Synthesis and studies of the antifungal activity of 2-anilino-/2,3-dianilino-/2-phenoxy- and 2,3-diphenoxy-1,4-naphthoquinones

Article abstract of DOI:10.1007/s11164-016-2732-3

Several synthetic and natural naphthoquinone derivatives have been associated with antifungal activity. Candida albicans is a fungus that is known to exist in the normal human flora, but under certain conditions it can cause mild to fatal infections. Its pathogenicity has been associated with fungus conversion from cellular yeast to filamentous form Y–M. Inhibition of this process by several anilino-, dianilino-, phenoxy-, and diphenoxy-1,4-naphthoquinones was investigated in order to find some correlation between structure, redox properties and biological activity.

Full text of DOI:10.1007/s11164-016-2732-3

Res Chem Intermed
DOI 10.1007/s11164-016-2732-3
Synthesis and studies of the antifungal activity of 2-
anilino-/2,3-dianilino-/2-phenoxy- and 2,3-diphenoxy-
1,4-naphthoquinones
Elisa Leyva1
Claudia G. Espinosa-Gonzalez
2
1
•
•
•
´ ´
´
Lluvia I. Lopez Ramon F. Garcıa de la Cruz
1
´
Received: 7 May 2016 / Accepted: 6 September 2016
Ó Springer Science+Business Media Dordrecht 2016
Abstract Several synthetic and natural naphthoquinone derivatives have been
associated with antifungal activity. Candida albicans is a fungus that is known to
exist in the normal human flora, but under certain conditions it can cause mild to
fatal infections. Its pathogenicity has been associated with fungus conversion from
cellular yeast to filamentous form Y–M. Inhibition of this process by several ani-
lino-, dianilino-, phenoxy-, and diphenoxy-1,4-naphthoquinones was investigated in
order to find some correlation between structure, redox properties and biological
activity.
Keywords 1,4-Naphthoquinone derivatives Á Candida albicans Á
Antifungal compounds Á Reduction potential
Introduction
Several synthetic and natural organic compounds containing a quinone moiety in
their structure have been associated with different biological activities. In some
cases, the biological activity of quinones has been related to their redox properties
and their capacity to accept one or two electrons to form the corresponding radical
anion (QÁÀ) and hydroquinone radical dianion (Q2-). These intermediate species
interact with crucial cellular molecules such as oxygen, DNA and proteins,
modifying their biological properties [1–3]. Therefore, studies on the synthesis,
&
Elisa Leyva
elisa@uaslp.mx
1
2
´
Facultad de Ciencias Quımicas, Universidad Autonoma de San Luis Potosı, Av. Manuel Nava
No. 6, Zona Universitaria, C.P. 78210, San Luis Potosı, S.L.P, Mexico
´
´
´
´
Facultad de Ciencias Quımicas, Universidad Autonoma de Coahuila, Blvd. V. Carranza e Ing.
Jose Cardenas s/n, Col Republica, C.P. 25250, Saltillo, Coahuila, Mexico
´
´
´
´
123

E. Leyva et al.
structure and physicochemical properties of novel quinone compounds with more
selective biological activity are basic for future medicinal applications.
1,4-Naphthoquinones, possessing a substituent like an anilino, phenoxy or
arylthio group in the two position, have been the subject of intensive research. Many
of them find use in a variety of medicinal and biological applications, such as
antituberculars [4], antimalarials [5, 6], antibacterials [7, 8], antitumor agents
[9, 10], larvicides, herbicides and fungicides [11–14]. It has been indicated that the
presence of a nitrogen, sulphur or oxygen atom allows modulation of the substituent
effects on the electronic properties of the quinone system.
Candida albicans is a dimorphic fungus that is known to exist in the normal
human flora [15]. However, it could cause mild to fatal opportunistic infections and
it is particularly dangerous in people with a compromised immune system such as
patients with cancer or AIDS [16, 17]. It is the most common fungal pathogen
associated with cancer. In general, the term candidiasis involves a wide range of
infections that range from minor vaginitis [18, 19] to systemic and quite life-
threatening diseases. There are three major forms of the disease: genital, oral, and
invasive or deep candidiasis. In this latter form, the fungus invades internal organs
and it is associated with a mortality rate of 40–60 % [20–24].
Its pathogenicity has been associated with fungus conversion from cellular yeast
to filamentous form Y–M [25]. Patients with severe candidiasis generally have
filaments of C. albicans penetrating the infected tissue [26, 27]. Filamentous
penetration through the gastrointestinal tract is most likely the origin of the majority
of deep or invasive candidiasis. In the laboratory, C. albicans can be converted from
the cellular yeast to the filamentous form Y–M under certain conditions. Therefore,
this in vitro experiment could provide a convenient methodology to test potential
compounds aimed to inhibit this highly pathogenic conversion [28–32]. Since there
are only a couple of reports on this type of study, we investigated the activity of
several anilino and phenoxy derivatives of 1,4-naphthoquinone (Scheme 1) to
inhibit C. albicans conversion to Y–M.
Results and discussion
In a general method to prepare 2-anilino-1,4-naphthoquinone, a substituted aniline
is added, in a 1,4-type manner, to the naphthoquinone structure [33, 34]. However,
this methodology is unpractical and requires tedious chromatographic purifications
since it gives low yields with aromatic amines containing electron-withdrawing
groups [35]. There are some reports on the use of Lewis acids to catalyze this
reaction [36, 37]. In recent studies, we reported the preparation of novel
2-fluoroanilino and 2-nitroanilino derivatives of 1,4-naphthoquinones in the
presence of a Lewis acid and strong oxidation agent such as CeCl3 or FeCl3
[38–41]. The catalyst facilitates an oxidative coupling between a given aniline and
naphthoquinone and, as a consequence, the reaction is cleaner. Several 2-anilino-
1,4-naphthoquinones 3a–k were prepared (Scheme 2) following the procedure
previously reported by us [38–41]. An ethanolic solution of 1,4-naphthoquinone and
a catalyst (CeCl3 or FeCl3) was prepared and stirred to activate the quinone. Then,
123

