A novel class of ethacrynic acid derivatives as promising drug-like potent generation of anticancer agents with established mechanism of action

Article abstract of DOI:10.1016/j.ejmech.2016.05.063

The well-known diuretic Ethacrynic acid (EA, Edecrin), showing low anti-proliferative activities, was chemically modified at different positions. The new EA derivatives have been tested in?vitro in anti-proliferative assays on both tumor KB (epidermal carcinoma) and leukemia HL60 (promyelocytic) cells suitable targets for anticancer activity. Reduction of the α-β double bond of EA completely abolished anti-cancer activities, whereas introduction of either 2-(4-substituted phenyl)ethanamine (series A) or 4-(4-substituted phenyl)piperazine (series B) moieties generated compounds showing moderate to strong anti-proliferative activities against human cancer cell lines. Several substitutions on the phenyl of these two moieties are tolerated. The mechanism of action of the EA derivatives prepared in this study is more complex than the inhibition of glutathione S-transferase π ascribed as unique effect to EA and might help to overcome tumor resistances.

Full text of DOI:10.1016/j.ejmech.2016.05.063

Accepted Manuscript
A novel class of ethacrynic acid derivatives as promising drug-like potent generation
of anticancer agents with established mechanism of action
Serge Mignani, Nabil El Brahmi, Saïd El Kazzouli, Laure Eloy, Delphine Courilleau,
Joachim Caron, Mosto M. Bousmina, Anne-Marie Caminade, Thierry Cresteil, Jean-
Pierre Majoral
PII:
S0223-5234(16)30471-8
10.1016/j.ejmech.2016.05.063
EJMECH 8655
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 March 2016
Revised Date: 9 May 2016
Accepted Date: 29 May 2016
Please cite this article as: S. Mignani, N. El Brahmi, S. El Kazzouli, L. Eloy, D. Courilleau, J. Caron,
M.M. Bousmina, A.-M. Caminade, T. Cresteil, J.-P. Majoral, A novel class of ethacrynic acid derivatives
as promising drug-like potent generation of anticancer agents with established mechanism of action,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.05.063.
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ACCEPTED MANUSCRIPT

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A Novel Class of Ethacrynic Acid Derivatives as Promising
Drug-like Potent Generation of Anticancer Agents with
Established Mechanism of Action.
Serge Mignani*,1 Nabil El Brahmi,2,3,6 Saïd El Kazzouli*,2 Laure Eloy,4 Delphine
Courilleau,5 Joachim Caron,2,3,6 Mosto M. Bousmina,2 Anne-Marie Caminade,3,6 Thierry
3,6
Cresteil*,4,5 and Jean-Pierre Majoral*r
1
Laboratoire de Chimie et de Biochimie pharmacologiques et toxicologique, CNRS UMR
860, Université Paris Descartes, PRES Sorbonne Paris Cité, 45, rue des Saints Pères, 75006
Paris, France
2 Euromed Research Institute, Engineering Division, Euro-Mediterranean University of Fes,
Fès-Shore, Route de Sidi Hrazem, 30070 Fès, Morocco
3 Laboratoire de Chimie de Coordination CNRS, 205 route de Narbonne, BP 44099, 31077
Toulouse cedex 4, France
4 Institut de Chimie des Substances Naturelles, CNRS UPR2301, Avenue de la terrasse,
91190 Gif sur Yvette, France
5 IPSIT Faculté de Pharmacie, Université Paris Sud, 92290 Chatenay-Malabry, France.
6 Université de Toulouse, UPS, INPT, F-31077 Toulouse Cedex4, France
r
Corresponding author: Jean Pierre Majoral. Email address: jean-pierre.majoral@lcc-
toulouse.fr
Keywords:
Anti-proliferative activities
Ethacrynic acid derivatives
Apoptosis pathway
Glutathione S-transferase π inhibitors

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Abstract: The well-known diuretic Ethacrynic acid (EA, Edecrin), showing low anti-
proliferative activities, was chemically modified at different positions. The new EA
derivatives have been tested in vitro in anti-proliferative assays on both tumor KB (epidermal
carcinoma) and leukemia HL60 (promyelocytic) cells suitable targets for anticancer activity.
Reduction of the α-β double bond of EA completely abolished anti-cancer activities, whereas
introduction of either 2-(4-substituted phenyl)ethanamine (series A) or 4-(4-substituted
phenyl)piperazine (series B) moieties generated compounds showing moderate to strong anti-
proliferative activities against human cancer cell lines. Several substitutions on the phenyl of
these two moieties are tolerated. The mechanism of action of the EA derivatives prepared in
this study is more complex than the inhibition of glutathione S-transferase π ascribed as
unique effect to EA and might help to overcome tumor resistances
.
Key words: Anticancer agents, ethacrynic acid, ethacrynic acid derivatives, glutathione S-
transferase π.
1. Introduction
Cancer is the second leading cause of death in North America and Europe [1]. Although
an armada of anticancer agents has received FDA approval over the past two decades [2], the
inefficiency of their discovery and development is no longer sustainable and the pipeline of
new cancer agents is slim. Today, the outcome of patients with advanced metastasis, for
instance regarding lung, colorectal, prostate and breast cancers, remains very poor [3]. The
development of anticancer agents for several decades was based on the identification of active
compounds, with cytostatic or cytotoxic activity on tumor cell lines, but with many side
effects. Consequently, new anticancer agents within new chemical families are urgently
needed to allow an extension of this cancer-fighting armada. In addition to the discovery and
development of new anticancer agents, an improved understanding of each cancer patient’s
needs is also essential to enhance the ability of standard treatments to kill cancer cells without
significantly affecting normal cells [4].
One important consideration in the development of cancer treatment regimens is resistance
against anticancer drugs, which remains a serious obstacle [5]. Indeed, microsomal
glutathione transferase 1 (mGST1) and glutathione transferase π (GST π) are often
overexpressed in tumors and confer resistance against a number of cytostatic drugs, such as
cisplatin and doxorubicin (DOX) [6]. To address this point, the diuretic drug ethacrynic acid
(EA, Edecrin) 1, an inhibitor of π class glutathione S-transferase, has been tested against
multiple myeloma, and as adjuvant in clinical trials [7]. On the basis of these considerations,
Dyson et al [8] and Osella et al [9] synthesized and characterized the bifunctional EA-Pt(IV)
complex
2
(ethacraplatin)
and
the
EA–Pt(II)
complex
2’
(cis-
diaminobis(ethacrynato)platinum(II) (Figure 1), both of them able to release a cytotoxic
platinum (II) agent (inducing apoptosis of cancer cell) and two EA moieties (inhibiting
glutathione S-transferase (GST) and overcoming drug resistance) via hydrolysis or reduction,
respectively. Ethacraplatin 2 resulted to be an excellent inhibitor of GST in several tumor cell
lines such as A549 lung adenocarcinoma epithelial cell line, MCF7 and T47D breast tumor
cell lines and HT29 colon cancer cell line albeit demonstrating a moderate anti-proliferative
activity. Unfortunately, the bifunctional conjugates 2 and 2’ did not offer any advantage over
cisplatin for the treatment of malignant pleural mesothelioma (MPM) cells, since the increase
of intracellular glutathione (GSH) counteracts the modest inhibition of GST [10]. Recently,
Yang et al. constructed biodegradable nanoparticles to codeliver EA and dichloro(1,2-

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diaminocyclohexane)platinum(II) (DACHPt) which is a precursor of oxaliplatin as a
promising approach to overcome the drug resistance in cancer chemotherapy [11]. In vitro
studies showed that these hybrid nanoparticles could release both EA and DACHPt enhancing
of up to ~5 fold of the anticancer efficacy versus DACHPt alone. Interestingly, in vivo studies
showed better anticancer activities than the simple combination of EA and DACHPt.
Figure 1.Chemical structure of ethacrynic acid (EA, 1), DACHPt, Pt(IV) and Pt(II)
ethacrynic derivatives (2 and 2’).
The development of new and potent anticancer agents based on EA will undoubtedly
offer new opportunities to tackle cancer and some reports in this direction appeared recently
reviewed by us and others [12,13]. In order to improve the poor anti-proliferative activity of
EA [14], we designed original EA derivatives based on modifications of the EA core
structure and tested the resulting derivatives for their capacity to inhibit cell growth in vitro.
For this initial screening, we tested the chemicals on three cell lines: two actively dividing cell
lines derived from human cancer, human KB carcinoma and human leukemia HL60, and the
non-dividing quiescent endothelial progenitor cells (EPC) from Cyprinus carpio. Cell number
and cellular NADH content evaluated after 72h chemical exposure provided relevant
information concerning viability. The two different tumor cell lines have been selected as
representative ‘models’ of the two common types of cancer: solid tumor cancers (e.g. breast,
lung, colon, etc.) and blood-based cancers (e.g. leukemia, lymphoma, myeloma etc.).
According to a recent analysis, the percentage of new patients annually diagnosed for solid
tumor cancers and blood-based cancers is 34% and 9%, respectively, whereas the percentage
of cancer related deaths is 43% and 9%, respectively [15].
Herein, we report the design, preparation and anti-proliferative activity of original EA
derivatives active upon cancer cell lines and exemplifying a promising class of potent
anticancer agents. The preparation of EA derivatives and their synthetic pathways are
described in Schemes 1-6 and their mode of action was proposed. The mechanism of action of
the EA derivatives prepared in this study is more complex that the potent inhibition of π class
glutathione S-transferase attributed to EA and could potentially overcome tumor resistances.
2. Chemistry
The initial strategic design of these EA derivatives can be divided into two parts as shown
in Figure 2: part 1: modification of the 3-methylenepentan-2-one unit; and part 2:

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modification of the carboxylic acid unit. In this study, we decided not to modify the core of
the EA.
Figure 2. EA chemical modifications performed in our study.
As a first strategic attempt to identify a strongly toxic EA derivative and to investigate
SAR, we targeted the α,β unsaturated carbonyl moiety of EA (part 1) as shown in Scheme 1.
Scheme 1.Selective reduction of EA to afford 3 and 4
.
The synthesis of compounds 3 and 4 was initiated from the commercially available
EA. The treatment of EA with hydrogen (3 bars) in the presence of Pd/C in isopropanol for
20 min provided compound 3 in quantitative yield. Compound 4 was prepared in three steps.
Thus, the treatment of EA with methanol in the presence of p-toluenesulfonic acid (PTSA) at
room temperature led to the corresponding methyl ester. This intermediate was subjected to
the Luche reduction with NaBH4 in presence of CeCl3 in a mixture of H2O/methanol. Then,
the reaction mixture was treated with sodium hydroxide in methanol to furnish the free acid 4.
Then, to further explore SAR in the EA series, we directed our efforts to the
preparation of two original EA derivative series based on modifications in the part 2 (Figure
3): series A (2-(4-substituted phenyl)ethanamine) and series B (4-(4-substituted
phenyl)piperazine) and for this purpose EA derivatives 5-22 were prepared.

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Figure 3. Representation of series A and B developed from EA.
The synthesis of EA derivatives 5-9, 19 and 22 is shown in Scheme 2, whereas the
synthesis of 10-17 is shown in Scheme 3. The preparation of original derivatives 5-9, 19 and
22 was achieved in one step synthesis from EA and various amines using conventional
conditions of peptide synthesis (amines in a mixture of dichloromethane (DCM)/N,N-
dimethylformamide
(DMF)
in
the
presence
of
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDCI) and 4-dimethylaminopyridine (DMAP) at room
temperature). The desired products were isolated after flash chromatography in moderate
yields (23-25 %). These low yields are essentially due to the formation of by-products of
Michael addition on the double bond.
Scheme 2. Synthesis of EA derivatives 5-9, 19 and 22.

