HM30181 Derivatives as Novel Potent and Selective Inhibitors of the

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HM30181 Derivatives as Novel Potent and Selective Inhibitors of the Breast Cancer Resistance Protein (BCRP/ABCG2) Sebastian C. Köhler and Michael Wiese* Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany ABSTRACT: The breast cancer resistance protein (BCRP, ABCG2) belongs to the superfamily of ATP binding-cassette (ABC) proteins. In addition to other physiological functions, it transports potentially cell-damaging compounds out of the cell using the energy from ATP hydrolysis. Certain tumors overexpressing BCRP were found to become resistant against various anticancer drugs. In previous work, we found that tariquidar analogues lacking the tetrahydroisoquinoline moiety selectively inhibit BCRP. In the present study, we synthesized 21 derivatives of the third-generation P-gp inhibitor HM30181, which is structurally related to tariquidar. The compounds were tested for their inhibitory activities against BCRP and screened against P-glycoprotein (P-gp, ABCB1) and multidrug resistance protein 1 (MRP1, ABCC1) to confirm the selectivity toward BCRP. The most potent compounds are selective toward BCRP and 2-fold more potent than the reference Ko143. Qualitative structure−activity relationship (SAR) analysis revealed that the presence of a methoxy group in the ortho or para position of at least one phenyl ring is beneficial for inhibitory activity. Furthermore, the cytotoxicity and multidrug resistance (MDR)-reversal ability of selected compounds were investigated. It was shown that they have a low cytotoxicity and the ability to reverse the BCRP-mediated SN-38 resistance.

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INTRODUCTION Members of the superfamily of ATP binding cassette (ABC) transporters are found in eukaryotes and prokaryotes.1 The ABC transporters actively transport their substrates using the energy derived from ATP hydrolysis. The first ABC transporter that was discovered was P-glycoprotein (P-gp, ABCB1). It was shown to confer resistance to cancer cells against several cytostatic agents used in cancer chemotherapy by actively transporting the chemotherapeutic agents out of the cells.2 As this resistance affects structurally unrelated cytostatics, it was named multidrug resistance (MDR). Cancer cells can be primarily resistant to chemotherapy or acquire MDR during treatment, making chemotherapy ineffective. Later it was found that P-gp is naturally expressed in several organs with barrier functions like the intestine, liver, and blood−brain barrier where it has a protective function for the body by transporting potentially cell-damaging compounds out of the cell.3,4 Besides P-gp, multidrug resistance associated protein 1 (MRP1, ABCC1) and breast cancer resistance protein (BCRP, ABCG2) are two other proteins that are associated with MDR in chemotherapy.5 Additionally, BCRP was found to be overexpressed in many untreated solid tumors.6 BCRP is a 72 kDa protein which was first discovered in 1998 in the multidrug resistant human breast cancer cell line MCF-7/AdrVp.7 It is a half-transporter consisting of six transmembrane helices and a N-terminal intracellular nucleotide binding domain and obtains its functionality via homo- or heterodimerization.8,9 It was shown that a broad variety of structurally different and important chemotherapeutic drugs such as methotrexate, camptothecin analogues, and tyrosine kinase inhibitors are substrates of BCRP. Efforts have been made to design potential © XXXX American Chemical Society

modulators in order to overcome BCRP-mediated MDR. The mycotoxin fumitremorgin C (FTC), isolated from Aspergillus f umigatus, was identified as a highly potent and specific inhibitor of BCRP.10,11 However, it has a high neurotoxic potential and is not suitable for therapeutic use. On the basis of the indolyl diketopiperazine scaffold of FTC, less toxic inhibitors such as Ko143 were developed.12 Other reported BCRP-specific inhibitors contain a chromone, chalcone, or quinazoline moiety as a basic structure.13 The well-known P-gp inhibitors elacridar (GF120918) and tariquidar (XR9576, Figure 1) were found to inhibit BCRP too, although with less efficacy.14 More recently, a new third-generation P-gp inhibitor, HM30181 (Figure 1), structurally related to tariquidar, but possessing a tetrazole ring instead of an amide linker, has been investigated.15 Furthermore, tariquidar derivatives lacking the tetrahydroisoquinoline moiety were found to selectively inhibit BCRP.16−18 Therefore, we decided to replace one of the amide linkers by a tetrazole linker in these derivatives (Figure 2). In this study, we present a new series of 21 compounds with different combinations of substituents. The best compounds were found to be highly potent and selective for BCRP and showing low toxicity in the MTT cell viability test.

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RESULTS AND DISCUSSION Chemistry. All new compounds of the present study were prepared as illustrated in Scheme 1. Sulfonyl hydrazide 1 was prepared from 2-nitrobenzaldehyde and benzenesulfonylhydra-

Received: February 2, 2015

A

DOI: 10.1021/acs.jmedchem.5b00188 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Structures of the third-generation P-gp inhibitors (A) tariquidar (XR9576), (B) XR9577, and (C) HM30181.

coupling of the differently substituted benzoyl chlorides with the amino group of 4a−g was performed in the presence of triethylamine to obtain the desired products 5−25 in the last step. The structures of intermediates 1, 3a−g, 4a−g, and the final compounds 5−25 were confirmed by NMR spectroscopy (1H and 13C). For verifying purity, elemental analyses and melting point determinations were performed. Biological Testing. Inhibition of BCRP (ABCG2). The final compounds 5−25 and the reference inhibitors XR9577 and Ko143 were investigated for their inhibitory activity against BCRP in the Hoechst 33342 accumulation assay using the MDCK II BCRP cell line. The bisbenzimidazole derivative Hoechst 33342 is a fluorescent dye and a substrate of BCRP. It was found that Hoechst 33342 shows much higher fluorescence

Figure 2. General structures of tariquidar-derived BCRP inhibitors and newly designed compounds containing a tetrazole moiety.

zine in ethanol. 1,5-Dipolar intramolecular cycloaddition of 1 with an aryldiazonium salt 2 resulted in the formation of the 2,5-diaryl tetrazoles 3a−g. The amino group in compounds 4a−g was obtained by catalytic hydrogenation of 3a−g. Amide Scheme 1. General Synthesis of the Tetrazoles 5−25a

a Reagents and conditions: (i) benzenesulfonylhydrazine, EtOH, T = 65 °C, yield 90%; (ii) NaNO2, HCl conc., EtOH, T < 0 °C; (iii) pyridine, T = −10 to −15 °C, yield 23−63%; (iv) Pd/C, H2, EtOH-THF (3:1), rt, yield quant.; (v) R2ArCOCl, TEA, THF, rt, yield 35−72%.

B



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higher inhibitory activity (IC50 = 0.184 μM) than the corresponding anthranilamide (IC50 of 0.94 μM18), which was the second most active compound in the series of anthranilamides. However, surprisingly 5 reached only a lower maximal response as compared to XR9577 (Figure 3). We

intensity when bound to DNA or being present in a lipophilic environment.19 Therefore, the observed fluorescence correlates with the increase of its intracellular concentration. The assay was used as previously described with minor modifications.14,16−18,20−27 The relative fluorescence intensity was measured in the presence of different concentrations of the modulator up to 10 μM for 2 h. For calculation of the IC50 values of the compounds 5−25, concentration−response curves were generated by plotting the averages of fluorescence values in the steady state against logarithmic concentrations of the modulators. The BCRP inhibitory activity data and maximal inhibition values are summarized in Table 1. Although tariquidar and Table 1. Inhibitory Activities of Compounds 5−25 and Reference Inhibitors against MDCK II BCRP Cells Using the Hoechst 33342 Assay

compd

R1

R2

IC50 ± SD [μM]a

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 XR9577 Ko143

4-nC3H7 4-CF3 4-CH3 4-OCH2CH3 2-OCH3 3-OCH3 4-OCH3 4-nC3H7 4-CF3 4-CH3 4-OCH2CH3 2-OCH3 3-OCH3 4-OCH3 4-CF3 4-OCH2CH3 4-OCH3 4-CF3 2-OCH3 4-nC3H7 4-OCH3

4-NO2 4-NO2 4-NO2 4-NO2 4-NO2 4-NO2 4-NO2 4-CF3 4-CF3 4-CF3 4-CF3 4-CF3 4-CF3 4-CF3 3,4-OCH3 3,4-OCH3 3,4-OCH3 4-OCH3 4-OCH3 4-OCH3 4-n-C3H7

0.184 ± 0.066 n.e.c 0.100 ± 0.038 n.e. 0.261 ± 0.015 0.0789 ± 0.0025 0.118 ± 0.036 n.e. n.e. 0.137 ± 0.027 n.e. 0.451 ± 0.095 11.2 ± 0.8 0.106 ± 0.017 0.269 ± 0.029 0.201 ± 0.056 0.117 ± 0.045 0.0730 ± 0.0208 0.180 ± 0.002 0.0642 ± 0.0065 0.0794 ± 0.0114 0.721 ± 0.254 0.128 ± 0.017

Imax ± SD [%]b

Figure 3. Representative concentration−response curves showing the reduced Imax of compound 5 (▲) in comparison to that of XR9577. Its corresponding analogue with an anthranilamide moiety instead of a tetrazole ring (●)18 and compound 22 (◆) reached the maximal response of XR9577 (■).

