hz, 1H, Ar–H5), 10.31 (t, J = 5.7 Hz, 1H, –NH–). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 46.26, 52.83, 60.80, 101.39, 108.33, 108.77, 109.75, 112.73, 114.39, 116.54, 119.79, 121.05, 122.53, 127.03, 128.75, 133.13, 133.29, 135.58, 139.01, 139.97, 146.93, 148.10, 153.48, 186.88 (C⚌O). HRMS (EI) m/z calcd for C24H19N3O4+ [M]+: 413.1376, found, 413.1375.
2.4 Sulforhodamine B assay
Sulforhodamine B was obtained from Sigma–Aldrich Corp. (St. Louis, MO, USA). Human hormone-refractory prostate cancer cell line PC-3 was from American Type Culture Collection (ATCC) (Rockville, MD). The cells were cultured in RPMI1640 medium with penicillin (100 units/mL)/streptomycin (0.1 mg/mL) and 5% fetal bovine serum (FBS) (v/v). Cultures were maintained in a humidified incubator at 37 °C in 95% air and 5% CO2. About 5 × 103 cells were seeded in 96-well plates in medium with 5% FBS. After 24 h, PC-3 cells were fixed with 10% trichloroacetic acid (TCA) to represent cell population at the time of compound addition (T0). After additional incubation of vehicle (0.1% DMSO) or the indicated compound for 48 h, cells were fixed with 10% TCA and SRB at 0.4% (w/v) in 1% acetic acid was added to stain cells. Unbound SRB was washed out by 1% acetic acid and SRB bound cells were solubilized with 0.01 M Trizma base. The absorbance was detected at a wavelength of 515 nm by spectrophotometer. Using the following absorbance measurements, such as time zero (T0), control growth (C), and cell growth in the presence of compound (Tx), the percentage growth was calculated at each of the compound concentrations levels. Percentage growth inhibition was calculated as: 100 − [(Tx − T0)/(C − T0)] × 100 for concentrations for which Tx ⩾ T0. The value of GI50 was determined at the drug concentration which results in 50% reduction of total protein increase in control cells during the compound incubation.
2.5 Telomeric repeat amplification protocol (TRAP) assays
The activity of telomerase was investigated by a modified version of the telomeric repeat amplification protocol (TRAP) protocol (Kim et al., 1994; Huang et al., 2008). Telomerase products were resolved by 10% polyacrylamide gel electrophoresis and visualized by staining with SYBER Green. As a source of telomerase, the total cell lysates derived from lung cancer cell line H1299 cells were used. Bovine serum albumin (BSA) was used as a standard to quantify protein. According to the BSA standard curve, the protein concentration of the lysates was assayed using Bio-Rad protein assay kit.
3 Biological evaluation
All of the synthesized compounds (1–32) were tested against PC-3 cell line by sulforhodamine B (SRB) assay. Simultaneously, the effects of compounds (1–32) for the inhibitory of telomerase by TRAP assay were also evaluated (Kim et al., 1994; Huang et al., 2008). Moreover, fifteen of our structures (1–6, 13, 14, 16, 17, 19, 21, 23, 28 and 31) were selected by NCI to be investigated against a panel of 60 human cancer cell lines. After a primary screening, compounds 1, 4 and 16 were chosen for further cell growth inhibition screening for the 50% growth inhibitory concentration (GI50), the total growth inhibition (TGI), and the 50% lethal concentration (LC50) by NCI.
3.1 Cytotoxicity evaluation
Cell survival rate or proliferation was determined by a common method, SRB assay. PC-3 cell line was used to evaluate the cell toxicity of each compound. Untreated cell was used as the control group, while compound-treated cell was used as the test group. All of the synthesized compounds were investigated and the values of IC50 were described as Table 1. Compounds with linear alkanes as side chain were exhibited the worse activities than others. Such as compounds 3/19 and 11/27, the IC50 values were higher than 30 μM. It is interesting about compounds 4, 12, 15, and 16 where the values of IC50 were less than 10 μM.
| Compound | IC50 (μM) | Compound | IC50 (μM) |
|---|---|---|---|
| 1 | 18.10 | 17 | 13.70 |
| 2 | 17.83 | 18 | 28.51 |
| 3 | >30 | 19 | >30 |
| 4 | 9.99 | 20 | >30 |
| 5 | 21.87 | 21 | >30 |
| 6 | 12.85 | 22 | >30 |
| 7 | 15.84 | 23 | 22.17 |
| 8 | 20.05 | 24 | 17.96 |
| 9 | 14.47 | 25 | 18.69 |
| 10 | 24.30 | 26 | >30 |
| 11 | >30 | 27 | >30 |
| 12 | 8.63 | 28 | >30 |
| 13 | >30 | 29 | 21.09 |
| 14 | >30 | 30 | >30 |
| 15 | 8.44 | 31 | >30 |
| 16 | 5.49 | 32 | >30 |
| SP600125 | N.D. | Mitoxantrone | 0.29 |
3.2 In vitro antiproliferative activity
The 7-substituted anthra[1,9-cd]pyrazol-6(2H)-one homologues were investigated for antiproliferative activity against human tumor cell lines in NCI Drug Screen Program. The tumor growth inhibition properties of the fifteen compounds 1, 2, 3, 4, 5, 6, 13, 14, 16, 17, 19, 21, 23, 28 and 31 with the NCI codes NSC764641, 764630, 764631, 764632, 764633, 764634, 764635, 764636, 764637, 764642, 764625, 764626, 764627, 764628 and 764629 (Fig. 2) selected among all submitted compounds were screened on human cancer cell lines at a primary single concentration (10 μM) at the NIH, Bethesda, Maryland, USA, under the drug discovery program of the NCI. The selected compounds were evaluated in NCI’s nine types of neoplastic cell lines and incubated for 48 h. And each subpanel has several neoplastic cell lines. The anticancer assay was executed in light of the protocol of developmental therapeutics program (one dose mean graph). Results of selected synthetic compounds were reported as the growth percent value of the treated cells (experimental group) when compared with that of the untreated cells (control group). The growth percentages at 10 μM for NCI 60 cancer cell lines are described in Table 2.
