DOI: 10.1002/cmdc.201402498

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Scaffold Identification of a New Class of Potent and Selective BCRP Inhibitors Federico Marighetti, Kerstin Steggemann, Maria Karbaum, and Michael Wiese*[a] the selected substituents led to compounds with low activity, but in some cases activity was retained. Among these, a phenolic hydroxy group proved to impart as much potency to the molecule as a hydroxyethyl side chain, initially considered necessary for activity. This derivative is one of the most active compounds in this class, maintaining an inhibitory activity similar to that of the reference compound; it is also selective for BCRP.

We recently reported the synthesis and quantitative structure– activity relationships of a new breast cancer resistance protein (BCRP) inhibitor class. In the study presented herein, we investigated the possibility to better define the scaffold of this compound class by removing or modifying the aromatic ring A with various substituents selected on the basis of their electronic and lipophilic properties. The results show that this aromatic ring is important, but not essential, for activity. Many of

Introduction ATP binding cassette (ABC) transporters are membrane proteins that are able to translocate a wide variety of substrates through the cell membrane using ATP hydrolysis as energy source. The overexpression of certain poly-specific ABC transporters in cancer cells is the most significant mechanism behind multidrug resistance (MDR). ABC transporters involved in MDR are P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance protein 1 (MRP1).[1] BCRP is a member of the ABCG subfamily, consisting of one N-terminal nucleotide binding domain followed by six transmembrane segments.[2, 3] BCRP has been suggested to function as a homodimer, with its two monomers connected by a disulfide bridge.[4] BCRP is expressed in many tissues of the human body, particularly in brain, placenta, liver, intestine, kidney, and stem cells.[5] Under physiological conditions, it plays a protective role against xenobiotics and is probably also involved in the transport of several physiological substrates such as porphyrins and vitamins.[6] BCRP is nonselective and is able to transport many kinds of molecules, both positively and negatively charged. Cytotoxic drugs transported by BCRP include mitoxantrone, topotecan, SN-38, camptothecin, flavopiridol, and methotrexate.[7] A possible strategy to overcome BCRPmediated resistance would be the use of potent and selective inhibitors or modulators. Among the limited number of BCRP inhibitors currently known are natural or synthetic compounds, and only a few of these are selective for this transporter.[8] Flavonoids and derivatives thereof are natural compounds reported to be potent but nonselective BCRP inhibitors.[9–11] Other natural products such as fumitremorgin C[12] and novobiocin[13]

were found to be selective BCRP inhibitors. Ko143, a synthetic analogue of fumitremorgin C, is a potent and selective inhibitor of BCRP.[14] Tyrosine kinase inhibitors have also been found to inhibit BCRP function.[15] It was recently reported that the efficacious third-generation P-gp modulator XR9576 (tariquidar) is also a BCRP inhibitor, although less potent.[16] Structural modification of XR9576, with deletion of the tetrahydroisoquinoline group, led to compounds that were shown to be selective BCRP inhibitors.[17] In our previous work[18] we investigated the structure–activity relationships for compounds of this new class with modifications on the aromatic rings B and C of the original scaffold (Figure 1). The aim of this study is to investigate the importance of modifications on aromatic ring A in order to better define a minimum scaffold for of this new BCRP inhibitor class. Herein we report the synthesis and bio-

[a] F. Marighetti, Dr. K. Steggemann, M. Karbaum, Prof. Dr. M. Wiese Pharmaceutical Institute, University of Bonn An der Immenburg 4, 53121 Bonn (Germany) E-mail: [email protected]

Figure 1. Structures of tariquidar (top), the initial series of derived BCRP inhibitors[18] (bottom left), and the current series of investigated compounds (bottom right).

Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201402498.

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Full Papers logical evaluation of 30 new derivatives with varying substituents on ring A and selected substituents on ring C.

Results and Discussion The general outline of the synthesis of the derivatives is shown in Scheme 1. As in our previous work[18] the presence of substituents on ring B (R’ and R’’; Figure 1) had been proven disad-

Figure 2. s–p diagram of selected active (*) and inactive (*) substituents. The substituent constants were taken from Ref. [31].

assays are listed in Table 1. Compounds show no inhibitory activity at concentrations up to 10 mm are reported as having no effect (NE). Considering the activity data and the s–p diagram of the substituents present on ring A (Figure 2), it can be concluded that substituents with small s-Hammett values are active, whereas there is no decisive influence of lipophilicity, probably except for very polar groups such as amide and sulfonamide, which lead to inactive compounds. In comparing the activity data and calculated log P values, there seems to be a tendency of higher activity with greater lipophilicity. Compounds 9, 14, and 27 are the most lipophilic and are among the most active. However, a plot of calculated log P values versus pIC50 values of the active derivatives does not confirm this conclusion. Although the regression line points upward, the squared correlation coefficient is close to zero (r2 = 0.05), both for all compounds and the subset of 4-nitro derivatives (data not shown). With regard to compounds with a nitro group at R1, compound 5, in which the terminal hydroxy group at R2 is replaced by a methyl ether, shows even slightly better inhibitory activity (IC50 = 1.23 mm) than the reference compound 4 (IC50 = 1.58 mm). Replacement of the hydroxy group with an acetyl ester also retains activity (compound 6) to a high degree, showing that a hydrogen bond donor is not necessary at this position. Replacement of the hydroxyethyl group with a phenolic hydroxy group (compound 8) leads to a derivative nearly as potent as the reference compound. However, compound 7, with a methoxy group at R2, is twofold less potent than compound 8. Interestingly, a nonpolar propyl group at R2 (compound 9) leads to the second most potent compound in the series (IC50 = 0.94 mm). This is unexpected, as derivatives containing an nonpolar methyl group at this position (19, 25, 26) show low activity or are even inactive. In contrast, compound 11, with no substituent at R2, has much lower inhibitory activity against BCRP (IC50 = 13.4 mm). Substitution with a benzoyl group (compound 14) gives a compound with an IC50 value of 1.63 mm. Compounds with bromine (10), polar amide (12), sulfonamide (13), or tertiary amine (15) substituents showed no inhibition up to 10 mm.

Scheme 1. Synthesis of the BCRP inhibitors. Reagents and conditions: a) THF, Et3N, RT, 12 h; b) H2, Pd/C, 2 bar, RT, 12 h; c) R-COCl, THF, Et3N, RT, 12 h.

vantageous, only compounds unsubstituted at these positions were synthesized in this study. The previously published compound 4 was used as a reference compound in this study for comparison. This compound has a hydroxyethyl group as R2, initially thought to be important for activity, and a nitro group as R1, which had been indicated in our precedent study as most favorable at this position. For this reason, many of the compounds in this study also have R1 as a nitro group. However, we also explored several other substituents at this position which had previously led to active compounds. To investigate the importance of the hydroxyethyl substituent on ring A it was replaced by several other groups. The selection was based on synthetic feasibility and the physicochemical properties of the substituents. They were selected from a s–p diagram so that at least two were present from each quadrant (Figure 2). We also considered hydrogen bond acceptor and donor properties as potentially important and included them in the selection. In two compounds, phenyl ring A was replaced by a 4-hydroxycyclohexyl group, which had been proven to be beneficial for activity. In one compound the entire phenyl ring A was removed and replaced by a small ethyl group, leading to an inactive derivative. The inhibitory activity of the compounds against BCRP was determined by a Hoechst 33342 assay in BCRP-overexpressing MCF-7 MX cells. To evaluate the selectivity as well, the inhibitory activity against P-gp was initially screened at a concentration of 10 mm using a calcein AM assay with A2780 Adr cells. For compounds showing > 25 % inhibition at 10 mm, full dose–response curves were measured to determine IC50 values. The structures of the derivatives and the results of the biological ChemMedChem 2015, 10, 742 – 751

