Structure–Activity Relationships, Ligand Efficiency, and Lipophilic Efficiency Profiles of Benzophenone-Type Inhibitors of the Multidrug Transporter P-Glycoprotein

Abstract

The drug efflux pump P-glycoprotein (P-gp) has been shown to promote multidrug resistance (MDR) in tumors as well as to influence ADME properties of drug candidates. Here we synthesized and tested a series of benzophenone derivatives structurally analogous to propafenone-type inhibitors of P-gp. Some of the compounds showed ligand efficiency and lipophilic efficiency (LipE) values in the range of compounds which entered clinical trials as MDR modulators. Interestingly, although lipophilicity plays a dominant role for P-gp inhibitors, all compounds investigated showed LipE values below the threshold for promising drug candidates. Docking studies of selected analogues into a homology model of P-glycoprotein suggest that benzophenones show an interaction pattern similar to that previously identified for propafenone-type inhibitors.

Introduction

Membrane transporters are increasingly recognized for playing a key role in safety profiles of drug candidates, predominantly by their involvement in drug–drug interactions.^1,2 One of the most intensively studied families in this context is the ATP-binding cassette (ABC) transporter superfamily.³⁻⁵ Several members of these ATP-driven transporters are expressed at tissue barriers and thus influence uptake and elimination of drugs and drug candidates.⁶ Originally they have been linked to development of multidrug resistance (MDR) in tumor therapy, as they transport a wide variety of natural product toxins such as anthracyclines, vincristine, and taxanes out of tumor cells.^7,8 Thus, P-glycoprotein (P-gp/ABCB1), discovered in 1976 and considered the paradigm ABC transporter,^9,10 shows a remarkably broad substrate pattern, transporting numerous structurally and functionally diverse compounds across cell membranes.³ P-gp is expressed at the blood–brain barrier (BBB), the blood–cerebrospinal fluid (B-CSF) barrier, and the intestinal barrier, thus modulating the absorption and excretion of xenobiotics across these barriers.⁶ P-gp and its ligands (substrates and inhibitors) are therefore extensively studied both with respect to reversing multidrug resistance in tumors and for modifying ADME-Tox properties of drug candidates,¹¹ such as central nervous system (CNS) active agents.^12,13 Within the past two decades, numerous modulators of P-gp mediated drug efflux have been identified^14,15 and several entered clinical studies up to phase III. However, up to now no compound achieved approval, which is mainly due to severe side effects and lack of efficacy. This further emphasizes the physiological role of efflux transporters in general and P-gp in particular¹⁶ and stresses the need for a more detailed knowledge on the structure and function of these proteins and the molecular basis of their interaction with small molecules.¹⁷ The latter has been approached by numerous SAR and QSAR studies, which revealed that high lipophilicity seems to be a general prerequisite for high P-gp inhibitory potency, valid across different chemical scaffolds. This is also in line with recent structure-based studies, which indicate an entry pathway via the membrane bilayer.^18,19

In recent years the concepts of “Binding energy of the ligand per atom” or ligand efficiency (LE)²⁰⁻²² and lipophilic efficiency (LipE),^23,24 which combines both “potency and lipophilicity,” have been shown to be useful tools in the lead optimization process.^25,26 In the light of our extensive SAR and QSAR studies on propafenone analogues^27,28 (Figure 1) and related compounds, we also utilized benzophenone-based probes, which contain a photoactive arylcarbonyl group as part of the pharmacophore. This led to the identification of key amino acid residues interacting with these ligands.^29,30 Within this study, we extended the set of benzophenones in order to identify compounds with higher potency, utilizing also the concepts of LE and LipE. In addition, docking studies of selected compounds into a homology model of P-gp were performed to shed light on the potential binding mode of these compounds and to compare it with the binding hypothesis derived for analogous propafenones.¹⁷

Figure 1.

Selected propafenone analogues used in this study.

Results and Discussion

Chemistry

Synthesis of benzophenone analogues 6–24 was carried out in analogy to the synthesis of propafenone derivatives.²⁷ Briefly, the respective ortho-, meta-, or para-hydroxy benzophenone (1a–c) was alkylated with epichlorohydrine yielding ortho-, meta-, and para-oxiranes 2a–c. Subsequent nucleophilic oxirane ring-opening with primary or secondary amines (R1) gave target compounds 6–19. Excessive amount of oxirane 2a upon nucleophilic ring-opening with piperazine yielded the homodimer 23, whereas equimolar amounts of both partners predominantly gave the piperazine analogue 5 (Scheme 1).

Scheme 1.

Reagents and conditions: (i) NaOH, epicholorohydrine, reflux for 24 h; (ii) methanol, respective amine (R1), reflux for 24 h (5–19, 23).

Further treatment of piperazine analogue 5 with phenylisocyanate and its thio-analogue as described by Pitha et al.³¹ yielded 21 and 22, respectively. O-Alkylation of 2-hydroxy-5-methyl acetophenone (3) with epichlorohydrine yielded 4, which, upon subsequent nucleophilic ring-opening by piperazine 5 yielded the heterodimer 24 (Scheme 2).

Scheme 2.

Reagents and conditions: (i) NaOH, epicholorohydrine, reflux for 24 h; (iv) p-tolyl isocyanate, CH₂Cl₂, stirring 2 h (X = O), p-tolylisothiocyanate, CH₂Cl₂, stirring 2 h (X = S); (v) methanol, reflux 5 h.

Biological Activity

Biological activity of target compounds 6–24 was assessed using the daunorubicin efflux protocol as described previously.³² Briefly, multidrug resistant CCRF-CEM vcr 1000 cells were preloaded with daunorubicin and efflux was monitored by time-dependent decrease in mean cellular fluorescence in the absence and presence of various concentrations of compounds. IC₅₀ values were calculated from concentration–response curves derived from first-order rate constant of transport (i.e.V_max/K_m) as a function of compound concentration.³² Thus, the effect of different modulators on the transport rate is measured in a direct functional assay. Values are given in Table 1 and are the mean of at least three independently performed experiments. Generally, interexperimental variation was below 20%.

Table 1. Chemical Structure, Ligand Efficiency (LE), Lipophilic Efficiency (LipE), and Pharmacological Activity of Compounds 6–24.

^aPosition of the side chain at central aromatic ring.

Structure–Activity Relationships

Table 1 shows the P-gp inhibitory potency of compounds 6–24. The IC₅₀ values cover a broad range, spanning from 0.05 μM for the dimer 23 up to 13.37 μM for the morpholine analogue 15. Besides the ortho-benzophenone dimer 23, also the ortho analogues showing an arylpiperazine moiety (6, 9) are highly active. Interestingly, the heterodimer 24 is one of the least active compounds in the data set, together with the morpholine derivatives 15 and 16. With respect to substitution pattern at the central aromatic benzene moiety, the rank order for arylpiperazine substituted compounds generally is ortho > meta > para. An analogous trend has also been observed for propafenone analogues.³³ However, for compounds bearing piperidine or morpholine moieties, this trend is partly reversed. In the case of piperidine derivatives, the para-derivative is slightly more active than the meta analogue (1.20 vs 3.55 vs 2.18). Interestingly, also for the morpholine analogues, the para-derivative is by a factor of 2 more active than ortho-derivative (P = 0.01). Thus, the influence of the substitution pattern at the central aromatic ring seems to be more pronounced if the vicinity of the nitrogen comprises large, lipophilic moieties. This is in line with our previous findings using hydrophobic moments as descriptors in QSAR studies.³⁴

To assess the role of lipophilicity as a general predictor for high potency, we also calculated logP values using the software Bio-Loom version 1.5³⁵ and correlated them with pIC₅₀ values (Figure 2). Boi-Loom, which calculates logP values by a fragment-based approach, was validated against experimental logP values by Sakuratani et al.³⁶ The r² value of 0.56 indicates that also in the series of benzophenones biological activity increases with the lipophilicity of the compounds. This is in agreement with the notion that compounds most probably enter the binding cavity of P-gp directly from the membrane bilayer.¹⁸ This is additionally supported by the recent X-ray structure of mouse P-gp, which shows a large inner cavity accessible from the membrane via putative entry ports composed of transmembrane helices 4/6 on one side and 10/12 on the other side.¹⁹

Figure 2.

