Natural product inspired library synthesis - Identification of 2,3-diarylbenzofuran and 2,3-dihydrobenzofuran based inhibitors of Chlamydia trachomatis

Article abstract of DOI:10.1016/j.ejmech.2017.11.099

A natural product inspired library was synthesized based on 2,3-diarylbenzofuran and 2,3-diaryl-2,3-dihydrobenzofuran scaffolds. The library of forty-eight compounds was prepared by utilizing Pd-catalyzed one-pot multicomponent reactions and ruthenium-catalyzed intramolecular carbenoid C-H insertions. The compounds were evaluated for antibacterial activity in a panel of test systems including phenotypic, biochemical and image-based screening assays. We identified several potent inhibitors that block intracellular replication of pathogenic Chlamydia trachomatis with IC₅₀ ≤ 3 μM. These new C. trachomatis inhibitors can serve as starting points for the development of specific treatments that reduces the global burden of C. trachomatis infections.

Full text of DOI:10.1016/j.ejmech.2017.11.099

Accepted Manuscript
Natural product inspired library synthesis - Identification of 2,3-diaryl benzofuran and
2,3-dihydrobenzofuran based inhibitors of Chlamydia trachomatis
Michael Saleeb, Sergio Mojica, Anna Eriksson, C. David Andersson, Åsa Gylfe,
Mikael Elofsson
PII:
S0223-5234(17)31002-4
10.1016/j.ejmech.2017.11.099
EJMECH 9974
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 May 2017
Revised Date: 23 November 2017
Accepted Date: 29 November 2017
Please cite this article as: M. Saleeb, S. Mojica, A. Eriksson, C.D. Andersson, Å. Gylfe, M.
Elofsson, Natural product inspired library synthesis - Identification of 2,3-diaryl benzofuran and 2,3-
dihydrobenzofuran based inhibitors of Chlamydia trachomatis, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2017.11.099.
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ACCEPTED MANUSCRIPT

