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Design, Synthesis and Structure-Activity Relationships of Novel Diaryl Urea Derivatives as Potential EGFR Inhibitors Nan Jiang 1 , Yanxin Bu 1 , Yu Wang 1 , Minhua Nie 1 , Dajun Zhang 2, * and Xin Zhai 1, * 1

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Key Lab. of New Drugs Design and Discovery of Liaoning Province, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, China; [email protected] (N.J.); [email protected] (Y.B.); [email protected] (Y.W.); [email protected] (M.N.) Department of Chemsitry, Shenyang Medical College, 146 Huanghe North Street, Huanggu District, Shenyang 110034, China Correspondence: [email protected] (D.Z.); [email protected] (X.Z.); Tel./Fax: +86-24-2398-6429 (X.Z.)

Academic Editor: Derek J. McPhee Received: 19 October 2016; Accepted: 15 November 2016; Published: 18 November 2016

Abstract: Two novel series of diaryl urea derivatives 5a–i and 13a–l were synthesized and evaluated for their cytotoxicity against H-460, HT-29, A549, and MDA-MB-231 cancer cell lines in vitro. Therein, 4-aminoquinazolinyl-diaryl urea derivatives 5a–i demonstrated significant activity, and seven of them are more active than sorafenib, with IC50 values ranging from 0.089 to 5.46 µM. Especially, compound 5a exhibited the most active potency both in cellular (IC50 = 0.15, 0.089, 0.36, and 0.75 µM, respectively) and enzymatic assay (IC50 = 56 nM against EGFR), representing a promising lead for further optimization. Keywords: diaryl urea; 4-aminoquinazolinyl; synthesis; cytotoxicity; EGFR inhibitors

1. Introduction Recently, small molecular multiple targeted drugs have played a crucial role in cancer therapy due to their high efficiency and low toxicity. Diaryl urea derivative sorafenib [1], the first oral multikinase inhibitor targeted Raf and receptor tyrosine kinases (RTKs), has been applied for the treatment of advanced renal cell carcinoma (RCC) [2], unresectable hepatocellular carcinoma (HCC) [3], and differentiated thyroid carcinoma (DTC) [4]. It is reported that the lipophilic diaryl urea moiety served as a key structural fragment for binding with the hydrophobic pocket of the kinase domain through hydrogen bonds and hydrophobic interactions [5]. Subsequently, diverse diaryl urea derivatives were developed successively in the past decades, such as linifanib [6], tivozanib [7], and ki8751 [8] (Figure 1). Therein, thieno[3,2-d]pyrimidine derivative S1 bearing diaryl urea moieties at the C-2 positon exhibited significant inhibition of tyrosine kinases, including c-Kit, orphan receptor tyrosine kinase (RET), and FLT3 for its prominent framework [9]. Similarly, the 4-aminoquinazolinyl skeleton, which competitively binds to the ATP binding pocket of intracellular kinase domains and blocks the conduction of downstream signaling networks mediated by tyrosine kinase, is also extensively used in the design of RTKs inhibitors (e.g., gefitinib, erlotinib and S2) [10,11]. In light of the abovementioned considerations, and as ongoing efforts to identify new potent antitumor agents, a novel series of 4-aminoquinazolinyl-diaryl urea derivatives 5a–i were designed according to bioisosterism theory to achieve synergistic antitumor effects (Figure 2). In addition, a variety of substituents and aliphatic amino were introduced into the terminal phenyl group and C-4 position on the quinazoline ring to explore the influence of electronic and Molecules 2016, 21, 1572; doi:10.3390/molecules21111572

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steric hindrance effect. Furthermore, another novel series of diaryl urea derivatives bearing effect. Furthermore, another novel series of diaryl urea derivatives 13a–l bearing a13a–l 5-pyridyl-4effect. Furthermore, another novel series of diaryl urea derivatives 13a–l bearing a 5-pyridyl-4a 5-pyridyl-4-aminopyrimidinyl motif were designed based on scaffold hopping principle, in the aminopyrimidinyl motif were designed based on scaffold hopping principle, in the hope of attaining aminopyrimidinyl motif were designed based on scaffold hopping principle, in the hope of attaining hope of attaining diversity optimal cellular potency improving water solubility. structural diversitystructural and optimal cellularand potency via improving water via solubility. All compounds were structural diversity and optimal cellular potency via improving water solubility. All compounds were All compounds were synthesized and evaluated for their cytotoxicity against HT-29, H-460, A549, and synthesized and evaluated for their cytotoxicity against HT-29, H-460, A549, and MDA-MB-231 cancer synthesized and evaluated for their cytotoxicity against HT-29, H-460, A549, and MDA-MB-231 cancer MDA-MB-231 cancer cell lines. Additionally, enzymatic assay of the most active compound 5a was cell lines. Additionally, enzymatic assay of the most active compound 5a was also presented in cell lines. Additionally, enzymatic assay of the most active compound 5a was also presented in also study. presented in this study. this this study.

Figure 1. Structures diaryl urea derivatives. Figure Structures of of Figure 1. 1. Structures of diaryl diaryl urea urea derivatives. derivatives.

Figure 2. Design of target compounds. Figure 2. 2. Design Design of of target target compounds. compounds. Figure

2. Results and Discussion 2. Results and Discussion 2. Results and Discussion 2.1. Chemistry 2.1. Chemistry Chemistry 2.1. The synthesis of target compounds 5a–i is depicted in Scheme 1. The synthesis of intermediates The synthesis synthesis of of target target compounds compounds 5a–i 5a–i is is depicted depicted in in Scheme Scheme 1. 1. The The synthesis synthesis of of intermediates intermediates The 3a–b have been described in detail in our previous work [12], so the synthetic method is not listed here. 3a–b have been described in detail in our previous work [12], so the synthetic method is not listedishere. 3a–b have been described in detail in our previous work [12], so the synthetic method not [12]. A Suzuki reaction of 3a,b with commercially available 4-aminophenylboronic acid pinacol ester [12]. Ahere. Suzuki reaction of 3a,breaction with commercially 4-aminophenylboronic acid pinacol ester listed [12]. A Suzuki of 3a,b withavailable commercially available 4-aminophenylboronic under nitrogen protection provided the key intermediates 4a,b [13]. A series of substituted aromatic underpinacol nitrogen protection provided protection the key intermediates 4a,b [13]. A series of substituted aromatic acid ester under nitrogen provided the key intermediates 4a,b [13]. A series isocyanates 6 and isothiocynates 7 were subsequently prepared by treating the corresponding arylamine isocyanates 6 and isothiocynates 7 were subsequently prepared by treating the corresponding arylamine of substituted aromatic isocyanates 6 and isothiocynates 7 were subsequently prepared by treating the with triphosgene (BTC) or 1,4-diazabicyclo[2.2.2]octane (DABCO) without further purification with triphosgene (BTC) with or 1,4-diazabicyclo[2.2.2]octane (DABCO) without further purification corresponding arylamine triphosgene (BTC) or 1,4-diazabicyclo[2.2.2]octane (DABCO) without [14,15]. Eventually, target compounds 5a–i were successfully obtained via the reaction of 4a,b with [14,15]. Eventually, target compounds 5a–i were successfully obtained via the reaction of 4a,b further purification [14,15]. Eventually, target compounds 5a–i were successfully obtained viawith the corresponding 6 or 7 in THF at 30 °C, respectively. corresponding or 7 corresponding in THF at 30 °C, respectively. reaction of 4a,b6with 6 or 7 in THF at 30 ◦ C, respectively.

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Scheme 1. Synthesis of target target compounds compounds5a–i. 5a–i. Reagents Reagentsand andconditions: conditions: (a) (a) urea, urea, 160 160 ◦°C, 12 h; h; (b) (b) POCl POCl33,, Scheme 1. Synthesis of C, 12 Scheme 1. Synthesis of target compounds 5a–i. 30 Reagents and conditions: (a) urea, 160 °C, 12 h;acid (b) POCl 3, 1,1 THF, TEA, ◦ ◦ DIPEA, 90 °C, 6 h; (c) NH 2 (CH 2 ) n R °C, 15 min; (d) 4-aminophenylboronic pinacol DIPEA, 90 C, 6 h; (c) NH2 (CH2 )n R1 , THF, TEA, 30 C, 15 min; (d) 4-aminophenylboronic acid pinacol DIPEA, 90 °C, 6 h; (c) NH 2(CH2)nR , THF, TEA, 30 °C, 15 min; (d) 4-aminophenylboronic acid pinacol ◦ ester, Na22CO ester, Na CO33,, Pd(PPh Pd(PPh33))22Cl Cl2,2THF, , THF,NN2,2r.f., , r.f.,55h;h;(e) (e)6a–d 6a–doror7a–b, 7a–b,THF, THF,30 30°C, C,66h; h;(f) (f)BTC, BTC, 1,4-dioxane, 1,4-dioxane, ester, Na2CO3, Pd(PPh3)2Cl2, THF, N2, r.f., 5 h; (e) 6a–d or 7a–b, THF, 30 °C, 6 h; (f) BTC, 1,4-dioxane, r.f., 8–12 h; h; (g) (g) (i) (i) DABCO, DABCO, CS CS22,, toluene, r.t., 12 12 h; h; (ii) (ii) BTC, BTC, CHCl CHCl33,, r.f., h. r.f., 8–12 toluene, r.t., r.f., 11 h. r.f., 8–12 h; (g) (i) DABCO, CS2, toluene, r.t., 12 h; (ii) BTC, CHCl3, r.f., 1 h.

The synthetic route routeofoftarget targetcompounds compounds13a–l 13a–l is outlined in Scheme 2. Dry HCl was bubbled The synthetic is outlined in in Scheme 2. Dry HCl was bubbled into The synthetic route of target compounds 13a–l is outlined Scheme 2. Dry HCl was bubbled into the mixture of 4-aminobenzonitrile in EtOH and 1,4-dioxane in an ice bath to generate compound theinto mixture of 4-aminobenzonitrile in EtOH andand 1,4-dioxane in in anan iceice bath the mixture of 4-aminobenzonitrile in EtOH 1,4-dioxane bathtotogenerate generatecompound compound 8, 8, which was further replaced with NHto3 to afford intermediate 9 [16]. 10 was prepared via reaction which was further replaced with drydry NH afford intermediate 9 [16]. 10 was prepared via reaction 8, which was further replaced with dry NH 3 to afford intermediate 9 [16]. 10 was prepared via reactionof 3 of 2-(pyridin-2-yl)acetonitrile and excess N,N-dimethylformamide dimethyl acetal (DMF-DMA) at 30 30 °C 2-(pyridin-2-yl)acetonitrile and excess N,N-dimethylformamide acetal(DMF-DMA) (DMF-DMA)atat of 2-(pyridin-2-yl)acetonitrile and excess N,N-dimethylformamide dimethyl dimethyl acetal 30 °C◦ C for 24 hh [17]. Condensation reaction of 9 and 10 in MeOH-H 2O solution in the presence of Na2CO3 gave forfor 2424 reaction and MeOH-H the presence h[17]. [17].Condensation Condensation reaction ofof9 9and 1010 in in MeOH-H 2O solution in theinpresence of Na2of CONa 3 gave 2 O solution 2 CO3 rise to intermediate 11 [18]. Subsequently, 11 condensed with corresponding aromatic isocyanate 6c–n riserise to intermediate 11 [18]. Subsequently, 11 condensed with corresponding aromatic isocyanate 6c–n gave to intermediate 11 [18]. Subsequently, 11 condensed with corresponding aromatic isocyanate in a similar manner as described for compounds 5a–i to furnish 12a–l. Finally, 12a–l were treated in ainsimilar manner as as described forfor compounds 5a–i to to furnish 12a–l. Finally, 12a–l were treated 6c–n a similar manner described compounds 5a–i furnish 12a–l. Finally, 12a–l were treated with 20%–30% hydrochloride ethanol solution to provide target compounds 13a–l. with 20%–30% hydrochlorideethanol ethanolsolution solutionto to provide provide target compounds compounds 13a–l. with 20%–30% hydrochloride 13a–l.

