Facile formation of methylenebis(chalcone)s through unprecedented methylenation reaction. Application to antiparasitic and natural product synthesis

Article abstract of DOI:10.1002/ejoc.201400104

The formation of methylenebis(chalcone)s has been discovered during deprotection with methoxymethyl groups from trihydroxychalcones. Studies on this methylenation reaction led to a mechanism hypothesis that was extended to other chalcones and to dihydrochalcone, acetophenone, benzophenone and flavone derivatives. This new method was applied to the rapid synthesis of natural product piperanduncin C. These original methylenebis compounds were also evaluated for their antiparasitic activity. Copyright

Full text of DOI:10.1002/ejoc.201400104

FULL PAPER
DOI: 10.1002/ejoc.201400104
Facile Formation of Methylenebis(chalcone)s through Unprecedented
Methylenation Reaction. Application to Antiparasitic and Natural Product
Synthesis
Marion Thévenin,[a] Elisabeth Mouray,[b] Philippe Grellier,[b] and Joëlle Dubois*[a]
Keywords: Synthetic methods / Natural products / Antibiotics / Protecting groups / Oxygen heterocycles
The formation of methylenebis(chalcone)s has been discov-
ered during deprotection with methoxymethyl groups from
trihydroxychalcones. Studies on this methylenation reaction
led to a mechanism hypothesis that was extended to other
chalcones and to dihydrochalcone, acetophenone, benzophe-
none and flavone derivatives. This new method was applied
to the rapid synthesis of natural product piperanduncin C.
These original methylenebis compounds were also evaluated
for their antiparasitic activity.
Introduction
Protection of hydroxyl groups is widely carried out in
multistep organic syntheses. Among common protective
groups, methoxymethyl (MOM) ether was described as an
easily prepared and removed group.[1] During the total syn-
thesis of a natural flavonoid possessing an antiparasitic ac-
tivity, we needed to protect and deprotect 2Ј,4Ј,6Ј-trihyd-
roxychalcone 1 (Scheme 1) with a MOM group. The MOM
group removal was classically performed with 10% HCl in
MeOH leading to desired deprotected chalcone 2 together
with two side products. The first side product was desig-
nated as flavanone 3, the formation of which from the 2Ј-
hydroxychalcone derivative in acidic medium is well
known.[2] The second compound was isolated and charac-
terized as methylenebis(chalcone) 4.
To the best of our knowledge, methylenation reaction be-
tween two trihydroxychalcone units has never been de-
scribed under conditions for MOM group removal. More-
over few syntheses of methylenebis(chalcone)s have been
published so far.[3] Notably, Reddy et al. obtained bis(chal-
cone)s from benzaldehydes in the presence of trioxane and
sulfuric acid.[4]
Nevertheless, some natural products that contain a meth-
ylene bridge were extracted from plants. For instance, bis-
(flavonoid)s were isolated from a western North America
fern, Pentagramma triangularis,[5] or a bis(dihydrochalcone)
Scheme 1. Deprotection of MOM group from chalcone 1.
from the slender tree Piper adundum has been described by
Orjala (piperaduncin C, Figure 1).[6] Recently, Anis and Ali
discovered methylenebis(santin) from the shrub Dodonaea
viscosa (Figure 1).[7]
Therefore, we decided that this original reaction deserved
further investigation to facilitate total synthesis of such nat-
ural compounds. We present here our study on the forma-
tion of methylenebis compounds under MOM group re-
moval conditions, and on its scope and limitations.
[a] CNRS UPR2301, Institut de Chimie des Substances Naturelles,
Centre de Recherche de Gif,
Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France
E-mail: joelle.dubois@cnrs.fr
http://www.icsn.cnrs-gif.fr/spip.php?article3
[b] Museum National d’Histoire Naturelle, UMR 7245 CNRS,
Département RDDM, CP52,
57 rue Cuvier, 75005 Paris, France
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201400104.
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Facile Formation of Methylenebis(chalcone)s
ture of deprotected chalcone 2 and flavanone 3. Therefore,
hydrogen chloride was required in the methylene reaction
mechanism. When p-toluenesulfonic acid (PTSA) in EtOH/
CH2Cl2 (7:1, v/v) was used deprotected chalcone 2 was
quantitatively produced in under 2 hours without any side
products (Scheme 2).
It was expected that a MOM degradation product would
be responsible for methylenation, therefore the reactivity of
chalcone 2 in the presence of MOMCl in acidic medium
was studied. Thus a mixture of chalcone 2 and MOMCl in
3 n HCl in MeOH was heated to reflux. After 10 min, all
starting material disappeared and methylenebis(chalcone) 4
was obtained in almost quantitative yield (Table 1, Entry 1).
Hence, chloromethyl methyl ether proved to be the methyl-
ene source of this reaction when HCl and methanol were
present in the reaction medium. As previously described for
compound 1, the reaction was carried out with various ac-
ids under these conditions. In all tests methylenebis com-
pound 4 was detected in the reaction mixture. In the pres-
ence of acetic acid or triflic acid a mixture of compounds
2, 3 and 4 was obtained. Nevertheless, trifluoroacetic acid
was the only other acid able to afford compound 4 in good
yield (74%) as the major product (Table 1, Entry 3). To
avoid the use of the very toxic commercial solution of
MOMCl, we generated it from dimethoxymethane in the
presence of acetyl chloride (Table 1, Entry 2).[8] As ex-
pected, the methylenation reaction turned out well.