Selected compounds for testing in the NCI screens.
| Panel/Cell line | Compounds/Growth percent (%)a | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Compd. No. | 1 | 2 | 3 | 4 | 5 | 6 | 13 | 14 | 16 | 17 | 19 | 21 | 23 | 28 | 31 |
| NCI No. | NSC764641 | NSC764630 | NSC764631 | NSC764632 | NSC764633 | NSC764634 | NSC764635 | NSC764636 | NSC764637 | NSC764642 | NSC764625 | NSC764626 | NSC764627 | NSC764628 | NSC764629 |
| Leukemia | |||||||||||||||
| CCRF-CEM | 72.25 | 69.66 | 77.17 | 38.99 | 50.81 | 69.02 | 83.68 | 95.35 | 10.17 | 90.58 | 93.22 | 89.87 | 69.41 | 92.80 | 80.93 |
| HL-60(TB) | 64.34 | 87.69 | 77.93 | 38.88 | 69.81 | 92.25 | 90.36 | 96.19 | 46.10 | 96.18 | 102.79 | 98.95 | 71.58 | 101.09 | 91.91 |
| K-562 | 60.92 | 80.19 | 79.91 | 47.34 | 69.93 | 64.66 | 94.72 | 93.14 | 48.87 | 83.40 | 95.59 | 88.19 | 33.24 | 73.94 | 50.06 |
| MOLT-4 | 43.42 | 55.89 | 64.13 | 18.61 | 48.82 | 60.33 | 88.72 | 80.18 | 33.14 | 77.04 | 94.56 | 81.97 | 71.19 | 87.22 | 88.99 |
| RPMI-8226 | 71.57 | 77.12 | 86.31 | 33.11 | 82.55 | 66.41 | 113.59 | 90.33 | 38.17 | 84.67 | 91.36 | 96.99 | 58.44 | 95.08 | 86.81 |
| SR | 45.92 | 69.67 | 67.94 | 17.73 | 62.97 | 55.34 | 85.91 | 76.95 | 3.65 | 65.84 | 78.75 | 78.79 | 74.70 | 64.40 | 39.24 |
| Non-small cell lung cancer | |||||||||||||||
| A549/ATCC | 52.33 | 90.46 | 80.32 | 48.31 | 58.41 | 76.21 | 90.63 | 84.45 | 17.90 | 86.72 | 90.38 | 95.83 | 82.33 | 93.54 | 89.26 |
| EKVX | N.T.b | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. |
| HOP-62 | 80.31 | 95.56 | 93.62 | 75.27 | 91.46 | 85.59 | 106.94 | 97.61 | 42.48 | 81.08 | 94.20 | 103.30 | 98.88 | 96.45 | 84.77 |
| HOP-92 | 71.18 | 68.14 | 69.27 | 30.65 | 73.24 | 71.31 | 105.09 | 101.06 | 58.85 | 84.53 | 64.09 | 85.26 | 60.17 | 76.63 | 81.80 |
| NCI-H226 | 55.98 | 70.79 | 68.72 | 50.07 | 77.88 | 73.32 | 101.95 | 91.47 | 50.65 | 84.76 | 96.27 | 100.24 | 103.60 | 90.18 | 90.46 |
| NCI-H23 | 65.41 | 80.11 | 72.41 | 61.77 | 83.86 | N.T. | 101.41 | 97.47 | 49.82 | N.T. | 86.16 | N.T. | 88.34 | 86.57 | 84.17 |
| NCI-H322M | 35.74 | 50.30 | 38.33 | 26.56 | 52.66 | 52.43 | 108.29 | 69.08 | 26.79 | 99.99 | 80.21 | 93.11 | 79.74 | 94.81 | 99.75 |
| NCI-H460 | 49.95 | 77.69 | 69.52 | 44.19 | 68.22 | 73.29 | 106.09 | 97.48 | 12.78 | 91.80 | 104.90 | 106.44 | 91.74 | 109.85 | 104.05 |
| NCI-H522 | 87.21 | 52.57 | 65.10 | 29.89 | 105.42 | 76.29 | 89.05 | 75.02 | 33.13 | 66.91 | 125.19 | 83.99 | 79.75 | 73.93 | 47.86 |
| Colon cancer | |||||||||||||||
| COLO 205 | 81.44 | 97.98 | 103.19 | 57.19 | 95.32 | 109.19 | 131.78 | 125.12 | 30.20 | 103.62 | 115.23 | 108.30 | 122.92 | 116.31 | 106.58 |
| HCC-2998 | 71.16 | 108.70 | 91.33 | 51.08 | 84.82 | 100.42 | 126.36 | 117.28 | 82.41 | 108.75 | 108.06 | 118.43 | 105.58 | 106.62 | 97.53 |
| HCT-116 | 47.10 | 68.36 | 68.42 | 31.53 | 51.51 | 74.44 | 102.62 | 96.34 | 2.72 | 69.77 | 73.87 | 98.77 | 88.67 | 85.21 | 78.36 |
| HCT-15 | 41.22 | 59.46 | 52.34 | 28.53 | 51.55 | 49.99 | 80.66 | 79.40 | 25.11 | 77.50 | 90.12 | 84.36 | 61.74 | 77.59 | 67.16 |
| HT29 | 69.39 | 72.99 | 78.69 | 44.13 | 72.31 | 67.23 | 90.01 | 99.40 | 56.29 | 81.60 | 102.62 | 91.38 | 62.63 | 108.53 | 95.72 |
| KM12 | 14.94 | 17.68 | 14.32 | 12.27 | 19.72 | 24.04 | 45.94 | 38.96 | 7.38 | 93.13 | 98.28 | 88.44 | 56.26 | 80.85 | 62.93 |
