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European Journal of Medicinal Chemistry 46 (2011) 3909e3916

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Probing the difference between BH3 groove of Mcl-1 and Bcl-2 protein:Implications for dual inhibitors design

Zhichao Zhang a,*, Hongna Yang a,1, Guiye Wu a,1, Zhiqiang Li b, Ting Song b, Xiang qian Li a

a State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian 116012, People’s Republic of Chinab School of Life Science and Technology, Dalian University of Technology, Dalian 116024, People’s Republic of China

a r t i c l e i n f o

Article history:Received 20 April 2011Received in revised form22 May 2011Accepted 24 May 2011Available online 31 May 2011

Keywords:ApoptosisMcl-1Bcl-2p2 pocketDual inhibitor

* Corresponding author. Tel./fax: þ86 411 8498603E-mail address: zczhang@dlut.edu.cn (Z. Zhang).

1 These authors contributed equally to this work.

0223-5234/$ e see front matter � 2011 Elsevier Masdoi:10.1016/j.ejmech.2011.05.062

a b s t r a c t

Based on our previous discovery of a dual inhibitor of Mcl-1 and Bcl-2, 3-thiomorpholin-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (1, S1), and guided by structure insight of 1 complex with Mcl-1and Bcl-2, we exploited the spatial orientation of BH3 groove of the two proteins by a series of analoguesof 1. These analogues contain substitutes with various steric hindrance designed to explore the widthand length of the p2 pocket. The structureeactivity relationships (SARs) studies together with dockingstudies and cell-based assays proved that the p2 pocket of Mcl-1 is relatively wider and shorter than thatof Bcl-2. A novel dual inhibitor 6 was obtained based on these new findings that it exhibited nanomalaraffinities toward Mcl-1 and Bcl-2, as well as nanomalar cytotoxicity activity against multiple cancer celllines.

� 2011 Elsevier Masson SAS. All rights reserved.

1. Introduction

Aberrant over-expression of anti-apoptotic B-cell lymphoma 2(Bcl-2)-like proteins combats tumor cell resistance by programmedcell death [1,2]. Therefore, strategies that target the functional BH3binding groove of these Bcl-2-like members may provide a selec-tive mechanism for killing tumor cells. Since there are more thanone member in Bcl-2-like family, the most versatile cell killer mustbe a broad-spectrum “pan” Bcl-2 inhibitor, capable of targeting theBH3 groove of multiple Bcl-2 family proteins [3].

Myeloid cell leukemia sequence 1 (Mcl-1) is one of the Bcl-2-likeproteins which has recently been revealed to play a critical anddistinct role in the regulation of apoptosis [4]. Furthermore, it hasbeen well established that a “pan-Bcl-2 inhibitor” should bea compound that may not bind to all of the Bcl-2-like proteins butthat can bind to at least Mcl-1 and Bcl-2 to disarm the apoptosiscapacities of these key targets [5]. Unfortunately, a few of smallmolecules described to date are dual inhibitors that bind to Mcl-1and Bcl-2. The most authentic BH3 mimetic ABT737, forexample, is not a pan-Bcl-2 inhibitor [6,7]. However, a recentpublication reported that the structural features in the antagonists

2.

son SAS. All rights reserved.

that subtly control the orientation of binding could perhaps beexploited in the design of more potent Mcl-1 inhibitors [8].

Recently, we have reported a small molecule 3-thiomorpholin-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (1, S1) as a dualnanomolar inhibitor of Mcl-1 and Bcl-2 (Ki¼ 58 and 310 nM,respectively) (Fig. 1) [9]. Further structureeactivity relationshipstudies revealed that 1 lies in the BH3 groove of Mcl-1 and Bcl-2,with the thiomorpholine extended into the p2 binding pocket ofMcl-1 and Bcl-2 [10].

To guide the design of Mcl-1/Bcl-2 dual inhibitors, especiallypredict the occupation of their p2 pockets, we further probed thedifference between the p2 pocket of Mcl-1 and Bcl-2 in this study.Based on two series potent pan-active inhibitors of structurallyrelated compounds with different binding profiles against Mcl-1and Bcl-2, we not only established the molecular determinantsgoverning the specificity of ligand engaging into the p2 of Mcl-1and Bcl-2, but also got a potent compound 6, binding to Mcl-1and Bcl-2 proteins with Ki values of 24 and 158 nM, respectively,which provide some fundamental insights into the future designand development of Mcl-1 and Bcl-2 inhibitors.

2. Chemistry

The 8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile deriva-tives 2aec, 3aec and 6were synthesized by the procedure reported

Fig. 1. Structures of compounds 1, 2aec, 3aec and 4e6.

Z. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3909e39163910

in Scheme 1. Compound 8 (5-bromoacenaphthenequinone) wasreadily accessible via the reaction between acenaphthenequinoneand bromine under reflux condition [11]. Compounds 9aeg wereobtained in high yields (85e95%) by the reaction between 8 andcorresponding phenol with K2CO3 in DMF. The key step in thesynthesis of target structures is the Knoevenagel condensationreaction [12,13] between substituted acenaphthenequinones(9aeg) and malononitrile, using silica gel in room temperature togive monoadducts (10aeg) with yields of 40e60% (Scheme 1). Thetarget compounds 2aec, 3aec and 6 were prepared by a “one-pot”base-catalyzed cyclization reaction of 10aeg, respectively [14]. Allsteps were in good yields and under mild conditions.