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Scheme 3. Synthesis of the phosphate based EA derivatives 10-17.
Starting from EA containing phenol groups 6 and 7 and either dimethyl
chlorophosphate (10 and 11), diethyl chlorophosphate (12 and 13), 2-chloro-1,3,2-
dioxaphosphorinane 2-oxide [16] (14 and 15) or 2-chloro-4-(3-chlorophenyl)-1,3,2-
dioxaphosphorinane 2-oxide [17] (16 and 17) in DCM in the presence of triethylamine, the
expected products 10-17 were isolated after purification by flash chromatography in yields
ranging between 36 and 67 %.
Based on the very interesting anti-proliferative activities of compounds 8 and 12 (see
Table 1, vide infra), we decided to prepare the disubstituted analogs 18, 19 and 20. Compound
18 was prepared using conventional conditions. Thus, as shown in Scheme 4, EA was treated
by 4-(2-amino-3-ethyl)-2-methoxyphenol in a mixture of DCM/DMF in the presence of N,N'-
dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) at room temperature
leading in moderate yield (48%) to the desired compound 18 which in turn was treated by
diethyl chlorophosphate in DCM in the presence of trimethylamine to lead to the expected
product 20 bearing phosphate moiety in moderate yield (47%). The derivative 19 was
obtained from EA and 2-(3,4-dimethoxyphenyl)ethanamine using conventional conditions of
peptide synthesis (EDCI, DMAP DCM/DMF, r.t.) in 45% yield (see Scheme 2, vide supra).

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Scheme 4. Synthesis of compounds 18, 19 and 20.
Then, to further investigate SAR within the chemical series A we turned our attention
to the replacement of the NH group of 6 by either an oxygen or a N-methyl group (Scheme 2
and 5). Compound 21 was directly obtained by treatment of EA with 4-(2-
hydroxyethyl)phenol in the presence of p-toluenesulfonic acid (APTS) in dichloromethane
(DCM) at room temperature with a 92% yield (Scheme 5). Compound 22 was prepared as
indicated in Scheme 2 by condensation of EA with 2-(4-methoxyphenyl)-N-
methylethanamine in a mixture of EDCI, DMAP/DCM/DMF, at room temperature with a
92% yield.
Scheme 5. Synthesis of compounds 21 and 22.
Finally, our attention has been directed towards the preparation of compounds 23-26
in order to confirm or not the crucial role of the α,β unsaturated carbonyl moiety in the
biologically active derivatives 6 (series A), 7 and 9 (series B). The synthetic route leading to
compounds 23-26, described in Scheme 6, was achieved from corresponding reduced EA
derivatives. Thus, the treatment of 3 or 4 (Scheme 1) in the presence of EDCI and a catalytic
amount of DMAP and either 4-(2-aminoethyl)phenol, 4-(piperazin-1-yl)phenol or 1-(4-

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methoxyphenyl)piperazine in a mixture of DCM/DMF (Scheme 6) led to the expected
products 23 (51% yield), 24 (15% yield), 25 (76% yield) and 26 (75 % yield).
Having in hand a wide range of new EA derivatives, we turned our attention to their
anti-proliferative properties and to the elucidation of their mechanisms of action.
Scheme 6. Synthesis of compounds 23, 24, 25 and 26.
3. Biological evaluation and discussion
First, based on a MTS (multiple target screening) assay, we examined the in vitro anti-
proliferative effects of the 26 EA derivatives prepared on both KB (epidermal carcinoma) and
HL60 (promyelocytic) cells. In addition, we tested these compounds against the non-dividing
quiescent endothelial progenitor cells (EPC) to figure a selectivity index (SI) and might
suggest that these chemicals are specifically acting on cells with a rapid proliferation.
Selective reduction of the ethylene double bond of EA (part 1, Figure 2) afforded the
compound 3 and totally abolished the moderate toxicity of EA, whatever the cell line exposed
(KB and HL60). Similarly, the simple reduction of the carbonyl moiety to the hydroxyl group
of EA gives the derivative 4, which was found also inactive.
Few papers described the transformation of the part 1 of EA. I. Janser et al. [12k]
highlighted the modification on the unsaturated double bond or the phenyl ring of EA
derivatives but not from EA itself. Analogues displayed no or very limited activities either in
anti-proliferative or anti-metastatic activities in wound healing assays at concentration ranged
50-100 µM. In another study performed by Cottam et al. [12c], the reduction of the α-β
double bond of EA led to a compound corresponding to our compound 3, and completely
abrogated the anti-proliferative activity against chronic lymphocytic leukemia (CLL) and the
inhibition of the canonical Wnt/β-catenin signaling pathway. Never in these studies the

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ACCEPTED MANUSCRIPT
carbonyl group has been reduced like in our study (compound 4). It can be concluded that, the
reduction of the α-β double bond of EA completely abolished anti-cancer activities.
A major urinary metabolite of EA results from the condensation of the ethylene double
bond to the thiol of GSH [18a]. The reactivity of the ethylene double bond was also postulated
to play a crucial role in the inhibition of GST π [18b]. Therefore the reduction of the ethylene
bond might severely reduce the reactivity of EA and its derivatives.
These results prompted us to consider modifications of the part 2 instead of the part 1
of the structure (Figure 2) including two series: series A (2-(4-substituted phenyl)ethanamine)
and series B (4-(4-substituted phenyl)piperazine), and for this purpose EA derivatives 5-22
were prepared.
The anti-proliferative activities of EA and 5-17 are shown in Table 1. Thus,
introduction of a 2-phenylethanamine chain (series A) affording the compound 5, provided
good anti-proliferative activity against both KB and HL60 cell lines with IC50 values of 0.6
µM and 1.3 µM, respectively. In addition, compound 5 showed low activities against EPC
quiescent cells, with an IC50 of 4.2 µM, suggesting its specific action on rapidly proliferating
cells, e.g. cancer cells. This encouraging result drove us to design other EA derivatives using
amide functions. Thus, as shown in Scheme 2, we prepared the derivatives 6 and 7 bearing 4-
hydroxy-2-phenylethanamine (series A) and 4-hydroxy-phenylpiperazine (series B) groups,
respectively.
Both amide derivatives (6 and 7) displayed anti-proliferative activity against KB and
HL60 cell lines with IC50s of 0.4 µM and 0.7 µM, and 0.4 µM and 0.8 µM, respectively. In
addition, both displayed a reduced activity against EPC cell lines, with IC50s around 3-4 µM.
Based on the promising results of these two active compounds, we decided to develop two
different chemical series: 2-(4-substituted phenyl)ethanamine and 4-(4-substituted
phenyl)piperazine. The simple substitution of the hydroxyl group of 6 and 7 by a methoxy
moiety, afforded the compounds 8 and 9, respectively. The compounds showed anti-
proliferative activity against both KB and HL60 cell lines, with IC50s of 0.8 µM and 1.3 µM,
and 0.4 µM and 0.8 µM, respectively. Interestingly, the replacement of the hydroxyl group of
6 and 7 by a dimethyl phosphate moiety afforded the compounds 10 and 11 with IC50s of 0.4
µM and 1.2 µM, and 0.17 µM and 0.8 µM against KB and HL60 cell lines, respectively. Its
substitution by a diethyl phosphate moiety (compounds 12 and 13), increased also the anti-
proliferative activity versus EA, and maintain the anti-proliferative activity versus 10 and 11,
with IC50s of 0.8 µM and 1.1 µM, and 0.3 µM and 0.5 µM against KB and HL60 cell lines,
respectively. Importantly, the introduction of a cyclic phosphate (2-hydroxy-1,3,2-
dioxaphosphinane 2-oxide) in position-4 on the phenyl groups of 6 and 7 instead of the
hydroxyl group, afforded the EA derivatives 14 and 15, displaying good anti-proliferative
activities, and showing IC50s of 0.5 µM and 1.2 µM, and 0.2 µM and 0.35 µM against KB and
HL60 cell lines, respectively. From these derivatives, we then decided to prepare the
derivatives 16 and 17 bearing a 3-chloro-phenyl group in position 4 of the 1,3,2-
dioxaphosphorinane ring as cytochrome CYP3A4-selective prodrugs of the hydroxyl group of
6 and 7. The EA derivatives 16 and 17 displayed similar anti-proliferative activities than 14
and 15.
It is noteworthy that two of the most potent EA analogs within the series B
(compounds 11 and 17), exhibited very promising SI. Compound 11 was respectively 17- and
3.6-fold more potent on KB and HL60 than on EPC. Compound 17 was respectively 21- and
8.7-fold more potent on KB and HL60 compared to EPC.
The very interesting anticancer activities of the compound 8 (series A) prompted us to
orientate our efforts towards the preparation of disubstituted EA derivatives 18, 19 and 20 in
order to evaluate the role of an additional group on the phenyl ring of 8 and 12.

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Table 1. Anti-proliferative activities of EA and compounds 5-17 (series A and B).
Selectivity
IC50 (µM)
Series
Compounds
R1
Index [a]
logP[b]
KB
HL60
EPC
KB/HL60
EA
5
11±2
46±12
1.3±0.3
0.7±0.1
1.3±0.1
1.2±0.1
1.1±0.1
>100
>9 / >2
7 / 3
3.0
4.6
4.3
4.5
4.7
5.4
A
A
A
A
A
H
0.6±0.1
0.4±0.05
0.8±0.1
0.4±0.1
0.8±0.1
4.2±0.7
3.6±0.1
3.7±0.1
0.7±0.2
3.2±0.2
6
HO
MeO
9 / 5
8
4.6 / 2.8
1.75 / 0.58
4 / 2.9
10
12
(MeO)2P(O)-O
(EtO)2P(O)-O
A
A
14
16
0.5±0.1
0.7±0.2
1.2±0.2
0.9±0.1
3.4±0.1
2.2±0.2
6.8 / 2.8
3.1 / 2.4
4.6
7.0
B
B
B
B
7
9
HO
0.4±0.1
0.4±0.2
0.8±0.1
0.8±0.1
0.8±0.1
0.5±0.1
3.2±0.1
3.6±0.1
2.9±0.1
2.8±0.2
8 / 4
4.3
4.6
4.9
5.6
MeO
9 / 4.5
11
13
(MeO)2P(O)-O
(EtO)2P(O)-O
0.17±0.02
0.3±0.1
17 / 3.6
9.3 / 5.6
B
B
15
17
0.2±0.1
0.4±0.1
0.35±0.05
0.9±0.1
1.25±0.05
7.8±1.4
6.25 / 3.6
21 / 8.7
4.7
7.1
[a] Selectivity Index (SI): IC50EPC/IC50KB or HL60. [b] Calculated partition coefficient (logP), see text
As shown in Table 2, we observed no improvement in the anti-proliferative activity of
18 and 19 versus 8. We then decided to introduce a methoxy group in position 3 of 12 due to
its excellent anti-proliferative activity to afford the compound 20. Also, we observed no
improvement in the anti-proliferative activity of 20 versus 12.

