Microwave-assisted synthesis of 3,5-disubstituted isoxazoles and evaluation of their anti-ageing activity

Article abstract of DOI:10.1016/j.ejmech.2014.06.046

One-pot uncatalysed microwave-assisted 1,3-dipolar cycloaddition reactions between in situ generated nitrile oxides and alkynes bearing protected antioxidant substituents, were regioselectively afforded 3,5-disubstituted isoxazoles. The yields were modera

Full text of DOI:10.1016/j.ejmech.2014.06.046

Accepted Manuscript
Microwave-assisted synthesis of 3,5-disubstituted isoxazoles and evaluation of their
anti-ageing activity
Maria Koufaki, Theano Fotopoulou, Marianna Kapetanou, Georgios A. Heropoulos,
Efstathios S. Gonos, Niki Chondrogianni
PII:
S0223-5234(14)00575-3
10.1016/j.ejmech.2014.06.046
EJMECH 7089
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 February 2014
Revised Date: 17 June 2014
Accepted Date: 23 June 2014
Please cite this article as: M. Koufaki, T. Fotopoulou, M. Kapetanou, G.A. Heropoulos, E.S. Gonos,
N. Chondrogianni, Microwave-assisted synthesis of 3,5-disubstituted isoxazoles and evaluation
of their anti-ageing activity, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.06.046.
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ACCEPTED MANUSCRIPT
Graphical abstract
Microwave-assisted synthesis of 3,5-disubstituted isoxazoles and evaluation
of their anti-ageing activity
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Maria Koufaki* , Theano Fotopoulou, Marianna Kapetanou, Georgios A. Heropoulos ,
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Efstathios S. Gonos, Niki Chondrogianni
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ACCEPTED MANUSCRIPT
Microwave-assisted synthesis of 3,5-disubstituted isoxazoles and evaluation
of their anti-ageing activity
#
#
Maria Koufaki* , Theano Fotopoulou, Marianna Kapetanou, Georgios A. Heropoulos ,
#
Efstathios S. Gonos, Niki Chondrogianni
National Hellenic Research Foundation, Institute of Biology, Medicinal Chemistry and
Biotechnology, 48, Vas. Constantinou Ave, Athens 11635, Greece
Corresponding author: Maria Koufaki
Tel: +302107273818
Fax: +302107273831
Email: mkoufa@eie.gr
#
these authors equally contributed to the manuscript

ACCEPTED MANUSCRIPT
Abstract
One-pot uncatalysed microwave-assisted 1,3-dipolar cycloaddition reactions between in situ
generated nitrile oxides and alkynes bearing protected antioxidant substituents, were
regioselectively afforded 3,5-disubstituted isoxazoles. The yields were moderate, based on the
starting aldehydes, while the reaction times were in general shorter than those reported in the
literature.
The cytoprotective and anti-ageing effect of the final deprotected compounds was evaluated
in vitro, on cellular survival following oxidative challenge and in vivo, on organismal
longevity using the nematode Caenorhabditis elegans. The activity of the isoxazole analogues
depends on the nature and the number of the antioxidant substituents. Analogue 17 bearing a
phenolic group and a 6-OH-chroman group is a promising anti-ageing agent, since it
increased survival of human primary fibroblasts following treatment with H O and extended
2
2
C. elegans lifespan.
Keywords: isoxazole, microwave, antioxidant, anti-ageing, C. elegans

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1
. Introduction
Isoxazoles, have attracted an increasing research interest, as non classical amide or ester
bioisosteres and potential pharmacophores endowed with anticancer [1], neuroprotective [2],
anti-obesity [3], antidepressant [4], insecticidal [5], antidiabetic [6] and anti-inflammatory [7]
activities. The major synthetic strategies to construct this heterocycle are: i) condensation of a
1
,3-dicarbonyl compound with hydroxylamine and ii) 1,3-dipolar cycloaddition of an alkyne
with a nitrile oxide, which is regioselective in the presence of copper(I), giving 3,5-
disubstituted isoxazoles [8].
Nitrile oxides can react with simple terminal alkynes without the need of a catalyst, because
of their increased reactivity, compared to azides. Very recently, several groups [1a,c,d,3,5,6,9-
1
2] accessed 3,5-disubstituted isoxazoles through a metal-free cycloaddition of alkynes with
nitrile oxides, usually in modest yields or long reaction times.
In general the 3,5-regioisomer was favored under uncatalyzed conditions. Use of
organocatalysts [7] or hypervalent iodine [13] reagents improved the yield/regioselectivity of
the reaction. Moreover, the regiospecific synthesis of novel isoxazolines and isoxazoles of N-
substituted saccharin derivatives, using a microwave oven, was also reported [14].
Our group has been involved in the synthesis of neuroprotective antioxidants and we reported
that the presence of isoxazole scafold results in higher in vitro neuroprotective activity,
compared to other nitrogen heteroaromatics. Isoxazole analogues were prepared by
conventional Cu catalyzed cycloadditions [2] or by using dual-frequency ultrasound
irradiation [15].
Although the isoxazole pharmacophore has been incorporated into a wide range of bioactive
agents, the effect of isoxazole analogues on the cellular or organismal lifespan has not yet
been reported.
Ageing is an inevitable natural biological process that is linked to the gradual deterioration of
organismal homeostasis and the increasing accumulation of damaged macromolecules [16].
The progression of ageing has been highly correlated with increased levels of reactive oxygen
species (ROS) and the extent of ROS formation and oxidative damage has been inversely
2