3. Results and discussion

3.1. Rationale

Our previous studies identified 1 lies along a BH3 hydrophobicbinding pocket of Mcl-1 and Bcl-2. A hydrogen bonding networkcould be formed between carbonyl group of 1 and R263 of Mcl-1and R146 of Bcl-2, respectively [10]. The similar positioning ofR263 of Mcl-1 and R146 of Bcl-2 in their three-dimensional struc-ture allows 1 to bind in similar orientations within their BH3binding grooves. As such, thiomorpholine extends to the p2 pocketwhen 1 binds to the two proteins. Further, we revealed that occu-pation of the two protein’s p2 pocket is crucial for a Mcl-1/Bcl-2inhibitor.

Scheme 1. Synthesis of compounds 2aec, 3aec and 6. Reagents and conditions: (a) Br2, 60reflux.

Since accommodation in p2 pocket of both Mcl-1 and Bcl-2could determine whether a molecule is a dual inhibitor of Mcl-1/Bcl-2, we aimed to further probe the character of this pocket inthe two proteins. Because the X-ray evidence of the BH3 groove ofBcl-2 protein was published very recently [15], at the beginning ofthis study we had to compare Mcl-1 with Bcl-xL, whose three-dimensional architecture is quite similar with that of Bcl-2 [16a].Either Bcl-2 or Bcl-xL composes of eight a-helices with a hydro-phobic groove on the surface. The overall backbone RMSD (rootmean square deviation) between them is only about 1.85�A,excluding the loop between a1 and a2 [16b]. Research showed helixa3 is well formed in the Mcl-1 but poorly so in the Bcl-xL, whichresults in the protein backbones of the p1 and p2 pockets are lesscontiguous in the Mcl-1 than Bcl-xL (Fig. 2a) [17,18]. Additionally,the BH3 groove of Bcl-xL is narrow than that of Mcl-1 [18]. Whencrystallized Bcl-2/Bak complex was reported, we compared Mcl-1with Bcl-2 directly by using AutoDock tools (ADT). Result identi-fied the BH3 groove of Bcl-2 is narrow than that of Mcl-1 (Fig. 2b).

Here, we focused synthetic two series analogues of 1 speciallydesigned to probe the spatial orientation of the p2 pocket thatthiomorpholine of 1 was replaced with substituent of various bulkand steric hindrance.

3.2. Structureeactivity relationship

We substituted the thiomorpholine using oxygen atoms asa linker, which renders flexibility to compounds, allowing them toengage well into the p2 pocket. Specifically, we examined the effectof various steric hindrance by using the methyl at the ortho-,meta-,and para-position of phenyl, yielding analogues 3-(o-tolyloxy)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (2a), 3-(m-tol-yloxy)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (2b),3-(p-tolyloxy)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile(2c), respectively (Fig. 1). The binding affinities Ki of the compoundswere evaluated using fluorescence polarization assays (FPAs) thatmeasure their abilities to competitively displace a Bid-derivedpeptide from Mcl-1 and Bcl-2 as described in the biological assay.The competitive binding curves of these compounds to Mcl-1 andBcl-2 are outlined in Fig. 3a and b, respectively. The para-methylsubstituted compound 2c exhibited potent inhibition against both

�C; (b) RePhOH, K2CO3, DMF, 60 �C; (c) CH2(CN)2, C2HCl2, silica gel; (d) K2CO3, CH3CN,

Fig. 2. Overlay of the crystal structures of (a) Mcl-1:Bcl-xL and (b) Mcl-1:Bcl-2 by using AutoDock Tools (ADT). Mcl-1, Bcl-xL, and Bcl-2 are shown in white, pink and blue,respectively. The significant difference in a3 of Mcl-1 and Bcl-xL is circled and the p1 and p2 pockets are labeled in (a). The dotted line in (b) represents the width of BH3 groove(vertical to the axis of the BH3 groove) formed largely by the a3, a4, and a5 helices (For interpretation of the references to color in this figure legend, the reader is referred to theweb version of this article.)

Z. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3909e3916 3911

Mcl-1 and Bcl-2 (174 nM and 445 nM, respectively). Interestingly,when shifting the methyl group from para-position to ortho/meta-position, different specificities for Mcl-1 and Bcl-2 were found. ForMcl-1, the Ki of ortho-substituted 2a (213 nM) is similar with that of2c, and nearly a 3-fold decrease was found for meta-substituted 2b(445 nM). A much more significant decrease of inhibition wasfound for Bcl-2. Compound 2a (1.43 mM) lost nanomolar affinity,while 2b (6.03 mM) even showed a remarkable 12-fold decrease.The data above indicated that the position of the methyl group iscrucial for occupying Bcl-2 over Mcl-1. This supported the differ-ence between BH3 groove of Mcl-1 and Bcl-2, especially in the p2pocket.