63 ± 4 71 ± 5 102 ± 7 58 ± 4 61 ± 5

decided to replace the propyl substituent on ring A and synthesized derivatives containing a 4-trifluoromethyl (6) or 4methyl group (7). Compound 6 exhibited no inhibitory effect up to 10 μM, but compound 7 achieved an IC50 of 0.100 μM against BCRP. Again, the compound did not reach the maximal response of the used standard, as compound 5. Thus, the spectrum of substituents on ring A was extended by a 4-ethoxy group (8) and methoxy groups in the ortho-, meta-, or paraposition (9, 10, 11). While compound 8 (4-OCH2CH3) exhibit no inhibiting effect at concentrations of up to 10 μM, we noticed that compounds 10 (3-OCH3) and 11 (4-OCH3) showed an inhibitory potency of 0.0789 μM and 0.118 μM, respectively. Both compounds showed reduced maximal responses similar to those of compounds 5 and 7. Only compound 9 (2-OCH3) reached a maximal response equivalent to that of XR9577 and a good IC50 value of 0.261 μM against BCRP. On the basis of these results, we decided to replace the 4nitro group on ring B by 4-trifluoromethyl and 3,4-dimethoxy substituents, which had also led to active derivatives in the series of anthranilamides.18 Compounds containing a 4trifluoromethyl moiety on ring B yielded results comparable to those of compounds with a 4-nitro substituent with the exception of compound 12. While the corresponding 4-nitro derivative 5 was found to be a potent inhibitor, compound 12 showed almost no effect up to 10 μM. As for the 4-nitro derivatives, a trifluoromethyl or ethoxy substituent at position R1 led to inactive compounds. For the other pairs of derivatives, the IC50 values were generally slightly higher for the trifluoromethyl derivatives with similar maximal responses. A slight exception is compound 16 (2-OCH3) that reaches the maximal response, but its inhibitory potency is decreased by

65 ± 7 100 ± 11 100d 93 ± 4 85 ± 15 106 ± 8 107 ± 3 102 ± 5 88 ± 10 88 ± 3 91 ± 7 100 103 ± 11

a

n = 3. bImax: maximum response of tested compounds in relation to standard XR9577. Ko143 was used as the positive control. cn.e. = no inhibitory effect up to 10 μM. dIC50 value obtained by constraining the maximal response to that of XR9577.

HM30181 (Figure 1) possess two methoxy groups at their middle phenyl ring, we synthesized compounds unsubstituted at this ring. Previous investigations had shown that in case of tariquidar-related anthranilamides substituents at this position decreased the inhibitory potency against BCRP.17 Initially, a nitro group at the para-position of ring B was selected as the substituent because this group had been proven to yield compounds with highest activity in the structurally related anthranilamides (Figure 2).17,18 Compound 5 showed a 5-fold C



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Figure 4. Effect of compounds 5−25 at a concentration of 10 μM on the accumulation of calcein AM in P-gp overexpressing A2780adr cells. Cyclosporin A (CsA, 10 μM) was used as a positive control leading to total inhibition. Data were obtained from three experiments and are expressed as a response in percentage of the positive control.

Thus, precipitation during the assays cannot be the reason for the reduced maximal effect of these compounds. If the substituent on ring B was 3,4-dimethoxy, potent inhibitors reaching maximal response were obtained. With this substituent on ring B also the trifluoromethyl derivative showed an acceptable IC50 value of 0.269 μM. Examining the upcoming inhibitory potency by introducing a 3,4-dimethoxy substituent, we synthesized another analogous derivative (20) of the inactive compounds 8 and 15 with the 4-ethoxy substituent on ring A, which yielded a similar potency (IC50 = 0.201 μM) and an increased Imax of 106%. Unfortunately, the replacement of 4trifluormethyl by 3,4-dimethoxy (21: IC50 = 0.117 μM) did not positively affect the inhibitory activity of this compound in comparison to that of compound 18 (IC50 = 0.106 μM). The presence of a methoxy substituent at the ortho- or paraposition seemed to be essential for the ability to inhibit BCRP without a decreased maximal response. Thus, a 4-methoxy group was introduced on ring B, which raised the inhibitory potency significantly. Compound 22 (IC50 = 0.0730 μM) showed a 3-fold higher activity than the corresponding 3,4dimethoxy derivative 19 (IC50 = 0.269 μM). This tendency was also seen for the compounds containing either a 2-methoxy (23: IC50 = 0.180 μM) or a 4-propyl substituent (24: IC50 = 0.0642 μM) on ring A. The best compounds 22, 24, and 25 are about twice more active in inhibiting BCRP than the standard Ko143. Compared to the anthranilamide series of inhibitors, the new derivatives seem generally more active. For the three compounds that can be compared directly, the new derivatives are between 5-fold (5) and 30-fold (11 and 21) more active than the corresponding anthranilamides.18 It seems that there have certain conditions have to be met regarding electronic influences and the substitution pattern of both substituents to yield potent inhibitors. If both rings contain electron withdrawing groups (-NO2 and -CF3), this leads to inactivity (6 and 13). Combining either of these substituents with electron donating groups such as alkyls (-CH3 and -C3H7) or ethers (-OCH3 and -OCH2CH3) on ring A, we obtained varying results. Compounds 9 and 16 bearing a methoxy group at the ortho-position at ring A are active and

around 2-fold compared to that of compound 9. For compound 17 (3-OCH3), a clear dose−response relationship was observed, but no plateau was reached up to 10 μM. Therefore, the inhibitory activity was calculated by constraining the maximal response to that of XR9577. This yielded a comparatively weak inhibitory activity of 11.2 μM, while the corresponding 4-nitro derivative was the most potent derivative in that series. Observing that the maximal response reached by some of the tested compounds did not always correspond to that of the used standards, we calculated the maximal inhibition (Imax) for each compound in relation to the standard. Compounds 5, 7, 10, 11, and 14 reached about two-thirds of the maximal response of XR9577 (Table 1 and Figure 3). This effect has also been recognized by Winter et al., who assumed that there might be a stimulation of basal ATPase activity by some of their tested compounds, contrary to their inhibitory ability.28 Kühnle et al. supposed that poor water solubility of compounds might be the reason for decreased Imax values.29 Also, our compounds 5−25 are poorly soluble in water but very soluble in DMSO (>10 mM), such as HM30181 or Tariquidar.30 We used methanol as additional solvent for preparing the 10 μM test solution to ensure that compounds with low solubility did not precipitate immediately. We investigated some compounds for slow precipitation during the measurement period of accumulation assays. Thus, 10 μM test solutions were prepared using doubly distilled water. This is the highest concentration used in the assays. The absorptions of the solutions were measured over a period of 3 h, which corresponds to more than the assay time. Three compounds with different Imax values (7, 71%; 9, 102%; 10, 58%) were selected. The absorbance of 9 began to decrease slightly after a few minutes, reaching about 75% at the end of the measurement. This result is in agreement with an observation in the Hoechst accumulation assay. Here, the highest concentration showed a decreased fluorescence as compared to lower concentrations of 9 which had already reached the Imax of XR9577. Compounds 7 and 10 behaved differently yielding constant absorptions over the testing time, showing that both compounds remained entirely in solution. D



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Figure 5. Effect of compounds 5−25 at a concentration of 10 μM on the accumulation of calcein AM in MRP1 overexpressing H69AR cells. Cyclosporin A (CsA, 10 μM) was used as a positive control leading to total inhibition. Data were obtained from three experiments and are expressed as a response in percentage of the positive control.

4 and 5 illustrate the percentage accumulation of calcein AM at 10 μM in A2780adr and H69AR cells, respectively. The broadspectrum MDR modulator cyclosporin A (CsA, 10 μM) was used as the positive control. Most compounds show only weak effects on P-gp without remarkable modulation of MRP1. The IC50 values for compounds 16, 20, 21, and 23 with an inhibition of ≥25% are presented in Table 2. Compared to

ensure maximal responses similar to those of the standards XR9577 and Ko143. The presence of a meta-methoxy substitutent lead to a decrease of either maximal response (10, 58%) or inhibitory effect (17, IC50 = 11.2 μM). The effects of the other selected substituents at the para-position on ring A varied as well. Derivatives with a 4-ethoxy group and in one case a 4-propyl group, lose their activity completely (8, 12, and 15), while other derivatives show activities in the high nanomolar range but a reduced Imax as compared to the standards (5, 7, 11, and 14). Generally, a 4-methoxy group led to derivatives with good inhibitory potency. This effect was confirmed by compounds 22−24 containing this substituent on ring B. The negative effect of a methoxy group in meta-position (see above) was also found in compounds with 3,4-dimethoxy substitution at phenyl ring B (19 and 20) having decreased activities. This influence of 3-methoxy is counterbalanced if there is a 4-methoxy substituent at the other phenyl ring (21). With regard to compound 22, the isomeric derivative 18 with exchanged substituents showed a similar potency. The isomeric counterpart 25 of the most active compound 24 was synthesized to verify whether there is only a small difference in the inhibitory activity by interchanging the both substituents at R1 and R2. This was confirmed by showing a similar IC50 value of 0.0794 μM. This result points to the possibility that mirror-like orientations of the phenyl rings A and B might occur upon binding to BCRP. Screening for P-gp (ABCB1) and MRP1 (ABCC1) Inhibition. All final compounds were investigated for their inhibition of other important multispecific ABC transporters such as P-gp and MRP1 by screening at a concentration of 10 μM. The assay was used as previously described with minor modifications.14,16−18,22,23,25−27 For this purpose, the P-gp overexpressing A2780adr cell line and the MRP1 overexpressing H69AR cell line were used in the calcein AM (acetoxymethyl ester) accumulation assay. After diffusion of calcein AM through the plasma membrane of cells, the ester moieties are hydrolyzed by unspecific esterases, and the corresponding calcein anion remains in cells and shows strong green fluorescence.31 Figures

Table 2. Inhibitory Activities of the Compounds Showing a Response of More than 25% in the P-gp Screening and the Reference Cyclosporin A against P-gp Overexpressing A2780adr Cells Using the Calcein AM Assay

a

compd

R1

R2

IC50 ± SD [μM]a

16 20 21 23 cyclosporin A

2-OCH3 4-OCH2CH3 4-OCH3 2-OCH3

4-CF3 3,4-OCH3 3,4-OCH3 4-OCH3

11.0 ± 0.6 32.8 ± 5.8 37.2 ± 1.7 7.73 ± 1.94 0.919 ± 0.165

n = 3.