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Full Papers and 5).[18] A possible explanation might be solubility problems due to the high lipophilicity of this compound. In the series of compounds bearing a 3,4-dimethoxy substitution at R1, the activity trends differed somewhat. Replacement of the hydroxyethyl side chain at R2 by a phenolic hydroxy group increased the inhibitory activity threefold (ref. [18] no. 27: IC50 = 3.61 mm, versus compound 17: IC50 = 1.16 mm). Again, Compd R1 R2 IC50 [mm][a] log Pcalcd compound 16, with a methoxy group at R2, is threeBCRP P-gp fold less active than the corresponding phenol. In 4 4-NO2 CH2CH2OH 1.58 œ 0.36[18] NE 3.34 contrast to compounds with a nitro group at position 5 4-NO2 CH2CH2OCH3 1.23 œ 0.40 NE 4.13 R1, the absence of a substituent at position R2 did CH2CH2OCOCH3 1.32 œ 0.28 NE 4.28 6 4-NO2 not dramatically decrease activity, and compound 20 OCH3 3.56 œ 0.84 NE 4.15 7 4-NO2 was found to be a BCRP inhibitor with an IC50 value 8 4-NO2 OH 1.76 œ 0.18 27.5 œ 1.84 3.46 CH2CH2CH3 0.94 œ 0.33 NE 5.73 9 4-NO2 of 1.75 mm. Chlorine (18) or a methyl group (19) led Br NE NE 5.37 10 4-NO2 to derivatives with low potency. H 13.4 œ 4.2 NE 4.20 11 4-NO2 Only a few derivatives were investigated that posCONH2 NE NE 3.16 12 4-NO2 sess a chlorine or methyl substituent at R1, as these SO2NH2 NE NE 3.01 13 4-NO2 (C=O)Ph 1.63 œ 0.33 5.17 œ 0.27 5.60 14 4-NO2 were found to be less active in the precedent study. N(CH3)2 NE NE 4.34 15 4-NO2 In agreement with the positive contribution of a phe16 3,4-OCH3 OCH3 3.34 œ 0.25 4.09 œ 0.50 4.33 nolic hydroxy group, compound 24 was found to be OH 1.16 œ 0.21 4.47 œ 1.00 3.64 17 3,4-OCH3 as active as the counterpart with a hydroxymethyl 18 3,4-OCH3 Cl 8.29 œ 3.10 NE 5.37 CH3 22.8 œ 2.8 13.5 œ 2.6 4.84 19 3,4-OCH3 side chain at this position (ref. [18] no. 20: IC50 = H 1.76 œ 0.64 3.98 œ 0.84 4.38 20 3,4-OCH3 3.08 mm). Whereas a methyl substituent at R2 led to 2.09 œ 0.20 NE 3.44 21 4-NHCOCH3 H inactive (26) or poorly active (19) derivatives, a benzoH 23.3 œ 8.9 NE 3.38 22 4-NH2 yl group had a strong positive effect, and compound NE NE 4.86 23 4-Cl OCH3 24 4-Cl OH 2.91 œ 0.88 NE 4.16 27 was found to be the most active derivative in the NE NE 5.37 25 4-Cl CH3 whole series, with an IC50 value of 0.56 mm. CH3 NE NE 5.06 26 4-CH3 Regarding compounds unsubstituted at R2, it is (C=O)Ph 0.56 œ 0.24 6.07 œ 1.48 6.00 27 4-CH3 possible to correlate their activity values expressed in CH2CH2OCH3 5.11 œ 0.87 NE 5.03 28 4-CF3 OH 1.80 œ 0.43 14.0 œ 3.4 4.37 29 4-CF3 logarithmic form with van der Waals surface area Cl NE NE 6.10 30 4-CF3 (VSA) of ring C containing the R1 substituent, calculatH NE NE 5.11 31 4-CF3 ed with the MOE software package.[19] Increasing the 32 4-NO2 4-OH-cHex 6.11 œ 2.58 NE 2.80 surface area of the R1 group leads to increased BCRP 4-OH-cHex 3.35 œ 1.06 20.8 œ 4.4 3.70 33 4-CF3 CH2CH3 NE NE 2.97 34 4-NO2 inhibitory activity. The two most active compounds – – tariquidar 0.68[30]/1.45[16] of this series without substituents at R2—compounds [a] Determined by Hoechst 33342 assay in MCF-7 MX cells and by calcein AM assay in 20 and 21—have the highest VSA values (164.3 and A2780 Adr cells; data are the mean œ SD (n = 3); NE: no effect for concentrations up to 154.1 æ2, respectively), whereas compounds 11 and 10 mm. 22 have low respective VSA values: 132.4 and 125.0 æ2. The correlation between activity and VSA suggests that a voluminous substituent at R1 increases the BCRP inhibitory activity of this compound class; this The trends observed for the compound series with a trifluocontribution to activity is increased by the absence of a subromethyl group at R1 are in agreement with those of the 4stituent at R2. This observation is in agreement with what has nitro series. This group was found to be slightly less active been suggested in our previous publication.[18] than the nitro derivative in the previously investigated series 2 with a hydroxyethyl group at R . Again, replacement of the hyThe structure–activity relationship of the substituents on ring A can be explained by Slog P, PM3 HOMO, and VSA dedroxyethyl group by a phenolic hydroxy group did not change scriptors (Figure 3). For compounds with a 4-nitro group on activity significantly (ref. [18] no. 22: IC50 = 2.20 mm, versus ring C, the active derivatives cluster in the descriptor space. As compound 29: IC50 = 1.80 mm). If no substituent is present at apparent in Figure 3, active derivatives must have a VSA R2, activity is largely decreased with a nitro group at position > 150 æ2 and a Slog P > 1. These compounds must also have R1, and is abolished in the case of a trifluoromethyl substituent values of PM3 HOMO (a descriptor highly correlated with s(compound 31). The only exception in the activity trends is Hammett) for the aromatic ring A between ¢9 and ¢10 eV. compound 28, with an IC50 value of 5.11 mm, which was found The two compounds (13 and 15) out of this range are not to be twofold less active than its analogue with a hydroxyethyl active against BCRP. Compounds with a cyclohexyl ring (32 group at R2 (ref. [18] no. 22: IC50 = 2.20 mm), whereas the activiand 33) in place of the phenyl ring A are less potent against ties of both derivatives are identical in the 4-nitro series (nos. 4 Table 1. Inhibitory activities of compounds 4–34.

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Full Papers significant inhibitory potency against P-gp, with an IC50 value of 5.17 mm. Given the presence of a benzophenone moiety, the latter compound could be expected to be a P-gp inhibitor, as other studies with compounds containing a benzophenone group had shown this to be a structural element recognized by P-gp.[20, 21] This is substantiated by the finding that the other derivative containing a benzophenone group (compound 27) showed very similar inhibitory activity against P-gp, with an IC50 value of 6.07 mm. As could be expected by the common presence of dimethoxy substitution in P-gp inhibitors, compounds with such a substitution pattern were found to be mostly non-selective for BCRP, and their inhibitory potency against P-gp was similar to that against BCRP. In particular, compounds 16, 17, and 20 possess similar IC50 values for P-gp, whereas compounds 18 and 19 show no or low activity. To investigate the capacity of the compounds to reverse the resistance of BCRP-expressing cells, the cytotoxicity of the BCRP substrate SN-38, the active metabolite of irinotecan, was determined in the presence and absence of a representative from the series of compounds. Compound 8 was selected as being equipotent to the reference 4 and with relatively good water solubility. Figure 5 illustrates the effect of compound 8

Figure 3. 3D plot of the Slog P, PM3 HOMO, and VSA descriptors of compounds with various substituents on aromatic ring A and a nitro group on ring C. Active derivatives are shown as black spheres, and inactive ones are in grey. Descriptor values were calculated for the substructure of a substituted benzene.

BCRP, but are still active, whereas an ethyl group at this position leads to loss of activity (compound 34). To investigate the possibility of a substrate-dependent pattern of BCRP inhibition, 12 derivatives and tariquidar were also tested with pheophorbide A as substrate. The inhibitory activities of the tested derivatives showed a high correlation, in agreement with the results obtained for a smaller set of derivatives containing a hydroxyethyl side chain.[17] The correlation between IC50 values in both test systems is shown in Figure 4. The calcein AM assay was used to investigate P-gp inhibition. Here compound 8 (IC50 = 27.5 mm) showed only weak inhibitory activity, whereas compound 14 was found to have

Figure 5. Representative shift in the dose–response curve of SN-38 cytotoxicity caused by increasing inhibitor (compound 8) concentration. Compound 8 was investigated at 5 mm (&) and 10 mm (~). MDCK BCRP cells without inhibitor (*) are more resistant than sensitive MDCK cells ( ! ). Shown is a representative example from three independent experiments with two replicates each.

on the EC50 of SN-38 in MDCK BCRP cells. Even at 5 mm the concentration effect curve is largely shifted to the left with a further shift occurring at the higher concentration of 10 mm. The MTT assay clearly shows that compound 8 is able to completely reverse the resistance of MDCK BCRP cells against SN-38.

Conclusions In summary, it is possible to assert that the hydroxyethyl substituent present in the original scaffold of this class of BCRP inhibitors is not necessary for the BCRP inhibitory activity of these compounds. Replacement of this substituent with a hydroxy group leads to selective BCRP inhibitors with IC50 values

Figure 4. Correlation of inhibitory potencies of selected compounds in the Hoechst 33342 and pheophorbide A assays of BCRP inhibition (r2 = 0.91).

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Full Papers similar to those of compounds with a hydroxyethyl group at the same position. Furthermore, the presence of a substituent at R2 is not necessary if position R1 is occupied by a group with a van der Waals surface area higher than ~ 150 æ2. Finally, the aromatic ring A was identified to be important for the activity of the compounds against BCRP, and its removal leads to compounds with low or no inhibitory activity against BCRP. Furthermore, methylation of the hydroxy group at R2 leads to compounds that are less active than non-methylated compounds. Compounds with a 3,4-dimethoxyphenyl group at R1 show a slightly different activity pattern from those with a 4-nitrophenyl substituent, and they also exhibit considerable inhibitory activity against P-gp.

night at room temperature under 2 bar H2 atmosphere in a Paar apparatus. The catalyst was filtered off, the solution was concentrated, and the product was dissolved in a little THF and precipitated with PE. N-(4-(2-Methoxyethyl)phenyl)-2-nitrobenzamide (2 a): According to method B, 4-(2-methoxyethyl)aniline (1.51 g, 10 mmol) and 4-nitrobenzoyl chloride (1.86 g, 10 mmol) were reacted, and compound 2 a was obtained as a pale-yellow powder (2.04 g, 68 %): Rf = 0.35 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 10.58 (s, 1 H), 8.13 (dd, J = 8.54, 1.02 Hz, 1 H), 7.87–7.83 (m, 1 H), 7.76–7.72 (m, 2 H), 7.56 (d, J = 8.45 Hz, 2 H), 7.21 (d, J = 8.47 Hz, 2 H), 3.51 (t, J = 6.85 Hz, 2 H), 3.24 (s, 3 H), 2.77 (t, J = 6.85 Hz, 2 H); 13C NMR (125 MHz, [D6]DMSO): d = 164.04, 146.67, 137.04, 134.88, 134.15, 132.89, 131.01, 129.39, 129.21 (2C), 124.34, 119.80 (2C), 72.98, 57.94, 34.97 ppm; Anal. calcd for C16H16N2O4 : C 63.99, H 5.32, N 9.33, found: C 64.03, H 5.32, N 9.54.

Experimental Section

(4-(2-Nitrobenzamido)phenethyl acetate (2 b): According to method B, 2-amino-N-[4-(2-hydroxyethyl)phenyl]benzamide (1.43 g, 5 mmol) and acetyl chloride (471 mg, 6 mmol) were reacted, and compound 2 b was obtained as a beige powder (1.54 g, 94 %): Rf = 0.16 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 10.59 (s, 1 H), 8.18–8.09 (m, 1 H), 7.88–7.83 (m,1 H), 7.76–7.72 (m, 2 H), 7.59 (d, J = 8.49 Hz, 2 H), 7.23 (d, J = 8.47 Hz, 2 H), 4.19 (t, J = 6.86 Hz, 2 H), 2.86 (t, J = 6.84 Hz, 2 H), 1.98 ppm (s, 3 H); 13C NMR (125 MHz, [D6]DMSO): d = 170.38, 164.05, 146.62, 137.31, 134.14, 133.69, 132.83, 131.01, 129.36, 129.24 (2C), 124.32, 119.85 (2C), 64.48, 33.92, 20.80 ppm; Anal. calcd for C16H16N2O3 : C 67.59, H 5.67, N 9.85, found: C 67.25, H 5.64, N 9.79.

The log P values were calculated with ACD log P, ver. 5.09 (Advanced Chemistry Development, Toronto, ON, Canada).