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correlated with longevity in different species [17]. Increased oxidative stress promotes the
deterioration of all biomolecules including DNA, lipids and proteins thus leading to a global
failure of cellular and organismal homeostasis [18]. There are various models used to study
ageing in vitro and in vivo. Human primary fibroblasts that age in vitro, the so called
replicative senescence model constitute the best accepted model to study human ageing in
vitro [19]. Moreover, human primary fibroblasts can easily be used in different assays to
reveal antioxidant properties on top of anti-ageing properties of different compounds. The
nematode Caenorhabditis elegans is also a prominent model to study organismal ageing due
to its short lifespan, the fast generation time and the multiple experimental applications [20,
2
1].
The discovery of agents which could slow down the deleterious effects of ageing in vitro
and/or in vivo has attracted an increasing research interest. Since ageing is associated with
increased incidence of diseases related to elevated levels of reactive oxygen species (ROS),
dietary phenolic antioxidants have emerged as promising candidates [22-27] while there is
one recent report on the anti-ageing properties of synthetic compounds applying the
aforementioned in vivo model [28]. Therefore bioactive isoxazoles bearing antioxidant groups
would represent an interesting approach towards the development of anti-ageing compounds.
Collectively, the aim of the present study was i) the green regiospecific microwave-assisted
one-pot synthesis of isoxazoles from in situ generated nitrile oxides and alkynes, in the
presence or absence of Cu(I) as catalyst and ii) the investigation of the effects of the derived
compounds on the cellular survival following oxidative challenge and on organismal
longevity.
2
. Results
We first investigated the cycloaddition reaction between the in situ generated 4-methoxy-
phenyl nitrile oxide and phenyl acetylene under conventional heating and microwave
irradiation in the presence or absence of Cu catalyst. The reaction was performed in a mixture
of tert-butanol/water (Scheme 1). Specifically, 4-methoxy-benzaldehyde was first converted
to the corresponding aldoxime via reaction with hydroxylamine. Without isolation, the
aldoxime was converted to the corresponding nitrile oxide using chloramine-T trihydrate
which acts as both a halogenating agent and a base. The results are shown in Table 1.
3

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Scheme 1
Table1
The Cu catalyzed reaction at ambient temperature gave 45% of the desired isoxazole after 24
h (entry 1). The yield was slightly improved when the temperature was increased to 90 °C
(entry 2) whereas the use of microwave irradiation significantly improved the yield and
shortened the reaction time (entry 7).
Concerning the amount of the catalyst, the use of 0.3 equivalents of CuSO and 0.6
4
equivalents of sodium ascorbate (entry 6) gave higher yields than lower catalyst loading
(entry 5).
On the contrary, the use of larger excess of chloramine-T (1.5 equivalents, entry 8) did not
affect the yield of the reaction.
The best results of the Cu catalysed reaction were obtained at 90 °C and 80 W. Lower (60 °C,
entry 6) or higher (100 and 110 °C) temperatures (entries 13 and 14) gave lower yields. The
yield was further decreased when 120 °C and 100 W were applied (entry 15). Solid additives
[
29] such as silica gel, Al O or NaCl had a detrimental effect on the reaction yield.
2 3
Although washing with NH OH, ensures quantitative removal of the copper salt during the
4
reaction work-up, Cu-free cycloaddition strategy, not requiring metals and additives, is a
promising approach. Thus, we set out to examine the feasibility and the regioselectivity of the
microwave-assisted 1,3-dipolar cycloaddition reactions, tert-butanol/water, under metal free
conditions.
Microwave irradiation increased the yield of the uncatalysed reaction (entries 4 and 16). The
optimum reaction time at 90 °C and 80 W was 45 min (entry 17) giving 68% of isoxazole.
Similar yields were obtained using 90 °C, 100 W for 22 min (entry 20).
Using the optimal conditions for the metal free reaction, we synthesized the compounds
depicted in Scheme 2 and Table 2. Although reaction time of 22 min and 100 W, did not
significantly affect the yield of the reaction in the case of phenyl acetylene (Table 1 entry 20),
when aliphatic alkyne was used (Table 2, entries 1, 4) the yield was decreased. The low yield
of the entry 6 of Table 2 (compound 10), is due to the removal of the 4-methoxybenzyl group,
under these reaction conditions. After purification by column chromatography, compound 10
and 3-(3,4-dimethoxyphenyl)-5-isoxazolyl-methanol were isolated.
4

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Scheme 2
Table 2
It should be noted that all the yields are based on the aldehydes and not on oximes or imidoyl
chlorides. Thus, the low to moderate yields of the Cu-free reactions are overall yields of a
three step reaction. As we have previously reported [15], in our experiments the in situ
generation of hydroximoyl chlorides and their conversion to nitrile oxides was fast, followed
by addition of terminal alkynes which are trapping agents to avoid the dimerization of nitrile
oxides to furoxans.
In general, the reaction times of the metal free reactions were shorter of those reported in the
literature (max 45 min instead of hours). In the case of analogue 8 the yield of the microwave
(80W/90 °C/45 min) uncatalysed reaction was comparable to that we have reported [2] for
.
this compound using CuSO 5H O/copper turnings, overnight and based on the oxime.
4
2
Since the synthesized methoxy analogues are not expected to possess antioxidant activity and
keeping in mind that anti-oxidant properties are usually linked to anti-ageing properties [23-
2
8], compounds 1, 2 and 6-9 were deprotected using BF .SMe as previously described [2]. In
3 2
the case of derivatives 6 and 7 the known alcohol (3-phenylisoxazol-5-yl) methanol 14 was
obtained due to the removal of the 4-methoxybenzyl or the 3,4-dimethoxybenzyl group under
these reaction conditions. The structures of the deprotected analogues 12-17 are depicted in
Figure 1.
Figure 1
Ageing is associated with increased levels of reactive oxygen species (ROS) thus, we sought
to test the anti-oxidant and/or anti-ageing properties of the isoxazole derivatives (12-17) in
two model systems; human primary fibroblasts (in vitro model) and the nematode C. elegans
(in vivo model). The first model was used to test cell survival following oxidative stress,
while the second one was used to reveal possible longevity-promoting effects of our
compounds in a eukaryotic multicellular organism. More specifically, we subjected young
human primary fibroblasts to oxidative stress (H O ) in the presence or absence of our
2
2
compounds and we then tested their survival ability.
5