| SW-620 | 63.57 | 83.61 | 80.67 | 45.29 | 67.02 | 85.62 | 114.80 | 110.14 | 30.53 | 80.26 | 99.26 | 107.11 | 110.89 | 108.30 | 91.88 |
| CNS cancer | |||||||||||||||
| SF-268 | 65.07 | 70.79 | 66.92 | 44.22 | 81.16 | 84.34 | 99.94 | 95.89 | 44.88 | 99.92 | 97.41 | 112.17 | 87.04 | 98.08 | 96.21 |
| SF-295 | 64.49 | N.T. | 98.80 | 41.04 | 67.97 | 79.06 | 94.31 | 97.94 | 42.56 | 80.19 | 80.18 | 91.93 | 83.96 | 95.34 | 79.77 |
| SF-539 | 68.94 | 82.10 | 73.48 | 49.85 | 70.18 | 83.73 | 111.58 | 87.65 | 50.65 | 78.46 | 88.23 | 93.71 | 79.25 | 85.44 | 85.77 |
| SNB-19 | 69.04 | 76.12 | 81.20 | 76.70 | 112.41 | 81.58 | 108.66 | 117.68 | 100.12 | 68.72 | 97.48 | 101.70 | 107.86 | 98.59 | 104.59 |
| SNB-75 | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. |
| U251 | 50.57 | 81.87 | 68.65 | 46.95 | 65.74 | 69.86 | 94.20 | 102.40 | 47.35 | 78.89 | 90.22 | 93.27 | 84.90 | 92.71 | 94.34 |
| Melanoma | |||||||||||||||
| LOX IMVI | 46.74 | 60.49 | 56.87 | 35.26 | 63.53 | 54.02 | 95.44 | 94.76 | 12.49 | 65.68 | 93.25 | 95.81 | 68.69 | 89.94 | 88.00 |
| MALME-3M | 76.88 | 91.98 | 84.27 | 47.42 | 92.31 | 90.01 | 114.64 | 136.43 | 47.89 | 99.26 | 119.47 | 38.24 | 117.84 | 117.85 | 93.65 |
| M14 | 61.17 | 76.76 | 69.15 | 32.70 | 65.29 | 76.87 | 104.93 | 96.82 | 32.43 | 112.09 | 111.61 | 96.41 | 107.26 | 97.10 | 88.70 |
| MDA-MB-435 | 79.19 | 87.77 | 87.90 | 49.40 | 75.77 | 93.96 | 101.32 | 104.19 | 29.09 | 100.25 | 104.77 | 104.26 | 95.20 | 74.17 | 37.73 |
| SK-MEL-2 | 46.42 | 81.43 | 76.34 | 19.69 | 77.84 | 87.27 | 95.15 | 86.33 | 10.31 | 127.18 | 101.84 | 105.67 | 108.46 | 88.33 | 86.66 |
| SK-MEL-28 | 107.22 | 89.27 | 103.68 | 62.00 | 98.33 | 99.55 | 111.78 | 113.85 | 48.10 | 116.68 | 118.63 | 108.10 | 103.12 | 101.72 | 88.85 |
| SK-MEL-5 | 71.84 | 97.41 | 99.13 | 58.22 | 98.33 | 91.74 | 103.10 | 95.71 | 36.82 | 79.74 | 96.90 | 96.71 | 99.11 | 81.37 | 93.41 |
| UACC-257 | 92.84 | 113.65 | 107.14 | 91.19 | 119.93 | 105.21 | 119.81 | 122.83 | 81.17 | 111.04 | 109.67 | 120.33 | 117.00 | 118.41 | 99.59 |
| UACC-62 | 65.64 | 80.42 | 77.31 | 59.19 | 76.52 | 80.31 | 90.89 | 91.33 | 59.70 | 102.26 | 96.78 | 88.99 | 93.71 | 85.51 | 78.23 |
| Ovarian cancer | |||||||||||||||
| IGROV1 | 38.56 | 72.95 | 56.79 | 28.99 | 71.93 | 63.04 | 98.92 | 124.74 | 42.86 | 90.68 | 110.15 | 110.96 | 106.75 | 133.22 | 91.80 |
| OVCAR-3 | 83.32 | 92.19 | 96.61 | 75.85 | 108.16 | 99.19 | 107.31 | 103.36 | 10.68 | 116.58 | 114.22 | 118.27 | 107.28 | 111.75 | 102.87 |
| OVCAR-4 | 56.40 | 66.67 | 66.24 | 55.45 | 77.62 | 57.51 | 93.80 | 78.56 | −2.57 | 92.86 | 114.07 | 94.39 | 98.07 | 87.29 | 90.44 |
| OVCAR-5 | 75.70 | 72.53 | 78.24 | 53.83 | 86.41 | 76.69 | 97.95 | 94.39 | 61.57 | 101.52 | 89.66 | 89.63 | 81.42 | 91.11 | 88.08 |
| OVCAR-8 | 74.44 | 79.36 | 81.21 | 44.95 | 90.68 | 81.38 | 99.23 | 94.67 | 58.26 | 81.99 | 91.78 | 99.25 | 86.92 | 93.72 | 99.52 |
| NCI/ADR-RES | 88.31 | 85.02 | 85.71 | 52.27 | 104.43 | 80.89 | 109.11 | 102.75 | 60.31 | 87.66 | 86.90 | 99.81 | 91.37 | 90.02 | 82.61 |
| SK-OV-3 | 40.75 | 81.11 | 70.98 | 12.71 | 85.74 | 92.52 | 124.84 | 105.21 | 44.75 | 96.10 | 97.40 | 115.11 | 108.40 | 100.74 | 98.07 |
| Renal cancer | |||||||||||||||
| 786-O | 70.65 | 98.05 | 95.79 | 49.43 | 102.03 | 93.17 | 109.16 | 108.24 | 46.54 | 82.52 | 100.26 | 109.34 | 91.59 | 104.28 | 102.84 |
| A498 | 23.72 | 47.85 | 21.97 | 46.78 | 81.12 | 66.82 | 108.64 | 96.94 | 39.52 | 73.74 | 104.91 | 78.80 | 91.23 | 86.12 | 108.92 |
