Marinopyrrole derivatives with sulfide spacers as selective disruptors of Mcl-1 binding to pro-apoptotic protein bim

Article abstract of DOI:10.3390/md12084311

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.

Full text of DOI:10.3390/md12084311

Mar. Drugs 2014, 12, 4311-4325; doi:10.3390/md12084311
OPEN ACCESS
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Marinopyrrole Derivatives with Sulfide Spacers as Selective
Disruptors of Mcl-1 Binding to Pro-Apoptotic Protein Bim
Chunwei Cheng 1, Yan Liu 2,3, Maria E. Balasis 2, Thomas P. Garner 4, Jerry Li 2,
Nicholas L. Simmons 5, Norbert Berndt 2, Hao Song 1, Lili Pan 1, Yong Qin 1,6,*, K. C. Nicolaou 5,7,
Evripidis Gavathiotis 4, Said M. Sebti 2,8 and Rongshi Li 2,3,8,
*
1
Key Laboratory of Drug Targeting and Drug Delivery Systems of the Ministry of Education and
State Key Laboratory of Biotherapy, Department of Medicinal Natural Products,
West China School of Pharmacy, Sichuan University, Chengdu 610041, China;
E-Mails: chengchunwei666@163.com (C.C.); haoright@163.com (H.S.);
pande179@163.com (L.P.)
2
Department of Drug Discovery and Chemical Biology and Molecular Medicine Program,
H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive,
Tampa, FL 33612, USA; E-Mails: yan.liu@unmc.edu (Y.L.); maria.balasis@moffitt.org (M.E.B.);
jerry.li@ucsf.edu (J.L.); Norbert.berndt@moffitt.org (N.B.); said.sebti@moffitt.org (S.M.S.)
Department of Pharmaceutical Sciences, Center for Drug Discovery, College of Pharmacy,
and Cancer Genes and Molecular Regulation Program, Buffett Cancer Center,
University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198, USA
Departments of Biochemistry and Medicine, Albert Einstein College of Medicine, Jack and Pearl
Resnick Campus, 1300 Morris Park Avenue, Forchheimer G46, Bronx, NY 10461, USA;
E-Mails: thomas.garner@einstein.yu.edu (T.P.G.); evripidis.gavathiotis@einstein.yu.edu (E.G.)
Department of Chemistry and The Skaggs Institute for Chemical Biology,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA;
E-Mails: nsimmons@scripps.edu (N.L.S.); kcn@rice.edu (K.C.N.)
3
4
5
6
7
The Innovative Drug Research Center, Chongqing University, Chongqing 400000, China
Department of Chemistry, BioScience Research Collaborative, Rice University, 6500 Main Street,
Houston, TX 77030, USA
8
Department of Oncologic Sciences, Morsani College of Medicine, University of South Florida,
12901 Bruce B. Downs, Tampa, FL 33612, USA
* Authors to whom correspondence should be addressed; E-Mails: qinyong@cqu.edu.cn (Y.Q.);
rongshi.li@unmc.edu (R.L.); Tel./Fax: +86-28-8550-3842 (Y.Q.); Tel.: +1-402-559-6609 (R.L.);
Fax: +1-402-559-5418 (R.L.).

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Received: 8 April 2014; in revised form: 23 May 2014 / Accepted: 14 July 2014 /
Published: 29 July 2014
Abstract: 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.
Keywords: marinopyrroles; protein-protein interaction disruptors; apoptosis; SAR
Abbreviations
ADME, absorption, distribution, metabolism and excretion; Bcl-2, B-cell lymphoma 2; Bcl-xL,
B-cell lymphoma extra large; BH3, Bcl-2 homology domain 3; calcd., calculated; m-CPBA,
m-chloroperbenzoic acid; DCM, dichloromethane; dd, double doublet; br, broad; DPPP,
bis(diphenylphosphino)propane; DIEA, diisopropylethylamine; DMF, dimethylformamide; DMSO,
dimethyl sulfoxide; EtOAc, ethyl acetate; ESI, electrospray ionization; HPLC, high performance liquid
chromatography; HRMS, high resolution mass spectrometry; HRP, horseradish peroxidase; IR,
infrared; KBr, potassium bromide; KF, potassium fluoride; LC, liquid chromatography; LiOH, lithium
hydroxide; Mcl-1, Myeloid cell leukemia 1; MeCN, acetonitrile; MeOH, methyl alcohol; MDA-MB-468,
breast cancer cell line; mp, melting point; MRSA, methicillin-resistant Staphylococcus aureus; NCS,
N-chlorosuccinimide; NaI, sodium iodide; NMR, nuclear magnetic resonance; PBS, phosphate-buffered
saline; RO5, Rule of Five; SAR, structure activity relationship; s, singlet; THF, tetrahydrofuran; TMB,
3,3′,5,5′-Tetramethylbenzidine; tox, toxicity; Pd2(dba)3, tris(dibenzylideneacetone)dipalladium.
1. Introduction
Marinopyrroles were first reported to show antibiotic activity against methicillin-resistant
Staphylococcus aureus (MRSA) in 2008 by the Fenical group [1]. Due to their novel molecular
structures and promising biological properties, marinopyrroles have attracted considerable
attention [2–16]. Following our finding that (±)-marinopyrrole A (1) antagonizes Mcl-1 and overcomes
resistance of human cancer cells to the Bcl-xL antagonist ABT-737 [10], we recently reported a series

