Nontoxic combretafuranone analogues with high in vitro antibacterial activity

Article abstract of DOI:10.1016/j.ejmech.2017.11.078

A library of thirty two 3,4-diphenylfuranones related to both combretastatin A-4 and antifungal 5-(acyloxymethyl)-3-(halophenyl)-2,5-dihydrofuran-2-ones was prepared. Cytotoxic effects on a panel of cancer and normal cell lines and antiinfective activity

Full text of DOI:10.1016/j.ejmech.2017.11.078

Accepted Manuscript
Nontoxic combretafuranone analogues with high in vitro antibacterial activity
P. Horký, M. Voráčová, K. Konečná, D. Sedlák, P. Bartůněk, J. Vacek, J. Kuneš, M.
Pour
PII:
S0223-5234(17)30981-9
10.1016/j.ejmech.2017.11.078
EJMECH 9953
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 August 2017
Revised Date: 27 November 2017
Accepted Date: 27 November 2017
Please cite this article as: P. Horký, M. Voráčová, K. Konečná, D. Sedlák, P. Bartůněk, J. Vacek, J.
Kuneš, M. Pour, Nontoxic combretafuranone analogues with high in vitro antibacterial activity, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.078.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Among 32 novel combretafuranones with substitution related to that of the
antifungal 5-acyloxymethyl-3-halofuranones, noncytotoxic antibacterial
compounds were identified.
HO
5
O
O
O
O
O
O
ZO
HO
3
H2N
MeO
OMe
OMe
Cl
this work
MeO
Z = H, COR
antifungal
Cl
H3C
Br
cytotoxic
MIC95 = 0.98 M S. aureus
noncytotoxic up to 40
ED50 3.3 - 5.3 nM
IC50 0.49 - 15.63
M
M

ACCEPTED MANUSCRIPT
NONTOXIC COMBRETAFURANONE ANALOGUES WITH
HIGH IN VITRO ANTIBACTERIAL ACTIVITY
HORKÝ P.1, VORÁČOVÁ M.1, KONEČNÁ K.2, SEDLÁK D.3, BARTŮNĚK P.3, VACEK
J.4, KUNEŠ J.1, POUR M.1*
1 Department of Bioorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,
Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
2 Department of Biological and Medical Sciences, Faculty of Pharmacy, Charles University,
Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
3 CZ-OPENSCREEN: National Infrastructure for Chemical Biology, Institute of Molecular
Genetics, Czech Academy of Sciences, Vídeňská 1083, 142 20 Praha 4, Czech Republic
4 Department of Medical Chemistry and Biochemistry, Palacký University, Hněvotínská 3,
775 15 Olomouc, Czech Republic
Corresponding Author: * Tel: +420 495067277, E-mail: pour@faf.cuni.cz
KEYWORDS: combretastatin analogue; combretafuranone; furanone; synthesis; cytotoxic;
antibacterial
ABSTRACT: A library of thirty two 3,4-diphenylfuranones related to both combretastatin A-4
and antifungal 5-(acyloxymethyl)-3-(halophenyl)-2,5-dihydrofuran-2-ones was prepared.
Cytotoxic effects on a panel of cancer and normal cell lines and antiinfective activity were
evaluated, and the data were complemented with tests for the activation of caspase 3 and 7.
High cytotoxicity was observed in some of the halogenated analogues, eg. 3-(3,4-
dichlorophenyl)-4-(4-methylphenyl)-2,5-dihydrofuran-2-one with IC50 0.12 – 0.23 µM, but
the compounds were also highly toxic against non-malignant control cells. More importantly,
notable antibacterial activity indicating G+ selectivity has been found in the 3,4-
diarylfuranone class of compounds for the first time. Hydroxymethylation of furanone C5
knocked out cytotoxic effects (up to 40 µM) while maintaining significant activity against
1

ACCEPTED MANUSCRIPT
Staphylococcus strains in some derivatives. MIC95 of the most promising compound, 3-(4-
bromophenyl)-5,5-bis(hydroxymethyl)-4-(4-methylphenyl)-2,5-dihydrofuran-2-one against S.
aureus strain ATCC 6538 was 0.98 µM (0.38 µg/mL) and 3.9 µM (1.52 µg/mL) after 24 and
48 hours, respectively.
1. Introduction
The cis-stilbene fragment is an established privileged structure in medicinal chemistry. The
archetypal compound of this family, combretastatin A-4 (CA4, Fig. 1, 1a), is a well-known
suppressor of tubulin assembly via interaction with the colchicine binding site, which results
in a strong inhibition of cell growth and angiogenesis [1]. In addition, CA-4 also belongs to
the family of vascular disrupting agents (VDAs) targeting tumor vasculature as well [2]. Its
poor water solubility has been resolved by conversion to a water soluble phosphate prodrug,
CA4P [1] (Fig 1, 1b), which is currently undergoing phase II/III clinical trials in combination
with bevacizumab [3]. Another issue of concern often mentioned in literature [4-6] is the
tendency of cis-stilbenes to isomerize to the more stable trans isomers. In response, a huge
number of analogs in which the olefinic linker was replaced by a rigid heterocyclic,
carbocyclic or aromatic motif (Fig. 1, general structure 2) allowing to maintain the positions
of the two vicinal substituted phenyl rings have been made [7-15]. The syntheses of the
compounds are generally straightforward and use low cost chemistry, and, for this reason,
novel bioactives of this class could rank among the least expensive anticancer drugs on the
market. However, despite a vast number of analogues of type 2 that have been prepared [7-
15], with some of them possessing truly amazing low nanomolar biological activities, we are
not aware of any synthetic analogue that would have advanced to clinical trials as of this date.
X
H
H
3
MeO
MeO
OZ
OMe
MeO
MeO
1a
OZ
A
B
4
OMe
OMe
OMe
2
Z = H CA4
1b Z = P(O)(OH)2 CA4P
X = rigid hetero/carbocyclic linker
Figure 1. CA4, CA4P and their restricted heterocylic analogues.
The design of the restricted heterocyclic analogues usually takes into consideration the
SARs developed around the substitution on the phenyl rings [2, 6, 16, 17] . First, the 3,4,5-
2

ACCEPTED MANUSCRIPT
trimethoxy substitution on ring A (Fig. 1, 1a,b) seems to be crucial for the activity, even
though no conclusive answer to this issue has been given. Second, the presence of the intact
4-methoxy group in ring B is also claimed to be important, while the 3-OH group can be
replaced without significant loss of antiproliferative effect [16]. Hence, the substitution of
most derivatives prepared to date follows the highly oxygenated substitution pattern of natural
combretastatine A4.
However, as early as in 1992, Cushman et al. [18] clearly demonstrated upon
extensive screening of a large library of cis-stilbenes that the replacement of the 4-methoxyl
in the B-ring with SMe, Me, Et and even Pr did not lead to a significant loss of activity.
Specifically, the values of ED50 for these compounds against the cell lines used remained
below 10 nmol/L, an undoubtedly excellent level of activity. Only the change for a butyl
group elevated ED50 values to a higher, but still interesting low micromolar range. A recent
paper [19] also describes 3,4-diaryl-1,2,5-selenadiazoles with excellent activity, and devoid of
the 4-OCH3 group in the B ring.
Figure 2. Structures of synthetic furanone analog 3.
Among the rigid heterocyclic components used, only one example using a
2,5-dihydrofuranone core has been reported. In 2002, Ahn et al. published [20] a small series
of 3,4-diphenyl-2,5-dihydrofuran-2-ones. Interestingly, even though the carbonyl group
makes the double bond highly polarized, only analogs with the trimethoxyphenyl ring
attached to the electron-rich α-position (C3) to carbonyl were prepared. Variation of
substitution on the other (C4) phenyl ring gave compounds with activities in low nanomolar
range (see structure 3, Fig. 2), but no mention has been made of their general cellular toxicity.
Our interest in the dihydrofuranone analogs stems from our previous work [21] on the
chemistry and antifungal activity of the structurally closely related 3-(substituted phenyl)-5-
acyloxymethyl-2,5-dihydrofuran-2-ones derived from the natural product, (-)incrustoporin.
We described the beneficial effect of halogens on antifungal activity, and identified
3

ACCEPTED MANUSCRIPT
halophenyl (4a, 4b, Fig. 3) analogues, the activities of which were comparable to that of
amphotericin B.
O
O
ZO
Cl
Cl
4a
4b
Z = H
Z = COR
ref 21
antifungal
IC50 0.49 - 15.63
M
Figure 3. Structures of halophenyl analogs 4a and 4b.
Prompted by the above observations, we became interested in expanding the 3,4-
diarylfuranone library by 4-trimethoxyphenyl derivative(s), halophenyl derivatives, and,
thirdly, those with a hydroxymethyl at C5. While a cytotoxic effect could be expected in all
compounds, we speculated that the addition of halogens and/or the hydroxymethyl group
might change the activity in favour of antifungal and/or antimicrobial. Accordingly, the goal
of this work was to investigate the impact of the following structural modifications on
cytotoxic and/or antiinfective activity of 3,4-diaryl furanones: 1. Attachment of the
trimethoxyphenyl moiety to the electron-deficient β-position (C4) to the carbonyl group. 2.
Variation of substitution towards halogens and other functional groups that could support
antifungal activity. 3. Introduction of hydroxymethyl/acyloxymethyl group(s) to furanone C5.
In this paper, we describe the synthesis of a library of such 3,4-diarylfuranones, and their
screening for biological activities including cytotoxicity, proapoptotic effects, antifungal and
antimicrobial activity.
2. Results and Discussion
2.1. Experimental Design
Having in mind the goals set forth in the Introduction and taking into consideration
commercial availability of substituted acetic acids and acetophenones as the starting materials
for the syntheses (vide infra), we projected three subseries of diarylfuranones. First, hitherto
unknown combretafuranone analogues with the trimethoxyphenyl ring β to carbonyl were
planned. Next, taking into account the supportive effect of halogens for antifungal activity of
the 3-aryl furanones 4, the preparation of a wide spectrum of compounds with halogen
4

