Superacid and thiol-ene reactions for access to psammaplin analogues with HDAC inhibition activities Dedicated to Professor Jean Marie Coustard, a creative chemist

Article abstract of DOI:10.1016/j.tet.2014.10.053

An innovative synthesis of psammaplin-like structure is proposed based on original methodologies using superacid, microwaves, and S-ene chemistry. The new compounds were evaluated as histone deacetylase inhibitors. The results highlight important consider

Full text of DOI:10.1016/j.tet.2014.10.053

Tetrahedron 70 (2014) 9702e9708
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Superacid and thiol-ene reactions for access to psammaplin
analogues with HDAC inhibition activities
a
b,
Fatima El Bahhaj , Jerome Desire , Christophe Blanquart c, Nadine Martinet d,
ꢀ ^
ꢀ
~
ꢀ a
e
e
e
b
ꢀ
Vincent Zwick , Claudia Simoes-Pires , Muriel Cuendet , Marc Gregoire ,
Philippe Bertrand a
,c,
*
a
ꢀ
Institut de Chimie des Milieux et Materiaux de Poitiers, UMR CNRS 7285, 4 rue Michel Brunet, B28, 86000 Poitiers, France
b Inserm, UMR 892, CNRS, UMR 6299, University of Nantes, 8 quai Moucousu, 44007 Nantes cedex 1, France
c
ꢀ
ꢀ ꢀ
ꢀ
^
Reseau Epigenetique du Canceropole Grand Ouest, France
d Institut de chimie, UMR CNRS 7272, UNSA, F-06108 Nice, France
e School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 30 Quai Ernest-Ansermet, 1211 Geneva, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 July 2014
Received in revised form 24 October 2014
Accepted 26 October 2014
Available online 29 October 2014
An innovative synthesis of psammaplin-like structure is proposed based on original methodologies using
superacid, microwaves, and S-ene chemistry. The new compounds were evaluated as histone deacetylase
inhibitors. The results highlight important considerations when using disulfide prodrugs activated under
reductive/oxidative conditions that must be carefully selected depending on tumor cell types.
Ó 2014 Elsevier Ltd. All rights reserved.
Dedicated to Professor Jean Marie Coustard,
a creative chemist
Keywords:
Superacid
Microwaves
S-Ene chemistry
Psammaplin
Cancer
1. Introduction
factors and DNA binding proteins to form multi-protein com-
plexes, in turn regulating genes transcription. Several human
In eukaryotic cells DNA is packed in a complex structure reg-
ulating transcriptional activity. The smaller level is the nucleo-
some,1 an octameric protein complex assembled from two copies
of four histone proteins (H2A, H2B, H3, and H4) with about 150
base pairs wrapped around this octamer. DNA transcription is
partially regulated by post translational modifications (PTM) on
histones and cytosine called epigenetic marks and defining the
histone code. 377 Enzymes are regulating these processes sorted
in three groups: the writers, erasers, and readers, respectively,
adding, removing or recognizing these marks on histones or DNA.
These enzymes participate in the recruitment of transcription
diseases such as cancers, neurodegenerative diseases or metabolic
disorders result from abnormal gene expression due to the dis-
equilibrium between epigenetic enzymes activities. As PTMs are
all reversible, renormalization of aberrant PTMs has been pursued
using epigenetic enzymes inhibitors. Histone N -3 lysine acetylation
was identified as a key player, a reaction controlled by histone
acetyl transferases (HAT)2 in balance with histone deacetylases
(HDAC)3 and recognized by bromodomain-containing proteins,4
other reactions on histones being known.5 HDAC have been par-
ticularly studied during the last years for their over expression in
several cancer cell lines, resulting in tumor suppressor gene
silencing.6
HDAC are grouped in four classes, classes I (HDAC 1e3, 8), II
(HDAC 4e7, 9, 10) and IV (HDAC 11) being zinc dependant and
class III (SIRT1-7) requiring NADPH. SAHA (1) (suberoyl anilide
hydroxy amide) and romidepsin (2), two HDAC inhibitors, are
* Corresponding author. Tel.: þ33 549454192; fax: þ33 549453702; e-mail ad-
dress: philippe.bertrand@univ-poitiers.fr (P. Bertrand).
http://dx.doi.org/10.1016/j.tet.2014.10.053
0040-4020/Ó 2014 Elsevier Ltd. All rights reserved.

F. El Bahhaj et al. / Tetrahedron 70 (2014) 9702e9708
9703
approved for treating cutaneous T-cell lymphoma7,8 and are cur-
rently investigated in clinical trials in single or combination ther-
apies9 (Fig. 1). In our search for new HDAC inhibitors, we were
interested in developing libraries of new thiol compounds with
innovative chemistry. Thiols are HDAC inhibitors supposed to bind
the zinc atom in the active site via the free sulfhydryl group. Natural
romidepsin (2), largazole (4),10 and psammaplins (3) illustrate this
family, with NHC-31 (5)11 as a synthetic example. Romidepsin12 is
in fact considered as a natural prodrug supposed to be cleaved in
cells to the free thiol, the real inhibitor. Largazole is also a prodrug,
the thioester group being probably cleaved by esterases in vivo.