BCRP, all 3,4-dimethoxy substituted compounds (20 and 21) possess only low inhibitory potency on P-gp. A 2-methoxysubstituent leads to a decrease of the selectivity toward BCRP with moderate inhibitory activities against P-gp (16, 11.0 μM; 23, IC50 = 7.73 μM). MTT Assay for Determining Cell Toxicity and the Ability to Reverse MDR. Whether or not the investigated compounds are usable for future in vivo experiments, MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity assay was used to determine their intrinsic cytotoxicity. The assay was used as previously described with minor modifications.18,22,26,27,32 Therefore, the most active compounds 22, 24, and 25 were selected as representatives for this class. MDCK II BCRP and wild-type cells were exposed to different concentrations of the compounds for a period of 72 h. They have a similar influence on the viability of the used cell lines (Table 3). Compound 22 showed the highest cytotoxicity E



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Table 3. Intrinsic Toxicity of the Most Active Compounds 22, 24, and 25 on MDCK II BCRP and MDCK II Wild-Type Cells Determined by the MTT Cytotoxicity Assay compd

R1

R2

MDCK II BCRP GI50 ± SD [μM]a

MDCK wild-type GI50 ± SD [μM]a

22 24 25

4-CF3 4-nC3H7 4-OCH3

4-OCH3 4-OCH3 4-nC3H7

40.2 ± 10.2 61.8 ± 13.9 60.9 ± 10.9

34.4 ± 6.5 63.2 ± 12.8 77.6 ± 10.4

a

n = 3; each experiment was performed in quadruplicate.

(GI50,BCRP = 40.2 μM; GI50,wild‑type = 34.4 μM), which is still around 500-fold higher than its IC50 value (0.0730 μM). A representative concentration−cell viability curve of compound 22 is shown in Figure 6. We observed that the both isomeric Figure 7. Representative concentration−cell viability curves of SN-38 (maximal concentration of 100 μM) showing the MDR reversal by compound 22 using the MTT cytotoxicity assay. The arrow illustrates the dose dependent sensitization of MDCK II BCRP cells by compound 22 toward SN-38. Triangles (SN-38 without 22), rhombi (SN-38 + 3 μM of 22), and circles (SN-38 + 5 μM of 22) indicate MDCK II BCRP cells. Squares indicate MDCK wild-type cells (SN-38, positive control).

substitution pattern in the outer phenyl rings on the BCRP modulation was investigated. In general, electron withdrawing groups led to inactive compounds or inhibitors showing a decreased maximal response, with the exception of three compounds (9, 16, and 18) that led to a maximal response comparable to that of the standards XR9577 and Ko143. The presence of electron donating groups on both rings A and B is optimal leading to compounds 22, 24, and 25 that are selective toward BCRP with about 2-fold higher inhibitory activities than the reference Ko143, which is the most potent BCRP inhibitor so far. We found that the interchange of a 4-methoxy group between the two rings had no significant influence on the IC50 values. Furthermore, it was shown that the most active compounds show low cytotoxicity leading to a beneficial ratio between cytotoxicity and inhibitory potency. We demonstrated the ability of the derivatives to reverse the BCRP-mediated SN38 resistance in MDCK II BCRP cells by coadministration of compound 22.

Figure 6. Representative concentration−cell viability curve of compound 22 obtained in the MTT cytotoxicity assay. Compounds were investigated up to a final concentration of 100 μM for a period of 72 h. Squares indicate MDCK wild-type cells and circles MDCK II BCRP cells.

compounds 24 and 25 which showed similar IC50 values on MDCK II BCRP cells (24, IC50 = 0.0642 μM; 25, IC50 = 0.0794 μM) have also a similar intrinsic cytotoxicity (24, GI50 = 61.8 μM; 25, GI50 = 60.9 μM). Moreover, the effect of these compounds on cell viability is around 1000-fold weaker than their inhibitory potency, and the GI50−IC50 ratios are even larger than the ratio of compound 22. Furthermore, the inhibitory potential of active class members was evaluated by investigating the antiproliferate effect of cytotoxic agents such as SN-38, the active metabolite of irinotecan, in the presence of compound 22 using the MTT assay. For this purpose, MDCK II BCRP and wild-type cells were exposed to the different concentrations of SN-38 in the absence and presence of 3 μM and 5 μM of 22 for a period of 72 h. We observed a decrease in cell viability and an increase of the cytotoxicity of SN-38, respectively, which indicates that 22 is able to reverse the BCRP-mediated SN-38 transport out of the cells (Figure 7).

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EXPERIMENTAL SECTION

Chemistry. Materials. The chemicals were purchased from various vendors (Acros Organics, Alfa Aesar, Merck Millipore or SigmaAldrich). The substituted anilines were purified by distillation or recrystallization before use. All other chemicals were used without further purification. Reactions were monitored by thin layer chromatography (TLC) using aluminum sheets coated with silica gel 60 F254 plates (Merck Millipore, Billerica, MA, USA) and chloroform/ toluene (3:1) as eluent. Compounds were visualized under UV light (254 nm). Column chromatography was performed on silica gel 60 (0.040−0.063 mm, Merck Millipore). Melting points were measured on a Sanyo Gallenkamp MPD350.BM3.5 melting point apparatus (Sanyo Gallenkamp PLC, Leicestershire, UK) and are uncorrected. The identity of intermediates and final compounds were confirmed by NMR. 1H- and 13C NMR spectra were recorded at room temperature (30 °C) either on a Bruker Advance 500 MHz (500/126 MHz) or Bruker Advance 600 MHz (600/151 MHz) NMR spectrometer. DMSO-d6 was used as a solvent as indicated below. Chemical shifts (δ) are expressed in parts per million (ppm), and the signal of the solvent was used as internal standard. Assignments of the 13C signals were performed using distortionsless enhancement by polarization

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CONCLUSIONS In conclusion, we demonstrated that HM30181 analogues lacking the tetrahydroisoquinoline moiety are selective inhibitors of BCRP. Structural optimization and SAR analyses led to the discovery of a series of tetrazole derivatives with high selectivity and potency toward BCRP. The influence of the F



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2-(4-Ethoxyphenyl)-5-(2-nitrophenyl)-2H-tetrazole (3d). The title compound was synthesized from 1 and 4-ethoxyaniline, recrystallized from EtOH/CHCl3 (1:1); yield 43%, colorless plates. 1H NMR (500 MHz, DMSO-d6) δ 8.14−8.10 (m, 1H), 8.04−8.00 (m, 2H), 7.93 (td, J = 7.6, 1.3 Hz, 1H), 7.87 (td, J = 7.8, 1.5 Hz, 1H), 7.24−7.20 (m, 1H), 4.15 (q, J = 7.0 Hz, 2H), 1.38 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 161.0, 160.0, 148.7, 133.3, 132.3, 131.1, 129.2, 124.5, 121.8 (2C), 119.9, 115.7 (2C), 63.9, 14.5. 2-(2-Methoxyphenyl)-5-(2-nitrophenyl)-2H-tetrazole (3e). The title compound was synthesized from 1 and 2-anisidine, recrystallized from acetone; yield 23%, colorless needles. 1H NMR (500 MHz, DMSO-d6) δ = 8.14−8.09 (m, 2H), 7.92 (td, J = 7.6, 1.3 Hz, 1H), 7.89−7.84 (m, 1H), 7.72−7.66 (m, 2H), 7.41 (dd, J = 8.4, 1.0 Hz, 1H), 7.22 (td, J = 7.7, 1.2 Hz, 1H), 3.86 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ = 160.6, 153.2, 148.7, 133.2, 133.0, 132.2, 130.9, 127.1, 125.1, 124.4, 120.8, 119.6, 113.5, 56.3. 2-(3-Methoxyphenyl)-5-(2-nitrophenyl)-2H-tetrazole (3f). The title compound was synthesized from 1 and 3-anisidine, recrystallized from EtOH/CHCl3 (1:1); yield 56%, off-white plates. 1H NMR (500 MHz, DMSO-d6) δ 8.15 (dd, J = 8.0, 1.1 Hz, 1H), 8.11 (dd, J = 7.6, 1.4 Hz, 1H), 7.94 (td, J = 7.6, 1.2 Hz, 1H), 7.88 (td, J = 7.8, 1.5 Hz, 1H), 7.71−7.67 (m, 1H), 7.64 (t, J = 2.3 Hz, 1H), 7.61 (t, J = 8.2 Hz, 1H), 7.25−7.20 (m, 1H). 13C NMR (126 MHz, DMSO-d6) δ 161.2, 160.2, 148.6, 136.8, 133.3, 132.3, 131.3, 131.1, 124.5, 119.7, 116.4, 112.1, 105.5, 55.7. 2-(4-Methoxyphenyl)-5-(2-nitrophenyl)-2H-tetrazole (3g). The title compound was synthesized from 1 and 4-anisidine, recrystallized from EtOH/CHCl3 (1:1); yield 45%, colorless needles. 1H NMR (500 MHz, DMSO-d6) δ 8.13 (dd, J = 8.0, 1.2 Hz, 1H), 8.11 (dd, J = 7.7, 1.4 Hz, 1H), 8.06−8.01 (m, 1H), 7.93 (td, J = 7.6, 1.3 Hz, 1H), 7.87 (td, J = 7.8, 1.5 Hz, 1H), 7.25−7.21 (m, 1H), 3.87 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 160.9, 160.6, 148.6, 133.2, 132.2, 131.0, 129.3, 124.4, 121.7 (2C), 119.8, 115.2 (2C), 55.7. General Procedure for the Preparation of 2-(2-Aryl-2H-tetrazol5-yl)anilines 4a−g. A solution of the corresponding compound 3 and palladium on charcoal (10 mol %) in ethanol/tetrahydrofuran (3:1, v/ v) was stirred in a Parr apparatus under 3 bar hydrogen atmosphere at room temperature overnight. After the removal of the catalyst by filtration through Celite, evaporation of the solvent under reduced pressure gave a white solid, which was used without further purification. 2-(2-(4-Propylphenyl)-2H-tetrazol-5-yl)aniline (4a). The title compound was synthesized from 3a; quantitative yield, white solid. 1 H NMR (500 MHz, DMSO-d6) δ 8.13−8.08 (m, 2H), 8.04 (dd, J = 7.9, 1.6 Hz, 1H), 7.53−7.47 (m, 2H), 7.27−7.21 (m, 1H), 6.92 (dd, J = 8.3, 1.1 Hz, 1H), 6.74−6.69 (m, 1H), 6.35 (s, 2H), 2.68 (t, J = 7.6 Hz, 2H), 1.66 (h, J = 7.4 Hz, 2H), 0.93 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO) δ 164.4, 146.9, 144.5, 134.1, 131.3, 129.8 (2C), 128.2, 119.8 (2C), 116.2, 115.7, 107.7, 23.8, 13.5. 2-(2-(4-(Trifluoromethyl)phenyl)-2H-tetrazol-5-yl)aniline (4b). The title compound was synthesized from 3b; quantitative yield, white solid. 1H NMR (500 MHz, DMSO-d6) δ 8.46 (d, J = 8.4 Hz, 2H), 8.09−8.04 (m, 3H), 7.28−7.23 (m, 1H), 6.93 (dd, J = 8.3, 1.1 Hz, 1H), 6.75−6.70 (m, 1H), 6.38 (s, 2H). 13C NMR (126 MHz, DMSO-d6) δ 164.7, 147.1, 138.7, 131.6, 129.8 (q, J = 32.5 Hz), 128.4, 127.3 (q, J = 3.4 Hz, 2C), 123.7 (d, J = 272.5 Hz), 120.5, 116.3, 115.7, 107.3. 2-(2-(4-Methylphenyl)-2H-tetrazol-5-yl)aniline (4c). The title compound was synthesized from 3c; quantitative yield, white solid. 1 H NMR (500 MHz, DMSO-d6) δ 8.09 (d, J = 8.5 Hz, 2H), 8.04 (dd, J = 7.9, 1.5 Hz, 1H), 7.49 (d, J = 8.1 Hz, 2H), 7.27−7.21 (m, 1H), 6.92 (dd, J = 8.3, 0.8 Hz, 1H), 6.74−6.68 (m, 1H), 6.35 (s, 2H), 2.43 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 164.4, 146.9, 139.9, 133.9, 131.3, 130.4 (2C), 128.2, 119.8 (2C), 116.2, 115.7, 107.7, 20.7. 2-(2-(4-Ethoxyphenyl)-2H-tetrazol-5-yl)aniline (4d). The title compound was synthesized from 3d; quantitative yield, white solid. 1 H NMR (500 MHz, DMSO-d6) δ 8.13−8.08 (m, 2H), 8.03 (dd, J = 7.9, 1.6 Hz, 1H), 7.25−7.18 (m, 3H), 6.91 (dd, J = 8.4, 1.1 Hz, 1H), 6.73−6.68 (m, 1H), 6.34 (s, 2H), 4.15 (q, J = 7.0 Hz, 2H), 1.38 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO) δ 164.3, 159.6, 146.8,

