PI3Kα inhibitors

European Journal of Medicinal Chemistry 93 (2015) 64e73

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Design, synthesis and docking studies of novel thienopyrimidine derivatives bearing chromone moiety as mTOR/PI3Ka inhibitors Wufu Zhu a, b, *, 1, Chen Chen a, 1, Chengyu Sun a, 1, Shan Xu a, Chunjiang Wu a, Fei Lei a, Hui Xia a, Qidong Tu a, Pengwu Zheng a, ** a

School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang, 330013, PR China Key Laboratory of Original New Drugs Design and Discovery of Ministry of Education, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, 110016, PR China

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 August 2014 Received in revised form 25 January 2015 Accepted 31 January 2015 Available online 31 January 2015

Two series of thienopyrimidine derivatives (10aek, 16aej) bearing chromone moiety were designed and synthesized. All the compounds were evaluated for inhibitory activity against mTOR kinase at a concentration of 10uM. Four selected compounds were further evaluated for the IC50 values against mTOR kinase, PI3Ka kinase and two cancer cell lines. Some of the target compounds exhibited moderate to excellent mTOR/PI3Ka kinase inhibitory activity and cytotoxicity. The most promising compound 16i showed good inhibitory activity against mTOR/PI3Ka kinase and good antitumor potency for H460 and PC-3 cell lines with IC50 values of 0.16 ± 0.03 mM, 2.35 ± 0.19 mM, 1.20 ± 0.23 mM and 0.85 ± 0.04 mM, which were 8.6, >5, 7.9 and 19.1 times more active than compound I (1.37 ± 0.07 mM, >10 mM, 9.52 ± 0.29 mM, 16.27 ± 0.54 mM), respectively. Structureeactivity relationships (SARs) and docking studies indicated that the chromone moiety is necessary for the potent antitumor activity and cytotoxicity of these compounds. Substitution of the chromone moiety at the 6-position has a significant impact to the inhibitory activity, in particular a carboxylic acid group, produced the best potency. © 2015 Elsevier Masson SAS. All rights reserved.

Keywords: Thienopyrimidine Chromone Synthesis Docking mTOR PI3Ka Cytotoxicity

1. Introduction The PI3K-Akt-mTOR signaling pathway plays a key role in cell proliferation, migration, survival, and angiogenesis, and developing mTOR/PI3Ka inhibitors is one of the research hotspots in molecular targeted therapy for the treatment of human cancer [1,2]. In recent years, a growing number of studies based on the (fused-)pyrimidine/triazine scaffolds towards antitumor activity have been carried out [3e7]. Among them, GDC-0941 (Fig. 1), which is a potent, orally bioavailable inhibitor of PI3K, exerted antitumor activity against an array of human tumor cell lines and is currently undergoing Phase II clinical trials [5]. Further optimization of GDC0941 led to several backup clinical candidates with similar structures, such as GNE-490 and GNE-493 [4] (Fig. 1). Compound I

* Corresponding author. School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang, 330013, PR China. ** Corresponding author. E-mail addresses: [email protected] (W. Zhu), [email protected] (P. Zheng). 1 These authors contribute equally to this work. http://dx.doi.org/10.1016/j.ejmech.2015.01.061 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

(Fig. 1) is a triazine-hydrazone derivative which exhibited potent anti-tumor activity with an mTOR IC50 value of 0.27 mM [6]. To our knowledge, few studies based on pyrimidine- or triazinehydrazones as mTOR inhibitors have been reported to date. Importantly, the hydrazinyl group or its mimics are key pharmacophores in the design of different potential anticancer agents [7]. Therefore, the research on this aspect seemed to be an attractive task. In our previous research [8e10], several series of 2-hydrazinyl4-morpholinothieno[3,2-d]pyrimidines which demonstrated potent antitumor activity were designed and synthesized, and SARs were discussed and summarized. The results showed that most of them exhibited excellent cytotoxicity but possessed disappointing in vivo antitumor activity, such as the representative compounds II [9] and III [10] (Fig. 1). This work also showed that introduction of indole-hydrazone to the target compound benefitted the antitumor activity. In continuation of our previous research towards compounds which possessed excellent antitumor activity, further indolehydrazone studies were carried out and are reported herein. Through analysis of the SARs and the results of docking with mTOR/

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Fig. 1. Structures of some reported (fused-)pyrimidine/triazines and compounds reported in previous research.

PI3Ka kinase, a new compound library was built with structural modifications at thienopyrimidine moiety and indole-hydrazone groups (Fig. 1). Firstly, a series of thieno[3, 2-d]pyrimidines 10aek (Fig. 2) were designed by replacing the N-benzyl indole or Nbenzyl benzimidazole moieties of compounds II and III with active pharmacophore chromone moiety which was proved in our previously research [11]. However, this series of compounds showed poor cytotoxicity [12] and moderate mTOR kinase inhibitory activity. Further investigations were carried out in detail to study the effects of the thienopyrimidine scaffold on the antitumor activity. Therefore, the fusion between the thienyl and pyrimidine in the scaffold was changed according to the theory of isostere principle, resulting in the series of thieno[2, 3-d]pyrimidines 16aej (Fig. 2). The inhibitory activity against mTOR kinase and cytotoxicity of compounds 16aej were better than that of compounds 10aek. Considering the high potency of GDC-0941 and its analogous on lung and prostate cancer cells, herein we disclose the design, synthesis and inhibitory activity of novel thieno[2,3-d]pyrimidine bearing a chromone moiety against mTOR kinase, as well as the

cytotoxicity against H460 (lung) and PC-3 (prostate) cancer cell lines. Further evaluation was carried out against PI3Ka kinase to confirm the types of target compounds, selective or mixed type inhibitors. Moreover, docking studies are presented providing a rationale for the observed activity. 2. Chemistry The preparation of target compounds 10aek and 16aej is described in Schemes 1 and 2. The structures and the predicted cLogp values of target compounds are shown in Table 1. The substituted 4-oxo-4H-chromene-3-carbaldehydes 4aek were synthesized through the procedures reported previously by our group (Scheme 1) [11,13]. The synthesis of compounds 10aek was similar to the procedures previously reported [8e10,12]. The key intermediate 9 condensed with substituted 4-oxo-4H-chromene-3-carbaldehydes 4aek to afford target compounds 10aek, respectively. Compounds 16aej were synthesized from methyl 2-

Fig. 2. Structures and design strategy for target compounds 10aek and 16aej.

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Scheme 1. Synthetic route of substituted 4-oxo-4H-chromene-3-carbaldehydes (4aek) Reagents and conditions: (a) Ac2O, conc. H2SO4, r.t.; (b) anhydrous AlCl3, 120
C; (c) DMF/ POCl3, r.t.

aminothiophene-3-carboxylate 11 through five steps. The commercially available methyl 2-aminothiophene-3-carboxylate 11 was condensed with urea at 180
C for 2 h to give thienopyrimidinedione 12. Chlorination of 12 with POCl3 and DMF (cat.) for

8 h at 115
C afforded 13 as yellow solid. Substitution reaction of 13 with morpholine at room temperature to yielded 14 which was then condensed with 4aej to furnish compounds 16aej, respectively.