Psammaplins are of particular interest because of their various
activities including antibacterial,13,14 chitinase inhibition,15 and
antiproliferative activities due to HDAC inhibition. Some psamma-
plins, such as psammaplins A16 and G,17 also inhibit DNA methyl-
transferases (DNMT) at the nanomolar level, DNA gyrase,18 and
aminopeptidase N, another zinc-dependent enzyme playing a key
role in angiogenesis.19 Psammaplins are also natural prodrugs, as
demonstrated in vitro on HDAC1 activity by incubation of psam-
maplin A with H2O2, dithiothreitol (DTT) or both.20 In cells, the
fluorescent.32 Pharmacokinetics of psammaplins has also been
determined.33 Psammaplins syntheses involve in general the
preparation of a protected oxime moiety from tyrosine derivatives
or oxazoles and final coupling with cystamine or analogues having
longer carbon chains. For psammaplin A itself, an improved syn-
thesis was reported by Godert et al., in four steps with an overall
43% yield.34 We describe in this article a novel and original access to
psammaplin analogues and some biological investigations.
addition of butionine sulfoximine (BSO), a specific inhibitor of g-
GCS, reduced the glutathione levels and was correlated with lower
histone acetylation. The disorganized microvasculature of solid
tumors led to hypoxic conditions being exploited with the de-
velopment of bioreductive compounds.21 In this respect, treating
hypoxic cancer cell lines such as malignant pleural mesothelioma
(MPM),22 an aggressive form of lung cancer with poor prognostic,
with bioreducible disulfide compounds should promote epigenetic
renormalization23 by higher cleavage of the disulfide prodrug.
The psammaplins family consists of thioethylamide bromotyr-
osine metabolites forming dimers by oxidative coupling of a thiol
group (Scheme 1). Psammaplin A was described for the first time in
1987, was isolated from various sponges, and has nanomolar
specificity toward class I HDACs. Several crystal structures of
psammaplin analogues were obtained24,25 in order to perform SAR
studies on various epigenetic targets.26e28 These studies (Scheme
1) showed that for HDAC inhibition (1) any halogen can replace
the bromine atom in the phenyl ring, (2) the phenyl ring should be
close to the oxime, (3) the oxime and the disulfide groups are es-
sential for the activity, and (4) the alkyl chain must be short. Mo-
lecular modeling using HDAC8 showed that the o-bromophenol
group is stabilized through interactions with Tyr100 and Phe152 at
the rim of the active site entrance, the oxime forming a hydrogen
bonding with Asp101. Equivalent results were obtained with
HDAC1 as model target. Additional syntheses or patents can be
found in the literature for the synthesis of modified monomers of
psammaplins or their analogues,28e31 some of them being
Scheme 1. Psammaplin pharmacophore and proposed new synthetic strategy
toward psammaplins.
2. Results and discussion
2.1. Synthetic strategy
During our research programs on innovative syntheses in
superacid media, we were able to develop an original way to obtain,
in one step, an ethoxycarboxyphenyloxime scaffold 8 (Scheme 1).35
This prompted us to investigate an innovative access to psamma-
plin analogues as zinc-dependent HDAC inhibitors, where the
methylene group between the aromatic ring and the oxime was
removed. Superacid chemistry has recently been also described for
the synthesis of another family of zinc-dependent enzymes: the
carbonic anhydrases.36 The scaffold 8 was reported in a dissym-
metric combinatorial synthesis of psammaplin analogues by Nic-
olaou’ group to be used against methicillin-resistant Staphylococcus
aureus.37 The oxime was not tested free but as an O-benzyl ether.
We report here the use of superacid media for the synthesis of free
oxime scaffolds (Scheme 1), introducing microwaves as an efficient
method to prepare an intermediate alkenylamide moiety (9). This
moiety was then converted by thioleene chemistry to a thio-
alkylamide (10) as a precursor of the expected dimer analogues
(11). Final spontaneous oxidative dimerization gave the target
compounds in four steps, a competitive strategy with the one of
Godert et al.34a Some key intermediates and the final thiol de-
rivatives were tested as HDAC inhibitors and for their toxic activi-
ties against MPM cells.
The superacid methodology developed by some of us is based on
the aromatic electrophilic substitution with a particular electro-
phile: the hydroxynitrilium ion. This ion is obtained by reacting
ethyl nitroacetate 7 in triflic acid, a superacid (Scheme 2). The
Fig. 1. SAHA and thiol-based HDAC inhibitors.

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F. El Bahhaj et al. / Tetrahedron 70 (2014) 9702e9708
hydroxynitrilium ion 12 is the result of water loss after several
protonations of the nitro group and is in mesomeric equilibrium
with the corresponding carbocation 13 that can be trapped by
nucleophiles such as activated aromatic rings. The final oxime can
exist as an E/Z mixture.38 SAR studies on psammaplins showed that
the aromatic ring can be substituted with no more than two groups.
