Enediynes bearing polyfluoroaryl sulfoxide as new antiproliferative agents with dual targeting of microtubules and DNA

Article abstract of DOI:10.1016/j.ejmech.2018.02.030

A novel series of enediynes possessing pentafluorophenylsulfoxide have been developed. The innovative compounds possess antiproliferative activity against a broad panel of human cancer cells originating from breast, blood, lung, kidney, colon, prostate, pancreas or skin with IC₅₀ ranging from 0.6 to 3.4 μM. The antiproliferative activity of enediynes in darkness is associated to their ability to compromise microtubule network. In addition, exposure to UV leads to double-stranded DNA cleavage caused by the newly synthesized molecules reducing further their IC₅₀ in nanomolar range against human tumor cells, including chemo-resistant pancreatic cancer cells. Taken together, the examined data demonstrate that enediynes possessing pentafluorosulfoxide are promising molecules in the cancer therapy.

Full text of DOI:10.1016/j.ejmech.2018.02.030

Accepted Manuscript
Enediynes bearing polyfluoroaryl sulfoxide as new antiproliferative agents with dual
targeting of microtubules and DNA
Cyril Borie, Shovan Mondal, Tanzeel Arif, Manon Briand, Hugo Lingua, Frédéric
Dumur, Didier Gigmes, Pierre Stocker, Bernadette Barbarat, Viviane Robert,
Cendrine Nicoletti, Daniel Olive, Marc Maresca, Malek Nechab
PII:
S0223-5234(18)30160-0
10.1016/j.ejmech.2018.02.030
EJMECH 10207
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 December 2017
Revised Date: 9 February 2018
Accepted Date: 10 February 2018
Please cite this article as: C. Borie, S. Mondal, T. Arif, M. Briand, H. Lingua, Fréé. Dumur, D. Gigmes,
P. Stocker, B. Barbarat, V. Robert, C. Nicoletti, D. Olive, M. Maresca, M. Nechab, Enediynes bearing
polyfluoroaryl sulfoxide as new antiproliferative agents with dual targeting of microtubules and DNA,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.02.030.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Enediynes Bearing Polyfluoroaryl Sulfoxide as New Antiproliferative Agents with Dual
Targeting of Microtubules and DNA.
Cyril Borie,a Shovan Mondal,a,b Tanzeel Arif,a Manon Briand,a Hugo Lingua,a Frédéric
Dumur,a Didier Gigmes,a Pierre Stocker,a Bernadette Barbarat,c,d Viviane Robert,e Cendrine
Nicoletti,e Daniel Olive,c,d Marc Maresca,e* Malek Nechab a*
a
Aix Marseille Univ, CNRS, Institut de Chimie Radicalaire UMR 7273, F-13390 Marseille,
France
b Department of Chemistry, Syamsundar College, Shyamsundar 713424, West Bengal, India
c
Centre de Recherche en Cancérologie de Marseille (CRCM) UMR-INSERM1068-IBiSA
Cancer Immunomonitoring Platform, Inserm, U1068, France
d
Institut Paoli Calmettes, 27, boulevard Lei Roure, BP30059, 13273, Marseille Cedex 09,
France
e
Aix Marseille Univ, CNRS, Centrale Marseille, iSm2 UMR 7313, F-13397, Marseille,
France
corresponding authors: m.maresca@univ-amu.fr; malek.nechab@univ-amu.fr
Abstract: A novel series of enediynes possessing pentafluorophenylsulfoxide have been
developed. The innovative compounds possess antiproliferative activity against a broad panel
of human cancer cells originating from breast, blood, lung, kidney, colon, prostate, pancreas
or skin with IC50 ranging from 0.6 to 3.4 µM. The antiproliferative activity of enediynes in
darkness is associated to their ability to compromise microtubule network. In addition,
exposure to UV leads to double-stranded DNA cleavage caused by the newly synthesized
molecules reducing further their IC50 in nanomolar range against human tumor cells,
including chemo-resistant pancreatic cancer cells. Taken together, the examined data
demonstrate that enediynes possessing pentafluorosulfoxide are promising molecules in the
cancer therapy.
Keywords: Enediynes, Anticancer, Microtubules, DNA cleavage, light activation.
1- Introduction
In the last decades, the enediynes derived from natural products, such as calicheamicins, and
their synthetic analogues have been used in DNA cleavage studies [1]. These molecules were
found as promising therapeutic agents against many types of cancers [2]. Their biological
activity stems from cyclization of the enediyne moiety to a highly reactive 1,4-benzenoid
diradical [1,3]. The latter is able to abstract hydrogen atoms from the sugar phosphate
backbone of adjacent strands of DNA causing scission of the DNA double helix. Despite the
great number of enediynes reported to display anti-tumor activities, there is a need of

