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

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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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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

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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

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

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

. 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

+

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

3

1

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

.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

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

3

1

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

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

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

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

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

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

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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

3

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

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

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

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

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

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

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

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

1

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

.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

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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M. Ritchie, D. Argentieri, M. Wachter, D. J. Hlasta, Synthesis and structure-activity

relationships of 3,5-diarylisoxazoles and 3,5-diaryl-1,2,4-oxadiazoles, novel classes of small

molecule interleukin-8 (IL-8) receptor antagonists. Bioorg. Med. Chem. Lett. 14 (2004)

4

307-4311. http://dx.doi.org/10.1016/j.bmcl.2004.05.080

[

35] Y.-J. Shang, Y.-G. Wan, Soluble polymer-supported synthesis of isoxazoles, Tetrahedron

Lett. 43 (2002) 2247–2249. http://dx.doi.org/10.1016/S0040-4039(02)00185-5

Scheme 1: One-pot isoxazole synthesis

Reagents and Conditions: a: NH OH·HCl, t-BuOH:H O (1:1), NaOH 1N, rt, b:

2

TsN(Cl)Na·3H O, tBuOH:H O (1:1), rt, c: CuSO ·5H O/Sodium ascorbate, phenylacetylene,

2

4

2

MW irradiation

2

1

ACCEPTED MANUSCRIPT

Table 1

Entry

b

Chloramine-T Catalyst

Method

Time

Yield %

#

1

2

3

4

5

6

7

8

1.05 eq

1.5 eq

a

room temperature

90 °C

24h

45

55

35

47

30

68

72

#

24h

#

a

90 °C

30 min

Cu free

a

90 °C

MW(60 °C, 80 W)

MW(90 °C, 80 W)

#

a

#

a

#

a

+

Silica gel

MW(90 °C, 80 W)

Al O

#

9

1.05 eq

a

30 min

Traces

45

+

2

3

#

1

0

a

MW(90 °C, 80 W)

1

.05 eq

Al O₃

#

1

a

45

+

2

#

12

13

14

1.05 eq

a +NaCl

MW(90 °C, 80 W)

MW(100 °C, 80 W)

MW(110 °C, 80 W)

30 min

50

58

62

#

a

#

a

#

1

5

6

7

8

1.05 eq

a

MW(120 °C, 100 W) 30 min

53

57

68

62

Cu free

MW(90 °C, 80 W)

30 min

45 min

60 min

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

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

52(34)

MeO

O

47

29

MeO

O

MeO

OMe

49(15)

O

N

OMe

O

MeO

F

O

5

6

H

30

18

O

9

O

F

O

H

O

MeO

OMe

O

H

7

N

26

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

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

ACCEPTED MANUSCRIPT

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

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

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

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

Article Doi

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

DOI: 10.1016/j.ejmech.2014.06.046

Source and publish data:

Authors:

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

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

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