Marinopyrrole Derivatives with Sulfide Spacers as Selective Disruptors of Mcl-1 Binding to Pro-Apoptotic Protein Bim

A series of novel marinopyrroles with sulfide and sulphone spacers were designed and synthesized. Their activity to disrupt the binding of the pro-apoptotic protein, Bim, to the pro-survival proteins, Mcl-1 and Bcl-xL, was evaluated using ELISA assays. Fluorescence-quenching (FQ) assays confirmed the direct binding of marinopyrroles to Mcl-1. Benzyl- and benzyl methoxy-containing sulfide derivatives 4 and 5 were highly potent dual Mcl-1/Bim and Bcl-xL/Bim disruptors (IC50 values of 600 and 700 nM), whereas carboxylate-containing sulfide derivative 9 exhibited 16.4-fold more selectivity for disrupting Mcl-1/Bim over Bcl-xL/Bim binding. In addition, a nonsymmetrical marinopyrrole 12 is as equally potent as the parent marinopyrrole A (1) for disrupting both Mcl-1/Bim and Bcl-xL/Bim binding. Some of the derivatives were also active in intact human breast cancer cells where they reduced the levels of Mcl-1, induced programd cell death (apoptosis) and inhibited cell proliferation.

Apoptosis evasion is one of the most important hallmarks that cells must acquire to become cancerous [17,18].One of the major mechanisms by which cancer cells evade apoptosis is by over expressing Bcl-xL, Bcl-2 and/or Mcl-1 contributing not only to tumorigenesis but also to tumor resistance to chemotherapy [18].Several small molecule inhibitors of the pro-survival Bcl-2 family of proteins have been identified [19][20][21].To date, the most extensively studied and promising small molecule BH3 mimetic is ABT-737 or its orally-available ABT-263.However, human tumors that overexpress Mcl-1 are resistant to Bcl-xL/Bcl-2-selective agents such as ABT-737 and ABT-263 [22][23][24].Fewer Mcl-1 antagonists have been reported, most are not highly selective for Mcl-1 and none have been developed enough to reach clinical trials [25][26][27][28][29][30][31].Here, we report on the design of a series of marinopyrroles with sulfide and sulphone spacers, some as dual Mcl-1 and Bcl-xL antagonists and others as selective disruptors of Mcl-1 binding to Bim.

Design of Marinopyrrole Derivatives
With the success of our synthetic and SAR studies on symmetrical, nonsymmetrical and cyclic marinopyrrole derivatives [3,6,7,[14][15][16] and based on our results that marinopyrrole A (1) binds to Mcl-1 in two regions according to chemical shift perturbations and docking studies [10], we focused our attention on a series ofsymmetrical derivatives with sulfide and sulphone spacers substituted in the para-position relative to the carbonyl group on both aromatic rings (3 to 10).We were particularly interested in exploring the structure activity relationships (SARs) of these series of marinopyrroles to probe: (i) whether the molecular geometries play a role; (ii) how large a functional group is tolerated; (iii) whether hydrophobic or hydrophilic groups are preferred; (iv) if hydrogen bond donors or hydrogen bond acceptors are desirable; (v) whether functional groups with ionic interactions are allowed.To evaluate the potential differences in activity between the nonsymmetrical marinopyrroles 11 and 12, both compounds [5] were included in this study (Figure 1).Some of the marinopyrroles designed for this study are potential candidates for improving aqueous solubility as the parent marinopyrrole A (1) exhibited poor solubility which hinders its further development.

