Synthesis of Potent and Selective Inhibitors of Aldo ... - ACS Publications

Article Cite This: J. Med. Chem. 2017, 60, 8441-8455

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Synthesis of Potent and Selective Inhibitors of Aldo-Keto Reductase 1B10 and Their Efficacy against Proliferation, Metastasis, and Cisplatin Resistance of Lung Cancer Cells Satoshi Endo,*,† Shuang Xia,‡ Miho Suyama,† Yoshifumi Morikawa,† Hiroaki Oguri,† Dawei Hu,‡ Yoshinori Ao,§ Satoyuki Takahara,‡ Yoshikazu Horino,§ Yoshihiro Hayakawa,∥ Yurie Watanabe,⊥ Hiroaki Gouda,⊥ Akira Hara,# Kazuo Kuwata,▽ Naoki Toyooka,*,‡,§ Toshiyuki Matsunaga,† and Akira Ikari† †

Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan Graduate School of Innovative Life Science, University of Toyama, Toyama 930-8555, Japan § Graduate School of Science and Engineering, University of Toyama, Toyama 930-8555, Japan ∥ Division of Pathogenic Biochemistry, Institute of Natural Medicine, University of Toyama, Toyama 930-0194, Japan ⊥ School of Pharmacy, Showa University, Tokyo 142-8555, Japan # Faculty of Engineering, Gifu University, Gifu 501-1193, Japan ▽ United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu 501-1193, Japan ‡

S Supporting Information *

ABSTRACT: Aldo-keto reductase 1B10 (AKR1B10) is overexpressed in several extraintestinal cancers, particularly in nonsmall-cell lung cancer, where AKR1B10 is a potential diagnostic marker and therapeutic target. Selective AKR1B10 inhibitors are required because compounds should not inhibit the highly related aldose reductase that is involved in monosaccharide and prostaglandin metabolism. Currently, 7hydroxy-2-(4-methoxyphenylimino)-2H-chromene-3-carboxylic acid benzylamide (HMPC) is known to be the most potent competitive inhibitor of AKR1B10, but it is nonselective. In this study, derivatives of HMPC were synthesized by removing the 4-methoxyphenylimino moiety and replacing the benzylamide with phenylpropylamide. Among them, 4c and 4e showed higher AKR1B10 inhibitory potency (IC50 4.2 and 3.5 nM, respectively) and selectivity than HMPC. The treatments with the two compounds significantly suppressed not only migration, proliferation, and metastasis of lung cancer A549 cells but also metastatic and invasive potentials of cisplatin-resistant A549 cells.

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INTRODUCTION Lung carcinoma is a leading cause of cancer deaths in Japan and worldwide for both men and women,1 of which non-small-cell lung carcinoma (NSCLC) accounts for approximately 85% of lung cancer cases. Most patients with NSCLC are diagnosed at advanced stages with an overall five-year survival rate of only 15%.2 Currently, several NSCLC markers, such as epithelial growth factor receptor (EGFR),3 anaplastic lymphoma kinase (ALK),4 mesenchymal−epithelial transition factor (MET)5 and KRAS,6 were identified. Molecular targeting therapies for NSCLC patients against gene mutations occurring in the genes encoding components of EGFR and the downstream mitogen-activated protein kinase and phosphatidylinositol 3kinase signaling pathways have been developed, but the overall prognosis remains poor.7 Fukumoto et al.8 identified a member of the aldo-keto reductase (AKR) superfamily, AKR1B10, as an overexpressing protein in NSCLC, in particular smokers’ NSCLC, and © 2017 American Chemical Society

suggested that the enzyme is a potential diagnostic marker. Subsequently, many researchers also verified AKR1B10 as a lung cancer biomarker.9−13 In addition, the enzyme has been reported to be overexpressed in hepatocarcinoma,14,15 breast cancer,16 pancreatic carcinoma,17 and oral squamous cell carcinomas.18 AKR1B10 is a monomeric cytosolic NADPHdependent reductase that catalyzes the reduction of various carbonyl compounds including reactive aldehydes, retinoids, and isoprenoids.14,19 Therefore, the enzyme has been thought to be involved in the development, progression, and survival of carcinomas through multiple mechanisms including detoxification of cytotoxic reactive carbonyls,14,20−23 modulation of retinoic acid level,24,25 regulation of cellular fatty acid synthesis and lipid metabolism,21,26,27 elevation of sphingosine-1phosphate,28 and integrin α5/δ-catenin mediated FAK/Src/ Received: June 12, 2017 Published: October 4, 2017 8441

DOI: 10.1021/acs.jmedchem.7b00830 J. Med. Chem. 2017, 60, 8441−8455

Journal of Medicinal Chemistry

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Figure 1. Chemical structures of representative AKR1B10 inhibitors and their inhibitory potency toward AKR1B10 (1B10) and its structurally similar enzyme AKR1B1 (1B1). Abbreviations: MHPC, (Z)-2-(4-methoxyphenylimino)-7-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-carboxamide; and BDMC, bisdemethoxycurcumin.

Rac1 signaling pathways.29 Furthermore, AKR1B10 is suggested to be implicated in acquiring cancer cell resistance to anticancer drugs such as mitomycin C, cisplatin (CDDP), anthracyclines (doxorubicin and idarubicin), and docetaxel.30−35 Thus, this enzyme has been recognized not only as a potential diagnostic or prognostic marker but also as a novel therapeutic target for the treatment of the above extraintestinal cancers and for chemoresistance. Due to its recognition as a therapeutic target, many inhibitors of AKR1B10 have been discovered in natural compounds and existing drugs, and synthesized until 2015 as described in recent reviews.33,36 Subsequently, two AKR1B10 inhibitors, polyhydroxysteroid derivatives 37 and 2-[5-chloro-2-[(2,3,4,5,6pentabromophenyl)methylcarbamoyl]phenoxy]acetic acid (MK204),38 have been reported. Figure 1 shows the

representative potent AKR1B10 inhibitors, which are divided into two groups, nonselective and selective ones, with respect to the inhibitory selectivity to AKR1B10 over human aldose reductase (AKR1B1), which is similar to AKR1B10 in both primary and tertiary structures.14,24 AKR1B1 and AKR1B10 show overlapped substrate specificity14,19 but differ in high prostaglandin F2α synthase39 and D-glucose reductase activities,19 which are exhibited only by AKR1B1. In addition, AKR1B1 has been shown to be involved in colon cancer growth,40 in contrast to a protective role of AKR1B10 in normal colon cells.41 Therefore, the inhibitory selectivity to AKR1B10 over AKR1B1 is ideally required for development of drugs in the treatment of the extraintestinal cancers and chemoresistance. Among the inhibitors shown in Figure 1, oleanolic acid42 is the most selective, then 3-(4-hydroxy-28442



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Figure 2. Design process of selective AKR1B10 inhibitor 4a from the lead compound HMCB.

Scheme 1. Synthesis of C1−C9

improve the poor selectivity of HMCB, its derivative (3a′) that replaced the benzylamide moiety with 3-hydroxyphenylpropylamide was designed and first synthesized (Figure 2). Condensation of 1a with cyanoacetic acid using EDC provided the amide 2a, which was converted to 3a via corresponding iminochromene. Deprotection of MOM group with acid afforded not desired 3a′ but 4a. To avoid the acid hydrolysis of the phenyliminochromene moiety, we conducted the deprotection of the MOM group first in the amide 2a to yield 2a′, which was converted to 3a′ as the same procedure for 3a. Unfortunately, the inhibitory effect of 3a′ was less potent and selective compared to HMCB, but very fortunately coumarin 4a showed good selectivity against AKR1B1 with acceptable inhibitory effect on AKR1B10. The coumarin 4a retained the inhibitory potency to AKR1B10 and showed low inhibition to AKR1B1, resulting in a 22-fold increase in the selectivity to AKR1B10 over AKR1B1. Thus, we aimed at developing the 4a-based derivatives (4b−l) as the new candidates for AKR1B10 inhibitors. The amides 2a−i were prepared by condensation with cyanoacetic acid with appropriate amines, which were prepared by hydrogenation of azides shown in Scheme 1, using EDC. The Knoevenagel condensation of 2a−i with 2-hydroxybenzaldehydes followed

methoxyphenyl)acrylic acid 3-(3-hydroxyphenyl) propyl ester (HAHE),43 and the most potent inhibitor is 7-hydroxy-2-(4methoxyphenylimino)-2H-chromene-3-carboxylic acid benzylamide (HMCB).44 In this study, we designed and synthesized novel analogues of HMCB by referring to the structure of the selective AKR1B10 inhibitor, HAHE,43 in order to develop more potent and selective AKR1B10 inhibitors. The analogues 4a−l were evaluated for inhibition of AKR1B10 and AKR1B1, and it was found that two (4c and 4e) of them are more potent and selective AKR1B10 inhibitors than HMCB. The structural reason for the increase in the inhibitory potency and selectivity was examined by docking simulation of 4c in AKR1B10. Additionally, 4c and 4e were evaluated in terms of inhibitory effects on migration, proliferation, and metastasis of lung cancer A549 cells, as well as ability to overcome resistance to CDDP.

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RESULTS AND DISCUSSION Design and Synthesis of Coumarin Derivatives. The HMCB structure except for its 4-methoxyphenylimino moiety is somewhat similar to the selective AKR1B10 inhibitor HAHE, but the benzylamide moiety of HMCB is shorter than the 3-(3hydroxyphenyl)propyl ester part of HAHE (Figure 1). To 8443



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Scheme 2. Synthesis of Coumarin Derivatives 4a−4l

Table 1. Inhibition of AKR1B10 and AKR1B1 by Coumarin Derivatives

IC50 (nM)

a

compd

n

R1

R2

R3

R4

4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l

3 3 3 3 3 3 3 3 4 3 3 2

3′-OH 2′-OH 4′-OH 3′-F 4′-F 3′,5′-diF 4′-Me 4′-OMe 4′-OH 4′-OH 4′-OH 4′-OH

H H H H H H H H H H OH H

OH OH OH OH OH OH OH OH OH H H OH

H H H H H H H H H H H H

AKR1B10

AKR1B1

selectivitya

± ± ± ± ± ± ± ± ± ± ± ±

180 ± 1 220 ± 19 204 ± 4 340 ± 42 277 ± 17 320 ± 49 200 ± 14 390 ± 40 314 ± 27 >10000b 9500 ± 750 98 ± 9.1

22 34 49 57 79 41 29 35 27 >4 6 5

8.3 6.4 4.2 6.0 3.5 7.8 7.0 11 11 2700 1500 20

1.4 0.4 0.2 1.4 0.1 0.3 0.6 0.9 0.2 400 70 1.0

Selectivity is expressed as a ratio of AKR1B1/AKR1B10. bInhibition percentage was 20% at 10000 nM.

by treatment of the resulting iminochromenes with pmethoxyaniline gave rise to phenyliminochromenes 3a−k. Acid-catalyzed hydrolysis of 3a−k provided the coumarins 4a−l (Scheme 2). Among the derivatives (4a−4c) hydroxylated at different positions on the phenyl ring, 4c with the 4′-hydroxy group was the most potent inhibitor and was approximately 10-

fold more selective toward AKR1B10 than HMCB (Table 1). Among the derivatives with fluorinated phenyl rings (4d−4f), 4e with 4′-F showed the highest inhibitory potency (IC50 3.5 nM) and selectivity (79-fold), which greatly exceeded that of HMCB. In contrast, the replacement of a methyl group (4g) and methoxy group (4h) instead of the 4′-hydroxy group of 4c 8444



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decreased both inhibitory potency and selectivity. These results suggest that the hydroxyl group or fluorine atom at the pposition on the phenylpropylamide moiety is a structural requisite for the selective and strong inhibition of AKR1B10. Other derivatives having a phenethyl moiety (4l)45 and a phenylbutyl moiety (4i) instead of the phenylpropyl moiety of 4c showed higher IC50 values and lower selectivity to AKR1B10 than those of 4c, suggesting that the 3-carbon chain length between the amide and phenyl ring of 4c is appropriate for fitting in the inhibitor-binding site of the enzyme. In addition, the derivatives without 7-OH (4j) and with 8-OH (4k) on the coumarin ring of 4c markedly reduced the inhibitory potency toward both AKR1B10 and AKR1B1. In particular, the decline observed in 4j was significant, suggesting that the 7-hydroxyl group is the most important structural prerequisite for the potent inhibition for the two enzymes. The synthetic route to 4a−l shown in Scheme 2 included the initial targets of iminochromenes 3a−k and was relatively long. Next we refined the synthetic route for the most potent inhibitor 4e. Known coumarin 546 was treated with 4fluorophenylpropylamine, prepared by reduction of corresponding amide,47 in the presence of EDC, HOBt, and Nmethylmorpholine to afford 4e in 78% yield (Scheme 3).