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Natural product inspired library synthesis -
Identification of 2,3-diaryl benzofuran and 2,3-
dihydrobenzofuran based inhibitors of Chlamydia
trachomatis
Michael Saleeb1, Sergio Mojica2, Anna Eriksson3, C. David Andersson1, Åsa Gylfe2,4,5
Mikael Elofsson*1, 5
,
2
1Department of Chemistry, Umeå University, Department of Clinical Microbiology, Umeå
3
4
University, Laboratories for Chemical Biology Umeå, Umeå University. Laboratory for
5
Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå Centre for
Microbial Research (UCMR), Umeå University, 901 87 Umeå, Sweden
Keywords: antibacterial • benzofuran • Chlamydia • 2,3-diaryl-benzofuran • 2,3-diaryl-2,3-
dihydrobenzofuran
Abstract
A natural product inspired library was synthesized based on 2,3-diarylbenzofuran and 2,3-
diaryl-2,3-dihydrobenzofuran scaffolds. The library of forty-eight compounds was prepared
by utilizing Pd-catalyzed one-pot multicomponent reactions and ruthenium-catalyzed
intramolecular carbenoid C-H insertions. The compounds were evaluated for antibacterial
activity in a panel of test systems including phenotypic, biochemical and image-based
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screening assays. We identified several potent inhibitors that block intracellular replication of
pathogenic Chlamydia trachomatis with IC50 ≤ 3 µM. These new C. trachomatis inhibitors
can serve as starting points for the development of specific treatments that reduces the global
burden of C. trachomatis infections.
1. Introduction
Natural products have been a valuable source to identify candidates for drug discovery and
development. [1-3] Our interest in biologically active natural products originates from the
identification of the plant derived resveratrol tetramer, (-)-hopeaphenol (Figure 1) as an
antibacterial agent that blocks the type III secretion system in Pseudomonas aeruginosa and
Yersinia pseudotuberculosis without affecting bacterial growth. [4, 5] In addition, we found
that (-)-hopeaphenol reduced cell entry of the obligate intracellular sexually transmitted
pathogen Chlamydia trachomatis. [4] (-)-Hopeaphenol, the first characterized resveratrol
oligomer, is based on a 2,3-trans-diaryl-2,3-dihydrobenzo[b]furan scaffold. Benzofuran and
2,3-disubstituted benzofuran moieties are widespread in nature and present in a large number
of natural products that display numerous biological activities, including antibacterial,
antifungal, antiviral, antioxidant, anti-inflammatory and anticancer activities (Figure 1). [6-
10] For example, 2-substiuted-3H-benzofurans and 3-benzoyl-2-aryl benzofurans were
reported as antibacterial against Mycobacterium tuberculosis H37Rv and methicillin-resistant
S. aureus (MRSA) respectively. [8]
In the present study, we designed, synthesized, and characterized a natural-product-like
screening library based on 2,3-diarylbenzo[b]furan and 2,3-diaryl-2,3-dihydrobenzo[b]furan
scaffolds to explore these scaffolds as a source for novel antibacterial compounds. This
compound collection was interrogated in a number of phenotypic and biochemical screening
assays leading to identification of potent inhibitors that block intracellular replication of the
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Gram-negative pathogen C. trachomatis. To the best of our knowledge there no benzofurans
besides (-)-hopeaphenol that have previously been reported to display antibacterial activity
against C. trachomatis.
C. trachomatis is a clinically important pathogen that causes over 100 million cases of
sexually transmitted disease annually, [11-13] which may give rise to infertility. [14]
Furthermore, trachoma, an ocular infection caused by C. trachomatis, is responsible for
preventable blindness. [15] More specific treatment of Chlamydia infections would be
beneficial compared to the broad-spectrum antibiotics used today, which have a long-lasting
effect on our commensal microbiota and contribute to the development of antibiotic
resistance. These new anti-chlamydial compounds constitute starting points for the
development of specific treatments that reduces the global burden of C. trachomatis
infections.
2. Results and discussion
2.1 Synthesis
2.1.1 An overview of benzofurans synthesis
While 2,3-disubstituted benzofurans can be readily prepared, [16-22] only few reports
describe efficient methods for the synthesis of 2,3-diaryl-2,3-dihydrobenzofurans and they
mostly focus on the total synthesis of their parental natural products. [23-26] Wai-Hung et al.
applied ruthenium porphyrins and in situ generated diazo compounds to synthesize cis-2,3-
disubstituted-2,3-dihydro-benzofurans via carbenoid C-H insertion. [23] However, only one
example of a non-substituted 2,3-diaryl-2,3-dihydrobenzofuran was disclosed. In a recent
report Soldi et al. [24] elegantly carried out enantioselective synthesis of resveratrol-based
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natural products containing the 2,3-dihydrobenzofuran scaffold by applying carbenoid C-H
insertion with chiral dirhodium catalyst. [24, 27] This approach was also applied for the total
synthesis of (-)-E-δ-viniferin. [26]
We have previously completed the total syntheses of (±)-ampelopsin B and (±)-ε-viniferin
[28] (Figure 1), and more recently we reported the total syntheses of other polyphenolic
nature products including viniferifuran, a resveratrol-picetannol hybrid and anigopreissin A.
[29] To extend the scope of our studies on benzofuran-based natural products, we recently
designed and synthesized a library based on 3-carboxy 2-aryl benzofuran and 3-carboxy 2-
aryl trans 2,3-dihydrobenzofuran scaffolds. [30]
2.1.2 Synthesis of 2,3-diarylbenzo[b]furans series
In this study, we thought to synthesize a set of 2,3-diarylbenzo[b]furans as precursors to
access cis- and trans-2,3-diaryl-2,3-dihydrobenzofuran via catalytic hydrogenation followed
by epimerization based on our earlier results (Scheme 1). [31] The synthesis of 2,3-
diarylbenzo[b]furan was achieved by utilizing efficient one-pot multicomponent reactions
reported by Cacchi [32], Flynn [16, 33], and Larock. [18] It starts with Sonogashira coupling
between an aryl alkyne and 2-iodophenol. Subsequently, one-pot heteroannulative coupling
with aryl iodides affords the 2,3-diarylbenzo[b]furan as depicted in Scheme 2. The library
was then designed by selecting nine commercially available aryl iodides (A1–A9), three 2-
iodophenols (B1–B3), and two aryl-alkynes (C1 and C2) to probe the chemistry outlined in
scheme 1 (Figure 2). The two selected aryl-alkynes, 1-ethynyl-4-methoxybenzene (C1) and
1-ethynyl-4-benzyloxybenzene (C2), carry para protected hydroxyl group that is
mechanistically required to assist the final epimerization under acidic condition as depicted in
Scheme 1. [28, 31] Synthesizing all combinations would have yielded fifty-four molecules.
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To evaluate the feasibility of the chemistry, a representative subset of twenty-eight molecules
was selected.
The microwave-assisted one-pot three-component reaction (Scheme 2) reported by Markina
et al. [18] allowed the synthesis of compounds 1–8 (Table 1) with the non-substituted 2-
iodophenol B1 in 35–91% yields. Unfortunately, this reaction resulted in low yield or no
product when using 2-iodo-5-methoxyphenol B2, or 4-fluoro-2-iodophenol B3. This is due to
that the initial Sonogashira coupling with both 4-methoxy phenyl acetylene C2 and 4-
benzyloxy phenyl acetylene C3 resulted in premature cyclization to give 2-substituted
benzo[b]furans (P-II, compound 9) as product instead of the intermediate 2-(1-alkynyl)
phenol (P-I) leading to the required target 2,3-diarylbenzofuran (P-III) (Scheme 3a). [16, 18]
We, therefore, turned to a method reported by Chaplin and Flynn [16] (Scheme 2) in which
two equivalents of Grignard reagent (CH3MgCl) was used to mask the phenol and to
eliminate the possibility of the premature cyclization. This method allowed the synthesis of
the remaining benzofuran series, compounds 10–16 in 45–91% yields (Table 1). To expand
our library further, variation of functional groups was achieved by demethyaltion of the
phenolic groups with BBr3 (Scheme 4) to yield the corresponding phenolic compounds 17–25
in 62–99% yields (Table 1).
2.1.3 Synthesis of cis- and trans-2,3-diaryl-2,3-dihydrobenzofurans series
We then used our knowledge from the total synthesis of (±)-ampelopsin B and (±)-ε-viniferin
[28] to synthesize the corresponding cis-dihydrobenzofurans via catalytic hydrogenation. We
were able to achieve 30–60% conversion of several benzofurans into the corresponding cis-
dihydrobenzofurans using high-pressure hydrogenation (5 bar) and Pd(OH)2 as a catalyst in
iPrOH. However, it was not possible to obtain full conversion of the starting material while
avoiding benzyl ether cleavage as a consecutive reaction (Scheme 3b). Various
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heterogeneous and homogenous catalysts as well as reaction conditions and solvents were
screened (data not shown). Nevertheless, we could not identify a reliable and efficient
hydrogenation method that can be employed for a divergent synthesis to complete the library.
These results are congruent with our findings during the attempts to synthesize 3-carboxy 2-
aryl cis-2,3-dihydrobenzofurans. [30] Thus, due to these limitations the strategy to obtain
dihydrobenzofurans outlined in Scheme 1 was abandoned.
We instead rerouted the synthetic strategy to synthesize dihydrobenzofurans via
intramolecular carbenoid C-H insertion using salicylaldehydes, aryl iodides and benzyl
halides as depicted in Scheme 5. We selected aryl iodides A5–A11, three salicylaldehydes
D1–D3 corresponding to iodophenols B1–B3, and two different benzyl bromides E1 and E2
as shown in Figure 2. From all possible forty-two combinations a representative subset of
fourteen compounds in the form of cis and trans isomers was selected.
We exploited the ruthenium porphyrin catalyst [23] to synthesize cis-trans diastereomeric
mixtures that can be readily separated (Scheme 5). The 2-hydroxydiarylketones (P-IV) are
key intermediates in this pathway and they are typically prepared by addition of
organolithium or Grignard reagent to aryl aldehyde followed by oxidation. [24, 26] However,
while this procedure is straightforward, it often requires protection and deprotection of
various base sensitive functionalities. In this work, we instead applied direct catalytic
arylation of aldehydic C-H using palladium chloride to obtain P-IV as pluripotent
intermediates. [34] The salicylaldehydes were coupled to the aryl iodides using palladium
chloride to afford the corresponding 2-hydroxylarylketones in 40–93% yields (Scheme 5).
Subsequently these were O-alkylated with two different benzyl bromides to afford
intermediates P-V in essentially quantitative yields (Scheme 5). Aryl tosylhydrazones (P-VI)
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were prepared from the corresponding ketone in 70–99% yields by condensation with p-
toluenesulfonylhydrazide, which was then used as precursors for the carbenoid C-H insertion.
Eventually, two-step one-pot ruthenium carbenoid intramolecular C-H insertion afforded a
racemic cis-trans mixture of the dihydrobenzofurans (P-VII) via in situ generation of diazo
compounds. In this study, we used commercially available ruthenium porphyrin
(RuII(TPP)CO), which yielded a racemic cis-trans mixture in a ratio of 1:1–1:3 and 27–70%
yields over two steps (Table 2). This diastereomeric mixture was subsequently resolved by
HPLC and the stereochemistry was verified through the J coupling constants (see supporting
information Figure S6). For compounds 28, 37, 44 and 48 only the trans isomer was isolated
in > 95% purity, while the cis isomer was only > 85% pure in a mix with the trans isomer.
We have thus explored the chemistry of 2,3-diarylbenzofurans and demonstrated a facile
construction of both 2,3-diarylbenzofurans as well as cis- and trans-2,3-diaryl-2,3-
dihydrobenzofurans libraries. These methods can be further expanded to construct different
libraries of natural products-like compounds based on benzofuran and 1H-indole scaffolds.
2.2 Biological evaluation
2.2.1 Biological screening
The 2,3-diarylbenzofurans and 2,3-diaryl-2,3-dihydrobenzofurans (Table 1 and 2) were then
evaluated in a number of biological assays. Biochemical screening was performed to identify
inhibitors of the ADP-ribose transferase activity of the P. aeruginosa toxin ExoS, [35] the Y.
pseudotuberculosis phosphatase YopH, [36] and the acetaldehydedehydrogenase activity of
AdhE from Escherichia coli. [37, 38] Phenotypic screening was executed with a Y.
pseudotuberculosis luciferase reporter-gene assay for identification of type III secretion
system inhibitors, [39] a lipase reporter assay for the twin arginine translocation (tat) system
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in P. aeruginosa, [40, 41] and image-based screening for compounds that block intracellular
replication of C. trachomatis in HeLa cells. [42] Active compounds were only identified in
the C. trachomatis screen indicating that the compound library does not contain frequent
hitters.
2.2.2 Identification of anti-chlamydial compounds
The C. trachomatis primary screen was performed in two independent experiments at 20 and
50 µM with Z’ factor of 0.6 and 0.8 respectively (see supporting information Figure S1). A
hit selection cut-off was set to two standard deviations based on inhibition of C. trachomatis
replication but also considering toxicity of the compounds based on cell counts (see
supporting information Figure S2 A). Seven compounds were found to be toxic to HeLa cells
at both concentrations with < 70% cell viability at 50 µM and were therefore not considered
for continued experimental investigation (see supporting information Figure S2 B).
2.2.3 Cytotoxicity profile of the identified anti-chlamydial
A toxic effect on the HeLa cells results in a reduction of Chlamydia inclusions and thus
obscures deduction of structure-activity relationships. The resulting twenty-six primary hits
were then subjected to extended cytotoxicity testing using the XTT reduction assay. [43] This
led to the exclusion of additional six compounds due to their dose-dependent toxicity (see
supporting information Table S1). The remaining non-toxic twenty compounds were
subjected to full dose-response analysis at eleven concentrations and subsequent
determination of IC50 values (i.e. 50% inhibition of control Chlamydia inclusion) (Table 3,
supporting information Figure S5). From the 2,3-diarylbenzofuran series 11 compounds
showed anti-chlamydial activity with a reproducible IC50 values between 2–27 µM (Table 3)
and hill-slope [44, 45] values of 1–2. Representative immunofluorescence images for dose-
response analysis of compounds 1, 3 and 44 are shown in Figure 3.
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2.2.4 Structure-activity relationships
In terms of structure-activity relationships, compounds 1, 3, 12, and 16, the top potent
inhibitors with IC50 below 5 µM all contain a pyridyl moiety as a substituent on position 3 of
the benzofuran. Moreover, compounds 3, 12 and 16, the most potent analogues with IC50 of
2.0, 2.7 and 4.2 µM respectively, all shared the 2-flouro-4-pyridyl and the 4-methoxyphenyl
substituents on position 3 and 2 of the benzofuran respectively, while their potency appear to
be less affected by substitution on the fused ring. This trend in activity is valid also for the
less potent analogues 7, 11, and 15, that carry a 4-methoxyphenyl substituent on position 3.
The 2,3-diarylbenzofurans with free phenolic groups were generally cytotoxic in the primary
screening (see supporting information Figure S2 B) and even the few compounds that passed
the primary screening cut-offs (i.e. 17, 18, 19, 23, and 25) were later eliminated in the
extended toxicity evaluation using the XTT assay (see supporting information Table S1).
This data indicate the need for protected phenols in order to reduce the toxicity. When
comparing compounds 1 and 3 with their benzyl-protected analogues, 6 and 4 respectively, it
is clear that the large benzyl group reduces potency possibly due to steric factors and/or
increased lipophilicity.
In the 2,3-diaryl-2,3-dihydrobenzofurans series the eight non-toxic hit compounds displayed
weak C. trachomatis inhibition with IC50 typically above 20 µM (Table 3). In contrast to the
2,3-diarylbenzofuran series, all compounds that belong to the 2,3-diaryl-2,3-
dihydrobenzofuran series featured steep dose-response curves and hill-slope values typically
exceeding 3.5 [44, 45] (see supporting information Figure S5). This group of inhibitors is
thus less attractive for further investigations. One intriguing exception is the non-toxic
compound 44, which showed a reproducible activity with IC50 of 0.9 µM. It cannot be
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excluded that this compound has a different mode of action than the potent 2,3-
diarylbenzofurans i.e. 1, 3, 12 and 16.
2.2.5 Comparable activity with clinically-used antibiotics
We then proceeded to benchmark the most potent compounds (1, 3, 12, 16 and 44) by
comparing them to ofloxacin and azithromycin, two antibiotics used in the clinic to treat C.
trachomatis infections. [46] Ofloxacin is a fluoroquinolone that inhibits bacterial DNA
gyrase, [47] while azithromycin is a macrolide antibiotic that inhibits bacterial proteins
synthesis by binding the ribosome 50S subunit. [48] The compounds demonstrated
comparable IC50 values to the antibiotics (Table 4) and the slope of the IC50 curves was less
steep for the compounds compared to the antibiotics (Figure 4). The most potent compound,
44, was however as potent as antibiotics, and thus constitute a promising starting point for
further development.
2.2.6 Efficacy against Chlamydia strains serovar A and serovar D
As a final step, compound 1, 3, 12, 16 and 44 were evaluated against two other different
Chlamydia strains, serovar D and serovar A. Serovar D is one of the most prevalent C.
trachomatis genital tract isolate worldwide, [49, 50] while serovar L2, which was used for the
primary evaluation, is less common and more virulent causing the locally disseminated
lymphogranuloma venerum infections. Both serovars are associated with variety of genital
infections, but also neonatal infections. [14, 50, 51] In contrast, serovar A associates with
ocular trachoma, which is a leading cause of infectious blindness worldwide. [14, 15, 51] We
found that the compounds are equally active against all strains with IC50 values in the range
of 0.079–5 µM (Table 4 and Figure 4).
2.2.7 Distinct mode of action compared to (-)-hopeaphenol
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Giving that this novel anti-chlamydial class are based on the scaffold of the natural product (-
)-hopeaphenol, we compared their inhibition profile with that of (-)-hopeaphenol. (-)-
Hopeaphenol displays anti-chlamydial activity but only if the bacteria are pretreated with 13–
100 µM of the compound 1 h before the infection of eukaryotic cells. [4] The lack of effect
when adding bacteria and compound simultaneously to the host cells is most likely due to
poor membrane permeability of (-)-hopeaphenol. Furthermore, it was established that (-)-
hopeaphenol irreversibly inhibits the T3S in Y. pseudotuberculosis. [4] In contrast, the 2,3-
diarylbenzofuran class does not block the T3S in Y. pseudotuberculosis and efficiently
inhibits the intracellular replication of Chlamydia without the need for pretreatment.
Altogether, this data suggest that the mechanism behind the anti-chlamydial activity of the
benzofurans is distinct from the mode of action of (-)-hopeaphenol.
2.2.8 In-silico assessment of pharmacological properties
At this point of early drug discovery process, it was then important to incorporate an
assessment of the pharmacological properties for those anti-chlamydial candidates.
Membrane permeability is one of the most important factors in drug absorption, but also it is
a crucial factor when it comes to the development of antibacterial. [52] Bypassing bacterial
membranes has always been a challenging aspect particularly in Gram-negative organisms
where the drug needs to penetrate outer membrane, inner membrane, and peptidoglycan
layer. [52] In case of C. trachomatis additional permeability barriers are formed since
Chlamydia is an obligate intracellular pathogen and a drug act intracellularly has to cross the
host cell plasma membrane and inclusion membrane. We therefore applied in silico
calculation of physicochemical properties including cell permeability and lipophilicity for the
top potent inhibitors 1, 3, 12, 16, and 44 using QikProp program (Schrödinger, LLC, New
York, NY, 2017) (see supporting information Table S2). We used two different in silico
models, Caco-2 [53] and MDCK [54], to predict permeability through the intestinal epithelial
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barriers. It appears that all compounds showed enhanced calculated Caco-2 and MDCK
membrane permeability as well as drug likeness properties (supporting information Table
S2). Clearly, the most potent inhibitor 44 appeared to have the highest permeability rate with
> 9900 nm/sec. Taken together this result support our hypothesis regarding the need for these
antibacterial compounds to cross the host cell membrane and maybe the bacterial inclusion
membrane if this class of inhibitors target proteins or other components residing inside the
inclusion body of C. trachomatis.
3. Conclusions
In conclusion, we have synthesized natural product-inspired compound libraries based on
2,3-diarylbenzofuran and 2,3-diaryl-2,3-dihydrobenzofuran. A phenotypic screening of this
library against Chlamydia infection allowed us to identify non-toxic and potent small-
molecules that inhibit strains representing all clinical presentations of C. trachomatis, serovar
L2, D and A infection with IC50 values in the range of 0.079–3 µM and thus similar to
ofloxacin and azithromycin. These small molecules are structurally distinct, yet more potent
or in the same range of potency as known anti-chlamydial compounds e.g.
salicylacylhydrazides, [55-58] 8-hydroxyquinoline based inhibitors, [59] N-acylated
derivatives of sulfamethoxazole and sulfafurazole, [42] salicylacylhydrazide sulfonamide
hybrids, [60] the Chlamydia protease inhibitor-peptide Boc-Val-Pro-ValP(OPh2) (JO146),
[61, 62] the isoflavone biochanin A, [63] dibenzocyclooctadiene lignans, [64] thiadiazinons
[65] and 2-pyridones. [66] These novel anti-chlamydial compounds can thus serve as starting
points for a continued medicinal chemistry programs, lead optimization, and the development
of a novel anti-chlamydial drug. Importantly, Chlamydia inhibitors, such as compounds 1, 3,
12, 16 and 44 can also serve as chemical probes since genetic systems for Chlamydia spp. are
inefficient and laborious.
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4. Experimental section
4.1 General chemistry
All reactions were carried out under inert atmosphere (N2 gas). Chemicals and reagents were
purchased from Aldrich, Alfa Aesar, AK Scientific, Matrix Scientific or Apollo Scientific.
5,10,15,20-tetraphenyl-21H,23H-porphine ruthenium(II) carbonyl (RuII(TPP)CO) was
purchased from Aldrich. Organic solvents were dried using the dry solvent system (Glass
Contour Solvent Systems, SG Water USA) except CH3CN, CH3OH, PhCH3 and DMSO,
which were dried over activated molecular sieves 3Å. Microwave reactions were performed
in Biotage Initiator. Flash chromatography was performed on Biotage Isolera One using
appropriate SNAP Cartridge KP-Sil or SNAP Ultra HP-Sphere 25 µm, and UV absorbance at
254 nm. TLC was performed on Silica gel 60 F254 (Merck) with detection by UV light unless
staining solution is mentioned. Preparative HPLC separation were performed on Gilson
System HPLC, using a VP 250/21 NUCLEODUR C18 column HTEC 5 µm, with a flow rate
18 mL/min, detection at 214 nm and the eluent system: A: aq. 0.005% HCOOH, and B:
0.005% HCOOH in CH3CN unless otherwise mentioned. The NMR spectra were recorded at
298 K on Bruker-DRX 400 MHz and 600 MHz using the residual peak of the solvent
DMSO-d6 (δH 2.50 ppm) or CDCl3 (δH 7.26 ppm) as internal standard for 1H, and DMSO-d6
(δc 39.50 ppm) and CDCl3 (δc 77.16 ppm) as internal standard for 13C. HRMS was performed
using a Bruker microtof II mass spectrometer with electrospray ionization (ES+); sodium
formate was used for calibration. LC-MS were recorded by detecting positive/negative ion
(ES+/ES-) with an electrospray Water Micromass ZG 2000 instrument using XTerra MS C18
(5 µm, 19x50 mm column) and H2O/CH3CN (0.2% HCOOH) as the eluent system, or with
Agilent 1290 infinity–6150 Quadrupole using YMC Triart C18 (1.9 µm, 20x50 mm column)
and H2O/CH3CN (0.1% HCOOH) as the eluent system. Melting points were measured on
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Electrothermal 1A9000 series Digital Melting Point Apparatus. All target compounds were
>95% pure according to HPLC UV-trace, 1H and 13C NMR.
4.2 Synthesis of 2,3-diarylbenzofuran series
4.2.1 General procedures for the synthesis of 2,3-diarylbenzofuran
Method A [18] (Exemplified by compound 1) (Scheme 2 A):
3-(2-(4-methoxyphenyl)benzofuran-3-yl)pyridine, 1: 2-Iodophenol (1.14 mmol, 250 mg) and
Pd(Ph3P)2Cl (5 mol%, 40 mg) were placed in a 20 mL microwave vial, and purged with N2.
Dry THF (0.8 mL) was added and the mixture was stirred until the iodophenol was
completely dissolved. Then dry triethylamine (2 mL) and a solution of CuI (3 mol%, 5.4 mg)
in triethylamine (1 mL) were added and the mixture was stirred for 10 min. 4-Ethynylanisole
(1.2 equiv., 180.3 mg) in THF (0.5 mL) was then added under N2, and the mixture was stirred
in the microwave reactor for 30 min at 40 °C. The corresponding 3-iodopyridine (1.2 equiv.,
279.6 mg) in dry CH3CN (3.7 mL) was added under N2, and the reaction mixture was heated
in the microwave reactor at 100 °C for 30 min. The reaction was monitored with TLC and
LC-MS. After cooling, the solvents were evaporated and the product was purified on silica
followed by preparative HPLC (25→100% B over 25 min).Method B[16] (Examplified by
compound 12) (Scheme 2 B):
2-fluoro-4-(6-methoxy-2-(4-methoxyphenyl)benzofuran-3-yl)pyridine, 12: To a solution of
the 2-Iodo-5-methoxyphenol (1 mmol, 252 mg) and the 4-Ethynylanisole (1.2 equiv., 166
mg) in dry THF (1.6 mL, 0.6 M of the Iodophenol), at 0 °C under N2, was added CH3MgBr
(THF solution, 2.3 M, 2 equiv., 0.792 mL) dropwise. The mixture was allowed to warm to
room temperature, and Pd(Ph3P)2Cl (3 mol%, 21.3 mg) was added before the mixture was
heated to 65 °C for 2 h under N2. The reaction mixture was then cooled to room temperature
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and the THF was removed under reduced pressure. DMSO (2 mL) was added to the residue
followed by addition of 2-Fluoro-4-iodopyridine (1.2 equiv., 269.7 mg) and the mixture was
heated to 80 °C for 4 h under N2. The mixture was cooled to room temperature and diluted
with EtOAc, washed with H2O and brine. The organic phase was dried over Na2SO4,
concentrated and purified on silica followed by preparative HPLC (25→100% B over 25
min).
4.2.2 General procedure for the deprotection of the permethylated compounds
Method C (Scheme 4):
To a solution of permethylated compound (0.53 mmol, 170 mg) in dry DCM (5 mL, 0.1 M)
at -78 °C was added dropwise BBr3 (0.136 mL, 1.5 equiv./OCH3). The mixture was stirred
and allowed to reach room temperature overnight. The reaction was monitored with TLC
(DCM/CH3OH 10%)/Heptane 7:3 and LC-MS, and quenched by addition of ice-cold
saturated aqueous solution of NaHCO3 dropwise. The mixture was diluted with EtOAc and
washed with H2O and brine. The organic phase was dried over Na2SO4, concentrated under
vacuum and purified on silica using (DCM/CH3OH 10%)/Heptane 7:3 to afford the
corresponding phenol in quantitative yield. The compounds were also purified on preparative
HPLC (25→100% B over 25 min).
4.3 Synthesis of cis- and trans-2,3-diaryl-2,3-dihydrobenzofuran series (Exemplified by
compound 44) (Scheme 5)
4.3.1 General procedure for the synthesis of 2’-hydroxybenzophenone
Method D:[34]
(3,4-difluorophenyl)(2-hydroxy-4-methoxyphenyl)methanone: A vial was charged with 2-
Hydroxy-4-methoxybenzaldehyde (1.97 mmol, 300 mg), PdCl2 (5 mol%, 17.5 mg), 1,2-
15