Scheme Synthesisofoftarget targetcompounds compounds5a–i. 5a–i. Reagents Reagents and and conditions: Scheme 2. 2.Synthesis conditions:(a) (a)HCl, HCl,EtOH-1,4-dioxane, EtOH-1,4-dioxane, Scheme 2. Synthesis of target compounds 5a–i. Reagents and conditions: (a) HCl, EtOH-1,4-dioxane, ◦ ◦ 3, EtOH, 0◦°C, 6 h, r.t., 24 h; (c) DMF-DMA, MeOH, 30 °C, 2CO3, 0 °C, 6 h, r.t., 48 h; (b) NH 0 C, 6 h, r.t., 48 h; (b) NH3 , EtOH, 0 C, 6 h, r.t., 24 h; (c) DMF-DMA, MeOH, 30 C,24 24h;h;(d) (d)Na Na 2 CO3 , 3, EtOH, 0 °C, 6 h, r.t., 24 h; (c) DMF-DMA, MeOH, 30 °C, 24 h; (d) Na2CO3, 0 °C, 6 h, r.t., 48 h; (b) NH MeOH-H 2O, 70◦ °C, 24 h; (e) 6c–n, THF, 30 ◦ °C, 6 h; and (f) HCl-EtOH, CHCl 3, 1 h. MeOH-H2 O, 70 C, 24 h; (e) 6c–n, THF, 30 C, 6 h; and (f) HCl-EtOH, CHCl3 , 1 h. MeOH-H2O, 70 °C, 24 h; (e) 6c–n, THF, 30 °C, 6 h; and (f) HCl-EtOH, CHCl3, 1 h.

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2.2. Biological Results and Discussion

