Studies on the photocyclization reaction of 8‐styryl‐substituted coralyne derivatives

Funding information Deutsche Forschungsgemeinschaft; Fonds der Chemischen Industrie; University of Siegen Abstract The photoreactivity of four 8-styryl-substituted coralyne derivatives was examined by UV/VISand H-NMR-spectroscopy. Except for the dimethylaminosubstituted derivative, these cationic azoniaheterocyclic dyes undergo photocyclization that most likely proceeds through an initial E-Z-isomerization of the double bond. Subsequent oxidation of the intermediates under aerobic conditions gave the pyrrolo-annelated quinolizinium derivatives as final products, thus providing a useful synthetic route to polycyclic azoniahetarene derivatives. The 6-(4-chlorophenyl)-substituted pyrroloquinolizinium derivative was isolated on a preparative scale, and the investigation of its photophysical properties revealed significantly red-shifted absorption (λabs = 465–480 nm) and emission bands (λfl = 550–562 nm) in comparison to the parent coralyne and the styryl-substituted derivatives.


| INTRODUCTION
Polycyclic cationic hetarenes have recently attracted much interest with regard to their synthesis and applications. Specifically, quinolizinium-type derivatives with a bridgehead quaternary nitrogen atom (azoniahetarenes) figure as promising lead structures for the development of DNA-binding and DNA-photodamaging agents, [1] enzyme inhibitors, [2] cystic fibrosis transmembrane conductance regulator activators, [3] fluorescent dyes and chemosensors, [4] or nonlinear optical materials. [5] As a result, several synthetic routes have been developed and explored for the synthesis of substituted or annelated quinolizinium derivatives. [6] Notably, a series of azonia aromatic natural products are known, for example, berberine, sanguinarine, sempervirine, or flavopereirine, which have favorable biological properties. [7] In this context, the synthetic berberine alkaloid coralyne (1) is of particular interest. This fully aromatic dibenzoquinolizinium derivative exhibits a very broad spectrum of biological and pharmacological activity, namely antimicrobial, antileukemic, antineoplastic and antitumor properties as well as enzyme inhibition. [8] Notably, the chemical and photophysical properties of the annelated quinolizinium are essentially maintained in coralyne, so that the synthesis of coralyne derivatives offers the opportunity to combine the desired quinolizinium properties with the latent biocompatibility and bioactivity of this alkaloid. Thus, we have demonstrated recently that the easily available 8-styrylsubstituted coralyne derivatives 2a-d are duplex and quadruplex DNA-binding ligands. [9] In particular, the biocompatibility of these compounds allowed to employ the aminostyryl-substituted derivative 2d as fluorescent probe for the detection of DNA in cells.
Having the coralyne derivatives 2a-d in hands we wished to explore their potential as substrates for further functionalization. Specifically, we were interested in the photo-induced cyclization reaction to give the pyrrolofused products.
Although pyrroloquinolizinium derivatives, that are members of the cyclazine family, [10] are known already, [11] there is, to the best of our knowledge, only one example reported for their formation through photocyclization of a styrylquinolizinium precursor. [12] Herein, we report our studies on the photoreactivity of the styrylcoralyne derivatives 2a-d, along with some mechanistic studies, and we present the absorption and emission properties of the resulting pyrrolo-annelated product 3.