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Difluoro-4-iodobenzene (2 equiv., 946.7 mg), Na2CO3 (2 equiv., 418.1 mg), LiCl (0.4 equiv.,
16.7 mg), and DMF (19.7 mL, 0.1 M of the aldehyde), purged with N2 and stirred at 110 °C
4–10 h. The reaction was monitored with LC-MS and TLC (TLC conditions: Aliquot was
diluted with CH3OH, eluted with EtOAc/heptane 1:3, and stained with 2,4-
dinitrophenylhydrazine solution). The reaction mixture was filtered over a pad of Celite,
diluted with EtOAc, washed 3 times with water, and the aqueous layers was acidified and
extracted twice with EtOAc. The combined organic layers was dried over Na2SO4,
concentrated and purified on silica using EtOAc/Heptane 1:20→1:9 step gradient) to afford
2’-hydroxybenzophenone in 69.3% yield. (NMR data is given in the supporting
information)4.3.2 General procedure for the synthesis of 2’-benzyloxybenzophenone
Method E:
(3,4-difluorophenyl)(4-methoxy-2-((3-methoxybenzyl)oxy)phenyl) methanone: To a solution
of (3,4-difluorophenyl)(2-hydroxy-4-methoxyphenyl)methanone (0.56 mmol) in acetone
(0.25 M, 2.4 mL) anhydrous K2CO3 (2 equiv., 157 mg) and KI (1 equiv., 94.3 mg) were
added followed by 3-Methoxybenzyl bromide (2 equiv., 0.159 mL), and the mixture was
refluxed and monitored with TLC (EtOAc/Heptane 1:3) till completion. After cooling to
room temperature, the mixture was filtered, concentrated and purified on silica using
EtOAc/Heptane (1:20→1:3 step gradient) to afford 2’-benzyloxybenzophenone in 96.7%
yield. (NMR data is given in the supporting information)4.3.3 General procedure for the
synthesis of tosylhydrazones
Method F:
(E/Z)-N'-((3,4-difluorophenyl)(4-methoxy-2-((3-methoxybenzyl) oxy)phenyl)methylene)-4-
methylbenzenesulfonohydrazide: To a rapidly stirred solution of p-toluenesulphonohydrazide
(2.08 mmol, 387.6 mg) in dry CH3OH (0.6 mL, 3 M) at 60 °C was added dropwise a solution
16

