Access to new antimicrobial 4-methylumbelliferone derivatives

Article abstract of DOI:10.1007/s12039-015-0927-6

Synthesis of some novel coumarin esters has been accomplished through iodine-catalyzed method using 4-methylumbelliferone as the starting material. Condensation of hydrazide, which was obtained in two steps from 4-methylumbelliferone, with some arylaldhydes provided hydrazone derivatives, while the reaction with phenylthioisocyanate leads to the thiosemicarbazide that evolved into two new compounds. Finally, condensation reaction of hydrazide with three diketones afforded new pyrrole and pyrazole derivatives. Structures of all synthesized compounds were established on the basis of spectroscopic methods including ¹H NMR, ¹³C NMR and ES-HRMS. They were finally tested for their antimicrobial activity and the structure-activity relationship was discussed.

Full text of DOI:10.1007/s12039-015-0927-6

c
J. Chem. Sci. ꢀ Indian Academy of Sciences.
DOI 10.1007/s12039-015-0927-6
Access to new antimicrobial 4-methylumbelliferone derivatives
MARWA ZAYANEa, ANIS ROMDHANEa,∗, MEJDA DAAMI-REMADIb and
HICHEM BEN JANNETa
aLaboratoire de Chimie Hétérocyclique, Produits Naturels et Réactivité, Equipe: Chimie Médicinale et
Produits Naturels, Faculté des Sciences de Monastir, Université de Monastir, Avenue de l’Environnement,
5019 Monastir, Tunisie
bUR13AGR09, Production Horticole Intégrée au Centre Est Tunisien, Centre Régional des Recherches en
Horticulture et Agriculture Biologique de Chott-Mariem, Université de Sousse, 4042, Chott-Mariem, Tunisie
e-mail: anis_romdhane@yahoo.fr
MS received 4 April 2015; revised 16 June 2015; accepted 21 June 2015
Abstract. Synthesis of some novel coumarin esters has been accomplished through iodine-catalyzed method
using 4-methylumbelliferone as the starting material. Condensation of hydrazide, which was obtained in two
steps from 4-methylumbelliferone, with some arylaldhydes provided hydrazone derivatives, while the reac-
tion with phenylthioisocyanate leads to the thiosemicarbazide that evolved into two new compounds. Finally,
condensation reaction of hydrazide with three diketones afforded new pyrrole and pyrazole derivatives. Struc-
tures of all synthesized compounds were established on the basis of spectroscopic methods including 1H NMR,
13C NMR and ES-HRMS. They were finally tested for their antimicrobial activity and the structure-activity
relationship was discussed.
Keywords. 7-hydroxy-4-methyl-coumarin; (4-methylumbelliferone); imine; triazole; thiadiazole; pyrazole;
pyrrole; antimicrobial activity.
antimicrobial,13 anticancer,14 antitubercular,15 antioxi-
dant and anti-inflammatory agents.16
1. Introduction
Coumarins occupy an important place in the realm
of natural products and organic synthesis. They con-
stitute an important class of oxygen heterocycles and
belong to the family of lactones having benzopyrane
system.1 The coumarin skeleton is found in many nat-
ural products and is also used as an intermediate for
the synthesis of specific and promising heterocyclic
compounds, possessing a wide spectrum of biological
activities such as anti-inflammatory,2 anti-tubercular,3
antioxidant,4 antimicrobial5 and anticoagulant agents.6
Many methods for the preparation of coumarin-based
compounds have been reported.7–9 One of the main rep-
resentative of this class is the 4-methylumbelliferone,
used as a useful precursor for the preparation of such
compounds.10–12
The study of nitrogen heterocyclic compounds has
led a considerable development due to the revelation
of their varied effects in diverse domains. In this con-
text, pyrroles, pyrazoles, thiadiazoles and triazoles, and
their derivatives are very attractive targets. They have
been the subject of many chemical and biological stud-
ies on account of their pharmacological activity, such as
In view of the above observations and as a contin-
uation of our previous work on the synthesis of new
coumarin derivatives,17 we report here the synthesis of
a precursor 2 from the 4-methylumbelliferone 1 and
its use as building blocks in the preparation of some
novel coumarin esters 4 and other new derivatives 6-13
incorporating the coumarin and pyrrole, pyrazole, thia-
diazole or triazole moieties and their antibacterial and
antifungal activity.
