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Journal of the Chilean Chemical Society

versión On-line ISSN 0717-9707

J. Chil. Chem. Soc. v.51 n.2 Concepción jun. 2006

http://dx.doi.org/10.4067/S0717-97072006000200005 

 

J. Chil. Chem. Soc., 51, Nº 2 (2006) , pags: 859-863

 

EVALUATION OF ANTIPARASITIC, ANTITUBERCULOSIS AND ANTIANGIOGENIC ACTIVITIES OF 3-AMINOQUINOLIN-2-ONE DERIVATIVES

 

Gisela C. Muscia, Mariela Bollini, Ana M. Bruno and Silvia E. Asís*

Centro de Síntesis y Estudio de Nuevos Compuestos Antineoplásicos y Antiparasitarios. Departamento de Química Orgánica. Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires. Ciudad Autónoma de Buenos Aires. Argentina.


ABSTRACT

Parasitic infections such as leishmaniasis, trypanosomiasis and malaria have significant impacts in third world countries and are the major reasons of mortality. In order to search a new class of antiprotozoal agent, a series of ten quinolin-2-one derivatives was tested in vitro against the parasites causative of malaria, leishmaniasis, sleeping sickness and Chagas´ disease. In general, these compounds exhibited moderate activity against Plasmodium falciparum and showed no activity against Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei. Facing to establish a possible mechanism of antimalarial activity their binding to hemin was assayed. Furthermore, the need of developing new therapeutic strategies for the effective control of tuberculosis prompted us to assay twelve of these compounds against Mycobacterium tuberculosis but they did not demonstrate inhibitory activity. In addition, three terms resulted also structurally related to linomide, a recognized drug against angiogenesis, an interesting target for designing new antineoplastic agents. So these compounds were selected at the National Cancer Institute for angiogenesis testing and they exhibited similar activity to those recently reported for linomide and its analogues.

Keywords: 3-Aminoquinolin-2-one Derivatives, Angiogenesis, Plasmodium falciparum, Tuberculosis


INTRODUCTION

Parasitic infections such as leishmaniasis, trypanosomiasis and malaria have significant impacts in third world countries and are the major reasons of mortality. Conventional therapies are often inadequate, toxic or are becoming less effective due to the emergence of numerous resistances1.

Recently we have reported the chemical properties of 3-amino-4-methyl (or phenyl) quinolin-2-ones which were obtained in high yields employing a general procedure (Scheme 1)2. A family of several derivatives of 3-amino-6-chloro-4-phenyl-quinolin-2(1H)-one 3 was formerly designed as a new class of antimalarial agents possessing a quinoline nucleous (compounds 1-15, Schemes 1-3)2. Then eight of the synthesized compounds were selected for a primary in vitro screening against different parasites, including P. falciparum, at Tropical Disease Research (TDR) Program, World Health Organization (WHO, Switzerland).


Scheme 1: Synthesis of 3-amino-quinolin-2-ones
a: benzene, reflux; b: pyridine, reflux; c: aniline, reflux


Scheme 2: Synthesis of selected 2-quinolone derivatives
a: 1. NaOH, acetone, r.t., 2. ICH3, 0-5 ºC; b: PhNCO, CH2Cl2, r.t.; c: Ac2O, reflux; d: Ac2O, AcOH, reflux; e: PhCH2OCONHCH2CH2COCl, CH2Cl2, reflux, f: HCOOH, Pd/C 5 %, MeOH, reflux.


Scheme 3: reaction of 7 with NBS and synthesis of compounds 11 and 12
a: NBS, benzoylperoxide, Cl4C, reflux, 6 h; b: acetone, reflux, 3 h; c: NaBH4, MeOH, reflux, 4 h.

On the other hand, tuberculosis (TB) continues to be a public heath threat. The resurgence of TB in association with human inmunodeficiency virus warrants the development of new therapeutic strategies for its effective control. In addition, the increase in Mycobacterium tuberculosis strains resistant to front-line antimycobacterial drugs such as rifampin and isoniazid has complicated the problem. However, no new chemotherapeutic agent specifically directed against TB has been introduced in the past 40 years. Recently, some quinoline derivatives initially synthesized as the precursors for targeted antimalarials have been reported as potential anti-tuberculosis agents3. Among the quinolone class of anti-bacterial agents, fluoroquinolones have shown promise in the treatment of TB. In addition, four novel 7-substituted quinolones were synthesized and evaluated in vivo against a multi-drug resistant strain4.

For the above exposed we were encouraged to suggest our 2-quinolone derivatives for in vitro screening at the Tuberculosis Antimicrobial Acquisition and Coordination Facility (TAACF, USA). Thus, twelve compounds were selected and submitted for primary testing.