2.2. Biological Results and Discussion For summarizing structure-activity relationships (SARs), all target compounds (5a–i and 13a–l) wereBiological evaluated for their cytotoxicity against HT-29 (human colon H-460 (human lung 2.2. Results and Discussion For structure-activity relationships (SARs), all cancer), target compounds (5a–i andcancer), Molecules 2016, 21,summarizing 1572 413a–l) of 12 A549 (human lung cancer), and MDA-MB-231 (human breast cancer) cell lines by 3-(4,5-dimethylthiazolwereFor evaluated for their cytotoxicity against HT-29 (human colon cancer), H-460 (human lung cancer), summarizing structure-activity relationships (SARs), all target compounds (5a–i and 13a–l) 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, taking sorafenib as the positive control. The results Molecules 2016, 21, 21, 1572cancer), and of 11 11 A549 (human lung MDA-MB-231 (human breast cancer) cell lines by 3-(4,5-dimethylthiazolMolecules 2016, 1572 44 of were evaluated for their cytotoxicity against HT-29 (human colon cancer), H-460 (human lung cancer), expressed as IC50 values (μM)bromide are presented in Tables 1 and 2. 2.2. Biological Results and Discussion 2-yl)-2,5-diphenyltetrazolium (MTT) assay, taking sorafenib as the positive control. The results Molecules 2016, 21, 1572cancer), and MDA-MB-231 (human breast cancer) cell lines by 3-(4,5-dimethylthiazol4 of 11 A549 (human lung Molecules 2016, 21, 1572 and Discussion 4 of 11 2.2. Biological Results expressed as IC 50 values (μM) are presented in Tables 1 and 2. 2.2. Biological Results and Discussion 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, taking sorafenib as the positive control. The results For summarizing structure-activity relationships (SARs), all H-460, targetA-549, compounds (5a–i and Table 1. Structures and cytotoxicity of compounds 5a–i against HT-29, and MDA-MB-231 2.2. Biological Results and Discussion For summarizing structure-activity relationships (SARs), all target compounds (5a–i and 13a–l) expressed as IC 50 values (μM) are presented in Tables 1 and 2. 13a–l) 2.2. were evaluated for their cytotoxicity against HT-29 (human colon cancer), H-460 (human Biological Results and Discussion For summarizing structure-activity relationships (SARs), all target compounds (5a–i and 13a–l) cells Tablein1.vitro. Structures and cytotoxicity of compounds 5a–i against HT-29, H-460, A-549, and MDA-MB-231 were evaluated for their cytotoxicity against HT-29 (human colon cancer), H-460 (human lung cancer), lung cancer), A549 (human lung cancer), and MDA-MB-231 (human cancer) cell lines by For summarizing structure-activity relationships (SARs), all cancer), targetbreast compounds (5a–i and 13a–l) wereFor evaluated for theirstructure-activity cytotoxicity against HT-29 (human colon H-460 (human lung cancer), cells in1.vitro. summarizing relationships (SARs), all target compounds (5a–i and 13a–l) a IC 50 (μM) Table Structures and cytotoxicity of compounds 5a–ibreast against HT-29, H-460, A-549, and MDA-MB-231 A549 (human lung cancer), and MDA-MB-231 MDA-MB-231 (human cancer) cell lines by 3-(4,5-dimethylthiazol2 cytotoxicity 1 against HT-29 were evaluated for their (human colon cancer), H-460 (human lung cancer), A549 (human lung cancer), and (human breast cancer) cell lines by 3-(4,5-dimethylthiazol3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, taking sorafenib as the Compd. X n R R were evaluated against HT-29 colon cancer), H-460 (human lung cells in vitro.for their cytotoxicity HT-29(human H-460 A-549 MDA-MB-231 a 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, taking sorafenib as50 the the positive control. Thecancer), results IC (μM) A549 (human lung cancer), and MDA-MB-231 (human breast cancer) cell lines by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, taking sorafenib as positive results positive control. results (µM) are presented in Tables 1 control. and 2. The 2 Molecules 2016,The 21, 1572 4 of 11 50 values Compd. Xlung n cancer), Rexpressed R1as IC A549 (human and MDA-MB-231 (human breast cancer) cell lines by 3-(4,5-dimethylthiazolexpressed as IC 50 values (μM) are presented in Tables 1 and 2. 5a 2-Cl, 3 0.15 ± 0.14 0.089 0.36 0.75 0.65 HT-29 H-460 A-549 MDA-MB-231 a ± 0.27 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, taking sorafenib as50 the the positive control. The± results results expressed asO IC50 2values (μM) are6-CH presented in Tables 1 and 2. ± 0.11 IC (μM) 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, taking sorafenib as positive control. The Molecules 2016, 21, 4 of 11 Compd. X 1572 n R2 R1 Table 1. Structures cytotoxicity of6-CH compounds 5a–i against HT-29, MDA-MB-231 expressed asO IC 50and are presented in Tables 1 and 2. ±±H-460, 5a 5b 2-Cl, 0.15 0.089 0.11 A-549, 0.36 0.27 0.75 ± 0.65 2values 3)23 1.09 ± 0.14 0.21 1.50 0.13 2.33 ±and 1.52 2.42 0.28 3,4-(CH 2.2. Biological Results and (μM) Discussion HT-29 H-460 A-549 MDA-MB-231 expressed as IC 50 values (μM) are presented in Tables 1 and 2. Table 1. Structures and cytotoxicity of compounds 5a–i against HT-29, H-460, A-549, and MDA-MB-231 Table and cytotoxicity of compounds 5a–i against HT-29, H-460, A-549, and MDA-MB-231 cells in vitro.1. Structures 5c 2-Cl, 6-CH 0.36 0.72 (SARs), 0.27 all ±±0.13 0.12 0.82 0.43 1.08 ± 0.65 0.15 OResults 5a 0.15 ± 0.14 0.089 0.11 0.36 ± 1.52 0.27 (5a–i 0.75 23 and 3)23 relationships 1.09 0.21 1.50 2.33 2.42 0.28 3,4-(CH 2.2. 5b Biological Discussion For structure-activity target compounds and 13a–l) cells summarizing in1.vitro. vitro. cells in Table Structures and cytotoxicity of compounds 5a–i against HT-29, H-460, A-549, and MDA-MB-231 Table 1. Structures and cytotoxicity of compounds 5a–i against HT-29, H-460, A-549, and MDA-MB-231 5d O 3 3 ) 2 1.72 ± 0.37 2.93 ± 0.62 5.46 ± 0.38 3.92 ± 1.24 3,4-(CH 5c 2-Cl, 6-CH 3 0.36 ± 0.72 0.27 ± 0.12 0.82 ± 0.43 1.08 ± 0.15 O 3 were5b evaluated cytotoxicity HT-290.21 (human colon cancer), H-460 (human lung 3)2 1.09 1.50 0.13 2.33 2.42 0.28 2 theirstructure-activity 3,4-(CHagainst a a 1.52 cells in vitro.for For summarizing relationships (SARs), allIC target compounds (5a–i andcancer), 13a–l) (µM) 50 50 IC (μM) a cells in vitro. IC 50 (μM) 22 and 11 3)2 5e O 3 4-F 2.64 ± 0.53 1.19 ± 0.96 3.41 ± 1.27 2.72 ± 0.42 Compd. n A549 (human lung cancer), MDA-MB-231 (human breast cancer) cell lines by 3-(4,5-dimethylthiazolX Compd. X n R 5d O 3 1.72 ± 0.37 2.93 ± 0.62 5.46 ± 0.38 3.92 ± 1.24 3,4-(CH R R 2 1 5cevaluated 2-Cl,R6-CH 3 0.36 ± 0.72 0.27 ± 0.12 0.82 ± 0.43 1.08 ± 0.15 O 3 their Compd. X for n R cytotoxicity were against HT-29 (human colon cancer), H-460 (human lung cancer), HT-29 H-460 A-549 MDA-MB-231 HT-29 H-460IC50 (μM) A-549 MDA-MB-231 a HT-29 H-460 A-549 MDA-MB-231 a ± 0.18 2-yl)-2,5-diphenyltetrazolium bromide assay, taking sorafenib as50 the positive The±± results 5f(humanO 4-Cl 0.24 0.17 0.77 0.22 2.30 2.59 0.36 Slung IC (μM) 5e 332 cancer), 4-F 2.64 0.53 1.19 0.96 3.41 1.27control. 2.72 0.42 Compd. X n R22 and R11 3)(MTT) 5d O 2 1.72 ±± 0.37 2.93 ±± 0.62 5.46 ± 3-(4,5-dimethylthiazol0.38 3.92 1.24 3,4-(CH A549 MDA-MB-231 (human breast cancer) cell lines by Compd. X n R R 5a 2-Cl, 6-CH 3 0.15 ± 0.14 0.089 ± 0.11 0.36 ± 0.27 0.75 ± 0.65 0.65 O 2 HT-29 H-460 A-549 MDA-MB-231 expressed as IC 50 2values (μM) are presented in Tables 1 and 2. 5a O 2 2-Cl, 6-CH 0.15 ± 0.089 ± 0.11 0.36 ± 0.27 0.75 ± 0.65 5a 2-Cl, 6-CH 3 0.15 ± 0.14 0.089 ± 0.11 0.36 ± 0.27 0.75 ± O 3 HT-29 H-460 A-549 MDA-MB-231 2-yl)-2,5-diphenyltetrazolium bromide assay, taking sorafenib positive The± results 5f 5g 4-Cl 0.24 0.17 0.77 ± 0.22 ± 0.18 2.59 0.36 4-OCF S 32 1.05 ±±0.012 1.28 0.68 2.81 0.73control. 3.02 0.51 5e O 4-F 3 (MTT) 2.64 0.53 1.19 0.96as the2.30 3.41 1.27 2.72 0.42 5a 5b 2-Cl, 6-CH 0.15 0.14 0.089 ± 0.11 0.36 0.27 0.75 0.65 O 2 3)23 1.09 ± 0.21 1.50 ± 0.13 2.33 ± 1.52 2.42 ± 0.28 3,4-(CH 5b 3 ) 2 1.09 ± 0.21 1.50 ± 0.13 2.33 ± 1.52 2.42 ± 0.28 O 2 3,4-(CH expressed (μM) are presented in Tables 2.±±±0.13 3,4-(CH 1.09 0.21 1 and 1.50 ± 1.52 2.420.75 ± 0.28 5b Oas SIC250 3 5a 2-Cl, 6-CH 0.15 0.14 0.089 0.11 2.33 0.36 33 )23 5h 0.65 0.33 1.02 0.74 1.44 0.39 3.13 0.85 5f 5g 4-Cl 0.24 ±0.012 0.17 0.77 0.22 ± 0.27 0.18 2.59 ± 0.65 0.36 4-OCF 2values 1.05 ±± 1.28 0.68 2.81 0.73 3.02 0.51 Table 1. Structures and cytotoxicity of compounds 5a–i against HT-29, H-460,2.30 A-549, and MDA-MB-231 5c 2-Cl, 6-CH 0.36 ±± 0.72 0.72 0.27 ± 0.12 0.12 0.82 ± 0.43 0.43 1.08 ±± 0.15 0.15 O 323 5b 3)233 1.09 0.21 1.50 0.13 2.33 1.52 2.42 0.28 3,4-(CH 5c 2-Cl, 6-CH 0.36 0.27 ± 0.82 ± 1.08 O 3)23 1.09 ±±0.012 0.21 1.50 0.13 2.33 ±± 1.52 ±± 0.28 O 3,4-(CH 5i S 4-OCF 2.41 2.58 0.74 4.86 0.56 4.07 1.02 5c 5b 2-Cl, 6-CH 0.36 ±± 0.72 0.27 ±± 0.82 ± 0.43 1.082.42 ± 0.15 5h 4-Cl 0.65 0.33 1.02 ±0.12 0.74 1.44 0.39 3.13 0.85 S 3 2332 cells O in vitro. 5g 4-OCF 3 1.05 1.28 0.68 2.81 0.73 3.02 0.51 Table 1. Structures and cytotoxicity of 5a–i against HT-29, H-460,5.46 A-549, and MDA-MB-231 5d O 3)2compounds 1.72 0.37 2.93 ±± 0.62 0.62 ±± 0.38 0.38 3.92 ±± 1.24 1.24 3,4-(CH 5c 2-Cl, 6-CH 0.36 ±±± 0.37 0.72 0.27 0.12 0.82 0.43 1.08 0.15 O 333 5d 3)23 1.72 2.93 5.46 3.92 3,4-(CH Sorafenib 3.27 0.32 2.15 0.43 4.47± 0.28 3.81± 0.50 5c 2-Cl, 6-CH 3 0.36 0.72 0.27 ± 0.12 0.82 ± 0.43 1.08 ± 0.15 O 5i S 4-OCF 2.41 2.58 0.74 4.86 0.56 3,4-(CH 1.72 ± 0.21 0.37 2.93 ± 0.62 ± 0.38 3.924.07 ± 1.241.02 5d 5h 33 )2 4-Cl 0.65 0.33 1.02 0.74 1.44 0.39 3.13 0.85 S 3 33 cells O in vitro. IC50 5.46 (μM) 5e O 4-F 2.64 ±±± 0.53 0.53 1.19of±±±three 0.96 3.41a ±±± 1.27 1.27 2.72 ±±± 0.42 0.42 5d 2 1.72 0.37 2.93 0.62 5.46 0.38 3.92 1.24 3,4-(CH a Results 2 1 3)± 5e O 33 expressed 4-F 2.64 1.19 0.96 3.41 2.72 are as means deviation) independent experiments. Compd. X n R R 5d O 3)2 SD (standard 1.72 ± 0.37 2.93 ± 0.62 5.46 ± 0.38 3.92 ± 1.24 3,4-(CH Sorafenib 3.27 0.32 2.15 0.43 4.47± 0.28 3.81± 0.50 4-OCF 2.41 ± 0.21 2.58 0.74 3.41 4.86 ± 0.56 2.72 4.07 ± 1.02 5e 5i O S 3 3 4-F 3 2.64 ± 0.53 1.19 ±±0.96 ± 1.27 ± 0.42 HT-29 H-460 A-549 MDA-MB-231 a ± 0.18 5f a 4-Cl 0.24 0.17 0.77 ± 0.22 0.22 2.30 2.59 0.36 S 232 IC50 (μM) 5e O 4-F 2.64 0.53 1.19 0.96 3.41 1.27 2.72 ±±± 0.42 0.42 5f 4-Cl 0.24 ±±±± 0.17 0.77 2.30 ±± 0.28 0.18 2.59 0.36 S 2 1 5e O 3 expressed 4-F 2.64 0.53 1.19 ±±three 0.96 3.41 1.27 2.72 Results are as means ± SD (standard deviation) of± independent experiments. Compd. X n R R Sorafenib 3.27 0.32 2.15 0.43 4.47± 3.81± 0.50 5f 5a S 2. Structures 22 4-Cl 0.24 ± 0.17 0.77 ± 0.22 2.30 ± 0.18 2.59 ± 0.36 2-Cl, 6-CH 3 0.15 ± 0.14 0.089 ± 0.11 0.36 ± 0.27 0.75 ± 0.65 O Table and cytotoxicity of compounds 13a–l against HT-29, H-460, A-549, and MDA-MB-231 HT-29 H-460 A-549 5f a 5g 4-Cl 33 0.24 0.17 0.77 ± 0.22 0.22 2.30 0.18 2.59 0.36 4-OCF S 22 1.05 0.012 1.28 0.68 2.81 0.73 MDA-MB-231 3.02 ±± 0.36 0.51 5g S 1.05 ±±±±0.012 1.28 0.68 2.81 0.73 3.02 0.51 5f 4-Cl 0.24 0.17 0.77 2.30 ±± 0.18 2.59 Results are expressed as4-OCF means ± SD (standard deviation) of± three independent experiments. cells in vitro. 3 ) 2 1.09 ± 0.21 1.50 ± 0.13 2.33 ± 1.52 2.42 ± 0.28 3,4-(CH 5g 5b S 2 4-OCF 1.05 ± 0.012 1.28 ± 0.68 2.81 ± 0.73 3.02 ± 0.51 5a 2-Cl, 6-CH 3 0.15 0.14 0.089 ± 0.11 0.36 0.27 0.75 0.65 O 2 Table 2. Structures and cytotoxicity compounds 13a–l HT-29, A-549, and MDA-MB-231 5h 4-Cl of 0.65±±±0.012 0.33 against 1.02 0.74 1.44 0.39 3.13 0.85 S 3 5g 4-OCF 33 1.05 1.28 0.68H-460,1.44 2.81 0.73 3.02 ±±± 0.85 0.51 5h 4-Cl 0.65 0.33 1.02 ±±± 0.74 ±±± 0.39 3.13 S 5g 4-OCF S 322 3 1.05 ± 0.012 1.28 0.68 2.81 0.73 3.02 0.51 cells in vitro. a 0.82 ± 0.43 5c 2-Cl, 6-CH 3 0.36 ± 0.72 0.27 ± 0.12 1.08 ± 0.15 O 3 IC 50 (μM) 3)2compounds 1.09 ±± 13a–l 0.21 1.50 0.13 2.33 ±± 1.52 ±± 0.28 O 3,4-(CH TableS 2. Structures and cytotoxicity HT-29, A-549, and MDA-MB-231 5i S 3 23 4-OCF 2.41 2.58 0.74H-460, 4.86 0.56 4.07 1.02 4-Clof 0.65 ± 0.33 against 1.02 ±± 1.44 ± 0.39 3.132.42 ± 0.85 5h 5b 5h 4-Cl 0.65 0.33 1.02 1.44 0.39 3.13 0.85 5i S 4-OCF 3 2.41 2.58 ±±0.74 0.74 4.86 4.07 Compd. R1 5h 4-Cl 0.65 ± 0.21 0.33 1.02 0.74 1.44 ± 0.56 0.39 3.13 ± 1.02 0.85 S 33 5d O 3 3HT-29 ) 2 1.72 ± 0.37 2.93 ± 0.62 ± 0.38 3.92 ± 1.24 3,4-(CH cells in vitro. H-460 A549 MDA-MB-231 a 5.46 Sorafenib 3.27 0.32 2.15 0.43 4.47± 0.28 3.81± 0.50 5c 2-Cl, 6-CH 3 0.36 ± 0.72 0.27 ± 0.12 0.82 ± 0.43 1.08 ± 0.15 O 3 IC 50 (μM) 4-OCF 2.41 0.21 2.58±±±0.74 0.74 4.86 4.86 0.56 4.074.07 4.07 1.02 5i 5i S S 3 4-OCF33 2.41 ± 2.58 ± 0.56 ± 1.020.50 Sorafenib 3.27 ±± 0.32 2.15 0.43 4.47± 3.81± 1 5i S 3 4-OCF 3 2.41 0.21 2.58 0.74 4.86 ± 0.28 0.56 1.02 Compd. R 13aaa Results H ± 2.56 28.64 ±2.15 1.08 44.83 ±4.47± 2.42 32.16 ± ±±±1.59 5e O 33 expressed 4-F30.52 2.64 0.53 1.19 ±0.43 0.96 3.41 1.27 2.72 0.42 are expressed as means means SD (standard deviation) of± three independent experiments. H-460 A549 MDA-MB-231 a 5.46 5d O are 3HT-29 )±±2 SD 1.72 ±± 0.37 2.93 0.62 ±± 0.28 0.38 3.92 1.24 3,4-(CH Sorafenib 3.27 ± ± 4.47 ± 0.28 3.81 ± 0.50 Sorafenib 3.27 0.32 2.15 0.43 3.81± 0.50 Results as (standard deviation) of three independent experiments. IC 50 (μM) Sorafenib 3.27 ± 0.32 NA2.15 ± 0.43 NA 4.47± 0.28 3.81± 0.50 1 Compd. R 13b 45.08 ±± 1.37 NA 2,4-diCH a Results a Results 5f 4-Cl 0.24 ±±deviation) 0.17 0.77 0.22 2.30 ±± 0.18 2.59 0.36 Sareare 2expressed 13a H as3 means 30.52 2.56 28.64 ±of1.08 44.83 ± 2.42 32.16 ± ±±1.59 5e O 3 4-F 2.64 0.53 1.19 ± 0.96 3.41 1.27 2.72 0.42 expressed as means ± SD (standard deviation) of± three independent experiments. ± SD (standard three independent experiments. HT-29 H-460 A549 MDA-MB-231 a Results are expressed as means SD (standard deviation) of three independent experiments. Table 2. 2. Structures Structures and cytotoxicity of±compounds compounds 13a–l against HT-29, H-460, A-549, and MDA-MB-231 13c 2-Cl, 6-CH 3 12.95 ± 1.37 0.68 24.66 ± 0.80 30.71 ±A-549, 1.73 and 25.42 ± 1.14 Table and cytotoxicity of 13a–l against HT-29, H-460, MDA-MB-231 13b 3 45.08 ± NA NA NA 2,4-diCH 5g 4-OCF S 2 3 1.05 1.28 ± 0.22 0.68 2.81 0.73 32.16 3.02± ±1.59 0.51 13a H 30.52 ± 2.56 28.64 ± 1.08 44.83 2.30 ± 2.42 5f 4-Cl 0.24±±0.012 0.17 0.77 ± 0.18 2.59 0.36 cells in in2.vitro. vitro. 13d 2,6-diCH 3 18.37 ±±compounds 0.45 24.62 ±± 2.75 ±±A-549, 0.95 28.12 ±± 2.44 cells Table Structures and cytotoxicity ofof compounds 13a–l against HT-29, 15.94 H-460, and A-549, MDA-MB-231 13c 2-Cl, 6-CH 3 12.95 0.68 24.66 0.80 30.71 1.73 25.42 1.14 Table 13b 2. Structures and cytotoxicity 13a–l against HT-29, H-460, and 3 45.08 ± 1.37 NA NA NA 2,4-diCH Table 2. Structures and cytotoxicity of compounds 13a–l HT-29, A-549, and MDA-MB-231 5h 4-Cl 0.65±±0.012 0.33 against ±± 0.74 1.44 ±± 0.39 3.13 0.85 S 5g 4-OCF S 32 2,6-diF 3 1.05 1.28 0.68H-460, 2.81 0.73 3.02± ±±1.63 0.51 13e 35.10 ±± 2.81 ND1.02 NA 20.94 cells in vitro. 