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Then, to further investigate SAR within the series A, we turned our attention to the
replacement of the NH group of 6 by either an oxygen or a N-methyl group affording
compounds 21 and 22, respectively. Both compounds 21 and 22 displayed good anti-
proliferative activities against KB and HL60 (Table 3). Given that 22 displayed similar
activities to 6, the replacement of a secondary amide group with a tertiary amide group did not
influence the cellular potency.
Table 2. Anti-proliferative activities of EA, 8, 12 and 18-20.
Selectivity
IC50 (µM)
Series Compounds
R1
R2
Index [a]
logP[b]
KB
HL60
EPC
KB / HL60
EA
11±2
46±12
>100
>9 / >2
4.6 / 2.8
4 / 2.9
3.0
4.5
5.4
4.6
4.4
5.1
A
A
A
A
A
8
MeO
0.8±0.1 1.3±0.1
0.8±0.1 1.1±0.1
0.8±0.1 1.6±0.1
0.5±0.1 0.8±0.1
0.7±0.1 0.8±0.1
3.7±0.1
3.2±0.2
4.5±0.3
3.4±0.1
2.3±0.1
12
18
19
20
(EtO)2P(O)-O
HO
MeO
MeO
OMe
5.6 / 2.8
6.8 / 4.2
3.2 / 2.8
MeO
O-P(O)(OEt)2
[a] Selectivity Index (SI): IC50EPC/IC50KB or HL60.[b] Calculated partition coefficient (logP), see text
Table 3. Anti-proliferative activities of EA, 6, 21 and 22.
Compounds
R
X
Selectivity
Index[a]
KB / HL60
IC50 (µM)
HL60
logP[b]
KB
EPC
EA
11±2
46±12
>100
>9 / >2
3.0
6
HO
NH
0.4±0.05
0.9±0.1
0.7±0.1
1.4±0.1
1.3±0.1
3.6±0.1
3.7±0.1
4±0.3
9 / 5
4.3
4.9
4.8
21
22
OH
O
4 / 2.65
5.7 / 3
OMe
N-Me
0.7±0.05
[a] Selectivity Index (SI):IC50EPC/IC50KB or HL60. [b] Calculated partition coefficient (logP), see text

12
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Finally, in order to fully confirm the essential role of the α,β unsaturated carbonyl
moiety of the derivatives 6 (series A), 7 and 9 (series B), we focused our attention on the
synthesis of compounds 23 and 24 (series A), and compounds 25 and 26 (series B) (Scheme
6). As previously observed for the compounds 3 and 4, the selective reduction of the α,β
unsaturated carbonyl moiety totally abolished the anti-proliferative activity of 6, 7 and 9 with
IC50s >100 µM against both KB, HL60 or EPC.
Then, we attempted to correlate the anti-proliferative activities of EA derivatives with
a classical physicochemical parameter such as calculated membrane-water partition
coefficient (logP). It is well known that lipophilicity, which associates both biological and
physicochemical properties, is one of the most important physical properties of a drug
conditioning its absorption, distribution, potency and elimination [19,20]. For each compound
prepared (Tables 2-4) we calculated the corresponding logP using Marvin application
(ChemAxon). No relationship can be reported between logP and IC50 but readily reveals that a
potent anti-proliferative activity was observed when logP is higher than 4 (ranging 4.3-7.1)
versus 3 for EA. It is important to note that the logP must be not too high in order to develop
drug-like compounds (e.g. solubility issue). Additionally, no correlation has been noticed
between IC50s and either pKa, MW, the number of hydrogen acceptors, or the number of
hydrogen donors using Marvin application (ChemAxon).
Based on the anti-proliferative activity and selectivity index, compounds 11 and 17 are
the most interesting derivatives. They showed a SI of 17 and 21, respectively (KB vs EPC)
and displayed similar but higher potency against KB in comparison with HL60. The most
promising is the compound 11 due to its lower logP (4.9 vs 7.1 for 17) and lower molecular
weight (529 vs 651).
For further investigations, we decided to explore the anti-proliferative activity of a
series of chemical diverse EA derivatives (series A and B) against a panel of cancer cell lines
representative of diverse tissues/organ tumors: human breast cancer cell line (MCF7), human
colon carcinoma cell line (HCT116), human prostate cancer cell line (PC3), ovarian
carcinoma cells (SK-OV3 and OVCAR8, Table 4).
Table 4. Anti-proliferative activity of EA and derivatives upon various cancer human
cells.
R1
IC50 µM)[a]
PC3
Series
Compounds
MCF7
41±9
HCT116
75±0
SK-OV3
>100
OVCAR8
72±3
75±0
EA
6
A
A
B
B
HO
1.8±0.2
0.9±0.1
0.8±0.1
0.5±0.1
1.7±0.6
1.5±0.6
2.0±0.7
0.8±0.5
1.0±0.5
0.8±0.2
3.9±3.1
1.1±0.1
4.5±1.8
2.8±0.2
2.3±1.2
2.2±1.3
1.5±0.3
1.5±0.2
0.8±0.3
1.4±0.2
10
7
(MeO)2P(O)-O
HO
11
(MeO)2P(O)-O

13
ACCEPTED MANUSCRIPT
B
15
0.9±0.4
1.0±0.6
0.5±0.1
3.5±0.5
1.9±0.6
EA retained a poor anti-proliferative activity against the five cell lines tested with IC50
above 50 µM, featuring its general lack of activity, while the EA derivatives 6, 7, 10, 11 and
15 displayed moderate to good anti-proliferative activities with IC50s ranking between 4.5 µM
and 0.5 µM. In the series A, the derivative with a trimethoxy phosphate group linked to the 4-
hydroxy-2-phenylethanamine moiety (10) was a potent anti-proliferative compound with IC50
slightly better than 6 having only the 4-hydroxy-2-phenylethanamine group (except against
OVCAR8 cell line). This suggests that the introduction of a trimethoxyphosphate group
moderately increased the anti-proliferative activity of EA derivatives. The replacement of the
4-hydroxy-2-phenylethanamine group by the 4-hydroxy-phenylpiperazine group (7 versus 6)
had low significant effect except against PC3 cell lines for which 6 is about 4-fold better than
7. Within the series B, the addition of the trimethoxyphosphate unit instead of the hydroxyl
group on the phenyl ring (11 versus 7) or the introduction of a cyclic phosphate (oxo 2, 1,3,2-
dioxaphosphorinane ring) also on the phenyl group (15 versus 7) moderately increases the
potency except for the SK-OV3 and OVCAR8 cell lines.
All together these data suggested that the nature, hydrophobicity and size of the
terminal group in position 4 of the phenyl unit linked to the EA molecule and the series (A
and B) had no noticeable importance for the anti-proliferative activity of EA derivatives. It is
noteworthy that the two phosphate derivatives 10 and 11 and the dioxaphosphorinane 15
showed good homogeneity related to their IC50s and higher activity towards MCF7, HCT116
and PC3 cell lines than towards the ovarian cancer cell lines SK-OV3 and OVCAR8. A
different sensibility to cisplatin has been reported in these two ovarian cell lines, SK-OV3
been considered as resistant compared to OVCAR8: a similar behavior can be observed with
EA and its derivatives [21].
The next step was the determination of the mechanism of action of EA derivatives and
was intended to confirm the modulation of anti-proliferative capacity by structural
modifications. Evaluations regarding vital cellular processes have been tested essentially in
HL60 cells for relevant comparison with a selection of 13 chemically diverse EA derivatives
selected in accordance with their structural design and bearing modification either in the part
A alleviating activity (23, 24, 25 and 26) or in the part B within the two series A and B:
amide-monosubstituted (5, 6, 7, 8 and 9), ester-monosubstituted (21), N-methyl amide-
monosubstituted (22), and disubstituted (18 and 19) showing IC50s against KB and HL60
between 0.4 µM and 1.6 µM.
To ensure the cytotoxicity of EA and its derivatives, the viability of HL60 cells was
investigated by Fluorescence Activated Cell Sorter (FACS) after a 24h exposure to a selection
of compounds chosen for their structure-activity determined previously. The viacount®
reagent used in these experiments is composed of two DNA binding dyes, one permeant to
viable cells and one impermeant allowing the discrimination between the number of viable
and the total number of cells remaining after chemical exposure. The IC50 of EA (measured
with the NADH reagent) was comparable at 24h and 72h in HL60 cells resulting from its poor
anti-proliferative activity. Although lower than at 72h, anti-proliferative activity of EA

14
ACCEPTED MANUSCRIPT
derivatives was higher than those of EA at 24h (Figure 4). Taking this into account,
concentrations used for testing were 50 µM for EA and 7 µM for derivatives.
HL60
EA
5
6
21
8
22
18
19
IC50
38±1
1.6±0.1 1.7±0.1 2.9±0.4 2.4±0.3 2.3±0.1 2.6±0.1 1.4±0.1
(µM)
Figure 4. Anti-proliferative activity of EA (50 µM) and derivatives (7 µM) upon HL60 cells
after a 24h exposure and cytotoxic activity measured by FACS. Results are expressed as the
percentage of viable cells (in black) and dead cells (in light grey). IC50 were determined with
the MTS test after a 24h exposure of HL60 cells.
The consequences of modification of the part 1 of the native EA molecule were not
tested due to the very low basal activity of the EA molecule. First, modifications on the α,β
unsaturated carbonyl moiety of EA derivatives were examined (Figure 2), the reduction of the
ethylene bond of the 4-hydroxy-phenylpiperazine (25, 26 vs 7) and 4-hydroxy-2-
phenylethanamine (23 vs 6) linked chain resulted in a noticeable increase in the cellular
viability to reach the value obtained with the native EA molecule. Reduction of the carbonyl
bond (24 vs 6) also resulted in the loss of toxicity. These data are in line with those collected
by measuring the reduced nicotinamide adenine dinucleotide (NADH) content of cells after
72h and therefore confirmed the critical role of the α,β unsaturated carbonyl moiety chain in
the cell death promoted by EA derivatives.
As expected, replacement of the amido by an ester linkage (21 vs 6) severely impaired the
toxicity of the derivative, in relation with the supposed hydrolysis of the ester bond (Table 3).
The combined presence of a hydroxy and a methoxy group on the phenyl ring of the
phenylethanamine chain (18) also augmented the IC50 at 24h and the viability of HL60 cells
compared to compounds having a phenyl (5), a phenyl ring with a single hydroxy or methoxy
group (6 and 8), or a dimethoxy (19) leading to potent cytotoxic molecules. The presence of a
-N(CH3)-CO- linkage instead of a -N(H)-CO- one (22 vs 8) had no effect.
Another marker to estimate the viability of cells consists in the determination of the
cellular reduced glutathione content, which is an essential cellular component protecting from

15
ACCEPTED MANUSCRIPT
damages caused by reactive oxygen species and electrophilic compounds [22]. Reduced
glutathione content was only moderately decreased after a 24h exposure to 50 µM EA,
whereas potent anti-proliferative derivatives at concentration 7 µM elicited a higher reduction
(Figure 5). Once more the replacement of the amido linkage by an ester linkage strongly
prevented the reduction of glutathione content (21 vs 6), as well as the presence of the 3-
methoxy-4-hydroxy-2-phenylethanamine chain (18), compared to single substitution
compounds (6 and 8) or to the dimethoxy 2-phenylethanamine derivative (19), potent inducers
of GSH depletion.
Figure 5. GSH content of HL60 cells exposed for 24h to EA (50 µM) and derivatives (7 µM).
Results are expressed as a percentage of the GSH content in cells treated with DMSO alone.
Actually the cell viability estimated by FACS and the endogenous glutathione content
were highly correlated (Figure 6), viable cells having a high glutathione content. This
suggests that after a 24h exposure to EA derivatives, the cell death is already proceeding and
results in the diminution of the reduced glutathione content, a reduction of the cellular NADH
content, and finally the decrease of cell viability.
120
% viability
100
80
y = 67,533x + 40,544
y = 67.533x + 40.544
2
R² = 0,8901
R = 0.8901
60
40
20
0
endogenous GSH
0,0
0.0
0,2
0.2
0,4
0.4
0,6
0.6
0,8
0.8
1,0
1.0
1,2
1.2
Figure 6. Relationship between cell viability and GSH content of HL60 cells exposed
to EA (50 µM) and derivatives (7 µM) for 24h. Results are expressed as a percentage of
values in cells treated with DMSO.