Scheme 2. Synthetic routes of target compounds 10aek and 16aej. Reagents and conditions: (a) 5 eq urea, 180
C, 2 h; (b) POCl3, DMF (cat.), reflux, 8 h; (c) 2.1 eq morpholine, MeOH, 0
C, 30 min, r.t., 1e2 h; (d) 80% NH2NH2$H2O,reflux, 8e10 h; (e) EtOH, reflux, 2e4 h.

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Table 1 Structures and inhibition rates of target compounds 10aek, 16aej against mTOR/PI3Ka kinase at 10 mM

Compd.

R

cLogP (predicted)d

(10 mM inhibitory %)a mTOR

PI3Ka

10a 10b 10c 10d 10e 10f 10g 10h 10i 10j 10k IIb,c IIIb,c

Fluoro Chloro Bromo Methyl Ethyl Tert-butyl Hydroxy Nitro Carboxyl H Isopropyl e e

4.2 4.8 4.9 4.6 5.1 5.9 4.0 3.8 4.0 4.1 5.5

<10% 28.8 ± 41.5 ± 49.5 ± 32.2 ± 25.4 ± 44.9 ± 54.3 ± 61.4 ± 36.2 ± 21.7 ± 36.9 ± 33.6 ±

e e e e e e e 58.8 ± 3.2 95.0 ± 7.9 e e e e

a b c d

6.6 9.4 0.5 3.4 3.1 2.8 0.6 0.5 3.3 2.7 2.8 3.3

Compd.

R

cLogP (predicted)d

(10 mM inhibitory %)a mTOR

PI3Ka

16a 16b 16c 16d 16e 16f 16g 16h 16i 16j

fluoro chloro bromo methyl ethyl tert-butyl hydroxy nitro carboxyl H

4.0 4.6 4.7 4.3 5.1 5.7 3.8 3.6 3.8 3.8

29.4 ± 5.5 30.8 ± 3.5 31.0 ± 0.3 41.5 ± 0.8 26.7 ± 1.4 34.9 ± 0.8 No active 81.0 ± 0.7 97.2 ± 0.4 42.9 ± 2.0

e e e e e e e 91.2 ± 11.7 90.6 ± 9.6 e

Ib PI-103b

e e

76.1 ± 0.3 98.6 ± 0.5

e 98.5 ± 3.8

The values are an average of two separate determinations. Used as a positive controls. Compounds reported in our previous research. cLogP values are predicted in sybyl 6.9.1.

The relative stereochemistry of the target compounds was easily confirmed as E isomeric form according to our previously published method [8e10,12].

3. Results and discussion 3.1. Biological evaluation The target compounds were evaluated for their inhibitory activity against mTOR kinase at a concentration of 10uM in vitro together with reference compounds I, II, III and PI103 by LANCE® Ultra time-resolved fluorescence resonance energy transfer (TRFRET) assay. Four selected compounds also measured the PI3Ka kinase inhibition rate at a concentration of 10 mM with reference PI103 by Kinase-Glo® Luminescent Kinase Assay. In addition, the four selected compounds 10hei and 16hei were further evaluated for their IC50 values against mTOR kinase, PI3Ka kinase and two cancer cell lines H460 (human lung cancer) and PC-3 (human prostate cancer). The results were expressed as percent inhibition at single concentration or IC50 values are summarized in Tables 1 and 2 and the values are the average of at least two independent experiments. As illustrated in Tables 1 and 2, most of the synthesized compounds showed moderate inhibitory activity against mTOR kinase and were more potent than the compounds (II and III) reported previously, suggesting that the replacement of N-benzyl indole or N-benzyl benzimidazole moiety with chromone moiety enhanced the anti-tumor activity. Four of them (compounds 10hei and 16hei) showed excellent inhibitory activity with IC50 values from 4.62 ± 0.54 mM to 0.16 ± 0.03 mM against mTOR kinase and from 1.80 mM to 2.35 mM against PI3Ka kinase. And overall their mTOR inhibition activity were little better than PI3Ka inhibition activity. The inhibitory activity against mTOR kinase of compounds 16h and 16i were 1.49 and 8.56 times more active than lead compound I.

In the first series, thieno[3, 2-d]pyrimidines 10aek were designed by replacing the N-benzyl indoles or N-benzyl benzimidazole moieties of compounds II and III with substituted chromone moieties. The inhibitory activity on mTOR kinase of compounds 10aek were increased in varying degrees. The results showed that variations in substitution at 6-position of the chromone moiety had a marked impact on the activity. For compounds 10aek, substitution of the chromone moiety at the 6-position was well tolerated and 6-NO2 (10h) and 6-COOH (10i) substitution were more preferred. Further investigations were carried out in detail to study the effects of the thienopyrimidine scaffold on the activity. Therefore, the fusion isomer of thienopyrimidine scaffold was changed, resulting in the other series thieno[2, 3-d]pyrimidines 16aej. The mTOR inhibition and cytotoxicity of this series of compounds were higher than that of compounds 10aek, but most of them the PI3Ka inhibition has little difference. It was noticeable that compound 16i

Table 2 mTOR/PI3Ka kinase activity and cytotoxicity of selected compounds and positive controls. Compounds no.

IC50 (mM)a mTOR

PI3Ka

H460

PC-3

10h 10i 16h 16i Ib IIb,c IIIb,c PI-103b

4.62 ± 0.54 3.31 ± 0.12 0.92 ± 0.14 0.16 ± 0.03 1.37 ± 0.07 16.62 ± 0.39 >30 0.019 ± 0.004

>10 1.81 ± 0.35 1.53 ± 0.21 2.35 ± 0.19 >10 e e 0.0075 ± 0.0018

ND ND 3.24 ± 1.12 1.20 ± 0.23 9.52 ± 0.29 ND ND ND

6.90 ± 1.22 6.26 ± 0.61 1.42 ± 0.19 0.85 ± 0.04 16.27 ± 0.54 ND ND ND

Bold values show IC50 of <1 mM. a The values are an average of two separate determinations. b Used as a positive controls. c Compounds reported in our previous research.

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(with negative charge group eCOOH at C-6 position) showed excellent antitumor activity with the inhibitory activity equal to the positive control PI-103 (97.2 ± 0.4% vs 98.6 ± 0.5%), which suggested that the negative charge character of the most active groups has a more predominant role. This significant increase in the mTOR potency could be ascribed to the increased hydrogen bonds between the 6-COOH and receptors necessary for activity or the increased polar interactions with the mTOR. The mTOR/PI3Ka kinase activity and cytotoxicity of the four selected compounds 10hei and 16hei as well as the four lead compounds are shown in Table 2. Obviously, these four compounds exhibited good activity towards mTOR/PI3Ka kinase and cytotoxicity. All of them display an IC50 for mTOR inhibition superior to that of lead compounds II and III, two of them (16hei) were superior to compound I. Three compounds (10i,16hei) are more active than compound I against PI3Ka kinase. The most promising compound 16i showed good inhibitory activity against mTOR, PI3Ka kinase and good cytotoxicity for H460 and PC-3 cell lines with IC50 values of 0.16 ± 0.03 mM, 2.35 ± 0.19 mM, 1.20 ± 0.23 mM and 0.85 ± 0.04 mM, which were 8.6, 7.9 and 19.1 times more active than compound I (1.37 ± 0.07 mM, 9.52 ± 0.29 mM, 16.27 ± 0.54 mM), respectively. As described above, chromone moieties were necessary for these thienopyrimidine derivatives. Variations in substitutions of the chromone moieties had a significant impact on the activity. Substitution of the chromone moieties at the 6-position with negatively charged groups was preferred and 6-COOH substitution produced the best potency.

