Synthesis of 3-tetrazolylmethyl-4H-chromen-4-ones via Ugi-azide and biological evaluation against Entamoeba histolytica, Giardia lamblia and Trichomona vaginalis

Article abstract of DOI:10.1016/j.bmc.2013.12.069

The synthesis of novel 3-tetrazolylmethyl-4H-chromen-4-ones via an Ugi-azide multicomponent reaction and their biological evaluation against Entamoeba histolytica, Giardia lamblia and Trichomona vaginalis are described. Reported yields are moderate to goo

Full text of DOI:10.1016/j.bmc.2013.12.069

Accepted Manuscript
Synthesis of 3-tetrazolylmethyl-4H-chromen-4-ones via Ugi-azide and biolog-
ical evaluation against Entamoeba histolytica, Giardia lamblia and Trichomona
vaginalis
Pedro A. Cano, Alejandro Islas-Jácome, Joaquín González-Marrero, Lilian
Yépez-Mulia, Fernando Calzada, Rocío Gámez-Montaño
PII:
S0968-0896(14)00021-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.12.069
BMC 11334
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 October 2013
19 December 2013
29 December 2013
Please cite this article as: Cano, P.A., Islas-Jácome, A., González-Marrero, J., Yépez-Mulia, L., Calzada, F.,
Gámez-Montaño, R., Synthesis of 3-tetrazolylmethyl-4H-chromen-4-ones via Ugi-azide and biological evaluation
against Entamoeba histolytica, Giardia lamblia and Trichomona vaginalis, Bioorganic & Medicinal Chemistry
(2014), doi: http://dx.doi.org/10.1016/j.bmc.2013.12.069
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evaluation against Entamoeba histolytica,
Giardia lamblia and Trichomona vaginalis
Pedro A. Cano, Alejandro Islas-Jácome, Joaquín González-Marrero, Lilian Yépez-Mulia, Fernando Calzada,
and Rocío Gámez-Montaño*
Departamento de Química, DCNE, Universidad de Guanajuato, Col. Noria Alta, 36050, Guanajuato, México.

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Synthesis of 3-tetrazolylmethyl-4H-chromen-4-ones via Ugi-azide and biological
evaluation against Entamoeba histolytica, Giardia lamblia and Trichomona vaginalis
Pedro A. Canoa, Alejandro Islas-Jácomea, Joaquín González-Marrerob, Lilian Yépez-Muliac, Fernando
Calzadad, and Rocío Gámez-Montañoa,*
aDepartamento de Química, DCNE, Universidad de Guanajuato, Noria Alta S/N, Guanajuato, C. P. 36050, Guanajuato, México. * Tel. (+52)-473-7320006 ext.
8191; Fax (+52)-473-7320006 ext. 8168; e-mail: rociogm@ugto.mx
bUnidad Profesional Interdisciplinaria de Ingeniería, Instituto Politécnico Nacional, Av. Mineral de Valenciana 200, Col. Fracc. Industrial Puerto Interior,
Silao de la Victoria, C. P. 36275, Guanajuato, México
cUnidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, UMAE, Hospital de Pediatría, Centro Médico Nacional Siglo XXI, IMSS, Av.
Cuauhtémoc No. 330, Col. Doctores, C. P. 06725, México DF, México.
dUnidad de Investigación Médica en Farmacología, UMAE, Hospital de Especialidades, 2do Piso, CORSE, Centro Médico Nacional Siglo XXI, IMSS, Av.
Cuauhtémoc No. 330, Col. Doctores, C. P. 06725, México DF, México.
ARTICLE INFO
ABSTRACT
Article history:
Received
The synthesis of novel 3-tetrazolylmethyl-4H-chromen-4-ones via an Ugi-azide multicomponent
reaction and their biological evaluation against Entamoeba histolytica, Giardia lamblia and
Trichomona vaginalis are described. Reported yields are moderate to good and biological results
show that these compounds could be considered as candidates to anti-parasitic drugs, especially
against Giardia lamblia.
Received in revised form
Accepted
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
3-substituted-chromen-4-ones
1,5-disubstituted-1H-tetrazoles
Ugi-azide
Half maximal inhibitory concentration
Anti-parasitic drug
1. Introduction
including giardiasis and amoebic dysentery. For decades, the use
of metronidazole has been noteworthy for its efficiency.
Parasitic infections are some of the most common human
health problems and are often caused by contaminated food or
water. Infections caused by intestinal protozoa are responsible for
high morbidity and mortality worldwide. The human intestine is
a major target for these pathogenic microorganisms resulting in
severe infections such as dysentery and diarrhea.1
However, potential carcinogenic, teratogenic, embryogenic
effects and clinical and laboratory-generated drug-resistance have
been described.6 Prolonged treatment or high doses of
metronidazole often cause side effects such as headache, dry
mouth, metallic taste, glossitis and urticaria.1, 3 Because of these
undesired side effects and considering the possible development
of new metronidazole resistant strains, there is a clear need for
new, effective and safer anti-protozoa agents.1 In an effort to
improve a theraphy for giardiasis and amoebic dysentery, the
creation of chemical libraries is necessary to obtain novel drugs
with high activity combined with low toxicity. In this context,
multicomponent reactions (MCR) provide products with the
needed diversity for the discovery of new leading compounds.
This diversity and easy accessibility to a large number of
compounds, combined with throughput screening techniques
make MCR a very important synthetic tool in modern drug
discovery processes.7
The parasitic disease known as amebiasis, which is caused by
the Entamoeba histolytica (a, Fig. 1) is actually the second
leading cause of death by parasites.2 The Giardia lamblia (b, Fig.