transfer (DEPT) and attached proton test (APT) techniques. Coupling constants J are given in hertz (Hz), and spin multiplicities are given as singulet (s), doublet (d), doublet of doublets (dd), triplet of doublets (td), triplet (t), doublet of triplets (dt), quartet (q), and multiplet (m). The purity of all biologically evaluated compounds was determined to be >95% by elemental analysis recorded on a Vario EL V24 CHN Elemental Analyzer (Elementar Analysensysteme GmbH, Hanau, Germany). All values are within 0.4% of the theoretical values. Calculated yields were referred to the amount of compound which was obtained after first crystallization. General Procedure for the Preparation of Substituted Benzoyl Chlorides. The corresponding acid was placed in a dry roundbottomed flask, and at least 5-fold excess of freshly distilled thionyl chloride was added. The reaction mixture was refluxed for 2−4 h under exclusion of moisture. After the removal of the excess of thionyl chloride at normal pressure, the residue was recrystallized from dry benzene or distilled under reduced pressure to give pure aryl acid chloride, which was stored in a desiccator at room temperature until use. Preparation of Ń -(2-Nitrobenzylidene)-benzenesulfonohydrazide (1). The title compound was synthesized according to the literature,33 recrystallized from EtOH;, yield 90%, yellow prisms. 1H NMR (500 MHz, DMSO-d6) δ 11.96 (s, 1H), 8.30 (s, 1H), 8.02 (dd, J = 8.2, 1.2 Hz, 1H), 7.91−7.87 (m, 2H), 7.81 (dd, J = 7.9, 1.5 Hz, 1H), 7.77− 7.72 (m, 1H), 7.70−7.61 (m, 4H). 13C NMR (126 MHz, DMSO-d6) δ 147.8, 142.5, 138.9, 133.7, 133.2, 130.7, 129.3 (2C), 127.9, 127.8, 127.1 (2C), 124.6. General Procedure for the Preparation of 2-Aryl-5-(2-nitrophenyl)-2H-tetrazoles 3a−g. Compounds were synthesized according to the literature with minor modifications.34 A cooled solution of sodium nitrite (15 mmol, 1 equiv) in demineralized water (4 mL) was added below 0 °C to a solution or suspension of a substituted aniline 2 (15 mmol, 1 equiv) and concentrated hydrochloric acid (45 mmol, 3 equiv) in an 1:1 mixture of EtOH/H2O (10 mL). After that, this solution was added dropwise to a stirred solution of 1 (15 mmol, 1 equiv) in dry pyridine (50 mL) between −10 °C and −15 °C using a sodium chloride−ice bath. The red reaction mixture was stirred until reaching room temperature and then extracted with chloroform (1 × 100 mL) and water (1 × 100 mL). The organic phase was washed with 1 N hydrochloric acid (50 mL) and water (50 mL), and dried over calcium chloride. After evaporation under reduced pressure, the resulting residue was purified by column chromatography on silica gel using toluene as eluent to give the desired tetrazole, which was recrystallized from an appropriate solvent. 5-(2-Nitrophenyl)-2-(4-propylphenyl)-2H-tetrazole (3a). The title compound was synthesized from 1 and 4-propylaniline, recrystallized from EtOH/CHCl3 (1:1); yield 63%, yellowish needles. 1H NMR (500 MHz, DMSO-d6) δ 8.14 (dd, J = 8.0, 1.0 Hz, 1H), 8.11 (dd, J = 7.7, 1.3 Hz, 1H), 8.02 (d, J = 8.5 Hz, 2H), 7.93 (td, J = 7.6, 1.2 Hz, 1H), 7.87 (td, J = 7.8, 1.4 Hz, 1H), 7.51 (d, J = 8.5 Hz, 2H), 2.68 (t, J = 7.6 Hz, 2H), 1.71−1.59 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 161.0, 148.6, 145.1, 133.9, 133.2, 132.2, 131.0, 130.0 (2C), 124.4, 119.9 (2C), 119.7, 36.7, 23.7, 13.4. 5-(2-Nitrophenyl)-2-(4-(trifluoromethyl)phenyl)-2H-tetrazole (3b). The title compound was synthesized from 1 and 4(trifluoromethyl)aniline, recrystallized from acetone; yield 51%, colorless prisms. 1H NMR (500 MHz, DMSO-d6) δ 8.36 (d, J = 8.4 Hz, 2H), 8.16 (d, J = 8.0 Hz, 1H), 8.13 (d, J = 7.6 Hz, 1H), 8.09 (d, J = 8.5 Hz, 2H), 7.95 (t, J = 7.5 Hz, 1H), 7.89 (t, J = 7.7 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 161.5, 148.6, 138.5, 133.3, 132.5, 131.1, 130.3 (q, J = 32.5 Hz), 127.6 (q, J = 3.3 Hz, 2C), 124.5, 123.5 (q, J = 272.5 Hz), 120.7 (2C), 119.4. 2-(4-Methylphenyl)-5-(2-nitrophenyl)-2H-tetrazole (3c). The title compound was synthesized from 1 and 4-toluidine, recrystallized from EtOH/CHCl3 (1:1); yield 62%, yellowish plates. 1H NMR (500 MHz, DMSO-d6) δ 8.14 (dd, J = 8.0, 1.1 Hz, 1H), 8.12 (dd, J = 7.7, 1.4 Hz, 1H), 8.01 (d, J = 8.5 Hz, 2H), 7.94 (td, J = 7.6, 1.3 Hz, 1H), 7.88 (td, J = 7.8, 1.5 Hz, 1H), 7.51 (d, J = 8.1 Hz, 2H), 2.44 (s, 3H), 2.44 (s, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 161.0, 148.6, 140.6, 133.7, 133.3, 132.3, 131.0, 130.6, 124.4, 119.9, 119.7, 20.7. G