Synthesis and studies of the antifungal activity of…
Scheme 1 Structure of 1,4-naphthoquinone derivatives
an ethanolic solution of aniline was added and the reaction mixture was refluxed for
several hours. The solid obtained was filtrated, washed with cold ethanol and
recrystallized.
A number of 5-hydroxy-1,4-naphthoquinones display different biological prop-
erties including antimalarial, antibacterial, and antifungal activity [11]. Therefore,
we also synthesized a series of 5-hydroxy-2-(anilino)-1,4-naphthoquinones 4a–
e following the same procedure (Scheme 2) previously described [38, 39]. In this
case, the 5-hydroxy substituent also favors the formation of one adduct with only
trace amounts of other secondary products.
Another method to prepare 2-anilino-1,4-naphthoquinones consists of the
nucleophilic displacement of an already halogenated 1,4-naphthoquinone. We
performed the preparation of 2-chloro-3-(anilino)-1,4-naphthoquinones 6a–6d
(Scheme 2) following the procedure reported in the literature [42] with some
modifications [43]. 2,3-Dichloro-1,4-naphthoquinone 5 (1 mol) was dissolved in
methanol and a large excess of aniline (2–4 mol) was added. The solution was
refluxed for several hours (2–10) to obtain a solid crystalline product.
Dianilino derivatives 8a–c were prepared in two steps (Scheme 2). 2-Anilino-3-
chloro-1,4-naphthoquinone 6a was nitrated with a mixture of concentrated acids
(HNO3 and H2SO4) to give the 2-chloro-3-(2,4-dinitroanilino)-1,4-naphthoquinone
7 [44]. To achieve the reaction of this latter quinone (1 mol) with aniline,
4-methoxy or 4-fluoroaniline, an excess of the amine (15 mol) was required with a
reflux in methanol for several hours (10–15) [45].
123

E. Leyva et al.
Scheme 2 Synthesis of 1,4-naphthoquinone derivatives
Since the presence of chlorine atoms and phenoxyl groups on the structure of
quinone has been demonstrated to improve antifungal activity [11], some
chlorophenoxy-naphthoquinone derivatives 10a–b and 11a–b (Scheme 2) were
prepared following the literature procedure with some modifications [46]. 2,3-
Dichloro-1,4-naphthoquinone was added to a mixture of a substituted phenol and
sodium carbonate in a minimal amount of DMSO. The reaction mixture was stirred
at room temperature (RT). The pure products were separated by column
chromatography.
Biological activity
Several naphthoquinone derivatives have shown potent antifungal activity against
pathogenic fungi [11]. However, there are no previous reports on their activity to
123

Synthesis and studies of the antifungal activity of…
inhibit morphological transition of C. albicans from yeast to mycelium Y–M [47].
The minimum concentration of 1,4-naphthoquinone derivatives required to inhibit
this Y–M conversion was determined by microscopic observation. C. albicans cells
were incubated in Lee’s medium [48] at regular human temperature (37 °C). At the
initial time (Fig. 1a), C. albicans cells showed an ovoid structure (5–7 lm in
diameter). After 30–45 min of incubation (Fig. 1b), blastospores showed the first
emission growth, that eventually gave rise to germ tube formation (Fig. 1c). Germ
tube growth was characterized by the appearance of septa and a continued
elongation (Fig. 1d) with a length 5–6 times the diameter of a blastospore at 3 h of
incubation (Fig. 1e).
The 1,4-naphthoquinone derivatives studied 3a–11b presented different degrees
of inhibition activity against Y–M conversion (Table 1). A series of 2-anilino
compounds 3a–3k were tested. For some of the compounds, having a substituent in
an ortho or para position (either electron donor or electron acceptor) had a very
minor or no effect on the inhibition since the minimum inhibitory concentration
(MIC) values were similar to the ones obtained with unsubstituted aniline 3a.
Exceptions are compounds 3b and 3j that have a fluorine atom in the ortho position
and have MIC values of 31.25 and 1.25, respectively.
These results on biological activity are quite interesting since they present some
correlation with our recent studies on spectral and electrochemical characterization of
2-(fluoroanilino)-1,4-naphthoquinones [38, 39]. UV–Vis spectral analysis of several
derivatives indicated the existence of an intramolecular electronic transfer from the
fluoro substituent on aniline to the 1,4-naphthoquinone moiety. In agreement with this
donor–acceptor character, the cyclic voltammograms gave two one-electron reduction
II
waves to the corresponding anion (EI1=2) and dianion (E ) intermediates.
1=2
In general, fluoro substituents reduced the electron density of the electroactive
naphthoquinone and facilitated the reduction process. In halogenated compounds,
Fig. 1 Candida albicans mycelia growth under microscopic observation (magnification 940).
Conditions 37 °C and 200 rpm shaking in Lee’s medium. a Initial time, b 30–45 min, c 1 h, d 2 h
and e 3 h of incubation
123

E. Leyva et al.
Table 1 Structures and inhibition of C. albicans Y–M conversion for 1,4-naphthoquinone derivatives
Compound
R2
R4
R5
R3
R5
MICa (lg/mL)
0
0
R3
0
0
0
R6
C. albicansb
3a
3b
3c
3d
3e
3f
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
500.00
31.25
500.00
500.00
nac
H
H
Br
H
H
CH3O
NO2
H
H
H
H
H
H
H
250.00
250.00
500.00
500.00
1.95
3g
3h
3i
H
Br
H
H
H
CH3O
H
H
H
NO2
F
H
H
3j
F
H
H
3k
4a
4b
4c
4d
4e
6a
6b
6c
6d
7
F
F
H
H
125.00
31.25
7.80
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
H
F
H
H
Br
H
H
H
250.00
125.00
62.50
62.50
15.62
250.00
125.00
125.00
31.25
31.25
62.50
3.90
F
H
F
F
H
H
H
Cl
Cl
Cl
Cl
Cl
R3d
R3d
R3d
Cl
Cl
R3e
R3f
F
H
H
H
F
H
F
F
H
NO2
H
NO2
H
H
8a
8b
8c
10a
10b
11a
11b
a
H
H
F
H
H
CH3O
H
H
Cl
H
H
CH3O
H
H
250.00
1.95
Cl
Cl
H
Cl
H
0.97
The MIC value was defined as the lowest concentration of the naphthoquinone derivative required to
inhibit the conversion of yeast to mycelium Y–M of Candida albicans
b
Fungi tested: Candida albicans ATCC 26555
c
na: No activity
d
R3: 2,4-dinitroanilino
e
R3: 2-chlorophenoxy
f
R3: 2,4-dichlorophenoxy
this effect is modulated by simultaneous electron attracting and donor properties of
the substituents [49]. Thus, strong electron-withdrawing substituents like fluoro and
chloro displaced the redox potentials to less negative values and made the reduction
of a substituted quinone easier than that of 1,4-naphthoquinone. Furthermore, a
slightly larger effect is observed when an ortho-fluoro substituent is present in the
123

