Chromene-based synthetic chalcones as potent antileishmanial agents: Synthesis and biological activity

Article abstract of DOI:10.1111/j.1747-0285.2010.00959.x

Two types of regioisomeric chromene-based chalcones namely, 1-(6-methoxy-2H-chromen-3-yl)-3-phenylpropen-1-ones and 3-(6-methoxy-2H-chromen- 3-yl)-1-phenylpropen-1-ones were prepared and investigated for their antileishmanial activity against promastigotes form of Leishmania major. The obtained results from in vitro biological assays indicated that chloro-substituted 1-(6-methoxy-2H-chromen-3-yl)-3-phenylpropen-1-ones exhibited excellent activity against Leishmania major at non-cytotoxic concentrations.

Full text of DOI:10.1111/j.1747-0285.2010.00959.x

ª 2010 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2010.00959.x
Chem Biol Drug Des 2010; 75: 590–596
Research Article
Chromene-Based Synthetic Chalcones as Potent
Antileishmanial Agents: Synthesis and Biological
Activity
Alireza Foroumadi1,2, Saeed Emami3,
limited efficacy, long-term treatment, cost expensive and severe
side effects (3–5). Thus, the necessity for the development of new,
efficient, affordable and safe drug is felt. Also, because of the high
toxicity associated with the currently used antileishmanial drugs,
efforts are being made to identify new structures from natural and
synthetic compounds.
Maedeh Sorkhi1, Maryam Nakhjiri1,
Zohreh Nazarian2, Samaneh Heydari2,
Sussan K. Ardestani4, Fatemeh Poorrajab4
and Abbas Shafiee1,*
1Department of Medicinal Chemistry, Faculty of Pharmacy and
Chalcones (1,3-diaryl-2-propen-1-ones) are natural or synthetic com-
pounds belonging to the flavonoid family with widespread distribu-
tion in vegetables, fruits, spices and tea and are present in a
variety of plant species (6). Chemically, they consist of open-chain
flavonoids in which the two aromatic rings are joined by a three-
carbon a,b-unsaturated carbonyl system. Chalcones have been
reported to possess many pharmacological activities (7), including
anti-inflammatory (8), immunomodulatory (9), antifungal (10), anti-
cancer (11,12), antioxidant (13), antibacterial (14), antimalarial
(15,16) and antileishmanial (17,18) properties.
Pharmaceutical Sciences Research Center, Tehran University of
Medical Sciences, Tehran 14174, Iran
2Drug Design & Development Research Center, Tehran University of
Medical Sciences, Tehran 14174, Iran
3Department of Medicinal Chemistry and Pharmaceutical Sciences
Research Center, Faculty of Pharmacy, Mazandaran University of
Medical Sciences, Sari, Iran
4Institute of Biochemistry and Biophysics, Department of
Biochemistry, University of Tehran, PO Box 13145-1384, Tehran, Iran
*Corresponding author: Abbas Shafiee, ashafiee@ams.ac.ir
In the past decade, synthetic or naturally occurring chalcones
emerged as a new class of antileishmanial compounds. The most
studied antileishmanial chalcones are licochalcone A (1) isolated
from the roots of Chinese liquorice, licochalcone C (2) and oxygen-
ated chalcones 3 (19). Recently, Narender et al. (20,21) have
described the promising antileishmanial activity of few naturally
occurring chromenochalcones 4, including crotaramosmin, crotaram-
in and crotin (Figure 1). These compounds contain a benzopyran sys-
tem, which is frequently found in many natural products.
Two types of regioisomeric chromene-based
chalcones namely, 1-(6-methoxy-2H-chromen-3-yl)-
3-phenylpropen-1-ones and 3-(6-methoxy-2H-chro-
men-3-yl)-1-phenylpropen-1-ones were prepared
and investigated for their antileishmanial activity
against promastigotes form of Leishmania major.
The obtained results from in vitro biological
assays indicated that chloro-substituted 1-(6-meth-
oxy-2H-chromen-3-yl)-3-phenylpropen-1-ones exhib-
ited excellent activity against Leishmania major at
non-cytotoxic concentrations.
In the search for new synthetic chromene-based chalcones (22), we
prepared two types of novel chromenochalcones 5a–e and 6a–e,
to investigate their antileishmanial activity against promastigotes
form of Leishmania major (Figure 1). The structure 5a–e possessing
the carbonyl group close to chromene ring is called Type A chal-
cones. The structure 6a–e possessing the carbonyl group away
from chromene ring is called Type B chalcones (Figure 1). The syn-
thesis and in vitro antileishmanial activity of both regioisomeric
chromene-based chalcones are reported in this article.
