Design, Synthesis and Anticancer Activity of a New Series of N-Aryl-N0-[4-(Pyridin-2-Ylmethoxy)Benzyl]Urea Derivatives

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Article Design, Synthesis and Anticancer Activity of a New Series of N-aryl-N0-[4-(pyridin-2-ylmethoxy)benzyl]urea Derivatives

Shicheng Hou 1, Shishao Liang 1, Chao Zhang 1, Yingmei Han 1, Jianhui Liang 1, Hongyu Hu 1, Xingeng Zhang 1, Chun Hu 1,* , Xiaoping Liu 1,* and Hong Zhang 2,*

1 Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China; [email protected] (S.H.); [email protected] (S.L.); [email protected] (C.Z.); [email protected] (Y.H.); [email protected] (J.L.); [email protected] (H.H.); [email protected] (X.Z.) 2 School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China * Correspondence: [email protected] (C.H.); [email protected] (X.L.); [email protected] (H.Z.); Tel.: +86-24-43520246 (C.H.)

Abstract: The development of cancer treatments requires continuous exploration and improvement, in which the discovery of new drugs for the treatment of cancer is still an important pathway. In this study, based on the molecular hybridization strategy, a new structural framework with an N-aryl-N’-arylmethylurea scaffold was designed, and 16 new target compounds were synthesized and evaluated for their antiproliferative activities against four different cancer cell lines A549, MCF7, HCT116, PC3, and human liver normal cell line HL7702. The results have shown seven compounds with 1-methylpiperidin-4-yl groups having excellent activities against all four cancer cell lines, and they exhibited scarcely any activities against HL7702. Among them, compound 9b and 9d showed

greatly excellent activity against the four kinds of cells, and the IC50 for MCF7 and PC3 cell lines Citation: Hou, S.; Liang, S.; Zhang, were even less than 3 µM. C.; Han, Y.; Liang, J.; Hu, H.; Zhang, X.; Hu, C.; Liu, X.; Zhang, H. Design, Synthesis and Anticancer Activity of Keywords: anticancer agent; urea derivative; synthesis; molecular hybridization; N-aryl-N’-arylmethy a New Series of N-aryl-N0-[4-(pyridin- -lurea; antiproliferative activity; cell cycle analysis 2-ylmethoxy)benzyl]urea Derivatives. Molecules 2021, 26, 3496. https:// doi.org/10.3390/molecules26123496 1. Introduction Academic Editor: Qiao-Hong Chen Nowadays, cancer has become a major challenge in human health, and a leading cause of death [1,2]. Cancer is caused by the uncontrolled proliferation of cells, a kind of Received: 23 April 2021 behavior unusual for cells, mostly related to some abnormal signal transduction regulation Accepted: 29 May 2021 mechanisms. In the diagnosis and treatment of cancer, many great developments have Published: 8 June 2021 been made, such as in the ﬁeld of surgery, drug therapy from toxic drugs to targeted drugs, etc. [3,4]. However, traditional chemotherapy drugs often have serious side effects and Publisher’s Note: MDPI stays neutral adverse reactions. The emergence of small-molecule targeted drugs has eased the severe with regard to jurisdictional claims in side effects of chemotherapy drugs to a certain extent, but targeted drugs are also prone to published maps and institutional afﬁl- drug resistance problems with the prolonged administration time [5–7]. Therefore, it is a iations. great challenge and opportunity to continuously develop new candidate drug molecules to bring new drugs to cancer treatment. Compared with earlier targeted drugs, multi-target inhibitors can act on a variety of different targets and inhibit different signal pathways at the same time [6,8,9]. The Copyright: © 2021 by the authors. multi-target inhibitors have a broader anti-tumor spectra and better prospects in clinical Licensee MDPI, Basel, Switzerland. practice [7]. Urea and urea isosteres are structures that possesses both a hydrogen bond This article is an open access article acceptor and a hydrogen bond donor, which makes it easy to form better interactions with distributed under the terms and drug target proteins [10]. These kinds of structures are an excellent pharmacodynamic conditions of the Creative Commons structure in drug molecules [11]. In many small-molecule targeted kinase inhibitors, urea Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ and urea isosteres, including aminopyrimidine, have been used in drug structures, some of 4.0/). which showed really favorable anti-cancer activity (Figure1)[12,13].

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isosteres, including aminopyrimidine, have been used in drug structures, some of which Molecules 2021, 26, 3496 showed really favorable anti-cancer activity (Figure 1) [12,13]. 2 of 14 isosteres, including aminopyrimidine, have been used in drug structures, some of which showed really favorable anti-cancer activity (Figure 1) [12,13].

Figure 1. The application of urea and its isosteres in anticancer drugs. FigureFigure 1. 1. TheThe application application of of urea urea and and its its isosteres isosteres in in anticancer anticancer drugs. drugs. Among the currently clinically used multi-target kinase inhibitors, sorafenib and its derivative regorafenib with a diaryl urea structure were the prime representatives, be- AmongAmong the the currently currently clinically clinically used used multi-target multi-target kinase kinase inhibitors, inhibitors, sorafenib sorafenib and and its its cause they had excellent inhibitory effects on a variety of solid tumors [14,15]. In sorafenib, derivativederivative regorafenib withwith aa diaryl diaryl urea urea structure structure were were the the prime prime representatives, representatives, because be- the rigidity of the diaryl urea structure causes the molecular rotation to not happen freely, causethey hadthey excellent had excellent inhibitory inhibitory effects effects on a on variety a variety of solid of solid tumors tumors [14, 15[14,15].]. In sorafenib, In sorafenib, the thus the poor solubility of sorafenib results in low bioavailability [16,17]. This study is therigidity rigidity of the of the diaryl diaryl urea urea structure structure causes causes the molecularthe molecular rotation rotation to not to happennot happen freely, freely, thus based on the structure of sorafenib and the retention of the urea scaffold, in which, in thusthe poor the poor solubility solubility of sorafenib of sorafenib results results in low bioavailabilityin low bioavailability [16,17]. This[16,17]. study This is basedstudy onis order to enhance the molecular flexibility, a carbon atom was inserted into the diaryl urea basedthe structure on the ofstructure sorafenib of and sorafenib the retention and the of theretention urea scaffold, of the inurea which, scaffold, in order in towhich, enhance in structure, and the basic urea scaffold was changed to an N-aryl-N’-benzylurea scaffold. orderthe molecular to enhance flexibility, the molecular a carbon flexibility, atom was a ca insertedrbon atom into was the diaryl inserted urea into structure, the diaryl and urea the Moreover, the diaryl ether fragment with a pyridyl group was also modified to a 4-(pyri- structure,basic urea and scaffold the basic was changedurea scaffold to an Nwas-aryl- changedN’-benzylurea to an N-aryl- scaffold.N’-benzylurea Moreover, thescaffold. diaryl dylmethoxy)phenyl fragment. The nitrogen atom in the pyridine ring is believed to con- Moreover,ether fragment the diaryl with ether a pyridyl fragment group with was a alsopyridyl modified group towas a 4-(pyridylmethoxy)phenylalso modified to a 4-(pyri- tinue to play a key role, for example, as a hydrogen bond acceptor with some proteins. dylmethoxy)phenylfragment. The nitrogen fragment. atom in The the nitrogen pyridine atom ring isin believed the pyridine to continue ring is to believed play a key to con- role, Meanwhile,for example, in as order a hydrogen to keep bond its position acceptor relati withve some to the proteins. core fragment Meanwhile, urea unchanged, in order to keep the tinue to play a key role, for example, as a hydrogen bond acceptor with some proteins. positionits position of the relative nitrogen to the atom core in fragment the pyridine urea unchanged, ring linked the to positionthe core ofurea the fragment nitrogen atomwas Meanwhile, in order to keep its position relative to the core fragment urea unchanged, the replacedin the pyridine from the ring 4-position linked to to the the core 2-position. urea fragment Validity was has replaced been verified from theby a 4-position simulation to position of the nitrogen atom in the pyridine ring linked to the core urea fragment was usingthe 2-position. the Discovery Validity Studio has 3.0 been software verified [18], by and a simulation the distances using between the Discovery the nitrogen Studio atom 3.0 replaced from the 4-position to the 2-position. Validity has been verified by a simulation insoftware the pyridine [18], andringthe and distances the urea betweenmoiety in the sorafenib nitrogen and atom a target in the compound pyridine ringare 10.386Å and the using the Discovery Studio 3.0 software [18], and the distances between the nitrogen atom andurea 10.604Å, moiety inrespectively sorafenib (Figure and a target 2). compound are 10.386Å and 10.604Å, respectively in the pyridine ring and the urea moiety in sorafenib and a target compound are 10.386Å (Figure2). and 10.604Å, respectively (Figure 2).

Figure 2. Spatial distance of the two nitrogen atoms in pyridine ring and urea moiety of sorafenib (left) and a target Figurecompound 2. Spatial (right ).distance of the two nitrogen atoms in pyridine ring and urea moiety of sorafenib (left) and a target compound (right). Figure 2. Spatial distance of the Furthermore,two nitrogen atoms in previous in pyridine reports, ring someand urea proton moiety pump of sorafenib inhibitors (left showed) and a anticancertarget compound (right). activity; lansoprazole was one of the drugs that performed well [19,20]. Based on the molecular hybridization and structural optimization strategies, the 3-methyl-4-(2,2,2- triﬂuoroethoxy)pyridin-2-ylmethyl moiety in lansoprazole and the N-aryl-N’-benzylurea

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Molecules 2021, 26, 3496 Furthermore, in previous reports, some proton pump inhibitors showed anticancer3 of 14 activity; lansoprazole was one of the drugs that performed well [19,20]. Based on the molecular hybridization and structural optimization strategies, the 3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-ylmethyl moiety in lansoprazole and the N-aryl-N’-benzylurea scaf- scaffoldfold were were retained retained into into the target the target compound compoundss (Figure (Figure 3) [21].3)[ Considering21]. Considering the extra the extraspace spacein some in someintracellular intracellular protein protein serine/threonine serine/threonine kinases kinases such as such BRAF as kinase BRAF binding kinase bind- with ingsorafenib, with sorafenib, a new series a new of N series-aryl-N of’-benzylureaN-aryl-N’-benzylurea derivatives derivatives modified with modiﬁed a 1-methylpi- with a 1-methylpiperidin-4-ylperidin-4-yl group on groupthe 3-position on the 3-position of the urea of thescaffold urea scaffoldwere designed, were designed, which are which ex- arepected expected to block to blockintracellular intracellular signal signal transduction transduction and enhance and enhance their antiproliferative their antiproliferative activ- activityity [22,23]. [22 ,23].

FigureFigure 3.3. DesignDesign ofof targettarget compoundscompounds withwith molecularmolecular hybridizationhybridization strategy.strategy.

2.2. ResultsResults andand discussiondiscussion 2.1.2.1. ChemistryChemistry AsAs shownshown in in Scheme Scheme1 ,1, the the target target molecules molecule weres were synthesized synthesized starting starting with with 2-(chlorome 2-(chloro- -thyl)-3-methyl-4-(2,2,2-triﬂuoromethoxy)pyridinemethyl)-3-methyl-4-(2,2,2-trifluoromethoxy)pyridine hydrochloride hydrochloride (1) and(1) and vanillin vanillin (2) via (2) avia Williamson a Williamson reaction reaction and obtainedand obtained the ether the ether compound compound3, which 3, which reacted reacted with hydroxy- with hy- lamine to convert the oxime 4. The oxime 4 was reduced by Ni-Al to obtain benzylamine droxylamine to convert the oxime 4. The oxime 4 was reduced by Ni-Al to obtain benzyl- 5. The reaction between benzylamine 5 and 1-methylpiperidin-4-one through reductive amine 5. The reaction between benzylamine 5 and 1-methylpiperidin-4-one through re- amination yielded the key intermediate 6. Finally, the different intermediate isocyanate ductive amination yielded the key intermediate 6. Finally, the different intermediate iso- 7, prepared by a reaction between substituting aniline or benzylamine, triphosgene, and cyanate 7, prepared by a reaction between substituting aniline or benzylamine, triphos- TEA, were mixed with compound 5 or compound 6 to yield the targeted compounds 8a–8i gene, and TEA, were mixed with compound 5 or compound 6 to yield the targeted com- and 9a–9g. pounds 8a–8i and 9a–9g. 2.2. Biological Activity Evaluation The target compounds were evaluated for their antiproliferative activity against different human cancer cell lines, including A549 (non-small cell lung cancer cell line), MCF-7 (breast cancer cell line), HCT116 (colon cancer cell line), PC-3 (prostate cancer cell line) and HL7702 (human liver normal cell line) by using the MTT assay with sorafenib as the control drug. The evaluated results as IC50 values are shown in Table1.

