Cytotoxic and Antifungal Activities of Diverse [alpha]-Naphthylamine Derivatives

Headnote

Abstract

Diverse α-naphthylamine derivatives were easily prepared from corresponding aldimines derived from commercially available α-naphthaldehyde and anilines or isomeric pyridinecarboxyaldehydes and α-naphthylamine. The secondary amines obtained were tested as possible antifungal and cytotoxic agents. The diverse N-aryl-N-[1-(1-naphthyl)but-3-enyl]amines obtained were active (IC^sub 50^ < 10 µg/mL) against breast (MCF-7), non-small cell lung (H-460), and central nervous system (SF-268) human cancer cell lines, while N-(pyridinylmethyl)-naphthalen-1-amines resulted in activity against (MIC 25-32 µg/mL) some human opportunistic pathogenic fungi including yeasts, hialohyphomycetes, and dermatophytes.

Keywords

α-Naphthylamines * Antifungal properties * Cytotoxic agents

Introduction

Secondary amines that include aromatic planar rings (e.g. aryl, naphthyl, etc.) are widely used throughout the chemical industry as basic intermediates to prepare pharmaceuticals, agrochemicals, and fine chemicals [1-5]. Moreover, they are one of the most common structural features of naturally occurring, biologically active compounds [6]. Due to their unique biological properties, these compounds have played an important role in chemo-therapeutic approaches in a variety of diseases, including antiparasitic infectious diseases [2]. However, it is known that naphthylamines could be carcinogenic for humans [7-9]. These compounds have still demonstrated interesting pharmacological models in biomedical studies [10-12]. As a result, the chemistry and biology of these compounds have received considerable attention from both the theoretical and practical points of view.

As a part of our drug discovery program, we have been actively involved in determining the features that are important for antifungal activities of different amines and we found that N-benzyl anilines (type A), or N-pyridinylmethylanilines (type B), and N-aryl-N-(1-pyridinylbut-3-enyl)amines ("homoallylamines") (type C) (Figure 1), easily accessible from aldimines, displayed significant activity against some pathogenic dermatophytes [13-18].

These facts encouraged us to develop other naphthylamine derivatives and evaluate them for their antifungal activity and in parallel their cytotoxic properties. So, we report herein the biological results of a new series of N-aryl-N-[1-(1-naphthyl)but-3-enyl]amines (type D) and N-(pyridinylmethyl)naphthalen-1-amines (type E) that could be considered as structural analogues of the antifungal drugs naftifine and terbinafine, called "allylamine derivatives" [19] (Fig. 1).

Results and Discussion

Chemistry

The desired naphthylamine derivatives 5 and 6 were prepared from commercially available aromatic aldehydes α-naphthaldehyde 1 and isomeric pyridinecarboxyaldehydes 2a-c and anilines 3 or α-naphthylamine 4 in easy two-step synthesis protocols via initial N-aryl aldimines formation.

These aldimines, either by nucleophilic addition of allylmagnesium bromide to the C=N bond or the reduction with an excess of NaBH4 in methanol, produced the corresponding secondary and N-aryl-N-[1-(1-naphthyl)but-3-enyl]amines 5a-f [18] ("homoallylamines", type D) (route a, Scheme 1) and N-(pyridinylmethyl)naphthalen-1-amines 6a-c (type E) (route b, Scheme 1), respectively.

These secondary amines were obtained as colored solids or viscous oils in 80-95% yields after purification using a SiO2 chromatography column (Table 1) and were strongly characterized by the spectral data.

Biological properties

These two series of compounds were tested for cytotoxic activities against breast (MCF-7), non-small cell lung (H-460), and central nervous system (SF-268) human cancer cell lines [20, 21] and for antifungal properties against a panel of 10 human opportunistic pathogenic fungi including yeasts, Aspergillus spp. and dermatophytes [22, 23] (Table 2).