1) causes the giardiasis, a highly contagious disease which can
lead to death.3 The Trichomona vaginalis (c, Fig. 1) causes the
trichomoniasis, which together with the candidiasis are the most
common women’s sexually transmitted infections.4 These three
anaerobic protozoa, despite the significant differences in their
life-cycle and pathogenic properties are customarily grouped
together based on their carbohydrate metabolism and lack of
mitochondria.5
Metronidazole (d, Fig. 1) is a 5-nitroimidazole that shows
activity against anaerobic microorganisms, which has been
considered as the drug of choice against protozoan infections

Some synthetic methodologies have been described to prepare
3-substituted-chromen-4-ones from the chromen-4-one, mainly
based on direct C-C couplings such as the Heck reaction,13a Pd-
catalized cross coupling reaction,13b Suzuki-Miyaura,13c-e
organolithium mediated alkylation13f-h and xanthate based
alkylation.13i
a)
b)
c)
Some 3-substituted-chromen-4-ones and particularly the 3-
formyl-chromen-4-one can be efficiently prepared using a
Vilsmeier-Haack reaction.14 This compound is an excellent 1,2
and 1,4 acceptor.15 There are only three reports using it as starting
material in multicomponent reactions.16
d)
N
Me
HO
NO2
N
On the other hand, 1,5-disubstituted-1H-tetrazoles (1,5-DS-
1H-T) are bioisosteres of the cis-amide bonds of peptides as a
result of the similarities in their physicochemical properties.17
This kind of heterocyclic systems are the core of several
synthetic compounds such as the in clinical phase drug BMS-
317180 (3) (Fig. 2), which was reported by Bristol-Myers Squibb
Company as a potent orally available ligand of the growth
hormone secretagogue (GHS).18
Figure 1.8 a) Entamoeba histolytica, b) Giardia lamblia, c) Trichomona
vaginalis, d) Metronidazole.
Chromen-4-ones are heterocyclic systems present in numerous
natural and synthetic products showing interesting biological
activities such as anti-asthmatic,9a anti-Parkinson’s disease,9b
mild anti-ulcer,9c anti-Alzheimer,9d anti-viral,9e anti-fungic,9f anti-
tumoral9g and anti-HIV.9h
Several reports describe the synthesis of compounds having
the 1,5-DS-1H-T ring system among the most important are those
based on both, [2+3]-dipolar cycloadditions of azides with
cyanides19 and in the Ugi-azide multicomponent reaction. 20
Among the most important chromen-4-ones, are the 2- and 3-
aryl-chromen-4-ones known as flavones and isoflavones
respectively, which often are excellent naturally ocurring
antioxidants.10
The synthesized compounds 1a-n have the 1,5-DS-1H-T and
the 3-substituted-chromen-4-one moieties. In this context, the
Ugi-azide reaction combined with suitably post-condensation
processes have been used to prepare various tetrazole containing
heterocyclic scaffolds such as ketopiperazine,21a azepine,21b
Several 3-substituted-chromen-4-ones are of great relevance
in medicinal chemistry because they present a variety of
privileged biological activities such as anti-microbial,11a-c anti-
bacterial,11b anti-allergenic,11d anti-anaphylactic,11e,f anti-tumoral9g
and anti-cancer,11g,h together in some cases with low toxicity.
benzodiazepine,21c
bis-pyrrolidinone,21d
quinoxaline,21e
indazoline,21f isoindolinone,21g amodiaquine,21h pyrrolidine,21i
azepinoindolone21j and tetrahydrocarboline-tetrazoles.21k In the
other hand, several methods have been described for the
synthesis of 3-substituted-chromen-4-one containing scaffolds
such as phenyl,22a bis-chromen,22b pyrimidin,22c indolinone,22c
pyrazol,22c imidazol22d and spiropyrolidinone-chromen-4-ones.16c
It is noteworthy that as far as we known, only three reports
describe the synthesis of compounds having the tetrazol-
chromen-4-one heterocyclic system based on stepwise synthetic
methods.11b, e, f.
In this context, the 3-substituted-chromen-4-one formononetin
(2), which was isolated from the Dalbergia frutescens, is a
stronger antiparasitic drug than metronidazole against Giardia
intestinalis12 (Figure 2).
chromen-4-ones
O
3-tetrazolylmethyl-4H-chromen-4-ones
5
4
N N
6
3
2
N
N
R5
O
O
In this work, we describe the first synthesis of 3-
tetrazolylmethyl-4H-chromen-4-ones by an Ugi-azide reaction in
moderate to good yields (55-90%) under mild conditions (room
temperature). Our hypothesis was “compounds having both
scaffolds, tetrazole and chromone in the same molecule, will
show antiprotozoal activity”. As we discussed below, tetrazoles
and chromones have been described to be independently active
against some parasites such as E. histolytica and Leishmania.
Thus, based on this idea and in the side effects caused by the
metronidazole, we are convinced it is really necessary increasing
the amount of available antiparasitic compounds that can be
prepared in a minimum of steps. The compounds 1a-n were
prepared in only one synthetic procedure with good yields.
7
O
1
8
NH
R1
R2
1,5-DS-1H-tetrazoles
3
N
N2
N1
R4
1a-n
4 N
R3
I
R
5
R1 = H, Br, I,NO2
R2 = H, OMe
R3 = H, Cl, I, OMe, NO2
R4 = H, OMe
R
Formononetin
O
OMe
R5 = t-Bu, Cy,2,6-diMePh
HO
O
2
BMS-317180
OH
2. Results and discussion
O
N
N
N
H
O
N
2.1. Synthesis of the 3-tetrazolylmethyl-4H-chromen-4-ones
N
3
O
The synthetic methodology involved a sequential combination
of the 3-formylchromen-4-one (4) with the anilines 5, the
trimethylsilylazide (6) and the isocyanides (7) to prepare the
series of 3-tetrazolylmethyl-4H-chromen-4-ones 1a-n (Table 1).
O
NH2
After an exhaustive exploration of the reaction conditions
varying the temperature, solvents and acidic catalysts, optimal
Figure 2. Objective compounds 1a-n, formononetin (2) and the BMS-
317180 (3).

parameters to prepare the 3-tetrazolylmethyl-4H-chromen-4-one
1a were found (Table 1).
