Design, synthesis and evaluation of marinopyrrole derivatives as selective inhibitors of Mcl-1 binding to pro-apoptotic Bim and dual Mcl-1/Bcl-xL inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.11.035

Inhibition of anti-apoptotic Mcl-1 is a promising anticancer strategy to overcome the survival and chemoresistance of a broad spectrum of human cancers. We previously reported on the identification of a natural product marinopyrrole A (1) that induces apoptosis in Mcl-1-dependent cells through Mcl-1 degradation. Here, we report the design and synthesis of novel marinopyrrole-based analogs and their evaluation as selective inhibitors of Mcl-1 as well as dual Mcl-1/Bcl-xL inhibitors. The most selective Mcl-1 antagonists were 34, 36 and 37 with 16-, 13-and 9-fold more selectivity for disrupting Mcl-1/Bim over Bcl-xL/Bim binding, respectively. Among the most potent dual inhibitors is 42 which inhibited Mcl-1/Bim and Bcl-xL/Bim binding 15-fold (IC₅₀ = 600 nM) and 33-fold (500 nM) more potently than (±)-marinopyrrole A (1), respectively. Fluorescence quenching, NMR analysis and molecular docking indicated binding of marinopyrroles to the BH3 binding site of Mcl-1. Several marinopyrroles potently decreased Mcl-1 cellular levels and induced caspase 3 activation in human breast cancer cells. Our studies provide novel lead marinopyrroles for further optimization as selective Mcl-1 inhibitors and dual Mcl-1 and Bcl-xL inhibitors.

Full text of DOI:10.1016/j.ejmech.2014.11.035

Accepted Manuscript
Design, synthesis and evaluation of Marinopyrrole derivatives as selective inhibitors
of Mcl-1 binding to pro-apoptotic Bim and dual Mcl-1/Bcl-xL inhibitors
Rongshi Li , Chunwei Cheng , Maria E. Balasis , Yan Liu , Thomas P. Garner ,
Kenyon G. Daniel , Jerry Li , Yong Qin , Evripidis Gavathiotis , Said M. Sebti
PII:
S0223-5234(14)01067-8
10.1016/j.ejmech.2014.11.035
EJMECH 7528
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 September 2014
Revised Date: 20 October 2014
Accepted Date: 19 November 2014
Please cite this article as: R. Li, C. Cheng, M.E. Balasis, Y. Liu, T.P. Garner, K.G. Daniel, J. Li, Y. Qin,
E. Gavathiotis, S.M. Sebti, Design, synthesis and evaluation of Marinopyrrole derivatives as selective
inhibitors of Mcl-1 binding to pro-apoptotic Bim and dual Mcl-1/Bcl-xL inhibitors, European Journal of
Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.11.035.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, Synthesis and Evaluation of Marinopyrrole Derivatives as
Selective Inhibitors of Mcl-1 Binding to Pro-apoptotic Bim and Dual Mcl-
1
/Bcl-xL Inhibitors
a,b,c,
‡,d
‡,b
‡,a
‡,e
Rongshi Li, * Chunwei Cheng, Maria E. Balasis, Yan Liu, Thomas P. Garner,
b
b
d,
e,
b,c
Kenyon G. Daniel, Jerry Li, Yong Qin, ** Evripidis Gavathiotis, ** Said M. Sebti
a
Department of Pharmaceutical Sciences, Center for Drug Discovery, College of
Pharmacy, Cancer Genes and Molecular Regulation Program, Fred and Pamela Buffett
Cancer Center, University of Nebraska Medical Center, 986805 Nebraska Medical Center,
Omaha, NE 68198, United States
b
Department of Drug Discovery, Chemical Biology & Molecular Medicine Program,
Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612, United States
c
Department of Oncologic Sciences, Morsani College of Medicine, University of South
Florida, 12901 Bruce B. Downs, Tampa, FL 33612, United States
d
Key Laboratory of Drug Targeting and Drug Delivery Systems of the Ministry of
Education, West China School of Pharmacy, and State Key Laboratory of Biotherapy,
Sichuan University, Chengdu 610041, China
e
Departments of Biochemistry and Medicine, Albert Einstein Cancer Center, Albert
Einstein College of Medicine, Jack and Pearl Resnick Campus, 1300 Morris Park Avenue,
Forchheimer G46, Bronx, NY 10461, United States
1

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ABSTRACT: Inhibition of anti-apoptotic Mcl-1 is a promising anticancer strategy to
overcome the survival and chemoresistance of a broad spectrum of human cancers. We
previously reported on the identification of a natural product marinopyrrole A (1) that
induces apoptosis in Mcl-1-dependent cells through Mcl-1 degradation. Here, we report the
design and synthesis of novel marinopyrrole-based analogues and their evaluation as
selective inhibitors of Mcl-1 as well as dual Mcl-1/Bcl-xL inhibitors. The most selective
Mcl-1 antagonists were 34, 36 and 37 with 16-, 13- and 9-fold more selectivity for
disrupting Mcl-1/Bim over Bcl-xL/Bim binding, respectively. Among the most potent dual
inhibitors is 42 which inhibited Mcl-1/Bim and Bcl-xL/Bim binding 15-fold (IC = 600
5
0
nM) and 33-fold (500 nM) more potently than (±)-marinopyrrole A (1), respectively.
Fluorescence quenching, NMR analysis and molecular docking indicated binding of
marinopyrroles to the BH3 binding site of Mcl-1. Several marinopyrroles potently
decreased Mcl-1 cellular levels and induced caspase 3 activation in human breast cancer
cells. Our studies provide novel “lead” marinopyrroles for further optimization as selective
Mcl-1 inhibitors and dual Mcl-1 and Bcl-xL inhibitors.
1
.
INTRODUCTION
Programmed cell death, or apoptosis, is a physiological mechanism that removes
damaged or unwanted cells during development and maintains tissue homeostasis [1,2].
Deregulation of apoptosis is a hallmark of malignant transformation as well as tumor
resistance to chemotherapy [3,4]. The B-cell lymphoma 2 (Bcl-2) family of proteins are
2

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critical regulators of the mitochondrial apoptotic pathway, comprising pro- and anti-
apoptotic members [5,6]. The c-terminal hydrophobic grooves of anti-apoptotic members
such as Bcl-2, Bcl-xL and Mcl-1 neutralize the activity of pro-apoptotic members by
binding the BH3 death helix of pro-apoptotic members Bax and Bak and the BH3-only
proteins [5,6]. The BH3-only proteins such as Bim, Bid, and Noxa, are a subgroup of pro-
apoptotic Bcl-2 proteins that have a single BH3 death helix that competes the inhibitory
activity of anti-apoptotic Bcl-2 grooves and also directly induces activation of Bax and Bak
[7]. Several structures of anti-apoptotic Bcl-2 proteins demonstrate how their c-terminal
hydrophobic grooves bind the pro-apoptotic BH3 death helices, defining the specific
protein interaction surfaces involved in apoptosis regulation. The inhibition of such
protein-protein interactions can restore apoptosis in cancer cells and it is a promising
therapeutic strategy for cancer therapy [8–10].
Effective targeting of anti-apoptotic Bcl-2 proteins with small molecules poses two
challenges. First, the anti-apoptotic/pro-apoptotic protein-protein interactions have large
and flexible interfaces that are more difficult to target than enzyme/substrate interactions
that involve smaller and more defined active sites [11]. Second, the potency and selectivity
of small molecule inhibitors require optimization for each anti-apoptotic Bcl-2 groove
despite the similarities among them. Despite these challenges, successful drug discovery
campaigns from academic and industry led to anti-apoptotic Bcl-2 inhibitors [12]. For
example, efforts from Abbot Laboratories have yielded two inhibitors undergoing clinical
evaluation, ABT-263 and ABT-199 [13,14]. ABT-263, the orally available analog of ABT-
7
37 [15], potently inhibits Bcl-xL and Bcl-2 whereas ABT-199 is a selective inhibitor of
Bcl-2. Several studies have shown that Bcl-2/Bcl-xL inhibitors have efficacy in select
3

