Synthesis and Biological Evaluation of New Eugenol Mannich Bases as Promising Antifungal Agents

Article abstract of DOI:10.1111/cbdd.12504

New Mannich base-type eugenol derivatives were synthesized and evaluated for their anticandidal activity using a broth microdilution assay. Among the synthesized compounds, 4-allyl-2-methoxy-6-(morpholin-4-ylmethyl) phenyl benzoate (7) and 4-{5-allyl-2-[(

Full text of DOI:10.1111/cbdd.12504

Received Date : 17-Oct-2014
Accepted Date : 15-Dec-2014
Article type : Research Article
Title: Synthesis and biological evaluation of new eugenol
Mannich bases as promising antifungal agents
Short running title: New antifungal eugenol Mannich bases
Key words: Eugenol, Mannich Bases, Antimicrobial Agents, Antifungal Action, Candida sp.
Pedro Henrique Oliveira Abrãoa, Rafael Brianti Pizia, Thiago Belarmino de Souzaa, Naiara
Chaves Silvab, Antonio Maciel Fregnana, Fernanda Negrão Silvaa, Luiz Felipe Leomil
Coelhob, Luiz Cosme Cotta Malaquiasb, Amanda Latercia Tranches Diasb, Danielle Ferreira
Diasc, Marcia Paranho Velosoa, Diogo Teixeira Carvalhoa,*
aLaboratório de Pesquisa em Química Farmacêutica, Faculdade de Ciências Farmacêuticas,
Universidade Federal de Alfenas, Rua Gabriel Monteiro da Silva, 700 Alfenas, MG, Brazil
bDepartamento de Microbiologia e Imunologia, Instituto de Ciências Biomédicas, Universidade
Federal de Alfenas, Rua Gabriel Monteiro da Silva, 700 Alfenas, Brazil
cLaboratório de Fitoquímica e Química Medicinal, Instituto de Química, Universidade Federal de
Alfenas, Rua Gabriel Monteiro da Silva, 700 Alfenas, MG, Brazil
*Corresponding author: Diogo Teixeira Carvalho.
Faculdade de Ciências Farmacêuticas,
Universidade Federal de Alfenas, Rua Gabriel Monteiro da Silva, 700, sala D-208e, Alfenas, MG,
Brazil. E-mail: diogo.carvalho@unifal-mg.edu.br. Phone number: +55 35 3299-1344
This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process
which may lead to differences between this version and the Version of Record. Please
cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12504
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Abstract
New Mannich base-type eugenol derivatives were synthesized and evaluated for
their anticandidal activity using a broth microdilution assay. Among the synthesized
compounds, 4-allyl-2-methoxy-6-(morpholin-4-ylmethyl) phenyl benzoate (7) and 4-{5-allyl-2-
[(4-chlorobenzoyl)oxy]-3-methoxybenzyl}morpholin-4-ium chloride (8) were found to be the
most effective antifungal compounds with low IC50 values, some of them well below those of
reference drug fluconazole. The most significant IC50 values were those of 7 against C.
glabrata (1.23 μM), C. albicans and C. krusei (both 0.63 μM). Additionally, the synthesized
compounds were evaluated for their in vitro cytotoxic effects on human mononuclear cells.
As result, the cytotoxic activity of eugenol in eukaryotic cells decreased with the introduction
of the morpholinyl group. Given these findings, we point out compounds 7 and 8 as the most
promising derivatives since they showed potency values greater than those for eugenol and
fluconazole and they also presented high selectivity indexes.
Introduction
In recent years, a significant increase in the incidence of opportunistic fungal
infections have been observed, especially among immunocompromised patients, especially
those undergoing chemotherapy, long treatment with antimicrobial agents and also patients
with immunosupressed conditions.The high toxicity, low bioavailability, the development of
resistance by many microorganisms and late diagnosis are the main causes of treatment
failure in these patients (1,2). In intensive care units around the world much effort has been
invested to reduce and control nosocomial and community-acquired infections (3). Although
a relatively small number of Candida species are pathogenic for humans, candidiasis are
considered the main causes of hospital infection, mainly due to modern medical procedures
specially the use of toxic and immunosuppressive drugs that can suppress the immune
system and alter the normal bacterial flora. These events can facilitate the development of
both skin infections and serious systemic diseases (4-6).