Since the Folkman research group suggested a novel concept of treatment solid cancer by inhibiting angiogenesis in 1971, clinical importance of therapeutic agents controlling it has been emphasized. Angiogenesis is the formation of new blood vessels from pre-existing ones. It has been shown that without angiogenesis a tumor can only reach the size of 1-2 mm, small enough to be easily resected or treated with conventional chemotherapeutic agents.

The antiangiogenesis activity of linomide (a ring substituted 1-methylquinolin-2-one) has been known for more than 10 years. Surprisingly, no structure-activity relationship studies of this activity have been reported until 2003. Six analogues of linomide were synthesized and their activities were evaluated using two established angiogenesis assays5. The first one was an in vivo chicken chorioallantoic membrane (CAM) assay. The other was the inhibition of human umbilical vein endothelial cell (HUVEC) proliferation, a standard line for antiangiogenesis screening, as set by the National Cancer Institute (NCI, USA).

Compounds 3a, 13 and 14 of our series resulted structurally similar to linomide and its analogues 16 and 17 (Table 5). So they were selected at the NCI to examine their anti-proliferative activity on HUVEC cells.

EXPERIMENTAL

Chemistry

NBS and benzoylperoxide were purchased from Fluka AG. Melting points were determined in a capillary Electrothermal 9100 apparatus and are given uncorrected. 1H and 13C NMR spectra were recorded using a Bruker 200 MHz spectrometer, at room temperature in DMSO as solvent, with TMS as the internal standard. The chemical shifts (d) are given in ppm. Infrared spectra were recorded on a FT Perkin Elmer Spectrum One from KBr discs. The mass spectrum was obtained on a Shimadzu QP 5000. Analytical TLC was carried out on DC-Alufolien Kiesegel 60 F254 Merck.

Compounds 1-9 and 13-15 were prepared according to a previous paper2. All new products exhibited satisfactory spectroscopic and analytical data.

3-(1-bromoethylideneamino)-6-chloro-4-phenylquinolin-2(1H)-one (10)

A mixture of 0.40 g (1.4 mmol) of oxazoloquinoline 7, 0.40 g (2.2 mmol) of NBS and a catalytic amount of dibenzoylperoxide in CCl4 (30 mL) was heated at reflux for 6 h. The reaction mixture turned orange. After cooling, a pale yellow solid was filtered off and washed with the same solvent.

Yield: 80 %, mp. 119-120 ºC; IR n cm-1: 3210, 3000, 1700, 1650, 1242, 810, 535. GC-MS m/z (%): 177 (27), 121 (2), 99 (62), 56 (100), 42 (17).1H NMR (DMSO-d6) d 11.08 (s, 1H, NHCO), 7.72-7.26 (m, 7H, Ar), 6.94 (d, 1H, Ar), 2.12 (s, 3H, CH3). Analysis for C17H12BrClN2O (375.65), calcd. C, 54.36; H, 3.22; N, 7.46; found C, 54.41; H, 3.23; N, 7.44.

3-(1-aza-2-methylprop-1-enyl)-6-chloro-4-phenyl-1 H-quinolin-2-one (11)

Compound 10 (0.40 g, 1.06 mmol) was heated at reflux temperature in acetone (30 mL) for 3 h. After cooling the solution, a white solid crystallized as needles. The product was collected by filtration and washed with acetone.

Yield: 25 %, mp. 244-247 ºC; IR n cm-1: 3147, 3000, 2677, 2618, 1653, 1241, 812, 741. 1H NMR (DMSO-d6): d 12.62 (s, 1H, NH), 7.72-7.47 (m, 5H, Ar), 7.31-7.26 (m, 2H, Ar), 6.94 (d, 1H, Ar), 2.12 (s, 6H, N(CH3)2). 13C NMR (DMSO-d6): 171.94, 158.21, 147.67, 137.15, 132.41, 131.24, 129.21, 129.16, 128.84, 127.42, 126.29, 125.75, 120.38, 117.75, 25.35. Analysis for C18H15ClN2O (310.78), calcd. C, 69.57; H, 4.86; N, 9.01; found C, 69.45; H, 4.87; N, 8.99.

6-chloro-3-[(methylethyl)amino]-4-phenyl-1 H-quinolin-2-one (12)

A suspension of compound 11 (0.15 g, 0.48 mmol) in MeOH (10 mL) was stirred in a water-ice bath and NaBH4 (50 mg, 1.26 mmol) was added in portions. The mixture was stirred for further 15 min. at 0-5 ºC, then it was allowed to warm to room temperature and heated at reflux for 4 h. The hot suspension was filtered and the solution obtained was concentrated under reduced pressure to give a white solid product.