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cancers as single agents when Mcl-1 levels are kept low, and demonstrated that resistance
to these agents can develop from the presence of overexpressed Mcl-1, therefore, limiting
the efficacy of these agents to broader spectrum of cancers [16,17]. Similarly, the
significance of Mcl-1 inhibition in cell survival of several hematological and solid tumors
has been demonstrated by indirect approaches that downregulate the expression or stability
of Mcl-1 [18,19]. Thus, selective and potent Mcl-1 inhibitors will be attractive agents for
the treatment of broad human cancers as single agents or in combination with other Bcl-2
family inhibitors [20].
Previously several Mcl-1 small molecules inhibitors and stapled peptides have been
discovered through structure-based design, high-throughput and fragment-based screening
approaches [21–31]. Most compounds are not highly selective for Mcl-1 or have not been
developed sufficiently for potent cellular and in vivo activity. We previously reported the
identification of a natural product marinopyrrole A (1) that induces apoptosis in Mcl-1-
dependent cells or ABT-737 resistant cells through targeting Mcl-1 degradation [25]. Here,
we report the design and synthesis of novel marinopyrrole-based analogues and their
evaluation as inhibitors of Mcl-1 and Bcl-xL-selective inhibitors as well as dual Mcl-1/Bcl-
xL inhibitors. Fluorescence quenching employed for measurement of direct binding affinity
and HSQC NMR analysis provided information about the binding mode of marinopyrrole-
based analogues. NMR-guided docking studies informed to rationalize the structure-
activity relationship studies and further design of analogues. The marinopyrrole analogues
were tested in cellular assays for their ability to decrease Mcl-1 levels, to inhibit tumor cell
survival and to induce apoptosis in human breast cancer cells.
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2
. RESULTS AND DISCUSSION
.1. Design of marynopyrole A derivatives that disrupt Mcl-1/Bim and Bcl-xL/Bim protein-
2
protein interactions
The unique molecular geometry of (±)-marinopyrrole A (1) offers excellent
opportunities to decorate this natural product-based bispyrrole system for desired activity
and selectivity. Previous NMR studies suggested that marinopyrrole A (1) most favorably
binds to Mcl-1 in a position centered at the p2 and p3 hydrophobic pockets formed by Mcl-
1
helices 4, 5 and 3 that bind the conserved hydrophobic residues Leu and Ile of the BIM
BH3 death helix [25]. The critical contributions of p2 and p3 pockets to the high affinity
and selectivity to Mcl-1 prompted the design of selective small molecule Mcl-1 inhibitors
based on these pockets [32–34]. The structural model of docked marinopyrrole A (1)
(Supplemental Figure 1) suggests that the marinopyrrole scaffold makes interaction
contacts with residues known to interact with Bim such as Phe 254, Val253, Met250,
Met231 in p2 and with Phe228, Ala227, N260, Arg263, Thr266 in p3. Therefore, the
molecular docking suggested that the marinopyrrole scaffold possesses favorable features
that mimic the BIM BH3 binding to the Mcl-1 groove and is amenable to further expansion
and optimization for the generation of more potent and selective binding to Mcl-1.
We undertook a structure-based design approach to better understand and explore
structure-activity relationships (SARs) using potential sites of marinopyrrole A amenable
for optimization as shown in Table 1. Our goal was to expand the interactions towards p1
and p4 pockets to increase binding affinity and selectivity. We aimed to target hydrophobic
interactions with Met231, Leu235, Val249 and hydrogen bonds with Lys234 or His252
towards the p1 pocket. We also aimed to target hydrophobic interactions with residues
5

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Val216, Val220 and potential hydrogen bonds with residues His224 and Asn223 in the
shallower p4 pocket. To this end, we designed a series of novel marinopyrrole derivatives
with substitution at the para-positions (4 and 4') of the two phenyl rings to the carbonyl
groups. Di-substitutions with hydrophobic groups on both phenyl rings furnished
compounds (23 to 28) while those with hydrophilic groups yielded derivatives 29, 30, 34
and 35. Tri-substitutions of “symmetrical” marinopyrroles on both phenyl rings provided
compounds 24, 30 and 36 to 38. Design of “nonsymmetrical” marinopyrroles included
compounds 46 to 52. Extension of functional groups in the para-position of the phenyl
groups with a sulfide spacer furnished compounds 31 to 34. Marinopyrroles with
bistriazole spacer, compounds 36 to 45, were designed to improve solubility and increased
binding to pockets p1 to p4. N-methyl analogues 53 and 54 were designed to investigate the
importance of the free NH group to Mcl-1 binding and selectivity.
2
.2. Chemistry
Starting from our previously reported compound 2 [35], mono-ketone 4 was
obtained in 73% yield over two steps by introduction of ortho-methoxy-para-methylphenyl
group (3 was not isolated) followed by IBX oxidation (Scheme 1). Removal of TBDMS
protecting group with TBAF gave alcohol 5 in 90% yield. Oxidation of 5 by IBX furnished
aldehyde 6 in 90% yield. Bisketone 8 was obtained in 54% yield after introduction of
second ortho-methoxy-para-methylphenyl group (without isolation of 7) followed by IBX
oxidation. Removal of para-toluenesulfonyl group with KOH generated 9 in 98% yield,
which was converted to 10 in 65% yield by chlorination with NCS [35]. The final
symmetrical marinopyrrole derivative 24 was obtained in 85% yield after demethylation
using BBr
3
/DCM [36]. Using our previously reported intermediate 11 [35] as a starting
6

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material, palladium-mediated substitution of the triflate 11 with ethynyltrimethylsilane
furnished 12 in 74% yield (Scheme 2). Demethylation of 12 using BBr /DCM gave 13 in
3
5
3% yield, which was converted to the final symmetrical marinopyrrole 1–3 in 78% yield.
Reduction of triple bonds in 1–3 with atmospheric H /Pd/BaSO provided para-vinyl
2
4
substituted marinopyrrole 26 in 60% yield, which was further reduced with atmospheric
H /Pd/BaSO to para-ethyl marinopyrrole 27 in 96% yield. Schemes 3–5 showed the
2
4
chemistry that we developed to synthesize bistriazole marinopyrrole derivatives. Start from
a common intermediate 14 [35], palladium-mediated substitution of the triflate 14 with
ethynyltrimethylsilane provided 15 in 92% yield, which was converted to 16 in 95% yield
after removal of tosyl group by KOH. Bistriazole marinopyrrole 17 in 78% yield was
constructed using “Click Chemistry” [37]. Chlorination of 17 with NCS generated 18. The
final product 36 was obtained in 50% yield after demethylation of 18 using BBr /DCM
3
(
Scheme 3). In order to improve overall yield, demethylation of 14 [35] using BBr /DCM
3
was performed first to give 19 in 90% yield as shown in Scheme 4. Palladium-mediated
substitution of the triflate 19 with ethynyltrimethylsilane furnished 20 in 98% yield.
Removal of tosyl group in 20 provided 21 in 95% yield. Intermediate 22 was obtained in
8
0% yield using “Click Chemistry” [37], which was subjected to chlorination with NCS to
give the final compound 37. The free carboxylic acid 38 was obtained in 65% yield after
removal of t-butyl group from 37. Compound 1–3 was used as a common starting material
by “Click Chemistry” to produce seven bistriazole marinopyrrole derivatives (39 to 45) as
shown in Scheme 5. The final compounds 39 to 44 were obtained in 55%, 70%, 52%, 48%,
5
2% and 83% yield, respectively. Removal of t-butyl group from 44 furnished 45 in 94%
yield. N-methyl analogue of marinopyrrole 53 was synthesized via N-methylation of a
7

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3
precursor that we reported previously followed by O-demethylation using BBr to generate
5
4 [36].
2
.3. Binding and Structural Characterization of Marinopyrroles to Mcl-1
To validate direct binding of marinopyrrole compounds to the BH3-binding groove
of Mcl-1 and investigate the protein-ligand interactions, we performed binding by
fluorescence-quenching and structural characterization by NMR and molecular docking.
We selected representative compound 35 from the list of symmetrical derivatives and
compound 38 from the list of compounds containing a bistriazole spacer (Table 1). We
first examined the binding affinity of compounds using a fluorescence-quenching (FQ)
assay based on the intrinsic Trp fluorescence of Mcl-1. Using this assay we have confirmed
direct binding of marinopyrrole analogues to Mcl-1 and calculated binding constants for 35
(
K = 3.5 ꢀM) and 38 (K = 2.5 ꢀM) from their corresponding binding isotherms (Figure 1).
d d
1
5
Next, we performed NMR analysis of N Mcl-1 upon titration of compounds 35 and 38.
1
5
The HSQC spectra of N Mcl-1 demonstrated well dispersed peaks characteristic of a
well-folded protein. Upon titration of marinopyrrole compounds Mcl-1 keeps its ordered
structure and does not undergo significant conformational changes (Figure 2, Supplemental
figure 2). The HSQC analysis confirmed binding to Mcl-1 for both compounds and
interaction of the compounds in an intermediate exchange regime, in agreement with the
low ꢀM binding affinity as suggested by FQ. The titration of compounds up to 2:1
compound-to-protein ratio induced significant chemical shift changes in several residues of
Mcl-1 located in the BH3-binding groove and specifically in residues from helices α2, α3,
α4, α5 (Figure 3A). Both compounds induced significant chemical changes in the same
residues of p1–p4 pockets although compound 38 induced significant broadening in several
8