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of novel cyclic marinopyrroles as disruptors of protein-protein interactions between the pro-apoptotic
protein, Bim, and the pro-survival proteins, Bcl-xL and Mcl-1 [16].
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 [19–21]. 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 [22–24].
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 [25–31]. 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.
2. Results and Discussion
2.1. Design of Marinopyrrole Derivatives
With the success of our synthetic and SAR studies on symmetrical, nonsymmetrical and cyclic
marinopyrrole derivatives [3,6,7,14–16] 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.
Figure 1. Marinopyrroles with sulfide and sulphone spacers and nonsymmetrical marinopyrroles.
R
Cl
Cl
1
HN
Cl
HN
HN
EtO
Cl
O
Cl
O
Cl
O
HO
O
O
OH
HO
3
N
N
1'
O
OH
Cl
Cl
N
OEt
Cl
Cl
Cl
3'
Cl
R
12
3 through 10 (R: see Figure 2)
11

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2.2. Synthesis of Marinopyrrole Derivatives
Starting from our previously reported compound 2 [14] as shown in Scheme 1, palladium-mediated
nucleophilic substitution of the triflate 2 with ethyl 2-mercaptoacetate, phenylmethanethiol and
(4-methoxyphenyl) methanethiol gave 3 (61%), 4 (96%) and 5 (85%), respectively. Sulfides 3, 4 and 5
were oxidized to the corresponding sulphones 6 (70%), 7 (75%) and 8 (65%) with m-chloroperbenzoic
acid (m-CPBA). State whether Baeyer-Villiger oxidation by-products are detected. The carboxylic
acids 9 and 10 were obtained by saponification of the corresponding esters 3 and 6 using LiOH in 85%
and 95% yields.
Scheme 1. Synthesis of marinopyrroles 3 to 10.
2.3. Physicochemical Properties and SAR of the Marinopyrroles
Both pKa and log p values were calculated using ChemAxon Software Version 5.12.3 [32,33]. The
pKa values of marinopyrrole A (1) are predicted to be 7.8 (pKa 1) and 8.4 (pKa 2), respectively (Figure 2
and Table 1). As reported previously [14–16], the difference in pKa values for the hydroxyl group in
ring A and ring B is presumably due to the axially chiral environment. The pKa values of 1 are
1.6–2.2 log units lower than that of phenol (pKa = 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 pKa 1 and pKa 2 values of marinopyrroles in Figure 2 range from 6.7 to
8.4. Compound 9 has pKa 3 (2.9) and pKa 4 (3.5) values, and 10 has a pKa 3 (2.2) and pKa 4 (2.9) due

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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.
Figure 2. Structure of marinopyrroles.
(pKa 4)
R
Cl
A
HN
Cl
O
(pKa 1)
HO
(pKa 2)
O
1'
OH
N
Cl
B
3'
Cl
(pKa 3)
R
Table 1. ELISA and physicochemical properties of marinopyrroles.
Compound Substituent (R) Mcl-1/Bim a Bcl-xL/Bim a pKa 1 b pKa 2 b pKa 3 b,c pKa 4 b,c Clog p b
1
3
4
H
8.9 ± 1.0 d
1.8 ± 0.3
0.7 ± 0.2
16.4 ± 3.3 d
1.2 ± 0.2
0.6 ± 0.2
7.8
7.8
7.8
8.4
8.4
8.4
-
-
-
-
-
-
5.6
6.1
SCH2CO2Et
SCH2Ph
10.2
SCH2
5
0.7 ± 0.1
0.6 ± 0.1
7.8
8.4
-
-
9.7
(p-MeOPh)
SO2CH2CO2Et
SO2CH2Ph
SO2CH2
6
7
37.3 ± 3.1
7.3 ± 1.4
>100
6.7
6.7
7.3
7.3
-
-
-
-
3.7
6.9
69.3 ± 15.8
8
17.4 ± 3.1
>100
6.7
7.3
-
-
6.4
(p-MeOPh)
SCH2CO2H
SO2CH2CO2H
e See Figure 1
e See Figure 1
9
10
1
6.1 ± 1.3
63.0 ± 5.4
25.1 ± 4.7
11.5 ± 1.9
>100
>100
7.8
6.7
-
8.4
7.3
8.1
-
2.9
2.2
-
3.5
2.9
-
5.3
2.9
4.5
4.5
96.6 (n = 2)
17.6 ± 4.5
12
8.1
-
-
a
IC50 in micromolar (average ± SEM, n ≥ 3); b calculated using ChemAxon Software Version 5.12.3; c pKa values from
carboxylic acid group; d ELISA data previously reported by us [16]; e Compounds were reported previously by us [5].
As reported previously [10,16], the IC50 value of racemic marinopyrrole A (1) to disrupt the binding
of Mcl-1 to Bim was 8.9 μM. The IC50 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 (3–5) 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 IC50
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

Mar. Drugs 2014, 12
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presumably due to different molecular geometries of the –S– and –SO2– 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 IC50 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 Quenching
To 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 (Kd = 2.7 μM, Figure 3), consistent with its IC50 value in the ELISA assay.
Figure 3. Direct binding of 9 to Mcl-1 measured by fluorescence quenching.
Kd = 2.7 µM
2.5. 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

