Design and synthesis of antimicrobial, anticoagulant, and anticholinesterase hybrid molecules from 4-methylumbelliferone

Article abstract of DOI:10.3109/14756366.2016.1158171

We designed and synthesized new series of diverse triazoles, isoxazoles, isoxazolines, and aziridines linked 4-methylumbelliferone 1 using intermolecular 1,3-dipolar cycloaddition reactions. Structures of these compounds were established on the basis of ¹H NMR, ¹³C NMR, and ESI-HRMS. All prepared compounds were evaluated for their antimicrobial, anticoagulant, and anticholinesterase activities. Interestingly, among the tested molecules, some of the analogs displayed better activities than the parent 4-methylumbelliferone 1 such as 6a and 6d for their antifungal properties. Moreover, compounds 4, 5, 6, and 7 showed the importance of the added fragments to 4-methylumbelliferone 1 via the linker methylene to have good activity.

Full text of DOI:10.3109/14756366.2016.1158171

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Design and synthesis of antimicrobial,
anticoagulant, and anticholinesterase hybrid
molecules from 4-methylumbelliferone
Marwa Zayane, Ameur Rahmouni, Mejda Daami-Remadi, Mohamed Ben
Mansour, Anis Romdhane & Hichem Ben Jannet
To cite this article: Marwa Zayane, Ameur Rahmouni, Mejda Daami-Remadi, Mohamed Ben
Mansour, Anis Romdhane & Hichem Ben Jannet (2016): Design and synthesis of antimicrobial,
anticoagulant, and anticholinesterase hybrid molecules from 4-methylumbelliferone, Journal
of Enzyme Inhibition and Medicinal Chemistry, DOI: 10.3109/14756366.2016.1158171
To link to this article: http://dx.doi.org/10.3109/14756366.2016.1158171
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–10
2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.3109/14756366.2016.1158171
!
RESEARCH ARTICLE
Design and synthesis of antimicrobial, anticoagulant, and
anticholinesterase hybrid molecules from 4-methylumbelliferone
Marwa Zayane1, Ameur Rahmouni1, Mejda Daami-Remadi2, Mohamed Ben Mansour3, Anis Romdhane1, and
Hichem Ben Jannet1
1
´ ´
´
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´
Laboratoire de Chimie Heterocyclique, Produits Naturels et Reactivite, Equipe: Chimie Medicinale et Produits Naturels, Faculte des Sciences de
2
Monastir, Universite de Monastir, Monastir, Tunisie, UR13AGR09, Production Horticole Integree au Centre Est Tunisien, Centre Regional des
3
Recherches en Horticulture et Agriculture Biologique de Chott-Mariem, Universite de Sousse, Chott-Mariem, Tunisie, and Laboratoire de
´
´
´
´
´
´
Pharmacologie 04/UR/01-09, Faculte de Medecine, Monastir, Tunisie
´
Abstract
Keywords
We designed and synthesized new series of diverse triazoles, isoxazoles, isoxazolines, and
aziridines linked 4-methylumbelliferone 1 using intermolecular 1,3-dipolar cycloaddition
reactions. Structures of these compounds were established on the basis of 1H NMR, 13C
NMR, and ESI-HRMS. All prepared compounds were evaluated for their antimicrobial,
anticoagulant, and anticholinesterase activities. Interestingly, among the tested molecules,
some of the analogs displayed better activities than the parent 4-methylumbelliferone 1 such
as 6a and 6d for their antifungal properties. Moreover, compounds 4, 5, 6, and 7 showed the
importance of the added fragments to 4-methylumbelliferone 1 via the linker methylene to
have good activity.
Biological activity, cycloaddition,
4-methylumbelliferone, hybrid molecules,
SAR
History
Received 18 December 2015
Revised 6 February 2016
Accepted 7 February 2016
Published online 23 March 2016
Introduction
Huisgen 1,3-dipolar cycloaddition of azides and alkynes15.
This cycloaddition reaction usually affords mixtures of 1,4 and
1,5-disubstituted 1,2,3-triazoles. Recently, Shakeel-u-Rehman and
collaborators16 reported, that 1,4-disubstituted 1,2,3-triazoles
linked with 6-hydroxycoumarin are specifically prepared from
azides and terminal alkynes under sharpless click chemistry
conditions (CuSO4 5H2O and sodium ascorbate). Also, the
regioisomeric 1,5-disubstituted triazoles are available from
azides and terminal alkynes by the use of either magnesium
acetylides or ruthenium catalysts17.
On the other hand, heterocyclic compounds substituted with
isoxazoles moieties have found increasing attention in organic
syntheses, biochemistry and medicinal chemistry research due to
their cytotoxic18, antimycobacterial19, antiviral20, and anticonvul-
sant21 activities.
In addition, heterocyclic compounds carrying isoxazolines
units have also a pharmaceutical importance and some of them
posse’s anticancer22, antimicrobial23, anticoagulant2, and anti
inflammatory24 activity.
It was revealed that aziridine ring is an element of a large
number of natural products type alkaloids25 and various biologic-
ally active compounds such as antibiotics26 and antitumor
agents27. Because of their broad spectrum of biological activities
and pharmacological properties28, many efficient and more
general synthetic strategies have been developed for the prepar-
ation of aziridines29.
The natural and synthetic coumarins have occupied an important
role in the drug discovery process and have considerable
pharmacological and chemical significance1. Literature survey
shows that a large number of coumarin derivatives are found to be
associated with diverse types of biological activities, such as
anticancer2, antioxidant3, antimicrobial4, anticoagulant2 and they
have proved to be promising anti-acetylcholinesterase5 agents.
The coumarins are extremely variable in structure, due to various
types of substitutions in their basic structure, for this reason we
can found many studies focus on the modification of coumarins
such as hydroxylated, alkoxylated, and alkylated to give more
bioactive molecules6.
Different types of 1,2,3-triazole derivatives have enabled
diverse applications in the fields of pharmaceuticals7, agrochem-
icals8, medicinal chemistry9, and material sciences10. Because of
their utility and numerous activities such as antitumoral11,
antifungal12, antiproliferative13, antioxidant and anti-inflamma-
tory14, several syntheses of 1,2,3-triazoles have been reported.
One of the synthetic methods to assemble 1,2,3-triazoles is the
Address for correspondence: Hichem Ben Jannet, Laboratoire de Chimie
Heterocyclique, Produits Naturels et Reactivite, Equipe: Chimie
Medicinale et Produits Naturels, Faculte des Sciences de Monastir,
Universite de Monastir, Avenue de l’Environnement, 5019 Monastir,
Tunisie. Tel: +21673500279. Fax: +21673500278. E-mail:
hich.benjannet@yahoo.fr
Taking into account the various biological activities of
triazoles, isoxazolines, isoxazoles, aziridines, and coumarins
cited, respectively, in the literature30–33, it appeared to us
interesting to think about the combination of these moieties

2
M. Zayane et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
hoping to access to more biologically effective compounds. In this removed under reduced pressure and the residue was loaded into a
context and as a continuation of our previous work on the silica gel column and eluted with a mixture of chloroform/ethyl
synthesis of new fused 4-methylumbelliferone scaffolds34, we acetate (9/1) to give pure1,4-triazoles 4a–e.
report here the synthesis of a diverse series of novel triazoles,
isoxazoles, isoxazolines, and aziridines linked to 4-methylumbel-
liferone through [3 + 2] cycloaddition in the side chain. The
synthetic compounds were then screened for their antimicrobial
(antibacterial + antifungal), anticoagulant and anticholinesterase
activities and the structure–activity relationship (SAR) has been
discussed.
4-Methyl-7-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)-2H-chro-
men-2-one (4a)
Yellow solid; 85 mg; Yield: 57%; m.p.: 170–172 ꢀC; 1H NMR
0
(CDCl3, 300 MHz) dH: 2.40 (s, 3H, H9), 5.36 (s, 2H, H6 ), 6.15 (s,
1H, H3), 6.95 (dd, 1H, H6, J ¼ 2.4 Hz, J ¼ 7.2 Hz), 6.97 (d, 1H,
H8, J ¼ 2.4 Hz), 7.46 (d, 1H, H5, J ¼ 7.2 Hz), 7.44–7.57 (m, 3H,
0
0
0
0
0
H9 + H10 + H11 ), 7.76 (2d, 2H, H8 + H12 , J ¼ 7.8 Hz), 8.13
(s, 1H, H5 ); 13C NMR (CDCl3, 75 MHz) dC: 18.1, 61.7, 101.6,
Materials and methods
0
111.8, 111.9, 113.6, 120.1 (C-80), 120.1 (C-120), 120.8, 125.2,
128.5, 129.3, 129.3, 136.3, 143.2, 151.9, 154.6, 160.5, 160.6. ESI-
HRMS [M + H]+ calcd. For (C19H16N3O3)+: 334.1192, found:
334.1196.