Correlation of P-gp inhibitory potency of compounds 6–24 (expressed as pIC₅₀ values) vs calculated logP values of the ligands.

The 4-hydroxy-4-phenyl-piperidine analogue 19 is located above the clogP/pIC₅₀ correlation line (pIC₅₀, 5.76 calcd vs 6.51 obs), which further confirms our previous results on the importance of the 4-hydroxy-4-phenyl-piperidine moiety for high biological activity of propafenone derivatives.³⁷ These results were recently supported by extensive docking studies of propafenone analogues.¹⁷ It is also interesting to note that the homodimer 23 is about one log unit more potent than predicted by the clogP/pIC₅₀ plot (pIC₅₀, 6.10 calcd vs 7.27 obs). A pairwise comparison of equilipophilic compounds 23 vs 21 (clogP, 4.27 vs 4.28; IC₅₀, 0.05 vs 0.48 μM) and 19 vs 20 (clogP, 3.65 vs 3.64; IC₅₀, 0.31 vs 1.21 μM) indicates that mutual activity differences might also be due to difference in molecular size. The dimer 23 (44 heavy atoms) is about 1 order of magnitude more active then 21 (35 heavy atoms). Similarly, 19 (32 heavy atoms) is about a factor of 4 more active than 20 (24 heavy atoms). This also points toward a commonly observed phenomenon in lead optimization programs, i.e., activity increases with the size of the molecules. Therefore, ligand efficiency (LE)²⁰⁻²² and lipophilic efficiency (LipE)^23,24 profiles of inhibitors/substrates of P-gp have been used to identify the derivatives with the best activity/size (or logP) ratio, which should provide further insights for the design of new ligands.^25,26

Ligand Efficiency (LE)

LE, most commonly defined as the ratio of free energy of binding to the number of heavy atoms, is a simple metric for assessing whether a ligand derives its potency from optimal fit with the target protein or simply by virtue of making many contacts.³⁸ To get more information on the most promising P-gp inhibitors and to compare them to well established P-gp inhibitors/substrates, we calculated ligand efficiency values of benzophenones 6–24, selected propafenone analogues (Figure 1), as well as P-gp inhibitors which entered clinical studies. Ligand efficiencies were calculated as described in the Materials and Methods section. For benzophenones, small ligands such as the N-propyl derivative 20 and the piperidine analogue 12 show higher efficiency values (0.35; 0.33) than the large dimers 23 and 24 (0.23; 0.18). For the whole data set, it can be observed that ligand efficiencies drop dramatically when the size of the ligands increases above 50 heavy atoms (Figure 3).

Figure 3.

Plot of ligand efficiency vs heavy atom count for benzophenone analogues, compounds which entered clinical studies and selected propafenones.

A similar trend has been observed in the literature, with LE showing generally a dependency on ligand size.²¹ As LE in principle is supposed to normalize for the size of the ligand, various proposals have been made to solve this problem.^22,39 As the heavy atom count of the ligands in our data set varies from 24 to 86 (20; valspodar), LE values were subsequently scaled as described by Reynolds et al.^21,22 to retrieve a size-independent ligand efficiency value (LE_Scale). This was achieved by fitting the top ligand efficiency versus heavy atom count to a simple exponential function, as outlined by Reynolds et al.,²¹ (eq 1; Figure 3). Subsequently, the ratio of ligand efficiency over normalized ligand efficiency scale gives a scoring function called “Fit Quality” (FQ) (eq 2). According to Reynolds et al., fit quality scores close to 1.0 or above indicate near optimal ligand binding, while low fit quality scores are indicative of suboptimal binding.

1

2

Use of this criterion shows that most of the compounds under clinical investigation show FQ scores above 1, including zosuquidar, ONT093, elacridar, and tariquidar, along with benzophenones 6 and 23, as well as propafenone and its analogues GPV062 and GPV576 (Figure 4; Table 2).

Figure 4.

Fit quality scores vs heavy atom counts of benzophenones 6–24, compounds which entered clinical studies, and selected propafenones. FQ score around 1 indicate a near optimal ligand binding affinity for a given number of heavy atoms.

Table 2. Pharmacological Activities, Ligand Efficiency (LE), and Lipophilic Efficiency (LipE) Profiles of Selected Propafenones and P-gp Inhibitors Which Entered in Clinical Studies.

compd	pIC50	HA	LE	clogP	LipE
Verapamil	6.24	33	0.27	4.47	1.77
Elacridar	7.14	42	0.24	4.21	2.93
Tariquidar	7.48	48	0.22	5.55	1.93
Zosuquidar	7.23	39	0.26	4.96	2.27
ONT093	7.50	37	0.29	7.30	0.19
Valspodar	6.30	86	0.10	15.09	–8.79
Cyclosporine A	6.99	85	0.12	14.36	–7.37
Niguldipine	6.15	45	0.20	7.80	–1.65
Propafenone	6.48	25	0.37	3.64	2.84
GPV576	8.25	35	0.33	6.02	2.23
GPV062	7.24	34	0.30	4.15	3.09
GPV005	6.22	27	0.33	4.38	1.84

It is interesting to note that especially those compounds which were specifically designed as P-gp inhibitors (ONT093, zosuquidar, elacridar, tariquidar) show higher FQ values than those originating from drug repurposing attempts (verapamil, cyclosporine, and its analogue valspodar). With respect to propafenone analogues, GPV576 is the hitherto most active analogue we synthesized showing a highly lipophilic but quite compact substituent at the nitrogen atom (4-tolylpiperazine). Interestingly, the top ranked benzophenone analogue 6 also has a 4-tolylpiperazine moiety. This might point toward the tolylpiperazine substituent for being a privileged substructure for P-gp inhibitors. GPV062 bears a 4-hydroxy-4-phenyl-piperidine moiety, which has been shown to influence biological activity independent of lipophilicity, resulting in an almost 10-fold increase of inhibitory potency when compared to compounds having other substituents at the nitrogen atom. This points toward a distinct additional interaction mediated by the 4-hydroxy group, most probably in the form of a hydrogen bond. Finally, propafenone itself shows a very good value, thus retrospectively demonstrating its validity as starting point for structural modifications. However, it should be noted that during its catalytic cycle the transporter undergoes a major conformational change and that it might well be that some inhibitors exert their effect by slow off kinetics rather than by strong binding to the apo state. In this case, LE values derived from IC₅₀ values for transport inhibition should be taken cautiously.

As already outlined, lipophilicity has been shown in numerous studies to be a general predictor for high P-gp inhibitory potency. This most probably is due to the proposed access path of the compounds, which seems to be directly from the membrane bilayer. On the other hand, high lipophilicity is very often associated with poor oral drug-like properties. This led to the assumption that clogP values between 2 and 3 are considered optimal in an oral drug program and prompted Leeson et al., to introduce the concept of lipophilic efficiency.²³

Liphophilic Efficiency (LipE)

LipE is a parameter that combines both potency and lipophilicity and is defined as a measure of how efficiently a ligand exploits its lipophilicity to bind to a given target. Briefly, in a lead optimization series, there is a greater likelihood of achieving good in vivo performance when potency can be increased without increasing logP or logD values. To explore this concept also for P-gp inhibitors, we calculated LipE values for the whole set of benzophenones as well as for the compounds used for the LE study (Table 2). The clogP values vary from 2.66 to 15.09, leading to a lipophilic efficiency range between −8.79 and +3.08. This is somewhat surprising, as it has been reported that a lipophilic efficiency greater than 5 combined with clogP values between 2 and 3 is considered optimal for a promising drug candidate.^23,24 None of the clinically tested P-gp inhibitors fulfils these requirements. Only the 4-hydroxy-4-phenyl-piperidine analogous propafenone GPV062 as well as the dimer 23 exhibit values slightly higher than 3. All other compounds show values lower than 3 (Figure 5). It is tempting to speculate whether this is due to the unique entrance pathway directly from the membrane bilayer, which requires a different logP profile than for compounds which access their binding site directly from the extracellular or intracellular aqueous compartment.

Figure 5.

Plot of clogP vs biological activity of inhibitors of P-gp; LipE values higher than 5 are considered to be the threshold for compounds of clinical interest; ● benzophenones, ■ propafenones, ▲ compounds which entered clinical trials.