Eugenol is a natural allyl phenol found in the essential oil of cloves (Syzygium
aromaticum), sassafras (Ocotea odorifera) e cinnamon (Cinnamomum zeylanicum) and has
attracted attention of a lot of researchers, either because of its wide range of biological
activities, either by chemical versatility of its structure (7). Among the biological activities
already described for eugenol one can cite analgesic and anti-inflammatory (8), anesthetic
(9), hypotensive (10), antioxidant (11), antitumoral (12), anti-parasitic (13), and antifungal
action (14).
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On the other hand, compounds known as Mannich bases were found to possess
biological activities. They come from Mannich reaction,
potent
a
condensation/aminomethylation involving a substrate containing an active hydrogen (as alkyl
ketones, phenols, N-H heterocycles, aldehydes, etc), a primary or secondary amine and
formaldehyde (or, occasionally, other aldehydes), resulting in the creation of the Mannich
bases (15, 16). These compounds are biologically active due to their basic character,
allowing its protonation under physiological conditions and favoring interactions between
ligand and receptor, besides improving its water solubility (17). Literature survey reveals that
Mannich bases are known for its several activities, among them anticancer (18), anti-
inflammatory (19), antimalarial (20), antiviral (21), antitubercular (22) and antimicrobial,
especially antifungal (23, 24, 25, 26, 27, 28, 29). It is also worth noting that piperazine or
morpholine bioisosteric rings play a crucial role in antimicrobial activity, as in drugs like
linezolid, eperezolid and itraconazole (23). The results presented in these studies indicate
that the Mannich base subunit seems to be directly involved in the antimicrobial good
results.
Our research group recently reported the anti-Candida activity of a glucoside
derivative of eugenol (14). Following our interest in eugenol derivatives and in view of the
potential of Mannich bases as antimicrobials we now devoted our attention to exploring the
antifungal activity of some Mannich bases of this natural product. So, we describe herein for
the first time, the synthesis and anti-Candida sp. activity of a morpholine-based Mannich
base of eugenol and of the esters thereof.
Results and discussion
Chemistry
Preparation of the target compounds is shown in Scheme 1. The esters 3-5, known
eugenol derivatives, were obtained as described according to literature with minor
adaptations (30, 31) and were synthesized to assess the influence of the Mannich base
subunit on anticandidal action of derivatives 2 and 6-8.
Eugenol Mannich base 2 was synthesized as previously reported (32) with some
modifications which enhanced product yield and facilitated its isolation. Its identity was
1
checked by IR, NMR and mass analysis. The H-NMR peaks at δ 3.73-3.72 and at δ 3.65
correspond respectively to the morpholinyl ring eight protons and to those protons of
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methylene group which bridges both rings. The derivatives 6-8 are new compounds and
were obtained in moderate yields by reaction of 2 with respective anhydrides or acyl chloride
using 4-dimethylaminopyridine as a catalyst. We were able to easily obtain these O-acylated
derivatives using an acyl chloride or anhydride, in spite of what reported Mazzei et al (20)
who achieved only methylene bis-derivatives when they treated Mannich bases of
hydroxycoumarin with anhydrides. The esters 7 and 8 were isolated as their hydrochloride
salts. NMR spectra showed clearly the expected peaks related to each inserted O-acyl
chain, despite the other peaks related to the rest of the structure. High resolution mass
spectrometry analysis showed the expected mass values for compounds 2 and 6-8. All of
the new compounds were isolated as crystalline solids after acid-base extraction or by
grinding raw product with light petroleum or by recrystallization from ethanol.