Key words: antileishmanial activity, chalcones, chromene, Leish-
mania major, promastigotes
Received 13 August 2009, revised 4 February 2010, accepted for publi-
cation 6 February 2010
Leishmaniasis comprises a group of parasitic diseases typically
transmitted by the bite of female phlebotomine sandfly and mani-
fest with visceral, cutaneous and mucutaneous forms (1). Human
leishmaniasis is mainly distributed in the tropic and subtropic area
with a prevalence of 12 million cases and approximate incidence of
0.5 million cases of visceral leishmaniasis and 1.5 million cases of
cutaneous leishmaniasis. Overall, 350 million individuals are at risk
of leishmaniasis infection in the 88 countries (2). Whereas no vac-
cine exists, the current chemotherapy for leishmaniasis possesses a
set of problems because of the emergence of drug resistant strains,
Experimental Section
All chemical and solvent used in this study were purchased from
Merck AG and Aldrich. Melting points were determined on a Kofler
hot stage apparatus and are uncorrected. The IR spectra were
obtained on a Shimadzu 470 spectrophotometer (potassium bromide
590

Chromene-Based Chalcones as Antileishmanial Agents
O
was added and stirred overnight in an ice-bath. The reaction mix-
ture was diluted with water and the precipitate was filtered
and crystallized from ethanol to give the corresponding chalcones
5a–e.
O
H3CO
HO
OH
H3CO
HO
OH
1, Licochalcone A
2, Licochalcone C
OH
O
(E)-1-(6-Methoxy-2H-chromen-3-yl)-3-phenylprop-
2-en-1-one (5a)
OH
O
R
IR (KBr, cm)1) mmax: 1649 (C=O), 1224 (C-O). 1H NMR (CDCl3) d:
7.74 (d, 1H, J = 15.6 Hz, H3 propenone), 7.63 (dd, 2H, J = 7.5 and
2 Hz, H2, H6 phenyl), 7.44 (s, 1H, H4 chromene), 7.42 (m, 3H, H3,
H4 and H5 phenyl), 7.38 (d, 1H, J = 15.6 Hz, H2 propenone), 6.84
(m, 2H, H7 and H8 chromene), 6.75 (d, 1H, J = 2.5 Hz, H5 chrom-
ene), 5.08 (s, 2H, OCH2), 3.80 (s, 3H, OCH3). MS (m ⁄ z, %): 293
(M + 1, 38), 291 (60), 273 (38), 270 (16), 159 (38), 130 (50), 100
(98), 74 (100). Anal. Calcd for C19H16O3: C, 78.06; H, 5.52. Found:
C, 77.87; H, 5.69.
O
H3CO
RO
OCH3
4
3
4a: Crotaramosmin: R = 4-OH
4b: Crotaramin: R = 4-OCH3
4c: Crotin: 3,4-(OH)2
3a: R = n-Butyl
3b: R = 1-Propen-3-yl
O
O
H3CO
H3CO
R
R
O
O
6a-e, Type B
5a-e, Type A
Figure 1: Structures of naturally occurring and synthetic antile-
ishmanial chalcones 1–4 and newly designed compounds (5 and 6)
as chromene-based antileishmanial chalcones.
(E)-3-(2-Chlorophenyl)-1-(6-methoxy-2H-chromen-
3-yl)prop-2-en-1-one (5b)
1
IR (KBr, cm)1) mmax: 1639 (C=O), 1219 (C-O). H NMR (CDCl3) d: 8.1
1
disks). H NMR spectra was recorded using a Bruker 500 MHz spec-
(d, 1H, J = 15.6 Hz, H3 propenone), 7.73 (dd, 1H, J = 7 and 2 Hz,
H6 phenyl), 7.44 (m,1H, H4 phenyl), 7.43 (s, 1H, H4 chromene), 7.33
(d, 1H, J = 15.6 Hz, H2 propenone),7.32 (m, 2H, H3 and H5 phenyl),
6.84 (m, 2H, H7 and H8 chromene), 6.74 (d, 1H, J = 2.5 Hz, H5
chromene), 5.07 (s, 2H, OCH2), 3.79 (s, 3H, OCH3). MS (m ⁄ z, %):
328 (M + 2, 15), 326 (M+, 45), 309 (40), 199 (11), 185 (100), 143
(33), 137 (94), 109 (80), 73 (46). Anal. Calcd for C19H15ClO3: C,
69.84; H, 4.63. Found: C, 70.03; H, 4.77.
trometer and chemical shifts are reported in parts per million (ppm)
relative to tetramethylsilane as internal standard. The mass spectra
were run on a Finigan TSQ-70 spectrometer (Finigan, San Jose, CA,
USA) at 70 eV. Elemental analyses were carried out on CHN-O rapid
elemental analyzer (Heraeus GmbH, Hanau, Germany) for C, H and
N, and the results are within €0.4% of the theoretical values.
Merck silica gel F254 plates were used for analytical TLC.
Synthesis of 1-(6-methoxy-2H-chromen-3-yl) eth-
anone (9)
(E)-3-(3-Chlorophenyl)-1-(6-methoxy-2H-chromen-
A mixture of 5-methoxy-2-hydroxybenzaldehyde (5 mmol) and potas-
sium carbonate (5 mmol) in 1,4-dioxane (5 mL) was treated with
methyl vinyl ketone (5 mmol). The mixture was heated at 100 ꢀC
for 4 h and allowed to cool. It was then diluted with water and
extracted several times with ether. The combined ether extracts
were dried (Na2SO4) and evaporated to give 9 as a yellow solid,
which was crystallized from ethyl acetate-hexane (23).