Chemistry
All reactions were monitored by TLC using aluminium sheets of
Merck silica gel 60 F254, 0.2 mm. Melting temperatures were
determined on an electrothermal 9002 apparatus and were
reported uncorrected. NMR spectra were recorded on a Bruker
AC-300 spectrometer at 300 MHz (1H) and 75 MHz (13C). All
chemical shifts were reported as d values (ppm) relative to
7-((1–(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-
methyl-2H-chromen-2-one (4b)
residual non deuterated solvent. ESI-HRMS were obtained with Yellow solid; 96 mg; Yield: 64%; m.p.: 169–171 ꢀC; 1H NMR
ESI-TOF (LCT, Waters) using the reflectron mode in the positive (CDCl3, 300 MHz) dH: 2.41 (s, 3H, H9), 3.88 (s, 3H, Ph-OCH3),
0
5.35 (s, 2H, H6 ), 6.16 (s, 1H, H3), 6.98–7.05 (m, 2H, H8 + H6),
ion mode.
0
0
0
0
,
7.05 (2d, 2H, H9 + H11 , J ¼ 8.4 Hz), 7.54 (2d, 2H, H8 + H12
J ¼ 8.4 Hz), 7.65 (d, 1H, H5, J ¼ 7.5 Hz), 8.02 (s, 1H, H5 ); 13C
NMR (CDCl3, 75 MHz) dC: 18.6, 55.6, 62.3, 77.2, 102.2, 112.3,
112.4, 114.1, 114.1, 114.8, 121.3, 122.3, 122.3, 125.7, 130.3,
143.5, 152.4, 155.1, 160.0, 161.1. ESI-HRMS [M + H]+ calcd. For
(C20H18N3O4)+: 364.1294, found: 363.1297.
0
General procedure for O-allylation and O-propargylation
of 4-methylumbelliferone (1)
The 4-methylumbelliferone 1 (2.6 g) was added in portions to a
stirred suspension of NaH (1.21 g) in dry DMF (15 mL) at 20 ꢀC.
After the addition was over, the mixture was stirred at 20 ꢀC for
30 min. A solution of propargyl bromide/allyl bromide (2 mL) in
DMF (5 mL) was added dropwise to the mixture at 20 ꢀC and the
stirring was continued for 30 min35,36. The whole reaction mixture
was poured into ice water and the solid separated was collected
with filtration, dried and recrystallized from ethanol affording
compounds 2 and 3, respectively.
7-((1–(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-
2H-chromen-2-one (4c)
1
Yellow solid; Yield: 100 mg; 67%; m.p.: 176–178 ꢀC; H NMR
0
(CDCl3, 300 MHz) dH: 2.42 (s, 3H, H9), 5.37 (s, 2H, H6 ), 6.17 (s,
1H, H3), 6.97–7.01 (m, 2H, H8 + H6), 7.51–7.56 (m, 4H,
0
0
0
0
H8 +H9 +H11 +H12 ), 7.73 (d, 1H, H5, J ¼ 8.4 Hz), 8.10 (s, 1H,
H5 ); 13C NMR (CDCl3, 75 MHz) dC: 18.1, 61.7, 101.7, 111.8,
0
4-Methyl-7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (2)
111.9, 113.7, 120.6, 121.2, 121.2, 125.2, 129.5, 129.5, 134.3,
134.8, 143.6, 151.8, 154.6, 160.4, 160.5. ESI-HRMS
[M + H]+ calcd. For (C19H15ClN3O3)+: 368.0802, found:
368.0808.
Brown solid; 1.95 g; yield: 75%; mp.: 153–155 ꢀC; 1H NMR
0
(CDCl3, 300 MHz) dH: 2.40 (s, 3H, H9), 2.58 (t, 1H, H4 ,
0
J ¼ 2.4 Hz), 4.77 (d, 2H, H2 , J ¼ 2.4 Hz), 6.15 (s, 1H, H3), 6.91–
6.95 (m, 2H, H8 + H6), 7.52 (d, 1H, H5, J ¼ 9.3 Hz); 13C NMR
(CDCl3, 75 MHz) dC: 18.1, 55.7, 75.9, 77.4, 101.9, 111.9, 112.1,
113.7, 125.1, 151.8, 154.5, 159.8, 160.5. ESI-HRMS
[M + H]+ calcd. For (C13H11O3)+: 215.0708, found: 215.0711.
4-Methyl-7-((1-(m-tolyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chro-
men-2-one (4d)
Yellow solid; 69 mg; Yield: 46%; m.p.: 168–170 ꢀC; 1H NMR
(CDCl3, 300 MHz) dH: 2.42 (s, 3H, Ph-CH3), 2.47 (s, 3H, H9),
7-(Allyloxy)-4-methyl-2H-chromen-2-one (3)
0
5.37 (s, 2H, H6 ), 6.17 (s, 1H, H3), 6.99–7.02 (m, 2H, H8 + H6),
0
Yellow solid; 1,69 g; yield: 65%; mp.: 149–151 ꢀC; 1H NMR 7.36–7.60 (m, 5H, H8 +H10 +H11 +H12 +H5), 8.08 (s, 1H, H5 );
0
0
0
0
(CDCl3, 300 MHz) dH: 2.37 (s, 3H, H9), 4.51 (m, 2H, H2 a,b), 5.25 13C NMR (CDCl3, 75 MHz) dC: 18.6, 21.3, 62.3, 101.8, 112.3,
0
0
0
,
(dd, 1H, H4a , J ¼ 10.5 Hz, J ¼ 1.2 Hz), 5.36 (dd, 1H, H4b
112.5, 112.7, 121.2, 125.5, 125.9, 129.5, 129.6, 129.8, 130.7,
0
J ¼ 17.4 Hz, J ¼ 1.2 Hz), 5.91–6.00 (m, 1H, H3 ), 6.03 (s, 1H, H3), 136.8, 143.6, 152.3, 155.1, 160.2, 161.1. ESI-HRMS
6.72 (d, 1H, H8, J ¼ 2.4 Hz), 6.80 (dd, 1H, H6, J ¼ 2.4 Hz, [M + H]+ calcd. For (C20H18N3O3)+: 348.1343, found: 347.1348.
J ¼ 8.7 Hz), 7.41 (d, 1H, H5, J ¼ 8.7 Hz); 13C NMR (CDCl3,
75 MHz) dC: 18.6, 68.7, 101.2, 111.5, 112.2, 113.2, 118.4, 125.5,
4-Methyl-7-((1-(naphthalen-1-yl)-1H-1,2,3-triazol-4-yl)methoxy)-
2H-chromen-2-one (4e)
131.7, 151.9, 154.7, 160.7, 161.0. ESI-HRMS [M + H]+ calcd. For
(C13H13O3)+: 217.0865, found: 217.0870.
Yellow solid; 79 mg; Yield: 53%; m.p.: 173–175 ꢀC; 1H NMR
0
(CDCl3, 300 MHz) dH: 2.41 (s, 3H, H9), 5.42 (s, 2H, H2 ), 6.16 (s,
General procedure for the synthesis of triazolyl
1H, H3), 7.00–7.05 (m, 2H, H8 +H6), 7.53–7.60 (m, 7H, Hnaph),
derivatives (4a–e)
7.96 (d, 1H, H5, J ¼ 7.5 Hz), 8.07 (s, 1H, H5 ); 13C NMR (CDCl3,
0
To a mixture of dipolarophile 2 (3.0 mmol) dissolved in toluene 75 MHz) dC: 18.1, 61.8, 101.7, 111.8, 111.9, 113.6, 121.6, 123.1,
(20 mL), the corresponding azides (3 eq), triethylamine (3 eq, 124.4, 125.2, 125.3, 126.6, 127.5, 127.8, 127.9, 130.1, 130.6,
0.3 mol%) and copper (I) iodide catalyzed (0.5 eq) were added. 132.9, 133.6, 142.5, 151.9, 154.6, 160.6. ESI-HRMS
This mixture was stirred at reflux for 12 h. The solvent was then [M + H]+ calcd. (C23H18N3O3)+: 384.1348, found: 384.1349.

DOI: 10.3109/14756366.2016.1158171
Design and synthesis of hybrid molecules from 4-methylumbelliferone
3
General procedure for the synthesis of isoxazoles (5a–e)
and isoxazolines (6a–e)
4-Methyl-7-((3–(4-nitrophenyl)isoxazol-5-yl)methoxy)-2H-chro-
men-2-one (5e)
A mixture of dipolarophile 2 or 3 (1.15 mmol) and arylnitrile Yellow solid; 90 mg; Yield: 60%; m.p.: 158–160 ꢀC; 1H NMR
0
oxides (3 eq) was dissolved in toluene (15 mL) in the presence of (CDCl3, 300 MHz) dH: 2.42 (s, 3H, H9), 5.32 (s, 2H, H2 ), 6.19 (s,
0
triethylamine (3 eq, 0.3 mol%), the solution was then refluxed for 1H, H3), 6.79 (s, 1H, H4 ), 6.94 (dd, 1H, H6, J ¼ 2.7 Hz,
12 h. The reaction was monitored with TLC after the completion J ¼ 8.7 Hz), 6.99 (d, 1H, H8, J ¼ 2.7 Hz), 7.58 (d, 1H, H5,
0
0
of the reaction, we stopped it and we noted that there was a good J ¼ 8.7 Hz), 8.01 (d, 2H, H7 + H11 , J ¼ 8.7 Hz), 8.33 (d, 2H,
amount of salt, so the salt was filtered off and the filtrate was H8 + H10 , J ¼ 8.7 Hz); 13C NMR (CDCl3, 75 MHz) dC: 18.6,
evaporated. The product formed in each case was purified using 19.6, 61.4, 101.9, 102.0, 112.3, 112.8, 114.7, 124.2, 124.2, 125.9,
precipitation with cyclohexane and ethyl acetate to yield com- 127.7, 127.7, 134.5, 148.8, 152.2, 155.1, 160.3, 160.8, 168.3. ESI-
0
0
pounds 5 or 6.