Results showed (Table 2) that the tested compounds of type D, and all N-aryl-N-[1-(naphth-1-yl)but-3-enyl]amines 5a-e, but not 5f, showed cytotoxicity with IC50 ≤ 10 µg/mL, making them potential anticancer agents. Of the three tested cell lines, MCF-7 and H-460 were the most susceptible ones. From these results, certain conclusions relating to the structure with the cytotoxic activity can be drawn: (i) the donor substituent OMe as R3 renders the most cytotoxic compound from both points of view with the lower IC50 value and the broader spectrum of action [compare activities of 5a (R3 = MeO); 5b (R3 = F); 5c (R3 = Cl); 5d (R3 = Br)]; (ii) the comparison of the activity of compounds possessing a halogen as R3 showed that a Br (5b, 5c and 5d) confers better activity than a chloro (Cl) atom, followed by a fluorine (F) atom; (iii) the addition of an extra Cl as R2 to 5c leads to the equipotent 5e, suggesting that a halogen in the meta position does not modify the activity; (iv) in contrast, the addition of an extra F as R1 to 5b leads to 5f, which shows a loss of activity in the three cell lines. This result could be related to the steric hindrance resulting of a substituent on the ortho position.

Regarding the results obtained from compounds type B, it appears that the position of the N atom in the pyridine ring of N-(pyridinylmethyl)naphthalen-1-amines 6a-c exert some influence in the activity since compounds possessing α- or β-pyridine rings (but not that γ-pyridine moiety) are inactive.

Results of the antifungal assays of type D molecules showed that the cytotoxic compounds 5a-e were completely devoid of antifungal properties, clearly suggesting a selective toxicity of these compounds against cancer cell lines.

For a better understanding of the antifungal results of compounds 6a-c, their cytotoxic and antifungal behaviors are shown in Table 3, in which the antifungal results of 6a-c in each fungal species is included.

Results showed that among compounds 6, molecule 6b displayed moderate antifungal activity with a broad spectrum of action, with T. rubrum being the most susceptible species (MIC = 6.25 µg/mL). In addition, it did not show cytoxicity at the concentration at which it was antifungal. Compound 6c showed moderate antifungal activity (MIC = 32-62 µg/mL) only against dermatophytes. However, at much lower concentrations (IC50 = 3.3-4.6 µg/mL) it was cytotoxic against the three tested cell lines.

Taking into account all of these results, we can say that among molecules 6, compounds 6b and 6c can be optimized in forthcoming works, considering that compound 6c is the best cytotoxic candidate and 6b the best antifungal against T. rubrum. This result is very interesting since this fungal species is responsible for approximately 80-93% of chronic and recurrent dermatophyte infections in human beings. They are the etiological agent of tinea unguium (producer of invasive nail infections), tinea manuum (palmar and interdigital areas of the hand infections), and tinea pedis (Athlete's foot), the last one being the most prevalent fungal infection in developed countries, and the first one accounting for 50% and 90% of all fingernail and toenail infections, respectively, and new agents with high activity against T. rubrum are highly welcomed.

In summary, we have analyzed the cytotoxic and antifungal properties of some α-naphthylamine derivatives prepared from available aldimines. N-Aryl-N-[1-(naphth-1-yl)but-3-enyl]amines 5a-e showed good selective cytotoxic activities against three cancer cell lines being the one containing an OMe as the most active substituent (5a). In turn, among the three N-(pyridinylmethyl)naphthalen-1-amines 6a-c, the one possessing the pyridine ring with N in the β-position (comp. 6b) showed moderate activity mainly against T. rubrum, while it is not cytotoxic at the same concentrations.

Experimental

Chemistry

IR spectra were recorded on a Lumex Infralum FT-02 spectrophotometer. 1H and 13C NMR spectra were measured on a Bruker AM-400 spectrometer (400 MHz 1H NMR and 100 MHz 13C NMR), using CDCl3 as the solvent. TMS was used as an internal standard. Chemical shifts (δ) and J values are reported in ppm and Hz, respectively. A Hewlett Packard 5890a Series II Gas Chromatograph interfaced to an HP 5972 Mass Selective Detector with an HP MS ChemStation Data system was used for MS identification at 70 eV using a 60 m capillary column coated with HP-5 [5% phenylpoly(dimethylsiloxane)]. Melting points were measured on a Fisher Johns melting point apparatus. The reaction progress was monitored using thin layer chromatography on Silufol UV 254 TLC aluminum sheets. Column chromatography was carried out using silica gel (230-400 mesh). All reagents were purchased from Sigma and Aldrich Chemical Co. and used without further purification.