3-yl)prop-2-en-1-one (5c)
1
IR (KBr, cm)1) mmax: 1644 (C=O), 1229 (C-O). H NMR (CDCl3) d: 7.81
(d, 1H, J = 15.6 Hz, H3 propenone), 7.73 (m, 1H, H6 phenyl), 7.58
(m, 2H, H2 and H4 phenyl), 7.43 (s,1H, H4 chromene), 7.32 (d, 1H,
J = 15.6 Hz, H2 propenone), 7.18 (m, 1H, H5 phenyl), 6.84 (m, 2H,
H7 and H8 chromene), 6.73 (d, 1H, J = 2.5 Hz, H5 chromene), 5.08
(s, 2H, OCH2), 3.80 (s, 3H, OCH3). MS (m ⁄ z, %): 328 (M + 2, 33),
326 (M+, 90), 309 (50), 201 (8), 189 (14), 159 (60), 146 (43), 101
(100), 88 (30). Anal. Calcd for C19H15ClO3: C, 69.84; H, 4.63. Found:
C, 69.71; H, 4.80.
Synthesis of 6-methoxy-2H-chromene-3-carbal-
dehyde (10)
A mixture of 5-methoxy-2-hydroxybenzaldehyde (7 mmol) and potas-
sium carbonate (7 mmol) in 1,4-dioxane (12.5 mL) was treated with
acrolein (0.5 mL). The mixture was heated at 100 ꢀC for 8 h and
allowed to cool. It was then diluted with water and extracted sev-
eral times with ether. The combined ether extracts were dried
(Na2SO4) and evaporated to give 10 as a yellow solid, which was
crystallized from ethyl acetate-hexane (23).
(E)-3-(4-Chlorophenyl)-1-(6-methoxy-2H-chromen-
3-yl)prop-2-en-1-one (5d)
IR (KBr, cm)1) mmax: 1649 (C=O), 1224 (C-O). 1H NMR (CDCl3) d:
7.68 (d, 1H, J = 15.6 Hz, H3 propenone), 7.56 (d, 2H, J = 8.2 Hz,
H2 and H6 phenyl), 7.43 (s, 1H, H4 chromene), 7.39 (d, 2H,
J = 8.2 Hz, H3 and H5 phenyl), 7.35 (d, 1H, J = 15.6 Hz, H2
propenone), 6.84 (m, 2H, H7 and H8 chromene), 6.75 (d, 1H,
J = 2.5 Hz, H5 chromene), 5.07 (s, 2H, OCH2), 3.80 (s, 3H, OCH3).
MS (m ⁄ z, %): 328 (M + 2, 30), 326 (M+, 90), 307 (42), 275 (17),
227 (8), 165 (50), 159 (63), 135 (67), 101 (100), 89 (58), 45 (50).
Anal. Calcd for C19H15ClO3: C, 69.84; H, 4.63. Found: 69.52; H,
4.49.
General procedure for the synthesis of (E)-1-(6-
methoxy-2H-chromen-3-yl)-3-phenyl prop-2-en-1-
one derivatives 5a–e
To a solution of compound 9 (1 mmol) and appropriate aldehyde
(1 mmol) in absolute ethanol (5 mL), NaOH solution (3.5 M, 2 mL)
Chem Biol Drug Des 2010; 75: 590–596
591

Foroumadi et al.
(E)-3-(2,4-Dichlorophenyl)-1-(6-methoxy-2H-chro-
J = 15.5 Hz, H2 propenone), 6.78 (m, 2H, H7, H8 chromene), 6.65 (d,
1H, J = 2.5 Hz, H5 chromene), 5.02 (s, 2H, OCH2), 3.78 (s, 3H,
OCH3). MS (m ⁄ z, %): 328 (M + 2, 28), 326 (M+, 84), 309 (76), 189
(11), 159 (60), 146 (45), 101 (100), 87 (28). Anal. Calcd for
C19H15ClO3: C, 69.84; H, 4.63. Found: C, 69.99; H, 4.48.
men-3-yl)prop-2-en-1-one (5e)
1
IR (KBr, cm)1) mmax: 1647 (C=O), 1222 (C-O). H NMR (CDCl3) d: 8.02
(d, 1H, J = 15.6 Hz, H3 propenone), 7.66 (d, 2H, J = 8.4 Hz H6 phe-
nyl), 7.47 (d, 1H, J = 2 Hz, H3 phenyl), 7.42 (s, 1H, H4 chromene),
7.32 (d, 1H, J = 15.6 Hz, H2 propenone), 7.28 (dd, 2H, J = 8.4 and
2 Hz, H5 phenyl), 6.86 (dd, 1H, J = 8.8 and 2.5 Hz, H7 chromene),
6.83 (d, 1H, J = 8.8 Hz, H8 chromene), 6.74 (d, 1H, J = 2.5 Hz, H5
chromene), 5.07 (s, 2H, OCH2), 3.79 (s, 3H, OCH3). MS (m ⁄ z, %):
364 (M + 4, 10), 362 (M + 2, 65), 361 (M+, 21), 360 (100), 325 (58),
309 (17), 260 (8), 202 (10), 198 (35), 161 (50), 118 (62), 89 (45), 63
(20). Anal. Calcd for C19H14Cl2O3: C, 63.18; H, 3.91. Found: C, 62.96;
H, 4.05.
