Synthesis of New 4-Methyl-2-( 4-pyridyl )-1 , 2 , 3 , 4-tetrahydroquinolines as Potent Antifungal Compounds

A síntese, a caracterização espectroscópica e os resultados biológicos de novas séries de 2-(4-piridil)-1,2,3,4-tetrahidroquinolinas e seus precursores, -N-aril-N-[1-(4-piridil)but-3-enil] aminas, são descritos. Foi encontrado que ambos, precursores substituídos g-piridil e os produtos finais, as tetrahidroquinolinas, demonstraram excelentes atividades antifúngicas contra Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Microsporum gypseun, Trichophyton rubrum e Trichophyton mentagrophytes.


Introduction
Both alicyclic and cyclic secondary amines are widely used throughout the chemical industry as basic intermediates to prepare fine chemicals, pharmaceuticals and agrochemicals. 1,2Moreover, they are one of the most common structural features of naturally occurring biologically active compounds.Due to their unique biological properties, these compounds have played an important role in chemotherapeutic approaches to a variety of diseases, including fungal infectious diseases.These compounds serve as interesting pharmacological models in biomedicine studies.So, chemistry and biology of these compounds have received considerable attention from both the theoretical and practical points of view.

Results and Discussion
The series of homoallylamines 6-10 were prepared using both standard Grignard and Barbier procedures 9 with prepared in situ allyl bromide magnesium in ether and with allyl bromide/indium/methanol system, respectively.Performing this later procedure, a mixture of allyl bromide (3 mmol), powder indio (1.5 mmol) and aldimines 1-5 (1 mmol) in dry MeOH was stirred at room temperature until the complete dissolution of metal, taking 1-4 h to obtain the desired products.Classical Grignard protocol consists in the addition of an ether solution of aldimines to preformed allyl magnesium bromide in ether that affords desired amine products.Thus, this nucleophilic addition was realized in ether (or THF) at 20 o C and, after stirring for 4 h, the reaction mixture was treated with saturated NH 4 Cl solution.The organic products were extracted affording the homoallylamines 6-10 in 53-81% yields (Scheme 1, Table 1).It is worth noting that the use of ether is limited by the solubility of utilized aldimines.
Each of these procedures demonstrated some advantages and disadvantages: traditional Grignard protocol needs to use commercial, expensive organometallic reagent and dry solvents that are toxic and expensive, but magnesium is an easily available and inexpensive metal.Barbier protocol does not need to form organometallic reagent and dry solvents, but indium is still a very expensive metal to perform scale-up preparation of precursor compounds.
Homoallylamines 6-10 were easily characterized by IR (ν NH 3273 and 3293 cm -1 ), 1 H-NMR, and Mass Spectra.This later technique demonstrated that molecular ions were of low intensity (< 5%) and the principal fragmentation of these compounds were due to the lost of allyl radical [M-41] + , that generates ArNHCHPy + -cations. 10 1H-NMR spectra of homoallylamines 6-10 were very similar one each other, and were characterized by the presence of three groups of signals, which resonated in different zones (Table 2).
Preparation of desired 1,2,3,4-tetrahydroquinolines 11-14 consisted in the simple treatment of obtained homoallylamines mainly with sulfuric acid (85%).This mixture was heated at 80-90 o C during 1-5 h.Final products were isolated, and then purified by alumina column chromatography.In the case of homoallylamine 8, PPA was used as mild cycling agent, because its treatment with sulfuric acid gave a complex mixture of inseparable products.It should be also noted that during the sulfuric acid catalysis of homoallylamine 10, quinoline sulfonic acid 15 was formed as a sulfonation and oxidation product in considerable yields (Table 3).The structure of each final product was strongly confirmed by NMR ( 1 H, 13 C), IR and GC-MS data.
Analysing GC-MS and NMR data, it was noted that final products 11-14 presented a mixture of two diastereomers (cis and trans) from different spatial orientation of the substituents at C-2 and C-4 positions.GC-MS data indicated that the cis:trans ratio for comp.11-14 were: 68:32; 79:21; 88:12 and 91:9, respectively.From NMR studies it was possible to assign cis-form for major isomer.This was corroborated by the protons 2-H, 3-H and 4-H coupling 2. sat.NH 4 Cl 1.