Synthesis of the secondary amines 5 and 6 was performed according to literature reports [17, 18]. Spectral data for known amines 5 were identical to those published in our work [18].

Spectral Data for unknown amines 6

Comp. 6a [24]: dark red viscous liquid. IR (neat), ν (cm-1): 3386, 3054, 2838, 1589, 1527, 771; EM (IE), m/z (%): 234 (M+., 100), 204 (5), 156 (40), 142 (4), 128 (20), 115 (28). 1H NMR (400 MHz, CDCl3, Me4Si) δ 8.57 (dd, J = 7.5, 1.6 Hz, 1H), 8.09-8.02 (m, 2H), 7.72 (m, 1H), 7.60-7.51 (m, 3H), 7.39-7.30 (m, 3H), 6.97 (d, J = 7.8, 1.4 Hz, 1H), 4.70 (s, 2H), 3.98 (br s, 1H); 13C NMR (100 MHz, CDCl3, Me4Si), δ (ppm): 157.8, 148.8, 142.9, 136.4, 134.1, 128.4, 126.5, 125.6, 124.5, 123.3, 121.9, 121.4, 120.2, 117.2, 104.5, 48.8. Anal. Calcd. for C16H14N2: C, 82.02; H, 6.02; N, 11.96. Found: C, 81.96; H, 6.13; N, 11.53.

Comp. 6b: red oil, IR (neat), ν (cm-1): 3355, 3039, 1581, 1527, 771; EM (IE); m/z (%): 234 (M+, 100), 204 (3), 142 (63), 115 (88), 92 (19), 65 (17). 1H NMR (400 MHz, CDCl3 Me4Si), δ (ppm): 8.65 (s, 1H), 8.50 (d, J = 7.5 Hz, 2H), 8.08-8.01 (m, 2H), 7.88 (dd, J = 7.5, 1.4 Hz, 1H), 7.58-7.51 (m, 3H), 7.37 (each d, J = 7.5 Hz, 2H), 6.98 (dd, J = 7.5, 1.6 Hz, 1H), 4.36 (s, 2H), 4.02 (br s, 1H); 13C NMR (100 MHz, CDCl3 Me4Si), δ (ppm): 156.7, 148.6, 142,4, 136.4, 134.3, 128.8, 126.5 (2C), 125.9, 123.3, 122.2 (2C), 119.9, 118.2, 104.8, 48.1. Calcd. for C16H14N2: C, 82.02; H, 6.02; N, 11.96. Found: C, 81.87; H, 6.16; N 12.01.

Comp. 6c: yellow oil, IR (neat), ν (cm-1): 3299, 3050, 2881, 1589, 1543, 767; EM (IE), m/z (%): 234 (M+., 100), 206 (2), 156 (13), 142 (45), 128 (18), 115 (67), 65 (7). 1H NMR (400 MHz, CDCl3, Me4Si) δ (ppm): 8.58 (each dd, J = 7.6, 1.4 Hz, 2H), 8.06-8.01 (m, 2H), 7.55-7.51 (m, 3H), 7.35-7.38 (m, 3H), 6.96 (dd, J = 7.5, 1.4 Hz, 1H), 4.35 (s, 2H), 4.00 (br s, 1H); 13C NMR (101 MHz, CDCl3, Me4Si) δ (ppm): 150.0 (2C), 148.7, 142.5, 134.4, 128.8, 126.5, 126.0, 125.0, 123.4, 122.2 (2C), 119.9, 118.1, 105.0, 47.2. Calcd. for C16H14N2: C, 82.02; H, 6.02; N, 11.96. Found: C, 81.78; H, 6.19; N, 11.85.