2. Experimental
2.1 General information
All reactions were monitored by TLC using aluminium
sheets of Merck silica gel 60 F254, 0.2 mm thick-
ness. Melting temperatures were determined on an
electrothermal 9002 apparatus and were reported as
uncorrected. NMR spectra were recorded on a Bruker
AC-300 spectrometer at 300 MHz (1H) and 75 MHz
(13C). All chemical shifts are reported as δ values (ppm)
relative to residual non- deuterated solvent. Mass spec-
tra were obtained with ESI-TOF (LCT, Waters) using
the reflectron mode in the positive ion mode.
∗For correspondence

Marwa Zayane et al.
A mixture of compound 5 (1 mmol) and phenyl isoth-
2.2 Synthesis
iocyanate (1 mmol) in dry dioxane (15 mL) was heated
to reflux for 24 h under argon atmosphere. The crude
product thus obtained was filtered to give compound 7.
2.2a Preparation of ethyl 2 -(4-methyl-2-oxo-2H-
chromen-7-yloxy) acetate 2: Equimolar solution (15
mmol) of 7-hydroxy-4-methylcoumarin 1, anhydrous
potassium carbonate and ethyl chloroacetate was
refluxed in dry DMF (40 mL) with continuous stirring
for 5 h under argon atmosphere. After addition of ice
water, the solid formed was collected by filtration, dried
and recrystallized from ethanol to give compound 2.
2.2g Preparation of 4-methyl-7-((4-phenyl-5-thioxo-
4,5-dihydro-1H-1,2,4-triazol-3-yl) methoxy)-2H-chromen-
2-one 8: A mixture of 7 (1 mmol) and a 2% aq. solu-
tion of NaOH (6 mL) was heated to reflux for 4 h
in open air. Then, the solution was neutralized with
AcOH, extracted by AcOEt and purified by silica gel
column to finally produce compound 8.
2.2b Preparation of 2-(4-methyl-2-oxo-2H-chromen-
7-yloxy) acetic acid 3: A solution of compound 2 (15
mmol) and NaOH 5% (15 mL) was refluxed in ethanol
(30 mL) for 2 h in open air. After cooling, the mixture
was acidified with HCl (6M). The precipitate obtained
was filtered, washed several times with cold water and
recrystallized from ethanol to give the acid 3.
2.2h Preparation of 4-methyl-7-((5-(phenylamino)-
1,3,4-thiadiazol-2-yl)methoxy)-2H-chromen-2-one 9:
A solution of 7 (1 mmol) in conc. H2SO4 (4 mL) was
kept at room temperature for one day in open air. After
neutralization of the solution with diluted NH4OH,
the solid product was filtered off and dried to give
compound 9.
2.2c General procedure for preparation of esters 4:
A mixture of compound 3 (1 mmol) and the appropri-
ate alcohol (10 mL) was refluxed in the presence of
a catalytic amount of iodine (I2) for 1h 30 min under
argon atmosphere. After removal of the excess of alco-
hol, the residue was extracted with diethyl ether and
the organic phase was washed with a sodium thiosul-
fate solution (0.1 M) and distilled water. After decanta-
tion, the organic phase was dried over sodium sulfate
and concentrated. The residue obtained was purified by
silica gel column to obtain the esters 4a-f.
2.2i General procedure for preparation of compounds
10-13: Equimolar solution of compound 5 (1.2 mmol)
and diketone was refluxed in dry ethanol (15 mL) for 1h
30 min under argon atmosphere. The precipitate formed
during the reaction was filtered and then purified by
silica gel column to finally produce the compounds
10-13.