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development of new compounds as potential drugs against chemoresistant cancers like
pancreatic tumors. To address this issue, design of novel molecules presenting a dual activity
is a challenging task [4].
During our recent interest in cascade reactions involving enediynes [5], we observed a high
reactivity of pentafluorophenylsulfone when compared to their non-fluorinated analogues.
Non-fluorinated compound 5 (Figure 1) has been reported to exhibit a moderate activity (IC50
~20 µM) against various tumor cells [6], Because, organic molecules containing fluorine
atoms are recognized to provide a high impact in medicinal chemistry [7], we report herein
the synthesis of a novel series of enediynes containing polyfluorinatedsulfoxides and their
actions against tumor cells related to their dual activity targeting microtubules and DNA
photocleavage.
2. Results and discussion
Synthesis
Inspired by a previously designed strategy [6,8], the syntheses of compounds have been
realized in 5 steps (Scheme 1). The first step consists of a Sonogashira coupling of protected
propargyl alcohol and 1,2-diiodobenzene derivatives using Pd(PPh3)4 [9]. A second
Sonogashira cross coupling afforded the enediynes moiety 2. The free alcohol was then
mesylated and immediately displaced, without any purification, by pentafluorothiophenol to
afford the corresponding thioether 3. The m-CPBA oxidation followed by a deprotection with
pyridinium p-toluenesulfonate (PPTS) afforded the desired products 4a-4k with satisfactory
overall yields (Figure 1).
Scheme 1 Synthetic route for enediynes 4
In-vitro antiproliferative activities
Propargyl sulfones constitute a class of bioactive compounds acting on DNA and cell division
[10]. To this end, we evaluated the capacity of compounds 4-7 to inhibit the proliferation of
cancer cells measured by the Alamar Blue assays. The in-vitro antiproliferative effect,
expressed as IC50 (50% inhibition of the cell proliferation), was first investigated using the
human pancreatic tumor cells MIAPaCa-2. These cells were chosen since pancreatic cancer is

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a highly aggressive malignant disease with high chemoresistance and thus considerable efforts
have to be furnished to solve this problem [11].
We have shown that the structural modification of the enediyne compounds has an influence
on the activity against tumor cells. We first demonstrated that the presence of fluorine atoms
(4a) has a crucial importance as it affords higher efficacy (IC50 of 2.6 µM, corresponding to a
6-fold increase compared to 5). The oxidation state of sulfur has also shown its importance,
the sulfoxide 4a provided a better IC50 when it is compared to the sulfone analogue 4b (IC50
of 3.9 µM). The elongation of side chain in 4c has however exhibited a detrimental effect
(IC50 of >10 µM). The same observation has been made with naphthyl analogue 4d (IC50 of
4.7 µM). The presence of free alcohol seems to be not imperative for the activity, the azide 4g
revealed the same IC50 as 4a (IC50 of 2.4 and 2.5 µM, respectively). However, the presence of
the two alkynes has been demonstrated to be important for the activity (IC50 of 7.8 µM for
compound 4e). On the other hand, the presence of ethyl ester group on the aromatic ring (4f)
allows a similar increase in the activity (IC50 of 1.6 µM). The thiophene derivative 6 afforded
an IC50 of 2.2 µM. Finally, the passage to pyridinyl compound 4i affords the best value of
IC50 (0.8 µM). Again, the synthesis intermediates 4h and 4j confirmed that the presence of
hydroxyl group is not essential. Based on these results, we then selected the heterocyclic
enediyne 4i to continue our studies. It is interesting to recall that clinically used antitumor
drug 5-fluoracil presents an IC50 6-fold higher than 4i [10b,12]. Gemcitabine, a widely used
difluoro-drug in the treatment of pancreatic cancers [13,14], was then evaluated as a positive
control in three independent experiments and showed an IC50 in the range 0.12 µM.

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Figure 1 Enediynes Structure and their IC50 in µM (in brackets) on the proliferation of human
pancreatic tumor cells MIAPaCa-2.
Encouraged by these results obtained with pancreatic cancer cells, we screened a broad panel
of human cancer cells originating from breast, blood, lung, kidney, colon, prostate or skin
(Table 1). Results showed that compound 4i is active against all cancer cell types tested,
exhibiting promising IC50 ranging from 0.6 to 3.4 µM, with K562 and MOLT-4 cells (blood
cancer) showing the highest efficacy whereas the renal cell line A498 revealed the lowest
sensitivity. This result suggests that this class of compounds can be used potentially to treat
various forms and types of cancers [15].
Table 1: IC50 values of 4i in different cells
IC (µM)
Cell lines
50
MIAPaCa-2 (pancreas)
MDA-MB 231 (breast)
K562 (blood)
MOLT-4 (blood)
A549 (lung)
A498 (kidney)
CaCo-2 (colon)
PC-3 (prostate)
0.8 ± 0.1
1.2 ± 0.1
0.7 ± 0.08
0.6 ± 0.06
1.6 ± 0.18
3.4 ± 0.2
1.0 ± 0.12
1.4 ± 0.1
1.5 ± 0.1
A431 (melanoma)

