Leishmanicidal evaluation of novel synthetic chromenes

Article abstract of DOI:10.1002/ardp.200800128

In the present paper, twelve chromenes were synthesized by coupling of 2,2,8,8-tetramethyl-8H-pyrano[2,3-f]chroman-4-one 1 with various aryl and benzylmagnesium chlorides. The synthetic compounds were examined for in-vitro activity against Leishmania majo

Full text of DOI:10.1002/ardp.200800128

Arch. Pharm. Chem. Life Sci. 2008, 341, 787–793
B. H. Alizadeh et al.
787
Full Paper
Leishmanicidal Evaluation of Novel Synthetic Chromenes
Babak Heidary Alizadeh1, Alireza Foroumadi2, Susan K. Ardestani3, Fatemeh Poorrajab3, and
Abass Shafiee2
1 Iranian Research Institute of Plant Protection (IRIPP), Tehran, Iran
2 Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center,
Tehran University of Medical Sciences, Tehran, Iran
3 Institute of Biochemistry and Biophysics, Department of Biochemistry, University of Tehran, Tehran, Iran
In the present paper, twelve chromenes were synthesized by coupling of 2,2,8,8-tetramethyl-8H-
pyrano[2,3-f]chroman-4-one 1 with various aryl and benzylmagnesium chlorides. The synthetic
compounds were examined for in-vitro activity against Leishmania major, and some of them dis-
played efficient anti-leishmanial activity. Among the compounds tested, compounds 9 (4-(2-
chloro-benzylidene)-2,2,8,8-tetramethyl-3,4-dihydro-2H,8H-pyrano[2,3-f]chromene 9a and 4-(2-
chloro-benzyl)-2,2,8,8-tetramethyl-2H,8H-pyrano[2,3-f]chromene 9b) were the most active with an
inhibitory activity of 73.4%.
Keywords: Chromene / Leishmanicidal activity / Synthesis /
Received: July 8, 2008; accepted: August 14, 2008
DOI 10.1002/ardp.200800128
bite of an infected sandfly, in the mammalian hosts they
differentiate to the amastigote stage, which lack flagella
and is an obligate intracellular pathogen infecting hema-
topoietic cells [3, 4]. Unfortunately, there are no effective
vaccines against the parasite yet. Thus, control of the dis-
ease relies primarily on chemical treatment. There are
over 17 species of Leishmania known to be infective to
humans. The species have been characterized on the
basis of biochemical and molecular differences and these
differences provide an explanation for variation in the
clinical responses and species' sensitivity to the pentava-
lent antimonials, sodium stibogluconate and meglumine
antimonate (Glucantime), in the treatment of leishma-
niasis over the past 50 years. Generally, the antileishma-
nial drugs presently on the market possess severe side
effects, are expensive, need long-term treatment, and do
not provide complete eradication of the disease. In addi-
tion, resistance to current drugs develops rapidly and a
large-scale resistance to pentavalent antimonials has
been reported in parts of the world, for example in India
and Sudan, so they are quickly becoming obsolete
[2, 5, 6]. Therefore, new efficacious and less toxic chemi-
cal agents for human are urgently needed.
Introduction
Protozoal parasitic diseases continue to pose a serious
problem for the public health in the world. Among them,
Leishmania spp. are causative agents of mortality and
morbidity in the human known as leishmaniasis, and it
was estimated that worldwide about twelve million peo-
ple are affected, with approximately 350 million individ-
uals at risk. During the last ten year, there has been this
global burden and extensive epidemic forms of the dis-
ease due to human migration, particularly problematic
is its coincidence with HIV and the capacity of Leishmania
to infect specialized immune cells [1, 2]. Leishmania spp.
are obligate intracellular parasites that proliferate and
develop in a virulent metacyclic stage into an inverte-
brate vector which can infect diverse vertebrate hosts.
The two distinct developmental stages of Leishmania are
recognized as promastigotes and amastigotes. The first
form which is found within the midgut of sandflies has
an elongated shape and long flagellum, but upon the
Correspondence: Abass Shafiee, Department of Medicinal Chemistry,
Faculty of Pharmacy and Pharmaceutical Sciences Research Center,
Tehran University of Medical Sciences, Tehran 14174, Iran.
E-mail: ashafiee@ams.ac.ir
There has been a revival of drug research and develop-
ment regarding neglected parasitic diseases compared to
Fax: +98 21 664-611-78
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B. H. Alizadeh et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 787–793
Table 1. Camparison of the inhibitory effect of the compounds
on the viability of L. major promastigotes under in-vitro condi-
tions.
Figure 1. Structure of synthetic leishmanicidal chromenes and
Glyasperin.
Compound
R
Inhibition %
after 72 h
the last 15 years, and novel chemical classes are being
pursued for the treatment. Natural products are known
as the chief source and are often identified as lead struc-
tures; the derivatives of such compounds may result in
highly effective agents with reduced toxicity and side
effects [7, 8].