(E)-1-(4-Chlorophenyl)-3-(6-methoxy-2H-chromen-
3-yl)prop-2-en-1-one (6d)
IR (KBr, cm)1) mmax: 1649 (C=O), 1229 (C-O). 1H NMR (CDCl3) d:
7.90 (d, 2H, J = 8.5 Hz, H2 and H6 phenyl), 7.52 (d, 1H,
J = 15.6 Hz, H3 propenone), 7.47 (d, 2H, J = 8.5 Hz, H3 and H5
phenyl), 6.83 (s,1H, H4 chromene), 6.80 (m, 2H, H7 and H8
chromene), 6.79 (d, 1H, J = 15.6 Hz, H2 propenone), 6.65 (d, 1H,
J = 2.5 Hz, H5 chromene), 5.02 (s, 2H, OCH2), 3.78 (s, 3H, OCH3).
MS (m ⁄ z, %): 328 (M + 2, 20), 326 (M+, 93), 323 (68), 311 (46),
308 (25), 245 (11), 202 (11), 187 (95), 184 (32), 139 (100), 111
(80), 109 (46), 75 (28). Anal. Calcd for C19H15ClO3: C, 69.84; H,
4.63. Found: C, 70.21; H, 4.49.
General procedure for the synthesis of 3-(6-meth-
oxy-2H-chromen-3-yl)-1-phenyl prop-2-en-1-one
derivatives 6a–e
To a solution of compound 10 (1 mmol) and appropriate acetophe-
none (1 mmol) in absolute ethanol (5 mL), NaOH solution (3.5 M,
2 mL) was added and stirred overnight in an ice-bath. The reaction
mixture was diluted with water and the precipitate was filtered
and crystallized from ethanol to give the corresponding chalcones
6a–e.
(E)-1-(2,4-Dichlorophenyl)-3-(6-methoxy-2H-chro-
men-3-yl)prop-2-en-1-one (6e)
1
IR (KBr, cm)1) mmax: 1662 (C=O), 1224 (C-O). H NMR (CDCl3) d: 7.47
(d, 1H, J = 2 Hz, H3 phenyl), 7.40 (d, 1H, J = 8 Hz, H6 phenyl), 7.35
(dd, 1H, J = 8 and 2 Hz, H5 phenyl), 7.19 (d, 1H, J = 16 Hz, H3
propenone), 6.77 (m, 2H, H7 and H8 chromene), 6.61 (d, 1H,
J = 2 Hz, H5 chromene), 6.46 (d, 1H, J = 16 Hz, H2 propenone), 4.96
(s, 2H, OCH2), 3.77 (s, 3H, OCH3). MS (m ⁄ z, %): 365 (M + 4, 7), 363
(M + 2, 42), 361 (M+, 63), 325 (46), 279 (60), 261 (18), 206 (8), 189
(23), 187 (100), 149 (100), 113 (48), 71 (64), 57 (70). Anal. Calcd for
C19H14Cl2O3: C, 63.18; H, 3.91. Found: C, 63.20; H, 4.04.
(E)-3-(6-Methoxy-2H-chromen-3-yl)-1-phenylprop-
2-en-1-one (6a)
1
IR (KBr, cm)1) mmax: 1649 (C=O), 1229 (C-O). H NMR (CDCl3) d: 7.96
(d, 2H, J = 7.5 Hz, H2 and H6 phenyl), 7.58 (m, 1H, H4 phenyl), 7.52
(d, 1H, J = 15.6 Hz, H3 propenone), 7.50 (m, 2H, H3 and H5 phenyl),
6.86 (d, 1H, J = 15.6 Hz, H2 propenone), 6.82 (s, 1H, H4 chromene),
6.80 (d, 1H, J = 8.7 Hz, H8 chromene), 6.76 (dd, 1H, J = 8.7 and
2.5 Hz, H7 chromene), 6.65 (d, 1H, J = 2.5 Hz, H5 chromene), 5.03
(s, 2H, OCH2), 3.78 (s, 3H, OCH3). MS (m ⁄ z, %): 292 (M+, 60), 277
(30), 187 (39), 142 (23), 113 (24), 105 (75), 75 (88), 45 (100). Anal.
Calcd for C19H16O3: C, 78.06; H, 5.52. Found: 78.32; H, 5.51.
Biological activity
Parasite and culture
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 passage
in susceptible BALB ⁄ c mice. The promastigote form of parasite was
grown in blood agar cultures at 25 ꢀC. The stationary parasite inoc-
ulation was 2 · 106 cells ⁄ mL. For the experiments described here,
the stationary phase of promastigotes was washed with phosphate-
buffered saline and recultured in RPMI 1640 medium (Sigma,
St. Louis, MO, USA) at 2 · 106 cells ⁄ mL density, supplemented
with 10% of heat-inactivated fetal bovine serum, 2-mM glutamine
(Sigma), pH approximately 7.2, 100 U ⁄ mL penicillin (Sigma) and
100 lg ⁄ mL streptomycin (Sigma).