ACCEPTED MANUSCRIPT
substitution was necessary. Finally, the introduction of 5-hydroxymethyl moiety to furanone
C5 was intended with a view to exploring the influence of this substitution on the biological
activity of the resultant 3,4-diphenylfuranones.
2.2. Synthesis
The target 3,4-diphenylfuranones were prepared via a modified synthetic route previously
employed for the preparation of rofecoxib [22, 23] (Scheme 1). To this end, a substituted
phenylacetic acid was esterified with a substituted α-bromoacetophenone, and the resultant
ester was subjected to a base-mediated cyclization followed by acidic dehydration. Instead of
DBU/DMF used in rofecoxib preparation [23], the cheaper NaH in THF was used in the
cyclization step.
Scheme 1. Synthesis of 3,4-diphenylfuranone analogs.
Overviews of furanones with natural combretastatin-like substitution and halogenated
derivatives are provided in Tables 1 and 2, respectively. The yields of the compounds
exceeded 50 % in all cases.
Pa
7a
7b
7c
7d
7e
7f
Yb
65
63
R1
H
R2
R3
H
R4
H
R5
H
R6
OCH2O
OCH3 OCH3
R7
R8
H
OCH3
OCH3
H
H
H
H
H
OCH3 OCH3
H
OCH3
H
H
OCH3 OCH3 OCH3 60
H
H
H
OH
H
H
OH
OH
H
H
H
70
69
60
OCH2O
OCH2O
H
H
OCH2O
H
OCH3 OCH3 OCH3
aproduct, byield (%)
Table 1. Overview of analogs with combretastatin substitution pattern.
5

ACCEPTED MANUSCRIPT
P
7g
7h
7i
R1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
R2
Br
Br
Br
Br
H
R3
H
R4
H
H
H
H
Cl
H
Cl
H
H
H
H
H
H
H
H
Cl
H
R5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
R6
H
R7
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
R8
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Y
62
69
57
59
61
62
57
55
58
63
59
75
70
65
62
55
50
H
CH3
CF3
H
H
CH2CH3
CH3
CH3
CH3
CH3
H
7j
H
7k
7l
H
Cl
H
Cl
Cl
F
7m
7n
7o
7p
7q
7r
7s
Cl
H
F
H
CH3
CH3
H
H
CF3
CF3
H
H
CF3
Br
Br
H
Cl
H
OCH3
OH
7t
H
7u
7v
7w
H
OCH3
SO2CH3
OCH3
H
aproduct, byield (%)
Table 2. Overview of halogenated derivatives.
In addition to diphenyl derivatives 7a-w, we also prepared analogs 10 and 13 (Scheme
2) in which the C3 phenyl ring was replaced with indolyl and pyridyl moiety. The former was
made as shown in Scheme 1, while the latter arose as the sole product from a one pot reaction
of pyridine-4-ylacetic acid and 4-trifluoromethyl-α-bromoacetophenone with KOtBu [22]
(Scheme 2).
6

ACCEPTED MANUSCRIPT
Scheme 2. Synthesis of heterocyclic derivatives 10 and 13.
Our next task was to develop the route towards the 5-hydroxymethyl derivatives. The
structure of furanones 7 offered enolization of γ-position to carbonyl followed by quench with
a formaldehyde source. To this end, deprotonization with LDA/THF followed by the addition
of paraformaldehyde provided only bis(hydroxymethyl) derivatives, milder conditions using
Na2CO3 and (CH2O)n in aqueous MeOH furnished mixtures of mono- and
bishydroxymethylated compounds easily separable by column chromatography. Thus,
selected compounds were subjected to this process (Scheme 3, Table 3).
Scheme 3. Hydroxymethylation of furanone C5.
SMa
7h
7h
7h
7b
7b
Pb R1
R2
Br
R3 R4 R5
R6
R7
H
R9
R10
Yc
14a
14b
14cd
14d
14e
14f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH2OH
H
50
Br
H
CH2OH CH2OH 47
CH2OAc CH2OAc 59*
Br
H
OCH3
OCH3
H
OCH3 OCH3 CH2OH
OCH3 OCH3 CH2OH CH2OH 43
Br CH2OH CH2OH 44
H
48
Cl Cl
H
7

ACCEPTED MANUSCRIPT
14g
H
H
Cl Cl
H
OCH3
H
CH2OH CH2OH 40
a starting material, bproduct, cyield (%), dobtained by standard acetylation
of 14b (AcCl, Pyridine, DCM)
Table 3. γ-Substituted 3,4-diphenylfuranones.
2.3. Screening for Biological Activities
Since some cytotoxicity could be expected with nearly 100 % certainty, screening of the
compounds on a panel of cancer and normal cell lines was performed first. For this purpose,
two non-malignant cell lines, i.e. retina epithelium cells RPE-1 and fibroblasts BJ, were used.
The compounds were tested in dose response, and the incubation lasted for 48 hours. Cell
viability assessment was based on the determination of the level of intracellular ATP using a
luminescent cell viability assay. The cytotoxic profiles obtained were compared to those
determined on cancer cell lines including two leukemia models K562 and HL-60, prostate
adenocarcinoma cells PC-3, breast adenocarcinoma cells MCF7, and hepatocellular
carcinoma Hep G2 cell line. In order to obtain an idea of the type of cell death, caspase 3/7
activity measurement was carried out. Results for all compounds are summarized in Table 4.
In addition, solubility and bioavailability parameters useful for the assessment of the
compounds as prospective drugs were calculated, see Table S1 in the Supporting Information
(SI).
Firstly, oxygenated derivatives 7a-7f were tested. No cytotoxic effects on RPE-1 and
BJ were found, with the exception of 7e that slightly decreased the viability of RPE-1.
Marginal activity against both leukemic lines was also observed in the same derivative. In the
next step, we focused on halogenated derivatives 7g-7s. Interesting cytotoxic effect in the
micromolar range against both leukemic cell lines K562 and HL60 with IC50 4.0 and 9.34
µM, respectively, was detected for the fluorophenyl compound 7p.
Cytotoxicity [IC50, µM]
Caspase 3/7 Activity [IC50, µM]
RPE-1 BJ K562 HL-60 PC-3 MCF7 Hep G2 RPE-1 K562 HL-60 PC-3 Hep G2
>40
>40
>40
>40
>40 15.65 15.46
>40
15.87
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
7a
7b
7c
7d
>40 >40
>40 22.81
>40 16.19
>40
>40
>40
>40
8

ACCEPTED MANUSCRIPT
35.53 >40 16.01 17.39
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
0.40
>40
>40
>40
>40
>40
>40
11.36
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
0.43
>40
>40
>40
8.16
>40
>40
13.29
>40
10.09
3.50
>40
>40
>40
>40
>40
56.42
>40
>40
>40
1.54
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
0.29
>40
>40
>40
>40
>40
>40
>40
3.23
0.31
>40
>40
>40
>40
>40
>40
>40
2.77
>40
>40
>40
>40
0.44
0.54
>40
7.53
2.66
>40
>40
2.18
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
4.37
7e
>40
>40
0.97
>40 >40
>40 >40
>40 0.30
>40
>40
0.65
>40
1.02
1.04
0.48
0.27
0.23
>40
9.34
0.66
>40
>40
0.83
>40
7f
>40
7g
14.80
6.37
13.00
>40
7h
7i
16.41 >40 >40
1.30
2.09
0.88
0.91
0.54
>40
>40
1.33
>40
>40 0.47
>40 0.78
>40 0.21
>40 0.19
>40 0.12
>40 >40
>40 4.00
>40 0.29
>40 >40
7j
7k
7l
>40
23.92
15.40
>40
7m
7n
7o
>40
7p
7q
7r
19.06
>40
14.86 >40 >40
12.34
14.28
>40
7s
3.35
>40
>40
>40
0.87
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40 0.53
>40 >40
7t
7u
7v
>40 14.43 15.08
>40
16.25 12.50
>40 >40
>40 0.26
>40 >40
>40
0.67
>40
>40
>40
>40
>40
46.68
>40
>40
>40
>40
>40
>40
>40
2.94
>40
>40
>40
>40
>40
>40
>40
>40
0.07
7w
10
>40
>40
13
>40 31.45 21.32
>40
14a
14b
14c
14d
14e
14f
14g
Camp
>40 >40
>40 >40
>40 >40
>40 >40
>40 >40
>40 >40
>40 0.09
>40
>40
>40
>40
>40
>40
0.02
>40
>40
>40
>40
>40
>40
0.34
27.20 0.711
Table 4. Cytotoxic and apoptotic profiling (activation of caspase 3/7) of all compounds. Cell types
used: RPE-1: human normal immortalized cells from pigmented epithelium in retina; BJ: human
9

ACCEPTED MANUSCRIPT
primary fibroblasts; Hep G2: human hepatocellular carcinoma; K562: human chronic myelogenous
leukemia; HL-60: human acute myeloid leukemia; PC-3: human prostate adenocarcinoma; MCF7:
human breast adenocarcinoma. Camp= (S)-(+)-Camptothecin.
At the same time, no cytotoxic effect was observed against the other cell lines tested,
indicating selectivity of 7p to leukemia cells. Since the compound also activated caspase 3/7
pathway at nearly the same concentration, triggering of apoptosis can be presumed as the
probable reason of cell death (Figure S1 in SI).
Of other compounds, 7v showed cytotoxic pattern for leukemic cells similar to that of
7p, albeit its efficiency is lower (see also Figure S2 in SI). On the other hand, high
submicromolar cytotoxic activities against both leukemic lines were found for the halogen
derivatives 7h, 7j, 7k, 7l, 7m, 7n, 7q and 7t. Unfortunately, the compounds were, without
exception, also highly cytotoxic against the non-malignant RPE-1 line. As regards
heterocyclic isosters 10 and 13, a notable difference was observed. While the only structural
difference was the presence of indolyl or pyridyl moiety at furanone C3, respectively, the
former demonstrated cytotoxic effects at submicromolar concentrations (including RPE-1),
but the latter was inactive.
Finally, the γ-substituted lactones were investigated without positive results. Only for
14a, marginal activities resulting in the suppression of leukemic K562 and HL-60 cell growth
were detected.
In summary, furanones 7f, 7g, 7o, 7r, 7u, 7w, 13 and 14b-14g were non-toxic for all
cell types up to 40 µM. In addition, the calculations of theoretical solubility and
bioavailability gave favourable values (see Table S1 in SI). Thus, given the antimicrobial
activities of compounds 7g, 14a, 14b, 14f and 14g (vide infra), their further development as
potential therapeutic agents is fully justified.
Bacterial strain
Compound [MIC95, µM]
7g 7l 14a 14b 14g Cipr
7.81 31.25 3.9 1.95 15.62 0.98 7.81 0.98
(incubation period)
7a
7c
24h
48h
24h
48h
SA
15.62 31.25 15.62 1.95 15.62 3.9 15.62 0.98
>500 62.5 31.25 15.62 62.5 250 62.5 500
MRSA
>500 62.5 62.5 15.62 62.5 500 125
500
10