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GERL 3.0 (melanoma)
0.9 ± 0.1
We have then investigated the antiproliferative and cytotoxic action of enediyne 4i regarding
normal / primary human cells and compared them to results obtained with MIAPaCa-2 (Table
2). The IC50 of enediyne 4i on cell proliferation on normal human cells ranged from 0.6 to
1.40 µM, demonstrating that the antiproliferative effect of enediyne 4i was not selective of
cancer cells. Similar results were found with enediyne 5 (Table 2). This absence of selectivity
of enediynes is not surprising as it was also observed with other antiproliferative drugs such
as doxorubicin and T138067 (Table 2). Importantly, when we measured the impact of
enediyne 4i on cell viability with confluent cells, we found that it caused cytotoxicity at much
higher doses in all cell tested (Table 2) with IC50 on cell viability ranging from 8.8 to 27.1
µM, corresponding to a 14 to 25-fold difference between the IC50 values on proliferation
versus on cell viability. T138067 and doxorubicin showed lower fold differences between
their antiproliferative and cytotoxic effects, except for the normal breast cells HBL-100 for
which the fold difference was >190 and 106-fold difference for T138067 and doxorubicin,
respectively, suggesting major difference in their toxic effect depending of cell types. Finally,
enediyne 5 caused also cell toxicity at doses much closer from the ones inhibiting cell
proliferation (0.8 to 7.8-fold difference). All together these results underlie the
antiproliferative nature of enediyne 4i and its relative safety in term of cell toxicity compared
to other antiproliferative drugs tested.
Table 2: Comparative effect of different drugs on proliferative and confluent human cells
(IC50 are expressed in µM).
Cell type
Drug
IC50 on cell
proliferation
1.0 ± 0.02
IC50 on cell
viability
19.6± 0.8
Fold
variation
18.6
Cell name
BEAS
4i
Normal (Airway)
29.5 ± 0.7
12.2 ± 0.4
2.4 ± 0.08
81.9 ± 3.4
> 40
2.8
5
> 3.2
7.8
T138067
Doxorubicin
18.6 ± 0.9
1.4 ± 0.01
19.0 ± 0.4
0.2 ± 0.01
0.08 ± 0.01
27.1 ± 0.8
35.4 ± 3.2
> 40
19.3
1.8
4i
Normal (Breast)
HBL-100
HUVEC
5
> 190
106
T138067
Doxorubicin
8.5 ± 2.1
0.6 ± 0.02
79.8 ± 3.2
6.3 ± 0.36
0.15 ± 0.03
8.8 ± 0.2
14
4i
Normal (Endothelium)
Cancer (Pancreas)
66.2 ± 1.6
16.4 ± 0.70
0.48 ± 0.06
0.8
2.6
3.2
5
T138067
Doxorubicin
0.8 ± 0.03
15.9 ± 0.7
1.3 ± 0.06
0.4 ± 0.02
20.5 ± 2.2
124 ± 9
25.3
7.8
4i
MIAPaCa-2
5
47.3 ± 4.5
3.1 ± 0.4
35.8
6.8
T138067
Doxorubicin

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Mechanism of action.
Based on their chemical structure, the mechanism by which compound 4 is giving biological
activity was first hypothesized to be related to DNA break. As already mentioned in the
introduction, biradicals issued from Bergman cyloaromatization (or Saito Myers cyclization)
are able to abstract hydrogen atom from DNA backbone [1b]. Moreover, propargyl sulfones
(or their allenic analogs) were already reported to act as alkylating agent [10b,16]. In order to
confirm such activity, we investigated the ability of Enediynes 4a and 4i to cleave DNA using
supercoiled plasmid pCDNA3 at 100 µM concentration of the compounds. Surprisingly,
intact DNA was recovered demonstrating that no cleavage is caused by these compounds.
Since DNA break was not involved in the anti-proliferative action of our compounds and
questioned by the result seen with compound devoid of enediyne moiety 4e (Figure 1), we
therefore turned our reflection to an alternative hypothesis. Indeed, a known anti-proliferative
compound called T138067 sharing with our compounds the perfluorinated moiety (Figure 2)
[17,18]. T138067 has been shown to act via a nucleophilic substitution in para position of its
perfluorinated moiety allowing the binding of the molecule to a cysteine residue (Cys239) of
tubulin resulting in microtubules depolymerisation and inhibition of the cell proliferation
[17c]. We thus investigated if compounds 4 containing also the perfluorinated moiety possess
anti-microtubules activity.
Indeed, pyridinyl compound 4i bears a parafluorobenzene sulfoxide, a molecular function that
could act as a binding site through the nucleophilic attack of a thiol. Action against
microtubules was thus investigated. A498 cells were treated with 4i, T138067 or with
colchicine (two molecules known to affect microtubules network) for 1, 3, 6 and 12h before
labelling of microtubules with antibodies directed against ß-tubulin (Figure 2). Results
showed that although the action of compound 4i is slower than T138067 or colchicine, it
results in a clear impact on microtubules integrity.

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4i
Figure 2: Time-dependent effect of compound 4i on microtubule network in A498 cells.
A498 cells were exposed for 1, 3, 6 or 12 h to compound 4i at 5 time its IC50 value
determined in proliferation assay. Cells were fixed and microtubule network (in green) was
immunolabelled with antibody directed against beta tubulin.
Similarly, 4i treatment leads to alteration of the microtubules network in A549 and
MIAPaCa-2 cells (Figure 3). The same result was obtained with other compounds of this
family, with the exception of the non perfluorinated compound 5 (Figure 4). This compound
bore a fully hydrogenated moiety and consequently showed no action on microtubules
integrity. This result supports the hypothesis of the involvement of the fluorinated part of this
family in an antimicrotubule activity, probably at the origin of their antiproliferative action on
cancer cells.