Figure 1. Methylene-bridged bis(flavonoid)s isolated from plants.
Results and Discussion
Synthesis
We first looked at the acidic conditions required to ob-
tain compound 4 from chalcone 1 that was prepared ac-
cording to Scheme 2. After protection of phloroaceto-
phenone by using MOMCl, acetophenone 5 reacted with
aldehyde 6 to afford chalcone 1 by base-catalyzed Claisen
condensation reaction in good yield. By optimizing the
methylenation reaction conditions, we noticed that an HCl
concentration lower than 3 n did not afford compound 4,
and deprotected chalcone 2 and flavanone 3 were mainly
obtained. On the contrary, if the HCl solution was more
concentrated than 3 n, degradation was observed. Finally,
we chose to continue our investigation by using a 3 n con-
centration. Then, to verify if hydrogen chloride was the only
acid that could be used for this reaction from protected
chalcone 1, various acids were tested. Surprisingly, tri-
fluoroacetic acid, acetic acid and triflic acid did not enable
CH2-bridge formation. Indeed, no methylenebis product 4
was observed but mostly starting material 1 and/or a mix-
Table 1. Reaction conditions to afford methylenebis(chalcone) 4
from trihydroxychalcone 2.
Entry Conditions
Yield
98%
1
MOMCl, 3 n HCl in MeOH, 70 °C,
15 min
2
(i) CH2(OMe)2, AcCl, ZnBr2 cat.,
CH2Cl2, room temp., 5 h;
(ii) 2, HCl in MeOH, 70 °C, 10 min
MOMCl, TFA, MeOH, 70 °C, 20 min
97%
3
4
74%[a]
HCHO, HCl, MeOH, room temp., 4 h 79%
[a] Yields calculated from NMR spectroscopic data.
Therefore all experiments for the formation of methyl-
ene-bridged compounds presented in Table 2 were per-
formed with in situ formed MOMCl.
Based on previous results[9] and on our work, we sug-
gested a possible mechanism for methylenation reaction in
three steps (Scheme 3). The first step, consisting of the re-
moval of the MOM group by HCl, leads to the formation
of a methoxymethylene cation.[9a] Then, this carbocation re-
acts with water in acidic medium to give methoxymethanol
by releasing HX (Scheme 3, Equation 1), In the presence of
any acid (H+X–), this molecule decomposes into methanol
and formaldehyde.[9b] In the early eighties, Bigi proposed a
mechanism in which phenol in the presence of formalde-
hyde formed a methylene-bridged dimer.[9c] Based on this
hypothesis, we could easily explain the formation of the
methylenebis compound [(Scheme 3, Equation 3). To con-
firm that formaldehyde was the CH2 source in our case, the
methylenation reaction was performed in the presence of
HCHO and HCl in MeOH. However, when the reaction
Scheme 2. Synthetic pathway to methylenebis(chalcone) 4. Rea-
gents and reaction conditions: (a) MOMCl, N,N-diisopropylethyl-
amine, CH2Cl2, 0 °C to room temp., 2.5 h, 72%; (b) KOH, EtOH,
70 °C, 4 d, 60%; (c) PTSA.H2O, EtOH/CH2Cl2 (7:1, v/v), 1.5 h,
100%; (d) See Table 1.
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Table 2. Methylenation reaction on phloroacetophenone, chalc- medium was heated at 70 °C under these conditions, degra-
ones, dihydrochalcones and benzophenones.[a]
dation was observed. Therefore the reaction was carried out
at room temperature and consequently required longer re-
action times to obtain compound 4 in good yield (Table 1,
Entry 4). Therefore we demonstrated that formaldehyde
could be the methylene source in this reaction, supporting
our proposed mechanism. An alternative mechanism could
be proposed in which the methoxymethylene cation reacts
directly with compound 7 before transformation into form-
aldehyde. However there is no evidence for either mecha-
nism. We think that the involvement of formaldehyde is
more probable because our reaction was not carried out
under anhydrous conditions and water is present in the re-
action medium because concentrated aqueous HCl was
used to make HCl 3 n in methanol.
To extend the method and to see its limits, some chalc-
ones, dihydrochalcones, benzophenones, phloroaceto-
phenone and a flavone were submitted to methylenation re-
action conditions. The structure of the methylene bridged-
derivatives was confirmed by X-ray analysis of methylene-
bis(chalcone) 8a (Figure 2). Then the role of hydroxyl
groups in 2Ј,4Ј and/or 6Ј positions was investigated by re-
placing them by one to three methoxy groups.
Figure 2. ORTEP representation of methylenebis(chalcone) 8a.