Octanol/water partition coefficients (LogPo/w) were calculated using QikProp program
(33) for eugenol and its derivatives. Morfolinyl group was clearly able to reduce the logP
values of derivatives 2 and 6-8 in relation to those of eugenol and simple esters, as shown in
Table 1. This finding may be important for the solubility profile of the derivatives during
biological evaluation and probably directly affects the antimicrobial potential of these
compounds.
Scheme 1: The synthesis of derivatives 2-8: a)morpholin, formaldehyde, toluene, 95°C. b)Ac2O
(derivatives 3 and 6) or Bz2O (derivatives 4 and 7) or 4-ClC6H4COCl (derivatives 5 and 8), DMAP,
CH2Cl2, r.t.
Antifungal and cytotoxic activity
Antimicrobial activity of phenolic components as eugenol against Candida spp. have
been reported in some studies with good antifungal potential (34, 35). Studies that evaluate
the antifungal activity of eugenol and its analogues have led to derivatives with moderate
activity against Candida species (36, 37) and indicated that the structural modification of
eugenol can generate optimized compounds. In this direction, supported by the biological
potential of eugenol and the reports of Mannich bases with antimicrobial action, we proposed
to obtain new derivatives of eugenol endowed with antifungal potential by associating the
morpholinyl group to it. In addition to that, we decided to explore the effect of having the
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phenolic hydroxyl group of these products free or masked as esters of different polarities and
volumes.
Most of the derivatives synthesized from eugenol showed significant antifungal
activities when evaluated against ATCC species of Candida spp., while the action of eugenol
was lower than that of the standard drug fluconazole on these same species (Table 1). The
Mannich bases 2, 7 and 8 showed IC50 values lower than those observed for fluconazole
against most of evaluated Candida species. Some of the derivatives exhibited values of
minimum inhibitory concentrations well below in relation to eugenol when tested against
species of non-albicans Candida. Derivatives 7 and 8 were respectively 166 and 90 times
more potent against C. krusei than that fungistatic drug. This is quite important when one
considers naturally fluconazole-resistant species as C. krusei. The addition of the
morpholinyl group seemed to be responsible for a significant impact on action against these
yeasts, since derivatives 2, 7 and 8 were all superior in activity compared to eugenol.
Derivatives 7 and 8 showed IC50 values generally quite small, like those in the range of 10.2
to 0.57 μM. The only exception was the derivative 4 which does not possess the morpholinyl
subunit but showed interesting activity against some species, but not as good as those of its
morpholinyl analogue.
It is interesting to note that while the 4-chlorobenzoic ester of eugenol (5) was not
active up to the highest concentration evaluated, its morphlolinyl analogue (8) reached IC50
values substantially lower than those of eugenol and fluconazole. Lal et al (38) reported that
similar modification was able to lead to a significant increase in the action of the antifungal
mulundocandin. This may be due to a balance between high lipophilicity and ionization
potencial for this compound which is important for this biological activity.
Table 1: In vitro antifungal activity (IC50, μM) and LogPo/w for eugenol (1) and its derivatives
(2-8)
table 1 and below its footnotes
a Calculated Log P o/w values using QikProp program
b Fluconazol
c Not active up to 100 μg.mL-1
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The cytotoxic activities of the most active compounds (2, 4, 7 and 8) were tested in
peripheral human blood mononuclear cells obtained from healthy donors and the results are
shown in Table 2. As can be seen most of eugenol derivatives showed higher CC50 values
than this parent compound.’
Mannich base derivative 2 was safer than eugenol and exhibited selective toxicity
towards all evaluated yeasts. Derivative 7 was 200-400 times less cytotoxic than eugenol
while its 4-chlorobenzoyl analogue 8 proved to be 1.4 to 316 times safer. These data
indicate that modifications done on the prototype have the potential to reduce the cytotoxic
activity of eugenol in eukaryotic cells and that the morpholinyl group may be involved in this.
Moreover, these observations corroborate that the anticandidal activities found may be due
to specific interactions in fungal cells, which is important in new antifungal drugs
development.