For further test the difference between p2 pocket of Mcl-1 andBcl-2, we replaced the methyl with a larger methoxy group at theortho-, meta- and para-position of phenyl, yielding another seriescompounds, 3-(2-methoxyphenoxy)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (3a), 3-(3-methoxyphenoxy)-8-oxo-8H-ace-naphtho[1,2-b]pyrrole-9-carbonitrile (3b), 3-(4-methoxyphenoxy)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (3c), respec-tively (Fig. 1). Excitingly, the same trend was found for these threecompounds that the position of the methyl group is crucial foroccupying Bcl-2 over Mcl-1. For Mcl-1, the Ki value of para-substituted 3c was 122 nM, which is similar with ortho-substituted3a (149 nM) and a little enhanced than meta-substituted 3b(375 nM). For Bcl-2, an about 3-fold lower affinity was found for 3a(886 nM, Fig. 3b) than 3c (304 nM), while a 50 times decrease of 3b(16.6 mM) revealed most of the affinities have been lost. These data

Fig. 3. Competitive binding curves of designed small-molecule inhibitor

suggested that the position change of the larger methoxy groupleads to more remarkable influence on affinity toward Bcl-2 thanthat of methyl. When the para-methoxy substituted phenyl moietycan be accommodated by both Mcl-1 and Bcl-2, the meta-methoxysubstituted phenyl can only be accepted by Mcl-1 and almostcompletely rejected by Bcl-2. This strongly supported that the p2pocket of Bcl-2 is narrow than that of Mcl-1.

To further investigate the effect of position of methyl/methoxyphenol moiety, we analyzed the predicted structures of the meta-methoxy substituted 3b and para-methoxy substituted 3c incomplex with human Mcl-1 and Bcl-2 proteins respectively, withthe help of computational modeling studies by using AutoDock 4.0.As shown in Fig. 4a and c, compounds 3b and 3c lie alonga hydrophobic binding pocket of Mcl-1 similar with the dockingresult of 1 [10]. It suggested whether methoxy group is attachedpara or meta-position, it could be accommodated by the p2 pocketof Mcl-1. By contrast, the p2 pocket of Bcl-2 could not endure themeta-methoxy substituted phenyl group of 3b so well as the para-methoxy substituted phenyl group of 3c (Fig. 4b and d). This furtheridentified the width (vertical to the long axis of BH3 groove) of thep2 pocket of Bcl-2 is narrow than that of Mcl-1. As such, the groupat meta-position is fatal for Bcl-2, which suggested that the para-substituent of phenyl was optimal for occupying the p2 of bothMcl-1 and Bcl-2.

Subsequently, we calculated the width of the substituted phenylgroup of different compounds by ChemBioDraw and AutoDocktools at the minimize energy of the structures. As shown in Table 1,

s 2aec and 3aec to (a) Mcl-l and (b) Bcl-2 as determined by FPAs.

Fig. 4. Predicted binding models of 3c in complex with (a) Mcl-1, (b) Bcl-2, and 3b in complex with (c) Mcl-1, (d) Bcl-2. Mcl-1 and Bcl-2 are shown in a surface representation, andthe carbon, oxygen, nitrogen, and sulfur atoms are shown in gray, red, blue, and yellow, respectively. The p1 and p2 pockets are labeled (For interpretation of the references to colorin this figure legend, the reader is referred to the web version of this article.)

Z. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3909e39163912

compounds 2c and 3c, which are most potent toward both Mcl-1and Bcl-2, exhibit 4.32�A in width. By comparison, 3b, whosewidth is 7.16�A, was completely excluded by the p2 pocket of Bcl-2,while it still can be accommodated by that of Mcl-1.

So far, we identified that the p2 pocket of Bcl-2 is indeed narrowthan that of Mcl-1. Surprisingly, it seems this result is not consistentwith the result of our previous study. In that study we found themolecule containing a t-amyl group at the para-position of phenylcould enter into the p2 of Bcl-2 but failed to enter into that of Mcl-1,which suggested that the p2 of Mcl-1 is more challenging than thatof Bcl-2. We hypothesized this is largely due to the discontinuous ofthe p1 and p2 pocket inMcl-1. In another word, there is a hindrancebetween the p1 and p2 pocket in Mcl-1, which is a challenge to thelength of an inhibitor (along the long axis of BH3 groove). Next,exploration of the proper length of group at the para-position ofphenyl was carried out, which may engage well in the p2 pocket ofBcl-2 without adversely affecting the occupation that of Mcl-1. Weused to identify that compound 4 containing isopropyl group at thepara-position of phenyl group is a dual nanomolar inhibitor of Mcl-1 and Bcl-2 (Ki¼ 99 nM and 232 nM, respectively). However,a more significant decrease of inhibitionwas found for compound 5

Table 1Thewidth of the substituted phenyl group of different compounds was calculated byChemBioDraw and AutoDock tools.

