European Journal of Medicinal Chemistry 46 (2011) 4448e4456
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Original article
Quinolinyl and quinolinyl N-oxide chalcones: Synthesis, antifungal and cytotoxic activities Luciana de Carvalho Tavares a, Susana Johann b, d, Tânia Maria de Almeida Alves d, Juliana Correia Guerra c, Elaine Maria de Souza-Fagundes c, Patrícia Silva Cisalpino b, Adailton J. Bortoluzzi a, Giovanni F. Caramori a, Rafael de Mattos Piccoli a, Hugo T.S. Braibante e, Mara E.F. Braibante e, **, Moacir G. Pizzolatti a, * a
Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil c Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil d Laboratório de Química dos Produtos Naturais, Centro de Pesquisas René Rachou, Fiocruz, Belo Horizonte, MG, Brazil e Departamento de Química, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 January 2011 Received in revised form 28 June 2011 Accepted 10 July 2011 Available online 3 August 2011
A series of new 6-quinolinyl and quinolinyl N-oxide chalcones were efficiently prepared. All chalcones were tested by minimal inhibitory concentration (MIC) against three species of Candida, Cryptococcus gattii and Paracoccidioides brasiliensis. The effect of these compounds was also tested on the survival and growth of the human cancer cell lines UACC-62 (melanoma), MCF-7 (breast), TK-10 (renal) and leukemic cells, Jurkat and HL60. The compounds tested presented strong activity against P. brasiliensis, most importantly compound 4e. C. gattii also presented interesting susceptibility for compounds 5b and 5f. The cytotoxic activity showed that compounds 3c and 4e, presented the best activity against MCF-7 and TK-10. For leukemic cells the compounds 4f, 3g, 4g and 5g have shown the best activity. Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords: Quinolinyl chalcones Quinolinyl N-oxide chalcones Cytotoxic activity Antifungal activity
1. Introduction Chalcones are an important class of natural compounds and have already been widely applied as synthons in synthetic organic chemistry. These a,b-unsaturated ketone derivatives have been reported to possess diverse and interesting biological properties such as anti-inflammatory [1e3], antioxidant [4], antiviral [5], antifungal [6], antitumor [7aec], antimalarial [8,9a,b], antileishmanial [10a,b], analgesic [11], antituberculosis [9a,12], antihyperglycemic [13] and anti-HIV [14]. In recent years, the necessity of effective therapies has led to research in novel biologically active agents. In these recent researches, the synthetic chalcones which have in its structure a heterocyclic ring have shown therapeutic potential, especially when the present heteroatom in the heterocyclic nucleus is the * Corresponding author. Tel.: þ55 48 3721 6844. ** Corresponding author. Tel.: þ55 55 3220 8759. E-mail addresses:
[email protected] (M.E.F. Braibante),
[email protected]. br (M.G. Pizzolatti). 0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2011.07.019
nitrogen. For example, was reported for the first time the antifilarial activity of benzotriazolyl, pyrrolidinyl and piperidinyl chalcones [15]. Another important example was the first study where the a,bunsaturated ketone derivatives, which have heterocyclic systems, were used as combination therapy against Plasmodium falciparum in vitro [16]. Among several synthetic chalcones with heterocyclic systems described in the literature, the derivatives that have in their structure the quinoline nucleus have been little studied in relation to its pharmacological effects. The 3, 4 or 7-quinolinylacrylophenones (quinolinyl chalcones) have been reported as antimalarial, antimicrobial, antileishmanial antiprotozoal, anticancer, anti-inflammatory and antidiabetic agents [17a,b,18aed]. On the other hand, no biological activity has been described for compounds 6-quinolinyl chalcones (Fig. 1). Our particular interest in the class of quinoline N-oxide derivatives lies in the fact that these species have been found in drugs exerting anticancer [19], antiviral [20] and antimalarial activities, microssomal Na, K-ATPase activity [21] or protein kinase inhibition [22]. Based on these aspects, we decided to report the synthesis and study of cytotoxic and antifungal activities of a series of 6-
L. de Carvalho Tavares et al. / European Journal of Medicinal Chemistry 46 (2011) 4448e4456
5
6 7
3
R2
2
R3
N 1
8
O
R1
4
Quinoline nucleus
IMe N
R4
R
N
R1 = H, Ph
a b c d e f g
O
R2 = H, Me, OMe R 3, R4 = H, Me Quinolinyl chalcones
quinolinylacrylophenones 3aeg as well as new derivatives of N-oxides 4aeg and salts 5aeg (Scheme 1 and 2). In the treatment of cancer, most of the chemotherapeutic agents demonstrate severe toxicity and cause many undesirable side effects. Besides, they are highly expensive, mutagenic, carcinogenic and teratogenic [23]. Therefore, it is necessary to find other drugs, and natural products are a good source for this. Mycoses have recently shown an important increase in their incidence because the general population is more exposed to factors that favor mycosis infection. In many cases, these predisposing factors are related to immunodeficiency. In the case of candidosis, these include treatment with antibiotics, steroids, cytostatic, and immunodepressant drugs; organ and bone marrow transplantation; diabetes; leukemia, lymphoma, and other types of cancer; AIDS; and malnutrition. Candidosis is the emerging mycosis that has the greatest effect due to its frequency and the severity of its complications [24]. In contrast, Cryptococcus gattii cause cryptococcal infections mostly in immunocompetent hosts [25]. The treatment of these mycosis is another important challenge. Despite new and more effective antimycotic drugs, therapies often fail because of ignorance regarding doses and therapeutic regimes or because of its increasing resistance to antifungal drugs [24]. Paracoccidioides brasiliensis, a dimorphic fungus occurring in Central and South America, is responsible for paracoccidioidomycosis (PCM), an endemic disease that may affect at least 10 million people in Latin America. The drugs most commonly used for treating patients with PCM are sulfonamides, ketoconazole, itraconazole and amphotericin B. These drugs can present toxicity with long periods of treatment or with high relapse incidence O 150-160 oC 24h
H N 1
O
O
O H
+
10% NaOH R
N 1
EtOH
acetone ref lux
N Me I
R 5a-g
R= H R=Me R=OMe R=NO2 R=F R=Br R= Cl
Scheme 2. Synthesis of the salts of the chalcones 5aeg.
Fig. 1. Structures of the quinoline nucleus and 3, 4 or 7-quinolinylacrylophenones.
SeO2
R 3a-g
R= H, Me, Cl,
N
O
O
Ar N
4449
N
[26e29]. These limitations emphasize the necessity to develop new and more effective antifungal agents.
2. Results and discussion 2.1. Chemistry A series of 6-quinolinyl chalcones 3aeg was synthesized by a condensation [3] of 6-quinolinecarboxaldehyde 1 with commercially available acetophenones 2aeg as shown in Scheme 1. The precursor 1 was obtained by reaction of oxidation of 6methylquinoline with selenium dioxide (SeO2) according reported procedure [30]. The structures of the products were unambiguously established based on the 1H and 13C NMR spectra, IR and elemental analysis. All the compounds presented the characteristic absorption in the IR (1654e1660 cm1 (C]O), 1570e1575 cm1 (C]C)). The 1H NMR spectra of these products 3aeg, in particular, showed two doublets around 7.60 ppm for Ha, and 8.00 ppm for Hb attributed to the a,b-unsaturated system, with a coupling constant between 15 and 16 Hz for their trans isomer. The 6-quinolinyl chalcones 3f (R ¼ Cl) and 3g (R ¼ F) were described for the first time. We also performed reaction of the systems 3aeg with the oxidizing agent m-CPBA in dichloromethane to generate efficiently the new 6-quinolinyl N-oxide chalcones 4aeg (Scheme 1). The structure of each product N-oxide was identified from spectroscopic data. All the compounds have shown the characteristic absorption in the IR (1204e1294 cm1 (NeO)). The salts of the chalcones 5aeg were obtained from the reaction of 6-quinolinyl chalcones 3aeg with methyl iodide in acetone as in Scheme 2. Besides elucidating the structures by the use of spectroscopic techniques, yellow crystals were obtained and the structures 3b and 4f were reconfirmed by crystallographic methods. These crystalline structures were obtained from slow evaporation of the solvent mixture CHCl3/diisopropyl ether (1:1) for derivative 3b and CHCl3/diisopropyl ether (1:2) for derivative 4f. The X-ray diffraction study confirmed the olefinic C9 ¼ C10 double bond has E configuration in both compounds (Figs. 2 and 3). 6-Quinolinyl group is quasi planar with respect to open chain (dihedral angles between
R 3a-g
2a-g
m-CPBA CH 2Cl2 a b c d e f g
R= H R=Me R=OMe R=NO2 R=F R=Br R= Cl
O
N O
R 4a-g
Scheme 1. Synthesis of the 6-quinolinyl chalcones 3aeg and N-oxide chalcones 4aeg.