To study in more detail whether the unique access path of P-gp inhibitors directly from the membrane bilayer is linked to this unexpectedly low LipE values, we studied the distribution of LipE profiles for a set of targets showing different access pathways of their ligands: P-glycoprotein (via the membrane bilayer), the serotonin transporter SERT (from the extracellular environment), and the hERG potassium channel (from the cytoplasm) (Figure 6). LipE values of inhibitors of SERT (extracted from the ChemBL database),⁴⁰ hERG blockers,⁴¹ and propafenone-type inhibitors of P-gp (in-house data) were calculated as described in the Materials and Methods section.

Figure 6.

Schematic representation of access of inhibitors/substrates to the binding sites of P-gp, SERT, and hERG along three different pathways. Ligands of P-gp approach the binding cavity via the membrane bilayer; in SERT the ligands get access from the extracellular environment, while in hERG this access occurs via the cytoplasm.

The LipE distribution profile of SERT inhibitors extracted from the ChemBL database identified about 13% of the compounds that cross the LipE threshold of 5 (Figure 7). These compounds cover a wide range of IC₅₀ (0.01 nM to 10 mM) and clogP (−3.42 to 4.66) (SM Figure 1). Moreover, 15 SERT inhibitors have been identified with clogP ∼ 2.5, LipE > 5, and IC₅₀ < 10 nM, none of them was listed as a marketed drug. In the case of hERG, only 2.5% of the compounds cross the LipE threshold of 5 that showed a potency distribution from 5 nM to 18 μM and clogP values between −0.77 and 2.21 (SM Figure 1). Only two compounds, almokalant and dofetilide, complied with the desired profile (clogP ∼ 2.5, LipE > 5, potency values <10 nM). Dofetilide is a registered class III antiarrhythmic agent, while almokalant is in phase II clinical investigations.^42,43

Figure 7.

LipE distribution profiles of ligands of P-gp, SERT, and the hERG potassium channel.

LipE profiles of P-gp inhibitors could not identify any compound that reaches the standard threshold value of 5. Most of the ligands fall in the LipE range of 1–2 (39%) or 2–3 (28%), with wide a range in distribution of their clogP (0.40 to 6.02) as well as IC₅₀ (5.60 nM to 1.20 mM) values (SM Figure 1). Thus, the LipE threshold for ligands of P-gp needs to be reconsidered. Nevertheless, from the benzophenone data set presented here, compounds 16, 19, 20, and 23 might be the most promising ones as their LipE values are between 2 and 3, a range where most of the compounds which in the past entered clinical trials are located.

Docking into a Homology Model of P-Glycoprotein

To get insights into the potential binding mode of propafenone-type benzophenones, we selected compounds 6, 19, and 20 and the dimer 23 for further in silico studies. Compounds 19, 20, and 23 were selected as they are ranked high both in LipE and FQ scores, and 6 was additionally included as it is top ranked with respect to FQ. Interestingly, this selection resembles the key features observed for propafenone analogues: compound 6 shows a 4-tolylpiperazine substituent (analogous to GPV576), compound 19 is analogous to GPV062 (4-hydroxy-4-phenyl-piperidine), and derivative 20 is the direct propafenone analogue (N-propyl). The docking protocol follows those previously published⁴⁴ and is provided in detail in the Materials and Methods section.

The analysis of the interaction pattern of selected docking poses indicates that the benzophenone scaffold interacts with F343 and F303 near the entry gate, whereas the lipophilic substituents in the vicinity of the basic nitrogen atom are surrounded by hydrophobic amino acid residues L724, I720, V981, I840, I836, and I765 located at TM 7, 8, 9, and 12 (Figure 8). This further supports the importance of high lipophilicity and also is in line with previous studies performed by Pajeva and Wiese, who showed that for a series of inhibitors of P-gp hydrophobicity represents a space directed molecular property rather than a simple overall descriptor.⁴⁵ The top ranked cluster of poses are in close vicinity of our previously purposed binding positions for benzopyrano[3,4-b][1,4]oxazines, where compounds having 4aS,10bR configuration interact mainly with amino acid residues of TM 4, 5, and 6 near the entry gate, while compounds having 4aR,10bS configuration are positioned deeper inside the binding cavity, being mainly surrounded by hydrophobic amino acid residues of TM 7, 8, 9, and 12.⁴⁴ Interestingly, the top scored dimer 23 is positioned in a way to bridge these two positions (Figure 8). Moreover, this pose might also aid in the explanation for the activity differences of homodimer 23 (0.05 μM) and heterodimer 22 (9.48 μM): The additional benzene ring in the best scored pose of homodimer 23 is surrounded by several hydrophobic amino acids (I836, L720, I840, and L724).

Figure 8.

Ligand–protein interaction profile of the best scored pose of benzophenone dimer 23. Blue circle represent the putative position of benzopyrano[3,4-b][1,4]oxazines having 4aS,10bR configuration, while the green circle indicates the position of diastereoisomers with 4aR,10bS configuration.

A representative docking pose of the 4-hydroxy-4-phenyl-piperidine derivative 19 showed an H-bond interaction between the 4-hydroxy group and A985 (Figure 9A). This further supports our SAR data and strengthens the importance of 4-hydroxy-4-phenyl-piperidine moieties for high inhibitory potency of propafenones and benzophenones. Furthermore, A985 was also identified as interacting with verapamil and the cyclic peptide (AQZ59-SSS) cocrystallized in mouse P-gp.¹⁹ A binding pocket of 4.5 Å around interacting amino acid residues of TM 7, 8, 9, and 12 showed two small hydrophobic cavities (encircled in Figure 9B), occupying the hydrophobic substituents at the basic nitrogen atom of the ligands. A closer look of the overlaid poses shows that the benzophenone substituent in dimer 23 fits well in the hydrophobic pockets, which might explain its high FQ score.

Figure 9.

(A) Ligand protein interaction pattern of clustered pose of 19. (B) Overlaid clustered poses (green, 23; blue, 19; yellow, 20; brown, 6). Two important hydrophobic binding cavities have been formed by amino acid residues of TM 7, 8, 9, and 12, indicated by two circles.

Overall, benzophenones shared a similar interaction profile as propafenones. Amino acids S952, F434, F336, L724, and Y307 have been identified as common interacting amino acid residues of all three classes of propafenone type inhibitors of P-gp (SM Figure 3). Selected benzophenone analogues have been previously used as photoaffinity ligands to characterize the drug-binding domain of propafenone-type analogues. In these studies, TM 3, 5, 6, 8, 10, 11, and 12 were identified as potential interacting helices.^30,46,47 This is well in line with our docking studies, which show main interactions with TM 5 and 6 near the entry gate and TM 7, 8, 9, and 12 deeper inside the cavity (SM Figure 4). No significant cluster of poses has been identified on the second wing (2/11 interface), which might be due to the asymmetry in the template used for building the homology model of P-gp, thus narrowing the available space at this side.

Conclusions

Calculation of ligand efficiency and lipophilic efficiency values for a set of P-gp inhibitors shows that ligands of P-gp exhibit LipE values below the threshold of 5 considered to be optimal for clinical candidates. This might be due to the unique entrance pathway of these classes of compounds, taking a route directly from the membrane bilayer. However, LipE and LE values of benzophenones 6, 19, and 20, as well as of the dimer 23, are close to compounds which entered clinical studies, thus qualifying them for further lead optimization cycles. Docking studies further strengthen the evidence provided by QSAR studies that the benzophenones bind to the same region as propafenone-type inhibitors. Moreover, the dimer 23 seems to bridge the two distinct binding sites recently proposed for benzopyrano[3,4-b][1,4]oxazines. This further supports the general assumption of a binding zone with distinct, but overlapping binding sites for individual scaffolds as a basis for the promiscuity of P-gp.