Compounds 2a 2b 2c 3a 3b 3cWidth/�A 5.273 5.903 4.320 6.230 7.159 4.317

(Ki¼ 1281 nM) toward Mcl-1, in which the isopropyl group wasreplaced with a longer phenyl group. Here, we tested whether 5could still occupy the p2 pocket of Bcl-2. FPA showed 5 exhibiteda potent affinity toward Bcl-2 with a Ki of 308 nM (Fig. 5b). Thisconfirmed our hypothesis that in the orientation along the long axisof BH3 groove Mcl-1 is more constricted than Bcl-2.

Consequently, we aimed to design a more potent dual inhibitorbased on our observation. We supposed that replacing phenylgroup of 5 with a shorter sec-butyl may occupy the p2 pocket ofboth Mcl-1 and Bcl-2 and then enhance the affinity to them.Therefore, we synthesized 3-(4-sec-butylphenoxy)-8-oxo-8H-ace-naphtho[1,2-b]pyrrole-9-carbonitrile (6) (Fig. 1). As expected, wedetected a significant improved affinity for 6 toward Mcl-1 and Bcl-2 (Ki¼ 24 nM and 158 nM, respectively, Fig. 5a). About 2e3 timesenhanced affinity than its parent 1 (58 and 310 nM toward Mcl-1and Bcl-2, respectively) was found. These data supported ourhypothesis that a proper spatial bulk, including both width andlength, is a determination for a molecule to occupy the p2 pocket ofboth Mcl-1 and Bcl-2.

Furthermore, we assessed the cytotoxicity of compounds 3b, 3cand 6 against MCF-7, K562 and HL-60 cell lines by using MTTmethod. The results are shown in Fig. 6. When 3b and 3c showedcomparable mediated cytotoxicity on MCF-7 and K562 cells, muchhigher cytotoxicity was found for 6. Of note, on HL-60 cells whoseBcl-2 level is much higher than that in MCF-7 and K562 cells, 3bexhibited much weaker killing ability than compound 3c. Thiscould be explained that 3b could not overcome the protection ofhigh level of Bcl-2 protein due to its poor affinity toward Bcl-2

-2 -1 0 1 2 3 4

0

20

40

60

80

100

a

0

20

40

60

80

100 b

-2 -1 0 1 2 3 4

inhibitor (Log[µM]) inhibitor (Log[µM])

Fig. 5. Competitive binding curves of designed compounds 4e6 to (a) Mcl-l and (b) Bcl-2 as determined by FPAs.

Fig. 6. Cytotoxicity evaluation of compounds 3b, 3c and 6 against tumor cell lines HL-60, K562, and MCF-7 (a). Histograms represent the IC50 values (mM, means� SD oftriplicate analyses). The levels of Mcl-1 and Bcl-2 expressionwere analyzed byWesternblot in the three cell lines (b). The levels of b-actin were determined to assure equalloading.

Z. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3909e3916 3913

protein. By contrast, over-expression of either Mcl-1 or Bcl-2 couldnot protect cells from compound 6. These data were in agreementwith our discovery in FP assay and further identify the strategyrepresented in this study for design of dual inhibitor.

4. Conclusions

In summary, we substituted the 3-position of compound 1 tofurther probe the spatial orientation of the p2 pocket of Mcl-1 andBcl-2. SAR studies together with docking studies revealed on theone hand that the p2 pocket of Bcl-2 could not enduremoiety at theortho or meta-position so well as the para-position, which toa much extent was different from Mcl-1. On the other hand,although the para-substituted could be accommodated well bybothMcl-1 and Bcl-2, the p2 pocket of Mcl-1 cannot endure relativelonger group than Bcl-2. It suggested that the p2 pocket of Mcl-1prefers wide and short moieties while that of Bcl-2 is apt tonarrow and long group.

With this regard, we synthesized compound 6 containing a sec-butyl group at para-position of phenyl. FP-based studies and cell-based studies identified it is a dual inhibitor with much improvedactivities than compound 1.

Taken together, we have successfully discovered a new potentand dual, nanomolar small-molecule inhibitor of Mcl-1 and Bcl-2based on the structural insight into the difference between the p2pocket of Mcl-1 and Bcl-2.

5. Experimental protocols

5.1. Chemistry

5.1.1. Materials and methodsAll commercial reagents were purchased and used without

further purification or distillation unless otherwise stated. Meltingpoints were measured on a Griffin apparatus and are uncorrected.NMR (1H, 13C) was obtained with Bruker AV-400 spectrometer withchemical shifts reported as parts per million (in CDCl3, TMS asinternal standard). The following abbreviations are used formultiplicity of NMR signals: s¼ singlet, d¼ doublet, t¼ triplet,q¼ quartet, m¼multiplet. High-resolution mass spectra (HRMS)were obtained on HPLC-Q-Tof MS (Micro) spectrometer. Columnchromatographywas performed on silica gel 200e300mesh. Purityof all final products was determined by analytical HPLC to be �95%.HPLC purity of compounds was measured with a reverse phaseHPLC (XBridge C18, 4.6�150 mm, 5 mm) with two diverse wave-length detection systems.

