Discovery of 5-Cyano-6-phenylpyrimidin Derivatives Containing an

Article Cite This: J. Med. Chem. 2018, 61, 5988−6001

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Discovery of 5‑Cyano-6-phenylpyrimidin Derivatives Containing an Acylurea Moiety as Orally Bioavailable Reversal Agents against P‑Glycoprotein-Mediated Mutidrug Resistance Bo Wang,†,§ Li-Ying Ma,†,§ Jing-Quan Wang,‡,§ Zi-Ning Lei,‡ Pranav Gupta,‡ Yuan-Di Zhao,† Zhong-Hua Li,† Ying Liu,† Xin-Hui Zhang,† Ya-Nan Li,† Bing Zhao,*,† Zhe-Sheng Chen,*,‡ and Hong-Min Liu*,† Downloaded via UNIV OF SUSSEX on July 28, 2018 at 05:04:28 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province; Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education; Key Laboratory of Henan Province for Drug Quality and Evaluation; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, P. R. China ‡ Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, New York 11439, United States S Supporting Information *

ABSTRACT: P-glycoprotein (ABCB1)-mediated multidrug resistance (MDR) has become a major obstacle in successful cancer chemotherapy, which attracted much effort to develop clinically useful compounds to reverse MDR. Here, we designed and synthesized a novel series of derivatives with a 5-cyano-6-phenylpyrimidine scaffold and evaluated their potential reversal activities against MDR. Among these compounds, 55, containing an acylurea appendage, showed the most potent activity in reversing paclitaxel resistance in SW620/AD300 cells. Further studies demonstrated 55 could increase accumulation of PTX, interrupt ABCB1-mediated Rh123 accumulation and efflux, stimulate ABCB1 ATPase activity, and especially have no effect on CYP3A4 activity, which avoid drug interaction caused toxicity. More importantly, 55 significantly enhanced the efficacy of PTX against the SW620/AD300 cell xenograft without obvious side effects for orally intake. Given all that, the pyrimidine-acylurea based ABCB1 inhibitor may be a promising lead in developing new efficacious ABCB1-dependent MDR modulator.

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everolimus) are substrates of ABCB1.9−13 Down-regulation or inhibition of ABCB1 can effectively enhance the anticancer efficacy of conventional chemotherapeutic agents.14 Because of its overexpression in cancer cells, ABCB1 has become a therapeutic target for getting around MDR.15 Several reviews concerning the discovery of reversal agents targeting ABCB1 protein have been published.16−19 In clinical work, combining ABCB1 modulators with chemotherapeutic agents has been acknowledged as a promising therapeutic strategy to get around ABCB1-mediated MDR. Therefore, over the past few decades, considerable efforts have been made in developing ABCB1 inhibitors.20−32 According to their specificity, affinity, and toxicity, ABCB1 modulators are classified into three generations. First-generation MDR reversal agents are represented by drugs in clinical use for other indications (e.g.,

INTRODUCTION Multidrug resistance (MDR) has become a major obstacle in successful cancer chemotherapy.1,2 Although it is reported that MDR is involved with multiple mechanisms, the overexpression of some members of the ATP-binding cassette (ABC) protein family is thought to be a major contributor to the development of MDR in cancer cells.3−5 P-glycoprotein (ABCB1), a main member of ABC transport proteins, is encoded by MDR1 gene and pumps specific chemotherapeutic agents out of the cancer cells by the energy of adenosine triphosphate (ATP) hydrolysis, thereby decreasing the intracellular drug accumulation and resulting in drug resistance.6,7ABCB1 was found to be widely overexpressed in human solid tumors and hematologic malignancies, such as acute lymphocytic leukemia, colon, liver, pancreas, and kidney cancers.8 It was shown that a broad variety of important chemotherapeutic drugs with different structures such as paclitaxel, camptothecin analogues, anthracyclines, vinca alkaloids, and tyrosine kinase inhibitors (e.g., imatinib, nilotinib, © 2018 American Chemical Society

Received: March 3, 2018 Published: July 5, 2018 5988

DOI: 10.1021/acs.jmedchem.8b00335 J. Med. Chem. 2018, 61, 5988−6001

Journal of Medicinal Chemistry

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Figure 1. Structures of PRA-1 and newly designed compounds containing an acylurea moiety.

Scheme 1. Synthesis of the Target Pyrimidine-Based Derivativesa

a Reagents and conditions: (a) absolute ethanol, absolute K2CO3, reflux, 10 h; (b) (i) propargyl bromide, dioxane, reflux; (ii) phosphorus oxychloride, reflux, 1 h; (c) CuSO4·5H2O, sodium ascorbate, THF−H2O (1:1), rt; (d) appropriate aniline, absolute ethanol, reflux, 6 h; (e) (i)oxalyl chloride, 1,2-dichloroethane, 90 °C; (ii) 2-aminopyridine or aniline, 0 °C; (f) NaN3, acetone−H2O.

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Table 1. PTX-Resistance Reversal Activity of 7−10 and 16−19 at 2 μM in SW620/AD300 Cellsa

compd

R1

IC50, PTX (μM)

RF

compd

R1

IC50, PTX (μM)

RF

7 8 9 10 VRP control

p-CH(CH3)2 p-CH3 m,p,m-tri-OCH3 p-Br

0.091 ± 0.003 0.193 ± 0.021 0.152 ± 0.011 1.235 ± 0.052 0.211 ± 0.012 4.186 ± 0.513

46.00 21.69 27.54 3.39 19.84 1.00

16 17 18 19

p-CH(CH3)2 p -CH3 m,p,m-tri-OCH3 p-Br

0.610 ± 0.052 0.241 ± 0.019 0.672 ± 0.073 2.978 ± 0.474

6.86 17.37 6.23 1.41

IC50 values were obtained from three independent repeats and represented as the mean ± SD.

a

sized based on series I to explore the effect of molecular size and lipophilicity on reversal activity. And then, after preliminarily reversal activity evaluations, compound 55, which showed the most potency, and two other compounds 52 and 60 with relative potent reversal activity were investigated for further mechanism of reversing ABCB1-mediated MDR.

quinidine, cyclosporine A, and verapamil) with limited selectivity and higher drug concentrations requirement.33 The second-generation agents are most analogues based on first generation drugs such as valspodar, dexverapamil, and elacridar. Despite undesirable toxic characteristics inherited limiting their pharmacological utilization, second-generation agents possessed higher activity and selectivity.34−36 In particular, the thirdgeneration ABCB1 inhibitors such as tariquidar possessed high affinity to ABCB1 at a nanomolar concentration to acquire more specific and effective inhibition against ABCB1 function. However, although several ABCB1 inhibitors have entered the clinical trial stage, there still have been no satisfactory results so far because of some limitations such as insignificant clinical effests, concerns about safely, or pharmacokinetic interaction.14,37,38 Therefore, developing more selective and effective novel ABCB1 inhibitors to reverse MDR has become an urgent requirement. In an effort to design and develop more selective and effective novel ABCB1 inhibitors to combat drug resistance, we previously conducted an activity test on our in-house structurally diverse molecular library (∼500 compounds) and identified the potent pyrimidine skeleton and subsequent extensive medicinal chemistry efforts leading to the discovery of potent triazole contained pyrimidine-based MDR mediator PRA-1 (Figure 1), which could effectively reverse PTX resistance in SW620/AD300 cells by increasing intracellular accumulation of PTX.39−41 Furthermore, lipophilicity has been identified as an important factor correlated to potent inhibitory activity against ABCB1, since the inhibitors majorly bind to the big hydrophobic pocket located within transmembrane portion of ABCB1 and lipophilicity helps them enter the cavity.42 Besides hydrophobicity, several features of ABCB1 inhibitors have been indicated as contributors of passive diffusion into cell membrane and/or inhibitory function, including halogen group, aromatic ring center, hydrogen bond acceptor, and positive ionizable groups.43,44 Our strategy of inhibitor design involved maximizing active site interactions, particularly by promoting a network of strong hydrogen bonding interactions with the large hydrophobic pocket for ABCB1. Therefore, in this work, we first evaluated the reversal activity of series I compounds with hydrogen bond donors and receptors properties (acetamide group) which have been identified from PRA-1.45 In addition, series II derivatives without 1,2,3-triazole group were synthe-