The scope of our strategy was determined with various activating
(hydroxy, alkyloxy, alkyl) or deactivating (halogen, nitro) sub-
stituents. Compounds 8 could add novelty to the current SAR
studies as such analogues were never reported in the context of
HDAC inhibitors.
irradiations as expected (1 h at 100 ꢁC under 300 W). In their re-
ported work, Ferroud et al.38 used ethyl acetate as a model for
amidation and with benzylamine, the corresponding amide was
obtained with a yield of 72%, at 200 ꢁC and 200 W for 2.5 min. For
vinylacetate, 1 h was required. Our conditions and yields appeared
similar. The less basic aniline did not give the amide 9c. Un-
fortunately, direct coupling of diamines such as cystamine (12) hy-
drochloride (treated or not to remove HCl) or diaminooctane (13)
with two esters 8a was found difficult both under heating and mi-
crowave conditions. Attempts to prepare 11a,b from the ester 8a by
peptide coupling via the acid 14 (obtained in 68% yield) were not
successful. This peptide coupling was described in psammaplin
syntheses with protected oxime, but in our case this could have
made the synthesis longer and not competitive. This suggests that
the oxime protection is critical for the peptide coupling strategy.
Scheme 2. Mechanism of benzaldoxime formation in superacid from ethyl nitro-
acetate and aromatics for the preparation of scaffold 8. Compounds 8a,b,d,k already
reported in Ref. 38. For 8a the yields have been improved with a new procedure.
Scheme 3. Transamidation: (i) amine, heating neat or microwave neat; (ii) LiOH 2.5 M
aq THF/MeOH; (iii) 12 or 13, EDC or DCC, NHS, CH2Cl2; (iv) hn, R2eSH; (v) NaOH,
MeOH; (vi) air.
2.2. Syntheses
The commercially available ethyl nitroacetate 7 was reacted
with various aromatic compounds 6, at ꢀ5 to ꢀ15 ꢁC depending on
the substrate reactivity in the presence of triflic acid (Scheme 2).
Monosubstituted halogenated substrates 6a,c gave the best yields
and selectively provided the para compounds as major products.
Nitrobenzene 6f did not react and was almost fully recovered while
the starting nitroacetate was not, indicating that the nitrilium ion
must be formed but the aromatic ring is not reactive enough to trap
it. As reported earlier, substrates 6d,k gave the ortho isomers as the
major product in good yield. In our hands, toluene did not react
properly, the reaction being sometimes violent and giving a com-
plex mixture from which the expected 8e was not isolated. With
phenols 6gej substituted with another halogen atom, the reaction
was carried out after optimization at ꢀ5 ꢁC instead of ꢀ15 ꢁC to give
moderate to good yields of compounds 8gej. The observed nega-
tive effect of the phenol group compared to monohalogenated
benzene was even higher with catechol 6i. The presence of free
hydroxyl groups probably increased the apparent water solubility,
making such derivatives difficult to extract from the complex
mixture. This will require further optimization.
Having in mind to maintain the overall synthesis as short as
possible, radical S-ene chemistry was investigated with allylamide 9a
to introduce the sulfur atom in a protected form (Scheme 3). para-
Methoxybenzylthiol and thioacetic acid gave access to the corre-
sponding thioether and ester 10a,b (UV irradiation lamp). The thio-
ester 10b was hydrolyzed to give the free thiol 10c and subsequent
oxidative dimerization gave the expected psammaplin analogue 11c.
Regarding the thioester 10b, this compound can be compared to
largazole (4) (Fig. 1). These two compounds 10b and 4 can be con-
sidered as prodrugs that can be hydrolyzed in cells.10 In this respect,
compound 10b was also evaluated in our biological models.
The reported configuration of natural psammaplins13,40 is E,
confirmed by X-ray analysis of various derivatives24,25 or found
during molecular modeling with epigenetic targets.24,27 Calculations
on isolated oximes 8 (MM2 refined by RHF/3-21G, Chembio3D tools)
and psammaplin A (2) showed that the Z oxime is always more
stable due to the six-membered ring formed by a hydrogen bonding
between the oxime hydrogen and carbonyl oxygen atoms. Similar
results were found for amides and esters. Indeed, it is possible that in
the HDAC active site, the compounds could be isomerized to gen-
erate better interactions with the zinc atom and additional residues.
Thus we assumed that the major isomers should be Z, which was
also explained by the reaction conditions or mechanism in super-
acids, where a trans attack on the carbocation is preferred (Scheme
2) as in the Beckmann rearrangement. Fortunately, in the amide 9
series it was possible to differentiate the oxime isomers for com-
pound 9b, the major one being partially separated by flash
From the esters 8, it was thought possible to prepare the psam-
maplin analogues 11 by transamidation of 8 with amines under
classical heating or under microwave neat conditions as previously
reported.39 This methodology was developed with ester 8a obtained
with the best yields for the preparation of derivatives 11 (Scheme 3).
Transamidation with basic monoamine (allyl- or benzylamine) un-
der heating gave the expected amides 9a,b in moderate to good
yields and the reaction times were shortened under microwaves

F. El Bahhaj et al. / Tetrahedron 70 (2014) 9702e9708
9705
Table 1
chromatography. The methylene group of the benzylamide part gave
HDAC inhibition (%)a in a nuclear extract and toward various isoforms
two distinct signals in proton NMR. For derivative 9b the amide
group in trans gave the more stable compound (Fig. 2). Two stable
isomers with different benzyl group orientations could be found and
could explain the two types of 1H NMR proton signals.