HRMS [M + H]+ calcd. For (C20H15N2O6)+: 379.0925, found:
379.0930.
4-Methyl-7-((3-phenylisoxazol-5-yl) methoxy)-2H-chromen-2-one
(5a)
4-Methyl-7-((3-phenyl-4,5-dihydroisoxazol-5-yl)methoxy)-2H-
chromen-2-one (6a)
Yellow solid; 52 mg; Yield: 35%; m.p.: 150–152 ꢀC; 1H NMR
Yellow solid; 48 mg; Yield: 32%; m.p.: 145–147 ꢀC; 1H NMR
0
(CDCl3, 300 MHz) dH: 2.41 (s, 3H, H9), 5.30 (s, 2H, H2 ), 6.18
0
(s, 1H, H3), 6.71 (s, 1H, H4 ), 6.95 (dd, 1H, H6, J ¼ 2.4 Hz,
0
(CDCl3, 300 MHz) dH: 2.38 (s, 3H, H9), 3.40 (dd, 1H, H4 a
,
J ¼ 8.7 Hz), 6.98 (d, 1H, H8, J ¼ 2.4 Hz), 7.46–7.48 (m,
0
J ¼ 16.5 Hz, J ¼ 7.5 Hz), 3.56 (dd, 1H, H4 b, J ¼ 16.8, J ¼ 10.8 Hz
0
0
0
3H, H8 + H9 + H10 ), 7.55 (d, 1H, H5, J ¼ 8.7 Hz), 7.80–7.83
0
Hz), 4.15 (dd, 1H, H2 a, J ¼ 14.1 Hz, J ¼ 7.5 Hz), 4.23 (dd, 1H,
(m, 2H, H7 + H11 ); 13C NMR (CDCl3, 75 MHz) dC: 18.1, 60.9,
101.4, 101.6, 111.8, 112.1, 114.0, 125.4, 126.3, 126.3, 128.0,
128.4, 128.4, 129.7, 151.8, 154.6, 160.0, 160.4, 162.1, 166.6.
ESI-HRMS [M + H]+ calcd. For (C20H16NO4)+: 334.1070, found:
334.1079.
0
0
0
0
H2 b, (J ¼ 14.1 Hz, J ¼ 5.1 Hz), 5.13–5.16 (m, 1H, H3 ), 6.13 (s,
1H, H3), 6.81 (d, 1H, H8, J ¼ 2.4 Hz), 6.86 (dd, 1H, H6,
0
0
0
J ¼ 2.4 Hz, J ¼ 8.1 Hz), 7.27–7.43 (m, 3H, H8 + H9 + H10 ), 7.47
(d, 1H, H5, J ¼ 8.1 Hz), 7.69–7.72 (m, 2H, H7 + H11 ); 13C NMR
(CDCl3, 75 MHz) dC: 18.1, 36.9, 68.6, 76.9, 77.9, 101.3, 111.7,
111.9, 113.6, 125.1, 126.2, 126.2, 128.2, 128.2, 129.8, 131.0,
151.6, 155.9, 160.5, 160.8. ESI-HRMS [M + H]+ calcd. For
(C20H18NO4)+: 336. 1231, found: 336.1236.
0
0
7-((3–(4-Methoxyphenyl) isoxazol-5-yl) methoxy)-4-methyl-2H-
chromen-2-one (5b)
Brown solid; 67 mg; Yield: 45%; m.p.: 148–150 ꢀC; 1H NMR
7-((3–(4-Methoxyphenyl)-4,5-dihydroisoxazol-5-yl)methoxy)-4-
methyl-2H-chromen-2-one (6b)
(CDCl3, 300 MHz) dH: 2.41 (s, 3H, H9), 3.86 (s, 3H, Ph-CH3),
0
5.26 (s, 2H, H2 ), 6.18 (s, 1H, H3), 6.64 (s, 1H, H4 ), 6.93 (dd, 1H,
0
Yellow solid; 67 mg; Yield: 45%; m.p.: 144–146 ꢀC; 1H NMR
H6, J ¼ 2.4 Hz, J ¼ 8.7 Hz), 6.97 (d, 1H, H8, J ¼ 2.4 Hz), 6.99 (d,
0
0
2H, H8 + H10 , J ¼ 8.1 Hz), 7.55 (d, 1H, H5, J ¼ 8.7 Hz), 7.75 (d,
0
(CDCl3, 300 MHz) dH: 2.28 (s, 3H, H9), 3.35 (dd, 1H, H4 a
,
2H, H7 + H11 , J ¼ 8.1 Hz); 13C NMR (CDCl3, 75 MHz) dC: 18.1,
54.8, 60.9, 101.2, 101.6, 111.8, 112.1, 113.8, 114.0, 114.0, 120.5,
125.3, 127.7, 127.7, 151.8, 154.6, 160.0, 160.4, 160.7, 161.7,
166.3. ESI-HRMS [M + H]+ calcd. (C21H18NO5)+: 364.1176,
found: 364.1185.
0
0
0
J ¼ 16.5 Hz, J ¼ 7.2 Hz), 3.43 (dd, 1H, H4 b
,
0
J ¼ 16.5 Hz,
J ¼ 10.5 Hz), 3.75 (s, 3H, Hb), 4.03 (dd, 1H, H2 a, J ¼ 14.2 Hz,
0
J ¼ 7.1 Hz), 4.12 (dd, 1H, H2 b, (J ¼ 14.2 Hz, J ¼ 5.2 Hz)), 5.00–
0
5.04 (m, 1H, H3 ), 6.03 (s, 1H, H3), 6.71 (d, 1H, H8, J ¼ 2.7 Hz),
0
0
,
6.79 (dd, 1H, H6, J ¼ 2.7 Hz, J ¼ 9 Hz), 6.85 (d, 2H, H8 + H10
J ¼ 9.6 Hz), 7.40 (d, 1H, H5, J ¼ 9 Hz), 7.55 (d, 2H,
7-((3–(4-Chlorophenyl) isoxazol-5-yl) methoxy)-4-methyl-2H-
chromen-2-one (5c)
H7 + H11 , J ¼ 9.6 Hz); 13C NMR (CDCl3, 75 MHz) dC: 18.1,
37.6, 55.3, 56.2, 68.6, 77.6, 101.3, 111.7, 111.9, 113.7, 113.7,
121.2, 125.6, 128.3, 128.3, 151.9, 154.6, 155.4, 160.5, 160.7,
160.9. ESI-HRMS [M + H]+ calcd. For (C21H20NO5)+: 366.1336,
found: 366.1341.
0
0
Brown solid; 60 mg; Yield: 40%; m.p.: 150–152 ꢀC; 1H NMR
0
(CDCl3, 300 MHz) dH: 2.41 (s, 3H, H9), 5.28 (s, 2H, H2 ), 6.18 (s,
0
1H, H3), 6.68 (s, 1H, H4 ), 6.93 (dd, 1H, H6, J ¼ 2.4 Hz,
J ¼ 8.7 Hz), 6.97 (d, 1H, H8, J ¼ 2.4 Hz), 7.44 (d, 2H,
0
0
H8 + H10 , J ¼ 8.4 Hz), 7.55 (d, 1H, H5, J ¼ 8.7 Hz), 7.76 (d,
7-((3–(4-Chlorophenyl)-4,5-dihydroisoxazol-5-yl)methoxy)-4-
methyl-2H-chromen-2-one (6c)
2H, H7 + H11 , J ¼ 8.4 Hz); 13C NMR (CDCl3, 75 MHz) dC: 18.1,
60.9, 101.3, 101.5, 111.8, 112.1, 114.1, 125.4, 126.5, 127.6,
127.6, 128.7, 128.7, 135.8, 151.7, 154.6, 159.9, 160.4, 161.1,
167.0. ESI-HRMS [M + H]+ calcd. For (C20H15ClNO4)+:
368.0798, found: 368.0802.