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p1–p4 residues which is consistent with its higher binding affinity compared to 35.
Interestingly, some residues in helices α1, α6 and α7 undergo chemical shifts effects
possibly from allosteric effects from the binding to the BH3-binding groove. Similar
allosteric effects have been also documented with NMR titrations of BH3 helices and Mcl-
1
[38]. Mapping of the chemical shift perturbation data onto the structure of Mcl-1 showed
the position of residues that are affected by binding of the compounds and conclusively
demonstrated that the compounds bind Mcl-1 protein at the same binding site where
conserved BIM BH3 residues bind Mcl-1 (Figure 3A). Taken together, NMR analysis and
mapping of the chemical shift perturbation data onto the structure of Mcl-1 are consistent
with a direct interaction of 35 and 38 with the BH3-binding groove of Mcl-1. Furthermore,
the data also show that 35 and 38 engage more residues in the p1 and p4 pockets than
compound 1, in agreement with our structure-based design approach (Figure 3B).
We next performed molecular docking using constraints from our NMR chemical
shift perturbation data analysis to investigate the compounds conformations and
interactions with the BH3-binding pocket of Mcl-1. The structural models suggest that
compounds 35 and 38 complement the BH3-binding site using an extended conformation
and making several hydrophophic and polar contacts (Figure 4). Both compounds bind with
the marinopyrrole scaffold positioned to the center of the BH3-binding site occupying p2
and p3 pockets as described above. Furthermore the symmetrical substitutions at either end
of the marinopyrrole core extend the interactions of these compounds further to p1 and
particularly for the larger 35 compound to p4, consistent with our analogue design
approach and NMR characterization (Figure 3). Several hydrophobic residues (Phe228,
Met231, L235, Leu246, Val249, Met250, Val253, Phe254, Val258, Gly262 and Leu267)
9

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within the p1, p2 and p3 pockets of the BH3-binding site are predicted to interact with 35
and 38. Furthermore, the marinopyrrole scaffold of 35 and 38 is predicted to form
hydrogen bond interactions with Arg263, Thr266 and Asn260 of helix α5. Interestingly,
the phosphate and carboxylate groups of 35 and 38, respectively, are predicted to form new
hydrogen bonds with the positively charged Lys234 in the p1 pocket. Moreover, 38
docking pose suggests that this compound extends its interactions in the p4 pocket with the
triazole group having contacts with Gly262 and Val216. The docking model suggests that
the aromatic rings of Phe318 and Phe319 are also in close distance with the carboxylate
and triazole groups, however chemical shifts are not observed for these phenylalanines in
helix α8 (Figure 3). Previous studies suggested that p4 pocket in Mcl-1 is shallower and
can accommodate smaller hydrophobic or polar groups in contrast to Bcl-xL [32–33].
Notably, a mutation of the Phe residue in BIM BH3 that binds p4 with a Val residue
changed the selectivity preference to Mcl-1 over Bcl-xL. Thus, the presence of the triazole
and carboxylate groups provides a possible explanation for the selectivity of 38 for Mcl-1
(Table 1). Taken together, the structural models of 35 and 38 bound to Mcl-1 may explain
the interactions of the new marinopyrrole analogues with residues of the BH3-binding
groove and suggest how increased affinity or selectivity can be achieved with interactions
in p1 and p4 pockets.
2
.4. Structure Activity Relationships of Marinopyrrole Derivatives
To investigate SARs of marinopyrrole derivatives, we selected the six sites
amenable for generating analogues as shown in Table 1. The design of analogues included
substitutions on the pyrrole nitrogen, two phenyl rings and substitution on the second
pyrrole ring. We evaluated potency and selectivity of the new analogues using an ELISA
1
0

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assay that evaluates competitive binding to Bim/Mcl-1 and Bim/Bcl-xL complexes (Table
1
). Marinopyrrole 23 [35], the bistrifluoromethyl derivative of 1, exhibited similar potency
to that of 1 against Mcl-1/Bim complex and was slightly more potent against Bcl-xL/Bim
complex. Nonsymmetrical marinopyrroles with either chlorine [39] or fluorine [40]
substitution in ring B (47 to 52) showed that chlorine in either 3' or 4' position improves
potency against both Mcl-1 and Bcl-xL. Chlorine in position 5' improves potency against
Mcl-1 but not Bcl-xL. The presence of fluorine in either 4' or 6' reduced potency against
both Mcl-1 and Bcl-xL. Compounds 24 to 27 with hydrophobic substitutions in the para-
position are two- to nine-fold more potent than 1 against both Mcl-1 and Bcl-xL, with the
ethyl substitution having the best interaction with Mcl-1 (IC = 2.1 µM). However the
5
0
trifluoromethanesulphonate analogue 28 further increased potency against Mcl-1 (IC =
5
0
1
.0 µM) and Bcl-xL (IC50 = 2.1 µM), nine- and eight-fold compared to 1, respectively.
Marinopyrroles with hydrophilic substitutions in 4' or 5' (29, 30 and 35) are less active than
against Bcl-xL/Bim, with 29 being less active against both Bcl-xL and Mcl-1.
1
Interestingly, these hydrophilic substitutions increased solubility of the compounds but
significantly reduced the potency against Bcl-xL [41]. Thus, the SAR results suggest that
hydrophobic substitutions at either end of the marinopyrrole scaffold lead to increased
affinity by reaching to p1 and p4 pockets, and that the trifluoromethanesulphonate moiety
may have the best fit compared to other hydrophobic substitutions. The presence of
hydrophobic groups in 4' position results in similar binding for Mcl-1 and Bcl-xL pocket as
expected from structural studies with BH3 helices [32,33,42].
Next, we tested symmetrical marinopyrroles with a triazole spacers attached to 4'
position of the phenyl rings (36 to 45). These substitutions increased the flexibility of the
1
1

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molecules; however, the hydrophobic nature of some substituents increased significantly
the potency for Mcl-1 and Bcl-xL. The most potent compound in this series is 42 which is
1
5- (IC = 0.6 µM) and 33-fold (IC = 0.5 µM) more potent than 1 against Mcl-1and Bcl-
50 50
xL, respectively. Furthermore, substituents like ethylester (36 and 43) or carboxylate (38
and 45) attached to the triazole group has somewhat similar activity to Mcl-1 but reduced
significantly potency against Bcl-xL. Compounds 36, 37, 38, 43 and 45 showed three- to
1
3-fold selectivity for Mcl-1 over Bcl-xL, consistent with the tolerability of Mcl-1 for
hydrophilic groups in the p4 pocket and in agreement with our SAR results and molecular
docking analysis.
Furthermore, we recently reported on marinopyrrole analogues with sulfide spacers
3
1–33 found that the most potent 32 and 33 inhibit potently Mcl-1/Bim and Bcl-xL/Bim
binding with IC values of 700 nM and 600 nM, respectively [41]. Interestingly, analogue
5
0
3
4 with the sulfide-acetic acid functional group in the para- positions lost some activity
(
IC = 6.1 µM) compared to 32 and 33 but exhibited 16.4-fold selectivity for Mcl-1 over
5
0
Bcl-xL in agreement with our observation in NMR and docking analysis regarding the
tolerance of hydrophilic group in p4 pocket.
The SAR results uncovered several selective Mcl-1 inhibitors as well as selective
Bcl-xL and dual Mcl-1/Bcl-xL inhibitors. The most selective Mcl-1 antagonist was the
above-mentioned 34 with the carboxylate-containing sulfide spacer having over 16-fold
more selectivity for disrupting Mcl-1/Bim over Bcl-xL/Bim binding. However, neutralizing
the carboxylate negative charge with an ethyl ester as in 31 or replacing the carboxylate
with either a phenyl (32) or a methoxyphenyl (1–11) not only greatly increased the potency
as discussed above, but also reversed the selectivity resulting in some of the most potent
1
2