16
ACCEPTED MANUSCRIPT
To ascertain the mode of action of EA derivatives, we intended to evaluate the
different biological pathways leading to cell death. A first attempt was to explore a possible
necrotic process [23] induced by EA derivatives in a set of experiments performed on HL60
cells. Necrosis results from the disruption of the plasma membrane and leads to the release of
the cytosolic content. Therefore, necrosis can be estimated by measuring the activity of lactate
dehydrogenase (LDH), released into the culture medium during treatment. Menadione, which
is known to induce necrosis in various cell lines, was used as a positive control. At 50 µM,
menadione elicited an increase of the LDH activity released in the culture medium up to 60%
after a 48h exposure (data not shown) and a reduction of the total cell number. Exposure of
HL60 cells to 50 µM EA had only a marginal effect on the LDH ratio after 24 and 48h,
confirming the poor intrinsic cytotoxicity of this molecule (Figure 7, upper panel). After a 24h
exposure to EA derivatives, the release of LDH was limited in HL60 cells and only a 48h
exposure to a few EA derivatives resulted in a significant but moderate release of LDH: the
dimethoxy 2-phenylethanamine linked EA (19) was the more active followed by the 4-
methoxy-2-phenylethanamine N-methyl amido EA (22) and the 4-methoxy-2-
phenylethanamine EA (8). A similar moderate effect was obtained within KB cells (Figure 7
middle panel) and also in HCT116 and MRC5 cells (data not shown).
Modifications on the α,β unsaturated carbonyl moiety of EA derivatives were also
examined (Figure 7 lower panel). The reduction of the ethylene double bond of the 4-
hydroxy-phenylpiperazine (26 and 25 vs 7) or 4-hydroxy-2-phenylethanamine (23 vs 6) linked
chain or reduction of the carbonyl of the 4-hydroxy-2-phenylethanamine linked chain (24) did
not modified substantially the LDH release. These results demonstrate that the necrotic
process is only marginally involved in the biological effect of EA derivatives.

17
ACCEPTED MANUSCRIPT
Figure 7. LDH release from cells treated for 24 and 48h with EA (50 µM) and
derivatives (7 µM). Results are the amount of LDH released in the culture medium from KB
and HL60 cells treated for 24 or 48h with EA and derivatives and expressed as a percentage
of LDH released by cells receiving DMSO alone.
Several markers can be utilized to characterize apoptosis which is a common pathway
used by anti-proliferative drugs to promote cell death [24]. Flow cytometry analysis (FACS) is
a reliable and fast method to demonstrate the entry of cells into the apoptotic process. An
early event in the apoptotic process is the externalization of phosphatidyl-serine from the
inner to the outer cell membrane, then accessible to annexin V. Later the plasma membrane is
disrupted during the late apoptosis/necrosis stage and cells can be classified as intact, in early
apoptosis or in late apoptosis or necrosis. To further analyze the biological effect of EA
derivatives, annexin-V-7 AAD staining was carried out and assessed by flow cytometry.
Therefore, HL60 cells were treated for 24h with EA and four of its derivatives selected for
their activity. Results are shown in Table 5. Control cells were treated with vehicle only. EA

18
ACCEPTED MANUSCRIPT
at low concentration had no effect after 24h, but induced the cell entry in early apoptosis at
high concentration.
Table 5. FACS analysis of cell death induced by EA and derivatives after 24h
exposure.
necrosis/late
apoptosis
intact
cells
early
apoptosis
Compound
Control
dead cells
0.1
0.1
0
0.6
91.6
87.8
57.9
12.8
33.7
47.3
7.7
11.2
41.3
65
EA (10 µM)
EA (50 µM)
5 (5 µM)
0.9
0.8
0.3
0.3
0.2
21.9
16.0
10.0
6 (5 µM)
50
21 (5 µM)
42.5
EA derivatives promoted early apoptosis at a lower concentration (5 µM instead of 10
or 50 µM) and also late apoptosis/necrosis: as expected from former data, the replacement of
the amido linkage by the ester linkage (21 vs 6) reduced the entry of cells in the apoptotic
process, especially in the late apoptosis/necrosis stage. The nature of the substituent on the
NH amino linkage had no effect since all derivatives displayed comparable capacity to
activate apoptosis (data not shown). Similar data were observed after a 48h exposure (data not
shown) indicating that the activation of the apoptotic pathway was a rapid answer to chemical
exposure. Therefore it is conceivable to conclude that EA derivatives induced cell death
through the activation of the apoptotic pathway.
Caspases (cysteine-aspartic acid proteases) are activated during apoptosis in many cells and
are known to play a vital role in both initiation and execution of apoptosis. It was reported
that caspase-3 is the terminal effector in the apoptotic cascade and is a major contributor to
cellular DNA fragmentation. To go further in the elucidation of the apoptotic pathway
stimulated by EA derivatives, the catalytic activity of caspase-3 was assayed in HL60 cells
treated for 24h with EA (50 µM) and the selected EA derivatives (5 µM). Doxorubicin (50
nM) was used as a positive control. At low concentration, EA failed to activate caspase
activity, in line with data obtained by FACS, but unexpectedly had also no effect at high
concentration (Figure 8). Consistent with the entry in early apoptosis observed above, EA
derivatives activated to a great extent caspase 3 after a 24h exposure: 2-phenylethanamine (5)
and 4-hydroxy-2-phenylethanamine (6) linked derivatives elicited comparable striking
activation, whereas the ester linked analog (21) was much less active.

19
ACCEPTED MANUSCRIPT
400%
300%
200%
100%
0%
dmso EA
EA
5
6
21
10µM 50µM
Figure 8. Activation of caspase 3/7 activity in HL60 cells by EA (50 µM) and
derivatives (5 µM) after 24 h exposure.
The Jun-JNK signaling pathway is a mechanism proposed to explain caspase
activation. JNK is a pro-apoptotic kinase [25a] associated to GST π in the cell cytosol then
impairing the kinase activity [25b]. The exposure to GST π inhibitors prevents the repression
of JNK activity and impacts apoptosis [25b,c]. Previously, EA at high doses was found to
synergize the arsenic oxide action in HL60 cells, through the activation of JNK and caspase 3
[26]. Therefore it is conceivable that the active EA derivatives might be potential inhibitors of
GST π, produce an activation of JNK to finally stimulate apoptosis.
Another mechanism involves the Wnt/β-catenin pathway. Overexpression of β-catenin
induces apoptosis in hematopoietic stem/progenitor cells and NIH 3T3 fibroblasts via the
elevation of caspase 3 [27a,b]. EA and EA amine derivatives have been reported as Wnt
inhibitors in a cell based Wnt reporter assay and selectively suppressed chronic lymphocytic
leukemia (CLL) cells survival although no strict relationship was observable between CLL
inhibition growth and Wnt inhibition [12c, 27c]. The inhibition of Wnt by active EA
derivatives described in this study might explain their antiproliferative capacity.
It has been assumed that anti-proliferative drugs triggering apoptosis and cell death,
ultimately promoted DNA fragmentation associated or not with prior cell cycle arrest in either
G1, S or G2/M phases. To ascertain whether or not EA and its derivatives are capable to
interact with the cell cycle, the impact of 2.5 µM EA derivatives was investigated in HL60
cells treated for 24 and 48h. For that purpose, we selected the EA derivatives 6, 7, 15
derivatives having high anti-proliferative activity, as well as two inactive derivatives 23, 24.
Cells were stained with propidium iodide (PI), and analyzed by FACS: a majority of control
cells contained 2n chromosomes (phase G0/G1) or participated in an active DNA synthesis
(phase S), with some cells already engaged in mitosis (phase G2/M). In apoptotic cells the
DNA was partly fragmented (phase sub G1) (Table 6). After a 24h exposure to either
derivative, no modification of the proportion of cells containing 2n, intermediate and 4n
chromosomes was noticeable. After 48h, 2.5 µM EA did not alter the partition of cells
between the cell cycle phases, whereas active anti-proliferative EA derivatives 6, 7 and 15
rose the proportion of apoptotic cells with a reduction of cells in S phase and an almost
complete disappearance of G2/M phase. As anticipated, the two inactive EA derivatives 23
and 24 did not raise the proportion of apoptotic cells, but unexpectedly reduced the number of

20
ACCEPTED MANUSCRIPT
cells in phases S and G2/M and augmented the G0/G1 phase. It could be concluded that EA
had no effect on the control of cell cycle, while its active derivatives prevented the DNA
synthesis and mitosis to favor resting cells before to promote apoptosis and DNA
fragmentation.
Table 6. Cell cycle analysis by FACS of HL60 cells treated for 24 and 48h with EA
and derivatives (2.5 µM)
Compounds
G0/G1
S Phase
G2/M
SubG1
Control 24h
43.5
45.4
43.2
41.7
40.7
42.1
42.3
48.9
43.6
47.3
49.3
47.1
46.7
50.5
5.1
8.3
6.2
6.1
9.6
7.9
5.2
2.6
2.7
3.3
2.9
2.6
3.3
2.0
EA
6
7
15
23
24
Control 48h
43.8
52.6
2.2
1.4
EA
6
41.5
44.3
49.3
41.7
51.7
51.9
54.1
45.8
33.8
30.7
41.9
41.5
1.3
0.1
0.4
0.0
2.2
0.6
3.1
9.9
7
16.5
27.6
4.2
15
23
24
6.1
4. Conclusions
The starting point of this original study is the diuretic drug ethacrynic acid which displays
by itself a modest anti-proliferative activity at high doses (>60 µM) [10a] and is believed to
have no direct effect on cell death but acts as inhibitor of π class glutathione S-transferase, in
line with its role of adjuvant in chemotherapy. This low intrinsic anti-proliferative activity
could be markedly enhanced through the introduction of an additional lateral chain affording
original EA derivatives. Clearly, our data feature several points:
- the presence of the α-β double bond of EA is essential for its cytotoxic activity, any
alteration of this residue resulting in a total loss of activity.
- addition of a lateral chain raising the logP results in a drastic increase of the anti-
proliferative capacity, reduces the intracellular glutathione content and finally the cell
viability.
- the replacement of the stable amido bond by a cleavable ester bond generates the release of
native EA and significantly lowers the anti-proliferative activity of derivatives.

21
ACCEPTED MANUSCRIPT
- when the α-β double bond of active EA derivatives is modified, the anti-proliferative
activity is totally lost confirming the crucial involvement of the α-β double bond in the cell
death process.
Therefore, the anti-proliferative activity of EA derivatives prepared during this study is the
combinational result of two elements: the basic toxicity due to the α-β double bond and its
potentiation by the addition of a hydrophobic lateral chain. Thus, it becomes possible to
modulate the poor anti-proliferative capacity of EA by introduction of relevant lateral chain
leading to the activation of the apoptotic cascade.
In summary, this study points out the potential of new EA derivatives as novel
efficient anticancer agents, with enhanced potency relative to EA itself. Basically and
interestingly, the active EA derivatives prepared in this study induce primarily the apoptosis
process without activation of necrosis, through the activation of the caspase cascade without
alteration of the cell division, while in marked contrast inactive EA derivatives promote cell
resting. This mechanism of action of the EA derivatives reported in this study is not single -
which potentially will avoid tumor resistances - than the unique mechanism of action
established for EA, which is an inhibitor of π class glutathione S-transferase.
Clearly our study demonstrates the optimization of EA which shows low anti-
proliferative activities (IC50 > 50 µM) with unclear/several mechanism(s) and affords potent,
original and simple EA derivatives (IC50 0.2-1.6 µM) through apoptotic process.
5. Experimental Section
Syntheses were carried out using standard high vacuum and dry-argon techniques. All
chemicals were purchased from Across, Aldrich, Fluka, and used without further purification.
The solvents were freshly dried and distilled according to standard procedures prior to use.
1H, 13C, and 31P NMR spectra were recorded with Bruker AV300, DPX300, AV400,
13
31
spectrometers. All C NMR and P NMR spectra were generally recorded decoupled {1H}.
The signal of the non-deuterated solvent served as internal standard. The following
abbreviations have been used for indicating the multiplicity of signals: s (singlet), d (doublet),
q (quadruplet), h (heptuplet), m (multiplet). Fourier transformed infrared (FTIR) spectra were
obtained with a Perkin–Elmer Spectrum 100 FT-IR spectrometer on neat samples (ATR FT-
IR) or in solutions. Mass spectrometry was carried out with a Thermo Fisher DS QII
(DCI/NH3 or DCI/CH4). Compounds 3 [12c], 4 [28] as well as compounds 6 and 7 [10] were
prepared following the previously reported procedure. The methyl ester of EA, which is the
intermediate for the preparation of compound 4, was prepared according to reference [29].
The chloro-2-oxo-2-dioxophosphorinase used for the synthesis of 14 and 15 was prepared
according to reference [13]. The 2-chloro-4-(3-chlorophenyl)-1,3,2-dioxaphosphorinane 2-
oxide used for the preparation of 16 and 17 was prepared according to reference [17], 2-(2,3-
dichloro-4-(2-methylbutanoyl)phenoxy)acetic acid (3) according to reference [12c] and 2-
(2,3-dichloro-4-(1-hydroxy-2-methylenebutyl)phenoxy)acetic acid (4) according to reference
[30].
General Procedure: Preparation of 5, 8, 9, 19 and 22. To a mixture of EDCI (1.2 equiv),
catalytic amount of DMAP and ethacrynic acid (1 equiv) in dry DMF (5 mL), amine (2-
phenylethanamine,
2-(4-methoxyphenyl)ethanamine,
2-(4-methoxyphenyl)-N-
methylethanamine, 4-(2-aminoethyl)-2-methoxyphenol, 2-(3,4-dimethoxyphenyl)ethanamine)
and 1-(4-methoxyphenyl)piperazine (1 equiv) was added at 0°C. The reaction mixture was
stirred overnight at room temperature. Then, ethyl acetate (100 mL) was added and the
organic layer was washed with water (2 × 50 mL) and brine (3 × 50 mL), dried over