5.1.2. General procedure for the preparation of 9aegA mixture of 5-bromoacenaphthenequinone (8) (260 mg,

1.0 mmol), corresponding phenol (1.2 mmol) and K2CO3 (165 mg,1.2 mmol) was stirred in DMF (10 mL) for 2 h at 50e60 �C. Thesolution was poured into a saturated NaCl aqueous solution(20 mL), followed by extraction with ethyl acetate (3� 20 mL). Theorganic layer was collected and dried with dry MgSO4. Afterevaporation under reduced pressure, the product was purified bycolumn chromatography on silica gel using dichloromethane/petroleum ether from 1:1 to 2:1.

5.1.2.1. 5-(p-Tolyloxy)acenaphthenequinone (9c). Yield: 0.270 g,94%. 1H NMR (400 MHz, CDCl3): d 8.48 (d, J¼ 8.4 Hz, 1H), 8.33 (d,J¼ 8.4 Hz, 1H), 8.18 (d, J¼ 7.6 Hz, 1H), 7.86 (t, J¼ 7.6 Hz, 1H), 7.45 (d,J¼ 8.4 Hz, 2H), 7.25 (d, J¼ 8.4 Hz, 2H), 7.08 (d, J¼ 7.6 Hz, 1H), 2.43(s, 3H). TOF MS (EIþ): C19H12O3, calcd 288.0786, found 288.0780.

5.1.2.2. 5-(4-Methoxyphenoxy)acenaphthenequinone (9f). Yield:0.276 mg, 91%. 1H NMR (400 MHz, CDCl3): d 8.44 (d, J¼ 8.4 Hz, 1H),8.35 (d, J¼ 9.2 Hz, 2H), 8.18 (d, J¼ 8.0 Hz, 1H), 8.11 (d, J¼ 8.0 Hz,1H), 7.89 (t, J¼ 8.0 Hz, 1H), 7.65 (t, J¼ 9.2 Hz, 1H), 7.30 (t, J¼ 9.2 Hz,2H), 7.25 (d, J¼ 8.4 Hz,1H). TOFMS (EIþ): C19H12O4, calcd 304.0736,found 304.0735.

Z. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3909e39163914

5.1.3. General procedure for the preparation of 10aegTo a solution was added the corresponding compounds 9aeg

(1 mmol) and malononitrile (1.5 mmol) in 3 mL CH2Cl2 in podgysilica gel containing with CH2Cl2 and then column chromatographyto yield a red power, which was used in the next step withoutfurther purification.

5.1.3.1. 2-[2-Oxo-2H-6-(o-tolyloxy)acenaphthylen-1-ylidene]-malo-nonitrile (10a). Yield: 0.14 g, 42%. Mp¼ 185e187 �C. 1H NMR(400 MHz, CDCl3): d 8.61 (d, J¼ 7.8 Hz, 1H), 8.00 (d, J¼ 8.0 Hz, 1H),7.88 (d, J¼ 7.6 Hz, 1H), 7.39 (d, J¼ 7.2 Hz, 2H), 7.33 (t, J¼ 7.6 Hz, 2H),7.30 (d, J¼ 7.6 Hz, 1H), 7.14 (d, J¼ 7.6 Hz, 1H), 2.21 (s, 3H). TOF MS(EIþ): C22H12N2O2, calcd 336.0899, found 336.0887.

5.1.3.2. 2-[2-Oxo-2H-6-(m-tolyloxy)acenaphthylen-1-ylidene]-malono-nitrile (10b). Yield: 0.16 g, 48%. Mp¼ 176e177 �C. 1H NMR (400MHz,CDCl3): d 8.59 (d, J¼ 8.0 Hz, 1H), 8.54 (d, J¼ 8.0 Hz, 1H), 8.03(d, J¼ 7.6 Hz, 1H), 7.86 (d, J¼ 8.4 Hz, 2H), 7.39 (t, J¼ 7.6 Hz, 1H), 7.16(d, J¼ 8.4 Hz, H), 7.02 (t, J¼ 7.6 Hz, 1H), 7.00 (d, J¼ 7.6 Hz, 1H), 2.43(s, 3H). TOF MS (EIþ): C22H12N2O2, calcd 336.0899, found 336.0890.

5.1.3.3. 2-[2-Oxo-2H-6-(p-tolyloxy)acenaphthylen-1-ylidene]-malo-nonitrile (10c). Yield: 0.18 g, 54%. Mp¼ 227e228 �C. 1H NMR(400 MHz, CDCl3): d 8.59 (d, J¼ 8.0 Hz, 1H), 8.57 (d, J¼ 8.0 Hz, 1H),8.01 (d, J¼ 8.0 Hz,1H), 7.87 (t, J¼ 8.0 Hz,1H), 7.31 (d, J¼ 8.0 Hz, 2H),7.11 (d, J¼ 8.0 Hz, 2H), 6.98 (d, J¼ 8.0 Hz, 1H), 2.43 (s, 3H). TOF MS(EIþ): C22H12N2O2, calcd 336.0899, found 336.0888.