Chemistry General methods: Commercial reagents and starting materials were purchased from Sigma–Aldrich, Fluka, Acros, or Alpha Aesar and were used without further purification. Reaction progress was monitored by TLC on alumina plates coated with silica gel (Merck silica gel 60 F254) and visualized by UV light (l = 254 nm). Spectral data were obtained as follows: 1H NMR, Bruker Advance 500 (500 MHz); 13C NMR, Bruker Advance 500 (125.8 MHz). Chemical shifts (d) are expressed in ppm by using solvent as an internal standard. Multiplicity of resonance peaks is indicated as singlet (s), doublet (d), triplet (t), quartet (q) and multiplet (m). J values are given in Hz, and the relative number of protons was determined by integration. The solvent used for each spectrum is reported. Elemental analyses were performed on a Vario EL (Elementar). Values were all within 0.4 % of theoretical, except when indicated. Spectroscopic data of published compounds are given in the Supporting Information for convenience.

2-Nitro-N-(4-propylphenyl)benzamide (2 e): According to method B, 4-propylaniline (676 mg, 5 mmol) and 4-nitrobenzoyl chloride (1.11 g, 6 mmol) were reacted, and compound 2 e was obtained as a white powder (1.27 g, 89 %): Rf = 0.48 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 10.54 (s, 1 H), 8.15–8.10 (m, 1 H), 7.89– 7.82 (m), 7.77–7.71 (m, 2 H), 7.56 (d, J = 8.5, 2 H), 7.16 (d, J = 8.5 Hz, 2 H), 2.55–2.50 (m, 2 H), 1.66–1.50 (m, 2 H), 0.88 ppm (t, J = 7.3, 3 H); 13 C NMR (125 MHz, [D6]DMSO): d = 163.96, 146.66, 137.88, 136.68, 134.11, 132.90, 130.97, 129.37, 128.68 (2C), 124.31, 119.80 (2C), 36.82, 24.24, 13.66 ppm; Anal. calcd for C16H16N2O3 : C 67.59, H 5.67, N 9.85, found: C 67.25, H 5.64, N 9.79.

General procedure for synthesis of acid chlorides (A): Aromatic carboxylic acid (1 equiv) was dissolved/suspended in dry THF or CH2Cl2 and a catalytic amount of dry DMF. Oxalyl chloride (1.2 equiv) was added dropwise, and the reaction mixture was stirred at room temperature for 1 h. Finally, the solvent and excess oxalyl chloride was removed under reduced pressure to yield the desired acid chloride, which was used immediately for the synthesis of amides.

2-Amino-N-(4-(2-methoxyethyl)phenyl)benzamide (3 a): According to method C N-(4-(2-methoxyethyl)phenyl)-2-nitrobenzamide (1.50 g, 5 mmol) was hydrogenated. The product was directly used for further synthesis. Rf = 0.86 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 9.92 (s, 1 H), 7.64–7.61 (m, 3 H), 7.20 (ddd, J = 1.9, 5.6, 8.6 Hz, 3 H), 6.76 (dd, J = 1.0, 8.3 Hz, 1 H), 6.61–6.57 (m, 1 H), 6.31 (s, 2 H), 3.53 (t, J = 6.9 Hz, 2 H), 3.26 (s, 3 H), 2.78 ppm (t, J = 6.9 Hz, 2 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.84, 149.82, 137.40, 134.23, 132.11, 128.89, 128.74, 120.70, 116.49, 115.53, 114.82, 73.04, 57.93, 34.98 ppm.

General procedure for synthesis of amides (B): Aromatic amine (1.2 equiv) and Et3N (3 equiv) were dissolved in dry THF and stirred at 0 8C. The acyl chloride (1.0 equiv) synthesized as per general method was dissolved in dry THF and added slowly. The solution was stirred for 12 h at room temperature. After completion of the reaction, the solvent was evaporated, and the residue was dissolved in H2O and extracted with EtOAc (3 Õ 15 mL). The organic phases were combined and washed with 1 n NaOH, 1 n HCl, and saturated brine, dried over MgSO4, and concentrated. The product was precipitated with THF/petroleum ether (PE) and purified by recrystallization with CH2Cl2/PE (1:1) or EtOH or by column chromatography with EtOAc/PE (1:1) as eluent.

4-(2-Aminobenzamido)phenethyl acetate (3 b): According to method C 4-(2-nitrobenzamido)phenethyl acetate (1.37 g, 4.2 mmol) was hydrogenated, and compound 3 b was obtained as a beige powder (937 mg, 78 %): Rf = 0.83 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 9.92 (s, 1 H), 7.65–7.59 (m, 3 H), 7.21–7.16 (m, 3 H), 6.74 (dd, J = 8.28, 1.07 Hz, 1 H), 6.58 (ddd, J = 8.13, 7.21, 1.19 Hz, 1 H), 6.28 (s, 2 H), 4.19 (t, J = 6.92 Hz, 2 H), 2.84 (t, J = 6.90 Hz, 2 H), 1.98 ppm (s, 3 H); 13C NMR (125 MHz, [D6]DMSO): d = 170.42, 167.89, 149.84, 137.75, 133.05, 132.14, 128.96 (2C), 128.78, 120.78 (2C), 116.50, 115.50, 114.83, 64.57, 33.97, 20.85 ppm; Anal. calcd for C17H18N2O3·0.33 H2O: C 67.09, H 6.18, N 9.20, found: C 67.47, H 6.01, N 9.16.

General procedure for hydrogenation of an aromatic nitro group (C): Nitro compound was dissolved in THF, and palladium on activated charcoal was added. The suspension was stirred overChemMedChem 2015, 10, 742 – 751

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Full Papers 2-Amino-N-(4-benzoylphenyl)benzamide (3 j): According to method C N-(4-benzoylphenyl)-2-nitrobenzamide (1.38 g, 4 mmol) was hydrogenated, and compound 3 j was obtained as a white powder (850 mg, 69 %): Rf = 0.87 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 10.32 (s, 1 H, 7.93 (d, J = 8.76 Hz, 2 H), 7.76 (d, J = 8.77 Hz, 2 H), 7.72 (dd, J = 8.18, 1.26 Hz, 2 H), 7.68–7.63 (m, 2 H), 7.55 (t, J = 7.58 Hz, 2 H), 7.22 (ddd, J = 8.43, 7.22, 1.47 Hz, 1 H), 6.78 (dd, J = 8.26, 0.91 Hz, 1 H), 6.60 (t, J = 8.06 Hz, 1 H), 6.37 ppm (s, 2 H); 13C NMR (125 MHz, [D6]DMSO): d = 194.76, 168.32, 150.14, 150.06, 143.81, 137.79, 132.64, 132.32, 131.53, 130.99 (2C), 129.49 (2C), 129.03, 128.60 (2C), 119.63 (2C), 116.63, 114.84 ppm; Anal. calcd for C20H16N2O2 : C 75.93, H 5.10, N 8.86, found: C 75.77, H 5.20, N 8.75.

7.49 (d, J = 8.82 Hz, 2 H), 7.28 (t, J = 7.28 Hz, 1 H), 6.78 ppm (d, J = 8.84 Hz, 2 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.00, 162.97, 154.54, 149.48, 140.29, 138.66, 132.23, 129.89, 128.93, 128.63 (2C), 124.14 (2C), 123.75, 123.41 (2C), 122.73, 121.38, 115.22 ppm (2C); HPLC: k’ = 0.53 (RP8 column, CH3CN/H2O (80:20)), purity 97.64 % 2-(4-Nitrobenzamido)-N-(4-propylphenyl)benzamide (9): According to method B, compound 3 e (87 mg, 0.5 mmol) and 4-nitrobenzoyl chloride (111 mg, 0.6 mmol) were reacted, and compound 9 was obtained as a yellow powder (187 mg, 93 %): Rf = 0.84 (EtOAc/ PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 8.37–8.33 (m, 3 H), 8.16 (d, J = 8.9 Hz, 2 H), 7.95 (d, J = 6.5 Hz, 1 H), 7.58–7.52 (m, 3 H), 7.22 (t, J = 7.3 Hz, 1 H), 7.13 (d, J = 8.5 Hz, 2 H), 2.52–2.49 (m, 2 H), 1.60– 1.51 (m, 2 H), 0.87 ppm (t, J = 7.3 Hz, 3 H); 13C NMR (125 MHz, [D6]DMSO): d = 166.80, 163.63, 149.14, 137.82, 136.61, 131.91, 129.16, 128.80 (2C), 128.53 (2C), 124.00, 123.91 (3C), 122.49, 120.96 (2C), 36.83, 24.23, 13.68 ppm; Anal. calcd for C23H21N3O4 : C 68.47, H 5.25, N 10.42, found: C 68.11, H 4.93, N 10.19.

N-(4-(2-Methoxyethyl)phenyl)-2-(4-nitrobenzamido)benzamide (5): According to method B, compound 3 a (405 mg, 1.5 mmol) and 4-nitrobenzoyl chloride (371 mg, 2 mmol) were reacted, and compound 5 was obtained as a beige powder (262 mg, 42 %): Rf = 0.79 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 11.84 (s, 1 H), 10.51 (s, 1 H), 8.40–8.36 (m, 3 H), 8.13 (d, J = 8.64 Hz, 2 H), 7.93 (d, J = 7.66 Hz, 1 H), 7.63–7.58 (m, 3 H), 7.31 (t, J = 7.50 Hz, 1 H), 7.20 (d, J = 8.42 Hz, 2 H), 3.51 (t, J = 6.83 Hz, 2 H), 3.23 (s, 3 H), 2.77 ppm (t, J = 6.81 Hz, 2 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.16, 163.26, 149.49, 136.66, 135.22, 132.22, 129.11, 129.06 (2C), 128.76 (2C), 127.97, 124.16 (2C), 123.88, 121.98, 121.27 (2C), 72.95, 57.93, 34.98 ppm; Anal. calcd for C23H21N3O5·0.5 H2O: C 64.48, H 5.18, N 9.81, found: C 64.27, H 5.01, N 9.70.