It is noteworthy that anilines, which commonly are not good
nucleophiles, were used as amino component in the Ugi-azide
reaction. It has been reported that Lewis acids favor the
condensation reactions between anilines with aldehydes or
ketones.23 Thus, a catalytic amount of InCl3 (5% mol) was added
to increase the electrophilicity of the 3-formyl-chromone (4)
promoting its condensation with the substituted anilines 5. The
catalyst-free classic Ugi-azide method usually works very well
when good nucleophilic amines were used.20b-c, 21f,k
Following with the results and discussion, cyclohexyl
isocyanide was used instead of t-butyl isocyanide to prepare the
compound 1b in 78% yield. Aniline was used instead of o-
iodoaniline to prepare the compounds 1c-d in 86 and 82% yields
respectively. p-chloro and p-iodoanilines were used to prepare
the compounds 1e-h obtaining 72 to 85% yields. Surprisingly,
when the poorly nucleophilic p-nitroaniline was used to prepare
the compound 1i, 70% yield was observed. When 2,6-
dimethylisocyanide was used, product 1j was obtained in 55%
yield. The latter experiment gave us the lowest yield probably
due to the high steric hindrance generated by the substituent of
the isocyanide (R5 = 2,5-diMePh). The amine having more ERG,
the 2,3,4-trimethoxyaniline was used to prepare the compound 1k
with the highest yield (90%). To complete the series, o-bromo
and o-nitroanilines were used in the Ugi-azide reaction to prepare
the products 1l-n with yields close to 70% (Table 1).
Figure 3. ORTEP diagram of compound 1a.
2.2. Biological evaluation
In 2008, Diwakar and co-workers designed and synthesized a
series of 3-(Z)-tetrazolylvinylchromen-4-one, which showed in
vitro both, antibacterial (E. faecalis, S. pneumonia and E. coli)
and antimicrobial (S. aureus) activity.11b
Diwakar’s work11b prompted us to synthesize our novel 3-
Table 1. Synthesis of the 3-tetrazolylmethyl-4H-chromen-4-ones 1a-n.
tetrazolylmethyl-4H-chromen-4-ones
1a-n,
which
were
biologically tested against E. histolytica, G. lamblia and T.
vaginalis (Table 2). These unicellular eukaryotes have become in
a serious worldwide health problem because they have been able
to resist the majority of the currently known drugs such as
metronidazole.25
N
N
NH2
N
N
R5
O
O
O
O
R1
R2
i
O
NH
R4
R1
R2
R3
Antiprotozoal activity of chromen-4-ones26a-c and tetrazoles26d
against parasites such as Leishmania has been practically
unexplored. In this context, the antiprotozoal activity of
tetrazoles27 particularly against E. histolytica has been described
only in one report, but the stepwise prepared compounds were
obtained in low overall yields. However, as far as we know, this
is the first report in which antiprotozoal activity of compounds
having both, tetrazole and chromen-4-one heterocyclic scaffolds
against three kinds of parasites, E. histolytica, G. lamblia and T.
vaginalis is described.
4
5
R4
R5NC
TMSN3
R3
1a-n
6
7
Prod.
R1
R2
R3
R4
R5
yield (%)a
1a
1b
I
I
85
78
t-Bu
H
H
H
H
H
H
H
H
Cy
86
H
H
H
H
1c
1d
1e
1f
t-Bu
Cy
82
76
H
H
H
H
H
Cl
Cl
t-Bu
Cy
H
H
Biological activity results are depicted in Table 2. The data is
presented in terms of the half-maximal inhibitory concentration
(IC50). The antiprotozoal activity of the synthesized compounds
1a-n was compared with the activity of metronidazole (positive
control), which is the most widely commercially available
antiamoebic drug.28 A total of 14 compounds were studied.
Among these compounds, 1k which has three methoxy groups in
R2, R3 and R4 positions of the amine moiety, showed the best
activity against E. histolytica (IC50 = 43.2 µg/mL) and G. lamblia
(IC50 = 84.2 µg/mL), while 1n was the most active against T.
vaginalis (IC50 = 83.9 µg/mL). All compounds showed selectivity
on G. lamblia. However, these values are considerable higher
than those presented by metronidazole (IC50 = 0.23 µg/mL
against E. histolytica, IC50 = 1.22 µg/mL against G. lamblia and
IC50 = 0.037 µg/mL against T. vaginalis) (Table 2).
72
85
79
70
55
H
H
H
H
H
H
1g
1h
1i
I
H
H
H
H
H
t-Bu
Cy
I
H
H
NO2
Cy
1j
2,6-diMePh
OMe
OMe
H
OMe
OMe
H
90
72
70
71
1k
1l
Cy
t-Bu
Cy
OMe
H
Br
Br
1m
1n
H
H
H
NO2
Cy
H
H
H
i) InCl3 (5% mol); i-PrOH; R. T.; 2 h
a Measured after recrystallization
The 3-tetrazolylmethyl-4H-chromen-4-ones 1a-n were
1
characterized by spectroscopic analysis: H and 13C-NMR, IR
and HRMS. Additionally, an adequate crystal of compound 1a
was obtained for X-ray analysis 24 (Figure 3).

Table 2. Half Maximal inhibitory concentration of the 3-tetrazolylmethyl-
4H-chromen-4-ones 1a-n against E. histolytica, G. lamblia and T. vaginalis.
iodine-containing compounds is observed. Another interesting
SAR study was made analyzing the effect of the high
electronegative nitro group in R3 and in R1 positions. Thus, the
activity of 1i (R3 = NO2, IC50 = 110.2 µg/mL) is lower than the
activity of 1n (R1 = NO2, IC50 = 86.5.4 µg/mL). Based on the
SAR study, one of the conclusions is that the G. lamblia is the
most susceptible parasite to the structural diversity. Other
interesting observations derived from the SAR study are
described below: i) compounds having o-bromide atom in the
aniline system (1l and 1m) show a considerable better activity
than those having o-iodine atom in the aniline system (1a and
1b); ii) compound 1k, which has three methoxy groups in R2, R3
and R4 positions of the amine moiety show better activity than
compound 1j, which has only two methoxy groups, in R2 and R3
positions of the amine moiety; iii) compound 1k, which is the
most active among them against E. histolytica and G. lamblia,
clearly shows a lower activity than metronidazole and a worse
activity against G. lamblia than the formononetin, which actually
is one of the most promising antiparasitic drug, especially against
G. lamblia; iv) compound 1n, which is the most active of all
compounds against T. vaginalis, shows also no better
antiprotozoal activity (Table 2).