3.2. Molecular docking study To explore the binding modes of target compounds with the active site of mTOR and PI3Ka, molecular docking simulation studies were carried out by using SURFLEX-DOCK module of SYBYL package version. Based on the in vitro inhibition results, we selected compound 16i, our best mTOR/PI3Ka inhibitor in this study, as ligand example, and the structures of mTOR (PDB ID code:4JT6 [15]) and PI3Ka (PDB ID code:4L23 [16]) were selected as the docking models. The binding modes of compound 16i and lead compounds were shown in Fig. 3aed. As depicted in Fig. 3a, b, compound 16i and PI103 can overlap in the position of morpholine group in the binding model and the eCOOH group of chromone moiety formed two hydrogen bonds with residues TRP2239 and ARG2348 in the docking model of mTOR, respectively. The morpholine group formed one hydrogen bond with residues VAL2240. Similarly, the hydrogen bonds were found in the docking model of PI3Ka in Fig. 3c, d, such as the hydrogen bonds between morpholine group and residue VAL851, the chromone moiety and the residues SER773, SER919. The activity against mTOR/PI3Ka kinase of compound 16i was difference, which was attributed to the difference of the hydrogen bonds between the chromone moiety and amino acid residues. According to the docking model of mTOR (Fig. 3b), eCOOH group of chromone moiety formed two hydrogen bonds with residues TRP2239 and ARG2348, respectively. However, only one hydrogen bond was formed between eCOOH group of chromone moiety and amino acid residues in the docking model of

Fig. 3. aed. Binding models of compounds 16i (shown in Capped Sticks) and native ligand PI 103 (shown in yellow lines) target into active site of mTOR （Fig. 3a, b） and PI3Ka （Fig. 3c, d）, respectively. Hydrogen bonds were showed in dashed lines (yellow). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

W. Zhu et al. / European Journal of Medicinal Chemistry 93 (2015) 64e73

PI3Ka(Fig. 3c). Analysis of compound 16i's binding mode in the active binding site and the results of activity suggested that the several hydrogen bonds, formed in morpholine group and the chromone moiety, really plays an important role in increasing the inhibitory potency of chromone derivatives against mTOR/PI3Ka kinase. The docking results indicated that these compounds have plausible binding modes to both enzymes. Furthermore, the docking results also give us a new direction to design new mTOR/PI3Ka inhibitors that can interact with VAL2240, TRP2239, ARG2348, VAL851 and SER773. The above-mentioned results of SAR analysis and molecular docking study may allow the rational design of more potent mTOR/PI3Ka inhibitors. 4. Conclusions In summary, two series of thieno-pyrimidine derivatives bearing chromone moieties were designed, synthesized and evaluated for inhibitory activity against mTOR kinase, PI3Ka kinase and cytotoxicity of two cancer cell lines in vitro. The pharmacological results indicated that most of the synthesized compounds displayed more mTOR inhibition than compounds reported in previous articles and two compounds (16hei) inhibited mTOR at lower doses and were more cytotoxic than lead compound I. And three compounds also displayed moderate to excellent PI3Ka kinase inhibition activity with IC50 values from 1.805 mM to 2.352 mM. The most promising compound 16i showed good inhibitory activity against mTOR/PI3Ka kinase and good antitumor potency for H460 and PC-3 cell lines with IC50 values of 0.16 ± 0.03 mM, 2.35 ± 0.19 mM, 1.20 ± 0.23 mM and 0.85 ± 0.04 mM, which were 8.6, >5, 7.9 and 19.1 times more active than compound I (1.37 ± 0.07 mM, >10 mM, 9.52 ± 0.29 mM, 16.27 ± 0.54 mM), respectively. The initial SARs and docking studies showed that the chromone moieties were necessary for the activity of these compounds. Variations in substitutions of the chromone moieties had a significant impact on the activity. Substitutions of chromone moiety at the 6-position have a significant impact to the inhibitory activity, in particular a carboxylic acid group, produced the best potency. 5. Experimental 5.1. Chemistry All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland) and were uncorrected. NMR spectra were performed using Bruker 400 MHz spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS as an internal standard. Mass spectra (MS) were taken in ESI mode on Agilent 1100 LCeMS (Agilent, Palo Alto, CA, USA). All the materials were obtained from commercial suppliers and used without purification, unless otherwise specified. Yields were not optimized. TLC analysis was carried out on silica gel plates GF254 (Qindao Haiyang Chemical, China). All the materials were obtained from commercial suppliers and used without purification, unless otherwise specified. Yields were not optimized. 5.2. General procedure for the preparation of compounds 4aek Compounds 4aek was synthesized from compounds 1aek according to the procedures in our previous research [11,13]. 5.3. Preparation of 4-(2-hydrazinylthieno[3,2-d]pyrimidin-4-yl) morpholine (9) Compound 9 was synthesized from compound 5 according to the procedures in our previous research [8e10,12].