Fig. 2. ORTEP plot of 6-quinolinyl chalcone 3b. Ellipsoids are shown at 40% probability level.
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Fig. 3. ORTEP plot of 6-quinolinyl N-oxide chalcone 4f. Ellipsoids are shown at 40% probability level.
main planes of these groups are 3.60(16)o for 3b and 7.27(22)o for 4f), whereas phenyl rings are twisted about 15 with respect to propenalyl mean plane. These small angles indicate high electronic conjugation involving aromatic rings and propenalyl chain. It is evidenced by the shortening of single bonds and lengthening of double bonds in the propenalyl fragment (Tables MS3 and MS4). 2.2. Biological activity In the present work all series of 6-quinolinyl chalcones (3aeg, 4aeg and 5aeg) were tested against five pathogenic fungi. P. brasiliensis was the most susceptible fungus to the compounds tested (Table 1). Only 6-quinolinyl chalcone 3f did not present activity against this fungus. The 6-quinolinyl N-oxide chalcone 4e revealed the best result against P. brasiliensis with MIC value of 1.90 mg/mL. C. gattii also presented interesting susceptibility to compounds tested. The salts 5b and 5f were the most potent compound against this fungus with MIC of 7.80 mg/mL. In contrast, the Candida spp. were more resistant against the series 3aeg and 4aeg. Candida spp. were sensible only to 5aeg with MIC value among 15.60e500.00 mg/mL. The salts of the chalcones 5aeg have shown activity against all the fungi tested, with MIC value among 7.80e500.00 mg/mL.
When comparing the antifungal activity of 6-quinolinyl chalcones with control drugs used, we observed that amphotericin B is more potent than chalcones, but trimethoprim-sulfamethoxazole is weaker than chalcones tested in the experimental conditions used for P. brasiliensis. Unfortunately, amphotericin B has been associated with substantial toxicity [27]. Sulfonamides were the first class of drugs available for treating patients with PCM, but long periods of treatment may be required (more than 2 years), with increasing concern about drug toxicity, cost of treatment, and unacceptable rates of noncompliance with therapy [26,28,29]. The fungal cell wall is an obvious target for development of antifungals since the biochemical machinery for its synthesis is not present in mammalian cells [31]. Therefore, in this work, we tested the action of the most active compounds (MIC 31.20 mg/mL) on cell walls of fungi used. According to literature [32] a distinctive feature of the specific inhibitors of the fungal cell wall is that the antifungal effect is reversed in media containing an osmotic stabilizer such as sorbitol. Osmotic stability has been used with Candida albicans and other fungi to study mode of action of several antibiotics. Aculeacin-treated C. albicans cells were viable if broth cultures were protected with sorbitol, but lysed if plated out on agar without an osmotic support [32]. Our results showed that the susceptibility to compounds 5bee of Candida tropicalis and C. gattii presented differences due to the presence of sorbitol in the culture medium (Table 2). MIC value of C. tropicalis in medium treated with 5b and supplemented with sorbitol produce an increase of MIC value (62.50 mg/mL) respective of those obtained without sorbitol (15.60 mg/mL). The compounds 5bee present difference the MIC value in presence of sorbitol against C. gattii is of only one dilution. These results suggested that the antifungal activity of 5b could affect fungal cell wall synthesis or assembly of C. tropicalis and 5bee of C. gattii. In order to achieve a possible structure activity relationship, the log ð1=MICðP: brasiliensisÞÞ and log ð1=MICðC:gattiiÞÞ were compared with different physicochemical descriptors such as SFlogP, clogP, p, sp, F, R, 3HOMO, 3LUMO, GAP(HOMO-LUMO), and equations of linear regression were calculated with the best correlated descriptors to describe the SAR (Structure Activity Relationship). Based on the inter-correlation matrix values, we can chose the most
Table 1 Antifungal activity of 6-quinolinyl chalcones (3aeg), 6-quinolinyl N-oxide (4aeg) and salts (5aeg) of the chalcones. Compounds
MIC (mg/mL) against five pathogenic fungi Paracoccidioides Brasiliensis (Pb18)
Candida albicans
Candida.tropicalis
Candida parapsilosis
Cryptococcus gattii
3a 3b 3c 3d 3e 3f 3g 4a 4b 4c 4d 4e 4f 4g 5a 5b 5c 5d 5e 5f 5g Amphotericin B Trimethoprim-sulfamethoxazole
7.80 7.80 62.50 500.00 7.80 >500.00 62.50 62.50 125.00 15.60 500.00 1.90 62.50 15.50 500.00 62.50 125.00 250.00 nt 31.20 125.00 0.006 300.00
>500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 125.00 62.50 >500.00 500.00 250.00 500.00 1.00 nt
>500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 125.00 15.60 62.50 250.00 62.50 15.60 31.20 0.25 nt
>500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 >500.00 125.00 >500.00 >500.00 125.00 >500.00 0.50 nt
125.00 >500.00 >500.00 >500.00 500.00 >500.00 >500.00 250.00 500.00 >500.00 >500.00 62.50 62.50 250.00 31.20 7.80 31.20 31.20 31.20 7.80 15.60 1.20 nt
nt ¼ not tested.
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Table 2 Effect of 1 M sorbitol in the Minimal inhibitory concentration of active compounds. -: MIC > 31.20 mg/mL. Compounds
3a 3b 3e 4c 4e 4g 5b 5c 5d 5e 5f 5g
MIC (mg/mL) Paracoccidioides brasiliensis (Pb18)
P. brasiliensis (Pb18) plus 1M sorbitol
Candida tropicalis
Candida tropicalis plus 1M sorbitol
Cryptococcus gattii
Cryptococcus gatii plus 1M sorbitol
7.80 7.80 7.80 15.60 1.90 15.60 e e e e 31.20 e
7.80 7.80 7.80 15.60 1.90 15.60 e e e e 31.20 e
e e e e e e 15.60 e e e 15.60 31.20
e e e e e e 62.50 e e e 15.60 31.20
e e e e e e 7.80 31.20 31.20 15.60 7.80 15.60
e e e e e e 15.50 62.50 62.50 62.50 7.80 15.60
Table 3 Correlation coefficient values (R2) for the inter-correlation between biological activities and physicochemical descriptors, including compounds 3aeg and 4aeg. n
Activities
Descriptor SFLogP
13 7
Log1/[MIC(P.brasiliensis)] Log1/[MIC(C.gattii)]
6 2
Log1/[MIC(P.brasiliensis)] Log1/[MIC(C.gattii)]
7 5
Log1/[MIC(P.brasiliensis)] Log1/[MIC(C.gattii)]
a b
cLogP
p
Compounds 3aeg and 4aeg 0.115 0.425 0.254 0.061 0.180 0.188 Compounds 3aeg 0.081 0.454 0.404 e e e Compounds 4aeg 0.272 0.425 0.163 0.322 0.181 0.118
sp
F
R
3Homo
3Lumo
Gap
Log MW
0.561a 0.535
0.086 0.441
0.566 0.102
0.195 0.097
0.607 0.648
0.301 0.344
0.383 0.514
0.081 e
0.544 e
0.330 e
0.853b e
0.874 e
0.864 e
0.897 e
0.272 0.764
0.271 0.934
0.772 0.648
0.271 0.727
0.506 0.731
0.575 0.636
0.177 0.627
R2 values in the following range (0.500 < R2 < 0.699) are depicted in italics. R2 values in the following range (0.700 < R2 < 0.999) are depicted in bold.