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o Scheme 1. Synthetic route of the target compounds. Reagents and conditions: a. K2CO3, DMF, 80oC, yield 97%; b. Scheme 1. Synthetic route of the target compounds. Reagents and conditions: a. K2CO3, DMF, 80oC, yield 97%; b. Scheme 1. Synthetic route of the target compounds. Reagents and conditions: a. K2CO3, DMF, 80oC, yield 97%; b. Scheme2 1. Synthetic route3 of the2 target compounds. Reagents and conditions:2 a. K2CO3, DMF, 80o C, yield 97%; b. SchemeNH2OH·HCl, 1. Synthetic NaHCO route3, EtOH, of theH2 O,target yield compou 98%; c.nds. Ni-Al, Reagents NaOH, and EtOH, conditions: H2O, yield a. K 95%;2CO3 ,d. DMF, 1-methylpiperidin-4-one, 80oC, yield 97%; b. SchemeNH2OH·HCl, 1. Synthetic NaHCO route3, EtOH, of theH2 O,target yield compou 98%; c.nds. Ni-Al, Reagents NaOH, and EtOH, conditions: H2O, yield a. K 95%;2CO3 ,d. DMF, 1-methylpiperidin-4-one, 80 C, yield 97%; b. NH2OH·HCl,3 NaHCO3, EtOH, H2O, yield 98%; c. Ni-Al, NaOH, EtOH, H2O, yield 95%; d. 1-methylpiperidin-4-one, NHNaBH2OH·HCl,3CN, AcOH, NaHCO MeOH3, EtOH, yield H77%;2O, e.yield amine, 98%; triphosgene, c. Ni-Al, NaOH,TEA, DCM; EtOH, f. isocyanateH2O, yield 7, 95%; DCM. d. 1-methylpiperidin-4-one, NHNaBH2OH·HCl,3CN, AcOH, NaHCO MeOH3, EtOH, yield H77%;2O, e.yield amine, 98%; triphosgene, c. Ni-Al, NaOH,TEA, DCM; EtOH, f. isocyanateH2O, yield 7, 95%;DCM. d. 1-methylpiperidin-4-one, NaBH3CN, AcOH, MeOH yield 77%; e. amine, triphosgene, TEA, DCM; f. isocyanate 7, DCM. NaBH3 CN, AcOH, MeOH yield 77%; e. amine, triphosgene, TEA, DCM; f. isocyanate 7, DCM. NaBH3CN, AcOH, MeOH yield 77%; e. amine, triphosgene, TEA, DCM; f. isocyanate 7, DCM. 2.2. Biological Activity Evaluation 2.2. Biological Activity Evaluation 2.2. BiologicalThe target Activity compounds Evaluation were evaluated for their antiproliferative activity against dif- The target compounds were evaluated for their antiproliferative activity against dif- Scheme 1. Synthetic route ofThe the target target compounds compounds. wereReagents evaluated and conditions: for their antiproliferativea. K2CO3, DMF, 80activityoC, ◦yield against 97%; dif-b. Scheme 1. Synthetic routeferentThe of human the target target cancer compounds compounds. cell lines, were Reagentsincluding evaluated and A549 conditions:for (non-small their antiproliferative a. cell K2CO lung3, DMF, cancer activity 80 cellC, againstline), yield MCF- 97%; dif- NH2OH·HCl, NaHCO3, ferentEtOH, humanH2O, yield cancer 98%; cell c. lines,Ni-Al, including NaOH, EtOH, A549 H (non-small2O, yield 95%; cell lungd. 1-methylpiperidin-4-one, cancer cell line), MCF- b. NH2OH·HCl, NaHCOferent7 (breast3, EtOH, human cancer H2O, cancer yieldcell line), 98%;cell lines, c.HCT116 Ni-Al, including NaOH, (colon A549 EtOH,cancer (non-small H cell2O, line), yield cell 95%;PC-3 lung d. (prostate 1-methylpiperidin-4-one, cancer cancercell line), cell MCF- line) NaBH3CN, AcOH, MeOH7 (breastyield 77%; cancer e. amine, cell line),triphosgene, HCT116 TEA, (colon DCM; cancer f. isocyanate cell line), 7, DCM. PC-3 (prostate cancer cell line) NaBH CN, AcOH, MeOH7and7 (breast(breast yield HL7702 77%; cancercancer (human e. amine, cellcell line),line), liver triphosgene, HCT116 HCT116normal TEA, cell (colon(colon line) DCM; cancercancer by f. isocyanateusing cellcell line),theline), 7,MTT DCM.PC-3PC-3 assay (prostate(prostate with sorafenibcancercancer cellcell as line)line) the 3 7and (breast HL7702 cancer (human cell line), liver HCT116 normal cell(colon line) cancer by using cell line),the MTT PC-3 assay (prostate with sorafenibcancer cell as line) the and HL7702 (human liver normal cell line)50 by using the MTT assay with sorafenib as the andcontrol HL7702 drug. (human The evaluated liver normal results cell as line)IC50 values by using are the shown MTT in assay Table with 1. sorafenib as the control2.2. Biological drug. The Activity evaluated Evaluation results as IC50 values are shown in Table 1. controlcontrol drug.drug. TheThe evaluatedevaluated resultsresults asas ICIC50 valuesvalues areare shownshown inin TableTable 1.1. Tablecontrol 1. The drug. chemical The structures evaluated and results inhibitory as IC activities50 values of are the shown target compounds. in Table 1. Table 1. The chemicalThe target structures compounds and inhibitory were activities evaluated of thefor target their compounds.antiproliferative activity against dif- Table 1. The chemical structures and inhibitory activities of the target compounds. Table 1. Theferent chemical human structures cancer and cell inhibitory lines, including activities A549 of the (non-small target compounds. cell lung cancer cell line), MCF- Table 1. The chemical structures and inhibitory activities of the target compounds.IC50(µM) IC50(μM) No. Structure7 (breast cancer cell line), HCT116 (colon cancer cellIC line),50(μ M)PC-3 (prostate cancer cell line) No. Structure A549 MCF7 HCT116IC50(μM) PC3 HL7702 No. Structure A549 MCF7 HCT116IC50(μ M) PC3 HL7702 No.No. StructureStructureand HL7702 (humanA549 liver normalMCF7 cell line) by usingHCT116IC the50( μMTT M) assayPC3 with sorafenibHL7702 as the No. Structure A549A549 MCF7MCF7 HCT116HCT116 PC3PC3 HL7702HL7702 control drug. TheA549 evaluated resultsMCF7 as IC 50 valuesHCT116 are shown in TablePC3 1. HL7702 8a 8a 5.305.30 ± 1.451.45 >50 7.25 7.25± ±0.87 0.87 >50 >50 >50 >50 8a 5.30 ± 1.45 >50 7.25 ± 0.87 >50 >50 8a Table 1. The chemical structures5.30 ± 1.45 and inhibitory >50 activities of the 7.25 target ± 0.87 compounds. >50 >50

8b 17.65 ± 5.65 10.98 ± 1.68 9.33 ± 1.38IC50 (μM) 29.13 ± 5.81 >50 8bNo. 8b Structure 17.6517.65 ±± 5.65 10.98 10.98 ± ± 1.681.68 9.33 9.33± ±1.38 1.38 29.13 29.13 ±± 5.815.81 >50 >50 8b8b 17.6517.65 ±±A549 5.655.65 10.98 10.98 ±MCF7± 1.681.68 9.33 9.33 HCT116 ±± 1.381.38 29.13 29.13 ±± PC3 5.815.81 >50 >50HL7702 8b 17.65 ± 5.65 10.98 ± 1.68 9.33 ± 1.38 29.13 ± 5.81 >50

8c 8a 4.885.30 ± 1.94 ± 1.45 >50 >50 11.38 7.25 ± 3.28 ± 0.87 >50 >50 >50 >50 8c 8c 4.884.88 ± 1.94 >50 11.38 11.38± ±3.28 3.28 >50 >50 >50 >50 8c8c 4.884.88 ±± 1.941.94 >50 >50 11.38 11.38 ±± 3.283.28 >50 >50 >50 >50 8c 4.88 ± 1.94 >50 11.38 ± 3.28 >50 >50

8d 8b 26.4717.65 ± 5.66 ± 5.65 10.98 >50 ± 1.68 9.44 9.33 ± 1.22 ± 1.38 29.13 >50 ± 5.81 >50 >50 8d 8d 26.4726.47 ±± 5.66 >50 9.44 9.44± ±1.22 1.22 >50 >50 >50 >50 8d 26.47 ± 5.66 >50 9.44 ± 1.22 >50 >50

8e 29.80 ± 5.09 >50 28.81 ± 3.11 >50 >50 8e 8c 29.804.88 ± 5.09 ± 1.94 >50 >50 28.81 11.38 ± 3.11 ± 3.28 >50 >50 >50 >50 8e 8e 29.8029.80 ± 5.095.09 >50 28.81± ±3.11 3.11 >50 >50 >50 >50

Molecules 2021, 26, x FOR PEER REVIEW 5 of 14 8f 12.40 ± 0.60 13.26 ± 2.27 13.35 ± 2.78 15.87 ± 0.73 >50 8f 8d 12.4026.47 ± 0.60 ± 5.66 13.26 ± >502.27 13.35 9.44 ± 2.78 ± 1.22 15.87 ± >500.73 >50 >50 8f 8f 12.4012.40 ± 0.600.60 13.26 13.26 ±± 2.272.27 13.35± ±2.78 2.78 15.87 15.87 ±± 0.730.73 >50 >50

8e 29.80 ± 5.09 >50 28.81 ± 3.11 >50 >50 ± 8g 8g >50>50 >50 7.84 7.84 ±1.40 1.40 >50 >50 >50 >50

8f 12.40 ± 0.60 13.26 ± 2.27 13.35 ± 2.78 15.87 ± 0.73 >50 8h 10.93 ± 2.02 >50 23.31 ± 3.12 >50 >50

N O N F C O 3 H H 8i N N 9.85 ± 4.40 >50 10.88 ± 2.85 >50 >50 O O

9a 17.53 ± 2.95 2.59 ± 0.29 4.41 ± 0.14 4.10 ± 0.19 >50

9b 4.93 ± 0.46 2.56 ± 0.07 2.90 ± 0.16 3.36 ± 0.17 >50

9c 15.76 ± 1.51 4.65 ± 0.73 12.90 ± 1.59 12.35 ± 1.75 >50

9d 3.17 ± 0.22 2.63 ± 0.08 2.56 ± 0.26 3.62 ± 0.27 >50

9e 5.48 ± 4.36 2.56 ± 0.16 5.82 ± 0.21 4.53 ± 0.33 >50

9f 6.09 ± 0.29 3.18 ± 0.30 5.18 ± 0.33 7.39 ± 0.63 >50

N N O O F C O 9g 3 H 6.04 ± 0.41 4.23 ± 0.47 2.93 ± 0.26 6.25 ± 0.43 >50 N N O O sorafenib 6.16 ± 0.46 3.54 ± 0.19 3.88 ± 0.36 5.26 ± 0.46 >50

