Synthesis and Fluorescence Spectroscopic Studies of Novel 9-phenylacridino-18-crown-6 Ether Type Sensor Molecules

The synthesis of two new 9-phenylacridino-18-crown-6 ether type sensor molecules [1 and (R,R)-2] was accomplished. The cation recognition ability of the achiral sensor molecule 1 towards various ions was studied in acetonitrile by UV/Vis and fluorescence spectroscopies. Our studies revealed the binding of Ag+, Cd2+, Ni2+, Pb2+, Zn2+ and NH4 + ions by the latter molecule. Selectivity of the chiral dimethyl-substituted analogue (R,R)-2 was studied toward the enantiomers of the hydrogen perchlorate salts of 1-phenylethylamine, 1-(1-naphthyl)ethylamine, phenylglycine methyl ester and phenylalanine methyl ester using fluorescence spectroscopy.

In this paper we report the preparation of two new 9-phenylacridino-18-crown-6 ether type sensors 1 and (R,R)-2 (Fig. 1). The cation recognition ability of the achiral sensor compound 1 toward various ions was studied by UV/Vis and fluorescence spectroscopies. The enantiomeric discrimination ability of the dimethyl-substituted analogue (R,R)-2 was studied towards the enantiomers of PEA, 1-NEA, PGME and PAME (Fig. 2) using fluorescence spectroscopy.
9-Phenylacridine-4,5-diol (6) was prepared in good yield by the O-demethylation of 4,5-dimethoxy-9-phenylacridine (9), using anhydrous aluminium chloride in dry and pure chlorobenzene (Scheme 2). Even higher yield was achieved for the latter transformation by using pyridinium chloride at elevated temperature (180°C). 4,5-Dimethoxy-9-phenylacridine (9) was obtained from 9-chloro-4,5-dimethoxyacridine (10) by the modification of the Kharasch reaction (other C-C coupling reactions such as Suzuki, Grignard and related reactions did not yield the desired product). The carbon-carbon coupling reaction was carried out with an excess of phenylmagnesium bromide in dry and pure THF and toluene. Tetrakis(triphenylphosphine) palladium(0) and dilithium tetrachlorocuprate were used as catalysts. By changing the order of additions of the reagents and using toluene instead of THF to dissolve chloro compound 10 a higher yield was achieved (see Experimental Section). Replacing tetrakis(triphenylphosphine)palladium(0) by palladium(II) acetate gave the highest yield (Scheme 2). The relatively low yields may be attributed to sterical hindrance. 4,5-Dimethoxy-9(10H)-acridinone (11a) was also isolated as a byproduct in all C-C coupling reactions, meaning that the dehalogenation and oxidation of the chloro compound (10) took place. 9-Chloro-4,5-dimethoxyacridine was prepared from 4,5-dimethoxy-9(10H)-acridinone 11a [28] applying phosphoryl chloride (Scheme 2). Macrocycle 1 was also synthesized from the parent acridono-18-crown-6 ether (12) [28]. The latter crown ether (12) was first reacted with phosphoryl chloride and the crude 9-chloro derivative (13), without purification, was treated with phenylmagnesium bromide to afford macrocycle 1 (Scheme 3). The overall yield of this multistep reaction was lower than the one outlined in Schemes 1 and 2, therefore it was not applied for the preparation of chiral macrocycle (R,R)-2. Scheme 3 An alternative synthesis of macrocycle 1.