Table 2. Inhibitory Effects of 4c and 4e on AKRs and CBR1 4c

4e

enzyme

substratea

IC50 (nM)

ratiob

IC50 (nM)

ratiob

AKR1B10

20 μM P3A 2.0 mM P3A* 7 μM STetralol 260 μM STetralol 1.4 mM STetralol 110 μM STetralol 50 μM Isatin

4.1 ± 0.3

1

3.4 ± 0.1

1

1500 ± 10

370

1700 ± 160

500

950 ± 25

230

12000 ± 600

3500

970 ± 57

240

610 ± 2

180

1600 ± 150

390

470 ± 19

140

820 ± 31

200

350 ± 19

100

4200 ± 400

1000

1100 ± 140

310

AKR1A1 AKR1C1 AKR1C2 AKR1C3 AKR1C4 CBR1 a

Substrate concentrations of AKRs correspond to the approximate Km values, which were reported19,66 and determined in this study (*). P3A, pyridine-3-aldehyde. bRatio to AKR1B10.

benzylamide moiety of HMCB is close to only Gln303, the 4-hydroxyphenylpropyl moiety of 4c was surrounded by eight residues (Phe116, Phe123, Pro124, Lys125, Ala131, Ile132, Val301, and Leu302) and its hydroxyl group formed an H-bond interaction with the main-chain carbonyl group of Pro124. These 4c-specific interactions might contribute to the higher inhibitory potency of 4c than HMCB, despite the lack of the 4methoxyphenylimino moiety in its structure. Among the above eight residues, Lys125, Ala131, Ile132, and Val301 differ from the corresponding residues (Leu, Val, Val and Leu, respectively) in AKR1B1, suggesting that these residue differences are related to the high selectivity of 4c to AKR1B10 over AKR1B1. To confirm the interactions predicted by the molecular docking of 4c, we prepared mutant AKR1B10s, in which Lys125, Val301, and Gln303 were replaced with the corresponding residues (Leu, Leu, and Ser, respectively) in AKR1B1. In addition, Trp220 was replaced by a smaller aromatic residue, Tyr, in order to evaluate the role of its hydrophobic interaction with 4c. Since 4c was a competitive inhibitor with respect to the alcohol substrate in the NADP+linked geraniol oxidation, the effects of the mutations on the Ki value for 4c were determined (Table 3). The Lys125Leu, Trp220Tyr, and Val301Leu mutant enzymes showed more than 3-fold higher Ki values than the wild-type enzyme. In contrast, the Gln303Ser mutation had no influence on the Ki value, probably because Gln303 is involved in the binding of HMCB, but not 4c (Figure 3). The results support the 4cbinding mode shown in its docking model. In order to investigate the selectivity of 4c, we compared AKR1B10 and AKR1B1 from the structural and energetic points of view. We assumed that 4c could bind to AKR1B1 in a manner similar to the binding mode of 4c with AKR1B10, because the binding site of AKR1B1 shows significant homology to that of AKR1B10. The structure of AKR1B1 docked with 4c was first generated by homology modeling method using the docking model of AKR1B10 with 4c (Figure 3A) as the template. All calculations for homology modeling were conducted using the Schrödinger suite 2015-4 (Schrödinger, LLC, New York, NY, USA). The interaction modes of 4c with residues of AKR1B10 and AKR1B1 were shown as a

Scheme 3. Improved Synthesis of 4e

AKR1A1, AKR1C1, AKR1C2, AKR1C3, and AKR1C4 are human members of the AKR superfamily, and their substrate specificity for aldehydes overlaps with that of AKR1B10.48 In addition, human carbonyl reductase 1 (CBR1) was reported to be inhibited by des-4-methoxyphenylimino derivatives of MHPC, such as 8-hydroxy-2-imino-2H-chromene-3-carboxylic acid (2-chlorophenyl)amide.49 Therefore, the inhibitory selectivity of 4c and 4e toward AKR1B10 was further examined by comparing their effects on the activities of these enzymes. Compounds 4c and 4e were more than 100-fold less inhibitory to these enzymes compared to AKR1B10 (Table 2). Thus, the two compounds are highly selective to this enzyme. Molecular Docking and Site-Directed Mutagenesis of Inhibitor Binding Residues. To elucidate the underlying structural reasons for the high AKR1B10-selective inhibition of the 7-hydroxycoumarin derivatives, models of docked 4c in the AKR1B10−NADP+ complex50 were constructed. In the model with the best docking score (Figure 3A), 4c was held in the substrate-binding site through a number of contacts including the catalytic residues, Tyr49 and His111. The 7-hydroxy group on the coumarin ring of 4c formed H-bond interactions with His111 and the nicotinamide of NADP+. The interactions are probably important for the high inhibitory potency of 4c, because the derivative without the 7-OH group (4j) showed extremely low inhibition. When this model was compared with the previously reported model with the nonselective AKR1B10 inhibitor HMCB docked44 (Figure 3B), the orientation of the coumarin ring of 4c in the binding site resembled that of the chromene ring of HMCB in its docking model, but those of the 4-hydroxyphenylpropyl moiety of 4c and the benzylamide moiety of HMCB were markedly different. While the 8445



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Figure 3. Docked models of 4c (A) and HMCB (B) in AKR1B10−NADP+ complex. The portion of NADP+ (yellow) and AKR1B10 residues (light gray) within 4.0 Å from the inhibitors are depicted with possible hydrogen bond interactions, which are shown in dotted lines with distances (Å).

Table 3. Effects of Mutations of AKR1B10 on Ki Value for 4c enzyme wild-type Lys125Leu Trp220Tyr Val301Leu Gln303Ser a

Ki (nM)

ratio

± ± ± ± ±

3.0 6.1 4.3 1.1

2.3 6.9 14 9.8 2.5

0.3 0.4 0.2 0.6 0.4

and AKR1B1 (Figure S1), the alkyl side chain of AKR1B10 Lys125 was in contact with 4c, but that of AKR1B1 Leu125 was away from 4c. Therefore, the hydrophobic interaction of the alkyl side chain of Lys125 with 4-hydroxyphenylpropyl moiety of 4c might mainly cause the selectivity of 4c for AKR1B10. Effects of 4c and 4e on Migration and Proliferation of Lung Cancer A549 Cells. The efficacies of 4c and 4e as AKR1B10 inhibitors were investigated by analyzing their effects on the migration and proliferation of A549 cells, since AKR1B10 was reported to enhance migratory and proliferative potentials of cancer cells through induction of matrix metalloproteinase 2 and regulation of isoprenoid homeostasis, respectively.32,52 When the effects of 4c and 4e on the migration of A549 cells were first examined in a wound-healing assay, the cells treated for 24 h with 4c or 4e showed significantly lower migration compared to the control group treated with vehicle alone (Figure 4A). Next, the effects of 4c and 4e on the cell growth were examined using previously established AKR1B10-stably overexpressing A549 cells (A549/ 1B10 cells)32,52 and the parental A549 cells (Figure 4B). The cell number of the 96 h cultured A549/1B10 cells was significantly greater than that of the parent A549 cells. Compounds 4c and 4e dose-dependently suppressed the growth of both A549 and A549/1B10 cells, and their suppressive effects were statistically significant at 20 μM. As the cell numbers of A549/1B10 cells treated with 20 μM inhibitors were the same as those of A549 cells under the same conditions, it is suggested that the inhibitors almost completely suppressed increased cell proliferation by the overexpressing AKR1B10 as well as the endogenous protein. In order to evaluate the significance of 4c and 4e as AKR1B10 inhibitors, their inhibitory effects on the metastatic potential of A549 cells were investigated by using an experimental lung metastasis model of A549-Luc2 cells (Figure 5). Compared to a group injected with the A549-Luc2 cells treated with vehicle alone, the groups injected with the 4c- and 4e-treated A549-Luc2 cells exhibited markedly reduced in vivo metastatic potential to the lung, indicating that 4c and 4e affect the behavior of lung cancer A549 cells not only in vitro but also in vivo. The results suggest that catalytic activity of AKR1B10 is related to the metastasis of A549 cells. These selective inhibitors would be useful in future studies that elucidate how AKR1B10-mediated

a

Ratio of mutant/wild type.

superimposed figure (Figure S1). Among residues near 4c, two residues different between AKR1B10 (Lys125 and Val301) and AKR1B1 (Leu125 and Leu301) were considered to cause the selectivity of 4c for AKR1B10. This is supported by the mutagenesis of the two residues (Table 3). We next performed binding free energy (ΔGbind) calculations based on molecular mechanics Poisson−Boltzmann surface area (MM-PBSA) methods, in order to investigate the selectivity of 4c from the energetic point of view. These calculations were conducted using the AMBER14 package,51 and Table S1 shows the resulting ΔGbind values calculated by MM-PBSA calculations. The ΔGbind (−42.95 kcal/mol) of 4c with AKR1B10 was more favorable than that of 4c with AKR1B1 (−41.25 kcal/mol), suggesting that the binding affinity of 4c was greater in AKR1B10 complex. This was consistent with the difference in IC50 values between the two enzymes, indicating that the MM-PBSA method could provide reliable ΔGbind values. Moreover, the contributions of Lys125 (Leu125) and Val301 (Leu301) of AKR1B10 (AKR1B1) to ΔGbind were estimated by the alanine scanning analysis, where the 125th or 301st residue was substituted by alanine in the computer. The ΔGbind values of 4c with the alanine-substituted AKR1B10s and AKR1B1s were calculated using the MM-PBSA python script within the AMBER14 package (Table S1). The ΔGbind values of Lys125Ala and Val301Ala mutants of AKR1B10 with 4c were −41.69 and −41.32 kcal/mol, respectively, and the respective binding free-energy changes (ΔΔGbind) were 1.26 and 1.62 kcal/mol. This suggested that Lys125 and Val301 of AKR1B10 contribute largely to the binding affinity for 4c. In contrast, the ΔΔGbind values for Leu125Ala and Leu301Ala mutants of AKR1B1 were −0.08 and 1.28 kcal/mol, respectively, suggesting that Leu301, but not Leu125, of AKR1B1 contributes largely to the binding affinity for 4c. In the superimposed 4c-docked models of AKR1B10 8446



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Figure 4. Inhibition of invasive and proliferative potential of A549 cells by 4c and 4e. (A) Wound-healing assay. The cells were scratched and then treated for 24 h with vehicle dimethyl sulfoxide (DMSO), 20 μM 4c, or 20 μM 4e. As representative photographs at 0 h and 24 h culture are shown, the cells migrated into the scratch wounds (two dotted lines). The migration distance of the inhibitor-treated cells is expressed as the percentage to that of the DMSO-treated control cells in the bar graph. Significant difference from the control cells, *p < 0.05 (n = 3). (B) Proliferative assay. A549 cells (left) and A549/1B10 cells (right) were cultured in the absence or presence of the indicated concentrations of 4c and 4e, and the viable cell numbers were estimated at 96 h. Significant differences (p < 0.05) from the inhibitorfree A549 cells and A549/1B10 cells are shown with * and #, respectively (n = 4).