Experimental Section

Chemistry

Material and Methods

The data set used consists of a set of previously published benzophenones 9(37) and 12, 19, and 20(29) as well as a series of newly synthesized analogues. Melting points were determined on Leica Galen III (ser. no. 1413 WT) and are uncorrected. Purity of the compounds was checked by elemental analysis, and all values were within ±0.3%. Elemental analysis was performed at Microanalytical Laboratory of the Institute of Physical Chemistry (Mag. Johannes Theiner), University of Vienna. The equipment used was a 2400 CHN-Elemental Analyzer from Perkin-Elmer. Mass spectra were recorded on a Maldi-TOF, Kratos Instruments, matrix assisted laser-desorption-ionization time-of-flight, reflection mass spectrometer. NMR spectra were recorded on a Bruker spectrospin for 200 MHz ¹H NMR and 50 MHz for ¹³C NMR. CDCl₃ and DMSO at room temperature were used as internal standards. Column chromatographic separations were performed by using silica gel 60 (Particle size 40–63 μm, 230–300 mesh) from J. T. Baker or Merck. Thin layer chromatography (TLC) was performed on silica gel 60F254 TLC plates from Merck.

General Procedure for the Preparation of (2-Oxiranylmethoxy-phenyl)-phenyl-methanone C₁₆H₁₄O₃ (2a)

First, 10 g (51 mmol) of 2-hydroxy-benzophenone was dissolved in epichlorohydrine (120 mL), treated with 2.04 g (51 mmol) of sodium hydroxide, and refluxed for 24 h. Then, after cooling, the residue was filtered off and washed with diethyl ether. Subsequently, the solvent was removed by rotary evaporation. The remaining oil was taken up in diethyl ether and washed with water (175 mL). The organic phase was then dried over anhydrous sodium sulfate. After removal of the solvent by rotary evaporation, yellow oil was obtained, yield 12.81 g (98.85%). ¹H NMR (CDCl₃) δ 2.37–2.66 (m, 2H, CH₂-O-CH), 2.96–3.00 (m, 1H, CH), 3.92–4.15 (m, 2H, Ar-O-CH₂), 6.97–7.81 (m, 9H, arom H). ¹³C NMR (CDCl₃) δ 44.03 (CH-CH₂-O), 49.59 (CH), 68.62 (Ar-O-CH₂), 112.72, 121.13, 128.05 (arom C), 129.00 (Ar-CO), 129.38, 129.69, 131.97, 132.66 (arom C), 137.93 (Ph-CO), 156.08 (Ar-O), 196.15 (CO).

General Procedure for the Preparation of (3-Oxiranylmethoxy-phenyl)-phenyl-methanone C₁₆H₁₄O₃ (2b)

First, 5 g (25.25 mmol) of 3-hydroxy-benzophenone was dissolved in epichlorohydrine (60 mL), treated with 1.01 g (25.25 mmol) of sodium hydroxide, refluxed for 6 h, and stirred overnight. Then the residue was filtered off and washed with diethyl ether. After removal of the solvents under reduced pressure, the resulting oil was taken up in diethyl ether and washed with water several times. The organic layers were combined, dried over anhydrous sodium sulfate, and evaporated to dryness yielding yellow oil. For further purification a column chromatography (silica gel, ether/petrol ether, 70 + 30) was performed. Subsequent removal of the solvents under reduced pressure gave white opalescent oil, yield 6 g (93.6%). ¹H NMR (CDCl₃) δ 2.71–2.74 (m, 1H, H_A), 2.86 (t, 1H, J = 4.42, H_B), 3.29–3.37 (m, 1H, CH), 3.93 (dd, 1H, J = 5.94/11.12, H_X), 4.28 (dd, H, J = 2.90/10.98, H_Y), 7.10–7.78 (arom H). ¹³C NMR (CDCl₃) δ 44.34 (CH₂–O), 49.81 (CH), 68.76 (Ar-O-CH₂), 114.93, 119.17, 123.10, 128.11, 129.18, 129.81, 132.31, (arom C), 137.28, 138.70 (C), 158.24 (Ar-O), 196.08 (CO).

General Procedure for the Preparation of (4-Oxiranylmethoxy-phenyl)-phenyl-methanone C₁₆H₁₄O₃ (2c)

first, 6 g (30.30 mmol) of 4-hydroxy-benzophenone was dissolved in epichlorohydrine (50 mL), treated with 2 g (50 mmol) of sodium hydroxide, refluxed for 5 h, and stirred overnight. The residue was filtered off and washed with diethyl ether. Subsequently, the solvent was removed by rotary evaporation. The remaining opalescent oil was taken up in diethyl ether and washed with water several times. The organic phase was then dried over anhydrous sodium sulfate. After removal of the solvents by rotary evaporation, a white solid was obtained, yield 6.7 g (87.54%). ¹H NMR (CDCl₃) δ 2.76 (dd, 1H, J = 2.66/4.8, H_A), 2.92 (t, 1H, J = 4.64, H_B), 3.35–3.40 (m, 1H, CH), 3.96–4.0 (dd, 1H, J = 5.81–11.12, H_X), 4.29–4.36 (dd, 1H, J = 2.91/11.11, H_Y), 6.95–7.00 (m, 2H, H-3, H-5), 7.42–7.56 (m, 5H, H-2, H-6, H-3′, H-4′, H-5′), 7.72–7.83 (m, 2H, H-2′, H-6′). ¹³C NMR (CDCl₃) δ 44.51 (CH₂-O), 49.84 (CH), 68.82 (Ar-O-CH₂), 114.07, 128.14, 129.65, 131.90, 132.46, (arom C), 130.54 (Ar-CO), 138.06 (Ph-CO), 161.93 (Ar-O), 195.41 (CO).

General Procedure for the Preparation of [2-(2-Hydroxy-3-piperazine-1-yl-propoxy)-phenyl]-phenyl-methanone (5)

First, 1.82 g (7.20 mmol) of (2-oxiranylmethoxy-phenyl)-phenyl-methanone (2a) was dissolved in 20–30 mL of methanol, 1.6 g (18.6 mmol) of piperazine was added, and then the reaction mixture was refluxed for 5 h. After removal of the solvent by rotary evaporation, a column chromatography was performed (silica gel, CH₂Cl₂/methanol/concentrated NH₃, 100/10/1) subsequent evaporation to dryness yielded 1.88 g (77.33%) of yellow oil which was solidified on cooling. ¹H NMR (CDCl₃) δ 2.01–2.42 (m, 6H, CH₂-N-(CH₂)₂), 2.77–2.81 (m, 4H, (CH₂)₂-N), 3.68–3.75 (m, 1H, CH), 3.90–3.94 (m, 2H, O–CH₂), 6.96–7.79 (m, 9H, arom H). ¹³C NMR (CDCl₃) δ 45.86 (N-(CH₂)₂), 54.30 ((CH₂)₂-NH), 60.79 (CH₂-N), 65.27 (CH), 70.76 (O-CH₂), 112.56, 121.05, 128.28, 129.51, 130.07, 132.33, 132.83 (arom C), 128.79 (Ar-CO), 138.36 (Ph-CO), 156.55 (arom C-O), 196.56 (CO).

(2-{3-[4-(2,3-Xylyl)-piperazine-1-yl]-2-hydroxy-propoxy}-phenyl)-phenyl-methanone (6)

First, 700 mg (2.75 mmol) of (2-oxiranylmethoxy-phenyl)-phenyl-methanone (2a) was dissolved in 15 mL of methanol and treated with 526 mg (2.75 mmol) of 1-(2,3-xylyl)-piperazine. The mixture was refluxed for 24 h. Subsequent removal of the solvent yielded yellow oil, crystallization from ethyl acetate/diethylether gave 904 mg (73.8%) white crystals; mp 116–118 °C. ¹H NMR (CDCl₃) δ 2.19–2.27 (m, 2H, CH₂), 2.21 (s, 3H, CH₃), 2.27 (s, 3H, CH₃), 2.42–2.47 (m, 2H, CH₂), 2.58–2.66 (m, 2H, CH₂), 2.83–2.88 (m, 4H, (CH₂)₂)-N-Xyl), 3.77–3.83 (m, 1H, CH), 3.91–4.02 (m, 2H, O-CH₂), 6.90 (dd, 2H, J = 2.90/7.83 Hz, arom H), 6.99–7.12 (m, 3H, arom H), 7.44–7.58 (m, 5H, arom H), 7.79–7.83 (m, 2H, arom H). ¹³C NMR (CDCl₃) δ 13.93 (CH₃-ortho), 20.61 (CH₃-meta), 51.97 (N-(CH₂)₂), 53.79 (CH₂-N), 60.36 ((CH₂)₂-N-Ph), 65.42 (CH), 70.75 (O-CH₂), 112.64, 116.56, 121.12, 125.01, 125.81, 128.33, 128.86, 129.58, 130.12, 132.37, 132.87 (arom C), 137.99 (Ar-CO), 138.43 (Ph-CO), 151.29 (Ar-N), 156.58 (Ar-O). MS m/e 556.25 (M⁺, 100%). Anal. Calcd for C₂₈H₃₂N₂O₃: C, 74.89; H, 7.29; N, 6.24. Found: C, 74.73; H, 7.59; N, 6.32.