Physicochemical Properties and SAR of the Marinopyrroles
Both pK a and log p values were calculated using ChemAxon Software Version 5.12.3 [32,33].The pK a values of marinopyrrole A (1) are predicted to be 7.8 (pK a 1) and 8.4 (pK a 2), respectively (Figure 2 and Table 1).As reported previously [14][15][16], the difference in pK a values for the hydroxyl group in ring A and ring B is presumably due to the axially chiral environment.The pK a values of 1 are 1.6-2.2log units lower than that of phenol (pK a = 9.98) [34].An equilibrium may exist between open and closed conformations in 1, similar to those observed in a recent report of intramolecular hydrogen bonding [35].The Fenical group reported the X-ray structure of marinopyrrole B (3′-Br analogue of 1) that indicated the preference for the formation of intramolecular hydrogen bonds between the peri-hydroxyl and the carbonyl group [1].These intramolecular hydrogen bond interactions contribute to increasing not only compound acidity, but also its lipophilicity [35].The calculated log p value of 1 is 5.6, which marginally violates the Rule of Five (RO5), drug-like properties formulated by Lipinski [36].The calculated pK a 1 and pK a 2 values of marinopyrroles in Figure 2 range from 6.7 to 8.4.Compound 9 has pK a 3 (2.9) and pK a 4 (3.5)values, and 10 has a pK a 3 (2.2) and pK a 4 (2.9) due to the carboxylic acid functional group.Clog p values of compounds 9 and 10 are 5.3 and 2.9, respectively.While the former marginally violates the RO5, the latter resides within the suggested range for drug-like compounds.Compound 6 also has a Clog p value of 3.7 whereas the remaining compounds 3, 4, 5, 7 and 8 violate RO5 with compounds 4 and 5 being five log unit higher than the desired limit of lipophilicity.Both nonsymmetrical marinopyrroles 11 and 12 have Clog p values of 4.5.As reported previously [10,16], the IC 50 value of racemic marinopyrrole A (1) to disrupt the binding of Mcl-1 to Bim was 8.9 μM.The IC 50 value of 1 to disrupt the binding of Bcl-xL to Bim (16.4 μM) was consistent with our recent report [16] but lower than we originally reported [10], due to the 2.5 times lower Bcl-xL (not Mcl-1) concentration utilized in our present assay (10 nM) vs. that originally used (25 nM).Symmetrical marinopyrroles with sulfide spacers (3)(4)(5) are five-to 13-fold and 20-to 27-fold more potent than 1 against Mcl-1/Bim and Bcl-xL/Bim, respectively (Figure 2).The sulfide substitutions greatly increased potency but did not alter selectivity as 3, 4 and 5 are also dual Mcl-1 and Bcl-xL antagonists (Figure 2).Compounds 4 and 5 are the most potent in the series with IC 50 values of 0.7 and 0.6 μM against Mcl-1/Bim and Bcl-xL/Bim, respectively.Marinopyrroles with a sulphone spacer (6-8) are at least 16-fold less active than their sulfide counterparts.This difference is

Activity in Intact Human Breast Cancer Cells
To determine if the marinopyrroles are active in intact cells, the human breast cancer MDA-MB-468 cells were treated with the marinopyrrole derivatives (40 μM for 16 h).The cells were then processed for Western blotting exactly as described by us previously [16,38].Figure 4 shows that treatment of the cells with 1 resulted in a significant decrease in the levels of Mcl-1.This is consistent with our previous reports [10,16].Figure 4 also shows that 3, 4, 5, 6, 10 and 11 were highly potent, whereas 8, 9 and 12 were moderately active, at decreasing Mcl-1 levels.We next evaluated the effects of the compounds on programed cell death (apoptosis) by determining their ability to induce cleaved caspase 3 in MDA-MB-468 cells.Figure 4 shows that 1, 3, 4, 5, 6, 7, 10 and 11 potently induced apoptosis, whereas the ability of 8, 9 and 12 to induce apoptosis was weak.None of the marinopyrroles affected the cellular levels of the control protein vinculin (Figure 4).In addition, the ability of the compounds to inhibit tumor growth of MDA-MB-468 cells was determined by MTT assays.To this end, MDA-MB-468 cells were treated for 48 h with the compounds at 0, 0.5, 5 and 50 μM, and the cells were processed for MTT assays as described by us [39].All compounds inhibited MDA-MB-468 cell

Synthesis of Marinopyrrole Derivatives
All chemicals were purchased from commercial suppliers and used without further purification.All solvents were dried and distilled before use.Tetrahydrofuran was distilled from sodium/benzophenone. Dichloromethane and acetonitrile were distilled over calcium hydride.Flash column chromatography was performed with silica gel (200-300 mesh). 1 H NMR spectra were recorded at either 400 MHz or 600 MHz at ambient temperature. 13C NMR spectra were recorded at either 100 or 150 MHz at ambient temperature.Infrared spectra were recorded on a Perkin-Elmer Spectrum 100 spectrometer.Copies of the NMR spectra of all the described compounds are provided in an Electronic Supporting Information (ESI) document.Melting points were determined with a melting point apparatus (Fukai X-4).High resolution mass spectra were performed by electrospray ionization (ESI) on an Agilent ESI-TOF LC-MS 6200 system.Analytical HPLC was performed on an Agilent 1100 series instrument with diode array detectors and auto samplers.All tested compounds possessed a purity of not less than 95%.