| ACHN | 37.56 | 61.12 | 49.48 | 32.24 | 65.34 | 53.75 | 85.00 | 79.94 | 5.18 | 69.30 | 101.63 | 100.26 | 90.02 | 100.54 | 93.55 |
| CAKI-1 | 14.82 | 61.44 | 43.54 | 22.08 | 54.26 | 50.21 | 85.41 | 81.95 | 10.20 | 64.42 | 90.08 | 101.74 | 88.81 | 106.01 | 100.36 |
| RXF 393 | 39.34 | 45.58 | 51.99 | 19.32 | 57.21 | 46.50 | 97.75 | 92.67 | −15.19 | 55.70 | 96.67 | 84.71 | 38.44 | 76.63 | 74.41 |
| SN12C | 59.11 | 70.36 | 69.18 | 36.27 | 80.41 | 69.76 | 97.18 | 101.67 | 45.04 | 80.23 | 93.61 | 103.07 | 82.64 | 96.04 | 97.13 |
| TK-10 | 40.75 | 82.27 | 77.73 | 40.70 | 76.94 | 74.04 | 106.29 | 93.91 | 6.44 | 54.51 | 90.16 | 108.19 | 99.06 | 109.57 | 109.16 |
| UO-31 | 24.18 | 30.63 | 34.36 | 20.06 | 58.39 | 20.02 | 77.73 | 117.61 | 14.35 | 48.97 | 90.54 | 97.36 | 75.33 | 94.90 | 88.47 |
| Prostate cancer | |||||||||||||||
| PC-3 | 71.00 | 82.65 | 74.17 | 51.65 | 84.22 | 80.22 | 102.99 | 97.07 | 60.59 | 103.57 | 94.65 | 102.57 | 96.50 | 99.14 | 101.38 |
| DU-145 | 63.43 | 55.93 | 57.60 | 40.21 | 64.89 | 77.60 | 92.14 | 93.75 | 28.00 | 101.80 | 110.96 | 101.76 | 90.10 | 97.82 | 93.81 |
| Breast cancer | |||||||||||||||
| MCF7 | 59.11 | 45.12 | 55.76 | 59.14 | 51.58 | 46.79 | 74.89 | 64.43 | 46.67 | 88.00 | 111.58 | 74.15 | 105.97 | 30.77 | 54.58 |
| MDA-MB-231/ATCC | 74.83 | 39.83 | 62.09 | 50.57 | 73.55 | 51.79 | 89.96 | 92.24 | 22.71 | 86.56 | 81.87 | 85.88 | 57.52 | 70.77 | 79.16 |
| HS 578T | 62.55 | 67.17 | 73.09 | 48.99 | 91.45 | 73.74 | 119.20 | 110.21 | 56.16 | 77.35 | 87.37 | 105.55 | 90.28 | 106.60 | 97.59 |
| BT-549 | 64.63 | 83.10 | 77.53 | 44.56 | 80.51 | 94.63 | 113.81 | 104.30 | 48.59 | 98.91 | 89.08 | 106.30 | 95.84 | 91.94 | 88.88 |
| T-47D | 66.36 | 53.70 | 51.52 | 32.27 | 73.81 | 55.39 | 92.13 | 80.54 | 31.08 | 89.44 | 87.23 | 63.96 | 81.80 | 81.08 | 84.12 |
| MDA-MB-468 | 56.11 | 28.79 | 49.69 | 52.10 | 36.51 | 43.93 | 52.99 | 50.44 | 25.53 | 69.13 | 97.49 | 47.43 | 80.55 | −15.55 | 9.29 |
| Mean | 60.29 | 72.42 | 71.24 | 43.37 | 75.19 | 72.42 | 72.89 | 98.98 | 36.17 | 86.67 | 96.96 | 95.54 | 87.26 | 91.19 | 85.74 |
| Delta | 45.47 | 54.74 | 56.92 | 31.10 | 55.47 | 54.74 | 52.87 | 53.04 | 51.36 | 37.70 | 32.87 | 57.30 | 54.02 | 106.74 | 76.45 |
| Range | 92.40 | 95.97 | 92.82 | 78.92 | 100.21 | 95.97 | 89.17 | 85.84 | 115.31 | 78.21 | 61.10 | 82.09 | 115.31 | 78.21 | 61.10 |
3.3 Telomerase inhibitory activities
Telomerase activity plays an important role of cell proliferation. In our previous studies, we pay attention to the potency of the synthetic derivatives against telomerase activities in the cell free extracts of H1299 cells. We evaluated the effects of the synthetic compounds on telomerase inhibition by TRAP assay. In the TRAP assay, if a compound can stabilize the telomeric sequences such that telomerase cannot act on telomere, then the effect of inhibiting the telomerase can be achieved. DMSO treatment represents as the positive control group (P), while a negative control group (N) used 5 μL of 100 μg/mL RNase A (CLONTECH) in the assay. It could be observed that a number of telomeric fragments were detected in the positive control group (P), while none was detected in the negative control group (N). 10 μM was selected as the concentration used to screen compounds.
4 Results and discussion