22
ACCEPTED MANUSCRIPT
anhydrous MgSO4 and concentrated under reduced pressure. The obtained residue was
purified by flash chromatography.
2-(2,3-dichloro-4-(2-methylenebutanoyl)phenoxy)-N-phenethylacetamide (5). Following
general procedure, the crude residue was purified by flash chromatography, using
DCM/EtOAc, 2:8 (v/v) to THF, as eluents, to furnish 5 as a white solid (155 mg, yield of
58%). 1H NMR (300 MHz, CDCl3) δ = 7.31-7.38 (m, 2H, Har), 7.24-7.30 (m, 3H, Har), 7.21
(d, J = 8.5 Hz, 1H, Har), 6.89 (d, J = 8.5 Hz, 1H, Har), 6.74 (s, 1H), 6.00 (s, 1H), 5.61 (s, 1H),
4.57 (s, 2H), 3.67 (q, J = 6.9 Hz, 2H), 2.91 (t, J = 6.9 Hz, 2H), 2.49 (q, J = 7.4 Hz, 2H), 1.18
13
(t, J = 7.4 Hz, 3H). C NMR (75 MHz, CDCl3) δ = 195.3 (C, C=O), 166.3 (C, C(=O)N),
154.6 (C, Car), 150.2 (C, Car), 138.6 (C, Car), 133.9 (C, Car), 131.0 (C, Car), 128.7 (2CH,
Car), 128.6 (2CH, Car), 128.7 (CH2, C=CH2), 127.2 (CH, Car), 126.6 (C, Car), 122,7 (C,
Car), 110.9 (CH, Car), 68.2 (CH2, OCH2), 39.9 (CH2), 35.4 (CH2), 23.4 (CH2), 12.2 (CH3). IR
(neat): ν = 3404 (NH), 1661(C=O) cm-1. HRMS [M+H]+ calculated for C21H21Cl2NO3:
406.0977, found: 406.0972.
2-(2,3-dichloro-4-(2-methylenebutanoyl)phenoxy)-N-(4-methoxyphenethyl)acetamide
(8). Following general procedure, the crude residue was purified by flash chromatography,
using DCM/EtOAc, 3:7 (v/v) to THF, as eluents, to furnish 8 as a white solid (181 mg, yield
of 63%). 1H NMR (300 MHz, CDCl3) δ = 7.18 (d, J = 8.6 Hz, 1H, Har), 7.13 (d, J = 8.7 Hz,
2H, Har), 6.87 (d, J = 8.7 Hz, 2H, Har), 6.83 (d, J = 8.6 Hz, 1H, Har), 5.97 (s, 1H), 6.75 (s,
1H, NH), 5.59 (s, 1H), 4.55 (s, 2H), 3.82 (s, 3H, OCH3), 3.65 (q, J = 6.6 Hz, 2H), 2.84 (t, J =
6.6 Hz, 2H), 2.49 (q, J = 7.4 Hz, 2H), 1.17 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ
= 195.5 (C, C(=O)), 166.5 (C, C(=O)N), 158.4 (C, Car), 154.4 (C, Car), 150.2 (C, Car), 134.1
(C, Car), 131.4 (C, Car), 130.1 (C, Car), 129.7 (2CH, Car), 128.7 (CH2, C=CH2), 127.2 (CH,
Car), 122.9 (C, Car),114.2 (2CH, Car), 110.7 (CH, Car), 68.1 (CH2), 55.3 (OCH3), 40.2
(CH2), 34.7 (CH2), 23.4 (CH2), 12.4 (CH3). IR (neat): ν = 3404 (NH), 1661(C=O) cm-1.
HRMS [M+H]+ calculated for C22H23Cl2NO4: 436.1082, found: 436.1094.
1-(2,3-dichloro-4-(2-(4-(4-methoxyphenyl)piperazin-1-yl)-2-oxoethoxy)phenyl)-2-
methylenebutan-1-one (9). Following general procedure, the crude residue was purified by
flash chromatography, using DCM/EtOAc, 8:2 (v/v) as eluent, to furnish 9 as a white solid
(180 mg, yield of 47%). 1H NMR (400 MHz, CDCl3) δ = 7.15 (d, J = 8.5 Hz, 1H, Har), 6.99
(d, J = 8.5 Hz, 1H, Har), 6.93-6.82 (m, 4H, Har), 5.93 (s, 1H), 5.59 (s, 1H), 4.86 (s, 2H), 3.83-
3.73 (m, 7H), 3.08 (t, J = 5.0 Hz, 2H), 3.03 (t, J = 5.0 Hz, 2H), 2.46 (q, J = 7.4 Hz, 2H), 1.14
13
(t, J = 7.4 Hz, 3H). C NMR (101 MHz, CDCl3) δ = 195.9 (C, C(=O)), 165.3 (C, C(=O)N),
155.4 (C, Car), 154.7 (C, Car), 150.3 (C, Car), 145.2 (C, Car), 133.9 (C, Car), 131.7 (C, Car),
128.8 (CH2, C=CH2), 127.3 (2CH, CHar), 123.0 (C, Car), 119.3 (CH, CHar), 114.7 (2CH,
CHar), 110.9 (CH, CHar), 68.9 (CH2), 55.7 (OCH3), 51.6 (CH2), 51.0 (CH2), 45.8 (CH2), 42.6
(CH2), 23.7 (CH2), 12.6 (CH3). IR (neat): ν = 1661 (C=O) cm-1. HRMS [M+H]+ calculated for
C24H26Cl2N2O4 477.1348, found 477.1324. Elemental analysis calculated (%) for
C24H26Cl2N2O4: C 60.38, H 5.49, N 5.85, found: C 60.42, H 5.24, N 5.69.
2-(2,3-dichloro-4-(2-methylenebutanoyl)phenoxy)-N-(3,4-dimethoxyphenethyl)acetamide
(19). Following general procedure, the crude residue was purified by flash chromatography,
using DCM/EtOAc, 2:8 (v/v) to THF, as eluents, to furnish 19 as a white solid (138 mg, yield
of 45%). 1H NMR (300 MHz, CDCl3) δ = 7.21 (d, J = 8.6 Hz, 1H, Har), 6.90 (d, J = 8.6 Hz,
1H, Har), 6.69-6.91 (m, 4H, Har), 6.00 (s, 1H), 5.60 (s,1H), 4.56 (s, 2H), 3.84 (s, 6H), 3.65 (q,
J = 6.6 Hz, 2H), 2.84 (t, J = 6.9 Hz, 2H), 2.49 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H).
13C NMR (75 MHz, CDCl3) δ = 195.3 (C, C(=O)), 166.3 (C, C(=O)N), 154.6 (CH, Car),
150.2 (CH, Car), 149.3 (C, Car), 148.0 (C, Car), 133.9 (C, Car), 131.0 (C, Car),130.9 (C,

23
ACCEPTED MANUSCRIPT
Car), 128.59 (CH, Car), 127.2 (CH, Car), 122.7 (C, Car), 120.7 (CH, Car), 112.1 (CH, Car),
111.7 (CH, Car), 110.9 (CH, Car), 68.3 (CH2), 55.8 (OCH3), 55.7 (OCH3), 40.1 (CH2), 35.0
(CH2), 23.4 (CH2), 12.2 (CH3). IR (neat): ν = 3404 (NH), 1661(C=O) cm-1. HRMS [M+H]+
calculated for C23H25Cl2NO5: 466.1188, found: 466.1188.
2-(2,3-dichloro-4-(2-methylenebutanoyl)phenoxy)-N-(4-methoxyphenethyl)-N-
methylacetamide (22). Following general procedure, the crude residue was purified by flash
chromatography, using DCM/EtOAc, 2:8 (v/v) to THF, as eluents, to furnish 22 as a white
foam (184 mg, yield of 58%).Two conformations (50% / 50%) were observed at room
temperature. 1H NMR (500 MHz, CDCl3) δ = 7.09-7.16 (m, 3H, Har), 7.03-7.09 (m, 3H, Har),
6.84-6.91 (m, 3H, Har), 6.05 (d, J = 1.6 Hz, 1H, Har), 5.95 (s, 1H), 5.94 (s, 1H), 5.63 (s, 5H),
5.61 (s, 1H), 4.80 (s, 1H), 4.44 (s, 1H), 3.80 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.61 (t, J =
6.8 Hz, 4H), 3.03 (s, 3H, NCH3), 2.98 (s, 3H, NCH3), 2.86 (t, J = 6.6 Hz, 2H), 2.80 (t, J = 7.2
Hz, 2H), 2.46-2.57 (m, 4H), 1.17 (t, J = 7.2 Hz, 6H). 13C NMR (126 MHz, C6D6) δ = 195.92
(d, C, C(=O)), 195.86 (d, C, C(=O)), 166.59 (C, C(=O)N),166.25 (C, C(=O)N), 158.34 (C,
Car), 157.79 (C, Car), 155.30 (C, Car), 155.22 (C, Car), 149.53 (C, Car), 132.88 (C, Car),
132.62 (C, Car), 131.15 (C, Car), 130.84 (C, Car), 130.45 (CH, Car), 130.43 (CH, Car),
130.36 (CH2, C=CH2), 130.26 (CH2, C=CH2), 130.04 (CH, Car), 129.81(C, Car), 127.17 (CH,
Car), 127.03 (CH, Car), 122.42 (C, Car), 122.04 (C, Car), 114.28 (CH, Car), 113.74 (CH,
Car), 110.32 (CH, Car), 110.21 (CH, Car), 67.42 (CH2), 66.77 (CH2), 55.46 (OCH3), 55.44
(OCH3), 50.76 (CH2), 50.41 (CH2), 35.49 (NCH3), 33.45 (NCH3), 32.92 (CH2), 32.53 (CH2),
23.27 (CH2), 23.24 (CH2), 12.4 (2CH3). IR (neat): ν = 1658 (C=O) cm-1. HRMS [M+H]+
calculated for C22H23Cl2NO4: 450.1239, found: 450.1235.
General Procedure: Preparation of 10, 11, 12, 13, 14, 15, 16, 17 and 20. To a mixture of the
corresponding phenol (1 equiv) and triethylamine (1.1 equiv) in dry DCM at 0 °C,
chlorophosphate was added dropwise (1 equiv). The reaction mixture was allowed to warm at
room temperature, then stirred at that temperature overnight. DCM (20 mL) was added and
the organic layer was washed with water (10 mL) and brine (10 mL), dried over anhydrous
MgSO4 and then concentrated under reduced pressure. The crude product was purified by
flash chromatography.
4-(2-(2-(2,3-dichloro-4-(2-methylenebutanoyl)phenoxy)acetamido)ethyl)phenyl dimethyl
phosphate (10). Following general procedure, the crude residue was purified by flash
chromatography, using DCM/EtOAc, 8:2 (v/v) as eluent, to furnish 10 as a colorless oil (50
mg, yield of 36%). 31P NMR (162 MHz, CDCl3) δ = -4.0 (s, 1P). 1H NMR (400 MHz, CDCl3)
δ = 7.12-7.22 (m, 5H, Har), 6.82 (d, J = 8.5 Hz, 1H, Har), 6.76 (t, J = 5.9 Hz, 1H, NH), 5.94
(t, J = 1.3 Hz, 1H, Har), 5.57 (s, 1H), 4.50-4.56 (m, 2H), 3.88 (s, 3H), 3.85 (s, 3H), 3.64 (q, J
= 6.6 Hz, 2H), 2.86 (t, J = 6.6 Hz, 2H), 2.46 (q, J = 7.4 Hz, 2H), 1.14 (t, J = 7.4 Hz, 3H). 13C
NMR (101 MHz, CDCl3) δ = 195.6 (C, C=O), 166.7 (C, C(=O)N), 154.5 (C, Car), 150.3 (C,
Car), 149.6 (d, J = 6.9 Hz, C, Car), 135.3 (C, Car), 134.3 (C, Car), 131.6 (C, Car), 130.2
(2CH, CHar), 128.9 (CH2, C=CH2), 127.3 (C, Car), 123.0 (C, Car), 120.3 (d, J = 4.8 Hz, 2CH,
Car), 110.9 (CH, Car), 68.2 (CH2, OCH2), 55.1 (d, J = 6.2 Hz, 2CH3), 40.2 (CH2), 35.0 (CH2),
23.5 (CH2), 12.5 (CH3). IR (neat): ν = 3416 (NH), 1666 (C=O) cm-1. HRMS [M+H]+
calculated for C23H26Cl2NO7P: 530.0902, found: 530.0909.
4-(4-(2-(2,3-dichloro-4-(2-methylenebutanoyl)phenoxy)acetyl)piperazin-1-yl)phenyl
dimethyl phosphate (11). Following general procedure, the crude residue was purified by
flash chromatography, using DCM/EtOAc/Et3N, 80:20:0.5 (v/v/v) as eluent, to furnish 11 as a
31
1
colorless oil (51 mg, yield of 39%). P NMR (162 MHz, CDCl3) δ = -3.6 (s, 1P). H NMR
(400 MHz, CDCl3) δ = 7.20-7.28 (m, 3H, Har), 6.99 (d, J = 8.6 Hz, 1H, Har), 6.87 (td, J =