Article

N-(2-(2-(4-Ethoxyphenyl)-2H-tetrazol-5-yl)phenyl)-4-nitrobenzamide (8). The title compound was synthesized from 4d and 4nitrobenzoyl chloride; yield 60%, yellow powder, recrystallized from EtOH/THF (4:3), mp 198−199 °C (decomposition). 1H NMR (600 MHz, DMSO-d6) δ 10.94 (s, 1H), 8.40 (d, J = 8.5 Hz, 2H), 8.29−8.23 (m, 3H), 8.16 (d, J = 8.1 Hz, 1H), 7.97 (d, J = 8.9 Hz, 2H), 7.66 (t, J = 7.7 Hz, 1H), 7.47 (t, J = 7.6 Hz, 1H), 7.18 (d, J = 8.9 Hz, 2H), 4.14 (q, J = 6.9 Hz, 2H), 1.37 (t, J = 6.9 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 163.8, 163.1, 159.8, 149.4, 140.2, 135.9, 131.2, 129.2, 129.1, 129.0 (2C), 125.8, 124.8, 123.8 (2C), 121.6 (2C), 119.1, 115.5 (2C), 63.7, 14.5. Anal. Calcd for C22H18N6O4: C, 61.39; H, 4.22; N, 19.53. Found: C, 61.66; H, 4.30; N, 19.30. N-(2-(2-(2-Methoxyphenyl)-2H-tetrazol-5-yl)phenyl)-4-nitrobenzamide (9). The title compound was synthesized from 4e and 4nitrobenzoyl chloride, recrystallized from EtOH/THF (10:1); yield 58%, yellow needles, mp 176−177 °C (decomposition). 1H NMR (500 MHz, DMSO-d6) δ 10.96 (s, 1H), 8.35−8.31 (m, 2H), 8.26− 8.19 (m, 4H), 7.71−7.63 (m, 3H), 7.46 (td, J = 7.6, 1.2 Hz, 1H), 7.40 (dd, J = 8.5, 1.0 Hz, 1H), 7.20 (td, J = 7.7, 1.1 Hz, 1H), 3.77 (s, 3H). 13 C NMR (126 MHz, DMSO) δ 163.6, 162.8, 153.1, 149.3, 140.2, 135.9, 132.8, 131.2, 128.9 (3C), 127.0, 125.7, 125.2, 124.5, 123.6 (2C), 120.7, 118.8, 113.5, 56.2. Anal. Calcd for C21H16N6O3: C, 60.57; H, 3.87; N, 20.18. Found: C, 60.54; H, 3.95; N, 19.05. N-(2-(2-(3-Methoxyphenyl)-2H-tetrazol-5-yl)phenyl)-4-nitrobenzamide (10). The title compound was synthesized from 4f and 4nitrobenzoyl chloride, recrystallized from EtOH/THF (2:1); yield 45%, yellow needles, mp 192−193 °C (decomposition). 1H NMR (500 MHz, DMSO-d6) δ 10.88 (s, 1H), 8.39 (d, J = 8.8 Hz, 2H), 8.28 (d, J = 8.4 Hz, 3H), 8.11 (d, J = 8.3 Hz, 1H), 7.70−7.64 (m, 2H), 7.57 (t, J = 8.2 Hz, 1H), 7.51−7.47 (m, 2H), 7.19 (dd, J = 8.3, 2.6 Hz, 1H), 3.81 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 163.8, 163.2, 160.1, 149.4, 140.2, 136.9, 135.9, 131.3, 131.2, 129.1, 129.0 (2C), 125.9, 125.1, 123.8 (2C), 119.2, 115.8, 111.8, 105.6, 55.6. Anal. Calcd for C21H16N6O4: C, 60.57; H, 3.87; N, 20.18. Found: C, 60.56; H, 3.83; N, 19.81. N-(2-(2-(4-Methoxyphenyl)-2H-tetrazol-5-yl)phenyl)-4-nitrobenzamide (11). The title compound was synthesized from 4g and 4nitrobenzoyl chloride, recrystallized from EtOH/THF (2:1); yield 58%, yellow fluff, mp 196−197 °C (decomposition). 1H NMR (500 MHz, DMSO-d6) δ 10.94 (s, 1H), 8.41−8.37 (m, 1H), 8.28−8.25 (m, 1H), 8.24 (dd, J = 7.9, 1.4 Hz, 1H), 8.17 (dd, J = 8.2, 0.7 Hz, 1H), 8.01−7.97 (m, 2H), 7.69−7.63 (m, 1H), 7.47 (td, J = 7.7, 1.1 Hz, 1H), 7.21−7.17 (m, 1H), 3.86 (s, 3H). 13C NMR (126 MHz, DMSO) δ 163.8, 163.1, 160.5, 149.4, 140.2, 135.9, 131.2, 129.4, 129.1, 129.0 (2C), 125.7, 124.7, 123.8 (2C), 121.6 (2C), 119.1, 115.12 (2C), 115.05, 55.7. Anal. Calcd for C21H16N6O4: C, 60.57; H, 3.87; N, 20.18. Found: C, 60.54; H, 4.01; N, 19.81. N - ( 2 - ( 2 - ( 4- P r o py l p h e n y l ) - 2 H- t e t r az o l - 5- y l ) ph e n y l ) - 4 (trifluoromethyl)benzamide (12). The title compound was synthesized from 4a and 4-(trifluoromethyl)benzoyl chloride, recrystallized from EtOH/THF (2:1); yield 62%, colorless fluff, mp 165−166 °C. 1 H NMR (500 MHz, DMSO-d6) δ 10.88 (s, 1H), 8.28−8.18 (m, 4H), 7.98−7.90 (m, 4H), 7.69−7.63 (m, 1H), 7.49−7.42 (m, 3H), 2.67 (t, J = 7.5 Hz, 2H), 1.69−1.60 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 164.2, 163.2, 144.8, 138.4, 136.1, 134.0, 131.7 (d, J = 32.0 Hz), 131.3, 129.8 (2C), 129.0, 128.4 (2C), 125.6 (d, J = 3.3 Hz, 2C), 125.6, 124.6, 119.8 (2C), 118.8, 36.6, 23.7, 13.4; one signal for −CF3 missing. Anal. Calcd for C24H20F3N5O: C, 63.85; H, 4.47; N, 15.51. Found: C, 63.88; H, 4,61; N, 15.40. 4-(Trifluoromethyl)-N-(2-(2-(4-(trifluoromethyl)phenyl)-2H-tetrazol-5-yl)phenyl)benzamide (13). The title compound was synthesized from 4b and 4-(trifluoromethyl)benzoyl chloride, recrystallized from EtOH; yield 50%, colorless needles, mp 182−183 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.81 (s, 1H), 8.28 (d, J = 8.5 Hz, 2H), 8.24 (dd, J = 7.9, 1.5 Hz, 1H), 8.22 (d, J = 8.1 Hz, 2H), 8.14 (dd, J = 8.2, 1.0 Hz, 1H), 8.03 (d, J = 8.6 Hz, 2H), 7.96 (d, J = 8.2 Hz, 2H), 7.71−7.65 (m, 1H), 7.48 (td, J = 7.6, 1.2 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 164.3, 163.7, 138.6, 138.3, 136.2, 131.7 (d, J = 32.0 Hz), 131.5, 130.1 (d, J = 32.7 Hz), 129.3, 128.4 (2C), 127.4 (q, J = 3.4 Hz, 2C), 125.73, 125.65 (q, J = 3.5 Hz, 2C), 125.1, 123.5 (d, J = 272.3 Hz), 120.5 (2C),