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Figure 2
As shown in Figure 2A, cells treated with compound 17 exhibited significantly enhanced
viability after oxidative insult as compared to the control cultures. In contrast, the rest of the
compounds (12-16) demonstrated low or no cytoprotective properties. Following the
identification of analogue 17 as the most potent, we then used a reference standard compound
namely quercetin, the most abundant dietary flavonol that has been shown previously to be a
potent antioxidant by others [30] as well by us [26], to compare its effects with the effects of
compound 17. As shown in Figure 2B, compound 17 was more potent as compared to
quercetin at the same concentrations (2 and 5 ꢀΜ). Therefore, the in vitro model revealed that
compound 17 enhances the ability of the cells to cope better with oxidative stressors while,
more interestingly, it does so better as compared to a standard antioxidant compound such as
quercetin, at lower concentrations. We then examined whether our antioxidant compounds
also exhibit anti-ageing properties in a multicellular organism. To this end, we fed wild type
nematode worms with different concentrations of each compound and the relative diluent
(DMSO). Compounds 12-16 did not promote any differences in the lifespan of the nematodes
(data not shown), thus coinciding with the results from the cell survival assays where no
cytoprotective effects were scored. In bright contrast, analogue 17 presented anti-ageing
activity. More specifically, as shown in Figure 3, treatment with various concentrations of 17
(ranging from 1 ꢀM to 20 ꢀM) resulted in significant extension of animal lifespan, with 20
ꢀ
M being the most effective concentration.
Figure 3
Given that we had previously shown the anti-ageing properties of quercetin using human
fibroblasts [26] while others have revealed its anti-ageing effects in C. elegans [31], we have
also compared the anti-ageing effects of compound 17 with the relative anti-ageing effects of
quercetin in C. elegans. We therefore fed wild type nematode worms with the most potent
concentrations of compound 17 that we have identified in our initial experiments, namely 10
and 20 ꢀM, and with equal concentrations of quercetin.
Figure 4
6

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As shown in Figure 4A, treatment of nematodes with 10 ꢀM compound 17 resulted in
significant extension of animal lifespan while treatment with 10 ꢀΜ quercetin did not
promote lifespan extension. Likewise, treatment of nematodes with 20 ꢀM compound 17
resulted in significant lifespan extension that was more enhanced as compared to the relative
extension induced by 20 ꢀM quercetin (Figure 4B). In total, compound 17 was revealed to act
protectively against oxidative stress in human primary fibroblasts and to promote lifespan
extension in C. elegans. More interestingly, 17 was identified to be more potent in lower
concentrations as compared to a well established natural antioxidant such as quercetin.
3
. Discussion
In this study, we have achieved the green regiospecific microwave-assisted one-pot synthesis
of bioactive isoxazoles bearing antioxidant groups and we have shown for the first time their
impact on cellular resistance to stress and on organismal ageing. More specifically, we have
revealed that the newly derived compounds possess anti-ageing properties depending on the
number and the nature of the antioxidant isoxazole substituents. The most potent of these
isoxazoles, namely analogue 17, confers resistance to oxidative stress in human primary
fibroblasts while in its presence an extended lifespan of the wild type C. elegans is observed.
The increased resistance to oxidative stress along with the anti-ageing effects are strongly
linked to the aforementioned antioxidant properties.
Compounds 12-15 and 17 bear the 3-(4-hydroxyphenyl)isoxazole moiety. The presence of an
alkyl group at 5-position of the isoxazole ring (compound 13) or a phenyl group (compound
1
2) had no impact on the antioxidant activity of the compounds. Isoxazoles 15 and 17 can be
considered as derivatives of 14. The 4-hydroxyphenyl-substituted isoxazoles 14 and 15
exhibited similar activity in cells indicating that the protected chroman does not affect the
possible cytoprotective activity of these compounds. In contrast, deprotection of the chroman
hydroxyl group gave the most active analogue 17. The significance of the antioxidant groups
is demonstrated by the fact that, the cytoprotective activity in the 6-OH-chroman derivative
1
6 that lacks a second antioxidant group is lost as compared to the relative activity of
compound 17 that carries both antioxidant groups. Thus the presence of two antioxidant
moieties, a phenolic and a 6-OH-chroman group in compound 17 results in significantly
elevated cell survival under oxidative stress. However, the number of hydroxyl groups does
not seem to be the only requisite for the cytoprotective properties against oxidative stress.
More specifically, quercetin which bears five free hydroxyl groups that contribute to its strong
7

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antioxidant activity, was nevertheless less active than compound 17 at the cellular level (in
vitro model).
Resistance to oxidative stress has been linked to longevity in C. elegans. More specifically,
many long-lived mutants exhibit increased resistance to a variety of stressors i.e. oxidants [32]
or heat [33]. Therefore, given that we detected resistance to oxidative stress in our cellular
model we sought to investigate the possible effects of our compounds on the lifespan of the C.
elegans. In accordance to the results reported in the cell assays, compound 17 was the only
one that exhibited lifespan-extending properties, thus further advocating for a conserved
positive effect of this isoxazole among species. Few natural antioxidants have been revealed
previously to promote extension of lifespan in C. elegans, i.e. epigallocatechin gallate [23],
the flavonoids myricetin, quercetin, kaempferol and naringenin [24] and tyrosol [25] among
others. However, it is noteworthy that our synthetic compound was able to promote longevity
at significantly lower doses as compared to the dosages of natural antioxidants that have been
shown to be necessary to exert beneficial effects on the ageing process in C. elegans [23-25,
3
1]. Therefore, compound 17 is more potent and efficient than other known natural
antioxidants both in vitro and in vivo, a highly advantageous characteristic in case of product
application.
Our results suggest that the antioxidant activities of our compounds are responsible for the
cellular stress resistance and the extension of organismal lifespan. Nonetheless, it is possible
that these isoxazole analogues may differentially affect various cellular signal cascades. Thus,
further studies are needed to determine the effects of the synthesized compounds on signalling
cascades in vivo.
4
. Conclusion
A series of 3,5-disubstituted isoxazoles were synthesized by microwave-assisted, Cu free, 1,3-
dipolar cycloaddition reaction between in situ generated nitrile oxides and alkynes bearing
protected antioxidant substituents. Uncatalysed reactions, in tert-butanol/water, were
regioselective giving low to moderate yields of the three step reaction, based on starting
aldehydes. The reaction times were significantly shorter compared to those reported in the
literature.
8