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of 2’-benzyloxybenzophenone (0.52 mmol, 200 mg) in dry CH3OH (1 mL, 0.5 M). The
reaction was stirred at reflux and monitored with TLC (EtOAc/Heptane 1:2) till completion.
The product was precipitated as crystalline, which was filtered off and washed with ice-cold
CH3OH. The remaining reaction mixture and the filtrate was concentrated and purified on
silica using (EtOAc/Heptane 1:2) to afford tosylhydrazone in 78.6% yield. (NMR data is
given in the supporting information)
4.3.4 General procedure for the one-pot, two-step synthesis of 2,3-dihydrobenzofuran
Method G: [23]
3-(3,4-difluorophenyl)-6-methoxy-2-(3-methoxyphenyl)-2,3-dihydrobenzofuran, 44: To a
solution of tosylhydrazone (0.4 mmol, 226 mg) in dry THF (2 mL, 0.2 M) LiHMDS (1.05
equiv., 1 M in THF, 0.450 mL) was added at -78 °C. The mixture was stirred and allowed to
warm to room temperature and stirred for additional 30 min before the solvent was
evaporated under reduced pressure. To the residue was added tetrabutylammonium
bromide (10 mol%, 13.2 mg), 4 Å crushed molecular sieve (0.2 g/mmol, 80 mg), dry toluene
(4 mL, 0.1 M of hydrazone) and RuII(TPP)CO (1 mol%, 6 mg), and the mixture was stirred
at 110 °C for 24 h under N2. The reaction mixture was then filtered over a pad of Celite,
concentrated and purified on silica using EtOAc/Heptane (99:1→95:5, linear gradient). The
diastereoisomers (i.e. cis and trans) were separated with HPLC (25→100% B over 25 min).
4.4 Analytical data for compound 1, 3 and 44
4.4.1 3-(2-(4-methoxyphenyl)benzofuran-3-yl)pyridine (1)
Synthesis: Methods A (300 mg, 88%, amorphous white solid, mp = 110–112 °C),
Chromatography: (10% CH3OH in DCM)/Heptane as the eluent (Step gradient 5→60%)
followed by HPLC purification. 1H NMR (600 MHz, DMSO-d6, see SI for mentioned proton
17