0
0
Brown solid; 87 mg; Yield: 58%; m.p.: 149–151 ꢀC; 1H NMR
0
(CDCl3, 300 MHz) dH: 2.40 (s, 3H, H9), 3.36 (dd, 1H, H4 a
,
0
J ¼ 16.8 Hz, J ¼ 7.2 Hz), 3.55 (dd, 1H, H4 b
,
J ¼ 16.8 Hz,
0
J ¼ 10.8 Hz), 4.18 (dd, 1H, H2 a, J ¼ 14.5 Hz, J ¼ 7.2 Hz), 4.21
0
0
(dd, 1H, H2 b, (J ¼ 14.5 Hz, J ¼ 5.1 Hz)), 5.14–5.19 (m, 1H, H3 ),
7-((3–(4-Ethylphenyl) isoxazol-5-yl) methoxy)-4-methyl-2H-chro-
men-2-one (5d)
6.16 (s, 1H, H3), 6.82 (d, 1H, H8, J ¼ 2.4 Hz), 6.89 (dd, 1H, H6,
0
0
J ¼ 2.4 Hz, J ¼ 8.7 Hz), 7.40 (d, 2H, H8 + H10 , J ¼ 8.4 Hz), 7.50
Yellow solid; 55 mg; Yield: 37%; m.p.: 155–157 ꢀC; 1H NMR
(CDCl3, 300 MHz) dH: 1.28 (t, 3H, Ph-CH2-CH3, J ¼ 7.5 Hz),
2.42 (s, 3H, H9), 2.73 (q, 2H, Ph-CH2-CH3, J ¼ 7.5 Hz), 5.30 (s,
(d, 1H, H5, J ¼ 8.7 Hz), 7.64 (d, 2H, H7 + H11 , J ¼ 8.4 Hz); 13C
NMR (CDCl3, 75 MHz) dC: 18.6, 37.3, 69.2, 78.7, 101.7, 112.4,
112.8, 114.1, 118.5, 125.5, 128.2, 128.2, 128.8, 128.8, 136.3,
152.3, 155.1, 155.5, 161.0, 161.2. ESI-HRMS [M + H]+ calcd. For
(C20H17NO4)+: 370.0846, found: 370. 0847.
0
0
0
0
2H, H2 ), 6.19 (s, 1H, H3), 6.68 (s, 1H, H4 ), 6.95 (dd, 1H, H6,
J ¼ 2.7 Hz, J ¼ 8.4 Hz), 6.99 (d, 1H, H8, J ¼ 2.7 Hz), 7.29 (d, 2H,
0
0
H8 + H10 , J ¼ 8.1 Hz), 7.56 (d, 1H, H5, J ¼ 8.4 Hz), 7.74 (d, 2H,
H7 + H11 , J ¼ 8.1 Hz); 13C NMR (CDCl3, 75 MHz) dC: 15.3,
18.6, 28.8, 61.5, 101.9, 102.1, 112.4, 112.6, 112.7, 114.5, 125.6,
126.8, 126.8, 128.4, 128.4, 146.7, 152.2, 155.1, 160.5, 160.9,
162.5, 166.9. ESI-HRMS [M + H]+ calcd. For (C22H20NO4)+:
362.1386, found: 362.1392.
0
0
7-((3–(4-Ethylphenyl)-4,5-dihydroisoxazol-5-yl)methoxy)-4-
methyl-2H-chromen-2-one (6d)
Brown solid; 30 mg; Yield: 20%; m.p.: 145–147 ꢀC; 1H NMR
(CDCl3, 300 MHz) dH: 1.27 (t, 3H, Ph-CH2-CH3, J ¼ 7.3 Hz),

4
M. Zayane et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
2.40 (s, 3H, H9), 2.70 (q, 2H, Ph-CH2-CH3, J ¼ 7.3 Hz), 3.35 7-((1–(4-Chlorophenyl) aziridin-2-yl)methoxy)-4-methyl-2H-
0
0
,
(dd, 1H, H4 a, J ¼ 16.5 Hz, J ¼ 6.6 Hz), 3.50 (dd, 1H, H4 b
chromen-2-one (7c)
0
0
J ¼ 16.8 Hz, J ¼ 10.8 Hz), 4.17–4.20 (m, 2H, H2 a+ H2 b), 5.13–
1
Yellow solid; 112 mg; Yield: 75%; m.p.: 146–148 ꢀC; H NMR
0
5.16 (m, 1H, H3 ), 6.14 (s, 1H, H3), 6.83 (d, 1H, H8, J ¼ 2.7 Hz),
0
(CDCl3, 300 MHz) dH: 2.13 (d, 1H, H4 a, J ¼ 6.6 Hz), 2.27 (d, 1H,
0
0
0
6.90 (dd, 1H, H6, J ¼ 2.7 Hz, J ¼ 9 Hz), 7.27 (d, 2H, H8 + H10
,
,
0
0
H4 b, J ¼ 3.3 Hz), 2.33 (s, 3H, H9), 2.45–2.49 (m, 1H, H3 ), 3.90
0
J ¼ 8.1 Hz), 7.50 (d, 1H, H5, J ¼ 9 Hz), 7.63 (d, 2H, H7 + H11
0
0
,
(dd, 1H, H2 a, J ¼ 7.5 Hz, J ¼ 10.5 Hz), 4.25 (dd, 1H, H2 b
J ¼ 3.9 Hz, J ¼ 10.5 Hz), 6.07 (s, 1H, H3), 6.80 (d, 1H, H8,
J ¼ 8.1 Hz); 13C NMR (CDCl3, 75 MHz) dC: 14.8, 18.1, 28.2,
37.1, 68.6, 77.7, 78.2, 101.8, 112.2, 113.6, 125.1, 126.0, 126.3,
126.3, 127.8, 127.8, 146.4, 151.9, 154.6, 155.9, 160.7, 160.9. ESI-
HRMS [M + H]+ calcd. For (C22H22NO4)+: 364. 1545, found:
363.1549.
0
0
J ¼ 2.4 Hz), 6.89 (2d, 2H, H7 + H11 , J ¼ 8.7 Hz), 6.90 (dd, 1H,
0
0
H6, J ¼ 2.4 Hz, J ¼ 8.7 Hz), 7.13 (2d, 2H, H8 + H10 , J ¼ 8.7 Hz),
7.44 (d, 1H, H5, J ¼ 8.7 Hz); 13C NMR (CDCl3, 75 MHz) dC:
18.1, 30.8, 37.6, 69.8, 101.2, 111.6, 112.1, 112.6, 121.4, 121.4,
125.1, 127.3, 128.5, 128.5, 151.6, 151.9, 154.7, 160.7, 161.1. ESI-
HRMS [M + H]+ calcd. For (C19H16ClNO3)+: 342.0896, found:
341.0897.
4-Methyl-7-((3–(4-nitrophenyl)-4,5-dihydroisoxazol-5-
yl)methoxy)-2H-chromen-2-one (6e)
Yellow solid; 82 mg; Yield: 55%; m.p.: 159–161 ꢀC; 1H NMR
0
(CDCl3, 300 MHz) dH: 2.47 (s, 3H, H9), 3.39 (dd, 1H, H4 a
,
4-Methyl-7-((1-(m-tolyl) aziridin-2-yl)methoxy)-2H-chromen-2-
one (7d)
0
J ¼ 16.8 Hz, J ¼ 7.2 Hz), 3.65 (dd, 1H, H4 b
,
J ¼ 16.8 Hz,
0
0
J ¼ 10.8 Hz), 4.24–4.33 (m, 2H, H2 a +H2 b), 5.14–5.20 (m, 1H,
Yellow solid; 72 mg; Yield: 48%; m.p.: 141–143 ꢀC; 1H NMR
0
H3 ), 6.16 (s, 1H, H3), 6.92 (d, 1H, H8, J ¼ 2.4 Hz), 6.96 (dd, 1H,
0
(CDCl3, 300 MHz) dH: 2.15 (d, 1H, H4 a, J ¼ 6.3 Hz), 2.21 (s, 3H,
H6, J ¼ 2.4 Hz, J ¼ 8.7 Hz), 7.65 (d, 1H, H5, J ¼ 8.7 Hz), 7.93 (d,
2H, H7 +H11 , J ¼ 9 Hz), 8.26 (d, 2H, H8 + H10 , J ¼ 9 Hz); 13C
NMR (CDCl3, 75 MHz) dC: 17.9, 36.1, 69.5, 79.7, 101.6, 111.3,
112.4, 113.5, 123.8, 123.8, 126.3, 127.7, 127.7, 135.4, 143.1,
153.0, 154.6, 155.7, 159.9, 161.3. ESI-HRMS [M + H]+ calcd. For
(C20H17N2O6)+: 381.1087, found: 381.1090.
0
Hb), 2.25 (d, 1H, H4 b, J ¼ 3.3 Hz), 2.38 (s, 3H, H9), 2.48–2.51
0
0
0
0
0
0
(m, 1H, H3 ), 4.08 (dd, 1H, H2 a, J ¼ 7.5 Hz, J ¼ 10.8 Hz), 4.35
0
(dd, 1H, H2 b, J ¼ 3.9 Hz, J ¼ 10.8 Hz), 6.16 (s, 1H, H3), 6.71–
0
0
0
0
6.78 (m, 3H, H7 + H9 +H11 ), 7.01–7.09 (m, 3H, H8 +H6 + H10 ),
7.65 (d, 1H, H5, J ¼ 9.3 Hz); 13C NMR (CDCl3, 75 MHz) dC:
17.9, 20.9, 30.6, 37.5, 70.3, 101.6, 111.2, 112.5, 113.3, 117.5,
121.0, 122.8, 126.3, 128.6, 138.1, 153.1, 153.8, 154.7, 159.9,
161.5. ESI-HRMS [M + H]+ calcd. For (C20H20NO3)+: 322. 1439,
found: 321.1443.