24
ACCEPTED MANUSCRIPT
9.3, 2.3 Hz, 2H, Har), 5.93 (t, J = 1.5 Hz, 1H), 5.58 (s, 1H), 4.86 (s, 2H), 3.86 (s, 3H), 3.83 (s,
3H), 3.75-3.81 (m, 4H), 3.14 (t, J = 5.0 Hz, 2H), 3.09 (t, J = 5.0 Hz, 2H), 2.46 (q, J = 7.4 Hz,
13
2H), 1.13 (t, J = 7.4 Hz, 3H). C NMR (101 MHz, CDCl3) δ = 195.9 (C, C=O), 165.3 (C,
C(=O)N), 155.3 (C, Car), 150.3 (C, Car), 148.4 (C, Car), 144.7 (d, J = 7.1 Hz, C, Car), 133.9
(C, Car), 131.6 (C, Car), 128.8 (CH2, C=CH2), 127.2 (CH, Car), 122.9 (C, Car), 120.7 (d, J =
4.7 Hz, 2CH, Car), 118.2 (2CH, Car), 110.8 (CH, Car), 68.9 (CH2, OCH2), 55.0 (d, J = 6.2
Hz, 2CH3), 50.6 (CH2), 50.0 (CH2), 45.6 (CH2), 42.3 (CH2), 23.6 (CH2), 12.5 (CH3). IR
(neat): ν = 3416 (NH), 1666 (C=O) cm-1. HRMS [M+H]+ calculated for C23H26Cl2NO7P:
530.0902, found: 530.0909.
4-(2-(2-(2,3-dichloro-4-(2-methylenebutanoyl)phenoxy)acetamido)ethyl)phenyl diethyl
phosphate (12). Following general procedure, the crude residue was purified by flash
chromatography, using DCM/EtOAc, 7:3 (v/v) as eluent, to furnish 12 as a colorless oil (103
1
mg, yield of 39%).31P NMR (121 MHz, CD2Cl2) δ = -6.32 (s, P). H NMR (300 MHz,
CD2Cl2) δ = 7.15-7.29 (m, 5H, Har), 6.91 (d, J = 8.5 Hz, 1H, Har), 6.77 (s, 1H, NH), 6.00 (s,
1H), 5.60 (s, 1H), 4.57 (s, 2H), 4.14-4.29 (m, 4H), 3.65 (q, J = 6.9 Hz, 2H), 2.89 (t, J = 6.9
Hz, 2H), 1.17 (t, J = 7.4 Hz, 3H), 2.48 (q, J = 7.4 Hz, 2H), 1.37 (td, J = 7.1, 1.0 Hz, 6H). 13C
NMR (75 MHz, CD2Cl2) δ = 195.5 (C, C=O), 166.4 (C, C(=O)N), 154.6 (C, Car), 150.2 (C,
Car), 149.6 (d, J = 6.8 Hz, Car), 135.3 (d, J = 1.2 Hz, Car), 133.9 (C, Car), 131.0 (2C, Car),
129.9 (C, Car), 128.6 (C, CH2), 127.3 (C, Car), 125.4 (C, Car), 120.1 (d, J = 4.8 Hz, 2C, Car),
111.0 (C, Car), 68.2 (CH2), 64.5 (d, J = 6.1 Hz, 2CH3), 40.0 (CH2), 34.8 (CH2), 23.4 (CH2),
15.9 (d, J = 6.6 Hz, 2CH2), 12.2 (CH3). IR (neat): ν = 3419 (NH), 1666 (C=O) cm-1. HRMS
[M+H]+ calculated for C25H30Cl2NO7P: 558.1215, found: 558.1227.
4-(4-(2-(2,3-dichloro-4-(2-methylenebutanoyl)phenoxy)acetyl)piperazin-1-yl)phenyl
diethyl phosphate (13). Following general procedure, the crude residue was purified by flash
chromatography, using DCM/EtOAc, 2:1 to 1:1 (v/v) as eluent, to furnish 13 as a white solid
1
(47 mg, yield of 46%).31P NMR (162 MHz, CDCl3) δ = -5.8 (s, 1P). H NMR (400 MHz,
CDCl3) δ = 7.09-7.18 (m, 3H, Har), 6.98 (d, J = 8.6 Hz, 1H, Har), 6.82-6.88 (m, 2H, Har),
5.93 (t, J = 1.5 Hz, 1H), 5.58 (s, 1H), 4.86 (s, 2H), 4.10-4.28 (m, 4H), 3.74-3.74 (m, 4H), 3.14
(t, J = 5.1 Hz, 2H), 3.09 (t, J = 5.1 Hz, 2H), 2.45 (q, J = 7.4 Hz, 2H), 1.34 (td, J = 7.1, 1.0 Hz,
13
6H), 1.13 (t, J = 7.4 Hz, 3H). C NMR (101 MHz, CDCl3) δ = 195.9 (C, C=O), 165.3 (C,
C(=O)N), 155.3 (C, Car), 150.3 (C, Car), 148.2 (C, Car), 144.9 (d, J = 7.0 Hz, C, Car), 133.9
(C, Car), 131.6 (C, Car), 128.8 (CH2, C=CH2), 127.2 (CH, Car), 122.9 (C, Car), 120.8 (d, J =
4.7 Hz, 2CH, Car), 118.2 (2CH, Car), 110.8 (CH, Car), 68.9 (CH2, OCH2), 64.6 (d, J = 6.1
Hz, 2CH2), 50.7 (CH2), 50.0 (CH2), 45.6 (CH2), 42.3 (CH2), 23.5 (CH2), 16.2 (d, J = 6.7 Hz,
2CH3), 12.5 (CH3). IR (neat): ν = 1663 (C=O) cm-1. HRMS [M+H]+ calculated for
C27H23Cl2N2O7P: 599.1481, found: 599.1486.
2-(2,3-dichloro-4-(2-methylenebutanoyl)phenoxy)-N-(4-((2-oxido-1,3,2-
dioxaphosphinan-2-yl)oxy)phenethyl)acetamide (14). Following general procedure, the
crude residue was purified by flash chromatography, using DCM/EtOAc, 8:2 to 1:1 (v/v) as
eluent, to furnish 14 as a colorless oil (75 mg, yield of 58%). 31P NMR (162 MHz, CD2Cl2) δ
= -13.4 (s, 1P). 1H NMR (400 MHz, CD2Cl2) δ = 7.13-7.23 (m, 5H, Har), 6.81 (d, J = 6.1 Hz,
1H, Har), 6.77 (t, J = 8.5 Hz, 1H, NH), 5.94 (t, J = 1.3 Hz, 1H, Har), 5.57 (s, 1H), 4.51-4.58
(m, 6H), 3.63 (q, J = 6.6 Hz, 2H), 2.85 (t, J = 6.6 Hz, 2H), 2.45 (q, J = 7.4 Hz, 2H), 2.31-2.42
(m, 1H), 1.77-1.81 (m, 1H), 1.13 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CD2Cl2) δ = 195.6
(C, C=O), 166.8 (C, C(=O)N), 154.6 (C, Car), 150.2 (C, Car), 149.4 (d, J = 6.7 Hz, C, Car),
135.2 (C, Car), 134.3 (C, Car), 131.5 (C, Car), 130.3 (2CH, CHar), 129.1 (CH2, C=CH2),
127.3 (C, Car), 123.1 (C, Car), 119.9 (d, J = 5.2 Hz, 2CH, Car), 110.9 (CH, Car), 69.5 (d, J =