13d 2,6-diCH 3 18.37 24.62 15.94 ±± 0.95 28.12 ±± 2.44 MDA-MB-231 cells 2-Cl, in vitro. IC±50 50 (μM) 13c 6-CH 3 12.95 ± 0.45 0.68 24.66 ±± 2.75 0.80 30.71aa 4.86 1.73 25.42 1.14 cells in vitro. IC (μM) 1 5i S 3 4-OCF 3 2.41 ± 0.21 2.58 0.74 ± 0.56 4.07 ± 1.02 Compd. R14-F 13f 6.33 ±±0.93 15.28 ± 1.19 10.49 ± 2.26 2.97 5h 4-Cl 0.65 ± 0.33 1.02 ± 0.74 1.44 ± 0.39 12.75 3.13±± ±1.63 0.85 S 3 3-Cl, Compd. R 13e 2,6-diF 35.10 ND NA 20.94 HT-29 H-460 A549 MDA-MB-231 a ± 0.95 13d 2,6-diCH 3 18.37 ± 2.81 0.45 24.62 ± 2.75 15.94 28.12 ± 2.44 IC±a 50 50 (μM) HT-29 H-460 A549 MDA-MB-231 Sorafenib 3.27 ± 0.32 2.15 0.43 4.47± 0.28 3.81± 0.50 a± 13g 3-Cl 10.07 ± 0.32 5.86 ± 1.34 15.43 1.02 21.95 ± 2.58 1 IC (μM) 5i S 3 3-Cl, 3 ± 0.93 2.41 ± 0.21 2.58 ± 0.74 4.86 ± 0.56 12.75 4.07± ±2.97 1.02 IC50± (µM) Compd. R 13f 6.33 15.28 1.19 10.49 ± 2.26 13a H14-F 4-OCF 30.52 2.56 28.64 1.08 44.83 2.42 32.16 1.59 13e 35.10 2.81 ND NA 20.94 1.63 Compd. 1R Compd. a Results are 2,6-diF HT-29 H-460 A549 MDA-MB-231 13a H 30.52 ±± 2.56 28.64 ±± 1.08 44.83 ±± 2.42 32.16 ±± 1.59 R expressed as means ±ND SD (standard deviation) of± three independent experiments. 13h 3-CF 3 ND ND ND HT-29 H-460 A549 MDA-MB-231 Sorafenib 3.27 ± 0.32 2.15 0.43 4.47± 0.28 3.81± 0.50 13g 3-Cl 10.07 ± 0.32 5.86 ± 1.34 15.43 ± 1.02 21.95 ± 2.58 HT-29 H-460 A549 MDA-MB-231 13b 3 45.08 1.37 NA NA NA 2,4-diCH 13f 3-Cl, 6.33 ±±±0.93 15.28 ± 1.19 10.49 ± 2.26 12.75 ± 2.97 13a H 4-F 30.52 2.56 28.64 1.08 44.83 2.42 32.16 1.59 13b 3 45.08 NA NA NA 13ia Results are2,4-diCH 4-OCF 3 26.58 1.27 23.46 2.29 16.54 0.70 30.65 3.06 13a H 30.52 ± 1.37 2.56 ±± 1.08 ±± 2.42 32.16 ±± 1.59 expressed as means ±ND SD (standard deviation) of three independent 13h 3-CF 3 ND ND ND 13a H 28.64 ±28.64 1.08 44.83 ±44.83 2.42 32.16 experiments. ± 1.59 ± 2.56 13c 2-Cl, 6-CH 3 30.52 10.07 12.95 0.68 24.66 ± 0.80 30.71 1.73 25.42 1.14 13g 3-Cl ± 0.32 5.86 ± 1.34 15.43 ± 1.02 21.95 ± 2.58 Table 2. Structures and cytotoxicity of compounds 13a–l against HT-29, H-460, A-549, and MDA-MB-231 13b 33 45.08 1.37 NA NA NA 2,4-diCH 13c 2-Cl, 6-CH 12.95 ± 0.68 24.66 ± 0.80 30.71 ± 1.73 25.42 ± 1.14 13j13b 4-F 3 3 3 45.08 45.08 17.95 0.94 ±± 2.87 ND 13b NA NA NA 2,4-diCH 2,4-diCH ± 1.37± 1.37 13i 4-OCF 26.58 1.27 NA32.28 23.46 2.29 NA15.94 16.54 ± 0.95 0.70 NA 28.12 30.65 ± 2.44 3.06 13d 2,6-diCH 18.37 0.45 24.62 2.75 13h 3-CF 3 3 ND ND ND ND cells in vitro.2-Cl, 13c13c 13d 2-Cl, 6-CH 12.95 0.6824.6613a–l 24.66 0.80 30.71 1.73 25.42 1.14 2,6-diCH 3 3 12.95 18.37 ±±± 0.45 ±±± 2.75 ±±±A-549, 0.95 28.12 ±±± 2.44 6-CH ±24.62 0.80against 30.71 ±15.94 1.73NA 25.42 ± 1.14 ± 0.68 13k 3-F 30.50 3.16 ND NA 3 Table 2. Structures and cytotoxicity of compounds HT-29, H-460, and MDA-MB-231 13c 2-Cl, 6-CH 3 12.95 0.68 24.66 0.80 30.71 1.73 25.42 1.14 13j 4-F 17.95 0.94 32.28 2.87 ND NA 13e 2,6-diF 35.10 ± 2.81 2.81 ND NA 20.94 ±± 1.63 1.63 13i13d 4-OCF 3 26.58 1.2724.62 ±24.62 23.46 ± 2.75 2.29 16.54 0.70 30.65 3.06 2.75ND 15.94 ±15.94 0.95NA 28.12 ± 2.44 ± 0.45± 13d 2,6-diCH 18.37 0.45 ±± 0.95 28.12 2.44 13e 2,6-diF 20.94 3 3 18.37 35.10 13l in vitro. 2,6-diCH 2-F 24.18 1.35 32.29 ±± 2.75 2.63 NA 35.56 ± 2.44 2.85 a ± 0.95 cells 13d 2,6-diCH 3 18.37 0.45 24.62 15.94 28.12 IC50NA (μM) 13k 3-F 30.50 ±±±0.93 3.16 ND NA NA 13e 2,6-diF ND 20.94 ± 1.63 35.10 ± 2.81 13f 3-Cl, 4-F 6.33 ± 15.28 1.19 10.49 ± 2.26 12.75 ± 2.97 13j 4-F 17.95 0.94 32.28 ± 2.87 ND NA 1 Compd. R 4-F 13e 2,6-diF 35.10 2.81 ND NA 20.94 1.63 13f 3-Cl, 6.33 ±±±±0.93 15.28 1.19 10.49 2.26 12.75 2.97 Sorafenib 3.27 0.32 2.15 ±±±0.43 4.47 ±±0.28 3.81 ±±±0.50 13e 2,6-diF 35.10 2.81 ND NA 20.94 1.63 HT-29 H-460 MDA-MB-231 3-Cl, 4-F 15.28 ±32.29 1.19 10.49 ± 2.26A549 12.75 ± 2.97 6.33 ± 0.93 13l13f 2-F 24.18 1.35 2.63 35.56 2.85 a ± 1.02 13g 3-Cl 10.07 0.32 5.86 ± 1.34 15.43 21.95 ± 2.58 13k 3-F 30.50 ± 3.16 ND NA NA IC 50 (μM) 3-Cl, 6.33 ±±(standard 0.93 15.28 1.19 10.49 2.26 12.75 ±±NA: 2.97 13g 3-Cl 0.325.86 ±deviation) 5.86 ±±±1.34 ± 1.02 21.95 2.58 a13f 14-Fas means 13g are expressed 3-Cl 1.34 15.43 ±15.43 1.02 21.95 ± 2.58 10.07 10.07 ± Results ±0.32 SD± of three independent experiments. Compd. R 13f 3-Cl, 6.33 15.28 1.19 10.49 2.26 12.75 2.97 Sorafenib 3.27 0.32 2.15 ±±0.43 4.47 ±±0.28 3.81ND ±±0.50 13a H 4-F 30.52 ±±0.93 2.56 28.64 1.08 2.42 32.16 1.59 13h 3-CF 3 ND ND ND 13l13h 2-F 24.18 1.35 32.29 2.63 ND44.83 NA 35.56 2.85 HT-29 H-460 A549 MDA-MB-231 13g 3-Cl 10.07 ± 0.32 5.86 ± 1.34 15.43 ± 1.02 21.95 ± 2.58 13h 3-CF 3 ND ND ND ND 3-CF ND ND ND 3 IC50 value > 10.07 compound showing 50± μM. ND: 13g 3-Cl 0.32 Not determined. 5.86NA ± 1.34 15.43 ± 1.02 experiments. 21.95 ± 2.58 a Results are 13b 3 means 1.37 NA 2,4-diCH expressed SD±± deviation) of three independent 13i13i 4-OCF 26.58 ±(standard 1.2723.46 ± 23.46 2.29 16.54 0.70 30.65 3.06 Sorafenib 3.27 0.32 2.15 ±±±0.43 4.47 ±±±0.28 3.81NA ±±±NA: 0.50 4-OCF 2.29ND 16.54 ±16.54 0.70 30.65 ± 3.06 26.58 45.08 ± 1.27 13a H3 3 33as 30.52 2.56 28.64 1.08 44.83 2.42 32.16 1.59 13h 3-CF ND ND ND 13i 4-OCF 26.58 ± 1.27 23.46 2.29 0.70 30.65 3.06 13h 3-CF ND ND ND ND 13c 2-Cl, 6-CH 3 17.95 ±(standard 0.68 24.66 ±± 0.80 ± 1.73 experiments. ±NA: 1.14 compound showing IC3 50asvalue > 12.95 50±0.94 μM. ND: Not ± determined. 13j 4-F 32.28 2.87NA ND30.71 NA 25.42 ± a13j 4-F 17.95 0.94 32.28 2.87 ND Results are expressed means SD deviation) of three independent 13b 3 45.08 ± 1.37 NA NA 2,4-diCH 13i13k 4-OCF 26.58 1.27 23.46 ± 2.87 2.29 16.54 ± 0.70 0.70 30.65 ± 3.06 3.06 cell 13j 4-F 3 30.50 17.95 0.94 32.28 ND As shown in Table demonstrated excellent activity towards four tested 3-F 1,3 compounds ND24.62 NA15.94 NA 28.12 ± 3.165a–i 13i 4-OCF 26.58 1.27 23.46 2.29 16.54 30.65 13d 2,6-diCH 18.37 ±± ND: 0.45 ±± 2.75 ±± 0.95 ±± 2.44 13k 3-F 30.50 3.16 ND NA NA compound showing IC50 3value > 30.50 50 μM. Not determined. 13c 2-Cl, 6-CH 3 12.95 0.68 24.66 0.80 30.71 1.73 25.42 1.14 13j 4-F 17.95 0.94 32.28 2.87 ND 3-F 3.16 ND NA NA 13l 2-F 32.29 ± 2.63 NA 35.56 ± 2.85 50 values ranging from 0.089 to 5.46 μM, and seven of them were more active against lines13k with IC 24.18 ± 1.35 13j 4-F 17.95 0.94 32.28 ±± 2.87 ND As Table 1, compounds 5a–i demonstrated excellent activity towards 20.94 fourNA tested 13e 2,6-diF 35.10 ±± 2.81 ND NA ±± 1.63 13l shown in 2,6-diCH 2-F 24.18 1.35 32.29 2.63 NA 35.56 2.85 cell 13d 3 18.37 0.45 24.62 2.75 ± 0.95 2.44 Sorafenib 3.27 ± 0.32 ± 2.15 ±32.29 0.43 4.47 ± 15.94 0.28 NA 3.81 ± 0.5028.12 13k 3-F 30.50 3.16 ND NA 13l 2-F 24.18 1.35 ± 2.63 35.56 ± 2.85 tested cell lines than the positive control sorafenib. It was noticeable that all compounds exhibited 13k 3-F 30.50 3.16 ND NA NA ranging from 0.089 todemonstrated 5.46 μM, and seven of them were more12.75 active against lines with IC50 values 13f 3-Cl, 4-F 6.33 ±±±0.93 15.28 ±0.43 1.19 10.49 ±0.28 2.26 ±0.50 2.97 As shown in Table 1, compounds excellent activity towards four tested cell 3.27 0.32 2.15 4.47 3.81 a Sorafenib 13e 35.10 2.81 ND NA 20.94 1.63 Results expressed2,6-diF as2-F means ± SD (standard deviation) of three experiments. NA: compound 13l are 24.18 1.35 32.29 2.63 35.56 2.85 Sorafenib 3.27 ±±5a–i 0.32 2.15 ±±±±independent 0.43 4.47 ±± 0.28 3.81 ±±±±0.50 prominent cytotoxicity against HT-29 superior to sorafenib, reflecting good selectivity for colon cancer. 13l 2-F 24.18 ± 1.35 32.29 2.63 NA 35.56 2.85 tested cell lines than the positive control sorafenib. It was noticeable that all compounds exhibited 13g 3-Cl 10.07 0.32 5.86 ± 1.34 15.43 ± 1.02 21.95 2.58 awith 50 values ranging from 0.089 to 5.46 μM, and seven of them were more active against lines IC showing IC value > 50 µM. ND: Not determined. Results as means means SD± (standard (standard deviation) of three independent experiments. NA: 50 are expressed 3-Cl, 4-Fas 6.33 0.93 15.28 ±0.43 1.19 10.49 ±0.28 2.26 12.75 2.97was a13f Sorafenib 3.27 0.32 2.15 4.47 3.81 ±±±NA: 0.50 Results are expressed ±± SD deviation) of independent In general, antitumor potency was related to thetoamino group R1three on side chain, andexperiments. dimethylamino Sorafenib 3.27 ± 0.32 2.15 ±± 0.43 4.47 ±± 0.28 3.81 0.50 prominent cytotoxicity against HT-29 superior sorafenib, reflecting good selectivity for colon cancer. 13h 3positive ND ND ND tested cell lines than3-CF the control sorafenib. It ND was noticeable that all compounds exhibited compound showing IC 50 value > 10.07 50 μM. ND: Not determined. 13g 3-Cl ± 0.32 5.86 ± 1.34 15.43 ± 1.02 21.95 ± 2.58 a compound showing IC50asvalue >(5a 50± μM. ND:5b Not Results are expressed means SD (standard deviation) of three independent experiments. NA: more than morpholinyl vs.(standard 5c, vs.determined. 5d,group 5g vs. which might bedimethylamino due to itsNA: smaller 15i), a effective In general, antitumor potency was related to the amino R on side chain, and was Results are expressed as means ± SD deviation) of three independent experiments. 13i 4-OCF 3 26.58 ± 1.27 23.46 ± 2.29 16.54 ± 0.70 30.65 ± 3.06 prominent against HT-29 superior to sorafenib, reflecting good selectivity for colon cancer. 13h ND ND ND exerted 2 on As shown incytotoxicity Table 1,3-CF compounds 5a–i demonstrated excellent activity towards four tested cell compound showing IC3 50 valueAdditionally, > 50 μM. ND: Not determined. bulk and stronger hydrophilia. the substituent R theND terminal phenyl ring more effective than morpholinyl vs. 5c, 5b vs. 5d, 5g vs. 5i), which might be due to its smaller compound showing IC 50 value >(5a 50 μM. ND: Not determined. 1 13j 4-F 17.95 ± 0.94 32.28 ± 2.87 ND NA As shown in Table Table 1,3 compounds compounds 5a–i demonstrated excellent activity towards four tested tested cell In general, antitumor potency was0.089 related the amino group R on side chain, and dimethylamino 13i 4-OCF 26.58to ± to 1.27 23.46 ±seven 2.29 16.54 ± 0.70 30.65 ±against 3.06was As shown in 1, 5a–i demonstrated excellent activity towards four cell lines with IC values ranging from 5.46 µM, and them were more active 50 2of bulk and stronger hydrophilia. Additionally, the substituent R onwhich the terminal phenyl ring exerted 13k 3-F 30.50 ± 3.16 ND NA NA 50 than values ranging from 0.089 to 5.46 μM, and seven of them were more active against lines with IC more effective morpholinyl (5a vs. 5c, 5b vs. 5d, 5g vs. 5i), might be due to its smaller 13j 4-F 17.95 ± 0.94 32.28 ± 2.87 ND NA As shown in Table 1, compounds compounds 5a–i demonstrated excellent activity towards four tested cell ranging from 0.089 todemonstrated 5.46ItμM, seven ofthat them morefour active against with IC50 values testedlines cell As lines than the positive control sorafenib. wasand noticeable allwere compounds exhibited shown in Table 1, 5a–i excellent activity towards tested cell 13l 2-F 24.18 ± 3.16 1.35 32.29 ± 2.63 NA 35.56 ±exhibited 2.85 tested cellstronger lines than the positive control sorafenib. It ND was noticeable that all compounds compounds bulk and hydrophilia. Additionally, the substituent R2 onofthe terminal phenyl ring exerted 13k 3-F 30.50 ± NA NA 50 values ranging from 0.089 to 5.46 μM, and seven them were more active against lines with IC tested cell lines than the positive control sorafenib. It was noticeable that all exhibited prominent cytotoxicity against HT-29from superior to reflecting selectivity for colon cancer. ranging 0.089 to sorafenib, 5.46 μM, seven good of4.47 them were more active against lines with IC50 values Sorafenib 3.27 ±±0.32 2.15and ±±0.43 ± 0.28 3.81 ±±0.50 prominent cytotoxicity against HT-29 superior to sorafenib, sorafenib, reflecting good selectivity for colon cancer. 2-F 24.18 1.35 32.29 2.63 NA 35.56 2.85 1 on side tested13l cell lines lines than the positive control sorafenib. It was was noticeable that all and compounds exhibited prominent cytotoxicity against HT-29 superior to reflecting good selectivity for colon cancer. In general, antitumor potency was related to the amino group R chain, dimethylamino tested cell than the positive control sorafenib. It noticeable that all compounds exhibited a Results are expressed as means ± SD (standard deviation) of 1three independent experiments. NA: InSorafenib general, antitumor antitumor potency was related to the thetoamino amino group R1 on on side side chain, and dimethylamino dimethylamino was 3.27 ±5c, 0.32 2.15 0.43 4.47 ± 0.28 3.81 0.50 prominent cytotoxicity againstwas HT-29 superior sorafenib, reflecting good selectivity for colon cancer. In general, potency related to group R chain, and was was more effective than morpholinyl (5a 5bNot vs.sorafenib, 5d, 5g± vs. 5i), which might be duefor tocolon its± smaller prominent cytotoxicity against HT-29 superior to reflecting good selectivity cancer. compound showing IC50 value >(5a 50vs. μM. ND: determined. more effective than morpholinyl vs. 5c, 5b vs. 5d, 5g vs. 5i), which might be due to its smaller 1 a In general, general, antitumor potency was related related to the amino group R15i), on which side chain, andexperiments. dimethylamino was more effective than morpholinyl (5a vs.(standard 5c,the 5bamino vs.deviation) 5d,group 5g vs. might bedimethylamino due to itsNA: smaller Results are expressed as means ± SD three independent 2R In antitumor potency was to on side chain, and was bulk and stronger hydrophilia. Additionally, the substituent Rof on the terminal phenyl ring exerted 2 on bulk and stronger hydrophilia. Additionally, the substituent R the terminal phenyl ring exerted 2 on more effective than morpholinyl vs. 5c, vs. 5d, 5g vs. 5i), which might be due to its smaller compound showing IC50 valueAdditionally, >(5a 50 μM. ND:5b Not determined. bulk and stronger hydrophilia. the substituent R the terminal phenyl ring exerted moreAs effective morpholinyl (5a vs.5a–i 5c, demonstrated 5b vs. 5d, 5g vs. 5i), which might be duefour to its smaller shown than in Table 1, compounds excellent activity towards tested cell bulk and and stronger stronger hydrophilia. hydrophilia. Additionally, Additionally, the the substituent substituent R R22 on on the the terminal terminal phenyl phenyl ring ring exerted exerted bulk ranging from 0.089 5.46 μM, andexcellent seven of activity them were morefour active against lines As with IC50 values shown in Table 1, compounds 5a–itodemonstrated towards tested cell tested cell lines than the positive control sorafenib. It was noticeable that all compounds exhibited lines with IC50 values ranging from 0.089 to 5.46 μM, and seven of them were more active against prominent cytotoxicity against HT-29 superior to sorafenib, good for colon cancer. tested cell lines than the positive control sorafenib. It wasreflecting noticeable thatselectivity all compounds exhibited 1 on side chain, and dimethylamino was In general, antitumor potency was related to the amino group R prominent cytotoxicity against HT-29 superior to sorafenib, reflecting good selectivity for colon cancer.