ACCEPTED MANUSCRIPT
125 125 31.25 15.62 62.5 62.5 250
500 125 62.5 15.62 62.5 500 250
250
250
24h
48h
24h
48h
24h
48h
24h
48h
24h
48h
24h
48h
SE
EF
>500 >500 >1000 >500 500 500 500 0.98
>500 >500 >1000 >500 >500 >500 >500 0.98
>500 >500 >1000 >500 >500 >500 >500 0.06
>500 >500 >1000 >500 >500 >500 >500 0.06
>500 >500 >1000 >500 >500 >500 >500 0.12
>500 >500 >1000 >500 >500 >500 >500 0.12
>500 >500 >1000 >500 >500 >500 >500 >500
>500 >500 >1000 >500 >500 >500 >500 >500
>500 >500 >1000 >500 >500 >500 >500 3.90
>500 >500 >1000 >500 >500 >500 >500 7.81
EC
KP
KP-E
PA
Table 5. Antimicrobial activities of selected derivatives, SA: Staphylococcus aureus ATCC 6538,
MRSA: Staphylococcus aureus HK5996/08, SE: Staphylococcus epidermidis HK6966/08, EF:
Enterococcus sp. HK14365/08, EC: Escherichia coli ATCC 8739, KP: Klebsiella pneumoniae
HK11750/08, KP-E: Klebsiella pneumoniae HK14368/08; and PA: Pseudomonas aeruginosa ATCC
9027. Cipr = Ciprofloxacin
Most of the above substances were subsequently tested for antifungal and antibacterial
activity. The former performed on a panel of fungal strains including both Candida species
and filamentous fungi showed no antifungal effect up to 1 mM concentration. However,
antibacterial screening against a panel based on standard reference and antibiotic-resistant
strains brought positive results. Of the oxygenated derivatives, compounds 7a and 7c
exhibited interesting activity against Staphylococcus aureus strain ATCC 6538 at the level of
minimum inhibitory concentration (MIC95) 7.8-31.2 µM (Table 5, Table S2 in SI).
Even higher activities were observed for the halogenated derivatives with selectivity to
Staphylococcus strains. Specifically, the lowest MIC95 values were found for compounds 7g
and 7l with MIC95 values, 3.9 and 1.9 µM, respectively, after incubation with Staphylococcus
aureus ATCC 6538 (Table 5, Table S2 in SI). In contrast to 7l, bromophenyl derivative 7g is
completely non-toxic against human normal cells RPE-1 and BJ up to 40 µM (Table 4), and
its activity against the methicillin-resistant clinical isolate is also notable. Other derivatives
involved in the study (7t, 7v, 7w, 10 and 13) were not active (Table S2 in SI). Some of the
11

ACCEPTED MANUSCRIPT
γ-substituted lactones (14a, b, f and g) were able to inhibit Staphylococcus strains at similar
MIC95 levels (0.98 - 31.25 µM, Table 5, Table S3 in SI). The best MIC95, comparable to that
of the standard ciprofloxacin, 0.98 µM, was found for compound 14b against Staphylococcus
aureus ATCC 6538 after 24 hour incubation. Derivatives 14a and 14g were also active, but at
somewhat higher concentrations than 14b (Table 5, Table S3 in SI). Considering the
nontoxicity of the γ-substituted lactones to the control human cells RPE-1 and BJ, and
favourable solubility and bioavailability parameters (Table S1 in SI), the structures are highly
promising for further development as novel antibacterials.
3. Conclusion
In summary, the results have shown that placing the 3,4,5-trimethoxyphenyl ring to
the β-position of the furanone core completely knocks out antiproliferative effects of the
resultant combretafuranone derivatives. Second, substitution of the phenyl ring with halogens
(as opposed to the highly oxygenated pattern typical of highly active combretastatin
analogues) does not necessarily decrease cytotoxic effects against leukemic cells, but the
compounds are also highly cytotoxic against non-malignant control cells. More interestingly,
significant antibacterial activity has been uncovered in this class of compounds for the first
time. Third, functionalization of furanone C5 with hydroxymethyl groups also leads to
compounds without cytotoxic effects. However, a significant antibacterial effect, worthy of
further development, is maintained. MIC95 of the most interesting derivative, 3-(4-
bromophenyl)-5,5-bis(hydroxymethyl)-4-(4-methylphenyl)-2,5-dihydrofuran-2-one against S.
aureus strain ATCC 6538 was 0.98 µM (0.38 µg/mL) after 24 hour incubation. This finding is
particularly important in view of the current state in the management of bacterial infections,
often referred to as the antibiotic resistance crisis.
4. Material and Methods
4.1. Chemistry
All reagents were purchased from Sigma–Aldrich and used without further purification.
Solvents (THF, DCM) were distilled prior to use. TLC analyses were performed using Merck
TLC Silica gel 60 F254 TLC plates and visualized by UV in combination with staining.
1
Column chromatography was carried out on Merck Silica gel 60 (0.040–0.063 mm). H and
13C NMR spectra were recorded with Varian Mercury VxBB 300 or VNMR S500
instruments. The chemical shifts were reported relative to TMS and referenced to the residual
12

ACCEPTED MANUSCRIPT
solvent peaks. IR spectra were recorded on a Nicolet 6700 FT-IR equipped with an ATR
device. MS data were measured on an Agilent Tech 500 Iontrap spectrometer. HR-MS data
were recorded on a QTOF mass spectrometer using the electrospray ionization mode.
Calculator Plugins were used for structure property prediction and calculation, Marvin
15.10.26, 2015, ChemAxon (http://www.chemaxon.com).
4.2. General Synthetic Methods
3,4-Diaryl-2,5-dihydrofuran-2-ones (Method A). A substituted 2-bromoketone (1 mmol) and
a substituted phenylacetic acid (1 mmol) were dissolved in dry THF (15 mL) and
triethylamine (1 mmol) under Ar. The reaction mixture was stirred for 24 hours at room
temperature; sodium hydride (60% oil suspension, 1 mmol) was then added, and the resultant
mixture stirred for 1 hour. Finally, concentrated aqueous hydrochloric acid was added (to
adjust pH to approx. 1), and the mixture stirred for 15 minutes. The reaction mixture was
quenched with water (15 mL), and extracted with ethyl acetate (3 × 20 mL). The organic
layers were dried over sodium sulfate, and concentrated under reduced pressure. The product
was purified by column chromatography using hexane: ethyl acetate 7:3 mixture as a mobile
phase.
Demethylation of aromatic methoxy groups (Method B). A 3,4-diphenyl furanone (2 mmol)
was dissolved in dry DCM (20 mL), and the solution was cooled to -50 °C. Boron tribromide
(1M solution in DCM, 6.0 mL) was then added dropwise, the resultant mixture was stirred for
1 hour, and allowed to warm to room temperature. Water (10 mL) was added, and the mixture
stirred for 30 min at RT. The reaction mixture was extracted with ethyl acetate (50 mL) and
water (100 mL). The organic layer was dried over sodium sulfate, and concentrated under
reduced pressure. The product was purified by silica gel column chromatography using
hexane: ethyl acetate 50:50 as a mobile phase.
Hydroxymethylation of furanone C5 (Method C). A 3,4-diphenyl furanone (2 mmol) and
sodium carbonate (2 mmol) were suspended in dry methanol (20 mL) under Ar atmosphere.
Paraformaldehyde (2 mmol) was then added, and the resultant mixture stirred for 12 hours at
room temperature. The mixture was extracted with ethyl acetate (50 mL) and water (100 mL).
The organic layer was dried over sodium sulfate, and concentrated under reduced pressure.
The product was purified by silica gel column chromatography using hexane: ethyl acetate
30:70 as a mobile phase.
13

ACCEPTED MANUSCRIPT
4-(3,4-methylenedioxophenyl)-3-(4-methoxyphenyl)-2,5-dihydrofuran-2-one (7a)
65% yield. Yellow liquid. The title compound was synthesized by Method A using 2-bromo-
1
1-(4-methoxyphenyl)ethan-1-one and 3,4-(methylenedioxy)phenylacetic acid. H NMR (500
MHz, CDCl3) δ 7.45–7.32 (m, 2H), 6.95–6.90 (m, 2H), 6.89–6.86 (m, 1H), 6.81–6.76 (m,
13
2H), 5.99 (s, 2H), 5.09 (s, 2H), 3.83 (s, 3H); C NMR (125 MHz, CDCl3) δ 173.9, 159.9,
154.2, 149.5, 148.1, 130.6, 124.9, 124.5, 122.4, 122.0, 114.3, 108.8, 107.6, 101.6, 70.5, 55.3;
IR (ATR) νmax 1057, 1172, 1294, 1307, 1335, 1353, 1379, 1445, 1466, 1489, 1514, 1571,
1605, 1634, 1727, 1747, 2926 cm-1. LRMS (APCI+): m/z (relative intensity) 311.0 [M+H]+ ,
+
292.9 (14); HRMS (TOF-ESI+) m/z calcd for C18H14O5 310.0814; found 310.0816. Anal.
Calc. for C18H14O5: C, 69.67; H, 4.55; found: C, 69.72; H, 4.59.
4-(3,4-dimethoxyphenyl)-3-(4-methoxyphenyl)- 2,5-dihydrofuran-2-one (7b)
63% yield. Yellow amorphous solid. The title compound was synthesized by Method A using
1
4-methoxyphenylacyl bromide and 3,4-dimethoxyphenylacetic acid. H NMR (500 MHz,
CDCl3) δ 7.46 – 7.38 (m, 2H), 7.00 – 6.91 (m, 3H), 6.89 – 6.82 (m, 2H), 5.16 (s, 2H), 3.90 (s,
13
3H), 3.83 (s, 3H), 3.59 (s, 3H); C NMR (125 MHz, CDCl3) δ 173.95, 159.84, 154.56,
151.01, 148.91, 130.76, 124.06, 123.48, 122.79, 120.38, 114.14, 111.02, 110.53, 70.30, 55.90,
55.57, 55.29; IR (ATR) νmax 1015, 1151, 1179, 1249, 1263, 1329, 1449, 1464, 1507, 1653,
+
1792, 1844, 1868, 2838, 2935, 3618, 3629 cm-1; HRMS (TOF-ESI+) m/z calcd for C19H18O5
327,1227; found 327,1234; Anal. Calc. for C19H18O5: C, 69.93; H, 5.56; found: C, 69.94; H,
5.59.
4-(2,3,4-trimethoxyphenyl)-3-(2,4,6-trimethoxyphenyl)-2,5-dihydrofuran-2-one (7c)
60% yield. White amorphous solid. The title compound was synthesized by Method A using
1
2,4,6-trimethoxyphenacyl bromide and 2,3,4-trimethoxyphenylacetic acid. H NMR (500
MHz, CDCl3) δ 7.78 – 7.49 (m, 1H), 6.80 – 6.59 (m, 1H), 6.15 (s, 2H), 5.23 (s, 2H), 4.01 (s,
13
3H), 3.92 (s, 3H), 3.86 (s, 3H), 3.82 (s, 6H), 3.81 (s, 3H); C NMR (125 MHz, CDCl3) δ
192.16, 172.07, 160.41, 159.03, 158.06, 154.31, 141.53, 126.00, 122.54, 107.33, 103.86,
90.56, 69.31, 61.25, 60.77, 56.11, 55.81, 55.29, 28.11; IR (ATR) νmax 1013, 1043, 1055,
1078, 1104, 1156, 1190, 1205, 1238, 1257, 1289, 1366, 1412, 1438, 1493, 1587, 1676, 1732,
2841, 2945 cm-1; LRMS (APCI+): m/z (relative intensity) 417.2 [M+H]+ ; Anal. Calc. for
C22H24O8: C, 63.45; H, 5.81; found: C, 63.46; H, 5.80.
4-(3,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)- 2,5-dihydrofuran-2-one (7d)
14