significant descriptors which were correlated together and separated for two series of the 6-quinolinyl chalcones and N-oxide derivatives as shown in Table 3. According to the r2 values for the individual groups 3aeg and 4aeg, these two series have distinctive properties. A biological and physicochemical correlation was also performed in the same way and an internal evaluation was made by the analysis of n (number of compounds), r (correlation coefficient), s (standard deviation), F (Fisher value) and Q2 (squared cross-
validation regression coefficient). Optimum correlation values were found between log ð1=MICðP: brasiliensisÞÞ and the physicochemical descriptors 3HOMO, 3LUMO, GAP(HOMO-LUMO) and log(MW) for 6-quinolinylacrylophenones series (3aeg). On the other hand, with the insertion of the N-oxide group in the quinoline nucleus (series 4aeg), the best correlation found was with the physicochemical descriptor R, suggesting the importance of the most electronegative substituent such as fluorine atom. In
Table 4 Anticancer activity of 6-quinolinyl, 6-quinolinyl N-oxide chalcones and salts of the chalcones. Compounds
3a 3b 3c 3e 3g 4a 4b 4c 4e 4f 4g 5a 5b 5c 5d 5e 5f 5g Etoposide e: not active.
IC50 against five cancer cell lines MCF-7
TK-10
UACC62
HL60
Jurkat
1.10 0.46 e 0.19 0.01 6.30 0.30 0.96 0.10 e 8.30 0.20 1.15 0.20 4.30 0.20 6.60 0.20 6.60 0.20 e e e e e e e 0.03 0.01
7.00 0.10 e e 1.00 0.05 e e 1.00 0.01 5.25 1.00 0.19 0.01 1.10 0.10 1.70 0.60 e e e e e e e 20.40 0.01
3.83 0.30 e 22.50 3.5 5.60 0.50 e e 5.30 0.50 4.30 0.90 2.75 0.30 4.70 0.90 4.90 0.90 e e e e e e e 1.40 0.01
e 8.28 0.80 12.60 0.80 20.80 3.40 8.80 0.70 4.90 0.40 20.30 5.7 14.80 2.10 e 2.60 1.90 33.80 0.80 e e 25.20 4.80 e e e 9.90 2.30 0.47 0.25
e e 19.60 7.50 26.50 1.70 15.00 3.50 e 32.50 4.50 36.30 0.60 71.90 18.00 65.50 4.30 4.90 0.40 e e 11.90 1.60 e e 7.30 1.70 4.90 0.40 2.80 0.86
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fact, the N-oxide with F substituent (4e) has the strongest antifungal activity against P. brasiliensis among the 20 compounds tested, followed by Cl and Br derivatives (Table 1). The effect of all series of 6-quinolinyl chalcones (3aeg, 4aeg and 5aeg) was investigated on the survival and growth of the human cancer cell lines UACC-62 (melanoma), MCF-7 (breast), and TK-10 (renal). Nine compounds presented more than 70% of inhibition to cancer cells utilized. These compounds were tested to determine the dose response curves and the IC50 (inhibition of 50% of growth). The results (Table 4) showed that compounds 3c and 4e, presented the best activity against MCF-7 and TK-10, respectively with IC50 of 0.19 0.01 mg/mL and 0.19 0.01 mg/mL. All the compounds analyzed against TK-10 showed values of IC50 minor than drug control tested (etoposide), which was of 20.40 0.01 mg/mL. The other compounds also presented good activity with IC50 among 1.00 0.01e22.50 3.50 mg/mL. The effect on the growth of leukemic cells Jurkat and HL60 were less effective than human cancer cell lines cited above. The compounds 4f, 4g and 5g were more effective with IC50 of 2.60 1.90, 4.90 0.40 and 4.90 0.40 mg/mL, respectively. The best activity for HL60 was observed for compounds 4a and 4f with IC50 of 4.90 0.40 and 2.60 1.90 mg/mL, respectively. We observed differences between the susceptibility of the leukemia and solid tumor cell lines showed by selectivity of the same compounds to HL60 leukemic cells. Among the series of 6-quinolinyl chalcones 3aeg, the compound 3b, which possesses the methyl substituent, was selective for HL60 cells with IC50 of 8.28 0.80 mg/mL. On the other hand, in the 6quinolinyl N-oxide chalcones 4aeg series, the compound 4a phenyl unsubstituted was selective for HL60 cells with IC50 of 4.90 0.40 mg/mL. While in the series of the salts 5aeg, the brominated derivative, compound 5f, was selective for Jurkat cells with IC50 of 7.30 1.70 mg/mL. The compound 5g that possesses the chlorine substituent displayed antiproliferative effects against the two leukemia cells (IC50 values of 9.90 2.30 and 4.90 0.40 mg/ mL to HL60 and Jurkat, respectively), without effect against the solid tumor cell lines. Consequently, we found no relationship between the substituents and selectivity for leukemia cells. 3. Conclusion In our study, we have prepared and characterized a series of new 6-quinolinyl N-oxide chalcones as well as the 6-quinolinyl chalcones and salts. The majority of chalcones tested against pathogenic fungi showed strong activity, mainly P. brasiliensis and C. gattii. Added, the results suggested the compounds 5bee could affect fungal cell wall synthesis or assembly of C. tropicalis (5b) and C. gattii (5bee). The series compounds also presented good activity against tumoral cells. These results suggest that new 6-quinolinyl chalcones, N-oxide and salts of the chalcones are attractive lead compounds that deserve further investigation aiming at developing new drugs to treat paracoccidioidomycoses, cryptococcosis and cancer. 4. Experimental 4.1. Chemistry Melting points were determined with a Microquímica APF-301 apparatus and are uncorrected. Infrared (IR) spectra were determined as KBr pellets on a Abb Bomen FTLA 2000 spectrometer. 1H and 13C NMR spectra were recorded in CDCl3 or DMSO-d6 on a Bruker DPX (200/400 MHz)/Varian Oxford AS-400 (400 MHz) spectrometers. The elemental analyses were carried out with a CHN EA 1110. Percentages of C and H were in agreement with the product formula (within 0.4% of theoretical values to C).