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number): δH 8.71-8.60 (m, 2H, H3a and H3c), 7.93 (dt, J = 8.0 Hz, 1.9 Hz, 1H, H3e), 7.69 (d,
J = 8.3 Hz, 1H, H4), 7.56 (dd, J = 8.0 Hz, 4.8 Hz, 1H, H3d), 7.50 (d, J = 8.9 Hz, 2H, H2b
and H2d), 7.47 (d, J = 7.7 Hz, 1H, H7), 7.39 (d, J = 7.7 Hz, 1H, H6), 7.30 (t, J = 7.7 Hz, 1H,
13
H5), 7.00 (appear as d, J = 8.9 Hz, 2H, H2a and H2e), 3.78 (s, 3H, OCH3) ppm. C NMR
(150 MHz, DMSO-d6): δC 159.88, 153.24, 151.13, 149.84, 148.79, 137.01, 129.15, 128.37,
128.28, 124.94, 124.16, 123.53, 121.79, 119.23, 114.44, 112.30, 111.20, 55.25 ppm. HRMS
m/z calcd for C20H15NO2 302.1176 [M+H+]; observed 302.1215
4.4.2 2-fluoro-4-(2-(4-methoxyphenyl)benzofuran-3-yl)pyridine (3)
Synthesis: Methods A (58 mg, 67%, amorphous yellowish solid, mp = 113–114 °C),
Chromatography: (1% CH3OH in DCM)/Heptane as the eluent (Step gradient elution
1
1→20%) followed by HPLC purification. H NMR (600 MHz, DMSO-d6, see SI for the
mentioned proton number): δH 8.35 (d, J = 5.2 Hz, 1H, H3b), 7.71 (d, J = 8.2 Hz, 1H, H3e),
7.59 (d, J = 7.6 Hz, 1H, H4), 7.55 (appear as d, J = 8.8 Hz, 2H, H2b and H2d), 7.48-7.39 (m,
2H, H3a and H7), 7.38-7.30 (m, 2H, H6 and H5), 7.04 (d, J = 8.7 Hz, 2H, H2a and H2e),
13
1
3.80 (s, 3H, OCH3) ppm. C NMR (150 M Hz, DMSO-d6): δC 163.73 (d, JCF = 235.3 Hz),
3
3
160.27, 153.30, 152.11, 148.45 (d, JCF = 15.7 Hz), 146.38 (d, JCF = 8.8 Hz), 128.95,
128.06, 125.22, 123.77, 122.57 (d, 4JCF = 3.1 Hz), 121.20, 119.38, 119.38, 114.55, 112.38 (d,
2
4JCF = 3.1 Hz), 111.33, 109.48 (d, JCF = 38 Hz), 55.31 ppm. HRMS m/z calcd for
C21H16FNO3 320.1074 [M+H+]; observed 320.1081
4.4.3 3-(3,4-difluorophenyl)-6-methoxy-2-(3-methoxyphenyl)-2,3-dihydrobenzofuran (44)
Synthesis: Methods D–G (20% yield, sticky colorless oil). Diastereoisomers were separated
1
by HPLC as described in general chemistry section. H NMR (600 MHz, DMSO-d6, see SI
for mentioned proton number): δH 7.41 (dt, J = 10.8 Hz, 8.5 Hz, 1H, H3b), 7.33-7.25 (m, 2H,
H3e and H2b), 7.08-7.02 (m, 1H, H3a), 6.94-6.90 (m, 1H, H2e), 6.90-6.86 (m, 2H, H2c and
18