As shown in Table 1, all the target compounds exhibited weak cytotoxic activities against HL7702, and most of the target compounds exhibit excellent antiproliferative activity against the A549 cell line and HCT116 cell line. The IC50 values of target compounds 8c, 9b, and 9d against the A549 cells line were less than 5 μM, and the IC50 values of compounds 9b, 9d, and 9g against the HCT116 cell line were less than 3 μM. The antiproliferative activities against the MCF7 cell line and the PC3 cell line of target compounds 9a–9g with the 1-methylpiperidin-4-yl group were significantly higher than that of compounds 8a–8i without the 1-methylpiperidin-4-yl group. The IC50 values of compounds 9a, 9b, 9d, and 9e against the MCF7 cell line and the PC3 cell line were less than 3 μM and 5 μM, respectively. Among them, target compounds 9b and 9d have shown a more potent antiproliferative activity against the four cancer cell lines with excellent IC50 values (under 5 μM) compared to the control drug sorafenib. The analyses of the structure-activity relationships of the target compounds with the 1-methylpiperidin-4-yl group were summarized as follows: (1) The introduction of fluorine atoms on the R1 substituent of the benzene ring was mostly beneficial to the antiproliferative activity. For example, Compounds 9a, 9b, 9d, and 9e with the fluorine atoms in substituent on the phenyl show a better antiproliferative activity against the MCF7 cell line and the PC3 cell line than the control drug sorafenib, especially in the MCF7 cell line. The inhibitory activities against several cell lines of compound 9f with the nitro group were relatively weaker than that of several compounds with fluorine atoms on the R1 substituent of the benzene ring. (2) The introduction of electron-withdrawing group sub-

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MoleculesMolecules 20212021,, 2626,, xx FORFOR PEERPEER REVIEWREVIEW 55 ofof 1414 Molecules 2021, 26, x FOR PEER REVIEW 5 of 14 Molecules 2021, 26, x FOR PEER REVIEW 5 of 14 Molecules Molecules 20212021,, 2626,, xx FORFOR PEERPEER REVIEWREVIEW 55 ofof 1414 Molecules 2021,, 26,, xx FORFOR PEERPEER REVIEWREVIEW Table 1. Cont. 5 of 14

IC50(µM) 8g No. Structure >50 >50 7.84 ± 1.40 >50 >50 8g A549>50 MCF7 >50 7.84 HCT116 ± 1.40 >50 PC3 >50 HL7702 8g >50 >50 7.84 ± 1.40 >50 >50 8g8g >50>50 >50 >50 7.84 7.84 ±± 1.401.40 >50 >50 >50 >50 8g >50 >50 7.84 ± 1.40 >50 >50 8h 8h 10.9310.93 ± 2.022.02 >50 23.31± ±3.12 3.12 >50 >50 >50 >50 8h 10.93 ± 2.02 >50 23.31 ± 3.12 >50 >50 8h 10.93 ± 2.02 >50 23.31 ± 3.12 >50 >50 8h N 10.93 ± 2.02 >50 23.31 ± 3.12 >50 >50 8h N 10.93 ± 2.02 >50 23.31 ± 3.12 >50 >50 8h O N 10.93 ± 2.02 >50 23.31 ± 3.12 >50 >50 8h F C O N 10.93 ± 2.02 >50 23.31 ± 3.12 >50 >50 8h 3 N O H H N 10.93 ± 2.02 >50 23.31 ± 3.12 >50 >50 F3C O O N 8i F C O N NH NH 9.85 ± 4.40 >50 10.88 ± 2.85 >50 >50 8i 8i 3 N O HN HN N 9.859.85 ± 4.40 >50 10.88 10.88± ±2.85 2.85 >50 >50 >50 >50 F3C O N O N 8i F C O O HN HN 9.85 ± 4.40 >50 10.88 ± 2.85 >50 >50 8i 3 N OO HN O HN N 9.85 ± 4.40 >50 10.88 ± 2.85 >50 >50 F3C O N O O N 8i F C O OO NH NH N 9.85 ± 4.40 >50 10.88 ± 2.85 >50 >50 3 O H O H 8i F3C O O N N N 9.85 ± 4.40 >50 10.88 ± 2.85 >50 >50 8i F C O O NH O NH 9.85 ± 4.40 >50 10.88 ± 2.85 >50 >50 8i 3 O HN O HN 9.85 ± 4.40 >50 10.88 ± 2.85 >50 >50 8i O N O N 9.85 ± 4.40 >50 10.88 ± 2.85 >50 >50 O O 9a O 17.53 ± 2.95 2.59 ± 0.29 4.41 ± 0.14 4.10 ± 0.19 >50 9a 9a O 17.5317.53 ±± 2.95 2.59 2.59 ±± 0.290.29 4.41 4.41± ±0.14 0.14 4.10 4.10 ±± 0.190.19 >50 >50 9a 17.53 ± 2.95 2.59 ± 0.29 4.41 ± 0.14 4.10 ± 0.19 >50 9a9a 17.5317.53 ±± 2.952.95 2.59 2.59 ±± 0.290.29 4.41 4.41 ±± 0.140.14 4.10 4.10 ±± 0.190.19 >50 >50 9a 17.53 ± 2.95 2.59 ± 0.29 4.41 ± 0.14 4.10 ± 0.19 >50

9b9b 4.934.93 ±± 0.460.46 2.56 2.56 ±± 0.070.07 2.90 2.90 ±± 0.160.16 3.36 3.36 ±± 0.170.17 >50 >50 9b 9b 4.934.93 ± 0.460.46 2.56 2.56 ±± 0.07 2.90 ± ±0.16 0.16 3.36 3.36 ±± 0.170.17 >50 >50 9b9b 4.934.93 ±± 0.460.46 2.56 2.56 ±± 0.070.07 2.90 2.90 ±± 0.160.16 3.36 3.36 ±± 0.170.17 >50 >50 9b9b 4.934.93 ±± 0.460.46 2.56 2.56 ±± 0.070.07 2.90 2.90 ±± 0.160.16 3.36 3.36 ±± 0.170.17 >50 >50 9b 4.93 ± 0.46 2.56 ± 0.07 2.90 ± 0.16 3.36 ± 0.17 >50

9c 15.76 ± 1.51 4.65 ± 0.73 12.90 ± 1.59 12.35 ± 1.75 >50 9c 15.76 ± 1.51 4.65 ± 0.73 12.90 ± 1.59 12.35 ± 1.75 >50 9c 9c 15.7615.76 ± 1.511.51 4.65 4.65 ±± 0.73 12.90 ± ±1.59 1.59 12.35 12.35 ±± 1.751.75 >50 >50 9c9c 15.7615.76 ±± 1.511.51 4.65 4.65 ±± 0.730.73 12.90 12.90 ±± 1.591.59 12.35 12.35 ±± 1.751.75 >50 >50 9c 15.76 ± 1.51 4.65 ± 0.73 12.90 ± 1.59 12.35 ± 1.75 >50

9d 3.17 ± 0.22 2.63 ± 0.08 2.56 ± 0.26 3.62 ± 0.27 >50 9d 3.17 ± 0.22 2.63 ± 0.08 2.56 ± 0.26 3.62 ± 0.27 >50 9d 3.17 ± 0.22 2.63 ± 0.08 2.56 ± 0.26 3.62 ± 0.27 >50 9d9d 9d 3.173.173.17 ±± 0.220.22 2.63 2.63 2.63 ± ± 0.080.08 2.56 2.56± ±±0.26 0.260.26 3.62 3.62 3.62 ±±± 0.270.270.27 >50 >50 >50 9d 3.17 ± 0.22 2.63 ± 0.08 2.56 ± 0.26 3.62 ± 0.27 >50

9e 5.48 ± 4.36 2.56 ± 0.16 5.82 ± 0.21 4.53 ± 0.33 >50 9e 5.48 ± 4.36 2.56 ± 0.16 5.82 ± 0.21 4.53 ± 0.33 >50 9e 5.48 ± 4.36 2.56 ± 0.16 5.82 ± 0.21 4.53 ± 0.33 >50 9e9e 9e 5.485.485.48 ±± 4.364.36 2.56 2.56 2.56 ± ± 0.160.16 5.82 5.82± ±±0.21 0.210.21 4.53 4.53 4.53 ±±± 0.330.330.33 >50 >50 >50 9e 5.48 ± 4.36 2.56 ± 0.16 5.82 ± 0.21 4.53 ± 0.33 >50