(E)-1-(2-Chlorophenyl)-3-(6-methoxy-2H-chromen-
3-yl)prop-2-en-1-one (6b)
1
IR (KBr, cm)1) mmax: 1659 (C=O), 1219 (C-O). H NMR (CDCl3) d: 7.45
(m, 2H, H4, H6 phenyl), 7.41 (m, 1H, H3 phenyl), 7.36 (m, 1H, H5
phenyl), 7.18 (d, 1H, J = 16 Hz, H3 propenone), 6.77 (m, 2H, H7, H8
chromene), 6.75 (s, 1H, H4 chromene), 6.61 (d, 1H, J = 2.5 Hz, H5
chromene), 6.48 (d, 1H, J = 16 Hz, H2 propenone), 4.97 (s, 2H,
OCH2), 3.76 (s, 3H, OCH3). MS (m ⁄ z, %): 328 (M + 2, 22), 326 (M+,
66), 311 (48), 236 (11), 201 (11), 185 (100), 141 (40), 139 (80), 109
(80), 67 (38), 55 (55). Anal. Calcd for C19H15ClO3: C, 69.84; H, 4.63.
Found: C, 69.83; H, 4.70.
In vitro antileishmanial activity
(E)-1-(3-Chlorophenyl)-3-(6-methoxy-2H-chromen-
The antileishmanial evaluation of compounds 5a–e and 6a–e was
performed using direct counting and MTT assay (24,25). The growth
curve of the L. major strain was determined daily under light micro-
scope and counting in a Neubauer's chamber. Then, parasites
(2 · 106 ⁄ mL) in the logarithmic phase were incubated with a serial
range of drug concentrations for 24 h at 25 ꢀC. To determine 50%
3-yl)prop-2-en-1-one (6c)
1
IR (KBr, cm)1) mmax: 1654 (C=O), 1219 (C-O). H NMR (CDCl3) d: 7.93
(s, 1H, H2 phenyl), 7.83 (d, 1H, J = 7.75 Hz, H4 phenyl), 7.56 (m,
1H, H6 phenyl), 7.53 (d, 1H, J = 15.5 Hz, H3 propenone), 7.44 (t, 1H,
J = 7.75 Hz, H5 phenyl), 6.84 (s, 1H, H4 chromene), 6.80 (d, 1H,
592
Chem Biol Drug Des 2010; 75: 590–596

Chromene-Based Chalcones as Antileishmanial Agents
inhibitory concentrations (IC50), the tetrazolium bromide salt (MTT)
assay was used. Briefly, promastigotes from early log phase of
growth were seeded in 96-well plastic cell culture trays, containing
serial dilution of drug and phenol red free RPMI 1640 medium, sup-
plemented with 10% of FCS, 2-mM glutamine, pH approximately
7.2 and antibiotics, in a volume of 200 lL. After 24 h of incubation
at 25 ꢀC, the media was renewed with 100 lg ⁄ well of MTT
(0.5 mg ⁄ mL) and plates were further incubated for 4 h at 37 ꢀC.
The plates were centrifuged (700 g · 5 min) and the pellets were
dissolved in 200 lL of DMSO. The samples were read using an
ELISA plate reader at a wavelength of 492 nm. Two or more inde-
pendent experiments in triplicate were performed for determination
of sensitivity to each drug, the IC50 was calculated by linear regres-
sion analysis, expressed in mean € SD. Control cells were incu-
bated with culture medium plus DMSO.
Table 1: Structures and physicochemical data of chromene-
based chalcones 5a–e and 6a–e
O
O
H3CO
H3CO
R
R
O
O
5a-e (Type A)
6a-e (Type B)
Compound
R
m.p. (ꢀC)
M.W.
Yield
Formula
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
H
73–75
87–89
54–56
111–113
124–125
156–158
91–93
146–148
187–189
137–138
292.33
326.77
326.77
326.77
361.22
292.33
326.77
326.77
326.77
361.22
76
62
59
34
89
55
61
64
78
97
C19H16O3
2-Cl
3-Cl
4-Cl
2,4-Cl2
H
2-Cl
3-Cl
4-Cl
2,4-Cl2
C19H15ClO3
C19H15ClO3
C19H15ClO3
C19H14Cl2O3
C19H16O3
C19H15ClO3
C19H15ClO3
C19H15ClO3
C19H14Cl2O3
Cytotoxicity against macrophages
In vitro toxicity against mouse peritoneal macrophages was
assessed with cells plated in 96-well plates at 2 · 105 cells ⁄ well.
After cell adherence, the medium was removed and replaced by the
media containing IC50 concentration of each compound. The plates
were incubated for 24 h at 37 ꢀC in a humidified incubator with
5% CO2. Control cells were incubated with culture medium plus
DMSO. Cell viability was determined using MTT colorimetric assay.