The reaction of 1,5-dichloroanthraquinone and hydrazine monohydrate was stirred at 120 °C in dimethyl sulfoxide (DMSO) and used N,N-diisopropylethylamine (DIPEA) as a basic catalyst for 2 h to obtain the key intermediate 1. Compound 17 is another key intermediate which was obtained from 1,5-dichloroanthraquinone, 2-hydroxyethylhydrazine and pyridine under the same condition. The reaction of appropriate primary amines with 1 or 17 in DMSO was stirred at 120 °C for 4 h and gained the corresponding 7-substituted anthra[1,9-cd]pyrazol-6(2H)-one (2–16) or 7-substituted 2-(2-hydroxyethyl)anthra[1,9-cd]pyrazol-6(2H)-one (18–32) (Scheme 1).
A series of 7-substituted anthra[1,9-cd]pyrazol-6(2H)-one derivatives were synthesized and the preparation involved various synthetic approaches with approximate yields (overall 14–75% in all steps) (Scheme 1). First, 1,5-dichloroanthraquinone with hydrazine monohydrate and DIPEA or with 2-hydroxyethylhydrazine and pyridine were dissolved in DMSO and heated at 120 °C for 2 h. Then, the key intermediate 1/17 with a series of primary amines and 4-dimethylaminopyridine (DMAP) yielded the corresponding side chain compounds 2–16 and 18–32, respectively. The quantity of all the synthetic compounds was dependent on substrates and the by-products isolated that purifies of the reaction mixtures required tedious recrystallization from ethyl acetate/n-hexane and/or ethanol. The protons and carbons from anthrapyrazole structures were also obvious from the 1H NMR and 13C NMR spectra, respectively. The exact molecular weight of all synthetic compounds 1–32 was determined using high resolution mass spectrometry (HRMS).
In SRB assay, four compounds (4, 12, 15, and 16) showed potent cell cytotoxicity with the IC50 values less than 10 μM. We found the pyrazole ring modified with a hydroxyethyl group was failed to grasp the main points. And the substituted side chains of anthrapyrazole derivatives were preferred benzylamine side chains which were exhibited the outstanding cytotoxicity. All the synthetic compounds were investigated but found none of the synthetic compounds showed significant repressive effects (data not shown).
According to the data from NCI 60 cancer cell lines, 28 could achieve inhibiting effects against breast cancer cell line MDA-MB-468, up to −15.55%. Compound 16 (NSC764637) was efficacious against renal cancer cell strain RXF 393, ACHN and TK-10, and could measure an inhibition effect on the growth of the said cancer cells up to −15.19%, 5.18% and 6.44%, respectively. Besides, 16 also has impactful against OVCAR-4 (ovarian cancer), SR (leukemia), HCT-116, and KM12 (colon cancer) cell lines that could attain a growth inhibition effect at −2.57%, 3.65%, 2.72% and 7.38%, severally. Compounds 4 and 16 were found that might have cytotoxic effect against Adriamycin (ADR)-resistance. The tested compounds, which decrease the average of growth percentage more than 33%, were picked out for further evaluation in the nine major panel of NCI’s 60 human cancer cell lines. As shown in Table 2, three compounds 1, 4 and 16 of the fifteen tested compounds in primary 60-cell panel assay showed salient cell growth inhibition were incubated for a advanced 60-cell panel assay at five logarithm concentrations (10−2, 10−1, 100, 101 and 102 μM) and the consequences were indicated with GI50, TGI and LC50.