5.1.3.4. 2-[2-Oxo-2H-6-(2-methoxyphenoxy)acenaphthylen-1-ylidene]-malononitrile (10d). Yield: 0.15 g, 43%. Mp¼ 194e195 �C. 1HNMR (400MHz, CDCl3): d 8.62 (d, J¼ 8.0 Hz, 1H), 8.59(d, J¼ 8.0 Hz, H), 8.00 (d, J¼ 8.0 Hz,1H), 7.88 (d, J¼ 8.0 Hz,1H), 7.35 (t,J¼ 8.0 Hz, 1H), 7.23 (t, J¼ 8.0 Hz, 1H), 7.13 (t, J¼ 8.4 Hz, 2H), 6.84 (d,J¼ 8.4 Hz,1H), 3.76 (s,1H). TOFMS (EIþ): C22H12N2O3, calcd 352.0848,found 352.0839.

5.1.3.5. 2-[2-Oxo-2H-6-(3-methoxyphenoxy)acenaphthylen-1-ylidene]-malononitrile (10e). Yield: 0.14 g, 40%. Mp¼ 178e179 �C. 1H NMR(400MHz, CDCl3): d 8.59 (d, J¼ 8.4 Hz, 1H), 8.54 (d, J¼ 8.4 Hz, 2H),8.04 (d, J¼ 8.0 Hz, 1H), 7.86 (d, J¼ 8.0 Hz, 1H), 7.40 (t, J¼ 8.0 Hz, 1H),7.06 (d, J¼ 8.0 Hz, 1H), 6.87 (d, J¼ 8.4 Hz, H), 6.80 (d, J¼ 8.4 Hz, 2H),3.84 (s, 1H). TOF MS (EIþ): C22H12N2O3, calcd 352.0848, found352.0841.

5.1.3.6. 2-[2-Oxo-2H-6-(4-methoxyphenoxy)acenaphthylen-1-ylidene]-malononitrile (10f). Yield: 0.19 g, 54%. Mp¼ 233e234 �C. 1H NMR(400MHz, CDCl3): d 8.44 (d, J¼ 8.4 Hz, 1H), 8.35(d, J¼ 9.2 Hz, 2H), 8.18 (d, J¼ 8.0 Hz, 1H), 8.11 (d, J¼ 8.0 Hz, 1H), 7.89(t, J¼ 8.0 Hz,1H), 7.65 (t, J¼ 9.2 Hz,1H), 7.30 (t, J¼ 9.2 Hz, 2H), 7.25 (d,J¼ 8.4 Hz, 1H). TOF MS (EIþ): C22H12N2O3, calcd 352.0848, found352.0839.

5.1.3.7. 2-[2-Oxo-2H-6-(4-sec-butylphenoxy)acenaphthylen-1-ylidene]-malononitrile (10g). Yield: 0.20 g, 53%. Mp¼ 176e177 �C. 1H NMR(400MHz, CDCl3): d 8.58 (d, J¼ 8.0 Hz,1H), 8.56 (d, J¼ 8.0 Hz,1H), 8.02(d, J¼ 8.0 Hz,1H), 7.85 (t, J¼ 8.0 Hz,1H), 7.32 (d, J¼ 8.4 Hz, 2H), 7.14 (d,J¼ 8.4 Hz, 2H), 7.00 (d, J¼ 8.0 Hz, 1H), 2.38 (m,1H), 1.64 (m, J¼ 8.0 Hz,2H), 1.29 (d, J¼ 8.0 Hz, 3H), 0.88 (t, J¼ 8.0 Hz, 3H). TOF MS (EIþ):C25H18N2O2, calcd 378.1368, found 378.1376.

5.1.4. General procedure for the preparation of 2aec, 3ae3c and 6To a solution was added the corresponding compounds 10aeg

(1 mmol) and K2CO3 (165 mg, 1.2 mmol) in CH3CN (5 mL) was

refluxed for 2 h. After the reaction was complete as monitored byTLC, the solvent was evaporated under reduced pressure and theproduct was purified by column chromatography on silica gel usingdichloromethane/petroleum ether (1:1).

5.1.4.1. 3-(o-Tolyloxy)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbo-nitrile (2a). Yield: 0.14 g, 42%. Mp¼ 210e211 �C. 1H NMR(400 MHz, CDCl3): d 8.96 (d, J¼ 8.0 Hz, 1H), 8.63 (d, J¼ 8.0 Hz, 1H),8.48 (d, J¼ 8.0 Hz, 1H), 8.01 (t, J¼ 8.0 Hz, 1H), 7.39 (d, J¼ 7.6 Hz, H),7.35 (d, J¼ 8.0 Hz, H), 7.32 (d, J¼ 7.6 Hz, 1H), 7.14 (d, J¼ 8.0 Hz, 1H),6.87 (d, J¼ 8.0 Hz, H), 2.19 (s, 3H). TOF MS (EIþ): C22H12N2O2, calcd336.0899, found 336.0890.