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RESULTS AND DISCUSSION Chemistry. The general synthesis route of the target pyrimidine-thiourea hybrids is depicted in Scheme 1. Benzaldehydes 1a−f, ethyl cyanoacetate 2, and thiourea 3 were prolonged heated in ethanol containing potassium carbonate to obtain 6-aryl-5-cyano-2-thiouracils 4a−f. Then, compounds 4a−d reacted with the propargyl bromide in dioxane to obtain the target derivatives 5a−d. Compounds 12 and 13 were readily synthesized from 11 and the corresponding arylamines following literature procedures. Compound 12 was allowed to react with sodium azide to yield 14. Compounds 6a− d and 15a−d were prepared via click reaction of compounds 5a−d with appropriately substituted benzyl azides and compound 14, respectively. Compounds 43a−h were prepared via reaction of compound 4a−f with appropriate 12, 13, respectively. Then, these highly activated intermediates (6a−d, 15a−d, and 43a−f) were reacted with appropriately substituted arylamines to obtain compounds 7−10, 16−42, and 44−72. Biological Testing. Effect of Target Compounds To Reverse PTX Resistance in SW620/AD300 Cells. In order to rule out the possibility that the potential toxic of compounds themselves may cause false positive results, we first evaluated the cytotoxicity of all tested compounds against sensitive SW620 cells and PTX-resistance SW620/AD300 cells, which overexpressed ABCB1. The data showed that almost all target compounds had no toxicity against both cell lines, and the survival rates of compounds 7−10, 16−42, and 44−72 at the concentration of 2.0 μM were all above 90%, which were suitable for testing their reversal acitivity in PTX-reisitance cell line SW620/AD300 (Tables S1, S2, and S3 in Supporting Information). Therefore, we chose 2 μM as the suitable incubation concentration for further study. The reversal-fold (RF) was usually used to represent the potency of MDR reversers, via the ratio of PTX IC50 values on SW620/AD300 cells between in the absence and in the presence of MDR modulators, and 0.1% DMSO was used as solvent 5990



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control. The nontoxic concentration of 2 μM was used to explore the effects of target compounds on reversal of PTXresistance in SW620/AD300 cells based on survival rate results. Data are depicted in Tables 1−4. PTX single-treatment

they displayed increased reversal activity compared with 26. In addition, the compounds 27−31 with electron-donating groups at R2 also showed increased activity compared with 20−25 with electron-withdrawing groups. As mentioned in the Introduction, lipophilicity has been identified as an important factor correlated to potent inhibitory activity against ABCB1. To explore the reasons for the decreased reversal activity of the series I compounds, the lipophilicity of compounds 7−10 and 16−19 has been tested and the results are shown in Figure S1, and compounds 16−19 containing acetamide group showed lower lipophilicity than the responding compounds 7−10 with higher reversal activity (Table 1). In addition, docking analysis found that the weaker activity of 18 may have resulted from more groups that solvate in water (such as the triazole group) and larger molecular size through docking analysis (Figure S2). Along with the lower binding energy score for 18 (XP Glide gscore, −6.833 kcal/mol) than 9 (XP Glide gscore, −8.453 kcal/ mol) at top-scoring poses, the weak binding affinity and reversal effect toward ABCB1 may result from lower lipophilicity and more groups that solvate in water and larger molecular size.43 However, two carbonyl oxygen atom of acetamide group of compound 18 were engaged in hydrogen bond with ABCB1 which inspired us to further optimization. On the basis of above results, to improve lipophilicity and reduce molecular size, compounds 44−69 without 1,2,3-triazole group were synthesized and their reversal effects are shown in Table 3. Unexpectedly, compounds 44 and 45 with electrondonating at R2 were less potent than corresponding compounds 17 and 27. In contrast, compound 47 with electron-withdrawing at R2 displayed significantly increased reversal activity, about 10fold more potent than 44−46. An opposite trend was observed at R1. Compounds 49, 50, and 64−68 with electron-withdrawing groups at R1 showed decreased reversal effect. These findings indicate that the electronic effects at R1 and R2 may be important contributors in determining activity. On the basis of above findings, further modifications were mainly focused on the position of substituent and the heteroatoms substitution. Compared with compounds (51, 55, and 56) at the 3substitution at R2, compounds (57, 53, and 54) at the 2,4substitution performed a relatively weak reversal effect. In particular, compound 55 with chlorine substitution in meta position at the phenyl ring in R2 displayed the most potent reversal activity compared to the ortho- and para-substituted compounds (53 and 54), about 40-fold more potent than verapamil. Surprisingly, compound 52 with bromine substitution in para position at the phenyl ring in R2 was about 10-fold more potent than compound 54 with chlorine substitution, which indicated that halogen substitutions at R2 may play an important role. During the SAR study, we also found the heteroatoms substitution at X was important for the reversal activity: the nitrogen atom substitution derivatives 58, 56, and 55 were more potent than the corresponding carbon heterosubstitution derivatives 59−61. In order to further investigate the relationship of strong activity and lipophilicity of series II, the hydrophilicity of representative compounds for series II has also been tested. Compounds 51, 54, 55, 48 without 1,2,3-triazole group exhibited higher lipophilicity, as well as much more potent reversal activity, than the responding compounds 33−35 and 40 (Figure S3). These results further indicated that the high reversal activity may be closely related to lipophilicity. Moreover, amide bond length is critical for MDR reversal activity, and compound 55 with acetamide group (n = 2) displayed the best reversal activity than 71 (n = 1) and 72 (n =

Table 2. PTX-Resistance Reversal Activity of 20−42 at 2 μM in SW620/AD300 Cellsa

compd

R1

R2

IC50, PTX (μM)

RF

20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 VRP control

p-CH(CH3)2 p-CH(CH3)2 p-CH(CH3)2 p-CH(CH3)2 p-CH(CH3)2 p-CH(CH3)2 p-CH(CH3)2 p-CH(CH3)2 p-CH(CH3)2 p-CH(CH3)2 p-CH(CH3)2 p-CH(CH3)2 m,p,m-tri-OCH3 m,p,m-tri-OCH3 m,p,m-tri-OCH3 m,p,m-tri-OCH3 p-CH3 p-CH3 p-CH3 p-CH3 p-CH3 p-Br p-Br

o-Cl p-F p-Cl m-Cl m-NO2 o-F H p-OCH3 o-CH3 o-OCH3 o-OH p-CH(CH3)2 p-CH(CH3)2 m-CF3 p-Cl m-Cl p-CH(CH3)2 p-OCH3 o-OCH3 m-Cl m-CF3 m-CF3 p-CH(CH3)2

0.882 ± 0.054 1.980 ± 0.297 2.523 ± 0.402 1.545 ± 0.189 1.402 ± 0.147 1.529 ± 0.184 2.532 ± 0.403 0.664 ± 0.078 0.893 ± 0.049 0.411 ± 0.038 0.307 ± 0.051 0.441 ± 0.035 0.318 ± 0.027 3.092 ± 0.490 2.445 ± 0.388 3.055 ± 0.485 3.535 ± 0.548 3.560 ± 0.545 0.391 ± 0.048 0.312 ± 0.050 2.222 ± 0.347 1.743 ± 0.241 2.462 ± 0.391 0.211 ± 0.012 4.186 ± 0.513

4.75 2.11 1.66 2.71 2.99 2.74 1.65 6.30 4.69 10.18 13.64 9.49 13.16 1.35 1.71 1.37 1.18 1.18 10.71 13.42 1.88 2.40 1.70 19.84 1.00

a


demonstrated little toxicity on SW620/AD300 cells (IC50 = 4.186 ± 0.513 μM). However, in the presence of compounds or verapamil (VRP), PTX showed improved toxic effects on SW620/AD300 cells to different extents, suggesting that a majority of the test compounds could reverse PTX-resistance. First, the reversal activity of 7−10 and 16−19 in PTX resistance was evaluated in SW620/AD300 cells by MTT (Table 1). Unexpectedly, the extension of the side chain by introducing the N-(pyridin-2-ylcarbamoyl)acetamide group (16−19) resulted in decreased activity, compared with the corresponding PRA-1-analogs 7−10. Further structure−activity relationship (SAR) studies found that the substitution pattern and electronic effect on the aromatic ring were important for the reverse activity. Compounds 16−18 with electron-donating groups at R1 have more potent reversal effect (RF 6.23−17.37) than compound 19 (RF 1.41) with electron-withdrawing groups. A similar trend was also observed (31, 36 vs 42). In terms of the substitution pattern at R2, whether they had an electron-withdrawing or -donating group (20−25 and 27−31), 5991



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compd

R1

R2

X

IC50, PTX (μM)