Table 2: Cytotoxic activity (CC50, μM) and selectivity index for antifungal compounds 2, 4, 7
and 8 on test microorganisms
table 2 and below its footnotes
a Not determined
b:The selective index was expressed as the ratio CC50/IC50.
Nosocomial infections can affect as many as 30% of patients admitted to an intensive
care unit and knowledge of the causative pathogen of infection is crucial in initiating and
managing the correct antimicrobial treatment (39). It is known that Candida spp. are among
the leading causes of nosocomial blood infections in world and the choice of studying
antifungal activity against Candida spp. was due to its great importance in the epidemiology
of fungal infections (40). It was observed a progressive increase in infections caused by non-
albicans Candida spp. and C. parapsilosis was the most frequent species, followed by C.
tropicalis and C. glabrata (41). It is suggested that the rise in the incidence of these
infections is associated with antimicrobial resistance and the restricted number of available
antifungal drugs (42). Observations like this support and confirm the importance of studying
new alternatives or complementary therapies to combat candidiasis.
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Conclusion
We reported herein the synthesis and antifungal evaluation of a morpholine-based
Mannich base of eugenol and the esters thereof. Results reveal clearly that the Mannich
base derivatives showed to be promising antifungal agents against Candida spp. since most
of them were more potent than fluconazole. Derivatives 7 and 8 are the most expressive
compounds for further optimization since they showed potency values generally greater than
those for eugenol and fluconazole, while they also presented high selectivity indexes.
Experimental
Chemistry
Thin-layer chromatography (TLC) on silica gel-G TLC plates (Merck) were used to
monitor reactions course. Melting points of the compounds were obtained on Microquímica
MOAs 301 melting-point apparatus and are uncorrected. IR spectra were recorded on
Shimadzu a FTIR-Affinity-1 spectrometer. NMR spectra were recorded on a Bruker AC-300
1
spectrometer (300 MHz for H-NMR and 75 MHz for 13C-NMR spectra). Chemical shifts are
expressed as values relative to TMS as internal standard. High resolution mass spectra
were acquired using a LCMS-IT-TOF mass spectrometer and the samples were solubilized
in MeOH + 0.1% formic acid, following manual injection.
Synthesis of 4-allyl-2-methoxy-6-(morpholin-4-ylmethyl)phenol (2)
A solution of eugenol (10mL, 65.16mmol), morpholin (11.24mL; 122mmol) and
formaldehyde (9.7mL; 49.8mmol) was stirred in toluene (100mL) at 95°C during 24h. After
the completion of the reaction, noticed by TLC (hexane/ethyl acetate 3.5:6.5 v/v), the mixture
was extracted with 1 mol.L-1 HCl (3x30mL). The pH of this aqueous phase was then raised
to 6 by addition of saturated NaHCO3 solution and then it was extracted with
dichloromethane (5x30mL). The organic layer was dried over anhydrous Na2SO4 and the
solvent removed under reduced pressure to give the product 2 (7.64g, 45%) as a white solid,
m.p. 48-49°C. IR (cm-1) v 2970, 2816, 1595, 1494, 1458, 1234. 1H-NMR (CDCl3, 300 MHz) δ
6.62 (s, 1H), 6.41 (s, 1H), 5.98-5.84 (m, 1H), 5.07-5.00 (m, 2H), 3.83 (s, 3H), 3.73-3.72 (m,
4H), 3.65 (s,2H), 3.25 (d, J = 6.7 Hz, 2H), 2.55 (m, 4H). 13C-NMR (CDCl3, 75 MHz) δ 147.6,
144.7, 137.6, 130.5, 120.4, 115.4, 111.4, 66.6, 61.4, 55.7, 52.7, 39.6. HRMS-ESI: m/z calcd.
for C15H21NO3 (M+H)+ 264.1594. Found 264.1487.
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Synthesis of derivatives 3-5
These compounds were synthesized as previously reported and their identities were
confirmed by comparing their infrared and nuclear magnetic resonance spectra to those in
the literature (30, 31).