4.1.1. General procedure for synthesis of 6-quinolinyl chalcones (3aeg) A mixture of 6-quinolinecarboxaldehyde 1 (2 mmol), the respective acetophenone 2aeg (2 mmol) was dissolved in 30 mL ethanol. To this mixture, sodium hydroxide (10%, 4 mL) was added at 0e5 C. The reaction mixture was stirred at room temperature for 15 h. The yellow solid thus obtained was filtered, washed with water and EtOH. The compounds were recrystallized from dichloromethane/diisopropyl ether to give analytically pure 6quinolinyl chalcones 3aeg. 4.1.1.1. (2E)-1-phenyl-3-(6-quinolinyl)-2-propen-1-one (3a). Yield 73%; yellow solid; m.p. 145e146 C; IR (KBr, cm1) 1660 (C]O), 1575 (C]C); 1H NMR (400 MHz, CDCl3, ppm): d 8.9 (dd, J ¼ 4.4, 1.7 Hz, 1H, Ar-Qn), 8.2 (dd, J ¼ 8.1, 1.5 Hz, 1H, Ar-Qn), 8.1 (d, J ¼ 8.8 Hz, 1H, Ar-Qn), 8.0 (m, 5H, (2 Ar-Qn, 2 Ar, 1Hb)), 7.7 (d, J ¼ 15.7 Hz, 1H, Ha), 7.6 (m, 1H, Ar), 7.5 (m, 2H, Ar), 7.4 (dd, J ¼ 8.3, 4.2 Hz, 1H, Ar-Qn); 13C NMR (100 MHz, CDCl3, ppm): d 190.09, 151.37, 149.13, 143.65, 138.00, 136.41, 133.06, 132.90, 130.26, 129.92, 128.64 (two C, Ar), 128.48 (two C, Ar), 128.26, 127.28, 123.09, 121.83; Anal. Calcd. for C18 H13NO: C,83.37; H, 5.05; N, 5.40, Found: C, 83.39; H, 5.06; N, 5.36. 4.1.1.2. (2E)-1-(4-Methylphenyl)-3-(6-quinolinyl)-2-propen-1-one (3b). Yield 62%; yellow solid; m.p. 161e162 C; IR (KBr, cm1) 1660 (C]O), 1571 (C]C); 1H NMR (400 MHz, CDCl3, ppm): d 8.9 (dd, J ¼ 4.3, 1.6 Hz, 1H, Ar-Qn), 8.1 (d, J ¼ 8.3 Hz, 1H, Ar-Qn), 8.1 (d, J ¼ 8.8 Hz, 1H, Ar-Qn), 8.0 (m, 1H, Ar-Qn), 8.0 (d, J ¼ 8.3 Hz, 2H, Ar), 7.9 (m, 2H, (1 Ar-Qn, 1Hb)), 7.6 (d, J ¼ 15.7 Hz, 1H, Ha), 7.4 (dd, J ¼ 8.3, 4.2 Hz, 1H, Ar-Qn), 7.3 (d, J ¼ 8.1 Hz, 2 H, Ar), 2.4 (s, 3H, CH3); 13 C NMR (100 MHz, CDCl3, ppm): d 189.41, 151.17, 148.95, 143.72, 143.10, 136.34, 135.33, 133.08, 130.06, 129.75, 129.26 (two C, Ar), 128.55 (two C, Ar), 128.17, 127.23, 122.97, 121.72, 21.54; Anal. Calcd. for C19 H15NO: C, 83.49; H, 5.53; N, 5.12. Found: C, 83.21; H, 5.83; N, 5.10. 4.1.1.3. (2E)-1-(4-Methoxyphenyl)-3-(6-quinolinyl)-2-propen-1-one (3c). Yield 58%; yellow solid; m.p. 134e136 C; IR (KBr, cm1) 1654 (C]O), 1570 (C]C); 1H NMR ( 200 MHz, CDCl3, ppm): d 8.9 (dd, J ¼ 4.2, 1.5 Hz, 1H, Ar-Qn), 8.2 (m, 5H, (3 Ar-Qn, 2 Ar)), 8.0 (m, 2H, (1 Ar-Qn, 1Hb)), 7.7 (d, J ¼ 15.7 Hz, 1H, Ha), 7.4 (dd, J ¼ 8.2, 4.3 Hz, 1H, Ar-Qn), 7.0 (d, J ¼ 8.8 Hz, 2 H, Ar), 3.9 (s, 3 H, OCH3); 13C NMR (50 MHz, CDCl3, ppm): d 188.12, 163.42, 151.17, 148.91, 142.70, 136.34, 133.12, 130.74 (three C, Ar), 130.04, 129.72, 128.16, 127.21, 122.72, 121.72, 113.7 (two C, Ar), 55.37; Anal. Calcd. For C19 H15NO2: C, 78.87; H, 5.23; N, 4.84. Found: C, 78.96; H, 5.55; N, 4.82. 4.1.1.4. (2E)-1-(4-Nitrophenyl)-3-(6-quinolinyl)-2-propen-1-one (3d). Yield 66%; yellow solid; m.p. 257e258 C; IR (KBr, cm1) 1660 (C]O), 1573 (C]C); 1H NMR (200 MHz, CDCl3, ppm): d 9.0 (dd, J ¼ 4.2, 1.5 Hz, 1H, Ar-Qn), 8.4 (d, J ¼ 9.05, 2H, Ar), 8.2 (m, 7H, (2 Ar, 4 Ar-Qn, 1Hb)), 7.6 (d, J ¼ 15.9 Hz, 1H, Ha), 7.5 (dd, J ¼ 8.3, 4.2 Hz, 1H, Ar-Qn); 13C NMR (100 MHz, CDCl3 þ CD3OD, ppm): d 189.01, 151.21, 150.24, 148.78, 145.43, 142.76, 137.13, 132.84, 130.39, 129.77, 129.37 (two C, Ar), 128.48, 127.60, 123.77 (two C, Ar), 122.74, 122.08; Anal. Calcd. for C18H12N2O3: C, 71.05; H, 3.97; N, 9.21. Found: C, 71.15; H, 4.01; N, 9.17. 4.1.1.5. (2E)-1-(4-Fluorophenyl)-3-(6-quinolinyl)-2-propen-1-one (3e). Yield 57%; yellow solid; m.p. 172e174 C; IR (KBr, cm1) 1660 (C]O), 1573 (C]C); 1H NMR (400 MHz, CDCl3, ppm): d 9.0 (dd, J ¼ 4.2, 1.7 Hz, 1H, Ar-Qn), 8.2 (d, J ¼ 8.3, 1H, Ar-Qn), 8.1 (m, 5H, (3 Ar-Qn, 2 Ar)), 8.0 (d, J ¼ 15.7 Hz, 1H, Hb), 7.70 (d, J ¼ 15.7 Hz, 1H, Ha), 7.5 (dd, J ¼ 8.3, 4.2 Hz, 1H, Ar-Qn), 7.2 (m, 2H, Ar); 13C NMR (100 MHz, CDCl3, ppm): d 188.45, 166.94, 164.41, 151.46, 149.17,
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143.91, 136.48, 134.37, 134.34, 132.96, 131.16, 131.06, 130.31, 130.08, 128.30, 127.25, 122.62, 121.91, 115.92, 115.70; Anal. Calcd. for C18 H12FNO: C, 77.97; H, 4.36; N, 5.05. Found: C, 77.53; H, 3.68; N, 5.11.
135.53, 135.16, 130.77, 129.53, 129.43 (two C, Ar), 128.69 (two C, Ar), 128.19, 125.98, 124.51, 121.85, 120.67, 21.67; Anal. Calcd. for C19 H15NO2: C, 78.87; H, 5.23; N, 4.84. Found: C, 78.38; H, 5.43; N, 4.88.
4.1.1.6. (2E)-1-(4-Bromophenyl)-3-(6-quinolinyl)-2-propen-1-one (3f). Yield 61%; yellow solid; m.p. 199e200 C; IR (KBr, cm1) 1657 (C]O), 1573 (C]C); 1H NMR (400 MHz, CDCl3, ppm): d 8.9 (dd, J ¼ 4.2, 1.5 Hz, 1H, Ar-Qn), 8.2 (d, J ¼ 7.8 Hz, 1H, Ar-Qn), 8.1 (d, J ¼ 8.8 Hz, 1H, Ar-Qn), 8.0 (m, 3H, (2 Ar-Qn, 1Hb)), 7.9 (d, J ¼ 8.6 Hz, 2H, Ar), 7.7 (d, J ¼ 8.3 Hz, 2 H, Ar), 7.6 (d, J ¼ 15.7 Hz, 1H, Ha), 7.5 (dd, J ¼ 8.3, 4.4 Hz, 1H, Ar-Qn); 13C NMR (100 MHz, CDCl3, ppm): d 189.01, 151.52, 149.24, 144.28, 136.76, 136.49, 132.91, 131.99 (two C, Ar), 130.38, 130.19, 130.02 (two C, Ar), 128.31, 128.11, 127.25, 122.51, 121.95; Anal. Calcd. for C18 H12BrNO: C, 63.92; H, 3.58; N, 4.14. Found: C, 63.29; H, 3.66; N, 4.09.
4.1.2.3. (2E)-1-(4-Methoxyphenyl)-3-(1-oxide-6-quinolinyl)-2propen-1-one (4c). Chromatography: 45% EtOAc-hexane; yield 73%; pale yellow solid; m.p. 187e189 C; IR (KBr, cm1) 1655 (C] O), 1568 (C]C), 1260 (N-O); 1H NMR (400 MHz, CDCl3, ppm): d 8.8 (d, J ¼ 9.0 Hz, 1H, Ar-Qn), 8.5 (d, J ¼ 5.9 Hz, 1H, Ar-Qn), 8.0 (m, 4 H, (2 Ar-Qn, 2 Ar)), 7.9 (d, J ¼ 15.7 Hz, 1H, Hb), 7.8 (d, J ¼ 8.3 Hz, 1H, ArQn), 7.7 (d, J ¼ 15.7 Hz, 1H, Ha), 7.3 (dd, J ¼ 8.3, 6.1 Hz, 1H, Ar-Qn), 7.0 (d, J ¼ 8.8 Hz, 2 H, Ar), 3.9 (s, 3 H, OCH3); 13C NMR (100 MHz, CDCl3, ppm): d 187.90, 163.72, 141.96, 141.45, 136.22, 135.62, 130.90 (two C, Ar), 130.77, 130.64, 129.42, 128.19, 126.00, 124.38, 121.83, 120.65, 113.96 (two C, Ar), 55.49; Anal. Calcd. for C19 H15NO3: C, 74.74; H, 4.95; N, 4.59. Found: C, 74.66; H, 5.01; N, 4.62.