Yield: 60 %, mp. 258-259 ºC; IR n cm-1 3200, 3000, 1620, 1460, 1500, 800, 680. 1H NMR (DMSO-d6): d 11.50 (s, 1H, NHCO), 8.3 (s, 1H, NH); 6.90-6.40 (m, 7H, Ar), 6.10 (s, 1H, Ar), 1.75 (br s, 1H, CH), 1.00 (s, 6H, (CH3)2). 13C NMR (DMSO-d6): 174.18, 164.33, 150.57, 141.49, 138.99, 135.14, 133.74, 133.54, 132.94, 130.99, 130.35, 125.80, 122.50, 27.47. Analysis for C18H17ClN2O (312.8), calcd. C, 69.12; H, 5.48; N, 8.96; found C, 69.23; H, 5.47; N, 8.92.

Antiprotozoal Activity

The in vitro protocols and activity criteria can be found at World Health Organization website.6 Summarizing, for antimalarial activity if the IC50 is > 5 mg/mL, the compound is classified as inactive. If the IC50 is 0.5-5 mg/mL, the compound is classified as moderately active. If the IC50 is < 0.5 mg/mL, the compound is classified as active and is further evaluated using two strains, K1 and NF54.

Hemin (FP) Binding Assay

The interaction of the compounds with hemin (Ferriprotoporphyrin IX, FP-Fe[III]) (Sigma) was examined spectrophotometrically under reducing and non reducing conditions by monitoring the Soret absorption band of FP. The solutions were prepared in DMSO and diluted with water to the final concentrations of 0.1, 1, 10, 100 and 1000 mM. Then they were diluted into non reducing (159 mM FP, 0.23 M sodium phosphate pH 7.4, 1 % SDS) or reducing (159 mM FP, 0.23 M sodium phosphate pH 7.4, 1 % SDS, 14 mM sodium dithionite) buffer. Chloroquine (CQ) was purchased from Parafarm and all other reagents were analytical grade. Absorption spectra were collected using a Jasco V-570 UV/Vis/NIR spectrophotometer. Data are average values of triplicate experiments.

In vitro evaluation of antimycobacterial activity

Primary screening was conducted at 6.25 mg/mL against Mycobacterium tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium using a broth microdilution assay, the Microplate Alamar Blue Assay (MABA). Compounds effecting < 90% inhibition in the primary screen (i.e. > 6.25 mg/mL) are not further evaluated7.

Growth inhibition assay

TNP-470 (NSC 642492) and paclitaxel (NSC 125973) are used as reference compounds in this assay as described (http://dtp.nci.nih.gov/. Their IC50 values are 3.16 and 1.65 nM, respectively.

RESULTS AND DISCUSSION

Compounds 1-5 and 7-9 were tested in vitro against P. falciparum, T. brucei and T. cruzi, causative agents of malaria, sleeping sickness and Chagas´ disease, respectively, and cytotoxicity was tested on the cell line MRC-5 (Table 1). Results are given as micromolar concentration that produces 50 % inhibition (IC50) in the assays used6. Compounds 1 and 2 showed some selective activity against P. falciparum Ghana, although the activity was not strong. The remaining compounds were inactive or showed only very weak antiparasitic activity. Compounds 1 and 2 were also assayed against P. falciparum K1 (chloroquine-resistant strain) and T. b. rhodesiense (Table 2). These compounds were weakly active on both parasites and were not recommended for secondary screening.



In the context of our on-going research, we wished to prepare the bromomethyl derivative from the oxazoloquinoline 7, employing N-bromosuccinimide (NBS) and dibenzoyl peroxide as activator, in carbon tetrachloride. It is assumed that the highly reactive bromomethyl intermediate led to compound 10, in moderate yield. Surprisingly, in an attempt to crystallize 10 from acetone, the imine 11 was obtained in good yield (Scheme 3).

In a previous attempt, when compound 3 was reacted with acetone, compound 11 could not be achieved. This behaviour implies that the nucleophilicity of the amino group has been diminished because of its partially enaminic nature. Then, the imine group in compound 11 was reduced employing sodium borohydride in methanol (Scheme 3). Compounds 11 and 12 were tested against T. cruzi, P. falciparum K1, L. donovani and T. b. rhodesiense and L-6 cells for cytotoxicity (Table 3).


Owing to most of the tested compounds exhibited activity against P. falciparum, the elucidation of a possible mechanism of action was intended. In malaria parasite metabolism, hemin (ferriprotoporphyrin IX, FP) is left after digestion of hemoglobin by the parasite. Then it is converted to hemozoin (malaria pigment), a polimerized form of heme devoid of immediate toxicity8.