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a major influence on pharmacological activity. Electrophilic groups are beneficial to the improvement of antitumor potency, as might be the reason of compounds 5a, 5c, 5f, and 5h with the Cl group showing eminent cytotoxicity. Conversely, a certain decrease in activity was observed once 3,4-(CH3 )2 were introduced, such as compounds 5b and 5d. Interestingly, 2-CH3 and 6-Cl analogue 5a showed best activity, with IC50 values of 0.15, 0.089, 0.36, and 0.75 µM, which were five- to 24-fold more potent than sorafenib respectively. The results indicated that steric hindrance in ortho-position was preferred. To further verify the optimized structural skeleton and explore the SARs, pharmacological data of compounds 13a–l bearing 4-aminopyrimidinyl moiety are illustrated in Table 2. Unexpectedly, the results were unsatisfied although multiple hydrophilic modifications were performed by introducing pyridine group and further salifying with hydrochloride. The contrast of antitumor potency between 4-aminoquinazoline and 4-aminopyrimidine derivatives suggested that further structural modification might have broken the binding affinity to intracellular kinase domains or switched the binding affinity to other targets, while the 4-aminoquinazolinyl moiety was an excellent skeleton for EGFR inhibitors. The activity toward EGFR kinase of compound 5a was surprised with the IC50 value of 56 nM, indicating that 4-aminoquinazolinyl-diaryl urea derivatives 5a–i were potent EGFR inhibitors (Table 3). Efforts to identify its mechanisms of action and further optimization are ongoing and will be reported in due course. Table 3. EGFR and VEGFR2/KDR kinases inhibitory activity of compound 5a in vitro.