Figure 6. Overcoming CDDP resistance of A549 cells by treatments with 4c and 4e. (A) Effects of 4c and 4e on CDDP sensitivity of CDDP-R-A549 cells. The CDDP-R-A549 cells and parental A549 cells were pretreated for 2 h without or with the indicated concentrations of 4c or 4e, and then treated for 24 h with 40 μM CDDP. The viability values are expressed as percentages to that of the parental cells treated without inhibitor. (B) Growth curves of A549 and CDDP-R-A549 cells in medium containing 0.5 μM CDDP (n = 3). (C) Effects of 4c and 4e on the growth of CDDP-R-A549 cells in the presence of 0.5 μM CDDP. The cells were cultured for 96 h in the absence or presence of 4c or 4e. The viable cell numbers are expressed as percentages to that of the parental A549 cells treated without inhibitor. Significant difference from CDDP-R-A549 cells treated without inhibitor, *p < 0.05 (n = 3).

viable after 24 h treatment with 40 μM CDDP in contrast to 36% viability of the parental A549 cells. Cotreatment with 4c or 4e and 40 μM CDDP decreased the cell viability of CDDP-RA549 cells in a dose-dependent manner, and this action was most obvious in the treatment of 40 μM 4e, which resulted in almost the same CDDP sensitivity as that of the parent A549 cells. Next, in order to reflect conditions of practical medical treatment, the effects of 4c or 4e on sensitivity of the CDDP-RA549 cells to a low concentration (0.5 μM) of CDDP were examined. Under the conditions, CDDP-R-A549 cells exponentially grew during 96 h culture in contrast to little growth of the parental A549 cells (Figure 6B) and were made CDDPsensitive by treatment with 4c or 4e (Figure 6C). Furthermore, we examined effects of 4c or 4e on metastatic and invasive potentials of CDDP-R-A549 cells (Figure 7). When the two potentials were analyzed by the Boyden chamber assays, the CDDP-R-A549 cells showed higher potentials than the parental A549 cells (data not shown). Compound 4e significantly inhibited the metastasis and invasion of the CDDP-resistant cells, although 4c was less effective. AKR1B10 was suggested to be implicated in acquiring cancer cell resistance to several

Figure 5. Inhibition of A549 cells metastasis into mouse lung by 4c and 4e. Cultured A549−Luc2 cells were treated with 20 μM 4c or 4e or vehicle DMSO (as a control) for 48 h and then injected into the tail vein of BALB/c nude mice (n = 5). After 4 days, luciferase luminescence in the lungs was evaluated. Significant difference from the control, *p < 0.05.

metabolism or pathways are responsible for the metastatic potential of A549 cells. Effects of 4c and 4e on CDDP-Resistant A549 Cells. AKR1B10 was reported to be involved in CDDP-resistance of A549 cells through its up-regulation.31 The CDDP-resistant A549 (CDDP-R-A549) cells were less sensitive to CDDP, as shown in Figure 6A, where almost all CDDP-R-A549 cells were 8447



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received unless otherwise noted. The purities (91 to >98%) of compounds 3a′ and 4a−l were analyzed by HPLC analysis. Analytical HPLC performed on JASCO PU-2082 plus intelligent HPLC pump with GL Science InertSustainC18 (5 μL, 4.6 mm × 250 mm) column and 100% MeCN as mobile phase with flow rate of 1−3 mL/min at 35−50 °C. General Procedure for the Preparation of Methanesulfonic Acid Ester (B). To a stirred solution of the alcohol (A1,53 A2,43 A3,54 A4,55 or A5,56 1 mmol) in CH2Cl2 (5 mL) were added MsCl (1.2 mmol) and Et3N (1.5 mmol) at 0 °C, and the resulting mixture was stirred for 2 h at room temperature. The reaction was quenched by satd. NH4Cl (aq), and the organic layer was separated. The aqueous mixture was extracted with CH2Cl2 (5 mL × 3), and the organic layer and extracts were combined, dried over Na2SO4, and evaporated. The residue was chromatographed on silica gel (15 g, hexane/ethyl acetate = 4:1) to give the corresponding methanesulfonic acid ester B, which was used in the next step immediately. Methanesulfonic Acid 3-(3-Methoxymethoxyphenyl)propyl Ester (B1). Yield 96%; 1H NMR (500 MHz, CDCl3) δ 2.08 (2H, quint, J = 7.5 Hz), 2.73 (2H, t, J = 7.5 Hz), 3.00 (3H, s), 3.48 (3H, s), 4.23 (2H, t, J = 7.5 Hz), 5.17 (2H, s), 6.83−6.91 (3H, m), 7.21 (1H, t, J = 8.0 Hz); 13C NMR (125 MHz, CDCl3) δ 30.2, 31.3, 37.0, 55.8, 69.0, 94.2, 113.8, 116.2, 121.8, 129.4, 141.8, 157.2. Methanesulfonic Acid 3-(2-Methoxymethoxyphenyl)propyl Ester (B2). Yield 94%; 1H NMR (500 MHz, CDCl3) δ 1.82 (2H, quin, J = 7.5 Hz), 2.71 (2H, t, J = 7.5 Hz), 3.15 (2H, t, J = 7.5 Hz), 3.49 (3H, s), 5.21 (2H, s), 6.84 (1H, t, J = 8.0 Hz), 7.08 (1H,d, J = 8.0 Hz), 7.10− 7.17 (1H, m); 13C NMR (125 MHz, CDCl3) δ 30.4, 31.9, 38.2, 55.8, 94.1, 113.5, 121.2, 127.3, 129.8, 123.1, 156.3. Methanesulfonic Acid 3-(4-Methoxymethoxyphenyl)propyl Ester (B3). Yield 96%; 1H NMR (500 MHz, CDCl3) δ 1.88 (2H, quint, J = 7.3 Hz), 2.65 (2H, t, J = 7.3 Hz), 3.28 (2H, t, J = 7.3 Hz), 3.48 (3H, s), 5.18 (2H, s), 6.97 (2H, d, J = 8.0 Hz), 7.09(2H, d, J = 8.0 Hz); 13C NMR (125 MHz, CDCl3) δ 30.6, 30.7, 30.9, 37.3, 55.9, 69.1, 94.5, 116.4, 129.3, 133.6, 155.7. Methanesulfonic Acid 4-(4-Methoxymethoxyphenyl)butyl ester (B4). Yield 96%; 1H NMR (500 MHz, CDCl3) δ 1.69−1.78 (4H, m), 2.60 (2H, t, J = 7.5 Hz), 2.98 (3H, s), 3.48 (3H, s), 4.23 (2H, t, J = 7.5 Hz), 5.15 (2H, s), 6.96 (2H, d, J = 8.5 Hz), 7.08 (2H, d, J = 8.5 Hz); 13 C NMR (125 MHz, CDCl3) δ 27.1, 28.3, 34.0, 36.9, 55.6, 69.8, 94.3, 116.0, 129.1, 134.7, 155.3. Methanesulfonic Acid 3-(3-Fluorophenyl)propyl Ester (B5). Yield 78%; 1H NMR (500 MHz, CDCl3) δ 2.89 (2H, quint, J = 7.5 Hz), 3.11 (2H, t, J = 7.5 Hz), 3.35 (3H, s), 4.58 (2H, t, J = 7.5 Hz), 7.11− 7.25 (3H, m), 7.61 (2H,d, J = 7.5 Hz); 13C NMR (125 MHz, CDCl3) δ 29.7, 30.5, 68.8, 112.31 (d, J = 19.4 Hz), 114.6 (d, J = 18.3 Hz), 123.7, 129.5 (d, J = 7.3 Hz), 142.5 (d, J = 9.6 Hz), 162.2 (d, J = 241.7 Hz). General Procedure for the Preparation of Azide (C). To a stirred solution of methanesulfonic acid ester (B1, B2, B3, B4, B5, or B6,57 1 mmol) in DMF/H2O (2:1) was added NaN3 (2 mmol), and then the reaction mixture was heated to 60 °C for 20 h. After cooling, the reaction was quenched by H2O. The aqueous mixture was extracted with CH2Cl2 (5 mL × 3). The organic extracts were combined, dried over Na2SO4, and evaporated. The residue was chromatographed on silica gel (15 g, hexane/acetone = 100:1) to give the corresponding azide C, which was used in the next step immediately. 1-(3-Azidopropyl)-3-methoxymethoxybenzene (C1). Yield 78%; 1 H NMR (500 MHz, CDCl3) δ 1.92 (2H, quint, J = 7.5 Hz), 2.68 (2H, t, J = 7.5 Hz), 3.29 (2H, t, J = 7.5 Hz), 3.49 (3H, s), 5.17 (2H, s), 6.83−6.90 (3H, m), 7.21 (1H, t, J = 8.0 Hz); 13C NMR (125 MHz, CDCl3) δ 30.2, 32.6, 50.5, 55.9, 94.3, 113.8, 116.3, 121.9, 129.4, 142.4, 157.3; IR (neat) 2422, 1652, 1634 cm−1. 1-(3-Azidopropyl)-2-methoxymethoxybenzene (C2). Yield 89%; 1 H NMR (500 MHz, CDCl3) δ 1.91 (2H, quint, J = 7.5 Hz), 2.73 (2H, t, J = 7.5 Hz), 3.29 (2H, t, J = 7.5 Hz), 3.49 (3H, s), 5.21 (2H, s), 6.94 (1H, t, J = 8.0 Hz), 7.09 (1H,d, J = 8.0 Hz), 7.13−7.18 (1H, m); 13C NMR (125 MHz, CDCl3) δ 27.4, 28.9, 50.7, 55.8, 94.1, 113.7, 121.48, 127.3, 129.7, 123.0, 155.0; IR (neat) 2421, 1652, 1634 cm−1.

Figure 7. Effects of 4c and 4e on metastatic and invasive potentials of CDDP-R-A549 cells. The cells were seeded on a polycarbonate membrane and Type I collagen-coated polycarbonate membrane in Boyden chambers for analyzing the metastatic (white bar) and invasive potentials (black bar), respectively, and then treated for 48 h without or with the indicated concentrations of 4c or 4e. The cells passed through pores of the membrane were counted microscopically, and their numbers are expressed as the percentages to those of the control cells treated without inhibitor. Significant difference from the control cells, *p < 0.05 (n ≥ 4).

anticancer drugs and enhancing metastatic and invasive potentials of doxorubicin-resistant gastric cancer cells32 through its ability to detoxify lipid peroxidation-derived aldehydes.30−35 The present results not only support the above roles of AKR1B10 in resistance to anticancer drugs but also demonstrate that 4c and 4e are effective agents for overcoming CDDP resistance of lung cancer cells.

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CONCLUSIONS Using HMCB as the lead, we newly synthesized its derivatives, 4c and 4e, that are currently the most potent AKR1B10 inhibitors with high selectivity to this enzyme over AKR1B1. The high selectivity was suggested to be brought about by interactions of the 4-hydroxyphenylpropyl moiety of 4c with the substrate-binding residues including Lys125 and Val301 that differ from the corresponding ones in AKR1B1. The two inhibitors significantly suppressed not only migration and proliferation of lung cancer A549 cells but also metastasis of the cells into mouse lung. Moreover, they increased CDDP sensitivity of CDDP-resistant A549 cells and inhibited the metastatic and invasive potentials of the CDDP-resistant cells. Accordingly, these ex vivo results suggest the effectiveness of the inhibitors as antiproliferative agents against AKR1B10-overexpressing cancers and adjuvant drugs for overcoming CDDP resistance of lung cancer.