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5.4. General procedure for the preparation of compounds 10aek A mixture of 4-(2-hydrazinylthieno[3,2-d]pyrimidin-4-yl)morpholine 9 (0.12 g, 0.48 mmol), substitued 4-oxo-4H-chromene-3carbaldehyde 4aek and a drop of glacial acetic acid in absolute ethanol (8 mL) was refluxed for 4e8 h. The mixture was cooled, separated by filtration and washed with EtOH to give off a white to light yellow solid. The crude product was recrystallized from ethanol or purified by chromatography on silica gel using MeOH/ CH2Cl2 to afford the solids 10aek. 5.4.1. (E)-6-Fluoro-3-((2-(4-morpholinothieno[3,2-d]pyrimidin-2yl)hydrazono)methyl)-4H-chromen-4-one (10a) This compound was obtained as yellow solid in 85% yield. m.p. 228e231
C. ESI-MS m/z:426.1 IR (KBr) cm1: 3432, 3086, 2992, 2958, 1629, 1428, 1382, 1255, 1196, 1174, 1115, 933, 881, 788. 1H NMR (400 MHz, DMSO) d 11.07 (s, 1H, NH), 8.73 (s, 1H, NCH), 8.24 (s, 1H, AreH), 8.05 (d, J ¼ 8.6 Hz,1H, AreH), 7.62 (d, J ¼ 7.9 Hz, 2H, AreH), 7.48 (d, J ¼ 6.2 Hz, 1H, AreH), 7.23 (d, J ¼ 6.1 Hz, 1H, AreH), 3.85 (s, 4H, OCH2), 3.75 (d, J ¼ 4.3 Hz, 4H, NCH2). 13C NMR (101 MHz, DMSO) d 163.1(C), 158.5(C), 157.6(C), 156.8(C), 154.5(CH), 154.1(CH), 151.9(C), 138.2(C),135.3(CH),133.8(CH),124.9(C),124.0(CH),120.2(CH),117.5(C), 111.6(CH), 106.7(C), 66.4(CH2), 66.3(CH2), 46.3(CH2), 45.8(CH2). Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SDC18 analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow, monitored by UV absorption at 325 nm, showed 96.5% purity. 5.4.2. (E)-6-Chloro-3-((2-(4-morpholinothieno[3,2-d]pyrimidin-2yl)hydrazono)methyl)-4H-chromen-4-one (10b) This compound was obtained as yellow solid in 88% yield. m.p. 236e238
C ESI-MS m/z:442.0 IR (KBr) cm1: 3434, 3092, 2998, 2986, 1651, 1632, 1561, 1545, 1467, 1429, 1379, 1116, 858, 824, 790. 1 H NMR (400 MHz, DMSO) d 11.11 (s, 1H, NH), 8.77 (s, 1H, NCH), 8.24 (d, J ¼ 18.4 Hz, 1H, AreH), 8.05 (d, J ¼ 2.4 Hz, 1H, AreH), 7.99e7.85 (m, 1H, AreH), 7.79 (d, J ¼ 8.9 Hz, 1H, AreH), 7.48 (d, J ¼ 6.2 Hz, 1H, AreH), 7.23 (d, J ¼ 6.1 Hz, 1H, AreH), 3.86 (s, 4H, OCH2), 3.75 (d, J ¼ 4.1 Hz, 4H, NCH2). 13C NMR (101 MHz, DMSO) d 188.2(C), 163.1(C), 162.6(C), 158.6(CH), 158.4(C), 157.6(CH), 155.4(C), 154.5(CH), 154.0(CH), 152.5(CH), 143.2(C), 135.9(CH), 135.4(C), 124.9(CH), 124.8(C), 106.7(C), 66.3(2CH2), 46.2(2CH2). Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow, monitored by UV absorption at 325 nm, showed 95.2% purity. 5.4.3. (E)-6-Bromo-3-((2-(4-morpholinothieno[3,2-d]pyrimidin-2yl)hydrazono)methyl)-4H-chromen-4-one (10c) This compound was obtained as yellow solid in 84% yield. m.p. 267e269
C. ESI-MS m/z:448.0 IR (KBr) cm1: 3430, 3112, 2962, 1644, 1464, 1377, 1305, 1161, 1116, 858, 822, 789. 1H NMR (400 MHz, DMSO) d 10.54 (s, 1H, NH), 8.68 (s, 1H, NCH), 8.21 (d, J ¼ 5.5 Hz, 1H, AreH), 8.04 (s, 1H, AreH), 7.76 (s, 1H, AreH), 7.62 (d, J ¼ 9.0 Hz, 1H, AreH), 7.37 (d, J ¼ 2.6 Hz, 1H, AreH), 7.16 (d, J ¼ 5.5 Hz, 1H, AreH), 3.82e3.76 (m, 4H, OCH2), 3.67 (d, J ¼ 3.9 Hz, 4H, NCH2). 13C NMR (101 MHz, DMSO) d 163.2(C), 158.4(C), 157.7(C), 155.0(CH), 154.0(C), 140.3(CH), 138.2(C), 138.0(CH), 135.8(CH), 135.3(CH), 133.2(C), 132.8(CH), 124.9(C), 117.9(CH), 111.6(C), 110.2(C), 66.4(2CH2), 46.3(2CH2). Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH (15% H2O) eluent at 1 mL/min flow, monitored by UV absorption at 325 nm, showed 91.9% purity. 5.4.4. (E)-6-Methyl-3-((2-(4-morpholinothieno[3,2-d]pyrimidin-2yl)hydrazono)methyl)-4H-chromen-4-one (10d) This compound was obtained as light yellow solid in 80% yield. m.p. 223e225
C. ESI-MS m/z:422.3 IR (KBr) cm1: 3412, 3100,

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2956, 2920, 1620, 1563, 1497, 1428, 1377, 1170, 1113, 825, 789. 1H NMR (400 MHz, DMSO) d 10.96 (s, 1H, NH), 8.74 (s, 1H, NCH), 8.22 (s, 1H, AreH), 8.10 (d, J ¼ 6.1 Hz, 1H, AreH), 7.92 (s, 1H, AreH), 7.66 (s, 1H, AreH), 7.24 (d, J ¼ 5.3 Hz, 1H, AreH), 7.06 (d, J ¼ 15.2 Hz, 1H, AreH), 3.89 (d, J ¼ 14.2 Hz, 4H, OCH2), 3.77 (s, 4H, NCH2), 2.45 (s, 3H, CH3). Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH (15% H2O) eluent at 1 mL/min flow, monitored by UV absorption at 325 nm, showed 96.2% purity. 5.4.5. (E)-6-Ethyl-3-((2-(4-morpholinothieno[3,2-d]pyrimidin-2yl)hydrazono)methyl)-4H-chromen-4-one (10e) This compound was obtained as yellow solid in 82% yield. m.p. 202e205
C. ESI-MS m/z:436.1 1H NMR (400 MHz, DMSO) d 10.62 (s, 1H, NH), 9.30 (s, 1H, NCH), 8.73 (s, 1H, AreH), 8.23 (s, 1H, AreH), 7.93 (s, 1H, AreH), 7.64 (s, 1H, AreH), 7.48 (d, J ¼ 5.3 Hz, 1H, AreH), 7.09 (s, 1H, AreH), 3.88 (s, 4H, OCH2), 3.77 (s, 4H, NCH2), 2.76 (d, J ¼ 6.7 Hz, 2H, CH2), 1.23 (dd, J ¼ 14.9, 7.4 Hz, 3H, CH3). Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow, monitored by UV absorption at 325 nm, showed 97.4% purity. 5.4.6. (E)-6-(Tert-butyl)-3-((2-(4-morpholinothieno[3,2-d] pyrimidin-2-yl)hydrazono)methyl)-4H-chromen-4-one (10f) This compound was obtained as light yellow solid in 79% yield. m.p. 225e227
C. ESI-MS m/z:464.1 1H NMR (400 MHz, DMSO) d 10.96 (s, 1H, NH), 8.74 (s, 1H, NCH), 8.22 (s, 1H, AreH), 8.08 (d, J ¼ 10.3 Hz, 2H, AreH), 7.63 (s, 2H, AreH), 7.26 (s, 1H, AreH), 3.91 (s, 4H, OCH2), 3.77 (s, 4H, NCH2), 1.36 (s, 9H, CH3). Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow, monitored by UV absorption at 325 nm, showed 94.5% purity. 5.4.7. (E)-6-Hydroxy-3-((2-(4-morpholinothieno[3,2-d]pyrimidin2-yl)hydrazono)methyl)-4H-chromen-4-one (10g) This compound was obtained as yellow solid in 85% yield. m.p. 250e251
C. ESI-MS m/z:424.1 IR (KBr) cm1: 3432, 3102, 2976, 1628, 1444, 1370, 1322, 1249, 1160, 1114, 879, 825, 788. 1H NMR (400 MHz, DMSO) d 10.89 (s, 1H, NH), 10.09 (s, 1H, OH), 8.66 (s, 1H, NCH), 8.21 (s, 1H, AreH), 8.08 (d, J ¼ 5.5 Hz, 1H, AreH), 7.58 (d, J ¼ 9.0 Hz, 1H, AreH), 7.38 (s, 1H, AreH), 7.24 (dd, J ¼ 13.4, 7.3 Hz, 2H, AreH), 3.90 (s, 4H, OCH2), 3.76 (s, 4H, NCH2). Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow, monitored by UV absorption at 325 nm, showed 100% purity. 5.4.8. (E)-3-((2-(4-Morpholinothieno[3,2-d]pyrimidin-2-yl) hydrazono)methyl)-6-nitro-4H-chromen-4-one (10h) This compound was obtained as yellow solid in 83% yield. m.p. 286e289
C. ESI-MS m/z:451.2 IR (KBr) cm1: 3418, 3100, 2956, 2920,1626,1545, 1431, 1380, 1116, 828, 789. 1H NMR (400 MHz, DMSO) d 10.66 (s, 1H, NH), 8.30 (d, J ¼ 5.4 Hz, 1H, NCH), 8.20e8.14 (m, 1H, AreH), 8.10 (d, J ¼ 5.5 Hz, 1H, AreH), 7.88 (s, 1H, AreH), 7.44 (d, J ¼ 5.5 Hz, 1H, AreH), 7.25 (d, J ¼ 5.4 Hz, 1H, AreH), 6.98 (d, J ¼ 9.0 Hz, 1H, AreH), 3.87 (s, 4H, OCH2), 3.76 (d, J ¼ 4.3 Hz, 4H, NCH2). 13C NMR (101 MHz, DMSO) d 185.5(C), 163.0(C), 162.2(C), 157.7(C), 149.1(CH), 135.8(CH), 135.4(C), 133.4(C), 128.5(CH), 127.1(CH), 126.3(CH), 124.8(C), 124.7(CH), 118.2(CH), 116.2(C), 106.5(C), 66.3(CH2), 66.1(CH2), 46.3(CH2), 45.8(CH2). ESI-HRMS m/ z: calcd for C20H16N6O5S [MþH]þ:453.0903; found 453.0975. Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow, monitored by UV absorption at 325 nm, showed 91.6% purity.