5.1.4.2. 3-(m-Tolyloxy)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbo-nitrile (2b). Yield: 0.18 g, 53%. Mp¼ 222e223 �C. 1H NMR(400 MHz, CDCl3): d 8.91 (d, J¼ 8.4 Hz, 1H), 8.65 (d, J¼ 8.4 Hz, 1H),8.46 (d, J¼ 7.6 Hz,1H), 7.87 (t, J¼ 7.6 Hz, 1H), 7.41 (d, J¼ 8.4 Hz, 2H),7.19 (d, J¼ 8.4 Hz, 1H), 7.03 (d, J¼ 7.6 Hz, 2H), 2.43 (s, 3H). TOF MS(EIþ): C22H12N2O2, calcd 336.0899, found 336.0894.

5.1.4.3. 3-(p-Tolyloxy)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbo-nitrile (2c). Yield: 0.20 g, 60%. Mp¼ 230e231 �C. 1H NMR(400 MHz, CDCl3): d 8.92 (d, J¼ 8.4 Hz, 1H), 8.63 (d, J¼ 8.4 Hz, 1H),8.45 (d, J¼ 7.6 Hz,1H), 7.86 (t, J¼ 7.6 Hz,1H), 7.32 (d, J¼ 8.4 Hz, 2H),7.09 (d, J¼ 8.4 Hz, 2H), 7.01 (d, J¼ 7.6 Hz, 1H), 2.44 (s, 3H). TOF MS(EIþ): C22H12N2O2, calcd 336.0899, found 336.0892.

5.1.4.4. 3-(2-Methoxyphenoxy)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (3a). Yield: 0.23 g, 65%. Mp¼ 250e251 �C. 1H NMR(400 MHz, CDCl3): d 8.97 (d, J¼ 8.0 Hz, 1H), 8.63 (d, J¼ 8.4 Hz, 1H),8.46 (d, J¼ 8.0 Hz, 1H), 7.87 (t, J¼ 8.0 Hz, 1H), 7.37 (t, J¼ 8.0 Hz, 1H),7.23 (s, H), 7.15 (d, J¼ 7.2 Hz, H), 7.11 (d, J¼ 8.0 Hz, H), 7.09 (d,J¼ 7.2 Hz, 1H), 3.75 (s, 3H). TOF MS (EIþ): C22H12N2O3, calcd352.0848, found 352.0840.

5.1.4.5. 3-(4-Methoxyphenoxy)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (3b). Yield: 0.24 g, 68%. Mp¼ 253e254 �C. 1H NMR(400 MHz, CDCl3): d 8.94 (d, J¼ 8.0 Hz, 1H), 8.63 (d, J¼ 8.4 Hz, 1H),8.46 (d, J¼ 8.0 Hz,1H), 7.88 (t, J¼ 8.0 Hz,1H), 7.26 (d, J¼ 7.2 Hz, 2H),7.13 (d, J¼ 7.2 Hz, 2H), 7.04 (d, J¼ 8.8 Hz, 1H), 3.83 (s, 3H). TOF MS(EIþ): C22H12N2O3, calcd 352.0848, found 352.0841.

5.1.4.6. 3-(4-Methoxyphenoxy)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (3c). Yield: 0.30 g, 85%. Mp¼ 257e258 �C. 1H NMR(400 MHz, CDCl3): d 8.92 (d, J¼ 8.0 Hz, 1H), 8.62 (d, J¼ 8.8 Hz, 1H),8.44 (d, J¼ 8.0 Hz,1H), 7.86 (t, J¼ 8.0 Hz,1H), 7.15 (d, J¼ 7.2 Hz, 2H),7.03 (d, J¼ 7.2 Hz, 2H), 7.00 (d, J¼ 8.8 Hz, 1H), 3.88 (s, 3H). TOF MS(EIþ): C22H12N2O3, calcd 352.0848, found 352.0839.

5.1.4.7. 3-(4-sec-Butylphenoxy)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (6). Yield: 0.31 g, 82%. Mp¼ 210e211 �C. 1H NMR(400 MHz, CDCl3): d 8.92 (d, J¼ 8.0 Hz, 1H), 8.65 (d, J¼ 8.8 Hz, 1H),8.46 (d, J¼ 8.0 Hz,1H), 7.87 (t, J¼ 8.0 Hz,1H), 7.33 (d, J¼ 8.4 Hz, 2H),7.14 (d, J¼ 8.4 Hz, 2H), 7.04 (d, J¼ 8.0 Hz, 1H), 2.70 (m, 1H), 1.65 (m,J¼ 8.0 Hz, 2H), 1.30 (d, J¼ 8.0 Hz, 3H), 0.88 (t, J¼ 8.0 Hz, 3H). 13CNMR (400 MHz, CDCl3): d 176.17, 163.64, 151.70, 147.53, 136.46,134.54,132.05,130.91,128.63,128.54,126.80,123.89,122.55,122.25,120.84, 120.55, 113.04, 112.87, 112.31, 41.53, 30.77, 21.32, 11.54. TOFMS (EIþ): C25H18N2O2, calcd 378.1368, found 378.1376.