sat. NH 4 Cl
constants (J 2ax,3ax 11.2-11.3Hz, J 2ax,3eq 2.3-2.9Hz and J 4ax,3eq 6.0-6.9Hz, and J 4ax,3ax 11.5-12.7 Hz), affirmation enough to indicate the equatorial-equatorial (cis) relationship to the case (Table 4).Figure 1 shows both diastereomeric forms.All our attempts in the separation of these diastereomers by gravity column chromatography failed in our hands, only the major diastereomer of comp.13 was isolated and characterized as a pure cis-form.
The antifungal properties of compounds 6-14 were evaluated by the microbroth dilution method following the guidelines of the Clinical and Laboratory Standard Institute (CLSI, formerly National Committee for Clinical and Laboratory Standards (NCCLS) against a panel of ten fungal species: four human opportunistic pathogenic yeasts, three hialohyphomycetes and three dermatophytes.To carry out the antifungal evaluation, concentrations of compounds up to 250 mg mL -1 were incorporated to growth media according to published procedures. 11,12The three most clinically used type of antifungal drugs were used as positive controls: the polyene Amphotericin B, the allylamine terbinafine and the azole ketoconazole.Biological results on fungal inhibition of the prepared molecules are summarized in Tables 5,6.
Regarding the activity displayed by homoallylamines 6-10 against the three most active fungal species, M. gypseum, T. rubrum and T. mentagrophytes, it appears that a substituent in the p-position produces an enhancement of the antifungal activity since 11 out the 12 MIC values of compounds 7-9 are lower than the corresponding MICs of compound 6.In addition, the electronic properties of the p-substituents could play a role in the antifungal activity since compound 9 with a electron-withdrawing substituent (Br) was more active (MIC range = 16-31.2µg mL -1 ) than compounds 7 and 8, which contain electron-donating groups (CH 3 and OCH 3 ) and their MICs range were 25-125 µg mL -1 .Another interesting observation is that methyl groups in both, the positions C-3 and C-5, do not significantly modify the activity (compare the activity of 6 with 10).
Regarding tetrahydroquinolines, the high activity displayed by compound 11, clearly showed that the closing of the ring dramatically enhanced the activity of the  unsubstituted homoallylamine 6, although the same was not observed for substituted homoallylamines 7-9.Compound 11 showed a broad spectrum of action inhibiting all fungi tested with MICs between 8 and 125 µg mL -1 .It was highly active against Aspergillus spp.(MIC = 31.2µg mL -1 ) and mainly against dermatophytes (MIC = 8 µg mL -1 ).
The fact that tetrahydroquinoline molecule 11 inhibited Aspergillus spp. is highly interesting since it is worthwhile to take in account that previous homoallylamine-derived tetrahydroquinolines did not show activity against Aspergillus species up to 50 µg mL -1 . 3,4t is well known 13 that invasive aspergillosis has emerged as a leading cause of infection-related mortality among immunocompromised individuals, in particular among recipients of hematopoietic stem cell transplants or solid organ transplants.Historically, the drug of choice for the treatment of invasive aspergillosis has been amphotericin B, followed by the newer medications voriconazole and the recently approved caspofungin, which appeared more effective and with fewer side effects than the polyene.However, although the last drugs have advanced the management of these fungal infections, failure rates remain high, and emergence of intrinsically resistant fungi is a growing problem. 14So, there is a general consensus that the discovery of new structures acting against Aspergillus spp. is highly necessary.Regarding the activity of compounds 9 and 11 against dermatophytes, the most important findings are that these compounds display low MICs (16-32 and 8 µg mL -1 , respectively) against both, T. rubrum and T. mentagrophytes.It is worthwhile to take in account that approximately 80-93% of chronic and recurrent dermatophyte infections (tineas) are estimated to be caused by Trichophyton spp. 15Although the treatment outcomes of these infections have now been greatly improved with the azole itraconazole or the allylamine terbinafine, there is unfortunately a considerable overlap of the strategies for treating the different tineas.This lack of specificity leads to chronic dermatophytoses or early re-infections after antifungal therapy and therefore, the superficial infections are very difficult to eradicate. 15he very low MIC displayed by compounds 9 and 11 against both Trichophyton spp.opens the possibility of using them as starting points for the development of new antifungal agents that selectively inhibit the most prevalent fungi in dermatomycoses.This hope is higher considering that both compounds not only inhibit but kill Trichophyton spp.(Minimum Fungicidal Concentration = 62 µg mL -1 for compound 9 and 32-62 µg mL -1 for compound 11) (Table 6) adding one of the needed requirements for being good candidates for future development.
In summary, we found that both g-pyridyl substituted precursors, homoallylamines, and final products, tetrahydroquinolines, showed very good antifungal activities against A. fumigatus, A. flavus, A. niger and the dermatophytes M. gypseum, T. rubrum and T. mentagrophytes.Among the compounds tested, the homoallylamine 9 and the tetrahydroquinoline 11 displayed the best activities against dermatophytes.Compound 11 showed also a very good activity against Aspergillus spp.In addition, 9 and 11 showed to be fungicide rather than fungistatic against dermatophytes, results that permit to consider them as good candidates for the further development of new antifungal agents.