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The biological evaluation of the deprotected compounds showed a correlation of their
antioxidant properties with stress resistance in human primary fibroblasts (in vitro model) and
with the extended longevity of the nematode C. elegans (in vivo model). Clearly, the activity
of our isoxazole analogues at the cellular and organismal level depends on the nature and the
number of the antioxidant substituents. Compound 17 bearing a phenolic group and a 6-OH-
chroman group was revealed to be a potent antioxidant against oxidative stress at a very low
dose in human primary fibroblasts and a promising anti-ageing agent as shown in the in vivo
model used. Additional studies using various strains of C. elegans bearing mutations in
molecular pathways that are key to the progression of ageing are required to reveal the exact
pathway through which analogue 17 functions as an anti-ageing agent.
5
5
5
. Experimental
.1. Chemistry
.1.1. Materials and methods
All starting materials and common laboratory chemicals were purchased from commercial
1
sources and used without further purification. H NMR spectra were recorded on Varian
1
3
spectrometers operating at 300 MHz or 600 MHz and C spectra were recorded at 75 MHz
using CDCl or (CD ) CO as solvent. Silica gel plates Macherey-Nagel Sil G-25 UV were
3
3 2
254
used for thin layer chromatography. Chromatographic purification was performed with silica
n
gel (200–400 mesh). Mass spectra were obtained on HPLC–MS Fleet-Thermo, in the ESI
n
mode. HRMS spectra were recorded, in the ESI mode, on UPLC-MS Orbitrap Velos-
Thermo. The microwave-assisted experiments were carried out with a CEM Discover 300W
monomode microwave instrument. The closed vessels used were special glass tubes with self-
sealing septa that controlled pressure with appropriate sensors on the top (outside the vial).
The temperature was monitored through a non-contact infrared sensor centrally located
beneath the cavity floor. Magnetic stirring was provided to assure complete mixing of the
reactants.
5
.1.2. General procedure for preparation of 3,5-disubstituted isoxazoles
To a solution of aldehyde (1 eq) and hydroxylamine hydrochloride (1.05 eq) in a mixture of t-
BuOH and H O (1:1) was added 1M aqueous NaOH (1.05 eq). The reaction mixture was
2
stirred at ambient temperature until thin-layer chromatography indicated consumption of the
aldehyde. After completion of oxime formation, 1.05 eq of chloramine-T [TsN(Cl)Na·3H O]
2
was added, followed (after 3 min) by the appropriate alkyne (1.05 eq), the pH of the reaction
9

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medium was adjusted to 6 (by addition of few drops of 1M aqueous NaOH) and the mixture
was microwave irradiated as indicated in Tables 1 and 2. The reaction mixture was extracted
with AcOEt, the organic layer was washed with saturated NaCl, dried over Na SO and
2
4
concentrated in vacuo. The crude residue was purified by column chromatography (pet.
ether/ethyl acetate, 90:10 to 80:20 affording products as colorless oils.
Compounds 1, 2, 8 were prepared according to the procedure described above and their
analytical data are in accordance with those previously reported [15].
5
.1.2. 1.5-(4-Methoxybenzyloxy)methyl-3-(4-methoxyphenyl)-isoxazole (6)
1
TLC (pet. ether/ethyl acetate, 85:15) R = 0.2, H NMR (600 MHz, CDCl ) δ: 7.73 (d, J=8.7
f
3
Hz, 2H, ArH), 7.29 (d, J=8.5 Hz, 2H, ArH), 6.96 (d, J=8.7 Hz, 2H, ArH), 6.89 (d, J=8.5 Hz,
2
3
1
H, ArH), 6.50 (s, 1H, H-isoxazole), 4.61 (s, 2H, CH ), 4.56 (s, 2H, CH ), 3.84(s, 3H, OCH ),
2 2 3
13
.80 (s, 3H, OCH ), C NMR (75 MHz, CDCl ) δ: 169.7, 162.0, 161.0, 159.5, 129.7, 129.2,
3
3
+
28.2, 121.5, 114.3, 113.9, 100.8, 72.6, 62.5, 55.3, 55.2, MS m/z: 326.36 (M+H) , 348.35
+
+
+
(
M+Na) , 672.96 (2M+Na) , HRMS: calcd for C H NO (M+H) 326.1387, C H NO Na
19 20 4 19 19 4
+
(
M+Na) 348.1206; found: 326.1381, 348.1200
5
.1.2. 2. 5-(3,4-Dimethoxybenzyloxy)methyl-3-(4-methoxyphenyl)-isoxazole (7)
1
TLC (pet. ether/ethyl acetate, 80:20) R = 0.15, H NMR (600 MHz, CDCl ) δ: 7.72 (d, J=8.8
f
3
Hz, 2H, ArH), 6.96 (d, J=8.8 Hz, 2H, ArH), 6.92-6.89 (m, 2H, ArH), 6.83 (d, J=8.0 Hz, 1H,
ArH), 6.50 (s, 1H, H-isoxazole), 4.62 (s, 2H, CH ), 4.56 (s, 2H, CH ), 3.88 (s, 3H, OCH ),
2
2
3
1
3
3
1
5
.86 (s, 3H, OCH ), 3.84 (s, 3H, OCH ), C NMR (75 MHz, CDCl ) δ: 169.6, 162.0, 161.0,
3 3 3
49.1, 148.9, 129.6, 128.2, 121.4, 120.7, 114.3, 111.2, 111.0, 100.9, 72.9, 62.4, 55.9, 55.8,
+
+
+
5.3, MS m/z: 356.25 (M+H) , 378.28 (M+Na) , 732.91 (2M+Na) , HRMS: calcd for
+
+
C H NO (M+H) 356.1492, C H NO Na (M+Na) 378.1312; found: 356.1492, 378.1307
20
22
5
20 21
5
5
.1.2.3. 5-{[(3,4-Dihydro-6-methoxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]
methyl}-3-(4-fluorophenyl)-isoxazole (9)
TLC (pet. ether/ethyl acetate, 90:10) R =0.3, H NMR (600 MHz, CDCl ) δ: 7.77-7.75 (m,
1
f
3
2
H, ArH), 7.13 (t, J=8.6 Hz, 2H, ArH), 6.46 (s, 1H, H-isoxazole), 4.74 (ABq, 2H, ꢁν =12.9
AB
Hz, J =13.9 Hz -O-CH -), 3.61 (s, 3H, -OCH ), 3.58 (ABq, 2H, ꢁν =27.7 Hz, J =10 Hz -
AB
2
3
AB
AB
CH -O-), 2.58 (t, J=6.8 Hz, 2H, -CH ), 2.17 (s, 3H, Ar-CH ), 2.12 (s, 3H, Ar-CH ), 2.08 (s,
2
2
3
3
1
3
3
H Ar-CH ), 2.02-1.97 (m, 1H, -CHH), 1.79-1.76 (m, 1H, -CHH), 1.30 (s, 3H, -CH ), C
3 3
NMR (75 MHz, CDCl ) δ: 170.2, 162.1, 161.4, 149.8, 147.3, 128.8, 128.7, 128.0, 125.9,
3
1
0