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Mcl-1 and Bcl-xL dual inhibitors. In the triazole series, 39 with a benzyl and 40 with a
phenyl were 2-fold more selective for Bcl-xL over Mcl-1. However, this selectivity was
reversed towards Mcl-1 when the benzyl or phenyl group was replaced with ethyl
carboxylate as in 43 (5.4 fold), carboxylate as in 45 (3.0 fold), cyclohexyl as in 41 (2.7
fold) or t-butyl carboxylate as in 44 (1.6 fold). Replacement with an octyl group as in 42
resulted in one of the most potent dual Mcl-1 and Bcl-xL antagonists, inhibiting Mcl-1/Bim
and Bcl-xL/Bim binding with IC50 values of 600 nM and 500 nM, respectively. In the
triazole series, adding chloro groups to the 5 and 5' positions increases selectivity for Mcl-1
over Bcl-xL [compare 36 (12.9 fold) to 43 (5.4 fold), 38 (9.6 fold) to 45 (3 fold) and 37
(8.8 fold) to 44 (1.6 fold)]. Moreover, substituting the 4 and 4' hydrogens in 1 with
hydrophobic groups such as vinyl (26), methyl and 5, 5'-Cl (24), ethyne (25), ethyl (27) and
CF (23) all lead to dual Mcl-1 and Bcl-xL antagonists. Taken together these SAR studies
3
provide further starting points to design selective or dual Mcl-1/Bcl-xL antagonists.
2
.5. Marinopyrrole derivatives are highly effective at decreasing the Mcl-1 levels and
inducing apoptosis in human breast cancer cells
To assess the ability of the marinopyrroles to reach their target and induce programmed
cell death (apoptosis) in human cancer cells, we treated MDA-MB-468 breast cancer cells
with the marinopyrrole derivatives at 3 µM and processed the cells for western blotting as
described by us previously [42]. Figure 4 shows that several symmetrical marinopyrrole
derivatives with hydrophobic substituents such as 23 (CF ), 25 (ethyne), 26 (vinyl), 24
3
(
para-CH and meta-Cl), 27 (ethyl) and 28 (trifluoromethanesulfonate) potently decreased
3
Mcl-1 and induced apoptosis as determined by activation of the protease caspase 3. This is
consistent with our previous data that shows that 1 binds Mcl-1 and induces its degradation
1
3

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.
in a proteasome-dependent manner which was associated with apoptosis induction [7]
Several non-symmetrical marinopyrrole derivatives were highly potent at decreasing Mcl-1
levels and inducing apoptosis of MDA-MB-468 breast cancer cells. These include 49, 50
and 51. In contrast, 48 and 52 which were active in vitro against Mcl-1were not active in
intact cells. Similarly, despite being highly potent in vitro, sulfides 31, 32 and 33 as well as
triazoles 37, 40 and 42 had little activity in intact cells (Figure 4). We do not know the
reason for these differences between in vitro and intact cells, but lower cellular uptake,
higher cellular efflux and/or metabolic inactivation are possible mechanisms.
Marinopyrrole derivatives, 29, 30, 34–36, 38, 39, 41, 43–46 were inactive at decreasing
Mcl-1 levels and at inducing caspase 3 cleavage at concentrations as high as 10 µM (data
not shown). As we have reported previously, none of the sulfides or sulphone derivatives
are active at the 3 µM concentration; however, at higher concentrations (40 µM) some of
the sulfide and sulphone derivatives are cell-active [40].
3
. CONCLUSIONS
This article describes the design, synthesis and SAR studies of novel marinopyrrole
derivatives. FQ supports binding of marinopyrroles to Mcl-1 and NMR with molecular
docking suggests their binding mode is within the p1–p4 pockets of the Mcl-1 BH3 groove
that bind the natural α-helical Bim peptide. Our comprehensive SAR studies clearly
demonstrated: i) symmetrical marinopyrroles with hydrophobic, but not hydrophilic,
substituents in the para-position to the carbonyl group are desirable for disrupting Mcl-
1
/Bim and Bcl-xL/Bim protein-protein interactions; ii) substituents with sulfide, but not
sulfone, spacers are tolerated and some greatly increase potency; iii) substituents with
triazole spacers are allowed in most cases against Mcl-1/Bim but not against Bcl-xL/Bim
1
4

ACCEPTED MANUSCRIPT
for 36, 38, 43 and 45; iv) substituents with chlorine or fluorine in B ring of non-
symmetrical marinopyrroles are tolerated in most cases; v) N-methylation of marinopyrrole
A (54) showed selectivity for Bcl-xL/Bim over Mcl-/Bim. In addition, we have identified
several symmetrical and non-symmetrical marinopyrrole derivatives that potently decrease
the levels of Mcl-1 and induce tumor cell apoptosis of human breast cancer cells. Our
studies generated a series of novel “lead” marinopyrroles for further optimization as highly
potent and selective Mcl-1 inhibitors as well as dual Mcl-1 and Bcl-xL inhibitors which can
induce apoptosis of human cancer cells.
4
. EXPERIMENTAL SECTION
4
.1. General
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
1
hydride. Flash column chromatography was performed with silica gel (200–300 mesh). H
13
NMR spectra were recorded at either 400 MHz or 600 MHz at ambient temperature. C
NMR spectra were recorded at either 100 or 150 MHz at ambient temperature. Infrared
spectra were recorded on a spectrophotometer (Perkin-Elmer Spectrum 100). Melting
points were determined with melting point apparatus (Fukai X-4). High resolution mass
spectra were performed by electrospray ionization (ESI) on an Agilent ESI-TOF LC-MS
6
200 system. Analytical HPLC was performed on an Agilent 1100 series with diode array
detectors and auto samplers. All tested compounds possessed a purity of not less than 95%.
1
5

ACCEPTED MANUSCRIPT
4
4
.1.1. (2-(((tert-Butyldimethylsilyl)oxy)methyl)-1'-tosyl-1'H-1,3'-bipyrrol-2'-yl)(2-methoxy-
-methylphenyl)methanol (3)
To a solution of 1-bromo-2-methoxy-4-methylbenzene (524 mg, 2.62 mmol) in anhydrous
THF (5 mL) at –78 °C under N was slowly added n-BuLi (1.15 mL, 2.5 M in n-pentane,
2
2
.88 mmol). After being stirred for 30 min, a solution of 2 [39] (600 mg, 1.31 mmol) in
anhydrous THF (1 mL) was added slowly via a syringe. The reaction was stirred for about
h and quenched by addition of a saturated aqueous NH Cl (15 mL) and extracted with
8
4
EtOAc (10 mL × 3). The combined organic layers were dried over anhydrous sodium
sulfate, filtered and concentrated in vacuum. The residue was purified quickly by column
chromatography (15% EtOAc/petroleum ether, R = 0.3) to yield 3 (unstable).
f
4
.1.2. (2-(((tert-Butyldimethylsilyl)oxy)methyl)-1'-tosyl-1'H-1,3'-bipyrrol-2'-yl)(2-
methoxy-4-methylphenyl)methanone (4)
To a solution of 3 in anhydrous DMSO (20 mL) was added IBX (618 mg, 2.20
mmol) at room temperature. The reaction was allowed to warm up to 30 °C and stirred
additionally for about 6 h. The reaction was quenched with water (30 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 column
chromatography (15% EtOAc/petroleum ether, R
f
= 0.2) to yield 4 (550 mg, 73% yield in
1
two steps) as a pale brown solid. mp 30.7–33.0 °C; H NMR (400 MHz, CDCl
3
) δ 0.006 (s,
6
2
H), 0.86 (s, 9H), 2.38 (s, 3H), 2.40 (s, 3H), 3.75 (s, 3H), 4.67 (s, 2H), 6.20 (dd, J = 4.0,
.4 Hz, 1H), 6.32 (d, J = 3.2 Hz, 1H), 6.67 (dd, J = 4.0, 1.6 Hz, 1H), 6.74 (s, 1H), 6.75 (d, J
7.6 Hz, 1H), 7.06 (dd, J = 2.4, 1.6 Hz, 1H), 7.18 (d, J = 3.6 Hz, 1H), 7.24–7.28 (m, 3H),
=
1
6