4.1.1.7. (2E)-1-(4-Chlorophenyl)-3-(6-quinolinyl)-2-propen-1-one (3g). Yield 51%; yellow solid; m.p. 196e198 C; IR (KBr, cm1) 1658 (C]O), 1570 (C]C); 1H NMR (400 MHz, CDCl3, ppm): d 9.0 (dd, J ¼ 4.3, 1.6 Hz, 1H, Ar-Qn), 8.2 (d, J ¼ 7.3 Hz, 1H, Ar-Qn), 8.1 (d, J ¼ 8.8 Hz, 1H, Ar-Qn), 8.0 (m, 5H, (2 Ar-Qn, 2 Ar, 1Hb)), 7.6 (d, J ¼ 15.7 Hz, 1H, Ha), 7.51 (d, J ¼ 8.6 Hz, 2H, Ar), 7.5 (dd, J ¼ 8.3, 4.2 Hz, 1H, Ar-Qn); 13C NMR (100 MHz, CDCl3, ppm): d 188.73, 151.48, 149.19, 144.16, 139.37, 136.45, 136.28, 132.86, 130.32, 130.14, 129.87 (two C, Ar), 128.97 (two C, Ar), 128.26, 127.21, 122.46, 121.90; Anal. Calcd. for C18 H12ClNO: C, 73.60; H, 4.12; N, 4.77. Found: C, 73.10; H, 4.13; N, 4.84. 4.1.2. General procedure for synthesis of 6-quinolinyl N-oxide chalcones (4aeg) To a solution of 6-quinolinyl chalcones 3aeg (2 mmol) in dichloromethane (CH2Cl2) (8 mL) at 0 C was added a solution of 77% m-chloroperoxybenzoic acid (m-CPBA) (0. 85 g, 3.69 mmol) in 4 mL of CH2Cl2 dropwise over a period of 10 min. After that, the reaction was stirred at room temperature for 13 h. To a mixture was added 1 mL of 10% aqueous sodium sulfite solution and was stirred for 1e2 min. The reaction was filtered and solid residue was washed with several small portions of CH2Cl2. The combined organic layers were washed with 10% Na2CO3 solution (3 15 mL). The solution was dried over anhydrous MgSO4. All the compounds were purified by column chromatography on silica gel to give the pure analytically 6-quinolinyl N-oxide chalcones 4aeg. 4.1.2.1. (2E)-3-(1-oxide-6-quinolinyl)-1-phenyl-2-propen-1-one (4a). Chromatography: 30% EtOAc-hexane; yield 65%; pale yellow solid; m.p. 148e150 C; IR (KBr, cm1) 1674 (C]O), 1571 (C]C), 1204 (N-O); 1H NMR (400 MHz, CDCl3, ppm): d 8.9 (d, J ¼ 5.4 Hz, 1H, Ar-Qn), 8.8 (d, J ¼ 9.0 Hz, 1H, Ar-Qn), 8.2 (d, J ¼ 9.3 Hz, 1H, Ar-Qn), 8.1 (m, 4 H, (2 Ar-Qn, 2 Ar)), 7.9 (d, J ¼ 15.7 Hz,1H, Hb), 7.7 (d, J ¼ 15.7 Hz, 1H, Ha), 7.6 (m, 1H, Ar), 7.5 (m, 2 H, Ar), 7.4 (m, 1H, ArQn); 13C NMR (100 MHz, CDCl3, ppm): d 189.83, 142.32, 137.80, 136.49, 135.56, 133.21, 129.63, 128.79, 128.70 (two C, Ar), 128.59 (two C, Ar), 128.37, 128.07, 126.37, 124.65, 121.92, 120.81. Anal. Calcd. for C18 H13NO2: C, 78.53; H, 4.76; N, 5.09. Found: C, 78.55; H, 4.74; N, 5.13. 4.1.2.2. (2E)-1-(4-Methylphenyl)-3-(1-oxide-6-quinolinyl)-2propen-1-one (4b). Chromatography: 30% EtOAc-hexane; yield 78%; pale yellow solid; m.p. 176e177 C; IR (KBr, cm1) 1661 (C]O), 1567 (C]C), 1294 (N-O); 1H NMR (400 MHz, CDCl3, ppm): d 8.8 (d, J ¼ 9.0 Hz, 1H, Ar-Qn), 8.5 (d, J ¼ 6.1 Hz, 1H, Ar-Qn), 8.1 (dd, J ¼ 9.0, 1.7 Hz, 1H, Ar-Qn), 8.0 (s, 1H, Ar-Qn), 8.0 (d, J ¼ 8.1 Hz, 2H, Ar), 7.9 (d, J ¼ 15.7 Hz, 1H, Hb), 7.8 (d, J ¼ 8.3 Hz, 1H, Ar-Qn), 7.7 (d, J ¼ 15.7 Hz, 1H, Ha), 7.3 (m, 3 H, (1 Ar-Qn, 2 Ar)), 2.5 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3, ppm): d 189.22, 144.14, 142.00, 141.87, 136.23,
4.1.2.4. (2E)-1-(4-Nitrophenyl)-3-(1-oxide-6-quinolinyl)-2-propen1-one (4d). Chromatography: 5% EtOH-CH2Cl2; yield 70%; yellow solid; m.p. 256e257 C; IR (KBr, cm1) 1664 (C]O), 1571 (C]C), 211 (NeO); 1H NMR (400 MHz, CDCl3, ppm): d 8.8 (d, J ¼ 9.5 Hz, 1H, Ar-Qn), 8.6 (d, J ¼ 6.1 Hz, 1H, Ar-Qn), 8.4 (d, J ¼ 8.8 Hz, 2H, Ar), 8.2 (d, J ¼ 8.6 Hz, 2H, Ar), 8.0 (m, 2H, Ar-Qn), 8.0 (d, J ¼ 15.7 Hz, 1H, Hb), 7.8 (d, J ¼ 8.3 Hz, 1H, Ar-Qn), 7.6 (d, J ¼ 15.7 Hz, 1H, Ha), 7.4 (dd, J ¼ 7.8, 6.4 Hz, 1H, Ar-Qn); 13C NMR (100 MHz, CDCl3 þ CD3OD, ppm): d 188.39, 150.33, 143.10, 142.09, 139.50, 136.01, 135.03, 131.08, 130.72, 129.97 (two C), 129.55 (two C, Ar), 124.83, 123.92 (two C, Ar), 122.20, 119.71. Anal. Calcd. for C18 H12N2O4: C, 67,50; H, 3.78; N, 8.75. Found: C, 67.88; H, 3.52; N, 8.55. 4.1.2.5. (2E)-1-(4-Fluorophenyl)-3-(1-oxide-6-quinolinyl)-2-propen1-one (4e). Chromatography: 30% EtOAc-hexane; yield 73%; pale yellow solid; m.p. 216e218 C; IR (KBr, cm1) 1668 (C]O), 1566 (C]C), 1222 (NeO); 1H NMR (400 MHz, CDCl3, ppm): d 8.8 (d, J ¼ 8.8 Hz, 1H, Ar-Qn), 8.6 (d, J ¼ 2.9 Hz, 1H, Ar-Qn), 8.1 (m, 4H, (2 Ar-Qn, 2 Ar)), 8.0 (d, J ¼ 15.7 Hz, 1H, Hb), 7.8 (d, J ¼ 8.8 Hz, 1H, ArQn), 7.7 (d, J ¼ 15.7 Hz, 1H, Ha), 7.4 (m, 1H, Ar-Qn), 7.2 (m, 2H, Ar); 13 C NMR (100 MHz, CDCl3, ppm): d 188.10, 167.17, 164.62, 142.44, 141.93, 136.87, 135.54, 134.14, 134.11, 131.29, 131.19, 130.81, 129.70, 128.58, 127.16, 124.29, 121.93, 120.78, 116.08, 115.87. Anal. Calcd. for C18 H12 FNO2: C, 73.71; H, 4.12; N, 4.78. Found: C,73.58; H, 4.23; N, 4.87. 4.1.2.6. (2E)-1-(4-Bromophenyl)-3-(1-oxide-6-quinolinyl)-2-propen1-one (4f). Chromatography: 30% EtOAc-hexane; yield 67%; pale yellow solid; m.p. 186e188 C; IR (KBr, cm1) 1663 (C]O), 1567 (C]C), 1208 (N-O); 1H NMR (400 MHz, CDCl3, ppm): d 8.8 (d, J ¼ 9.0 Hz, 1H, Ar-Qn), 8.6 (d, J ¼ 6.1 Hz, 1H, Ar-Qn), 8.1 (m, 1H, ArQn), 8.0 (m, 3H, (1Hb, 2 Ar)), 7.8 (d, J ¼ 8.6 Hz, 1H, Ar-Qn), 7.7 (m, 3H, (1Ha, 2 Ar)), 7.4 (dd, J ¼ 8.3, 6.1 Hz, 1H, Ar-Qn); 13C NMR (100 MHz, CDCl3, ppm): d 188.68, 142.91, 142.19, 136.46, 136.39, 135.24, 132.09 (two C, Ar), 130.80, 130.06 (two C, Ar), 129.85, 128.41, 128.11, 125.91, 123.89, 122.00, 120.87; Anal. Calcd. for C18 H12BrNO2: C, 61.04; H, 3.41; N, 3.95. Found: C, 61.13; H, 3.36; N, 3.93. 4.1.2.7. (2E)-1-(4-Chlorophenyl)-3-(1-oxide-6-quinolinyl)-2-propen1-one (4g). Chromatography: 30% EtOAc-hexane; yield 77%; pale yellow solid; m.p. 180e182 C; IR (KBr, cm1) 1668 (C]O), 1566 (C]C), 1222 (NeO); 1H NMR (400 MHz, CDCl3, ppm): d 8.8 (d, J ¼ 8.8 Hz, 1H, Ar-Qn); 8.6 (d, J ¼ 5.9 Hz, 1H, Ar-Qn), 8.0 (m, 4H (2 ArQn, 2 Ar)), 7.9 (d, J ¼ 15.7 Hz, 1H, Hb), 7.8 (d, J ¼ 8.6 Hz, 1H, Ar-Qn), 7.7 (d, J ¼ 15.7 Hz, 1H, Ha), 7.5 (d, J ¼ 8,55, 2H, Ar), 7.4 (dd, J ¼ 8.6, 6.1 Hz, 1H, Ar-Qn); 13C NMR (100 MHz, CDCl3, ppm): d 188.36, 142.75, 142.09, 139.62, 136.30, 135.97, 135.16, 130.72, 129.90 (two C, Ar), 129.77, 129.02 (two C, Ar), 128.04, 125.84, 123.84, 121.93,