RF

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 VRP control

p-CH(CH3)2 p-CH(CH3)2 p-CH(CH3)2 p-CH(CH3)2 p -CH3 p-Cl m,p-diF m,p,m-tri-OCH3 m,p,m-tri-OCH3 m,p,m-tri-OCH3 m,p,m-tri-OCH3 m,p,m-triOCH3 m,p,m-tri-OCH3 m,p,m-tri-OCH3 m,p,m-tri-OCH3 m,p,m-tri-OCH3 m,p,m-tri-OCH3 m,p,m-tri-OCH3 m,p,m-tri-OCH3 m,p,m-tri-OCH3 m,p-diF m,p-diF m,p-diF m,p-diF p-Cl H

p-CH3 p-OCH3 m-CH3 m-CF3 m-CF3 m-CF3 m-CF3 m-CF3 p-Br o-Cl p-Cl m-Cl m-NO2 p-CF3 o-OCH3 o-OCH3 m-Cl m-NO2 p-F p-CH(CH3)2 o-Cl m-Cl o-OCH3 p-Cl p-Cl p-CH3

N N N N N N N N N N N N N N N C C C C C N N N N N N

2.547 ± 0.406 1.926 ± 0.285 1.598 ± 0.204 0.113 ± 0.011 0.691 ± 0.061 1.466 ± 0.166 1.345 ± 0.129 0.072 ± 0.002 0.022 ± 0.001 0.110 ± 0.008 0.332 ± 0.021 0.005 ± 0.000 0.051 ± 0.003 1.177 ± 0.071 0.483 ± 0.052 1.144 ± 0.058 0.016 ± 0.000 0.199 ± 0.003 0.165 ± 0.014 0.167 ± 0.021 0.294 ± 0.031 0.958 ± 0.019 0.312 ± 0.029 1.195 ± 0.077 1.808 ± 0.257 2.506 ± 0.399 0.211 ± 0.012 4.186 ± 0.513

1.64 2.17 2.62 37.04 6.06 2.86 3.11 58.14 190.27 38.05 12.61 837.20 82.08 3.56 8.67 3.66 261.63 21.04 25.37 25.07 14.24 4.37 13.42 3.50 2.32 1.67 19.84 1.00

IC50 values were obtained from three independent repeats and represented as mean ± SD.

a


compd

R1

R2

X

n

IC50, PTX (μM)

RF

54 70 55 71 72 VRP control

m,p,m-tri-OCH3 m,p,m-tri-OCH3 m,p,m-tri-OCH3 m,p,m-tri-OCH3 m,p,m-tri-OCH3

p-Cl p-Cl m-Cl m-Cl m-Cl

N H N N H

2 0 2 1 0

0.332 ± 0.021 3.375 ± 0.264 0.005 ± 0.000 0.759 ± 0.032 3.986 ± 0.126 0.211 ± 0.012 4.186 ± 0.513

12.61 1.24 837.20 5.51 1.05 19.84 1.00


a

0), respectively, as well as 54 vs 70, which may be related to the stronger hydrogen bonds interaction with ABCB1 (Table 4). SAR was summarized and indicated in Figure 2. Molecular Docking Analysis. The top-scoring docked pose of compound 55 (IFD Glide gscore, −15.084 kcal/mol) into the

drug binding pocket of human homology ABCB1 is presented in Figure 3. The Glide gscore is an index that indicates the approximate ligand binding free energy.46 The core structure of 55 was mainly stabilized in a large hydrophobic cavity formed on the basis of a number of aromatic and hydrophobic and residues, 5992



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Figure 2. SAR for MDR reversal activity of series II against ABCB1-mediated MDR.

Figure 3. IFD predicted binding mode of compound 55 to human ABCB1 homology model. (A) Ribbon diagram of 3D structural in-ward facing conformation of a homology model of human ABCB1 ground on the crystal structure counterparts of mouse ABCB1. The location of 55 (orange) molecule, as illustrated by a ball and stick model, is shown within the ABCB1 internal cavity. (B) The docked conformation of 55 (ball and stick model) is shown within the ABCB1 drug-binding cavity, with the atoms colored as follows: carbon, orange; hydrogen, white; oxygen, red; nitrogen, blue; sulfur, yellow; chloride, green. Important amino acid residues are depicted (sticks model) with the same color scheme as above for all atoms but carbon atoms in gray. Dotted yellow lines represent hydrogen-bonding interactions, whereas dotted blue lines represent π−π stacking interactions. The values of the relevant distances are indicated in Å. (C) Schemic diagram of ligand−receptor interaction between 55 and human ABCB1. The amino acids within 4 Å are depicted as colored bubbles, polar residues are in blue color, and hydrophobic residues are in green color. Gray circles indicate solvent exposure. Purple arrows represent H-bonds, and green lines represent π−π stacking aromatic interactions.

of Phe983. In addition, several hydrogen bonds were predicted between compound 55 and the surrounding residues in the drug binding pocket of human homology ABCB1. Gln725 was involved in two hydrogen bonding interactions by one of the carbamoyl hydrogen atom interacting with the carbonyl oxygen atom of acetamide group of 55 (-CO···H2N-Gln725, 2.05 Å)

including Ile299, Ala302, Phe303, Ile306, Tyr307, Tyr310, Phe335, Phe336, Leu339, Ile340, Phe343, Leu724, Phe728, Ala729, Phe732, Phe770, Phe983, Met 986, and Ala987. From the in silico modeling analysis, two π−π stacking interactions were found between the trimethoxyphenyl ring of 55 and the phenyl ring of hydroxyphenyl ring of Tyr310 and the phenyl ring 5993



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and the other carbamoyl hydrogen atom interacting with the nitrogen atom in the pyridine ring of 55 (-N···H2N-Gln725, 2.72 Å). Similarly, the hydrogen atom in the carbamoyl group of Gln990 was engaged in a hydrogen bond with the N3 atom in the pyrimidine ring of 55 (-N···H2N-Gln990, 2.28 Å). The carbamoyl oxygen in Gln990 also interacted, by hydrogen bonding, with the two secondary amides in the pyridin-2ylcarbamoyl group (-NH···OC-Gln990, 1.96 Å) and the acetamide group (-NH···OC-Gln990, 2.02 Å) of 55, respectively. Moreover, hydrogen bonds were formed between the carbamoyl oxygen in 55 and the phenolic group of Tyr307 (-CO···HO-Tyr307, 1.82 Å), as well as between the 4-methoxy group in the trimethoxyphenyl ring of 55 and the phenolic group of Tyr310 (CH3O···HO-Tyr310, 1.65 Å). Docking analysis predicted relatively high binding affinity of compound 55 to human ABCB1, which supports the observations from cell-based reversal activity test. The high binding affinity of compound 55 to ABCB1 central hydrophobic drug-binding cavity may be closely related to its pharmacophoric features such as hydrophobicity, H-bond acceptor, aromatic ring center, and halogen group.43,44 Effect of Compounds 52, 55, and 60 at Lower Concentration on Reversing PTX Resistance. In order to investigate the dose-dependent relationship, we chose compounds 52, 55, and 60 with relatively more potent reversal activity than others at 2 μM to evaluate their effects at lower concentrations. The results (Table 5) showed that effects of

Figure 4. Effect of compound 55 on ABCB1 expression and subcellular localization. (A) Protein levels of ABCB1 in SW620 and SW620/ AD300 cell lines, with the GAPDH used as a loading control. (B) The protein level of ABCB1 in SW620/AD300 cells treated with compound 55 for 48 h was examined, and the GAPDH was used as a loading control. (C) Effect of compound 55 on the subcellular localization of ABCB1 (green) after treatment for 48 h. Hoechst 33258 (blue) was stained for the cell nuclei.