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growth with 1, 3 and 11 being the most potent with estimated IC50 values of 2, 3 and 2 μM,
respectively. Compounds 4, 5, 6, 7, 8, 9, 10 and 12 were less potent and had estimated IC50 values of
28, 50, 50, 20, 50, 29, 20 and 16 μM, respectively. Finally, the effects of the compounds on
nuclear and cellular morphology were assessed by DAPI nuclear staining and phase imaging.
Figure S1 in Supplementary Information shows that the compounds had little effects on nuclear and
cellular morphology.
Figure 4. Effect of marinopyrroles on Mcl-1 levels in human breast cancer cells.
DMSO 1
4
7
5
8
9
10
3
6
11
12
Mcl-1
Cleaved Caspase 3
Vinculin
3. Experimental Section
3.1. 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
1
was performed with silica gel (200–300 mesh). 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%.
(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis(((2-hydroxy-4-(β-ethylacetate)thiophenyl)
methanone) (3). Under N2, a mixture of 2 [14] (50 mg, 0.06 mmol), ethyl 2-mercaptoacetate (33 mg,
0.24 mmol), Pd2(dba)3 (2 mg, 0.003 mmol), xantphos (4 mg, 0.006 mmol) and i-Pr2NEt (31 mg,
0.24 mmol) was dissolved in 1,4-dioxane (5 mL). The reaction was heated to reflux and stirred for
about 10 h. The reaction was quenched with water (10 mL) and extracted with EtOAc (15 mL × 3).
The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated in
vacuum. The residue was purified by flash column chromatography (33% EtOAc/petroleum ether,
1
Rf = 0.3) to yield 3 (28 mg, 61%) as a yellow solid. mp 51.8–53.3 °C; H NMR (400 MHz, CDCl3) δ

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1.24–1.29 (m, 6H), 3.71 (s, 2H), 3.76 (s, 2H), 4.19–4.25 (m, 4H), 6.11 (s, 1H), 6.46 (d, J = 8.4 Hz,
1H), 6.73 (d, J = 8.0 Hz, 1H), 6.80 (d, J = 9.6 Hz, 2H), 7.20 (br s, 1H), 7.37 (br s, 1H), 10.51 (br s,
1H), 10.93 (s, 1H), 11.58 (s, 1H) ppm; 13C NMR (CDCl3, 100 MHz) δ 14.01, 14.01, 33.92, 34.17,
62.05, 62.09, 108.68, 111.90, 113.64, 114.13, 116.27, 116.27, 116.47, 116.80, 116.93, 118.74, 121.32,
122.17, 124.17, 124.62, 130.62, 133.37, 147.17, 148.83, 161.82, 162.94, 168.61, 168.70, 185.14,
186.83 ppm; HRMS ESI [M + H]+ calcd for C30H25Cl4N2O8S2 744.9806, found 744.9812; IR (KBr)
3671, 3368, 3080, 2960, 2920, 1919, 1735, 1618, 1585, 1439, 1216, 1145, 1030, 745, 703 cm−1. HPLC
purity, 96.8% (Flow rate: 1.0 mL/min; Column: Agilent ZORBAX 300SB-C8, 5 μm, 150 × 4.6 mm;
Wavelength: UV 254 nm; Temperature: 25 °C; Mobile phase: MeOH:H2O = 90:10; tR = 20.0 min).
(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis(((2-hydroxy-4-benzylthio)phenyl)methanone) (4).
Under N2, a mixture of 2 [14] (50 mg, 0.06 mmol), phenylmethanethiol (32 mg, 0.24 mmol), Pd2(dba)3
(2 mg, 0.003 mmol), Xantphos (4 mg, 0.006 mmol) and i-Pr2NEt (31 mg, 0.24 mmol) was dissolved in
1,4-dioxane (5 mL). The reaction was heated to reflux and stirred for about 4 h. The reaction was
quenched with water (10 mL) and extracted with EtOAc (15 mL × 3). The combined organic layers
were dried over anhydrous sodium sulfate, filtered and concentrated in vacuum. The residue was
purified by flash column chromatography (15% EtOAc/petroleum ether, Rf = 0.3) to yield 4 (45 mg,
1
96%) as a yellow solid. mp 82.4–83.7 °C; H NMR (400 MHz, CDCl3) δ 4.17 (s, 2H), 4.22 (s, 2H),
6.11 (s, 1H), 6.47 (d, J = 8.4 Hz, 1H), 6.61 (br, s, 1H), 6.74 (s, 1H), 6.80 (s, 1H), 7.27–7.41 (m, 12H),
9.90 (br s, 1H), 10.95 (s, 1H), 11.63 (s, 1H) ppm; 13C NMR (CDCl3, 100 MHz) δ 36.24, 36.43, 108.77,
111.98, 113.35, 113.85, 115.84, 116.15, 116.26, 116.79, 117.01, 121.27, 122.15, 124.21, 124.72,
127.65, 127.65, 127.68, 127.68, 128.68, 128.68, 128.68, 128.78, 128.78, 128.78, 128.78, 130.46,
133.14, 135.34, 135.49, 149.49, 151.08, 161.84, 163.04, 185.08, 186.71 ppm; HRMS ESI [M + H]+
calcd for C36H25Cl4N2O4S2 753.0010, found 753.0005; IR (KBr) 3410, 3236, 3061, 3028, 2923, 1616,
1581, 1483, 1448, 1391, 1327, 1223, 1073, 928, 780 cm−1. HPLC purity, 96.9% (Flow rate: 1.0 mL/min;
Column: Agilent ZORBAX 300SB-C8, 5 μm, 150 × 4.6 mm; Wavelength: UV 254 nm; Temperature:
25 °C; Mobile phase: MeOH:H2O = 95:5; tR = 6.6 min).
(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis(((2-hydroxy-4-(4-methoxybenzylthio)phenyl)
methanone) (5). Under N2, a mixture of 2 [14] (20 mg, 0.02 mmol), (4-methoxyphenyl) methanethiol
(15 mg, 0.10 mmol), Pd2(dba)3 (0.8 mg, 0.001 mmol), Xantphos (1.6 mg, 0.002 mmol) and i-Pr2NEt
(12 mg, 0.10 mmol) was dissolved in 1,4-dioxane (3 mL). The reaction was heated to reflux and stirred
for about 4 h. The reaction was quenched with water (10 mL) and extracted with EtOAc (10 mL × 3).
The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated in
vacuum. The residue was purified by flash column chromatography (20% EtOAc/petroleum ether,
1
Rf = 0.3) to yield 5 (17 mg, 85%) as a yellow solid. mp 85.4–86.7 °C; H NMR (400 MHz, CDCl3)
δ 3.78 (s, 3H), 3.79 (s, 3H), 4.13 (s, 2H), 4.18 (s, 2H), 6.11 (s, 1H), 6.46 (d, J = 8.4 Hz, 1H), 6.62 (s,
1H), 6.74 (s, 1H), 6.80 (s, 1H), 6.86 (dd, J = 8.4, 3.2 Hz, 5H), 7.31 (t, J = 8.4 Hz, 5H), 9.71 (br s, 1H),
13
10.99 (s, 1H), 11.64 (s, 1H) ppm; C NMR (CDCl3, 100 MHz) δ 35.72, 35.90, 53.66, 55.25, 108.83,
111.94, 113.30, 113.83, 114.18, 114.18, 114.18, 114.18, 114.18, 115.78, 116.11, 116.24, 116.72,
117.01, 121.23, 121.96, 124.08, 124.77, 127.11, 127.29, 129.85, 129.85, 129.96, 129.96, 130.40,
133.12, 149.68, 151.31, 159.01, 159.06, 161.90, 163.05, 185.02, 186.71 ppm; HRMS ESI [M + H]+
calcd for C38H29Cl4N2O6S2 813.0221, found 813.0228; IR (KBr) 3421, 3253, 2929, 2836, 1702, 1615,