1
3
4
–
h
4-Cl
H
4-Cl
3-Cl
2-Cl
2,6-diCl
2,3-diCl
2,4-diCl
4-F
3-OMe
4-OMe
49.4
38.0
53.2
38.0
41.8
48.1
73.4
65.8
29.1
60.8
59.5
36.7
30.4
67.2
6a, b
7a, b
8a, b
9a, b
10a, b
11a, b
12a, b
13a, b
14a, b
15a, b
Glucantime
Recently, Glyasperin, a chromene isolated from Smirno-
via iranica, has been reported to be highly effective
against the Leishmania parasite (Fig. 1). Chromenes are
structurally simple compounds belonging to a large class
of molecules known as benzopyranes, and the chromane-
4-one moiety is an integral part of many natural products
[9]. These compounds and related derivatives have
diverse biological activities, including antitumor, anti-
bacterial, and leishmanicidal potency [10] that make
them attractive agents for screening novel therapeutic
drugs as do further backbone substitutions [11, 12]. In
order to find new drugs with more potent activity against
Leishmania parasite, we have been engaged in a program
to investigate some new synthetic compounds [13–16].
Moreover, the biological potency of heterocyclic chro-
menes has been widely documented and considerable
efforts have been made to explore new routes for the syn-
thesis of therapeutically more efficient heterocyclic chro-
menes. In an investigation to find new anti-leishmanial
agents which are structurally related to 2,2-dimethyl-2H-
chromenes, a series of synthetic chromenes was prepared
by a condensation reaction of chromene 1 and Grignard
reagents [17, 18]. The prepared compounds, namely com-
pounds 1, 3, 4, and 6–15 were tested against Leishmania
major promastigotes. Some of the derivatives were mod-
erately active against L. major, whereas the others dis-
played good activity against the parasite (Table 1).
by the reaction of compound 1 with appropriate
Grignard reagents and an subsequent acidic dehydration
reaction in refluxing 1 N HCl [22] (Scheme 1). Com-
pounds 6(a, b) to 15(a, b) were a mixture of two regio-iso-
1
mers and their structures were established by H-NMR
analysis (compounds 3, 4, 6(a, b) to 8(a, b); they were
reported previously [23]). In addition, exo-isomers (9b to
15b) were a mixture of E- and Z-isomers. It should be
noted that none of these isomers could be separated by
conventional methods [24]. Differentiation between exo-
and endo-isomers as well as the E- and Z-form of exo-iso-
mers were based on the H-NMR, 13C-NMR, and NOESY
1
experiments. In 1H-NMR, the observation of a doublet at
2.27 to 2.72 ppm (3-CH2 of compound 9a to 15a) and a sin-
glet at 3.63 to 3.93 ppm (benzylic CH2 of compound 9a to
15a) showed the existence of both exo- and endo-isomers
in the ratio of the 46 : 54 and 75 : 25, respectively. More-
over, the exo-product appeared to be a mixture of E- and
Z-steroisomers which were assigned based on the aniso-
tropic effect of the substituted phenyl ring on H5 of the Z-
isomer. In the E-isomer, H5 of compound 10a appeared as
a doublet at 7.47 (J = 8.5 Hz) and in the Z-isomer the H5
appeared at 7.45 ppm (J = 8.5 Hz). However, in most com-
pounds the Z-isomers were a minor and an undetectable
product. The configuration of the E-isomer as a major
product was confirmed through 2D-NOESY NMR spectro-
scopy and the assignment of stereochemistry of com-
Chemistry
Compound 1 (Scheme 1) was prepared by thermal cycliza-
tion of 2,2-dimethyl-7-(2-methylbut-3-yn-2-yloxy)-chro-
man-4-one in N,N-dimethyl-aniline at 1808C [19–21].
Compounds 3, 4, and 6(a, b) to 15(a, b) were synthesized
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Arch. Pharm. Chem. Life Sci. 2008, 341, 787–793
Leishmanicidal Synthetic Chromenes
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Scheme 1. Synthesis of compounds 3, 4, 6(a, b)-15(a, b).
pound 10a is consisted with the observed strong cross-
peak between H5 and vinylic-H (Ar-CH=C) and the rela-
tively weak cross-peak between H3 and the ortho-H phenyl
ring. In addition, in 13C-NMR the 3-CH2 of exo and vinylic
CH2 of the endo-isomers appeared as two separate signals
at 32.7–37.3 and 37.1–37.9 ppm. The vinylic CH (exo-iso-
mer) and olefinic CH (endo-3-CH) appeared as two sepa-
rate singlets at 117.2–120.5 ppm and 123.2–123.6 ppm,
respectively. The Dept-135 and Dept-90 experiments con-
firmed the mentioned CH2 and CH groups.
Results and discussion
Promastigotes were seeded at approximately 26106 cells/mL and during 8 days. The
cell concentrations were determined daily by Neubauer`s chamber. (0) The growth
phases of untreated cells, (G) Promastigote growth rate after treatment with Glucan-
time as a standard. Each time point represents the mean value of three replicates with
the standard deviation.