Bioassays

Cytotoxic susceptibility testing

The cytotoxic activity was determined according to the method of Monks et al. Briefly, the three human cells lines [breast (MCF-7), non-small cell lung (H-460), and central nervous system (SF-268), obtained from the U.S. National Cancer Institute] were counted, diluted with fresh medium, and added to 96-well microtiter plates(100 µL/well) containing test materials (1 mg in 100 µL in DMSO). Test plates were incubated for 2 days at 37 °C in a 5% CO2 incubator. All treatments were performed in duplicate. After the incubation periods, cells were fixed by addition of 50 µL of cold 50% aqueous TCA solution (4°C for 60 min.), washed 4-5 times with tap water, and air-dried. The fixed cells were stained with 100 µL sulforhodamine B (SRB) (0.4% wt/vol. in 1% acetic acid) for 15 min. Free SRB solution was then removed by rinsing with 1% acetic acid (x 5). The plates were then air-dried, the bound dye was solubilized with 100 µL of 10 mM tris-base, and absorbance was determined at 515 nm using an ELISA plate reader (Bio-Tek Instruments, Inc. Model ELX-800). Finally, the absorbance values obtained with each of the treatment procedures were averaged, and the averaged value obtained with the zero day control was subtracted, measuring in this way the relative cell growth or unviability in treated and untreated cells. From the curves, growth inhibition (or growth stimulation) and 50% inhibition of growth (IC50) was calculated [21,22]. Adriamycin was used as the reference compound. The procedure for cell viability measurement was evaluated by a colorimetric method with resazurin. The macrophages J774 were seeded (5 × 104 cells/well) in 96-well flat-bottom microplates with 100 µL of RPMI 1640 medium. The cells were allowed to attach for 24 h at 37 °C, 5% CO2 and the medium was replaced by different concentrations of the drugs in 200 µL of medium, and exposed for another 24 h. Growth controls were also included. Afterwards, a volume of 20 µL of the 2mM resazurin solution was added and plates were returned to the incubator for another 3 h to evaluate cell viability. The reduction of resazurin was determined by dual wavelength absorbance measurement at 490 nm and 595 nm. The background was subtracted. Each concentration was assayed in triplicate. The medium and drug controls were used as blanks in each test. Compounds with IC50 ≥ 10 µg/mL were considered inactive (not cytotoxic).

Antifungal activity

For the antifungal evaluation, standardized strains from the American Type Culture Collection (ATCC), Rockville, MD, USA, and CEREMIC (CCC), Centro de Referencia en Micología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Suipacha 531-(2000)-Rosario, Argentina, were used: C. albicans ATCC 10231, C. tropicalis CCC 191, S. cerevisiae ATCC 9763, C. neoformans ATCC 32264, A. flavus ATCC 9170, A. fumigatus ATTC 26934, A. niger ATCC 9029, T. rubrum CCC 110, T. mentagrophytes ATCC 9972 and M. gypseum CCC 115. Strains were grown on Sabouraud-chloramphenicol agar slants for 48 h at 30 °C, maintained on slopes of Sabouraud-dextrose agar (SDA, Oxoid) and sub-cultured every 15 days to prevent pleomorphic transformations. Inocula of spore suspensions were obtained according to reported procedures [22, 23] and adjusted to 1-5 x103 spores with colony forming units (CFU)/mL.

Antifungal susceptibility testing

Broth microdilution techniques were performed following the guidelines of the CLSI for yeasts [22] and for filamentous fungi [23]. MIC values were determined in RPMI-1640 (Sigma) buffered to pH 7.0 with MOPS (Sigma). Microtiter trays were incubated at 35 °C for yeasts and hyalohyphomycetes and at 28 °C for dermatophyte strains in a moist, dark chamber; MICs were recorded at 48 h for yeasts, and at a time according to the amount of control fungus growth, for the rest of fungi. The susceptibilities of the standard drugs ketoconazole, terbinafine, and amphotericin B were defined as the lowest concentration of drug which resulted in total inhibition of fungal growth. For the assay, compound stock solutions were diluted two-fold with RPMI-1640 from 100 to 0.24 µg/mL (final volume = 100 µL) and a final DMSO (Sigma) concentration <1%. A volume of 100 µL of the inoculum suspension was added to each well with the exception of the sterility control where sterile water was added to the well instead. MIC was defined as the minimum inhibitory concentration of the compound, which resulted in total inhibition of fungal growth. Compounds with MICs ≥ 100 µg/mL were considered inactive.

Acknowledgement

Financial support from Patrimonio Autónomo Fondo Nacional de Financiamiento para la Ciencia, la Tecnología y la Innovación, Francisco José de Caldas, contract RC-0572-2012, is gratefully acknowledged (VVK). LYVM thanks COLCIENCIAS for PhD fellowship, SZ and MS acknowledge ANPCyT PICT 0608-2010 and PICT 0049-2010. MPG acknowledges the National Secretariat of Science, Technology and Innovation of Panama (SENACYT) for the Supporting National System of Investigators.