Experimental
The melting points (uncorrected) were determined on a Fisher-Johns melting point apparatus.The IR spectra were recorded on a Lumex Infralum FT-02 spectrophotometer in KBr. 1 H NMR spectra were recorded on Bruker AM-400 spectrometer.Chemical shifts are reported in ppm (d) relative to the solvent peak (CHCl 3 in CDCl 3 at 7.24 ppm for protons).Signals are designated as follows: s, singlet; d, doublet; dd, doublet of doublets; ddd, doublet of doublets of doublets; t, triplet; td, triplet of doublets; q, quartet; sp, septet; m, multiplet; b, broad.On DEPT-135 spectra, the signals of CH 3 , CH 2 and CH carbons are shown as positive (+) and negative (-), respectively.Quaternary carbons are not shown.A Hewlett Packard 5890a series II Gas Chromatograph interfaced to an HP 5972 Mass Selective Detector (MSD) 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(dimethyl-siloxane)].Elemental analyses were performed on a Perkin Elmer 2400 Series II analyzer and were within ±0.4 of theoretical values.The reaction progress was monitored using thin layer chromatography on a silufol UV254 TLC aluminum sheet.

Grignard protocol
To an Et 2 O solution (200 mL) of allyl magnesium bromide, prepared from allyl bromide (36.3 g, 0.30 mol) and magnesium (12.15 g, 0.50 mol), were added slowly aldimines 1-5 (0.10 mol), dissolved in absolute THF (50 mL).The mixture was kept during 4 h at room temperature, and then cooled to 0 o C to treat rapidly with water, and finally with saturated ammonium chloride solution.The organic layer was separated and the aqueous layer was extracted with ether (4 × 50 mL).The combined organic extracts were dried (Na 2 SO 4 ) and concentrated.The residue was purified by Al 2 O 3 column chromatography (eluent -hexanes/ ethyl acetate) to give new homoallylamines 6-10, those physicochemical characteristics are shown in Tables 1,2.