ACCEPTED MANUSCRIPT
70% yield. Yellow liquid. The title compound was synthesized by Method A and
subsequently Method B. 1H NMR (500 MHz, CD3OD) δ 7.33 – 7.13 (m, 2H), 6.87 – 6.80 (m,
13
4H), 6.75 – 6.72 (m, 1H), 5.20 (s, 2H); C NMR (125 MHz, CD3OD) δ 177.01, 158.99,
158.06, 149.38, 146.61, 131.83, 123.89, 123.82, 123.20, 121.26, 116.52, 116.47, 115.71,
72.16; IR (ATR) νmax 1043, 1101, 1127, 1164, 1226, 1263, 1437, 1489, 1559, 1570, 1606,
1653, 1716, 1792, 1868, 2853, 2923, 3314, 3566, 3629 cm-1; LRMS (APCI+): m/z (relative
intensity) 345 [M+H]+ , 346 (20), 347 (2); Anal. Calc. for C16H12O5: C, 67.60; H, 4.26; found:
C, 67.62; H, 4.29.
3-(3,4-methylenedioxophenyl)-4-(3,4-dimethoxyphenyl) )-2,5-dihydrofuran-2-one (7e)
69% yield. White amorphous solid. The title compound was synthesized by Method A using
1
3,4-dimethoxyphenacyl bromide and 3,4-methylenedioxophenylacetic acid. H NMR (500
MHz, CDCl3) δ 6.99 – 6.91 (m, 3H), 6.90 – 6.83 (m, 3H), 5.98 (s, 2H), 5.15 (s, 2H), 3.91 (s,
13
3H), 3.64 (s, 3H); C NMR (125 MHz, CDCl3) δ 173.72, 155.06, 151.12, 148.94, 147.88,
147.83, 124.13, 124.03, 123.46, 123.20, 120.54, 111.06, 110.55, 109.77, 108.67, 101.21,
70.27, 55.92, 55.64; IR (ATR) νmax 1018, 1071, 1124, 1163, 1250, 1272, 1335, 1441, 1548,
1640, 1728, 1754, 1778, 1820, 1900, 1950, 2031, 2855, 2927, 3019 cm-1; HRMS (TOF-
ESI+) m/z calcd for C19H16O6+ 341,1020; found 341,1024; Anal. Calc. for C19H16O6 : C,
67.06; H, 4.74; ; found: C, 67,44 ; H, 5.75.
3-(3,4-methylenedioxophenyl)-4-(3,4,5-trimethoxyphenyl)-2,5-dihydrofuran-2-one (7f)
60% yield. White amorphous solid. The title compound was synthesized by Method A using
1
3,4,5-trimethoxyphenacyl bromide and 3,4-methylenedioxophenylacetic. H NMR (500
MHz, CDCl3) δ 7.03 – 6.69 (m, 3H), 6.56 (s, 2H), 5.95 (s, 2H), 5.12 (s, 2H), 3.85 (s, 3H),
3.68 (s, 6H); 13C NMR (125 MHz, CDCl3) δ 173.4, 155.1, 153.4, 147.9, 147.8, 140.1, 125.8,
125.1, 123.8, 123.5, 109.7, 108.6, 104.9, 101.2, 70.4, 60.9, 56.1; LRMS (APCI+): m/z (rel.
intensity) 371 [M+H]+ , 372 (20); IR (ATR) νmax 1078, 1175, 1199, 1236, 1344, 1368, 1416,
1457, 1494, 1508, 1581, 1635, 1744, 2929 cm-1; Anal. Calc. for C20H18O7: C, 64.86; H, 4.90;
found: C, 64.90; H, 4.98.
3-(4-bromophenyl)-4-phenyl-2,5-dihydrofuran-2-one (7g)
62% yield. White amorphous solid. The title compound was synthesized by Method A using
phenylacyl bromide and 4-bromophenylacetic acid. 1H NMR (500 MHz, CDCl3) δ 7.54–7.49
13
(m, 2H), 7.47–7.41 (m, 1H), 7.41–7.36 (m, 2H), 7.35–7.30 (m, 4H), 5.17 (s, 2H); C NMR
15

ACCEPTED MANUSCRIPT
(125 MHz, CDCl3) δ 173.0, 156.7, 131.9, 130.9, 130.8, 130.5, 129.1, 129.0, 127.4, 125.0,
123.1, 70.6; IR (ATR) νmax 1086, 1099, 1158, 1230, 1338, 1368, 1395, 1452, 1485, 1497,
1575, 1589, 1652, 1746, 3075 cm-1; LRMS (APCI+): m/z (rel. intensity) 315 [M+H]+ , 317
(97), 236 (50); HRMS (TOF-ESI+) m/z calcd for C16H11BrO2+ 315.0015; found 315.0030;
Anal. Calc. for C18H14O5: C, 60.98; H, 3.52; found: C, 60.69; H, 3.50.
3-(4-bromophenyl)-4-(4-methylphenyl)-2,5-dihydrofuran-2-one (7h)
69% yield. Yellow amorphous solid. The title compound was synthesized by Method A using
1
4-methylphenacyl bromide and 4-bromophenylacetic acid. H NMR (500 MHz, CDCl3) δ
7.54–7.48 (m, 2H), 7.36–7.30 (m, 2H), 7.24 – 7.20 (m, 2H), 7.20 – 7.15 (m, 2H), 5.15 (s, 2H),
13
2.38 (s, 3H); C NMR (125 MHz, CDCl3) δ 173.2 156.8, 141.5, 131.9, 130.9, 129.8, 129.3,
127.6, 127.3, 124.2, 123.2, 70.6, 21.5; IR (ATR) νmax 1064, 1112, 1125, 1186, 1241, 1337,
1367, 1379, 1450, 1488, 1514, 1586, 1609, 1650, 1752, 2920 cm-1; LRMS (APCI+): m/z
(relative intensity) 329 [M+H]+ , 332 (18); HRMS (TOF-ESI+) m/z calcd for C17H13BrO2
+
329.0170; found 329.0182; Anal. Calc. for C17H13BrO2: C, 62.03; H, 3.98; found: C, 62.15;
H, 4.02.
3-(4-bromophenyl)-4-(4-(trifluoromethyl)phenyl) -2,5-dihydrofuran-2-one (7i)
57% yield of yellow amorphous solid . The title compound was synthesized by Method A
using phenacyl bromide and phenylacetic acid. 1H NMR (500 MHz, CDCl3) δ 7.71 – 7.61 (m,
13
2H), 7.57 – 7.49 (m, 2H), 7.48 – 7.39 (m, 2H), 7.34 – 7.28 (m, 2H), 5.18 (s, 2H). C NMR
(125 MHz, CDCl3) δ 172.5, 154.9, 134.3, 132.6 (q, J = 33.02 Hz), 132.3, 130.9, 128.4, 128.0,
127.2, 126.3 (q, J = 3.42 Hz), 123.9, 123.6 (q, J = 272.55 Hz), 70.7. LRMS (APCI+): m/z
(relative intensity) 383.1 [M+H]+ ; 385 (97). Anal. Calc. for C17H10BrF3O2: C, 53.29; H, 2.63;
found: C, 53.15; H, 2.52.
3-(4-bromophenyl)-4-(4-ethylphenyl)- 2,5-dihydrofuran-2-one (7j)
59% yield. Yellow amorphous solid. The title compound was synthesized by Method A using
1
4ethylphenacyl bromide and 4-bromophenylacetic acid. H NMR (500 MHz, CDCl3) δ 7.56
– 7.47 (m, 2H), 7.36 – 7.31 (m, 2H), 7.26 – 7.23 (m, 2H), 7.22 – 7.18 (m, 2H), 5.16 (s, 2H),
2.75 – 2.60 (m, 2H), 1.25 (t, J = 7.6 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 173.17, 156.74,
147.64, 131.88, 130.92, 130.91, 129.29, 128.60, 127.77, 127.40, 122.97, 70.59, 28.73, 15.07;
IR (ATR) νmax 1055, 1067, 1133, 1167, 1191, 1340, 1390, 1366, 1434, 1458, 1484, 1519,
1559, 1609, 1647, 1698, 1737, 2933, 2969 cm-1; HRMS (TOF-ESI+) m/z calcd for
16