General procedure for the synthesis of derivatives 6-8
To a solution of 2 (0.5g, 1.9mmol) in dichloromethane (25mL), the appropriate
acylating agent (2.28mmol) and 4-(dimethylamino)pyridine (0,23mmol) were added. The
reaction mixture was stirred at room temperature for a period of 1,5-48h and the progress of
the reaction was checked by TLC (hexane/ethyl acetate 3.5:6.5 v/v). After its completion ice
cold water was added and the organic phase was washed with 0.5 mol.L-1 NaOH (6x10mL)
followed by water until pH 7. The organic phase was then dried over anhydrous Na2SO4 and
the solvent removed under reduced pressure which led to the directly to the desired products
or after crystallization procedures.
4-allyl-2-methoxy-6-(morpholin-4-ylmethyl)phenyl acetate (6)
From acetic anhydride. White solid; yield: 56%; mp 64-65°C; IR (cm-1) v 3003, 2970,
2870, 1761, 1597, 1490, 1197, 1112. 1H-NMR (CDCl3, 300 MHz) δ 6.78 (s, 1H), 6.72 (s, 1H),
6.02-5.88 (m, 1H), 5.13-5.08 (m, 2H), 3.80 (s, 3H), 3.68-3.65 (m, 4H), 3.40-3.35 (m, 4H),
2.41 (s, 4H), 2.30 (s, 3H). 13C-NMR (CDCl3, 75 MHz) δ 168.4, 151.0, 138.0, 136.9, 136.7,
122.2, 115.9, 111.4, 66.6, 57.8, 55.6, 53.2, 39.8, 20.4. HRMS-ESI: m/z calcd. for C17H23NO4
(M+H)+ 306.1700. Found 306.1599.
4-allyl-2-methoxy-6-(morpholin-4-ylmethyl)phenyl benzoate (7)
From benzoic anhydride. White solid after treatment with light petroleum; yield: 49%;
1
mp 84-85°C; IR (cm-1) v 3074, 2976, 2864, 1732, 1602, 1494, 1460, 1269, 1109. H-NMR
(CDCl3, 300 MHz) δ 8.23-8.20 (m, 2H), 7.64-7.60 (m, 1H), 7.53-7.48 (m, 2H), 6.82-6.78 (m,
2H), 6.05-5.91 (m, 1H), 5.15-5.08 (m, 2H), 3.80 (s, 3H), 3.48-3.38 (m, 6H), 2.34 (s, 4H). 13C-
NMR (CDCl3, 75 MHz) δ 164.3, 151.6, 138.3, 137.4, 137.0, 133.2, 130.1, 129.8, 128.4,
122.5, 116.1, 111.9, 66.5, 58.2, 22.9, 53.2, 40.1. HRMS-ESI: m/z calcd. for C22H25NO4
(M+H)+ 368.1856. Found 368.1803.
4-{5-allyl-2-[(4-chlorobenzoyl)oxy]-3-methoxybenzyl}morpholin-4-ium chloride (8)
From 4-chlorobenzoyl chloride. White solid after recrystallization from ethanol. yield:
1
70%; mp 200-201°C; IR (cm-1) v 3014, 2980, 2362, 1749, 1597, 1494, 1261, 1124. H-NMR
(CDCl3, 300 MHz) δ 13.19 (s, 1H), 8.12-8.07 (m, 2H), 7.54-7.47 (m, 3H), 6.89 (s, 1H), 6.04-
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5.90 (m, 2H), 5.17-5.10 (m, 2H), 4.32-4.24 (m, 2H), 4.12 (d, J = 4.6 Hz, 2H), 3.89 (dd, J2 =
13 Hz, J3 = 3.2 Hz, 2H), 3.44 (d, J = 6 Hz, 2H), 3.32 (d, J = 13 Hz, 2H), 2.89-2.76 (m, 2H).
13C-NMR (CDCl3, 75 MHz) δ 163.5, 151.1, 140.9, 140.3, 137.9, 136.1, 131.6, 129.2, 126.5,
124.3, 120.9, 116.9, 114.4, 63.5, 56.0, 54.2, 51.2, 39.8. HRMS-ESI: m/z calcd. for
C22H25Cl2NO4 (M-Cl)+ 402.1467 Found 402.1257.