ACCEPTED MANUSCRIPT
1
3
7
2
1
1
5
1
3
.84 (d, J = 8.8 Hz, 2H) ppm; C NMR (CDCl , 100 MHz) δ –5.70, –5.70, 18.33, 21.46,
1.71, 25.80, 25.80, 25.80, 53.45, 55.45, 108.90, 111.46, 112.05, 120.34, 121.16, 122.99,
26.70, 126.86, 126.86, 127.20, 129.50, 129.64, 129.64, 129.81, 132.40, 132.62, 136.66,
+
41.84, 144.60, 157.22, 184.12 ppm; HRMS ESI (M+H ) calcd for C H N O SSi
3
1
39
2
5
79.2349, found 579.2358; IR (KBr) 3434, 3443, 2954, 2930, 2856, 1916, 1708, 1936,
–
1
608, 1498, 1409, 1368, 1256, 1179, 1035, 839, 772, 670, 602 cm .
4
.1.3. (2-(Hydroxymethyl)-1'-tosyl-1'H-1,3'-bipyrrol-2'-yl)(2-methoxy-4-
methylphenyl)methanone (5)
To a solution of 4 (550 mg, 0.95 mmol) in anhydrous THF (10 mL) was added
TBAF (745 mg, 2.85 mmol) at room temperature. The reaction was allowed to stir
additionally for about 5 h at room temperature. 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 column chromatography (20% EtOAc/petroleum ether, R = 0.3) to yield 5 (397 mg,
f
1
9
3
0% yield) as a brown-red solid. mp 29.9–31.7 °C; H NMR (400 MHz, CDCl ) δ 2.38 (s,
3
H), 2.40 (s, 3H), 2.97 (t, J = 6.8 Hz, 1H), 3.74 (s, 3H), 4.55 (d, J = 6.8 Hz, 2H), 6.23 (dd,
J = 4.0, 2.4 Hz, 1H), 6.34 (d, J = 3.6 Hz, 1H), 6.63 (dd, J = 4.0, 1.6 Hz, 1H), 6.73 (s, 1H),
6
.76 (d, J = 7.6 Hz, 1H), 7.00 (dd, J = 2.4, 2.0 Hz, 1H), 7.22 (d, J = 7.6 Hz, 1H), 7.28 (d, J
1
3
=
3
3.6 Hz, 1H), 7.32 (d, J = 8.0 Hz, 2H), 7.85 (d, J = 8.4 Hz, 2H) ppm; C NMR (CDCl ,
1
1
1
00 MHz) δ 21.50, 21.70, 55.02, 55.48, 109.61, 110.85, 112.06, 120.41, 121.03, 123.68,
26.61, 127.08, 127.08, 128.22, 128.72, 129.27, 129.95, 129.95, 132.60, 132.82, 135.73,
+
41.93, 145.21, 157.09, 185.16 ppm; HRMS ESI (M+Na ) calcd for C H N NaO S
2
5
24
2
5
1
7

ACCEPTED MANUSCRIPT
4
87.1304, found 487.1297; IR (KBr) 3445, 3141, 2956, 2930, 1771, 1702, 1631, 1609,
–
1
1
498, 1461, 1410, 1366, 1177, 1138, 1034, 1014, 931, 720 cm .
4
.1.4. 2'-(2-Methoxy-4-methylbenzoyl)-1'-tosyl-1'H-1,3'-bipyrrole-2-carbaldehyde (6)
To a solution of 5 (413 mg, 0.89 mmol) in DMSO (20 mL) was added IBX (374
mg, 1.33 mmol) at room temperature. The reaction was allowed to warm up to 50 °C and
stirred for about 3 h. The reaction was quenched with water (30 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 column
chromatography (15% EtOAc/petroleum ether, R = 0.3) to yield 6 (370 mg, 90% yield) as
f
1
a white solid. mp 116.4–118.0 °C; H NMR (400 MHz, CDCl ) δ 2.38 (s, 3H), 2.42 (s, 3H),
3
3
6
7
.73 (s, 3H), 6.27 (s, 1H), 6.45 (d, J = 3.2 Hz, 1H), 6.68 (d, J = 2.0 Hz, 1H), 6.72 (s, 1H),
.76 (d, J = 7.6 Hz, 1H), 6.99 (s, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 2H),
1
3
3
.68 (d, J = 3.2 Hz, 1H), 7.89 (d, J = 8.0 Hz, 2H), 9.62 (s, 1H) ppm; C NMR (CDCl , 100
MHz) δ 21.53, 21.69, 55.41, 109.99, 111.43, 112.02, 120.41, 123.04, 125.22, 125.99,
1
27.50, 127.94, 127.94, 129.80, 129.83, 129.83, 131.70, 133.68, 134.72, 139.12, 142.26,
+
1
45.71, 157.32, 176.91, 184.11 ppm; HRMS ESI (M+H ) calcd for C H N O S 463.1328,
2
5
23
2
5
found 463.1336; IR (KBr) 3449, 3150, 3129, 2957, 2924, 2854, 1690, 1671, 1606, 1565,
–
1
1
408, 1357, 1263, 1170, 1073, 1009, 773 cm .
4
.1.5. (2-(Hydroxy(2-methoxy-4-methylphenyl)methyl)-1'-tosyl-1'H-1,3'-bipyrrol-2'-yl)(2-
methoxy-4-methylphenyl)methanone (7)
To a solution of 1-bromo-2-methoxy-4-methylbenzene (378 mg, 1.89 mmol) in
anhydrous THF (5 mL) at –78 °C under N was slowly added t-BuLi (1.46 mL, 1.3 M, 1.89
2
mmol). After being stirred for 30 min, a solution of 6 (350 mg, 0.76 mmol) in anhydrous
1
8

ACCEPTED MANUSCRIPT
THF (1 mL) was added slowly via a syringe. The reaction was stirred for about 8 h and
quenched by addition of a saturated aqueous NH Cl (15 mL) and extracted with EtOAc (10
4
mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered
and concentrated in vacuum. The residue was purified quickly by column chromatography
(
12% EtOAc/petroleum ether, R = 0.3) to yield 7 (unstable).
f
4
.1.6. (1'-Tosyl-1'H-1,3'-bipyrrole-2,2'-diyl)bis((2-methoxy-4-methylphenyl)methanone)
(8)
To a solution of 7 in anhydrous DMSO (20 mL) was added IBX (275 mg, 0.98
mmol) at room temperature. After being stirred for about 3 h, the reaction was quenched
with water (30 mL) and extracted with EtOAc (15 mL × 3). The combined organic layers
were dried over anhydrous sodium sulfate, filtered and concentrated in vacuum. The
f
residue was purified by column chromatography (12% EtOAc/petroleum ether, R = 0.2) to
1
yield 8 (240 mg, 54% yield two steps) as a pale brown solid. mp 71.0–72.7 °C; H NMR
(
400 MHz, CDCl ) δ 2.27 (s, 3H), 2.37 (s, 3H), 3.43 (s, 3H), 3.65 (s, 3H), 3.75 (s, 3H), 5.85
3
(t, J = 3.2 Hz, 1H), 6.29 (dd, J = 4.0, 1.6 Hz, 1H), 6.46–6.48 (m, 2H), 6.53 (d, J = 8.0 Hz,
1
1
H), 6.70 (d, J = 8.0 Hz, 1H), 6.73 (s, 2H), 6.96 (d, J = 7.6 Hz, 1H), 7.28 (d, J = 8.0 Hz,
1
3
H), 7.33 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 3.6 Hz, 1H), 7.94 (d, J = 8.0 Hz, 2H) ppm; C
NMR (CDCl , 100 MHz) δ 21.50, 21.62, 21.79, 55.39, 55.39, 108.52, 111.61, 111.99,
3
112.04, 119.88, 120.37, 122.95, 123.41, 124.79, 126.25, 128.07, 128.07, 128.61, 129.41,
1
29.41, 129.41, 129.73, 131.48, 132.17, 132.29, 135.78, 141.65, 144.24, 144.81, 157.25,
+
1
58.29, 183.15, 184.47 ppm; HRMS ESI (M+H ) calcd for C33
H
31
N
2
O
6
S, found 583.1903,
found 583.1890; IR (KBr) 3356, 3006, 2958, 2851, 1631, 1612, 1463, 1408, 1262, 1157,
–
1
1
088, 1033, 859, 746 cm .
1
9