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120.75; Anal. Calcd. for C18 H12ClNO2: C, 69.80; H, 3.90; N, 4.52. Found: C, 69.92; H, 3.79; N, 4.51. 4.1.3. General procedure for synthesis of salts of the chalcones (5aeg) To a solution of 6-quinolinyl chalcones 3aeg (3 mmol) in acetone (15 mL) was added CH3I (1 mL, 15.4 mmol). After that, the reaction was stirred under reflux for 10 h. The resulting yellow powder was filtered and washed with acetone to give analytically pure salts of the chalcones 5aeg. 4.1.3.1. 6-[(1E)-3-oxo-3-phenyl-1-propen-1-yl] 1-methylquinolinium iodide (5a). Yield 77%; yellow solid; m.p. 222e224 C; 1H NMR (400 MHz, DMSO-d6, ppm): d 9.5 (d, J ¼ 5.6 Hz, 1H, Ar-Qn), 9.2 (d, J ¼ 8.6 Hz, 1H, Ar-Qn), 8.9 (s, 1H, Ar-Qn), 8.9 (dd, J ¼ 9.2, 1.8 Hz, 1H, Ar-Qn), 8.6 (d, J ¼ 9.3 Hz, 1H, Ar-Qn), 8.3 (d, J ¼ 15.7 Hz, 1H, Hb), 8.2 (d, J ¼ 7.1 Hz, 2H, Ar), 8.0 (d, J ¼ 15.9 Hz, 1H, Ha), 7.7 (m, 1H, Ar-Qn), 7.6 (m, 2H, Ar), 7.5 (m, 1H, Ar), 4.7 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6, ppm): d 189.00, 150.47, 147.10, 140.78, 137.10, 135.94, 134.08, 133.57, 130.91, 129.44, 128.89 (two C, Ar), 128.69 (two C, Ar), 127.85, 126.11, 122.77, 119.89, 45.49; Anal. Calcd. for C19H16INO: C, 56.87; H, 4.02; N, 3.49. Found: C, 56.81; H, 4.04; N, 3.52. 4.1.3.2. 6-[(1E)-3-(4-Methylphenyl)-3-oxo-1-propen-1-yl]-1methylquinolinium iodide (5b). Yield 78%; yellow solid; m.p. 244,6246,0 C; 1H NMR (400 MHz, DMSO-d6, ppm): d 9.5 (d, J ¼ 5.6 Hz, 1H, Ar-Qn), 9.2 (d, J ¼ 8.1 Hz, 1H, Ar-Qn), 8.9 (s, 1H, Ar-Qn), 8.8 (dd, J ¼ 9.2, 1.8 Hz, 1H, Ar-Qn), 8.5 (d, J ¼ 9.3 Hz, 1 H, Ar-Qn), 8.2 (m, 2H, (1 Ar-Qn, 1Hb)), 8.1 (d, J ¼ 8.1 Hz, 2H, Ar), 7.9 (d, J ¼ 15.9 Hz, 1H, Ha), 7.4 (d, J ¼ 8.1 Hz, 2H, Ar), 4.7 (s, 3H,), 2.4 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6, ppm): d 188.25, 150.15, 146.84, 143.79, 140.02, 138.68, 135.86, 134.46, 134.05, 130.37, 129.29, 129.17 (two C, Ar), 128.58 (two C, Ar), 126.19, 122.48, 119.54, 45.34, 20.96; Anal. Calcd. for C20H18INO: C, 57.85; H, 4.37; N, 3.37. Found: C, 57.76; H, 4.71; N, 3.46. 4.1.3.3. 6-[(1E)-3-(4-Methoxyphenyl)-3-oxo-1-propen-1-yl]-1methylquinolinium iodide (5c). Yield 82%; yellow solid; m.p. 245e247 C; IR (KBr, cm1) 1656 (C]O), 1571 (C]C); 1H NMR (400 MHz, DMSO-d6, ppm): d 9.5 (d, J ¼ 5.6 Hz, 1H, Ar-Qn), 9.2 (d, J ¼ 8.3 Hz, 1H, Ar-Qn), 8.9 (s, 1H, Ar-Qn), 8.8 (dd, J ¼ 9.2, 1.8 Hz, 1H, Ar-Qn), 8.5 (d, J ¼ 9.3 Hz, 1H, Ar-Qn), 8.2 (m, 4H, (1Hb, 2 Ar, 1 ArQn)), 7.9 (d, J ¼ 15.7 Hz, 1H, Ha), 7.1 (d, J ¼ 8.8 Hz, 2H, Ar), 4.7 (s, 3H, Nþ CH3), 3.9 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO-d6, ppm): d 187.02, 163.40, 150.14, 146.88, 139.60, 138.68, 136.01, 134.05, 130.93 (two C, Ar), 130.32, 129.92, 129.34, 126.24, 122.50, 119.56, 113.98 (two C, Ar), 55.49, 45.35; Anal. Calcd. for C20H18INO2: C, 55,70; H, 4,21; N, 3,25. Found: C, 55.98; H, 4.29; N, 3.36. 4.1.3.4. 6-[(1E)-3-(4-Nitrophenyl)-3-oxo-1-propen-1-yl]-1-methylquinolinium iodide (5d). Yield 81%; yellow solid; m.p. 256e257 C; 1 H NMR (400 MHz, DMSO-d6, ppm): d 9.5 (d, J ¼ 5.6 Hz, 1H, Ar-Qn), 9.2 (d, J ¼ 8.3 Hz, 1H, Ar-Qn), 8.9 (s, 1H, Ar-Qn), 8.8 (dd, J ¼ 9.3, 1.7 Hz, 1H, Ar-Qn), 8.6 (d, J ¼ 9.0 Hz, 1H, Ar-Qn), 8.4 (s, 4H, Ar), 8.2 (m, 2H, (1 Ar-Qn, 1Hb)), 8.0 (d, J ¼ 15.7Hz, 1H, Ha), 4.7 (s, 3H, Nþ CH3); 13C NMR (100 MHz, DMSO-d6, ppm): d 188.29, 150.43, 149.93, 147.02, 141.87, 141.75, 138.91, 135.55, 133.96, 130.96, 129.82 (two C, Ar), 129.28, 125.96, 123.65 (two C, Ar), 122.61, 119.70, 45.33; Anal. Calcd. for C19H15IN2O3: C, 51.14; H, 3.39; N, 6.28. Found: C, 51.26; H, 3.35 N, 6.21. 4.1.3.5. 6-[(1E)-3-(4- fluorophenyl)-3-oxo-1-propen-1-yl]-1-methylquinolinium iodide (5e). Yield 79%; yellow solid; m.p. 251e252 C; 1H NMR (400 MHz, DMSO-d6, ppm) d 9.5 (d, J ¼ 5.4 Hz, 1H, Ar-Qn), 9.2 (d, J ¼ 8.3 Hz, 1H, Ar-Qn), 8.9 (s, 1H, Ar-Qn), 8.8 (d,
J ¼ 9.3 Hz, 1H, Ar-Qn), 8.6 (d, J ¼ 9.3 Hz, 1H, Ar-Qn), 8.3 (m, 2H, (1 ArQn, 1Hb)), 8.2 (m, 2H, Ar), 7.9 (d, J ¼ 15.7 Hz, 1H, Ha), 7.4 (m, 2H, Ar), 4.7 (s, 3H, Nþ CH3); 13C NMR (100 MHz, DMSO-d6, ppm): d 187.40, 166.27, 163.76, 150.22, 146.86, 140.52, 138.74, 135.73, 133.97, 133.66, 131.52, 131.43, 130.56, 129.26, 125.94, 122.51, 119.58, 115.73, 115.51, 45.33; Anal. Calcd. for C19H15 FINO: C, 54.43; H, 3.61; N, 3.34. Found: C, 54,24; H, 3.91; N, 3.46. 4.1.3.6. 