N-(4-Bromophenyl)-2-(4-nitrobenzamido)benzamide (10): According to method B, 2-amino-N-(4-bromophenyl)benzamide (146 mg, 0.5 mmol) and 4-nitrobenzoyl chloride (111 mg, 0.6 mmol) were reacted, and compound 10 was obtained as a pale-yellow powder (75 mg, 34 %): Rf = 0.74 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 8.35 (d, J = 9.0 Hz, 2 H), 8.26 (d, J = 7.3 Hz, 1 H), 8.14 (d, J = 8.9 Hz, 2 H), 7.91 (d, J = 7.8 Hz, 1 H), 7.65 (d, J = 8.9 Hz, 2 H), 7.56 (t, J = 7.1 Hz, 1 H), 7.50 (d, J = 8.8 Hz, 2 H), 7.25 ppm (t, J = 7.5 Hz, 1 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.10, 163.81, 149.63, 138.51, 131.96, 131.55 (2C), 129.19, 128.84 (2C), 124.52, 123.92 (2C), 124.40, 122.74 (2C), 115.60 ppm; Anal. calcd for C20H14BrN3O4·0.6 H2O: C 53.23, H 3.42, N 9.29, found: C 53.23, H 3.48, N 8.85.

4-(2-(4-Nitrobenzamido)benzamido)phenethyl acetate (6): According to method B, compound 3 b (119 mg, 0.4 mmol) and 4-nitrobenzoyl chloride (111 mg, 0.6 mmol) were reacted, and compound 6 was obtained as a pale-yellow powder (120 mg, 67 %): Rf = 0.74 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 11.71 (s, 1 H), 11.01 (s, 1 H), 8.39–8.33 (m, 3 H), 8.14 (d, J = 8.86 Hz, 2 H), 7.94 (dd, J = 7.81, 1.17 Hz, 1 H), 7.62–7.56 (m, 3 H), 7.27 (t, J = 7.38 Hz, 1 H), 7.21 (d, J = 8.48 Hz, 2 H), 4.18 (t, J = 6.88 Hz, 2 H), 2.85 (t, J = 6.87 Hz, 2 H), 1.97 ppm (s, 3 H); 13C NMR (125 MHz, [D6]DMSO): d = 170.42, 167.03, 163.50, 149.34, 141,26, 139,44, 137.17, 133.83, 132.06, 129.16, 129.12 (2C), 128.81 (2C), 124.07, 124.05 (2C), 123.56, 122.32, 121.17 (2C), 64.52, 33.97, 20.84 ppm; Anal. calcd for C24H21N3O6·0.5 H2O: C 63.15, H 4.86, N 9.21, found: C 63.26, H 4.72, N 8.95.

2-(4-Nitrobenzamido)-N-phenylbenzamide (11): According to method B, 2-amino-N-phenylbenzamide (318 mg, 1.5 mmol) and 4nitrobenzoyl chloride (334 mg, 1.8 mmol) were reacted, and compound 11 was obtained as a cream-colored powder (310 mg, 57 %): Rf = 0.89 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 11.37 (s, 2 H), 8.38–8.35 (m, 2 H), 8.33 (dd, J = 8.22, 0.84 Hz, 1 H), 8.17–8.12 (m, 2 H), 7.95 (dd, J = 7.82, 1.29 Hz, 1 H), 7.69 (d, J = 7.61 Hz, 2 H), 7.58 (t, J = 7.35 Hz, 1 H), 7.35–7.31 (m, 2 H), 7.27 (t, J = 7.45 Hz, 1 H), 7.10 ppm (t, J = 7.39 Hz, 1 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.05, 163.57, 149.28, 138.90, 132.00, 129.23, 128.83 (2C), 128.74 (2C), 124.30, 124.08, 123.99 (2C), 123.47, 122.50, 121.05 ppm (2C); Anal. calcd for C20H15N3O4 : C 66.48, H 4.18, N 11.63, found: C 66.31, H 4.48, N 11.45.

N-(4-Methoxyphenyl)-2-(4-nitrobenzamido)benzamide (7): According to method B, 2-amino-N-(4-methoxyphenyl)benzamide (111 mg, 0.6 mmol) and 4-nitrobenzoyl chloride (111 mg, 0.6 mmol) were reacted, and compound 7 was obtained as a yellow powder (113 mg, 58 %): Rf = 0.70 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 11.96 (s, 1 H), 10.49 (s, 1 H), 8.41 (d, J = 8.14 Hz, 1 H), 8.38 (d, J = 8.82 Hz, 2 H), 8.13 (d, J = 8.87 Hz, 2 H), 7.94 (dd, J = 7.81, 1.05 Hz, 1 H), 7.62–7.58 (m, 3 H), 7.30 (t, J = 7.41 Hz, 1 H), 6.92 (d, J = 9.05 Hz, 2 H), 3.74 ppm (s, 3 H); 13C NMR (125 MHz, [D6]DMSO): d = 166.95, 163.23, 156.17, 149.48, 132.18, 131.58, 129.02, 128.73 (2C), 124.16 (2C), 123.51, 122.96, 121.84, 113.95 (4C), 55.37 ppm; Anal. calcd for C21H17N3O5·0.33 H2O: C 63.47, H 4.48, N 10.57, found: C 63.59, H 4.60, N 10.50.

N-(4-Carbamoylphenyl)-2-(4-nitrobenzamido)benzamide (12): According to method B, 2-amino-N-(4-carbamoylphenyl)benzamide (128 mg, 0.5 mmol) and 4-nitrobenzoyl chloride (111 mg, 0.6 mmol) were reacted, and compound 12 was obtained as a yellow powder (173 mg, 79 %): Rf = 0.30 (EtOAc); 1H NMR (500 MHz, [D6]DMSO): d = 11.52 (s, 1 H), 10.67 (s, 1 H), 8.38 (d, J = 8.7 Hz, 2 H), 8.25 (d, J = 8.2 Hz, 1 H), 8.13 (d, J = 8.6 Hz, 2 H), 7.92–7.83 (m, 4 H), 7.78 (d, J = 8.6 Hz, 2 H), 7.63 (t, J = 7.7 Hz, 1 H), 7.34 (t, J = 7.5 Hz, 1 H), 7.24 ppm (s, 2 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.48, 167.32, 163.43, 149.50, 141.51, 140.34, 137.74, 132.23, 129.72, 129.21, 128.85 (2C), 128.31 (2C), 125.00, 124.29, 124.10 (2C), 122.59, 120.00 ppm (2C); Anal. calcd for C20H16N3O6S·0.66 H2O: C 60.57, H 4.20, N 13.46, found: C 60.17, H 3.97, N 13.07.

(N-(4-Hydroxyphenyl)-2-(4-nitrobenzamido)benzamide (8): According to method B, 2-amino-N-(4-hydroxyphenyl)benzamide (137 mg, 0.6 mmol) and 4-nitrobenzoyl chloride (92.8 mg, 0.5 mmol) were reacted, and compound 8 was obtained as a yellow powder (143 mg, 76 %): Rf = 0.44 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 12.15 (s, 1 H), 10.33 (s, 1 H), 9.34 (s, 1 H), 8.49 (d, J = 8.16 Hz, 1 H), 8.36 (d, J = 8.81 Hz, 2 H), 8.11 (d, J = 8.83 Hz, 2 H), 7.95 (dd, J = 7.72, 0.85 Hz, 1 H), 7.61–7.56 (m, 1 H),

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2-(4-Nitrobenzamido)-N-(4-sulfamoylphenyl)benzamide (13): According to method B, 2-amino-N-(4-sulfamoylphenyl)benzamide (146 mg, 0.5 mmol) and 4-nitrobenzoyl chloride (111 mg, 0.6 mmol) were reacted, and compound 13 was obtained as a pale-yellow powder (183 mg, 83 %): Rf = 0.84 (EtOAc); 1H NMR (500 MHz,

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Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers (d, J = 8.86 Hz, 2 H), 3.83 (s, 3 H), 3.83 ppm (s, 3 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.23, 164.23, 154.46, 152.11, 148.85, 139.32, 132.26, 129.95, 128.86, 126.98, 123.26 (2C), 122.92, 122.04, 120.78, 120.01, 115.19 (2C), 111.59, 110.74, 55.87, 55.63 ppm; Anal. calcd for C22H20N2O5·0.66 H2O: C 65.34, H 5.32, N 6.93, found: C 65.00, H 5.31, N 6.86.

[D6]DMSO): d = 8.37 (d, J = 8.8 Hz, 2 H), 8.21 (d, J = 7.4 Hz, 1 H), 8.13 (d, J = 8.9 Hz, 2 H), 7.90 (d, J = 6.6 Hz, 1 H), 7.86 (d, J = 8.8 Hz, 2 H), 7.78 (d, J = 8.8 Hz, 2 H), 7.61 (t, J = 7.3 Hz, 1 H), 7.32 (t, J = 7.5 Hz, 1 H), 7.24 ppm (s, 2 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.35, 163.61, 149.47, 141.96, 139.09, 132.18, 129.25, 128.89 (2C), 126.61 (2C), 125.23, 124.03 (2C), 122.87, 120.38 ppm (2C); HPLC: k’ = 3.88 (RP18 column, CH3OH/H2O (70:30)), purity 99.8 %.

N-(2-(4-Chlorophenylcarbamoyl)phenyl)-3,4-dimethoxybenzamide (18): According to method A, 3,4-dimethoxybenzoic acid (109 mg, 0.6 mmol) was reacted to yield 3,4-dimethoxybenzoyl chloride, which was then, according to method B, reacted with 2amino-N-(4-chlorophenyl)benzamide (123 mg, 0.5 mmol) to obtain compound 18 as a beige powder (84 mg, 41 %): Rf = 0.59 (EtOAc/ PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 11.51 (s, 1 H), 10.60 (s, 1 H), 8.43 (dd, J = 8.34, 0.93 Hz, 1 H), 7.90 (dd, J = 7.88, 1.42 Hz, 1 H), 7.76 (d, J = 8.88 Hz, 2 H), 7.62–7.58 (m, 1 H), 7.49 (td, J = 3.58, 2.07 Hz, 2 H), 7.42 (d, J = 8.90 Hz, 2 H), 7.26 (dt, J = 7.70, 1.16 Hz, 1 H), 7.12 (d, J = 8.26 Hz, 1 H), 3.83 (s, 3 H), 3.82 ppm (s, 3 H); 13 C NMR (125 MHz, [D6]DMSO): d = 167.65, 164.37, 152.11, 148.80, 138.96, 137.71, 132.45, 129.09, 128.68 (2C), 128.03, 126.91, 123.19, 122.87, 122.66 (2C), 121.41, 120.18, 111.53, 110.78, 55.87, 55.64 ppm; Anal. calcd for C22H19ClN2O4 : C 64.31, H 4.66, Cl 8.63, N 6.82, found: C 64.01, H 4.77, N 7.04.