Synthesis and studies of the antifungal activity of…
aniline due to an intramolecular bifurcated or three-center hydrogen bond
(Scheme 3) [50]. Therefore, the biological activity of 2-(fluoroanilino)- and
2-(2,4-difluoroanilino)-1,4-naphthoquinones could be related to their redox prop-
erties (Table 2) and the presence of an intermolecular hydrogen bond. These
physicochemical properties enhance their capacity to form intermediate species
(radical anion or dianion) that easily interact with different cellular molecules such
as oxygen, DNA and proteins.
It has been reported that the presence of a hydroxyl group adjacent to the
carbonyl group on 1,4-naphthoquinone affects its biological properties [51]. In
general, 5-hydroxy-1,4-naphthoquinone derivatives 4a–d showed improved activity
as inhibitors of Y–M transition in comparison with non-hydroxylated analogues 3a–
c and 3f. As an example, 4b gave a smaller MIC (7.80 lg/mL) relative to the one
observed (31.25 lg/mL) for 3b. Once again, an intramolecular hydrogen bond
between the phenolic hydrogen and the quinone oxygen might explain some of these
findings. The introduction of a hydroxyl group at the C5 position of the 1,4-
naphthoquinone structure increases its reduction potential by means of an
intramolecular hydrogen bond that favors the formation of an anion or dianion
[51]. This physicochemical effect might be responsible for the observed enhance-
ment in biological activity.
In order to determine the effect of a chlorine atom in the inhibition of Y–
M transition, several 2-(anilino)-3-chloro-1,4-naphthoquinone derivatives were
prepared 6a–d. In compound 6a, the simple substitution of a hydrogen by a chlorine
atom on 1,4-naphthoquinone ring, induced the activity to increase (MIC of
62.50 lg/mL) relative to compound 3a (MIC of 500.00 lg/mL). Furthermore, an
increased in the activity by 50 % was observed for ortho-fluoro derivative 3b with
an MIC of 31.25 lg/mL upon addition of a chlorine atom to give 6b with an MIC of
15.62 lg/mL. However, the addition of a chlorine atom on 3j derivative reversed
the biological activity for this compound, increasing the MIC from 1.95 lg/mL with
3j to 125.00 lg/mL with 6d. This latter observation indicates that the introduction
of an electron-withdrawing chloro substituent at the quinone does not improve
biological activity.
In previous reports, 2,3-disubstituted-1,4-naphthoquinone with arylthio and
arylamino substituents demonstrated some antifungal activity on diverse pathogenic
fungi [11–14]. In this instance, 2-(anilino)-3-(2,4-dinitroanilino)-1,4-naphtho-
quinone derivatives 8a–c were prepared and their inhibitory activity on germ tube
formation Y–M of C. albicans was evaluated. The presence of an additional
Scheme 3 Structure of 2-(2-
fluoroanilino)-1,4-
naphthoquinone showing the
effect of fluorine through a
bifurcated or three-center
hydrogen bond
123

E. Leyva et al.
Table 2 Correlation of electrochemical parameters of 1,4-naphthoquinone derivatives with inhibition of
Y–M Candida albicans transition
Compound
Substituent
Activity MICd
cEII
‘
(lg/mL)
3h
3a
3g
3b
3j
a
4-CH3O
H
500.00
500.00
250.00
31.25
1.95
-1809b
-1685b
-1642b
-1636a
-1636a
4-Br
2-F
2,4-F
Determined by cyclic voltammetry at 100 mV/s [38]
b
c
d
Determined by cyclic voltammetry at 100 mV/s [49]
EII is the second wave reduction potential
‘
The MIC value was defined as the lowest concentration of naphthoquinone derivative required to
inhibit the conversion of yeast to mycelium Y–M of Candida albicans
nitroaniline moiety on the 1,4-naphthoquinone in compound 8a gave an enhanced
activity (MIC of 31.25 lg/mL), relative to 2-chloro-3-(2,4-dinitroanilino)-1,4-
naphthoquinone 7 (MIC of 125.00 lg/mL). Nitro groups are known to have strong
electron-withdrawing properties due to inductive and resonance effects and favor
reduction of NQ [49].
2-Chloro-3-phenoxy-1,4-naphthoquinone derivatives have been used as synthetic
intermediates for heterocyclic compounds with promising biological activity [46].
Several phenoxy-1,4-naphthoquinone 10a–b and 2,3-bis(phenoxy)-1,4-naphtho-
quinone 11a–b were prepared. Experimental studies (Table 1) indicated a
remarkable activity for 2-chloro-3-(2-chlorophenoxy)-1,4-naphthoquinone 10a,
2,3-bis(2-chlorophenoxy)-1,4-naphthoquinone 11a and 2,3-bis(2,4-dichlorophe-
noxy)-1,4-naphthoquinone 11b with MIC values of 3.90, 1.95 and 0.97 lg/mL,
respectively. Therefore, these compounds were shown to be the most active
inhibitors against Y–M transition on C. albicans. In these latter cases, a
chlorophenoxyl substituent must exert a strong electron-withdrawing effect
reducing the electron density on the quinone ring, thus favoring reduction to
generate intermediate radical anions. A similar effect has been previously reported
for chloroanilino substituents [49].
Experimental
Materials and methods
All chemicals were reagent grade and used without further purification. Melting
points were measured with a Fisher Johns apparatus and are uncorrected. UV–Vis
spectra were obtained on a Shimadzu UV-2401 PC spectrophotometer. IR spectra
were recorded on a Nicolet Nexus 470 FT-IR spectrophotometer using KBr pellets,
and values are given in cm-1. NMR spectra were obtained on a Varian Mercury
123