% HDAC inhibition (300
HeLa nuclear extract
mM)
HDAC 1
HDAC 2
HDAC 6
10a
10b
10c
11c
0.3
5.7
13.2
10.4
NDb
ND
19.4
0
ND
ND
13.3
8.1
ND
ND
29.0
38.9
2.3. Biology
a
Designed to be HDAC inhibitors, compounds 8e11 were first
tested for their ability to restore histone acetylation. In order to
verify if the arylcarbamoyloxime group can be isosteric to the
hydroxamic acid or benzamide groups found in HDAC inhibitors,
we selected intermediates 8a,h,j and 9a,b for preliminary tests. No
HDAC inhibition was observed with these compounds, even at high
Results are the means of two independent experiments.
ND: not determined.
b
signal was observed associated with a dose dependent mild toxicity
(Fig. 3B and D). This toxicity is dose dependent and could be re-
sponsible for the absence of BRET signal induction. The use of
compounds 10c and 11c combined with NAC was then investigated.
We first confirmed that the treatment with NAC alone did not
change the BRET signal and cell viability as shown in Fig. 3AeD.
However, the BRET signal induction by 10c and 11c in the presence
of NAC was strongly increased (Fig. 3A and C, right): 5-fold for 11c
and 6-fold for 10c, implying an increased histone H3 acetylation.
This increase of HDAC inhibition activity was associated with a de-
crease of toxicity (Fig. 3B and D, right). The effect of NAC is specific
to psammaplin derivatives given that it did not change HDACi ac-
tivity of SAHA (Supplementary data Fig. S1A and B). These results
highlight the importance of the oxidative process in cancer cells
when dealing with biomolecules reacting to oxidative or reducing
environment, illustrated by the activity of the disulfide prodrug 11c
in the context of HDAC inhibition.
300 mM concentrations, indicating that the carbamoylbenzaldox-
ime group is not a hydroxamic acid or benzamide substitute for
HDAC inhibition, possibly due to the steric hindrance of the con-
jugate phenyl ring.
In contrast, the final thiol derivatives 10aec and 11c showed
some HDAC inhibition in vitro (Table 1). If compound 10a, protected
as a PMB ether was expected to give no inhibition, more surpris-
ingly compounds 10c and 11c gave a moderate HDAC inhibition
despite their similarities to psammaplins. The percentage in-
hibition was determined for compound 11c as 10% at 300
mM.
Compound 11c seems to have some selectivity for HDAC 6 com-
pared to HDAC 2 (about 5-fold) while 10c was only two fold more
selective for HDAC 6 compared to HDAC1-2.
In order to evaluate the effect of the compounds on histone H3
acetylation in living cells, a bioluminescent resonance energy
transfer (BRET)-based assay was used.41 This assay consists in the
transfection of cells with an expression vector coding for a bromo-
domain (BrD), which recognizes acetylated histones, fused to
Renilla luciferase (Rluc) and with an expression vector coding for
histone H3 fused to the yellow fluorescent protein (YFP). In the
presence of an HDACi, the histone H3eYFP is acetylated and then
recruits the BrD-Rluc. In the presence of the Rluc substrate, coe-
lenterazine, an energy transfer from Rluc to YFP occurs due to their
close proximity. This results in the emission of a fluorescent signal
at 530 nm. Thus, an increase of the BRET signal reflects an increase
of histone H3 acetylation. From enzymatic results, only compounds
10c and 11c were evaluated in the BRET assay.
3. Conclusion
In conclusion, an innovative synthetic strategy to access psam-
maplin analogues that could be used to prepare new derivatives
without protecting chemistry is proposed. Interestingly, anti-
proliferative micromolar activities were observed in the mesothe-
lioma cell line M56 for compounds 8a, 9a, and 9b (data not shown).
In contrast, other fluoro compounds including a phenol group like
8h,j were inactive. Some oxime intermediates 8 showed toxic ac-
tivities not correlated with HDAC inhibition. The moderate HDAC
inhibition observed for final thiol derivatives when used in
Enzymatic HDAC inhibition by some psammaplins and their
analogues can reach the nanomolar level but usually in cells higher
concentrations are required to obtain the same effect, resulting in
toxicity. For psammaplins this could be due to the necessary con-
version of disulfide to free thiols,20 limiting the amount of bioactive
compound at a given time. Hypoxic or normoxic environments in
tumors can also alter the formation of free thiols from disulfides.
Hypoxic cancer cells such as MPM overexpress several cytochrome
oxidases and various dehydrogenases42 or reactive oxygen species
(ROS) that may also modify the thiol formation from disulfides.
To take these elements into consideration we compared the
BRET results for HDAC inhibition for compounds used alone or to-
gether with N-acetylcystein (NAC). NAC is a powerful antioxidant
molecule that should favor the free thiol formation and diminish
oxidative processes. This is illustrated in Fig. 3A and C. When cells
were treated with compounds 10c and 11c a poor induction of BRET
Fig. 3. Biological characterization of compounds 10c and 11c using BRET. MeT-5A cells
were transfected with phRluc-C1 BrD and pEYFP-C1 histone H3. Then, cells were
treated for 24 h with the indicated compound concentrations in the presence or not of
NAC. (A, C) Induced-BRET signal obtained with the various conditions. (B, D) Viability
of cells measured with the various conditions. Viability was determined by measuring
luciferase activity and is expressed as the percent of luciferase activity in the control
condition. Results are the meansꢂS.E.M of three independent experiments.