ACCEPTED MANUSCRIPT
C18H15BrO2+ 343,0328; found 343,0329; Anal. Calc. for C18H15BrO2: C, 62.99; H, 4.41;
found: C, 62.95; H, 4.37.
3-(2-chlorophenyl)-4-(4-methylphenyl)-2,5-dihydrofuran-2-one (7k)
61% yield. White amorphous solid. The title compound was synthesized by Method A using
1
4-methylphenacyl bromide and 2-chlorophenylacetic acid. H NMR (500 MHz, CDCl3) δ
7.54 – 7.46 (m, 1H), 7.43 – 7.32 (m, 2H), 7.34 – 7.28 (m, 1H), 7.16 – 7.08 (m, 4H), 5.31 (d, J
= 12.3 Hz, 2H), 2.35 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 172.92, 157.65, 141.75, 133.91,
131.28, 130.35, 130.19, 130.07, 129.74, 127.58, 127.28, 127.00, 123.54, 70.52, 21.49; IR
(ATR) νmax 1036, 1059, 1128, 1164, 1337, 1364, 1438, 1472, 1516, 1611, 1651, 1750 cm-1;
LRMS (APCI+): m/z (relative intensity) 285.2 [M+H]+ HRMS (TOF-ESI+) m/z calcd for
C17H13ClO2+ 285,0677; found 285,0682; Anal. Calc. for C17H13ClO2: C, 71.71; H, 4.60;
found: C, 71.75; H, 4.65.
3-(3-chlorophenyl)-4-(4-methylphenyl)-2,5-dihydrofuran-2-one (7l)
62% yield. White amorphous solid. The title compound was synthesized by Method A using
1
4-methylphenacyl bromide and 3-chlorophenylacetic acid. H NMR (500 MHz, CDCl3) δ
13
7.48–7.45 (m, 2H), 7.38 –7.29 (m, 4H), 7.23 – 7.15 (m, 2H), 5.18 (s, 2H), 2.38 (s, 3H); C
NMR (125 MHz, CDCl3) δ 173.07, 157.22, 141.59, 134.57, 132.22, 129.93, 129.83, 129.27,
128.87, 127.42, 127.37, 124.02, 70.55, 21.49, 21.48; IR (ATR) νmax 1023, 1038, 1063, 1103,
1158, 1236, 1338, 1358, 1434, 1477, 1560, 1612, 1652, 1764, 2913 cm-1; HRMS (TOF-ESI+)
m/z calcd for C17H13ClO2+ 285,0677; found 285,0686; Anal. Calc. for C17H13ClO2: C, 71.71;
H, 4.60; found: C, 71.78; H, 4.66.
3-(2,4-dichlorophenyl)-4-(4-methylphenyl)-2,5-dihydrofuran-2-one (7m)
57% yield. White amorphous solid. The title compound was synthesized by Method A using
1
4-methylphenacyl bromide and 2,4-dichlorophenylacetic acid. H NMR (500 MHz, CDCl3) δ
7.52 (d, J = 2.0 Hz, 1H), 7.38 – 7.31 (m, 1H), 7.28 – 7.24 (m, 1H), 7.18 – 7.14 (m, 2H), 7.13 –
13
7.09 (m, 2H), 5.55 – 5.04 (m, 2H), 2.37 (s, 3H); C NMR (125 MHz, CDCl3) δ 172.61,
158.32, 142.04, 135.52, 134.76, 132.17, 130.04, 129.84, 128.92, 127.73, 127.35, 126.95,
122.44, 70.58, 21.49; IR (ATR) νmax 1044, 1062, 1070, 1163, 1188, 1337, 1368, 1476, 1558,
1610, 1646, 1683, 1746, 1792, 1844, 1868, 1922, 2929, 3674 cm-1; HRMS (TOF-ESI+) m/z
calcd for C17H12Cl2O2+ 319,0287; found 319,0295; Anal. Calc. for C17H12Cl2O2: C, 63.97; H,
3.79; found: C, 64,27 ; H, 3,77.
17

ACCEPTED MANUSCRIPT
3-(3,4-dichlorophenyl)-4-(4-methylphenyl)-2,5-dihydrofuran-2-one (7n)
55% yield. White amorphous solid. The title compound was synthesized by Method A using
4-methylphenacyl bromide and 3,4-dichlorophenylacetic acid. 1H NMR (500 MHz, CDCl3) δ
7.60 (d, J = 2.0 Hz, 1H), 7.45 (d, J = 8.3 Hz, 1H), 7.33 – 7.24 (m, 1H), 7.24 – 7.16 (m, 4H),
13
5.17 (s, 2H), 2.39 (s, 3H); C NMR (125 MHz, CDCl3) δ 172.79, 157.67, 141.79, 132.92,
132.89, 131.10, 130.63, 130.33, 129.92, 128.62, 127.30, 127.19, 122.97, 70.61, 21.48; IR
(ATR) νmax 1070, 1137, 1166, 1190, 1230, 1321, 1344, 1368, 1437, 1457, 1473, 1489, 1517,
1541, 1558, 1610, 1637, 1652, 1684, 1717, 1737, 1934, 3082 cm-1; HRMS (TOF-ESI+) m/z
calcd for C17H22Cl2O2+ 319,0287; found 319,0294; Anal. Calc. for C17H22Cl2O2: C, 63.97; H,
3.79; found: C, 63,64 ; H, 3,77.
3-(4-fluorophenyl)-4-phenyl-2,5-dihydrofuran-2-one (7o)
58% yield. White amorphous solid. The title compound was synthesized by Method A using
1
phenacyl bromide and 4-fluorophenylacetic acid. H NMR (500 MHz, CDCl3) δ 7.47 – 7.40
13
(m, 3H), 7.40 – 7.34 (m, 2H), 7.34 – 7.30 (m, 2H), 7.12 – 7.03 (m, 2H), 5.18 (s, 2H); C
NMR (125 MHz, CDCl3) δ 173.4, 163.2 (d, J = 249.0 Hz), 156.4, 131.3 (d, J = 8.3 Hz),
130.8, 130.8, 129.2, 127.6, 126.2 (d, J = 3.5 Hz), 125.3, 115.9 (d, J = 21.6 Hz), 70.8; IR
(ATR) νmax 1002, 1028, 1067, 1085, 1100, 1163, 1217, 1338, 1365, 1408, 1447, 1498, 1510,
1541, 1575, 1596, 1640, 1707, 2922, 3051 cm-1; HRMS (APCI+): m/z (rel. Intensity) 255.1
[M+H]+.
3-(4-fluorophenyl)-4-(4-methylphenyl)-2,5-dihydrofuran-2-one (7p)
63% yield. White amorphous solid. The title compound was synthesized by Method A using
4-methylphenacyl bromide and 4-fluorophenylacetic acid. The spectral data were in
1
accordance with those in the literature [24]. H NMR (500 MHz, CDCl3) δ 7.47 – 7.40 (m,
13
2H), 7.24 – 7.14 (m, 4H), 7.08 (s, 2H), 5.16 (s, 2H), 2.38 (s, 3H); C NMR (125 MHz,
CDCl3) δ 173.6, 163.0 (d, J = 248.8 Hz), 156.5, 141.4, 131.3 (d, J = 8.2 Hz), 129.9, 127.9,
127.5, 126.5 (d, J = 3.4 Hz), 124.5, 115.9 (d, J = 21.7 Hz), 70.7, 21.6; IR (ATR) νmax 1020,
1068, 1117, 1129, 1193, 1337, 1364, 1407, 1444, 1505, 1518, 1598, 1611, 1645, 1708, 1745,
1915, 2864, 2922, 3067 cm-1. LRMS (APCI+): m/z (relative intensity) 269.1 [M+H]+ ; 267
(5); Anal. Calc. for C17H13FO2: C, 76.11; H, 4.88; found: C, 76.12 ; H, 4.90.
4-(4-methylphenyl)-3-(3-(trifluoromethyl)phenyl)- 2,5-dihydrofuran-2-one (7q)
59% yield. White amorphous solid. The title compound was synthesized by Method A using
1
3-(trifluoromethyl)phenacyl bromide and 4-methylphenylacetic acid. H NMR (500 MHz,
18

ACCEPTED MANUSCRIPT
CDCl3) δ 7.79 – 7.71 (m, 1H), 7.67 – 7.58 (m, 2H), 7.56 – 7.44 (m, 1H), 7.24 – 7.07 (m, 4H),
13
5.20 (s, 2H), 2.38 (s, 3H); C NMR (125 MHz, CDCl3) δ 173.2, 157.8, 141.9, 132.9, 131.4,
131.2 (q, J = 32.55), 130.3, 129.3, 127.5, 127.4, 126.5 – 126.3 (m), 126.0 (q, J = 3.7 Hz),
124.0 (q, J = 272.47 Hz), 70.8 (t, J = 5.91), 21.6; IR (ATR) νmax 1074; 1128; 1135; 1157;
1180; 1280; 1322; 1348; 1445; 1611; 1649; 1703; 1741; 2926; 3070 cm-1; HRMS (TOF-
ESI+) m/z calcd for C18H13F3O2+ 319,0940; found 319,0952; LRMS (APCI+): m/z (relative
intensity) 319.5 [M+H]+ 320 (20); Anal. Calc. for C18H13F3O2: C, 67.92; H, 4.12; found: C,
;
67.94; H, 4.20.
4-phenyl-3-[3-(trifluoromethyl)phenyl]-2,5-dihydrofuran-2-one (7r)
75% yield. Yellow liquid. The title compound was synthesized by Method A using 3-
(trifluoromethyl)phenacyl bromide and phenylacetic acid. 1H NMR (500 MHz, CDCl3) δ 7.75
– 7.70 (m, 1H), 7.70 – 7.60 (m, 2H), 7.56 – 7.48 (m, 1H), 7.48 – 7.42 (m, 1H), 7.41 – 7.36 (m,
13
2H), 7.34 – 7.28 (m, 2H), 5.22 (s, 2H); C NMR (125 MHz, CDCl3) δ 173.0. 157.8. 132.8,
131.3 (q, J = 32.64 Hz) 131.2, 131.1, 130.4, 129.4, 127.6, 126.5 – 126.3 (q≈m, J = 1.71 Hz),
125.7 (q, J = 3.80 Hz), 124.9, 123.9 (q, J = 272.57 Hz), 70.9 (t, J = 4.98 Hz); LRMS (APCI+):
m/z (relative intensity) 305.5 [M+H]+ ; Anal. Calc. for C17H11F3O2: C, 67.11; H, 3.64; found:
C, 67.15; H, 3.72.
4-(3-chlorophenyl)-3-[4-(trifluoromethyl)phenyl]- 2,5-dihydrofuran-2-one (7s)
70% yield. White liquid. The title compound was synthesized by Method A using 4-
1
(trifloromethyl)phenacyl bromide and 3-chlorophenylacetic acid. H NMR (500 MHz,
CDCl3) δ 7.65 (d, J = 8.3 Hz, 2H), 7.48 – 7.42 (m, 3H), 7.41 – 7.36 (m, 1H), 7.35 – 7.30 (m,
13
1H), 7.29 – 7.23 (m, 1H), 5.20 (s, 2H); C NMR (125 MHz, CDCl3) δ 172.4, 155.5, 135.0,
134.0 (q͍≈d , J = 1.3 Hz), 132.6 (q, J = 33.0 Hz), 131.3, 130.3, 129.6, 129.3,128.1, 127.5,
127.0, 126.3 (q, J = 3.76 Hz), 123.6 (q, J = 272.6), 70.6; LRMS (APCI+): m/z (relative
intensity) 339.4 [M+H]+; HRMS (TOF-ESI+) m/z calcd for C17H10ClF3O2+ 339,0394; found
339,0396.
3-(4-bromophenyl)-4-(4-methoxyphenyl)-2,5-dihydrofuran-2-one (7t)
65% yield. White amorphous solid. The title compound was synthesized by Method A using
1
4-bromophenacyl bromide and 4-methoxyphenylacetic acid. H NMR (500 MHz, CDCl3) δ
13
7.53 (m, 2H), 7.31 (m, 4H ), 6.87 (m, 2H), 5.15 (s, 2H), 3.83 (s, 3H); C NMR (125 MHz,
CDCl3 δ δ 173.3, 161.6, 156.3, 131.9, 130.9, 129.5, 129.1, 123.0, 122.9, 122.7, 114.5, 70.4,
19