9f 6.09 ± 0.29 3.18 ± 0.30 5.18 ± 0.33 7.39 ± 0.63 >50 9f 6.09 ± 0.29 3.18 ± 0.30 5.18 ± 0.33 7.39 ± 0.63 >50 9f 6.09 ± 0.29 3.18 ± 0.30 5.18 ± 0.33 7.39 ± 0.63 >50 9f9f 6.096.09 ±± 0.290.29 3.18 3.18 ±± 0.300.30 5.18 5.18 ±± 0.330.33 7.39 7.39 ±± 0.630.63 >50 >50 9f 9f N 6.096.09 ± 0.29 3.18 3.18 ±± 0.300.30 5.18 5.18± ±0.33 0.33 7.39 7.39 ±± 0.630.63 >50 >50 9f N N 6.09 ± 0.29 3.18 ± 0.30 5.18 ± 0.33 7.39 ± 0.63 >50 N N N O N O F3C O N O O 9g F C O O N H O 6.04 ± 0.41 4.23 ± 0.47 2.93 ± 0.26 6.25 ± 0.43 >50 3 N NN NH 9g F3C O N O N O 6.04 ± 0.41 4.23 ± 0.47 2.93 ± 0.26 6.25 ± 0.43 >50 9g F C O O N HN O 6.04 ± 0.41 4.23 ± 0.47 2.93 ± 0.26 6.25 ± 0.43 >50 3 N O N HN 9g F3C O N O N O O 6.04 ± 0.41 4.23 ± 0.47 2.93 ± 0.26 6.25 ± 0.43 >50 9g F C O OO N HN O 6.04 ± 0.41 4.23 ± 0.47 2.93 ± 0.26 6.25 ± 0.43 >50 3 N O O 9g F3C O O N NH O 6.04 ± 0.41 4.23 ± 0.47 2.93 ± 0.26 6.25 ± 0.43 >50 9g F C O O N O HN O 6.04 ± 0.41 4.23 ± 0.47 2.93 ± 0.26 6.25 ± 0.43 >50 9g F 3C O O N O NH 6.04 ± 0.41 4.23 ± 0.47 2.93 ± 0.26 6.25 ± 0.43 >50 sorafenib9g 9g 3 O N O HN 6.166.046.04 ± 0.460.410.41 3.544.23 4.23 ±± 0.470.19 2.933.88± ±0.26 0.360.26 5.266.25 6.25 ±± 0.460.430.43 >50 >50 sorafenib O N O N 6.16 ± 0.46 3.54 ± 0.19 3.88 ± 0.36 5.26 ± 0.46 >50 sorafenib O O 6.16 ± 0.46 3.54 ± 0.19 3.88 ± 0.36 5.26 ± 0.46 >50 sorafenib O 6.16 ± 0.46 3.54 ± 0.19 3.88 ± 0.36 5.26 ± 0.46 >50 sorafenib O 6.16 ± 0.46 3.54 ± 0.19 3.88 ± 0.36 5.26 ± 0.46 >50 sorafenibsorafenib 6.166.16 ±± 0.460.46 3.54 3.54 ±± 0.190.19 3.88 3.88 ±± 0.360.36 5.26 5.26 ±± 0.460.46 >50 >50 sorafenibsorafenib As shown in 6.16Table6.16 ± 0.460.461, all the 3.54target 3.54 ±± 0.190.19comp ounds 3.88 3.88 exhibited± ±0.36 0.36 weak 5.26 5.26 cytotoxic±± 0.460.46 activities >50 >50 As shown in Table 1, all the target compounds exhibited weak cytotoxic activities againstAs HL7702,shown in and Table most 1, ofall the the target target comp compoundsounds exhibit exhibited excellent weak antiproliferative cytotoxic activities ac- againstAsAs HL7702,shownshown inin and TableTable most 1,1, ofallall the thethe target targettarget comp compcompoundsoundsounds exhibit exhibitedexhibited excellent weakweak antiproliferative cytotoxiccytotoxic activitiesactivities ac- againstAs HL7702,shown in and Table most 1, ofall the the target target comp compoundsounds. exhibit exhibited 50excellent weak antiproliferative cytotoxic activities ac- tivityagainsttivityAs againstagainst HL7702,shown thethe in and A549A549 Table most cellcell 1, line lineofall the andtheand target target HCT116HCT116 comp comp cellcelloundsounds lineline .exhibit The Theexhibited ICIC excellent50 valuesvalues weak of ofantiproliferative cytotoxic targettarget compoundscompounds activities ac- againsttivityagainst against HL7702,HL7702,As shown the and andA549 in mostmost Tablecell lineofof1 ,thethe alland targettarget the HCT116 target compcomp cell compoundsoundsounds line . exhibitexhibit The exhibitedIC excellent50excellent values weak of antiproliferativeantiproliferative target cytotoxic compounds activities ac-ac- tivityagainst against HL7702, the andA549 most cell lineof the and target HCT116 comp cellounds line. exhibit The IC 50excellent values of antiproliferative target50 compounds ac- 8ctivityagainst8c,, 9b,9b, against andand HL7702, 9d9d the againstagainst andA549 most the thecell A549A549 lineof the and cellscells target HCT116 lineline comp werewere cellounds lessless line thanthan.. exhibit The 55 μICμM,M, 50excellent values andand thethe of antiproliferative ICIC target50 valuesvalues compounds ofof com-com- ac- 8ctivity, 9b,against against and 9d HL7702, the against A549 and the cell most A549 line of and cells the HCT116 targetline were compounds cell less line than. The exhibit 5 μICM,5050 valuesand excellent the of IC target antiproliferative50 values compounds of com- activ- tivity8ctivity, 9b, againstagainst and 9d thethe against A549A549 the cellcell A549 lineline andand cells HCT116HCT116 line were cellcell less lineline than. TheThe 5 ICμICM,50 values valuesand the ofof IC targettarget50 values compoundscompounds of com- pounds8ctivitypounds, 9b, against and 9b9b, , 9d 9d,9d, the against andand A549 9g9g the againstcellagainst A549 line thetheand cells HCT116HCT116 HCT116 line were cellcell cell less lineline line than were.were The 5 lesslessICμM,50 than than valuesand 33the μ μofM.M. IC target The50The values antiprolifer-antiprolifer-compounds of com- pounds8c, 9b,ity and against 9b, 9d9d, against theand A549 9g theagainst cell A549 line the cells and HCT116 line HCT116 were cell celllessline line.than were The5 less μM, IC than and50 values 3the μM. IC of50The50 targetvalues antiprolifer- compounds of com- 8cpounds, 9b, and 9b ,9d 9d, against and 9g the against A549 the cells HCT116 line were cell less line than were 5 lessμM, than and 3the μM. IC The50 values antiprolifer- of com- ativepounds8c,, 9b, activities andand 9b, 9d9d, againstagainst and 9g the theagainst A549A549MCF7 the cellscells cell HCT116 lineline werewereand cell the lessless line PC3 thanthan were cell 55 less μlineM, than ofand target 3the μM. IC compounds The50 valuesvalues antiprolifer- ofof 9a com-com-–9g poundsativepounds8c activities, 9b, 9b9b,, and9d,9d, against andand9d against 9g9g againsttheagainst MCF7 the thethe A549 cell HCT116HCT116 line cells and linecellcell the lineline were PC3 werewere less cell lessless thanline thanthan of 5 targetµ 3M,3 μμM.M. and compounds TheThe the antiprolifer-antiprolifer- IC50 9avalues–9g of withativepounds theactivities 9b 1-methylpiperidin-4-yl,, 9d, against andand 9g theagainstagainst MCF7 thethe groupcell HCT116HCT116 line were and cellcell si thegnificantly lineline PC3 werewere cell lessless higherline thanthan of targetthan 33 μ M.that compounds The of antiprolifer-compounds 9a–9g ativewithativecompounds activitiestheactivities 1-methylpiperidin-4-yl againstagainst9b, 9d, thetheand MCF7MCF79g against group cellcell lineline thewere andand HCT116 si thethegnificantly PC3PC3 cell cellcell line linehigherline were ofof target targetthan less thanthat compoundscompounds 3ofµ compoundsM. The 9a9a antipro-––9g9g withative theactivities 1-methylpiperidin-4-yl against the MCF7 group cell line were and si thegnificantly PC3 cell50 higherline of targetthan that compounds of compounds 9a–9g 8awithative8a––8i8i theactivities withoutwithout 1-methylpiperidin-4-yl the theagainst 1-methylpipe1-methylpipe the MCF7ridin-4-ylridin-4-yl groupcell line were andgroup.group. si thegnificantly The ThePC3 ICIC cell50 values valueslinehigher of of oftargetthan compoundscompounds that compounds of compounds 9a9a,, 9b9b 9a,, – 9d9d9g,, 8awith–8iliferative thewithout 1-methylpiperidin-4-yl activitiesthe 1-methylpipe against theridin-4-yl group MCF7 were group. cell si linegnificantly The and IC the50 values higher PC3 cell of than compounds line that of targetof compounds 9a compounds, 9b, 9d, with8awith–8i the thewithout 1-methylpiperidin-4-yl1-methylpiperidin-4-yl the 1-methylpiperidin-4-yl groupgroup werewere group. sisignificantlygnificantly The IC50 values higherhigher of thanthan compounds thatthat ofof compoundscompounds 9a, 9b, 9d, and8awithand–8i 9a9e 9ethe without– against9gagainst 1-methylpiperidin-4-ylwith the the the 1-methylpipe MCF7 1-methylpiperidin-4-ylMCF7 cellcell linelineridin-4-yl group andand werethethe group. PC3 groupPC3 significantly cellcellThe were line lineIC50 significantly werevalueswere higher lessless of than compoundsthanthan higher that 33 μ μofMM than compounds and and9a that, 9b 55 μ,μ of9dM,M, com-, and8a–8i 9e without against the the 1-methylpipe MCF7 cell lineridin-4-yl and the group. PC3 cell The line IC5050 werevalues less of compoundsthan 3 μM and 9a, 9b5 μ, 9dM,, 8aand–8i 9e without against the the 1-methylpipe MCF7 cell lineridin-4-yl and the group. PC3 cellThe lineIC 50 werevalues less of compoundsthan 3 μM and9a, 9b 5 ,μ 9dM,, respectively.and8a–8i 9e withoutwithout against Among thethe the 1-methylpipe1-methylpipe MCF7 them, cell target lineridin-4-yl compounds and the group. PC3 9b cellThe and line IC 9d50 werehavevaluesvalues shownless ofof compoundscompoundsthan a more3 μM potent and 9a,, 9b5 anti-μ,, 9dM,,, andrespectively.and pounds 9e9e againstagainst8a Among – the8ithewithout MCF7MCF7 them, cellcell the target line 1-methylpiperidin-4-ylline compounds andand thethe PC3PC3 9b cellcell andand lineline 9d group. were werehavehave The lessshownshownless IC thanthan50 aa values more more33 μμMM potent potent and ofand compounds 55 anti-anti-μμM,M, respectively.and 9e against Among the MCF7 them, cell target line compounds and the PC3 9b cell and line 9d werehave shownless than a 50more 3 μM potent and 5 anti-μM, proliferativerespectively.andproliferative 9a9e, 9bagainst, 9d activityAmongactivity, andthe 9eMCF7 againstthem,againstagainst cell target thethethe line fourfour MCF7 compounds and cancercancer the cell PC3 line cellcell 9b andcell lines linesand theline 9dwithwithPC3 werehave excellentexcellent cell shownless line than wereICIC a 50more3 values valuesμ lessM potent thanand (under(under 5 3 anti-μµM,M 55 and proliferativerespectively. activityAmong againstthem, target the four compounds cancer cell 9b lines and with9d have excellent shown IC a 50more values potent (under anti- 5 respectively.proliferativerespectively. AmongactivityAmong them,againstthem, targettarget the four compoundscompounds cancer cell 9b9b lines andand 9dwith9d havehave excellent shownshown IC aa 50moremore values potentpotent (under anti-anti- 5 μproliferativerespectively.μM)M)5 comparedcomparedµM, respectively. activityAmong toto thethe againstthem, controlcontrol Among target the drugdrug them, four compounds sorafenib.sorafenib. targetcancer compounds cell 9b lines and 9dwith9b have andexcellent shown9d have IC a 50more shown values potent a (under more anti- potent 5 μproliferativeM) compared activity to the againstcontrol thedrug four sorafenib. cancer cell lines with excellent IC5050 values (under 5 proliferativeμM) compared activity to the against control the drug four sorafenib. cancer cell lines with excellent IC 50 values (under 5 μproliferativeM)antiproliferative Thecompared analyses activity to ofthe activitytheagainst control structure-activity against thedrug four sorafenib. the cancer four rela cancer celltionships lines cell with linesof the excellent with target excellent compounds IC50 valuesvalues IC values (under(underwith the (under 55 μμM)M) comparedThecompared analyses toto theofthe the controlcontrol structure-activity drugdrug sorafenib.sorafenib. rela tionshipstionships ofof thethe targettarget compoundscompounds50 withwith thethe 1-methylpiperidin-4-ylμM)5 ThecomparedµM) analyses compared to ofthe the to control group thestructure-activity control drug were sorafenib. drugsummarized sorafenib. rela tionships as follows: of the (1) target The introductioncompounds with of fluo- the 1-methylpiperidin-4-ylTheThe analysesanalyses ofof thethe group structure-activitystructure-activity were summarized relarelationshipstionships as follows: ofof thethe (1) targettarget The compoundsintroductioncompounds with withof fluo- thethe 1-methylpiperidin-4-ylThe analyses of1 the group structure-activity were summarized relationships as follows: of the (1) target The introductioncompounds with of fluo- the rine1-methylpiperidin-4-ylrine atomsatomsThe Theanalyses onon analyses thethe RofR1 substituentthesubstituent ofgroup structure-activity the structure-activitywere ofof the thesummarized benzenebenzene relationships ringring relationships as follows: waswas of mostlymostly the (1) oftarget The beneficialbeneficial the compoundsintroduction target toto compounds thethe withantipro- antipro-of fluo- the with rine1-methylpiperidin-4-yl atoms on the R1 substituent group were of the summarized benzene ring as follows:was mostly (1) Thebeneficial introduction to the antipro- of fluo- 1-methylpiperidin-4-ylrine1-methylpiperidin-4-yl atoms on the R1 substituent groupgroup werewere of the summarizedsummarized benzene ring asas follows: follows:was mostly (1)(1) The Thebeneficial introductionintroduction to the antipro- ofof fluo-fluo- liferativerine1-methylpiperidin-4-ylliferative theatoms 1-methylpiperidin-4-yl activity.activity. on the RForFor1 substituent example,example, group were Compounds groupCompoundsof the summarized werebenzene summarized9a9a, , ring 9b9b as,, 9dfollows:9dwas,, andand mostly as 9e 9e follows:(1) withwith Thebeneficial thetheintroduction (1) fluorinefluorine The to the introduction atoms atomsantipro- of fluo- inin of liferativerine atoms activity. on the ForR11 substituent example, Compounds of the benzene 9a, ring9b, 9d was, and mostly 9e with beneficial the fluorine to the atoms antipro- in rineliferative atoms activity. on the RFor1 substituent example, Compounds of the benzene 9a ,ring 9b, 9dwas, and mostly 9e with beneficial the fluorine to the atomsantipro- in substituentliferativerine fluorineatoms activity. onon atoms the the RFor phenyl1 on substituentsubstituent example, the show R substituent Compounds ofofa betterthethe benzenebenzene anti of 9aproliferative the, ringring9b benzene, 9d waswas, and mostlymostly activity ring 9e withwas beneficialbeneficial against the mostly fluorine totheto beneficial thethe MCF7 atoms antipro-antipro- cell toin the liferativesubstituentliferative activity.activity. on the ForFor phenyl example,example, show1 CompoundsCompounds a better anti 9a9aproliferative,, 9b9b,, 9d9d,, andand activity 9e9e withwith against thethe fluorinefluorine the MCF7 atomsatoms cell inin linesubstituentliferative antiproliferativeand the activity. onPC3 the cell For phenyl line activity. example, than show Forthe Compounds a control example,better druganti Compounds 9aproliferative sorafenib,,, 9b,, 9d,, andand 9aespecially activity , 9e9b withwith, 9d against, inthethe and the fluorinefluorine9e MCF7 thewith MCF7 atomsatomscell the line. fluorinecell inin substituentlinelinesubstituent andand thethe on onPC3PC3 thethe cellcell phenylphenyl lineline thanthan showshow thethe aa control control betterbetter antidrugantidrugproliferativeproliferative sorafenib,sorafenib, especially especially activityactivity againstagainst inin thethe MCF7 MCF7thethe MCF7MCF7 cellcell line.line. cellcell Thelinesubstituent atomsandinhibitory the in onPC3 substituent activitiesthe cell phenyl line againstthan on show the the severalaphenyl control better cell showantidrug linesproliferative sorafenib, a better of compound antiproliferative especially activity 9f against within the the activity MCF7 the nitro MCF7 cell against group line. cell the lineTheline andandinhibitory thethe PC3PC3 activities cellcell lineline thanagainstthan thethe several controlcontrol cell drugdrug lines sorafenib,sorafenib, of compound especiallyespecially 9f inwithinwith thethe thethe MCF7MCF7 nitronitro cellcell groupgroup line.line. wereTheline MCF7andinhibitory relatively the cell PC3 line activitiesweaker cell and line the than againstthan PC3 that the cell several controlof line severa than cell druglthe compoundslines sorafenib, control of compound drug especiallywith sorafenib, fluorine 9f within the especially atoms the MCF7 nitro on cell in thegroup the line. R1 MCF7 ThewereThe inhibitoryinhibitory relatively activities activitiesweaker than againstagainst that severalseveral of severa cellcellll linescompoundscompoundslines ofof compoundcompound withwith fluorinefluorine 9f9f withwith atomsatoms thethe nitronitro onon thethegroupgroup R1R1 substituentwereThe inhibitory relatively of the activitiesweaker benzene than against ring that. (2) several of The severa introduction celll compoundslines of ofcompound el ectron-withdrawingwith fluorine 9f withwith atoms thethe nitro nitrogroup on thegroupgroup sub- R1 weresubstituentwere relativelyrelatively of the weakerweaker benzene thanthan ring thatthat.. (2)(2) ofof TheThe severasevera introductionintroductionll compoundscompounds ofof elel ectron-withdrawingwithwith fluorinefluorine atomsatoms group onon thethe sub- R1R1 substituentwere relatively of the weaker benzene than ring that. (2) of The severa introductionl compounds of el ectron-withdrawingwith fluorine atoms group on the sub- R1 substituentsubstituent ofof thethe benzenebenzene ringring.. (2)(2) TheThe introductionintroduction ofof elelectron-withdrawingectron-withdrawing groupgroup sub-sub- substituent of the benzene ring.. (2)(2) TheThe introductionintroduction ofof elelectron-withdrawing group sub-