5.4.9. (E)-3-((2-(4-Morpholinothieno[3,2-d]pyrimidin-2-yl) hydrazono)methyl)-4-oxo-4H-chromene-6-carboxylic acid (10i) This compound was obtained as yellow solid in 87% yield. m.p. 280e282
C. ESI-MS m/z:452.1 IR (KBr) cm1: 3424, 3106, 2962, 2926, 1626, 1545, 1428, 1377, 1161, 1113, 828, 789. 1H NMR (400 MHz, DMSO) d 10.59 (s, 1H, COOH), 10.46 (s, 1H, NH), 8.27 (d, J ¼ 5.5 Hz, 1H, NCH), 8.11 (s, 1H, AreH), 8.06 (d, J ¼ 5.1 Hz, 1H, AreH), 7.86 (d, J ¼ 8.3 Hz, 1H, AreH), 7.79 (d, J ¼ 6.2 Hz, 1H, AreH), 7.44 (d, J ¼ 5.6 Hz, 1H, AreH), 7.23 (d, J ¼ 5.3 Hz, 1H, AreH), 3.85 (s, 4H, OCH2), 3.72 (s, 4H, NCH2). 13C NMR (101 MHz, DMSO) d 167.3(C), 163.1(C), 159.6(C), 158.4(C), 157.6(C), 154.0(CH), 138.2(CH), 135.2(C), 133.6(CH), 132.8(CH), 132.0(CH), 124.9(CH), 124.0(C), 122.0(C), 120.2(CH), 119.1(C), 115.8(C), 66.4(CH2), 66.2(CH2), 46.2(CH2), 45.7(CH2). ESI-HRMS m/z: calcd for C21H17N5O5S [MþH]þ:452.0950; found 452.0567. Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/ MeOH (39%H2O) eluent at 1 mL/min flow, monitored by UV absorption at 325 nm, showed 97.4% purity. 5.4.10. (E)-3-((2-(4-Morpholinothieno[3,2-d]pyrimidin-2-yl) hydrazono)methyl)-4H-chromen-4-one (10j) This compound was obtained as light yellow solid in 79% yield. m.p. 280e282
C. ESI-MS m/z:430.2 IR (KBr) cm1: 3424, 3112, 2962, 2914, 1647, 1563, 1545, 1464, 1158, 852, 789. 1H NMR (400 MHz, DMSO) d 11.12 (s, 1H, NH), 8.79 (s, 1H, NCH), 8.27 (s, 1H, AreH), 8.16 (d, J ¼ 7.9 Hz, 1H, AreH), 7.89 (d, J ¼ 7.8 Hz, 1H, AreH), 7.76 (d, J ¼ 8.4 Hz, 1H, AreH), 7.58 (t, J ¼ 7.4 Hz, 1H, AreH), 7.51 (d, J ¼ 6.1 Hz, 1H, AreH), 7.26 (d, J ¼ 5.9 Hz, 1H, AreH), 3.89 (s, 4H, OCH2), 3.78 (s, 4H, NCH2). Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow, monitored by UV absorption at 325 nm, showed 98.4% purity. 5.4.11. (E)-6-Isopropyl-3-((2-(4-morpholinothieno[3,2-d] pyrimidin-2-yl)hydrazono)methyl)-4H-chromen-4-one (10k) This compound was obtained as light yellow solid in 87% yield. m.p. 163e165
C. ESI-MS m/z:450.1 IR (KBr) cm1: 3442, 3076, 2962, 1638, 1542, 1482, 1110, 825, 786, 702. 1H NMR (400 MHz, DMSO) d 10.92 (s, 1H, NH), 8.69 (s, 1H, NCH), 8.20 (s, 1H, AreH), 8.07 (d, J ¼ 5.6 Hz, 1H, AreH), 7.93 (s, 1H, AreH), 7.79e7.69 (m, 1H, AreH), 7.64 (s, 1H, AreH), 7.21 (d, J ¼ 5.4 Hz, 1H, AreH), 3.89 (s, 4H, OCH2), 3.74 (s, 4H, NCH2), 3.07e2.99 (m, 1H, CH), 1.24 (d, J ¼ 7.0 Hz, 6H, CH3). 13C NMR (101 MHz, DMSO) d 175.1(C), 162.8(C), 158.1(C), 157.2(CH), 154.2(C), 152.7(CH), 146.1(C), 133.4(CH), 133.1(CH), 132.1(CH), 123.6(C), 123.0(CH), 121.6(C), 119.2(CH), 118.6(C), 118.5(C),65.8(2CH2), 45.8(2CH2), 32.9(C), 23.7(2CH3). Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH (15% H2O) eluent at 1 mL/min flow, monitored by UV absorption at 325 nm, showed 90.1% purity. 5.5. Thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (12) Urea (30 g, 0.5 mol) was added to methyl 2-aminothiophene-3carboxylate (11, 10 g, 0.06 mol). Heated to 180
C and stirred for 2 h. After cooling to 140
C, reactant was dissolved with 1 N sodium hydroxide solution. After filtration, 2 N hydrochloric acid was added to the filtrate until pH 7. After filtration, the filtrate was refrigerated for 12 h and white solid appeared in the filtrate. The product was filtered off, washed with water and dry to afford 6.0 g thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (12). Yield:56.1%,1H NMR (400 MHz, DMSO) d 11.94 (s, 1H,CONH), 11.18 (s, 1H, CONH), 7.11 (d, J ¼ 16.0, 5.6 Hz, 2H,2 AreH) ESI-MS [MH] m/z:167.2.