General procedure for the synthesis of aziridine (7a–e)
A mixture of dipolarophile 3 (1 eq) and arylazides (3 eq)
catalyzed (0.5 eq) was dissolved in toluene (15 mL) and the
solution was then refluxed for 6 h. The reaction was monitored
with TLC after the completion of the reaction; the reaction
mixture was concentrated and cooled. The product formed in each
case was purified by column chromatography using 9:1 mixture of
chloroform and ethylacetate to yield compounds 7a–e.
4-Methyl-7-((1-(naphthalen-1-yl) aziridin-2-yl)methoxy)-2H-
chromen-2-one (7e)
Yellow solid; 67 mg; Yield: 45%; m.p.: 143–145 ꢀC; 1H NMR
0
(CDCl3, 300 MHz) dH: 2.38 (d, 1H, H4 a, J ¼ 6.3 Hz), 2.42 (s, 3H,
0
0
H9), 2.58 (d, 1H, H4 b, J ¼ 3.3 Hz), 2.81–2.85 (m, 1H, H3 ), 4.27
0
0
,
(dd, 1H, H2 a, J ¼ 6.9 Hz, J ¼ 10.2 Hz), 4.47 (dd, 1H, H2 b
4-Methyl-7-((1-phenylaziridin-2-yl)methoxy)-2H-chromen-2-one
(7a)
J ¼ 3.9 Hz, J ¼ 10.2 Hz), 6.17 (s, 1H, H3), 6.95 (d, 1H, H8,
0
J ¼ 2.4 Hz), 6.99–7.03 (m, 2H, H6 +H7 ), 7.40 (d, 1H, H5,
Yellow solid; 97 mg; Yield: 65%; m.p.: 142–144 ꢀC; 1H NMR
0
0
0
0
0
0
J ¼ 8.1 Hz), 7.55–8.50 (m, 6H, H8 +H9 +H10 +H11 +H12 +H13 );
13C NMR (CDCl3, 75 MHz) dC: 18.1, 31.9, 38.0, 70.6, 101.3,
111.7, 112.0, 113.4, 114.2, 122.2, 122.9, 125.0, 125.1, 125.2,
125.6, 127.5, 127.8, 133.7, 148.1, 151.9, 154.7, 160.6, 161.1. ESI-
HRMS [M + H]+ calcd. For (C23H20NO3)+: 358. 1443, found:
358. 1448.
0
(CDCl3, 300 MHz) dH: 2.15 (d, 1H, H4 a, J ¼ 6.3 Hz), 2.25 (d, 1H,
0
0
H4 b, J ¼ 3.3 Hz), 2.31 (s, 3H, H9), 2.48–2.51 (m, 1H, H3 ), 3.94
0
0
,
(dd, 1H, H2 a, J ¼ 7.5 Hz, J ¼ 10.5 Hz), 4.25 (dd, 1H, H2 b
J ¼ 3.9 Hz, J ¼ 10.5 Hz), 6.05 (s, 1H, H3), 6.80 (d, 1H, H8,
J ¼ 2.4 Hz), 6.91 (dd, 1H, H6, J ¼ 2.4 Hz, J ¼ 8.7 Hz), 6.93–7.19
0
0
0
0
0
(m, 5H, H7 +H8 +H9 +H10 +H11 ), 7.44 (d, 1H, H5, J ¼ 8.7 Hz);
13C NMR (CDCl3, 75 MHz) dC: 18.1, 30.7, 37.3, 70.1, 101.3,
111.6, 112.1, 113.3, 120.1, 120.1, 122.2, 125.1, 128.5, 128.5,
151.9, 153.0, 154.7, 160.6, 161.2. ESI-HRMS [M + H]+ calcd. For
(C19H18NO3)+: 308.1287, found: 307.1290.
Biological evaluation
Antibacterial activity37
The purified products were screened for their antibacterial
activity by using the agar disc diffusion method toward
Pseudomonas syringae pv, Syringae, Pseudomonas savatanoi,
and Agrobacterium tumefasciens. Nutrient agar (NA) medium
cooled at 45 ꢀC was supplemented with a bacterial suspension
(106 CFU/mL) and poured into Petri plates. After solidification,
sterile Whatman paper discs (diameter 6 mm) were placed at the
surface of the culture medium and 20 mL (1000 mg/mL) of the
product dissolved in dimethyl sulfoxide (DMSO) was dropped
onto each disc. The negative control plates had no product added
to the filter paper whereas in the positive control plates, discs
were impregnated with the same volume of ampicillin solution
(5 mg/mL). The treated Petri dishes were incubated at 25 ꢀC for
48 h. The antibacterial activity was evaluated by measuring the
diameter of the inhibitory zones formed around the discs. The
experiment was replicated twice.
7-((1–(4-Methoxyphenyl) aziridin-2-yl)methoxy)-4-methyl-2H-
chromen-2-one (7b)
Yellow solid; 82 mg; Yield: 55%; m.p.: 137–139 ꢀC; 1H NMR
0
(CDCl3, 300 MHz) dH: 2.15 (d, 1H, H4 a, J ¼ 6.6 Hz), 2.26 (d, 1H,
0
0
H4 b, J ¼ 3.3 Hz,), 2.38 (s, 3H, H9), 2.46–2.49 (m, 1H, H3 ), 3.74
0
(s, 3H, Hb), 3.99 (dd, 1H, H2 a, J ¼ 7.5 Hz, J ¼ 10.5 Hz), 4.29 (dd,
0
1H, H2 b, J ¼ 3.9 Hz, J ¼ 10.5 Hz), 6.12 (s, 1H, H3), 6.78 (2d, 2H,
0
0
H7 + H11 , J ¼ 9 Hz), 6.87 (dd, 1H, H6, J ¼ 2.4 Hz, J ¼ 8.7 Hz),
0
0
6.93 (d, 1H, H8, J ¼ 2.4 Hz), 6.97 (2d, 2H, H8 + H10 , J ¼ 9 Hz),
7.49 (d, 1H, H5, J ¼ 8.7 Hz); 13C NMR (CDCl3, 75 MHz) dC:
18.1, 30.9, 37.5, 55.0, 70.1, 101.3, 111.6, 112.1, 113.3, 113.8,
113.8, 120.9, 120.9, 125.1, 148.1, 151.9, 154.7, 154.9, 160.7,
161.2. ESI-HRMS [M + H]+ calcd. For (C20H20NO4)+: 337.1390,
found: 338.1392.

DOI: 10.3109/14756366.2016.1158171
Design and synthesis of hybrid molecules from 4-methylumbelliferone
5
Antifungal activity38
The sample concentration providing 50% inhibition (IC50) was
calculated by plotting inhibition percentages against concentra-
tions of the sample.
Aspergillus flavus, Aspergillus niger, and Penicillium italicum
were used for the screening of antifungal activity of the products
tested by using the disc diffusion method. A conidial suspension
of the tested fungi was prepared (104–105 CFU/mL) and added to
potato dextrose agar (PDA) medium cooled at 45 ꢀC and poured First, our key intermediate was the dipolarophiles 2 and 3, which
uniformly into Petri plates (diameter 90 mm). Sterilized paper can easily be obtained by O-propargylation and O-allylation41,42
Results and discussion
,
discs (6 mm, Whatman No. 1 filter paper) were impregnated with respectively of 4-methylumbelliferone 1 in one step (Scheme 1).
20 mL (1000 mg/mL) of the product dissolved in DMSO and Indeed, in our investigation, dimethylformamide was found to be
placed on the culture plates whereas the negative control plates an excellent solvent for the reaction of allyl and propargylbromide
had no product added to the filter paper. In the positive control with 4-methylumbelliferone 1, in the presence of NaH. The
plates, discs were imbibed with the same volume of a dipolarophiles 2 and 3 were obtained in 75% and 65% yield,
carbendazim suspension (0.5 mg/mL). The diameter of the respectively and which were identified by the spectroscopic
inhibition zone (mm) around the disc was measured after means (1H, 13C, and ES-HRMS)43,35
.
incubation at 25 ꢀC for 4 d and compared with control. The test
was performed in triplicate.
In recent years, the click chemistry is a powerful tool, allowing
synthetic chemists to obtain selectivity, simple experimental set-
up, tolerance to various functional groups and quantitative
yields36. The click reaction between azide and terminal alkyne
catalyzed by Cu(I) has extensively facilitated the practical and
efficient regiospecific formation of 1,4-disubstituted 1,2,3-tri-
azoles44. With this method, the reaction of the dipolarophile 2
with different arylazides generated in situ and copper (I) iodide
catalyzed, in the presence of catalytic amount of triethylamine (3
eq) in refluxing toluene for 6–12 h, led to the formation of new
triazoles linked 4-methylumbelliferone (Scheme 2). The isolated
product yields were in the range of 50–55%. The structures of
these compounds were confirmed according to their spectral data,
where the 1H NMR spectra of compounds 4a–e indicated the
Anticoagulant activity39
Activated partial thromboplastin time (aPTT) was performed
using Platelin LS reagent (Trinity Biotech PLC, Cowicklow,
Ireland) on a STAR analyzer (Diagnostica Stago, Asnieres,
France). Coagulation test was performed on 4-methylumbellifer-
one derivatives 2, 3, 4a–e, 5a–e, 6a–e, and 7a–e samples at
various concentrations diluted in a pool of frozen normal plasma.