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Molecules 2021, 26, x FOR PEER REVIEW 6 of 14

cell line. The inhibitory activities against several cell lines of compound 9f with the nitro group were relatively weaker than that of several compounds with fluorine atoms on the R1stituent substituent R1 on ofthe the benzene benzene ring ring. results (2) Thein an introduction increase in antiproliferative of electron-withdrawing activity. The group an- substituenttiproliferative R1 on activities the benzene against ring all results four cancer in an increasecell lines inof antiproliferative the target compounds activity. 9b The and antiproliferative9d with a trifluoromethyl activities group against were all foursignificantly cancer cell higher lines than ofthe compound target compounds 9a with a meth-9b andoxy9d groupwith and a trifluoromethyl 9c with a trifluoromethoxy group were significantly group. higher than compound 9a with a methoxyThe group antiproliferative and 9c with activities a trifluoromethoxy of the target group. compounds without the 1-methylpiperidin-4-ylThe antiproliferativegroup have shown activities similar of structure-activity the target compounds relationships. without the For 1-methylpiperidin- example, the anti- 4-ylproliferative group have activity shown against similar the structure-activity HCT116 cell line relationships. of compounds For example, 8a, 8b, 8c the, and antiprolif- 8d with erativefluorine activity atoms against in substituent the HCT116 on the cell phenyl line of compoundswere also better8a, 8b than, 8c, that and of8d thewith other fluorine com- atomspounds in without substituent fluorine on the atoms, phenyl and were the alsoIC50 value better of than compound that ofthe 8c was other less compounds than 5 μM withoutagainst fluorinethe A549 atoms, cell line. and the IC50 value of compound 8c was less than 5 µM against the A549 cell line. 2.3. Cell Cycle Analysis 2.3. Cell Cycle Analysis The effect of compound 9b on the cell cycle was assayed. After treatment of MCF-7 The effect of compound 9b on the cell cycle was assayed. After treatment of MCF- cells with compound 9b for 72 h at different concentrations (2.5, 5, 10, 20 μM), the per- 7 cells with compound 9b for 72 h at different concentrations (2.5, 5, 10, 20 µM), the centages of cells in the G2/M phase were 16.7%, 23.5%, 27.7%, and 39.3%, respectively (Fig- percentages of cells in the G /M phase were 16.7%, 23.5%, 27.7%, and 39.3%, respec- ure 4), indicating that compound2 9b could cause an obvious G2/M arrest in a concentra- tively (Figure4) , indicating that compound 9b could cause an obvious G2/M arrest in a tion-dependent manner with a concomitant decrease in terms of the number of cells in concentration-dependent manner with a concomitant decrease in terms of the number of other phases of the cell cycle. cells in other phases of the cell cycle.

0 μM 2.5 μM 5 μM

20 μM 10 μM

(a)

Figure 4. Cont.

MoleculesMolecules 20212021, 26, 26, x, 3496FOR PEER REVIEW 7 7of of 14 14

80 G1 S 60 G2/M

% of cell cycle distribution 0

M M μ μ 0μM 5μM 2.5μM 10 20 concentrations of treatment

(b)

FigureFigure 4. Effects 4. Effects of compound of compound9b on 9b MCF7on MCF7 cell cell cycle cycle progress progress for for 24 h.24 (h.a) ( Treatmenta) Treatment of MCF7of MCF7 cells cells with with compound compound9b 9bat differentat different concentrations (0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) for 24 h. (b) Quantitative analysis of cell cycle. concentrations (0 µM, 2.5 µM, 5 µM, 10 µM, 20 µM) for 24 h. (b) Quantitative analysis of cell cycle.

3.3. Materials Materials and and Methods Methods 3.1.3.1. Chemistry Chemistry 3.1.1.3.1.1. General General Information Information AllAll reagents reagents were were obtained obtained from from commerci commercialal suppliers suppliers and andused used without without further further purification.purification. The Theprogress progress of the ofthe reactions reactions was was monitored monitored by by thin-layer thin-layer chromatography chromatography (TLC)(TLC) on on silica silica gel gel plates plates and and the the spots spots visualized visualized under under ultraviolet ultraviolet (UV) (UV) light light (254 (254 nm). nm). TheThe column column chromatography chromatography was was performed performed using using 200–300 200–300 mesh mesh silica silicagel (Qingdao gel (Qingdao Hai- yang,Haiyang, Qingdao, Qingdao, China). China). Mass Mass spectra spectra were weremeasured measured with withan electrospray an electrospray (ESI-MS) (ESI-MS) on an on 1 Agilentan Agilent 1100 1100Series Series LC/MSD LC/MSD Trap (Agilent Trap (Agilent Corporation, Corporation, Santa Clara, Santa Clara,CA, USA). CA, 1 USA).H-NMRH- 13 andNMR 13C-NMR and C-NMR spectra spectra were wererecorded recorded on Bruker on Bruker NMR NMR spectrophotometers spectrophotometers (Karlsruhe, (Karlsruhe, d Germany)Germany) using using DMSO- DMSO-d6 as6 as the the solvent. solvent. The The IR IR spectra spectra were were measured measured using using a aBruker Bruker 1 FourierFourier number number infrared infrared spectrometer spectrometer (Agi (Agilentlent Corporation, Corporation, Santa Santa Clara, Clara, CA, CA, USA). USA). 1H-H- 13 NMR,NMR, 13C-NMR,C-NMR, ESI-MS ESI-MS and and HRMS HRMS spectra spectra of of the the target target compounds compounds are are available available in in the the SupplementarySupplementary Material Material (Figures (Figures S1–S80). S1–S80). 3.1.2. Synthesis of 3-methoxy-4-[(3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy] 3.1.2.benzaldehyde Synthesis of (3 )3-methoxy-4-[(3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy] benzaldehyde (3) A mixture of 2-(chloromethyl)-3-methyl-4-(2,2,2-trifluoroethoxy)pyridine hydrochlo- A mixture of 2-(chloromethyl)-3-methyl-4-(2,2,2-trifluoroethoxy)pyridine hydro- ride 1 (27.6 g, 100 mmol), vanillin 2 (15.22 g, 100 mmol), K2CO3 (69 g, 0.5 mol) and chlorideDMF(100 1 (27.6 mL) g, was 100 stirred mmol), at vanillin 80 ◦C overnight.2 (15.22 g, 100 The mmol), reaction K2CO was3 (69 monitored g, 0.5 mol) by and TLC DMF(100(PE:EA =mL) 3:1). was The stirred mixture at 80 was°C overnight. poured into The 750 reaction mL of was water monitored and stirred by TLC for (PE:EA 30 min 2 3 = until3:1). The the Kmixture2CO3 was was completely poured into dissolved. 750 mL of Thewater solids and thatstirred precipitated for 30 min outuntil were the K filtered,CO waswashed completely with 2 dissolved. mol/L NaOH The solids aqueous that solution precipitated and water, out were then filtered, dried to washed obtain with a white 2 mol/Lsolid NaOH 34.54 g. aqueous The yield solution was 97%, and ESI-MS: water, then 356.3([M dried + to H] obtain+). a white solid 34.54 g. The yield was 97%, ESI-MS: 356.3([M + H]+). 3.1.3. Synthesis of (E)-3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl) 3.1.3.methoxy)benzaldehyde Synthesis of (E)-3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl) oxime (4) methoxy)benzaldehydeA mixture of hydroxylamine oxime (4) hydrochloride (3.06 g, 44 mmol) and NaHCO3 (3.69 g,

44 mmol)A mixture in water of hydroxylamine (50 mL) was stirred hydrochloride at room temperature (3.06 g, 44 until mmol) no gas and was NaHCO released.3 (3.69 A solu- g, 44tion mmol) of 3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzaldehyde in water (50 mL) was stirred at room temperature until no gas was released. A 3 solution(14.21 g,40 of mmol)3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)ben- in EtOH (50 mL) was added into the mixture and continued string for 3 h. zaldehydeThe progress 3 (14.21 of the g,40 reaction mmol) can in EtOH be conﬁrmed (50 mL) bywa TLC.s added EtOH into was the removedmixture and under continued reduced

Molecules 2021, 26, 3496 8 of 14

pressure, and the white solid was precipitated, ﬁltered off with suction, and washed with water. After drying, the white solid obtained was 14.6 g, with a yield of 98%, ESI-MS: 371.3([M + H]+).