Results of the research spread the initial in vitro observation described in the data above and confirm the importance and significance of anticancer activity. Compound 1 carrying with a chlorine atom at 7-position exhibited relatively growth inhibition activity but not cytotoxic activity. SP600125 (NSC75890), a well-known anthrapyrazole, has no substitution at 7-position and the average of GI50 over all NCI’s cell lines is about 20.3 μM (from NCI Cancer Screen Data, 10/2009). Comparison to 4 with side chain –NH(CH2)4CH2OH displayed relatively growth inhibition activity but not cytotoxic activity. Compound 4 had also the best potent activity against colon cancer KM12 with GI50 as little as 0.54 μM, but LC50 and TGI with >100 μM. The nine dose–response curves displayed that the renal cancer subpanel was the most sensitive part for 4, with GI50 of seven renal cancer cell lines ranging from 1.01–14.9 μM. The prostate cancer subpanel was the second most sensitive with GI50 between 4.35 and 7.50 μM. Moreover, it is especially notable that 16 with a bulky side chain (piperonylamine) was quite sensitive for all subpanels (GI50 < 10 μM), except leukemia. Compound 16 was also found that might have cytotoxic effect against Adriamycin (ADR)-resistance cell line (NCI/ADR-RES) at 5.66 μM. For all antitumor activity parameter in NCI Drug Screen Program, the averaged values of mean graph midpoint (MG-MID) were calculated over 60 cell lines.
As shown in Table 3 for compound 1, the average concentration required to inhibit GI50 was 10.50 μM with a range of 2.50 μM (colon cancer: KM12) to >100 μM (melanoma: SK-MEL-28). The average concentration of 4 required to inhibit GI50 was 20.83 μM with a range of 0.54 μM (colon cancer: KM12) to >100 μM (colon cancer: HCC-2998; CNS cancer: SNB-19; melanoma: SK-MEL-28 and UACC-257; ovarian cancer: OVCAR-3 and OVCAR-4). And the average concentration of 16 required to inhibit GI50 was 4.52 μM with a range of 0.96 μM (colon cancer: KM12) to 56.4 μM (leukemia: HL-60). In addition, >100 μM (10−4 M) was required to achieve LC50 in the great majority of 60-human tumor cell lines, cell line sensitive to 1 and 4, exhibited that both the values of LC50 and TGI are more than 100 μM (10−4 M) (colon cancer: KM12). And cell line sensitive to the other 16, exhibited an LC50 with as little as 16.6 μM and also TGI with >100 μM (colon cancer: KM12). Compounds 1, 4 and 16 showed dose-dependent inhibition of proliferation in 60 tumor cell lines. All the data were summarized in Table 3 and used for further analysis. According to the ratio obtained by distinguishing the full panel MID (the average sensitivity of all cell lines toward the test agent), the selected compounds showed different selectivity (Noolvi et al., 2011). The values of selectivity ratio greater than 6 refer to high selectivity; values between 3 and 6 indicate moderately toward the homologous cell line, while compounds are not in line with either of these criteria accounted non-selective (Acton et al., 1994; Rostom, 2006). On the basis of the selectivity ratio, 4 in the study was found to be moderate selectivity for growth inhibition regarding renal and prostate cancer subpanels with selectivity ratio of 4.797 and 3.516, otherwise it was found lower selectivity against other cell panels (Table 3).
| Panel/Cell line (μM) | 1 (NSC764641) | 4 (NSC764632) | 16 (NSC764637) | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| GI50 | TGI (μM) | LC50 (μM) | GI50 | TGI (μM) | LC50 (μM) | GI50 | TGI (μM) | LC50 (μM) | |||||||
| (μM) | Subpanel MIDb | Selectivity ratio | (μM) | Subpanel MIDb | Selectivity ratio | (μM) | Subpanel MIDb | Selectivity ratio | |||||||
| Leukemia | |||||||||||||||
| CCRF-CEM | 6.32 | 11.000 | 0.954 | >100 | >100 | 6.51 | 23.580 | 0.883 | >100 | >100 | 3.31 | 13.508 | 0.336 | >100 | >100 |