ACCEPTED MANUSCRIPT
4
.1.7. 1'H-1,3'-Bipyrrole-2,2'-diylbis((2-methoxy-4-methylphenyl)methanone) (9)
To a solution of 8 (210 mg, 0.36 mmol) in a mixture of MeOH/THF (1:1, 5 mL)
was added KOH (60 mg, 1.08 mmol) at room temperature. After being stirred for 15 min,
the reaction was adjusted to pH 7.0 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 column chromatography (33%
f
EtOAc/petroleum ether, R = 0.3) to yield 9 (151 mg, 98% yield) as a light white solid. mp
1
1
3
6
7
2
1
1
4
8
71.1–172.3 °C; H NMR (400 MHz, CDCl ) δ 2.25 (s, 3H), 2.38 (s, 3H), 3.67 (s, 3H),
3
.77 (s, 3H), 5.80–5.82 (m, 1H), 6.30 (t, J = 2.8 Hz, 1H), 6.35 (dd, J = 4.0, 1.6 Hz, 1H),
.46–6.48 (m, 2H), 6.62 (t, J = 2.8 Hz, 1H), 6.73–6.74 (m, 2H), 7.02 (t, J = 2.8 Hz, 1H),
1
3
.08 (dd, J = 9.6, 7.6 Hz, 2H), 9.40 (br s, 1H) ppm; C NMR (CDCl , 100 MHz) δ 21.78,
3
1.81, 55.33, 55.58, 108.32, 110.60, 111.35, 112.20, 119.99, 120.63, 122.76, 122.98,
25.23, 125.92, 126.77, 129.01, 129.89, 131.13, 132.22, 132.34, 141.51, 141.55, 156.69,
+
57.43, 183.43, 183.90 ppm; HRMS ESI (M+H ) calcd for C26
H
25
N
2
O
4
429.1814, found
29.1811; IR (KBr) 3357, 3006, 2957, 2852, 1631, 1612, 1462, 1408, 1264, 1127, 1033,
–
1
59, 747 cm .
4
.1.8. (4,4',5,5'-Tetrachloro-1'H-1,3'-bipyrrole-2,2'-diyl)bis((5-chloro-2-methoxy-4-
methylphenyl)methanone) (10)
To a solution of 9 (10 mg, 0.02 mmol) in anhydrous MeCN (1 mL) at room
temperature was added NCS (18.7 mg, 0.14 mmol) slowly. After being stirred for about 20
min at room temperature,the reaction was quenched with water (5 mL) and extracted with
EtOAc (5 mL × 3). The combined organic layers were dried over anhydrous sodium
2
0

ACCEPTED MANUSCRIPT
sulfate, filtered and concentrated in vacuum. The residue was purified by column
chromatography (12% EtOAc/petroleum ether, R = 0.2) to yield 10 (9 mg, 65% yield) as a
f
1
pale brown solid. mp 78.3–80.0 °C; H NMR (400 MHz, CDCl ) δ 2.30 (s, 3H), 2.32 (s,
3
3
1
H), 3.72 (s, 3H), 3.76 (s, 3H), 6.44 (s, 1H), 6.67 (s, 1H), 6.80 (s, 1H), 7.06 (s, 1H), 7.20 (s,
1
3
H), 9.90–10.10 (br s, 1H) ppm; C NMR (CDCl , 100 MHz) δ 20.54, 20.65, 55.80, 55.89,
3
110.91, 111.82, 113.10, 114.04, 120.46, 120.80, 124.20, 124.75, 124.87, 125.07, 125.31,
1
26.30, 128.27, 128.66, 129.86, 130.65, 139.95, 140.16, 155.25, 156.06, 180.59, 180.86
+
ppm; HRMS ESI (M+H ) calcd for C26
H19Cl
6
N
2
O
4
632.9476, found 632.9492; IR (KBr)
–
1
3
232, 2955, 2918, 2849, 1736, 1644, 1604, 1462, 1428, 1401, 1172, 1039, 871, 678 cm .
4
.1.9. (4,4',5,5'-Tetrachloro-1'H-1,3'-bipyrrole-2,2'-diyl)bis((5-chloro-2-hydroxy-4-
methylphenyl)methanone) (24)
To a solution of 10 (47 mg, 0.07 mmol) in anhydrous CH
2
Cl
2
(5 mL) was slowly
added a solution of BBr
3
(75 mg, 0.30 mmol) in anhydrous CH
2
Cl
2
(1 mL) via a syringe
under N at –78 °C. After being stirred for 0.5 h, the reaction was quenched by addition of
2
water (10 mL) and extracted with CH Cl (10 mL × 3). The combined organic layers were
2
2
dried over anhydrous sodium sulfate, filtered and concentrated in vacuum. The residue was
purified by column chromatography (12% EtOAc/petroleum ether, R = 0.2) to yield 24 (38
f
1
mg, 85% yield) as a pale brown solid. mp 84.7–86.0 °C; H NMR (400 MHz, CDCl ) δ
3
2
1
.32 (s, 3H), 2.39 (s, 3H), 6.76 (s, 1H), 6.80 (s, 1H), 6.90 (s, 1H), 7.39 (s, 2H), 9.89 (br s,
1
3
H), 10.29 (s, 1H), 10.98 (s, 1H) ppm; C NMR (CDCl
3
, 100 MHz) δ 20.72, 20.80,
113.28, 117.53, 117.77, 119.80, 119.82, 120.40, 120.44, 120.74, 120.76, 123.34, 123.43,
1
24.38, 124.72, 128.60, 129.65, 130.91, 145.51, 145.71, 159.52, 160.76, 184.34, 185.20
+
ppm; HRMS ESI (M+H ) calcd for C H Cl N O 604.9163, found 604.9168; IR (KBr)
2
4
15
6
2
4
2
1

ACCEPTED MANUSCRIPT
–
1
3
415, 3238, 2955, 2927, 2856, 1628, 1595, 1479, 1430, 1215, 1027, 871, 690 cm . HPLC
purity, 95.6% (Flow rate: 1.0 mL/min; Column: Agilent ZORBAX 300SB-C8, 5 µm, 150 ×
4
2
.6 mm; Wavelength: UV 254 nm; Temperature: 25 °C; Mobile phase: MeOH : H O = 75 :
2
R
5; t = 9.0 min).
4
.1.10. (4,4',5,5'-Tetrachloro-1'-tosyl-1'H-1,3'-bipyrrole-2,2'-diyl)bis((2-methoxy-4-
((trimethylsilyl)ethynyl)phenyl)methanone) (12)
Under N , a mixture of 11 [12] (300 mg, 0.30 mmol), ethynyltrimethylsilane (176 mg, 1.80
2
3 4 3
mmol), Pd(PPh ) (70 mg, 0.06 mmol) and Et N (30 mg, 0.30 mmol) was dissolved in
anhydrous DMF (5 mL). The reaction was allowed to stir for about 16 h at room
temperature. The reaction was quenched with water (15 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 column chromatography (10%
EtOAc/petroleum ether, R = 0.3) to yield 12 (199 mg, 74% yield) as a light yellow solid.
f
1
mp 118.7–120.0 °C; H NMR(400 MHz, acetone-d ) δ 0.25 (s, 18H), 2.54 (s, 3H), 3.56 (s,
6
3
H), 3.63 (s, 3H), 6.52 (s, 1H), 6.69 (d, J = 8.0 Hz, 1H), 6.93 (d, J = 2.8 Hz, 2H), 7.04 (dd,
J = 8.0, 1.2 Hz, 1H), 7.13 (d, J = 7.6 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz,
1
3
2
H), 7.96 (d, J = 8.4 Hz, 2H) ppm; C NMR (CDCl
.14, -0.14, -0.14, 21.72, 56.11, 56.11, 96.70, 97.96, 104.84, 105.05, 114.43, 114.66,
15.16, 116.20, 117.25, 118.20, 120.38, 121.80, 123.55, 124.09, 124.78, 127.79, 127.98,
3
, 100 MHz) δ -0.18, -0.18, -0.18, -
0
1
1
1
8
1
29.23, 129.23, 129.80, 130.33, 131.02, 131.02, 131.10, 131.64, 134.42, 134.99, 147.58,
+
58.07, 159.35, 181.79, 183.11 ppm; HRMS ESI (M+H ) calcd for C H Cl N O SSi
4
1
39
4
2
6
2
83.0821, found 883.0812; IR (KBr) 3445, 2960, 2857, 2159, 1654, 1600, 1556, 1456,
–
1
400, 1268, 1250, 1192, 1034, 951, 851 cm .
2
2