The presence of at least one free hydroxyl group is re-
quired to undergo methylenation as shown by the absence
of conversion observed with 2Ј,3Ј,4Ј-trimethoxychalcone 7d
[a] Reactions were carried out with (i) CH2(OMe)2 (4 equiv.), AcCl,
ZnBr2 cat., CH2Cl2, room temp., 5 h; (ii) 7, 3 n HCl in MeOH, 70 °C,
(Table 2, Entry 4). In cases in which there was at least one
hydroxyl group available, a methylene bridge was created
5 min to 2 h. [b] Yields calculated from NMR spectroscopic data.
between the two monomer units under our conditions. Gen-
Scheme 3. Plausible mechanism for the methylenation reaction from MOM-protected acetophenones, chalcones, dihydrochalcones or
benzophenones.
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Facile Formation of Methylenebis(chalcone)s
erally, good to excellent yields were obtained with 2Ј,4Ј,6Ј- piperaduncin C (8f; Scheme 5). Hence the total synthesis of
trisubstituted substrates bearing two OH groups in relative this antibacterial methylenebis(dihydrochalcone)[6] was per-
meta position (65–91%, Table 2, Entries 1, 2, 5 and 8) ex- formed in only three steps with 37% overall yield.
cept for dihydrochalcones 7g and 7j which bear free hy-
droxyl groups on the B cycle and for phloroacetophenone
7k (Table 2, Entries 7, 10, 11). However, when two out of
three hydroxyl groups were methylated, much lower conver-
sion was noted (Table 2, Entry 3). Likewise, when 4Ј-OH
involved in our proposed mechanism was absent, methylen-
ation yield decreased (Table 2, Entry 8 relative to Entry 9).
In the case of chalcone 6c, the only available OH group
was located at the ortho position relative to the carbonyl
moiety and was thus involved in a hydrogen bound, making
it less reactive. That is why, we suggested that formaldehyde
preferentially reacts with the OH group at the 4Ј-position
and in its absence, the reaction would occur at the other
positions but more sluggishly. Consequently, methylenebis
products were obtained in lower yields as observed for
methylenebis(chalcone)s 8c and 8i (Table 2, Entries 3 and
9) relative to their trihydroxy analogues 4 and 8h (Table 1,
Entry 1, and Table 2, Entry 8), respectively. Although
methylenation readily took place with dihydrochalcones 7e
and 7f (Table 2, Entries 5 and 6), modest yields were ob-
Scheme 5. Total synthesis of piperaduncin C (8f). Reagents and re-
action conditions: (a) MeI (1.1 equiv.), K2CO3 (1.1 equiv.), dimeth-
ylformamide, room temp., 24 h, 66%; (b) NH4HCO2 (9 equiv.),
tained for the synthesis of bis(dihydrochalcone)s 8g and 8j
(Table 2, Entries 7 and 10) or bis(acetophenone) 8k
(Table 2, Entry 11). Indeed, in those cases, the reaction was
not complete but longer reaction times only led to degrada-
tion product formation. Likewise, the conversion was low
with commercially available 2Ј,4Ј-dihydroxychalcone 7l
(Table 2, Entry 12) or chrysin 7m (Scheme 4), partly owing
to starting material poor solubility. Conditions would have
10% Pd/C, reflux, 2 h, 92%; (c) (i) CH2(OMe)2 (4 equiv.), AcCl,
ZnBr2 cat., CH2Cl2, room temp., 5 h; (ii) 7f, 3 n HCl in MeOH,
70 °C, 1.5 h, 61%.
Biological Evaluation
As part of our work on antiparasitic agents, these new
to be optimized in those cases. Hence the presence of three derivatives deserved to be evaluated for their inhibitory ac-
free hydroxyl groups seemed to greatly facilitate the methyl- tivity against parasitic growth. Therefore, some of them
ene bridge formation. Besides, it should be noted that solu- were assayed on the intraerythrocytic stages of Plasmodium
bility issues of final products led to challenging purification falciparum,[9] responsible for malaria and in the blood-
and consequently were responsible for significant differ- stream forms of Trypanosoma brucei gambiense, the patho-
ences between the calculated NMR spectroscopic and iso- genic agent of African sleeping sickness.[10] To see whether
lated yields.
methylenation would be beneficial to activity, the corre-
sponding monomers were also evaluated. Results are sum-
marized in Table 3.
In general, bis(benzophenone) derivatives 8h–8j and bi-
s(acetophenone) 8k along with their respective monomers
displayed no antiparasitic activity. On the contrary, chalc-
one, dihydrochalcone and flavone derivatives were generally
active against both parasites, except chalcone 7a and chry-
sin 7m. Therefore, a longer link between A and B cycles
seemed to be required to display inhibition. Moreover, we
observed that for all chalcones, the most potent compounds
of this series, the formation of the methylene bridge be-
tween two monomer units was beneficial to activity on both
parasites. Thus methylenebis(chalcone)s 4, 8a and 8b dis-
played better results than their corresponding precursors 2,
7a and 7b (Table 3). Notably, dimer 8a became a potential
Scheme 4. Methylenation reaction from chrysin 7m. Reagents and
reaction conditions: (i) CH2(OMe)2 (4 equiv.), AcCl, ZnBr2 cat.,
CH2Cl2, room temp., 5 h; (ii) Chrysin 7m, 3 n HCl in MeOH, MW
(70 °C, 10 min), 1% and 10% isolated and calculated NMR spec-
troscopic yields, respectively.