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5.6. 2,4-Dichlorothieno[2,3-d]pyrimidine (13) Thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (12) (2.5 g, 0.012 mol) and a drop of DMF were added to 20 mL POCl3. Heated to 115
C for 8 h. After cooling to room temperature, the reactant was added to 500 g ice-water mixture slowly. The product was filtered off and dissolved in CH2Cl2. The insoluble was filtered off and the filtrate was distilled under reduced pressure to afford 1.7 g 2,4dichlorothieno[2,3-d]pyrimidine (13). Yield: 56.0%, 1H NMR (400 MHz, DMSO) d 8.16 (d, J ¼ 3.4 Hz, 1H, AreH), 7.62 (d, J ¼ 3.4 Hz, 1H, AreH). ESI-MS m/z:205.1. 5.7. 4-(2-Chlorothieno[2,3-d]pyrimidin-4-yl)morpholine (14) 2,4-Dichlorothieno[2,3-d]pyrimidine (13, 2.8 g, 0.014 mol) was dissolved in 20 mL CH3OH. 2.8 mL morpholine was added slowly at room temperature. Stirred for 1.5 h. The product was filtered off and dry to afford 3.1 g 4-(2-chlorothieno[2,3-d]pyrimidin-4-yl)morpholine (14). Yield: 86.1%. 1H NMR (400 MHz, DMSO) d 7.70 (s, 1H, AreH), 7.69e7.65 (m, 1H, AreH), 3.96e3.86 (m, 4H, OCH2), 3.75 (d, J ¼ 4.4 Hz, 4H, NCH2). ESI-MS [MþH] m/z:256.1. 5.8. 4-(2-Hydrazinylthieno[2,3-d]pyrimidin-4-yl)morpholine (15) Compound 15 was synthesized from compound 14 according to the procedures in our previous research [6,7]. 1H NMR (400 MHz, DMSO) d 7.73 (s, 1H,NH), 7.38 (d, J ¼ 6.2 Hz, 1H, AreH), 7.07 (d, J ¼ 6.1 Hz, 1H, AreH), 4.11 (s, 2H,NH2), 3.76 (d, J ¼ 4.9 Hz, 4H, OCH2), 3.71 (d, J ¼ 4.8 Hz, 4H, NCH2).ESI-MS [MþH] (m/z):252.1. 5.9. General procedure for the preparation of compounds 16aej A mixture of 4-(2-hydrazinylthieno[2,3-d]pyrimidin-4-yl)morpholine 15 (50 mg, 0.20 mmol), substitued 4-oxo-4H-chromene-3carbaldehyde 4aej (0.20 mmol) and a drop of glacial acetic acid in absolute ethanol (5 mL) was refluxed for 4e8 h. The mixture was cooled, separated by filtration and washed with EtOH to afford the solids 16aej. 5.9.1. (E)-6-Fluoro-3-((2-(4-morpholinothieno[2,3-d]pyrimidin-2yl)hydrazono)methyl)-4H-chromen-4-one (16a) This compound was obtained as light yellow solid in 83% yield. m.p. 228.3e229.1
C. ESI-MS m/z:426.1. IR (KBr) cm1: 3380, 3288,3018, 2964, 1640, 1530, 1480, 1318, 1188, 887, 783, 725.1H NMR (400 MHz, DMSO) d 11.13 (s, 1H, NH), 8.80 (s, 1H, NCH), 8.23 (s, 1H, AreH), 7.96e7.66 (m, 3H, AreH), 7.49 (d, J ¼ 6.2 Hz, 1H, AreH), 7.24 (d, J ¼ 6.2 Hz, 1H, AreH), 3.86 (d, J ¼ 4.4 Hz, 4H, OCH2), 3.76 (d, J ¼ 4.5 Hz, 4H, NCH2). 13C NMR (101 MHz, DMSO) d 174.9(C), 171.7(C), 160.7(C), 159.1(C), 156.3(CH), 153.7(CH), 152.7(C), 132.6(C), 123.1(CH), 122.9(CH), 121.9(C), 119.2(CH), 117.7(CH), 111.0(C), 110.3(CH), 110.1(C), 66.5(2CH2), 47.0(2CH2). Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH(15%H2O) eluent at 1 mL/min flow, monitored by UV absorption at 335 nm, showed 93.9% purity. 5.9.2. (E)-6-Chloro-3-((2-(4-morpholinothieno[2,3-d]pyrimidin-2yl)hydrazono)methyl)-4H-chromen-4-one (16b) This compound was obtained as yellow solid in 80% yield. m.p.207.4~208.2
C. ESI-MS m/z:442.0 IR (KBr) cm1: 3416, 3054, 2958, 1628, 1537, 1481, 1391, 1336, 1199, 1020, 865, 783, 720. 1H NMR (400 MHz, DMSO) d 11.09 (s, 1H, NH), 8.77 (s, 1H, NCH), 8.22 (s, 1H, AreH), 8.05 (s, 1H, AreH), 7.87 (t, J ¼ 10.1 Hz, 1H, AreH), 7.80 (d, J ¼ 9.1 Hz, 1H, AreH), 7.47 (s, 1H, AreH), 7.22 (d, J ¼ 6.2 Hz, 1H, AreH), 3.85 (s, 4H, OCH2), 3.74 (s, 4H, NCH2). 13C NMR (101 MHz, DMSO) d 174.5(C), 171.6(C), 159.0(C), 156.2(CH), 154.8(C), 153.7(CH),