N-(4-Benzoylphenyl)-2-(4-nitrobenzamido)benzamide (14): According to method B, compound 3 j (158 mg, 0.5 mmol) and 4-nitrobenzoyl chloride (111 mg, 0.6 mmol) were reacted, and compound 14 was obtained as a pale-yellow powder (177 mg, 76 %): Rf = 0.86 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 11.40 (s, 1 H), 10.83 (s, 1 H), 8.42–8.35 (m, 2 H), 8.19 (d, J = 8.27 Hz, 1 H), 8.15–8.11 (m, 2 H), 7.92–7.88 (m, 3 H), 7.78–7.75 (m, 2 H), 7.71 (td, J = 8.38, 1.78, 1.78 Hz, 2 H), 7.68–7.62 (m, 2 H), 7.55 (t, J = 7.61 Hz, 2 H), 7.36 ppm (dt, J = 7.64, 1.02 Hz, 1 H); 13C NMR (125 MHz, [D6]DMSO): d = 194.75, 167.43, 163.52, 149.49, 143.15, 140.30, 137.59, 137.50, 132.43, 132.24, 132.17, 130.97 (2C), 129.51 (2C), 129.25, 128.89 (2C), 128.61 (2C), 125.54, 124.43, 124.06 (2C), 122.86, 119.98 ppm (2C); Anal. calcd for C27H19N3O5 : C 69.67, H 4.11, N 9.03, found: C 69.34, H 3.91, N 8.83. N-(4-(Dimethylamino)phenyl)-2-(4-nitrobenzamido)benzamide (15): According to method B, 2-amino-N-(4-(dimethylamino)phenyl)benzamide (128 mg, 0.5 mmol) and 4-nitrobenzoyl chloride (111 mg, 0.6 mmol) were reacted, and compound 15 was obtained as an orange powder (86 mg, 42 %): Rf = 0.75 (EtOAc/PE (1:1)); 1 H NMR (500 MHz, [D6]DMSO): d = 12.18 (s, 1 H), 10.30 (s, 1 H), 8.49 (d, J = 8.1 Hz, 1 H), 8.39 (d, J = 8.9 Hz, 2 H), 8.12 (d, J = 8.9 Hz, 2 H), 7.95 (d, J = 6.8 Hz, 1 H), 7.61 (t, J = 7.2 Hz, 1 H), 7.49 (d, J = 9.0 Hz, 2 H), 7.30 (t, J = 7.4 Hz, 1 H), 6.72 (d, J = 9.1 Hz, 2 H), 2.87 ppm (s, 6 H); 13C NMR (125 MHz, [D6]DMSO): d = 166.78, 162.84, 149.22, 147.91, 140.29, 138.54, 132.14, 128.87, 128.63 (2C), 127.81, 124.19 (2C), 123.78, 123,28, 122.88 (2C), 121.35, 112.44 (2C), 40.47 ppm (2C); Anal. calcd for C22H20N4O4 : C 65.34, H 4.98, N 13.85, found: C 65.38, H 4.99, N 13.58.

3,4-Dimethoxy-N-(2-(p-tolylcarbamoyl)phenyl)benzamide (19): According to method A, 3,4-dimethoxybenzoic acid (656 mg, 3.6 mmol) was reacted to yield 3,4-dimethoxybenzoyl chloride, which was then, according to method B, reacted with 2-amino-(N4-methylphenyl)benzamide (679 mg, 3.0 mmol) to obtain compound 19 as a white powder (1077 mg, 92 %): Rf = 0.82 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 11.74 (s, 1 H), 10.43 (s, 1 H), 8.50 (dd, J = 0.9, 8.3 Hz, 1 H), 7.92 (dd, J = 1.3, 7.9 Hz, 1 H), 7.62–7.56 (m, 3 H), 7.52–7.48 (m, 2 H), 7.28–7.22 (m, 1 H), 7.17 (d, J = 8.2 Hz, 2 H), 7.13 (d, J = 8.8 Hz, 1 H), 3.83 (s, 3 H), 3.82 (s, 3 H), 2.28 ppm (s, 3 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.50, 164.28, 152.10, 148.82, 139.16, 136.04, 133.52, 132.34, 129.16 (2C), 129.0, 126.93, 123.03, 122.43, 121.31 (2C), 121.03, 120.09, 111.56, 110.72, 55.87, 55.62, 20.65 ppm; Anal. calcd for C23H22N2O4 : C 70.75, H 5.68, N 7.17, found: C 70.42, H 5.59, N 7.12.

3,4-Dimethoxy-N-(2-(4-methoxyphenylcarbamoyl)-phenyl)benzamide (16): According to method A, 3,4-dimethoxybenzoic acid (109 mg, 0.6 mmol) was reacted to yield 3,4-dimethoxybenzoyl chloride, which was then, according to method B, reacted with 2amino-N-(4-methoxyphenyl)benzamide (123 mg, 0.5 mmol) to obtain compound 16 as a cream-colored powder (103 mg, 51 %): Rf = 0.65 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 11.84 (s, 1 H), 10.41 (s, 1 H), 8.53 (d, J = 8.05 Hz, 1 H), 7.93 (d, J = 7.08 Hz, 1 H), 7.65–7.56 (m, 3 H), 7.51–7.48 (m, 2 H), 7.27–7.22 (m, 1 H), 7.13 (d, J = 8.98 Hz, 1 H), 6.94 (d, J = 9.02 Hz, 2 H), 3.83 (s, 3 H), 3.82 (s, 3H), 3.75 ppm (s, 3 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.33, 164.25, 156.23, 152.11, 148.84, 139.26, 132.32, 131.53, 128.92, 126.96, 122.97 (2C), 122.23, 122.16, 120.89, 120.04, 113.95 (2C), 111.57, 110.74, 55.87, 55.64, 55.38 ppm; Anal. calcd for C23H22N2O5·0.25 H2O: C 67.22, H 5.52, N 6.82, found: C 66.82, H 5.23 N 7.07

3,4-Dimethoxy-N-(2-(phenylcarbamoyl)phenyl)benzamide (20): According to method A, 3,4-dimethoxybenzoic acid (109 mg, 0.6 mmol) was reacted to yield 3,4-dimethoxybenzoyl chloride, which was then, according to method B, reacted with 2-amino-Nphenylbenzamide (106 mg, 0.5 mmol) to obtain compound 20 as a white powder (127 mg, 67 %): Rf = 0.62 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 11.66 (s, 1 H), 10.51 (s, 1 H), 8.49 (dd, J = 8.36, 0.83 Hz, 1 H), 7.93 (dd, J = 7.85, 1.42 Hz, 1 H), 7.73 (d, J = 7.51 Hz, 2 H), 7.60 (t, J = 7.07 Hz, 1 H), 7.52–7.49 (m, 2 H), 7.39–7.33 (m, 2 H), 7.26 (dt, J = 7.66, 1.19 Hz, 1 H), 7.15–7.12 (m, 2 H), 3.83 (s, 3 H), 3.82 ppm (s, 3 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.65, 164.32, 152.11, 148.82, 139.12, 138.65, 132.38, 129.08, 128.76 (2C), 126.94, 124.40, 123.09, 122.65, 121.25 (2C), 121.18, 120.11, 111.56, 110.76, 55.86, 55.63 ppm; Anal. calcd for C22H20N2O4 : C 70.20, H 5.36, N 7.44, found: C 69.99, H 5.46, N 7.37.

N-(2-(4-Hydroxyphenylcarbamoyl)phenyl)-3,4-dimethoxybenzamide (17): According to method A, 3,4-dimethoxybenzoic acid (73 mg, 0.4 mmol) was reacted to yield 3,4-dimethoxybenzoyl chloride, which was then, according to method B, reacted with 2amino-N-(4-hydroxyphenyl)benzamide (114 mg, 0.5 mmol) to obtain compound 17 as a cream-colored powder (40 mg, 25 %): Rf = 0.79 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 11.91 (s, 1 H), 10.31 (s, 1 H), 9.31 (s, 1 H), 8.54 (dd, J = 8.30, 0.62 Hz, 1 H), 7.92 (dd, J = 7.88, 1.18 Hz, 1 H), 7.60–7.56 (m, 1 H), 7.51–7.47 (m, 4 H), 7.23 (dt, J = 7.74, 1.10 Hz, 1 H), 7.13 (d, J = 8.98 Hz, 1 H), 6.76

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2-(4-Acetamidobenzamido)-N-phenylbenzamide (21): Compound 22 (99 mg, 0.3 mmol) was dissolved in dry THF and 10 drops of Et3N were added. A solution of acetyl chloride (39 mg, 0.5 mmol) in THF was added dropwise. The solution was stirred for 3 h and afterward the solvent was evaporated under reduced pressure. Water was added to the residue, followed by extraction with EtOAc (3 Õ 10 mL). The organic phase was washed with 1 n NaOH and 1 n HCl and evaporated under reduced pressure. The crude compound was dissolved in THF and precipitated with PE. Com-

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Full Papers pound 21 was obtained as a cream-colored powder (84 mg, 74 %): Rf = 0.54 (EtOAc); 1H NMR (500 MHz, [D6]DMSO): d = 10.34 (s, 1 H), 8.46 (dd, J = 8.28, 0.76 Hz, 1 H), 7.94 (dd, J = 7.84, 1.34 Hz, 1 H), 7.86 (d, J = 8.73 Hz, 2 H), 7.76–7.69 (m, 4 H), 7.60–7.55 (m, 1 H), 7.38–7.33 (m, 2 H), 7.23 (t, J = 7.43 Hz, 1 H), 7.12 (t, J = 7.39 Hz, 1 H), 2.08 ppm (s, 3 H); 13C NMR (125 MHz, [D6]DMSO): d = 168.96, 167.54, 164.34, 142.74, 138.85, 132.21, 128.97 (2C), 128.76, 128.12 (2C), 124.23, 122.93, 122.89, 121.49, 121.28 (2C), 118.67 (2C), 24.24 ppm; HPLC: k’ = 2.34 (RP18 column, CH3OH/H2O (80:20)), purity 98.0 %.