The optimization of this methylenation reaction was ap- growth inhibitor of P. falciparum and T. brucei after methyl-
plied to the rapid synthesis of a natural compound, pipera- enation of inactive monomer 7a. Likewise, bis(flavone) 8m
duncin C (Figure 1). After methylation of chrysin, flavone showed antiplasmodial activity whereas chrysin 7m demon-
9 was submitted to hydrogenation to obtain dihydro- strated no significant inhibition of Plasmodium falciparum
chalcone 7f that was submitted to methylenation affording proliferation. Finally, methylenebis(chalcone) 8b possessing
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M. Thévenin, E. Mouray, P. Grellier, J. Dubois
FULL PAPER
ried out on pre-coated silica gel aluminum plates (SDS TLC plates,
silica gel 60F254). Column chromatography was performed with
pre-packed Redisep columns. Preparative TLC (PLC) was per-
formed with Merck TLC with silica gel 60F254. NMR spectra, in-
Table 3. Inhibitory activity of monomer units and their methyl-
enebis derivatives.
IC50 on T. brucei [μm] IC50 on P. falciparum [μm]
1
cluding H, 13C (HMQC and HMBC) experiments, were recorded
2
8.2Ϯ0.3
5.8Ϯ0.5
Ͼ 50
27.4Ϯ3.2
7.7[a]
4
with a Bruker Avance 300 (300 MHz) and Avance 500 (500 MHz)
spectrometers. Chemical shifts are given in ppm relative to CDCl3
(δ =7.26 ppm; 77.2 ppm), [D6]acetone (δ =2.05 ppm; 30.5 ppm) or
[D6] Dimethyl sulfoxide (DMSO; δ = 2.50 ppm; 39.5 ppm). Split-
ting patterns are designed as: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet and combinations thereof. IR spectra were
recorded with a Perkin–Elmer Spectrum BX. Mass spectra were
recorded with Thermoquest AQA Navigator with a TOF detection
(ESI-HRMS). UHPLC analyses were realized with Waters Acquity
UPLC. Melting points were measured with a Büchi b-450 instru-
ment.
7a
Ͼ 50
8a
12.7Ϯ1.1
5.6Ϯ0.2
1.0 Ϯ0.1
17.4Ϯ1.0
32.5Ϯ2.1
7.8Ϯ0.2
1.3Ϯ0.1
30.1Ϯ0.2
52.4Ϯ1.1
Ͼ 50
Ͼ 50
Ͼ 50
Ͼ 50
Ͼ 50
12.3[a]
7b
34.8Ϯ3.1
8.4Ϯ1.7
9.8Ϯ0.3
9.6Ϯ0.2
27Ϯ2.3
7.8Ϯ0.2
9.6Ϯ0.5
29.8Ϯ0.9
Ͼ 50
Ͼ 50
Ͼ 50
Ͼ 50
Ͼ 50
8b
7e
8e
7f
8f
7g
8g
7h
8h
7j
8j
The purity of all target compounds was measured by using re-
versed-phase UHPLC (HSS C-18, 2.1ϫ50 mm column): com-
pounds were eluted with 95:5 A/B for 0.5 min then with a gradient
of 5–100% B/A for 3.5 min followed by 0:100 isocratic for 1 min at
a flow rate of 0.6 mL/min, in which solvent A was 0.1% formic
acid in H2O, and solvent B was 0.1% formic acid in CH3CN. Purity
was determined with TAC (total absorbance current from 200 to
400 nm).
7k
8k
Ͼ 50
14.4Ϯ2.0
30.9Ϯ1.2
Ͼ 50
7m
Ͼ 50
8m
31.3[a]
Chloroquine
0.072Ϯ0.0074
Pentamidine 0.011Ϯ0.0017
[a] Determined from only 2 experiments.
General Procedure for the Methylenation Reaction: Acetyl chloride
(4 mmol) and ZnBr2 (0.01 mol-%) were added under argon to a
solution of dimethoxymethane (4 mmol) in CH2Cl2 (0.25 mL). The
solution was stirred at room temperature under argon for 5 h. The
resulting solution of in situ generated chloromethyl methyl ether
(4 mmol) was then added to an HCl solution (3 n in MeOH,
12 mL) of the phenolic compound (1 mmol). The reaction mixture
was stirred at 70 °C (unless otherwise indicated) until the reaction
was complete. The resulting solution was cooled to room tempera-
ture. After addition of water to the reaction mixture, the precipitate
(if formed, unless indicated) was filtered, washed with water and
dried to yield the desired methylenebis product.
a methoxy group on 2Ј-position displayed the highest activi-
ties with IC50 = 1.0Ϯ0.1 μm against T. brucei gambiense.
The most potent antiplasmodial agent was bis(chalcone) 4
with IC50 = 7.7 μm.