Table 5. PTX-Resistance Reversal Activity of 52, 55, 60 in SW620/AD300 Cellsa compd

IC50, PTX (μM)

RF

PTX +55 (0.5 μM) +55 (1 μM) +55 (2 μM) +52 (0.5 μM) +52 (1 μM) +52(2 μM) +60 (0.5 μM) +60 (1 μM) +60 (2 μM) +verapamil (2 μM)

4.186 ± 0.513 0.058 ± 0.002 0.031 ± 0.001 0.005 ± 0.000 0.552 ± 0.003 0.130 ± 0.002 0.022 ± 0.001 0.113 ± 0.018 0.159 ± 0.022 0.016 ± 0.000 0.211 ± 0.012

1.00 72.17 135.03 837.20 8.019 32.20 190.27 37.04 26.33 261.63 19.84

What’s more, compound 55 did not alter the subcellular localization compared to the control. Compounds 52 and 60 gave similar results (Figure S4). These findings indicated that the reversal activity of compound 55 was not due to a downregulation of ABCB1 expression but its functional inhibition. Inhibitory Effect of ABCB1-Mediated Rhodamine 123 Accumulation and Efflux. Because of the unchanged expression of ABCB1 after treatment of compound 55, we hypothesized that it may be involved in the function of ABCB1. ABCB1dependent fluorescent rhodamine 123 (Rh123) efflux assay was widely used to evaluate the reversal activity of ABCB1-mediated MDR modulators.47 As shown in Figure 5A, the intracellular Rh123 was notably lower in ABCB1-overexpressed SW620/ AD300 than parental cells, and the VRP was used as positive control. Compound 55 could significantly increase the accumulation of Rh 123 in a dose-dependent pattern in ABCB1-overexpressed SW620/AD300 cells but not in ABCB1-low-expressed SW620 cells, which may be related to the specific interaction with ABCB1. Furthermore, Rh123 efflux assay showed (Figure 5B) that after removing Rh123 incubation, the intracellular Rh123 in SW620/AD300 decreased much faster than parental cells over time, suggesting a ABCB1-mediated positive process of drug efflux. In ABCB1-low-expressed SW620 cells, compound 55 did not significantly affect its intracellular Rh123 efflux, while in ABCB1-overexpressed SW620/AD300 cell line, compound 55 could inhibit Rh123 efflux in a dose-dependent manner, which is also more potent than VRP. Compounds 52 and 60 gave similar

a


compounds 55, 52, and verapamil followed a concentrationdependent pattern where higher concentration showed more potent reversal activity. The effects of compound 60 at 0.5 and 1 μM had no significant difference, but when at 2 μM, compound 60 showed more effective activity. These findings suggested that compounds 52, 55, and 60 could reverse the PTX resistance in a dose-dependent manner. Effect of Compound 55 on ABCB1 Expression and Subcellular Localization. Down-regulating the expression of ABCB1 or inhibiting its function may also cause the reversal of ABCB1-mediated MDR. We first evaluated if compound 55 could effectively be regulated on the expression level of ABCB1 expression. The SW620/AD300 cells possessed much higher level of ABCB1 than SW620 cells (Figure 4A) and no significant changes on the level of ABCB1 under treatment of compound 55 (0, 1, 2, or 4 μM) for 48 h (Figure 4B). Further immunofluorescence assay showed similar results (Figure 4C). 5994



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Figure 5. Effect of compound 55 and VRP on accumulation (A) and efflux (B) of Rh 123 in SW620 and SW620/AD300 cells. Data are presented as the mean ± SD of three independent experiments, and a representative experiment is shown.

Figure 6. Effects of compound 55 and VRP on accumulation and maintenance of PTX. (A) The exact amount of PTX in SW620 or SW620/AD300 cell lines was measured by HPLC. (B) Effect of compound 55 and VRP on accumulation of [3H]-paclitaxel in SW620 and SW620/AD300 cell lines. (C) After 0, 30, 60, or 120 min, the same numbers of SW620 (left) and SW620/Ad300 (right) were measured for the radioactivity with the scintillation fluid. Data were obtained from three independent assays and shown as the mean ± SD.

Effect of Compound 55 on PTX Accumulation. For further investigation of the reversal mechanism, we first evaluated the exact amount of PTX in SW620 or ABCB1-overexpressed SW620/AD300 cell lines after treatment of compound 55 and VRP by HPLC. The results were depicted in Figure 6A. PTX

results (Figure S5). These data may suggest that compound 55 sensitized SW620/AD300 to PTX by increasing its intracellular accumulation and inhibiting its efflux, which may due to the inhibition of ABCB1 activity. 5995



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accumulation in SW620 cells was higher than that of SW620/ AD300 cells in the absence of inhibitors, which was due to the ABCB1 mediated drug efflux. Importantly, the intracellular PTX was maintained at a significantly higher amount compared to the control in SW620/AD300 cells under treatment with compound 55 or VRP. A similar trend was also observed by measuring the accumulation of tritium-labeled PTX ([3H]-PTX) in SW620/ AD300 cells (Figure 6B). Moveover, [3H]-PTX efflux assay was also tested. As is shown in Figure 6C, after 120 min of efflux, 29% and 69% normalized loss of [3H]-paclitaxel occurred in SW620 and SW620/Ad300 cells, respectively. Also, at the end of 120 min efflux, 36% and 69% of normalized [3H]-paclitaxel was removed in SW620/Ad300 cells with or without co-incubation with compound 55. Moreover, effects of compound 55 followed a concentration-dependent pattern where 2 and 4 μM compound 55 exhibited significant increase of [3H]-paclitaxel in ABCB1-overexpressing cells (Figure 6C, right). Efflux pattern of parental cells were not significantly altered by compound 55 (Figure 6C, left). These findings further suggested that 55 was a potent ABCB1 inhibitor by increasing PTX intracellular accumulation and inhibiting its efflux. Effect of Compound 55 on the ABCB1 ATP Hydrolysis. The activity of ATPase could be reflected by ATP consumption because the ABCB1 transporter takes advantage of energy from ATP hydrolysis to pump its substrates across the cell membrane against concentration gradient. Therefore, we measured ATP hydrolysis mediated by ABCB1 in the presence of compound 55 at gradient concentrations from 0 to 40 μM. The result shows that compound 55 could stimulate the ATPase activity of ABCB1 with a maximal stimulation of 4.56-fold, and the concentration of compound 55 to obtain 50% stimulation is 0.97 μM (Figure 7).

Figure 8. Dose−response curves after different treatment: the canonical competitive CYP3A4 inhibitor ketoconazole, positive reversal modulator of ABCB1-mediated resistance verapamil, compound 55.

compounds has the potential as leading compounds to further develop safe ABCB1 inhibitors. Cellular Thermal Shift Assay (CETSA). As ABCC1 is the second most prevalent ABC transporter in MDR, most ABCB1 inhibitors have been evaluated their inhibitory activity against ABCC1 and usually exhibited potent inhibitory activity.48 Furthermore, compounds containing pyrimidine scaffold have been proved as potent dual inhibitors of ABCB1 and ABCC1.49−52 Therefore, the binding affinity of 55 with ABCB1 and ABCC1 was tested by CETSA, respectively.53 In brief, the lysate of SW620/AD300 was incubated with compound 55 and then heated at different temperatures. The target protein would remain stable after heating once it was bound by compound compared to the DMSO control group. As shown in Figure 9, compound 55 efficiently could stabilize

Figure 9. Compound 55 engaged to ABCB1 (upper) and ABCC1 (lower) in SW620/AD300.

Figure 7. Effect of compound 55 on the ABCB1 ATP hydrolysis. The ATPase assay was carried out using PREDEASY ATPase kit.

ABCB1 but not ABCC1 at higher temperatures. These data suggested that the reversal activity of compound 55 may be associated with its specific target to ABCB1. Vincristine-Resistance Reversal Activity of Compound 55 on ABCB1-Overexpressed and ABCC1-Overexpressed Cell Lines. Considering the fact that drug selected cell line may possess other drug resistance factors, we established the ABCB1 and ABCC1 selected overexpressing cell system HEK293/ ABCB1 and HEK293/ABCC1 to investigate the predominant role of ABCB1 in reversal activity of compound 55. Correspondingly, we also used other two drug selected cancer cell lines KB-C2 and KB-CV60 overexpressed ABCB1 and ABCC1, respectively. What’s more, other than PTX, we used another clinical antitumor drug vincristine, which is the common substrate for ABCB1 and ABCC1. The data in Table 6 showed that compound 55 could reverse vincristine resistance

Effect of Compound 55 on Cytochrome P3A4 (CYP3A4). The overlapped substrate specificity between ABCB1 and CYP3A4 often generates unexpected pharmacokinetic parameters when combining ABCB1 inhibitors with anticancer agents. In order to identify whether compound 55 was a safe ABCB1 modulator at effective dose, we evaluated the inhibition activity of compound 55 against CYP3A4 (Figure 8). The widely used specific CYP3A4 inhibitor ketoconazole could notably inhibit the activity of CYP3A4 in a dose-dependent pattern with IC50 = 0.065 ± 0.007 μM, while compound 55 possessed a poor inhibitory effect against CYP3A4 with IC50 = 17.243 ± 1.237 μM, even poorer than verapamil (IC50 = 8.193 ± 0.913 μM). And compounds 52 and 60 had weaker activity against CYP3A4 (Figure S6) than 55. These data indicated that this series of 5996



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Table 6. Vincristine-Resistance Reversal Activity of Compound 55 on ABCB1-Overexpressed and ABCC1-Overexpressed Cell Lines.a IC50 (μM) treatment

KB-C2

RF

HEK293/ABCB1

RF

vincristine +55, 1 μM +verapamil, 3 μM

1.138 ± 0.012 0.295 ± 0.032 0.064 ± 0.018

1.00 3.85 17.92

1.095 ± 0.071 0.112 ± 0.030 0.051 ± 0.016

1.00 9.75 21.52

IC50 (μM) treatment

KB-CV60

RF

HEK/ABCC1

RF

vincristine +55, 1 μM +MK571, 25 μM

0.249 ± 0.050 0.221 ± 0.025 0.056 ± 0.014

1.00 1.13 4.45

0.113 ± 0.023 0.099 ± 0.019 0.029 ± 0.009

1.00 1.14 3.90


a

Figure 10. In vivo antitumor effects after single or combined treatment of compound 55 and PTX. (A) Representative tumor size after single or combined treatment of compound 55 and PTX. Tumor volume (B), body weight (C), tumor weight (D) of the animals with the indicated treatment.