131.3, 129.4, 128.2, 121.6 (2C), 116.2, 115.7, 115.4 (2C), 107.8, 63.7, 14.5. 2-(2-(2-Methoxyphenyl)-2H-tetrazol-5-yl)aniline (4e). The title compound was synthesized from 3e; quantitative yield, white solid. 1 H NMR (500 MHz, DMSO-d6) δ 8.00 (dd, J = 7.9, 1.6 Hz, 1H), 7.72 (dd, J = 7.8, 1.7 Hz, 1H), 7.70−7.66 (m, 1H), 7.40 (dd, J = 8.5, 1.2 Hz, 1H), 7.26−7.19 (m, 2H), 6.90 (dd, J = 8.3, 1.2 Hz, 1H), 6.73−6.68 (m, 1H), 6.29 (s, 2H), 3.85 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 164.1, 153.4, 146.8, 132.6, 131.2, 128.1, 127.3, 125.4, 120.7, 116.2, 115.8, 113.4, 107.8, 56.3. 2-(2-(3-Methoxyphenyl)-2H-tetrazol-5-yl)aniline (4f). The title compound was synthesized from 3f; quantitative yield, white solid. 1 H NMR (500 MHz, DMSO-d6) δ 8.05 (dd, J = 8.0, 1.6 Hz, 1H), 7.80−7.76 (m, 1H), 7.73 (t, J = 2.3 Hz, 1H), 7.60 (t, J = 8.2 Hz, 1H), 7.27−7.22 (m, 1H), 7.20−7.17 (m, 1H), 6.92 (dd, J = 8.2, 1.1 Hz, 1H), 6.74−6.69 (m, 1H), 6.35 (s, 2H), 3.90 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 164.5, 160.2, 146.9, 137.0, 131.4, 131.0, 128.3, 116.3, 115.75, 115.72, 112.0, 107.6, 105.5, 55.7. 2-(2-(4-Methoxyphenyl)-2H-tetrazol-5-yl)aniline (4g). The title compound was synthesized from 3g; quantitative yield, white solid. 1H NMR (500 MHz, DMSO-d6) δ 8.15−8.09 (m, 2H), 8.03 (dd, J = 7.9, 1.6 Hz, 1H), 7.25−7.19 (m, 3H), 6.91 (dd, J = 8.3, 1.1 Hz, 1H), 6.74− 6.68 (m, 1H), 6.34 (s, 2H), 3.87 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 164.3, 160.3, 146.9, 131.3, 129.5, 128.2, 121.6 (2C), 116.2, 115.7, 115.0 (2C), 107.8, 55.7. General Procedure for the Preparation of N-(2-(2-Aryl-2Htetrazol-5-yl)phenyl)arylamides 5−25. An equimolar amount of a substituted benzoyl chloride in dry tetrahydrofuran (10 mL) was added dropwise to a solution of 2-(2-aryl-2H-tetrazol-5-yl)aniline and triethylamine (1.4 equiv) in dry tetrahydrofuran (10 mL) at room temperature under moisture exclusion. The reaction mixture was stirred overnight. After the removal of the precipitated triethylamine hydrochloride by filtration, the solvent was evaporated under reduced pressure and the residue recrystallized from an appropriate solvent. 4-Nitro-N-(2-(2-(4-propylphenyl)-2H-tetrazol-5-yl)phenyl)benzamide (5). The title compound was synthesized from 4a and 4nitrobenzoyl chloride, recrystallized from EtOH/THF (2:1); yield 57%, yellowish needles, mp 174−175 °C (decomposition). 1H NMR (500 MHz, DMSO-d6) δ 10.92 (s, 1H), 8.41−8.36 (m, 2H), 8.29− 8.23 (m, 3H), 8.15 (dd, J = 8.2, 0.9 Hz, 1H), 7.99−7.94 (m, 2H), 7.69−7.64 (m, 1H), 7.50−7.44 (m, 3H), 2.70−2.64 (m, 2H), 1.69− 1.60 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, DMSOd6) δ 163.8, 163.2, 149.4, 144.8, 140.2, 136.0, 134.0, 131.3, 129.9 (2C), 129.1, 129.0 (2C), 125.8, 124.9, 123.7 (2C), 119.8 (2C), 119.2, 36.6, 23.7, 13.4. Anal. Calcd for C23H20N6O3: C, 64.48; H, 4.71; N, 19.62. Found: C, 64.46; H, 4.76; N, 19.25. 4-Nitro-N-(2-(2-(4-(trifluoromethyl)phenyl)-2H-tetrazol-5-yl)phenyl)benzamide (6). The title compound was synthesized from 4b and 4-nitrobenzoyl chloride, recrystallized from EtOH/THF (2:1); yield 46%, yellow powder, mp 209−210 °C (decomposition). 1H NMR (500 MHz, DMSO-d6) δ 10.86 (s, 1H), 8.39 (t, J = 9.7 Hz, 2H), 8.30 (d, J = 8.5 Hz, 2H), 8.25 (d, J = 8.6 Hz, 3H), 8.12−8.04 (m, 3H), 7.69 (t, J = 7.3 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 164.0, 163.9, 149.6, 140.3, 138.7, 136.2, 131.7, 130.3 (d, J = 32.5 Hz), 129.5, 129.1 (2C), 127.7 (q, J = 3.5 Hz, 2C), 126.1, 125.5, 123.9 (2C), 120.7, 119.5 (2C); One signal for −CF3 missing. Anal. Calcd for C21H13F3N6O3: C, 55.51; H, 2.88; N, 18.50. Found: C, 55.14 ; H, 2.96; N, 18.10. N-(2-(2-(4-Methylphenyl)-2H-tetrazol-5-yl)phenyl)-4-nitrobenzamide (7). The title compound was synthesized from 4c and 4nitrobenzoyl chloride, recrystallized from EtOH/THF (2:1), yield 64%, yellowish needles, mp 187−188 °C (decomposition). 1H NMR (500 MHz, DMSO-d6) δ 10.91 (s, 1H), 8.42−8.37 (m, 2H), 8.29− 8.25 (m, 2H), 8.24 (dd, J = 8.0, 1.4 Hz, 1H), 8.14 (dd, J = 8.2, 0.8 Hz, 1H), 7.97−7.92 (m, 2H), 7.69−7.64 (m, 1H), 7.50−7.44 (m, 1H), 2.42 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 163.8, 163.2, 149.4, 140.3, 140.2, 135.9, 133.8, 131.3, 130.5 (2C), 129.1, 129.0 (2C), 125.8, 124.9, 123.8 (2C), 119.7 (2C), 119.2, 20.7. Anal. Calcd for C21 H16 N6 O3: C, 62.99; H, 4.03; N, 20.99. Found: C, 63.00; H, 4.16; N, 20.87. H



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1H), 7.40 (td, J = 7.7, 1.2 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 3.86 (s, 3H), 3.80 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 164.7, 163.9, 152.0, 148.6, 138.6, 137.0, 131.5, 130.1 (q, J = 32.4 Hz), 129.0,127.4 (d, J = 3.6 Hz, 2C), 126.7, 124.6, 123.6 (d, J = 272.7 Hz), 123.4, 120.7, 120.7 (2C), 116.9, 111.2, 110.8, 55.7, 55.5. Anal. Calcd for C23H18F3N5O3: C, 58.85; H, 3.87; N, 14.92. Found: C, 58.69 ; H, 3.94; N, 14.75. N-(2-(2-(4-Ethoxyphenyl)-2H-tetrazol-5-yl)phenyl)-3,4-dimethoxybenzamide (20). The title compound was synthesized from 4d and 3,4-dimethoxybenzoyl chloride; yield 64%, recrystallized from EtOH/ THF (3:2), off-white fluff, mp 165−167 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.82 (s, 1H), 8.48 (dd, J = 8.3, 1.0 Hz, 1H), 8.26 (dd, J = 7.9, 1.5 Hz, 1H), 8.03−7.98 (m, 2H), 7.70 (dd, J = 8.4, 2.2 Hz, 1H), 7.63 (d, J = 2.1 Hz, 1H), 7.63−7.59 (m, 1H), 7.39−7.33 (m, 1H), 7.21−7.16 (m, 2H), 7.13 (d, J = 8.4 Hz, 1H), 4.18 (q, J = 7.0 Hz, 2H), 3.89 (s, 3H), 3.84 (d, J = 3.6 Hz, 3H), 1.39 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO) δ 164.3, 163.1, 159.7, 152.0, 148.6, 136.8, 130.8, 129.1, 128.4, 126.9, 123.9, 122.3, 121.5 (2C), 120.5, 116.2, 115.4 (2C), 111.5, 111.2, 63.6, 55.6, 55.6, 14.0. Anal. Calcd for C24H23N5O4: C, 64.71; H, 5.20; N, 15.72. Found: C, 64.41; H, 5.29; N, 15.68. 3,4-Dimethoxy-N-(2-(2-(4-methoxyphenyl)-2H-tetrazol-5-yl)phenyl)benzamide (21). The title compound was synthesized from 4g and 3,4-dimethoxybenzoyl chloride, recrystallized from EtOH/THF (1:1); yield 72%, colorless powder, mp 150−151 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.86 (s, 1H), 8.45−8.41 (m, 1H), 8.25 (dd, J = 7.9, 1.5 Hz, 1H), 8.05−8.00 (m, 2H), 7.70 (dd, J = 8.4, 2.1 Hz, 1H), 7.64−7.59 (m, 2H), 7.37 (td, J = 7.8, 1.1 Hz, 1H), 7.23−7.18 (m, 2H), 7.13 (d, J = 8.5 Hz, 1H), 3.873 (s, 3H), 3.869 (s, 3H), 3.82 (s, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 164.6, 163.3, 160.6, 152.0, 148.6, 136.9, 131.2, 129.4, 128.8, 126.8, 124.4, 122.8, 121.7 (2C), 120.7, 116.7, 115.1 (2C), 111.2, 110.8, 55.7 (2C), 55.5. Anal. Calcd for C23H21N5O4: C, 64.03; H, 4.91; N, 16.23. Found: C, 63.90; H, 4.86; N, 16.17. 4-Methoxy-N-(2-(2-(4-(trifluoromethyl)phenyl)-2H-tetrazol-5-yl)phenyl)benzamide (22). The title compound was synthesized from 4b and 4-(methoxy)benzoyl chloride, recrystallized from EtOH/THF (1:1); yield 35%, colorless needles, mp 175−176 °C (decomposition). 1 H NMR (600 MHz, DMSO-d6) δ 10.69 (s, 1H), 8.34 (d, J = 8.3 Hz, 3H), 8.26 (d, J = 7.8 Hz, 1H), 8.09 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 8.6 Hz, 2H), 7.65 (t, J = 7.8 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.12 (d, J = 8.6 Hz, 2H), 3.86 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 164.7, 163.9, 162.2, 138.6, 137.0, 131.5, 130.2 (q, J = 32.4 Hz), 129.4 (2C), 129.0, 126.6, 127.5 (q, J = 3.6 Hz, 2C), 124.7, 123.60 (q, J = 272.4 Hz), 123.59, 120.7 (2C), 117.1, 114.0 (2C), 55.5. Anal. Calcd for C22H16F3N5O2: C, 60.41; H, 3.67; N, 15.49. Found: C, 60.16 ; H, 3.73; N, 15.83. 4-Methoxy-N-(2-(2-(2-methoxyphenyl)-2H-tetrazol-5-yl)phenyl)benzamide (23). The title compound was synthesized from 4e and 4methoxybenzoyl chloride, recrystallized from EtOH/THF (15:1); yield 65%, yellowish needles, mp 172−174 °C (decomposition). 1H NMR (500 MHz, DMSO-d6) δ 10.89 (s, 1H), 8.49 (dd, J = 8.4, 1.0 Hz, 1H), 8.27 (dd, J = 7.9, 1.5 Hz, 1H), 8.03−7.99 (m, 2H), 7.74− 7.68 (m, 2H), 7.64−7.60 (m, 1H), 7.43 (dd, J = 8.5, 1.0 Hz, 1H), 7.39−7.35 (m, 1H), 7.24 (td, J = 7.7, 1.2 Hz, 1H), 7.05−7.01 (m, 2H), 3.84 (s, 3H), 3.81 (s, 3H). 13C NMR (126 MHz, DMSO) δ 164.4, 163.0, 162.2, 153.2, 136.9, 132.9, 131.3, 129.2 (2C), 128.6, 127.1, 126.6, 125.2, 124.3, 122.5, 120.7, 116.2, 113.9 (2C), 113.4, 56.2, 55.4. Anal. Calcd for C22H19N5O3: C, 65.83; H, 4.77; N, 17.45. Found: C, 65.57; H, 4.86; N, 17.12. 4-Methoxy-N-(2-(2-(4-propylphenyl)-2H-tetrazol-5-yl)phenyl)benzamide (24). The title compound was synthesized from 4a and 4methoxybenzoyl chloride, recrystallized from EtOH; yield 68%, offwhite fluff, mp 110−111 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.79 (s, 1H), 8.41 (dd, J = 8.3, 1.0 Hz, 1H), 8.26 (dd, J = 7.9, 1.6 Hz, 1H), 8.06−8.03 (m, 2H), 8.03−7.99 (m, 2H), 7.64−7.59 (m, 1H), 7.51− 7.47 (m, 1H), 7.40−7.35 (m, 1H), 7.12−7.07 (m, 2H), 3.87 (s, 3H), 2.70−2.66 (m, 2H), 1.69−1.61 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 164.7, 163.5, 162.4, 145.1, 137.1, 134.2, 131.5, 130.0 (2C), 129.5 (2C), 128.9, 126.9, 124.6, 123.2, 120.1