ACCEPTED MANUSCRIPT
55.4; IR (ATR) νmax 1165, 1100, 1165, 1181, 1261, 1306, 1369, 1426, 1454, 1488, 1518,
1571, 1605, 1641, 1727, 1915, 2852, 2925 cm-1; LRMS (APCI+): m/z (relative intensity) 345
+
[M+H]+ , 348 (20), 349 (1,5); HRMS (TOF-ESI+) m/z calcd for C17H13BrO3 345,0131;
found 345,0130; Anal. Calc. for C17H13BrO3: C, 59.15; H, 3.80; found: C, 59.37; H, 3.78.
3-(4-bromophenyl)-4-(4-hydroxyphenyl)- 2,5-dihydrofuran-2-one (7u)
62% yield. White liquid. The title compound was synthesized by Method A and subsequently
1
Method B. H NMR (500 MHz, CDCl3) δ 7.61 – 7.48 (m, 2H), 7.35 – 7.28 (m, 2H), 7.29 –
13
7.22 (m, 2H), 6.84 – 6.70 (m, 2H), 5.28 (s, 2H); C NMR (125 MHz, CDCl3) δ 176.00,
161.60, 159.99, 132.94, 132.42, 131.72, 130.70, 123.56, 122.84, 122.70, 116.85, 72.27; IR
(ATR) νmax 1037, 1043, 1064, 1171, 1251, 1344, 1396, 1540, 1608, 1636, 1709, 1792, 1869,
3237 cm-1; HRMS (TOF-ESI+) m/z calcd for C16H11BrO3+ 330,9964; found 330,9961; Anal.
Calc. for C16H11BrO3: C, 58.03; H, 3.35; found: C, 58.12; H, 3.42.
3-(2,6-dichlorophenyl)-4-(4-methoxyphenyl)-2,5-dihydrofuran-2-one (7v)
55% yield. Yellow amorphous solid. The title compound was synthesized by Method A using
4-methoxyphenacyl bromide and 2,6-dichlorophenylacetic acid. 1H NMR (500 MHz, CDCl3)
δ 7.45 – 7.41 (m, 2H), 7.36 – 7.29 (m, 1H), 7.19 – 7.13 (m, 2H), 6.91 – 6.73 (m, 2H), 5.35 (s,
13
2H), 3.81 (s, 3H); C NMR (125 MHz, CDCl3) δ 172.0, 162.1, 158.2, 135.7, 130.6, 130.0,
128.5, 128.4, 122.7, 120.2, 114.7, 70.6, 55.4; IR (ATR) νmax 1028, 1063, 1098, 1181, 1261,
1335, 1431, 1518, 1606, 1650, 1711, 1748, 2850, 2928, 3075 cm-1; LRMS (APCI+): m/z
(relative intensity) 345 [M+H]+ , 348 (20), 349 (1,5); Anal. Calc. for C17H12Cl2O3: C, 60.92;
H, 3.61; found: C, 60.99; H, 3.66.
3-(4-methoxyphenyl)-4-[4-(methylsulfonyl)phenyl]-2,5-dihydrofuran-2-one (7w)
50% yield. White amorphous solid. The title compound was synthesized by Method A using
1
4-methylsulfonylphenacyl bromide and 4-methoxyphenylacetic acid. H NMR (500 MHz,
CDCl3) δ 8.04 – 7.89 (m, 2H), 7.62 – 7.49 (m, 2H), 7.44 – 7.32 (m, 2H), 6.98 – 6.65 (m, 2H),
13
5.16 (s, 2H), 3.84 (s, 3H), 3.08 (s, 3H); C NMR (125 MHz, CDCl3) δ 172.7, 160.5, 151.8,
141.7, 136.7, 130.6, 128.5, 128.4, 128.1, 121.2, 114.4, 70.3, 55.3, 44.3; IR (ATR) νmax 1024;
1114; 1126; 1178; 1253; 1287; 1310; 1365; 1458; 1541; 1607; 1753; 1922; 2894; 2952; 2958;
3009 cm-1; LRMS (APCI+): m/z (relative intensity) 345 [M+H]+ , 346 (20), 347 (2); Anal.
Calc. for C18H16O5S: C, 62.78; H, 4.68; found: C, 62.84; H, 4.73.
20

ACCEPTED MANUSCRIPT
3-(pyridin-4-yl)-4-[4-(trifluoromethyl)phenyl]- 2,5-dihydrofuran-2-one (13)
45% yield. White amorphous solid. 4-pyridylacetic acid hydrochloride (1 mmol) was
dissolved in dry methanol (10 mL), and potassium tert-butoxide (2 mmol) was added. The
resultant mixture was stirred for 15 minutes under Ar atmosphere. The solvent was then
removed, and the remaining solid redissolved in dry DMF (10 mL). 4-
(Trifluoromethyl)phenacyl bromide (1 mmol) was added to the solution, and the reaction
mixture was stirred for 1 hour. The mixture was extracted with ethyl acetate (50 mL) and
water (100 mL). The organic layer was dried over sodium sulfate, and the solvent removed.
The product was purified by silica gel column chromatography using hexane: ethyl acetate
1
40:60 as a mobile phase. H NMR (500 MHz, CDCl3) δ 8.71 – 8.59 (m, 2H), 7.75 – 7.60 (m,
13
2H), 7.46 – 7.39 (m, 2H), 7.36 – 7.32 (m, 2H), 5.19 (s, 2H); C NMR (125 MHz, CDCl3) δ
171.4, 157.2, 150.4, 137.8, 133.8, 133.5, 133.2, 132.9, 132.7, 129.1, 127.9, 126.7, 126.4,
126.4, 126.4, 126.3, 125.9, 124.5, 123.5, 122.4, 70.7; HRMS (TOF-ESI+) m/z calcd for
C16H10F3NO2+ 306,0736; found 306,0742; LRMS (APCI+): m/z (relative intensity) 306.4
[M+H]+ ; Anal. Calc. for C16H10F3NO2: C, 50.03; H, 2.36; N, 3.65; found: C, 50.04; H, 2.39,
N, 3.57.
3-(1H-indol-3-yl)-4-(4-methylphenyl)-2,5-dihydrofuran-2-one (10)
56% yield. White amorphous solid. The title compound was synthesized by Method A using
1
4-methylphenacyl bromide and indol-3-yl acetic acid. H NMR (500 MHz, acetone) δ 10.67
(s, 1H), 7.79 – 7.73 (m, 1H), 7.52 – 7.46 (m, 1H), 7.42 – 7.36 (m, 2H), 7.25 – 6.99 (m, 3H),
13
6.90 – 6.74 (m, 2H), 5.34 (s, 2H), 2.33 (s, 3H); C NMR (125 MHz, acetone) δ 174.60,
153.15, 140.86, 137.49, 130.37, 129.96, 128.34, 127.85, 125.82, 122.48, 121.63, 120.03,
119.85, 112.57, 106.45, 71.27, 21.37; IR (ATR) νmax 1108; 1246; 1295; 1342; 1350; 1380;
1438; 1459; 1506; 1559; 1616; 1652; 1683; 2928; 3277 cm-1. LRMS (APCI+): m/z (relative
intensity) 290.2 [M+H]+ 291.1 (20). Anal. Calc. for C19H15NO2: C, 78.87; H, 5.23; N, 4.84;
;
+
found: C, 79.86; H, 5.25; N, 4.82. HRMS (TOF-ESI+) m/z calcd for C19H15NO2 290,1176;
found 290,1181.
3-(4-bromophenyl)-5-(hydroxymethyl)-4-(4-methylphenyl)-2,5-dihydrofuran-2-one (14a)
50% yield. Yellow amorphous solid. The title compound was synthesized by Method A and
1
subsequently by Method C. H NMR (500 MHz, CDCl3) δ 7.53 – 7.39 (m, 2H), 7.36 – 7.28
(m, 2H), 7.24 – 7.10 (m, 4H), 5.60 – 5.46 (m, 1H), 4.02 (dd, J = 12.5, 2.7 Hz, 1H), 3.68 (dd, J
= 12.5, 4.8 Hz, 1H), 2.38 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 172.1, 157.9, 141.0, 131.7,
21