a
IC50
E. histolytica
G. lamblia
T. vaginalis
µg/mL µM
Product
µM
µg/mL
µg/mL
µM
67.3
64.3
103.8
104.1
118.7
134.3
122.0
1a
1b
207.2
197.5
140.2
279.8
281.6
432.3
148.4
162.2
187.6
182.7
161.1
175.6
70.4
73.3
65.9
76.4
316.4
1c
1d
1e
1f
114.7
110.0
285.9
268.9
150.2
151.7
374.4
370.8
94.9
218.1
123.7
284.3
71.1
61.9
51.8
53.8
141.9
117.4
116.1
111.3
87.9
98.7
1g
1h
1i
197.0
226.5
247.0
240.5
171.4
242.3
253.0
193.9
123.4
116.9
91.4
246.3
221.8
204.9
191.6
175.5
234.4
194.7
188.0
119.4
110.2
116.2
84.2
1j
92.6
43.2
56.7
1k
1l
86.2
109.8
125.1
114.0
124.4
106.2
93.3
121.2
86.5
54.6
55.5
1m
1n
83.9
Met.b
For.c
1.22
0.03
0.037
0.23
3. Conclusions
This work describes the first one pot synthesis of novel 3-
tetrazolylmethyl-4H-chromen-4-ones and their evaluation against
E. histolytica, G. lamblia and T. vaginalis. Compound 1k shows
the best antiprotozoal activity against E. histolytica and G.
lamblia, while the compound 1n shows the best activity against
T. vaginalis. Compounds 1a-n showed the best activity against
G. lamblia among the three parasites, but none of them was
better than metronidazole. These compounds could represent a
suitable alternative when the studied parasites become resistant
to the majority of the current antiparasitic drugs.29 As main
conclusion, it can be considered that our hypothesis was true
because the 3-tetrazolylmethyl-4H-chromen-4-ones 1a-n, which
have both scaffols, chromone and tetrazole, showed a moderate
antiprotozoal activity.
a
antiprotozoal activity of compounds 1a-n.
In vitro
b Met. = metronidazole
n = 3
c For. = formononetin
2.3. Structure Activity Relationship
The SAR study revealed that the isocyanide substituent (R5)
does not significantly affect to the antiprotozoal activity against
E. histolytica as can be seen comparing the value for 1a (R5 = t-
Bu, IC50 = 67.3 µg/mL) with the value of 1b (R5 = Cy, IC50
=
64.3 µg/mL) or the value of 1c (R5 = t-Bu, IC50 = 70.4 µg/mL)
with the value of 1d (R5 = Cy, IC50 = 73.3 µg/mL). The same
behavior was observed for these compounds against the other
two studied protozoa. Another brief variation in the activity
against E. histolytica could be observed analyzing the effect of
the iodine atom position as can be seen comparing the value of
1a (R1 = I, IC50 = 67.3 µg/mL) with the value of 1g (R3 = I, IC50
= 71.1 µg/mL) or 1b (R1 = I, IC50 = 64.3 µg/mL) with 1h (R3 = I,
IC50 = 61.9 µg/mL). Respect to G. lamblia, comparing the value
of 1a (R1 = I, IC50 = 103.8 µg/mL) with the value of 1g (R3 = I,
IC50 = 98.7 µg/mL), the difference of the activities was kept to a
minimum, but, if a comparison is made between the activity of
1b (R1 = I, IC50 = 104.3 µg/mL) with the activity of 1h (R3 = I,
IC50 = 119.4 µg/mL), a considerable difference can be observed,
which signifies the position of iodine atom really affect to G.
lamblia when R5 = Cy. A comparison of values for both, 1a (R1 =
I, IC50 = 140.2 µg/mL) with 1g (R3 = I, IC50 = 123.4 µg/mL) and
1b (R1 = I, IC50 = 148.4 µg/mL) with 1h (R3 = I, IC50 = 116.9
µg/mL) allows to observe a considerable difference in the
activity against T. vaginalis. Another revelation based on the
SAR study can be seen by analyzing the effect of the substitution
of chlorine by iodine in position R3. The value for the activity
against E. histolytica of compound 1e (R3 = Cl, IC50 = 65.9
µg/mL) respect to the value of 1g (R3 = I, IC50 = 71.1 µg/mL)
gave a small advantage for the compound having chlorine. This
behavior is reversed making a comparison between the values of
compound 1e (R3 = Cl, IC50 = 110.0 µg/mL) with the value of
compound 1g (R3 = I, IC50 = 98.7 µg/mL) against G. lamblia and
also between the values of compound 1e (R3 = Cl, IC50 = 151.7
µg/mL) with the value of compound 1g (R3 = I, IC50 = 123.7
µg/mL) against T. vaginalis, in which an advantage for the
4. Experimental section
4.1 General Information, instrumentation and chemicals
Commercially available starting materials were purchased
from Sigma-Aldrich and were used without further purification.
The solvents were distilled and dried using common procedures.
IR spectra were recorded on a Perkin Elmer 100 FT-IR
1
spectrometer ( in cm-1). H and 13C NMR spectra were acquired
in Bruker (500, 400, 300 MHz) spectrometers. CDCl3 was used
as solvent and chemical shifts were reported in ppm. Coupling
1
constants were reported in Hz. Internal reference for H-NMR
spectra is respect to TMS at 0.0 ppm. Internal reference for 13C-
NMR spectra is respect to CDCl3 at 77.23 ppm. HRMS were
recorded on a JEOL GC-mate spectrometer by EI+ ionization
mode. Reaction progress was monitored by TLC on precoated
silica gel Kieselgel 60 F254 plates. The spots were visualized
under UV light (254 and 365 nm). Cold anhydrous diethyl ether
was used as recrystallization solvent. Melting points were
determined on a Fisher-Johns apparatus and were uncorrected.
The Purity of the synthesized compounds was supposed nearly to
100% due to the fact that perfect crystals of them were used for
characterization and to prepare the samples for biological tests.
4.2. General procedure for the synthesis of the 3-
tetrazolylmethyl-4H-chromen-4-ones 1a-n (GP).