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H2a), 6.84 (d, J = 8.2 Hz, 1H, H4), 6.61 (d, J = 2.2 Hz, 1H, H7), 6.47 (dd, J = 8.2 Hz, 2.3
Hz, 1H, H5), 5.63 (d, J = 7.5 Hz, 1H, H2), 4.60 (d, J = 7.5 Hz, 1H, H3), 3.74 (s, 3H, OCH3),
3.73 (s, 3H, OCH3) ppm. 13C NMR (150 MHz, DMSO-d6): δC 160.61, 160.19, 159.33, 149.40
(dd, 1,2JCF = 246 Hz, 13 Hz), 148.52 (dd, 1,2JCF = 245 Hz, 13 Hz), 141.62, 139.74 (t, 3,3JCF
=
4 Hz), 129.79, 125.13, 124.73 (m), 113.62, 111.47, 107.00, 95.99, 91.71, 55.36, 55.05, 54.58
ppm. HRMS m/z calcd for C22H18F2O3 391.1116 [M+Na+]; observed 391.1105
4.5 Biology
4.5.1 Cell culture and chlamydial infection
To determine compound IC50 values, HeLa299 cells (ATCC, Manassas, VA, USA) were
inoculated into 96 well plates (Corning, New York, USA) with a density of 15,000 cells per
well and cultured overnight at 37° C with 5% CO2 in RPMI 640 (Sigma Aldrich, St. Louis,
MO, USA), supplemented with 10% Fetal Bovine Serum (FBS) and 2 mM L-glutamate. The
cells were infected the following day with C. trachomatis serovar L2 454/Bu (ATCC,
Manassas, VA, USA) at a multiplicity of infection (MOI) of approximately 0.3 in Hank's
Balanced Salt Solution (HBSS) (Life Technologies, Carlsbad, CA, USA). At 1 h post
infection, the inoculum was replaced with RPMI media supplemented as above and
containing 0.5% DMSO and serial 1:1 dilutions of the tested compounds, starting at 50 µM
and reaching 0.1953 µM. The infection was allowed to proceed for 44–48 h before fixation
by aspiration of the media and adding methanol for 5 min. Chlamydial inclusions were
stained with an in-house generated primary rabbit anti-Chlamydia antibody [42] and a
secondary donkey anti-rabbit FITC-labeled antibody (Jackson ImmunoResearch, West
Grove, PA, USA). The DNA of the cells and Chlamydia was stained by DAPI. The number
of inclusions exceeding 130 square microns in size were counted by an ArrayScan VTI HCS
automated reader (Thermo Scientific, Waltham, PA, USA). Inhibition of Chlamydia was
19