3.1.4. Synthesis of (3-methoxy-4-((3-methyl-4-(2,2,2-triﬂuoroethoxy)pyridin-2- yl)methoxy)phenyl)methanamine (5)

An aqueous solution of NaOH (5 mol/L, 60 mL) was added into the solution of 3- methoxy-4-((3-methyl-4-(2,2,2-triﬂuoroethoxy)pyridin-2-yl)methoxy)benzaldehyde oxime 4 (11.10 g,30 mmol) in EtOH (50 mL) under an ice bath. Nickel-aluminum alloy (10 g) was slowly added into the mixture in several times, during which a lot of gas was generated. Then slowly returned to room temperature and stirred overnight. The progress of the reaction was monitored by TLC. After removing the solid by suction ﬁltration, EtOH was distilled off under reduced pressure. The residual solution was extracted by EA, and the organic phase was washed by water and brine, then dried by Na2SO4. After the solvent was removed under reduced pressure, the white solid obtained was 10.14 g, with a yield of 95%, ESI-MS: 357.2([M + H]+).

3.1.5. Synthesis of N-(3-methoxy-4-((3-methyl-4-(2,2,2-triﬂuoroethoxy)pyridin-2-yl) methoxy)benzyl)-1-methylpiperidin-4-amine (6) A mixture of 1-methylpiperidin-4-one (2.26 g, 20 mmol), (3-methoxy-4-((3-methyl- 4-(2,2,2-triﬂuoroethoxy)pyridin-2-yl)methoxy)phenyl)methanamine(7.12 g, 20 mmol) 5, AcOH and MeOH was stirred for 1h at room temperature. NaBH3CN was added in 3 times, during which a lot of gas was generated and stirring was continued for 3 h. The progress of the reaction was monitored by TLC. After MeOH was distilled off under reduced pressure, a paste mixture was obtained. An aqueous solution of NaOH (2 mol/L) was added to the mixture and stirred until the paste dissolved. The solution was extracted by EA, and the organic phase was washed by water and brine, then dried by Na2SO4. After the solvent was removed under reduced pressure, a yellowish oil of 7.02 g was obtained, with a yield of 77%, 454.2([M + H]+).

3.1.6. General Procedure for the Synthesis of the Target Urea Derivatives Triphosgene (0.20 g, 0.67 mmol) was dissolved in 10 mL DCM, a solution of substituted aniline or benzylamine (2 mmol) in 10 mL DCM was slowly dropped in during stirring. There were solids that gradually precipitated out. Then a solution of TEA (0.4 g, 4 mmol) in DCM (10 mL) was slowly dropped into the mixture, the solids gradually dissolved, and the solution of substituted isocyanate 7 was obtained. The solution of 5 or 6 (2 mmol) in 10 mL DCM was added. After the reaction was completed, the mixture was washed by water and brine and dried by Na2SO4. After DCM was distilled off under reduced pressure, the mixture was purified by silica gel chromatography (DCM:EA = 5:1, v/v) to afford 8a–8i and silica gel chromatography (DCM:MeOH = 20:1, v/v) to afford 9a–9g. 1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-3-(4-(trifluorom ethoxy)phenyl)urea (8a), white powder 0.99 g, yield 89%; m.p.: 158.8–159.3 ◦C; MS: 560.4([M + 1 + H] ); H NMR (400 MHz, DMSO-d6) δ 8.73 (s, 1H), 8.34 (d, J = 5.7 Hz, 1H), 7.54–7.47 (m, 2H), 7.23 (d, J = 8.6 Hz, 2H), 7.14 (d, J = 5.7 Hz, 1H), 7.05 (d, J = 8.2 Hz, 1H), 6.94 (d, J = 2.0 Hz, 1H), 6.81 (dd, J = 8.2, 2.0 Hz, 1H), 6.59 (t, J = 5.9 Hz, 1H), 5.14 (s, 2H), 4.92 (q, J = 8.8 Hz, 2H), 4.23 (d, J = 5.7 Hz, 2H), 3.74 (s, 3H), 2.22 (s, 3H).; 13C NMR (101 MHz, DMSO-d6) δ 161.82, 155.94, 155.51, 149.63, 148.02, 147.18, 142.55, 140.26, 133.67, 124.25 (q, J = 277.9 Hz), 122.08, 121.99, 120.69 (q, J = 254.9 Hz), 119.69, 119.19, 114.27, 112.07, 108.02, 71.56, 65.11 (q, J = 34.7 Hz), 55.95, 43.10, 10.30; IR: 3399, 3053, 3008, 2971, 2829, 1706, 1556, 1507, 1454, 1416, 1256, 1220, 1194, 1154, 1015, 911, 847, 796, 672, 645, 544; HRMS: − + 558.146914([M − H] ) for C25H22F6N3O5, 560.161467([M + H] ) for C25H24F6N3O5. 1-(3-chloro-4-fluorophenyl)-3-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy) pyridin-2-yl)meth oxy)benzyl)urea (8b), white powder 0.86 g, yield 82%; m.p.: 167.8–169.1 ◦C; MS:528.3([M + H]+); Molecules 2021, 26, 3496 9 of 14

1 H NMR (400 MHz, DMSO-d6) δ 8.72 (s, 1H), 8.34 (d, J = 5.7 Hz, 1H), 7.77 (dd, J = 6.8, 2.4 Hz, 1H), 7.32–7.18 (m, 2H), 7.14 (d, J = 5.7 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.93 (d, J = 2.0 Hz, 1H), 6.80 (dd, J = 8.2, 1.9 Hz, 1H), 6.63 (t, J = 5.8 Hz, 1H), 5.13 (s, 2H), 4.91 (q, J = 8.7 Hz, 2H), 4.22 (d, J = 5.7 Hz, 2H), 3.74 (s, 3H), 2.22 (s, 3H).; 13C NMR (101 MHz, DMSO-d6) δ 161.81, 155.94, 155.44, 152.33 (d, J = 240.0 Hz), 149.59, 148.04, 147.15, 138.26 (d, J = 2.8 Hz), 124.27 (q, J = 277.4 Hz), 122.05, 119.69, 119.44 (d, J = 18.9 Hz), 119.31, 118.23 (d, J = 6.5 Hz), 117.13 (d, J = 21.3 Hz), 114.26, 112.08, 108.06, 71.55, 65.08 (q, J = 34.2 Hz), 56.00, 43.09, 10.35; IR: 3313, 2944, 2883, 1641, 1564, 1500, 1477, 1420, 1390, 1308, 1258, 1209, 1164, 1131, 1008, 970, 911, 862, 800, 757, 647, 576, 445; HRMS: 526.116220([M − H]−) for + C24H21ClF4N3O4, 528.130773([M + H] ) for C24H23ClF4N3O4. 1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-3-(4-(trifluorom ethyl)phenyl)urea (8c), white powder 0.78 g, yield 72%; m.p: 169.9–171.0 ◦C; MS:544.5([M + + + 1 H] ), 566.1([M + Na] ); H NMR (400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.34 (d, J = 5.7 Hz, 1H), 7.59 (q, J = 8.8 Hz, 4H), 7.14 (d, J = 5.7 Hz, 1H), 7.05 (d, J = 8.2 Hz, 1H), 6.95 (d, J = 2.0 Hz, 1H), 6.82 (dd, J = 8.2, 1.9 Hz, 1H), 6.69 (t, J = 5.8 Hz, 1H), 5.14 (s, 2H), 4.91 (q, J = 8.7 Hz, 2H), 13 4.24 (d, J = 5.7 Hz, 2H), 3.74 (s, 3H), 2.22 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 161.81, 155.93, 155.25, 149.61, 148.03, 147.19, 144.64, 133.47, 126.37, 125.09 (q, J = 270.6 Hz), 124.26 (q, J = 277.5 Hz), 122.06, 119.73, 117.72, 114.26, 112.10, 108.05, 71.55, 65.09 (q, J = 35.0 Hz), 55.98, 43.10, 10.31; IR: 3414, 3376, 2940, 2886, 1703, 1686, 1581, 1534, 1512, 1477, 1408, 1321, 1256, 1220, 1180, 1155, 1135, 1102, 1063, 1008, 979, 862, 842, 812, 595, 554;HRMS: 542.151999 − + + ([M − H] ) for C25H22F6N3O4, 544.166552([M + H] ) for C25H24F6N3O4. 1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-3-(3-(trifluorom ethyl)phenyl)urea (8d), white powder 0.83 g, yield 76%; m.p.: 153.2–154.2 ◦C; MS:544.2([M + + − 1 H] ), 542.0([M − H] ); H NMR (400 MHz, DMSO-d6) δ 8.90 (s, 1H), 8.34 (d, J = 5.7 Hz, 1H), 7.98 (s, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.45 (t, J = 7.9 Hz, 1H), 7.23 (d, J = 7.6 Hz, 1H), 7.14 (d, J = 5.7 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.94 (d, J = 1.9 Hz, 1H), 6.81 (dd, J = 8.2, 1.9 Hz, 1H), 6.68 (t, J = 5.9 Hz, 1H), 5.13 (s, 2H), 4.91 (q, J = 8.7 Hz, 2H), 4.23 (d, J = 5.8 Hz, 2H), 3.74 13 (s, 3H), 2.22 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 161.82, 155.94, 155.46, 149.62, 148.02, 147.18, 141.80, 133.59, 130.11, 124.73 (q, J = 272.3 Hz), 124.25 (q, J = 277.4 Hz), 122.07, 121.62, 119.69, 117.68, 114.28, 114.06, 112.08, 108.02 (d, J = 4.4 Hz), 71.56, 65.10 (q, J = 34.2 Hz), 55.97 (d, J = 3.2 Hz), 43.08, 10.33; IR: 3412, 3374, 2940, 2876, 1702, 1582, 1551, 1514, 1477, 1442, 1380, 1341, 1312, 1255, 1221, 1182, 1158, 1111, 1067, 1028, 1007, 979, 891, 864, 813, 796, 702, − + + 664, 597, 552; HRMS: 542.151999([M − H] ) for C25H22F6N3O4 , 544.161552 ([M + H] ) for C25H24F6N3O4. 1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-3-(4-methoxyph enyl)urea (8e), white powder 0.59 g, yield 58%; m.p.: 179.5–180.3 ◦C; MS:566.2 ([M + H]+), − 1 504.0([M − H] ); H NMR (400 MHz, DMSO-d6) δ 8.28 (s, 1H), 7.29 (d, J = 8.9 Hz, 2H), 7.14 (d, J = 5.7 Hz, 1H), 6.93 (d, J = 1.9 Hz, 1H), 6.81 (dd, J = 9.0, 7.1 Hz, 3H), 6.40 (t, J = 5.9 Hz, 1H), 5.13 (s, 2H), 4.92 (q, J = 8.8 Hz, 2H), 4.20 (d, J = 5.8 Hz, 2H), 3.70 (s, 3H), 2.22 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 161.80, 155.96, 155.86, 154.40, 149.57, 148.05, 147.08, 134.08, 133.97, 124.28 (d, J = 277.9 Hz), 122.05, 119.91, 119.64, 114.33, 114.27, 112.06, 108.07, 71.58, 65.08 (q, J = 34.5 Hz), 55.99, 55.59, 43.09, 10.36; IR: 3312, 2940, 2839, 1631, 1571, 1508, 1467, 1417, 1376, 1363, 1308, 1271, 1241, 1160, 1136, 1030, 973, 862, 827, 669, 578, 524, 423; HRMS: − + 504.175179 ([M − H] ) for C25H25F3N3O5, 506.189732 ([M + H] ) for C25H27F3N3O5. 1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-3-(3-nitropheny l)urea (8f), yellow powder 0.31 g, yield 30%; m.p.: 168.9–170.5 ◦C; MS: 521.2 ([M + H]+), − 1 519.0([M − H] ); H NMR (400 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.52 (t, J = 2.2 Hz, 1H), 8.33 (d, J = 5.8 Hz, 1H), 7.75 (d, J = 8.3 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.50 (dd, J = 9.1, 7.3 Hz, 1H), 7.13 (d, J = 5.9 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.96–6.92 (m, 1H), 6.85–6.71 (m, 2H), 5.13 (s, 2H), 4.91 (q, J = 8.8 Hz, 2H), 4.24 (d, J = 5.9 Hz, 2H), 3.74 (s, 3H), 2.21 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 161.83, 155.93, 155.35, 149.63, 148.59, 148.02, 147.20, 142.28, 133.51, 130.31, 124.26 (q, J = 277.8 Hz), 124.16, 122.07, 119.74, 115.95, 114.32, 112.14, 112.06, 108.05, 71.56, 65.11 (q, J = 34.4 Hz), 56.02, 43.11, 10.34; IR: 3410, 3010, 2943, 2882, 2832, 1701, Molecules 2021, 26, 3496 10 of 14