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EXPERIMENTAL SECTION

Chemistry: General. Melting points were determined with an AS ONE micro melting point apparatus and are uncorrected. Flash chromatography was performed with Kanto Kagaku silica gel 60N (63−210 mm). NMR spectra were recorded on a JEOL JNM-A 400, JEOL JNM-ECX 500 spectrometer in the solvent indicated. Chemical shifts (δ) are given in ppm downfield from TMS and referenced with CHCl3 (7.26 ppm) or DMSO (2.49 ppm) as an internal standard. Peak multiplicities are designated by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Coupling constants (J) are given in hertz. Infrared spectra were obtained with a SHIMADZU FTIR-8400 spectrometer using film KBr pellet technique. High resolution mass spectral data was obtained on a JEOL MStation JMS-700. All commercial reagents were used as 8448



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2-Cyano-N-[3-(4-fluorophenyl)propyl]acetamide (2e). Yield 42%; mp 73−75 °C; 1H NMR (500 MHz, CDCl3) δ 1.87 (2H, quit, J = 7.5 Hz), 2.64 (2H, t, J = 7.5 Hz), 3.30−3.36 (4H, m), 6.96−7.00 (2H, m), 7.12−7.15 (2H, m); 13C NMR (125 MHz, CDCl3) δ 25.7, 30.5, 30.7, 32.8, 39.6, 114.8 (d, J = 3.6 Hz), 115.0, 128.2 (d, J = 19.5 Hz), 129.4 (d, J = 7.3 Hz Hz), 136.6 (d, J = 2.5 Hz), 161.0 (d, J = 242.0 Hz), 161.7; IR (KBr) 3323, 1660, 1587, 1554 cm−1; MS (EI) m/z 220 (M+); HRMS Calcd for 220.1012, Found 220.1013. 2-Cyano-N-[3-(3,5-difluorophenyl)propyl]acetamide (2f). Yield 48%; mp 100−102 °C; 1H NMR (500 MHz, DMSO-d6) δ 1.70 (2H, quint, J = 7.5 Hz), 2.60 (2H, t, J = 7.5 Hz), 3.60 (2H, q, J = 7.5 Hz), 3.60 (2H, s), 6.94−7.03 (3H, m), 8.24 (1H, br); 13C NMR (125 MHz, CDCl3) δ 26.0, 30.3, 32.9, 39.8, 101.7 (t, J = 25.5 Hz), 111.1 (d, J = 4.8 Hz), 111.2 (d, J = 4.8 Hz), 115.0, 145.0 (t, J = 8.5 Hz), 162.3, 163.1 (d, J = 248 Hz), 163.2 (d, J = 247.0 Hz); IR (KBr) 3301, 1647, 1599 cm−1; MS (EI) m/z 238 (M+); HRMS Calcd for C12H13F2N2O 238.0918, Found 238.0918. 2-Cyano-N-(3-p-tolylpropyl)acetamide (2g). Yield 66%; mp 69− 71 °C; 1H NMR (500 MHz, CDCl3) δ 1.88 (2H, quint, J = 7.5 Hz), 2.32 (3H, s), 2.64 (2H, t, J = 7.5 Hz), 3.34(4H, q, J = 7.5 Hz), 7.11 (2H, q, J = 7.83 Hz); 13C NMR (125 MHz, CDCl3) δ 20.6, 25.6, 30.3, 32.2, 39.6, 115.0, 127.9, 128.8, 135.2, 137.7, 161.9; IR (KBr) 3295, 1652, 1558 cm−1; MS (EI) m/z 216 (M+); HRMS Calcd for C13H16N2O 216.1263, Found 216.1264. 2-Cyano-N-[3-(4-methoxyphenyl)propyl]acetamide (2h). Yield 45%; mp 61−62 °C; 1H NMR (500 MHz, CDCl3) δ 1.88 (2H, quint, J = 7.5 Hz), 2.62 (2H, t, J = 7.5 Hz), 3.31 (2H, s), 3.33 (2H, q, J = 7.5 Hz), 3.80 (3H, s), 5.99 (1H, br), 6.84 (2H, d, J = 9.0 Hz), 7.10 (2H, d, J = 9.0 Hz); 13C NMR (125 MHz, CDCl3) δ 25.7, 30.6, 31.9, 39.7, 55.0, 113.7, 114.9, 129.0, 132.9, 157.7, 161.6; IR (KBr) 3355, 1655, 1559, 1516 cm−1; MS (EI) m/z 232 (M+); HRMS Calcd for C13H16N2O2 232.1212, Found 232.1218. 2-Cyano-N-[4-(4-methoxymethoxyphenyl)butyl]acetamide (2i). Yield 49%; mp 59−60 °C; 1H NMR (500 MHz, CDCl3) δ 1.56− 1.65 (4H, m), 2.59 (2H, t, J = 7.5 Hz), 3.32 (2H, q, J = 7.5 Hz), 3.35 (2H, s), 3.45 (3H, s), 5.15 (2H, s), 6.05 (1H, br), 6.97 (2H, d, J = 8.5 Hz), 7.07 (2H, d, J = 8.5 Hz); 13C NMR (125 MHz, CDCl3) δ 25.7, 28.47, 28.50, 34.3, 40.0, 55.7, 94.4, 114.8, 116.1, 129.1, 135.2, 155.2, 161.3; IR (KBr) 3448, 1653, 1558, 1507 cm−1; MS (EI) m/z 276 (M+); HRMS Calcd for C14H18N2O3 276.1474, Found 276.1474. 2-Cyano-N-[3-(3-hydroxyphenyl)propyl]acetamide (2a′). To a stirred solution of 2a (262 mg, 1 mmol) in THF (5 mL) was added 10% HCl (5 drops), and the resulting mixture was heated at 40 °C for 12 h. After cooling, H2O was added to the reaction mixture, and the aqueous mixture was extracted with EtOAc (5 mL × 3). The organic extracts were combined, dried over Na2SO4, and evaporated. The residue was chromatographed on silica gel (15 g, CH2Cl2/MeOH = 100:1) to give 2a′ (174 mg, 81%) as a colorless oil. mp 100−101 °C; 1 H NMR (400 MHz, CDCl3) δ 1.89 (2H, q, J = 7.7 Hz), 2.63 (2H, t, J = 7.7 Hz), 3.32 (2H, s), 3.34 (2H, t, J = 7.7 Hz), 6.09 (1H, br), 6.68 (1H, s), 6.69 (1H, d, J = 7.3 Hz), 6.75 (1H, d, J = 7.3 Hz), 7.16 (1H, t, J = 7.3 Hz); 13C NMR (125 MHz, DMSO-d6) δ 25.3, 30.4, 32.4, 38.7, 112.8, 115.2, 116.3, 118.9, 129.2, 142.9, 157.3, 162.0; IR (KBr) 3387, 1649, 1556 cm−1; MS (EI) m/z 218 (M+); HRMS Calcd for C12H14N2O2 218.1055, Found 218.1050. General Procedure for the Preparation of Amide (3a−k). To a stirred solution of amide (2a−i, 1 mmol) in EtOH (3 mL) were added catalytic amount of piperidine (2 drops) and benzaldehyde (1 mmol), and the reaction mixture was stirred at room temperature for 12 h. The suspension was filtered to give iminochromene, which was used directly in the next step. To a stirred solution of the iminochromene obtained above in acetic acid (2 mL) was added pmethoxyaniline (1 mmol), and the resulting mixture was stirred at room temperature for 12 h. The solvent was removed under reduced pressure, and the residue was chromatographed on silica gel (15 g, CH2Cl2) to give the corresponding phenyliminochromene (3a−k) as the yellow solid. 7-Hydroxy-2-(4-methoxyphenylimino)-2H-chromene-3-carboxylic Acid [3-(3-Methoxymethoxyphenyl)propyl]amide (3a). Yield 67%; mp 144−146 °C; 1H NMR (500 MHz, CDCl3) δ 1.96 (2H,