2.3 Biological activity
2.3a Antibacterial activity: The purified products
were screened for their antibacterial activity by the
agar disc diffusion method.18 NA medium cooled at
45◦C was supplemented with a bacterial suspension
(106 CFU/mL) and poured into Petri plates. After solid-
ification, sterile Whatman paper discs (diameter 6 mm)
were placed at the surface of the culture medium and 20
μL (1000 μg/mL) of the product dissolved in 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.
2.2d Preparation of 2-(4-methyl-2-Oxo-2H-chromen-
7-yloxy) acetohydrazide 5: Compound 2 (15 mmol)
was dissolved in a solution containing ethanol (30 mL)
and hydrazine hydrate (5 mL) and the mixture was left
under reflux for 2 h in open air. The precipitate formed
during the reaction was filtered hot and then recrys-
tallized in ethanol/water (90/10) to give compound
5.
2.2e General procedure for preparation of imines 6:
Equimolar solution (1.2 mmol) of 2-(4-methyl-2-Oxo-
2H-chromen-7-yloxy)acetohydrazide 5 and the aro-
matic aldehyde was refluxed in dry ethanol (15 mL)
for 1 h under argon atmosphere. The white precipitate
formed during the reaction was filtered hot and then
recrystallized in ethanol to afford compounds 6a-c.
2.3b Antifungal activity: Aspergillus flavus, Aspergillus
2.2f Preparation of 2-(2-((4-methyl-2-oxo-2H-chromen- niger and Penicillium italicum were used for the screen-
7-yl)oxy)acetyl)-N-phenylhydrazinecarbothioamide 7: ing of antifungal activity of the products tested by using

Transformation of 4-methylumbelliferone
the disc diffusion method.19 A conidial suspension of
the tested fungi was prepared (104-105 CFU/mL) and
added to PDA medium cooled at 45◦C and poured
uniformly into Petri plates (diameter 90 mm). Steril-
ized paper discs (6 mm, Whatman No. 1 filter paper)
were impregnated with 20 μL (1000 μg/mL) of the
product dissolved in DMSO and placed on the culture
plates whereas the negative control plates had no prod-
uct added to the filter paper. In the positive control
plates, discs were imbibed with the same volume of a
carbendazim suspension (0.5 mg/mL). The diameter of
the inhibition zone (mm) around the disc was measured
after incubation at 25◦C for 4 days and compared with
control. The test was performed in triplicate.
compounds exhibited the presence of new signals at
(9.6-78.9 ppm) relative to the carbons also introduced
by the used alcohol.
Mechanistically, being Lewis acid, I+ makes a strong
covalent bond with carbonyl function of acid 3 and
then, the non-isolable intermediate 3’ is subjected to
nucleophilic attack by the alcohol used on the electron-
deficient carbon, followed by an intramolecular rear-
rangement to give new coumarin esters 4a-f along with
regeneration of the catalyst (scheme 2).
Our approach to the target coumarin compounds 6-
13 was started with the synthesis of the precursor 5 via
the condensation between the coumarin ester 2 and an
excess of hydrazine hydrate under reflux of ethanol.24
The above-mentioned precursor 5 which contains the
hydrazide moiety represents a class of intermediates
3. Results and Discussion
OR'
OH
3.1 Chemistry
O
I
R
O
R
OR'
HI
According to the literature,20 the reaction of 4-
methylumbelliferone 1 with ethyl chloroacetate upon
refluxing in dry DMF using potassium carbonate
afforded the key intermediate 2 which was treated with
a solution of NaOH (5%) in ethanol and followed by
protonation,21 to give 2-(4-methyl-2-oxo-2H-chromen-
7-yloxy)-acetic acid 3. The structure of this compound
O
3''
O
4
R'
+ H2O
O
OH
R
O
H
I2
O
3'
I
I
R
OH
O
was identified by H and 13C NMR spectra. Esterifi-
cation of compound 3 with a series of alcohols using
iodine as catalyst,22,23 led to the formation of new esters
4a-f (scheme 1).