Barbier protocol
To a mixture of aldimines 1, 4 and 5 (1 mmol) and powder indium (170 mg, 1.5 mmol) in methanol (10 mL) was added allyl bromide (365 mg, 3 mmol).The reaction mixture was stirred vigorously at room temperature until all the indium had dissolved (1 h to 4 h), at which time TLC indicated complete reaction.The reaction mixture was diluted with sat.NH 4 Cl solution and extracted with ethyl acetate.The extract was washed with brine and dried (Na 2 SO 4 ).The product was purified by flash column chromatography on alumina, using hexanes/ethyl acetate as eluent.Physicochemical characteristics of the obtained homoallylamines 6, 9 and 10 were in agreement with those obtained by Grignard method.
General procedure for the preparation of 4-methyl-2-(4-pyridyl)-1,2,3,4-tetrahydroquinolines 85% (v/v) sulfuric acid (4.0 mL) was added dropwise at 0 o C to the homoallylamines 6-10 (2.0 g) in minimal CH 2 Cl 2 amount, and the resulting mixture was heated at 80-90 o C for 3 h while stirring vigorously.The reaction progress was monitored via TLC.At the end of the reaction the mixture was cooled down to room temperature and concentrated ammonium hydroxide solution was added to pH 10.Four 25 mL extractions with dichloromethane were performed.The organic layers were combined, dried (Na 2 SO 4 ) and concentrated.The oily residue was purified by column chromatography over alumina to give new THQ 11-14 and quinoline sulfonic acid 15.The synthesis of THQ 13 was realized using PPA.Physicochemical characteristics of the obtained compounds are given in Table 3.

Microorganisms and media
For the antifungal evaluation, standardized strains from the American Type Culture Collection (ATCC), Rockville, MD, USA, and CEREMIC (C), Centro de Referencia en Micología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Suipacha 531-( 2000 Strains were grown on Sabouraud-chloramphenicol agar slants for 48 h at 30 o C, maintained on slopes of Sabourauddextrose agar (SDA, Oxoid) and subcultured every 15 days to prevent pleomorphic transformations.Inocula of cell or spore suspensions were obtained according to reported procedures and adjusted to 1-5 ×10 3 cells/spores with colony forming units (CFU) mL -1 . 11,12

Antifungal susceptibility testing
Minimal Inhibitory Concentration (MIC) of each compound was determined by using broth microdilution techniques according to the guideliness of the CLSI (formerly NCCLS) M27-A2 11 and for filamentous fungi M 38 A. 12 MIC values were determined in RPMI-1640 (Sigma, St Louis, Mo, USA) buffered to pH 7.0 with MOPS.Microtiter trays were incubated at 35 o C for yeasts and hialohyphomycetes and at 28-30 o C for dermatophyte strains in a moist, dark chamber, and MICs were visually recorded at 48 h for yeasts, and at a time according to the control fungus growth, for the rest of fungi.
Endpoints were defined as the lowest concentration of compound resulting in total inhibition (MIC 100 ) of visual growth compared to the growth control wells which contain culture medium and fungal inoculum but no antifungal drug.
For the assay, stock solutions of pure compounds were two-fold diluted with RPMI from 250-0.95 mg mL -1 (final volume = 100 mL) and a final DMSO concentration ≤ 1%.A volume of 100 mL of inoculum suspension was added to each well with the exception of the sterility control where sterile water was added to the well instead.Ketoconazole (Sigma Chem.Co.), Terbinafine (Novartis, Bs.As.) and Amphotericin B (Sigma Chem.Co.) were used as positive controls.All tests were made by duplicate.
The minimum fungicidal concentration (MFC) of each compound against each strain was determined as follows: After determining the MIC, an aliquot of 5 mL was withdrawn from each clear well of the microtiter tray and plated onto a 150-mm RPMI-1640 agar plate buffered with MOPS (Remel, Lenexa, Kans.).Inoculated plates were incubated at 30 °C, and MFC was recorded after 48 h.The MFC was defined as the lowest concentration of each compound that resulted in total inhibition of visible growth in the agarized plate.