| HL-60(TB) | 36.9 | >100 | >100 | 51.2 | >100 | >100 | 56.4 | >100 | >100 | ||||||
| K-562 | 7.80 | >100 | >100 | 69.6 | >100 | >100 | 9.08 | >100 | >100 | ||||||
| MOLT-4 | 4.41 | >100 | >100 | 6.88 | >100 | >100 | 6.32 | 77.9 | >100 | ||||||
| RPMI-8226 | 7.48 | >100 | >100 | 4.18 | >100 | >100 | 3.00 | 17.3 | >100 | ||||||
| SR | 3.09 | >100 | >100 | 3.11 | >100 | >100 | 2.94 | 53.1 | >100 | ||||||
| Non-small cell lung cancer | |||||||||||||||
| A549/ATCC | 4.87 | 6.477 | 1.621 | >100 | >100 | 9.09 | 12.940 | 1.610 | >100 | >100 | 2.76 | 3.201 | 1.419 | 10.4 | 78.6 |
| EKVX | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | ||||||
| HOP-62 | 12.3 | >100 | >100 | 7.00 | >100 | >100 | 3.18 | 8.37 | 30.7 | ||||||
| HOP-92 | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | ||||||
| NCI-H226 | 7.77 | >100 | >100 | 18.00 | >100 | >100 | 2.52 | 10.4 | 49.7 | ||||||
| NCI-H23 | 6.65 | >100 | >100 | 41.7 | >100 | >100 | 4.72 | 30.2 | >100 | ||||||
| NCI-H322M | 5.62 | >100 | >100 | 4.52 | >100 | >100 | 3.2 | 12.8 | >100 | ||||||
| NCI-460 | 4.15 | >100 | >100 | 6.01 | >100 | >100 | 2.52 | 7.44 | >100 | ||||||
| NCI-H522 | 3.98 | >100 | >100 | 4.26 | >100 | >100 | 3.51 | 20.9 | >100 | ||||||
| Colon cancer | |||||||||||||||
| COLO 205 | 12.2 | 9.763 | 1.075 | >100 | >100 | 18.4 | 19.830 | 1.051 | >100 | >100 | 2.83 | 3.517 | 1.291 | 6.33 | 39.8 |
| HCC-2998 | 30.9 | >100 | >100 | >100 | >100 | >100 | 6.77 | >100 | >100 | ||||||
| HCT-116 | 5.42 | >100 | >100 | 3.87 | >100 | >100 | 2.19 | 4.92 | 14.1 | ||||||
| HCT-15 | 4.34 | >100 | >100 | 2.02 | >100 | >100 | 3.44 | >100 | >100 | ||||||
| HT29 | 7.84 | >100 | >100 | 7.56 | >100 | >100 | 4.61 | 20.4 | >100 | ||||||
| KM12 | 2.50 | >100 | >100 | 0.54 | >100 | >100 | 0.96 | 16.6 | >100 | ||||||
| SW-620 | 5.14 | >100 | >100 | 6.42 | >100 | >100 | 3.82 | 19.7 | >100 | ||||||
| CNS cancer | |||||||||||||||
| SF-268 | 8.71 | 8.053 | 1.304 | >100 | >100 | 7.95 | 28.398 | 0.734 | >100 | >100 | 5.44 | 4.358 | 1.042 | 29.4 | >100 |
| SF-295 | 7.79 | >100 | >100 | 6.19 | >100 | >100 | N.T. | N.T. | N.T. | ||||||
| SF-539 | 14.2 | >100 | >100 | 40.9 | >100 | >100 | 3.68 | 15.7 | >100 | ||||||
| SNB-19 | 8.27 | >100 | >100 | >100 | >100 | >100 | 7.73 | 40.1 | >100 | ||||||
| SNB-75 | 4.53 | >100 | >100 | 4.35 | >100 | >100 | 1.89 | 6.87 | 81.8 | ||||||
| U251 | 4.82 | >100 | >100 | 11.0 | >100 | >100 | 3.05 | 15.0 | 55.5 | ||||||
| Melanoma | |||||||||||||||
| LOX IMV1 | 4.74 | 19.894 | 0.528 | >100 | >100 | 5.50 | 26.642 | 0.782 | >100 | >100 | 3.99 | 3.480 | 1.305 | 18.6 | >100 |
| MALME-3M | 8.87 | >100 | >100 | 5.70 | >100 | >100 | 2.92 | 7.81 | >100 | ||||||
| M14 | 7.49 | >100 | >100 | 5.23 | >100 | >100 | 4.19 | 23.0 | >100 | ||||||
| MDA-MB-435 | 7.01 | >100 | >100 | 7.53 | >100 | >100 | 3.07 | 23.5 | >100 | ||||||
| SK-MEL-2 | 7.36 | >100 | >100 | 2.91 | >100 | >100 | 4.17 | 18.8 | >100 | ||||||
| SK-MEL-28 | >100 | >100 | >100 | >100 | >100 | >100 | 2.42 | 6.11 | 59.5 | ||||||
| SK-MEL-5 | 4.10 | >100 | >100 | 6.97 | >100 | >100 | 3.10 | 12.9 | >100 | ||||||
| UACC-257 | 35.1 | >100 | >100 | >100 | >100 | >100 | 4.57 | 46.6 | >100 | ||||||
| UACC-62 | 4.38 | >100 | >100 | 5.94 | >100 | >100 | 2.89 | 12.6 | 62.1 | ||||||
| Ovarian cancer | |||||||||||||||
| IGROV1 | 5.94 | 13.591 | 0.772 | >100 | >100 | 4.45 | 41.433 | 0.503 | >100 | >100 | 4.18 | 4.390 | 1.035 | 16.7 | 86.8 |
| OVCAR-3 | 12.8 | >100 | >100 | >100 | >100 | >100 | 4.08 | 15.4 | 72.0 | ||||||
| OVCAR-4 | 6.39 | >100 | >100 | >100 | >100 | >100 | 3.15 | 17.9 | >100 | ||||||
| OVCAR-5 | 34.5 | >100 | >100 | 10.5 | >100 | >100 | 5.19 | 27.0 | >100 | ||||||
| OVCAR-8 | 8.66 | >100 | >100 | 17.6 | >100 | >100 | 5.23 | 31.0 | >100 | ||||||
| NCI/ADR-RES | 22.5 | >100 | >100 | 52.3 | >100 | >100 | 5.66 | 47.0 | >100 | ||||||
| SK-OV-3 | 4.35 | >100 | >100 | 5.18 | >100 | >100 | 3.24 | 8.18 | 44.9 | ||||||
| Renal cancer | |||||||||||||||