1
R
OH
I
O
O
3
O
I
The beginning of
new catalytic cycle
The structures of compounds 4a-f were character-
ized by H NMR showing new signals in the region
1
(0.85-4.97 ppm) attributed to protons introduced by the
Scheme 2. Proposed mechanism for iodine catalyzed
used alcohol. Further, the 13C NMR spectra of these esterification.
Scheme 1. Synthetic route of esters 4a-f.

Marwa Zayane et al.
which are known to be highly reactive and used for the A survey of the literature indicates that the reaction of
synthesis of heterocyclic compounds which incorporate hydrazides with isothiocyanates is a typical procedure
five- and six-membered rings containing nitrogen.25 applied for the preparation of 1,2,4-triazole-3-thiones or
The 1H NMR spectrum of compound 5 showed the dis- 1,3,4-thiadiazoles.19 The two-step reactions led initially
appearance of the signals relative to the alkoxy pro- to the thiosemicarbazide 7, which subsequently under-
tons of the ester and the appearance of new signals, goes cyclocondensation under acidic or basic reaction
attributable to the NH and NH2 protons at 9.45 and 4.38 conditions.27 Using these methods, the intermediate 7
ppm, respectively.
was converted into triazolthione 8 (35%) by heating in
Firstly, refluxing 5 with different aromatic aldehydes aqueous NaOH solution and into thiadiazole 9 (82%)
in ethanol26 was conducted until TLC indicated that the by stirring in the presence of a solution of conc. H2SO4
starting materials have been completely converted into at room temperature (scheme 4).
1
products 6a-c which were confirmed by their H and
The 1H NMR spectra of compounds 8 and 9 exhibited
a singlet relative to NH proton at 7.01 and 4.03 ppm,
13C NMR data (scheme 3).
Compounds 6a-c were established by their 1H NMR respectively, and these signals were in good agreement
spectra showing the disappearance of NH2 protons at with the expected cyclization reaction. The observation
4.38 ppm and the appearance of a new singlet at 8.50- of a new signal at 166.9 ppm in the 13C NMR spectrum
8.58 ppm attributable to the N=CH proton in addi- of compound 8 attributable to the thiocarbonyl function
tion to the signals relative to protons introduced by the added to the disappearance of those corresponding to
coumarin hydrazide 5 and the used aromatic aldehydes. the amide and thiosemicarbazide carbonyl functions in
The 13C NMR spectra of compounds 6a-c dis- the precursor 7 confirmed the identified structure.
played in particular a new signal at 144.1-146.8 ppm
The disappearance of the amide carbonyl and the
attributable to the iminic carbon CH=N in addition thiocarbonyl signals from the 13C NMR spectrum of
to the new signals at 109.2-153.3 ppm relative to the compound 9 was in concordance with the proposed
aromatic carbons introduced by the used aldehydes.
According to the literature,26,27 reaction of compound
structure.
With the aim to continue the study of the reactiv-
5 with phenylisothiocyanate in refluxing dioxane gave ity of the hydrazide 5, we reacted it with a series of
the thiosemicarbazide 7 (scheme 4). diketones (pentane-2,4-dione, hexane-2,5-dione and 1-
1
The H NMR spectrum of the compound 7 exhib- phenyl-1,3-butanedione) in refluxing ethanol according
ited essentially two new signals due to -NHCSNH- at to the literature13 to produce the corresponding pyrrole
9.69-10.33 ppm. Further, the 13C NMR spectrum of 10 and pyrazole 11-13 derivatives, respectively. Despite
this compound showed the presence of new signals the low yield (50%) of the formation of the 5-hydroxy-
corresponding to the aromatic carbons introduced by 3,5-dimethyl-4,5-dihydro-1H-pyrazole 11, we did not
the phenylisothiocyanate and a signal at 181.1 ppm notice its dehydration that normally leads to the corre-
assigned to the C=S carbon.
sponding 3,5-dimethyl-1H-pyrazol. This finding can be
Scheme 3. Synthetic route of compounds 6-7.