The quinoline antimalarial drugs are thought to exert their activities by inhibiting FP detoxification, causing a builtup of toxic FP molecules that eventually inhibit parasites enzymes and destroy the integrity of the membranes. The binding of drugs such as chloroquine (CQ) to FP modifies its spectral characteristics, producing a decrease in the absorption and a broadening of the main Soret band at 400 nm9. The absorbance ratio at 430 and 400 nm (A430/A400) is a useful indicator of changes to the structure of the Soret band in the presence of compounds. In this way, the affinity of compounds 1-12 to FP was determined and CQ was used as reference drug. Only the interaction of compounds 1, 2, 4 and 5 with hemin draws some parallels with CQ-hemin under non reducing conditions but not under reducing conditions (Figure 1). Soret band is not modified by CQ in the presence of sodium dithionite, meanwhile the named compounds interact also with ferroporphyrin IX. Our results show that these derivatives somehow bind to both FP-Fe(III) and FP-Fe(II) species, so an alternative mode of action may be suggested.


  Figura 1: FP-Binding under non-reducing (top) and reducing (bottom) conditions of compounds 1-5 and CQ.

The primary screening against M. tuberculosis of a variety of compounds (Table 4) showed no inhibition activity. Only compound 4 with N1-methyl group displayed a minimum percentage (5 %). Neither the substituted 2-quinolones nor the quinoline derivatives were selected for further evaluation.


Regarding the angiogenesis assay, selected compounds 3a, 13 and 14 have the same scaffold and carry similar moieties of linomide and its analogues 16 and 17. Although the hydroxy group seems to be important for linomide anti-proliferative activity, compounds 3a and 14 exhibited IC50 values quite close to linomide derivatives (Table 5). We suggest that the inversion of the functional group in 3-position as well as the introduction of chloro and phenyl or methyl groups are promising strategies for designing potential antiangiogenesis agents. In addition, these results support the relationship between this activity and certain antimalarial drugs because of their effect in decreasing tumor necrosis factor (TNF-a), as it was proposed for chloroquine in case of cerebral malaria10. Other example is the natural sesquiterpene endoperoxide artemisinin, a lead compound in the development of antimalarial agents. Recently, The Chen and Lee research groups have examined that artemisinin, dihydroartemisinin and several semisynthetic derivatives have the antiangiogenic activity11,12.


CONCLUSIONS

The tested compounds showed weak or moderate activity against P. falciparum. N1-methyl group increased the antimalarial activity but the substitution with alkyl or alkylamino groups in 3-position did not improve this activity as expected.

The anti-proliferative activity exhibited by our quinolin-2-one derivatives will let us design more effective small molecules that might be suitable as clinical therapies.

Efforts are currently underway towards further optimisation of the antimalarial and anti-tuberculosis activities through the replacement of carbonyl group with other selective moieties and the synthesis of novel quinoline derivatives. Some of these new compounds are under biological testing.

 

REFERENCES

1. Fakhfakh, M. A.; Fournet, A.; Prina, E. Bioorg. Med. Chem. 2003, 11, 5013-5023.         [ Links ]

2. Asís, S. E.; Bruno, A. M.; Dominici, D.; Bollini, M.; Gaozza, C. H. J. Heterocyclic Chem. 2003, 40, 107-112.         [ Links ]

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6. www.who.int/tdr, for in vitro protocols and activity criteria.         [ Links ]

7. www.taacf.org, for description of TAACF assays.         [ Links ]

8. Paitayatat, S.; Tarnchompoo, B.; Thebtaranonth, Y.; Yuthavong, Y. J. Med. Chem. 1997, 40, 633-638.         [ Links ]

9. Taylor, D. K.; Avery, T. D.; Greatrex, B. W. Tiekink, E. R.; Macreadie, I. G.; Macreadie, P. I.; Humphries, A. D; Kalkanidis, M.; Fox, E. N.; Klonis, N.; Tilley, L. J. Med. Chem. 2004, 47, 1833-1839.         [ Links ]

10. Foley, M.; Tilley, L. Pharmacol. Ther. 1998, 79, 55-87.         [ Links ]

11. Oh, S.; Jeong, I.; Shin, W.; Lee, S. Bioorg. Med. Chem. Lett. 2003, 13, 3665-3668.         [ Links ]

12. Oh, S.; Jeong, I.; Shin, W.; Lee, S. Bioorg. Med. Chem. Lett. 2004, 14, 3683-3686.         [ Links ]

ACKNOWLEDGEMENTS

We wish to thank Dr. J. R. L. Pink and Dr. Foluke Fakorede, TDR, WHO (Switzerland). We are grateful to TAACF (USA) and to NCI, Developmental Therapeutics Program (USA).

 

e-mail: elizabet@ffxb.uba.ar