Complexation studies
The complexation ability of achiral sensor 1 was first studied by UV/Vis spectroscopy in acetonitrile toward the perchlorate salts of various metal ions (Ag + , Ca 2+ , Cd 2+ , K + , Mg 2+ , Na + , Ni 2+ , Pb 2+ , Zn 2+ ), ammonium perchlorate and benzylammonium perchlorate. The UV/Vis spectra of ligand 1 did not show any changes upon addition of a twentyfold excess of Ca 2+ , K + , Mg 2+ , Na + and benzylammonium ions. This suggests the lack of complexation or the formation of complexes with low stabilities. However, a bathochromic shift of the absorption spectra was observed in the cases of Ag + , Cd 2+ , Ni 2+ , Pb 2+ , Zn 2+ and NH 4 + . The absorption spectra of ligand 1 and its complexes are represented in Fig. 3.
Fluorescence titration was also performed in order to determine the stability constants (K S ) and stoichiometry of the complexes. In all cases, with the exception of Cd 2+ , the fluorescence emission spectra showed a decrease upon addition of the salts, which means that the fluorescence was quenched by complex formation, as a typical example see Fig. 4. In the case of Cd 2+ red shift of the spectra was observed (Fig. 5). In the case of Cd 2+ ions the complex formed by the achiral crown ether and the Cd 2+ ions may have caused significant conformational changes, which resulted in different photophysical behavior compared to the other complexed ions. Upon being treated with different ions the fluorescence changes of the sensor followed the Benesi-Hildebrand equation [29,30] therefore we could assume the formation of complexes with 1:1 ligand to metal ion ratios. The changes in the spectra were further analyzed using nonlinear regression analysis, all of the titration series of the spectra could be fitted satisfactorily using a complex form with 1:1 stoichiometry. The logK values determined by the global nonlinear regression analysis are represented in Table 1. Sensor 1 formed the most stable complexes with Cd 2+ , Pb 2+ , Zn 2+ and NH 4 + ( Table 1). Pb 2+ , Zn 2+ and NH 4 + caused the fluorescence emission to decrease, as shown for Ag + in Fig. 4, but in the case of Cd 2+ a significant bathochromic shift could be observed (Fig. 5).   The enantiomeric discrimination of the dimethyl-substituted acridino-18-crown-6 ethers (R,R)-3, (S,S)-5 and the diisobutylsubstituted analogue (R,R)-4 toward the enantiomers of the perchlorate salts of primary aralkylamines and α-amino acid esters was studied by Kertész and coworkers [16]. We anticipated that the substitution of the acridine ring at position 9 with an aromatic unit may further increase the degree of enantiomeric recognition. Thus, the enantioselectivity of the novel dimethylsubstituted 9-phenyl-acridino-18-crown-6 sensor molecule (R,R)-2 was studied toward the enantiomers of PEA, 1-NEA, PGME and PAME using UV/Vis and fluorescence spectroscopies. The absorbances of ligand (R,R)-2 were essentially unchanged upon titration with the enantiomers of the above optically active salts. However, the fluorescence emission spectra showed a relatively large decrease upon addition of the guest molecules. This means that the fluorescence was significantly quenched in the complexes (Figs. 6 and 7).  These fluorescence changes were used to determine the stability constants of the complexes ( Table 2) and the degree of enantiomeric differentiation (∆logK = logK (S) -logK (R) , Table 2). In all cases global nonlinear regression analysis was used for evaluation of the spectral data. Table 2 Stability constants and enantioselectivity of the complexes of (R,R)-2 with the enantiomers of PEA, 1-NEA, PGME and PAME in MeCN.