Mar. Drugs 2014, 12
4319
1511, 1447, 1393, 1329, 1248, 1177, 1075, 1034, 943 cm−1. HPLC purity, 96.5% (Flow rate:
1.0 mL/min; Column: Agilent ZORBAX 300SB-C8, 5 μm, 150 × 4.6 mm; Wavelength: UV 254 nm;
Temperature: 25 °C; Mobile phase: MeOH:H2O = 95:5; tR = 6.4 min).
(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis(((2-hydroxy-4-(β-ethylacetate)sulfonylphenyl)
methanone) (6). To a solution of 3 (22 mg, 0.03 mmol) in CH2Cl2 (2 mL) was added a solution of
m-CPBA (31 mg, 0.18 mmol) in CH2Cl2 (1 mL) at room temperature. After being stirred for about
20 h, the reaction was quenched by addition water (10 mL) and extracted with CH2Cl2 (10 mL × 3).
The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated in
vacuum. The residue was purified by flash column chromatography (20% acetone, 27% EtOAc, 53%
1
petroleum ether, Rf = 0.2) to yield 6 (16.7 mg, 70%) as a yellow solid. mp 90.0–91.7 °C; H NMR
(400 MHz, acetone-d6) δ 1.16–1.18 (m, 6H), 4.09–4.14 (m, 4H), 4.33 (s, 2H), 4.42 (s, 2H), 6.48
(s, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.45–7.52 (m, 3H), 7.61 (d, J = 8.0 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H),
10.48 (br s, 2H), 12.33 (br s, 1H) ppm; 13C NMR (acetone-d6, 100 MHz) δ 14.14, 14.14, 60.98, 61.02,
62.58, 62.58, 110.42, 112.21, 117.11, 117.38, 118.94, 119.29, 119.65, 122.89, 124.02, 126.06, 127.21,
128.65, 128.75, 129.08, 131.08, 132.87, 144.04, 144.64, 157.98, 158.59, 163.00, 163.05, 183.41,
184.48 ppm; HRMS ESI [M + H]+ calcd for C30H25Cl4N2O12S2 808.9603, found 808.9599; IR (KBr)
2918, 2850, 2490, 1741, 1637, 1589, 1441, 1407, 1330, 1269, 1215, 1151, 1029, 751, 703, 637 cm−1.
HPLC purity, 98.1% (Flow rate: 1.0 mL/min; Column: Waters C18, 5 μm, 150 × 4.6 mm; Wavelength:
UV 254 nm; Temperature: 25 °C; Mobile phase: MeOH:H2O = 70:30; tR = 30.4 min).
(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis(((2-hydroxy-4-benzylsulfonyl)phenyl)methanone)
(7). To a solution of 4 (30 mg, 0.04 mmol) in CH2Cl2 (2 mL) was added a solution of m-CPBA
(42 mg, 0.24 mmol) in CH2Cl2 (1 mL) at room temperature. After being stirred for about 5 h, the
reaction was quenched by addition water (10 mL) and extracted with CH2Cl2 (10 mL × 3). The
combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated in
vacuum. The residue was purified by flash column chromatography (20% acetone, 27% EtOAc, 53%
1
petroleum ether, Rf = 0.2) to yield 7 (24 mg, 75%) as a yellow solid. mp 130.9–132.7 °C; H NMR
(400 MHz, CDCl3) δ 4.26 (s, 2H), 4.34 (s, 2H), 6.12 (s, 1H), 6.99 (d, J = 8.4 Hz, 1H), 7.05 (d,
J = 8.4 Hz, 1H), 7.14–7.17 (m, 4H), 7.29–7.37 (m, 8H), 7.58 (d, J = 8.4 Hz, 1H), 7.68 (d, J = 8.0 Hz,
1H) ppm; 13C NMR (CDCl3, 100 MHz) δ 62.17, 62.35, 110.30, 110.34, 112.10, 117.23, 117.51,
118.83, 119.18, 119.80, 124.03, 126.02, 127.12, 127.83, 128.40, 128.75, 129.07, 129.15, 129.15,
129.19, 129.19, 129.34, 129.40, 129.45, 131.25, 131.88, 131.88, 131.88, 131.98, 131.98, 132.82,
144.07, 144.21, 158.24, 183.74, 184.70 ppm; HRMS ESI [M + H]+ calcd for C36H25Cl4N2O8S2
816.9806, found 816.9800; IR (KBr) 3078, 3030, 2959, 2920, 2851, 2583, 1730, 1636, 1591, 1142,
1407, 1319, 1149, 1125, 879, 750, 701, 628 cm−1. HPLC purity, 97.1% (Flow rate: 1.0 mL/min;
Column: Agilent ZORBAX 300SB-C8, 5 μm, 150 × 4.6 mm; Wavelength: UV 254 nm; Temperature:
25 °C; Mobile phase: MeOH:H2O = 60:40; tR = 11.9 min).
(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis(((2-hydroxy-4-(4-methoxybenzylsulfonyl)phenyl)
methanone) (8). To a solution of 5 (60 mg, 0.07 mmol) in CH2Cl2 (5 mL) was added a solution of
m-CPBA (128 mg, 0.74 mmol) in CH2Cl2 (2 mL) at room temperature. After being stirred for about 20 h,
the reaction was quenched by adding water (10 mL) and extracted with CH2Cl2 (10 mL × 3).
The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated in