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1
25.2, 122.8, 117.4, 116.2, 115.9, 100.7, 74.9, 64.6, 60.4, 28.4, 22.0, 20.2, 12.6, 11.9, 11.7,
1
9
+
+
F NMR δ: -110.6, MS m/z: 426.17 (M+H) , 872.75 (2M+Na) , HRMS: calcd for
+
+
C H FNO (M+H) 426.2075, C H FNO Na (M+Na) 448.1895; found: 426.2079,
25
29
4
25 28
4
4
48.1895
5
.1.2. 4. 5-(4-Methoxybenzyloxy)methyl-3-(3,4-dimethoxyphenyl)-isoxazole (10)
1
TLC (pet. ether/ethyl acetate, 85:15) R = 0.1, H NMR (600 MHz, CDCl ) δ: 7.40 (s, 1H,
f
3
ArH), 7.31-7.28 (m, 1H, ArH), 6.91 (d, J=8.3 Hz, 1H, ArH), 6.52 (s, 1H, H-isoxazole), 4.61
s, 2H, -CH -O-), 4.57 (s, 2H, -OCH -), 3.93 (s, 3H, OCH ), 3.91 (s, 3H, OCH ), 3.80 (s, 3H,
(
2
2
3
3
13
OCH ), C NMR (75 MHz, CDCl ) δ: 169.8, 162.1, 159.5, 150.6, 150.2, 149.3, 129.7, 129.1,
3
3
1
26.4, 121.6, 119.9, 113.9, 111.0, 109.3, 100.9, 72.6, 62.5, 56.1, 56.0, 55.9, MS m/z: 356.31
+
+
+
+
(
M+H) , 378.28 (M+Na) , 732.84 (2M+Na) , HRMS: calcd for C H NO (M+H)
20 22 5
+
3
56.1492, C H NO Na (M+Na) 378.1312; found: 356.1489, 378.1307
20 21 5
5
.1.3. General procedure for deprotection of methoxy groups
A solution of the appropriate protected compound (1, 2 and 6-9, 1 equiv) in anhydrous
CH Cl (3 mL), was cooled at 0 ºC and BF ·SMe (10 equiv for each methoxy group) was
2
2
3
2
added. After stirring for 24 h, the solvent and excess reagent were evaporated under argon
stream. The residue was taken up in EtOAc and washed with H O and saturated NaCl. The
2
organic layer was dried over Na SO and concentrated in vacuo.
2
4
5
.1.3.1. 3-(4-Hydroxyphenyl)-5-phenyl-isoxazole (12) [34]
Compound 1 was treated as described in the general procedure. Purification by column
chromatography (pet. ether/ethyl acetate, 90:10 to 85:15), Yield: 84%, white solid, mp 178-
1
1
80 °C, TLC (pet. ether/ethyl acetate, 85:15) R = 0.2, H NMR (600 MHz, CDCl ) δ: 7.87 (d,
f
3
J=7.1 Hz, 2H, ArH), 7.77 (d, J=8.7 Hz, 2H, ArH), 7.51-7.45 (m, 3H, ArH), 6.95 (d, J=8.7 Hz,
1
3
2
H, ArH), 6.78 (s, 1H, H-isoxazole), 5.20 (bs, 1H, -OH), C NMR (75 MHz, CDCl ) δ:
3
1
70.1, 162.8, 158.5, 130.1, 128.9, 128.3, 127.4, 125.8, 120.6, 115.8, 97.3, MS m/z: 236.08
-
+
+
(
M-H) , HRMS: calcd for C H NO (M+H) 238.0863, C H NO Na (M+Na) 260.0682;
15 12 2 15 11 2
found: 238.0860, 260.0680
5
.1.3.2. 3-(4-Hydroxyphenyl)-5-propyl-isoxazole (13)
Compound 13 was obtained by deprotection of 2. Purification by column chromatography
pet. ether/ethyl acetate, 80:20), Yield: 75%, white solid, mp 107-109 °C, TLC (pet.
(
1
1