Results and discussion
Chemistry
As illustrated in Scheme 1, the routes to synthesis of both
target compounds 5a–e and 6a–e was started from phenolic
H3CO
OH
7
a
H3CO
CHO
OH
8
d
b
H3CO
H3CO
CHO
COCH3
O
O
9
10
c
e
O
O
H3CO
H3CO
R
R
O
O
5a-e (Type A)
6a-e (Type B)
R= H; 2-Cl; 3-Cl; 4-Cl; 2,4-Cl2
Scheme 1: Synthesis of chromene-based chalcones 5a–e and 6a–e. Reagents and conditions: (a) NaOH, CHCl3, H2O, reflux (b) methyl
vinyl ketone, 1,4-dioxane, K2CO3, reflux (c) appropriate aldehyde, NaOH, EtOH, (d) acrolein, 1,4-dioxane, K2CO3, reflux (e) appropriate acetophe-
none, NaOH, EtOH.
Chem Biol Drug Des 2010; 75: 590–596
593

Foroumadi et al.
Table 2: In vitro activities of chromene-based chalcones 5a–e
and 6a–e against promastigote form of L. major
results indicate that compounds 5b–e (Type A chalcones) exhibited
excellent activity against Leishmania (100% inhibition) at the con-
centration of 10 lM. Compound 5a that showed no inhibition at
the first day of exposure exhibited potent inhibitory activity after
3 days (% inhibition = 93%). In addition, remaining compounds
(Type B chalcones) showed weak to moderate inhibitory activity at
this level of concentration.
O
O
H3CO
H3CO
R
R
O
O
5a-e (Type A)
6a-e (Type B)
% Inhibition at the
concentration of 10 lM
a
The IC50 values of type A and type B chalcones against L. major in
comparison with meglumine antimonate (Glucantimeꢁ, Aventis, Paris,
France) are presented in Table 2. The IC50 values of the test
compounds against L. major indicate that most compounds possessed
good leishmanicidal activity (IC50 £ 50 lM) with respect to reference
drugs. The most potent compounds against the promastigote form of
L. major were found to be chloro-substituted Type A chalcones 5c–e
with IC50 values less than 1.0 lM. The activity profile of these
compounds (5c–e) against promastigotes demonstrated that there
are no significant differences in their IC50 values.
Compound
R
Day 1
Day 2
Day 3
IC50 (lM)b
5a
5b
H
0.0
100
100
100
98.1
0.0
0.0
0.0
0.0
4.3
18.8
100
100
100
100
12.8
0.0
0.0
0.0
4.3
93
100
100
100
100
62.8
1.0
75.2
5.4
8.4
30 € 0.06
5 € 0.14
0.8 € 0.26
0.7 € 0.3
0.75 € 0.23
45 € 0.15
>50
2-Cl
3-Cl
4-Cl
2,4-Cl2
H
2-Cl
3-Cl
4-Cl
2,4-Cl2
5c
5d
5e
6a
6b
6c
45 € 0.22
>50
6d
6e
>50
The effect of positional substitution was investigated by preparing
all three possible chloro-substitutions (2-Cl, 3-Cl or 4-Cl) and 2,4-
dichloro-substitutions on phenyl ring attached to propenone scaf-
fold. Although chlorine substitution on phenyl ring increases the
activity in Type A chalcones, (compounds 5b–e in comparison with
5a) this alteration in Type B compounds cannot improve antileish-
manial activity (compounds 6b–e in comparison with 6a). The bet-
ter results were achieved with 3-chloro- or 4-chloro-containing
analogues in the Type A series.
Glucantimeꢁ
81.97 € 3.85c
aThe growth inhibitory effect of compounds on Leishmania parasite in three
consecutive days.
bThe values represent mean € SD.
cIC50 in mM.
compound 7. Compound 7 was converted to salicylaldehyde
derivative 8 according to the general literature method (26,27).
Then, compound 8 was reacted with methyl vinyl ketone, as a
Michael acceptor, in refluxing dioxane in the presence of K2CO3
to give 3-acetylchromene 9. Claisen-Schmidt condensation of 3-
acetylchromene 9 with different aldehydes in ethanolic solution
of NaOH yielded corresponding type A chalcones 5a–e. For
obtaining type B chalcones 6a–e, a slightly different strategy
was used. Treatment of salicylaldehyde 8 with acrolein in reflux-
ing dioxane in the presence of K2CO3 afforded chromene-3-carb-
aldehyde 10. Subsequently, condensation of aldehyde 10 with
appropriate acetophenone in ethanolic solution of NaOH afforded
type B chalcones 6a–e. The structures of desired products were
established with IR, NMR, mass spectrometry and elemental
analysis. 1H NMR spectra showed that only (E)-isomer of chal-
cones were obtained. The structures and physicochemical data of
target compounds are listed in Table 1.
The cytotoxicity of target compounds was also assessed using MTT
colorimetric assay on macrophage cells. Macrophage cells were
treated with synthesized compounds at the concentration equal to
IC50 values for 24h, side by side with the reference drug Glucan-
timeꢁ (25). The results showed that these compounds display an-
tileishmanial activity at non-cytotoxic concentrations.