Fig. 2. Relative stability of 9b conformers.

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F. El Bahhaj et al. / Tetrahedron 70 (2014) 9702e9708
combination with NAC warrant further synthetic investigations to
improve the pharmacophore model for this series of analogues. The
redox properties of the tumoral environment can be very important
when analyzing the activities of compounds such as the final thiol
derivatives 10c and 11c. Our work complements results described
to decipher psammaplin derivatives behavior in cells with the use
of an alternative ROS scavenger contributing to the improvement of
histone acetylation in cells.12,20,21 With cancer cell lines developing
hypoxic conditions, the use of bioreducible/oxidable compounds
should be carefully investigated.
atmosphere and allowed to warm to room temperature, then
poured into DCM/MeOH (4:1, 50 ml) preliminary cooled between
ꢀ30 ꢁC and ꢀ40 ꢁC. Saturated solutions of NaCl (10 ml) and NaHCO3
(55.8 mmol) were added while at ꢀ40 ꢁC and allowed to warm to
room temperature. After pH control (pH 5), the resulting mixture
was extracted (DCM/MeOH 10:1, 4ꢃ20 ml), the combined organic
layers dried (MgSO4), and concentrated under vacuum. The crude
product was recrystallized from DCM to give 8g as a light yellow
powder (695 mg, 28%). Rf: 0.5 DCM/MeOH (10:1); 1H NMR
(400 MHz, CD3OD)
6.93 (d, 1H, J¼8.4 Hz), 7.34 (dd, 1H, J¼2, 8.4 Hz), 7.70 (d, 1H, J¼2 Hz);
d
: 1.33 (t, 3H, J¼7.1 Hz), 4.31 (q, 2H, J¼7.1 Hz),
4. Experimental section
4.1. General methods
13C NMR (100 MHz, CD3OD)
135.5, 156.6, 148.9, 165.9.
d: 14.4, 62.9, 110.0, 116.3, 123.3, 131.3,
4.5. Ethyl 2-(3-fluoro-4-hydroxy-phenyl)-2-hydroxyimino-ac-
All commercial chemicals and solvents are reagent grade and
were used without further purification. Reaction were monitored
by thin layer chromatography on 0.25 mm silica gel plates (60F-
254, E. Merck) and visualized with UV light or 5% phosphomolybdic
acid in 95% ethanol. 1H NMR and 13C were recorded on a Bruker
400 MHz Avance DPX. Chemical shifts are reported in parts per
etate (8h)
In a flask was added CF3SO3H (5 ml, 55.8 mmol) and after
cooling at ꢀ5 ꢁC with an acetone bath a mixture of acetate 7 (1 ml,
8.75 mmol) and 2-fluorophenol (0.7 ml, 7.84 mmol) was added
drop wise. The reaction mixture was then stirred for 6 h under
nitrogen atmosphere and allowed to warm to room temperature,
then poured into DCM/MeOH (4:1, 50 ml) preliminary cooled be-
tween ꢀ30 ꢁC and ꢀ40 ꢁC. Saturated solutions of NaCl (10 ml) and
NaHCO3 (55.8 mmol) were added while at ꢀ40 ꢁC and allowed to
warm to room temperature. After pH control (pH 5), the resulting
mixture was extracted (DCM/MeOH 10:1, 4ꢃ20 ml), the combined
organic layers dried (MgSO4), and concentrated under vacuum. The
crude product was recrystallized from DCM to give 8h as a white
powder (457 mg, 23%). Rf: 0.54 DCM/MeOH (10:0.5); 1H NMR
million (ppm,
d units). Abbreviations used: PE: petroleum ether;
EtOAc: ethyl acetate; DMF: dimethylformamide; DCM: dichloro-
methane; THF: tetrahydrofuran.
4.2. Ethyl 2-(4-fluorophenyl)-2-hydroxyimino-acetate (8a)
In a flask was added CF3SO3H (5 ml, 55.8 mmol) and after
cooling at ꢀ15 ꢁC with an acetone bath a mixture of acetate 7
(0.8 ml, 7 mmol) and PhF (2 ml, 7 mmol) was added drop wise. The
reaction mixture was stirred for 7 h under nitrogen atmosphere
and allowed to warm to room temperature. Ice (10 g), NaHCO3
(4.7 g, 55.9 mmol), and NaCl for aqueous phase saturation were
then added. The resulting mixture was extracted (DCM, 4ꢃ20 ml)
and the combined organic layers dried (MgSO4) and concentrated
under vacuum. The crude product was recrystallized from PE/DCM
to give 8a as a beige powder (1.3 g, 88%). Rf: 0.86 DCM/MeOH
(400 MHz, CD3OD)
d
: 1.33 (t, 3H, J¼7.1 Hz), 4.32 (q, 2H, J¼7.1 Hz),
6.95 (t, 1H), 7.18 (dt, 1H, J¼2, 8 Hz), 7.35 (dd, 1H, J¼2, 12 Hz); 13C
NMR (100 MHz, CD3OD)
147.4, 150.7, 165.9.
d: 14.4, 62.9, 153.1, 117.9, 122.2, 127.5, 118.7,
4.6. Ethyl 2-(3,4-dihydroxy-phenyl)-2-hydroxyimino-acetate
(8i)
(10:1); 1H NMR (400 MHz, acetone-d6)
d
: 1.31 (t, 3H, J¼7.1 Hz), 4.29
(q, 2H, J¼7.1 Hz), 7.22 (t, 2H, J¼8.9 Hz), 7.59 (m, 2H),11.47 (s,1H); 19
F
In a flask was added CF3SO3H (5 ml, 55.8 mmol) and after cooling
at ꢀ5 ꢁC with an acetone bath a mixture of acetate 7 (1 ml,
8.75 mmol) and catechol (700 mg, 6.36 mmol) was added drop wise.