To a stirred solution of 3-formylchromone (4) (1.0 mmol, 1.0
equiv.) in anhydrous isopropanol with InCl3 (5% mol) at room
7.69 (ddd, J = 8.6, 7.1, 1.7 Hz, 1 H, ArH), 7.48-7.40 (m, 2 H,
ArH), 7.22-7.15 (m, 2 H, ArH), 6.83-6.76 (m, 1 H, ArH), 6.73-
6.68 (m, 2 H, ArH), 6.18 (d, J = 7.0 Hz, 1 H, CH), 4.98 (d, J =
7.1 Hz, 1 H, NH), 4.75-4.66 (m, 1 H, CH), 2.09-1.88 (m, 8 H, 4
CH2), 1.51-1.38 (m, 2 H, CH2); 13C-NMR (75 MHz, CDCl3):
176.7, 156.4, 155.4, 154.2, 145.0, 134.2, 129.6, 125.6, 123.5,
121.4, 119.4, 118.4, 113.8, 58.4, 44.6, 33.3, 32.7, 29.7, 25.2,
24.9; HRMS (EI+) calcd. for C23H23N5O2, 401.1852, found:
401.1850.
temperature,
anilines
5
(1.0
mmol,
1.0
equiv.),
azidotrimethylsilane (6) (1.0 mmol, 1.0 equiv.) and isocyanides 7
(1.0 mmol, 1.0 equiv) were sequentially added. After 2 h at room
temperature, the solvent was removed until dryness. Then, the
crude was dissolved in DCM (20 mL) and washed with a
saturated solution of NaHCO3 (10 mL), followed by a treatment
with an excess of brine. The organic layer was dried over
anhydrous Na2SO4 and concentrated under reduced pressure.
Finally a recrystallization using cold anhydrous diethyl ether was
made to afford the corresponding 3-tetrazolylmethyl-4H-
chromen-4-ones 1a-n.
3-((1-(tert-butyl)-1H-tetrazol-5-yl)((4-
chlorophenyl)amino)methyl)-4H-chromen-4-one (1e)
Yield = 76%; white solid; mp = 200-202 °C; Rf = 0.34 (Hexanes-
AcOEt, 7:3 V/V); FT-IR (ATR)νmax/cm-1: 3316 (N-H), 1646
(C=O), 1287 (N-N=N), 1225 (C–O–C); 1H-NMR (300 MHz,
4.3. Characterization of the 3-tetrazolylmethyl-4H-chromen-4-
ones 1a-n.
CDCl3):
8.29 (d, J = 0.9 Hz, 1 H, CH), 8.18-8.15 (m, 1 H,
3-((1-(tert-butyl)-1H-tetrazol-5-yl)((2-
iodophenyl)amino)methyl)-4H-chromen-4-one (1a)
ArH), 7.69 (ddd, J = 8.6, 7.0, 1.6 Hz, 1 H, ArH), 7.49-7.46 (m, 1
H, ArH), 7.42 (ddd, J = 8.1, 7.1, 1.1 Hz, 1 H, ArH), 7.10 (d, J =
8.9 Hz, 2 H, ArH), 6.62 (d, J = 9.9 Hz, 2 H, ArH), 6.41 (d, J =
6.1 Hz, 1 H, CH), 4.94 (d, J = 6.5 Hz, 1 H, NH), 1.83 (s, 9 H, 3
CH3); 13C-NMR (75 MHz, CDCl3): 176.8, 156.5, 155.7, 153.9,
143.6, 134.2, 129.5, 125.6, 124.0, 123.5, 121.4, 118.3, 114.6,
62.4, 45.3, 30.0; HRMS (EI+) calcd. for C21H20N5O2Cl: 409.1306,
found: 409.1319.
Yield: 85%; white solid; mp = 150-152 °C; Rf = 0.39 (Hexanes-
AcOEt, 7:3 V/V); FT-IR (ATR)νmax/cm-1: 3334 (N-H), 1640
(C=O), 1283 (N-N=N), 1230 (C–O–C); H-NMR (500 MHz,
1
CDCl3): 8.27 (s, 1 H, CH), 8.17 (dd, J = 8.0, 1.5 Hz, 1 H, ArH),
7.72-7.68 (m, 2 H, ArH), 7.49 (d, J = 8.4 Hz, 1 H, ArH), 7.45-
7.42 (m, 1 H, ArH), 7.22-7.18 (m, 1 H, ArH), 6.63 (d, J = 8.1 Hz,
1 H, ArH), 6.57-6.53 (m, 2 H, ArH and CH), 4.80 (d, J = 8.1 Hz,
1 H, NH), 1.85 (d, J = 10.7 Hz, 9 H, 3 CH3); 13C-NMR (125
MHz, CDCl3): 176.8, 156.5, 155.7, 153.6, 144.0, 139.5, 134.2,
129.9, 125.6, 123.4, 120.9, 118.4, 111.4, 85.7, 62.3, 45.4, 30.0;
HRMS (EI+) calcd. for C21H20N5O2I: 501.0662, found: 501.0662.
3-((4-chlorophenyl)amino)((1-cyclohexyl-1H-tetrazol-5-
yl)methyl)-4H-chromen-4-one (1f)
Yield = 72%; yellow solid; mp = 210-212 °C; Rf = 0.37
(Hexanes-AcOEt, 7:3 V/V); FT-IR (ATR)νmax/cm-1: 3344 (N-H),
1640 (C=O), 1285 (N-N=N), 1227 (C–O–C); 1H-NMR (300
MHz, CDCl3): 8.27 (d, J = 0.7 Hz, 1 H, CH), 8.19 (ddd, J = 8.0,
1.9, 0.5 Hz, 1 H, ArH), 7.70 (ddd, J = 8.7, 7.1, 1.7 Hz, 1 H, ArH),
7.48-7.41 (m, 2 H, ArH), 7.15-7.09 (m, 2 H, ArH), 6.66-6.61 (m,
2 H, ArH), 6.15 (d, J = 7.7 Hz, 1 H, CH), 5.15 (d, J = 7.8 Hz,
3-((1-cyclohexyl-1H-tetrazol-5-yl)((2-iodophenyl)amino)
methyl)-4H-chromen-4-one (1b)
Yield: 78%; white solid; mp = 156-158 °C; Rf = 0.42 (Hexanes-
AcOEt, 7:3 V/V); FT-IR (ATR)νmax/cm-1: 3355 (N-H), 1637
(C=O), 1290 (N-N=N), 1227 (C-O-C). H-NMR (300 MHz,
NH), 4.73-4.61 (m, 1 H, CH), 2.02-1.78 (m, 8 H, 4 CH2), 1.53-
1.38 (m, 2 H, CH2); C-NMR (75 MHz, CDCl3): 176.6, 156.4,
1
13
CDCl3): 8.25 (d, J = 0.6 Hz, 1 H, CH), 8.21 (ddd, J = 8.0, 1.7,
0.4 Hz, 1 H, ArH), 7.73-7.67 (m, 2 H, ArH), 7.49-7.41 (m, 2 H,
ArH), 7.21-7.15 (m, 1 H, ArH), 6.63-6.51 (m, 1 H, ArH), 6.27 (d,
J = 6.9 Hz, 1 H, CH), 5.45 (d, J = 7.2 Hz, 1 H, NH), 4.77-4.66
(m, 1 H, CH), 2.06-1.86 (m, 8 H, 4 CH2), 1.54-1.41 (m, 2 H,
CH2); 13C-NMR (75 MHz, CDCl3): 176.6, 156.4, 155.7, 153.7,
144.3, 139.6, 134.3, 129.7, 125.8, 125.7, 123.4, 121.0, 120.8,
118.4, 111.7, 85.8, 58.5, 44.9, 33.3, 32.9, 25.28, 25.23, 24.9;
HRMS (EI+) calcd. for C23H22N5O2I: 527.0819, found: 527.0834.