Guest
logK ∆logK The results clearly demonstrate that in the cases of the enantiomers of PEA, 1-NEA and PGME the enantiomeric recognition ability of the dimethyl-substituted sensor (R,R)-2 is similar to that of the earlier reported [16] macrocycles [(R,R)-3, (R,R)-4 and (S,S)-5] containing methyl and isobutyl groups at their stereogenic centers. For the enantiomers of PAME the stability constants of (R,R)-2 are smaller. However, the degree of enantiomeric recognition (∆logK values in Table 2) are higher in the cases of PEA and 1-NEA for (R,R)-2. Moreover, the selectivity toward the enantiomers of PGME of the dimethyl substituted (R,R)-2 is comparable to that of the reported [16] diisobutyl-substituted (R,R)-4. This can be attributed to the increased bulkiness of (R,R)-2, meaning that sterical hindrance probably play a vital role in enantioselectivity. It was found for the studied primary amines (PEA and 1-NEA) that in all cases the (S)-enantiomer formed a more stable complex with (R,R)-2 than the (R)-enantiomer, demonstrating the generally observed higher stability of heterochiral complexes [(R,R)-crown ether-(S)-ammonium salt] compared to that of homochiral complexes [(R,R)-crown ether-(R)-ammonium salt]. This behavior is in unison with the earlier observations using acridino-crown ether based sensors and selectors [16,24,25]. Kertész and coworkers showed [16] that in the case of PGME the homochiral complex had higher stability, which was also observed by us. This phenomenon was attributed [16] to the fact that, based on the Cahn-Ingold-Prelog naming system, (R)-PEA, (R)-1-NEA, (S)-PGME and (S)-PAME have the same spatial arrangement. In our case PAME showed unusual behavior, the complex formed by (R,R)-2 and (S)-PAME had higher stability than the complex of (R,R)-2 and (R)-PAME, this behavior can again [16] be attributed to the structural difference of PAME compared to the other salts, namely the presence of an additional methylene unit between the stereogenic centre and the aromatic ring (Fig. 2).

Experimental 3.1 Chemicals and solvents
Starting materials and reagents were purchased from Sigma-Aldrich Corporation unless otherwise noted. Silica Gel 60 F 254 (Merck) and aluminium oxide 60 F 254 neutral type E (Merck) plates were used for TLC. Aluminium oxide (neutral, activated, Brockman I) and Silica Gel 60 (70-230 mesh, Merck) were used for column chromatography. Ratios of solvents for the eluents are given in volumes (cm 3 /cm 3 ). Solvents were dried and purified according to well established methods [31]. Evaporations were carried out under reduced pressure unless otherwise stated.

Apparatus
Melting points were taken on a Boetius micro-melting point apparatus. Infrared spectra were recorded on a Bruker Alpha-T FT-IR spectrometer using KBr pastilles. Optical rotations were taken on a Perkin-Elmer 241 polarimeter that was calibrated by measuring the optical rotations of both enantiomers of menthol. NMR spectra were recorded on a Bruker 300 Avance spectrometer (at 300 MHz for 1 H and at 75.5 MHz for 13 C spectra). HRMS analyses were performed on a LTQ FT Ultra (Thermo Fisher Scientific, Bremen, Germany) system. The ionization method was ESI and operated in positive ion mode. The protonated molecular ion peaks were fragmented by CID at a normalized collision energy of 45-65%. The samples were solved in methanol. Data acquisition and analysis were accomplished with Xcalibur software version 2.0 (Thermo Fisher Scientific). UV/Vis spectra were taken on a Multiskan Spectrum Microplate Spectrophotometer controlled by SkanIt Software for Multiscan version 2.1. Fluorescence spectra were recorded on a BMG Labtech CLARIOstar spectrophotometer. Spectrophotometric titrations were carried out according to the literature [27]. The stability constants of the complexes were determined by global nonlinear regression analysis using the ReactLab TM Equilibria spectral analyses suite (Jplus Consulting, www. jplusconsulting.com). The concentrations of the solutions of sensors 1 and (R,R)-2 were 50 µM for the UV-vis measurements and 10 µM in the case of ligand 1 or 20 µM in the case of (R,R)-2 for the fluorescence titrations. 3.3.1 23-Phenyl-6,7,9,10,12,13,15,16-octahydro-1,21-methenobenzo[n,q][1,4,7,10

Procedure B)
4,5-Dimethoxy-9-phenylacridine 9 (220 mg, 0.7 mmol) and pyridinium chloride (4.8 g, 41 mmol) was stirred at 180°C for 2h. After the reaction was completed, the mixture was cooled down to rt and it was mixed with water (100 cm 3 ). The queous solution was extracted with CH 2 Cl 2 (3 × 100 cm 3 ). The combined organic phase was dried over MgSO 4 , filtered and the solvent was removed. The crude product was purified by column chromatography on silica gel using 1:10 MeOH/ CH 2 Cl 2 mixture as an eluent to give 6 (180 mg, 90%), which had the same physical properties and spectroscopic data as the one prepared with the above described (A) procedure.