ACCEPTED MANUSCRIPT
130.8, 129.9, 128.8, 127.8, 127.5, 126.1, 123.0, 82.6, 62.4, 21.4; IR (ATR) νmax 1039, 1105,
1072, 1170, 1233, 1334, 1356, 1396, 1418, 1456, 1473, 1507, 1540, 1653, 1684, 1699, 1736,
2855, 2926, 3402 cm-1; LRMS (APCI+): m/z (relative intensity) 359 [M+H]+ , 361 (97), 362
(20), 360 (17); Anal. Calc. for C18H15BrO3: C, 60.19; H, 4.21; found: C, 60.24; H, 4.25;
+
HRMS (TOF-ESI+) m/z calcd for C18H15BrO3 359,0277; found 359,0283.
3-(4-bromophenyl)-5,5-bis(hydroxymethyl)-4-(4-methylphenyl)-2,5-dihydrofuran-2-one (14b)
47% overall yield White liquid. The title compound was synthesized by Method A and
1
subsequently by Method C. H NMR (500 MHz, CDCl3) δ 7.40 – 7.34 (m, 2H), 7.32 – 7.17
13
(m, 6H), 3.89 (d, J = 12.3 Hz, 2H), 3.73 (d, J = 12.3 Hz, 2H), 2.34 (s, 3H); C NMR (125
MHz, CDCl3) δ 173.6, 163.0, 140.7, 132.2, 132.1, 130.7, 130.5, 130.2, 130.1, 129.1, 123.4,
94.3, 62.1, 21.4; LRMS (APCI+): m/z (relative intensity) 389 [M+H]+ , 391 (97), 391 (17);
HRMS (TOF-ESI+) m/z calcd for C19H17BrO4+ 389,0383; found 389,0393; Anal. Calc. for
C19H17BrO4: C, 58.63; H, 4.40; found: C, 58.71; H, 4.49.
5,5-bis(acetoxymethyl)-3-(4-bromophenyl)-4-(4-methylphenyl)-2,5-dihydrofuran-2-one (14c)
59% overall yield. White liquid. Lactone 14b (0.5 mmol) was dissolved in dry DCM (10 mL).
Pyridine (1,5 mmol), and acetyl chloride (1,5 mmol) were added to the solution. After 1 hour,
the solution was diluted with water and stirred for 30 min at RT. The resultant mixture was
extracted with ethyl acetate (50 mL) and water (100 mL). The organic layer was dried over
sodium sulfate and concentrated under reduced pressure. The product was purified by column
chromatography using hexane: ethyl acetate 70:30 mixture as a mobile phase. 1H NMR (500
MHz, CDCl3) δ 7.44 – 7.37 (m, 2H), 7.32 – 7.25 (m, 2H), 7.25 – 7.19 (m, 2H), 7.05 – 6.98
13
(m, 2H), 4.55 (d, J = 12.1 Hz, 2H), 4.30 (d, J = 12.1 Hz, 2H), 2.38 (s, 3H), 2.05 (s, 6H); C
NMR (125 MHz, CDCl3) δ 170.05, 169.94, 158.28, 140.25, 131.57, 130.65, 130.24, 129.05,
128.01, 127.75, 127.45, 123.38, 86.28, 62.76, 21.32, 20.51; Anal. Calc. for C23H21BrO6: C,
58.37; H, 4.47; found: C, 58.40; H, 4.48; HRMS (TOF-ESI+) m/z calcd for C23H21BrO6+
472,0500; found 472,0610.
4-(3,4-dimethoxyphenyl)-5-(hydroxymethyl)-3-(4-methoxyphenyl)- 2,5-dihydrofuran-2-one
(14d)
48% overall yield. White amorphous solid. The title compound was synthesized by Method A
and subsequently by Method C using paraformaldehyde. 1H NMR (500 MHz, CDCl3) δ 7.44
– 7.36 (m, 2H), 6.95 – 6.85 (m, 4H), 6.81 – 6.77 (m, 1H), 5.55 – 5.40 (m, 2H), 4.08 – 4.00 (m,
22

ACCEPTED MANUSCRIPT
13
2H), 3.91 (s, 3H), 3.82 (s, 3H), 3.73 – 3.66 (m, 2H), 3.62 (s, 3H); C NMR (125 MHz,
CDCl3) δ 172.72, 159.84, 155.29, 150.74, 149.09, 130.70, 125.99, 123.29, 122.28, 121.00,
113.97, 111.22, 111.13, 82.15, 63.15, 55.91, 55.68, 55.27;. HRMS (TOF-ESI+) m/z calcd for
C20H20O6+ 356,1300; found 356,1300; Anal. Calc. for C20H20O6: C, 67.41; H, 5.66 found: C
68.00; H, 5.67.
4-(3,4-dimethoxyphenyl)-5,5-bis(hydroxymethyl)-3-(4-methoxyphenyl)- 2,5-dihydrofuran-2-
one (14e)
43% overall yield. Yellow amorphous solid. The title compound was synthesized by Method
1
A and subsequently by Method C using paraformaldehyde. H NMR (500 MHz, CDCl3) δ
7.46 – 7.32 (m, 2H), 6.90 – 6.87 (m, 2H), 6.83 – 6.74 (m, 3H), 4.02 – 3.95 (m, 2H), 3.90 (s,
13
3H), 3.87 – 3.79 (m, 2H), 3.77 (s, 3H), 3.73 (s, 3H). C NMR (125 MHz, CDCl3) δ 172.48,
159.67, 158.21, 149.75, 149.39, 130.50, 128.73, 124.19, 121.99, 120.66, 113.62, 111.55,
111.16, 91.98, 62.54, 55.90, 55.84, 55.17. HRMS (TOF-ESI+) m/z calcd for C21H22O7+
386,1400; found 386,1400. Anal. Calc. for C21H22O7: C, 65.28; H, 5.74 found: C 65.30; H,
5.75.
4-(4-bromophenyl)-3-(3,4-dichlorophenyl)-5,5-bis(hydroxymethyl)-2,5-dihydrofuran-2-one
(14f)
44% overall yield. Yellow amorphous solid. The title compound was synthesized by Method
1
A and subsequently by Method C using paraformaldehyde. H NMR (500 MHz, CDCl3) δ
7.55 – 7.46 (m, 1H), 7.45 – 7.36 (m, 2H), 7.31 – 7.19 (m, 3H), 7.17 – 7.06 (m, 1H), 4.02 –
3.88 (m, 2H), 3.79 – 3.68 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 170.9, 157.6, 134.2, 133.9,
131.7, 131.5, 131.4, 130.6, 130.3, 129.6, 127.5, 127.4, 123.6, 92.0, 61.7; LRMS (APCI+): m/z
(relative intensity) 443.0 [M+H]+ , 447 (70); IR (ATR) νmax 1013, 1030, 1080, 1137, 1159,
1202, 1271, 1296, 1321, 1352, 1375, 1403, 1448, 1469, 1485, 1497, 1572, 1585, 1641, 1658,
1721, 1758, 1819, 1839, 1852, 1903, 1962 cm-1; Anal. Calc. for C18H13BrCl2O4: C, 48.68; H,
2.95; found: C, 48.73; H, 3.09.
3-(3,4-dichlorophenyl)-5,5-bis(hydroxymethyl)-4-(4-methoxyphenyl)- 2,5-dihydrofuran-2-one
(14g)
40% overall yield. White amorphous solid. The title compound was synthesized by Method A
and subsequently by Method C using paraformaldehyde. 1H NMR (500 MHz, CDCl3) δ 7.67
– 7.53 (m, 1H), 7.30 – 7.23 (m, 1H), 7.22 – 7.16 (m, 2H), 7.16 – 7.07 (m, 1H), 6.98 – 6.86 (m,
23

ACCEPTED MANUSCRIPT
2H), 4.02 – 3.92 (m, 2H), 3.85 – 3.75 (m, 5H), 3.07 (bs, 1H); 13C NMR (125 MHz, CDCl3) δ
171.90, 162.04, 160.65, 132.69, 132.41, 130.95, 130.13, 129.57, 129.27, 128.46, 127.24,
122.84, 114.86, 92.84, 62.05, 55.32; IR (ATR) νmax 1030, 1053, 1092, 1179, 1252, 1292,
1349, 1473, 1512, 1570, 1607, 1645, 1736, 2840, 2934, 3401 cm-1; Anal. Calc. for
C19H16Cl2O5: C, 57.74; H, 4.08; found: C, 57.78; H, 4.13.
4.3. Biological testing
4.3.1. Cell Lines
The cell lines used throughout this study are included in the standard testing panel at CZ
OPENSCREEN, and cover several major oncological diseases. Cellular profiling was carried
out on a panel of cell lines of diverse tissue origin: RPE-1 – human normal immortalized
cells from pigmented epithelium in retina; BJ – human primary fibroblasts; Hep G2 – human
hepatocellular carcinoma; K562 – human chronic myelogenous leukemia; HL-60 – human
acute myeloid leukemia; PC-3 – human prostate adenocarcinoma; MCF7 – human breast
adenocarcinoma.
4.3.2. Cell Culture
Cell lines were purchased from the American Type Culture Collection (ATCC). The cells
were propagated in a monolayer in the cell culture medium supplemented with 10% fetal
bovine serum, 2 mM glutamax, 1 mM non-essential amino acids (NEAA) and
penicillin/streptomycin (Thermo Fisher Scientific, MA, USA) and incubated in a 5% CO2
humidified atmosphere at 37 °C. DMEM was used as a culture medium for RPE-1, Hep G2
and BJ cells, F-12K for PC3 and IMDM for K562 and HL-60. BJ cells were propagated in the
medium supplemented with 10 ng/ml of bFGF.
4.3.3. Cell Viability and Apoptosis Assay
Cells were propagated in the cell growth medium to the amount needed for the experiment.
The cells were harvested, counted and diluted in the growth medium to the final concentration
of 0.1x106/ml and dispensed with a liquid dispenser Multidrop (Thermo Fisher Scientific,
MA, USA), to the cell culture treated, white solid 1536-well plates (Corning Inc., NY, USA)
at 500 cells/well in 5 µl of the total media volume. The test compounds were diluted in
DMSO and transferred to the cells using acoustic dispenser Echo 520 (Labcyte) integrated in
the fully automated robotic HTS station Cell∷Explorer (Perkin Elmer). The compounds were
24