5.2. Biological assay

5.2.1. Reagents, plasmid and antibodiesA 21-residue Bid BH3 peptide (residues 79e99) [19,20] bearing

a6-carboxyfluorescein succinimidyl esterfluorescence tag (FAM-Bid)

Z. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3909e3916 3915

was synthesized at HD Biosciences (ShangHai, China). Recombinanthuman Bcl-2 protein was purchased from Santa Cruz Biotechnology(sc-4096, Santa Cruz) [21]. For expression of human Mcl-1 as ourreport described [9]. Antibodies were Bcl-2 antibody (sc-783, SantaCruz) and rabbit antibody against Mcl-1 (BS1220, Bioworld).

5.2.2. Cell linesThe human leukemia cell line K562, human breast adenocarci-

noma cell line MCF-7, and human myeloid leukemia cell line HL-60were purchased from China Center for Type Culture Collection(Wuhan, China). Cells were cultured in 75 cm2 Falcon flasks (BectonDickinson, Franklin Lakes) in phenol-red-free RPMI medium 1640(Gibco/BRL) supplemented with 10% fetal bovine serum, HEPES(10 mM), sodium bicarbonate (24 mM), sodium pyruvate (1 mM),2-mercaptoethanol (0.05 mM), penicillin (60 mg/mL) and strepto-mycin (100 mg/mL). Cell culture was kept in a humidified incubatorwith 5% CO2 at 37 �C.

5.2.3. Fluorescence polarization-based binding assayFor the competitive binding assay for Bcl-2 protein, FAM-Bid

peptide (10 nM) and Bcl-2 protein (40 nM) were preincubated inthe assay buffer (100 mM potassium phosphate, pH 7.5; 100 mg/mLbovine gamma globulin; 0.02% sodium azide). The detergent TritonX-100 was freshly prepared daily as a 1% (v/v) stock in 50 mM KPi.For each experiment, a control containing Bcl-2 and FAM-Bidpeptide (equivalent to 0% inhibition), and another control con-taining only FAM-Bid, were included on each assay plate. Next,serial dilutions of compounds with 0.01% Triton X-100 were added.After 30 min incubation, the polarization values were measuredusing the Spectra Max M5 Detection System in a black 96-wellplate. Saturation experiments determined that FAM-Bid binds tothe Bcl-2 protein with a Kd value of 8 nM. For Mcl-1, assays wereperformed in the same manner as that for Bcl-2 with the followingexceptions: 50 nMMcl-1 and 10 nM FAM-Bid peptide were used inthe assay buffer (25 mM Tris, pH 8.0; 150 mM NaCl). FAM-Bidpeptide binds to the Mcl-1 protein with a Kd value of 1.9 nM. TheKi value for each inhibitor was calculated using the equation wehave developed for FP-based assays [9].

5.2.4. Western blotCellswere lysed indigitonin lysisbuffer. (62.5 mMTriseHCl,pH6.8,

2%w/vSDS,10%glycerol, 50mMDTT,1 mMPMSF) [9]. Electrophoresisand transfer tomembranewere performed routinely. Themembraneswere blocked and probed with indicated antibody, followed byperoxidase-conjugated IgG. The expected proteins were detected bythe ECL method (Amersham Pharmacia Biotech, Pittsburgh, PA).

5.2.5. MTT assayStandard procedures were used [22]. Tumor cells were plated in

96-well (1�104 cells per well) plates, and were cultured in thepresence of various concentrations of compounds (0.01e100 mM)from DMSO stock (1 mM or 10 mM) for 48 h. The experiments wereperformed in triplicates. Control cells were treated with DMSO equalto the highest percentage of solvent used in the experimental condi-tions. At the end of the exposure time, themediumwas removed and3-[4,5-dimethylthiazol-2-yl]-2,5-diphentetrazoliumbromide (MTT)wasadded toeachwell. Theplateswere incubatedat 37 �C for 4 h. Thesolution was removed and DMSO was added to each well. The IC50value was obtained from plots of % viability against the dose of thecompound added.

5.3. Molecular docking

The 3D structures of the human Mcl-1 (hMcl-1; PDB ID: 2NLA)and human Bcl-2 (Bcl-2; PDB ID: 1GJH) were obtained from the

protein bank in the RCSB [23] The 3D structures of the inhibitorswere generated using Chembio3D Ultra 11.0 followed by energyminimization. AutoDock 4.0 program equipped with ADT was usedto perform the automated molecular docking [24]. Grid mapscovering residues that were perturbed more than the thresholdvalue of 0.1 ppm in the BH3 binding groove of the proteins weredefined for all inhibitors in the AutoDock calculations using a gridspacing of 0.375�A. The GA-LS algorithmwas adopted using defaultsettings. For each docking job, 100 hybrid GA-LS runs were carriedout. A total of 100 possible binding conformations were generatedand grouped into clusters based on a 1.0�A cluster tolerance. Thedocking models were analyzed and represented using ADT.

Acknowledgments

This work was supported by the Fundamental Research Fundsfor the Central Universities and partly supported by the NationalNatural Science Foundation of China (Grant 30772622).

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