0.5 mmol) and 4-methylbenzoyl chloride (73 mg, 0.6 mmol) were reacted, and compound 26 was obtained as a white powder (139 mg, 80 %): Rf = 0.86 (EtOAc/PE (1:2)); 1H NMR (500 MHz, [D6]DMSO): d = 11.77 (s, 1 H), 10.70 (s, 1 H), 8.49 (d, J = 8.23 Hz, 1 H), 7.94 (dd, J = 7.78, 1.02 Hz, 1 H), 7.82 (d, J = 8.13 Hz, 2 H), 7.57 (dd, J = 11.69, 8.55 Hz, 3 H), 7.35 (d, J = 7.97 Hz, 2 H), 7.22 (t, J = 7.50, 7.50 Hz, 1 H), 7.15 (d, J = 8.25 Hz, 2 H), 2.37 (s, 3 H), 2.28 ppm (s, 3 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.38, 164.78, 141.99, 136.26, 133.30, 132.15, 129.48 (2C), 129.15 (2C), 129.11, 127.20 (2C), 122.86, 122.74, 121.42, 121.34 (2C), 21.13, 20.65 ppm; Anal. calcd for C22H20N2O2·1.4 H2O: C 71.49, H 6.22, N 7.58, found: C 71.36, H 5.72, N 7.35.

2-(4-Aminobenzamido)-N-phenylbenzamide (22): Compound 11 0.6 (217 mg, 0.6 mmol) was reacted according to method C to yield compound 22 as a white powder (119 mg, 60 %): Rf = 0.77 (EtOAc); 1H NMR (500 MHz, [D6]DMSO): d = 11.47 (s, 1 H), 10.49 (s, 1 H), 8.54 (d, J = 8.35 Hz, 1 H), 7.91 (dd, J = 7.85, 1.43 Hz, 1 H), 7.72 (d, J = 8.61 Hz, 2 H), 7.61 (d, J = 8.66 Hz, 2 H), 7.58–7.54 (m, 1 H), 7.40–7.35 (m, 2 H), 7.20 (dt, J = 1.11 Hz, 1 H), 7.14 (t, J = 7.39 Hz, 1 H), 6.62 (d, J = 8.65 Hz, 2 H), 5.83 ppm (s, 2 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.81, 164.61, 152.77, 139.78, 138.58, 132.35, 129.03, 128.81 (2C), 128.78 (2C), 124.39, 122.35, 121.72, 121.35 (2C), 120.84, 120.67, 113.08 ppm (2C); HPLC: k’ = 1.63 (RP18 column, CH3OH/H2O (80:20)); purity 98.4 %

N-(4-Benzoylphenyl)-2-(4-methylbenzamido)benzamide (27): According to method B, 2-amino-N-(4-benzoylphenyl)benzamide (158 mg, 0.5 mmol) and 4-methylbenzoyl chloride (93 mg, 0.6 mmol) were reacted, and compound 27 was obtained as a white powder (170 mg, 78 %): Rf = 0.58 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 11.42 (s, 1 H), 10.87 (s, 1 H), 8.40 (dd, J = 8.32, 0.97 Hz, 1 H), 7.94–7.90 (m, 3 H), 7.82 (d, J = 8.19 Hz, 2 H), 7.80– 7.76 (m, 2 H), 7.74–7.71 (m, 2 H), 7.68–7.64 (m, 1 H), 7.64–7.59 (m, 1 H), 7.55 (t, J = 7.65 Hz, 2 H), 7.35 (d, J = 7.91 Hz, 2 H), 7.29 (dt, J = 7.67, 1.12 Hz, 1 H), 2.37 ppm (s, 3 H); 13C NMR (125 MHz, [D6]DMSO): d = 194.77, 167.89, 164.81, 143.18, 142.23, 138.77, 137.63, 132.44 (2C), 132.24, 131.89, 130.98 (2C), 129.55 (2C), 129.50 (2C), 129.29, 128.62 (2C), 127.26 (2C), 123.55, 123.41, 121.82, 120.21 (2C), 21.13 ppm; Anal. calcd for C28H22N2O3 : C 77.40, H 5.10, N 6.45, found: C 76.98, H 5.24, N 6.34.

2-(4-Chlorobenzamido)-N-(4-methoxyphenyl)benzamide (23): According to method B, 2-amino-N-(4-methoxyphenyl)benzamide (123 mg, 0.5 mmol) and 4-chlorobenzoyl chloride (88 mg, 0.6 mmol) were reacted, and compound 23 was obtained as a light-grey powder (113 mg, 58 %): Rf = 0.74 (EtOAc/PE (1:1)); 1 H NMR (500 MHz, [D6]DMSO): d = 11.88 (s, 1 H), 10.41 (s, 1 H), 8.48 (d, J = 8.19 Hz, 1 H), 7.92 (t, J = 9.13 Hz, 3 H), 7.65–7.57 (m, 5 H), 7.28 (t, J = 7.40 Hz, 1 H), 6.94 (d, J = 8.94 Hz, 2 H), 3.74 ppm (s, 3 H); 13 C NMR (125 MHz, [D6]DMSO): d = 167.17, 163.64, 156.23, 138.76, 136.99, 133.42, 132.27, 131.45, 129.13 (2C), 129.02 (2C), 128.96, 123.48, 123.08 (2C), 122.73, 121.34, 113.94 (2C), 55.36 ppm; Anal. calcd for C21H17ClN2O3 : C 66.23, H 4.50, N 7.36, found: C 66.53, H 4.91, N 6.98.

N-(4-(2-Methoxyethyl)phenyl)-2-(4-(trifluoromethyl)benzamido)benzamide (28): According to method A, 4-trifluoromethylbenzoic acid (228 mg, 1.2 mmol) was reacted with 4-trifluoromethylbenzoyl chloride and then, according to method B, with compound 3 a (203 mg, 0.75 mmol), and compound 28 was obtained as a beige powder (96 mg, 29 %): Rf = 0.81 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 11.83 (s, 1 H), 10.47 (s, 1 H), 8.43 (d, J = 8.19 Hz, 1 H), 8.11–8.07 (m, 2 H), 7.96–7.91 (m, 3 H), 7.64–7.57 (m, 3 H), 7.31 (t, J = 7.46 Hz, 1 H), 7.21 (d, J = 8.43 Hz, 2 H), 3.51 (t, J = 6.84 Hz, 2 H), 3.23 (s, 3 H), 2.77 ppm (t, J = 6.82 Hz, 2 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.28, 163.63, 138.49, 138.42, 136.60, 135.28, 132.29, 131.85, 129.08, 129.06 (2C), 128.12 (2C), 126.08 (2C), 123.97, 123.83, 123.48, 121.72, 121.33 (2C), 72.95, 57.93, 34.98 ppm; Anal. calcd for C21H21F3N2O3·0.25 H2O: C 64.50, H 4.85, N 6.27, found: C 64.61, H 4.80, N 6.27.

2-(4-Chlorobenzamido)-N-(4-hydroxyphenyl)benzamide (24): According to method B, 2-amino-N-(4-hydroxyphenyl)benzamide (114 mg, 0.5 mmol) and 4-chlorobenzoyl chloride (70 mg, 0.4 mmol) were reacted, and compound 24 was obtained as a white powder (140 mg, 95 %): Rf = 0.28 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 11.97 (s, 1 H), 10.32 (s, 1 H), 9.33 (s, 1 H), 8.50 (d, J = 8.17 Hz, 1 H), 7.94–7.89 (m, 3 H), 7.64 (d, J = 8.53 Hz, 2 H), 7.61–7.57 (m, 1 H), 7.46 (d, J = 8.80 Hz, 2 H), 7.27 (t, J = 7.62 Hz, 1 H), 6.76 ppm (d, J = 8.85 Hz, 2 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.09, 163.63, 154.49, 138.87, 137.00, 133.46, 132.25, 129.86, 129.17 (2C), 129.02 (2C), 128.93, 123.39 (3C), 122.53, 121.20, 115.20 ppm (2C); Anal. calcd for C21H17ClN2O3·0.66 H2O: C 63.41, H 4.35, N 7.40, found: C 63.37, H 4.27, N 7.22.

N-(4-Hydroxyphenyl)-2-(4-(trifluoromethyl)benzamido)benzamide (29): According to method A, 4-trifluoromethylbenzoic acid (133 mg, 0.7 mmol) was reacted with 4-trifluoromethylbenzoyl chloride and then, according to method B, with 2-amino-N-(4-hydroxyphenyl)benzamide (114 mg, 0.86 mmol), and compound 29 was obtained as a beige powder (179 mg, 64 %): Rf = 0.99 (EtOAc); 1 H NMR (500 MHz, [D6]DMSO): d = 12.06 (s, 1 H), 10.33 (s, 1 H), 9.30 (s, 1 H), 8.50 (d, J = 7.8 Hz, 1 H), 8.09 (d, J = 8.2 Hz, 2 H), 7.98–7.90 (m, 3 H), 7.61 (t, J = 7.8 Hz, 1 H), 7.47 (d, J = 8.9 Hz, 2 H), 7.29 (t, J = 7.6 Hz, 1 H), 6.76 ppm (d, J = 8.8 Hz, 2 H); 13C NMR (125 MHz, [D6]DMSO): d = 166.99, 163.50, 154.47, 138.63, 138.49, 132.23, 132.20–131.28 (m),129.87, 128.93, 128.06 (2C), 126.09 (2C), 123.68, 123.34 (2C), 127.31–120.58 (m), 122.81, 121.34, 115.18 ppm (2C); Anal. calcd for C21H15F3N2O3·0.25 H2O: C 62.30, H 3.86, N 6.92, found: C 62.29, H 4.03, N 6.97.

2-(4-Chlorobenzamido)-N-p-tolylbenzamide (25): According to method B, 2-amino-(N-4-methylphenyl)benzamide (113 mg, 0.5 mmol) 4-chlorobenzoyl chloride (94 mg, 0.6 mmol) were reacted, and compound 25 was obtained as a white powder (51 mg, 28 %): Rf = 0.90 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 11.79 (s, 1 H), 10.53 (s, 1 H), 8.43 (d, J = 8.18 Hz, 1 H), 7.94–7.90 (m, 3 H), 7.62 (d, J = 8.49 Hz, 2 H), 7.58 (m, 3 H), 7.26 (t, J = 7.48 Hz, 1 H), 7.16 (d, J = 8.19 Hz, 2 H), 2.28 ppm (s, 3 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.27, 163.77, 136.87, 136.09, 133.41, 132.20, 129.14 (2C), 129.08 (5C), 123.40, 123.18, 121.63, 121.34 (2C), 20.63 ppm; Anal. calcd for C21H17ClN2O2·0.33 H2O: C 68.02, H 4.80, N 7.55; found: C 67.91, H 4.63, N 7.31.