4-Methylumbelliferone 1 was used as reference.
`
BChE inhibitory activity assay40
Butyrylcholinesterase (BChE) was used to hydrolyze butyrylthio- existence of the triazolic moieties by showing essentially the
1
choline (colorless) to butyrate and thiocholine. Then, thiocholine resonance of the protons introduced by the arylazides. The H
reacts with 5,50-dithiobis[2-nitrobenzoic acid] (DTNB) to form 5- NMR spectrum for 4a as an example, showed a singlet at dH 5.36
0
mercapto-2-nitrobenzoic acid (5-MNBA), a yellow product. The relative to the methylenic protons H6 and another singlet at dH
rate of 5-MNBA formation, measured spectrophotometrically at 8.13 corresponding to the H5 triazole proton. The 13C NMR
0
405 nm is proportional to the enzymatic activity of BChE in the spectrum showed a signal at dH 61.7 for the –O–CH2 (C6’) carbon
sample. Human plasma (pool plasma from samples designated for and two signals at dC 120.1 and 143.2 for the two characteristic
0
0
biochemical analysis) was used as a source of BChE. 100 mL of triazole carbons C5 and C4 , respectively. The mass spectrum
compound (concentration of 1000 mg/mL) were added to 100 mL recorded in ES-HRMS showed a pseudo-molecular ion peak
of plasma and the mixture was incubated at 37 ꢀC for a [M + H]+ at m/z 334.1192 which is consistent with its molecular
determined time. After incubation, the enzyme activity was formula C19H16N3O3. The resulting 1,4-regioisomers were
measured by Konelab 30Õ UV apparatus. The control (plasma and evidenced from the NOEs between H5 (triazole)/H6 (1.85 A)
˚
0
0
˚
distilled water) was treated in the same conditions. All assays and H5 (triazole)/Harom (1.30 A) and the absence of any NOE
0
0
were carried out in duplicate. The anticholinesterase activity was between H6 /Harom
.
calculated by the following formula:
The 1,3-dipolar cycloaddition has became widely used as a
highly efficient method for the synthesis of isoxazoles45. Using
this method, the reaction of the dipolarophile 2 with different
aylnitrile oxides in refluxing toluene with a catalytic amount of
ÂÀ
% Inhibition ¼ activity of control ꢁ activity of sampleÞ=
Ã
activity of control ꢂ 100:
9
5
4
3
6
Br
2'
2
7
1'
O
3'
O
O
1
8
2 (75%)
4'
4'
DMF/NaH
20 °C
O
O
OH
1
Br
2'
1'
O
O
O
3'
3 (65%)
Scheme 1. Synthetic route of dipolarophiles 2 and 3.

6
M. Zayane et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
a
Scheme 2. Synthetic route of triazoles 4.
5
4
6
7
3
3'
6'
2
Ar-N3
N
4'
O
O
O
O
O
O
N 2'
Cu(I)/ Toluene
8
1
3
N
1'
Ar
4a-e (50-55%)
5'
Ar=a: C H , b:p-(OCH )-C H , c:p-Cl-C H , d:m-CH -C H , e:naphthyl
6
5
3
6
4
6
4
3
6
4
a
5
8
4
Ar
Cl
6
7
3
N
2
OH
O
5'
3'
O
O
O
1'
O
O
O
2'
N
1
Toluene/ TEA
2
5a-e (35-60%)
4'
Ar
a
5
Ar
4
6
3
N
2
7
Cl
OH
Toluene/ TEA
O
5'
3'
O
O
1
O
2'
O
O
O
8
N
1'
3
4'
6a-e (20-58%)
Ar: a: Ph, b: Ph-OCH , c:Ph-Cl, d:Ph-Et, e:Ph-NO
Ar
3
2
Scheme 3. Synthetic route of isoxazoles 5a–e and isoxazolines 6a–e.
triethylamine (3 eq) for 12 h led to the formation of new The structures of cycloadducts 5a–e and 6a–e were further
isoxazoles 5a–e (Scheme 3) in good to moderate yields (35–60%). confirmed by mass spectrometry.
The 1H NMR spectrum of compound 5a as an example showed in
addition of the signals corresponding to the protons introduced by according to the 1,3-dipolar cycloaddition with various arylazides
Following the same way, the dipolarophile 3 was treated
the dipole the appearance of a singlet at dH 5.30 relative to the as a dipole in refluxing toluene for 6 h, providing with complete
0
proton H2 and another singlet at dH 6.71 corresponding to the regiosepecificity the awaited 1,2,3-triazoles I which lose a
proton H4 . The 13C NMR spectrum of this cycloadduct 5a was molecule of nitrogen to yield the corresponding aziridines 7a–e
also a good support for the proposed structure which exhibited (Scheme 4) in excellent to moderate yields (65–75%).
0
characteristic signals at dC 60.9 corresponding to O–CH2 carbon,
The structure of compounds 7a–e has been evidenced via their
1
and at dC 101.4, 160.0, and 166.6 relative to isoxazoline carbons 1H and 13C NMR spectra. The H NMR spectrum of compound
0
C4 , C3 , and C5 , respectively.
0
0
7a as an example showed in addition to the protons introduced by
In another hand, we studied the behavior of the dipolarophile 3 the dipole the appearance of two doublets at dH 2.15 (J ¼ 6.3 Hz)
with some arylnitrile oxides under conventional conditions by and at dH 2.25 (J ¼ 3.3 Hz) attributable to the methylenic protons
0
0
refluxing in toluene in the presence of a catalytic amount of H4 a and H4 b of the aziridine ring, two doublets of doublets at dH
triethylamine for 12 h. The reaction led to the formation of new 3.94 (J ¼ 10.5 Hz, 7.5 Hz) and at dH 4.25 (J ¼ 10.5 Hz, 3.9 Hz)
0
corresponding to the diastereotopic methylenic protons H2 a and
cycloadducts 6a–e (20–58%) (Scheme 3).
0
Their structures were established on the basis of their spectral H2 b and a multiplet centered at dH 2.50 attributable to the proton
data (1H, 13C NMR, and ES-HRMS). In fact, the 1H NMR H3 of the stereogenic center. Further, the 13C NMR spectrum of
0
spectrum of compound 6a, as an example, showed the presence of the same compound showed the presence of signals at dC 30.7 and
0
0
two doublets of doublets at dH 3.40 (J ¼ 16.8 Hz, J ¼ 7.2 Hz) and dC 37.3 relative to aziridine carbons C4 and C3 , respectively and
at dH 3.56 (J ¼ 16.8 Hz, J ¼ 10.8 Hz) corresponding to the protons a signal at dC 70.1 corresponding to –O–CH2 carbon. The
0
0
H4 a and H4 b, two other doublets of doublets at dH 4.15 structures of compound 7a–e were further confirmed by their
(J ¼ 14.1 Hz, J ¼ 7.5 Hz) and dH 4.23 (J ¼ 14.1 Hz, J ¼ 5.1 Hz) mass spectra.
0
0
attributable to protons H2 a and H2 b. The same spectrum showed a
0
multiplet centered at dH 5.13 attributable to the H3 of the
Antibacterial activity
stereogenic center. Further, the 13C NMR spectrum of this
compound showed in addition to the carbons introduced by the
dipole, the presence of a signal at dC 68.6 relative to –O–CH2
carbon and two signals at dC 36.9 and 77.9 relative to the
isoxazoline carbons C4 and C3 , respectively.
The resulting regiochemistry was evidenced from the NOEs
The antibacterial activity of the newly synthesized compounds 2,
3, 4, 5, 6, and 7 were evaluated against some bacteria, namely
P. syringae pv, Syringae, P. savatanoi, and A. tumefasciens. The
results mentioned in Table 1 showed the inactivity of compound 1
against all the bacteria used. The antibacterial effect of some
synthesized hybride molecules from the series 4, 5, 6, and 7
showed the importance of the added fragments to
0
0
˚
between H4 (isoxazoline)/H2 (1.88 A) and H4 (isoxazoline)/
0
0
0
˚
Harom (1.32 A) and the absence of any NOE between H2 /Harom
0
.

DOI: 10.3109/14756366.2016.1158171
Design and synthesis of hybrid molecules from 4-methylumbelliferone
7
9
5
4
Ar-N
6
7
3
2
3
Ar
N
2'
O
Toluene/ TEA
O
O
O
O
O
3'
N
-N
N
2
O
O
1
O
1'
3
8
N
5'
4'
Ar
7a-e (45-75%)
Ar= a: C H , b: p-(OCH )-C H , c: p-Cl-C H , d: m-CH -C H , e: naphthyl
6
5
3
6
4
6
4
3
6 4
Scheme 4. Synthetic route of aziridines 7a-e.
compound 6a with a phenyl group in its isoxazoline moiety.