Authors' Statement

Competing Interests

The authors declare no conflict of interest

References

References

[1] Seayad A, Ahmed M, Klein H, Jackstell R, Gross T, Beller M.

Internal Olefins to Linear Amines.

Science. 2002; 297: 1676-1678.

http://dx.doi.org/10.1126/science.1074801

[2] Kleemann A, Engel J.

Pharmaceutical Substances,

Thieme: New York, 1999.

[3] Evers A, Hessler G, Matter H, Klabunde T.

Virtual Screening of Biogenic Amine-Binding G-Protein Coupled Receptors: Comparative Evaluation of Protein- and Ligand-Based Virtual Screening Protocols.

J Med Chem. 2005; 48: 5448-5465.

http://dx.doi.org/10.1021/jm050090o

[4] Salvatore RN, Yoon CH, Jung KW.

Synthesis of secondary amines.

Tetrahedron. 2001; 57: 7785-7811.

http://dx.doi.org/10.1016/S0040-4020(01)00722-0

[5] Henkel T, Brunne RM, Mueller H, Reichel F.

Statistical Investigation into the Structural Complementarity of Natural Products and Synthetic Compounds.

Angew Chem Int Ed. 1999; 38: 643-747.

http://dx.doi.org/10.1002/(SICI)1521-3773(19990301)38:5<643::AID-ANIE643>3.0.CO;2-G

[6] Abdel-Magid AF, Mehrman SJ.

A Review on the Use of Sodium Triacetoxyborohydride in the Reductive Amination of Ketones and Aldehydes.

Org Process Res Dev. 2006; 10: 971-1031.

http://dx.doi.org/10.1021/op0601013

[7] Miller EC.

Some Current Perspectives on Chemical Carcinogenesis in Humans and Experimental Animals: Presidential Address.

Cancer Res. 1978; 38: 1479-1496.

http://www.ncbi.nlm.nih.gov/pubmed/348302

[8] Butler MA, Iwasaki M, Guengerich FP, Kadlubar FF.

Human cytochrome P-450PA (P-450IA2), the phenacetin O-deet-hylase, is primarily responsible for the hepatic 3-demethylation of caffeine and N-oxidation of carcinogenic arylamines.

Proc Nat Acad Sci U S A. 1989; 86: 7696-7700.

http://dx.doi.org/10.1073/pnas.86.20.7696

[9] Hecht SS.

Cigarette smoking: cancer risks, carcinogens, and mechanisms.

Langenbecks Arch Surg. 2006; 391: 603-613.

http://dx.doi.org/10.1007/s00423-006-0111-z

[10] Taboada MC, Rodriguez B, Millán R, Míguez I.

Role of dietary L-arginine supplementation on serum parameters and intestinal enzyme activities in rats fed an excess-fat diet.

Biomed Pharm. 2006; 60: 10-13.

http://dx.doi.org/10.1016/j.biopha.2005.07.014

[11] Wilson ZMP, Bowman RE, Smith HJ, Nicholls PJ, Hewlins MJE.

Potential Protein Kinase C Inhibitors. 8,9,10,11α:-Tetrahydro-7αH-7,11-methano-12,12-dimethylcycloocta[de]naphthyl-9-amines.

J Pharm Pharmacol. 1996; 48: 160-171.

http://dx.doi.org/10.1111/j.2042-7158.1996.tb07116.x

[12] Kovacic P, Somanathan R.

Novel, unifying mechanism for aromatic primary-amines (therapeutics, carcinogens and toxins): electron transfer, reactive oxygen species, oxidative stress and metabolites.

Med Chem Commun. 2011; 2: 106-112.

http://dx.doi.org/10.1039/c0md00233j

[13] Urbina JMG, Cortés JC, Palma A, López SN, Zacchino SA, Enriz DR, Ribas JC, Kouznetsov V.

Inhibitors of the Fungal Cell Wall. Synthesis of 4-Aryl-4-N-arylamine-1-butenes and Related Compounds with Inhibitory Activities on β(1±3) Glucan and Chitin Synthases.