6-[(1E)-3-(4-Bromophenyl)-3-oxo-1-propen-1-yl]-1methylquinolinium iodide (5f). Yield 91%; yellow solid; m.p. 244e245 C; IR (KBr, cm1) 1659 (C]O), 1561 (C]C); 1H NMR (400 MHz, DMSO-d6, ppm): d 9.5 (d, J ¼ 5.4 Hz, 1H, Ar-Qn), 9.2 (d, J ¼ 8.8 Hz, 1H, Ar-Qn), 8.9 (s, 1H, Ar-Qn), 8.8 (dd, J ¼ 8.9, 1.6 Hz, 1H, Ar-Qn), 8.6 (d, J ¼ 9.3 Hz, 1H, Ar-Qn), 8.2 (m, 2H, (1 Ar-Qn, 1Hb)), 8.1 (d, J ¼ 8.6 Hz, 2H, Ar), 8.0 (d, J ¼ 15.7 Hz, 1H, Ha), 7.8 (d, J ¼ 8.6 Hz, 2H, Ar), 4.7 (s, 3H, Nþ CH3); 13C NMR (100 MHz, DMSO-d6, ppm): d 188.12, 150.30, 146.94, 140.90, 138.80, 136.00, 135.74, 133.98, 131.71 (two C, Ar), 130.66, 130.43 (two C, Ar), 129.30, 127.44, 125.89, 122.55, 119.61, 45.30; Anal. Calcd. for C19 H15BrINO: C, 47.53; H, 3.15; N, 2.92. Found: C, 47.12; H, 3.42; N, 2.89. 4.1.3.7. 6-[(1E)-3-(4-Chlorophenyl)-3-oxo-1-propen-1-yl]-1methylquinolinium iodide (5g). Yield 84%; yellow solid; m.p. 260e262 C; IR (KBr, cm1) 1659 (C]O), 1566 (C]C); 1H NMR (400 MHz, DMSO-d6, ppm): d 9.5 (d, J ¼ 4.6 Hz, 1H, Ar-Qn), 9.2 (d, J ¼ 8.3 Hz, 1H, Ar-Qn), 8.9 (s, 1H, Ar-Qn), 8.8 (d, J ¼ 8.6 Hz, 1H, ArQn), 8.6 (d, J ¼ 9.0 Hz, 1H, Ar-Qn), 8.2 (m, 4H (1 Ar-Qn, 2 Ar, 1Hb)), 7.9 (d, J ¼ 15.9 Hz, 1H, Ha), 7.7 (d, J ¼ 7.3 Hz, 2H, Ar), 4.7 (s, 3H, Nþ CH3); 13C NMR (100 MHz, DMSO-d6, ppm): d 187.83, 150.26, 146.88, 140.83, 138.76, 138.26, 135.67, 135.60, 133.98, 130.64, 130.33 (two C, Ar), 129.26, 128.72 (two C, Ar), 125.82, 122.53, 119.59, 45.34; Anal. Calcd. for C19 H15ClINO: C, 52.38; H, 3.47; N, 3.21. Found: C, 52.07; H, 3.78; N, 3.23. 4.2. Biological assays 4.2.1. Cell lines UACC-62 (melanoma), MCF-7 (breast), and TK-10 (renal). These cell lines were purchased from the National Cancer Institute, (Maryland, USA). Leukemia cells used in our studies include HL60 cells (human promyelocytic leukemia HL-60 cells) and human immortalized line of T lymphocyte (Jurkat cells) were a kind gift from Dr. Gustavo Amarante-Mendes (Universidade de São Paulo, São Paulo, Brazil). All lineages were cultivated in the logarithmic phase of growth in RPMI 1640 SigmaeAldrich (St. Louis, MO) supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin (GIBCO BRL, Grand Island, NY), enriched with 2 mM of L-glutamine and 10% of fetal bovine serum. All cultures were maintained at 37 C in a humidified incubator with 5% CO2. 4.2.2. Assays with human cancer cell lines The effect of compounds on the survival and growth of the solid human cancer cell lines UACC-62 (melanoma), MCF-7 (breast), and TK-10 (renal) was determined using a colorimetric method developed at the National Cancer Institute- USA [33,34]. Briefly, the cells were inoculated in 96-well plates and incubated at 37 C for 24 h in 5% CO2 atmosphere. The solutions of the test samples were added to the culture wells to attain the desired concentrations, and the plates incubated for further 48 h. Trichloroacetic acid was added to each well to precipitate the proteins, which were stained with sulforhodamine B. After washing out the unbound dye, the stained protein was dissolved with 10 mM Tris, and the absorbance measured at the wavelength of 515 nm. Results were calculated using the absorbance measured in the test wells (T) in comparison with that of the control wells corresponding to the initial cell
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inoculum (Ti) and cells grown for 48 h without drug (Tf), using the formula: [(T Ti)/(Tf Ti)] 100. This formula allows the quantification of both growth inhibition (values between zero and 100) and cell death (values smaller than zero). Each sample was tested in duplicate in two independent experiments. Experiments to determine the dose response curves and half maximal inhibitory concentration (IC50) were run as above, using 1:2 serial dilutions of the test compounds to reach the appropriate concentrations. The experiments were run in duplicate and repeated at least three times. The leukemia cell lines were cultured in 96 plate wells at densities of 50,000 cells/well and 100,000 cells/well, respectively, in a final volume of 200 mL/well. The plates were pre-incubated in a 5% CO2/95% air-humidified atmosphere at 37 C for 24 h to allow adaptation of cells prior to the addition of the test compounds. All compounds were dissolved in dimethyl sulfoxide (DMSO), prior to dilution. The half maximal inhibitory concentration (IC50) was determined over a range of concentrations (0.1e100 mg/mL). All cell cultures were incubated in a 5% CO2/95% air-humidified atmosphere at 37 C for 48 h. Cell viability was estimated measuring the rate of mitochondrial reduction of yellow tetrazolium salt MTT (3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SigmaeAldrich, St. Louis, MO) to insoluble purple formazan crystals [35]. After incubation with the test compounds, MTT solution (20 mL; 5 mg/mL) was added to each well and incubated for 4 h. At the end of this incubation, the supernatant was removed and 200 mL of 0.04 M HCl in isopropyl alcohol were added to dissolve the formazan crystal. The optical densities (OD) were measured with a spectrophotometer at 590 nm. Results were normalized with DMSO control (0.05%) and expressed as percentage of cell viability inhibition. Interactions of compounds and media were estimated on the basis of the variations between drug-containing medium and drug-free medium to control for false-positive or false-negative results. The IC50 values were obtained graphically from dose-effect curves using Prism 5.0 (GraphPad Software Inc.). For comparison, the cytotoxicity of cisplatin was evaluated under the same experimental conditions. The experiments were run in triplicate and performed in three different days. 4.2.3. Antifungal activity 4.2.3.1. Culture and maintenance. For an antifungal evaluation, strains from the American Type Culture Collection (ATCC, Rochville, MD, USA) were used: C. albicans ATCC 18804, C. tropicalis ATCC 750, Candida parapsilosis ATCC 22019 and C. gattii ATCC 32608. All fungal strains were maintained on Sabouraud Dextrose Agar (SDA, Oxoid, Basingstoke, UK) at 4 C and transfers were done at three-month intervals. P. brasiliensis strain, Pb18 (Faculty of Medicine, Universidade de São Paulo, São Paulo, Brazil) was maintained by continuous passages in YPD (yeast, peptone and dextrose) medium at 37 C. The P. brasiliensis was used after 710 days of growth. 