Synthesis and studies of the antifungal activity of…
400 MHz model spectrometer using CHCl3 or DMSO as a solvent and TMS as
internal standard. Mass spectra were recorded on a Finnigan MAT 8200
spectrometer at 70 eV.
General procedures for the synthesis of 1,4-naphthoquinone derivatives
General procedure for 2-(anilino)-1,4-naphthoquinone 3a–k and 2-(anilino)-5-
hydroxy-1,4-naphthoquinone 4a–e These compounds were prepared by a method
previously reported in the literature by us [38–41]. 1,4-Naphthoquinone or
5-hydroxy-1,4-naphthoquinone (1 mmol) was dissolved in ethanol (10 mL) and
an amount (0.1 mmol) of catalyst (FeCl3 or CeCl3) was added. The reaction mixture
was stirred for 15 min to allow the reaction between the Lewis base (1,4-
naphthoquinone) and catalyst. A solution of the substituted aniline (1 mmol) in
ethanol (10 mL) was slowly added and the mixture was refluxed for 4 h. TLC
analysis indicated only one product was formed. The resulting solid was filtered,
washed with cold ethanol and recrystallized from ethanol. Total characterization of
compounds 3a–k has been previously reported [38–41].
2-(Anilino)-5-hydroxy-1,4-naphthoquinone 4a Orange-red solid (73 %), mp:
224 °C; UV–Vis, kmax (log e): 413 (3.74), 271 (4.29), 205 (4.40); IR (KBr,
cm-1): 3278 (N–H), 3052 (C–H), 1632 (C=O), 1578 (C=C), 1515 (N–H), 1273 (C–
1
N), 1238 (C–CO–C), 731, 693 (C–H); H NMR (400 MHz, DMSO-d6) d (ppm):
11.53 (s, 1H, O–H), 9.28 (s, 1H, N–H), 7.72 (t, J = 7.6 Hz, 1H, H7), 7.44 (t,
J = 7.2 Hz, 2H, H30, H50), 7.42 (d, J = 7.4 Hz, 1H, H6), 7.37 (d, J = 7.8 Hz, 2H,
H20, H60), 7.24 (t, J = 7.2 Hz, 1H, H40), 7.21 (d, J = 7.4 Hz, 1H, H8), 6.02 (s, 1H,
H3); EM (m/z): 265 [M]? (100 %). The exact mass was 265.0739 amu, the mass
observed was 265.0711 amu for C16H11NO3.
2-(2-Fluoroanilino)-5-hydroxy-1,4-naphthoquinone 4b Orange-red solid (100 %),
mp: 238 °C; UV–Vis, kmax (log e): 412 (3.72), 267 (4.19), 204 (4.36); IR (KBr,
cm-1): 3300 (N–H), 3053 (C–H), 1628 (C=O), 1596 (C=C), 1504 (N–H), 1373 (C–
1
N), 1264 (C–CO–C), 1174 (C–F), 761 (C–H); H NMR (400 MHz, DMSO-d6) d
(ppm): 11.46 (s, 1H, O–H), 9.16 (s, 1H, N–H), 7.72 (dt, J = 7.8 Hz, J = 0.8 Hz,
1H, H7), 7.41 (dd, J = 7.4 Hz, J = 0.9 Hz, 1H, H6), 7.35 (m, 3H, H30, H40, H50),
7.31 (m, 1H, H60), 7.25 (dd, J = 7.4 Hz, J = 0.8 Hz, 1H, H8), 5.47 (s, 1H, H3); 19F
NMR: -120.115 ppm; EM (m/z): 283 [M]? (100 %). The exact mass was
283.0645 amu, the mass observed was 283.0611 amu for C16H10FNO3.
2-(2-Bromoanilino)-5-hydroxy-1,4-naphthoquinone
4c Yellow-orange
solid
(86 %), mp: 186 °C; UV–Vis, kmax (log e): 413 (3.72), 267 (4.19), 205 (4.40); IR
(KBr, cm-1): 3306 (N–H), 1641 (C=O), 1612, 1586 (C=C), 1465 (N–H), 1250 (C–
1
CO–C), 739 (C–H); 1248 (C–N), 694 (C–Br), H NMR (400 MHz, DMSO-d6) d
(ppm): 11.46 (bs, 1H, O–H), 9.14 (bs, 1H, N–H), 7.78 (dd, J = 8.20 Hz, J = 1.5 Hz
1H, H30), 7.72 (dt, J = 7.8 Hz, J = 1.2 Hz, 1H, H7), 7.51 (dd, J = 7.4 Hz,
J = 1.2 Hz, 1H, H6), 7.42 (m, 2H, H40, H50), 7.30 (dd, J = 7.4 Hz, J = 1.5 Hz,
1H, H60), 7.26 (dd, J = 7.4 Hz, J = 1.2 Hz, 1H, H8), 5.37 (s, 1H, H3); EM (m/z):
123