71

134.6(C), 132.5(CH), 130.7(CH), 124.8(CH), 124.6(C), 121.8(CH), 121.6(C), 119.8(CH), 117.8(C), 111.0(C), 66.5(2CH2), 47.0(2CH2). Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow, monitored by UV absorption at 335 nm, showed 92.4% purity. 5.9.3. (E)-6-Bromo-3-((2-(4-morpholinothieno[2,3-d]pyrimidin-2yl)hydrazono)methyl)-4H-chromen-4-one (16c) This compound was obtained as yellow solid in 72% yield. m.p. 222.8e223.9
C. ESI-MS m/z:488.0 IR (KBr) cm1: 3426, 3086, 2960, 1650, 1571, 1536, 1462, 1364, 1195, 1016, 866, 776. 1H NMR (400 MHz, DMSO) d 11.12 (s, 1H, NH), 8.78 (s, 1H, NCH), 8.20 (d, J ¼ 9.4 Hz, 2H, AreH), 8.00 (d, J ¼ 9.9 Hz, 1H, AreH), 7.73 (d, J ¼ 9.0 Hz, 1H, AreH), 7.48 (s, 1H, AreH), 7.22 (s, 1H, AreH), 3.85 (s, 4H, OCH2), 3.74 (s, 4H, NCH2). Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH (10% H2O) eluent at 1 mL/min flow, monitored by UV absorption at 335 nm, showed 89.5% purity. 5.9.4. (E)-6-Methyl-3-((2-(4-morpholinothieno[2,3-d]pyrimidin-2yl)hydrazono)methyl)-4H-chromen-4-one (16d) This compound was obtained as light yellow solid in 88% yield. m.p.231.7~233.2
C. ESI-MS m/z:422.1 IR (KBr) cm1: 3424, 3104, 2964, 1638, 1545, 1482, 1444, 1367, 1318, 1179, 1109, 861, 810, 719. 1H NMR (400 MHz, DMSO) d 11.07 (s, 1H, NH), 8.73 (s, 1H, NCH), 8.23 (s, 1H, AreH), 7.92 (s, 1H, AreH), 7.65 (dd, J ¼ 18.4, 8.7 Hz, 2H, AreH), 7.48 (d, J ¼ 6.1 Hz, 1H, AreH), 7.22 (d, J ¼ 6.1 Hz, 1H, AreH), 3.85 (s, 4H, OCH2), 3.74 (s, 4H, NCH2), 2.45 (s, 3H, CH3). 13C NMR (101 MHz, DMSO) d 175.5(C), 171.7(C), 159.1(C), 156.3(CH), 154.5(C), 153.3 (2C), 135.9(CH), 133.1(C), 124.8(CH), 123.5(C), 121.9(CH), 119.6(C), 118.9(CH), 117.7(CH), 111.0(C), 66.5(2CH2), 47.0(2CH2), 20.9(CH3). Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow, monitored by UV absorption at 335 nm, showed 96.8% purity. 5.9.5. (E)-6-Ethyl-3-((2-(4-morpholinothieno[2,3-d]pyrimidin-2yl)hydrazono)methyl)-4H-chromen-4-one (16e) This compound was obtained as yellow solid in 77% yield. m.p. 215.8e216.8
C. ESI-MS m/z:435.5 IR (KBr) cm1: 3428, 3258, 2958, 2854, 1621, 1527, 1480, 1308, 1159, 1114, 862, 822, 782, 720. 1H NMR (400 MHz, DMSO) d 11.08 (s, 1H, NH), 8.74 (s, 1H, NCH), 8.24 (s, 1H, AreH), 7.93 (s, 1H, AreH), 7.72 (d, J ¼ 8.6 Hz, 1H, AreH), 7.66 (s, 1H, AreH), 7.47 (s, 1H, AreH), 7.23 (d, J ¼ 6.3 Hz, 1H, AreH), 3.86 (s, 4H, OCH2), 3.74 (s, 4H, NCH2), 2.09 (s, 2H, CH2), 1.24 (t, J ¼ 7.5 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO) d 175.0(C), 171.2(C), 158.6(C), 157.2(CH), 155.9(CH), 154.2(C), 152.8(C), 141.6(C), 134.4(CH), 132.7(CH), 123.1(CH), 121.4(C), 119.1(C), 118.5(CH), 117.2(CH), 110.5(C), 66.0(2CH2), 46.5(2CH2), 27.5(CH2), 15.3(CH3). Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow, monitored by UV absorption at 335 nm, showed 90.6% purity. 5.9.6. (E)-6-(Tert-butyl)-3-((2-(4-morpholinothieno[2,3-d] pyrimidin-2-yl)hydrazono)methyl)-4H-chromen-4-one (16f) This compound was obtained as yellow solid in 79% yield. m.p. 131.2e132.5
C. ESI-MS m/z:464.2 IR (KBr) cm1: 3440, 3032, 2960, 1642, 1532, 1484, 1366, 1116, 866, 706. 1H NMR (400 MHz, DMSO) d 11.10 (s, 1H, NH), 8.74 (s, 1H, NCH), 8.24 (s, 1H, AreH), 8.04 (s, 1H, AreH), 7.91 (s, 1H, AreH), 7.65 (d, J ¼ 8.7 Hz1, H, AreH), 7.47 (d, J ¼ 5.6 Hz, 1H, AreH), 7.22 (d, J ¼ 5.6 Hz, 1H, AreH), 3.84 (s, 4H, OCH2), 3.73 (s, 4H, NCH2), 1.34 (s, 9H, CH3). 13C NMR (101 MHz, DMSO) d 175.6(C), 171.1(C), 159.0(C), 156.1(CH), 154.4(CH), 153.4(C), 148.9(C), 133.4(C), 132.7(CH), 123.1(CH), 121.9(CH), 120.8(C), 119.5(C), 118.8(CH), 117.8(CH), 111.0(C), 66.5(2CH2), 47.0(2CH2), 35.0(C), 31.4(3CH3). Analytical HPLC on an Agilent (1100) using a

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250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH (15% H2O) eluent at 1 mL/min flow, monitored by UV absorption at 335 nm, showed 99.3% purity.

(15%H2O) eluent at 1 mL/min flow, monitored by UV absorption at 335 nm, showed 97.6% purity. 5.10. mTOR Kinase Assay [10]

5.9.7. (E)-6-Hydroxy-3-((2-(4-morpholinothieno[2,3-d]pyrimidin2-yl)hydrazono)methyl)-4H-chromen-4-one (16g) This compound was obtained as light yellow solid in 86% yield. m.p. 265.3e266.3
C. ESI-MS m/z:446.1 IR (KBr) cm1: 3428, 3096, 2960, 2896, 1628, 1546, 1476, 1366, 1320, 1188, 1114, 1022, 870, 820, 784, 730. 1H NMR (400 MHz, DMSO) d 11.03 (s, 1H, NH), 10.11 (s, 1H, OH), 8.68 (s, 1H,NCH), 8.21 (s, 1H, AreH), 7.57 (d, J ¼ 9.5 Hz, 1H, AreH), 7.46 (d, J ¼ 4.0 Hz, 1H, AreH), 7.36 (s, 1H, AreH), 7.22 (dd, J ¼ 16.6, 7.7 Hz, 2H, AreH), 3.83 (s, 4H, OCH2), 3.73 (s, 4H, NCH2). Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH (15% H2O) eluent at 1 mL/ min flow, monitored by UV absorption at 335 nm, showed 100% purity. 5.9.8. (E)-3-((2-(4-Morpholinothieno[2,3-d]pyrimidin-2-yl) hydrazono)methyl)-6-nitro-4H-chromen-4-one (16h) This compound was obtained as yellow solid in 79% yield. m.p. 254.7e255.4
C. ESI-MS m/z:251.1 IR (KBr) cm1: 3424, 3104, 2952, 2852, 1628, 1546, 1422, 1336, 1116, 878, 716. 1H NMR (400 MHz, DMSO) d 10.68 (s, 1H, NH), 8.20 (d, J ¼ 2.3 Hz, 1H, NCH), 7.88 (s, 1H, AreH), 7.63 (d, J ¼ 3.4 Hz, 2H, AreH), 7.45 (d, J ¼ 6.1 Hz, 1H, AreH), 7.24e7.15 (m, 1H, AreH), 7.01 (d, J ¼ 9.0 Hz, 1H, AreH), 3.81 (d, J ¼ 4.2 Hz, 4H, OCH2), 3.73 (d, J ¼ 4.3 Hz, 4H, NCH2). 13C NMR (101 MHz, DMSO) d 168.1(C), 162.1(C), 161.5(C), 159.0(C), 156.7(CH), 139.4(CH), 137.9(C), 127.1(C), 126.4(CH), 121.9(CH), 121.5(C), 120.7(C), 120.5(CH), 117.6(CH), 115.7(CH), 66.3(2CH2), 47.0(2CH2). ESI-HRMS m/z: calcd for C20H16N6O5S [MþH]þ:453.0903; found 453.3333. Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH (25% H2O) eluent at 1 mL/min flow, monitored by UV absorption at 335 nm, showed 92.4% purity. 5.9.9. (E)-3-((2-(4-Morpholinothieno[2,3-d]pyrimidin-2-yl) hydrazono)methyl)-4-oxo-4H-chromene-6-carboxylic acid (16i) This compound was obtained as light yellow solid in 85% yield. m.p. 237.7e238.8
C. ESI-MS m/z:452.1 IR (KBr) cm1: 3420, 3100, 2960, 2924, 1698, 1620, 1544, 1422, 1198, 1116, 866, 772. 1H NMR (400 MHz, DMSO) d 12.88 (s, 1H, COOH), 10.70 (s, 1H, NH), 8.78 (s, 1H, NCH), 8.67 (d, J ¼ 11.0 Hz, 1H, AreH), 8.17 (s, 1H, AreH), 7.89 (d, J ¼ 8.5 Hz, 1H, AreH), 7.50e7.42 (m, 2H, AreH), 7.19 (d, J ¼ 5.6 Hz, 1H, AreH), 3.82 (s, 4H, OCH2), 3.74 (s, 4H, NCH2). 13C NMR (101 MHz, DMSO) d 175.3(C), 171.6(C), 166.6(C), 159.0(C), 158.2(C), 155.7(CH), 153.7(CH), 151.9(C), 134.8(CH), 132.5(CH), 132.2(CH), 131.4(C), 127.5(C), 121.9(CH), 120.2(C), 119.8(CH), 117.8(C), 66.51(2CH2), 46.99(2CH2).ESI-HRMS m/z: calcd for C21H17N5O5S [MþH]þ:452.0950; found 452.1018. Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/ MeOH (50%H2O) eluent at 1 mL/min flow, monitored by UV absorption at 335 nm, showed 95.9% purity. 5.9.10. (E)-3-((2-(4-Morpholinothieno[2,3-d]pyrimidin-2-yl) hydrazono)methyl)-4H-chromen-4-one (16j) This compound was obtained as light yellow solid in 78% yield. m.p.221.2~222.0
C. ESI-MS m/z:430.1 IR (KBr) cm1: 3444, 3072, 1718, 1640, 1546, 1474, 1186, 1114, 1024, 864, 774, 726. 1H NMR (400 MHz, DMSO) d 11.08 (s, 1H, NH), 8.75 (s, 1H, NCH), 8.23 (s, 1H, AreH), 8.12 (d, J ¼ 7.9 Hz, 1H, AreH), 7.84 (t, J ¼ 7.9 Hz, 1H, AreH), 7.71 (d, J ¼ 8.4 Hz, 1H, AreH), 7.53 (t, J ¼ 7.4 Hz, 1H, AreH), 7.47 (d, J ¼ 6.1 Hz, 1H, AreH), 7.21 (d, J ¼ 5.9 Hz, 1H, AreH), 3.84 (s, 4H, OCH2), 3.73 (s, 4H, NCH2). Analytical HPLC on an Agilent (1100) using a 250 mm  4.6 mm SD-C18 analytical column, H2O/MeOH