155.6, 154.0, 143.5, 134.3, 129.5, 125.8, 125.6, 124.2, 123.4,
121.1, 118.4, 114.9, 58.4, 44.5, 33.3, 32.8, 25.2, 24.8; HRMS
(EI+) calcd. for C23H22N5O2Cl: 435.1462, found: 435.1462.
3-((1-(tert-butyl)-1H-tetrazol-5-yl)((4-
iodophenyl)amino)methyl)-4H-chromen-4-one (1g)
Yield = 85%; white solid; mp = 166-168 °C; Rf = 0.26 (Hexanes-
AcOEt, 7:3 V/V); FT-IR (ATR)νmax/cm-1: 3316 (N-H), 1641
(C=O), 1286 (N-N=N), 1224 (C–O–C); H-NMR (300 MHz,
1
3-((1-(tert-butyl)-1H-tetrazol-5-yl)(phenylamino)methyl)-4H-
chromen-4-one (1c)
CDCl3): 8.27 (d, J = 0.8 Hz, 1 H, CH), 8.17 (dd, J = 8.0, 1.3 Hz,
1 H, ArH), 7.73-7.67 (m, 1 H, ArH), 7.50-7.40 (m, 4 H, ArH),
6.51-6.46 (m, 2 H, ArH), 6.44 (d, J = 8.3 Hz, 1 H, CH), 4.78 (d, J
= 8.3 Hz, 1 H, NH), 1.84 (s, 9 H, 3 CH3); 13C-NMR (75 MHz,
CDCl3): 176.7, 156.5, 155.7, 153.9, 144.5, 138.3, 134.2, 125.6,
121.4, 118.4, 115.6, 80.5, 62.3, 44.9, 30.0; HRMS (EI+) calcd.
for C21H20N5O2I: 501.0662, found: 501.0662.
Yield: 86%; white solid; mp = 220-222 °C; Rf = 0.32 (Hexanes-
AcOEt, 7:3 V/V); FT-IR (ATR)νmax/cm-1: 3345 (N-H), 1640
(C=O), 1285 (N-N=N), 1227 (C–O–C); H-NMR (500 MHz,
1
CDCl3):
8.31 (s, 1 H, CH), 8.16 (d, J = 8.0 Hz, 1 H, ArH),
7.70-7.66 (m, 1 H, ArH), 7.47 (d, J = 8.4 Hz, 1 H, ArH), 7.43-
7.39 (m, 1 H, ArH), 7.19-7.15 (m, 2 H, ArH), 6.80-6.75 (m, 1 H,
ArH), 6.68 (d, J = 7.9 Hz, 2 H, ArH), 6.46 (d, J = 7.6 Hz, 1 H,
CH), 4.79-4.76 (m, 1 H, NH), 1.85 (s, 9 H, 3 CH3); 13C-NMR
(125 MHz, CDCl3): 176.9, 156.5, 155.6, 134.1, 129.6, 125.6,
125.5, 123.5, 118.4, 113.3, 62.3, 45.2, 30.0; HRMS (EI+) calcd.
for C21H21N5O2: 375.1695, found: 375.1679.
3-((1-cyclohexyl-1H-tetrazol-5-yl)((4-
iodophenyl)amino)methyl)-4H-chromen-4-one (1h)
Yield = 79%; white solid; mp = 162-164 °C; Rf = 0.29 (Hexanes-
AcOEt, 7:3 V/V); FT-IR (ATR)νmax/cm-1: 3318 (N-H), 1645
(C=O), 1286 (N-N=N), 1230 (C–O–C); H-NMR (300 MHz,
1
CDCl3): 8.26 (s, 1 H, CH), 8.18 (dd, J = 8.0, 2.0 Hz, 1 H, ArH),
7.73-7.67 (m, 1 H, ArH), 7.47-7.40 (m, 4 H, ArH), 6.53-6.47 (m,
2 H, ArH), 6.15 (d, J = 7.6 Hz, 1 H, CH), 5.31 (d, J = 7.8 Hz, 1
H, NH), 4.72-4.60 (m, 1 H, CH), 1.99-1.74 (m, 8 H, 4 CH2),
1.51-1.38 (m, 2 H, CH2); 13C-NMR (75 MHz, CDCl3): 176.6,
156.4, 155.6, 153.9, 144.7, 138.2, 134.3, 125.7, 125.6, 123.3,
121.0, 118.4, 115.8, 80.5, 58.5, 44.1, 33.2, 32.8, 25.25, 25.19,
3-((1-cyclohexyl-1H-tetrazol-5-yl)(phenylamino)methyl)-4H-
chromen-4-one (1d)
Yield = 82%; white solid; mp = 218-220 °C; Rf = 0.37 (Hexanes-
AcOEt, 7:3 V/V); FT-IR (ATR)νmax/cm-1 3373 (N-H), 1640
(C=O), 1290 (N-N=N), 1226 (C–O–C); H-NMR (300 MHz,
CDCl3): 8.30 (s, 1 H, CH), 8.19 (dd, J = 8.0, 1.4 Hz, 1 H, ArH),
1

24.9; HRMS (EI+) calcd. for C23H22N5O2I: 527.0819, found:
527.0854.