N-(4-Chlorophenyl)-2-(4-(trifluoromethyl)benzamido)benzamide (30): According to method A, 4-trifluoromethylbenzoic acid (114 mg, 0.6 mmol) was reacted with 4-trifluoromethylbenzoyl chloride and then, according to method B, with 2-amino-N-(4-chlo-

2-(4-Methylbenzamido)-N-p-tolylbenzamide (26): According to method B, 2-amino-(N-4-methylphenyl)benzamide (113 mg,

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Full Papers rophenyl)benzamide (123 mg, 0.5 mmol), and compound 30 was obtained as a cream-colored powder (56 mg, 23 %): Rf = 0.85 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 11.57 (s, 1 H), 10.61 (s, 1 H), 8.33 (d, J = 7.7 Hz, 1 H), 8.09 (d, J = 8.1 Hz, 2 H), 7.93 (d, J = 8.3 Hz, 2 H), 7.89 (d, J = 6.6 Hz, 1 H), 7.74 (d, J = 8.9 Hz, 2 H), 7.62 (m, 1 H), 7.40 (d, J = 8.9 Hz, 2 H), 7.32 ppm (td, J = 1.0, 7.7 Hz, 1 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.31, 163.78, 138.50, 138.07, 137.74, 132.28, 129.12, 128.66 (2C), 128.18 (2C), 127.95, 125.99 (2C), 124.02, 123.96, 122.61 (2C), 122.20 ppm; Anal. calcd for C21H14ClF3N2O2 : C 60.23, H 3.37, N 6.69, found: C 60.29, H 3.26, N 6.62.

1 H), 8.89 (s, 1 H), 8.59 (dd, J = 8.35, 1.04 Hz, 1 H), 8.41 (d, J = 8.85 Hz, 2 H), 8.14 (d, J = 8.87 Hz, 2 H), 7.85 (dd, J = 7.94, 1.43 Hz, 1 H), 7.59–7.55 (m, 1 H), 7.23 (dt, J = 7.82, 1.15 Hz, 1 H), 3.33 (dq, J = 7.22, 5.63 Hz, 2 H), 1.14 ppm (t, J = 7.22 Hz, 3 H); 13C NMR (125 MHz, [D6]DMSO): d = 168.37, 162.83, 149.57, 140.32, 139.14, 132.34, 128.59 (2C), 128.31, 124.28 (2C), 123.50, 120.76, 120.66, 34.36, 14.54 ppm; Anal. calcd for C16H15N3O4·0.2 H2O: C, 60.64; H, 4.90; N, 13.26, found: C, 60.90; H, 4.84; N, 13.13.

N-Phenyl-2-(4-(trifluoromethyl)benzamido)benzamide (31): According to method A, 4-trifluoromethylbenzoic acid (91 mg, 0.48 mmol) was reacted with 4-trifluoromethylbenzoyl chloride and then, according to method B, with 2-amino-N-phenylbenzamide (85 mg, 0.4 mmol), and compound 31 was obtained as a white powder (81 mg, 42 %): Rf = 0.63 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 11.74 (s, 1 H), 10.51 (s, 1 H), 8.40 (dd, J = 8.31, 0.87 Hz, 1 H), 8.10 (d, J = 8.10 Hz, 2 H), 7.96–7.91 (m, 3 H), 7.70 (dd, J = 8.55, 1.02 Hz, 2 H), 7.65–7.60 (m, 1 H), 7.37–7.30 (m, 3 H), 7.13 ppm (m, 1 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.36, 163.68, 138.64, 138.48, 138.28, 132.29, 133.00–130.10 (m), 129.14, 128.75 (2C), 128.14 (2C), 126.06 (2C), 124.37, 127.01–123 (m), 123.92, 123.80, 121.89, 121.27 ppm (2C); Anal. calcd for C21H15F3N2O2 : C 65.62, H 3.93, N 7.29, found: C 65.38, H 4.27, N 7.42.

Materials: All chemicals were purchased from Sigma–Aldrich Chemicals (Taufkirchen, Germany) unless otherwise specified. For cellbased assays, 10 mm stock solutions of the compounds were prepared in DMSO. Various dilutions of the test compounds were prepared in sterile filtered Krebs-HEPES buffer (KHB). For some compounds with low solubility, methanol (50 %) was used for preparing 1 mm concentrations. The amount of methanol did not exceed 0.5 % at the highest concentration assayed. The highest concentration of DMSO present was not more than 0.1 %.

Biological studies

Cell culture: The BCRP-expressing cell line MCF-7 MX and the parental cell line MCF-7 were grown in RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS), 50 mg mL¢1 streptomycin, 50 U mL¢1 penicillin G, and 2 mm l-glutamine. MDCK wild-type and MDCK BCRP cell lines were received as a generous gift from Dr. A. Schinkel (The Netherlands Cancer Institute, Amsterdam). MDCK BCRP cells were generated by transfection of the canine kidney epithelial cell line MDCKII with the human wild-type BCRP cDNA C-terminally linked to the cDNA of green fluorescent protein (GFP). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10 % FBS, 50 mg mL¢1 streptomycin, 50 U mL¢1 penicillin G, and 2 mm l-glutamine.

N-(4-Hydroxycyclohexyl)-2-(4-nitrobenzamido)benzamide (32): According to method B, 2-amino-N-(4-hydroxycyclohexyl)benzamide (94 mg, 0.5 mmol) and 4-nitrobenzoyl chloride (93 mg, 0.5 mmol) were reacted, and compound 32 was obtained as a pale-yellow powder (31 mg, 20 %): Rf = 0.52 (EtOAc); 1H NMR (500 MHz, [D6]DMSO): d = 12.68 (s, 1 H), 8.59–8.53 (m, 2 H), 8.42 (d, J = 8.82 Hz, 2 H), 8.13 (d, J = 8.83 Hz, 2 H), 7.85 (dd, J = 7.93, 1.33 Hz, 1 H), 7.59–7.55 (m, 1 H), 7.25–7.19 (m, 1 H), 4.54 (d, J = 4.41 Hz, 1 H), 3.82–3.72 (m, 1 H), 3.42–3.34 (m, 1 H), 1.88–1.78 (m, 4 H), 1.44–1.34 (m, 2 H), 1.28–1.19 ppm (m, 2 H); 13C NMR (125 MHz, [D6]DMSO): d = 167.85, 162.89, 149.58, 140.33, 138.92, 132.27, 128.61 (3C), 124.32 (2C), 123.50, 121.21, 120.74, 68.38, 48.17, 34.25 (2C), 30.09 ppm (2C); Anal. calcd for C20H21N3O5·0.66 H2O: C 60.75, H 5.69, N 10.63, found: C 60.59, H 5.50, N 10.39.

The human ovarian carcinoma cell line A2780 and the corresponding MDR1-overexpressing doxorubicin-resistant A2780 Adr cell line were purchased from ECACC (nos. 93112519 and 93112520). The cell lines were grown in RPMI-1640 medium supplemented with 10 % FBS, 50 mg mL¢1 streptomycin, 50 U mL¢1 penicillin G, and 2 mm l-glutamine. To maintain P-gp overexpression in the A2780 Adr cell line, periodically (every 5 to 7 passages) cells were incubated with 1 mm doxorubicin for one passage. All cells were maintained under a 5 % CO2 humidified atmosphere at 37 8C. After reaching a confluence of 80–90 %, cells were harvested with trypsin-EDTA (0.05 % trypsin/0.02 % EDTA), centrifuged (266 g, 4 8C, 4 min) and re-suspended in fresh culture medium. Cell counting was performed with a CASY1 model TT cell counter (Schaerfe System GmbH, Reutlingen, Germany) with 150 mm capillary to determine constant cell densities in the various cell-based assays.

N-(4-Hydroxycyclohexyl)-2-(4-(trifluoromethyl)benzamido)benzamide (33): According to method A, 4-trifluoromethylbenzoic acid (95 mg, 0.5 mmol) was reacted with 4-trifluoromethylbenzoyl chloride and then, according to method B, 2-amino-N-(4-hydroxycyclohexyl)benzamide (94 mg, 0.5 mmol), and compound 33 was obtained as a beige powder (26 mg, 16 %): Rf = 0.14 (EtOAc/PE (1:1)); 1 H NMR (500 MHz, [D6]DMSO): d = 12.64 (s, 1 H), 8.59–8.56 (m, 2 H), 8.11 (d, J = 8.10 Hz, 2 H), 7.98 (d, J = 8.23 Hz, 2 H), 7.84 (dd, J = 7.93, 1.35 Hz, 1 H), 7.58–7.54 (m, 1 H), 7.21 (dt, J = 7.89, 1.09 Hz, 1 H), 4.54 (d, J = 4.35 Hz, 1 H), 3.81–3.73 (m, 1 H), 3.42–3.34 (m, 1 H), 1.83 (dd, J = 10.85, 7.90 Hz, 4 H), 1.44–1.34 (m, 2 H), 1.28–1.19 ppm (m, 2 H); 13 C NMR (125 MHz, [D6]DMSO): d = 167.91, 163.36, 139.08, 138.55, 132.27, 132.16–131.42 (m), 128.59, 128.02 (2C), 126.22 (2C), 125.08, 123.33, 121.08, 120.66, 68.39, 48.17, 34.27 (2C), 30.11 ppm (2C); Anal. calcd for C21H21F3N2O3·H2O: C 59.43, H 5.46, N 6.60, found: C 59.51, H 5.29, N 6.55.