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against both ABCB1-overexpressed cell lines, KB-C2 and HEK293/ABCB1, while in ABCC1-overexpressed cell lines, KB-CV60 and HEK293/ABCC1, compound 55 did not exhibit reversal activity. These findings indicated that ABCB1 may play an important and selective role in resistance reversal activity of compound 55. Xenograft Study. On the basis of the potent reversal activity of ABCB1-mediated MDR, the reversal activity in vivo was also examined after single or combined treatment of compound 55 and PTX on xenograft model bearing ABCB1-overexpressed SW620/AD300 cells by subcutaneous implantation. After the treatment of single or combined treatment of compound 55 and PTX, the tumor weight and the tumor volume were measured and recorded every 3 days. As shown in Figure 10, neither single treatment of compound 55 nor PTX affects the tumor growth significantly, while after the combined treatment of compound 55 and PTX, the tumor growth was apparently inhibited with the precondition of unchanged body weight.

CONCLUSIONS

In this study, our efforts have yielded a series of compounds with a 5-cyano-6-phenylpyrimidin scaffold as ABCB1-mediated MDR modulators. The reversal activities of all the compounds against ABCB1-mediated MDR were evaluated, and one of these compounds, 55, showed the most potency and selective activity. Further mechanism studies, including increasing accumulation of PTX, interrupting ABCB1-mediated Rh123 accumulation and efflux, and stimulating the ABCB1 ATPase activity, demonstrated that the MDR reversal by compound 55 was not due to down-regulation of ABCB1 expression but its functional inhibition. In particular, the activity of CYP3A4 was not influenced by compound 55, and the growth of ABCB1overexpressed SW620/AD300 cell line in vivo was significantly inhibited by PTX combined with compound 55 compared to single treatment of PTX and compound 55. Our findings indicate that the pyrimidine-acylurea based ABCB1 inactivators may serve as leading compounds targeting ABCB1-dependent MDR. 5997



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5. Lipophilicity Assay. Lipophilicity assay was performed as previous described.55 In brief, add the excess powder of each compound to phosphate buffer (pH 7.4) until heterogeneous suspension was obtained. Then the heterogeneous suspension was sonicated in a water bath for 30 min and shakem for 24 h at room temperature to reach thermodynamic equilibrium. Then, the suspension was centrifuged at 15 000 rpm for 15 min and the supernatant was filtered through 0.45 μm membrane. The filtrate was measured by UV-2700 spectroscopy (Shimadzu, Japan). 6. Docking Analysis. The structure of compound 55 was built using the 2D building sketcher of Maestro v11.1 and energy minimized by Macromodel v11.5 (Schrödinger, LLC, New York, USA). The ligand structure was then prepared to become a low-energy 3D structure using LigPrep v4.1 (Schrödinger, LLC, New York, USA). The homology modeled human ABCB1 was kindly provided by S. Aller, which was established on the basis of the refined mouse ABCB1 protein (PDB code 4M1M). The receptor docking grid with length of 25 Å was generated using Glide v7.4 (Schrödinger, LLC, New York, USA) program by selecting all amino acid residues demonstrated previously to interact with drugs or other small molecules.56 The flexible docking was performed with the XP (extra precision) mode in Glide v7.4 to simulate the binding of 55 into the homology model of human ABCB1 at the drug-binding site. In order to get the optimal ligand−receptor binding simulation, induced-fit docking (IFD) was conducted by Glide v7.4. The receptor grid for the IFD simulation was generated based on the binding position of 55 obtained from the Glide XP docking process.The IFD protocol was run with default parameters, and the docking scores were calculated and expressed as kcal/mol. 7. Rhodamine 123 Accumulation and Efflux Assay. For the rhodamine 123 accumulation, about 2 × 105 cells per well were seeded into 6-well plate. Once the cells attached, the medium was replaced by fresh medium containing compound at nontoxic concentration for 4 h. Then, the cells were added to 1 μg/mL rhodamine 123 and incubated for 30 min at 37 °C. Then, the cells were harvested and immediately detected by flow cytometric at excitation wavelength 488 nm and emission wavelength 530 nm, while for efflux assay, after rhodamine 123 incubation, the cells were further cultured in rhodamine 123-free medium with or without reversal reagents for another 30, 60, 90, 120 min at 37 °C. Then the cells were harvested and analyzed separately as accumulation assay. 8. HPLC. Same number of SW620/AD300 cells were incubated by 6 μg/mL PTX 2 h and then further cultured in PTX-free medium with or without compounds for 30 min at 37 °C. The cells were harvested, broken by ultrasound, and extracted by ethyl acetate. Next, after a centrifuge, the layer of ehyl acetate was separated from the extraction system and dried by nitrogen purging equipment. Each sample was dissolved using mobile phase (MeOH, CH3CN, and H2O, 27:43:30, v/ v/v) before detection by HPLC. The signal was detected at a wavelength of 227 nm with a UV detector, and the flow rate was set as 1.0 mL/min.. 9. [3H]-Paclitaxel Accumulation and Efflux Assay. The [3H]paclitaxel accumulation in SW620 and SW620/AD300 was evaluated in the presence or absence of inhibitors (verapamil or compound 55). Cells were incubated previously with or without compound at different concentrations for 4 h at 37 °C. Subsequently, 0.1 μM [3H]-paclitaxel was added for 2 h in the presence of the above treatment. After being washed with cold PBS three times, cells were lysed and placed in 5 mL of scintillation liquid. Radioactivity was measured in the Packard TRICARB 1900CA liquid scintillation analyzer (Packard Instrument, Downers Grove, IL, USA). For [3H]-paclitaxel efflux assay, after incubation of 0.1 μM [3H]paclitaxel for 2 h for drug accumulation as accumulation assay, cells were cultured in [3H]-free medium incubation for 2 h as efflux phase. During this efflux phase, each sample was taken and analyzed at 0, 30, 60, and 120 min. Radioactivity was measured as described above. 10. Western Blotting. After being treated with candidate compound, cells were harvested and lysed. Then, the lysate was isolated by ultracentrifuge and quantified by BCA assay according to the protocol. After being denatured, equivalent protein of each sample was separated by SDS−PAGE gel and wet-transferred onto 0.22 μm