1584, 1527, 1503, 1480, 1383, 1347, 1318, 1264, 1207, 1161, 1138, 1122, 1033, 1000, 980, 868, − 814, 794, 735, 671, 612, 585, 557, 445; HRMS: 519.149693 ([M − H] ) for C24H22F3N4O6, + 521.164246([M + H] ) for C24H24F3N4O6. 1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-3-(4-meth oxy- benzyl)urea (8g), white powder 0.39 g, yield 38%; m.p.: 153.0–154.2 ◦C; MS: 520.2([M + H]+), − 1 517.9([M − H] ); H NMR (400 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.52 (t, J = 2.2 Hz, 1H), 8.33 (d, J = 5.8 Hz, 1H), 7.75 (d, J = 8.3 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.50 (dd, J = 9.1, 7.3 Hz, 1H), 7.13 (d, J= 5.9 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.96–6.92 (m, 1H), 6.85–6.71 (m, 2H), 5.13 (s, 2H), 4.91 (q, J = 8.8 Hz, 2H), 4.24 (d, J = 5.9 Hz, 2H), 3.74 (s, 3H), 2.21 (s, 13 3H); C NMR (101 MHz, DMSO-d6) δ 161.82, 158.54, 158.46, 155.99, 149.57, 148.04, 146.99, 134.47, 133.30, 124.28 (q, J = 277.7 Hz), 122.06, 119.46, 114.26, 114.07, 111.86, 108.08, 71.61, 65.10 (q, J = 34.9 Hz), 55.92, 55.51, 43.25, 42.90, 10.36; IR: 3349, 3301, 2949, 2925, 2884, 2832, 1605, 1579, 1561, 1515, 1468, 1424, 1363, 1363, 1363, 1275, 1251, 1169, 1156, 1133, 1103, 1038, − 975, 863, 816, 728, 637, 561; HRMS: 518.190829 ([M − H] ) for C26H27F3N3O5, 520.205382 + ([M + H] ) for C26H29F3N3O5. 1-(4-ethoxybenzyl)-3-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy) benzyl)urea (8h), white powder 0.57 g, yield 53%; m.p.: 148.9–149.9 ◦C; MS: 534.3 ([M + H]+); 1H NMR (400 MHz, DMSO-d6) δ 8.34 (d, J = 5.7 Hz, 1H), 7.20–7.11 (m, 3H), 7.02 (d, J = 8.2 Hz, 1H), 6.90–6.81 (m, 3H), 6.75 (dd, J = 8.3, 1.9 Hz, 1H), 6.30 (td, J = 6.1, 2.3 Hz, 2H), 5.13 (s, 2H), 4.92 (q, J = 8.8 Hz, 2H), 4.15 (d, J = 5.8 Hz, 4H), 3.99 (q, J = 7.0 Hz, 2H), 3.71 (s, 13 3H), 2.22 (s, 3H), 1.31 (t, J = 7.0 Hz, 3H); C NMR (101 MHz, DMSO-d6) δ 161.82, 158.48, 157.77, 155.96, 149.54, 148.02, 146.96, 134.46, 133.16, 128.76, 124.28 (q, J= 277.7, 277.3 Hz), 122.06, 119.44, 114.56, 114.21, 111.81, 108.08, 71.57, 65.08 (q, J = 34.5 Hz), 63.39, 55.89, 43.24, 42.89, 15.11, 10.36; IR: 3331, 2978, 2927, 2883, 1609, 1579, 1556, 1516, 1471, 1423, 1390, 1275, 1249, 1148, 1025, 974, 861, 815, 753, 728, 639, 574, 548; HRMS: 532.206479 ([M − H]−) for + C27H29F3N3O5, 534.221032([M + H] ) for C27H31F3N3O5. 1-(4-(dimethylamino)benzyl)-3-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2yl)meth oxy)benzyl)urea (8i), white powder 0.73 g, yield 69%; m.p.: 159.4–161.4 ◦C; MS:533.9 ([M + + 1 + H] ), 555.4([M + Na] ); H NMR (400 MHz, DMSO-d6) δ 8.34 (d, J = 5.6 Hz, 1H), 7.14 (d, J = 5.7 Hz, 1H), 7.11–7.06 (m, 2H), 7.02 (d, J = 8.2 Hz, 1H), 6.87 (d, J = 2.0 Hz, 1H), 6.74 (dd, J = 8.2, 1.9 Hz, 1H), 6.69–6.65 (m, 2H), 6.22 (dt, J = 21.9, 5.9 Hz, 2H), 5.12 (s, 2H), 4.92 (q, J = 8.7 Hz, 2H), 4.15 (d, J = 5.9 Hz, 2H), 4.10 (d, J = 5.8 Hz, 2H), 3.71 (s, 3H), 2.85 (s, 13 6H), 2.22 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 161.81, 158.46, 155.98, 149.97, 149.55, 148.04, 146.96, 134.50, 128.77, 128.49, 124.28 (q, J = 277.7 Hz), 122.05, 119.44, 114.22, 112.85, 111.82, 108.08, 71.59, 65.08 (q, J = 34.3 Hz), 55.92, 43.23, 43.06, 10.36; IR: 3336, 2940, 2918, 2879, 1613, 1570, 1517, 1468, 1421, 1308, 1256, 1233, 1175, 1137, 1039, 1011, 969, 922, 854, 810, − + 736, 650, 564; HRMS: 531.222464 ([M − H] ) for C27H30F3N4O4, 522.237017 ([M + H] ) for C27H32F3N4O4. 1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-1-(1-methylpip eridin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea (9a), white powder 0.79 g, yield 60%; m.p.: ◦ + 127.3–129.4 C; MS:657.2([M + H] ); 1H NMR (400 MHz, DMSO-d6) δ 8.52 (s, 1H), 8.33 (d, J = 5.6 Hz, 1H), 7.58–7.51 (m, 2H), 7.23 (d, J = 8.6 Hz, 2H), 7.14 (d, J = 5.7 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 2.0 Hz, 1H), 6.75 (dd, J = 8.2, 1.9 Hz, 1H), 5.11 (s, 2H), 4.91 (q, J = 8.7 Hz, 2H), 4.52 (s, 2H), 4.10 (tt, J = 12.0, 4.0 Hz, 1H), 3.70 (s, 3H), 2.83 (d, J = 11.6 Hz, 2H), 2.20 (d, J = 5.4 Hz, 6H), 2.06 (d, J = 10.0 Hz, 2H), 1.70 (tt, J = 12.4, 6.7 Hz, 13 2H), 1.55 (d, J = 14.4 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 161.81, 155.73, 149.48, 148.05, 146.93, 143.15, 140.30, 133.64, 124.26(q, J =276.1Hz), 121.50, 118.77, 120.66 (q, J = 253.5 Hz), 111.28, 108.07, 71.55, 65.11 (q, J = 34.8, 34.0 Hz), 55.95, 55.08, 53.09, 45.99, 45.70, 45.16, 39.69, 29.90, 10.36; IR: 3397, 2950, 2848, 2799, 1648, 1584, 1513, 1470, 1416, 1377, 1293, 1256, 1227, 1204, 1159, 1132, 1031, 982, 921, 847, 825, 800, 754, 536; HRMS: 655.236063 ([M − H]−) for + C31H33F6N4O5, 657.250616 ([M + H] ) for C31H35F6N4O5. 1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-1-(1-methylpipe ridin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea (9b), white powder 0.73 g, yield 57%; m.p.: 138.4– Molecules 2021, 26, 3496 11 of 14