Conclusions
In conclusion, we discovered a previously unreported,
new reaction that occurs during MOM group removal. We
synthesized various chalcone, dihydrochalcone, benzo-
phenone, acetophenone and flavone derivatives to study the
reactivity of these monomers and understand the mecha-
nism that we herein propose. During deprotection with the
MOM group, formaldehyde is formed. The next step
seemed to require at least one free OH group in the mono-
mer to occur. Though reaction conditions should be opti-
mized for each derivative, this reaction provides a new
method to synthesize bis(methylene) natural products, as
was demonstrated by the rapid total synthesis of piperadun-
cin C. Furthermore, application of this new reaction on
various flavonoids afforded some new methylene-bridged
compounds that were evaluated for their inhibitory activity
on parasite growth. All methylenebis(chalcone)s displayed
higher activities than their monomer homologues. Finally,
the most potent trypanocidal compound of this series,
methylenebis(chalcone) 8b, showed a promising activity
with an IC50 value of 1.0 μm. Accordingly these encourag-
ing results are paving the way for further investigations on
antiparasitic activity of methylenebis(chalcone)s.
(2E,2ЈE)-1,1Ј-[Methylenebis(2,4,6-trihydroxy-3,1-phenylene)]bis{3-
[4-(benzyloxy)-3-methoxyphenyl]prop-2-en-1-one} (4): General pro-
cedure (15 min at 70 °C) applied to chalcone 2 (50.0 mg) to afford
methylenebis(chalcone) 4 (50.0 mg, 98%) as an orange amorphous
1
solid. H NMR ([D6]DMSO): δ = 14.4 (s, 2 H), 10.8 (s, 2 H), 10.1
(s, 2 H), 8.04 (d, J = 15.5 Hz, 2 H), 7.66 (d, J = 15.5 Hz, 2 H),
7.47–7.34 (m, 10 H), 7.28 (d, J = 1.8 Hz, 2 H), 7.23 (dd, J = 8.4,
1.8 Hz, 2 H), 7.11 (d, J = 8.4 Hz, 2 H), 5.96 (s, 2 H), 5.15 (s, 4 H),
3.83 (s, 6 H), 3.64 (s, 2 H) ppm. 13C NMR ([D6]DMSO): δ = 191.7,
164.2, 163.0, 159.7, 149.8, 149.2, 141.6, 136.7, 128.4, 128.3, 127.9,
127.9, 125.6, 122.2, 113.4, 111.1, 105.7, 104.1, 94.6, 69.9, 55.5,
15.5 ppm. MS (ESI–, MeOH + CH2Cl2): m/z = 795.2 [M – H]–.
HRMS: calcd. for C47H41O12+ [M + H]+ 797.2598; found 797.2609.
IR (neat): ν = 3240, 1719, 1604, 1250, 1220, 1086, 1026 cm–1, m.p.
˜
200 °C. UPLC method (H2O/MeCN): room temp., 5.99 min, 94%.
(2E,2ЈE)-1,1Ј-[Methylenebis(2,4,6-trihydroxy-3,1-phenylene)]bis-
(3-phenylprop-2-en-1-one) (8a): General procedure (10 min at 70 °C)
applied to chalcone 7a (100 mg). Purification by flash chromatog-
raphy on silica gel [gradient heptane to heptane/EtOAc (6:4, v/v)
in 25 min] to afford methylenebis(chalcone) 8a (6.0 mg, isolated
yield: 8 %; NMR yield before purification: 65 %) as an orange
1
amorphous solid. H NMR ([D6]acetone): δ = 10.8 (s, 2 H), 8.32
(d, J = 15.5 Hz, 2 H), 7.87 (d, J = 15.5 Hz, 2 H), 7.73–7.71 (m, 4
H), 7.47–7.45 (m, 6 H), 6.12 (s, 2 H), 3.81 (s, 2 H) ppm. 13C NMR
([D6]acetone): δ = 193.7, 163.7, 143.9, 136.4, 131.3, 129.9, 129.4,
127.9, 106.7, 105.5, 97.1, 16.1 ppm. MS (ESI+, MeOH + CH2Cl2):
Experimental Section
General: All commercial reagents were used as received without
further purification. Analytical thin-layer chromatography was car-
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Facile Formation of Methylenebis(chalcone)s
m/z = 523.1 [M + H]+. HRMS: calcd. for C31H25O8 [M + H]+
523.1453; found 523.1393. IR (neat): ν = 3249, 1625, 1592, 1285,
[Methylenebis(2,4,6-trihydroxy-3,1-phenylene)]bis(phenylmethanone)
(8h): General procedure (4 days at room temperature) applied to
+
˜
1225, 1180, 1087 cm–1, m.p. 145 °C. UPLC method (H2O/MeCN): benzophenone 7h (16.0 mg) to afford methylenebis(benzophenone)
room temp., 5.52 min, 82%.