Antifungal activity evaluation
Eugenol 1 and its derivatives 2-8 were evaluated in vitro for their activities against the
fungi through a Mueller Hinton broth microdilution method and with the methodology and
interpretative criteria proposed by document M27A3 (43). The stock solutions of all the
compounds were prepared in DMSO 1% (v/v) at final concentration and tested at the
following concentrations (µg.mL-1): 100; 62.5; 31.2; 15.6; 7.8; 3.9; 1.95; 0.48; 0.24 and 0.06.
The standard drug fluconazole was applied as control of fungistatic action at the following
concentration (µg/mL): 64; 32; 16; 8; 4; 2; 1; 0.5; 0.25; 0.125; 0.0625 and 0.03125. The
microplates were incubated at 37oC for 24h. Results were visualized and analyzed by
spectrophotometry. The inhibitory concentration of microbial growth was determined at 50%
(IC50) in µM and compared for each compound and microorganism. The tests were all done
in duplicates and the results obtained from the replicas were coincident.
Cytotoxic activity evaluation
The cytotoxicity to peripheral human blood mononuclear cells (PBMCs) of eugenol
and their derivatives 2, 4, 7 and 8 (concentration range 100 to 0.78 μg/mL) was determined
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method (44).
The PBMCs were obtained from healthy volunteers by Ficoll-Hypaque density gradient
centrifugation. The cell suspension of PBMCs at a concentration of 2.4 × 106 cells/mL was
distributed in a 96-well plate, 90 μL in each well with 10 μL of test compounds at different
concentrations, incubated at 37 °C in an incubator at 5% CO2 for 48 h. After, it was added
10 μL of MTT dye (5 mg.mL-1) and the cells were incubated again for an additional 4 hours
period. Then, the medium was carefully removed and added to 100 μL of DMSO for
solubilization of formazan crystals. The plates were shaken for 5 min and absorbance for
each sample was measured in a spectrophotometric microplate reader at 560 nm. The
percentage of cytotoxicity was calculated as [(A-B)/Ax100)], where A and B are the
absorbances of control and treated cells, respectively. Data were analyzed using linear
regression to obtain values for CC50 (cytotoxic concentration for 50%). Selectivity indexes
were expressed as the ratio CC50/IC50.
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Acknowledgements
This work was supported by grants from Fapemig, Brazil (APQ-01641-12). The authors are
also grateful to Rede Mineira de Química, Brazil.
Conflict of interest
The authors declare no conflict of interest.
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a
Compound LogPo/w
Microrganism
C. albicans
C. tropicalis
C. krusei
C. parapsilosis
C. glabrata
ATCC10231
ATCC750
ATCC 6258
ATCC 22019
ATCC 90030
1
2.675
2.349
2.621
3.938
4.579
2.075
3.208
3.717
0.450
365.80
0.88
609.8
380.0
485.3
86.0
182.9
28.5
182.9
28.5
609.8
0.88
2
3
291.0
0.86
145.6
1.75
145.6
14.0
485.3
14.0
4
c
c
c
c
c
5
6
-
-
-
-
-
c
c
c
c
983.0
0.63
74.7
3.27
-
-
-
-
c
7
-
0.63
1.17
10.2
74.7
13.1
1.23
4.67
13.1
8
0.57
3.27
FLCb
104.5
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Compound
CC50
Selectivity indexb
C. albicans
C. tropicalis
C. krusei
C. parapsilosis
C. glabrata
ATCC 10231
ATCC 750
ATCC 6258
ATCC 22019
ATCC 90030
1
2
4
7
8
549.13
1030.80
459.28
378.50
161.85
1.5
1170.0
534.0
600.8
2.17
0.9
2.7
5.3
3.0
3.0
36.0
32.8
37.1
2.2
0.9
1170.0
32.8
36.0
262.4
600.8
138.3
a
-
307.7
34.7
283.9
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