(3-{3-[4-(2,3-Xylyl)-piperazine-1-yl]-2-hydroxy-propoxy}-phenyl)-phenyl-methanone (7)

First, 560 mg (2.20 mmol) of 3-oxiranylmethoxy-phenyl)-phenyl-methanone (2b) was dissolved in 20 mL of methanol and treated with 418 mg (2.20 mmol) of 1-(2,3-xylyl)-piperazine. Then the mixture was refluxed for 6 h. After removal of the solvent under reduced pressure, a yellow oil was obtained which was crystallized from ethyl acetate/diethyl ether, yielding 960 mg (98%) white crystals; mp 92–99 °C. ¹H NMR (CDCl₃) δ 2.23, 2.27 (2s, 6H, 2CH₃), 2.62–2.66 (m, 4H, N-(CH₂)₂), 2.83–2.93 (m, 6H, CH₂-N, (CH₂)₂-N-Ph), 4.08–4.18 (m, 3H, O-CH₂-CH), 6.92 (d, 2H, J = 7.07, H-4, H-6), 7.05–7.20 (m, 2H, arom H), 7.37–7.79 (m, 6H, arom H), 7.80–7.83 (m, 2H, arom H). ¹³C NMR (CDCl₃) δ 13.92 (CH₃-ortho), 20.60 (CH₃-meta), 52.15 (N-(CH₂)₂), 53.77 ((CH₂)₂-N), 60.38 (CH₂-N), 65.38(CH), 70.49(O-CH₂), 115.07, 116.58, 119.30, 123.09, 125.03, 125.81, 128.24, 129.26, 130.00 (arom C), 131.19 (C), 132.42 (arom C), 137.52, 137.97, 138.86, 151.31 (C), 158.70 (Ar-O), 196.41(CO). MS m/e 444.68 (M, 90%). Anal. Calcd for C₂₈H₃₂N₂O₃·0.3H₂O: C, 74.64; H, 7.31; N, 6.22. Found: C, 74.76; H, 7.56; N, 6.08.

(4-{3-[4-(2,3-Xylyl)-piperazine-1-yl]-2-hydroxy-propoxy}-phenyl)-phenyl-methanone (8)

First, 700 mg (2.76 mmol) of (4-oxiranylmethoxy-phenyl)-phenyl-methanone (2c) was dissolved in 15 mL of methanol, treated with 523.6 mg (2.756 mmol) of 1-(2,3 xylyl)-piperazine, and the mixture was refluxed for 5 h. Then, removal of the solvent under reduced pressure yielded white crystals which were recrystallized from methanol giving 1.06 g (94.62%) white crystals; mp 122–126 °C. ¹H NMR (CDCl₃) δ 2.23–2.27 (2s, 6H, 2CH₃), 2.55–2.70 (m, 4H, (CH₂)₂-N-Ph), 2.70–3.00 (m, 6H, CH₂-N-(CH₂)₂), 3.40–3.70 (bs, 1H, OH), 4.10–4.17 (m, 3H, O-CH₂-CH), 6.90–7.03 (m, 5H, arom H), 7.44–7.51 (m, 3H, arom H), 7.74–7.81 (m, 4H, arom H). ¹³C NMR (CDCl₃) δ 13.90 (CH₃-ortho), 20.58 (CH₃-meta), 52.15, 53.76 (CH₂-N-(CH₂)₂), 60.38 ((CH₂)₂-N), 65.31 (CH), 70.44 (O-CH₂), 114.08, 116.57, 125.05, 125.81, 128.15, 129.69 (arom C), 130.37, 131.88 (C), 131.88, 132.50 (arom C), 137.97, 138.18 (C), 151.29 (Ar-N), 162.34 (Ar-O), 195.50 (CO). MS m/e 445.4 (M⁺, 100%). Anal. Calcd for C₂₈H₃₂N₂O₃: C, 75.65; H, 7.26; N, 6.30. Found: C, 75.61; H, 7.48; N, 6.33.

(3-{3-[4-(4-Fluoro-phenyl)-piperazine-1-yl]-2hydroxy-propoxy}-phenyl)-phenyl-methanone (10)

First, 500 mg (1.97 mmol) of (3-oxiranylmethoxy-phenyl)-phenyl-methanone (2b) was dissolved in 15 mL of methanol, treated with 360 mg (2 mmol) of p-F-phenyl-piperazine, and the mixture was refluxed for 6 h. Then the solution was allowed to cool down and stirred at room temperature overnight. The obtained solid was filtered off and washed with diethyl ether, giving 690 mg (80.76%) white crystals; mp 131–133 °C. ¹H NMR (CDCl₃) δ 2.60–2.85 (m, 6H, CH₂-N-(CH₂)₂), 3.12–3.17 (m, 4H, (CH₂)₂-N-Ph), 4.05–4.18 (m, 3H, O-CH₂-CH-OH), 6.87–6.97 (m, 4H, arom H), 7.19–7.25 (m, 1H, arom H), 7.37–7.60 (m, 6H, arom H), 7.78–7.82 (m, 2H, arom H). ¹³C NMR (CDCl₃) δ 50.18, 53.28, 60.30 (CH₂-N-(CH₂)₄), 65.46 (CH), 70.36 (O-CH₂), 115.04, 115.29, 115.73, 117.81, 117.97, 119.28, 123.15, 128.24, 129.99, 132.43 (arom C), 137.48 (Ar-CO), 138.87 (Ph-CO), 147.71 (Ar-N), 157.22 (J = 239 Hz, C-F), 158.64 (Ar-O), 196.39 (CO). MS m/e 434.77 (M, 100%). Anal. Calcd for C₂₆H₂₇FN₂O₃: C, 71.87; H, 6.26; N,6.45. Found: C, 71.65; H, 6.45; N, 6.46.

(4-{3-[4-(4-Fluoro-phenyl)-piperazine-1-yl]-2-hydroxy-propoxy}-phenyl)-phenyl-methanone (11)

First, 700 mg (2.75 mmol) of (4-oxiranylmethoxy-phenyl)-phenyl-methanone (2c) was dissolved in 15 mL of methanol, treated with 496 mg (2.75 mmol) of p-F-phenyl-piperazine, and refluxed for 5 h. Then removal of solvent under reduced pressure left a yellow oil which was crystallized from isopropyl alcohol, yielding 1 g (83.6%) white crystals; mp 96–100 °C. ¹H NMR (CDCl₃) δ 2.61–2.69 (m, 4H, N-(CH₂)₂), 2.80–2.91 (m, 2H, CH₂-N), 3.12–3.17 (m, 4H, (CH₂)₂-N-Ph), 3.56 (br, 1H, OH), 4.11–4.19 (m, 3H, CH₂-CH), 6.89–7.85 (m, 13H, arom H). ¹³C NMR (CDCl₃) δ 50.24, 53.28, 60.28 (CH₂-N-(CH₂)₄), 65.40 (CH), 70.33 (O-CH₂), 114.07, 115.31, 115.75, 117.81, 117.96, 128.17, 129.71, (arom C), 130.42 (Ar-CO), 131.92, 132.52 (arom C), 138.15 (Ph-CO), 147.77 (Ar-N), 162.28 (Ar-O), 195.50 (CO). MS m/e 435.3 (M⁺, 100%). Anal. Calcd for C₂₆H₂₇FN₂O₃: C, 71.87; H, 6.26; N, 6.45. Found: C, 71.61; H, 6.43; N, 6.41.