1-(3-Azidopropyl)-4-methoxymethoxybenzene (C3). Yield 94%; H NMR (500 MHz, CDCl3) δ 1.91 (2H, quint, J = 7.3 Hz), 2.73 (2H, t, J = 7.3 Hz), 3.29 (2H, t, J = 7.3 Hz), 3.49 (3H, s), 5.21 (2H, s), 6.94 (1H, t, J = 8.0 Hz), 7.09 (1H,d, J = 8.0 Hz), 7.10−7.18 (1H, m); 13C NMR (125 MHz, CDCl3) δ 30.3, 31.6, 50.3, 55.6, 94.3, 116.1, 129.13, 133.9, 155.5; IR (neat) 2422, 1652, 1635 cm−1. 1-(4-Azidobutyl)-4-methoxymethoxybenzene (C4). Yield 81%; 1H NMR (500 MHz, CDCl3) δ 1.62−1.68 (4H, m), 2.59 (2H, t, J = 7.5 Hz), 3.28 (2H, t, J = 7.5 Hz), 3.48 (3H, s), 5.15 (2H, s), 6.96 (2H, dd, J = 4.5, 2.0 Hz), 7.08 (2H, dd, J = 4.5, 2.0 Hz); 13C NMR (125 MHz, CDCl3) δ 28.4, 28.6, 34.5, 51.3, 55.9, 94.6, 116.3, 129.3, 135.2, 155.5; IR (neat) 2384, 1652, 1635 cm−1. 1-(3-Azidopropyl)-3-fluorobenzene (C5). Yield 72%; 1H NMR (500 MHz, CDCl3) δ 1.90 (2H, quint, J = 7.5 Hz), 2.71 (2H, t, J = 7.5 Hz), 3.29 (2H, t, J = 7.5 Hz), 6.82−6.95 (3H, m), 7.25 (2H, q, J = 7.0 Hz); 13C NMR (125 MHz, CDCl3) δ 29.9, 32.2, 50.2, 112.8 (d, J = 20.6 Hz), 115.0 (d, J = 20.75 Hz), 123.9, 129.7 (d, J = 7.4 Hz), 143.3 (d, J = 7.3 Hz), 162.8 (d, J = 244.3 Hz); IR (neat) 2424, 1654, 1635 cm−1. 1-(3-Azidopropyl)-4-methylbenzene (C6). Yield 75%; 1H NMR (500 MHz, CDCl3) δ 1.90 (2H, quint, J = 7.5 Hz), 2.33 (3H, s), 2.67 (2H, t, J = 7.5 Hz), 3.28 (2H, t, J = 7.5 Hz), 7.09 (2H, d, J = 8.7 Hz); 13 C NMR (125 MHz, CDCl3) δ20.5, 30.2, 31.9, 50.1, 127.9, 128.8, 135.0, 137.4; IR (neat) 2385, 1652, 1635 cm−1. General Procedure for the Preparation of Amide (2a−i). A stirred suspension of C1, C2, C3, C4, C5, C6, C7,58 C8,59 or C960 (1 mmol) and 10% Pd−C (3 mg) in MeOH (5 mL) was hydrogenated under hydrogen atmosphere (1 atm.) for 12 h. The catalyst was removed, and the filtrate was evaporated to give the corresponding amine (1a−i), which was used directly in the next step. To a stirred solution of cyanoacetic acid (1 mmol) in CH2Cl2 (5 mL) were added the corresponding amine (1a−i, 1 mmol), EDC (2 mmol), and DMAP (0.2 mmol), and the resulting mixture was stirred at room temperature for 20 h. The reaction mixture was evaporated, and the residue was chromatographed on silica gel (15 g, hexane/acetone = 5:1) to give the corresponding amide (2). 2-Cyano-N-[3-(3-methoxymethoxyphenyl)propyl]acetamide (2a). Yield 54%; 1H NMR (500 MHz, CDCl3) δ 1.90 (2H, quint, J = 7.5 Hz), 2.65 (2H, t, J = 7.5 Hz), 3.32 (2H, s), 3.34 (2H, q, J = 7.5 Hz), 3.48 (3H, s), 5.17 (2H, s), 6.82−6.90 (3H, m), 7.22 (1H, t, J = 8.0 Hz); 13C NMR (125 MHz, CDCl3) δ 25.7, 30.3, 33.0, 39.9, 55.9, 94.3, 113.9, 114.8, 116.2, 121.8, 129.5, 142.6, 157.3, 161.2; IR (neat) 3421, 1652, 1635 cm−1; MS (EI) m/z 262 (M+); HRMS Calcd for C14H18N2O3 262.1317, Found 262.1315. 2-Cyano-N-[3-(2-methoxymethoxyphenyl)propyl]acetamide (2b). Yield 70%; mp 80−83 °C; 1H NMR (500 MHz, CDCl3) δ 1.86 (2H, quint, J = 7.0 Hz), 2.72 (2H, t, J = 7.0 Hz), 3.33 (4H, q, J = 7.0 Hz), 3.49 (3H, s), 5.25 (2H, s), 6.39 (1H, br), 6.96 (1H, t, J = 8.0 Hz), 7.09(2H,d, J = 8.0 Hz), 7.14−7.18(2H,m); 13C NMR (125 MHz, CDCl3) δ 25.6, 27.1, 29.2, 30.8, 56.0, 94.4, 113.9, 114.8, 121.7, 127.3, 129.9, 129.9, 154.8, 161.2; IR (KBr) 3294, 1653, 1559 cm−1; MS (EI) m/z 262 (M+); HRMS Calcd for C14H18N2O3 262.1317, Found 262.1314. 2-Cyano-N-[3-(4-methoxymethoxylphenyl)propyl]acetamide (2c). Yield 49%; mp 78−81 °C; 1H NMR (500 MHz, CDCl3) δ 1.86 (2H, quint, J = 7.3 Hz), 2.62 (2H, t, J = 7.3 Hz), 3.32 (4H, q, J = 7.3 Hz), 3.47 (3H, s), 5.15 (2H, s), 6.03 (1H, br), 6.97 (2H, d, J = 8.5 Hz), 7.09 (2H,d, J = 8.5 Hz); 13C NMR (125 MHz, CDCl3) δ 25.7, 30.6, 32.1, 39.8, 55.8, 94.4, 114.8, 116.3, 129.1, 134.3, 155.4, 161.3; IR (KBr) 3420, 1652, 1558 cm−1; MS (EI) m/z 262 (M+); HRMS Calcd for C14H18N2O3 262.1317, Found 262.1314. 2-Cyano-N-[3-(3-fluorophenyl)propyl]acetamide (2d). Yield 58%; mp 68−69 °C; 1H NMR (500 MHz, CDCl3) δ 1.89 (2H, quint, J = 7.5 Hz), 2.67 (2H, t, J = 7.5 Hz), 3.32−3.36 (4H, m), 6.10 (1H, br), 6.90−6.96 (3H, m), 7.26 (1H, m); 13C NMR (125 MHz, CDCl3) δ 25.7, 30.0, 32.4, 39.4, 112.6 (d, J = 20.6 Hz), 114.8 (d, J = 20.8 Hz), 123.8 (d, J = 2.5 Hz), 129.6 (d, J = 8.5 Hz), 143.5 (d, J = 7.3 Hz), 161.2 (d, J = 6.1 Hz), 162.5 (d, J = 243.0 Hz); IR (KBr) 3328, 1661, 1554 cm−1; MS (EI) m/z 220 (M+); HRMS Calcd for C12H13FN2O 220.1012, Found 220.1013. 1

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3447, 1653, 1559, 1506 cm−1; MS (EI) m/z 446 (M+); HRMS Calcd for C26H23FN2O4 446.1642, Found 446.1641. 7-Hydroxy-2-(4-methoxyphenylimino)-2H-chromene-3-carboxylic Acid [3-(3,5-Difluorophenyl)propyl]amide (3f). Yield 85%; mp 230−232 °C; 1H NMR (500 MHz, DMSO-d6) δ 1.86 (2H, quint, J = 7.5 Hz), 2.68 (2H, t, J = 7.5 Hz), 3.29−3.56 (2H, m), 3.77 (3H, s), 6.52 (1H, d, J = 2.0 Hz), 6.71 (1H, dd, J = 6.5, 2.0 Hz), 6.94−7.02 (5H, m), 7.30 (2H, d, J = 9.0 Hz), 7.61 (1H, d, J = 8.5 Hz), 8.39 (1H, s), 10.18 (1H, t, J = 5.5 Hz), 10.65 (1H, br); 13C NMR (125 MHz, DMSO-d6) δ; 30.6, 32.8, 38.6, 55.7, 101.8 (t, J = 25.5 Hz), 111.5, 111.9 (dd, J = 24.3, 4.9 Hz), 113. 5, 114.5, 117.4, 125.3, 131.6, 137.5, 141.1, 146.7 (t, J = 9.8 Hz), 148.8, 155.1, 162.8 (d, J = 244.3 Hz), 156.3 (d, J = 237.0 Hz), 163.0, 169.3; IR (KBr) 3458, 1652, 1558, 1506 cm−1; MS (EI) m/z 464 (M+); HRMS Calcd for C26H22F2N2O4 464.1548, Found; 464.1552. 7-Hydroxy-2-(4-methoxyphenylimino)-2H-chromene-3-carboxylic Acid (3-p-Tolylpropyl)amide (3g). Yield 82%; mp 220−222 °C; 1 H NMR (500 MHz, DMSO-d6) δ 1.81 (2H, quint, J = 7.3 Hz), 2.28 (3H, s), 2.49 (2H, t, J = 7.3 Hz), 3.25−3.39 (2H, m), 3.76 (3H, s), 6.52 (1H, s), 6.73 (1H, dd, J = 2.0, 8.5 Hz), 6.49 (2H, d, J = 8.5 Hz), 7.06 (4H, q, J = 8.5 Hz), 7.30 (2H, d, J = 8.5 Hz), 7.59 (1H, d, J = 8.5 Hz), 8.38 (1H, s), 10.20 (1H, t, J = 5.5 Hz); 13C NMR (125 MHz, DMSO-d6) δ 58.9, 68.2, 69.7, 75.8, 92.7, 138.8, 150.5, 151.5, 162.2, 165.7, 166.4, 168.6, 172.2, 175.7, 178.1, 185.9, 192.1, 192.1, 193.8, 199.2; IR (KBr) 3227 1662, 1559, 1504 cm−1; MS (EI) m/z 442 (M+); HRMS Calcd for C27H26N2O4 442.1893, Found 442.1888. 7-Hydroxy-2-(4-methoxyphenylimino)-2H-chromene-3-carboxylic Acid [3-(4-Methoxyphenyl)propyl]amide (3h). Yield 87%; mp 198−200 °C; 1H NMR (500 MHz, DMSO-d6) δ 1.92 (2H, quint, J = 7.5 Hz), 2.67 (2H, t, J = 7.5 Hz), 3.45 (2H, q, J = 7.5 Hz), 3.75 (3H, s), 3.82 (3H, s), 6.64 (1H, s), 6.73 (1H, d, J = 8.3 Hz), 6.78 (2H, d, J = 8.5 Hz), 6.90 (2H, d, J = 9.0 Hz), 7.08 (2H, d, J = 8.5 Hz), 7.24−7.27 (3H, m), 7.32 (1H, d, J = 8.3 Hz), 8.43 (1H, s), 10.65 (1H, br); 13C NMR (125 MHz, DMSO-d6) δ; 31.3, 32.2, 38.9, 55.4, 55.7, 101.8, 111.5, 113.5, 114.2, 114.5, 117.4, 125.2, 129.7, 131.6, 133.7, 137.5, 141.1, 148.8, 155.1, 156.8, 157.9, 162.2, 162.8; IR (KBr) 3441, 1652, 1558, 1506 cm−1; MS (EI) m/z 458 (M+); HRMS Calcd for C27H26N2O5 458.1842, Found 458.1843. 7-Hydroxy-2-(4-methoxyphenylimino)-2H-chromene-3-carboxylic Acid [4-(4-Methoxymethoxyphenyl)butyl]amide (3i). Yield 83%; mp 179−181 °C; 1H NMR (500 MHz, CDCl3) δ 1.62−1.75 (4H, m), 2.60 (2H, t, J = 7.5 Hz), 3.46−3.49 (5H, m), 3.83 (3H, s), 5.14 (2H, s), 6.63 (1H, s), 6.72 (1H, d, J = 8.5 Hz), 6.89−6.93 (4H, m), 7.06 (2H, d, J = 8.5 Hz), 7.22 (2H, d, J = 8.5 Hz), 7.32 (1H, d, J = 8.5 Hz), 8.42 (1H, s), 10.59 (1H, t, J = 5.0 Hz); 13C NMR (125 MHz, CDCl3) δ 28.9, 29.2, 34.7, 35.0, 39.9, 55.5, 56.0, 78.5, 94.7, 102.5, 112.0, 114.1, 116.3, 124.8, 129.4, 130.8, 135.7, 137.2, 141.2, 155.2, 155.4, 156.8, 162.0, 163.6, 185.7; IR (KBr) 3442, 1652, 1558, 1506 cm−1; MS (EI) m/z 502 (M+); HRMS Calcd for C29H30N2O6 502.2104, Found 502.2099. 2-(4-Methoxyphenylimino)-2H-chromene-3-carboxylic Acid [3(4-Methoxymethoxyphenyl)propyl]amide (3j). Yield 94%; mp 110− 112 °C; 1H NMR (500 MHz, CDCl3) δ 1.94 (2H, quint, J = 7.5 Hz), 2.70 (2H, t, J = 7.5 Hz), 3.46−3.50 (5H, m), 3.85 (3H, s), 5.14 (2H, s), 6.94 (4H, d, J = 8.5 Hz), 7.12 (3H, d, J = 8.5 Hz), 7.20 (1H, t, J = 8.5, Hz), 7.30 (2H, t, J = 8.5 Hz), 7.44 (1H, t, J = 8.5 Hz), 7.51 (1H, dd, J = 2.0, 8.5 Hz), 8.52 (1H, s), 10.52 (1H, t, J = 5.5 Hz); 13C NMR (125 MHz, CDCl3) δ 32.8, 32.2, 28.8, 55.1, 55.5, 94.2, 113.7, 114.4, 114.9, 116.0, 118.8, 121.2, 124.0, 124.6, 128.9, 129.1, 132.0, 134.5, 134.0, 147.9, 153.0, 155.2, 156.6, 161.8; IR (KBr) 3447, 1653, 1558, 1506 cm−1; MS (EI) m/z 472 (M+); HRMS Calcd for C28H28N2O5 472.1998, Found 472.2006. 8-Hydroxy-2-(4-methoxyphenylimino)-2H-chromene-3-carboxylic Acid [3-(4-Methoxymethoxyphenyl)propyl]amide (3k). Yield 89%; mp 192−194 °C; 1H NMR (500 MHz, CDCl3) δ 1.94 (2H, quint, J = 7.5 Hz), 2.68 (2H, t, J = 7.5 Hz), 3.45−3.84 (5H, m), 3.85 (3H, s), 5.15 (2H, s), 6.93−6.97 (5H, m), 7.06−7.13 (5H, m), 7.20− 7.23 (2H, m), 8.53 (1H, s), 10.33 (1H, br); 13C NMR (125 MHz, CDCl3) δ 20.7, 31.0, 32.4, 39.3, 55.4, 55.9, 94.5, 114.2, 116.3, 119.2, 119.4, 120.6, 121.0, 124.4, 129.3, 134.7, 136.9, 138.8, 141.0, 143.0,