Compd. 5a Sorafenib

IC50 (nM) VEGFR2/KDR

EGFR

>3000 93

56 -

3. Experimental Section 3.1. Chemistry Melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland) and were uncorrected. Mass spectra (MS) were taken in electrospray ionization (ESI) mode on an Agilent 1100 LC-MS (Agilent, Palo Alto, CA, USA). Proton (1H) nuclear magnetic resonance spectroscopy were performed using a Bruker ARX-300, 300 or 400 MHz spectrometers (Bruker Bioscience, Billerica, MA, USA) with tetramethylsilane (TMS) as an internal standard. Thin-layer chromatography (TLC) analysis was carried out on silica gel plates GF254 (Qingdao Haiyang Chemical, Qingdao, China). Unless otherwise noted, all common reagents and solvents were used as obtained from commercial suppliers without further purification. 3.2. General Procedure for Preparation of 2-(4-Aminophenyl)-4-aminoquinazolines (4a–b) Dioxane (100 mL) was added to a solution of sodium carbonate (3.2 g, 0.03 mol) in water (25 mL). Under argon, 2-chloro-4-aminoquinazoline 3 (0.01 mol), 4-aminophenylboronic acid pinacol ester (2.4 g, 0.011 mol) and Pd(PPh3 )2 Cl2 (0.7 g, 1 mmol) were added to the mixture successively. After refluxing for 6 h, water (50 mL) was added to the reaction mixture. The mixture was extracted by dichloromethane (50 mL × 3). The organic phase was washed by brine (50 mL × 1), dried, and evaporated to yield 4a–b. N1 -(2-(4-aminophenyl)quinazolin-4-yl)-N2 ,N2 -dimethylethane-1,2-diamine (4a). Yield: 73.5%; MS (ESI) m/z: 308.2 [M + H+ ]. 2-(4-Aminophenyl)-N-(3-morpholinopropyl)quinazolin-4-amine (4b). Yield: 78.1%; MS (ESI) m/z: 364.5 [M + H+ ]. 3.3. General Procedure for Preparation of Componds 5a–i A mixture of intermediate 4 (1 mmmol) and corresponding aromatic isocyanate 6 or isothiocyanate 7 (1.1 mmol) in dry THF (15 mL) was stirred at 30 ◦ C for 6 h and monitored by TLC. The precipitate was

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collected by filtration, washed with ether, and purified by silica gel chromatography (MeOH:CH2 Cl2 = 20:1) to afford target compounds 5a–i. 1-(2-Chloro-6-methylphenyl)-3-(4-(4-((2-(dimethylamino)ethyl)amino)quinazolin-2-yl)phenyl)urea (5a). Yield: 62%; m.p.: 144.5–147.0 ◦ C; MS (ESI) m/z: 474.9 [M + H+ ]; 1 H-NMR (400 MHz, DMSO-d6 ) δ (ppm): 9.08 (s, 1H), 8.2.4 (d, J = 8.8 Hz, 2H ), 8.12 (t, J = 5.2, 4.8 Hz, H), 8.04 (d, J = 8.0 Hz, 1H), 7.95 (s, 1H), 7.56 (m, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.29 (m, 1H), 7.23 (d, J = 8.0 Hz, 1H), 7.11 (d, J = 7.6 Hz, 1H), 7.05 (m, 1H), 3.17 (q, 2H), 2.47 (t, J = 8.0 Hz, 2H), 2.38 (s, 3H), 2.21 (s, 6H). Anal. Calcd for C26 H27 ClN6 O (%): C, 65.74; H, 5.73; N, 17.69; Found (%): C, 65.71; H, 5.76; N, 17.68. 1-(4-(4-((2-(Dimethylamino)ethyl)amino)quinazolin-2-yl)phenyl)-3-(3,4-dimethylphenyl)urea (5b). Yield: 68%; m.p.: 132.0–134.0 ◦ C; MS (ESI) m/z: 455.2 [M + H+ ]; 1 H-NMR (400 MHz, DMSO-d6 ) δ (ppm): 10.04 (s, 1H), 8.39 (t, J = 7.6 Hz, 1H), 8.24 (d, J = 7.2 Hz, 3H), 8.10 (d, J = 8.4 Hz, 1H), 7.55 (m, 3H), 7.46 (d, J = 8.4 Hz, 2H), 7.28 (t, J = 7.2 Hz, 1H), 6.89 (d, J = 7.2 Hz, 1H), 6.60 (d, J = 7.2 Hz, 1H), 3.17 (q, 2H), 2.47 (t, J = 8.0 Hz, 2H), 2.23 (s, 3H), 2.22 (s, 3H), 2.19 (s, 6H). Anal. Calcd for C27 H30 N6 O (%): C, 71.34; H, 6.65; N, 18.49; Found (%): C, 71.36; H, 6.62; N, 18.47. 1-(2-Chloro-6-methylphenyl)-3-(4-(4-((3-morpholinopropyl)amino)quinazolin-2-yl)phenyl)urea (5c). Yield: 70%; m.p.: 152.5–154.5 ◦ C; MS (ESI) m/z: 531.1 [M + H+ ]; 1 H-NMR (400 MHz, DMSO-d6 ) δ (ppm): 9.20 (s, 1H), 8.38 (d, J = 8.4 Hz, 2H ), 8.28 (t, J = 4.8, 5.2 Hz, H), 8.18 (d, J = 8.4 Hz, 1H), 8.07 (s, 1H), 7.71 (m, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.42 (m, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.24 (d, J = 7.6 Hz, 1H), 7.19 (m, 1H), 3.70 (m, 2H), 3.57 (t, J = 8.4 Hz, 4H), 2.40 (m, 6H), 2.27 (s, 3H), 1.87(m, 2H). Anal. Calcd for C29 H31 ClN6 O2 (%): C, 65.59; H, 5.88; N, 15.83; Found (%): C, 65.61; H, 5.85; N, 15.88. 1-(3,4-Dimethylphenyl)-3-(4-(4-((3-morpholinopropyl)amino)quinazolin-2-yl)phenyl)urea (5d). Yield: 56%; m.p.: 140.7–143.0 ◦ C; MS (ESI) m/z: 510.9 [M + H+ ]; 1 H-NMR (400 MHz, DMSO-d6 ) δ (ppm): 10.18 (s, 1H), 8.54 (t, J = 7.2 Hz, 1H), 8.37 (d, J = 7.2 Hz, 3H), 8.23 (d, J = 8.0 Hz, 1H), 7.69 (m, 3H), 7.59 (d, J = 8.0 Hz, 2H), 7.41 (t, J = 7.2 Hz, 1H), 7.01 (d, J = 7.2 Hz, 1H), 6.73 (d, J = 7.2 Hz, 1H), 3.70 (m, 2H), 3.57 (t, J = 7.2 Hz, 4H), 2.42 (t, J = 7.2 Hz, 2H), 2.37 (t, J = 7.2 Hz, 4H), 2.24 (s, 3H), 2.23 (s, 3H), 1.86 (m, 2H). Anal. Calcd for C30 H34 N6 O2 (%): C, 70.56; H, 6.71; N, 16.46; Found (%): C, 70.55; H, 6.76; N, 16.42. 1-(4-Fluorophenyl)-3-(4-(4-((3-morpholinopropyl)amino)quinazolin-2-yl)phenyl)urea (5e). Yield: 52%; m.p.: 137.0–139.5 ◦ C; MS (ESI) m/z: 501.2 [M + H+ ]; 1 H-NMR (400 MHz, DMSO-d6 ) δ (ppm): 9.91 (s, 2H), 8.51 (t, J = 5.2 Hz, 1H), 8.39 (d, J = 8.8 Hz, 2H), 8.30 (d, J = 8.4 Hz, 1H), 7.72 (m, 4H), 7.60 (d, J = 8.8 Hz, 2H), 7.43 (m, 1H), 7.36 (d, J = 8.4 Hz, 2H), 4.15 (t, J = 6.8 Hz, 2H ), 3.72 (t, J = 6.4 Hz, 4H), 2.43 (t, J = 6.8 Hz, 2H), 2.38 (t, J = 6.4 Hz, 4H), 1.86 (m, 2H). Anal. Calcd for C28 H29 FN6 O2 (%): C, 67.18; H, 5.84; N, 16.79; Found (%): C, 67.15; H, 5.88; N, 16.75. 1-(4-Chlorophenyl)-3-(4-(4-((2-(dimethylamino)ethyl)amino)quinazolin-2-yl)phenyl)thiourea (5f). Yield: 65%; m.p.: 131.5–134.0 ◦ C; MS (ESI) m/z: 477.1 [M + H+ ]; 1 H-NMR (400 MHz, DMSO-d6 ) δ (ppm): 10.75 (s, 2H), 8.31 (d, J = 8.4 Hz, 3H), 8.07 (d, J = 8.1 Hz, 1H), 7.68–7.59 (m, 4H), 7.45 (d, J = 8.4 Hz, 2H), 7.37–7.26 (m, 3H), 3.86–3.77 (q, 2H), 2.73 (t, J = 6.4 Hz, 2H), 2.33 (s, 6H). Anal. Calcd for C25 H25 ClN6 S (%): C, 62.95; H, 5.28; N, 17.62; Found (%): C, 62.94; H, 5.27; N, 17.64. 1-(4-(4-((2-(Dimethylamino)ethyl)amino)quinazolin-2-yl)phenyl)-3-(4-(trifluoromethoxy)phenyl)thiourea (5g). Yield: 60%; m.p.: 150.8–153.0 ◦ C; MS (ESI) m/z: 527.2 [M + H+ ]; 1 H-NMR (400 MHz, DMSO-d6 ) δ (ppm): 10.12 (s, 1H), 10.03 (s, 1H), 8.25 (d, J = 8.4 Hz, 2H), 8.18 (t, J = 6.8 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.58 (m, 3H), 7.47 (d, J = 8.8 Hz, 2H), 7.31 (m, 3H), 6.96 (d, J = 8.4 Hz, 1H), 3.38 (q, 2H), 2.56 (t, J = 7.2 Hz, 2H), 2.23 (s, 6H). Anal. Calcd for C26 H25 F3 N6 OS (%): C, 59.30; H, 4.79; N, 15.96; Found (%): C, 59.32; H, 4.76; N, 15.94. 1-(4-Chlorophenyl)-3-(4-(4-((3-morpholinopropyl)amino)quinazolin-2-yl)phenyl)thiourea (5h). Yield: 63%; m.p.: 145.5–148.0 ◦ C; MS (ESI) m/z: 533.8 [M + H+ ]; 1 H-NMR (400 MHz, DMSO-d6 ) δ (ppm): 10.90 (s, 2H), 8.42 (d, J = 8.8 Hz, 3H), 8.24 (d, J = 8.4 Hz, 1H), 7.73 (m, 4H), 7.65 (d, J = 8.4 Hz, 2H), 7.44 (m, 1H), 7.38 (d, J = 8.8 Hz, 2H), 3.71 (m, 2H), 3.58 (t, J = 8.4 Hz, 4H), 2.43 (t, J = 7.2 Hz, 2H), 2.38 (t, J = 8.4 Hz,