Hoechst 33342 accumulation assay: To investigate the inhibitory effect of the synthesized compounds on BCRP, a Hoechst 33342 accumulation assay was performed as described earlier with a Hoechst 33342 concentration of 1 mm in the fluorescence assay.[16, 22–26] The increase in cellular fluorescence due to Hoechst 33342 accumulation was determined in the absence and presence of various inhibitor concentrations. Briefly, after reaching a confluence of 80–90 %, cells were harvested by gentle trypsin treatment (0.05 % trypsin/0.02 % EDTA) and then transferred to a 50 mL tube followed by centrifugation (266 g, 4 8C, 4 min). The cell pellet obtained was re-suspended in fresh culture medium, and the cell density was determined with a CASY1 model TT cell counter (Schaerfe System GmbH). Cells were again centrifuged and re-suspended in KHB to yield a cell density of 3 Õ 106 cells per mL; 90 mL of this final suspension, containing ~ 27 000 cells, were seeded into

N-Ethyl-2-(4-nitrobenzamido)benzamide (34): According to method B, 2-amino-N-ethylbenzamide (82 mg, 0.5 mmol) and 4-nitrobenzoyl chloride (130 mg, 0.7 mmol) were reacted, and compound 34 was obtained as a cream-colored powder (105 mg, 67 %): Rf = 0.65 (EtOAc/PE (1:1)); 1H NMR (500 MHz, [D6]DMSO): d = 12.84 (s,

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Full Papers black 96-well plates (Greiner, Frickenhausen, Germany). Test compounds (10 mL) at various concentrations were added to a total volume of 100 mL. The prepared 96-well plate was incubated under 5 % CO2 at 37 8C for 30 min. After this pre-incubation period, a solution of Hoechst 33342 (20 mL, 6 mm; protected from light) was added to each well. Fluorescence was measured immediately at constant intervals of 60 s up to 120 min at an excitation wavelength of 355 nm and an emission wavelength of 460 nm using a BMG POLARstar microplate reader (BMG Labtech, Offenburg, Germany) held at 37 8C. For assay data analysis, the fluorescence from KHB was initially subtracted from the fluorescence reading obtained from MCF-7 MX cells. The upper plateau value (ymax) of each fluorescence–time curve was determined based on a one-phase exponential association curve fit. From these data, concentration– response curves were generated by nonlinear regression using the four-parameter logistic equation with variable Hill slope (Prism 5.0, GraphPad Software Inc., San Diego, CA, USA).

centration in a final volume of 200 mL. After incubation for 72 h, MTT was added (20 mL of a 5 mg mL¢1 solution) to each well. Plates were further incubated for 1 h and then the assay was terminated by removing supernatants and lysing the cells with 100 mL DMSO per well. Cell viability was determined spectrophotometrically by measuring absorbance at 544 nm and background correction at 710 nm using a BMG POLARstar microplate reader.

Keywords: ABCG2 · ABC transporter · BCRP · inhibitors · multidrug resistance [1] K. F. K. Ejendal, C. Hrycyna, Curr. Protein Pept. Sci. 2002, 3, 503 – 511. [2] L. Doyle, W. Yang, L. V. Abruzzo, T. Krogmann, Y. Gao, K. Rishi, D. D. Ross, Proc. Natl. Acad. Sci. USA 1998, 95, 15665 – 15670. [3] H. Wang, E.-W. Lee, X. Cai, Z. Ni, L. Zhou, Q. Mao, Biochemistry 2008, 47, 13778 – 13787. [4] K. Kage, T. Fujita, Y. Sugimoto, Cancer Sci. 2005, 96, 866 – 872. [5] F. Staud, P. Pavek, Int. J. Biochem. Cell Biol. 2005, 37, 720 – 725. [6] M. L. H. Vlaming, J. S. Lagas, A. H. Schinkel, Adv. Drug Delivery Rev. 2009, 61, 14 – 25. [7] B. Sarkadi, L. Homolya, G. Szak‚cs, A. V‚radi, Physiol. Rev. 2006, 86, 1179 – 1236. [8] A. Ahmed-Belkacem, A. Pozza, S. Macalou, J. M. P¦rez-Victoria, A. Boumendjel, A. Di Pietro, Anti-Cancer Drugs 2006, 17, 239 – 243. [9] S. Zhang, X. Yang, M. E. Morris, Mol. Pharmacol. 2004, 65, 1208 – 1216. [10] K. Katayama, K. Masuyama, S. Yoshioka, H. Hasegawa, J. Mitsuhashi, Y. Sugimoto, Cancer Chemother. Pharmacol. 2007, 60, 789 – 797. [11] A. Pick, H. Mìller, R. Mayer, B. Haenisch, I. K. Pajeva, M. Weigt, H. Bçnisch, C. E. Mìller, M. Wiese, Bioorg. Med. Chem. 2011, 19, 2090 – 2102. [12] S. K. Rabindran, D. D. Ross, L. A. Doyle, W. Yang, L. M. Greenberger, Cancer Res. 2000, 60, 47 – 50. [13] K. Shiozawa, M. Oka, H. Soda, M. Yoshikawa, Y. Ikegami, J. Tsurutani, K. Nakatomi, Y. Nakamura, S. Doi, T. Kitazaki, Y. Mizuta, K. Murase, H. Yoshida, D. D. Ross, S. Kohno, Int. J. Cancer 2004, 108, 146 – 151. [14] J. D. Allen, A. Van Loevezijn, J. M. Lakhai, M. van der Valk, O. van Tellingen, G. Reid, J. H. M. Schellens, G.-J. Koomen, A. H. Schinkel, Mol. Cancer Ther. 2002, 1, 417 – 425. [15] C. Lemos, G. Jansen, G. J. Peters, Br. J. Cancer 2008, 98, 857 – 862. [16] A. Pick, H. Mìller, M. Wiese, Bioorg. Med. Chem. 2008, 16, 8224 – 8236. [17] A. Pick, W. Klinkhammer, M. Wiese, ChemMedChem 2010, 5, 1498 – 1505. [18] F. Marighetti, K. Steggemann, M. Hanl, M. Wiese, ChemMedChem 2013, 8, 125 – 135. [19] Molecular Operating Environment (MOE), 2012.10; Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC, H3A 2R7 (Canada). [20] I. Jabeen, K. Pleban, U. Rinner, P. Chiba, G. F. Ecker, J. Med. Chem. 2012, 55, 3261 – 3273. [21] P. Chiba, S. Burghofer, E. Richter, B. Tell, A. Moser, G. Ecker, J. Med. Chem. 1995, 38, 2789 – 2793. [22] H. Mìller, W. Klinkhammer, C. Globisch, M. U. Kassack, I. K. Pajeva, M. Wiese, Bioorg. Med. Chem. 2007, 15, 7470 – 7479. [23] H. Mìller, I. K. Pajeva, C. Globisch, M. Wiese, Bioorg. Med. Chem. 2008, 16, 2456 – 2470. [24] K. Juvale, M. Wiese, Bioorg. Med. Chem. Lett. 2012, 22, 6766 – 6769. [25] K. Juvale, K. Stefan, M. Wiese, Eur. J. Med. Chem. 2013, 67, 115 – 126. [26] K. Juvale, J. Gallus, M. Wiese, Bioorg. Med. Chem. 2013, 21, 7858 – 7873. [27] A. Pick, M. Wiese, ChemMedChem 2012, 7, 650 – 662. [28] K. Juvale, V. F. Pape, M. Wiese, Bioorg. Med. Chem. 2012, 20, 346 – 355. [29] H. Mueller, M. U. Kassack, M. Wiese, J. Biomol. Screening 2004, 9, 506 – 515. [30] A. Pick, H. Mìller, M. Wiese, Bioorg. Med. Chem. Lett. 2010, 20, 180 – 183. [31] K.-J. Schaper, J. K. Seydel, Chemische Struktur und biologische Aktivit•t von Wirkstoffen, Verlag Chemie, Weinheim, 1979.

Pheophorbide A assay: This assay was performed as described earlier.[16, 17, 27] Cells were prepared as described for the Hoechst 33342 assay. Approximately 45 000 cells per well were seeded into Ushaped clear 96-well plates (Greiner) in a volume of 160 mL. To this, test compounds (20 mL) at various concentrations were added. The prepared 96-well plate was kept under 5 % CO2 at 37 8C for 30 min. After this pre-incubation period, a solution of pheophorbide A (20 mL, 5 mm, protected from light) was added to each well, and plates were incubated for a further 120 min. Fluorescence was measured by flow cytometry (FACSCalibur, Becton Dickinson, Heidelberg, Germany). Pheophorbide A was excited at l 488 nm, and emission was detected in the FL3 channel (Š 670 nm). Concentration–response curves were generated by nonlinear regression using the four-parameter logistic equation with variable Hill slope (GraphPad Prism 5.0). Calcein AM assay: To determine the selectivity of compounds toward BCRP, they were further tested for their P-gp inhibition by calcein AM assay using A2780 Adr cells, as described previously.[16, 28] After reaching a confluence of 80–90 %, cells were harvested with trypsin-EDTA (0.05 % trypsin/0.02 % EDTA), centrifuged (266 g, 4 8C, 4 min) and re-suspended in fresh culture medium. Cells were prepared as described above and seeded into clear 96-well plates (Greiner) at a density of ~ 30 000 cells in a volume of 90 mL per well. Test compounds (10 mL) were then added, resulting in a final volume of 100 mL per well. The prepared 96-well plates were preincubated for 30 min. After this pre-incubation period, calcein AM (33 mL, 1.25 mm; protected from light) was added to each well. The fluorescence was measured immediately at constant time intervals (60 s) up to 60 min using an excitation wavelength of 485 nm and an emission wavelength of 520 nm with a BMG POLARstar microplate reader held at 37 8C. The slope of the initial linear portion of each fluorescence–time curve was calculated by linear regression. From the slopes, concentration–response curves were generated by nonlinear regression using the four-parameter logistic equation with variable Hill slope (GraphPad Prism 5.0). MTT cytotoxicity assay: To investigate the MDR-reversal capacity of selected compounds, the MTT cytotoxicity assay was used. The assay was performed as described earlier with minor modifications.[26, 27, 29] In brief, MDCK BCRP and parental MDCK cells were seeded into 96-well tissue culture plates (Sarstedt, Newton, USA) at a density of 2500 cells per well in a volume of 160 mL and kept under 5 % CO2 at 37 8C for 6 h. Attachment of cells was monitored by microscope, and SN-38 (20 mL), the active metabolite of irinotecan, was added at various concentrations. Finally, test compounds (20 mL) were added to the cells to achieve the required final con-

ChemMedChem 2015, 10, 742 – 751

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Received: November 21, 2014 Revised: January 26, 2015 Published online on March 3, 2015

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