quint, J = 7.5 Hz), 2.70 (2H, t, J = 7.5 Hz), 3.45−3.50 (5H, m), 3.82 (3H, s), 5.13 (2H, s), 6.65 (1H, s), 6.75 (1H, d, J = 7.5 Hz), 6.82−6.94 (4H, m), 7.14−7.18 (1H, m), 7.25−7.27 (3H, m), 7.30 (1H, d, J = 7.5 Hz), 8.42 (1H, s), 10.75 (1H, br); 13C NMR (125 MHz, CDCl3) δ 30.9, 33.4, 39.6, 55.5, 56.1, 94.5, 102.4, 111.8, 113.4, 113.8, 114.1, 116.5, 122.2, 124.8, 129.5, 130.8, 137.3, 141.4, 143.1, 148.8, 155.3, 156.8, 157.4, 161.8, 162.5, 163.8, 171.8; IR (KBr) 3442, 1653, 1559, 1506 cm−1; MS (EI) m/z 488 (M+); HRMS Calcd for C28H28N2O6 488.1947, Found 488.1943. 7-Hydroxy-2-(4-methoxyphenylimino)-2H-chromene-3-carboxylic Acid [3-(3-Hydroxyphenyl)propyl]amide (3a′). Yield 65%; mp 235−236 °C; 1H NMR (500 MHz, DMSO-d6) δ 1.80 (2H, quint, J = 7.6 Hz), 2.57 (2H, t, J = 7.6 Hz), 3.29−3.30 (2H, m), 3.77 (3H, s), 6.53 (1H, d, J = 2.3 Hz), 6.56 (1H, dd, J = 8.3, 2.3 Hz), 6.59 (1H, s), 6.60 (1H, d, J = 8.9 Hz), 6.72 (1H, dd, J = 8.3, 2.3 Hz), 6.95 (2H, d, J = 8.6 Hz), 7.04 (1H, t, J = 8.3 Hz), 7.32 (2H, d, J = 8.6 Hz), 7.60 (1H, d, J = 8.9 Hz), 8.39 (1H, s), 9.24 (1H, br), 10.22 (1H, t, J = 5.6 Hz); 13 C NMR (125 MHz, DMSO-d6) δ 31.1, 33.2, 39.0, 55.7, 101.8, 111.5, 113.3, 113.5, 114.5, 115.7, 117.5, 119.5, 125.3, 129.8, 131.7, 137.5, 141.1, 143.3, 148.9, 155.1, 156.8, 157.9, 162.2, 162.9; IR (KBr) 3190, 1666, 1566, 1448 cm−1; MS (EI) m/z 444 (M+); HRMS calcd for C26H24N2O5 444.1685, found 444.1689; Purity 97% (HPLC). 7-Hydroxy-2-(4-methoxyphenylimino)-2H-chromene-3-carboxylic Acid [3-(2-Methoxymethoxyphenyl)propyl]amide (3b). Yield 63%; mp 198−199 °C; 1H NMR (500 MHz, CDCl3) δ 1.95 (2H, quint, J = 7.5 Hz), 2.77 (2H, t, J = 7.5 Hz), 3.41 (3H, s), 3.50 (2H, q, J = 7.5 Hz), 3.83 (3H, s), 5.13 (2H, s), 6.65 (1H, d, J = 2.5 Hz), 6.74 (1H, dd, J = 6.0, 2.5 Hz), 6.89−6.92 (3H, m), 7.05 (1H, d, J = 8.0 Hz), 7.12−7.16 (2H, m), 7.26−7.29 (3H, m), 7.33 (1H, d, J = 8.5 Hz), 8.44 (1H, s), 10.68 (1H, t, J = 5.5 Hz); 13C NMR (125 MHz, CDCl3) δ 27.9, 29.5, 39.6, 55.4, 55.9, 56.0, 94.2, 102.3, 107.1, 111.8, 113.2, 113.8, 114.0, 121.7, 124.7, 127.3, 129.6, 130.1, 130.3, 130.6, 137.2, 141.2, 155.09, 155.12, 156.7, 163.5; IR (KBr) 3348, 1652, 1558, 1506 cm−1; MS (EI) m/z 488 (M+); HRMS Calcd for C28H28N2O6 488.1947, Found 488.1943. 7-Hydroxy-2-(4-methoxyphenylimino)-2H-chromene-3-carboxylic Acid [3-(4-Methoxymethoxyphenyl)propyl]amide (3c). Yield 76%; mp 100−103 °C; 1H NMR (500 MHz, DMSO-d6) δ 1.93 (2H, quint, J = 7.5 Hz), 2.68 (2H, t, J = 7.5 Hz), 3.35−3.46 (5H,m), 3.84 (3H, s), 5.13 (2H, s), 6.61 (1H, s), 6.70 (2H, d, J = 8.5 Hz), 6.93 (4H, t, J = 8.5 Hz), 7.11 (2H, d, J = 8.5 Hz), 7.37 (1H, d, J = 8.5 Hz), 7.62 (1H, d, J = 8.5 Hz), 8.42−8.50 (1H, m), 10.50 (1H, t, J = 4.75 Hz); 13 C NMR (125 MHz, DMSO-d6) δ; 31.0, 32.4, 39.3, 55.4, 55.9, 88.6, 94.5, 102.3, 113.2, 114.0, 116.3, 124.6, 129.4, 130.7, 134.7, 141.2, 148.6, 155.2, 155.5, 156.7, 161.9, 163.6, 177.9; IR (KBr) 3443, 1653, 1559, 1507 cm−1; MS (EI) m/z 488 (M+); HRMS Calcd for C28H28N2O6 488.1947, Found 488.1943. 7-Hydroxy-2-(4-methoxyphenylimino)-2H-chromene-3-carboxylic Acid [3-(3-Fluorophenyl)propyl]amide (3d). Yield 75%; mp 208− 210 °C; 1H NMR (500 MHz, DMSO-d6) δ 1.85 (2H, quint, J = 7.5 Hz), 2.68 (2H, t, J = 7.5 Hz), 3.17−3.29 (2H, m), 3.77 (3H, s), 6.53 (1H, s), 6.72 (1H, dd, J = 2.0, 8.5 Hz), 6.84−6.97 (3H, m), 7.05 (2H, d, J = 8.5 Hz), 7.30 (3H, d, J = 8.5 Hz), 7.60 (1H, d, J = 8.5 Hz), 8.38 (1H, s), 10.21 (1H, t, J = 5.5 Hz); 13C NMR (125 MHz, DMSO-d6) δ 30.9. 32.8, 38.9, 55.8, 101.9, 111.5, 113.1 (d, J = 20.6 Hz), 113.5, 114.5, 115.5, 115.6, 117.5, 125.0 (d, J = 2.5 Hz), 125.2, 130.7 (d, J = 8.5 Hz), 131.7, 137.5, 141.1, 145.0 (d, J = 7.38 Hz), 148.9, 155.1, 156.8, 162.3, 162.9, 162.8 (d, J = 241.75 Hz); IR (KBr) 3447, 1662, 1559, 1506 cm−1; MS (EI) m/z 446 (M+); HRMS Calcd for C26H23FN2O4 446.1642, Found 446.1641. 7-Hydroxy-2-(4-methoxyphenylimino)-2H-chromene-3-carboxylic Acid [3-(4-Fluorophenyl)propyl]amide (3e). Yield 68%; mp 207− 209 °C; 1H NMR (500 MHz, DMSO-d6) δ 1.94 (2H, quint, J = 7.5 Hz), 2.70 (2H, t, J = 7.5 Hz), 3.46 (2H, t, J = 7.5 Hz), 3.84 (3H, s), 6.62 (1H, s), 6.71 (1H, d, J = 8.5 Hz), 6.93 (4H, t, J = 8.5 Hz), 7.13 (2H, t, J = 8.5 Hz), 7.24 (1H, s), (1H, d, J = 8.5 Hz), 8.452 (1H, s), 10.54 (1H, s); 13C NMR (125 MHz, DMSO-d6) δ 35.2, 36.2, 42.9, 59.7, 105.8, 115.5, 117.5, 118.5, 119.4 (d, J = 21.9 Hz), 121.4, 129.2, 132.8 (d, J = 3.8 Hz), 134.9 (d, J = 8.5 Hz), 135.8, 141.5, 142.0 (d, J = 2.4 Hz), 145.1, 152.8, 165.1 (d, J = 240.6 Hz), 166.2, 166.8; IR (KBr) 8450