Inhibitory effects of the synthesized compounds in a con-
centration of 10 lg/mL were determined by counting the
number of treated promastigotes remaining alive in the
cultures, and by analyzing the growth inhibition of the
parasit as described above. The growth kinetics of L. major
for a period of 8 days is shown in Fig. 2. The chromenes
exhibited a clear time-dependent inhibitory effect on L.
major (Fig. 3). Five compounds (4, 9, 10, 12, and 13) were
shown to be effective after 72 h incubation, with a
growth inhibition A 50% (Table 1).
The chromanone intermediate 1 was moderately active
(Inh.% = 49.4). The target compounds 3 and 6 having no
substitution in phenyl ring had the same inhibitory
activity (Inh % = 38.0). Insertion of a Cl in the 4-position of
phenyl ring in compound 3, increased the inhibitory
effect to 53.2% (compound 4). As can be deduced from the
Figure 2. The growth curve of L. major.
data in Table 1, substitution of a chlorine atom in phenyl
ring of compound 6 enhanced the biological activity of
the compounds in the following order: 2-Cl A 3-Cl A 4-Cl.
In fact, chromene 9 having 2-chloro substitution was the
most active compound showing 73.4% inhibitory activ-
ity. Addition of the second Cl in the 3-position of the phe-
nyl ring in compound 9 (namely compound 11) resulted
in a large decrease in inhibitory activity (Inh. % = 29.1).
However, compounds 10 and 12 having 2,6- and 2,4-
dichloro substitutions showed 65.8% and 60.8% inhibi-
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Arch. Pharm. Chem. Life Sci. 2008, 341, 787–793
0.8 M) under argon. The resulting mixture was stirred and
refluxed for 18 hours. After cooling, the organic phase was
washed with 1 N HCl (365 mL). The solvent was evaporated
under reduce pressure to give an oil. To this oily residue was
added 2 N HCl (15 mL) and the mixture was refluxed for 12 hours.
After cooling, it was extracted with ethyl acetate (3620 mL). The
organic layer was washed with 1 N NaOH and saturated aqueous
NaHCO3 and was dried with Na2SO4. After filtration, the solvent
was evaporated under reduce pressure. The residue was purified
byflashcolumnchromatography(silicagel= 25 g, hexane /EtOAc
= 5 : 1) to give 9a, b (0.33 g, 0.92 mmol, 79%) as a colorless oil as
mixture ofexo-and endo-isomers (48 : 52).
9a (exo-isomer)
1H-NMR (CDCl3) d [ppm]: 1.27 (s, 6H, 2-CH3), 1.45 (s, 6H, 8-CH3),
2.56 (d, 2H, J = 1.2 Hz, 3-CH2), 5.54 (d, 1H, J = 9.9 Hz, H9), 6.68 (d,
1H, J = 8.4 Hz, H6), 6.69 (d, 1H, J = 9.9 Hz, H10), 7.01 (s, 1H, 2-Cl-Ph-
CH=C), 7.16–7.40 (m, 4H, Ar), 7.46 (d, 1H, J = 8.4 Hz, H5).
The parasites were cultured for 24 h (white bar), 48 h (grey bar), and 72 h (black bar)
in the presence of the compounds as described in Experimental, Section 4. The
results are given as the percentage of inhibition (mean l SD).
Figure 3. The effects of the synthetic agents on the in vitro
growth rate of L. major promastigotes.
9b (endo-isomer)
1H-NMR (CDCl3) d [ppm]: 1.40 (s, 6H, 2-CH3), 1.41 (s, 6H, 8-CH3),
3.76 (s, 2H, 2-ClPh-CH2), 5.08 (s, 1H, 3-CH), 5.57 (d, 1H, J = 9.9 Hz,
H9), 6.31 (d, 1H, J = 8.4 Hz, H6), 6.71 (d, 1H, J = 9.9 Hz, H10), 6.86 (d,
1H, J = 8.4 Hz, H5), 7.16–7.40 (m, 4H, Ar).
tory activity, respectively. Replacement of Cl in com-
pound 7 with F increased the inhibitory action to 59.5%.
In contrast, substitution with a methoxy group, compare
compounds 14 and 15, decreased the inhibitory percent-
age to 36.7 and 30.4, respectively.
9a/9b (exo-/endo-isomers)
13C-NMR (CDCl3) d [ppm]: 27.8 (q), 27.9 (q), 28.0 (q), 35.0 (t), 37.4 (t),
75.7 (s), 76.0 (s), 76.1 (s), 108.3 (d), 109.1 (d), 110.1 (s), 110.2 (s),
114.3 (s), 115.2 (s), 116.9 (d), 117.0 (d), 118.4 (d), 123.4 (d), 124.6
(d), 126.1 (d), 126.8 (d), 127.7 (d), 127.8 (d), 127.9 (d), 128.2 (d),
128.5 (d), 129.0 (d), 129.4 (d), 130.5 (d), 131.1 (d), 131.3 (s), 131.9
(s), 134.4 (s), 134.5 (s), 135.9 (s), 136.7 (s), 148.9 (s), 149.3 (s), 153.7
(s), 154.2 (s). IR (KBr) [cm– 1]: 2975 (s), 2930 (sh), 1632 (s), 1610 (sh),
1472 (m), 1250 (w), 1170 (w), 1114 (s), 1055 (s), 815 (s). MS m/z [%]:
368 [M+ + 2] (13), 366 [M+] (40), 354 (18), 251 (22), 339 (87), 336
(100), 163 (13). Anal. Calcd. for C23H23ClO2: C, 75.30; H, 6.32.