[3-(2-Hydroxy-3-piperidine-1-yl-propoxy)-phenyl]-phenyl-methanone (13)

First, 700 mg (2.75 mmol) of (3-oxiranylmethoxy-phenyl)-phenyl-methanone (2b) was dissolved in 15 mL of methanol and treated with 234 mg (2.75 mmol) of piperidine. Then the mixture was refluxed for 4 h and stirred at room temperature overnight. After removal of the solvent under reduced pressure, 336 mg (36%) yellow oil was obtained; mp 64 °C. ¹H NMR (CDCl₃) δ 1.45–1.59 (m, 6H, CH₂), 2.37–2.50 (m, 4H, N-(CH₂)₂), 2.56–2.62 (m, 2H, CH₂-N), 4.02–4.13 (m, 3H, O-CH₂-CH), 7.13–7.81 (m, 9H, arom H). ¹³C NMR (CDCl₃) δ 24.17, 26.05 (CH₂), 54.67 (N-(CH₂)₂), 60.89 (CH₂-N), 65.18 (CH), 70.64 (O-CH₂), 115.11, 119.28, 122.99, 128.23, 129.22 130.00, 132.39 (arom C), 137.54, 138.82 (C), 158.75 (Ar-O), 196.44 (CO). MS m/e 340.43 (M⁺, 100%). Anal. Calcd for C₂₁H₂₅NO₃: C, 73.53; H, 7.46; N, 4.08. Found: C, 73.51; H, 7.71; N, 4.11.

[4-(2-Hydroxy-3-piperidine-1-yl-propoxy)-phenyl]-phenyl-methanone (14)

First, 700 mg (2.75 mmol) of (4-oxiranylmethoxy-phenyl)-phenyl-methanone (2c) was dissolved in 10 mL of piperidine and refluxed for 2.5 h. Then after removal of piperidine under reduced pressure, a yellow oil was obtained. For further purification, a column chromatography was performed (silica gel, CH₂Cl₂/methanol/concentrated NH₃, 95/5/1 after removal of piperidine the percentage of methanol was increased to 80/20/1). Solvents were removed by rotary evaporation to yield light-yellow oil, which crystallized from ethyl acetate. Further recrystallization from methanol/diethyl ether yielded 400 mg (42.8%) white crystals; mp 106 °C. ¹H NMR (CDCl₃) δ 1.49–1.72 (m, 6H, CH₂), 2.58–2.73 (m, 6H, CH₂-N-(CH₂)₂), 4.05–4.08 (m, 2H, O-CH₂), 4.20–4.29 (m, 1H, CH), 4.32 (s, 1H, OH), 6.97 (d, 2H, J = 8.85, H-3, H-5), 7.45–7.82 (m, 7H, arom H). ¹³C NMR (CDCl₃) δ 23.65, 25.33 (CH₂), 54.78 (N-(CH₂)₂), 61.21 (CH₂-N), 64.89 (CH), 70.64 (O-CH₂), 114.06, 128.15, 129.67 (arom C_r), 130.38 (Ar-O), 131.89, 132.48 (arom C), 138.13 (Ph-CO), 162.21 (Ar-O), 195.49 (CO). MS m/e 340.3 (M⁺, 100%). Anal. Calcd for C₂₁H₂₅NO₃: C, 71.77; H, 7.55; N, 3.99. Found: C, 71.68; H, 7.51; N, 4.02.

[2-(2-Hydroxy-3-morpholine-4-yl-propoxy)-phenyl]-phenyl-methanone (15)

First, 1 g (4.33 mmol) of (2-oxiranylmethoxy-phenyl)-phenyl-methanone (2a) was dissolved in 20 mL of methanol and treated with 500 mg (5.75 mmol) of morpholine. Then the mixture was refluxed for 4 h. Removal of the solvents gives yellow oil, which was purified via column chromatography (silica gel, CH₂Cl₂/methanol/concentrated NH₃, 200/10/1). Subsequent removal of the solvents under reduced pressure yielded 400 mg (27%) of light-yellow oil; mp 185–193 °C. ¹H NMR (CDCl₃) δ 1.97–2.46 (m, 6H, CH₂-N-(CH₂)₂), 3.73 (t, 2H, J = 4.67, O-CH₂), 3.71–3.80 (m, 4H, O-(CH₂)₂), 6.95–7.10 (m, 2H, arom H), 7.41–7.58 (m, 5H, arom H), 7.75–7.79 (m, 5H, arom H). ¹³C NMR (CDCl₃) δ 53.57, 60.68 (CH₂-N-(CH₂)₂), 62.24 (CH), 66.68 (O-(CH₂)₂), 70.60 (O-CH₃), 112.54, 121.07, 128.25 (arom C), 128.72 (Ar-CO), 129.48, 130.04, 132.31, 132.80 (arom C), 138.31 (Ph-CO), 156.45 (Ar-O), 196.41 (CO). MS m/e 341.50 (M, 20%). Anal. Calcd for C₂₀H₂₃NO₄: C, 68.55; H, 6.90; N, 4.00. Found: C, 68.30; H, 6.62; N, 4.29.

[4-(2-Hydroxy-3-morpholine-4-yl-propoxy)-phenyl]-phenyl-methanone (16)

First, 700 mg (2.75 mmol) of (4-oxiranylmethoxy-phenyl)-phenyl-methanone (2c) was dissolved in 15 mL of methanol, treated with 240 mg of morpholine, and refluxed for 7 h. Then subsequent removal of the solvent under reduced pressure left 910 mg (96.8%) of yellow oil; mp 135–141 °C. ¹H NMR (CDCl₃) δ 2.44–2.71 (m, 6H, CH₂-N-(CH₂)₂), 3.73 (t, 4H, J = 4.56, (CH₂)₂-O), 4.06–4.17 (m, 3H, O-CH₂), 6.98 (d, 2H, J = 8.82, H-3, H-5), 7.42–7.56 (m, 3H, arom H), 7.72–7.84 (m, 4H, arom H). ¹³C NMR (CDCl₃) δ 53.69, 60.83 (CH₂-N-(CH₂)₂), 65.18 (CH), 66.94 ((CH₂)₂-O), 70.28 (O-CH₂), 114.04, 128.15, 129.68 (arom C), 130.40 (Ar-CO), 131.90, 132.49 (arom C), 138.13 (Ph-CO), 162.24 (Ar-O), 195.48 (CO). MS m/e 342.30 (M⁺, 95%). Anal. Calcd for C₂₀H₂₃NO₄: C, 69.15; H, 6.87; N, 4.03. Found: C, 69.16; H, 7.07; N, 4.18.

{3-[2-Hydroxy-3-(4-o-tolyl-piperazine-1-yl)-propoxy]-phenyl}-phenyl-methanone (17)

First, 700 mg (2.75 mmol) of (3-oxiranylmethoxy-phenyl)-phenyl-methanone (2b) was dissolved in 15 mL of methanol and treated with 500 mg (2.84 mmol) of o-tolyl-piperazine, and the mixture was refluxed for 6 h. Then subsequent removal of the solvent yielded 1.07 g (90.29%) of yellow oil; mp 173–179 °C. ¹H NMR (CDCl₃) δ 2.31 (s, 3H, CH₃), 2.61–2.66 (m, 4H, N(CH₂)₂), 2.28–2.96 (m, 6H, CH₂-N, (CH₂)₂)-N-Phe), 4.05–4.18 (m, 3H, O-CH₂-CH), 6.99–7.05 (m, 2H, arom H), 7.37–7.60 (m, 6H, arom H), 7.79–7.83 (m, 2H, arom H). ¹³C NMR (CDCl₃) δ 17.82 (CH₃), 51.71 (N-(CH₂)₂), 53.74 (CH₂-N), 60.34 ((CH₂)₂-N-Ph), 65.37(CH), 70.46 (O-CH₂), 115.05, 118.92, 119.28, 123.07, 123.18, 126.53, 128.23, 129.23, 131.02, 132.40 (arom C), 132.40 (Ar-CO), 138.83 (Ph-CO), 151.23 (Ar–N), 158.68 (Ar-O), 196.40 (CO). MS m/e 430.52 (M, 20%). Anal. Calcd for C₂₇H₃₀N₂O₃: C, 73.78; H, 7.11; N, 6.37. Found. C, 73.71; H, 7.31; N, 6.24.