Bioorg Med Chem. 2000; 8: 691-698.

http://dx.doi.org/10.1016/S0968-0896(00)00003-1

[14] Vargas MLY, Castelli MV, Kouznetsov VV, Urbina GJM, López SN, Sortino M, Enriz RD, Ribas JC, Zacchino SA.

In Vitro Antifungal Activity of New Series of Homoallylamines and Related Compounds with Inhibitory Properties of the Synthesis of Fungal Cell Wall Polymers.

Bioorg Med Chem. 2003; 11: 1531-1550.

http://dx.doi.org/10.1016/S0968-0896(02)00605-3

[15] Kouznetsov VV, Rivero Castro J, Ochoa Puentes C, Stashenko EE, René Martínez J, Ochoa C, Montoro Pereira D, Nogal Ruiz JJ, Fernández Portillo C, Muelas Serrano S, Gómez Barrio A, Bahsas A, Amaro-Luis J.

Synthesis and Antiparasitic Properties of New 4-N-Benzylamino-4-Hetarylbut-1-enes.

Arch Pharm. 2005; 338: 32-37.

http://dx.doi.org/10.1002/ardp.200400909

[16] Suvire DF, Sortino M, Kouznetsov VV, Vargas MLY, Zacchino SA, Mora Cruz U, Enriz RD.

Structure-activity relationship study of homoallylamines and related derivatives acting as antifungal agents.

Bioorg Med Chem. 2006; 14: 1851-1862.

http://dx.doi.org/10.1016/j.bmc.2005.10.036

[17] Kouznetsov VV, Astudillo Saavedra L, Vargas Méndez LY, Cazar Ramírez ME.

Synthesis of some secondary amine derivatives bearing a heteroaryl fragment.

J Chil Chem Soc. 2004; 49: 197-203.

http://dx.doi.org/10.4067/S0717-97072004000400010

[18] Kouznetsov V, Rodríguez W, Stashenko E, Ochoa C, Vega C, Rolón M, Montero Pereira D, Escario JA, Gómez Barrio A.

Transformation of schiffbases derived from alpha-naphthaldehyde. Synthesis, spectral data and biological activity of new-3-aryl-2-(α-naphthyl)-4-thiazolidinones and N-aryl-N-[1-(α-naphthyl)but-3-enyl]amines.

J Heterocycl Chem. 2004; 41: 995-999.

http://dx.doi.org/10.1002/jhet.5570410624

[19] Fuglseth E, Otterholt E, Høgmoen H, Sundby E, Charnock C, HoffBH.

Chiral derivatives of Butenafine and Terbinafine: synthesis and antifungal activity.

Tetrahedron. 2009; 65: 9807-9813.

http://dx.doi.org/10.1016/j.tet.2009.09.067

[20] Monks A, Scudiero DA, Johnson GS, Pauli KD, Sausville EA.

Mini-review. The NCI anti-cancer drug screen: a smart screen to identify effectors of novel targets.

Anticancer Drug Des. 1997; 12: 533-541.

http://www.ncbi.nlm.nih.gov/pubmed/9365500

[21] Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, Hose C, Langley J, Cronise P, Vaigro-WolffA, Gray-Goodrich M, Campbell H, Mayo J, Boyd M.

Feasibility of high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines.

J Nat Cancer Inst. 1991; 83: 757-766.

http://dx.doi.org/10.1093/jnci/83.11.757

[22] Clinical and Laboratory Standards Institute (CLSI, formerly National Committee for Clinical and Laboratory Standards NCCLS) In Method M27-A2, 2nd ed, Wayne Ed.; NCCLS: Pennsylvania, 2002, Vol. 22, no. 15, pp 1-29.

[23] Clinical and Laboratory Standards Institute (CLSI, formerly National Committee for Clinical and Laboratory Standards NCCLS) In Method M-38A, 2nd ed, Wayne Ed.; NCCLS: Pennsylvania, 2002, Vol. 22, no. 16, pp 1-27.

[24] Czuba W, Kowalski P, Grzegozek M.

Synthesis of ethylenediamine derivatives.

Polish J Chem. 1980; 54: 1573-1578.