The reaction mixture was then stirred for 6 h under nitrogen at-
mosphere and allowed to warm to room temperature, then poured
into DCM/MeOH (4:1, 50 ml) preliminary cooled between ꢀ30 ꢁC
and ꢀ40 ꢁC. Saturated solutions of NaCl (10 ml) and NaHCO3
(55.8 mmol) were added while at ꢀ40 ꢁC and allowed to warm to
room temperature. After pH control (pH 5) the resulting mixture was
extracted (DCM/MeOH 10:1, 4ꢃ20 ml), the combined organic layers
dried (MgSO4), and concentrated under vacuum. The crude product
was purified by flash chromatography (silica gel, DCM/MeOH gra-
dient 100:0 to 90:10) to give 8i as a brown powder (101 mg, 7%),
which is a mixture of two inseparable isomers. Rf: 0.4 DCM/MeOH
NMR (376 MHz, acetone-d6)
d
: ꢀ113.1, ꢀ115.1; 13C NMR (100 MHz,
CDCl3) d: 14.4, 62.5, 115.4/115.6, 126.2/126.3, 132.65/132.7, 149.2,
162.4, 164.6/164.9.
4.3. Ethyl 2-(4-bromophenyl)-2-hydroxyimino-acetate (8c)
In a flask was added CF3SO3H (4.5 ml, 51 mmol) and after cooling
at ꢀ15 ꢁC with an acetone bath a mixture of acetate 7 (0.72 ml,
6 mmol) and PhBr (2 ml, 7 mmol) was added drop wise. The reaction
mixture was stirred for 7 h under nitrogen atmosphere and allowed
to warm to room temperature. Ice (10 g), NaHCO3 (4.7 g, 55.9 mmol),
and NaCl for aqueous phase saturation were then added. The
resulting mixture was extracted (DCM, 4ꢃ20 ml) and the combined
organic layers dried (MgSO4) during 1.5 h and concentrated under
vacuum. The crude product was purified (flash chromatography,
silica gel, DCM/MeOH 95:5) to give 8c as a white solid (0.86 g, 51%).
(10:0.5). Isomer 1 1H NMR (400 MHz, acetone-d6)
d: 1.16 (t, 3H,
J¼7.1 Hz), 4.28 (q, 2H, J¼7.1 Hz), 6.80 (m, 1H), 6.92 (dd, 1H, J¼2,
8.3 Hz), 7.06 (d,1H, J¼2 Hz). Isomer 2 1H NMR (400 MHz, acetone-d6)
Rf: 0.84 DCM/MeOH (9:1); 1H NMR (400 MHz, CDCl3)
d: 1.32 (t, 3H,
d
: 1.30 (t, 3H, J¼7.1 Hz), 4.38 (q, 2H, J¼7.1 Hz), 6.80 (m, 1H, H-5), 6.92
J¼8 Hz), 4.35 (q, 2H, J¼7.1 Hz), 7.39 (m, 4H); 13C NMR (100 MHz,
(dd, J¼2, 8.3 Hz,1H, H-6), 7.09 (d, J¼2 Hz,1H, H-2). Isomer 113C NMR
CDCl3)
d: 14.1, 62.4, 77.2, 128.5, 128.6, 131.0, 131.3, 132.0, 152.1.
(100 MHz, acetone-d6) d: 14.3, 61.6, 113.1, 115.4, 122.2, 123.0, 145.0,
147.2, 149.9, 165.0. Isomer 2 13C NMR (100 MHz, acetone-d6)
d: 14.5,
4.4. Ethyl 2-(3-bromo-4-hydroxy-phenyl)-2-hydroxyimino-
acetate (8g)
62.1, 116.2, 117.4, 123.0, 123.9, 146.1, 148.1, 151.8, 165.4.