ACCEPTED MANUSCRIPT
1
ether/ethyl acetate, 90:10) R = 0.25, H NMR (600 MHz, (CD ) CO) δ: 8.85 (bs, 1H, -OH),
f
3 2
7
.70 (d, J=8.0 Hz, 2H, ArH), 6.93 (d, J=8.0 Hz, 2H, ArH), 6.50 (s, 1H, H-isoxazole), 2.74 (t,
J=7.5 Hz, 2H, CH CH CH -), 1.75-1.72 (m, 2H, CH CH CH -), 0.98 (t, J=7.4 Hz, 3H,
3
2
2
3
2
2
1
3
CH CH CH -), C NMR (75 MHz, (CD ) CO) δ: 173.6, 161.8, 158.9, 128.1, 120.9, 115.8,
3
2
2
3 2
+
+
9
8.3, 28.2, 20.8, 13.1, MS m/z: 204.12 (M+H) , 428.80 (2M+Na) , HRMS: calcd for
+
+
C H NO (M+H) 204.1019, C H NO Na (M+Na) 226.0838; found: 204.1018, 226.0839
12
14
2
12 13
2
5
.1.3.4. 3-(4-Hydroxyphenyl isoxazol-5-yl)methanol (14) [35]
Compound 6 was treated as described in the general procedure. Purification by column
chromatography (pet. ether/ethyl acetate, 85:15), Yield: 18%, white solid, mp 148-150 °C,
1
TLC (pet. ether/ethyl acetate, 90:10) R = 0.2, H NMR (600 MHz, (CD ) CO) δ: 8.87 (bs,
f
3 2
1
H, -OH), 7.71 (d, J=8.7 Hz, 2H, ArH), 6.93 (d, J=8.7 Hz, 2H, ArH), 6.66 (s, 1H, H-
1
3
isoxazole), 4.70 (s, 2H, -CH OH), C NMR (75 MHz, (CD ) CO) δ: 173.1, 161.7, 128.1,
2
3 2
-
-
1
20.7, 115.7, 99.1, 55.5, MS m/z: 190.09 (M-H) , 380.79 (2M) , HRMS: calcd for C H NO
10
10
3
+
(
M+H) 192.0655; found: 192.0657
5
.1.3.5.
5-{[(3,4-Dihydro-6-methoxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-
yl)methoxy] methyl}-3-(4-hydroxyphenyl)-isoxazole (15) [2]
Compound 8 was treated according to the general procedure described above giving a mixture
of analogues 15 and 17 which was purified by column chromatography (pet. ether/ethyl
1
acetate, 70:30). Yield: 27% yellowish foam, H NMR (600 MHz, CDCl ) δ: 7.67 (d, J=8.3
3
Hz, 2H, ArH), 6.90 (d, J = 8.3 Hz, 2H, ArH), 6.46 (s, 1H, H-isoxazole), 5.53 (bs, 1H, -OH),
4
.72 (ABq, 2H, ꢁν =14.9 Hz, J =13.9 Hz, -O-CH -), 3.63 (s, 3H, OCH ), 3.57 (ABq, 2H,
AB AB 2 3
ꢁ
ν =29.9 Hz, J =9.8 Hz -CH -O-), 2.60 (t, J=6.7 Hz, 2H, -CH -), 2.18 (s, 3H, Ar-CH ),
AB AB 2 2 3
2
.13 (s, 3H, Ar-CH ), 2.09 (s, 3H, Ar-CH ), 2.03-1.98 (m, 1H, -CHH), 1.80-1.75 (m, 1H, -
3 3
1
3
CHH), 1.31 (s, 3H, -CH ), C NMR (75 MHz, CDCl ) δ: 169.8, 161.9, 157.3, 149.7, 147.3,
3
3
1
1
4
28.4, 128.0, 125.9, 122.8, 121.5, 117.5, 115.8, 100.7, 76.2, 75.0, 64.6, 60.4, 28.3, 22.1, 20.1,
-
-
+
2.6, 11.9, 11.7, MS m/z: 422.14 (M-H) , 844.31 (2M) HRMS: calcd for C H NO (M+H)
25
30
5
24.2118; found: 424.2103
5
1
.1.3.6. 2-{[(3-(4-Fluorophenyl)isoxazol-5-yl)methoxy]-methyl}-2,5,7,8-tetramethyl-2H-
-benzopyran-6-ol (16)
Compound 16 was obtained by deprotection of 9. Purification by column chromatography
pet. ether/ethyl acetate, 90:10 to 80:20), Yield: 50%, yellowish oil, TLC (pet. ether/ethyl
(
1
2

ACCEPTED MANUSCRIPT
1
acetate, 90:10) R = 0.1, H NMR (600 MHz, CDCl ) δ: 7.77-7.75 (m, 2H, ArH), 7.13 (t, J =
f
3
8
.7 Hz, 2H, ArH), 6.45 (s, 1H, H-isoxazole), 4.73 (ABq, 2H, ꢁν =12.5 Hz, J =14 Hz, -O-
AB AB
CH -), 4.20 (bs, 1H, -OH), 3.58 (ABq, 2H, ꢁν =22.1 Hz, J =10 Hz, -CH -O-), 2.59 (t,
2
AB
AB
2
J=6.8 Hz, 2H, -CH -), 2.17 (s, 3H, Ar-CH ), 2.12 (s, 3H, Ar-CH ), 2.08 (s, 3H, Ar-CH ),
2
3
3
3
13
2
.03-1.97 (m, 1H, -CHH), 1.79-1.75 (m, 1H, -CHH), 1.30 (s, 3H, -CH ), C NMR (75 MHz,
3
CDCl ) δ: 170.3, 161.4, 145.0, 144.9, 128.8, 128.7, 122.5, 121.2, 118.6, 117.2, 100.6, 76.2,
3
1
9
+
7
4
4.7, 64.6, 28.6, 22.0, 20.3, 12.2, 11.9, 11.3, F NMR δ: -110.6, MS m/z: 412.17 (M+H) ,
+
+
+
34.33 (M+Na) , 457.08 (M+2Na) , 844.92 (2M+Na) , HRMS: calcd for C H FNO
4
24
27
+
+
(
M+H) 412.1919, C H FNO Na (M+Na) 434.1738; found: 412.1916, 434.1731
24 26 4
5
2
.1.3.7.
2-{[(3-(4-Hydroxyphenyl)isoxazol-5-yl)methoxy]-methyl}-2,5,7,8-tetramethyl-
H-1-benzopyran-6-ol (17)
1
Yield: 57% white foam, H NMR (600 MHz, CDCl ) δ: 7.64 (d, J=8.7 Hz, 2H ArH), 6.90 (d,
3
J = 8.7 Hz, 2H, ArH), 6.44 (s, 1H, H-isoxazole), 5.03 (bs, 1H, -OH), 4.72 (ABq, 2H,
ꢁ
ν =13.4 Hz, J =13.9 Hz, -O-CH -), 4.20 (bs, 1H, -OH), 3.57 (ABq, 2H, ꢁν =23.5 Hz,
AB AB 2 AB
J =10 Hz, -CH -O-), 2.62 (t, J=6.8 Hz, 2H, -CH -), 2.15 (s, 3H, Ar-CH ), 2.11 (s, 3H, Ar-
AB
2
2
3
CH ), 2.10 (s, 3H, Ar-CH ), 2.02-1.95 (m, 1H, -CHH), 1.80-1.75 (m, 1H, -CHH), 1.30 (s, 3H,
3
3
1
3
-
CH ), C NMR (75 MHz, CDCl ) δ: 169.8, 162.0, 157.4, 145.1, 144.9, 128.4, 122.5, 121.3,
3
3
1
4
4
18.6, 117.3, 115.9, 100.6, 76.2, 74.7, 64.6, 28.6, 22.0, 20.3, 12.2, 11.9, 11.3,, MS m/z:
-
-
+
08.16 (M-H) , 816.44 (2M) , HRMS: calcd for C H NO (M+H) 410.1962; found:
2
4
28
5
10.1959
5
5
.2. Biology
.2.1. Cell culture
HFL-1 human embryonic fibroblasts were obtained by the European Collection of Cell
Cultures and were maintained in Dulbecco's modified Eagle's medium (DMEΜ; Invitrogen)
supplemented with 10% fetal bovine serum (v/v; Invitrogen), 2 mM glutamine, and 1% non-
ο
essential amino-acids. HFL-1 cells were subcultured at 37 C, 5% CO and 95% humidity and
2
were fed approximately 16 h prior to each assay.
5
.2.2. Cell survival
Cell survival was assessed by counting the number of cells after treatment with 300 ꢀM H O2
2
4
for 2.5 h. Briefly, 10 HFL-1 cells were plated in 6-well plates and 24 h later were treated
with 2 ꢀM compound 17 and quercetin (Sigma Aldrich) and 5 ꢀM compounds 12-17 and
1
3