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4i
Figure 3: Effect of compound 4i on microtubule network of different cancer cells. A498,
A549 and MIAPaCa-2 cells were exposed for 12 h to compound 4i at 5 time its IC50 value on
each cell type determined in proliferation assay. After 12 h, cells were fixed and microtubule
network (in green) was immunolabelled with antibody directed against beta tubulin.
Colchicine
T138067
4d
4a
5
DMSO
A498
100 µm
1549
MIAPaCa-2
Figure 4: Effect of compounds 4d, 4a and 5 on microtubule network of different cancer
cells. A498, A549 and MIAPaCa-2 cells were exposed for 12 h to compounds Colchincine,
T138067 4d, 4a and 5, respectively at 5 time their IC50 value determined in proliferation
assay. After 12 h, cells were fixed and microtubule network (in green) was immunolabelled
with antibody directed against beta tubulin.

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Photocleavage:
Since drug resistance is the major cause of failure of chemotherapeutics for further
development, we thought that a drug possessing a dual activity will be more promising.
Settling of the sulfoxide moiety was judiciously planned to this issue. Indeed, sulfoxide is
known to be photochemically sensible [19] and more interestingly, it has been demonstrated
that this entity is able to trigger DNA photocleavage [20]. The above enediynes were naturally
investigated regarding their ability for DNA cleavage, upon excitation by light, using a
plasmid relaxation assays [21]. So, although our compounds do not cause DNA break in the
dark (Figure 6), we investigated if excitation by light leads to DNA cleavage [22]. The control
experiments in the dark confirm the results of Figure 5 that the compounds are not able to
cleave DNA. On another hand, when the compounds were irradiated, a full DNA cleavages
were observed and more importantly, a double-stranded cleavage (form III) was detected on
gel. This break is known to be more difficult to repair by enzymes and thus provides a better
chance to initiate a cell death [23].
DNA
in dark
DNA+ 4i DNA + DNA +4i
in dark UV-365 + UV-365
Ladder
II
III
I
Figure 5. Photochemical cleavage (λ = 365 nm during 1h) of pCDNA3 supercoiled DNA (20
µM/bp) by 4i (100 µM) in TE buffer.
Table 3: Antiproliferative activity, expresse as IC50 (in µM), of Enediyne 4i in the absence or
presence of light (10 minutes at 365 nm)
Cell-line
IC50 (in µM) in dark IC50 (in µM) with Selectivity factor
light
MIAPaCa-2
MDA-MB 231 1.2 ± 0.1
MOLT-4
A549
A498
CaCo-2
PC-3
A431
0.8 ± 0.1
0.2 ± 0.05
0.5 ± 0.1
0.4 ±0.05
0.8 ± 0.1
3.2 ± 0.3
0.5 ± 0.17
0.5 ± 0.14
1.1 ± 0.07
0.6 ± 0.07
3.7
2.2
1.6
2
1.1
2
2.5
1.3
1.4
0.6 ± 0.06
1.6 ± 0.18
3.4 ± 0.2
1.0 ± 0.12
1.4 ± 0.1
1.5 ± 0.1
0.9 ± 0.1
GERL 3.0
We then investigated if photo-activation of compound 4i increases its anti-proliferative effect
on cancer cells. The Alamar Blue assay was used in the same conditions as above to

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determine the relative viability upon irradiation at 365 nm during 10 minutes with low energy
of 0.6 J/cm2. The results are summarized in Table 3. By comparing the dark and light-exposed
conditions, we calculated an enhancement factor as the selectivity ratio of activity of
compound 4i in the presence and absence of light. Results showed an increased efficiency in
the presence of light in all of cell lines tested, particularly in the case of MIAPaCa-2, with a
factor of 3.7 being reached with an anti-proliferative activity of 0.23 µM after light exposure.
We then screened the pyridinyl derivatives 4h, 4j and 4k regarding their ability to
phototrigger cytotoxicity against MIAPaCa-2. Interestingly, the ester 4k has shown the best
selectivity of factor 6 (IC50 of 3 µM in the dark and 0.5 µM in the presence of light).
Conclusion
In this study, we have synthesized a dozen of enediynes that have been evaluated against
pancreatic tumor cells (MIAPaca-2). The pyridinyl enediyne 4i revealed the highest
antiproliferative activity (IC50 of 0.8 µM). This compound was also active against the
proliferation of a broad panel of human cancer cells like breast, blood, lung, kidney, colon,
prostate and skin. Mechanistic study showed that 4i affects microtubules network which was
unprecedented for such family of compound. Additionally, thanks to the presence of the
sulfoxide moiety, we have succeeded in a photoactivation process which increases the
antiproliferative activity up to 3.71 times (IC50 reduced from 0.861 to 0.232 µM). DNA
plasmid assays, confirmed the ability of this enediyne to trigger a double-strand cleavage.
Importantly, although the antiproliferative effect of enediyne 4i is poorly selective, affecting
the division of both normal and cancer cells, its limited cell toxicity (14 to 25-fold difference
between the IC50 values on proliferation and cell viability) make it a potential new drug to
fight cancer.
3. Experimental Section
Chemical synthesis of enediynes
All reactions were carried out in dry glassware using magnetic stirring and a positive pressure
of argon. Solvents are commercially available, most of them were used as purchased
(analytical
grade), without further purification. DCM was collected from a solvent purication system.
THF
and Et2O were distilled over sodium benzophenone ketyl prior to use. Dry state adsorption
conditions and purification were performed on silica gel 60 Å (70-230 mesh). Analytical thin
layer chromatography was performed on pre-coated silica gel plates. Visualization was
accomplished by UV (254 nm) and with phosphomolybdic acid in ethanol. 1H NMR spectra
were recorded on 400 MHz and 13C spectra were recorded at 100 MHz or 75 MHz. Chemical
shifts (δ) are reported in ppm. Signals due to residual protonated solvent (1H NMR) or to the
solvent (13C NMR) served as the internal standard: CDCl3 (7.26 ppm and 77.16 ppm).
Multiplicity is indicated by one or more of the following: s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), br (broad). The lists of coupling constants (J) correspond to the order
of multiplicity assignment and are reported in Hertz (Hz). Whenever necessary, APT, HMQC
and HMBC sequences (when necessary) were used for 13C spectra assignment. All melting