25
ACCEPTED MANUSCRIPT
7.4 Hz, 2CH2, OCH2), 68.3 (CH2, OCH2), 40.3 (CH2), 34.9 (CH2), 26.2 (d, J = 7.3 Hz, CH2),
23.5 (CH2), 12.52 (CH3). IR (neat): ν = 3418 (NH), 1664 (C=O) cm-1. HRMS [M+H]+
calculated for C24H26Cl2NO7P: 542.0902, found: 542.0911.
1-(2,3-dichloro-4-(2-(4-(4-((2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)phenyl)piperazin-1-
yl)-2-oxoethoxy)phenyl)-2-methylenebutan-1-one (15). Following general procedure, the
crude residue was purified by flash chromatography, using DCM/EtOAc, 2:1 to 1:1 (v/v) as
eluent, to furnish 14 as a white solid (60 mg, yield of 59%). 31P NMR (162 MHz, CDCl3) δ =
1
-13.0 (s, 1P). H NMR (400 MHz, CDCl3) δ = 7.12-17.19 (m, 3H, Har), 6.99 (d, J = 8.6 Hz,
1H, Har), 6.84-6.91 (m, 2H, Har), 5.93 (s, 1H), 5.59 (s, 1H), 4.86 (s, 2H), 4.42-4.55 (m, 4H),
3.74-3.82 (m, 4H), 3.14 (t, J = 5.1 Hz, 2H), 3.09 (t, J = 5.1 Hz, 2H), 2.46 (q, J = 7.4 Hz, 2H),
2.30-2.42 (m, 1H), 1.75-1.86 (m, 1H), 1.14 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3)
δ = 195.9 (C, C=O), 165.4 (C, C(=O)N), 155.4 (C, Car), 150.3 (C, Car), 148.5 (C, Car), 144.5
(d, J = 6.8 Hz, C, Car), 133.9 (C, Car), 131.7 (C, Car), 128.7 (CH2, C=CH2), 127.3 (CH, Car),
122.9 (C, Car), 120.6 (d, J = 5.0 Hz, 2CH, Car), 118.5 (2CH, Car), 110.9 (CH, Car), 69.4 (d, J
= 7.3 Hz, 2CH2), 68.9 (CH2, OCH2), 50.7 (CH2), 50.1 (CH2), 45.7 (CH2), 42.3 (CH2), 26.1 (d,
J = 7.3 Hz, CH2), 23.6 (CH2), 12.6 (CH3). IR (neat): ν = 1661 (C=O) cm-1. HRMS [M+H]+
calculated for C26H29Cl2N2O7P: 583.1168, found: 583.1177.
N-(4-((4-(3-chlorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)phenethyl)-2-(2,3-
dichloro-4-(2-methylenebutanoyl)phenoxy)acetamide (16). Following general procedure,
the crude residue was purified by flash chromatography, using DCM/EtOAc, 2:1 to 1:1 (v/v)
as eluent, to furnish 16 as a white foam (40 mg, yield of 50%). 31P NMR (162 MHz, CDCl3)
δ= -11.3 (s, 1P). 1H NMR (400 MHz, CDCl3) δ = 7.29-7.31 (m, 2H, Har), 7.25-7.28 (m, 1H,
Har), 7.15-7.24 (m, 6H, Har), 6.76-6.83 (m, 2H, Har), 5.94 (t, J = 1.5 Hz, 1H, Har), 5.60-5.74
(m, 1H), 5.56 (s, 1H), 4.62-4.78 (m, 1H), 4.45-4.56 (m, 3H), 3.65 (q, J = 6.7 Hz, 2H), 2.88 (t,
J = 6.7 Hz, 2H), 2.46 (q, J = 7.4 Hz, 2H), 2.24-2.38 (m, 1H), 2.06-2.18 (m, 1H), 1.14 (t, J =
7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ = 195.7 (C, C=O), 166.8 (C, C(=O)N), 154.5 (C,
Car), 150.4 (C, Car), 149.6 (d, J = 7.5 Hz, C, Car), 140.7 (d, J = 7.9 Hz, C, Car), 135.7 (d, J =
1.3 Hz, C, Car), 134.9 (C, Car), 134.4 (C, Car), 131.6 (C, Car), 130.2 (CH, CHar), 130.2
(2CH, CHar), 129.1 (CH, CHar), 128.9 (CH2, C=CH2), 127.3 (CH, Car), 125.9 (CH, Car),
123.8 (CH, Car), 123.1 (C, Car), 120.7 (d, J = 4.7 Hz, 2CH, Car), 110.9 (CH, Car), 80.1 (d, J
= 5.5 Hz, CH), 68.3 (CH2, OCH2), 67.3 (d, J = 5.8 Hz, CH2), 40.3 (CH2), 35.0 (CH2), 33.4 (d,
J = 7.4 Hz, CH2), 23.6 (CH2), 12.5 (CH3). IR (neat): ν = 3419 (NH), 1666 (C=O) cm-1. HRMS
[M+H]+ calculated for C30H29Cl3NO7P: 652.0825, found: 652.0851.
1-(2,3-dichloro-4-(2-(4-(4-((4-(3-chlorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-
yl)oxy)phenyl)piperazin-1-yl)-2-oxoethoxy)phenyl)-2-methylenebutan-1-one
(17).
Following general procedure, the crude residue was purified by flash chromatography, using
31
DCM/EtOAc, 1:1 (v/v) as eluent, to furnish 17 as a white foam (60 mg, yield of 67%). P
1
NMR (162 MHz, CDCl3) δ = -10.9 (s, 1P). H NMR (400 MHz, CDCl3) δ = 7.27-7.33 (m,
2H, Har), 7.24-7.27 (m, 1H, Har), 7.11-7.18 (m, 4H, Har), 6.99 (d, J = 8.6 Hz, 1H, Har), 6.86-
6.91 (m, 2H, Har), 5.93 (t, J = 1.5 Hz, 1H, Har), 5.61-5.69 (m, 1H), 5.58 (s, 1H), 4.86 (s, 2H),
4.61-4.77 (m, 1H), 4.40-4.54 (m, 1H), 3.78 (q, J = 5.3 Hz, 4H), 3.06-3.22 (m, 4H), 2.45 (q, J
= 7.4 Hz, 2H), 2.15-2.37 (m, 1H), 2.02-2.20 (m, 1H), 1.13 (t, J = 7.4 Hz, 3H). 13C NMR (101
MHz, CDCl3) δ = 195.9 (C, C=O), 165.4 (C, C(=O)N), 155.4 (C, Car), 150.3 (C, Car), 148.7
(C, Car), 144.6 (d, J = 7.8 Hz, C, Car), 140.9 (d, J = 8.0 Hz, C, Car), 134.9 (C, Car), 133.9 (C,
Car), 131.6 (C, Car), 130.3 (2CH, CHar), 129.1 (CH, CHar), 128.8 (CH2, C=CH2), 127.3
(CH, Car), 125.9 (CH, Car), 123.8 (CH, Car), 122.9 (C, Car), 121.1 (d, J = 4.5 Hz, 2CH, Car),
118.2 (CH, Car), 110.8 (CH, Car), 80.1 (d, J = 5.5 Hz, CH), 68.9 (CH2, OCH2), 67.4 (d, J =

26
ACCEPTED MANUSCRIPT
5.8 Hz, CH2), 50.5 (CH2), 49.9 (CH2), 45.5 (CH2), 42.3 (CH2), 33.5 (d, J = 7.2 Hz, CH2), 23.5
(CH2), 12.6 (3). IR (neat): ν = 1663 (C=O) cm-1. HRMS [M+H]+ calculated for
C32H32Cl3N2O7P: 693.1091, found 693.1124. Elemental analysis calculated (%) for
C32H32Cl3N2O7P: C 55.39, H 4.65, N 4.04, found: C 55.49, H 4.38, N 3.77.
4-(2-(2-(2,3-dichloro-4-(2-methylenebutanoyl)phenoxy)acetamido)ethyl)-2-
methoxyphenyl diethyl phosphate (20). Following general procedure, the crude residue was
purified by flash chromatography, using DCM/EtOAc, 2:8 (v/v) as eluent, to furnish 20 as a
white foam (122 mg, yield of 47%). 31P NMR (121 MHz, CD2Cl2) δ = -5.85 (s, P). 1H NMR
(300 MHz, CD2Cl2) δ = 7.22 (d, J = 8.6 Hz, 1H, Har), 7.21 (dd, J = 8.1, 1.9 Hz, 1H, Har),
6.91 (d, J = 8.6 Hz, 1H, Har), 6.89 (s, 1H), 6.84 (s, 1H, NH), 6.80 (dd, J = 8.1, 1.9 Hz, 1H),
5.99 (s, 1H), 5.60 (s, 1H), 4.57 (s, 2H), 4.16-4.31 (m, 4H), 3.88 (s, 3H, OCH3), 3.66 (q, J =
6.9 Hz, 2H), 2.89 (t, J = 7.0 Hz, 2H), 2.48 (q, J = 7.4 Hz, 2H), 1.37 (td, J = 7.1, 1.0 Hz, 6H),
1.17 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CD2Cl2) δ = 195.3 (C, C=O), 166.4 (C,
C(=O)N), 154.6 (C, Car), 150.7 (d, J = 5.3 Hz, C, Car), 150.2 (C, Car), 138.6 (d, J = 7.1 Hz,
C, Car), 136.4 (d, J = 1.5 Hz, C, Car), 134.0 (C, Car), 131.0 (C, Car), 128.6 (CH2, C=CH2),
127.3 (CH, Car), 122.7 (C, Car), 121.2 (d, J = 2.8 Hz, CH, Car),120.6 (d, J = 1.3 Hz, CH,
Car), 113.1 (CH, Car), 111.1 (CH, Car), 68.3 (CH2), 64.5 (d, J = 6.1 Hz, 2CH2), 55.9 (OCH3),
39.9 (CH2), 35.3 (CH2), 23.4 (CH2), 15.9 (d, J = 6.8 Hz, 2CH3),12.2 (C, CH3). IR (neat): ν =
3419 (NH), 1666 (C=O) cm-1.
4-hydroxyphenethyl 2-(2,3-dichloro-4-(2-methylenebutanoyl) phenoxy)acetate (21).To a
mixture of paratoluenesulfonic acid (APTS) (1.1 equiv) and ethacrynic acid (1 equiv) in DCM
(10 mL) was added at 0°C 4-(2-hydroxyethyl)phenol (1 equiv). The reaction mixture was
heated to 50 °C for 12h and then evaporated to dryness. The crude residue was purified by
flash chromatography using hexane/EtOAc, 4:6 (v/v) as eluent, to furnish 21 as a white solid
(256 mg, yield of 92%). 1H NMR (400 MHz, CD2Cl2) δ = 7.08 (d, J = 8.6 Hz, 1H, Har), 7.01
(d, J = 8.4 Hz, 2H, Har), 6.69 (d, J = 8.4 Hz, 2H, Har), 6.62 (d, J = 8.6 Hz, 1H, Har), 6.02 (s,
1H), 5.63 (s, 1H), 5.54 (s, 1H, OH), 4.76 (s, 2H), 4.40 (t, J = 6.5 Hz, 2H), 2.91 (t, J = 6.5 Hz,
13
2H), 2.49 (q, J = 7.4 Hz, 2H), 1.18 (t, J = 7.4 Hz, 3H). C NMR (101 MHz, CD2Cl2) δ =
196.3 (C, C=O), 167.7 (C, C(=O)N), 155.5 (C, Car), 154.6 (C, Car), 150.3 (C, Car), 133.5 (C,
Car), 131.1 (C, Car), 129.8 (2CH, Car), 129.3 (C, Car), 129.2 (CH2, C=CH2), 126.8 (CH,
Car), 122.7 (C, Car), 115.4 (2CH, Car), 110.4 (CH, Car), 66.2 (CH2), 66.1 (CH2), 33.9 (CH2),
23.4 (CH2), 12.2 (CH3). IR(neat): ν = 3438 (NH), 1750 (C=O) cm-1. HRMS [M+H]+
calculated for C21H20Cl2O5: 423.0766, found: 423.0773.
2-(2,3-dichloro-4-(2-methylenebutanoyl)phenoxy)-N-(4-hydroxy-3-methoxyphenethyl)
acetamide (18).To a mixture of DCC (1.1 equiv), HOBt (1.1 equiv) and ethacrynic acid (1
equiv) in dry DMF (5 mL) were added at 0°C, 4-(2-aminoethyl)-2-methoxyphenol (1 equiv).
The reaction mixture was stirred overnight and then the solution was filtered and evaporated.
The product was purified by flash chromatography using DCM/EtOAc, 3:7 (v/v) as eluent, to
furnish 18 as a white solid (143 mg, 48%). 1H NMR (300 MHz, CD2Cl2) δ = 7.20 (d, J = 8.5
Hz, 1H, Har), 7.00 (s, 1H, Har), 6.66-6.91 (m, 4H, Har and NH), 5.99 (s, 1H, C=CH2), 5.60
(s, 1H, C=CH2), 5.20 (s, 1H, OH), 4.56 (s, 2H, OCH2), 3.85 (s, 3H, OCH3), 3.62 (dd, J = 12.3,
6.3 Hz, 2H, CH2), 2.82 (t, J = 6.9 Hz, 2H, CH2), 2.48 (q, J = 7.4 Hz, 2H, CH2), 1.17 (t, J = 7.4
Hz, 1H, CH3). 13C NMR (75 MHz, CDCl3) δ = 195.39 (C, C=O), 166.39 (C, C(=O)N), 154.59
(CH, Car), 150.16 (C, Car), 147.10 (C, Car), 144.98 (C, Car), 135.82 (C, Car), 133.92 (C,
Car), 131.02 (C, Car), 129.87 (C, Car), 128.66 (CH2, C=CH2), 127.24 (CH, Car), 121.29 (CH,
Car) , 114.71 (CH, Car), 111.41 (CH, Car), 110.98 (CH, Car), 68.28 (CH2), 55.79 (OCH3),
40.20 (CH2), 35.05 (CH2), 23.38 (CH2), 12.20 (CH3).