ACCEPTED MANUSCRIPT
quercetin or DMSO (solvent control) for 24 h. Cells were then incubated with 300 ꢀM
hydrogen peroxide (H O ) for 2.5 h in the presence of each compound and were then washed
2
2
thoroughly with PBS. Treated cultures were left to recover in complete medium for 5 days
and their numbers were determined in triplicates using a Coulter Z counter. Each experiment
2
was performed at least two times with the exception of quercetin that was repeated once in 5-
plicates.
5
.2.3. C. elegans strains, culture conditions and compounds treatments
The N2 (wt Bristol isolate) strain was used. It was provided by the Caenorhabditis Genetics
Center, supported by the NIH National Center for Research Resources (NCRR). We followed
standard procedures for C. elegans strain maintenance at 20 °C. The nematodes were grown
on solid nematode growth medium (NGM) seeded with E. coli (OP50) for food. For
compounds treatments, UV-irradiated NGM/OP50 plates were supplemented with different
compounds concentrations (solubilized in DMSO) as indicated. DMSO was included as a
solvent control in control cultures. UV-killing was used to avoid any adverse effects of live E.
coli on the compounds. The compounds or DMSO were diluted in M9 reaching a volume of
2
00 ꢀl and added over the agar and dead bacteria right after the UV irradiation. Plates were
allowed to dry for 24 h before use.
5
.2.4. Lifespan analysis
Gravid N2 worms were allowed to lay eggs for 3h to produce synchronized populations. At
L4 larval stage, 80-120 animals/condition were transferred to fresh plates containing either the
compound or DMSO. Day 1 of adulthood was set as t=0. Animals were maintained at 20 °C,
were transferred to fresh plates containing the compound or DMSO every 2 days to avoid
confounding of generation and starvation and were examined every day for touch-provoked
movement and pharyngeal pumping until death. Animal populations were maintained in the
respective compounds throughout their lifespans. Each survival assay was repeated at least
twice unless otherwise indicated and representative assays are shown. Survival curves were
created using the product-limit method by Kaplan and Meier. The log-rank (Mantel–Cox) test
was used to evaluate differences between survivals and to determine P values for all available
independent data. The n in lifespan figures is the number of animals that died/total where total
equals the animals number that died plus the number of censored animals (due to internally
hatched eggs, extruded gonad or desiccation due to crawling off the plates). Median lifespan
values are expressed as mean±SEM.
1
4

ACCEPTED MANUSCRIPT
5
.2.5. Statistical analysis
Statistical analyses were performed using Prism (GraphPad Software, San Diego, California
USA) and Microsoft Office 2003 Excel (Microsoft Corporation) software packages.
Acknowledgments
This work was supported by the European Union's Seventh Framework Programme (FP7-
REGPOT-2009-1) under grant agreement no. 245866 to MK and GAH as well as by a
Research Funding Program: Thales “GenAge”, co-financed by the European Union (European
Social Fund – ESF) and Greek national funds through the Operational Program "Education
and Lifelong Learning" of the National Strategic Reference Framework (NSRF) and a
SYNERGASIA project (11ΣΥΝ_1_1112) to ESG and NC. Nematode strains used in this
study were provided by the Caenorhabditis Genetics Center, supported by the NIH National
Center for Research Resources (NCRR).
1
5

ACCEPTED MANUSCRIPT
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Scheme 1: One-pot isoxazole synthesis
Reagents and Conditions: a: NH OH·HCl, t-BuOH:H O (1:1), NaOH 1N, rt, b:
2
2
TsN(Cl)Na·3H O, tBuOH:H O (1:1), rt, c: CuSO ·5H O/Sodium ascorbate, phenylacetylene,
2
2
4
2
MW irradiation
2
1