E. Leyva et al.
343/345 [M]? (24/24 %). The exact mass was 342.9844 amu, the mass observed
was 342.9813 amu for C16H10BrNO3.
2-(4-Fluoroanilino)-5-hydroxy-1,4-naphthoquinone 4d Wine-red solid (87 %),
mp: 246 °C; UV–Vis, kmax (log e): 413 (3.73), 269 (4.26), 225 (4.08), 205
(4.39); IR (KBr, cm-1): 3216 (N–H), 3000 (C–H), 1629 (C=O), 1606, 1585 (C=C),
1
1509 (N–H), 1327 (C–N), 1312 (C–CO–C), 1228 (C–F), 811 (C–H); H NMR
(400 MHz, DMSO-d6) d (ppm): 11.45 (s, 1H, O–H), 9.23 (s, 1H, N–H), 7.71 (dt,
J = 7.8 Hz, J = 0.8 Hz, 1H, H7), 7.44 (dd, J = 7.4 Hz, J = 0.9 Hz 1H, H6), 7.39
(dd, J = 8.6 Hz, J = 5.1 Hz, 2H, H20, H60), 7.26 (dd, J = 8.20 Hz, J = 5.1 Hz,
2H, H30, H50) 7.22 (d, J = 7.8 Hz, 1H, H8), 5.90 (s, 1H, H3); 19F NMR:
-119.858 ppm; EM (m/z): 283 [M]? (100 %). The exact mass was 283.0645 amu,
the mass observed was 283.0611 amu for C16H10FNO3.
2-(2,4-Difluoroanilino)-5-hydroxy-1,4-naphthoquinone 4e Orange solid (73 %),
mp: 236 °C; UV–Vis, kmax (log e): 412 (3.73), 264 (4.17), 235 (4.09), 205 (4.35); IR
(KBr, cm-1): 3344 (N–H), 1635 (C=O), 1600, (C=C), 1488 (N–H), 1251 (C–N),
1
1137 (C–F), 863, 824 (C–H); H NMR (400 MHz, DMSO-d6) d (ppm): 11.44 (bs,
1H, O–H), 9.12 (bs, 1H, N–H), 7.72 (dt, J = 7.8 Hz, J = 0.8 Hz, 1H, H7), 7.51 (dd,
J = 7.4 Hz, J = 0.9 Hz, 1H, H6), 7.42 (m, 2H, H50, H60), 7.25 (dd, J = 8.2 Hz,
J = 0.8 Hz, 1H, H-8), 7.19 (dt, J = 8.9 Hz, J = 3.1 Hz, 1H, H30), 5.42 (s, 1H, H3);
19F NMR: -111.565, -115.301 ppm; EM (m/z): 301 [M]? (100 %). The exact
mass was 301.0550 amu, the mass observed was 301.525 amu for C16H9F2NO3.
General synthetic procedure for 2-chloro-3-(anilino)-1,4-naphthoquinones6a–d These
compounds were prepared following the literature procedure [42] with some
modifications [43]. A mixture of 2,3-dichloro-1,4-naphthoquinone (5 mmol) and
several equivalents of the corresponding aniline (aniline and 4-fluoroaniline,
10 mmol; 2-fluoroaniline and 2,4-difluoroaniline, 20 mmol) in 50 mL of methanol
was stirred and refluxed for several hours. The reaction progress was monitored by
TLC. After the reaction was finished, the mixture was kept in an ice-water bath. The
precipitated product was collected by filtration, washed with cold methanol and
purified by recrystallization from CH2Cl2.
2-Chloro-3-(2-fluoroanilino)-1,4-naphthoquinone 6b Red solid (90 %), mp:
176 °C; UV–Vis, kmax (log e): 464 (3.64), 274 (4.53), 208 (4.59); IR (KBr,
cm-1): 3255 (N–H), 1671 (C=O), 1596, 1567 (C=C), 1503 (N–H), 1332 (C–N),
1292 (C–CO–C), 1264 (C–F), 1141 (C–Cl), 762 (C–H); 1H NMR (400 MHz,
DMSO-d6) d (ppm): 9.23 (s, 1H, N–H), 8.01 (dd, J = 7.6 Hz, J = 1.4 Hz, 2H, H5,
H8), 7.86 (td, J = 7.6 Hz, J = 1.4 Hz, 1H, H6), 7.78 (td, J = 7.6 Hz, 1.4 Hz, 1H,
H7), 7.33 (td, J = 9.2 Hz, J = 6.6 Hz, 1H, H30), 7.25 (m, 2H, H40, H50), 7.18 (td,
J = 9.2 Hz, J = 7.1 Hz, 1H, H60); 19F NMR: -122.81 ppm; EM (m/z): 301/303
[M]? (100/35 %). The exact mass was 301.0306 amu, the mass observed was
301.0300 amu for C16H9ClFNO2.
2-Chloro-3-(4-fluoroanilino)-1,4-naphthoquinone 6c Red solid (98 %), mp:
235 °C; UV–Vis, kmax (log e): 474 (3.69), 275 (4.53), 208 (4.67); IR (KBr,
123

Synthesis and studies of the antifungal activity of…
cm-1): 3259 (N–H), 1674 (C=O), 1599, 1567 (C=C), 1491 (N–H), 1332 (C–N),
1290 (C–CO–C), 1215 (C–F), 1140 (C–Cl), 829 (C–H); 1H NMR (400 MHz,
DMSO-d6) d (ppm): 9.35 (s, 1H, N–H), 8.05 (dd, J = 7.6 Hz, J = 1.4 Hz, 2H, H5,
H8), 7.85 (td, J = 7.6 Hz, J = 1.4 Hz, 1H, H6), 7.75 (td, J = 7.6 Hz, J = 1.4 Hz,
1H, H7), 7.15 (dd, J = 8.6 Hz, J = 5.0 Hz, 4H, H20, H30, H50, H60); 19F NMR:
-116.24 ppm; EM (m/z): 301/303 [M]? (73/30 %). The exact mass was
301.0306 amu, the mass observed was 301.0300 amu for C16H9ClFNO2.
2-Chloro-3-(2,4-difluoroanilino)-1,4-naphthoquinone 6d Red solid (92 %), mp:
187–188 °C; UV–Vis, kmax (log e): 459 (3.71), 272 (4.60), 209 (4.76); IR (KBr,
cm-1): 3237 (N–H), 1680 (C=O), 1606, 1569 (C=C), 1498 (N–H), 1331 (C–N),
1293 (C–CO–C), 1246 (C–F), 1138 (C–Cl), 846 (C–H); 1H NMR (400 MHz,
DMSO-d6) d (ppm): 9.18 (s, 1H, N–H), 8.01 (dd, J = 7.8 Hz, J = 1.5 Hz, 2H, H5,
H8), 7.86 (td, J = 7.6 Hz, J = 1.4 Hz, 1H, H6), 7.79 (td, J = 7.6 Hz, J = 1.4 Hz,
1H, H7), 7.38 (dd, J = 8.9 Hz, J = 6.2 Hz, 1H, H60), 7.31 (td, J = 10.6, J = 8.4,
1H, H50), 7.08 (td, J = 10.0 Hz, J = 2.74 Hz, 1H, H30); 19F NMR: -113.05,
-117.69 ppm; EM (m/z): 319/321 [M]? (100/39 %). The exact mass was
319.0212 amu, the mass observed was 319.0210 amu for C16H8ClFNO2.
General synthetic procedure for 2-chloro-3-(2,4-dinitroanilino)-1,4-naphtho-
quinone 7 This compound was prepared by direct nitration of 2-anilino-3-
chloro-1,4-naphthoquinone 6a according to literature procedure [42] with some
modifications [43]. A solution of concentrated acids HNO3/H2SO4 (8/2 mL) was
slowly added to 6a (9 mmol). The resulting mixture was stirred at RT for 4 h. To
induced precipitation, a volume of cold water (100 mL) was added. The precipitate
was collected by filtration, washed with NaHCO3 solution (5 %), cold water and
ethanol.
General synthetic procedure for 2-(anilino)-3-(2,4-dinitroanilino)-1,4-naphtho-
quinone 8a–c Preparation and characterization of these compounds has been
recently reported by us. [45]. A solution of 7 (1 mmol) in 50 mL of ethanol and a
large amount of substituted aniline (15 mmol) were placed in a flask. The mixture
was refluxed vigorously for several hours (10–15). Cold distilled water was added
and the reaction mixture was placed in an ice-bath. The product was separated by
filtration and purified by column chromatography (silica gel/CHCl3).
General synthetic procedure for 2-chloro-3-(phenoxy)-1,4-naphthoquinone 10a–
b and 2,3-bis(phenoxy)-1,4-naphthoquinone 11a–b These compounds were
obtained from the reaction between 2,3-dichloro-1,4-naphthoquinone 5 and a given
phenol 9 according to the procedure reported in the literature [46] with several
modifications [43]. A mixture of substituted phenol (4 mmol) and Na2CO3
(4 mmol) was stirred in 15 mL of DMSO at RT for 15 min. An amount of 5
(2 mmol) was slowly added. The reaction was monitored by TLC. When the
reaction was finished, 20 mL of cold distilled H2O was added and the precipitate
was filtered and washed with cool methanol. The crude product was purified by
silica gel column chromatography (silica gel/CHCl3:Hex, 1:1).
123