140.5 ◦C; MS: 641.1([M + H]+), 321.5 ([M + 2H]2+), 639.5([M − H]−); 1H NMR (400 MHz, DMSO-d6) δ 8.73 (s, 1H), 8.33 (d, J = 5.6 Hz, 1H), 7.67 (d, J = 8.6 Hz, 2H), 7.58 (d, J = 8.7 Hz, 2H), 7.14 (d, J = 5.7 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 6.89 (d, J = 2.0 Hz, 1H), 6.75 (dd, J = 8.3, 2.0 Hz, 1H), 5.10 (s, 2H), 4.91 (q, J = 8.7 Hz, 2H), 4.54 (s, 2H), 4.16–4.07 (m, 1H), 3.70 (s, 3H), 2.84 (d, J = 10.9 Hz, 2H), 2.19 (s, 6H), 2.06 (t, J = 10.0 Hz, 2H), 1.72 (q, J = 12.6, 11.8 Hz, 13 2H), 1.57 (d, J = 11.4 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 161.81, 155.92, 155.50, 149.49, 148.05, 146.97, 144.86, 133.47, 125.91 (q, J = 3.9 Hz), 125.08 (q, J = 270.9 Hz), 124.26 (q, J = 277.6, 277.1 Hz), 122.16 (q, J = 31.8 Hz), 122.08, 118.76, 114.21, 111.26, 108.06, 71.54, 65.11 (q, J = 34.4 Hz), 55.94, 55.06, 53.24, 45.70, 45.25, 29.88, 10.34; IR: 3387, 2943, 2846, 2802, 1650, 1584, 1513, 1468, 1416, 1378, 1313, 1250, 1224, 1162, 1131, 1063, 1030, 1016, 981, 862, − + 843, 811, 753, 577; HRMS: 639.241148 ([M − H] ) for C31H33F6N4O4, 641.255701([M + H] ) for C31H35F6N4O4. 1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-3-(4-methoxyph enyl)-1-(1-methylpiperidin-4-yl)urea (9c), white powder 0.71 g, yield 59%; m.p.: 179.1–180.8; + 1 MS:603.2([M + H] ); H NMR (400 MHz, DMSO-d6) δ 8.33 (d, J = 5.7 Hz, 1H), 8.12 (s, 1H), 7.33–7.26 (m, 2H), 7.14 (d, J = 5.7 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 2.0 Hz, 1H), 6.84–6.74 (m, 3H), 5.11 (s, 2H), 4.91 (q, J = 8.7 Hz, 2H), 4.49 (s, 2H), 4.08 (tt, J = 11.3, 3.9 Hz, 1H), 3.71 (d, J = 1.9 Hz, 6H), 2.80 (d, J = 11.0 Hz, 2H), 2.21 (s, 3H), 2.17 (s, 3H), 2.06–1.95 (m, 13 2H), 1.67 (qd, J = 12.1, 3.9 Hz, 2H), 1.54 (d, J = 12.0 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 161.81, 156.08, 155.96, 155.07, 149.46, 148.06, 146.87, 133.99, 133.88, 124.27 (q, J = 278.0 Hz), 122.70, 122.06, 118.80, 114.19, 113.89, 111.29, 108.08, 71.57, 65.10 (q, J = 33.8 Hz), 55.96, 55.59, 55.28, 52.98, 46.03, 45.03, 30.19, 10.38; IR: 3403, 2943, 2836, 2788, 2759, 1642, 1583, 1511, 1467, 1446, 1416, 1373, 1295, 1250, 1220, 1159, 1128, 1034, 1011, 962, 860, 824, 737, 666, 576, − + 542, 441; HRMS: 637.241006 ([M + Cl] ) for C31H37ClF3N4O5, 603.278881 ([M + H] ) for C31H38F3N4O5. 1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methoxy)benzyl)-1-(1-methylpip eridin-4-yl)-3-(3-(trifluoromethyl)phenyl)urea (9d), white powder 0.75 g, yield 59%; m.p.: ◦ + 1 160.4–162.2 C; MS: 641.2([M + H] ); H NMR (400 MHz, DMSO-d6) δ 8.69 (s, 1H), 8.33 (d, J = 5.7 Hz, 1H), 7.91 (d, J = 2.0 Hz, 1H), 7.74 (dd, J = 8.2, 2.0 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.27 (d, J = 7.8 Hz, 1H), 7.14 (d, J = 5.7 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 2.0 Hz, 1H), 6.76 (dd, J = 8.3, 2.0 Hz, 1H), 5.11 (s, 2H), 4.91 (q, J = 8.7 Hz, 2H), 4.54 (s, 2H), 4.11 (tt, J = 12.1, 3.9 Hz, 1H), 3.70 (s, 3H), 2.84 (d, J = 11.2 Hz, 2H), 2.20 (s, 6H), 2.05 (d, J = 13.2 Hz, 2H), 1.72 (tt, J = 12.4, 6.8 Hz, 2H), 1.57 (d, J = 11.8 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 161.81, 155.93, 155.63, 149.48, 148.06, 146.94, 141.89, 133.50, 129.78, 129.51 (q, J = 32.3, 31.7 Hz), 124.76 (d, J = 272.5 Hz), 124.26 (q, J = 278.9, 278.5 Hz), 123.84, 118.75, 118.39 (d, J = 3.9 Hz), 116.39 (d, J = 4.4 Hz), 114.22, 111.25, 108.08, 71.54, 65.10 (q, J = 34.6 Hz), 55.94, 55.05, 53.16, 46.04, 45.68, 45.21, 29.82, 10.35; IR: 3385, 2940, 2839, 2791, 2771, 2735, 1645, 1583, 1513, 1493, 1468, 1444, 1376, 1326, 1247, 1222, 1152, 1122, 1030, 1000, − 972, 909, 835, 788, 749, 701, 667, 540, 458; HRMS: 639.241148 ([M − H] ) for C31H33F6N4O4, + 641.255701 ([M + H] ) for C31H35F6N4O4. 3-(3-chloro-4-fluorophenyl)-1-(3-methoxy-4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)meth oxy)benzyl)-1-(1-methylpiperidin-4-yl)urea (9e), white powder 0.69 g, yield 55%; m.p.: 176.2– ◦ + 1 177.7 C; MS:625.2([M + H] ); H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 1H), 8.33 (d, J = 5.6 Hz, 1H), 7.74 (dd, J = 6.9, 2.6 Hz, 1H), 7.41 (ddd, J = 9.2, 4.4, 2.6 Hz, 1H), 7.27 (t, J = 9.1 Hz, 1H), 7.14 (d, J = 5.7 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.89 (d, J = 2.0 Hz, 1H), 6.75 (dd, J = 8.2, 1.9 Hz, 1H), 5.11 (s, 2H), 4.91 (q, J = 8.7 Hz, 2H), 4.51 (s, 2H), 4.08 (tt, J = 12.1, 3.9 Hz, 1H), 3.71 (s, 3H), 2.83 (d, J = 11.2 Hz, 2H), 2.20 (d, J = 2.9 Hz, 6H), 2.05 (d, J = 11.7 Hz, 2H), 1.71 (qd, J = 12.3, 3.7 Hz, 2H), 1.59–1.51 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 161.81, 155.93, 155.62, 152.83 (d, J = 240.9 Hz), 149.46, 148.06, 146.93, 138.30, 133.52, 124.27 (q, J = 277.7 Hz), 122.06, 121.79, 120.59 (d, J = 6.5 Hz), 119.01 (d, J = 18.2 Hz), 118.75, 116.71 (d, J = 21.4 Hz), 114.20, 111.26, 108.08, 71.54, 65.10 (q, J = 34.6 Hz), 55.97, 55.09, 53.16, 45.77, 45.19, 29.89, 10.37; IR: 3403, 3365, 2940, 2881, 2822, 1702, 1598, 1515, 1448, Molecules 2021, 26, 3496 12 of 14

1407, 1370, 1319, 1260, 1217, 1151, 1107, 1063, 965, 906, 842, 785, 713, 593, 509, 455; HRMS: − + 623.205307 ([M − H] ) for C30H33ClF4N4O4, 625.219923 ([M + H] ) for C30H35ClF4N4O4. 1-(3-methoxy-4-((3-methyl-4-(2,2,2-triﬂuoroethoxy)pyridin-2-yl)methoxy)benzyl)-1-(1-methylpipe ridin-4-yl)-3-(3-nitrophenyl)urea (9f), yellow powder 0.79 g, yield 64%; m.p.: 157.5–159.1 ◦ + 1 C; MS:618.2([M + H] ); H NMR (400 MHz, DMSO-d6) δ 8.86 (s, 1H), 8.47 (t, J = 2.3 Hz, 1H), 8.33 (d, J = 5.7 Hz, 1H), 7.92 (dd, J = 8.2, 2.0 Hz, 1H), 7.79 (dd, J = 8.1, 2.2 Hz, 1H), 7.51 (t, J = 8.2 Hz, 1H), 7.13 (d, J = 5.7 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.91 (d, J = 2.0 Hz, 1H), 6.76 (dd, J = 8.3, 2.0 Hz, 1H), 5.10 (s, 2H), 4.91 (q, J = 8.7 Hz, 2H), 4.55 (s, 2H), 4.10 (tt, J = 11.9, 4.0 Hz, 1H), 3.71 (s, 3H), 2.80 (d, J = 11.0 Hz, 2H), 2.20 (s, 3H), 2.16 (s, 3H), 2.00 (t, J = 11.3 Hz, 2H), 1.71 (qd, J = 12.1, 3.8 Hz, 2H), 1.61–1.52 (m, 2H); 13C NMR (101 MHz, DMSO-d6) δ 161.80, 155.93, 155.52, 149.48, 148.28, 148.06, 146.95, 142.46, 133.45, 129.97, 126.24, 124.27 (q, J = 277.6 Hz), 122.06, 118.74, 116.58, 114.26, 114.21, 111.25, 108.07, 71.54, 65.10 (q, J = 34.4 Hz), 55.97, 55.21, 53.48, 46.01, 45.24, 30.08, 10.37; IR: 3366, 2940, 2842, 2781, 1657, 1585, 1512, 1467, 1426, 1376, 1343, 1248, 1222, 1161, 1131, 1032, 1011, 967, 861, 824, 737, − + 667, 584, 454; HRMS: 616.238842 ([M − H] ) for C30H33F3N5O6, 618.253395 ([M + H] ) for C30H35F3N5O6. 3-(4-ethoxybenzyl)-1-(3-methoxy-4-((3-methyl-4-(2,2,2-triﬂuoroethoxy)pyridin-2-yl)methoxy)ben zyl)-1-(1-methylpiperidin-4-yl)urea (9g), white powder 0.41 g, yield 33%; m.p.: 119.0–121.2 ◦C; + 1 MS 631.3([M + H] ); H NMR (400 MHz, DMSO-d6) δ 8.34 (d, J = 5.7 Hz, 1H), 7.13 (dd, J = 15.2, 7.1 Hz, 3H), 7.01 (d, J = 8.3 Hz, 1H), 6.84–6.75 (m, 4H), 6.72 (dd, J = 8.3, 2.0 Hz, 1H), 5.11 (s, 2H), 4.92 (q, J = 8.7 Hz, 2H), 4.36 (s, 2H), 4.18 (d, J = 5.6 Hz, 2H), 3.98 (q, J = 6.9 Hz, 3H), 3.63 (s, 3H), 2.79 (d, J = 11.0 Hz, 2H), 2.21 (s, 3H), 2.17 (s, 3H), 2.00 (t, J = 11.8 Hz, 2H), 1.61 (qd, J = 12.2, 3.8 Hz, 2H), 1.48 (d, J = 9.9 Hz, 2H), 1.32 (d, J = 7.0 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 161.82, 157.98, 157.62, 155.99, 149.46, 148.06, 146.82, 134.24, 133.60, 128.63, 124.28 (q, J = 277.9 Hz), 122.08, 118.80, 114.39, 114.08, 111.14, 108.09, 71.61, 65.12 (q, J = 34.4 Hz), 63.37, 55.79, 55.18, 52.47, 45.79, 44.88, 43.52, 29.99, 15.12, 10.38; IR: 3348, 2937, 2881, 2837, 2793, 1612, 1584, 1509, 1477, 1447, 1395, 1374, 1292, 1253, 1162, 1131, 1033, 1003, − 971, 916, 804, 772, 577; HRMS: 665.272306 ([M + Cl] ) for C33H41ClF3N4O5, 631.310182 + ([M + H] ) for C33H42F3N4O5.

3.2. Biological Evaluation 3.2.1. Antiproliferative Activity Assays The antiproliferative activities of target compounds were determined using a standard MTT assay [24–27]. Exponentially growing cells A549 (1.5 × 103 cells/well), MCF-7 (2.2 × 103 cells/well), HCT-116 (800 cells/well), PC-3 (2.0 × 103 cells/well) and HL7702 (5.0 × 103 cells/well) were seeded into 96-well plates and incubated for 24 h to allow the cells to attach. Then, a fresh medium containing various concentrations of the candidate compounds was added to each well. The cells were then incubated for 96 h, thereafter MTT assays were performed, and cell viability was assessed at 570 nm by a microplate reader (ThermoFisher Scientiﬁc (Shanghai) Instrument Co., Ltd., Shanghai, China). The optical densities (OD) at 570 nm were measured, and the IC50 of the target compounds was calculated by using GraphPad Prism 5.0 software to perform nonlinear ﬁtting with the cell survival rate under different concentrations of the compounds.

3.2.2. Cell Cycle Analysis As for the flow cytometric analysis of DNA content, 1 × 105 MCF-7 cells in exponential growth were treated with different concentrations of compound 9b for 24 h. After an incubation period, the cells were collected, centrifuged, and fixed with ice-cold ethanol (70%). The cells were then treated with buffer containing RNAse A and 0.1% Triton X-100 and then stained with the propidium iodide (PI). The samples were analyzed on a flow cytometer (Becton, Dickinson, Franklin Lakes, NJ, USA) [28]. Data were analyzed using Flowjo software v9.0. Molecules 2021, 26, 3496 13 of 14

4. Conclusions In summary, based on the structure of sorafenib and lansoprazole, 16 target N-aryl-N’- arylmethylurea derivatives were designed with molecular hybridization and synthesized, and their antiproliferative activities were assayed. The target compounds 9b and 9d have shown excellent antiproliferative activities against all four kinds of tumor cell lines (non-small cell lung cancer A549, breast cancer MCF-7, colon cancer HCT116, prostate cancer PC-3). All target compounds have demonstrated weak cytotoxic activities against human liver normal cell line HL7702. The biological assay results showed these target compounds with the 1-methylpiperidin-4-yl group on the 3-position of urea in the target compounds and substituents containing ﬂuorine atoms on the phenyl ring exhibit potently antiproliferative activities. The cell cycle evaluation has shown that compound 9b could cause an obvious G2/M arrest in a concentration-dependent manner.

Supplementary Materials: The following are available online, 1H-NMR, 13C-NMR, ESI-MS and HRMS of the target compounds (Figures S1–S80). Author Contributions: Proposal for the subject: C.H. and X.L.; synthetic work and the characterization of all target compounds: S.H., S.L., C.Z., Y.H., J.L., H.H. and X.Z.; biological assays: S.H., X.Z. and H.Z.; preparation of the manuscript: S.H. and C.Z.; review and correction for the manuscript: C.H., X.L. and H.Z. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the National Science Foundation of China (Grant No. 21342006), the Program for Innovative Research Team of the Ministry of Education of China (Grant No. IRT_14R36). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding authors. Conﬂicts of Interest: The authors declare no conﬂict of interest. Sample Availability: Samples of all target compounds are available from the authors.

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