EXPERIMENTAL SECTION

1. General Chemistry. Chemicals and solvents were obtained from standard suppliers and used directly without further purification. Melting points were taken on an X-5 micromelting apparatus and were uncorrected. 1H and 13C NMR spectra were respectively determined with a 400 and 100 MHz spectrometer. High resolution mass spectra (HRMS) were obtained with a Waters Q-TOF electrospray mass spectrometer (Waters, Milford, MA). The spectra data of compounds are provided in Supporting Information. Final products were of >95% purity as analyzed by HPLC analysis (Phenomenex column, C-18, 5.0 μm, 4.6 mm × 150 mm) on Dionex UltiMate 3000 UHPLC instrument from ThermoFisher. The signal was monitored at 278 nm with a UV detector. A flow rate of 1.0 mL/min was used with a eluent of methanol in H2O (80:20, v/v), and the column temperature was 25 °C. The purities of compounds 52, 55, and 60 were also determined by binary gradient. Analytical conditions are as follows: eluent A, H2O; eluent B, methanol; flow rate, 1 mL/min; gradient program, 1−3 min % B = 5− 95 gradient, 3−7 min % B = 95, 7−9 min % B = 95−5 gradient, 9−10 min % B = 5; column temperature, 25 °C; flow rate, 1.0 mL/min. Besides, PAINS screening of the synthesized compounds was carried out by employing the online program (http://www.cbligand.org/ PAINS/),54 and all the tested compounds passed the filter. 2. General Procedure for the Synthesis of Compound 43−72. The detailed information on synthesis and characterization of compounds 7−10, 16−42 was reported in published articles and in the Supporting Information.40,41,45 A mixture of the appropriate 2-mercaptodihydropyrimidine derivatives 4a−j (1 mmol), 12a−c or 13 (1.5 mmol), and anhydrous potassium carbonate (1 mmol) was refluxed in dry dioxane. Upon completion, as judged by thin-layer chromatography (TLC), phosphorus oxychloride was added dropwise with stirring while maintaining the temperature of the reaction mixture. Stirring was continued for an additional 1−2 h. The cooled reaction mixture was poured on crushed ice, and the separated solid was filtered off, washed with water, dried, and crystallized from aqueous ethanol to yield the products 43a−h which were used for next steps without further purfication. To a well stirred solution of the appropriate amine (2 mmol) in absolute ethanol (10 mL), a solution of compounds 43a−f (1 mmol) in absolute ethanol (10 mL) was added. The reaction mixture was stirred for 1.5 h at room temperature and then heated under reflux for additional 5 h. Upon completion, as judged by thin-layer chromatography (TLC), the precipitated product was filtered off, washed with ethanol to afford the crude product. The crude product was recrystallized from ethanol to yield the pure product. 3. Cell Lines and Cell Culture. The ABCB1-overexpressed MDR cell line KB-C2 and ABCC1-overexpressed MDR cell line KB-CV60 were both established from human epidermoid carcinoma cell line KB3-1 cell line by drug selection. KB-C2 was selected by 2 μg/mL vincristine, while KB-CV60 was by 1 μg/mL of cepharanthine and 60 ng/mL of vincristine. Both cell lines were kindly provided by Dr. Shinichi Akiyama (Kagoshima University, Japan). Both SW620 and SW620/AD300 were kindly provided by Drs. Susan E. Bates and Robert W. Robey (NCI, NIH, Bethesda, MD). HEK293/pcDNA3.1, HEK293/ABCB1, HEK293/ABCC1 cells were established by transfecting with empty vector pcDNA3.1 or pcDNA3.1 vector with ABCB1 or ABCC1 coding gene and cultured in medium containing 2 mg/mL G418. All cell lines were cultured with DMEM culture medium (Hyclone Co., Omaha, NE) containing 10% bovine serum at 37 °C and 5% CO2. 4. MTT Assay. About 5 × 103 cells per well were seeded in 96-well plate and treated by gradient concentration of compounds for 72 h. Then, to each well was added 20 μL of 5 mg/mL MTT solution and the plate was incubated for another 4 h. Discard the medium, and the dark blue crystal on the bottom was dissolved completely with 150 μL of DMSO. The plates were measured with an ELx 800 Universal microplate reader (Bio-Tek, Inc., USA) at a wavelength of 490 nm. IC50 represented as the mean ± SD was obtained by three dependent performs. 5998



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*Z.-S.C.: e-mail, [email protected]; phone, 1-718-99-1432. *H.-M.L.: e-mail, [email protected]; phone, 86-371-67781739.

nitrocellulose membrane. After being blocked by PBS containing 5% skim milk, the membrane was incubated with primary antibody at 4 °C overnight and secondary antibody at room tempreture for 2 h and then visualized by enhanced chemiluminescence kit (Thermo Fisher, USA), sequentially. 11. Immunofluorescence Assay. About 1 × 104 exponentially growing cells were seeded on coverslip placed in 24-well plate and incubated with candidate compounds at different concentrations for 48 h. Then, remove the medium and wash the cells with cold PBS three times. Cells were next fixed by 4% paraformaldehyde for at least 20 min, blocked with PBS containing 5% BSA at room temperature for 2 h, and incubated with primary antibody overnight at 4 °C. After being washed with PBS three times, cells were incubated with Alexa Fluor 488 rabbit anti-goat IgG (H+L) for 2 h and stained cell nuclei using Hoechst 33258 for 20 min. Images were obtained by laser scanning confocal microscopy (Olympus, FV10i, Olympus Corporation, Tokyo, Japan). 12. Cellular Thermal Shift Assay (CETSA). Generally, enough SW620/AD300 cells were harvested and lysed by freeze−thawing cycle using liquid nitrogen three times. Then, the supernatant was obtained after lutracentrifuge and divided into two aliquots, of which one was treated with candidate compound and another was solvent control. After 30 min incubation at 37 °C, aliquot the lysate into 8 PCR tubes and heat them to the tempretures of 40, 43, 46, 49, 52, 55, 58, and 61 °C separately for 3 min. Then, the lysates were analyzed as Western blotting analysis. 13. ATPase Assay. Vi-sensitive ATPase activity of ABCB1 was measured using membrane vesicles of High Five insect cells by PREDEASY ATPase kits with modified protocols, as previously described.57 14. CYP3A4 Assay. The effect of compounds against CYP3A4 was performed exactly according to the instruction of cytochrome P450 3A4 (CYP3A4) inhibitor screening kit (Fluorometric) (Biovison, USA). Ketoconazole provided in the kit was used as a positive CYP3A4 inhibitor control. 15. Xenograft Studies. Animals experiment was carried out according to the approved guidelines of the Institutional Animal Care and Use Committee. Female BALB/c nude mice weighing 18−20 g and aged 5−6 weeks were purchased from Hunan Slack Scene of Laboratory Animal Co., Ltd. (Hunan, China) and established xenograft model using human MDR cell line SW620/AD300. Once the tumor volume reached 100 mm3, the mice were divided into four groups: NS (normal saline, 0.2 mL/kg/day, intraperitoneal injection), PTX (5 mg/kg/3 day, intraperitoneal injection), compound 55 (20 mg/kg/day, orally), and 55-PTX. The body weight and tumor volume of each mice were measured at 2-day intervals during the period of 21 days. The mice were euthanized, and the tumors were isolated and weighed after the last day.

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ORCID

Hong-Min Liu: 0000-0001-6771-9421 Author Contributions §

B.W., L.-Y.M., and J.-Q.W. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (Projects 81430085, 21372206, 81773562, and 81703328); National Key Research Program of Proteins (Grant 2016YFA0501800); Key Research Program of Henan Province (Grant 1611003110100); the Starting Grant of Zhengzhou University (Grant 32210535); Scientific Program of Henan Province (Grant 182102310070). We are thankful to Dr. Stephen Aller (The University of Alabama at Birmingham, Birmingham, AL, U.S.) for kindly providing human ABCB1 homology model. We thank Tanaji T. Talele (St. John’s University, New York, NY, U.S.) for providing the computing resources for docking analysis. We thank Drs. Susan E. Bates and Robert W. Robey (NCI, NIH, Bethesda, MD, U.S.) for providing cell lines SW620 and SW620/AD300. We thank Dr. Shin-Ich Akiyama (Kagoshima University, Kagoshima, Japan) for the KB-3-1, KB-C2, and KB-CV60 cells. ABBREVIATIONS USED

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REFERENCES

ATP, adenosine triphosphate; ABC, ATP-binding cassette; ABCB1, ATP-binding cassette subfamily B member 1; P-gp, Pglycoprotein; MDR, multidrug resistance; PTX, paclitaxel; VRP, verapamil; CYP3A4, cytochrome P450 3A4; RF, reversal-fold; SAR, structure−activity relationship; XP, extra precision; IFD, induced-fit docking; Rh123, rhodamine 123; HPLC, highperformance liquid chromatography; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMSO, dimethyl sulfoxide; SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonyl fluoride; BCA, bicinchoninic acid; SDS− PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; BSA, bovine serum albumin

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00335. Molecular formula strings and some data (CSV) Human-Pgp-homology-model (PDB) Solubility of compounds 7−10 and 16−19; XP Glide docking predicted binding mode of compound 18 to human ABCB1 homology model; solubility of compounds 33−35, 40, 48, 51, 54, 55; associated assay about ABCB1 of compounds 52 and 60; IC50 and survival rate (at 2 μM) of all the target compounds toward SW620 and SW620/AD300 cells; characterization of compounds 44−72; 1H and 13C NMR spectra and HPLC chromatograms of compounds 44−72 (PDF)

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(1) Gottesman, M. M. Mechanisms of cancer drug resistance. Annu. Rev. Med. 2002, 53, 615−627. (2) Holohan, C.; Van Schaeybroeck, S.; Longley, D. B.; Johnston, P. G. Cancer drug resistance: an evolving paradigm. Nat. Rev. Cancer 2013, 13, 714−726. (3) Gottesman, M. M.; Fojo, T.; Bates, S. E. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat. Rev. Cancer 2002, 2, 48−58. (4) Eckford, P. D. W.; Sharom, F. J. ABC Efflux pump-based resistance to chemotherapy drugs. Chem. Rev. 2009, 109, 2989−3011. (5) Kartal-Yandim, M.; Adan-Gokbulut, A.; Baran, Y. Molecular mechanisms of drug resistance and its reversal in cancer. Crit. Rev. Biotechnol. 2016, 36, 716−726. (6) Aller, S. G.; Yu, J.; Ward, A.; Weng, Y.; Chittaboina, S.; Zhuo, R.; Harrell, P. M.; Trinh, Y. T.; Zhang, Q.; Urbatsch, I. L.; Chang, G. Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. Science 2009, 323, 1718. (7) Yang, X.; Liu, K. P-gp inhibition-based strategies for modulating pharmacokinetics of anticancer drugs: an update. Curr. Drug Metab. 2016, 17, 806−826.