119.0; one signal for -CF3 missing. Anal. Calcd for C22H13F6N5O: C, 55.35; H, 2.75; N, 14.67. Found: C, 55.55; H, 2.94; N, 14.53. N-(2-(2 -(4-Methylphenyl) -2H-tetrazol -5-yl )phenyl)-4(trifluoromethyl)benzamide (14). The title compound was synthesized from 4c and 4-(trifluoromethyl)benzoyl chloride, recrystallized from EtOH; yield 60%, white needles, mp 157−158 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.87 (s, 1H), 8.26−8.22 (m, 3H), 8.19 (dd, J = 8.2, 0.9 Hz, 1H), 7.97−7.91 (m, 4H), 7.68−7.63 (m, 1H), 7.49− 7.41 (m, 3H), 2.41 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 164.2, 163.2, 140.3, 138.4, 136.1, 133.8, 131.7 (d, J = 31.9 Hz), 131.3, 130.4 (2C), 129.0, 128.4 (2C), 125.63 (q, J = 3.8 Hz, 2C), 125.55, 124.6, 123.8 (q, J = 272.5 Hz), 119.7 (2C), 118.8, 20.7. Anal. Calcd for C22 H16 F3 N5 O: C, 62.41; H, 3.81; N, 16.54. Found: C, 62.14; H, 3.90; N, 16.51. N- (2-( 2-(4 -Ethoxyphe nyl )-2H- tetra zol-5 -yl)p he nyl)- 4(trifluoromethyl)benzamide (15). The title compound was synthesized from 4d and 4-(trifluoromethyl)benzoyl chloride, recrystallized from EtOH/THF (10:1); yield 62%, off-white powder, mp 146−147 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.90 (s, 1H), 8.26−8.21 (m, 4H), 7.98−7.93 (m, 4H), 7.68−7.62 (m, 1H), 7.45 (td, J = 7.7, 1.1 Hz, 1H), 7.17−7.13 (m, 2H), 4.13 (q, J = 7.0 Hz, 2H), 1.37 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 164.2, 163.1, 159.8, 138.4, 136.1, 131.7 (d, J = 31.9 Hz), 131.2, 129.2, 129.0, 128.4 (2C), 125.7 (q, J = 3.6 Hz, 2C), 125.5, 124.4, 123.8 (d, J = 272.8 Hz), 121.6 (2C), 118.6, 115.4 (2C), 63.7, 14.4. Anal. Calcd for C23H18F3N5O2: C, 60.93; H, 4.00; N, 15.45. Found: C, 60.88; H, 4.08; N, 15.38. N-(2-(2-(2-Methoxyphenyl)-2H-tetrazol-5-yl)phenyl)-4(trifluoromethyl)benzamide (16). The title compound was synthesized from 4e and 4-(trifluoromethyl)benzoyl chloride, recrystallized from EtOH; yield 45%, colorless needles, mp 146−147 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.94 (s, 1H), 8.28−8.23 (m, 2H), 8.21 (d, J = 8.1 Hz, 2H), 7.88 (d, J = 8.2 Hz, 2H), 7.70−7.63 (m, 3H), 7.47− 7.43 (m, 1H), 7.40 (dd, J = 8.5, 1.0 Hz, 1H), 7.19 (td, J = 7.7, 1.2 Hz, 1H), 3.77 (s, 3H). 13C NMR (126 MHz, DMSO) δ 164.1, 162.9, 153.1, 138.4, 136.1, 132.8, 131.6 (d, J = 31.9 Hz), 131.2, 128.9 (2C), 128.3, 127.0, 125.5 (q, J = 3.5 Hz, 2C), 125.4, 125.2, 124.1, 123.8 (d, J = 272.4 Hz), 120.7, 118.3, 113.4, 56.2. Anal. Calcd for C22H16F3N5O2: C, 60.14; H, 3.67; N, 15.94. Found: C, 59.81; H, 3.88 ; N, 15.68. N-(2-(2-(3-Methoxyphenyl)-2H-tetrazol-5-yl)phenyl)-4(trifluoromethyl)benzamide (17). The title compound was synthesized from 4f and 4-(trifluoromethyl)benzoyl chloride, recrystallized from EtOH/THF (10:1); yield 48%, colorless needles, mp 189−190 °C (decomposition). 1H NMR (500 MHz, DMSO-d6) δ 10.84 (s, 1H), 8.28 (dd, J = 7.8, 1.5 Hz, 1H), 8.25 (d, J = 8.1 Hz, 2H), 8.18 (dd, J = 8.2, 0.9 Hz, 1H), 7.94 (d, J = 8.2 Hz, 2H), 7.69−7.63 (m, 2H), 7.58−7.52 (m, 2H), 7.47 (td, J = 7.6, 1.2 Hz, 1H), 7.21−7.18 (m, 1H), 3.79 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 164.2, 163.2, 160.2, 138.4, 136.9, 136.1, 131.7 (d, J = 31.9 Hz), 131.3, 131.1, 129.1, 128.4 (2C), 125.6 (q, J = 3.7 Hz, 2C), 125.6, 124.8, 123.8 (d, J = 272.5 Hz), 118.8, 115.9, 111.8, 105.5, 55.5. Anal. Calcd for C22 H16 F3 N5 O3: C, 60.14; H, 3.67; N, 15.94. Found: C, 59.98; H, 3.74; N, 15.97. N-(2-(2-(4-Methoxyphenyl)-2H-tetrazol-5-yl)phenyl)-4(trifluoromethyl)benzamide (18). The title compound was synthesized from 4c and 4-(trifluoromethyl)benzoyl chloride, recrystallized from EtOH; yield 56%, white needles, mp 150−152 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.90 (s, 1H), 8.26−8.20 (m, 4H), 7.99− 7.92 (m, 4H), 7.67−7.61 (m, 1H), 7.44 (td, J = 7.6, 1.2 Hz, 1H), 7.19−7.14 (m, 2H), 3.86 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 164.2, 163.1, 160.5, 138.4, 136.1, 131.7 (q, J = 32.0 Hz), 131.2, 129.4, 128.9, 128.4 (2C), 125.7 (q, J = 3.3 Hz, 2C), 125.4, 124.3, 123.8 (q, J = 272.5 Hz), 121.5 (2C), 118.5, 115.0 (2C), 55.7. Anal. Calcd for C22 H16 F3 N5 O3: C, 60.14; H, 3.67; N, 15.94. Found: C, 59.88; H, 3.74; N, 15.63. 3,4-Dimethoxy-N-(2-(2-(4-(trifluoromethyl)phenyl)-2H-tetrazol-5yl)phenyl)benzamide (19). The title compound was synthesized from 4b and 3,4-dimethoxybenzoyl chloride, recrystallized from EtOH; yield 63%, colorless powder, mp 167−168 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.73 (s, 1H), 8.36 (dd, J = 8.3, 0.8 Hz, 1H), 8.33 (d, J = 8.5 Hz, 2H), 8.25 (dd, J = 7.9, 1.5 Hz, 1H), 8.07 (d, J = 8.6 Hz, 2H), 7.69 (dd, J = 8.4, 2.1 Hz, 1H), 7.66−7.62 (m, 1H), 7.59 (d, J = 2.1 Hz, I