ACCEPTED MANUSCRIPT
4
.1.11. (4,4',5,5'-Tetrachloro-1'-tosyl-1'H-1,3'-bipyrrole-2,2'-diyl)bis((2-hydroxy-4-
((trimethylsilyl)ethynyl)phenyl)methanone) (13)
To a solution of 12 (43 mg, 0.05 mmol) in anhydrous CH Cl (5 mL) was slowly
2
2
added a solution of BBr (61 mg, 0.24 mmol) in anhydrous CH Cl (1 mL) via a syringe
3
2
2
under N at –78 °C. After being stirred for 30 min, the reaction was quenched by addition
2
2 2
of water (10 mL) and extracted with CH Cl (10 mL × 3). The combined organic layers
were dried over anhydrous sodium sulfate, filtered and concentrated in vacuum. The
residue was purified by column chromatography (10% EtOAc/petroleum ether, R = 0.2) to
f
1
yield 13 (22 mg, 53% yield) as a pale brown solid. mp 97.3–99.7 °C; H NMR (400 MHz,
acetone-d ) δ 0.27 (s, 18H), 2.52 (s, 3H), 6.86 (s, 1H), 6.91-6.93 (m, 2H), 6.96 (d, J = 8.8
6
Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.56 (d, J = 8.0 Hz, 3H), 7.90
1
3
(
d, J = 8.4 Hz, 2H) ppm; C NMR (CDCl , 100 MHz) δ -0.23, -0.23, -0.23, -0.23, -0.23, -
3
0
1
1
.23, 21.84, 99.67, 103.39, 103.72, 106.30, 112.25, 113.64, 113.86, 120.07, 120.83, 121.20,
21.25, 121.35, 122.60, 122.62, 122.80, 124.44, 128.25, 128.25, 128.26, 130.14, 130.14,
31.68, 131.94, 131.98, 133.23, 133.80, 146.95, 146.95, 162.00, 162.00, 180.55, 188.94
+
ppm; HRMS ESI (M+H ) calcd for C H Cl N O SSi 855.0508, found 855.0502; IR
3
9
35
4
2
6
2
(
KBr) 2957, 2923, 2852, 2159, 1728, 1624, 1547, 1382, 1343, 1245, 1191, 973, 850, 662
–
1
cm .
4
.1.12. (4,4',5,5'-Tetrachloro-1'H-1,3'-bipyrrole-2,2'-diyl)bis((4-ethynyl-2-
hydroxyphenyl)methanone) (25)
To a solution of 13 (22 mg, 0.03 mmol) in a mixture of MeOH/THF (1:1, 3 mL)
was added KOH (7.2 mg, 0.13 mmol) at room temperature. After being stirred for 15 min,
2
3

ACCEPTED MANUSCRIPT
the reaction was adjusted to pH 7.0 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 column chromatography (15%
EtOAc/petroleum ether, R = 0.2) to yield 25 (13 mg, 91% yield) as a light yellow solid. mp
f
1
8
6
1
8
1
1
3.3–84.1 °C; H NMR (400 MHz, acetone-d ) δ 3.90 (s, 1H), 4.00 (s, 1H), 6.49 (s, 1H),
6
.81 (d, J = 8.0 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 7.05 (s, 1H), 7.54 (t, J = 8.4 Hz, 3H),
1
3
3
0.50 (s, 1H), 10.82 (s, 1H) 12.39 (br s, 1H) ppm; C NMR (CDCl , 100 MHz) δ 78.81,
2.37, 82.99, 83.19, 109.91, 120.99, 121.00, 121.09, 121.19, 121.81, 122.40, 122.75,
23.54, 124.80, 125.28, 129.32, 130.71, 131.35, 134.03, 137.85, 141.50, 142.85, 161.00,
+
61.22, 185.62, 186.50 ppm; HRMS ESI (M+H ) calcd for C H Cl N O 556.9629,
2
6
13
4
2
4
found 556.9632; IR (KBr) 3405, 3295, 2969, 2929, 2108, 1701, 1624, 1594, 1551, 1448,
–
1
1
393, 1332, 1246, 1120, 965, 788, 675 cm . HPLC purity, 99.1% (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 : H O = 70 : 30; t = 5.7 min).
2
R
4
.1.13. (4,4',5,5'-Tetrachloro-1'H-1,3'-bipyrrole-2,2'-diyl)bis((2-hydroxy-4-
vinylphenyl)methanone) (26)
2 4
Under 1 atm H , 25 (100 mg, 0.18 mmol) and Pd/BaSO (5 mg) were dissolved in
MeOH (3 mL). The reaction was allowed to cool to 10 °C and stirred for about 30 min. The
suspension was filtered and the filtrate was washed with EtOAc (50 mL). The combined
organic layers were concentrated in vacuum and the residue was purified by column
chromatography (20% EtOAc/petroleum ether, R = 0.2) to yield 26 (61 mg, 60% yield) as
f
1
a yellow solid. mp 76.4–77.7 °C; H NMR (400 MHz, acetone-d ) δ 5.41 (d, J = 10.8 Hz,
6
1
H), 5.49 (d, J = 11.2 Hz, 1H), 5.95 (d, J = 17.6 Hz, 1H), 6.04 (d, J = 17.6 Hz, 1H), 6.50 (s,
2
4

ACCEPTED MANUSCRIPT
1
1
1
H), 6.95-6.79 (m, 2H), 6.81 (dd, J = 8.0, 1.2 Hz, 1H), 6.98 (d, J = 1.2 Hz, 1H), 7.03 (s,
H), 7.07 (d, J = 8.4 Hz, 1H), 7.49 (d, J = 7.2 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 11.16 (s,
1
3
H) ppm; C NMR (acetone-d , 100 MHz) δ 90.24, 107.07, 109.66, 111.20, 115.52,
6
115.60, 117.12, 118.04, 118.27, 119.04, 119.89, 128.70, 130.80, 131.83, 134.74, 136.58,
1
36.60, 137.88, 145.64, 147.00, 152.18, 162.22, 171.91, 172.95, 185.92, 186.38 ppm;
+
HRMS ESI (M+H ) calcd for C H Cl N O 560.9942, found 560.9952; IR (KBr) 3423,
2
6
17
4
2
4
3
7
3
275, 2961, 2926, 1920, 1847, 1737, 1626, 1575, 1499, 1450, 1390, 1353, 1216, 887, 797,
–
1
23 cm . HPLC purity, 99.2% (Flow rate, 1.0 mL/min; Column, Agilent ZORBAX
00SB-C8, 5 µm, 150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25 °C; Mobile
phase: MeOH : H O = 90 : 10; t = 5.1 min).
2
R
4
.1.14. (4,4',5,5'-Tetrachloro-1'H-1,3'-bipyrrole-2,2'-diyl)bis((4-ethyl-2-
hydroxyphenyl)methanone) (27)
Under 1 atm H , 26 (50 mg, 0.09 mmol) and Pd/BaSO (5 mg) were dissolved in
2
4
MeOH (3 mL). The reaction was allowed to stir for about 3 h at room temperature. The
suspension was filtered and the filtrate was washed with EtOAc (50 mL). The combined
organic layers were concentrated in vacuum and the residue was purified by column
chromatography (12% EtOAc/petroleum ether, R
f
= 0.2) to yield 27 (48 mg, 96% yield) as
) δ 1.17–1.24 (m, 6H),
1
a yellow solid. mp 90.3–92.0 °C; H NMR (400 MHz, acetone-d
6
2
8
1
1
1
.59 (dd, J = 15.2, 7.6 Hz, 2H), 2.65 (dd, J = 15.2, 7.6 Hz, 2H), 6.47 (s, 1H), 6.53 (dd, J =
.4, 2.8 Hz, 1H), 6.77–6.80 (m, 3H), 7.35 (br s, 1H), 7.47 (d, J = 8.0 Hz, 1H), 10.80 (br s,
1
3
H), 11.24 (s, 1H), 12.27 (br s, 1H) ppm; C NMR (acetone-d , 100 MHz) δ 15.01, 15.01,
6
5.59, 15.59, 109.53, 117.00, 117.16, 118.24, 118.34, 119.10, 120.31, 121.72, 124.79,
26.30, 128.10, 131.56, 131.56, 134.54, 154.47, 154.47, 156.17, 156.17, 163.15, 163.41,
2
5