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evaluated as the number of inclusions in compound treated infection compared to the
numbers of inclusions in DMSO treated control infections (% of control). IC50 values are
representative of at least three independent experiments. The data analysis was performed
using nonlinear regression (curve fit) in GraphPad Prism v.5. For microscopy, HeLa299 cells
were inoculated onto glass coverslips in 24 well plates. Cells were infected with Chlamydia,
treated with compounds, fixed and stained as described, but using a rhodamine-labelled anti-
rabbit antibody (Jackson ImmunoResearch, West Grove, PA, USA). Infected cells were
observed on a Nikon 90i Fluorescent Microscope using a 20x objective.
4.5.2 XTT reduction assay
HeLa299 cells were inoculated into 96 well plates with a density of 7,000 cells per well and
cultured overnight at 37 °C with 5% CO2 in RPMI 640 supplemented with 10% FBS and 2
mM L-glutamate. The following day, the RPMI was replaced with DMEM without Phenol
Red (Life Technologies, Carlsbad, CA, USA) containing 0.5% DMSO with or without test
compounds at 25 and 50 µM concentrations, with at least three replicate wells for each
compound concentration, and incubated at 37 °C with 5% CO2 for 48 hours. Wells containing
no cells were used as blank controls. Following this incubation with compounds, the XTT
Cell Proliferation Assay kit was used according to the manufacturer’s instructions (ATCC,
Manassas, VA, USA). Briefly, activation reagent and XTT reagent were thawed at 37 °C,
then mixed in a 1:50 ratio with enough volume to add 50 µL to each well. The plates were
placed in the 37 °C incubator for 2.5 hours. The absorbance of each well was then read at 475
nm and 660 nm with a Tecan Infinite m200 plate reader (Tecan, Männedorf, Switzerland). To
analyze the data, the 660 nm reading was subtracted from the 475 nm reading to help
eliminate non-specific readings from the assay results. Average blank control well readings
were subtracted from those containing cells. Final readings from wells containing compound
were compared to DMSO-only control wells and expressed as a percent (% of control).
20

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A)
B)
Figure 1 A) Structures of natural oligomeric resveratrol based on benzofuran and 2,3-diaryl-benzofuran scaffolds including
(-)-hopeaphenol.[67, 68] B) An overlay of energy-minimized conformers of cis-2,3-diaryl-2,3-dihydrobenzofuran, trans-2,3-
diaryl-2,3-dihydrobenzofuran and 2,3-diarylbenzofuran (created in Schrödinger Maestro version 10.5.014).
21

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Aryl Iodides
N
I
F I
I
I
CF3
CN
A1
A2
A4
A3
CN
F
I
F
I
O
I
I
N
A5
A6
A7
A8
F
O
I
I
F
O
F
I
A9
A10
A11
2-Iodophenols
I
I
I
O
O
OH
OH
OH
B1
B2
B3
Alkynes
O
C1
C2
Salicylaldehydes
O
H
O
H
O
H
OH
OH
OH
F
D2
O
D1
D3
Benzyl bromides
Br
O
Br
E1
E2
Figure 2. Building blocks selected for the design and the synthesis of the compound library.
A)
ID
3.12 µM
6.25 µM
12.5 µM
25 µM
50 µM
1
3
Infected control
0.5% DMSO
Uninfected control
0.5% DMSO
B)
ID
1.56 µM
3.13 µM
6.25 µM
12.5 µM
22

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44
Infected control
0.5% DMSO
Uninfected control
0.5% DMSO
Figure 3. Immunofluorescence images for dose-response analysis of A) compounds 1 and 3 and B) compound 44 against C.
trachomatis L2-454/Bu infected HeLa cells fixed with methanol at 48 h post infection and stained using antibodies specific
for Chlamydia inclusions in red and DAPI for DNA (host cell nuclei) in blue. Epifluorescence images were obtained on a
Nikon 90i Fluorescent Microscope (20× objective).
A)
B)
C)
Figure 4. Dose-response curves of most potent compounds against A) C. trachomatis compared to antibiotics ofloxacin and
azithromycin. B) Chlamydia serovar D. C) Chlamydia serovar A.
23