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points were uncorrected and were recorded in open capillary tubes using a Buchi melting
point apparatus. High resolution mass spectra were obtained on QStar Elite (Applied
Biosystems SCIEX).
The enediyne 4a (250 mg) was prepared by using the procedure detailed in supporting
1
information. white solid. m.p.=100 ºC. H NMR (400 MHz, CDCl3) δ: 7.41 (1H, dd, J=6.5
and 1.5 Hz, CHar), 7.32-7.24 (3H, m, CHar), 4.52 (1H, d, A part of AB system, J=15.8 Hz,
CH2), 4.49 (2H, s, superimposed CH2), 4.36 (1H, d, B parto f AB system, J=15.7 Hz, CH2),
2.75 (1H, bs, OH). 13C NMR (100 MHz, CDCl3) δ: 132.0 (CHar), 131.9 (CHar), 129.1 (CHar),
128.3 (CHar), 126.1 (Car), 124.0 (Car), 92.6 (CC≡C), 87.9 (CC≡C), 83.5 (CC≡C), 80.6 (CC≡C), 51.6
(CH2), 46.3 (CH2). 19F NMR (376 MHz, CDCl3) δ: -137.9 (2F), -145.2 (1F), -158.2 (2F).
HRMS (ESI): m/z: calcd for [M+Na]+ C18H9O2SF5Na: 407.0135, found: 407.0136.
The enediyne 4b (130 mg) was prepared by using the procedure detailed in supporting
information. 1H NMR (400 MHz, CDCl3) δ: 7.42 (1H, d, J=7.4 Hz, CHar), 7.36-7.24 (3H, m,
CHar), 4.51 (4H, s, 2xCH2), 2.23 (1H, ls, OH). 13C NMR (100 MHz, CDCl3) δ: 132.1 (CHar),
132.0 (CHar), 129.4 (CHar), 128.2 (CHar), 126.3 (Car), 123.5 (Car), 92.7 (CC≡C), 88.3 (CC≡C),
83.2 (CC≡C), 77.8 (CC≡C), 51.6 (CH2), 51.2 (CH2). 19F NMR (376 MHz, CDCl3) δ: -134.5
(2F), -141.4 (1F), -157.4 (2F). HRMS (ESI): m/z: calcd for [M+NH4]+ C18H13NF5O3S :
418.0531, found: 418.0526.
The enediyne 4c (350 mg) was prepared by using the procedure detailed in supporting
information. 1H NMR (400 MHz, CDCl3) δ: 7.36 (1H, d, J=7.8 Hz), 7.27-7.16 (3H, m), 4.56
(1H, d, A part of AB, J=15.6 Hz), 4.50 (1H, d, B part of AB, J=15.6 Hz), 3.71 (2H, t, J=6.3
Hz), 2.48 (2H, t, J=6.8 Hz), 1.82-1.65 (5H, m). 13C NMR (100 MHz, CDCl3) δ: 132.2
(CHar), 132.1 (CHar), 129.0 (CHar), 127.5 (CHar), 126.9 (Car), 123.5 (Car), 94.9 (CC≡C), 88.2
(CC≡C), 79.5 (CC≡C), 79.2 (CC≡C), 62.5 (CH2), 45.8 (CH2), 31.9 (CH2), 25.0 (CH2), 19.4 (CH2).
19F NMR (400 MHz, CDCl3) δ: -137.6 (2F), -145.5 (1F), -158.5 (2F). HRMS (ESI):
m/z:calcd for [M+Na]+ C21H15O2SF5Na: 449.0605, found: 449.0605.
The enediyne 4d (57 mg) was prepared by using the procedure detailed in supporting
information. 1H NMR (400 MHz, CDCl3) δ: 7.94 (1H, s, CHar), 7.86 (1H, s, CHar), 7.75 (2H,
m, CHar), 7.51 (2H, m, CHar), 4.57 (1H, A part of an AB system, d, J=15.8 Hz, CH2), 4.54
13
(2H, s, CH2), 4.39 (1H, B part of an AB system, d, J=15.6 Hz, CH2), 2.81 (1H, ls, OH). C
NMR (100 MHz, CDCl3) δ: 132.8 (Car), 132.5 (CHar), 132.2 (Car), 132.1 (CHar), 128.0
(CHar), 127.8 (2xCHar), 127.7 (CHar), 121.9 (Car), 120.5 (Car), 91.9 (CC≡C), 88.1 (CC≡C), 83.6
19
(CC≡C), 80.2 (CC≡C), 51.6 (CH2), 46.4 (CH2). F NMR (376 MHz, CDCl3) δ: -137.9 (2F), -
145.1 (1F), -158.1 (2F). HRMS (ESI): m/z: calcd for [M+H]+ C22H12F5O2S : 435.0473,
found: 435.0468.
The compound 4e (288 mg) was prepared by using the procedure detailed in supporting
1
information. H NMR (400 MHz, CDCl3) δ: 7.38-7,25 (5H, m, CHarom), 4,51 (1H, d, A part
of AB pattern, J=15.6 Hz, CH2) 4,36 (1H, d, B part of AB pattern, J=15.6 Hz, CH2). 13C
NMR (100 MHz, CDCl3) δ: 133.3 (CFar), 131.8 (CHar), 129.7 (CFar), 129.4 (CHar), 128.7
(CFar), 128.65 (CHar) 125.2 (C-Car), 121.3 (C-S(O)), 86.4 (CC≡C), 76.0 (CC≡C), 45.5 (CH2). 19F