E. Leyva et al.
2-Chloro-3-(2-chlorophenoxy)-1,4-naphthoquinone 10a Orange solid (50 %), mp:
162 °C; UV–Vis, kmax (log e): 335 (3.44), 276 (4.07), 251 (4.12), 240 (4.11), 203
(4.43); IR (KBr, cm-1): 1669 (C=O), 1611 (C=C), 1249, 1229 (C–O), 1054 (C–Cl),
1
753, 690 (C–H); H NMR (400 MHz, DMSO-d6) d (ppm): 8.11 (dd, J = 7.6 Hz,
J = 1.5 Hz, 1H, H5), 7.96 (dd, J = 7.4 Hz, J = 1.5 Hz, 1H, H8), 7.92 (td,
J = 7.4 Hz, J = 1.5 Hz, 2H, H6), 7.87 (td, J = 7.4 Hz, J = 1.5 Hz, 2H, H7), 7.57
(dd, J = 8.0 Hz, J = 1.5 Hz, H30), 7.30 (dd, J = 8.2, J = 1.4 Hz, 1H, H60), 7.23
(td, J = 7.9 Hz, J = 1.5 Hz, 1H, H50), 7.14 (td, J = 7.6 Hz, J = 1.4 Hz, 1H, H40);
EM (m/z): 318/320/322 [M]? (14/10/2 %). The exact mass was 317.9850 amu, the
mass observed was 317.9810 amu for C16H8Cl2O3.
2-Chloro-3-(4-methoxyphenoxy)-1,4-naphthoquinone 10b Purple solid (90 %),
mp: 127 °C; UV–Vis, kmax (log e): 336 (3.31), 277 (4.17), 244 (4.22), 222 (4.23),
206 (4.40); IR (KBr, cm-1): 3076 (=C–H), 2923 (C–H), 1676 (C=O), 1590 (C=C),
1
1241, 1204 (C–O), 717 (C–Cl), 854 (C–H); H NMR (400 MHz, DMSO-d6) d
(ppm): 8.19 (dd, J = 7.6 Hz, J = 1.7 Hz; 1H, H5), 8.03 (dd, J = 7.4 Hz,
J = 1.6 Hz, 1H, H8), 7.78 (td, J = 7.4 Hz, J = 1.5 Hz, 1H, H6), 7.74 (td,
J = 7.6 Hz, J = 1.6 Hz, 1H, H7), 6.96 (dd, J = 9.2 Hz, J = 2.5 Hz, 2H, H20, H60),
6.84 (dd, J = 9.12, J = 2.5 Hz, 2H, H30, H50); EM (m/z): 314/316 [M]? (100/
37 %). The exact mass was 314.0346 amu, the mass observed was 314.0312 amu
for C17H11ClO4.
2,3-Bis-(2-chlorophenoxy)-1,4-naphthoquinone 11a Yellow solid (48 %), mp:
153 °C; UV–Vis, kmax (log e): 335 (3.52), 270 (4.10), 250 (4.26), 204 (4.55); IR
(KBr, cm-1): 2923 (C–H), 1669 (C=O), 1611 (C=C), 1249, 1229 (C–O), 1054 (C–
Cl), 753, 690 (C–H); 1H NMR (400 MHz, DMSO-d6) d (ppm): 8.01 (dd,
J = 5.5 Hz, J = 3.3 Hz, 2H, H5, H8), 7.89 (dd, J = 5.5 Hz, J = 3.3 Hz, 2H,
H6, H7), 7.42 (dd, J = 8.0 Hz, J = 1.0 Hz, 2H, H3a0, H3b0), 7.33 (dd, J = 8.0 Hz,
J = 1.0 Hz, 2H, H6a0, H6b0), 7.15 (td, J = 7.6 Hz, J = 1.0 Hz, 2H, H5a0, H5b0),
7.04 (td, J = 7.6 Hz, J = 1.0 Hz, 2H, H4a0, H4b0); EM (m/z): 410/412/414 [M]?
(4.6/4/1.3 %). The exact mass was 410.0113 amu, the mass observed was
410.0100 amu for C22H12Cl2O4.
2,3-Bis-(2,4-dichlorophenoxy)-1,4-naphthoquinone 11b Yellow solid (85 %), mp:
153 °C; UV–Vis, kmax (log e): 334 (3.55), 274 (4.15), 250 (4.34), 205 (4.68); IR
(KBr, cm-1): 3087, 3027 (C–H), 1670, 1665 (C=O), 1592 (C=C), 1264, 1234 (C–
1
O), 1102 (C–Cl), 866, 821 (C–H); H NMR (400 MHz, DMSO-d6) d (ppm): 8.01
(dd, J = 5.6 Hz, J = 3.3 Hz, 2H, H5, H8), 7.89 (dd, J = 5.6 Hz, J = 3.3 Hz, 2H,
H6, H7), 7.63 (d, J = 2.5 Hz, 2H, H3a0, H3b0), 7.42 (d, J = 8.9 Hz, 2H, H6a0,
H6b0), 7.27 (dd, J = 8.8 Hz, J = 2.54 Hz, 2H, H5a0, H5b0); EM (m/z): 478/480/482
[M]? (13.8/17.8/9.9 %). The exact mass was 477.9333 amu, the mass observed was
477.9305 amu for C22H10Cl4O4.
Inhibition of Y–M C. albicans transition
Germ tube inhibition assay The 1,4-naphthoquinone derivatives were dissolved in
DMSO as 10 mg/mL (stock) and diluted appropriately in sterile Lee’s medium [48]
123