| RESULTS AND DISCUSSION
The absorption spectra of the styryl-substituted coralyne derivatives 2a-d exhibit characteristic long-wavelength absorption bands with maxima at 436 nm (2a-c) or 469 nm (2d) in MeCN ( Figure 1). [9] The irradiation of the dyes 2a-c under aerobic conditions with 315 nm or 360 nm light did not lead to significant changes in the absorption spectra, whereas the use of 420 nm light caused a decrease of the absorption accompanied by a bathochromic shift. While the dye 2a was irradiated for more than 3 hours until no more changes in the absorption spectra were detected ( Figure 1A), the complete photoreaction only took approximately 30 minutes for 2b and 2c ( Figure 1B,C). Hence, the substitution with a methyl or chlorine substituent apparently enhances the photoreactivity of the substrate, since both substituents increase the electron density of the conjugated π system either through an inductive effect (methyl group) or, in the case of the chloro substituent, through resonance, as the latter operates as a π donor in conjugation with the electron withdrawing coralyne unit. The significant changes of the absorption spectra were, however, not in agreement with the expected E-Z-isomerization of the double bond. But the similar course of the spectral changes led to the conclusion that 2a-c form analogous photoproducts upon irradiation. In fact, the normalized absorption spectra after an irradiation time of 210 minutes (2a) or 60 minutes (2b and 2c), respectively, are almost superimposable and show a broad longwavelength absorption band at 463 nm with a red-shifted shoulder between 500 and 540 nm as well as a distinct absorption band at 327 nm ( Figure S1A). In contrast, the irradiation of 2d only caused minor changes in the absorption spectra providing no clear indication of a light-induced reaction ( Figure 1D). Most likely, 2d forms a charge-transfer excited state that returns to the ground state by competing radiative [9] and radiationless transitions before a photoreaction can take place.
The irradiation of 2c was followed exemplarily by 1 H-NMR spectroscopy in order to obtain an insight into the underlying mechanism and a detailed analysis of the photoproduct ( Figure  The normalized absorption spectrum of the solution after an irradiation time of 30 minutes showed the same absorption bands in comparison to the one obtained upon irradiation at a concentration of c L = 20 μM ( Figure S1B). This indicates that the formation of the photoproduct is essentially independent of the concentration in the range between 20 μM and 2 mM. Furthermore, the mass-spectrometric analysis (ESI) of the solution revealed the loss of two hydrogen atoms from substrate 2c (m/z = 484), and the photoproduct 3 was identified as 6-(4 0 -chlorophenyl)-2, 3,9,10-tetramethoxydibenzo [a,g]pyrrolo [2,1,5-de]quinolizinium (3) by additional NMRspectroscopic investigations [correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC)]. The formation of this pyrroloquinolizinium is in agreement with F I G U R E 2 1 H-NMR spectra (6.7-10.1 ppm) of 8-(4-chlorostryryl) coralyne (2c) (c L = 2.0 mM) upon irradiation with 420 nm light in CD 3 CN (ν = 600 MHz) (cf. Scheme 1, conditions a) S C H E M E 1 Synthesis of the pyrroloquinolizinium derivative the known photocyclization-oxidation reaction of a 4-styrylsubstituted benzo[a]quinolizinium. [12] In order to synthesize the pyrroloquinolizinium 3 on a preparative scale, a 200 μM solution of 2c was irradiated with a high pressure Hg mercury lamp (λ irr > 390 nm) (Scheme 1, conditions b), and the reaction was monitored by absorption spectroscopy (Figure 3). After an irradiation time of 8.5 hours, the product 3 was obtained in 26% yield, and its structure was confirmed by NMR spectroscopy ( 1 H, 13 C, COSY, HSQC, HMBC), electrospray ionization mass spectrometry (ESI-MS), and elemental analysis. It should be noted that the 1 H-NMR signals of a saturated solution of 3 in CD 3 CN are not only much broader but also significantly shifted upfield ( Figure S3) in comparison to the signals that were detected after the irradiation of a 2 mM solution of 2c (Figure 2, 30 minutes). We attribute this to the formation of dye aggregates at higher concentrations, as commonly observed for positively charged, heterocyclic systems. [13] As revealed by the 1 H-NMR spectroscopic reaction monitoring, the mechanism of the formation of 3 most likely proceeds through an initial E-Z-isomerization of the double bond and a subsequent photo-induced electrocyclization to give intermediate A (Scheme 2). After the deprotonation of A, the resulting intermediate C is readily oxidized under the employed aerobic conditions to achieve aromatization and formation of product 3. In this context, it has to be noted that the intermediate C might also be formed after deprotonation of the tertiary carbenium ion B that results from a [1,2]-H shift of A.
The pyrroloquinolizinium derivative 3 is hardly soluble in various nonpolar, polar protic, and polar aprotic solvents. Nevertheless, we determined its absorption and emission properties in representative solvents ( Figure 4, Table 1). Thus, the long-wavelength absorption maximum of 3 ranges from 465 nm in MeCN to 480 nm in CHCl 3 (ε max = 19 100-22 500 M -1 cm -1 ) ( Figure 4A). Thereby, the red-shift in a chlorinated solvent such as CHCl 3 was previously attributed to the high polarizability of these solvents. [4g] Furthermore, the dye 3 is fluorescent in the investigated solvents and shows broad emission bands at λ fl = 550-562 nm with moderate emission quantum yields (Φ fl = 0.19-0.27) ( Figure 4B). In comparison to the parent compound coralyne (1) and the styryl dyes 2a-c (λ abs = 434-436 nm and λ fl = 473-484 nm in MeCN), [9] both the absorption and the emission bands of the pyrrolo-annelated compound 3 are significantly redshifted due to its more extended aromatic system.