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NMR (400 MHz, CDCl3) δ: -138.8 (2F), -146.8 (1F) -159.8 (2F). HRMS (ESI): m/z: calcd
for [M+H]+ C15H7F5SO : 331.0211, found: 331.0209.
The enediyne 4f (83 mg) was prepared by using the procedure detailed in supporting
1
information. H NMR (400 MHz, CDCl3) δ: 7.99 (1H, s, CHar), 7.96 (1H, d, J=8.1 Hz,
CHar), 7.48 (1H, d, J=8.1 Hz, CHar), 4.56-4.52 (1H, d, A part of AB system, J=16.0 Hz, CH2),
4.52 (2H, s, superimposed CH2), 4.40-4.35 (2H, q, J=7.1 Hz, CH2), 4.35-4.32 (1H, d, B part
of AB system, J=15.8 Hz, CH2), 2.91 (1H, lt, J=6.1 Hz, OH), 1.39 (3H, t, J=7.1 Hz, CH3).
13C NMR (100 MHz, CDCl3) δ: 165.2 (C=O), 133.0 (CHar), 131.9 (CHar), 130.2 (2xCar),
129.9 (CHar), 124.2 (Car), 95.7 (CC≡C), 87.0 (CC≡C), 83.0 (CC≡C), 81.5 (CC≡C), 61.6 (CH2), 51.6
19
(CH2), 46.3 (CH2), 14.4 (CH3). F NMR (376 MHz, CDCl3) δ: -137.9 (2F), -144.8 (1F), -
157.8 (2F). HRMS (ESI): m/z: calcd for [M+Na]+ C21H13O4SF5Na : 479.0347, found:
479.0345.
The enediyne 4g (278 mg) was prepared by using the procedure detailed in supporting
1
information. H NMR (400 MHz, CDCl3) δ: 7.45-7.44 (1H, m, CHar), 7.33-7.26 (3H, m,
CHar), 4.55 (1H, A part of an AB system, d, J=15.6 Hz, CH2), 4.44 (1H, B part of an AB
system, d, J=15.6 Hz, CH2). 13C NMR (100 MHz, CDCl3) δ: 132.6 (CHar), 132.4 (CHar),
129.2 (CHar), 128.8 (CHar), 124.9 (Car), 124.0 (Car), 87.5 (CC≡C), 85.44 (CC≡C), 85.41 (CC≡C),
80.5 (CC≡C), 45.8 (CH2), 40.7 (CH2). 19F NMR (376 MHz, CDCl3) δ: -137.7 (2F), -145.4
(1F), -158.4 (2F). HRMS (ESI): m/z: calcd for [M+H]+ C18H9N3OSF5 : 410.0381, found:
410.0383.
The enediyne 5 (115 mg) was prepared by using the procedure detailed in supporting
1
information. H NMR (400 MHz, CDCl3) δ: 7.77-7.74 (2H, m, CHar), 7.54-7.52 (3H, m,
CHar), 7.43 (1H, d, J=7.5 Hz, CHar), 7.34-7.21 (3H, m, CHar), 4.53 (2H, s, CH2), 4.21 (1H, ls,
OH), 3.97 (1H, d, A part of AB system, J=16.1 Hz, CH2), 3.80 (1H, d, B part of AB system,
J=16.1 Hz, CH2). 13C NMR (100 MHz, CDCl3) δ: 142.5 (Car), 131.9 (CHar), 131.6 (CHar),
131.4 (CHar), 129.4 (2xCHar), 128.6 (CHar), 127.9 (CHar), 126.5 (Car), 125.0 (Car), 124.5
(2xCHar), 93.3 (CC≡C), 87.0 (CC≡C), 83.6 (CC≡C), 82.2 (CC≡C), 51.5 (CH2), 49.4 (CH2). HRMS
(ESI): m/z: calcd for [M+Na]+ C18H14O2SNa : 317.0607, found: 317.0608.
The enediyne 4h (285 mg) was prepared by using the procedure detailed in supporting
1
information. H NMR (400 MHz, CDCl3) δ: 8.46 (1H, dd, J=4.8 and 1.5 Hz, CHar), 7.52
(1H, dd, J=7.9 and 1.6 Hz, CHar), 7.13 (1H, dd, J=7.9 and 4.9 Hz, CHar), 4.89 (1H, s, CH),
4.52-4.36 (4H, m, CH2), 3.82-3.76 (1H, m, CH2), 3.52-3.49 (1H, m, CH2), 1.79-1.50 (6H, m,
CH2). 13C NMR (100 MHz, CDCl3) δ: 149.7 (CHar), 144.9 (Car), 139.3 (CHar), 122.4 (CHar),
121.1 (Car), 96.6 (CH), 90.1 (CC≡C), 85.4 (CC≡C), 83.6 (CC≡C), 82.9 (CC≡C), 61.9 (CH2), 54.3
19
(CH2), 45.7 (CH2), 30.3 (CH2), 25.5 (CH2), 18.9 (CH2). F NMR (376 MHz, CDCl3) δ: -
137.7 (2F), -145.0 (1F), -158.2 (2F). HRMS (ESI): m/z: calcd for [M+H]+ C22H17F5NO3S :
470.0844, found: 470.0845.
The enediyne 4i (74 mg) was prepared by using the procedure detailed in supporting
1
information. H NMR (400 MHz, CDCl3) δ: 8.53 (1H, dd, J=5.0 and 1.5 Hz, CHar), 7.65
(1H, dd, J=7.9 and 1.5 Hz, CHar), 7.21 (1H, dd, J=7.9 and 5 Hz, CHar), 4.54 (1H, A part of an
AB system, d, J=16.1 Hz, CH2), 4.52 (2H, s, superimposed CH2), 4.34 (1H, B part of an AB