On the other hand, only compounds 6a and 6d bearing a phenyl
and m-CH3-C6H4, respectively, as substituents at their isoxazo-
line system, exhibited the same activity toward P. savatanoi
(IZ ¼ 10.5 mm), while against P. syringae pv, Syringae only
compound 6a (C6H5) showed an activity (IZ ¼ 11.5 mm). These
finding showed that the noted activities of compounds 6a–e
against the three bacteria used varies depending on the aryl
group introduced by each aldehyde used in their preparation
and the sensitivity against these compounds depends of the
strain used.
Table 1. Antibacterial activity of 4-methylumbelliferone and its
synthesized derivatives 2, 3, 4a–e, 5a–e, 6a–e, and 7a–e.
Inhibition zone diameter (mm)
Pseudomonas
syringae pv, Syringae
Pseudomonas
savatanoi
Agrobacterium
tumefasciens
Productsa
1
2
3
4a
-
-
10.5
12
9
-
10
11
-
8
11.5
-
4b
4c
8
-
8
-
-
-
The most significant activity of isoxazolines 6 compared to
those of the isoxazoles 5 bearing the same aryl groups especially
against A. tumefaciens allowed to conclude the importance of the
spatial arrangement of the isoxazoline ring in compounds 6 with
4d
4e
-
7.5
-
-
-
-
5a
5b
-
-
-
-
-
-
0
the creation of a stereocenter (C3 ), which certainly influenced the
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
-
-
-
-
-
-
-
10
9
activity in one direction or another. We cite as an example the
case of compounds 6a and 6d which have been found active
against P. savatanoi (IZ ¼ 10.5 mm) while the corresponding
isoxazoles 5a and 5d were found to be inactive against the same
bacteria and unlike isoxazoles 5d (IZ ¼ 10 mm) and 5e
(IZ ¼ 9 mm) are slightly more active than the corresponding
iosoxazolines 6d (IZ ¼ 8.5 mm) and 6e (IZ ¼ 8.5 mm) against A.
tumefaciens.
11.5
-
-
-
-
9.5
10.5
11
8.5
10.5
22
-
10.5
-
-
10.5
-
13
-
11.5
11
10
18
-
-
10
8.5
8.5
8.5
11
11
7.5
7.5
-
Most compounds 7a–e displayed an antibacterial activity
against the microorganisms used.
Therefore, compound 7a bearing a phenyl in its aziridine
moiety exhibited a good activity (IZ ¼ 13 and 11 mm) against
P. savatanoi and A. tumefasciens, respectively. This result
compared to those of the other derivatives from the same series
shows that the presence of the non substituted phenyl group
introduced by the arylazide used seems at the origin of this
relatively high activity.
7e
Ampicillin
DMSO
25
-
aConcentration used ¼ 1 mg/mL.
- no inhibition zone observed.
4-methylumbelliferone 1 via the linker methylene to have activity.
Compounds 7c with a p-Cl-C6H4 (IZ ¼ 11 mm) exhibited a
The same results showed that the propargylated compound 3 is good activity against P. syringae pv, Syringae compared to the rest
slightly more active than the allylated derivative 2 against the of aziridines. This result can be explained by the presence of a
three bacterial strains used. This slight difference in activity may chlorine atom in para position of the N-linked phenyl group in the
be explained by the linearity of the propargyl moiety in aziridine fragment.
comparison to the planar structure of the allyl group in
compound 2.
On the other hand, it has been noticed that the arylation of the
nitrogen atom in the aziridine moiety in compound 7e by a
Most 1,4-triazoles 4 and isoxazoles 5 did not display any naphthyl group resulted in the loss of activity toward A.
activity against the used bacteria. However, compound 4b bearing tumefasciens whereas this substituent was found in favor of the
a p-MeO-C6H4 as a substituent at its triazole moiety which activity of the same derivative against both P. syringae pv,
exhibited a moderate activity toward P. syringae pv, Syringae, and Syringae, and P. savatanoi (IZ ¼ 10.5 and 10 mm, respectively).
P. savatanoi (IZ ¼ 8 mm), also compound 4e with a p-NO2-C6H4 This finding reinforces the independence of action of this
in the trizaole ring displayed activity only against P. syringae pv, compound against three strains used.
Syringae (IZ ¼ 7 mm). In the series 5, only derivatives 5d and 5e Overall, from all the results explained above and given in
bearing a m-CH3-C6H4 and p-NO2-C6H4, respectively, as sub- Table 1, we found that the aziridine system introduced with
stituents at the isoxazole moiety, were found to be active toward various substituents on its nitrogen atom in compounds 7 brings
A. tumefasciens (IZ ¼ 10 and 9 mm, respectively). more antibacterial efficiency than isoxazoles (5) and isoxazolines
Most isoxazolines 6 displayed an acceptable antibacterial (6) and even propargyl (2) and allyl (3) fragments and appears to
activity against A. tumefasciens (IZ ¼ 8.5–10 mm) except act independently of the bacterial strain.

8
M. Zayane et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Antifungal activity
favor of the anticoagulant activity. On the other hand, it was found
that the atom of chlorine (in compound 4c) was the substituent
which brought more activity compared to the others from the
same series. Also we noticed that the naphthyl group in the
compound 4e (aPTT ¼ 59.7 s) has not remarkably improved the
activity compared to that of 4a (aPTT ¼ 55.6 s) where we have a
phenyl group. The extension of the field of conjugation of the
nitrogen doublet with the naphthyl group may be at the origin of
this slight improvement of the noted activity.
Similarly, compounds 5a–e acted almost in the same way as
compounds 4. Their activities ranged from 54.6 to 59.9 s and the
compound 5a (aPTT ¼ 54.6 s) with an unsubstituted phenyl group
was found the least active in the series while compound 5d
(aPTT ¼ 59.9 s) bearing a m-CH3-C6H4 proved the most active.
For compounds 6a–e, the anticoagulant activity varies between
As it can be concluded from the data given in Table 2, only
compounds 6a and 6d showed activity against P. italicum
(IZ ¼ 9.5 mm) and A. flavus (IZ ¼ 9.5 mm), respectively. Despite
the presence of the coumarin moiety in all tested molecules 2, 3,
4, 5, and 7, no significant activity was obtained. This finding let
us to think that the linkage of proargyl, allyl, triazoles, isoxazoles,
and aziridines systems to 4-methylumbelliferone 1, was not
probably in favor of this activity but the antifungal potent of
compounds 6 appears due to the nature of the aryl group attached
to the isoxazoline ring.
Anticoagulant activity
The in vitro anticoagulant activity of the 4-methylumbelliferone 1
and its synthesized derivatives 2, 3, 4a–e, 5a–e, 6a–e, and 7a–e aPTT ¼57.2 and 63.5 s. Compound 6e with a p-NO2-C6H4
were assessed by measuring the activated partial thromboplastin showed the highest activity (aPTT ¼ 63.5 s), while the less
time at three different concentrations 200, 500, and 1000 mg/mL. active compound in this series is 6d (aPTT ¼ 57.2 s) whose
The results given in Table 3 showed that compounds tested at the phenyl borne by the isoxazoline ring is para-ethylated. On the
concentration of 1000 mg/mL exhibited moderate to significant other hand, the comparable activity of compounds 6b (p-OCH3-
anticoagulant activities (aPTT ¼ 41.7–74.3 s).
C6H4, aPTT ¼ 63.2 s) and 6e (p-NO2-C6H4, aPTT ¼ 63.5 s) shows
As shown in Table 3, the aPTT assay indicated that the starting that the donor or attractor electronic effect exerted by the
material 4-methylumbelliferone 1 (aPTT ¼ 41 s) was found to be substituent on the aromatic ring attached to the isoxazoline moiety
significantly less active than all the synthesized hybride mol- does not appear to affect considerably the activity but the nature
ecules (aPTT ¼ 54.6–74.3 s). This finding showed the contribu- of the substituent seems behind the difference in activity between
tion of all the fragments introduced (propargyl, allyl, triazole, the various compounds. The relatively low activity of isoxazoles 5
isoxazole, isoaxazoline, and aziridine) each react in its own way to compared to that of most of isoxazolines 6 can only be explained
0
by the appearance of the new seterocenter C3 in the structure
improve the anticoagulant activity of 4-methylumbelliferone 1.
As can be seen in Table 3, the O-allylated derivative 3 of the latters.