27
ACCEPTED MANUSCRIPT
General Procedure: Preparation of 23, 25 and 26. To a solution of ethacrynic acid analog 3
(1 equiv) and amine (1-(4-methoxyphenyl)piperazine, (1-(4-hedroxyphenyl)piperazine or
tyramine) (1 equiv) in a mixture of DCM/DMF (5 ml), EDCI (1.5 equiv) and a catalytic
amount of DMAP were added. The reaction mixture was stirred overnight at room
temperature, then, ethyl acetate (50 ml) was added and the organic layer was washed with
water (2 x 50 ml) and brine (3 x 50 ml). The combined organic layers were dried over MgSO4
and concentrated under reduced pressure. The obtained residue was purified by column
chromatography.
2-(2,3-dichloro-4-(2-methylbutanoyl)phenoxy)-N-(4-hydroxyphenethyl)acetamide (23).
Following general procedure, the crude residue was purified by column chromatography,
using DCM/EtOAc, 8:2 (v/v) as eluent, to furnish 23 as a white foam (50 mg, yield of 51%).
1H NMR (400 MHz, CDCl3) δ = 7.22 (d, J = 8.6 Hz, 1H, Har), 7.00 (td, J = 8.6, 1.4 Hz, 2H,
Har), 6.68-6.75 (m, 3H), 6.61 (t, J = 5.8 Hz, 1H, NH), 5.78 (br s, 1H, OH), 4.54 (s, 2H), 3.62
(q, J = 6.6 Hz, 2H), 3.12-3.23 (m, 1H), 2.79 (t, J = 6.6 Hz, 2H), 1.72-1.88 (m, 1H), 1.38-1.51
(m, 1H), 1.17 (d, J = 6.9 Hz, 3H), 0.93 (t, J = 7.4, Hz, 3H). 13C NMR (100 MHz, CDCl3) δ =
206.8 (C, C=O), 166.9 (C, C(=O)N), 154.9 (C, Car), 154.8 (C, Car), 135.3 (C, Car), 131.3 (C,
Car), 129.84 (C, Car), 129.79 (2CH, Car), 127.2 (CH, Car), 123.3 (C, Car), 115.9 (2CH, Car),
110.8 (CH, Car), 68.1 (CH2, OCH2), 47.1 (CH), 40.3 (CH2), 34.4 (CH2), 26.0 (CH2), 15.7
(CH3), 11.7 (CH3). IR (neat): ν = 3402 (OH), 1664 (C=O) cm-1. HRMS [M+H]+ calculated for
C21H23Cl2NO4: 424.1082, found: 424.1075.
1-(2,3-dichloro-4-(2-(4-(4-hydroxyphenyl)piperazin-1-yl)-2-oxoethoxy)phenyl)-2-
methylbutan-1-one (25). Following general procedure, the crude residue was purified by
column chromatography, using DCM/EtOAc, 8:2 to 5:5 (v/v) as eluent, to furnish 25 as a
white foam (58 mg, yield of 76%). 1H NMR (400 MHz, CDCl3) δ = 7.26 (d, J = 8.6 Hz, 1H,
Har), 6.98 (d, J = 8.6 Hz, 1H, Har), 6.74-6.85 (m, 4H, Har), 5.31 (br s, 1H, OH), 4.86 (s, 2H),
3.77 (t, J = 5.0 Hz, 4H), 3.17 (h, J = 6.8 Hz, 1H), 3.03 (td, J = 21.0, 5.0 Hz, 4H), 1.77 (dqd, J
= 13.5, 7.5, 6.0 Hz, 1H), 1.43 (pd, J = 14.5, 7.5 Hz, 1H), 1.16 (d, J = 6.9 Hz, 3H), 0.91 (t, J =
7.4, Hz, 3H). 13C NMR (100 MHz, CDCl3) δ = 206.3 (C, C=O), 165.4 (C, C(=O)N), 155.7 (C,
Car), 150.8 (C, Car), 145.1 (C, Car), 135.0 (C, Car), 131.4 (C, Car), 127.3 (CH, Car), 123.3
(C, Car), 119.4 (2CH, Car), 116.1 (2CH, Car), 111.0 (CH, Car), 68.8 (CH2, OCH2), 51.7
(CH2), 51.0 (CH2), 46.9 (CH), 45.8 (CH2), 42.6 (CH2), 26.1 (CH2), 15.8 (CH3), 11.7 (CH3). IR
(neat): ν = 3320 (OH), 1692 (C=O) cm-1. HRMS [M+H]+ calculated for C23H26Cl2N2O4
465.1348, found 465.1339. Elemental analysis calculated (%) for C23H26Cl2N2O4: C 59.36, H
5.63, N 6.02, found: C 59.36, H 5.46, N 5.84.
1-(2,3-dichloro-4-(2-(4-(4-methoxyphenyl)piperazin-1-yl)-2-oxoethoxy)phenyl)-2-
methylbutan-1-one (26). Following general procedure, the crude residue was purified by
column chromatography, using DCM/EtOAc, 8:2 (v/v) as eluent, to furnish 26 as a white
foam (64 mg, yield of 75%). 1H NMR (400 MHz, CDCl3) δ = 7.27 (d, J = 8.7 Hz, 1H, Har),
6.99 (d, J = 8.7 Hz, 1H, Har), 6.82-6.92 (m, 4H, Har), 4.87 (s, 2H), 3.75-3.80 (m, 7H), 3.14-
3.25 (m, 1H), 3.01-3.13 (m, 4H), 1.72-1.86 (m, 1H), 1.37-1.52 (m, 1H), 1.16 (d, J = 6.9 Hz,
13
3H), 0.92 (t, J = 7.4, Hz, 3H). C NMR (100 MHz, CDCl3) δ = 206.2 (C, C=O), 165.2 (C,
C(=O)N), 155.8 (C, Car), 154.7 (C, Car), 145.2 (C, Car), 135.0 (C, Car), 131.4 (C, Car),
127.3 (2CH, Car), 123.3 (C, Car), 119.2 (CH, Car), 114.7 (2CH, Car), 111.0 (CH, Car), 68.9
(CH2, OCH2), 55.7 (CH3, OCH3), 51.6 (CH2), 51.0 (CH2), 46.9 (CH), 45.8 (CH2), 42.5 (CH2),
26.1 (CH2), 15.8 (CH3), 11.8 (CH3). IR (neat): ν = 1692 (C=O) cm-1. HRMS [M+H]+
calculated for C24H28Cl2N2O4: 479.1504, found: 479.1499.

28
ACCEPTED MANUSCRIPT
2-(2,3-dichloro-4-(1-hydroxy-2-methylenebutyl)phenoxy)-N-(4-
hydroxyphenethyl)acetamide (24). To a mixture of compound 4 (1 equiv) and tyramine (1
equiv) in a mixture of DCM/DMF (30 ml), EDCI (1.5 equiv) and a catalytic amount of
DMAP were added. The reaction mixture was stirred overnight at room temperature, then,
ethyl acetate (50 ml) was added and the organic layer was washed with water (2 x 50 ml) and
brine (3 x 50 ml). The combined organic phases were dried over MgSO4 and concentrated
under reduced pressure. The obtained residue was purified by column chromatography using
DCM/EtOAc, 8:2 to 5.5 (v/v) as eluent, to furnish 24 as a white foam (70 mg, yield of 15%).
1H NMR (400 MHz, CDCl3) δ = 7.32 (d, J = 8.7 Hz, 1H, Har), 6.97 (d, J = 8.5 Hz, 2H, Har),
6.64-6.73 (m, 4H), 6.51 (br s, 1H, OH), 5.55 (s, 1H), 5.15 (s, 1H), 5.03 (s, 1H), 4.49 (d, J =
14.8 Hz, 1H), 4.43 (d, J = 14.8 Hz, 1H), 3.50-3.70 (m, 2H), 2.68-2.87 (m, 3H), 1.85-2.04 (m,
2H), 1.02 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ = 167.7 (C, C(=O)N), 154.9 (C,
Car), 153.0 (C, Car), 151.0 (C, Car), 134.9 (C, Car), 133.1 (C, Car), 129.8 (2CH, CHar),
129.7 (C, Car), 126.8 (CH, Car), 122.1 (C, Car), 115.9 (2CH, Car), 111.3 (CH, Car), 110.4
(CH2, C=CH2), 73.4 (CH), 68.0 (CH2, OCH2), 40.4 (CH2), 34.3 (CH2), 25.3 (CH2), 12.2
(CH3). IR (neat): ν =3400 (OH), 1662 (C=O) cm-1. HRMS [M+H]+ calculated for
C21H23Cl2NO4: 424.1082, found: 424.1066.
Cell culture. The human cell lines KB (nasopharyngeal epidermis carcinoma) and SK-OV-3
(ovary adenocarcinoma) were originated from the NCI, HCT116 (colon adenocarcinoma) and
PC3 (prostate adenocarcinoma) were purchased from ECACC (Salisbury, UK), EPC (carp
skin epithelium), MRC5-SV2 (human fetal lung) and HL60 (promyeocytic leukaemia) cells
from ATCC. MCF7 (breast adenocarcinoma) were kindly provided by Dr M. Kassack (Bonn
University, Germany) and OVCAR8 (ovary carcinoma) by Dr M. Liscovitch (Rehovot,
Israel). KB and MRC5 cells were grown in D-MEM medium supplemented with 10% fetal
calf serum, in the presence of penicillin, streptomycin and fungizone in 75 cm² flask under
5% CO2, whereas all other cell lines were grown in complete RPMI medium.
Cell proliferation assay. Cells were plated in 96-well tissue culture plates in 200 µL medium
and treated 24 h later with compounds dissolved in DMSO. Control cells received the same
volume of DMSO (1% final volume). After 24 or 72h exposure to the drug, inhibition of cell
proliferation was determined as previously reported [21]. For IC50 determinations (50%
inhibition of cell proliferation) experiments were performed in duplicate with concentrations
ranged 0.5 nm to 100 µm.
Cell viability. Cell viability and GSH content were measured in HL60 cells treated with
compounds for 24h. 100,000 cells were grown in 500 µl medium in 24-well microplates:
viability was monitored on a 20 µl aliquot by flow cytometry after addition of the Viacount
reagent following the recommendations of the manufacturer and fluorescence was measured
with a Guava EasyCyte plus cytometer (Guava, Merck-Millipore, Billerica, USA). Remaining
cells were pelleted by gentle centrifugation, resuspended in 250 µl phosphate buffered
solution and sonicated. Lysate (20 µl) was transferred to a 384-well black microplate and
incubated in a 50 µl final volume of 100 mM sodium phosphate (pH 7.5) with 100 µM
monochlorobimane and 0.4 µg recombinant human GST π (Oxford Biomedical Research,
Oxford, USA). Fluorescence was monitored (excitation 380 nm, emission 460 nm) over a 60
min period.
Necrosis. Necrosis was estimated through the release of LDH in the culture medium. 20,000
HL60 cells were incubated for 24 and 48 h in the presence of chemicals in 96-well [21,31].
Fluorescence was recorded (exc 560 nm, em 590 nm): results are expressed as the residual