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4H), 1.88 (m, 2H). Anal. Calcd for C28 H29 ClN6 OS (%): C, 63.09; H, 5.48; N, 15.76; Found (%): C, 63.05; H, 5.47; N, 15.79. 1-(4-(4-((3-Morpholinopropyl)amino)quinazolin-2-yl)phenyl)-3-(4-(trifluoromethoxy)phenyl)thiourea (5i). Yield: 59%; m.p.: 164.5–166.5 ◦ C; MS (ESI) m/z: 583.9 [M + H+ ]; 1 H-NMR (400 MHz, DMSO-d6 ) δ (ppm): 10.25 (s, 1H), 10.16 (s, 1H), 8.45 (d, J = 8.8 Hz, 2H), 8.35 (t, J = 7.2 Hz, 1H), 8.22 (d, J = 8.4 Hz, 1H), 7.74 (m, 3H), 7.64 (d, J = 8.8 Hz, 2H), 7.48 (m, 3H), 7.11 (d, J = 8.0 Hz, 1H), 3.73 (m, 2H), 3.59 (t, J = 4.4 Hz, 4H), 2.44 (t, J = 7.2 Hz, 2H), 2.39 (t, J = 4.4 Hz, 4H), 1.89 (m, 2H). Anal. Calcd for C29 H29 F3 N6 O2 S (%): C, 59.78; H, 5.02; N, 14.42; Found (%): C, 59.75; H, 5.01; N, 14.47. 3.4. General Procedure for Preparation of Aromatic Isocyanates (6a–n) Appropriate aromatic amine (0.02 mol) was added slowly to a stirred solution of BTC (3.0 g, 0.01 mol) in 1,4-dioxane (30 mL) at room temperature. After refluxing for 8-12 h, excess 1,4-dioxane was evaporated. The residue was distilled under reduced pressure to afford compounds 6a–n. 1-Chloro-4-isocyanatobenzene (6a). Purity: 78.2%; b.p.: 110–114 ◦ C (30–40 mmHg). 4-Isocyanato-1,2-dimethylbenzene (6b). Purity: 80.5%; b.p.: 59–65 ◦ C (30–40 mmHg). 1-Chloro-2-isocyanato-3-methylbenzene (6c). Purity: 80.8%; b.p.: 116–118 ◦ C (30–40 mmHg). 1-Fluoro-4-isocyanatobenzene (6d). Purity: 84.2%; b.p.: 94–95 ◦ C (30–40 mmHg). 1-Fluoro-3-isocyanatobenzene (6e). Purity: 83.6%; b.p.: 85–91 ◦ C (30–40 mmHg). 1-Fluoro-2-isocyanatobenzene (6f). Purity: 84.3%; b.p.: 76–80 ◦ C (30–40 mmHg). 1-Chloro-3-isocyanatobenzene (6g). Purity: 80.1%; b.p.: 125–128 ◦ C (30–40 mmHg). 2-Chloro-1-fluoro-4-isocyanatobenzene (6h). Purity: 85.2%; b.p.: 93–98 ◦ C (30–40 mmHg). 1,3-Difluoro-2-isocyanatobenzene (6i). Purity: 80.9%; b.p.: 67–71 ◦ C (30–40 mmHg). 2-Isocyanato-1,3-dimethylbenzene (6j). Purity: 76.9%; b.p.: 67–71 ◦ C (30–40 mmHg). 1-Isocyanato-2,4-dimethylbenzene (6k). Purity: 77.5%; b.p.: 71–77 ◦ C (30–40 mmHg). 1-Isocyanato-3-(trifluoromethyl)benzene (6l). Purity: 83.2%; b.p.: 105–111 ◦ C (30–40 mmHg). 1-Isocyanato-4-(trifluoromethoxy)benzene (6m). Purity: 81.0%; b.p.: 113–116 ◦ C (30–40 mmHg). Isocyanatobenzene (6n). Purity: 80.2%; b.p.: 45–50 ◦ C (30–40 mmHg). 3.5. General Procedure for Preparation of Aromatic Isothiocyanates (7a,b) Appropriate aromatic amine (0.02 mol) and DABCO (6.7 g, 0.06 mol) were dissolved in toluene (25 mL) at room temperature. Under stirring, CS2 (4.6 g, 0.06 mol) was added dropwise to the solution within 20 min. After stirring at room temperature for 12 h, the precipitate was collected by filtration and washed with toluene. The residue was dried and suspended in CHCl3 (25 mL), and BTC (19.6 g, 0.066 mol) in CHCl3 (25 mL) was added dropwise to the mixture slowly in an ice bath. Then the mixture was stirred for 1 h at room temperature and refluxed for 1 h. The mixture was filtered and evaporated to give compounds 7a,b. 1-Chloro-4-isothiocyanatobenzene (7a). Purity: 77.5%. 1-Isothiocyanato-4-(trifluoromethoxy)benzene (7b). Purity: 79.8%. 3.6. Ethyl 4-aminobenzimidate Dihydrochloride (8) Dry hydrogen chloride was bubbled into the mixture of 4-aminobenzonitrile (15 g, 0.127 mol), 1,4-dioxane (100 mL) and anhydrous ethanol (100 mL) for 6 h in an ice bath. Then the mixture was

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sealed and stirred at room temperature for 48 h. After completion, the solution was concentrated under reduced pressure and the semisolid residue was diluted with anhydrous ether (200 mL). The suspension was filtered, washed with anhydrous ether and dried to give the title compound 8 as a white solid (29.3 g, 97.7%). MS (ESI) m/z: 164.9 [M + H+ ]. 3.7. 4-Aminobenzimidamide Hydrochloride (9) Dry ammonia was bubbled into the mixture of compound 8 (29.3 g, 0.123 mol) and anhydrous ethanol (200 mL) for 6 h in an ice bath. Then the mixture was sealed and stirred at room temperature for 24 h. Then the solution was concentrated under a reduced pressure and the semisolid residue was diluted with anhydrous ether (200 mL). The suspension was filtered, washed with anhydrous ether, and dried to give the title compound 9 as a white solid (20.2 g, 95.3%). MS (ESI) m/z: 135.9 [M + H+ ]. 3.8. 3-(Dimethylamino)-2-(pyridin-2-yl)acrylonitrile (10) A mixture of 2-(pyridin-2-yl)acetonitrile (15.0 g, 0.127 mol), DMF-DMA (30 mL, 0.229 mol) and methanol (36 mL) was stirred at 30 ◦ C for 24 h. The solvent was evaporated, moderate methanol and ethyl acetate was added in. The solution was concentrated under a reduced pressure to remove excess DMF-DMA completely. The residue was poured into petroleum ether (100 mL) and stirred for 6 h. The suspension was separated by filtration and washed with ether to give compound 10 as a dark red solid (18.3 g, 83.2%). MS (ESI) m/z: 174.0 [M + H+ ]. 3.9. 2-(4-Aminophenyl)-5-(pyridin-2-yl)pyrimidin-4-amine (11) A mixture of compound 9 (20.2 g, 0.118 mol ), compound 10 (17.0 g, 0.098 mol), sodium carbonate (12.5 g, 0.0118 mol), methnol (200 mL) and water (60 mL) was heated to 70 ◦ C for 24 h. The solution was concentrated and added to water (200 mL). The precipitate was collected by filtration, washed with ether, and recrystallized with ethnol to afford compound 11 as a pale white solid (13.9 g, 53.8%). MS (ESI) m/z: 264.1 [M + H+ ]. 3.10. General Procedure for Preparation of Compounds 12a–l and 13a–l A mixture of intermediate 11 (1 mmmol) and corresponding aromatic isocyanate 6 (1.1 mmol) in dry THF (15 mL) was stirred at 30 ◦ C for 6 h and monitored by TLC. The precipitate was collected by filtration, washed with ether, and dried to afford compounds 12a–l without further purification. At room temperature, 20%–30% hydrochloride ethanol solution (1 mL) was added dropwise to a stirred solution of compound 12 dissolved in CHCl3 (10 mL). The suspension was stirred for 1 h, filtered and dried to give the corresponding compounds 13a–l. 1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-phenylurea dihydrochloride (13a). Yield: 79%; m.p.: 303.0–306.0 ◦ C; MS (ESI) m/z: 383.1 [M + H+ ]; 1 H-NMR (300 MHz, DMSO-d6 ) δ (ppm): 9.41 (s, 1H), 9.11 (s, 1H), 8.93 (s, 2H), 8.77 (s, 1H), 8.68 (d, J = 4.4 Hz, 1H), 8.32 (d, J = 8.4 Hz, 2H), 8.10 (d, J = 8.0 Hz, 1H), 7.94 (t, J = 7.9 Hz, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.4 Hz), 7.38–7.32 (m, 1H), 7.30 (t, J = 7.7 Hz, 2H), 7.00 (t, J = 6.9 Hz). Anal. Calcd for C22 H20 Cl2 N6 O (%): C, 58.03; H, 4.43; N, 18.46; Found (%): C, 58.07; H, 4.46; N, 18.42. 1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(2,4-dimethylphenyl)urea dihydrochloride (13b). Yield: 65%; m.p.: 286.0–288.5 ◦ C; MS (ESI) m/z: 411.3 [M + H+ ]; 1 H-NMR (300 MHz, DMSO-d6 ) δ (ppm): 9.77 (s, 1H), 8.89 (s, 1H), 8.74 (d, J = 4.6 Hz, 1H), 8.25(d, J = 8.6 Hz, 2H), 8.22 (d, J = 7.8 Hz, 1H), 8.05 (t, J = 7.9 Hz, 1H), 7.72(d, J = 8.6 Hz, 2H), 7.66 (d, J = 8.1 Hz, 1H), 7.52 (t, J = 4.8 Hz, 1H), 7.02 (s, 1H), 6.98 (d, J = 8.2 Hz, 2H), 2.24 (s, 6H). Anal. Calcd for C24 H24 Cl2 N6 O (%): C, 59.63; H, 5.00; N, 17.39; Found (%): C, 59.60; H, 5.06; N, 17.34. 1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(6-chloro-2-methylphenyl)urea dihydrochloride (13c). Yield: 72%; m.p.: 252.0–254.0 ◦ C; MS (ESI) m/z: 431.0 [M + H+ ]; 1 H-NMR (300 MHz, DMSO-d6 ) δ