8h (12.0 mg, 75%) as a pale yellow amorphous solid. 1H NMR
([D6]acetone): δ = 12.1 (s, 2 H), 9.81 (s, 2 H), 9.65 (s, 2 H), 7.63 (d,
J = 7.5 Hz, 4 H), 7.51 (t, J = 7.5 Hz, 2 H), 7.42 (t, J = 7.5 Hz, 4
H), 6.11 (s, 2 H), 3.82 (s, 2 H) ppm. 13C NMR ([D6]acetone): δ =
200.2, 162.5, 161.1, 160.7, 142.1, 132.0, 129.1, 128.5, 106.6, 96.9,
16.3 ppm. MS (ESI+, MeCN + CH2Cl2): m/z = 473.1 [M + H]+.
(2E,2ЈE)-1,1Ј-[Methylenebis(2,4-dihydroxy-6-methoxy-3,1-phenyl-
ene)]bis(3-phenylprop-2-en-1-one) (8b): General procedure (2 h at
70 °C) applied to chalcone 7b (31.0 mg) to afford methylene-
bis(chalcone) 8b (28.0 mg, 91%) as an orange amorphous solid. 1H
NMR ([D6]DMSO): δ = 13.9 (s, 2 H), 9.91 (s, 2 H), 7.46 (d, J =
15.5 Hz, 2 H), 7.26 (d, J = 7.5 Hz, 4 H), 7.22 (d, J = 15.5 Hz, 2
H), 7.00–6.99 (m, 6 H), 5.57 (s, 2 H), 3.41 (s, 6 H), 3.27 (s, 2
H) ppm. 13C NMR ([D6]DMSO): δ = 191.6, 165.1, 163.4, 160.4,
141.2, 135.0, 130.2, 129.0, 128.3, 127.6, 106.8, 104.4, 91.0, 55.7,
15.6 ppm. MS (ESI+, MeCN + CH2Cl2): m/z = 553.2 [M + H]+.
HRMS: calcd. for C27H21O8 [M + H]+ 473.1236; found 473.1235.
+
IR (neat): ν = 3295, 1739, 1596, 1287, 1175, 1113, 1079 cm–1, m.p.
˜
243 °C.
[Methylenebis(2,6-dihydroxy-4-methoxy-3,1-phenylene)]bis(phenyl-
methanone) (8i): General procedure (16 h at room temperature) ap-
plied to benzophenone 7i (8.0 mg) to afford methylenebis(benzo-
phenone) 8i (4.0 mg, 50%) to as a pale yellow amorphous solid.
1H NMR ([D6]acetone): δ = 10.3 (s, 2 H), 10.1 (s, 2 H), 7.62 (d, J
= 7.5 Hz, 4 H), 7.51 (t, J = 7.5 Hz, 2 H), 7.42 (t, J = 7.5 Hz, 4 H),
6.17 (s, 2 H), 3.86 (s, 6 H), 3.81 (s, 2 H) ppm. 13 C NMR
([D6]acetone): δ = 200.0, 164.7, 161.5, 159.6, 142.3, 132.1, 129.2,
128.5, 107.9, 106.2, 92.5, 56.4, 16.2 ppm. MS (ESI+, MeCN +
HRMS: calcd. for C33H29O8 [M + H]+ 553.1862; found 553.1843.
+
IR (neat): ν = 1741, 1624, 1591, 1285, 1232, 1118 cm–1, m.p.
˜
259 °C. UPLC method (H2O/MeCN): room temp., 6.72 min, 92%.
1,1Ј-[Methylenebis(2,4,6-trihydroxy-3,1-phenylene)]bis[3-(4-
hydroxy-3-methoxyphenyl)propan-1-one] (8e): General procedure
(40 min at 70 °C) applied to dihydrochalcone 7e (20.0 mg). Purifi-
cation by flash chromatography on silica gel [gradient heptane to
heptane/EtOAc (4:6, v/v) in 15 min] to afford methylenebis(dihyd-
rochalcone) 8e (2.0 mg, isolated yield: 10%; NMR yield before pu-
rification: 80 %) as a pale yellow amorphous solid. 1H NMR
([D6]acetone): δ = 14.5 (br. s, 2 H), 10.7 (s, 2 H), 9.53 (br. s, 2 H),
7.26 (s, 2 H), 6.88 (s, 2 H), 6.74–6.70 (m, 4 H), 6.07 (s, 2 H), 3.82
(s, 6 H), 3.76 (s, 2 H), 3.40 (t, J = 8.0 Hz, 4 H), 2.91 (t, J = 8.0 Hz,
4 H) ppm. 13C NMR ([D6]acetone): δ = 206.7, 163.1, 162.7, 162.5,
148.2, 145.7, 133.9, 121.6, 115.7, 113.0, 106.4, 105.3, 96.8, 56.2,
46.8, 31.1, 15.9 ppm. MS (ESI+, MeCN + CH2Cl2): m/z = 621.2 [M
+
CH2Cl2): m/z = 501.2 [M + H]+. HRMS: calcd. for C29H25O8 [M
+ H]+ 501.1549; found 501.1595. IR (neat): ν = 3338, 1737, 1624,
˜
1594, 1239, 1201, 1124, 1100, 1077, 1025 cm–1, m.p. 174 °C.