Mar. Drugs 2014, 12
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vacuum. The residue was purified by flash column chromatography (20% acetone, 27% EtOAc, 53%
1
petroleum ether, Rf = 0.2) to yield 8 (42 mg, 65%) as a yellow solid. mp 104.3–106.3 °C; H NMR
(400 MHz, CDCl3) δ 3.77 (s, 6H), 4.20 (s, 2H), 4.31 (s, 2H), 6.17 (s, 1H), 6.80 (t, J = 7.2 Hz, 4H), 6.97
(d, J = 8.4 Hz, 1H), 7.05 (d, J = 8.4 Hz, 5H), 7.16 (s, 1H), 7.36 (s, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.67
13
(d, J = 8.0 Hz, 1H), 10.44 (br s, 1H), 11.00 (br s, 1H) ppm; C NMR (CDCl3, 100 MHz) δ 55.26,
55.26, 61.84, 61.84, 109.45, 112.63, 114.18, 114.18, 114.18, 114.22, 114.22, 117.59, 118.33, 118.57,
118.57, 118.82, 118.93, 122.16, 122.80, 123.45, 124.62, 125.25, 126.44, 131.09, 132.02, 132.02,
132.02, 132.09, 132.09, 133.78, 143.91, 144.84, 160.15, 160.22, 160.63, 161.77, 184.85, 187.06 ppm;
HRMS ESI [M + Na]+ calcd for C38H28Cl4N2NaO10S2 898.9837, found 898.9838; IR (KBr) 2961,
2920, 2850, 1730, 1632, 1592, 1512, 1444, 1257, 1148, 1099, 1030, 798 cm−1. HPLC purity, 95.1%
(Flow rate: 1.0 mL/min; Column: Agilent ZORBAX 300SB-C8, 5 μm, 150 × 4.6 mm; Wavelength:
UV 254 nm; Temperature: 25 °C; Mobile phase: MeOH:H2O = 65:35; tR = 6.0 min).
(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis(((2-hydroxy-4-(β-acetic
acid)thiophenyl)
methanone) (9). To a solution of 3 (20 mg, 0.03 mmol) in a mixture of H2O/THF (1:2, 3 mL) was
added LiOH (15 mg, 0.35 mmol) at room temperature. The reaction was allowed to warm up to 50 °C
and stirred for about 10 h. The reaction was adjusted to pH 5.0 with 0.5 N HCl and extracted with
EtOAc (5 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered
and concentrated in vacuum. The residue was purified by reverse-phase column chromatography (C18
reverse silica gel, 7% AcOH, 22% H2O, 71% MeOH, Rf = 0.2) to yield 9 (15.7 mg, 85%) as a brown
1
solid. mp 137.9–139.7 °C; H NMR (400 MHz, DMSO-d6) δ 3.46 (s, 2H), 3.65 (s, 2H), 6.03 (s, 1H),
13
6.53–6.60 (m, 3H), 6.64 (s, 1H), 7.38 (d, J = 7.6 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H) ppm; C NMR
(DMSO-d6, 100 MHz) δ 36.10, 36.80, 108.30, 109.39, 112.71, 112.94, 115.56, 116.02, 116.84, 118.22,
120.95, 122.60, 123.09, 124.39, 128.56, 131.13, 132.65, 133.61, 144.10, 149.87, 159.26, 160.70,
171.50, 172.90, 181.57, 186.55 ppm; HRMS ESI [M + Na]+ calcd for C26H16Cl4N2NaO8S2 710.9000,
found 710.9009; IR (KBr) 3392, 2955, 2918, 2849, 1592, 1382, 1223, 1023, 671 cm−1. HPLC purity,
98.7% (Flow rate: 1.0 mL/min; Column: Agilent ZORBAX 300SB-C8, 5 μm, 150 × 4.6 mm;
Wavelength: UV 254 nm; Temperature: 25 °C; Mobile phase: MeOH:H2O = 60:40; tR = 8.0 min).
(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis(((2-hydroxy-4-(β-acetic acid)sulfonylphenyl)
methanone) (10). To a solution of 6 (70 mg, 0.09 mmol) in a mixture of H2O/THF (1:2, 5 mL) was
added LiOH (49 mg, 1.13 mmol) at room temperature. The reaction was allowed to warm up to 50 °C
and stir for about 3 h. The reaction was adjusted to pH 5.0 with 0.5 N HCl and extracted with EtOAc
(10 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and
concentrated in vacuum. The residue was purified by reverse column chromatography (C18 reverse
silica gel, 4% AcOH, 38% H2O, 58% MeOH, Rf = 0.2) to yield 10 (62 mg, 95%) as a yellow solid. mp
1
143.2–144.7 °C; H NMR (400 MHz, DMSO-d6) δ 4.12 (s, 2H), 4.18 (s, 2H), 6.22 (s, 1H), 7.15 (d,
J = 8.0 Hz, 1H), 7.27–7.29 (m, 4H), 7.36–7.40 (m, 1H) ppm; 13C NMR (acetone-d6, 100 MHz)
δ 52.09, 52.09, 100.10, 100.18, 100.98, 105.79, 107.20, 108.26, 108.65, 112.73, 113.40, 115.44,
118.90, 118.91, 120.25, 120.40, 120.53, 121.64, 133.09, 146.18, 146.89, 154.58, 163.09, 163.09,
171.58, 172.34 ppm; HRMS ESI [M + Na]+ calcd for C26H17Cl4N2O12S2 752.8977, found 752.8981; IR
(KBr) 3395, 2957, 2923, 1628, 1445, 1407, 1313, 1147, 1026, 1000, 906, 825, 701 cm−1. HPLC purity,