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(2C), 117.0, 114.1 (2C), 55.6, 36.8, 23.9, 13.6. Anal. Calcd for C24H23N5O2: C, 69.72; H, 5.61; N, 16.94. Found: C, 69.51; H, 5.80; N, 16.84. N-(2-(2-(4-Methoxyphenyl)-2H-tetrazol-5-yl)phenyl)-4-propylbenzamide (25). The title compound was synthesized from 4g and 4propylbenzoyl chloride, recrystallized from EtOH; yield 58%, off-white powder, mp 103−104 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.86 (s, 1H), 8.42 (d, J = 8.0 Hz, 1H), 8.26 (dd, J = 7.8, 1.4 Hz, 1H), 8.05− 8.01 (m, 2H), 7.98 (d, J = 8.2 Hz, 2H), 7.65−7.59 (m, 1H), 7.42−7.36 (m, 3H), 7.22−7.17 (m, 2H), 3.87 (s, 3H), 2.67 (t, J = 7.5 Hz, 2H), 1.70−1.61 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.1, 163.2, 160.5, 146.6, 136.8, 132.1, 131.2, 129.4, 128.7, 128.6 (2C), 127.4 (2C), 124.5, 123.0, 121.7 (2C), 116.9, 115.0 (2C), 55.7, 37.0, 23.8, 13.5. Anal. Calcd for C24H23N5O2: C, 69.72; H, 5.61; N, 16.94. Found: C, 69.42; H, 5.60; N, 16.81. Biological Investigation. Chemicals. Reference compound Ko143 ((3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2methylpropyl)-1,4-dioxopyrazino[1′,2′:1,6]pyrido[3,4-b]indole-3propanoic acid 1,1-dimethylethyl ester) was purchased from Tocris Bioscience (Bristol, UK). Reference compound XR9577 (N-(2-((4-(2(3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)carbamoyl)phenyl)quinoline-3-carboxamide) was synthesized according to the literature.35,36 All other chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany). Stock solutions of the compounds (10 mM) in DMSO were used to prepare the test samples for all cell-based assays. Krebs−HEPES buffer (KHB) water solution was prepared from 118.6 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 4.2 mM NaHCO3, 2.5 mM CaCl2, 1.2 mM MgSO4, 11.7 mM D-glucose monohydrate, and 10.0 mM HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid). The buffer solution was adjusted to pH 7.4 at 37 °C using sodium hydroxide solution and then sterilized by filtration through a membrane filter (size 0.2 μM). Cell Culture. MDCK II BCRP cells were generated by transfection of the canine kidney epithelial cell line MDCK II with the human wildtype cDNA C-terminally linked to the cDNA of the green fluorescent protein (GFP) and were received as a generous gift from Dr. A. Schinkel (The Netherlands Cancer Institute, Amsterdam, The Netherlands). Cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) with 10% FCS (fetal calf serum), 50 μg/mL streptomycin, 50 U/mL penicillin G, and 2 mM L-glutamine. Human ovarian carcinoma cell line A2780adr, which is doxorubicin resistant and overexpresses P-gp, was purchased from European Collection of Animal Cell Culture (ECACC, No 93112520). The cell line was grown in RPMI-1640 medium supplemented with 10% FCS, 50 μg/ mL streptomycin, 50 U/mL penicillin G, and 2 mM L-glutamine. To ensure P-gp overexpression in the A2780adr cell line, intermittently cells were incubated with 0.1 μM doxorubicin for one passage. The small cell lung cancer cell line H69AR expressing MRP1 was purchased from American Type Culture Collection (ATCC, CRL11351). This cell line was grown in RPMI-1640 medium supplemented with 20% FCS, 50 μg/mL streptomycin, 50 U/mL penicillin G, and 2 mM L-glutamine. All cells were incubated under a 5% CO2-humidified atmosphere at 37 °C. After reaching confluence of at least 90%, cells were harvested for subculturing and using in cellbased assays. The cells were treated gently with a 0.05% trypsin/0.02% EDTA solution to detach them from the inner surface of the culture flask and transferred into a 50 mL tube followed by a centrifugation (266g, 4 °C, 4 min). After aspiration of the supernatant, the cell pellet was resuspended in fresh culture medium, and the cell density was determined using a CASY1 model TT cell counter with a 150 μM capillary (Schaerfe System GmbH, Reutlingen, Germany). Then the volume of suspension containing the required quantity of cells for an assay (see below) was transferred into a vial and further centrifuged, and the cells were washed three times with KHB. UV Spectroscopy for the Investigation of Precipitation Behavior during Fluorescent Accumulation Assays. Ten micromolar test solutions were prepared from the stock solutions using double distilled water. Absorbance of the samples was measured at 254 nm in constant time intervals (60 s) for a period of 3 h at room temperature using a Ultrospec 4000 UV/visible spectrophotometer (Pharmacia Biotech

Ltd., UK). The absorbance of double distilled water containing 0.1% DMSO was used as reference and subtracted automatically from the values of samples. Hoechst 33342 Accumulation Assay. To investigate the inhibitory effect on BCRP, the Hoechst 33342 accumulation assay was performed as described earlier with small modifications.14,16−18,20−27 Different dilutions of compounds were prepared in KHB. For some compounds with low solubility, methanol was used as additional solvent to prepare 10 μM test solutions. The amount of methanol was chosen so that it did not exceed 5% in the highest concentration used in the assay. The highest concentration of DMSO in dilutions for the assays was not more than 0.1%. After the preparation of different concentrations of test compounds, 20 μL of each concentration was placed into black 96-well plates (Greiner, Frickenhausen, Germany). Black plates were chosen as they yielded much smaller background fluorescence than colorless plates when irradiated in the UV. The prepared cells were added into each well of the plate at a density of approximately 30,000 cells per well to a total volume of 180 μL. The 96-well plate was stored under 5% CO2 at 37 °C for 30 min. After this preincubation period, 20 μL of a 10 μM Hoechst 33342 solution (protected from light) was added to each well. Fluorescence was measured immediately at constant intervals (60 s) for a period of 120 min with an excitation of 355 nm and an emission wavelength of 460 nm at 37 °C using a BMG POLARstar microplate reader (BMG Labtech, Offenburg, Germany). For the analysis of the data obtained from the assay, first the fluorescence of KHB was subtracted from the total fluorescence detected for MDCK II cells. Average of fluorescence values in the steady state (from 100 up to 109 min) was calculated for each concentration. These values were plotted against logarithmic concentrations of tested compounds. Concentration−response curves were generated by nonlinear regression using the four-parameter logistic equation with variable Hill slope (GraphPad Prism, version 6.0, San Diego, CA, USA). Calcein AM Assay. For determining the selectivity toward BCRP, compounds were further tested for their P-gp and MRP1 inhibition using the calcein AM assay as described earlier with small modifications.14,16−18,22,23,25−27 The assay was performed in the same way as the Hoechst 33342 accumulation assay. Twenty microliters of the prepared solutions containing a test compound (see above) were pipetted into colorless 96-well plates (Greiner, Frickenhausen, Germany). Then, A2780adr cells for P-gp or H96AR cells for MRP1 were seeded into each well of the plate at a density of approximately 30,000 cells in a volume of 160 μL per well. After a preincubation time of 30 min, 20 μL of a 3.125 μM calcein AM solution (protected from light) was added to each well. The fluorescence was measured immediately at constant time intervals (60 s) for a period of 60 min at 37 °C with an excitation of 485 nm and an emission of 520 nm using a BMG POLARstar microplate reader. For calculation of inhibitory effects, the first linear part of the fluorescence time curves was used for calculating slopes. These slopes were plotted against logarithmic concentrations of tested compounds. Concentration−response curves were generated by nonlinear regression using the four-parameter logistic equation with variable Hill slope (GraphPad Prism). MTT Assay for Determining Cytotoxicity. Intrinsic cytotoxicity of selected compounds was determined in MDCK II BCRP and MDCK II wild-type cells using the MTT cytotoxicity assay. The assay was performed as described earlier with modifications.18,22,26,27,31 Cells were seeded into 96-well tissue-culture treated plates (Starlab GmbH, Hamburg, Germany) at a density of 3000 cells per well in a volume of 180 μL and kept under 5% CO2-atmosphere at 37 °C for 6 h. Attachment of cells was controlled under a microscope. Different concentrations of test compounds were made in culture medium. The highest concentration of DMSO in dilutions used for the assays was not more than 1.0%. Then, 20 μL of test compound was added to achieve the required final concentration in a volume of 200 μL. Additionally, wells were prepared containing only medium (negative control) and 10% (v/v) of DMSO (positive control). After an incubation period of 72 h, a solution of MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) in phosphate buffered J



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saline (5 mg/mL) was added to each well (20 μL). Plates were further incubated for 1 h, after which MTT is reduced to a water insoluble formazan. Then, the supernatants were removed, and the cells were lysed with 100 μL of DMSO per well. The color intensity of the formed formazan was determined spectrophotometrically by measuring absorbance at 570 nm and background correction at 690 nm using a Multiscan Ex microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA). The cell viability was calculated as percentage of the negative controls. GI50 values were calculated by nonlinear regression analysis, assuming a sigmoidal concentration−response curve with variable Hill slope (GraphPad Prism). MTT Assay for Determining the Ability of MDR Reversal. The MTT assay was also used to investigate the effect of the inhibitors on the reversal of resistance against cytotoxic agents such as SN-38 (7ethyl-10-hydroxy-camptothecin) in BCRP expressing cells. The cytotoxic effect of SN-38 was determined in the absence and presence of different concentrations of selected compounds. For this purpose, MDCK II wild-type and MDCK II BCRP cells were seeded into 96well tissue-culture treated plates at a density of 3000 cells per well in a volume of 160 μL and kept under a 5% CO2-atmosphere at 37 °C for 6 h. Attachment of cells was controlled under a microscope. Different concentrations of SN-38 were prepared in culture medium, and 20 μL of them were added to the cell containing wells. Then, 20 μL of culture medium (negative control) and 3 μM or 5 μM of test compound (dissolved in culture medium) were added to the wells containing MDCK II BCRP cells. Furthermore, 20 μL of culture medium was added to the MDCK II wild-type cell containing wells to achieve the required final concentration in a volume of 200 μL. The wild-type cells were used as the positive control. The following steps were the same as mentioned above.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 228 735213. Fax: +49 228 737929. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Cell lines MDCK II wild-type and MDCK II BCRP were received as a generous gift from Dr. A. Schinkel, The Netherlands Cancer Institute (Amsterdam, The Netherlands). We thank Dr. N. Tzvetkov, Pharmaceutical Institute (University of Bonn, Germany), for discussions and generous advice.

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ABBREVIATIONS USED ABC, ATP-binding cassette; BCRP, breast cancer resistance protein (ABCG2); CsA, cyclosporin A; FTC, fumitremorgin C; GC50, half-maximal growth inhibition; Imax, maximal inhibition; MDCK, Madin Darby Canine Kidney; MDR, multidrug resistance; MRP1, multidrug resistance protein 1 (ABCC1); MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide; Pd/C, palladium on charcoal; P-gp, P-glycoprotein (ABCB1); TEA, triethylamine

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