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Scheme 1. Preliminary synthetic strategy for the generation of the 2,3-diarylbenzofurans library.
Scheme 2. Three-component one-pot reactions for the synthesis of 2,3-diarylbenzofurans as reported by Markina et al.[18]
(top) and Chaplin and Flynn[16] (bottom).
a)
b)
Scheme 3. Synthetic challenges faced during the syntheses of 2,3-diarylbenzofuran and 2,3-dihydrobenzofuran. a) Synthesis
of 2,3-diarylbenzofurans via 2-(1-alkynyl) phenol P-I showing the premature cyclized product P-II. b) Hydrogenation
reaction showing the inevitable benzyl ether cleavage and ring opening.
24

ACCEPTED MANUSCRIPT
Scheme 4. Demethylation of protected phenolic compound via BBr3.
Scheme 5.Synthetic sequence for the synthesis of 2,3-diaryl-2,3-dihydrobenzofurans.
Table 1. Synthesized and characterized compounds in the
Table 2. Synthesized and characterized compounds in the 2,3-
diaryl-2,3-dihydrobenzofurans series.
2,3-diarylbenzofurans series.
Structurea
Yieldb
23%
46%
ID
Structure
Yield
88%
ID
Structure
Yield
32%
ID
Structurea
Yieldb
22%
22%
ID
1
26
(cis)
38
(cis)
9
(R=CH3)
17
(R=H)
88%
27
(trans)
39
(trans)
40
17%
17%
(cis)
28
(trans)
60%
73%
40%
2
10
41
(trans)
3
11
29
42
67%
99%
62%
92%
14%
14%
22%
22%
(R=CH3)
(R=CH3)
(cis)
(cis)
18
(R=H)
21
(R=H)
30
(trans)
43
(trans)
12
31
78%
85%
15%
35%
(R=CH3)
(cis)
44
61%
76%
35%
20%
27%
4
5
6
(trans)
22
(R=H)
32
(trans)
33
13%
25%
(cis)
45%
13
45c
34
(trans)
14
(R=CH3)
35
(cis)
46
(cis)
67%
62%
16%
35%
24%
46%
23
(R=H)
36
(trans)
47
(trans)
25

ACCEPTED MANUSCRIPT
7
15
58%
96%
50%
(R=CH3)
(R=CH3)
37
(trans)
48
(trans)
28%
20%
19
(R=H)
24
(R=H)
99%
8
16
[a] All cis and trans compounds are racemic. [b] Isolated yield over two-
step, one-pot reaction and after column chromatography and/or HPLC
separation of the diastereomeric mixture. [c] A single isomer was isolated
as the other isomer was not stable, thus the relative stereochemistry could
not be assigned unambiguously.
91%
89%
58%
92%
(R=CH3)
(R=CH3)
20
(R=H)
25
(R=H)
Table 3. Anti-chlamydial activity and toxicity profile of hit compounds.
HeLa cell
viability %
at 25 µM
82 ± 5
90 ± 8
84 ± 3
76 ± 7
92 ± 8
79 ± 2
106 ± 15
68 ± 7
96 ± 11
82 ± 7
90 ± 19
90 ± 4
88 ± 2
80 ± 4
83 ± 12
84 ± 7
76 ± 6
93 ± 9
72 ± 10
75 ± 6
HeLa cell
viability %
at 50 µM
103 ± 4
96 ± 8
a
ID
IC50 (µM)a
log IC50
1
2
3
6
7
8
11
12
13
15
16
28
30
33
37
38
41
44
46
48
2.4
8.7
2.0
-5.612
-5.059
-5.690
-4.824
-4.937
-4.655
-4.892
-5.564
-4.568
-4.819
-5.376
-4.602
-4.589
-4.702
-4.604
-4.631
-4.974
-6.001
-4.595
-4.566
97 ± 8
82 ± 9
94 ± 5
84 ± 3
15.0
11.6
22.1
12.8
2.7
27.0
15.2
4.2
25.0
25.8
19.8
24.9
23.4
10.6
1.0
100 ± 2
93 ± 10
101 ± 5
99 ± 2
103 ± 5
96 ± 5
103 ± 3
118 ± 4
84 ± 4
102 ± 1
90 ± 3
95 ± 1
88 ± 10
87 ± 8
25.4
27.2
[a] IC50 represents 50% inhibition of control Chlamydia inclusion.
Table 4. IC50 values for the most potent compounds, ofloxacin and azithromycin against C. trachomatis, C. serovar D and C.
serovar A.
IC50 (µM)
C. trachomatis
serovar L2
(IC50 95% CI)a
1.0
(0.925–1.25)
0.965
(0.876–1.06)
1.81
(1.45–2.25)
4.73
(3.92–5.71)
0.581
(0.415–0.795)
0.666
(0.577–0.764)
0.727
IC50 (µM)
C. trachomatis
serovar D
(IC50 95% CI)a
2.44
(2.14–2.80)
2.04
(1.85–2.25)
2.19
(1.87–2.58)
5.04
(4.46–5.70)
0.700
IC50 (µM)
C. trachomatis
serovar A
(IC50 95% CI)
2.63
(1.96–3.46)
1.60
(1.39–1.84)
1.34
(1.1–1.63)
1.84
(1.15–2.75)
0.079
ID
1
3
12
16
44
(0.673–0.727)
(0.074–0.084)
Ofloxacin
Azithromycin
ndb
ndb
ndb
ndb
(0.670–0.784)
[a] IC50 range calculated with 95% confidence interval (CI). [b] not determined
26

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Corresponding Author
Mikael Elofsson, Ph.D Eng, e-mail: mikael.elofsson@umu.se
Department of Chemistry, Umeå University, Linnaeus vägen, 90187, Umeå, Sweden
Conflict of interest
All authors have given approval to the final version of the manuscript and declared no
potential conflicts of interest with respect to the research, authorship, and/or publication of
this article
Acknowledgment
We would like to thank the Swedish Foundation for Strategic Research and the Swedish
Research Council for funding, (SSF, SB12-0022) and (VR, 621-2014-4670) respectively. We
also thank Dr. Caroline Zetterström, Pia Uusitalo, Dr. Jonas Eriksson, Dr. Francesco Massai
for testing the compounds in different screening assays.
Supporting Information
The
supporting
information
is
available
on
the
publication
website.
SI contents: Primary screening statistics and scattar plots, immunofluorescence images and
1
hit selection critiera, cytotoxicity and dose-response curves for all hit compounds, H NMR,
13C NMR, HRMS or LC-MS and melting points for all final compounds and synthesized
1
intermediates, and H NMR and 13C NMR spectra for the reported compounds and
intermediates (file type: PDF).
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