Chemically, chalcones consist of open-chain flavonoids in which the
two aromatic ring A and ring B are joined by a three-carbon a,b-
unsaturated carbonyl linker. Ring A is attached to the b-position
respect to the carbonyl group and ring B is aryl moiety connected
to the carbonyl group. Based on previous studies on antileishmanial
chalcones, ring A and its substitution pattern are generally consid-
ered less important for antileishmanial activity compared to ring B
(28,29). Our results with chromene-based chalcones revealed that
very good antileishmanial activity was observed when ring B is 6-
methoxy-2H-chromen-3-yl and ring A is 3- or 4-chlorophenyl moiety
(compounds 5c–e). In this work, the mechanisms by which the
chromene-based chalcones showed antileishmanial activity were
not addressed but based on the literature, it can be predicted that
chalcones could potentially interfere with the function of parasite
mitochondria and inhibit the activity of fumarate reductase, succi-
nate dehydrogenase, NADH dehydrogenase, or succinate- and
NADH-cytochrome c reductases (30–32).
Antileishmanial activity
The life cycle of Leishmania parasites consists of two evolutionary
stages: promastigotes, flagellated extracellular parasites and am-
astigotes, non-flagellated, non-motile stages that is more sensitive
and live in macrophages. In this study, the chromene-based chal-
cones 5a–e and 6a–e were evaluated for their in vitro activity
against the promastigote form of the Leishmania major using MTT
assay. In primary screening assay, the Leishmania parasite was
affected by 10 lM concentration of the synthesized compounds
5a–e and 6a–e for three consecutive days and the growth inhibi-
tory effect of these compounds was monitored during Day 1, Day 2
and Day 3 and the results are reported in Table 2. The obtained
In conclusion, we prepared two types of regioisomeric chromene-based
chalcones namely, 1-(6-methoxy-2H-chromen-3-yl)-3-phenylpropen-1-
ones and 3-(6-methoxy-2H-chromen-3-yl)-1-phenylpropen-1-ones and
investigated their antileishmanial activity against promastigotes
594
Chem Biol Drug Des 2010; 75: 590–596

Chromene-Based Chalcones as Antileishmanial Agents
form of Leishmania major. The obtained results from in vitro biologi-
cal assays indicated that chloro-substituted 1-(6-methoxy-2H-chro-
men-3-yl)-3-phenylpropen-1-ones exhibited excellent activity against
Leishmania major at non-cytotoxic concentrations. The marked activ-
ity and simple synthesis of these chalcones suggest that they are
potential leads for the development of antileishmanial compounds
and further work is in progress to improve the potency of these
compounds.
12. Lawrence N.J., Rannison D., McGown A.T., Ducki S., Gul L.A.,
Hadfield J.A., Khan N. (2001) Linked parallel synthesis and MTT
bioassay screening of substituted chalcones. J Comb Chem;
3:421–426.
13. Yaylı N., ꢁÅꢂncꢂ O., Yas¸ ar A., Gçk Y., KꢂÅꢂk M., Kolaylı S.
(2004) Stereoselective photochemistry of methoxy chalcones
in solution and their radical scavenging activity. Turk J Chem;
28:515–521.
14. Sivakumar P.M., Priya S., Doble M. (2009) Synthesis, biological
evaluation, mechanism of action and quantitative structure-activ-
ity relationship studies of chalcones as antibacterial agents.
Chem Biol Drug Des;73:403–415.
Acknowledgment
15. Xue C.X., Cui S.Y., Liu M.C., Hu Z.D., Fan B.T. (2004) 3D QSAR
studies on antimalarial alkoxylated and hydroxylated chalcones
by CoMFA and CoMSIA. Eur J Med Chem;39:745–753.
16. Liu M., Wiliarat P., Go M.L. (2001) Antimalarial alkoxylated and
hydroxylated chalones: structure-activity relationship analysis.
J Med Chem;44:4443–4452.
This work was supported by grants from the research council of
Tehran University of Medical Sciences and Iran National Science
Foundation (INSF).
References
17. Liu M., Wilairat P., Croft S.L., Tan A.L., Go M.L. (2003) Struc-
ture–activity relationships of antileishmanial and antimalarial
chalcones. Bioorg Med Chem;11:2729–2738.
1. Guerin P.J., Olliaro P., Sundar S., Boelaert M., Croft S.L., Desjeux
P., Wasunna M.K., Bryceson A.D. (2002) Visceral leishmaniasis:
current status of control, diagnosis, and treatment, and a pro-
posed research and development agenda. Lancet Infect
Dis;2:494–501.
2. PiÇero J., Temporal R.M., Silva-Goncalves A.J., Jimꢀnez I.A.,
Bazzocchi I.L., Oliva A., Perera A., Leonb L.L., Valladares
B. (2006) New administration model of trans-chalcone biode-
gradable polymers for the treatment of experimental leishmania-
sis. Acta Trop;98:59–65.
3. Croft S.L., Coombs G.H. (2003) Leishmaniasis-current chemother-
apy and recent advances in the search for novel drugs. Trends
Parasitol;19:502–508.
4. Hemmateenejad B., Miri R., Niroomand U., Foroumadi A.,
Shafiee A. (2007) A mechanistic QSAR study on the leishmani-
cidal activity of some 5-substituted-1,3,4-thiadiazole derivatives.
Chem Biol Drug Des;69:435–443.
18. Chen M., Christensen S.B., Theander T.G., Kharazmi A. (1994)
Antileishmanial activity of licochalcone A in mice infected with
Leishmania major and in hamsters infected with Leishmania
donovani. Antimicrob Agents Chemother;38:1339–1344.