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Table 1
Entry
b
Chloramine-T Catalyst
Method
Time
Yield %
#
1
2
3
4
5
6
7
8
1.05 eq
1.05 eq
1.05 eq
1.05 eq
1.05 eq
1.05 eq
1.05 eq
1.5 eq
a
a
room temperature
90 °C
24h
45
55
35
47
30
68
72
72
#
24h
#
a
90 °C
30 min
30 min
30 min
30 min
30 min
30 min
Cu free
a
90 °C
MW(60 °C, 80 W)
MW(60 °C, 80 W)
MW(90 °C, 80 W)
MW(90 °C, 80 W)
#
a
#
a
#
a
+
Silica gel
MW(90 °C, 80 W)
Al O
#
9
1.05 eq
1.05 eq
a
30 min
30 min
30 min
Traces
45
+
2
3
#
1
0
a
MW(90 °C, 80 W)
MW(90 °C, 80 W)
1
.05 eq
Al O3
#
1
1
a
45
+
2
#
12
13
14
1.05 eq
1.05 eq
1.05 eq
a +NaCl
MW(90 °C, 80 W)
MW(100 °C, 80 W)
MW(110 °C, 80 W)
30 min
30 min
30 min
50
58
62
#
a
#
a
#
1
1
1
1
5
6
7
8
1.05 eq
1.05 eq
1.05 eq
1.05 eq
a
MW(120 °C, 100 W) 30 min
53
57
68
62
Cu free
MW(90 °C, 80 W)
MW(90 °C, 80 W)
MW(90 °C, 80 W)
30 min
45 min
60 min
Cu free
Cu free
Cu free
1
9
1.05 eq
MW(90 °C, 80 W)
30 min
26
+
NaCl
2
0
1.05 eq
Cu free
MW(90 °C, 100 W)
22 min
65
2
1
1.05 eq
Cu free
MW(90 °C, 100 W)
30 min
45
#
Solvent: t-BuOH:H O (1:1), (a) CuSO ·5H O/Sodium ascorbate (0.05 eq/0.1 eq), (0.3
2
4
2
b
eq/0.6eq). isolated yields based on 4-methoxy-benzaldehyde, after column chromatography
2
2

ACCEPTED MANUSCRIPT
Scheme 2 Uncatalysed synthesis of isoxazoles
a
Table 2 Copper free examples of the optimized conditions (80W/ 90 °C/ 45 min)
Entry
Aldehyde
Acetylene
Isoxazole
Yield (%)
O
1
2
3
4
H
H
H
H
52(34)
MeO
O
O
O
O
47
29
MeO
MeO
MeO
MeO
O
MeO
MeO
OMe
49(15)
O
O
O
N
OMe
O
MeO
F
O
5
6
H
30
18
O
9
O
O
F
O
H
O
MeO
MeO
OMe
O
H
7
N
26
O
O
(a) In parentheses are the yields of isoxazoles using 100W/ 90°C/ 22 min
2
3

ACCEPTED MANUSCRIPT
Figure 1: Structures of the tested compounds
2
4

ACCEPTED MANUSCRIPT
Figure 2: Treatment with compound 17 increases cellular resistance to oxidative stress.
Number of cells treated with (A) 2 ꢀM compound 17 or 5 ꢀM of compounds 12 - 17 or
DMSO (solvent control) and (B) 2 and 5 ꢀM compound 17 or quercetin or DMSO (solvent
control) for 24 h following treatment with 300 ꢀM H O for 2.5 h and a five-day recovery
2
2
period. Results with p-values, p < 0.05, p < 0.01 or p < 0.001 are denoted in graphs by a
single (*), double (**) or triple (***) asterisk, respectively.
A
3
00ꢀΜ H O , 2.5h
2 2
1
50000
00000
*
**
*
*
1
50000
0
B
3
00ꢀΜ H O , 2.5h
2 2
150000
*
**
100000
**
*
50000
0
2
5

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Figure 3: Treatment with compound 17 extends the lifespan of wt C. elegans. (A-C)
Survival curves of wt N2 worms treated with (A) 1ꢀM, (B) 10 ꢀM and (C) 20 ꢀM compound
1
7 as compared to the relative control cultures (DMSO).The percentage of animals remaining
alive is plotted against animal age. (A) Control: mean=15±0.85, n=471/489 (number of
animals that died/total; see Materials and Methods), compound 17 1 ꢀM: mean=18±0.33,
n=304/313, P<0.0001, 17 10 ꢀM: mean=18±0.5, n=319/325, P<0.0001, 17 20 ꢀΜ, 20±0.33,
n=297/308, P<0.0001.
Α 100
control
7 1ꢀΜ
1
80
60
***
40
20
0
0
10 20
Adult age (days)
30
Β
100
control
7 10ꢀΜ
1
80
60
40
20
0
***
0
10
20
30
Adult age (days)
C
100
control
7 20ꢀΜ
1
80
60
40
20
***
0
0
10
20
30
Adult age (days)
2
6

ACCEPTED MANUSCRIPT
Figure 4: Comparative treatment of C. elegans with compound 17 and quercetin. (A-B)
Survival curves (1 experiment) of wt N2 worms treated with (A) 10 ꢀM and (B) 20 ꢀM of
compound 17, quercetin or the relevant amount of DMSO (control). The percentage of
animals remaining alive is plotted against animal age. (A-B) Control: mean=17, n=106/109
(number of animals that died/total; see Materials and Methods), (A) compound 17 10 ꢀM:
mean=19, n=107/111, P<0.0001 compared with control, 10 ꢀM quercetin: mean=18,
n=106/108, P=0,1927 compared with control, P<0.0001 10 ꢀM compound 17 compared with
1
0 ꢀM quercetin: (B) 20 ꢀM compound 17: mean=20, n=111/121, P<0.0001 compared with
control, 20 ꢀM quercetin: mean=19, n=111/115, P=0,0051 compared with control, P= 0,0004
2
0 ꢀM compound 17 compared with 20 ꢀM quercetin.
A
100
control
80
17 10ꢀΜ
Quercetin 10ꢀΜ
60
40
20
0
0
10
20
30
Adult age (days)
B
100
control
80
60
40
20
0
17 20ꢀΜ
Quercetin 20ꢀΜ
0
10
20
30
Adult age (days)
2
7