{4-[2-Hydroxy-3-(4-o-tolyl-piperazine-1-yl)-propoxy]-phenyl}-phenyl-methanone (18)

First, 700 mg (2.75 mmol) of (4-oxiranylmethoxy-phenyl)-phenyl-methanone (2c) was dissolved in 15 mL of methanol and treated with 500 mg (2.84 mmol) of o-tolyl-piperazine, and the mixture was refluxed for 5 h. Then subsequent removal of the solvent under reduced pressure yield 1.1 g (92.83%) of white crystals, which were recrystallized from methanol; mp 107–114 °C. ¹H NMR (CDCl₃) δ 2.31 (s, 3H, CH₃), 2.63–2.67 (m, 4H, N(CH₂)₂), 2.84–2.97 (m, 6H, CH₂-N, (CH₂)₂)-N-Ph), 4.10–4.22 (m, 3H, O-CH₂-CH), 6.99–7.03 (m, 4H, arom H), 7.15–7.21 (m, 2H, arom H), 7.48–7.58 (m, 3H, arom H), 7.74–7.86 (m, 4H, arom H). ¹³C NMR (CDCl₃) δ 17.82 (CH₃), 51.74 (N-(CH₂)₂), 53.74 (CH₂-N), 60.36 ((CH₂)₂-N-Ph), 65.29 (CH), 70.42 (O-CH₂), 114.09, 118.94, 123.24, 126.57, 128.17, 129.71, (arom C), 130.38 (Ar-CO), 131.06, 131.90, 132.52 (arom C), 138.18 (Ph-CO), 151.22 (Ar-N), 162.34 (Ar-O), 195.51 (CO); MS m/e 431.4 (M⁺, 100%). Anal. Calcd for C₂₇H₃₀N₂O₃: C, 75.32; H, 7.02; N, 6.51. Found: C, 75.11; H, 7.12; N, 6.39.

4-[3-(2-Benzoyl-phenoxy)-2-hydroxy-propyl]-piperazine-1-carboxylic Acid p-Tolylamide (21)

First, 186 mg (0.54 mmol) of [2-(2-hydroxy-3-piperazine-1-yl-propoxy)-phenyl]-phenyl-methanone (5) was dissolved in 15 mL of dichloromethane, and then a solution of 75 mg (0.57 mmol) of 4-methyl phenyl isocyanate in dichloromethane was added dropwise. The mixture was stirred at room temperature and monitored by TLC. After evaporation to dryness, a yellow oil was obtained which was purified by column chromatography (silica gel, CH₂Cl₂/methanol/concentrated NH₃, 100/10/1). Subsequent removal of the solvents under reduced pressure yielded 136 mg (52.56%) of a colorless oil. ¹H NMR (CDCl₃) δ 2.41–2.53 (m, 6H, CH₂-N-(CH₂)₂), 2.43 (s, 3H, CH₃), 3.13 (s, 1H, OH), 3.51–3.53 (m, 4H, (CH₂)₂-N), 3.86–3.95 (m, 1H, CH), 4.09 (d, 2H, J = 4.80, O-CH₂), 7.08–7.96 (m, 14H, arom H, NH). ¹³C NMR (CDCl₃) δ 20.57 (CH₃), 43.70 ((CH₂)₂-N-CO), 52.76 (N-(CH₂)₂), 59.97 (CH₂-N), 65.60 (CH), 70.60 (O-CH₂), 112.56, 120.32, 120.96, 128.20, 29.07, 129.42, 129.88, 132.27, 132.85 (arom C), 128.54 (Ar-CO), 132.30 (Ar-CH₃), 136.38 (NH-Ar), 138.08 (Ph-CO), 155.24 (N-CO-NH), 156.39 (arom C–O), 196.54 (CO). MS m/e 473.8 (M, 18%). Anal. Calcd for C₂₈H₃₁N₃O₄: C, 71.02; H, 6.60; N, 8.87. Found: C, 70.77; H, 6.89; N, 8.64.

4-[3-(2-Benzoyl-phenoxy)-2-hydroxy-propyl]-piperazine-1-carbothioic Acid p-Tolylamide (22)

First, 200 mg (0.58 mmol) of [2-(2-hydroxy-3-piperazine-1-yl-propoxy)-phenyl]-phenyl-methanone (5) was dissolved in 10 mL of dichloromethane, and then a solution of 91 mg (0.61 mmol) of 4-methyl phenyl isothiocyanate in dichloromethane was added dropwise. The mixture was stirred at room temperature and monitored by TLC. After evaporation to dryness, yellow oil was obtained, which was further purified by column chromatography (silica gel, CH₂Cl₂/methanol/concentrated NH₃, 100/10/1). Subsequent removal of the solvents under reduced pressure yielded 181 mg (62.92%) of a light-yellow oil. ¹H NMR (CDCl₃) δ 2.12–2.20 (m, 2H, CH₂-N), 2.33 (s, 3H, CH₃), 3.39–2.47 (m, 4H, N-(CH₂)₂), 3.74–3.79 (m, 5H, (CH₂)₂-N, CH), 3.96 (d, 2H, J = 4.9, O-CH₂), 6.96–7.80 (m, 14H, arom H, NH). ¹³C NMR (CDCl₃) δ 20.89 (CH₃), 49.02 (N-(CH₂)₂), 52.55 ((CH₂)₂-N), 59.78 (CH₂-N), 65.88 (CH), 70.74 (O-CH₂), 112.73, 121.15, 123.47, 125.45, 128.31, (arom C), 128.71 (Ar-CO), 129.53, 129.63, 130.12, 132.38, 132.93 (arom H), 135.11 (Ar-CH₃), 137.34 (NH-Ar), 138.25 (Ph-CO), 156.51 (arom CO), 183.26 (CS), 196.47 (CO). MS m/e 489.5 (M, 15%). Anal. Calcd for C₂₈H₃₁N₃O₃S: C, 68.69; H, 6.38; N, 8.58. Found: C, 68.60; H, 6.53; N, 8.47.

[2-(3-{Benzoyl-phenoxy)-2-hydroxyl-propyl]-piperazine-1-yl}-2-hydroxy-propoxy)-phenyl]-phenyl-methanone (23)

First, 356 mg (1.40 mmol) of (2-oxiranylmethoxy-phenyl)-phenyl-methanone (2a) was dissolved in 20–30 mL of methanol and then 50.8 mg (0.59 mmol) of piperazine was added and the reaction mixture was refluxed for 5 h. Then after removal of the solvent by rotary evaporation, a column chromatography was performed (silica gel, CH₂Cl₂/methanol/concentrated NH₃, 100/10/1). Subsequent evaporation to dryness gave yellow oil, which crystallized from isopropyl alcohol to leave 424 mg (51%) of white solid; mp 125–135 °C. ¹H NMR (CDCl₃) δ 1.98–2.42 (m, 12H, CH₂-N-(CH₂)₂-N-CH₂), 3.10 (s, 1H, OH), 3.70–3.76 (m, 2H, 2CH), 3.94–4.07 (m, 5H, O-CH₂, OH), 6.99–7.83 (m, 18H, arom H). ¹³C NMR (CDCl₃) δ 53.13 (N-(CH₂)₄-N), 60.06 (CH₂-NH), 65.47 (CH), 70.78 (O-CH₂), 112.62, 121.10, 128.28, 129.53, 130.09, 132.33, 132.82 (arom C), 128.81 (Ar-CO), 138.34 (Ph-CO), 156.55 (Ar-O), 196.46 (CO). MS m/e 594.7 (M, 100%). Anal. Calcd for C₃₆H₃₈N₂O₆: C, 70.57; H, 7.14; N, 4.22. Found: C, 70.91; H, 7.02; N, 4.26.