Synthesis and studies of the antifungal activity of…
just before use to obtain the required concentrations. Identical amounts of DMSO
were added to control the solvent effect. C. albicans ATCC strain 26,555 was used
in this study. Cells were maintained and subcultivated on Sabouroud dextrose agar
at 28 °C. The final pH was adjusted to 5.6. Inhibition assays were performed in
liquid cultures using Lee’s medium. The composition of medium (per liter) was as
follows: 2.5 g anhydrous K2PO4H; 0.2 g MgSO4Á7H2O; 5 g anhydrous (NH4)2SO4;
5 g NaCl; 12.5 g glucose; 0.5 g prolina; 0.001 g biotin. The final pH was adjusted to
6.8. To study the effect of 1,4-naphthoquinone derivatives on the Y–M transition of
C. albicans, stationary-phase cells were collected by centrifugation
(5000g 9 10 min) and inoculated into appropriate volume of Lee’s medium and
the initial concentration was regulated adjusting the optical density at 600 nm
between 0.10 and 0.15. A volume of 475 lL of yeast suspension was transferred to
5-mL tubes and incubated at 37 °C and 200 rpm in an orbital incubator (New
Brunswick Sci., Co.). 25 lL of a naphthoquinone derivative solution was inoculated
into yeast suspension in incubation with decreasing concentrations of derivative (1:2
serial dilutions) after 30–45 min of incubation since this is the time required for
germ tube formation (Fig. 1b). Samples were observed in an optical microscope
(409 magnification) after 1–3 h. All the experiments were performed in triplicate
and twice independently. All tests were run in duplicate.
Growth conditions C. albicans yeast cells were propagated in Lee’s medium as
follows: an aliquot of 50 mL with 7 lg of cells (dried weight per mL of medium)
was dispensed into a 250-mL Erlenmeyer flask. The culture was incubated at 28 °C
and 200 rpm in an orbital incubator (New Brunswick Sci., Co.). Cellular growth was
monitored by measuring the optical density at 600 nm using a Shimadzu UV-160-A
spectrophotometer. The cellular amount was determined using absorbance–cellular
dried weight/ml of the medium relation curve. The culture was incubated by
12–15 h to obtain 0.6 absorbance, a value that corresponds to medium point
exponential growth. To obtain a saturated culture containing synchronized cells, the
yeast cells were collected by centrifugation (3500 rpm, 10 min), washed with sterile
distilled water two times and re-suspended in 25 mL of sterile distilled water to a
final concentration of 1 mg/mL. The culture was incubated at 28 °C and 200 rpm in
an orbital incubator for 1–2 h and then stored for several hours (12–24) at 4 °C
(metabolic repose) [47]. In this form, yeast cells must be kept a maximum of 48 h
before inhibition germ tube and inhibition growth assays.
Conclusions
Structural analysis of several 1,4-naphthoquinone derivatives revealed that their
capacity to inhibit C. albicans Y–M transition is related to the position and nature of
the substituent groups and the presence of intramolecular hydrogen bonds on the
naphthoquinone structure.
Having a chlorophenoxyl substituent or an intramolecular hydrogen bond due to
the presence of a 2-fluoroanilino or 5-hydroxyl substituent on 1,4-naphthoquinone
structure enhances its biological activity. These structural features facilitate quinone
reduction to give a reactive intermediate that could easily interact with several
123