4.2.3.2. Determination of minimal inhibitory concentrations (MIC). The bioassay with all fungi was performed following CLSI M27-A2 guidelines [36]. For assay with P. brasiliensis were done modifications suggested by Nakai et al. [37] and Johann et al. [38]. Amphotericin B (Sigma, St Louis, USA) and trimethoprim/sulfamethoxazole (SMT/TMP) (Ducto, Brazil) were included as positive antifungal controls. Their stock solutions were prepared in dimethyl sulfoxide and water, respectively, from which twofold serial dilutions were prepared as described in the CLSI document M27-A2 [36]. 4.2.3.3. Sorbitol protection assay. This assay was realized only for compounds that present the better results against fungi tested (MIC value 31.20 mg/mL). MIC values were determined using
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P. brasiliensis isolate Pb18, C. tropicalis (ATCC 750) and C. gattii (ATCC 32608) by the standard broth microdilution procedure described above. Duplicate plates were prepared: one containing twofold dilutions (from 1000 to 7.8 mg/mL) of compounds and the other containing compounds plus 1 M sorbitol, as an osmotic support. MICs were read after 2 days for C. tropicalis, 3 days for C. gattii and 10 days for P. brasiliensis at 37 C [39]. Acknowledgments We are grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for fellowships to L.C.T., S.J. thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG), G.F.C. and R.M.P. thank FAPESC for financial support grant 17413/2009-0, Brazil. Appendix. Supplementary material X-ray data collection and final refinement parameters including anisotropic thermal parameters and full list of the bond lengths and angles have been deposited with the Cambridge Crystallographic Data Center in the CIF format as supplementary publication No.: CCDC 809335 (3b) and 809336 (4f). Copies of these information can be obtained free of charge from the Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (Fax: þ44 1223 336033; email: deposit@ ccdc.cam.ac.uk). 1H NMR and 13C NMR spectra. Appendix. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ejmech.2011.07.019. References [1] H.H. Ko, L.T. Tsao, K.L. Yu, C.T. Liu, J.P. Wang, C.N. Lin, Bioorg. Med. Chem. 11 (2003) 105e111. [2] H. Matsuda, T. Morikawa, S. Ando, T. Iwao, Y. Masayuki, Bioorg. Med. Chem. 11 (2003) 1995e2000. [3] F. Herencia, M.L. Ferrandiz, A. Ubeda, J.N. Dominguez, J.E. Charris, G.M. Lobo, M.J. Alcaraz, Bioorg. Med. Chem. Lett. 8 (1998) 1169e1174. [4] S. Vogel, M. Barbic, G. Jurgenliemk, J. Heilmann, Eur. J. Med. Chem. 45 (2010) 2206e2213. [5] J.C. Trivedi, J.B. Bariwal, K.D.U. Naliapara, Y.T. Joshi, S.K. Pannecouque, E.D. Cleroq, A.K. Shah, Tetrahedron Lett. 48 (2007) 8472e8474. [6] S.N. Lopez, M.V. Castelli, S.A. Zacchino, J.N. Dominguez, G. Lobo, C.C. Jaime, J.C.G. Cortes, J.C. Ribas, C. Devia, M.R. Ana, D.E. Ricardo, Bioorg. Med. Chem. 9 (2001) 1999e2013. [7] (a) B.P. Bandgar, S.S. Gawande, Bioorg. Med. Chem. 18 (2010) 2206e2213; (b) K.V. Sashidhara, A. Kumar, M. Kumar, J. Sarkar, S. Sinha, Bioorg. Med. Chem. Lett. 20 (2010) 7205e7211; (c) Y. Na, J.M. Nam, Bioorg. Med. Chem. Lett. 21 (2011) 211e214. [8] R. Li, G.L. Kenyon, F. Cohen, X. Chen, B. Gong, J.N. Dominguez, E. Davidson, G. Kurzban, R.E. Millar, E.O. Nuzum, P.J. Rosenthal, J.H. Mckerrow, J. Med. Chem. 38 (1995) 5031e5037. [9] (a) M. Liu, P. Wilairat, M.L. Go, J. Med. Chem. 44 (2001) 4443e4452; (b) R.H. Hans, E.M. Guantai, C. Lategan, P.J. Smith, B. Wan, S.G. Franzblau, J. Gut, P.J. Rosenthal, K. Chibale, Bioorg. Med. Chem. Lett. 20 (2010) 942e944. [10] (a) S.F. Nielsen, S.B. Christensen, G. Cruciani, A. Kharazmi, T. Liljefors, J. Med. Chem. 41 (1998) 4819e4832; (b) J.C. Aponte, D. Castillo, Y. Estevez, G. Gonzalez, J. Arevalo, G.B. Hammonda, M. Sauvain, Bioorg. Med. Chem. Lett. 20 (2010) 100e103. [11] G.S. Viana, M.A. Bandeira, F.J. Matos, Phytomedicine 10 (2003) 189e195. [12] Y.M. Lin, Y. Zhou, M.T. Flavin, L.M. Zhou, W. Nie, F.C. Chen, Bioorg. Med. Chem. 10 (2002) 2795e2802. [13] P. Shukla, A.B. Singh, A.K. Srivastava, R. Pratap, Bioorg. Med. Chem. Lett. 17 (2007) 799e802. [14] H. Sharma, S. Patil, T.W. Sanchez, N. Neamati, R.F. Schinazi, J.K. Buolamwini, Bioorg. Med. Chem. 19 (2011) 2030e2045. [15] S.K. Awasthi, N. Mishra, S.K. Dixit, A. Singh, M. Yadav, S.S. Yadav, S. Rathaur, Am. J. Trop. Med. Hyg. 80 (2009) 764e768. [16] A. Bhattacharya, L.C. Mishra, M. Sharma, S.K. Awasthi, V.K. Bhasin, Eur. J. Med. Chem. 44 (2009) 3388e3393.
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