| CONCLUSIONS
The 8-styryl-substituted coralyne derivatives 2a-c undergo a photocyclization-oxidation reaction upon irradiation under aerobic conditions to form pyrroloquinolizinium derivatives. As revealed by 1 H-NMR-spectroscopy, the photocyclization most likely proceeds through an initial E-Z-isomerization of the double bond. Since this reaction is straightforward and should be applicable to similar cationic hetarenes, the irradiation of such styryl derivatives offers a useful general synthetic access to novel polycyclic azoniahetarene derivatives, specifically those containing the important coralyne lead structure. In addition, the synthesis of the respective chlorophenyl-substituted pyrrolo-annelated quinolizinium 3 by irradiation of 2c on a preparative scale enabled for the first time the analysis of the absorption and emission properties of this class of compounds. Hence, the derivative 3 exhibits significantly red-shifted absorption and emission bands in comparison to coralyne (1) and the styrylcoralyne derivatives 2a-c.

| Equipment
NMR spectra were recorded with a Varian VNMR-S 600 ( 1 H: 600 MHz, 13 C: 150 MHz) at 25 C. Spectra were processed with the software MestReNova (version: 12.0.1) and referenced to the respective solvent (CD 3 CN: δ H = 1.94, δ C = 1.34). The chemical shifts are given in ppm. Absorption spectra were recorded with a Cary 100 Bio spectrophotometer in Hellma quartz cells 110-QS (10 mm) with baseline correction at 20 C. Emission spectra were collected with a Cary Eclipse Fluorescence spectrophotometer in Hellma quartz cells 114F-QS (10 mm × 4 mm) at 20 C. Elemental analyses data were determined with a HEKAtech EUROEA combustion analyzer by Mr. Rochus Breuer (Universität Siegen, Organische Chemie I). Mass spectra (ESI) were recorded on a Finnigan LCQ Deca (U = 6 kV; working gas: Argon; auxiliary gas: Nitrogen; temperature of the capillary: 200 C). The melting points were measured with a BÜCHI 545 (BÜCHI, Flawil, CH) and are uncorrected. Solutions were irradiated with a diode-array light apparatus (Atlas Photonics LUMOS 43) or with a high-pressure Hg-lamp (Heraeus TQ 150-56001725).

| Materials
The styryl-substituted coralyne derivatives 2a-d were synthesized from coralyne (1) by a Knoevenagel type reaction as previously described. [9] The commercially available chemicals were reagent-grade and used without further purification. Absorption and emission spectra were recorded from solutions prepared with spectroscopic grade solvents. Fluorescence quantum yield relative to Coumarin 153 (Φ fl = 0.38); [14] estimated error for fluorescence quantum yields: ±10%. determined in a range between 200 and 700 nm (260-700 nm for dimethyl sulfoxide and 240-700 nm for CHCl 3 ) and subsequently smoothed in the Origin software with the function "adjacent-averaging" (factor of 10).

| Methods
For the photochemical studies, air-saturated solutions of 2a-d in MeCN were irradiated with a diode-array light apparatus (Atlas Photonics LUMOS 43) at λ irr = 420 nm either in Hellma quartz cells 110-QS (10 mm) or in 3 mm NMR tubes.