ACCEPTED MANUSCRIPT
tested at 12 different concentration points in the concentration range from 40 µM to 1 nM, in
triplicates. Cell viability was assessed after 48h incubation with the compounds by
determining the level of intracellular ATP using the ATPlite™ (Perkin Elmer, USA)
luminescent assay. Induction of apoptosis was assessed after 24h of incubation with the
compounds by measuring the activity of caspase-3 and -7 using Caspase-Glo® 3/7
luminescent assay (Promega, USA). In both cases, luciferase signal was measured on the
multimode plate reader Envision (Perkin Elmer, USA). The data were collected and processed
with an in-house developed LIMS system ScreenX. Here, the data were processed,
normalized, and ED50 values were calculated using a nonlinear regression function (dose
response, variable slope).
4.3.4. Antimicrobial Activity Testing
Antifungal activities of the compounds were evaluated on a panel of four ATCC strains
(Candida albicans ATCC 44859, Candida albicans ATCC 90028, Candida parapsilosis
ATCC 22019, Candida krusei ATCC 6258) and eight clinical isolates of yeasts (Candida
krusei E28, Candida tropicalis 156, Candida glabrata 20/I, Candida lusitaniae 2446/I,
Trichosporon asahii 1188) and filamentous fungi (Aspergillus fumigatus 231, Absidia
corymbifera 272, Trichophyton mentagrophytes 445) from the collection of fungal strains
deposited at the Department of Biological and Medical Sciences, Faculty of Pharmacy,
Charles University, Hradec Králové, Czech Republic. Three ATCC strains were used as the
quality control strains. All the isolates were maintained on Sabouraud dextrose agar prior to
being tested.
Minimum inhibitory concentrations (MICs) were determined by modified CLSI standard
of microdilution format of the M27-A3 and M38-A2 documents [25, 26]. Dimethyl sulfoxide
(100 %) served as a diluent for all compounds; the final concentration did not exceed 2 %.
RPMI 1640 (Sevapharma, Prague) medium supplemented with L-glutamine and buffered with
0.165 M morpholinepropanesulfonic acid (Serva) to pH 7.0 by 10 M NaOH was used as the
test medium. The wells of the microdilution tray contained 200 µl of the RPMI 1640 medium
with 2-fold serial dilutions of the compounds (2000 to 0.488 µmol/l for the new compounds)
and 10 µl of inoculum suspension. Fungal inoculum in RPMI 1640 was prepared to give a
final concentration of 5 × 103 ± 0.2 cfu.ml-1. The trays were incubated at 35°C and MICs were
read visually after 24 h and 48 h. The MIC values for the dermatophytic strain (T.
mentagrophytes) were determined after 72 h and 120 h. The MICs were defined as 80 %
inhibition (IC80) of the growth of control for yeasts and as 50 % inhibition (IC50) of the
25

ACCEPTED MANUSCRIPT
growth of control for filamentous fungi. MICs were determined twice and in duplicate. The
deviations from the usually obtained values were no higher than the nearest concentration
value up and down the dilution scale.
In vitro antibacterial activities [27] of the compounds were evaluated on a panel of three
ATCC strains (Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739,
Pseudomonas aeruginosa ATCC 9027) and five clinical isolates (Staphylococcus aureus
MRSA HK5996/08, Staphylococcus epidermidis HK6966/08, Enterococcus sp. HK14365/08,
Klebsiella pneumoniae HK11750/08, Klebsiella pneumoniae ESBL HK14368/08) from the
collection of fungal strains deposited at the Department of Biological and Medical Sciences,
Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic. The above-
mentioned ATCC strains also served as the quality control strains. All the isolates were
maintained on Mueller-Hinton agar prior to being tested.
Dimethyl sulfoxide (100 %) served as a diluent for all compounds; the final
concentration did not exceed 2 %. Mueller-Hinton agar (MH, HiMedia, Čadersky-Envitek,
Czech Republic) buffered to pH 7.4 (±0.2) was used as the test medium. The wells of the
microdilution tray contained 200 µl of the Mueller-Hinton medium with 2-fold serial dilutions
of the compounds (2000 to 0.488 µmol/l) and 10 µl of inoculum suspension. Inoculum in MH
medium was prepared to give a final concentration of 0.5 McFarland scale (1.5 × 108 cfu.ml-
1). The trays were incubated at 37°C and MICs were read visually after 24 h and 48 h. The
MICs were defined as 95 % inhibition of the growth of control. MICs were determined twice,
and in duplicate. The deviations from the usually obtained values were no higher than the
nearest concentration value up and down the dilution scale.
Acknowledgement
This study was supported by the Czech Science Foundation (project No. 15-07332S).
Graduate student (P. Horký) acknowledges support from Charles University (project No.
1906214). Ms Ida Dufková is gratefully acknowledged for technical assistance with
antifungal and antibacterial activity determination. CZ-OPENSCREEN: National
infrastructure for chemical biology is supported by the Ministry of Education, Youth and
Sports of the Czech Republic (LO1220 and LM2015063). We thank Olga Martínková a
Žaneta Peikerová for their excellent technical assistance with cell cultures and cellular
profiling.
26

ACCEPTED MANUSCRIPT
Appendix A. Supplementary data
Supplementary data (Tables S1-S2 and Figures S1-S2) related to this article can be found at...
27

ACCEPTED MANUSCRIPT
References
[1] G.M. Cragg, D.G.I. Kingston, D.J. Newman, Anticancer agents from natural products. 2nd ed. Boca
Raton: CRC Press, ISBN 978-1-4398-1382-9, (2011).
[2] G. Nagaiah, S.C. Remick, Combretastatin A4 phosphate: a novel vascular disrupting agent, Future
Oncology, 6 (2010) 1219-1228.
[3] B.J. Monk, M.W. Sill, J.L. Walker, C.J. Darus, G. Sutton, K.S. Tewari, L.P. Martin, J.M. Schilder, R.L.
Coleman, J. Balkissoon, C. Aghajanian, Randomized phase II evaluation of bevacizumab versus
bevacizumab plus fosbretabulin in recurrent ovarian, tubal, or peritoneal carcinoma: An NRG
oncology/gynecologic oncology group study, J. Clin. Oncol., 34 (2016) 2279-2286.
[4] G.R. Pettit, M.R. Rhodes, Antineoplastic agents 389. New syntheses of the combretastatin A-4
prodrug, Anti-Cancer Drug Design, 13 (1998) 183-191.
[5] G.R. Pettit, J.W. Lippert, M.R. Boyd, P. Verdier-Pinard, E. Hamel, Antineoplastic agents 442.
Synthesis and biological activities of dioxostatin, Anti-Cancer Drug Design, 15 (2000) 361-371.
[6] N.H. Nam, Combretastatin A-4 analogues as antimitotic antitumor agents, Current Medicinal
Chemistry, 10 (2003) 1697-1722.
[7] M.S. Gerova, S.R. Stateva, E.M. Radonova, R.B. Kalenderska, R.I. Rusew, R.P. Nikolova, C.D.
Chanev, B.L. Shivachev, M.D. Apostolova, O.I. Petrov, Combretastatin A-4 analogues with
benzoxazolone scaffold: Synthesis, structure and biological activity, Eur. J. Med. Chem., 120 (2016)
121-133.
[8] R. Kaur, G. Kaur, R.K. Gill, R. Soni, J. Bariwal, Recent developments in tubulin polymerization
inhibitors: An overview, Eur. J. Med. Chem., 87 (2014) 89-124.
[9] Y. Lu, J. Chen, M. Xiao, W. Li, D.D. Miller, An overview of tubulin inhibitors that interact with the
colchicine binding site, Pharm. Res., 29 (2012) 2943-2971.
[10] P. Mowery, F. Banales Mejia, C.L. Franceschi, M.H. Kean, D.O. Kwansare, M.M. Lafferty, N.D.
Neerukonda, C.E. Rolph, N.J. Truax, E.T. Pelkey, Synthesis and evaluation of the anti-proliferative
activity of diaryl-3-pyrrolin-2-ones and fused analogs, Bioorg. Med. Chem. Lett., 27 (2017) 191-195.
[11] A. Siebert, M. Gensicka, G. Cholewinski, K. Dzierzbicka, Synthesis of combretastatin A-4 analogs
and their biological activities, Anti-Cancer Agents Med. Chem., 16 (2016) 942-960.
[12] D.V. Tsyganov, V.N. Khrustalev, L.D. Konyushkin, M.M. Raihstat, S.I. Firgang, R.V. Semenov, A.S.
Kiselyov, M.N. Semenova, V.V. Semenov, 3-(5-)-Amino-o-diarylisoxazoles: Regioselective synthesis
and antitubulin activity, Eur. J. Med. Chem., 73 (2014) 112-125.
[13] X. Wu, Q. Wang, W. Li, Recent advances in heterocyclic Tubulin inhibitors targeting the
colchicine binding site, Anti-Cancer Agents Med. Chem., 16 (2016) 1325-1338.
[14] S. Zheng, Q. Zhong, M. Mottamal, Q. Zhang, C. Zhang, E. Lemelle, H. McFerrin, G. Wang, Design,
synthesis, and biological evaluation of novel pyridine-bridged analogues of combretastatin-A4 as
anticancer agents, J. Med. Chem., 57 (2014) 3369-3381.
[15] B.L. Flynn, E. Hamel, M.K. Jung, One-pot synthesis of benzo b furan and indole inhibitors of
tubulin polymerization, Journal of Medicinal Chemistry, 45 (2002) 2670-2673.
[16] G.C. Tron, T. Pirali, G. Sorba, F. Pagliai, S. Busacca, A.A. Genazzani, Medicinal chemistry of
combretastatin A4: Present and future directions, J. Med. Chem., 49 (2006) 3033-3044.
[17] R. Kaur, G. Kaur, R.K. Gill, R. Soni, J. Bariwal, Recent developments in tubulin polymerization
inhibitors: An overview, European Journal of Medicinal Chemistry, 87 (2014) 89-124.
[18] M. Cushman, D. Nagarathnam, D. Gopal, H.M. He, C.M. Lin, E. Hamel, Synthesis and evaluation
of analogues of (Z)-1-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethene as potential cytotoxic
and antimitotic agents, J. Med. Chem., 35 (1992) 2293-2306.
[19] Q. Guan, F. Yang, D. Guo, J. Xu, M. Jiang, C. Liu, K. Bao, Y. Wu, W. Zhang, Synthesis and biological
evaluation of novel 3,4-diaryl-1,2,5-selenadiazol analogues of combretastatin A-4, Eur. J. Med.
Chem., 87 (2014) 1-9.
[20] Y. Kim, N.H. Nam, Y.J. You, B.Z. Ahn, Synthesis and cytotoxicity of 3,4-diaryl-2(5H)-furanones,
Bioorg. Med. Chem. Lett., 12 (2002) 719-722.
28