Found: C, 75.28; H, 6.36.
This research was supported by a grant from INSF (Iran
National Science Foundation). The author is indebted to Miss
Javidnia and Mr. Abdi for their help in taking Mass, NMR, and
IR spectra, respectively.
The authors have declared no conflict of interest.
The other compounds 10(a, b) to 15(a, b) were prepared by the
same method. In addition, analytical data of compounds 3, 4,
6(a, b) to 8(a, b) were reported previously [19].
Experimental
General
All chemicals and reagents were obtained from Merck Chemical
Company (Darmstadt, Germany) and Sigma-Aldrich Chemical
Company (Steinhein, Germany). 1H-NMR spectra were measured
using a Bruker 500 spectrometer (Brucker, Rheinstetten, Ger-
many) and chemical shifts are expressed as d (ppm) with tetra-
methylsilane as internal standard. The IR spectra were taken
using a Nicolet FT-IR Magna 550 spectrographs (KBr disks) (Nico-
let, Madison, WI, USA). MS spectra were obtained with a Finni-
gan MAT TSQ-70 spectrometer (Finnigan Mat, Bremen, Ger-
many). The purity of a compound was confirmed by TLC using
different mobile phases. The results of elemental analyses (C, H,
N) were within l 0.4% of theoretical values for C, H, and N.
4-(2,6-di-Chloro-benzylidene)-2,2,8,8-tetramethyl-3,4-
dihydro-2H,8H-pyrano[2,3-f]chroman (10a-exo) and 4-
(2,6-di-Chloro-benzyl)-2,2,8,8- tetramethyl-2H,8H-
pyrano[2,3-f]chromene (10b-endo)
A colorless oil (69%) as mixture of exo- (E and Z, 97 : 3) and endo-
isomers (63 : 37).
10a (exo-isomer)
1H-NMR (CDCl3) d [ppm]: 1.25(Z) and 1.26(E) (2s, 6H, 2-CH3), 1.46 (s,
6H, 8-CH3), 2.27 (d, 2H, J = 1.4 Hz, 3-CH2), 5.56 (d, 1H, J = 9.9 Hz,
H9), 6.44 (d, 1H, J =8.4 Hz, H6), 6.67 (d, 1H, J = 9.9 Hz, H10), 6.71 (s,
1H, 2,6-di-Cl-Ph-CH=C), 7.07–7.34 (m, 3H, Ar), 7.45(Z) (d, 1H, J =
8.5 Hz, H5), 7.47(E) (d, 1H, J = 8.5 Hz, H5).
Chemistry
4-(2-Chloro-benzylidene)-2,2,8,8-tetramethyl-3,4-
dihydro-2H,8H-pyrano[2,3-f] chroman 9a and 4-(2-
Chloro-benzyl)-2,2,8,8-tetramethyl- 2H,8H- pyrano[2,3-
f]chromene 9b
To a stirred solution of 1 (0.3 g, 1.2 mmol) in dry ether (3 mL) was
added 2-chlorobenzylmagnesium chloride (1.25 mL, 1 mmol,
10b (endo-isomer)
1H-NMR (CDCl3) d [ppm]: 1.31 (s, 6H, 2-CH3), 1.43 (s, 6H, 8-CH3),
3.93 (s, 2H, 2,6-di-Cl-Ph-CH2), 5.07 (s, 1H, 3-CH), 5.58 (d, 1H, J =
9.9 Hz, H9), 6.42 (d, 1H, J = 8.5 Hz, H6), 6.70 (d, 1H, J = 9.9 Hz, H10),
7.12 (d, 1H, J = 8.5 Hz, H5), 7.07–7.34 (m, 3H, Ar).
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Arch. Pharm. Chem. Life Sci. 2008, 341, 787–793
Leishmanicidal Synthetic Chromenes
791
10a/10b (exo-/endo-isomers)
12b (endo-isomer)
13C-NMR (CDCl3) d [ppm]: 26.6 (q), 27.9 (q), 28.0 (q), 32.7 (t), 38.0 (t),
75.8 (s), 76.0 (s), 76.0 (s), 76.5 (s), 108.1 (d), 108.6 (d), 110.0 (s),
110.2 (s), 113.6 (s), 115.5 (s), 116.9 (d), 117.0 (d), 122.4 (d), 122.9
(d), 124.5 (d), 127.3 (d), 127.8 (d), 127.9 (d), 128.1 (d), 128.2 (d),
128.6 (d), 129.0 (d), 131.7 (s), 131.9 (s), 133.3 (s), 134.8 (s), 135.2 (s),
135.7 (s), 136.4 (s), 148.7 (s), 149.3 (s), 153.7 (s), 154.3 (s). IR (KBr)
[cm– 1]: 2980 (s), 2930 (sh), 1638 (vs), 1625 (sh), 1410 (sh), 1435 (s),
1365 (m), 1207 (m), 1118 (vs), 1054 (m), 777 (m). MS m/z [%]: 404
[M+ + 4] (4), 403 [M+ + 3] (7), 402 [M+ + 2] (26), 401 [M+ + 1] (10), 400
[M+] (40), 386 (100), 323 (10), 239 (11), 149 (14). Anal. Calcd. for
C23H22Cl2O2: C, 68.83; H, 5.53. Found: C, 68.73; H, 5.45.