AUTHOR INFORMATION

Corresponding Authors

*B.Z.: e-mail, [email protected]. 5999



Article

(8) Nobili, S.; Landini, I.; Mazzei, T.; Mini, E. Overcoming tumor multidrug resistance using drugs able to evade P-glycoprotein or to exploit its expression. Med. Res. Rev. 2012, 32, 1220−1262. (9) Nobili, S.; Landini, I.; Giglioni, B.; Mini, E. Pharmacological strategies for overcoming multidrug resistance. Curr. Drug Targets 2006, 7, 861−879. (10) Mahon, F.-X.; Hayette, S.; Lagarde, V.; Belloc, F.; Turcq, B.; Nicolini, F.; Belanger, C.; Manley, P. W.; Leroy, C.; Etienne, G.; Roche, S.; Pasquet, J.-M. Evidence that resistance to nilotinib may be due to BCR-ABL, Pgp, or Src kinase overexpression. Cancer Res. 2008, 68, 9809−9816. (11) Eadie, L. N.; Hughes, T. P.; White, D. L. Interaction of the efflux transporters ABCB1 and ABCG2 with imatinib, nilotinib, and dasatinib. Clin. Pharmacol. Ther. 2014, 95, 294−306. (12) Cui, H.; Zhang, A. J.; Chen, M.; Liu, J. J. ABC transporter inhibitors in reversing multidrug resistance to chemotherapy. Curr. Drug Targets 2015, 16, 1356−1371. (13) Rosti, G.; Castagnetti, F.; Gugliotta, G.; Baccarani, M. Tyrosine kinase inhibitors in chronic myeloid leukaemia: which, when, for whom? Nat. Rev. Clin. Oncol. 2017, 14, 141−154. (14) Szakács, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.; Gottesman, M. M. Targeting multidrug resistance in cancer. Nat. Rev. Drug Discovery 2006, 5, 219. (15) Binkhathlan, Z.; Lavasanifar, A. P-glycoprotein inhibition as a therapeutic approach for overcoming multidrug resistance in cancer: current status and future perspectives. Curr. Cancer Drug Targets 2013, 13, 326−346. (16) Baumert, C.; Hilgeroth, A. Recent advances in the development of P-gp inhibitors. Anti-Cancer Agents Med. Chem. 2009, 9, 415−436. (17) Mayur, Y. c.; Peters, G. J.; Prasad, V. V. S. R.; Lemos, C.; Sathish, N. K. Design of new drug molecules to be used in reversing multidrug resistance in cancer cells. Curr. Cancer Drug Targets 2009, 9, 298−306. (18) Dewanjee, S.; Dua, T.; Bhattacharjee, N.; Das, A.; Gangopadhyay, M.; Khanra, R.; Joardar, S.; Riaz, M.; Feo, V.; Zia-UlHaq, M. Natural products as alternative choices for P-glycoprotein (Pgp) inhibition. Molecules 2017, 22, 871. (19) Joshi, P.; Vishwakarma, R. A.; Bharate, S. B. Natural alkaloids as P-gp inhibitors for multidrug resistance reversal in cancer. Eur. J. Med. Chem. 2017, 138, 273−292. (20) Shi, Z.; Peng, X. X.; Kim, I. W.; Shukla, S.; Si, Q. S.; Robey, R. W.; Bates, S. E.; Shen, T.; Ashby, C. R., Jr.; Fu, L. W.; Ambudkar, S. V.; Chen, Z. S. Erlotinib (Tarceva, OSI-774) antagonizes ATP-binding cassette subfamily B member 1 and ATP-binding cassette subfamily G member 2-mediated drug resistance. Cancer Res. 2007, 67, 11012− 11020. (21) Tang, S. J.; Chen, L. K.; Wang, F.; Zhang, Y. K.; Huang, Z. C.; To, K. K.; Wang, X. K.; Talele, T. T.; Chen, Z. S.; Chen, W. Q.; Fu, L. W. CEP-33779 antagonizes ATP-binding cassette subfamily B member 1 mediated multidrug resistance by inhibiting its transport function. Biochem. Pharmacol. 2014, 91, 144−156. (22) Jiao, L.; Qiu, Q.; Liu, B.; Zhao, T.; Huang, W.; Qian, H. Design, synthesis and evaluation of novel triazole core based P-glycoproteinmediated multidrug resistance reversal agents. Bioorg. Med. Chem. 2014, 22, 6857−6866. (23) Liu, B.; Qiu, Q.; Zhao, T.; Jiao, L.; Hou, J.; Li, Y.; Qian, H.; Huang, W. Discovery of novel P-glycoprotein-mediated multidrug resistance inhibitors bearing triazole core via click chemistry. Chem. Biol. Drug Des. 2014, 84, 182−191. (24) Kim, J. C.; Kim, K. S.; Kim, D. S.; Jin, S. G.; Kim, D. W.; Kim, Y. I.; Park, J. H.; Kim, J. O.; Yong, C. S.; Youn, Y. S.; Woo, J. S.; Choi, H. G. Effect of HM30181 mesylate salt-loaded microcapsules on the oral absorption of paclitaxel as a novel P-glycoprotein inhibitor. Int. J. Pharm. 2016, 506, 93−101. (25) Chen, C. Y.; Liu, N. Y.; Lin, H. C.; Lee, C. Y.; Hung, C. C.; Chang, C. S. Synthesis and bioevaluation of novel benzodipyranone derivatives as P-glycoprotein inhibitors for multidrug resistance reversal agents. Eur. J. Med. Chem. 2016, 118, 219−229.