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(ppm): 9.78 (s, 1H), 8.85 (s, 1H), 8.74 (d, J = 4.2 Hz, 1H), 8.56 (s, 1H), 8.26 (d, J = 8.7 Hz, 2H), 8.22 (d, J = 8.1 Hz, 1H), 8.04 (t, J = 6.7 Hz, 1H), 7.72 (d, J = 8.9 Hz, 2H), 7.54–7.50 (m, 1H), 7.36 (d, J = 5.4 Hz, 1H), 7.27–7.18 (m, 2H), 2.28 (s, 3H). Anal. Calcd for C23 H21 Cl3 N6 O (%): C, 54.83; H, 4.20; N, 16.68; Found (%): C, 54.84; H, 4.23; N, 16.65. 1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(2,6-dimethylphenyl)urea dihydrochloride (13d). Yield: 76%; m.p.: 271.0–274.0 ◦ C; MS (ESI) m/z: 411.2 [M + H+ ]; 1 H-NMR (300 MHz, DMSO-d6 ) δ (ppm): 9.83 (s, 1H), 8.85 (s, 1H), 8.74(d, J = 4.2 Hz, 1H), 8.34 (s, 1H), 8.23 (d, J = 9.3 Hz, 2H), 8.21 (d, J = 8.1 Hz, 1H), 8.05(t, J = 7.8 Hz, 1H), 7.72 (d, J = 9.0 Hz, 2H), 7.54–7.50 (m, 1H), 7.08 (s, 3H), 2.23 (s, 6H). Anal. Calcd for C24 H24 Cl2 N6 O (%): C, 59.63; H, 5.00; N, 17.39; Found (%): C, 59.67; H, 5.02; N, 17.36. 1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(2,6-difluorophenyl)urea dihydrochloride (13e). Yield: 68%; m.p.: 279.5–281.0 ◦ C; MS (ESI) m/z: 419.1 [M + H+ ]; 1 H-NMR (300 MHz, DMSO-d6 ) δ (ppm): 9.78 (s, 1H), 8.88 (s, 1H), 8.73 (d, J = 4.7 Hz, 1H), 8.53 (s, 1H), 8.40 (s, 2H), 8.24 (d, J = 8.8 Hz, 2H), 8.20 (d, J = 9.3 Hz, 1H), 8.04 (t, J = 7.8 Hz, 1H), 7.72 (d, J = 8.6 Hz, 2H), 7.54–7.49 (m, 1H), 7.32–7.25 (m, 3H). Anal. Calcd for C22 H18 Cl2 F2 N6 O (%): C, 53.78; H, 3.69; N, 17.10; Found (%): C, 53.75; H, 3.72; N, 17.08. 1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(3-chloro-4-fluorophenyl)urea dihydrochloride (13f). Yield: 80%; m.p.: 289.5–291.5 ◦ C; MS (ESI) m/z: 435.0 [M + H+ ]; 1 H-NMR (300 MHz, DMSO-d6 ) δ (ppm): 9.69 (s, 1H), 9.55 (s, 1H), 8.88 (s, 1H), 8.74 (d, J = 4.4 Hz, 1H), 8.25 (d, J = 8.8 Hz, 2H), 8.21 (d, J = 8.2 Hz, 1H), 8.05 (t, J = 7.8 Hz, 1H), 7.85–7.82 (m, 1H), 7.72 (d, J = 8.8 Hz, 2H), 7.54–7.49 (m, 1H), 7.40–7.34 (m, 2H). Anal. Calcd for C22 H18 Cl3 FN6 O (%): C, 52.04; H, 3.57; N, 16.55; Found (%): C, 52.05; H, 3.52; N, 16.56. 1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(3-chlorophenyl)urea dihydrochloride (13g). Yield: 71%; m.p.: 270.0–272.5 ◦ C; MS (ESI) m/z: 417.0 [M + H+ ]; 1 H-NMR (300 MHz, DMSO-d6 ) δ (ppm): 9.89 (s, 1H), 9.71 (s, 1H), 8.86 (s, 1H), 8.74 (d, J = 4.5 Hz, 1H), 8.25 (d, J = 8.7 Hz, 2H), 8.21 (d, J = 8.2 Hz, 1H), 8.05 (t, J = 7.9 Hz, 1H), 7.73 (d, J = 8.8 Hz, 2H), 7.72 (s, 1H), 7.55–7.51 (m, 1H), 7.33–7.30 (m, 2H), 7.04 (d, J = 5.8 Hz, 1H). Anal. Calcd for C22 H19 Cl3 N6 O (%): C, 53.95; H, 3.91; N, 17.16; Found (%): C, 53.94; H, 3.92; N, 17.14. 1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(4-(trifluoromethyl)phenyl)urea dihydrochloride (13h). Yield: 65%; m.p.: 267.0–269.5 ◦ C; MS (ESI) m/z: 451.1 [M + H+ ]; 1 H-NMR (300 MHz, DMSO-d6 ) δ (ppm): 9.78 (s, 1H), 9.74 (s, 1H), 8.89 (s, 1H), 8.74 (d, J = 4.5 Hz, 1H), 8.26 (d, J = 8.8 Hz, 2H), 8.21 (m, 2H), 8.05 (s, 2H), 7.74 (d, J = 8.8 Hz, 2H), 7.61–7.50 (m, 4H), 7.35 (d, J = 7.6 Hz, 1H). Anal. Calcd for C23 H19 Cl2 F3 N6 O (%): C, 52.79; H, 3.66; N, 16.06; Found (%): C, 52.75; H, 3.69; N, 16.05. 1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea dihydrochloride (13i). Yield: 78%; m.p.: 308.5–310.5 ◦ C; MS (ESI) m/z: 467.1 [M + H+ ]; 1 H-NMR (300 MHz, DMSO-d6 ) δ (ppm): 9.50 (s, 1H), 9.35 (s, 1H), 8.89 (s, 1H), 8.74 (d, J = 4.5 Hz, 1H), 8.24 (d, J = 8.7 Hz, 2H), 8.19 (d, J = 8.4 Hz, 1H), 8.04 (t, J = 6.7 Hz, 1H), 7.71 (d, J = 8.7 Hz, 2H), 7.59 (d, 2H), 7.60–7.51 (m, 1H), 7.32 (d, J = 8.1 Hz, 2H). Anal. Calcd for C23 H19 Cl2 F3 N6 O2 (%): C, 51.22; H, 3.55; N, 15.58; Found (%): C, 51.24; H, 3.59; N, 15.54. 1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(4-fluorophenyl)urea dihydrochloride (13j). Yield: 75%; m.p.: 264.0–267.0 ◦ C; MS (ESI) m/z: 401.0 [M + H+ ]; 1 H-NMR (300 MHz, DMSO-d6 ) δ (ppm): 9.80 (s, 1H), 9.47 (s, 1H), 8.87 (s, 1H), 8.75 (d, J = 4.6 Hz, 1H), 8.26 (d, J = 8.7 Hz, 2H), 8.22 (d, J = 7.8 Hz, 1H), 8.07 (t, J = 7.9 Hz, 1H), 7.73 (d, J = 8.8 Hz, 2H), 7.56–7.52 (m, 2H), 7.49 (t, J = 4.8 Hz, 1H), 7.15 (t, J = 8.8 Hz, 2H). Anal. Calcd for C22 H19 Cl2 FN6 O (%): C, 55.82; H, 4.05; N, 17.76; Found (%): C, 55.84; H, 4.01; N, 17.79. 1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(3-fluorophenyl)urea dihydrochloride (13k). Yield: 67%; m.p.: 228.5–231.0 ◦ C; MS (ESI) m/z: 401.1 [M + H+ ]; 1 H-NMR (300 MHz, DMSO-d6 ) δ (ppm): 9.89 (s, 1H), 9.73 (s, 1H), 8.86 (s, 1H), 8.74 (d, J = 4.5 Hz), 8.26 (d, J = 8.9 Hz, 2H), 8.22 (d, J = 8.4 Hz, 1H), 8.05 (t, J = 7.8 Hz, 1H), 7.72 (d, J = 8.9 Hz, 2H), 7.55 (s, 1H), 7.53–7.50 (m, 1H), 7.37–7.29 (m, 1H), 7.14 (d,

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J = 8.1 Hz, 1H), 6.82 (t, J = 8.4 Hz, 1H). Anal. Calcd for C22 H19 Cl2 FN6 O (%): C, 55.82; H, 4.05; N, 17.76; Found (%): C, 55.81; H, 4.06; N, 17.72. 1-(4-(4-Amino-5-(pyridin-2-yl)pyrimidin-2-yl)phenyl)-3-(2-fluorophenyl)urea dihydrochloride (13l). Yield: 73%; m.p.: 258.5–261.5 ◦ C; MS (ESI) m/z: 401.0 [M + H+ ]; 1 H-NMR (300 MHz, DMSO-d6 ) δ (ppm): 10.01 (s, 1H), 8.94 (s, 1H), 8.87 (s, 1H), 8.74 (d, J = 4.5 Hz, 1H), 8.27 (d, J = 9.0 Hz, 2H), 8.22 (d, J = 8.1 Hz, 1H), 8.16–8.03 (m, 1H), 7.74 (d, J = 8.7 Hz, 2H), 7.55–7.50 (m, 1H), 7.29–7.03 (m, 3H). Anal. Calcd for C22 H19 Cl2 FN6 O (%): C, 55.82; H, 4.05; N, 17.76; Found (%): C, 55.86; H, 4.03; N, 17.75. 3.11. Evaluation of the Biological Activity The antitumor activity of compounds 5a–i and 13a–l was evaluated with HT-29, H-460, A549, and MDA-MB-231 by the MTT method in vitro, with sorafenib as positive control. The cancer cells were cultured in minimum essential medium (MEM) supplemented 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 were added to the culture medium at the indicated final concentrations and the cell cultures were continued for 72 h. Fresh MTT was added to each well at a final concentration of 5 µg/mL and incubated with cells at 37 ◦ C for 4 h. The formazan crystals were dissolved in 100 µL DMSO per each well, and the absorbency at 492 nm (for the absorbance of MTT formazan) and 630 nm (for the reference wavelength) was measured with the ELISA reader. All of the compounds were tested twice in each of the cell lines. The results expressed as IC50 (inhibitory concentration of 50%) were the averages of two determinations and were calculated by using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software. 3.12. EGFR and VEGFR2/KDR Kinases Assay In Vitro The target compound 5a was tested for its activity against EGFR and VEGFR2/KDR kinases through the mobility shift assay. All kinase assays were performed in 96-well plates in a 50 µL reaction volume. The kinase buffer contains 50 mM HEPES, pH 7.5, 10 mM MgCl2 , 0.0015% Brij-35 and 2 mM DTT. The stop buffer contains 100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, and 50 mM ethylene diamine tetraacetic acid (EDTA). Compounds were diluted to 500 µM by 100% DMSO, then 10 µL of the compounds were transfered to a new 96-well plate as the intermediate plate, and 90 µL kinase buffer was added to each well. Five microliters of each well of the intermediate plate was transferred to a 384-well plate. The following amounts of enzyme and substrate were used per well: kinase base buffer, FAM-labeled peptide, ATP, and enzyme solution. Wells containing the substrate, enzyme, and DMSO without compound were used as the DMSO control. Wells containing just the substrate without enzyme were used as the low control. The compounds were incubated at room temperature for 10 min. Ten microliters of peptide solution was added to each well and incubated at 28 ◦ C for a specified period of time and the reaction stopped by 25 µL of stop buffer. Finally, data was collected using the Caliper program, which converted conversion values to inhibition values. Percent inhibition = (max − conversion)/(max − min) × 100,

(1)

where “max” stands for DMSO control; “min” stands for low control. 4. Conclusions Taken as a whole, two novel series of diaryl urea derivatives were synthesized and evaluated for their cytotoxicity against four human cancer cell lines (H-460, HT-29, A549, and MDA-MB-231). Our strategy that 4-aminoquinazolinyl moiety was incorporated into the diaryl urea scaffold conveyed cellular potency and resulted in analogues 5a–i demonstrating excellent activity in the single-digit µM range, superior to sorafenib. The preliminary SARs showed that electrophilic groups on terminal phenyl ring and steric hindrance in ortho-position were beneficial to improved antitumor potency. The exploration of detailed SARs led to the identification of EGFR inhibitor 5a as a valuable lead,

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which possessed prominent activity both in cellular (IC50 = 0.15, 0.089, 0.36, and 0.75 µM, respectively) and enzymatic assay (IC50 = 56 nM against EGFR). Further optimization and investigation based on these structures are in progress. Acknowledgments: This work was supported by National Natural Science Foundation of China (21002065), Project of Education Department of Liaoning (L2013382), Development Project of Ministry of Education Innovation Team (IRT1073) and Science and Technology Program of Shenyang (No. F15-139-9-02). Author Contributions: N.J. and D.Z. conceived and designed the experiments; Y.B. and M.N. performed the experiments; Y.W. analyzed the data; X.Z. contributed reagents/materials/analysis tools; N.J. wrote the paper. Conflicts of Interest: The authors declare no conflict interest.

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Sample Availability: Samples of the compounds are available from the authors. © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).