1H-NMR (CDCl3) d [ppm]: 1.37 (s, 6H, 2-CH3), 1.41 (s, 6H, 8-CH3),
3.71 (s, 2H, 2,4-di-Cl-Ph-CH2), 5.08 (s, 1H, 3-CH), 5.58 (d, 1H, J =
9.8 Hz, H9), 6.28 (d, 1H, J = 8.4 Hz, H6), 6.70 (d, 1H, J = 9.8 Hz, H10),
6.80 (d, 1H, J = 8.4 Hz, H5), 7.08–7.42 (m, 3H, Ar).
12a/12b (exo-/endo-isomers)
13C-NMR (CDCl3) d [ppm]: 27.2 (q), 27.9 (q), 28.0 (q), 34.5 (t), 37.4 (t),
75.6 (s), 76.1 (s), 76.5 (s), 108.4 (d), 109.2 (d), 110.2 (s), 110.4 (s),
114.0 (s), 114.9 (s), 116.8 (d), 116.9 (d), 117.2 (d), 123.2 (d), 124.6
(d), 126.4 (d), 126.5 (d), 127.2 (d), 127.9 (d), 128.2 (d), 128.5 (d),
129.0 (d), 129.1 (d), 129.3 (d), 131.3 (d), 131.7 (d), 132.7 (s), 132.8
(s), 134.5 (s), 134.9 (s), 135.2 (s), 135.4 (s), 135.9 (s), 136.5 (s), 148.8
(s), 149.3 (s), 153.8 (s), 154.4 (s). IR (KBr) [cm– 1]: 2971 (s), 2894 (m),
1629 (vs), 1571 (s), 1475 (s), 1430 (m), 1369 (s), 1272 (m), 1180 (s),
1108 (s), 1150 (s), 1059 (m), 942 (m), 894 (w), 813 (s), 721 (s). MS
m/z [%]: 404 [M+ + 4] (6), 403 [M+ + 3] (10), 402 [M+ + 2] (40), 401 [M+ +
1] (16), 400 [M+] (60), 386 (100), 382 (24), 240 (10), 224 (13), 169
(15), 161 (27), 150 (22), 142 (15), 79 (18). Anal. Calcd. for
C23H22Cl2O2: C, 68.83; H, 5.53. Found: C, 68.73; H, 5.48.
4-(2,3-di-Chloro-benzylidene)-2,2,8,8-tetramethyl-3,4-
dihydro-2H,8H-pyrano[2,3-f]chroman (11a) and 4-(2,3-di-
Chloro-benzyl)-2,2,8,8-tetramethyl-2H,8H- pyrano[2,3-
f]chromene (11b)
A colorless oil (67%) as mixture of exo- and endo-isomers
(46 : 54).
11a (exo-isomer)
1H-NMR (CDCl3) d [ppm]: 1.28 (s, 6H, 2-CH3), 1.45 (s, 6H, 8-CH3),
2.53 (d, 2H, J = 1.4 Hz, 3-CH2), 5.52 (d, 1H, J = 9.8 Hz, H9), 6.45 (d,
1H, J = 8.4 Hz, H6), 6.69 (d, 1H, J = 9.8 Hz, H10), 6.97 (s, 1H, 2,3-di-Cl-
Ph-CH=C), 7.10–7.36 (m, 3H, Ar), 7.45 (d, 1H, J = 8.6 Hz, H5).
4-(4-Fluorobenzylidene)-2,2,8,8-tetramethyl-3,4-dihydro-
2H,8H-pyrano[2,3-f] chroman (13a) and 4-(4-Fluoro
benzyl)-2,2,8,8-tetramethyl-2H,8H-pyrano[2,3-
f]chromene (13b)
A colorless oil (70%) as mixture of exo- and endo-isomers
(53 : 47).
11b (endo-isomer)
1H-NMR (CDCl3) d [ppm]: 1.39 (s, 6H, 2-CH3), 1.40 (s, 6H, 8-CH3),
3.79 (s, 2H, 2,3-di-Cl-Ph-CH2), 5.10 (s, 1H, 3-CH), 5.58 (d, 1H, J =
9.8 Hz, H9), 6.28 (d, 1H, J = 8.4 Hz, H6), 6.71 (d, 1H, J = 9.8 Hz, H10),
6.80 (d, 1H, J = 8.4 Hz, H5), 7.10–7.36 (m, 3H, Ar).