HRMS (EI+) calcd. for C21H20N5O2Br: 453.0800, found:
453.0804.
3-((1-cyclohexyl-1H-tetrazol-5-yl)((4-
nitrophenyl)amino)methyl)-4H-chromen-4-one (1i)
3-((2-bromophenyl)amino)(1-cyclohexyl-1H-tetrazol-5-
yl)methyl)-4H-chromen-4-one (1m)
Yield = 70%; yellow solid; mp = 210-212 °C; Rf = 0.14
(Hexanes-AcOEt, 7:3 V/V); FT-IR (ATR)νmax/cm-1: 3330 (N-H),
1650 (C=O), 1281 (N-N=N), 1235 (C–O–C); 1H-NMR (500
Yield = 70%; yellow solid; mp = 184-186 °C; Rf = 0.47
(Hexanes-AcOEt, 7:3 V/V); FT-IR (ATR) νmax/cm-1: 3300 (N-H),
1640 (C=O), 1283 (N-N=N), 1232 (C–O–C); 1H-NMR (300
MHz, CDCl3):
8.26 (s, 1 H, CH), 8.23 (d, J = 8.0 Hz, 1 H,
MHz, CDCl3):
8.26 (d, J = 0.7 Hz, 1 H, CH), 8.23-8.19 (m,
ArH), 8.06 (d, J = 9.1 Hz, 2 H, ArH), 7.75-7.71 (m, 1 H, ArH),
7.50-7.46 (m, 2 H, ArH), 6.72 (d, J = 9.1 Hz, 2 H, ArH), 6.35 (d,
J = 7.2 Hz, 1 H, CH), 6.32 (d, J = 7.2 Hz, 1 H, NH), 4.70-4.63
(m, 1 H, CH), 2.07-2.01 (m, 2 H, CH2), 2.00-1.88 (m, 4 H, 2
CH2), 1.79-1.73 (m, 1 H, H of CH2), 1.52-1.41 (m, 2 H, CH2),
1.36-1.25 (m, 1 H, H of CH2); 13C-NMR (125 MHz, CDCl3):
175.7, 155.7, 155.2, 152.7, 149.5, 139.0, 133.8, 125.5, 125.2,
124.8, 122.4, 119.8, 117.5, 111.3, 57.4, 42.1, 31.8, 31.4, 23.7,
23.6, 23.3; HRMS (EI+) calcd. for C23H22N6O4: 446.1703, found:
446.1710.
1H), 7.70 (ddd, J = 8.6, 7.1, 1.7 Hz, 1 H, ArH), 7.50-7.41 (m, 3
H, ArH), 7.18-7.12 (m, 1 H, ArH), 6.71-6.64 (m, 2 H, ArH), 6.28
(d, J = 7.6 Hz, 1 H, CH), 5.57 (d, J = 7.6 Hz, 1 H, NH), 4.77-4.66
(m, 1 H, CH), 2.06-1.86 (m, 8 H, 4 CH2), 1.52-1.41 (m, 2 H,
CH2); 13C-NMR (75 MHz, CDCl3): 176.6, 156.4, 155.6, 153.7,
141.9, 134.3, 132.9, 128.8, 125.8, 125.7, 123.4, 121.0, 120.0,
118.4, 112.3, 110.4, 58.5, 44.5, 33.3, 32.9, 25.3, 25.2, 24.9;
HRMS (EI+) calcd. for C23H22N5O2Br: 479.0957, found:
479.0956.
3-((1-cyclohexyl-1H-tetrazol-5-yl)((2-
nitrophenyl)amino)methyl)-4H-chromen-4-one (1n)
3-((3,4-dimethoxyphenyl)amino)((1-(2,6-dimethylphenyl)-1H-
tetrazol-5-yl)methyl)-4H-chromen-4-one (1j)
Yield = 71%; Yellow solid; mp = 180-182 °C; Rf = 0.28
(Hexanes-AcOEt, 7:3 V/V); FT-IR (ATR) νmax/cm-1: 3338 (N-H),
1640 (C=O), 1263 (N-N=N), 1227 (C–O–C); 1H-NMR (500
MHz, CDCl3): 9.0 (d, J = 6.5 Hz, 1 H, NH), 8.31 (s, 1 H, CH),
8.25 (d, J = 7.7 Hz, 2 H, ArH), 7.76-7.71 (m, 1 H, ArH), 7.51-
7.44 (m, 3 H, ArH), 6.85 (d, J = 8.51 Hz, 1 H, ArH), 6.83-6.79
(m, 1 H, ArH), 6.48 (d, J = 6.7 Hz, 1 H, CH), 4.73-4.65 (m, 1 H,
CH), 2.08-2.01 (m, 2 H, CH2), 1.99-1.85 (m, 4 H, 2 CH2), 1.79-
1.73 (m, 1 H, 1 H of CH2), 1.54-1.39 (m, 2 H, CH2), 1.36-1.25
(m, 1 H, CH2); 13C-NMR (125 MHz, CDCl3): 176.1, 156.4,
156.3, 153.2, 142.0, 136.6, 134.6, 133.6, 127.2, 126.0, 125.8,
123.3, 121.1, 118.5, 117.6, 113.9, 58.5, 43.0, 33.2, 33.0, 25.3,
25.2, 24.8; HRMS (EI+) calcd. for C23H21N6O4: 445.0397, found:
445.0399.
Yield = 55%; brown solid; mp = 171-173 °C; Rf = 0.11
(Hexanes-AcOEt, 7:3 V/V); FT-IR (ATR)νmax/cm-1: 3330 (N-H),
1648 (C=O), 1289 (N-N=N), 1222 (C–O–C); 1H-NMR (500
MHz, CDCl3):
8.26 (s, 1 H, CH), 8.00 (d, J = 8.0 Hz, 1 H,
ArH), 7.71-7.66 (m, 1 H, ArH), 7.44 (d, J = 8.5 Hz, 1 H, ArH),
7.43-7.38 (m, 1 H, ArH), 7.36-7.32 (m, 1 H, ArH), 7.17 (d, J =
7.6 Hz, 1 H, ArH), 7.13 (d, J = 7.6 Hz, 1 H, ArH), 6.61 (d, J =
8.5 Hz, 1 H, ArH), 6.23-6.21 (m, 1 H, ArH), 6.08 (d, J = 8.5 Hz,
1 H, CH), 5.83 (d, J = 8.9 Hz, 1 H, ArH), 4.56 (d, J = 9.0 Hz, 1
H, NH), 3.75 (s, 3 H, OCH3), 3.71 (s, 3 H, OCH3), 1.90-1.86 (m,
13
6 H, 2 CH3); C-NMR (125 MHz, CDCl3): 175.9, 156.2, 156.1,
155.3, 150.0, 143.1, 139.4, 136.2, 136.1, 134.1, 131.5, 131.1,
128.9, 128.8, 125.8, 125.6, 123.5, 121.3, 118.2, 112.8, 105.7,
100.3, 56.4, 55.8, 45.2, 17.4, 17.2; HRMS (EI+) calcd. for
C27H25N5O4: 483.1907, found: 483.1922.