4.7. Ethyl 2-(2-fluoro-4-hydroxy-phenyl)-2-hydroxyimino-ac-
In a flask was added CF3SO3H (5 ml, 55.8 mmol) and after
cooling at ꢀ15 ꢁC with an acetone bath a mixture of acetate 7 (1 ml,
8.75 mmol) and 2-bromophenol (1 ml, 8.62 mmol) was added drop
wise. The reaction mixture was then stirred for 6 h under nitrogen
etate (8j)
In a flask was added CF3SO3H (5 ml, 55.8 mmol) and after
cooling at ꢀ25 ꢁC with an acetone bath a mixture of acetate 7

F. El Bahhaj et al. / Tetrahedron 70 (2014) 9702e9708
9707
(0.8 ml, 21 mmol) and 3-fluorophenol (2 ml, 21 mmol) was added
drop wise. The reaction mixture was then stirred for 24 h under
nitrogen atmosphere and allowed to warm to room temperature,
then poured into ice (10 g). Saturated NaCl (10 ml) and NaHCO3
(4.7 g, 55.8 mmol) were then added. The resulting mixture was
extracted (DCM/MeOH, 95:5, 4ꢃ20 ml), the combined organic
layers dried (MgSO4) for 1 h, and concentrated under vacuum. The
crude product was purified (flash chromatography, silica gel EtOAc/
PE gradient 30:70 to 90:100) to give 8j as a beige powder (128.1 mg,
and purified by silica gel flash chromatography (DCM/MeOH, 98:2)
to give 10a as a white solid (255 mg, 75%). Rf¼0.4 (DCM/MeOH,
98:2); 1H NMR (400 MHz, CDCl3)
d (ppm): 1.77 (m, 2H), 2.41 (t, 2H),
3.37 (q, 2H), 3.65 (s, 2H), 3.78 (s, 3H), 6.85 (d, 2H), 7.06 (t, 2H), 7.20
(d, 2H), 7.45 (m, 2H), 8.92 (s, 1H); 13C NMR (100 MHz, CDCl3)
d
: 28.1,
28.5, 35.5, 38.4, 55.5, 114.1, 114.8, 115.1, 124.1, 130.0, 130.4, 131.9,
150.3, 158.5, 163.3; HRMS for C18H19FN2O3SNa, m/z calcd 399.1154
[MþNa]þ, found 399.1153.
8%). 1H NMR (400 MHz, acetone-d6)
d
: 1.28 (t, 3H, J¼7.1 Hz), 4.26 (t,
4.12. S-(3-(2-(4-Fluorophenyl)-2-(hydroxyimino)acetamido)-
propyl) ethanethioate (10b)
2H, J¼7.1 Hz), 6.66 (dd, 1H, J¼2.3, 11.6 Hz), 6.76 (dd, 1H, J¼0.4,
8.6 Hz), 7.33 (t, 1H, J¼8.3 Hz), 9.11 (s, 1H), 11.55 (s, 1H); 13C NMR
(100 MHz, acetone-d6) d: 30, 100, 105, 110, 113, 130, 206.2.
A solution of 8a (250 mg, 1.12 mmol) in acetonitrile (15 ml) was
degassed under Ar for 10 min, then DPAP (62 mg, 20 mol %) and
4.8. 2-(4-Fluorophenyl)-2-hydroxyiminoacetic acid (14)
thioacetic acid (240
mL, 3 equiv, 3.37 mmol) were added. The
mixture was stirred under irradiation for 1 h30, concentrated under
vacuum, and purified by silica gel flash chromatography (DCM/
MeOH, 98:2) to give 10b as a white solid (190 mg, 56%). Rf¼0.4
Ester 8a (0.5 g, 2.37 mmol) and 2.5 M aqueous LiOH (5.4 ml)
were added to a solution of THF/MeOH (27:5.4 ml (5:1)). After
overnight stirring at ambient temperature, the reaction mixture
was diluted with EtOAc (100 ml) and 6 N aqueous HCl was added
until pH¼2. The layers were separated and the organic one washed
with saturated aqueous NaCl, dried (MgSO4), and concentrated
under vacuum. The crude compound was recrystallized from
DCM/MeOH to give acid 14 as a white solid (0.41 g, 94%). Rf: 0.08
(DCM/MeOH, 98:2); 1H NMR (400 MHz, CDCl3)
d: 1.82 (m, 2H), 2.34
(s, 3H), 2.92 (t, 2H), 3.37 (q, 2H), 6.67 (s, 1H), 7.07 (t, 2H), 7.49 (m,
2H); 13C NMR (100 MHz, CDCl3)
d
: 26.3, 29.5, 30.7, 37.8, 114.8, 115.9,
124.2, 129.2, 131.9; HRMS for C12H13FN2O3SNa, m/z calcd 321.0685
[MþNa]þ, found 321.0683.
(DCM/MeOH 9:1). 1H NMR (400 MHz, DMSO-D6)
d: 6.93 (d, 2H,
J¼J¼8 Hz), 7.15 (d, 2H, J¼8 Hz), 12.06 (s, 1H), 12.79 (s, 1H).
4.13. 2-(4-Fluorophenyl)-2-(hydroxyimino)-N-(3-
mercaptopropyl)acetamide (10c)
4.9. N-Allyl-2-(4-fluorophenyl)-2-hydroxyimino-acetamide
(9a)
A solution of 10b (190 mg, 0.64 mmol) in methanol (10 ml) was
degassed under argon for 10 min, then NaSMe (45 mg, 1 equiv) was
added and the mixture stirred at room temperature under argon for
45 min. The resulting solution was poured into 0.1 M aqueous HCl
(20 ml) and extracted with DCM (3ꢃ15 ml). The organic layers were
washed with brine (20 ml), dried (MgSO4), and concentrated under
Fluoroester 8a (2.82 g, 13 mmol) was dissolved in allylamine
(20 ml, 268 mmol) and irradiated under microwaves at 100 ꢁC for
1 h at 300 W (25 W consumption most of the time). After cooling,
the excess allylamine was evaporated under vacuum and the
resulting oil purified (flash chromatography DCM/MeOH 99:1) to
give amide 9b as a solid (2.66 g, 92%). Rf: 0.67 (DCM/MeOH 9:1); 1H
vacuum.