Procedure B)
A modification of the above described (A) procedure gave a better yield. A solution of 10 (750 mg, 2.74 mmol) and dry and pure toluene (200 cm 3 ) was added dropwise to a stirred suspension of phenylmagnesium bromide (3M in THF, 5.5 cm 3 , 16.5 mmol), Pd(PPh 3 ) 4 (30 mg, 0.025 mmol), dilithium tetrachloro cuprate (0.1M in THF, 0.3 cm 3 , 0.03 mmol) and dry and pure toluene (40 cm 3 ) at rt under Ar. The reaction mixture was stirred for 5 h at rt, then the solvent was removed. The residue was taken up in EtOAc (200 cm 3 ) and water (200 cm 3 ). The phases were shaken well and separated. The aqueous phase was extracted with EtOAc (3 × 150 cm 3 ). The combined organic phase was dried over MgSO 4 , filtered and the solvent was evaporated. The crude product was purified by column chromatography on silica gel using EtOAc as an eluent to give 9 (260 mg, 30%). This product (9) had the same physical properties and spectroscopic data as the one prepared by the above described (A) procedure.

Procedure C)
The highest yield was achieved by using Pd(OAc) 2 instead of Pd(PPh 3 ) 4 . A solution of 10 (750 mg, 2.74 mmol) and in dry and pure toluene (200 cm 3 ) was added dropwise to a stirred suspension of phenylmagnesium bromide (3M in THF, 5.5 cm 3 , 16.5 mmol), Pd(OAc) 2 (15 mg, 0,066 mmol), dilithium tetrachloro cuprate (0.1M in THF, 0.3 cm 3 , 0.03 mmol) and dry and pure toluene (40 cm 3 ) at rt under Ar. The reaction mixture was stirred for 5 h at rt, then the solvent was removed. The residue was taken up in EtOAc (200 cm 3 ) and water (200 m 3 ). The phases were shaken well and separated. The aqueous phase was extracted with EtOAc (3 × 150 cm 3 ). The combined organic phase was dried over MgSO 4 , filtered and the solvent was evaporated. The crude product was purified by column chromatography on silica gel using EtOAc as an eluent to give 9 (400 mg, 46%), which had the same physical properties and spectroscopic data as the one prepared by the above described (A) procedure.

Conclusion
The synthesis and characterization of two new 9-phenylacridino-18-crown-6 ether type sensors [1 and (R,R)-2] was accomplished. The cation recognition ability towards various ions of the achiral sensor compound 1 was studied in acetonitrile by UV-vis and fluorescence spectroscopies. Our studies revealed the binding of Ag + , Cd 2+ , Ni 2+ , Pb 2+ , Zn 2+ and NH 4 + ions by this sensor compound. We examined the enantiomeric recognition properties of the dimethyl-substituted ligand (R,R)-2 toward the enantiomers of protonated primary amines (PEA, 1-NEA) and amino acid derivatives (PGME, PAME) using fluorescence spectroscopy.
Experiments are in progress to prepare derivatives of sensor 1, which contain lipophilic side chains in order to prepare ion-selective electrodes. Thus, apart from the detection of ions, the measurement of their quantity may also be possible.
Also our further aim is to synthesize an analogue of (R,R)-2, which contains a carboxylic group at position 4 of the phenyl group. This functional group is suitable for a condensation reaction with 3-aminopropyltriethoxysilane, which gives the crown ether containing a triethoxysilane end group. The latter macrocycles can easily be attached to silica gel with covalent bonds to produce a new CSP.