13a (exo-isomer)
11a/11b (exo-/endo-isomers)
1H-NMR (CDCl3) d [ppm]: 1.28 (s, 6H, 2-CH3), 1.45 (s, 6H, 8-CH3),
2.66 (d, 2H, J = 1.4 Hz, 3-CH2), 5.55 (d, 1H, J = 9.9 Hz, H9), 6.28 (d,
1H, J = 8.4 Hz, H6), 6.70 (d, 1H, J = 9.9 Hz, H10), 6.98 (s, 1H, 4-F-Ph-
CH=C), 6.99–7.24 (m, 4H, Ar), 7.40 (d, 1H, J = 8.5 Hz, H5).
13C-NMR (CDCl3) d [ppm]: 26.7 (q), 27.9 (q), 28.0 (q), 36.0 (t), 37.4 (t),
75.7 (s), 76.0 (s), 76.1 (s), 76.5 (s), 108.4 (d), 109.1 (d), 110.1 (s),
110.3 (s), 114.0 (s), 114.9 (s), 116.8 (d), 116.9 (d), 118.3 (d), 123.3
(d), 124.6 (d), 126.5 (d), 126.6 (d), 127.0 (d), 127.2 (d), 128.5 (d),
128.6 (d), 129.1 (d), 129.2 (d), 129.4 (d), 132.5 (s), 132.6 (s), 132.7
(s), 133.0 (s), 133.2 (s), 138.2 (s), 139.2 (s), 148.9 (s), 149.4 (s), 153.8
(s), 154.4 (s). IR (KBr) cm– 1: 2974 (vs), 1634 (s), 1576 (s), 1480 (s),
1450 (sh), 1368 (m), 1271 (m), 1213 (s), 1174 (s), 1150 (s), 1112 (vs),
1058 (s), 942 (w), 894 (w), 812 (w), 725 (m). MS m/z (%): 404 [M+ + 4]
(5), 403 [M+ + 3] (8), 402 [M+ + 2] (32), 401 [M+ + 1] (13), 400 [M+] (50),
386 (100), 380 (12), 223 (10), 184 (16), 141 (13), 99 (31), 61 (50).
Anal. Calcd. for C23H22Cl2O2: C, 68.83; H, 5.53. Found: C, 68.76; H,
5.58.
13b (endo-isomer)
1H-NMR (CDCl3) d [ppm]: 1.40 (s, 6H, 2-CH3), 1.41 (s, 6H, 8-CH3),
3.63 (s, 2H, 4-F-Ph-CH2), 5.16 (s, 1H, 3-CH), 5.57 (d, 1H, J = 9.9 Hz,
H9), 6.42 (d, 1H, J = 8.4 Hz, H6), 6.70 (d, 1H, J = 9.9 Hz, H10), 6.85 (d,
1H, J = 8.5 Hz, H5), 6.99–7.24 (m, 4H, Ar).
4-(2,4-di-Chloro-benzylidene)-2,2,8,8-tetramethyl-3,4-
dihydro-2H,8H-pyrano[2,3-f]chroman (12a) and 4-(2,4-di-
Chloro-benzyl)-2,2,8,8-tetramethyl- 2H,8H- pyrano[2,3-
f]chromene (12b)
A colorless oil (64%) as mixture of exo-and endo-isomers (56 : 44).
13a/13b (exo-/endo-isomers)
13C-NMR (CDCl3) d [ppm]: 26.7 (q), 27.9 (q), 28.1 (q), 37.0 (t), 37.1 (t),
75.4 (s), 75.9 (s), 76.0 (s), 76.4 (s), 108.2 (d), 109.0 (d), 110.1 (s),
110.2 (s), 114.5 (s), 115.0 (d), 115.1 (d), 115.2 (d), 115.3 (d), 116.8
(d), 117.0 (d), 120.0 (d), 123.4 (d), 124.1 (d), 126.2 (d), 128.5 (d),
128.9 (d), 130.1 (d), 130.2 (d), 130.5 (s), 130.7 (d), 130.8 (d), 133.5
(s), 133.6 (s), 134.5 (s), 134.6 (s), 149.1 (s), 153.6 (s), 153.9 (s), 160.4
(s), 160.5 (s), 162.3 (s), 162.4 (s). IR (KBr) [cm– 1]: 2974 (s), 2930 (m),
1592 (vs), 1506 (vs), 1471 (s), 1366 (s), 1270 (m), 1215 (s), 1114 (s),
1054 (s), 818 (m). MS m/z [%]: 351 [M+ + 1] (24), 350 [M+] (40), 285
(25), 267 (100), 174 (10), 108 (28), 68 (41), 66 (57). Anal. Calcd. for
C23H23FO2: C, 78.83; H, 6.62. Found: C, 78.78; H, 6.68.