The mTOR kinase activity of all the compounds were determined using LANCE® Ultra time-resolved fluorescence resonance energy transfer (TR-FRET) assay following the manufacturer's instructions, with compounds I, II, III and PI-103 as positive controls. Briefly, mTOR enzyme (10 nM), ATP (21.6 uM), ULight-4E-BP1 Peptide (100 nM) and test compounds were diluted in kinase buffer (50 mM HEPES pH 7.5.1 mM EGTA, 3 mM MnCl2, 10 mM MgCl2, 2 mM DTT and 0.01%Tween-20). The reactions were performed in white 384-well Optiplates (PerkinElmer, MA, USA) at room temperature for 1 h and stopped by adding EDTA to 16 mM. Eu-antiphospho-4E-BP1 (Thr37/46) Antibody (PerkinElmer, MA, USA) was then added to each well to a final concentration of 2 nM. The intensity of the light emission was measured with an EnVision® Multilabel Reader (PerkinElmer, MA, USA) in TR-FRET mode (excitation at 320 nm and emission at 665 nm). All of the compounds were tested two times. The results expressed as IC50 (inhibitory concentration 50%) were the averages of two determinations. 5.11. PI3Ka Kinase Assay [14] The selected compounds (10h, 10i, 16h, 16i) are tested for their activity against PI3Ka using a Kinase-Glo® Luminescent Kinase Assay, with compounds I and PI103 as positive controls. The kinase reaction is done in 384-well black plate. Each well is loaded with 50 mL of test items (in 90% DMSO) and 5 mL reaction buffer containing 10 mg/mL PI substrate (L-a-phosphatidylinositol; Avanti Polar Lipids; prepared in 3% octyl-glucoside) and the PI3Ka protein 10 nM is then added to it. The reaction is started by the addition of 5 mL of 1 mM ATP prepared in the reaction buffer and is incubated for 60 min for p110a. It is terminated by the addition of 10 mL Kinase-Glo buffer. The plates are then read in a Synergy 2 reader for luminescence detection. All of the compounds were tested two times. 5.12. Cytotoxicity assay in vitro The cytotoxic activity of four selected compounds (10hei, 16hei) were evaluated with H460, PC-3 cell lines by the standard MTT assay in vitro, with compound I as positive control. The cancer cell lines were cultured in minimum essential medium (MEM) supplement with 10% fetal bovine serum (FBS). Approximately 4  103 cells, suspended in MEM medium, were plated onto each well of a 96-well plate and incubated in 5% CO2 at 37
C for 24 h. The test compounds at indicated final concentrations were added to the culture medium and the cell cultures were continued for 72 h. Fresh MTT was added to each well at a terminal concentration of 5 mg/mL and incubated with cells at 37
C for 4 h. The formazan crystals were dissolved in 100 mL DMSO each well, and the absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm (for the reference wavelength) was measured with the ELISA reader. All of the compounds were tested three times in each of the cell lines. The results expressed as inhibition rates or IC50 (half-maximal inhibitory concentration) were the averages of two determinations and calculated by using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software. 5.13. Docking studies For docking purposes, the three-dimensional structures of the mTOR (PDB code: 4JT6) and PI3Ka (PDB code: 4L23) were obtained from RCSB Protein Data Bank [15,16]. Hydrogen atoms were added to the structure allowing for appropriate ionization at physiological

W. Zhu et al. / European Journal of Medicinal Chemistry 93 (2015) 64e73

pH and waters were removed. The protonated state of several important residues, such as GLY2238, ASP2357, VAL2240 and TYR2225, were adjusted by using SYBYL6.9.1 (Tripos, St. Louis, USA) in favor of forming reasonable hydrogen bond with the ligand. Molecular docking analysis was carried out by the SURFLEX-DOCK module of SYBYL 6.9.1 package to explore the binding model for the active site of mTOR with its ligand. All atoms located within the range of 5.0 Å from any atom of the cofactor were selected into the active site, and the corresponding amino acid residue was, therefore, involved into the active site if only one of its atoms was selected. Other default parameters were adopted in the SURFLEXDOCK calculations. All calculations were performed on Silicon Graphics workstation. Acknowledgments We gratefully acknowledge the generous support provided by The National Natural Science Funds of China (No. 80140357), Project supported by the Natural Science Foundation of Jiangxi Province of China (No. 20142BAB215020), Doctoral Scientific Research Foundation of Jiangxi Science & Technology Normal University and Program of Key Laboratory of Drug Design and Optimization,Jiangxi Science & Technology Normal University (300098010306). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2015.01.061. References [1] J. Rodon, R. Dienstmann, V. Serra, J. Tabernero, Nat. Rev. Clin. Oncol. 10 (2013)

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