ACCEPTED MANUSCRIPT
+
1
5
9
86.65, 188.42 ppm; HRMS ESI (M+H ) calcd for C26
H21Cl
4
N
2
O
4
565.0255, found
65.0261; IR (KBr) 3420, 3251, 2967, 2932, 1628, 1590, 1500, 1450, 1393, 1258, 1124,
–
1
44, 792, 531cm . HPLC purity, 97.5% (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 : H O = 75 : 25; t = 9.4 min).
2
R
4
.1.15. (1'-Tosyl-1'H-1,3'-bipyrrole-2,2'-diyl)bis((2-methoxy-4-
((trimethylsilyl)ethynyl)phenyl)methanone) (15)
Under N
2
, a mixture of 14 [12] (50 mg, 0.06 mmol), ethynyltrimethylsilane (34 mg,
(15 mg, 0.01 mmol) and Et N (18 mg, 0.18 mmol) was dissolved in
0
.35 mmol), Pd(PPh
3
)
4
3
anhydrous DMF (5 mL). The reaction was heated to 60 °C and stirred for 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 column chromatography (10%
EtOAc/petroleum, R = 0.3) to yield 15 (40 mg, 92% yield) as a brown solid. mp 105.3–
f
1
1
06.7 °C; H NMR (400 MHz, CDCl ) δ 0.07 (s, 6H), 0.26 (s, 6H), 0.27 (s, 6H), 2.44 (s,
3
3
H), 3.65 (s, 3H), 3.77 (s, 3H), 5.90 (t, J = 2.8 Hz, 1H), 6.29 (dd, J = 4.0, 1.2 Hz, 1H), 6.44
(
d, J = 3.2 Hz, 1H), 6.71 (br s, 2H), 6.79 (d, J = 7.6 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 6.99
s, 1H), 7.04 (d, J = 7.6 Hz, 1H), 7.21 (d, J = 7.6 Hz, 1H), 7.35 (d, J = 8.0 Hz, 2H), 7.56 (d,
(
1
3
J = 3.2 Hz, 1H), 7.94 (d, J = 8.4 Hz, 2H) ppm; C NMR (CDCl , 100 MHz) δ –0.13, –0.13,
3
–
0.13, –0.13, –0.13, –0.13, –21.71, 55.67, 55.67, 95.75, 96.80, 104.23, 104.31, 109.13,
111.89, 114.16, 114.39, 123.46, 123.55, 123.55, 124.47, 125.81, 127.61, 127.65, 128.31,
1
28.31, 128.52, 129.06, 129.63, 129.63, 129.90, 130.86, 131.92, 132.76, 133.02, 135.74,
+
1
45.16, 156.89, 157.58, 182.54, 184.05 ppm; HRMS ESI (M+H ) calcd for
2
6

ACCEPTED MANUSCRIPT
C
41
H
43
N
2
O
6
SSi
2
747.2380, found 747.2382; IR (KBr) 3443, 3145, 2959, 2857, 2158, 1649,
–
1
1
600, 1556, 1405, 1377, 1272, 1253, 1175, 1135, 1034, 952, 852, 667 cm .
.1.16. 1'H-1,3'-Bipyrrole-2,2'-diylbis((4-ethynyl-2-methoxyphenyl)methanone) (16)
To a solution of 15 (300 mg, 0.40 mmol) in a mixture of MeOH/THF (1:1, 10 mL)
4
was added KOH (113 mg, 2.0 mmol) at room temperature. After being stirred for 2 h, the
reaction was adjusted to pH 7.0 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 column chromatography (33%
EtOAc/petroleum ether, R = 0.3) to yield 16 (171 mg, 95% yield) as a brown solid. mp
f
1
1
3
78.7–180.0 °C; H NMR (400 MHz, CDCl ) δ 3.10 (s, 1H), 3.15 (s, 1H), 3.69 (s, 3H),
3
.79 (s, 3H), 5.88 (dd, J = 4.0, 2.4 Hz, 1H), 6.31 (t, J = 2.4 Hz, 1H), 6.36 (dd, J = 4.0, 1.6
Hz, 1H), 6.64 (t, J = 2.0 Hz, 1H), 6.78 (s, 1H), 6.79 (d, J = 7.6 Hz, 1H), 7.04 (s, 1H), 7.08
1
3
(
t, J = 2.8 Hz, 1H), 7.10–7.12 (m, 3H), 9.43 (br s, 1H) ppm; C NMR (DMSO-d
6
+acetone-
d6, 100 MHz) δ 55.39, 55.65, 80.75, 80.98, 83.21, 83.38, 108.79, 109.96, 114.08, 114.94,
1
1
22.74, 123.51, 123.68, 123.76, 124.20, 124.45, 126.25, 128.65, 129.17, 129.75, 130.70,
+
31.13, 132.10, 133.06, 156.16, 156.69, 181.92, 182.70 ppm; HRMS ESI (M+H ) calcd for
C H N O 449.1501, found 449.1494; IR (KBr) 3339, 3281, 3259, 3130, 2942, 2855,
28
21
2
4
–
1
1
645, 1612, 1559, 1494, 1409, 1263, 1121, 938, 749 cm .
4
.1.17. Diethyl 2,2'-(4,4'-((1'H-1,3'-bipyrrole-2,2'-dicarbonyl)bis(3-methoxy-4,1-
phenylene))bis(1H-1,2,3-triazole-4,1-diyl))diacetate (17)
Under N , a mixture of 16 (50 mg, 0.11 mmol), ethyl 2-azidoacetate (58 mg, 0.44
2
mmol), and CuCl (10 mg, 0.11 mmol) was dissolved in THF (5 mL). The reaction was
2
7

ACCEPTED MANUSCRIPT
allowed to warm up to reflux and stirred for about 10 h. The suspension was filtered and
the filtrate was concentrated in vacuum. The residue was purified by column
chromatography (33% EtOAc/petroleum ether, R = 0.3) to yield 17 (61 mg, 78% yield) as
f
1
a light yellow solid. mp 136.3–137.7 °C; H NMR (400 MHz, CDCl ) δ 1.31–1.41 (m, 6H),
3
3
6
.78 (s, 3H), 3.87 (s, 3H), 4.27–4.33 (m, 4H), 5.22 (s, 4H), 5.87 (dd, J = 4.0, 2.8 Hz, 1H),
.33 (t, J = 2.4 Hz, 1H), 6.36 (dd, J = 4.0, 1.6, 1H), 6.71 (t, J = 2.4 Hz, 1H), 7.01 (d, J = 7.6
Hz, 1H), 7.07 (t, J = 3.2 Hz, 1H), 7.17 (d, J = 7.6 Hz, 1H), 7.22–7.24 (m, 2H), 7.36 (s, 1H),
1
3
7
.55 (s, 1H), 7.89 (s, 1H), 8.00 (s, 1H), 9.45 (br s, 1H) ppm; C NMR (DMSO-d
00 MHz) δ 14.51, 14.59, 51.80, 51.80, 56.04, 56.28, 63.15, 63.15, 108.73, 109.47, 110.08,
11.05, 117.76, 118.22, 124.40, 124.50, 124.55, 124.59, 124.59, 127.38, 129.49, 130.13,
6 3
+CD OD,
1
1
1
1
7
1
30.54, 131.28, 132.62, 133.40, 134.38, 134.48, 134.55, 147.93, 158.25, 158.82, 168.38,
+
68.43, 184.34, 184.86 ppm; HRMS ESI (M+H ) calcd for C H N O 707.2578, found
3
6
35
8
8
07.2588; IR (KBr) 3420, 3265, 3139, 2986, 2942, 2852, 1745, 1634, 1614, 1562, 1412,
–
1
247, 1226, 1132, 1028, 932, 783 cm .
4
.1.18. Diethyl 2,2'-(4,4'-((4,4',5,5'-tetrachloro-1'H-1,3'-bipyrrole-2,2'-dicarbonyl)bis(2-
chloro-5-methoxy-4,1-phenylene))bis(1H-1,2,3-triazole-4,1-diyl))diacetate (18)
To a solution of 17 (40 mg, 0.06mmol) in AcOH (3 mL) at room temperature was
added NCS (54 mg, 0.40 mmol) slowly. The reaction was allowed to stir for about 8 h at
room temperature. The reaction was quenched with water (15 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 column
chromatography (25% EtOAc/petroleum ether, R = 0.2) to yield 18 (5 mg, 10% yield) as a
f
1
brown solid. mp 118.7–120.3 °C; H NMR (400 MHz, CDCl ) δ 1.28-1.36 (m, 6H), 3.84
3
2
8