(DCM/MeOH, 98:2) gave 10c (113 mg, 69%) as a white solid. Rf¼0.4
(DCM/MeOH, 98:2); 1H NMR (400 MHz, CD3OD)
: 1.75 (m, 2H),
2.44 (t, 2H), 3.20 (t, 1H), 3.31 (t, 2H), 7.03 (t, 2H), 7.44 (m, 2H); 13C
NMR (100 MHz, CD3OD) : 22.4, 34.7, 39.0, 115.5, 116.4, 127.0, 129.3,
11H13FN2O2SNa, m/z calcd 279.0579
A purification by silica gel flash chromatography
NMR (400 MHz, acetone-d6)
d
: (major isomer) 3.95 (m, 2H), 5.09
d
(qd, 1H, J¼10.3, 1.5 Hz), 5.23 (qd, 1H, J¼17.2, 1.8 Hz), 5.92 (m, 1H),
7.19 (m, 2H), 7.63 (m, 2H), 11.1 (s, 1H); 13C NMR (100 MHz, DMSO-
d
d6)
d
: 42.2, 115.1/115.3, 115.7, 126.9, 129.3, 133.1/133.2, 135.9, 150.8,
133.1, 151.0; HRMS for C
164.1.
[MþNa]þ, found 279.0578.
4.10. N-Benzyl-2-(4-fluorophenyl)-2-hydroxyimino-acet-
amide (9b)
4.14. N,N0-(Disulfanediylbis(propane-3,1-diyl))bis(2-(4-fluo-
rophenyl)-2-(hydroxyimino)acetamide) (11c)
Fluoroester 8a (100 mg, 0.47 mmol) was dissolved in benzyl-
amine (1.04 ml, 9.48 mmol) and irradiated under microwaves at
100 ꢁC for 1 h at 300 W. After cooling, the reaction mixture was
diluted with DCM (1 ml) and washed several times with 1 M
aqueous HCl until no more excess benzylamine was detected by
TLC. The resulting viscous oil was recrystallized from DCM/PE to
give amide 9b as a mixture of two isomers in a 1:3.3 ratio as white
solid (69.5 mg, 54%). Rf: 0.8 (DCM/MeOH 95:5); 1H NMR (400 MHz,
An aqueous solution of NH4OH (2 ml) was added to a solution of
10c (100 mg, 0.41 mmol) in MeOH (20 ml), then the mixture was
stirred at room temperature under air atmosphere for 24 h. After
concentration, the crude was diluted with water (10 ml) and
extracted with dichloromethane (3ꢃ20 ml). The organic layers
were dried (MgSO4) and concentrated under vacuum to give 11c
without further purification. Rf¼0.4 (DCM/MeOH, 98:2); 1H NMR
(400 MHz, CD3OD)
d
(ppm): 1.87 (m, 4H), 2.66 (t, 4H), 3.31 (t, 4H),
: 30.1,
CDCl3)
d: (isolated major isomer) 4.39 (d, 2H, J¼6.1 Hz), 7.20 (t, 2H,
7.03 (m, 4H), 7.44 (m, 4H); 13C NMR (100 MHz, acetone-d6)
d
J¼7.0 Hz), 7.26 (t, 1H, J¼7.0 Hz), 7.35 (m, 4H), 7.65 (dd, 2H, J¼5.6,
8.8 Hz), 8.12 (s, 1H), 11.15 (s, 1H); (signals for minor isomer in the
mixture) 4.50 (d, 2H, J¼6.3 Hz), 7.32 ((t, 2H, J¼8.3 Hz), 7.56 (dd, 2H,
J¼2.7, 4.6 Hz).
36.4, 38.8, 115.0, 115.3, 116.1, 116.4, 127.0, 129.2, 133.1, 162.4, 164.5;
HRMS for C22H24F2N4O4S2Na, m/z calcd 533.1104 [MþNa]þ, found
533.1108.
4.11. 2-(4-Fluorophenyl)-2-(hydroxyimino)-N-(3-((4-
Acknowledgements
methoxybenzyl)thio)propyl)acetamide (10a)
Authors thank the Centre National de la Recherche Scientifique,
A solution of 8a (200 mg, 0.90 mmol) in acetonitrile (15 ml) was
degassed under Ar for 10 min, then DPAP (46 mg, 20 mol %) and
University of Poitiers, Region Poitou-Charentes (F.E.B. grant), the
ꢀ
Ligue Contre le Cancer: Committees of Vendee, et Charente-
PMBSH (376
mL, 3 equiv, 2.70 mmol) were added. The mixture was
Maritime, ARSMeso44, COST Actions TD0905, and CM 1106 for fi-
stirred under UV irradiation for 1 h30, concentrated under vacuum,
nancial support.

9708
F. El Bahhaj et al. / Tetrahedron 70 (2014) 9702e9708
25. Shearman, J. W.; Myers, R. M.; Beale, T. M.; Brenton, J. D.; Ley, S. V. Tetrahedron
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