Compounds
(aPTT ¼ 72.7 s) was found to be more active than the
7a–e
displayed
anticoagulant
activity
O-propargylated one 2 (aPTT ¼ 63.9 s). This finding may be (aPTT ¼ 54.9–74.3 s) comparable to that of the other tested
interpreted by the planar structure of the allyl group in compound derivatives except compound 7d bearing a m-CH3-C4H6 substitu-
3 in comparison to the linearity of the propargyl moiety in ent in its aziridine ring (aPTT ¼ 74.3 s). The comparison of this
compound 2.
activity to that of the rest of the compounds of the same series
The results obtained show that compounds 4a–e displayed shows clearly the certain contribution of the m-CH3-C4H6
remarkable anticoagulant activity with aPTT values ranging from fragment in the noted activity. This derivative was found to be
55.6 s (compound 4a) to 60.8 s (compound 4c). The variation of the most active among all the tested compounds.
this activity is easily explained by the nature of the aromatic As shown in Table 4, the most active compounds 3 and 7d at
system attached to the triazole ring. In fact, the slightly important the concentration of 1000 mg/mL exhibited remarkable anti-
activity of compounds 4b (p-OCH3-C4H6, aPTT ¼ 58.6 s), 4c coagulant activity as indicated by the significant prolongation of
(p-Cl-C6H4,
aPTT ¼ 60.8 s),
and
4d
(m-CH3-C6H4, aPTT in a dose-dependent manner.
aPTT ¼ 56.8 s) compared to that of 4a (C6H5, aPTT ¼ 55.6 s)
showed that the substitution of the phenyl system could be in Anticholinesterase activity
These results were expressed as IC50 values showing the activity
of the synthesized of 4-methylumbelliferone 1 and most of its
synthesized derivatives as shown in Table 5. The results
showed that the dipolarophile 3 (IC50 ¼0.3266 0.01 mg/mL)
Table 2. Antifungal activity of compounds 6a and 6d.
Inhibition zone diameter (mm)
Productsa Aspergillus flavus Aspergillus niger Penicillium italicum
Table 4. Anticoagulant activity of compounds 3 and 7d determined by
measuring the activated partial thromboplastin time (s) at different
concentrations.
6a
6d
FONG
DMSO
-
9.5
37
-
-
9.5
-
32.5
-
-
40.5
-
[ ] (mg/mL)
0
250
500
1000
FONG: carbendazime 0.5 mg/mL.
aConcentration used ¼ 1 mg/mL.
- no inhibition zone observed.
3
7d
31.6
31.6
39.2
39.3
41.9
42.8
72.7
74.3
Table 3. Anticoagulant activity of 4-methylumbelliferone 1 and its synthesized derivatives 2, 3, 4a–e, 5a–e, 6a–e, and 7a–e.
Compoundsa
1
2
3
4a
4b
4c
4d
4e
5a
5b
5c
5d
aPTT (s)
Compounds
aPTT (s)
41.7
5e
58.3
63.9
6a
60.2
72.7
6b
63.2
55.6
6c
61.2
58.6
6d
57.2
60.8
6e
63.5
56.8
7a
54.9
59.7
7b
54.9
54.6
7c
61.2
59.1
7d
74.3
55.2
7e
60.4
59.9
aConcentration used ¼ 1000 mg/mL.

DOI: 10.3109/14756366.2016.1158171
Design and synthesis of hybrid molecules from 4-methylumbelliferone
9
Table 5. Anticholinesterase activity of 4-methylumbelliferone 1 and most
of its synthesized derivatives 2, 3, 4, 5, 6, and 7.
6d (IC50 ¼0.415 0.018 mg/mL), and 6e (IC50 ¼0.183
0.020 mg/mL) have been shown to be more active than the
starting material 1 (IC50 ¼0.692 0.055 mg/mL). This finding
still shows the effect of the NO2 group observed in the case of 5e
Compounds
IC50 (mg/mL)
0
which is added that of the new stereocenter C3 . The same
phenomena was noted with compound 6a (IC50
1
2
3
4a
4b
4d
5a
5b
5c
5d
5e
6a
6b
6d
6e
7a
7b
7c
7d
7e
0.692 0.055
0.413 0.021
0.327 0.012
0.326 0.010
0.360 0.012
0.334 0.021
0.556 0.010
0.553 0.030
0.634 0.010
0.390 0.012
0.495 0.022
0.338 0.011
0.865 0.015
0.415 0.018
0.183 0.020
0.243 0.023
0.427 0.012
0.151 0.012
0.454 0.015
0.292 0.023
0.38 ꢂ 10ꢁ3 0.002 ꢂ 10ꢁ3
¼
0.338 0.010 mg/mL) when compared to its isoxazole analogous
5a (IC50 ¼0.556 0.010 mg/mL). In the case of the ethyl group as
substituent, our results did not show any remarkable effect of the
newly formed stetreocenter to improve or to reduce the anti-BChE
activity of compound 6d (IC50 ¼0.415 0.018 mg/mL) compared
to that of its analogous 5d (IC50 ¼0.390 0.012 mg/mL).
In the contrary, in the case of compound 6b
(IC50 ¼0.865 0.015 mg/mL), the co-presence of the p-OCH3-
0
C6H4 and the new stereocenter C3 in the isoxazoline moiety
reduce considerably the anti-BChE activity of 4-methylumbelli-
ferone 1. The above results allowed concluding that the activity of
compounds 6 depends along the new asymmetric center and the
aryl group fixed at the isoxazoline moiety.
Compounds 7a–e were found considerably more active than 4-
methylumbelliferone 1. It is clear that independently of the aryl
group attached, the aziridine moiety has improved the anti-BChE
activity of 4-methylumbelliferone 1. Our results showed that
the activity was improved more than 4.5 times when we joined
the 4-methylumbelliferone 1 to an aziridine moiety bearing a
p-chlorophenyl ring through the spacer methylene (compound 7c,
IC50 ¼0.151 0.010 mg/mL). The activity of this series varies
depending on the aryl group attached to the aziridine, it
decreases in the order 7c (p-Cl-C6H4, IC50 ¼0.151 0.010 mg/
mL), 7a (C6H5, IC50 ¼0.243 0.020 mg/mL), 7e (p-Cl-C6H4,
IC50 ¼0.292 0.020 mg/mL), 7d and 7b (p-OCH3-C6H4,
IC50 ¼0.427 0.012 mg/mL). This finding shows clearly the
effect of the aryl group attached to the aziridine ring on the
anti-BChE activity.
Galanthamine
aConcentration used ¼ 1 mg/mL.
was found to be more active than its analogous
2
(IC50 ¼0.4131 0.015 mg/mL). This finding could probably be
due to the planar structure of the allyl group in compound 3
compared to the linearity of the propargyl moiety in compound 2.
Moreover, the high activity of these alkylated derivatives 2 and 3
compared
to
that
of
the
starting
material
1
(IC50 ¼0.692 0.055 mg/mL) showed the contribution of the
two alkyl fragments introduced to the improvement of the activity
of the latter.
On the other hand, the obtained results showed the certain
intervention of the triazole ring introduced in the compounds 4a
(IC50 ¼0.326 0.010 mg/mL), 4b (IC50 ¼0.360 0.012 mg/mL),
and 4d (IC50 ¼0.334 0.021 mg/mL) to practically double the
activity of compound 1 (IC50 ¼0.692 0.055 mg/mL). The com-
parable inhibitory potency of compounds 4a (C6H5), 4b (p-OCH3-
C6H4), and 4d (m-CH3-C6H4) allows to think that the nature of the
aryl fragment attached to the corresponding triazole in these three
cases seems without effect. The limited number of tested
derivatives (only three) fails to consider unambiguously that the
aromatic system attached at C2 of the triazole ring manages the
behavior of these compounds against BChE. The diversification
of fragments borne by the triazole ring in a larger series of
derivatives could lead to a plausible conclusion.
All the isoxazoles derivatives 5a–e (IC50 ¼0.634–0.390 mg/
mL) proved to inhibit more the BChE than the starting compound
1 (IC50 ¼0.692 0.055 mg/mL). This finding showed the import-
ance of the isoxazole moiety to improve the anti-BChE potential
of compounds 5. The derivatives 5d and 5e bearing a p-Et-C6H4
and a p-NO2-C6H4 borne by their isoxazoline rings were found to
be the most active (IC50 ¼0.390 0.012 and 0.495 0.020 mg/
mL, respectively). This result shows the role of the substitutent
fixed in para position of the aromatic ring, indeed the ethyl group
(electron-donating) in compound 5d seems to have more effect to
enhance the contribution of the isoxazole to improve the activity
of compound 1.
Conclusion
The present work constitutes a contribution to the valorization
of 4-methylumbelliferone 1 as a coumarin derivative to access
to new biaocative hybride molecules. This coumarin has been
transformed into two propargylated and allylated dipolarophiles 2
and 3, respectively to be used as precursors for synthesizing the
target coumarin-containing triazole, isoxazoles, isoxazolines, and
aziridines via intermolecular 1,3-dipolar cycloaddition reactions.
The newly prepared cycloadducts have been tested for their
possible antimicrobial, anticoagulant and anticholinesterase
activities. The study of the SAR permitted to conclude in most
cases the contribution of the added heterocyle (triazle, isoxazole,
isoxazoline, and aziridine) to the starting coumarin via the
methylene group as a linker to improve or even to bring activity.
The nature of the aryl fragment attached to the added heterocyles
and the created stereocenter in the case of the isoxazolines 6 have
been the origin of some obtained results.
Declaration of interest
The authors declare no conflicts of interests. The authors alone
are responsible for the content and writing of this article.
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