12a (exo-isomer)
1H-NMR (CDCl3) d [ppm]: 1.28 (s, 6H, 2-CH3), 1.48 (s, 6H, 8-CH3),
2.52 (d, 2H, J = 1.3 Hz, 3-CH2), 5.54 (d, 1H, J = 9.8 Hz, H9), 6.45 (d,
1H, J = 8.4 Hz, H6), 6.68 (d, 1H, J = 9.8 Hz, H10), 6.92 (s, 1H, 2,4-di-Cl-
Ph-CH=C), 7.08–7.42 (m, 3H, Ar), 7.43 (d, 1H, J = 8.4 Hz, H5).
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792
B. H. Alizadeh et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 787–793
4-(3-Methoxy-benzylidene)-2,2,8,8-tetramethyl-3,4-
dihydro-2H,8H-pyrano[2,3-f]chroman (14a) and 4-(3-
Methoxy-benzyl)-2,2,8,8-tetramethyl-2H,8H- pyrano[2,3-
f]chromene (14b)
A colorless oil (70%) as mixture of exo- and endo-isomers
(54 : 46).
15a/15b (exo-/endo-isomers)
13C-NMR (CDCl3) d [ppm]: 26.7 (q), 27.9 (q), 28.1 (q), 36.9 (t), 37.3 (t),
55.2 (q), 55.3 (q), 75.4 (s), 76.0 (s), 76.5 (s), 108.2 (d), 109.0 (d), 110.1
(s), 113.64 (d), 113.7 (s), 113.8 (d), 114.9 (s), 116.9(d), 117.2 (d),
120.5 (d), 123.6 (d), 124.1 (d), 126.0 (d), 128.4 (d), 128.9 (d), 129.1
(d), 129.5 (d), 129.7 (d), 130.2 (s), 130.6 (s), 130.5 (s), 133.7 (s), 134.0
(s), 149.0 (s), 153.5 (s), 153.7 (s), 157.8 (s), 158.1 (s). IR (KBr) [cm– 1]:
2965 (m), 2931 (m), 2830 (m), 1607 (s), 1509 (vs), 1456 (s), 1370
(m), 1260 (vs), 1174 (s), 1116 (s), 1035 (s), 829 (s), 723 (w). MS m/z
[%]: 363 [M+ + 1] (23), 362 [M+] (85), 348 (100), 243 (28), 164 (14),
120 (100), 75 (57). Anal. Calcd. for C24H26O3: C, 79.53; H, 7.23.
Found: C, 79.56; H, 7.25.
14a (exo-isomer)
1H-NMR (CDCl3) d [ppm]: 1.30 (s, 6H, 2-CH3), 1.45 (s, 6H, 8-CH3),
2.72 (d, 2H, J = 1.3 Hz, 3-CH2), 3.85 (2s, 3H, 3-MeOPh), 5.57 (d, 1H, J
= 10.0 Hz, H9), 6.44 (d, 1H, J = 8.4 Hz, H6), 6.71 (d, 1H, J = 10.0 Hz,
H10), 6.80–7.30 (m, 4H, Ar), 7.01 (s, 1H, 3-MeO-Ph-CH=C), 7.44 (d,
1H, J = 8.4 Hz, H5).
Biological activity
The strain of L. major used in this study was the vaccine strain
(MRHO/IR/75/ER) obtained from Pasteur Institute, Tehran, Iran.
The infectivity of the parasites was maintained by regular pas-
sage in susceptible BALB/C mice. The promastigote form of the
parasite was grown in NNN-blood agar medium at 258C. The sta-
tionary parasite inoculation was 26106 cells/mL. For the experi-
ments described here, the stationary-phase promastigotes were
washed with phosphate-buffered saline (PBS) and recultured in
RPMI 1640 medium, pH l 7.2, at 26106 cells/mL density, supple-
mented with 10% of heat-inactivated fetal bovine serum (FBS),
2 mM glutamine, 100 U/mL penicillin) and 100 lg/mL strepto-
mycin [3, 4].
14b (endo-isomer)
1H-NMR (CDCl3) d [ppm]: 1.42 (s, 6H, 2-CH3), 1.43 (s, 6H, 8-CH3),
3.66 (s, 2H, 3-MeO-Ph-CH2), 3.79 (s, 3H, 3-MeOPh), 5.23 (s, 1H, 3-
CH), 5.58 (d, 1H, J = 10.0 Hz, H9), 6.29 (d, 1H, J = 8.4 Hz, H6), 6.71 (d,
1H, J = 10.0 Hz, H10), 6.80–7.30 (m, 4H, Ar), 6.90 (d, 1H, J = 8.4 Hz,
H5).