[Methylenebis(2,4,6-trihydroxy-3,1-phenylene)]bis[(3,4-dihydroxy-
phenyl)methanone] (8j): General procedure (overnight at room tem-
perature) applied to commercially available maclurin 7j (200 mg).
No precipitate was formed. Water was added to the reaction mix-
ture that was extracted twice with ethyl acetate. The combined or-
ganic layers were washed twice with brine, dried with MgSO4 and
concentrated under vacuum. The crude product was then purified
by flash chromatography on silica gel [gradient heptane to heptane/
EtOAc/2-propanol (3:6:1, v/v/v) in 25 min] to afford methylene-
bis(maclurin) 8j (59.0 mg, 29%) as a pale yellow amorphous solid.
1H NMR ([D6]acetone): δ = 9.43 (s, 2 H), 8.54 (br. s, 2 H), 8.22
(br. s, 2 H), 7.27 (d, J = 2.5 Hz, 2 H), 7.18 (dd, J = 8.0; 2.5 Hz, 2
H), 6.84 (d, J = 8.0 Hz, 2 H), 6.12 (s, 2 H), 3.80 (s, 2 H) ppm. 13C
NMR ([D6]acetone): δ = 197.8, 160.7, 159.5, 158.9, 150.3, 145.0,
133.3, 123.7, 117.1, 115.1, 106.8, 106.5, 96.7, 16.6 ppm. MS (ESI+,
MeCN + CH2Cl2): m/z = 537.1 [M + H]+. HRMS: calcd. for
+ H]+. HRMS: calcd. for C33H33O12 [M + H]+ 621.1972; found
+
621.2018. IR (neat): ν = 3272, 2923, 2853, 1603, 1515, 1452, 1365,
˜
1259, 1207, 1123, 1081, 1032 cm–1. UPLC method (H2O/MeCN):
room temp., 4.36 min, 100%.
Piperaduncin C (8f): General procedure (1.5 h at 70 °C) applied to
dihydrochalcone 7f (50.0 mg) to afford piperaduncin C (8f;
31.0 mg, 61%) as a beige amorphous solid. 1H NMR ([D6]acetone):
δ = 12.1 (br. s, 2 H), 11.5 (s, 2 H), 7.27–7.15 (m, 10 H), 6.11 (s, 2
H), 3.86 (s, 6 H), 3.80 (s, 2 H), 3.41 (t, J = 8.0 Hz, 4 H), 2.97 (t, J
= 8.0 Hz, 4 H) ppm. 13C NMR ([D6]acetone): δ = 206.1, 165.0,
163.6, 161.6, 142.9, 129.3, 129.2, 126.7, 107.5, 105.5, 92.3, 56.2,
46.6, 31.4, 15.8 ppm. MS (ESI+, MeCN + CH2Cl2): m/z = 557.2
[M + H]+. HRMS: calcd. for C33H33O8+ [M + H]+ 557.2175; found
C27H21O12 [M + H]+ 537.1033; found 537.1033. IR (neat): ν =
+
˜
3258, 1686, 1591, 1514, 1436, 1282, 1236, 1161, 1113, 1082 cm–1,
m.p. 219 °C; UPLC method (H2O/MeCN): room temp., 2.60 min,
100%.
557.2171. IR (neat): ν = 1622, 1422, 1218, 1140, 699 cm–1, m.p.
˜
1,1Ј-[Methylenebis(2,4,6-trihydroxy-3,1-phenylene)]diethanone (8k):
General procedure (overnight at room temperature) applied to
phloroacetophenone 7k (100 mg) to afford methylenebis(aceto-
phenone) 8k (25.0 mg, 24%) as a beige amorphous solid. 1H NMR
([D6]acetone): δ = 14.5 (br. s, 2 H), 10.6 (s, 2 H), 9.50 (s, 2 H), 6.06
(s, 2 H), 3.73 (s, 2 H), 2.66 (s, 6 H) ppm. 13C NMR ([D6]acetone): δ
= 204.8, 163.2, 162.7, 106.2, 105.5, 96.7, 32.6, 15.8 ppm. MS (ESI+,
169 °C; UPLC method (H2O/MeCN): room temp., 6.07 min, 91%.
1,1Ј-[Methylenebis(2,4,6-trihydroxy-3,1-phenylene)]bis[3-(4-
hydroxyphenyl)propan-1-one] (8g): General procedure (overnight at
room temperature) applied to commercially available phloretin 7g
(10.0 mg). Purification by flash chromatography on silica gel [gra-
dient heptane to heptane/EtOAc (65:35, v/v) in 15 min] to afford
methylenebis(phloretin) 8g (1.0 mg, isolated yield: 10 %; NMR
yield before purification: 29 %) as a beige amorphous solid. 1H
NMR ([D6]acetone): δ = 14.5 (br. s, 2 H), 10.7 (s, 2 H), 8.07 (s, 2
H), 7.10 (d, J = 8.0 Hz, 4 H), 6.75 (d, J = 8.0 Hz, 4 H), 6.06 (s, 2
MeCN + CH2Cl2): m/z = 349.1 [M + H]+. HRMS: calcd. for
+
C17H17O8 [M + H]+ 349.0923; found 349.0935. IR (neat): ν =
˜
3198, 1608, 1588, 1448, 1365, 1262, 1099, 1073 cm–1, m.p. 210 °C;
UPLC method (H2O/MeCN): room temp., 3.77 min, 97%.