19. Nowakowska Z. (2007) A review of anti-infective and anti-
inflammatory chalcones. Eur J Med Chem;42:125–137.
20. Kumar J.K., Narender T., Rao M.S., Rao P.S., Toth G., Balazs B.,
Duddeck H. (1999) Further Dihydrochalcones from Crotolaria
ramosissima. J Braz Chem Soc;10:278–280.
21. Narender T., Shweta, Gupta S. (2004) A convenient and bioge-
netic type synthesis of few naturally occurring chromeno dihydr-
ochalcones and their in vitro antileishmanial activity. Bioorg
Med Chem Lett;14:3913–3916.
22. Nazarian Z., Emami S., Heydari S., Ardestani S.K., Nakhjiri M.,
Poorrajab F., Shafiee A., Foroumadi A. (2010) Novel antileishma-
nial chalconoids: synthesis and biological activity of 1- or 3-(6-
chloro-2H-chromen-3-yl)propen-1-ones. Eur J Med Chem; In
press: doi:10.1016/j.ejmech.2009.12.046.
5. Singh S., Sivakumar R.J. (2004) Challenges and new discoveries
in the treatment of leishmaniasis. J Infect Chemother;10:
307–315.
23. Sorkhi M., Forouzani M., Dehghan G., Abdolahi M., Shafiee A.,
Foroumadi A. (2008) Synthesis and evaluation of antioxidant
activity of 6-methoxy-2H-chromenes. Asian J Chem;20:2151–
2155.
24. Behrouzi-Fardmoghadam M., Poorrajab F., Ardestani S.K., Emami
S., Foroumadi A., Shafiee A. (2008) Synthesis and in vitro
anti-leishmanial activity of 1-[5-(5-nitrofuran-2-yl)-1,3,4-thia-
diazol-2-yl]- and 1-[5-(5-nitrothiophen-2-yl)-1,3,4-thiadiazol-2-yl]-4-
aroylpiperazines. Bioorg Med Chem;16:4509–4515.
6. Di Carlo G., Mascolo N., Izzo A.A., Capasso F. (1999) Flavonoids:
old and new aspects of a class of natural therapeutic drugs.
Life Sci;65:337–353.
7. Go M.L., Wu X., Liu X.L. (2005) Chalcones: an update on cyto-
toxic and chemoprotective properties. Curr Med Chem;12:481–
499.
8. Hsieh H.K., Lee T.H., Wang J.P., Wang J.J., Lin C.N. (1998) Syn-
thesis and anti-inflammatory effect of chalcones and related
compounds. Pharm Res;15:39–46.
25. Dutta A., Bandyopadhyay S., Mandal C., Chatterjee M. (2005)
Development of a modified MTT assay for screening antimonial
resistant field isolates of Indian visceral leishmaniasis. Parasitol
Int;54:119–122.
26. Nielsen A.T., Houlihan W.J. (1968) The aldol condensation. Org
React;16:1–438.
9. Barford L., Kemp K., Hansen M., Kharazmi A. (2002) Chalcones
from Chinese liquorice inhibit proliferation of T cells and produc-
tion of cytokines. Int Immunopharmacol;2:545–555.
10. Sivakumar P.M., Muthu Kumar T., Doble M. (2009) Antifungal
activity, mechanism and QSAR studies on chalcones. Chem Biol
Drug Des;74:68–79.
27. Fine S.A., Pulaski P.D. (1973) Reexamination of the Claisen-
Schmidt condensation of phenylacetone with aromatic alde-
hydes. J Org Chem;38:1747–1749.
11. Nam N.H., Kim Y., You Y.J., Hong D.H., Kim H.M., Ahn B.Z.
(2003) Cytotoxic 2¢,5¢-dihydroxychalcones with unexpected anti-
angiogenic activity. Eur J Med Chem;38:179–187.
Chem Biol Drug Des 2010; 75: 590–596
595

Foroumadi et al.
28. Nielsen S.F., Christensen S.B., Cruciani G., Kharazmi A., Liljefors
T. (1998) Antileishmanial chalcones: statistical design, synthesis,
and three- dimensional quantitative structure-activity relationship
analysis. J Med Chem;41:4819–4832.
31. Zhai L., Blom J., Chen M., Christensen S.B., Kharazmi A. (1995)
The antileishmanial agent licochalcone A interferes with the
function of parasite mitochondria. Antimicrob Agents Chemo-
ther;39:2742–2748.
29. Kayser O., Kiderlen A.F. (2001) In vitro leishmanicidal activity of
naturally occurring chalcones. Phytother Res;15:148–152.
30. Chen M., Zhai L., Brogger S., Christensen S.B., Theander T.G.,
Kharazmi A. (2001) Inhibition of fumarate reductase in Leish-
mania major and L. donovani by chalcones. Antimicrob Agents
Chemother;45:2023–2029.
32. Zhai L., Chen M., Blom J., Christensen S.B., Theander T.G., Khar-
azmi A. (1999) The antileishmanial activity of novel oxygenated
chalcones and their mechanism of action. J Antimicrob Chemo-
ther;43:793–803.
596
Chem Biol Drug Des 2010; 75: 590–596