14a/14b (exo-/endo-isomers)
13C-NMR (CDCl3) d [ppm]: 26.8 (q), 27.9 (q), 28.1 (q), 37.3 (t), 37.9 (t),
55.1 (q), 55.2 (q), 75.5 (s), 76.0 (s), 76.1 (s), 76.4 (s), 108.2 (d), 109.0
(d), 110.1 (s), 110.2 (s), 111.5 (s), 111.6 (d), 114.5 (d), 114.6 (s), 115.1
(d), 115.4 (s), 116.9 (d), 117.1 (d), 120.8 (d), 120.9 (d), 121.2 (d),
122.0 (d), 123.6 (d), 124.2 (d), 126.3 (d), 128.5 (d), 128.9 (d), 129.1
(d), 129.3 (d), 129.4 (d), 130.5 (s), 130.9 (s), 139.1 (s), 140.7 (s), 143.4
(s), 148.9 (s), 149.2 (s), 153.5 (s), 153.9 (s), 159.4 (s), 159.7 (s). IR
(KBr) [cm– 1]: 2981 (s), 2932 (s), 1648 (s), 1591 (vs), 1460 (s), 1370
(m), 1277 (s), 1220 (w), 1161 (s), 1108 (vs), 1054 (s), 777 (m), 727 (s).
MS m/z [%]: 363 [M+ + 1] (18), 362 [M+] (63), 346 (100), 240 (14), 165
(16), 120 (54), 77 (14). Anal. Calcd. for C24H26O3: C, 79.53; H, 7.23.
Found: C, 79.50; H, 7.24.
Analysis of L. major growth kinetics
At first, the growth curve of the parasite was determined by daily
counting, using a Neubauer's chamber and light microscopy.
26106 cells/mL log-phase promastigotes were inoculated into
5 mL fresh RPMI 1640 medium in T25 culture flasks, besides, the
current anti-leishmanial drug Glucantime was used as a refer-
ence. Assessment of growth by cell counting (Neubauer Cham-
ber) was determined at 24-hours intervals for 8 days. The values
obtained were used to determine the relative growth rate and
the results were expressed as the mean values of at least three
experiments.
4-(4-Methoxy-benzylidene)-2,2,8,8-tetramethyl-3,4-
dihydro-2H,8H-pyrano[2,3-f]chroman (15a) and 4-(4-
Methoxy-benzyl)-2,2,8,8-tetramethyl-2H,8H- pyrano[2,3-
f]chromene (15b)
A colorless oil (72%) as mixture of exo- and endo-isomers
(75 : 25).
In-vitro L. major promastigote culture and compound
toxicity assays
The in-vitro growth inhibition of the chromenes was evaluated
by direct counting under a light microscope. Briefly, promasti-
gotes in the logarithmic growth phase were incubated at an
average of 26106 cells/mL in RPMI 1640 medium supplemented
with 10% FBS as described above, in the presence of chromene
compounds dissolved in dimethylsulphoxide (DMSO), and incu-
bated for 24, 48, and 72 h at 258C. Promastigotes in the control
groups were incubated with DMSO alone or with the antimonial
drug Glucantime. Finally, the number of promastigotes was
recorded for at least three independent experiments and the
results are expressed as the mean percentage reduction of para-
site numbers compared with the untreated controls. The per-
centage of growth inhibition was calculated as (1 – (Cell number
of drug-treated culture / Cell number of control culture))6100.
15a (exo-isomer)
1H-NMR (CDCl3) d [ppm]: 1.30 (s, 6H, 2-CH3), 1.46 (s, 6H, 8-CH3),
2.72 (d, 2H, J = 1.5 Hz, 3-CH2), 3.81 (2 s, 3H, 4-MeOPh), 5.58 (d, 1H, J
= 10.0 Hz, H9), 6.43 (d, 1H, J = 8.4 Hz, H6), 6.72 (d, 1H, J = 10.0 Hz,
H10), 6.84 (d, 2H, J = 8.4 Hz, Ar), 6.99 (s, 1H, 4-MeO-Ph-CH=C), 7.12
(d, 2H, J = 8.4 Hz, Ar), 7.43 (d, 1H, J= 8.4 Hz, H5).
15b (endo-isomer)
1H-NMR (CDCl3) d [ppm]: 1.41 (s, 6H, 2-CH3), 1.43 (s, 6H, 8-CH3),
3.68 (s, 2H, 4-MeO-Ph-CH2), 3.85 (s, 3H, 4-MeOPh), 5.23 (s, 1H, 3-
CH), 5.59 (d, 1H, J = 10.0 Hz, H9), 6.29 (d, 1H, J = 8.4 Hz, H6), 6.71 (d,
1H, J = 10.0 Hz, H10), 6.93 (d, 2H, J = 8.5 Hz, Ar), 7.17 (d, 1H, J =
8.5 Hz, H5), 7.25 (d, 2H, J = 8.5 Hz, Ar).
Growth curve of L. major
A general procedure was used in the current study to identify
three different growth phases of promastigotes. The growth
kinetics for a period of 8 days is shown in Fig. 3. The logarithmic
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Arch. Pharm. Chem. Life Sci. 2008, 341, 787–793
Leishmanicidal Synthetic Chromenes
793
phase of the promastigotes lasted until day 3, thereafter, the par-
asites gradually entered the stationery phase. The maximum
cell concentration of 1.16107/mL was achieved after 96 hours.
Besides, we assessed the growth curve of the promastigotes
treated with Glucantime which showed gradually reduction
during 5 days. The determinations of the growth inhibition
achieved by the drug were made when the parasites were still in
the log phase, prior to entering the stationary phase.
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