H), 3.75 (s, 2 H), 3.39 (t, 4 H), 2.90 (t, 4 H) ppm. 13C NMR 6,6Ј-Methylenebis(5,7-dihydroxy-2-phenyl-4H-chromen-4-one) (8m):
([D6]acetone): δ = 206.7, 163.2, 162.8, 162.6, 156.6, 133.3, 130.3,
General procedure (10 min at 70 °C under microwave irradiation)
applied to commercially available chrysin 7m (50.0 mg). Purifica-
tion by preparative TLC (eluent: heptane/EtOAc/2-propanol, 5:4:1,
v/v/v) afforded methylenebis(flavone) 8m (0.6 mg, isolated yield:
116.1, 106.4, 105.3, 96.9, 46.9, 30.6, 16.0 ppm. MS (ESI+, MeCN
+ CH2Cl2): m/z = 561.2 [M + H]+. HRMS: calcd. for C31H29O10
+
[M + H]+ 561.1760; found 561.1756. IR (neat): ν = 3197, 1603,
˜
1515, 1454, 1259, 1091, 1015, 796 cm–1, m.p. 225 °C. UPLC 1%; NMR yield before purification: 10%) as a pale yellow amorph-
1
method (H2O/MeCN): room temp., 4.24 min, 100%.
ous solid. H NMR ([D6]acetone): δ = 13.0 (d, 2 H), 8.00 (d, J =
Eur. J. Org. Chem. 2014, 2986–2992
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2991

M. Thévenin, E. Mouray, P. Grellier, J. Dubois
FULL PAPER
7.00 Hz, 4 H), 7.54–7.48 (m, 6 H), 6.73 (s, 2 H), 6.33 (s, 2 H), 4.47
(s, 2 H) ppm. 13C NMR ([D6]DMSO): δ = 182.3, 163.2, 162.8,
159.3, 154.9, 131.9, 131.2, 129.1, 126.4, 104.8, 105.2, 103.7, 98.5,
16.7 ppm. MS (ESI+, MeCN + CH2Cl2): m/z = 521.1 [M + H]+.
was tested in triplicate and the experiment repeated twice. The per-
centage of inhibition of parasite growth rate was calculated by com-
paring the fluorescence of parasites maintained in the presence of
drug to that in the absence of drug. DMSO was used as a control.
IC50 values were determined from the dose-response curves with
drug concentrations ranging from 100 μm to 50 nm. IC50 value is
the mean Ϯ standard deviation of three independent experiments.
Pentamidine was used as anti-trypanosomal drug control.
+
HRMS: calcd. for C31H21O8 [M + H]+ 521.1236; found 521.1265.
IR (neat): ν = 2961, 1737, 1643, 1604, 13701, 1259, 1096,
˜
1032 cm–1. UPLC method (H2O/MeCN): room temp., 4.41 min,
91%.
Supporting Information (see footnote on the first page of this arti-
cle): Experimental procedures, characterization data and NMR
spectra of all compounds.
Biological Evaluation
Assay for in Vitro Inhibition of P. Falciparum Growth: Chloroquine-
resistant strain FcB1/Colombia of Plasmodium falciparum was
maintained in vitro on human erythrocytes in RPMI 1640 reaction
mixture supplemented by 8% (v/v) heat-inactivated human serum,
at 37 °C, under an atmosphere of 3% CO2, 6% O2, and 91% N2.
In vitro drug susceptibility assays were measured by [3H]-hypoxan-
thine incorporation as described.[9,10] Drugs were prepared in
DMSO at 10 mm. Compounds were serially diluted twofold with
100 μL culture medium in 96-well plates. Asynchronous parasite
cultures (100 μL, 1% parasitaemia and 1% final hematocrite) were
then added to each well and incubated for 24 h at 37 °C prior to
the addition of 0.5 μCi of [3H]-hypoxanthine (GE Healthcare,
France, 1 to5 Ci·mmol/mL) per well. After a further incubation
period of 24 h, plates were frozen and thawed. Cell lysates were
then collected onto glass-fiber filters and counted in a liquid scintil-
lation spectrometer. The growth inhibition for each drug concen-
tration was determined through comparison of the radioactivity
incorporated in the treated culture with that in the control culture
maintained on the same plate. The concentration which resulted in
50% growth inhibition (IC50) was obtained from the drug concen-
tration-response curve and the results were expressed as the mean
values Ϯ standard deviations determined from several independent
experiments. Chloroquine was used as antimalarial drug control.
Acknowledgments
The authors thank O. Thoison and her co-workers for UHPLC
analyses, P. Retailleau for X-ray analysis, ICSN and Centre
National de la Recherche Scientifique (CNRS) for financial sup-
port.
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Received: January 21, 2014
Published Online: March 27, 2014
2992
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 2986–2992