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95.2% (Flow rate: 1.0 mL/min; Column: Agilent ZORBAX 300SB-C8, 5 μm, 150 × 4.6 mm; Wavelength:
UV 254 nm; Temperature: 25 °C; Mobile phase: MeOH:H2O = 60:40; tR = 4.0 min).
3.2. Fluorescence Quenching, Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting
Following Treatment of Intact Human Breast Cancer Cells
3.2.1. Fluorescence Quenching
Recombinant His-tagged Mcl-1 (residues 172–327) was purified as previously described [37].
Intrinsic Trp fluorescence of Mcl-1 was measured with a TECAN M1000 plate reader at excitation
wavelength of 285 nm and emission wavelengths of 310–400 nm. Serial dilutions of compound 9
(starting from 20 μM) in 20 mM K-phosphate pH 7.6, 150 nM KCl was used to assay compound
binding using 2.5 μM of Mcl-1. Each condition is run at triplicate and binding constants are calculated
by nonlinear regression analysis of dose response curves using Prism software 6.0 (Graphpad, La Jolla,
CA, USA). Control experiments included titration of DMSO solvent and fluorescence quenching of
denatured Mcl-1 in the presence of compound.
3.2.2. Enzyme-Linked Immunosorbent Assay
ELISAs were performed using a similar procedure as previously described [16]. Briefly, 40 nM of
biotinylated Bim BH3 peptide (Biomatik, Wilmington, DE, USA) in SuperBlock blocking buffer
(Thermo Scientific Pierce, Rockford, IL, USA) was incubated in high-binding capacity streptavidin
coated plates (Thermo Scientific Pierce, Rockford, IL, USA) for 2 h. Compounds were diluted in
120 µL of PBS containing 10 nM of GST-Mcl-1 or 10 nM GST-Bcl-xL in 1.5-mL tubes for 15 min.
Wells were washed with wash buffer (PBS containing 0.05% Tween-20) and then 100 µL of the
compound/GST-protein mixture was transferred to the Bim-containing wells. The plates were
incubated for 2 h, and then, the wells were washed with wash buffer. HRP-conjugated anti-GST
antibody (Bethyl Laboratories, Montgomery, TX, USA) was diluted 1:2000 in SuperBlock, and
100 µL were transferred to each well. The plate was incubated for 1 h, and then, the wells were washed
with wash buffer followed by PBS. One hundred microliters of SureBlue TMB Microwell Peroxidase
Substrate (VWR International, Radnor, PA, USA) was added to each well, and the plates were
developed for 5–10 min. One hundred microliters of 1 N HCl was added to each well to stop the
reaction, and the absorbance was read at 450 nm using a µQuant plate reader (Bio-Tek Instruments,
Winooski, VT, USA).
3.2.3. Western Blotting Following Treatment of Intact Human Breast Cancer Cells
Treatment of the human breast cancer (MDA-MB-468) cells and Western blotting were performed
using the methods described by us previously [16,38].
3.2.4. MTT Assays, Nuclear and Cellular Morphology
MDA-MB-468 cells were treated and processed for MTT, DAPI nuclear staining and phase
imaging of cells as described previously by us [39].

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4. 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 17.6-fold (3 vs. 1) for disrupting Mcl-1/Bim and Bcl-xL/Bim binding, respectively (Figure 2);
(ii) symmetrical marinopyrroles with CH2Ph and/or CH2(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
(IC50 = 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 (IC50 = 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.
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China
(21321061 and 21132006), the National Science and Technology Major Project of China
(2011ZX09401-304) and the National Basic Research Program of China (973 program, 2010CB833200) to
Yong Qin, as well as funds from the Skaggs Institute for Chemical Biology, a grant from the National
Institutes of Health (USA) to K.C. Nicolaou, a graduate fellowship from the National Science
Foundation to Nicholas L. Simmons, NIH grant R00 HL095929 and Developmental award
P30CA013330-39S1 to E.G., and start-up funds (partial support) from the Moffitt Cancer Center and
the University of Nebraska Medical Center to Rongshi Li. We are grateful to ChemAxon Software for
pKa and log p calculations.
Author Contributions
Conceived and designed the experiments: RL, SMS, YQ. Performed the experiments: CC, HS, JL,
LP, MEB, NB, TPG, YL. Analyzed the data: EG, RL, SMS, YQ. Contributed materials/reagents:

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KCN, NLS. Wrote the first draft of the manuscript: RL. Contributed to the writing of the manuscript:
CC, EG, JL, NLS, RL, SMS.
Conflicts of Interest
The authors declare no conflicts of interest.
References
1. Hughes, C.C.; Prieto-Davo, A.; Jensen, P.R.; Fenical, W. The marinopyrroles, antibiotics of an
unprecedented structure class from a marine Streptomyces sp. Org. Lett. 2008, 10, 629–631.
2. Hughes, C.C.; Kauffman, C.A.; Jensen, P.R.; Fenical, W. Structures, reactivities, and antibiotic
properties of the marinopyrroles A–F. J. Org. Chem. 2010, 75, 3240–3250.
3. Cheng, C.; Pan, L.; Chen, Y.; Song, H.; Qin, Y.; Li, R. Total synthesis of (±)-marinopyrrole a and
its library as potential antibiotic and anticancer agents. J. Comb. Chem. 2010, 12, 541–547.
4. Kanakis, A.A.; Sarli, V. Total synthesis of (±)-marinopyrrole A via copper-mediated N-arylation.
Org. Lett. 2010, 12, 4872–4875.
5. Nicolaou, K.C.; Simmons, N.L.; Chen, J.S.; Haste, N.M.; Nizet, V. Total synthesis and biological
evaluation of marinopyrrole A and analogues. Tetrahedron Lett. 2011, 52, 2041–2043.
6. Liu, Y.; Haste, N.M.; Thienphrapa, W.; Nizet, V.; Hensler, M.; Li, R. Marinopyrrole derivatives
as potential antibiotic agents against methicillin-resistant Staphylococcus aureus (I). Mar. Drugs
2012, 10, 953–962.
7. Pan, L.; Cheng, C.; Song, H. Optimization of synthetic method of marinopyrrole A derivatives.
Chem. J. Chin. Univ. 2012, 33, 1476–1480.
8. Hughes, C.C.; Yang, Y.L.; Liu, W.T.; Dorrestein, P.C.; la Clair, J.J.; Fenical, W. Marinopyrrole A
target elucidation by acyl dye transfer. J. Am. Chem. Soc. 2009, 131, 12094–12096.
9. Yamanaka, K.; Ryan, K.S.; Gulder, T.A.; Hughes, C.C.; Moore, B.S. Flavoenzyme-catalyzed
atropo-selective N,C-bipyrrole homocoupling in marinopyrrole biosynthesis. J. Am. Chem. Soc.
2012, 134, 12434–12437.
10. Doi, K.; Li, R.; Sung, S.S.; Wu, H.; Liu, Y.; Manieri, W.; Krishnegowda, G.; Awwad, A.; Dewey, A.;
Liu, X.; et al. Discovery of marinopyrrole A (Maritoclax) as a selective Mcl-1 antagonist that
overcomes ABT-737 resistance by binding to and targeting Mcl-1 for proteasomal degradation.
J. Biol. Chem. 2012, 287, 10224–10235.
11. Cheng, P.; Clive, D.L.; Fernandopulle, S.; Chen, Z. Racemic marinopyrrole B by total synthesis.
Chem. Commun. 2013, 49, 558–560.
12. Clive, D.L.J.; Cheng, P. The marinopyrroles. Tetrahedron 2013, 69, 5067–5078.
13. Cheng, P.; Shao, W.; Clive, D.L. A general route to 1,3′-bipyrroles. J. Org. Chem. 2013, 78,
11860–11873.
14. Cheng, C.; Liu, Y.; Song, H.; Pan, L.; Li, J.; Qin, Y.; Li, R. Marinopyrrole derivatives as potential
antibiotic agents against methicillin-resistant Staphylococcus aureus (II). Mar. Drugs 2013, 11,
2927–2948.