(26) Srinivas, N. R. Understanding the role of tariquidar, a potent Pgp inhibitor, in combination trials with cytotoxic drugs: What is missing? Cancer Chemother. Pharmacol. 2016, 78, 1097−1098. (27) Hsiao, S.-H.; Lu, Y.-J.; Yang, C.-C.; Tuo, W.-C.; Li, Y.-Q.; Huang, Y.-H.; Hsieh, C.-H.; Hung, T.-H.; Wu, C.-P. Hernandezine, a bisbenzylisoquinoline alkaloid with selective inhibitory activity against multidrug-resistance-linked ATP-binding cassette drug transporter ABCB1. J. Nat. Prod. 2016, 79, 2135−2142. (28) Zhou, Z. Y.; Wan, L. L.; Yang, Q. J.; Han, Y. L.; Li, D.; Lu, J.; Guo, C. Nilotinib reverses ABCB1/P-glycoprotein-mediated multidrug resistance but increases cardiotoxicity of doxorubicin in a MDR xenograft model. Toxicol. Lett. 2016, 259, 124−132. (29) Qiu, Q.; Liu, B.; Cui, J.; Li, Z.; Deng, X.; Qiang, H.; Li, J.; Liao, C.; Zhang, B.; Shi, W.; Pan, M.; Huang, W.; Qian, H. Design, synthesis, and pharmacological characterization of N-(4-(2 (6,7-Dimethoxy-3,4dihydroisoquinolin-2(1H)yl)ethyl)phenyl)quinazolin-4-amine derivatives: novel inhibitors reversing P-glycoprotein-mediated multidrug resistance. J. Med. Chem. 2017, 60, 3289−3302. (30) Paterna, A.; Kincses, A.; Spengler, G.; Mulhovo, S.; Molnár, J.; Ferreira, M.-J. U. Dregamine and tabernaemontanine derivatives as ABCB1 modulators on resistant cancer cells. Eur. J. Med. Chem. 2017, 128, 247−257. (31) Qiu, Q.; Shi, W.; Li, Z.; Zhang, B.; Pan, M.; Cui, J.; Dai, Y.; Huang, W.; Qian, H. Exploration of 2-((Pyridin-4-ylmethyl)amino)nicotinamide derivatives as potent reversal agents against Pglycoprotein-mediated multidrug resistance. J. Med. Chem. 2017, 60, 2930−2943. (32) Zou, W.; Sarisozen, C.; Torchilin, V. P. The reversal of multidrug resistance in ovarian carcinoma cells by co-application of tariquidar and paclitaxel in transferrin-targeted polymeric micelles. J. Drug Target. 2017, 25, 225−234. (33) Thomas, H.; Coley, H. M. Overcoming multidrug resistance in cancer: an update on the clinical strategy of inhibiting P-glycoprotein. Cancer Control. 2003, 10, 159−165. (34) Kang, M. H.; Figg, W. D.; Ando, Y.; Blagosklonny, M. V.; Liewehr, D.; Fojo, T.; Bates, S. E. The P-glycoprotein antagonist PSC 833 increases the plasma concentrations of 6α-hydroxypaclitaxel, a major metabolite of paclitaxel. Clin. Cancer Res. 2001, 7, 1610−1617. (35) Kuppens, I. E. L. M.; Witteveen, E. O.; Jewell, R. C.; Radema, S. A.; Paul, E. M.; Mangum, S. G.; Beijnen, J. H.; Voest, E. E.; Schellens, J. H. M. A phase I, randomized, open-label, parallel-cohort, dose-finding study of elacridar (GF120918) and oral topotecan in cancer patients. Clin. Cancer Res. 2007, 13, 3276−3285. (36) Lhommé, C.; Joly, F.; Walker, J. L.; Lissoni, A. A.; Nicoletto, M. O.; Manikhas, G. M.; Baekelandt, M. M. O.; Gordon, A. N.; Fracasso, P. M.; Mietlowski, W. L.; Jones, G. J.; Dugan, M. H. Phase III study of valspodar (PSC 833) combined with paclitaxel and carboplatin compared with paclitaxel and carboplatin alone in patients with stage IV or suboptimally debulked stage III epithelial ovarian cancer or primary peritoneal cancer. J. Clin. Oncol. 2008, 26, 2674−2682. (37) Shukla, S.; Wu, C.-P.; Ambudkar, S. V. Development of inhibitors of ATP-binding cassette drug transporters − present status and challenges. Expert Opin. Drug Metab. Toxicol. 2008, 4, 205−223. (38) Shukla, S.; Ohnuma, S.; Ambudkar, S. V. Improving cancer chemotherapy with modulators of ABC drug transporters. Curr. Drug Targets 2011, 12, 621−630. (39) Wang, B.; Zhao, B.; Chen, Z.-S.; Pang, L.-P.; Zhao, Y.-D.; Guo, Q.; Zhang, X.-H.; Liu, Y.; Liu, G.-Y.; Hao-Zhang; Zhang, X.-Y.; Ma, L.Y.; Liu, H.-M. Exploration of 1,2,3-triazole-pyrimidine hybrids as potent reversal agents against ABCB1-mediated multidrug resistance. Eur. J. Med. Chem. 2018, 143, 1535−1542. (40) Ma, L.-Y.; Pang, L.-P.; Wang, B.; Zhang, M.; Hu, B.; Xue, D.-Q.; Shao, K.-P.; Zhang, B.-L.; Liu, Y.; Zhang, E.; Liu, H.-M. Design and synthesis of novel 1,2,3-triazole-pyrimidine hybrids as potential anticancer agents. Eur. J. Med. Chem. 2014, 86, 368−380. (41) Ma, L.-Y.; Zheng, Y.-C.; Wang, S.-Q.; Wang, B.; Wang, Z.-R.; Pang, L.-P.; Zhang, M.; Wang, J.-W.; Ding, L.; Li, J.; Wang, C.; Hu, B.; Liu, Y.; Zhang, X.-D.; Wang, J.-J.; Wang, Z.-J.; Zhao, W.; Liu, H.-M. Design, Synthesis and structure−activity relationship of novel LSD1 6000



Article

drug resistance in cancer cells. Cell. Physiol. Biochem. 2018, 45, 1515− 1528.

inhibitors based on pyrimidine−thiourea hybrids as potent, orally active antitumor agents. J. Med. Chem. 2015, 58, 1705−1716. (42) Klopman, G.; Shi, L. M.; Ramu, A. Quantitative structure-activity relationship of multidrug resistance reversal agents. Mol. Pharmacol. 1997, 52, 323−334. (43) Raub, T. J. P-glycoprotein recognition of substrates and circumvention through rational drug design. Mol. Pharmaceutics 2006, 3, 3−25. (44) Pajeva, I. K.; Globisch, C.; Wiese, M. Combined pharmacophore modeling, docking, and 3D QSAR studies of ABCB1 and ABCC1 transporter inhibitors. ChemMedChem 2009, 4, 1883−1896. (45) Ma, L.-Y.; Wang, B.; Pang, L.-P.; Zhang, M.; Wang, S.-Q.; Zheng, Y.-C.; Shao, K.-P.; Xue, D.-Q.; Liu, H.-M. Design and synthesis of novel 1,2,3-triazole-pyrimidine-urea hybrids as potential anticancer agents. Bioorg. Med. Chem. Lett. 2015, 25, 1124−1128. (46) Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein−ligand complexes. J. Med. Chem. 2006, 49, 6177−6196. (47) Ludescher, C.; Thaler, J.; Drach, D.; Drach, J.; Spitaler, M.; Gattringer, C.; Huber, H.; Hofmann, J. Detection of activity of pglycoprotein in human tumour samples usingrhodamine 123. Br. J. Haematol. 1992, 82, 161−168. (48) Boumendjel, A.; Baubichon-Cortay, H.; Trompier, D.; Perrotton, T.; Di Pietro, A. Anticancer multidrug resistance mediated by MRP1: recent advances in the discovery of reversal agents. Med. Res. Rev. 2005, 25, 453−472. (49) Wang, S.; Ryder, H.; Pretswell, I.; Depledge, P.; Milton, J.; Hancox, T. C.; Dale, I.; Dangerfield, W.; Charlton, P.; Faint, R.; Dodd, R.; Hassan, S. Studies on quinazolinones as dual inhibitors of Pgp and MRP1 in multidrug resistance. Bioorg. Med. Chem. Lett. 2002, 12, 571− 574. (50) Wang, S.; Folkes, A.; Chuckowree, I.; Cockcroft, X.; Sohal, S.; Miller, W.; Milton, J.; Wren, S. P.; Vicker, N.; Depledge, P.; Scott, J.; Smith, L.; Jones, H.; Mistry, P.; Faint, R.; Thompson, D.; Cocks, S. Studies on pyrrolopyrimidines as selective inhibitors of multidrugresistance-associated protein in multidrug resistance. J. Med. Chem. 2004, 47, 1329−1338. (51) Wang, S.; Wan, N. C.; Harrison, J.; Miller, W.; Chuckowree, I.; Sohal, S.; Hancox, T. C.; Baker, S.; Folkes, A.; Wilson, F.; Thompson, D.; Cocks, S.; Farmer, H.; Boyce, A.; Freathy, C.; Broadbridge, J.; Scott, J.; Depledge, P.; Faint, R.; Mistry, P.; Charlton, P. Design and synthesis of new templates derived from pyrrolopyrimidine as selective multidrug-resistance-associated protein inhibitors in multidrug resistance. J. Med. Chem. 2004, 47, 1339−1350. (52) Schmitt, S. M.; Stefan, K.; Wiese, M. Pyrrolopyrimidine derivatives as novel inhibitors of multidrug resistance-associated protein 1 (MRP1, ABCC1). J. Med. Chem. 2016, 59, 3018−3033. (53) Martinez Molina, D.; Jafari, R.; Ignatushchenko, M.; Seki, T.; Larsson, E. A.; Dan, C.; Sreekumar, L.; Cao, Y.; Nordlund, P. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 2013, 341, 84−87. (54) Baell, J. B.; Holloway, G. A. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J. Med. Chem. 2010, 53, 2719− 2740. (55) Zhou, H.; Zhou, W.; Zhou, B.; Liu, L.; Chern, T. R.; Chinnaswamy, K.; Lu, J.; Bernard, D.; Yang, C. Y.; Li, S.; Wang, M.; Stuckey, J.; Sun, Y.; Wang, S. High-affinity peptidomimetic inhibitors of the DCN1-UBC12 protein-protein interaction. J. Med. Chem. 2018, 61, 1934−1950. (56) Aller, S. G.; Yu, J.; Ward, A.; Weng, Y.; Chittaboina, S.; Zhuo, R.; Harrell, P. M.; Trinh, Y. T.; Zhang, Q.; Urbatsch, I. L.; Chang, G. Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. Science 2009, 323, 1718−1722. (57) Gupta, P.; Zhang, Y. K.; Zhang, X. Y.; Wang, Y. J.; Lu, K. W.; Hall, T.; Peng, R.; Yang, D. H.; Xie, N.; Chen, Z. S. Voruciclib, a potent CDK4/6 inhibitor, antagonizes ABCB1 and ABCG2-mediated multi6001


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