Conclusions
This article describes general synthetic routes to access novel marinopyrrole derivatives with sulfide and sulphone spacers and an evaluation of their in vitro activity against the binding of the pro-survival proteins, Mcl-1 and Bcl-xL, to the pro-apoptotic protein, Bim.Our efforts were focused on improving anti-Mcl-1/Bim and Bcl-xL/Bim potency and selectivity.SAR studies of these marinopyrrole derivatives have clearly demonstrated that: (i) symmetrical marinopyrrole 3 with ethyl mercaptoacetate extended in the para-position to the carbonyl group with a sulfide spacer improved the potency by five-and 17
presumably due to different molecular geometries of the -S-and -SO 2 -bonds which might result in desired and undesired orientation of the substituents in the binding pockets.Interestingly, compound 9 demonstrated 16.4-fold selectivity for Mcl-1/Bim over Bcl-xL/Bim with an IC 50 value of 6.1 μM and >100 μM, respectively.Nonsymmetrical marinopyrrole 12 exhibited similar potencies to 1 against both Mcl-1/Bim and Bcl-xL/Bim although another nonsymmetrical marinopyrrole 11 is much less active than the parent marinopyrrole 1 against Mcl-1/Bim and Bcl-xL/Bim.2.4.Direct Binding Measurement by Fluorescence QuenchingTo confirm direct binding of the compounds to Mcl-1, we have established a fluorescence-quenching assay based on the intrinsic Trp fluorescence of Mcl-1[37].Using this assay we have confirmed direct binding of marinopyrrole analogue 9 to Mcl-1 by generating binding isotherms and calculated the binding constant for 9 (K d = 2.7 μM, Figure3), consistent with its IC 50 value in the ELISA assay.
FigureS1in Supplementary Information shows that the compounds had little effects on nuclear and cellular morphology.

Figure 4 .
Figure 4. Effect of marinopyrroles on Mcl-1 levels in human breast cancer cells.
.6-fold (3 vs. 1) for disrupting Mcl-1/Bim and Bcl-xL/Bim binding, respectively (Figure 2); (ii) symmetrical marinopyrroles with CH 2 Ph and/or CH 2 (p-MeOPh) extended in the para-position to the carbonyl group with sulfide spacers (4 and 5) are not only 12.7-fold and 27.3-fold more potent than the parent marinopyrrole 1 against Mcl-1/Bim and Bcl-xL/Bim but also potent dual inhibitors (IC 50 = 0.6 µM and 0.7 µM, respectively); (iii) Potency was decreased dramatically when the sulfide was replaced by sulphone spacers (Figure 2: 3 vs.6, 4 vs. 7 and 5 vs. 8); (iv) Although marinopyrroles with sulphone spacers are less potent than their sulfide counterparts against both Mcl-1/Bim and Bcl-xL/Bim, they are in general more selective for Mcl-1/Bim over Bcl-xL/Bim (Figure 2); (v) the symmetrical marinopyrrole 9 with mercaptoacetic acid in the para-position to the carbonyl group is an excellent "lead" for further optimization of Mcl-1 selective inhibitors (IC 50 = 6.1 µM, 16.4-fold more selective for Mcl-1 over Bcl-xL, Figure 2); (vi) nonsymmetrical marinopyrrole 12 exhibited the same potency as 1 against both Mcl-1/Bim and Bcl-xL/Bim.Furthermore, most of the derivatives were cell active as demonstrated by their ability to decrease the levels of Mcl-1, induce apoptosis and inhibit tumor cell growth of human breast cancer cells.In summary, we have designed and synthesized a series of novel symmetrical and nonsymmetrical marinopyrroles with improved potency against both Mcl-1 and Bcl-xL.Further optimization is actively ongoing, and the activity, selectivity and absorption, distribution, metabolism and excretion (ADME)/tox data of these compounds will be published in due course.

Table 1 .
ELISA and physicochemical properties of marinopyrroles.

Substituent (R) Mcl-1/Bim a Bcl-xL/Bim a pK a 1 b pK a 2 b pK a 3 b,c pK a 4 b,c Clog p b
[5]]ISA data previously reported by us[16]; e Compounds were reported previously by us[5].