Transformation of 4-methylumbelliferone
Scheme 4. Synthetic route of triazolethione 8 and thiadiazole 9.
Scheme 5. Synthetic route of compounds 10-13.
ꢁ
explained by the hydrogen bond between the OH group to CH2 group (H6 ) and the disappearance of the sig-
and the carbonyl function leading to the formation of a nals related to the NH and NH2 protons at 9.45 and
stable pseudo-six-membered ring.
On the other hand, the treatment of the hydrazide same compound showed the appearance of new signals
4.38 ppm, respectively. The 13C NMR spectrum, the
ꢁ
ꢁ
5 with 1-phenyl-1,3-butanedione led to primarily the related to carbons C6 and C7 at 51.2 and 144.1 ppm,
5-hydroxy-3,5-dimethyl-4,5-dihydro-1H-pyrazole 12 to- respectively.
1
gether with its di-dehydrated analogue 13. This result
Similarly, the H NMR spectrum of compound 10
can be explained by the presence of the phenyl group showed the appearance of a singlet related to the NH
which causes a steric hindrance inhibiting the forma- proton at 8.69 ppm and the disappearance of the signal
tion of a hydrogen bond and stabilizes the molecule related to the NH2 protons at 4.38 ppm. Analysis of 13C
by the extensive conjugation with the pyrazole system NMR spectrum of this compound showed the appear-
(scheme 5).
The structure of compound 11 was evidenced via its equivalent carbons (C6 +C7 ) at 120.6 ppm.
1H NMR spectrum showing the appearance of a new
The 1H NMR spectrum of compound 12 showed the
ance of new signals attributable to the magnetically
ꢁ
ꢁ
signal attributable to the OH proton at 4.26 ppm which disappearance of the signals of the NH and NH2 pro-
provides no correlation in the CHcorr spectrum and two tons at 9.45 and 4.38 ppm, respectively and the appear-
doublets at 2.82 and 3.03 ppm (J = 18.6 Hz) related ance of a singlet assigned to the OH proton at 7.03 ppm

Marwa Zayane et al.
and two doublets in the region 3.03-3.18 ppm corre- P. savatanoi. Thus, compounds 4b (IZ=11 mm) and 4e
ꢁ
sponding to the non-equivalent methylene protons (H6 a, (IZ=13 mm) were most active against this strain. We
H6 b). The 13C NMR spectrum of this compound dis- noticed that the presence of two free hydroxyl groups in
ꢁ
played new signals at 55.2 and 143.4 ppm attributable to compound 4a has not improved this activity. The results
ꢁ
ꢁ
C6 and C7 , respectively. Compound 13 obtained after showed that only compounds 6a, 6b and 6c exhibited
1
dehydratation of pyrazole 12 was confirmed by its H activity against P. huttiensis with IZ values of 11, 9 and
NMR spectrum showing the disappearance of the sin- 8.5 mm, respectively. This finding shows that the pres-
glet relative to the OH proton at 7.03 ppm and the ence of the dimethylamino group in para position of the
appearance of a new singlet at 6.97 ppm relative to aromatic system introduced by the aldehyde used seems
13
ꢁ
H6 . Further, the C NMR spectrum of the pyrazole 13 at the origin of this relatively high activity.
showed essentially two new signals at 108.2 and 144.5
It was found that the conversion of compound 7 into
ꢁ
ꢁ
ppm attributable to the carbons C6 and C7 , respectively. triazole thione 8 and thiadiazole 9 via the thiosemicar-
The ESI-HRMS of all the synthesized compounds bazide function did not improve its activity towards P.
showed the correct protonated molecular ion peaks savatanoi (IZ=11, 9 and 9.5 mm, respectively). The
[M+H]+ which are compatible with the proposed struc- thiosemicarbazide function in compound 7 could con-
tures.
tribute to its activity. On the other hand, these com-
pounds did not show any activity against P. huttiensis
and only compound 8 exhibited a low activity towards
A. tumefasciens (IZ=7.5 mm).
3.2 Biological activity
Pyrazole and pyrrole derivatives 10-12 were found to
be active towards A. tumefasciens. The activity of com-
pounds 11 and 12 could be explained by the presence
in each one of the tertiary alcohol and of the asym-
All the synthesized compounds have been evaluated for
their antibacterial and antifungal activities activities.