Mar. Drugs 2014, 12
4324
15. Liu, Y.; Haste, N.H.; Thienphrapa, W.; Li, J.; Nizet, V.; Hensler, M.; Li, R. Marinopyrrole
derivatives as potential antibiotic agents against methicillin-resistant Staphylococcus aureus (III).
Mar. Drugs 2014, 12, 2458–2470.
16. Cheng, C.; Liu, Y.; Balasis, M.E.; Simmons, N.L.; Li, J.; Song, H.; Pan, L.; Qin, Y.;
Nicolaou, K.C.; Sebti, S.M.; et al. Cyclic marinopyrrole derivatives as disruptors of Mcl-1 and
Bcl-xL binding to Bim. Mar. Drugs 2014, 12, 1335–1348.
17. Hanahan, D.; Weinberg, R.A. Hallmarks of Cancer: The Next Generation. Cell 2011, 144, 646–674.
18. Kelly, P.N.; Strasser, A. The role of Bcl-2 and its pro-survival relatives in tumourigenesis and
cancer therapy. Cell Death Differ. 2011, 18, 1414–1424.
19. Mohammad, R.M.; Goustin, A.S.; Aboukameel, A.; Chen, B.; Banerjee, S.; Wang, G.;
Nikolovska-Coleska, Z.; Wang, S.; Al-Katib, A. Preclinical studies of TW-37, a new nonpeptidic
small-molecule inhibitor of Bcl-2, in diffuse large cell lymphoma xenograft model reveal drug
action on both Bcl-2 and Mcl-1. Clin. Cancer Res. 2007, 13, 2226–2235.
20. Paoluzzi, L.; Gonen, M.; Gardner, J.R.; Mastrella, J.; Yang, D.; Holmlund, J.; Sorensen, M.;
Leopold, L.; Manova, K.; Marcucci, G.; et al. Targeting Bcl-2 family members with the BH3
mimetic AT-101 markedly enhances the therapeutic effects of chemotherapeutic agents in in vitro
and in vivo models of B-cell lymphoma. Blood 2008, 111, 5350–5358.
21. Petros, A.M.; Olejniczak, E.T.; Fesik, S.W. Structural biology of the Bcl-2 family of proteins.
Biochim. Biophys. Acta 2004, 1644, 83–94.
22. Konopleva, M.; Contractor, R.; Tsao, T.; Samudio, I.; Ruvolo, P.P.; Kitada, S.; Deng, X.; Zhai, D.;
Shi, Y.X.; Sneed, T.; et al. Mechanisms of apoptosis sensitivity and resistance to the BH3
mimetic ABT-737 in acute myeloid leukemia. Cancer Cell 2006, 10, 375–388.
23. Warr, M.R.; Shore, G.C. Unique biology of Mcl-1: Therapeutic opportunities in cancer.
Curr. Mol. Med. 2008, 8, 138–147.
24. Akgul, C. Mcl-1 is a potential therapeutic target in multiple types of cancer. Cell. Mol. Life Sci.
2009, 66, 1326–1336.
25. Tang, G.; Ding, K.; Nikolovska-Coleska, Z.; Yang, C.Y.; Qiu, S.; Shangary, S.; Wang, R.; Guo, J.;
Gao, W.; Meagher, J.; et al. Structure-based design of flavonoid compounds as a new class of
small-molecule inhibitors of the anti-apoptotic Bcl-2 proteins. J. Med. Chem. 2007, 50, 3163–3166.
26. Bernardo, P.H.; Sivaraman, T.; Wan, K.F.; Xu, J.; Krishnamoorthy, J.; Song, C.M.; Tian, L.;
Chin, J.S.; Lim, D.S.; Mok, H.Y.; et al. Structural insights into the design of small molecule
inhibitors that selectively antagonize Mcl-1. J. Med. Chem. 2010, 53, 2314–2318.
27. Rega, M.F.; Wu, B.; Wei, J.; Zhang, Z.; Cellitti, J.F.; Pellecchia, M. SAR by interligand nuclear
overhauser effects (ILOEs) based discovery of acylsulfonamide compounds active against
Bcl-x(L) and Mcl-1. J. Med. Chem. 2011, 54, 6000–6013.
28. Zhang, Z.; Liu, C.; Li, X.; Song, T.; Wu, Z.; Liang, X.; Zhao, Y.; Shen, X.; Chen, H. Fragment-based
design, synthesis, and biological evaluation of N-substituted-5-(4-isopropylthiophenol)-2-hydroxy
nicotinamide derivatives as novel Mcl-1 inhibitors. Eur. J. Med. Chem. 2013, 60, 410–420.
29. Friberg, A.; Vigil, D.; Zhao, B.; Daniels, R.N.; Burke, J.P.; Garcia-Barrantes, P.M.; Camper, D.;
Chauder, B.A.; Lee, T.; Olejniczak, E.T.; et al. Discovery of potent myeloid cell leukemia 1 (Mcl-1)
inhibitors using fragment-based methods and structure-based design. J. Med. Chem. 2013, 56, 15–30.

Mar. Drugs 2014, 12
4325
30. Ding, X.; Li, Y.; Lv, L.; Zhou, M.; Han, L.; Zhang, Z.; Ba, Q.; Li, J.; Wang, H.; Liu, H.; et al.
De novo design, synthesis and evaluation of benzylpiperazine derivatives as highly selective
binders of Mcl-1. ChemMedChem 2013, 8, 1986–2014.
31. Abulwerdi, F.; Laio, C.; Liu, M.; Azmi, A.S.; Aboukameel, A.; Mady, A.S.A.; Gulappa, T.;
Cierpicki, T.; Owens, S.; Zhang, T.; et al. A novel small-molecule inhibitor of Mcl-1 blocks
pancreatic cancer growth in vitro and in vivo. Mol. Cancer Ther. 2014, 13, 565–575.
32. Dixon, S.L.; Jurs, P.C. Estimation of pKa for organic oxyacids using calculated atomic charge.
J. Comput. Chem. 1993, 14, 1460–1467.
33. Csizmadia, F.; Tsantili-Kakoulidou, A.; Panderi, I.; Darvas, F. Prediction of distribution
coefficient from structure. 1. Estimation method. J. Pharm. Sci. 1997, 86, 865–871.
34. Liptak, M.D.; Gross, K.C.; Seybold, P.G.; Feldgus, S.; Shields, G.C. Absolute pK(a)
determinations for substituted phenols. J. Am. Chem. Soc. 2002, 124, 6421–6427.
35. Kuhn, B.; Mohr, P.; Stahl, M. Intramolecular hydrogen bonding in medicinal chemistry.
J. Med. Chem. 2010, 53, 2601–2611.
36. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational
approaches to estimate solubility and permeability in drug discovery and development settings.
Adv. Drug Deliv. Rev. 2001, 46, 3–26.
37. Fire, E.; Gulla, S.V.; Grant, R.A.; Keating, A.E. Mcl-1-Bim complexes accommodate surprising
point mutations via minor structural changes. Protein Sci. 2010, 19, 507–519.
38. Balasis, M.E.; Forinash, K.D.; Chen, Y.A.; Fulp, W.J.; Coppola, D.; Hamilton, A.D.; Cheng, J.Q.;
Sebti, S.M. Combination of farnesyltransferase and Akt inhibitors is synergistic in breast cancer
cells and causes significant breast tumor regression in ErbB2 transgenic mice. Clin. Cancer Res.
2011, 17, 2852–2862.
39. Kazi, A.; Sun, J.; Doi K.; Sung, S.S.; Takahashi, Y.; Yin, H.; Rodriguez, J.M.; Becerril, J.;
Berndt, N. ; Hamilton, A.D.; et al. The BH3 alpha-helical mimic BH3-M6 disrupts Bcl-X(L),
Bcl-2, and MCL-1 protein-protein interactions with Bax, Bak, Bad, or Bim and induces apoptosis
in a Bax- and Bim-dependent manner. J. Biol. Chem. 2011, 18, 9382–9392.
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