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147.6, 155.4, 156.9, 162.4; IR (KBr) 6424, 1662, 1558, 1506 cm−1; MS (EI) m/z 488 (M+); HRMS Calcd for C28H28N2O6 488.1947, Found 488.1952. General Procedure for the Synthesis of Coumarin (4a−l). To a stirred solution of phenyliminochromene (3a−k, 1 mmol) in THF (5 mL) was added 10% HCl (5 drops), and the resulting mixture was heated at 40 °C for 12 h. After cooling, H2O was added to the reaction mixture, and the aqueous mixture was extracted with EtOAc (5 mL × 3). The organic extracts were combined, dried over Na2SO4, and evaporated. The residue was chromatographed on silica gel (15 g, CH2Cl2/MeOH = 100:1) to give the corresponding coumarin (4a−l) as solid. The compounds for the measurement of mp, HPLC analysis, and biological tests were used after recrystallization from MeOH or EtOH, which is indicated after mp. 7-Hydroxy-2-oxo-2H-chromene-3-carboxylic Acid [3-(3Hydroxyphenyl)propyl]amide (4a). Yield 71%; mp 243−245 °C (MeOH); 1H NMR (500 MHz, DMSO-d6) δ 1.91 (2H, quint, J = 7.5 Hz), 2.64 (2H, t, J = 7.5 Hz), 3.44 (2H, q, J = 7.5 Hz), 6.65−6.66 (1H, m), 6.71−6.74 (2H, m), 6.85 (1H, d, J = 2.5 Hz), 6.97 (1H, dd, J = 6.0, 2.5), 7.09 (1H, t, J = 8.0 Hz), 7.79 (1H, d, J = 8.5 Hz), 8.74 (1H, br), 8.80 (1H, s); 13C NMR (125 MHz, DMSO-d6) δ 31.1, 33.0, 102.3, 111.6, 113.3, 114.3, 114.9, 115.7, 119.4, 129.8, 132.5, 143.4, 148.4, 156.8, 157.8, 161.6, 164.2; IR (KBr) 3340, 1700, 1545 cm−1; MS (EI) m/z 339 (M+); HRMS Calcd for C19H17NO5 339.3420, Found 339.1106; Purity >98% (HPLC). 7-Hydroxy-2-oxo-2H-chromene-3-carboxylic Acid [3-(2Hydroxyphenyl)propyl]amide (4b). Yield 64%; mp 220−222 °C (MeOH); 1H NMR (500 MHz, DMSO-d6) δ 1.92 (2H, quint, J = 7.5 Hz), 2.72 (2H, t, J = 7.5 Hz), 3.44 (2H, q, J = 7.5 Hz), 6.83−6.85 (2H, m), 6.96−7.03 (2H, m), 7.14 (1H, d, J = 7.5 Hz), 8.80 (1H, s), 8.81 (1H, br); 13C NMR (125 MHz, DMSO-d6) δ 27.5, 29.8, 39.2, 102.3, 111.7, 114.3, 114.8, 115.4, 119.4, 127.4, 128.1, 130.3, 132.4, 148.4, 155.6, 156.8, 161.6, 162.0, 164.1; IR (KBr) 3420, 1700, 1540, 1227 cm−1; MS (EI) m/z 339 (M+); HRMS Calcd for C19H17NO5 339.3420, Found 339.1106; Purity 97% (HPLC). 7-Hydroxy-2-oxo-2H-chromene-3-carboxylic Acid [3-(4Hydroxyphenyl)propyl]amide (4c). Yield 95%; mp 220−222 °C (MeOH); 1H NMR (500 MHz, acetone-d6) δ 1.84 (2H, quint, J = 7.5 Hz), 2.58 (2H, t, J = 7.5 Hz), 3.38 (2H, q, J = 7.5 Hz), 6.72 (2H, d, J = 8.5 Hz), 6.82 (1H, d, J = 2.0 Hz), 6.94 (1H, dd, J = 6.5, 2.0 Hz), 6.04 (2H, d, J = 8.5 Hz), 7.76 (1H, d, J = 8.5 Hz), 8.69 (1H, br), 8.77 (1H, s); 13C NMR (125 MHz, DMSO-d6) δ 31.5, 32.2, 39.1, 102.3, 111.6, 114.3, 114.9, 115.6, 129.6, 132.0, 132.5, 148.4, 155.9, 156.8, 161.6, 162.1, 164.1; IR (KBr) 3442, 1734, cm−1; MS (EI) m/z 339 (M+); HRMS Calcd for C19H17NO5 339.3420, Found 339.1106; Purity >98% (HPLC). 7-Hydroxy-2-oxo-2H-chromene-3-carboxylic Acid [3-(3Fluorophenyl)propyl]amide (4d). Yield 90%; mp 209−210 °C (MeOH); 1H NMR (500 MHz, DMSO-d6) δ 1.83 (2H, quint, J = 7.5 Hz), 2.64 (2H, t, J = 7.5 Hz), 3.21−3.35 (2H, m), 6.79 (1H, s), 6.86 (1H, dd, J = 2.0, 8.5 Hz), 6.98 (1H, t, J = 8.5 Hz), 7.05 (2H, d, J = 8.5 Hz), 7.30 (1H, q, J = 8.5 Hz), 7.80 (2H, d, J = 8.5 Hz), 8.66 (1H, t, J = 5.8 Hz), 8.75 (1H, s); 19F NMR (376 MHz, DMSO-d6) δ −113.67; 13 C NMR (125 MHz, DMSO-d6) δ 30.9, 32.7, 39.0, 102.3, 111.7, 113.1(d, J = 20.63 Hz), 114.4, 114.9, 115.5 (d, J = 20.6 Hz), 125.0 (d, J = 2.5 Hz), 125.3, 130.7 (d, J = 8.6 Hz), 125.3, 130.7 (d, J = 10.7 Hz), 132.5, 145.1 (d, J = 6.0 Hz), 148.4, 156.8, 161.6, 163.1 (d, J = 246.8 Hz), 163.8; IR (KBr) 3562, 1705, 1550, 1226 cm−1; MS (EI) m/z 341 (M+); HRMS Calcd for C19H16FNO4 341.1063, Found 341.1067; Purity 98% (HPLC). 7-Hydroxy-2-oxo-2H-chromene-3-carboxylic Acid [3-(4Fluorophenyl)propyl]amide (4e). Yield 96%; mp 207−209 °C (EtOH); 1H NMR (500 MHz, DMSO-d6) δ 1.95 (2H, quint, J = 7.5 Hz), 2.70 (2H, t, J = 7.5 Hz), 3.49 (2H, q, J = 7.5 Hz), 6.88−6.91 (2H, m), 6.94−6.98 (2H, m), 7.14−7.18 (2H, m), 7.20−7.23 (1H, m), 7.57 (1H, d, J = 8.5 Hz), 8.83 (1H, s), 8.87 (1H, br); 19F NMR (376 MHz, DMSO-d6) δ −117.54; 13C NMR (125 MHz, DMSO-d6) δ; 31.3, 32.1, 39.0, 102.3, 111.6, 114.3, 114.8, 115.4 (d, J = 20.6 Hz), 128.8 (d, J = 3.6 Hz), 130.5 (d, J = 8.6 Hz), 132.4, 138.1, 148.4, 156.7, 156.7, 161.1 (d, J = 239.9 Hz), 164.6, IR (KBr) 3572, 1717, 1558,

1267 cm−1; MS (EI) m/z 341 (M+); HRMS Calcd for C19H16FNO4 341.1063, Found 341.1065; Purity >98% (HPLC). 7-Hydroxy-2-oxo-2H-chromene-3-carboxylic Acid [3-(3,5Difluorophenyl)propyl]amide (4f). Yield 92%; mp 213-214 °C (MeOH); 1H NMR (500 MHz, DMSO-d6) δ 1.85 (2H, quint, J = 7.5 Hz), 2.67 (2H, t, J = 7.5 Hz), 3.27−3.41 (2H, m), 6.81 (1H, s), 6.88 (2H, d, J = 8.5 Hz), 6.99−7.02 (3H, m), 7.82 (1H, d, J = 8.5 Hz), 8.67 (1H, t, J = 5.5 Hz), 8.78 (1H, s); 19F NMR (376 MHz, DMSOd6) δ −110.54; 13C NMR (100 MHz, DMSO-d6) δ 30.5, 32.6, 38.9, 101.7 (t, J = 28.3 Hz), 102.3, 111.6, 111.9 (dd, J = 4.9, 19.4 Hz), 114.2, 114.8, 132.4, 146.8 (t, J = 9.1 Hz), 148.4, 156.7, 161.5, 162.8 (d, J = 244.3 Hz), 162.1, 162.9 (d, J = 244.3 Hz), 164.1; IR (KBr) 3227, 1718, 1549, 1226 cm−1; MS (EI) m/z 359 (M+); HRMS Calcd for C19H15F2NO4 359.0969, Found 359.0968; Purity 95% (HPLC). 7-Hydroxy-2-oxo-2H-chromene-3-carboxylic Acid (3-pTolylpropyl)amide (4g). Yield 90%; mp 220−222 °C (MeOH); 1H NMR (500 MHz, DMSO-d6) δ 1.79 (2H, quint, J = 7.5 Hz), 2.23 (3H, s), 2.56 (2H, t, J = 7.5 Hz), 3.25−7.37 (2H, m), 6.78 (1H, s), 6.80 (1H, dd, J = 2.0, 8.5 Hz), 7.05 (4H, q, J = 8.5 Hz), 7.79 (1H, d, J = 8.5 Hz), 8.64 (1H, t, J = 5.8 Hz), 8.74 (1H, s); 13C NMR (125 MHz, DMSO-d6) δ 21.1, 31.3, 32.6, 39.1, 102.3, 111.7, 114.4, 114.8, 128.7, 129.4, 132.5, 135.1, 138.9, 148.4, 156.8, 161.6, 162.0, 164.1; IR (KBr) 3214, 1715, 1549 cm−1; MS (EI) m/z 337 (M+); HRMS Calcd for C20H19NO4 337.1314, Found 337.1311; Purity >98% (HPLC). 7-Hydroxy-2-oxo-2H-chromene-3-carboxylic Acid [3-(4Methoxyphenyl)propyl]amide (4h). Yield 95%; mp 205−207 °C (MeOH); 1H NMR (500 MHz, acetone-d6) δ 1.95 (2H, quint, J = 7.5 Hz), 2.69 (2H, t, J = 7.5 Hz), 3.47 (2H, q, J = 7.5 Hz), 3.79 (3H, s), 6.87−6.89 (3H, m), 7.02 (1H, dd, J = 6.0, 2.5 Hz), 7.21 (2H, d, J = 6.5 Hz), 7.84 (1H, d, J = 8.5 Hz), 8.76 (1H, br), 8.84 (1H, s); 13C NMR (125 MHz, DMSO-d6) δ 31.4, 32.1, 39.1, 55.4, 102.3, 111.6, 114.2, 114.3, 114.8, 129.7, 132.4, 133.8, 148.4, 156.7, 157.9, 161.6, 162.0, 164.1; IR (KBr) 3318, 1706, 1540 cm−1; MS (EI) m/z 353 (M+); HRMS Calcd for C20H19NO5 353.1263, Found 353.1262; Purity 95% (HPLC). 7-Hydroxy-2-oxo-2H-chromene-3-carboxylic Acid [4-(4Hydroxyphenyl)butyl]amide (4i). Yield 66%; mp 225−226 °C (MeOH); 1H NMR (500 MHz, DMSO-d6) δ 1.59−1.63 (4H, m), 2.55 (2H, t, J = 7.5 Hz), 3.45 (2H, q, J = 7.5 Hz), 6.75 (2H, d, J = 8.5 Hz), 6.82 (1H, d, J = 2.0 Hz), 6.95 (1H, dd, J = 6.5, 2.0 Hz), 7.01 (2H, d, J = 8.5 Hz), 7.76 (1H, d, J = 8.5 Hz), 8.10 (1H, br), 8.76 (1H, s); 13 C NMR (125 MHz, DMSO-d6) δ 29.2, 29.2, 34.4, 39.3, 102.3, 111.6, 114.2, 114.8, 115.5, 129.6, 132.4, 132.6, 148.4, 155.8, 156.7, 161.6, 161.9; IR (KBr) 3312, 1707, 1550 1227 cm−1; MS (EI) m/z 353 (M+); HRMS Calcd for C20H19NO5 353.1263, Found 353.1262; Purity >98% (HPLC). 2-Oxo-2H-chromene-3-carboxylic Acid [3-(4-Hydroxyphenyl)propyl]amide (4j). Yield 67%; mp 194−196 °C (MeOH); 1H NMR (500 MHz, DMSO-d6) δ 1.92 (2H, quint, J = 7.5 Hz), 2.65 (2H, t, J = 7.5 Hz), 3.27−3.41 (2H, m), 6.76 (2H, d, J = 8.5 Hz), 7.06 (2H, d, J = 8.5 Hz), 7.37−7.42 (2H, m), 7.67 (2H, m), 8.92 (1H, s); 13C NMR (125 MHz, DMSO-d6) δ 31.4, 32.1, 39.8, 115.6, 116.6, 119.0, 119.7, 125.6, 129.6, 130.7, 131.9, 134.5, 147.7, 154.3, 155.9, 160.9, 161.6; IR (KBr) 3345, 1717, 1608, 1569 cm−1; MS (EI) m/z 323 (M+); HRMS Calcd for C19H17NO4 23.1158, Found 323.1156; Purity >98% (HPLC). 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic Acid [3-(4Hydroxyphenyl)propyl]amide (4k). Yield 86%; mp 225−227 °C (MeOH); 1H NMR (500 MHz, acetone-d6) δ 1.93 (2H, quint, J = 7.5 Hz), 2.67 (2H, t, J = 7.5 Hz), 3.45 (2H, q, J = 7.5 Hz), 6.80 (2H, d, J = 8.5 Hz), 7.12 (2H, d, J = 8.5 Hz), 7.29−7.41 (2H, m), 7.45 (1H, t, J = 5.0 Hz), 8.14 (1H, s), 8.82 (1H, br), 8.89 (1H, s); 13C NMR (125 MHz, DMSO-d6) δ 31.4, 32.1, 39.2, 115.6, 119.5, 119.9, 120.5, 125.6, 129.6, 131.9, 143.0, 144.9, 148.1, 155.8, 160.8, 161.7; IR (KBr) 3390, 1718, 1610 cm−1; MS (EI) m/z 339 (M+); HRMS Calcd for C19H17NO5 339.1107, Found 339.1108; Purity >98% (HPLC). 7-Hydroxy-2-oxo-2H-chromene-3-carboxylic Acid [2-(4Hydroxyphenyl)ethyl]amide (4l).45 Yield 65%; mp 271−273 °C (MeOH); 1H NMR (500 MHz, DMSO-d6) δ 2.81 (2H, t, J = 7.5 Hz), 3.61 (2H, q, J = 7.5 Hz), 6.78 (2H, dd, J = 4.5, 2.0 Hz), 7.11 (2H, dd, J 8451