4.4. Evaluation of antiprotozoal activity. Antiprotozoal assays:
Entamoeba histolytica strain HM1-IMSS used in all experiments
was grown axenically at 37 ºC in TYI-S-33 medium
supplemented with 10% heat inactivated bovine serum. In the
case of Giardia lamblia, strain IMSS: 8909:1 was grown in TYI-
S-33 modified medium supplemented with 10% calf serum and
bovine bile. Trophozoites of Trichomona vaginalis strain GT3
were maintained in TYI-S-33 medium supplemented with10%
bovine serum. The trophozoites were axenically maintained and
were employed for assays in the log phase of growth. In vitro
susceptibility tests were performed using the subculture method
previously described.30 Briefly, E. histolytica (6 × 103) or G.
lamblia (5 × 104) trophozoites were incubated for 48 h at 37 ºC in
the presence of different concentrations (2.5-200 µg//mL) of pure
compounds 1a-n in dimethyl sulfoxide (DMSO). Each test
included standard amoebicidal and giardicidal drugs, a control
(culture medium plus trophozoites and DMSO) and a blank
(culture medium). After incubation, the trophozoites were
detached by chilling and 50 µL samples of each tube were
subcultured in fresh medium for another 48 h, without
antiprotozoal samples. The final number of parasites was
determined with a hemocytometer and the percentages of
trophozoites growth inhibition were calculated by comparison
with the control culture. The results were confirmed by a
colorimetric method: the trophozoites were washed and
incubated for 45 min at 37 ºC in a saline phosphate buffer with
3-((1-cyclohexyl-1H-tetrazol-5-yl)((3,4,5-
trimethoxyphenyl)amino)methyl)-4H-chromen-4-one (1k)
Yield = 90%; yellow solid; mp = 160-162 °C; Rf = 0.14
(Hexanes-AcOEt, 7:3 V/V); FT-IR (ATR) νmax/cm-1: 3332 (N-H),
1650 (C=O), 1281 (N-N=N), 1235 (C–O–C); 1H-NMR (400
MHz, CDCl3):
8.21 (s, 1 H, CH), 8.04 (d, J = 7.9 Hz, 1 H,
ArH), 7.59-7.54 (m, 1 H, ArH), 7.35-7.28 (m, 2 H, ArH), 6.06 (d,
J = 7.9 Hz, 1 H, CH), 5.86 (s, 2 H, ArH), 5.01 (d, J = 8.1 Hz, 1
H, NH), 4.53-4.45 (m, 1 H, CH), 3.61 (s, 6 H, 2 OCH3), 3.59 (s, 3
H, OCH3), 1.87-1.72 (m, 8 H, 4 CH2), 1.36-1.24 (m, 2 H, CH2);
13C-NMR (100 MHz, CDCl3): 175.3, 155.0, 154.6, 153.0, 152.7,
140.4, 133.0, 129.9, 124.5, 124.2, 122.0, 120.5, 117.1, 90.4, 63.0,
59.7, 57.0, 54.7, 43.2, 31.8, 31.5, 24.0, 23.9, 23.5; HRMS (EI+)
calcd. for C26H29N5O5: 491.2169, found: 491.2164.
3-((2-bromophenyl)amino)((1-(tert-butyl)-1H-tetrazol-5-
yl)methyl)-4H-chromen-4-one (1l)
Yield = 72%; yellow solid; mp = 180-182 °C; Rf = 0.37
(Hexanes-AcOEt, 7:3 V/V); FT-IR (ATR)νmax/cm-1: 3300 (N-H),
1650 (C=O), 1281 (N-N=N), 1235 (C–O–C); H-NMR (300
1
MHz, CDCl3): 8.28 (d, J = 0.8 Hz, 1 H, CH), 8.17 (dd, J = 8.0,
1.6 Hz, 1 H, ArH), 7.72-7.65 (ddd, J = 8.6, 7.1, 1.7 Hz, 1 H,
ArH), 7.49-7.39 (m, 3 H, ArH), 7.20-7.14 (m, 1 H, ArH), 6.74-
6.65 (m, 2 H, ArH), 6.56 (d, J = 8.2 Hz, 1 H, CH), 4.96 (d, J =
8.2 Hz, 1 H, NH), 1.85 (s, 9 H, 3 CH3); 13C-NMR (75 MHz,
CDCl3): 176.7, 156.5, 155.7, 153.7, 141.7, 134.2, 132.9, 129.0,
125.6, 123.4, 121.3, 120.1, 118.4, 112.1, 110.2, 62.3, 45.0, 30.0;
MTT
(3-[4,5-dimethylhiazol-2-il]-2,5-diphenyl
tetrazolium
bromide) and phenazine methosulfate. The dye produced
(formazan) was extracted and the absorbance was determined at

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Acknowledgments
R. G.-M. Thanks CONACYT for financial support (Project:
CB-2011-166747-Q), also to Dr. Francisco Delgado (ENCB-
IPN), Dr. Erick Cuevas (CCIQS-UAEMex) and Dr. Luis D.
Miranda (IQ-UNAM) for technical support respect to HRMS,
NMR and X-ray analysis respectively. Post-doctoral CONACYT
scholarship awarded to Dr. Alejandro Islas Jácome (CVU
176745, 290662_UG, 290735_UG) is gratefully acknowledged.
Pedro A. Cano acknowledges the CONACYT scholarship
(370144/304476).
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Supplementary Material
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submitted along with the manuscript and graphic files to the
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