ACCEPTED MANUSCRIPT
system, d, J=16.1 Hz, CH2), 3.03 (1H, s, OH). 13C NMR (100 MHz, CDCl3) δ: 149.7 (CHar),
145.3 (Car), 139.1 (CHar), 122.3 (CHar), 121.1 (Car), 92.9 (CC≡C), 85.5 (CC≡C), 83.6 (CC≡C),
83.2 (CC≡C), 51.4 (CH2), 46.3 (CH2). 19F NMR (376 MHz, CDCl3) δ: -138.0 (2F), -144.7 (1F),
-157.8 (2F). HRMS (ESI): m/z: calcd for [M+H]+ C17H9F5NO2S : 386.0269, found:
386.0265.
The enediyne 4j (97 mg) was prepared by using the procedure detailed in supporting
1
information. H NMR (400 MHz, CDCl3) δ: 8.52 (1H, dd, J=4.9 and 1.6 Hz, CHar), 7.59
(1H, dd, J=7.9 and 1.6 Hz, CHar), 7.20 (1H, dd, J=7.9 and 4.9 Hz, CHar), 4.88 (2H, d, J=1.5
Hz, CH), 4.56 (1H, A part of AB pattern, d, J=15.8 Hz, CH2), 4.50 (2H, s, CH2), 4.47 (1H, B
part of AB pattern, d, J=15.8 Hz, CH2), 3.75 (2H, m, CH2), 3.58 (2H, m, CH2), 3.39 (3H, s,
13
CH3). C NMR (100 MHz, CDCl3) δ: 149.8 (CHar), 144.8 (Car), 139.4 (CHar), 122.5 (Car),
121.1 (CHar), 93.8 (CH2), 89.5 (CC≡C), 85.3 (CC≡C), 83.9 (CC≡C), 83.1 (CC≡C), 71.8 (CH2), 67.4
19
(CH2), 59.1 (CH3), 54.6 (CH2), 45.7 (CH2). F NMR (376 MHz, CDCl3) δ: -137.8 (2F), -
145.0 (1F), -158.2 (2F). HRMS (ESI): m/z: calcd for [M+H]+ C21H17NO4SF5 : 474.0793,
found: 474.0792.
4. Material and Methods
Cell culture
Human cancer cell lines and normal cells were obtained from American Type Culture
Collection (ATCC). HUVEC cells were grown in defined all-in-one ready to use endothelial
cell growth medium (Sigma Aldrich). Other cells were cultured in Dulbecco's modified
Eagle’s medium (DMEM, Gibco Life Technologies) supplemented with 10% heat-inactivated
fetal calf serum (FCS, Lonza) at 37 °C in a 100% humidified atmosphere containing 5% CO2.
Cells were sub-cultured in new media every 3-4 days and were regularly checked for
mycoplasma contamination and remained negative throughout the experiments.
Cell proliferation and cell viability assay
Antiproliferative and cytotoxic effect were measured using alamar blue. Briefly, for the
antiproliferative assay, cells were detached from growing flask using trypsin, cells were
counted using the Cellometer Auto T4 apparatus (Nexcelom Bioscience LLC) automated cell
counting technique and 0.2% trypan blue reagent (Aldrich) and were seeded in 96 well-plates
in the growth media overnight at an initial cell density of 2000 cells/well. For the cell viability
assay, cells were detached from growing flask using trypsin, counted and seeded in 96 well-
plates in the growth media at an initial cell density of 10,000 cells/well. Cells were then left to
grown until they reached complete confluence (generally 2-3 days) before exposure to drugs.
Stock solutions of molecules were prepared in DMSO. Proliferative and confluent cells were
exposed to increasing concentrations of compounds, in all case resulting in the presence of
0.1% (v:v) DMSO, control cells receiving only DMSO. Cells were then incubated for 70 h,
and the cell viability was assessed with CellTiter-Blue® Cell Viability Assay (Promega). 20
µL of resazurin stock solution was added to the treatment media per well. After 8 h
incubation, the fluorescence was recorded at an excitation/emission wavelength of 560/590
nm, with a microplate reader (FLUOstar OPTIMA, BMG Labtech). The fluorescence