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= 4.5, 2.0 Hz), 7.79 (1H, d, J = 8.5 Hz), 8.73 (1H, br), 8.80 (1H, s); 13 C NMR (125 MHz, DMSO-d6) δ 34.7, 41.4, 102.3, 111.6, 114.0, 114.9, 115.7, 129.7, 130.0, 132.5, 148.5, 156.2, 161.5, 161.9, 164.2; IR (KBr) 3313, 1709, 1539 cm−1; MS (EI) m/z 325 (M+); HRMS Calcd for C19H17NO5 325.3154, Found 325.0947; Purity 96% (HPLC). Improved Synthesis of 4e from 5. To a stirred solution of pfluorobenzenepropanamide47 (200 mg, 1.2 mmol) in THF (10 mL) was added LiAlH4 (136 mg, 3.6 mmol), and the resulting suspension was refluxed for 18 h. After cooling, the reaction mixture was diluted with AcOEt (10 mL) and then quenched with 10% NaOH aq. at 0 °C. The insoluble materials were filtered off using Celite, and the filtrate was dried over Na2SO4 and concentrated in vacuo to yield corresponding amine, which was used for the next reaction without further purification. To a stirred solution of the amine obtained above in THF (6 mL) were added 7-hydroxycoumarin-3-carboxylic acid (190 mg, 0.9 mmol)46 and EDC (265 mg, 1.4 mmol) at room temperature, and the resulting mixture was stirred at room temperature for 1 h. To the reaction mixture was added N-methylmorpholine (0.2 mL, 1.8 mmol), and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with AcOEt (10 mL) and 10% HCl aq., and the organic phase was separated; the aqueous mixture was extracted with AcOEt (5 mL × 3). The organic phase and extracts were combined, dried over Na2SO4, and concentrated in vacuo. The residue was chromatographed on SiO2 (20 g, CH2Cl2/MeOH = 100:1) to give 4e (314 mg, 78%) as a yellow solid. Enzyme Purification. Homogenous recombinant AKR1B10,19 AKR1B1,61 AKR1A1,49 AKR1C isoforms (1C1−1C4),62,63 CBR1,64 and mutant enzymes of AKR1B10 (K125L, W220Y, V301L, and Q303S)43,65,66 were prepared and purified to homogeneity as previously described. All enzymes were stored at −20 °C in 10 mM potassium phosphate buffer, pH 7.0, containing 20% glycerol, 1 mM EDTA, and 5 mM 2-mercaptoethanol. Enzyme Activity Assay. The potency of the compounds was determined by measuring their ability to inhibit the reductase or dehydrogenase activities of the enzymes, which were assayed at 25 °C by measuring the rate of change in NADPH absorbance at 340 nm. The reaction mixture (total volume of 2.0 mL) consisted of 0.1 M potassium phosphate, pH 7.4, coenzyme (0.1 mM NADPH or 0.25 mM NADP+), substrate, enzyme, and inhibitor.19 The inhibitors were dissolved in methanol and added to the reaction mixture, not to exceed 2% methanol concentration. The reaction was started by the addition of the enzyme. The enzyme concentrations of AKR1B10 and AKR1B1 were 3−6 μg/mL. The IC50 values for inhibitors were determined in the NADPH-linked reductase activities toward 0.2 mM pyridine-3-aldehyde (for AKR1B10 and AKR1B1),19,44 2 mM pyridine-3-aldehyde (for AKR1A1), and 50 μM isatin (for CBR1).49 In the IC50 determination of AKR1C1−1C4, their NADP+-linked dehydrogenase activities were assayed using (S)-(+)-1,2,3,4-tetrahydro-1-naphthol (S-tetralol) as the substrate.67 Kinetic studies of AKR1B10 and its mutant enzymes in the presence of inhibitors were carried out in NADP+-linked geraniol oxidation over a range of five substrate concentrations [(0.5−5) × Km] at a saturating concentration (0.25 mM) of the coenzyme.19 The inhibition patterns were analyzed from the Lineweaver−Burk plots (double reciprocal plots of the initial velocities versus concentrations of substrate), and Ki (inhibition constant) for the competitive inhibitor was estimated from replots of the slopes of the Lineweaver−Burk plots versus inhibitor concentrations as described previously.19,44 Molecular Modeling. The modeling of binding mode of 4c with AKR1B10 was performed using the Schrö dinger suite 2013-2 (Schrödinger, LLC, New York, NY, 2013). The 3D structure of 4c was first generated using the LigPrep 2.7 program. The conformational search of 4c was then performed using the ConfGen 2.5 program, and the resulting conformers were used in the following docking calculations. The X-ray structure of the AKR1B10 in complex with caffeic acid phenethyl ester (CAPE) (PDB ID 4GQ0) was used as a receptor for docking. Because CAPE possesses a similar chemical structure to 4c, it would be more adequate to use the receptor structure derived from the CAPE−AKR1B10 complex. The X-ray structure was minimized using force-field OPLS 2005 through the

Protein Preparation Wizard in Maestro 9.5. We removed H2O molecules before docking calculations. The docking calculation of 4c against AKR1B10 was performed using the Glide 6.0 SP mode. Box center for the “Receptor Grid Generation” protocol was set to a centroid of CAPE binding to AKR1B10. The van der Waal radius scaling of 1.0 was used for both protein and ligand, and the maximum number of poses per conformer was set to 2. The generated poses were ranked according to docking score, and we finally selected a docked complex with the lowest docking score as the interaction model. In order to test the docking procedure, we applied it for the CAPE−AKR1B10 complex. The positional and conformational rootmean-square deviations between X-ray and docked poses were 0.34 and 0.17 Å, respectively. This result suggested that the procedure is adequate for producing a reliable binding model between CAPE and AKR1B10. Cell Culture. Lung cancer A549 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C in a humidified incubator containing 5% CO2. The A549/1B10 cells68,69 and CDDP-R-A549 cells31 were established as reported previously. For establishment of the subpopulation resistant to CDDP, the A549 cells were continuously treated in the growth medium supplemented with CDDR whose concentration was increased in a stepwise manner (0.05−5 μM). Measurement of Metastatic and Invasive Potential. Migrating potential of the cells was determined by wound-healing and Boyden chamber assays as described previously.32 Briefly, the cells were seeded into a 6-well culture plate at a density of 3 × 105 cells/well. After reaching a 90% confluence of the cells, the cell monolayer was scratched with a 10-μL pipet tip, washed twice with the growth medium, and then incubated for 24-h in serum-free DMEM supplemented with AKR1B10 inhibitors or the vehicle DMSO, in order to allow the cells to migrate from the wound surface to the cellfree area. The migrating potential was evaluated by microscopically measuring the width of the cell-free area. In case of the Boyden chamber assay, the cells were seeded on a polycarbonate membrane (8-μm pore) at a density of 1 × 104 cells/well and cultured for 36 h. After medium in the upper layer of the membrane was replaced with fresh growth medium, the cells were incubated for 48 h in serum-free DMEM supplemented with AKR1B10 inhibitors or the vehicle DMSO. The cells that migrated to the underside of the membrane were fixed with methanol and stained with Giemsa solution, and their number was counted microscopically. Invasive activity of cells was similarly analyzed using the polycarbonate membrane coated with type-I collagen (Nitta Gelatin, Osaka, Japan). Measurement of Proliferation Rate and CDDP Sensitivity. In proliferation assay, A549 cells and CDDP-R-A549 cells were seeded into a 48-well multiplate at a density of 2 × 104 cells/well, and then treated for 24 or 96 h with AKR1B10 inhibitors. To evaluate CDDP sensitivity of A549 cells and CDDP-R-A549 cells, the cells were seeded into a 96-well multiplate at a density of 2 × 104 cells/well, and then treated for 24 h with AKR1B10 inhibitors in the presence or absence of 0.5 or 40 μM CDDP. Proliferation rate and drug sensitivity of the cells were estimated by monitoring the cell viability, which was evaluated by tetrazolium dye-based cytotoxicity assay using 2-(4iodophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium.70 Experimental Metastasis Assay by iv Inoculation. Female 6week-old C.B-17/lcrHsd-Prkdcscid (SCID) mice were purchased from Japan SLC (Hamamatsu, Japan). All experiments were approved and carried out according to the guidelines of the Care and Use of Laboratory Animals of University of Toyama (Toyama, Japan). The experimental lung metastasis of A549/Luc2 cells was performed as previously described.71 Briefly, A549/Luc2 were precultured in the absence or presence of 20 μM 4c or 4e for 48 h and inoculated intravenously (2 × 106 cells/200 μL PBS/mouse) into mice. Four days after the tumor inoculation, mice were intraperitoneally injected with 200 μL of 1.5 mg/mL luciferin (VivoGlo; Promega, Madison, WI, USA) 10 min prior to removal of the lungs for bioluminescent assay 8452



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using an in vivo imaging system (IVIS Lumina II, Caliper Life Sciences, Hopkinton, MA, USA). Statistical Analysis. Data are expressed as the means ± SD of at least three independent experiments, unless otherwise noted. Statistical evaluation of the data was performed by using the unpaired Student’s t test and ANOVA followed by Fisher’s test.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00830. Data of 4c-binding modes in AKR1B10 and AKR1B1 and effect of alanine scanning on binding free energy for 4c (PDF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*S.E.: e-mail, [email protected]; phone, +81-58-230-8100; fax, +81-58-230-8105. *N.T.: e-mail, [email protected]; phone, +81-76445-6859; fax, +81-76-445-6703. ORCID

Satoshi Endo: 0000-0003-0578-9672 Yoshikazu Horino: 0000-0002-8916-6298 Author Contributions

S.E. and N.T. participated in research design and mainly contributed to writing the paper. S.X., D.H., Y.A., S.T., and Y.H. synthesized novel compounds. M.S., Y.M., H.O., and T.M. performed the biological analyses. Y.H. performed the in vivo study. Y.W. and H.G. contributed to the molecular docking. H.G., A.H, K.K., T.M., and A.I. contributed to the experiment plan and discussion of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was partially supported by JSPS Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (MEXT KAKENHI Grant Number JP26460149).

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ABBREVIATIONS USED AKR, aldo-keto reductase; AKR1B1, human aldose reductase; A549/1B10 cells, A549 cells stably overexpressing AKR1B10; BDMC, bisdemethoxycurcumin; CAPE, caffeic acid phenethyl ester; CBR1, carbonyl reductase 1; CDDP, cisplatin; CDDP-RA549 cells, CDDP-resistant A549 cells; DMAP, 4-dimethylaminopyridine; DMEM, Dulbecco’s modified Eagle medium; DMSO, dimethyl sulfoxide; EDC, 1-ethyl-3-(3dimethylaminopropyl)carbodiimide; EGFR, epithelial growth factor receptor; HAHE, 3-(4-hydroxy-2-methoxyphenyl)acrylic acid 3-(3-hydroxyphenyl) propyl ester; HMCB, 7-hydroxy-2(4-methoxyphenylimino)-2H-chromene-3-carboxylic acid benzylamide; MHPC, (Z)-2-(4-methoxyphenylimino)-7-hydroxyN-(pyridin-2-yl)-2H-chromene-3-carboxamide; MM-PBSA, molecular mechanics Poisson−Boltzmann surface area; NSCLC, non-small-cell lung carcinoma 8453



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NOTE ADDED AFTER ASAP PUBLICATION After this paper was published ASAP on October 13, 2017, Scheme 2 was corrected. The revised version was reposted October 17, 2017.

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