1-[2-(3-{4-[3-(2-Benzoyl-phenoxy)-2-hydroxyl-propyl]-piperazine-1-yl}-2-hydroxy-propoxy)-5-methyl-phenyl]-ethanone (24)

First, 500 mg (2.43 mmol) of 1-(5-methyl-2-oxiranylmethoxy-phenyl)-ethanone (4) was dissolved in 10 mL of methanol, treated with 857.1 mg (2.52 mmol) of [2-(2-hydroxy-3-piperazine-1-yl-propoxy)-phenyl]-phenyl-methanone (5), and refluxed for 5 h. Then after removal of solvent under reduced pressure, a column chromatography was performed (silica gel, CH₂Cl₂/methanol/concentrated NH₃, 120/10/1). Subsequent removal of the solvents under reduced pressure yielded 1.20 g (90.67%) of an orange oil: mp 89–93 °C. ¹H NMR (CDCl₃) δ 2.05–2.13 (m, 2H, CH₂-N), 2.29 (s, 3H, CH₃), 2.33–2.55 (m, 10H, N-(CH₂)₄-N-CH₂), 2.62 (s, 3H, CO-CH₃), 2.98–3.01 (m, 1H, OH), 3.69–4.10 (m, 6H, 2O-CH₂-CH), 6.83–7.80 (m, 12H, arom H). ¹³C NMR (CDCl₃) δ 20.22 (CH₃-Ar), 31.81 (CH₃-CO), 53.20 (N-(CH₂)₂), 60.03, 60.49 (N-CH₂), 65.43, 65.51 (CH), 70.75, 71.12 (O-CH₂), 128.03, 128.82 (Ar-CO), 130.25 (CH₃-Ar), 112.61, 112.79, 121.10, 128.27, 129.51, 130.08, 130.59, 132.32, 132.81, 134.12 (arom C), 138 (Ph-CO), 156.00, 156.55 (Ar-O), 196.46 (CO), 199.88 (Ar-CO-CH₃). MS m/e 594.7 (M+, 100%). Anal. Calcd for C₃₂H₃₈N₂O₆: C, 61.82; H, 6.65; N, 4.51. Found: C, 61.60; H, 6.91; N, 4.37.

Computational Studies

Ligand Efficiency (LE)

Ligand efficiency (LE = Δg) values of the data were calculated by normalizing binding free energy of a ligand for number of heavy atoms. Free energy calculation was carried out as described by Hopkins et al. (eq 3). According to Hopkins et al., IC₅₀ from percentage inhibition can be substituted for Kd (dissociation constant potency)⁴⁸ which was further confirmed by experimental results of Kuntz and co-workers.³⁸

3

Ligand efficiency calculations was done for a temperature of 310 K and given in kcal per heavy atom (eq 4).

4

A size independent fit quality score was obtained as described by Reynolds et al.²¹ by fitting the maximum LE over a large range of molecular size. All calculations regarding ligand efficiency were done by using Excel spreadsheet. IC₅₀ values of the propafenone type inhibitors (GPV576, GPV005, GPV062, and propafenone) were determined experimentally by a daunorubicin efflux essay.^37,49 Inhibition of rhodamine 123 efflux in the transfectant mouse lymphoma line L5178 VMDR1 C.06 were used to characterize the MDR-modulating activity values of verapamil, niguldipine, and cyclosporine A. IC₅₀ values of tariquidar,⁵⁰ elacridar,^51,52 valdapodar,⁵³ zosuquidar,^54,55 and ONT-093⁵⁶ were taken from literature (Table 2). IC₅₀ values for most of the compounds in clinical studies were reported by using rhodamine 123 efflux essays. We use these values, as there is a direct correlation between the IC₅₀ values from daunorubicin and rhodamine 123 efflux essays.⁵⁷

Lipophilic Efficiency (LipE)

LipE of benzophenones were calculated (eq 5) and compared with the compounds which reached clinical studies (verapamil, tariquidar, valspodar, elacridar, zosuquidar, ONT-093, niguldipine, and cyclosporine A) as well as with selected propafenone analogues.

5

clogP values of the data set were computed by using the Bio-Loom software package,³⁵ and the LipE calculations were performed by using Excel spreadsheet. To compare the standard threshold of LipE along three different entry pathways of ligands into respective binding pockets of P-gp, hERG and SERT, a data set from literature was used. It includes 744 SERT inhibitors extracted from the ChEMBL database,⁴⁰ 313 hERG blockers⁴¹ from literature, and 372 inhibitors of P-gp mediated daunorubicin efflux (in-house data). The data sets are available at our homepage (pharminfo.univie.ac.at) and from Chemspider (www.chemspider.com).

Docking

Compounds 6, 19, 20, and 23 were docked in their neutral form into an open state homology model of human P-gp¹⁷ based on the X-ray structure of mouse P-gp (PDB: 3G5U)¹⁹ by using the software package GOLD. To avoid any bias, we considered the whole transmembrane domain region as binding pocket. Then 100 poses per ligand were obtained, and finally ligand protein complexes were minimized by LigX, a minimization tool implemented in MOE, by using the MMFF94 force field.

A complete work flow of poses selection has been provided in Supporting Information (SM Workflow 1). Briefly, agglomerative Hierarchical Cluster analysis of the consensus rmsd matrix based on the common scaffold of the ligands identified two interesting clusters of poses containing all four ligands. However, additional five clusters have been identified containing three out of four ligands. All seven clusters were occupying the center of the binding cavity mainly interacting with amino acid residues of TM 1, 5, 6, 7, 8, 10, and 11 (SM Figure 2A). For a more detailed analysis of the ligand–protein interaction profiles of selected ligands, we used the two clusters containing all four ligands (SM Figure 2B).

To prioritize among the two clusters, a rescoring of all docking poses by using four different scoring functions in MOE (ASE, affinity dG, Alpha HB, London dG) was performed. Subsequently, for each ligand, the top 10 ranked poses according to consensus scoring were taken and analyzed. Out of these 40 poses, seven poses were present in cluster 1 while only one showed up in cluster 2. In addition, taking only the top ranked pose per ligand, two (6, 23) out of four ligands were located in cluster 1 (SM Figure 2B). Additionally, cluster 1 was supported by photo affinity labeling and SAR data of the series. Therefore interaction position of cluster 1 was supposed to be the most likely one for benzophenones.

Biological Assay

Cell Lines

The resistant CCRF vcr1000 cell line was maintained in RPMI 1640 medium containing 10% fetal calf serum (FCS) and 1000 ng/mL vincristine. The selecting agent was washed out 1 week before the experiments. This cell line was selected due to its distinct P-gp expression.

Inhibition of Daunorubicin Efflux

IC₅₀ values for daunorubicin efflux inhibition were determined as reported.³² Internal concentration (3 μM) of daunorubicin in the efflux inhibition assays was identified at least an order of magnitude below the K_m values published for P-gp.^59,60

Briefly, cells were sedimented, the supernatant was removed by aspiration, and the cells were resuspended at a density of 1 × 10⁶/mL in RPMI 1640 medium containing daunorubicin (Sigma Chemical Co., St. Louis, MO) at a final concentration of 3 μmol/L. Cell suspensions were incubated at 37 °C for 30 min. Tubes were chilled on ice and centrifuged at 500g in an Eppendorf 5403 centrifuge (Eppendorf, Hamburg, Germany). Supernatants were removed, and the cell pellet was resuspended in medium prewarmed to 37 °C containing either no inhibitor or compounds at various concentrations ranging from 20 to 200 μM, depending on the solubility and expected potency of the inhibitor. Eight concentrations (serial 1:3 dilution) were tested for each inhibitor. After 60, 120, 180, and 240 s, aliquots of the incubation mixture were transferred to tubes containing an equal volume of ice-cold stop solution (RPMI medium containing GPV31 at a final concentration of 5 μmol/L). Zero time points were determined by immediately pipetting daunorubicin-preloaded cells into ice-cold stop solution. Samples drawn at the respective time points were kept in an ice water bath and measured within 1 h on a Becton Dickinson FACS Calibur flow cytometer (Becton Dickinson, Vienna, Austria). Viable cells were selected by setting appropriate gates for forward and side scatter. The excitation and emission wavelengths were 482 and 558 nm, respectively. Five thousand gated events were accumulated for the determination of mean fluorescence values.

Glossary

Abbreviations Used

P-gp

P-glycoprotein

LipE

lipophilic efficiency

LE

ligand efficiency

MDR

multidrug resistance

ABC

ATP binding cassette

QSAR

quantitative structure–activity relationship

Supporting Information Available

LipE profiles of inhibitors of P-gp, SERT, and hERG; docking workflow; clustered docking poses; overlap of interacting amino acid residues of propafenone-type inhibitors of P-gp; photolabeled drug binding domains of propafenone-type inhibitors of P-gp. This material is available free of charge via the Internet at http://pubs.acs.org.

The authors declare no competing financial interest.

Accession Codes

PDB ID: 3G5U.