E. Leyva et al.
biomolecules. These results could lead to the generation of new selective
compounds as an alternative treatment for infections caused by fungi or C. albicans.
Acknowledgments This work was supported by CONACyT (Grant 155678). C.G.E.G. acknowledges
financial support by CONACyT (Scholarship 219858-290917).
References
´
´
1. L.I. Lopez, E. Leyva, R. Garcıa de la Cruz, Rev. Mex. Cienc. Farm. 42, 6 (2011)
2. N. El-Najjar, H. Gali-Muhtasib, R.A. Ketola, P. Vuorela, A. Urtti, H. Vuorela, Phytochem. Rev. 10,
353 (2011)
3. S.E. Loredo-Carrillo, E. Leyva, Naftoquinonas metodolog´ıas de s´ıntesis y propiedades fotof´ısicas
(Verlag/Publicia, Berlin, 2015)
´
4. T. Tran, E. Saheba, A.V. Arcerio, V. Chavez, Q. Li, L.E. Martınez, T.P. Primm, Bioorg. Med. Chem.
12, 4809 (2004)
5. G.J. Kapadia, M.A. Azuine, V. Balasubramanian, R. Sridhar, Pharmacol. Res. 43, 363 (2001)
6. M.H. Khraiwesh, C.M. Lee, Y. Brandy, E.S. Akinboye, S. Berhe, G. Gittens, M.M. Abbas, F.R.
Ampy, M. Ashraf, O. Bakare, Arch. Pharm. Res. 35, 27 (2012)
7. C. Ibis, A. Fatih Tuyun, Z. Ozsoy-Gunes, H. Bahar, M.V. Stasevych, R. Musyanovych, O.
Komarovska-Porokhnyavets, V. Novikov, Eur. J. Med. Chem. 46, 5861 (2011)
8. R.R. Kitagawa, C. Bonacorsi, L.M. da Fonseca, W. Vilegas, M.S.G. Raddi, Rev. Bras. Farmacogn.
Braz. J. Pharmacogn. 22, 53 (2012)
9. T. Kayashima, M. Mori, H. Yoshida, Y. Mizushina, K. Matsubara, Cancer Lett. 278, 34 (2009)
10. N. Pradidphol, N. Kongkathip, P. Sittikul, N. Boonyalai, B. Kongkathip, Eur. J. Med. Chem. 49, 253
(2012)
11. C.-K. Ryu, J.-Y. Shim, M.-J. Chae, I.-H. Choi, J.-Y. Han, O.-J. Jung, J.-Y. Lee, S.-H. Jeong, Eur.
J. Med. Chem. 40, 438 (2005)
12. V.K. Tandon, H.K. Maurya, M.K. Verma, R. Kumar, P.K. Shukla, Eur. J. Med. Chem. 45, 2418
(2010)
13. A.L. Lourenco, P.A. Abreu, B. Leal, E.N. da Silva Jr., A.V. Pinto, M.C.F.R. Pinto, A.M.T. Souza,
J.S. Novais, M.B. Paiva, L.M. Cabral, C.R. Rodrigues, V.F. Ferreira, H.C. Castro, Curr. Microbiol.
62, 684 (2011)
14. V.K. Tandon, H.K. Maurya, N.N. Mishra, P.K. Shukla, Bioorg. Med. Chem. Lett. 21, 6398 (2011)
15. P. Sudbery, N. Gow, J. Berman, J. Trends Microbiol. 12, 317 (2004)
16. A. Pugliese, D. Torre, F.M. Baccino, G. di Perri, C. Cantamessa, L. Gerbaudo, A. Saini, V. Vidotto,
Cell Biochem. Funct. 18, 235 (2002)
17. A. Mane, S. Gaikwad, S. Bembalkar, A. Risbud, J. Med. Microbiol. 61, 285 (2012)
18. R. Faraji, M.A. Rahimi, F. Rezvanmadani, M. Hashemi, Afr. J. Microbiol. Res. 6, 2773 (2012)
19. M.E.L. Consolaro, T.A. Albertoni, A.E. Svidzinski, R.M. Peralta, Mycopathologia 159, 501 (2005)
20. M.A. Pfaller, D.J. Diekema, Clin. Microbiol. Rev. 20, 133 (2007)
21. C.A. Sable, K.M. Strohmaier, J.A. Chodakewitz, Annu. Rev. Med. 59, 361 (2008)
22. F.C. Odds, Future Microbiol. 5, 67 (2010)
23. A. MacCallum, Chapter 5. Candida albicans: new insights in infection, disease, and treatment, in
New Insights in Medical Mycology, ed. by K. Kavanagh (Springer, Berlin, 2007)
24. S.K. Pitman, R.H. Drew, J.R. Perfect, Expert Opin. Emerg. Drugs 16, 559 (2011)
25. S.P. Saville, A.L. Lazzell, C. Monteagudo, J.L. Lopez-Ribot, Eukaryot. Cell 2, 1053 (2003)
26. M. Whiteway, U. Oberholzer, Curr. Opin. Microbiol. 7, 350 (2004)
27. M. Whiteway, C. Bachewich, Annu. Rev. Microbiol. 61, 529 (2007)
28. E. Manavathu, C. Duncan, Q. Porte, M. Gunasekaran, Mycopathologia 135, 79 (1996)
29. S.K. Biswas, K. Yokoyama, K. Kamei, K. Nishimura, M. Miyaji, Med. Mycol. 39, 283 (2001)
30. P. McGeady, D.L. Wansley, D.A. Logan, J. Nat. Prod. 65, 953 (2002)
31. C.A. Baker, K. Desrosiers, J.W. Dolan, Antimicrob. Agents Chemother. 46, 3617 (2002)
32. T.G. Brayman, J.W. Wilks, Antimicrob. Agents Chemother. 47, 3305 (2003)
33. M. Matsuoka, T. Takei, T. Kitao, Chem. Lett. 8, 627 (1979)
34. J.W. Macleod, R.H.J. Thomson, Org. Chem. 25, 36 (1960)
123

Synthesis and studies of the antifungal activity of…
35. A.A. Kutyrev, Tetrahedron 47, 8043 (1991)
36. Y.T. Pratt, J. Org. Chem. 27, 3905 (1962)
´
37. E. Leyva, L.I. Lopez, E. Moctezuma, H. de Lasa, Top. Catal. 49, 281 (2008)
´ ´
38. E. Leyva, L.I. Lopez, S.E. Loredo-Carrillo, M. Rodrıguez-Kessler, A. Montes-Rojas, J. Fluor. Chem.
132, 94 (2011)
´
´
´
39. L.I. Lopez, J.J. Vaquera-Garcıa, A. Saenz-Galindo, S.Y. Silva-Belmares, Lett. Org. Chem. 11, 573
(2014)
40. E. Leyva, S.J.S. Sobeck, S.E. Loredo-Carrillo, D.A. Magaldi-Lara, J. Mol. Struct. 1068, 1 (2014)
´
41. E. Leyva, K.M. Baines, C.G. Espinosa-Gonzalez, D.A. Magaldi-Lara, S.E. Loredo-Carrillo, T.A. De
´
´
Luna-Mendez, L.I. Lopez, J. Fluor. Chem. 180, 152 (2015)
42. T. Win, S. Bittner, Tetrahedron Lett. 46, 3229 (2005)
´
´
43. L.I. Lopez, Doctorate thesis, University of San Luis Potosı (2008)
44. T. Win, S. Yerushalmi, S. Bittner, Synthesis 10, 1631 (2005)
´ ´
45. E. Leyva, K.M. Baines, C.G. Espinosa-Gonzalez, L.I. Lopez, D.A. Magaldi-Lara, S. Leyva, Tetra-
hedron Lett. 56, 5248 (2015)
46. H.-X. Chang, T.-C. Chou, N. Savaraj, L.F. Liu, C. Yu, C.C. Cheng, J. Med. Chem. 42, 405 (1999)
47. M.V. Elorza, H. Rico, D. Gozalbo, R. Sentandreu, R. Antonie van Leeuwenhoek, J. Microbiol. Serol.
49, 457 (1983)
48. K.L. Lee, H.R. Buckley, C.C. Campbell, Sabouraudia 13, 148 (1975)
´
Ruvalcaba, J. Org. Chem. 64, 3684 (1999)
´ ´ ´
49. M. Aguilar-Martınez, G. Cuevas, M. Jimenez-Estrada, I. Gonzalez, B. Lotina-Hennsen, N.J. Macıas-
´
50. S. Vega-Rodrıguez, R. Jimenez-Catan˜o, E. Leyva, S.E. Loredo-Carrillo, J. Fluor. Chem. 145, 58
´
(2013)
51. C. Frontana, I. Gonzalez, J. Braz. Chem. Soc. 16, 299 (2005)
´
123