3.2a Antibacterial activity: The antibacterial activ-
ity of these compounds was performed against a panel
of microorganisms: Pseudomonas savatanoi, Pseu-
domonas huttiensis and Agrobacterium tumefasciens.
The results given in table 1 shows that most compounds
have displayed acceptable antibacterial activity against
ꢁ
metric center C8 , whereas the activity of compound 10
against the same strain (IZ=9 mm) could be due to
the pyrrole and the acetamide moieties. The presence
of the pyrazole and the extent of its conjugation with
the phenyl group in compound 13 could explain the
loss in activity against the same bacterium. The activity
of compounds 12 (IZ=10.5 mm) and 13 (IZ=9.5 mm)
against P. savatanoi could be due to the presence of the
common N-2-oxoethoxy and phenyl systems (table 1).
Table 1. Antibacterial activity of the synthesized
compounds.
Inhibition zone diameter (mm)
3.2b Antifungal activity: The antifungal activity of
the synthesized compounds was tested against three
fungal strains: Aspergillus flavus,A. niger and Penicil-
lium italicum. According to the results given in table 2,
most synthesized compounds showed variable degrees
of inhibition againstA. niger. However, compounds 4a-
d showed practically the most relatively high activity
against this fungus (IZ=10-13 mm). This finding could
be explained by the nature of the alcohols used in the
preparation of these esters comparatively to 4f (IZ=9.5
mm) and 4e (not active). Similarly, the activity of com-
pounds 6a-c against the same fungus varies depending
on the aryl group introduced by each aldehyde used in
their preparation.
The results mentioned in table 2 showed that com-
pound 7 and its derivative 8 (triazole thione) display the
same activity (IZ=9.5 mm) against A. niger, whereas
compound 9 (thiadiazole) was found to be inactive.
This result could be explained by the loss of the
thiosemicarbazide skeleton in this compound but
Product
Pseudomonas Pseudomonas Agrobacterium
savatanoi
huttiensis
tumefasciens
4a
4b
4c
4d
4e
4f
6a
6b
6c
7
8
9
10
11
12
13
8.5
11
10
–
13
10
–
12
–
11
9
–
–
–
–
–
–
11
9
8.5
–
–
–
10
–
12
–
–
–
–
–
–
7.5
–
9.5
–
–
–
–
–
9
10.5
11
–
25
–
10.5
9.5
22
–
–
–
18
–
Ampicilin
DMSO
- no inhibition zone observed.

Transformation of 4-methylumbelliferone
Table 2. Antifungal activity of the synthesized
compounds.
Supplementary Information
1H and 13C NMR spectra and analytical data for all new
synthesized compounds are available at www.ias.ac.in/
chemsci.
Inhibition zone diameter (mm)
Product
Aspergillus
flavus
Aspergillus
niger
Penicillium
italicum
Acknowledgements
4a
4b
4c
4d
4e
4f
9
–
–
9
10
9
12
10
13
12
–
9.5
9
7.5
–
10
–
–
–
The authors are grateful to Miss Amna Benzarti, NMR
service at the Faculty of Monastir, University of Mona-
stir for the NMR analysis and to the Ministry of Higher
Education and Scientific Research of Tunisia for finan-
cial support.
6a
–
–
6b
6c
7
8
9
10
11
12
13
–
–
7
7.5
–
–
–
–
10.5
11
9.5
9.5
–
10
9.5
11
11
32.5
–
–
9.5
–
–
–
–
–
–
–
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