| 786-O | 23.5 | 6.370 | 1.648 | >100 | >100 | 14.9 | 4.343 | 4.797 | >100 | >100 | 2.44 | 2.361 | 1.923 | 5.31 | 60.5 |
| A498 | 2.63 | >100 | >100 | 2.35 | >100 | >100 | 1.36 | 4.43 | >100 | ||||||
| ACHN | 3.14 | >100 | >100 | 1.37 | >100 | >100 | 1.84 | 4.70 | 17.5 | ||||||
| CAKI-1 | 2.52 | >100 | >100 | 1.01 | >100 | >100 | 1.74 | 4.30 | 12.8 | ||||||
| RXF 393 | 4.55 | >100 | >100 | 3.47 | >100 | >100 | 1.94 | 4.15 | 8.88 | ||||||
| SN12C | 5.42 | >100 | >100 | 4.35 | >100 | >100 | 5.11 | 27.9 | >100 | ||||||
| TK-10 | N.T. | N.T. | N.T. | N.T. | N.T. | N.T. | 2.46 | 8.08 | 48.0 | ||||||
| UO-31 | 2.83 | >100 | >100 | 2.95 | >100 | >100 | 2.00 | 5.04 | 24.6 | ||||||
| Prostate cancer | |||||||||||||||
| PC-3 | 5.93 | 5.360 | 1.959 | >100 | >100 | 4.35 | 5.925 | 3.516 | >100 | >100 | 2.93 | 3.295 | 1.378 | 10.2 | 50.5 |
| DU-145 | 4.79 | >100 | >100 | 7.50 | >100 | >100 | 3.66 | 15.5 | 61.4 | ||||||
| Breast cancer | |||||||||||||||
| MCF7 | 5.42 | 6.813 | 1.541 | >100 | >100 | 12.7 | 12.350 | 1.687 | >100 | >100 | 3.88 | 3.580 | 1.269 | 70.3 | >100 |
| MDA-MB-231/ATCC | 7.23 | >100 | >100 | 4.31 | >100 | >100 | 3.64 | 17.1 | 89.1 | ||||||
| HS 578T | 4.77 | >100 | >100 | 5.58 | >100 | >100 | 4.25 | 95.2 | >100 | ||||||
| BT-549 | 10.6 | >100 | >100 | 40.8 | >100 | >100 | 4.32 | 19.7 | 85.6 | ||||||
| T-47D | 8.44 | >100 | >100 | 4.57 | >100 | >100 | 3.92 | 39.7 | >100 | ||||||
| MDA-MB-468 | 4.42 | >100 | >100 | 6.14 | >100 | >100 | 1.47 | 4.73 | 48.6 | ||||||
| MIDa | 10.498 | 20.832 | 4.542 | ||||||||||||
MIDa = Average sensitivity of all cell line in μM; MIDb = Average sensitivity of all cell line of a particular subpanel in μM; selectivity ratio = MIDa:MIDb; N.T. = No test.
5 Conclusion
As a summary, several heterocycle-fused anthrapyrazolone derivatives, derived from 1 or 17, have been prepared by the addition or substitution nucleophilic reactions, using N-nucleophiles. Those anthrapyrazolone, with different substituents at the C-7 position, were further transformed into the extended tetracyclic systems bearing nitrogen-heterocyclic rings fused to the quinone moiety. The compounds synthesized have been evaluated for their cytotoxic of cell growth inhibitory effect and telomerase inhibitory activity. As expected, analysis of tetracyclic core-structure derivatives might accord further insight into designing better leading compounds for anti-tumor therapies. Among the compounds tested as antitumor in this work, the average GI50 of compounds 1 and 16 were 10.498 and 4.542 μM over 60 cell lines. Based on the structure of anthrapyrazolone, we found that particularity for optimum antiproliferative activities are: (1) the 2-hydroxyethyl group at N-2 is decreased potency; (2) substituent at C-7 is fond of the side chain which contains oxygen atoms in the end but not an alkane one. However, the pyrazole-fused anthraquinone system has shown no inhibitory effect at the micromolar concentrations. Obviously, the overall synthetic compounds showed no significant correlation between cytotoxicity and telomerase inhibition. In general, the heterofused anthrapyrazolone were less active as cytotoxic than their corresponding AQ precursors or intermediates with the exception of the pyrazole-fused compounds that showed positive results on the three biological tests. The compounds might have the potential to be the leading compounds as candidate drugs for anti-cancer agents even if the mechanism of action of the anthrapyrazoles is still controversial now.
Acknowledgments
The present study was supported by National Science Council Grants of Taiwan (NSC100-2113-M-016 and NSC101-2314-B-016-005), Ministry of Science and Technology (MOST103-2113-M-038-002), Taipei Medical University (TMUTOP103003-1, TMU102-AE1-B32), and Chi-Mei Medical Center (CMNDMC-10002). We are grateful to thank NIH-NCI for their supports.