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measurement was normalized using culture medium without cells. Values measured were
adjusted to negative control value (exposure to 0.1% DMSO alone). Data are expressed as the
means ± standard deviation (SD) of three independent experiments, each with triplicate
measurements. Data were analyzed with Origin 6.0 software. The IC50 values of each
compound on cell proliferation and cell viability were obtained from the dose-dependent
curve fit.
Cell proliferation assays upon photoactivation
The cells were prepared with the same protocol as in the dark experiments. After 4h
incubation with the enediynes, light activation was achieved by irradiating the cells, at room
temperature for 10 min using a transilluminator (8W 365 nm tubes, 30% intensity ie ≈0,6
J/cm² energy, Biostep). As a control, the antiproliferative activity of the tested compounds
was also assessed without UV light activation, the plate being protected with aluminium foil
during UV irradiation. The intensity of UV light irradiation was determined with a UV
radiometer (VLX-3W, Vilber Lourmat) with a 365 nm sensor. After irradiation, cells were
incubated for 70 h, and the cell viability was assessed with CellTiter-Blue®. Data are
expressed as the means ± standard deviation (SD) of three independent experiments, each
with triplicate measurements. Data were analyzed with Origin 6.0 software. The IC50 values
of each compound were obtained from the dose-dependent curve fit.
Plasmid DNA Photocleavage cleavage
Compound 4 in DMSO (1 mM) was added to the solution of supercoiled PcDNA3 (5446 pb,
0.5 µg) in TE buffer (tris-HCl + EDTA, 20 µL, pH 8) to reach 100 µM of 4 as a final
concentration. Samples were placed at a distance of 20 cm from LED source (Shimamatzu
LC-L5, 365 nm) and irradiated at 37 °C during 1 hour. Crude mixtures were then subjected to
gel electrophoresis (0.8% agarose containing SYBR® Safe DNA gel Stain) in TAE 0.5X for 1
h at 100 V, then pictures were taken thanks to FluorChem-Q apparatus equipped with filter.
Microtubules immunofluorescence
Cells were seeded in 24-well plates at 10.000 cells/cm2 on glass coverslips. After 48 h,
compounds dissolved in DMSO were added. After pre-defined times, cells were fixed with
0.5 mL 4% PFA for 20 min at room temperature. After three washes with PBS, cells were
saturated and permeabilized for 1 h at room temperature using PBS containing BSA at 2%
and Triton X100 at 0.1%. Primary antibody directed against beta-tubulin was then added
(mouse anti-human beta tubulin, sc-398937 from Santa Cruz, diluted at 1:100 in PBS/BSA
2% accordingly to manufacturer’s instructions). After three washes with PBS, cells were
treated with secondary antibody directed against mouse IgG (goat anti-mouse IgG, coupled to
Alexa488 Green fluorochrome from Invitrogen diluted at 1:200 in PBS/BSA 2%. After 1 h
incubation at room temperature, cells were washed three times with PBS and coverslips were
mounted with vectashield liquid, cealed with nailpolish and observed by optical
epifluorescence microscope (objective 40x, magnification factor x1.6, Leica DMRB,
equipped with Leica DFC 450C camera).

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Acknowledgement
Financial assistance from ANR JCJC MOCER2, Région PACA, Aix-Marseille University is
acknowledged.
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Highlights
- Novel Enediynes as a potential antiproliferative drugs.
- Dual Targeting of Microtubules and DNA.
- IC50 ranging from 0.6 to 3.4 µM against a braod panel of human cancer cells.
- Enhanced activity upon photoactivation.
- Compound 4i showed double-stranded DNA cleavage.