A Facile Oxidative Opening of the C-Ring in Luotonin A and Derivatives

An oxidative ring opening reaction of the central ring C in the alkaloid Luotonin A and two of its derivatives was found to occur upon heating with an excess amine and potassium carbonate in dimethylsulfoxide (DMSO) solution in the presence of air oxygen. The structure of the novel amide-type products was elucidated and a possible mechanism for this reaction is proposed. Four of the new compounds show moderate in vitro anticancer activity towards human colon adenocarcinoma cells.


Introduction
In search of new anticancer agents, structural modifications of the alkaloid Luotonin A (first isolated in 1997 [1]) have found considerable interest during the past two decades [2][3][4][5][6][7][8][9][10][11][12]. Like its more prominent (and more potent) naturally occurring relative, Camptothecin (CPT) [13][14][15], Luotonin A can act as a topoisomerase 1 poison by stabilizing the DNA/Topo1 complex [2,16]. Due to its aromatic E-ring (see Figure 1), Luotonin A is principally not associated with bladder toxicity which is a specific adverse effect of CPT-derived drugs like Topotecan or Irinotecan with their labile lactone structure as ring E [14]. On the other hand, the antineoplastic activity of the Luotonin A core structure is markedly lower than that of CPT [16] and thus has stimulated efforts to improve topoisomerase 1 toxicity by various modifications of the lead structure.

Introduction
In search of new anticancer agents, structural modifications of the alkaloid Luotonin A (first isolated in 1997 [1]) have found considerable interest during the past two decades [2][3][4][5][6][7][8][9][10][11][12]. Like its more prominent (and more potent) naturally occurring relative, Camptothecin (CPT) [13][14][15], Luotonin A can act as a topoisomerase 1 poison by stabilizing the DNA/Topo1 complex [2,16]. Due to its aromatic E-ring (see Figure 1), Luotonin A is principally not associated with bladder toxicity which is a specific adverse effect of CPT-derived drugs like Topotecan or Irinotecan with their labile lactone structure as ring E [14]. On the other hand, the antineoplastic activity of the Luotonin A core structure is markedly lower than that of CPT [16] and thus has stimulated efforts to improve topoisomerase 1 toxicity by various modifications of the lead structure. Several different strategies have been described for the total synthesis of Luotonin A and related compounds, as summarized in a review article [17]. Among the approaches aiming at variation of the substitution pattern of ring A, there have been some recent investigations of our group in which we made use of two orthogonal cycloaddition routes [18][19][20]: the first route is based on a pathway that was previously explored by Zhou et al. [21] for the synthesis of the parent compound and we found it to be well suited for the introduction of substituents into positions 2 or 4, whereas the second route offers a convenient access to 1-or 3-substituted congeners. Although a Several different strategies have been described for the total synthesis of Luotonin A and related compounds, as summarized in a review article [17]. Among the approaches aiming at variation of the substitution pattern of ring A, there have been some recent investigations of our group in which we made use of two orthogonal cycloaddition routes [18][19][20]: the first route is based on a pathway that was previously explored by Zhou et al. [21] for the synthesis of the parent compound and we found it to be well suited for the introduction of substituents into positions 2 or 4, whereas the second route offers a convenient access to 1-or 3-substituted congeners. Although a number of new Luotonin A derivatives could be made available via these two routes directly, additional modifications by replacement of existing substituents were envisaged in order to further extend our compound library. In particular, nucleophilic substitution of a fluoro functionality with amines appeared quite promising in view of the reported facile transformation of 8-fluoroquinolines into the corresponding 8-(di)alkylaminoquinolines via nucleophilic substitution [22,23]. Herein, we report on the synthesis of a hitherto unknown 4-fluoro derivative of Luotonin A (containing the 8-fluoroquinoline motif) and the observation of an unprecedented oxidative opening of ring C on attempted nucleophilic substitution reactions with primary or secondary amines.

Results and Discussion
Among the four possible sites at ring A, position 4 (corresponding to position 8 of the quinoline core) appeared most interesting for the envisaged nucleophilic replacement of a fluorine atom with an amino substituent, as there have been several reports describing the preferential attack of nitrogen nucleophiles at this position in various di-and trifluoroquinolines [24][25][26][27]. We therefore commenced our investigations by preparing the hitherto unknown 4-fluoro derivative of Luotonin A via the pathway depicted in Scheme 1, starting from the quinazolinonecarboxylic acid ester 1 [21,28]. Weinreb amidation [29] with 2-fluoroaniline/trimethylaluminium gave the N-arylamide 2 in good yield, selective propargylation at the quinazoline N-3 afforded the key intermediate 3. On treatment of the latter with bis(triphenyl)oxodiphosphonium triflate (Hendrickson's reagent [30]) in dry dichloromethane at room temperature, an intramolecular [4+2] cycloaddition smoothly afforded the pentacyclic compound 4 in excellent overall yield. number of new Luotonin A derivatives could be made available via these two routes directly, additional modifications by replacement of existing substituents were envisaged in order to further extend our compound library. In particular, nucleophilic substitution of a fluoro functionality with amines appeared quite promising in view of the reported facile transformation of 8-fluoroquinolines into the corresponding 8-(di)alkylaminoquinolines via nucleophilic substitution [22,23]. Herein, we report on the synthesis of a hitherto unknown 4-fluoro derivative of Luotonin A (containing the 8-fluoroquinoline motif) and the observation of an unprecedented oxidative opening of ring C on attempted nucleophilic substitution reactions with primary or secondary amines.

Results and Discussion
Among the four possible sites at ring A, position 4 (corresponding to position 8 of the quinoline core) appeared most interesting for the envisaged nucleophilic replacement of a fluorine atom with an amino substituent, as there have been several reports describing the preferential attack of nitrogen nucleophiles at this position in various di-and trifluoroquinolines [24][25][26][27]. We therefore commenced our investigations by preparing the hitherto unknown 4-fluoro derivative of Luotonin A via the pathway depicted in Scheme 1, starting from the quinazolinonecarboxylic acid ester 1 [21,28]. Weinreb amidation [29] with 2-fluoroaniline/trimethylaluminium gave the N-arylamide 2 in good yield, selective propargylation at the quinazoline N-3 afforded the key intermediate 3. On treatment of the latter with bis(triphenyl)oxodiphosphonium triflate (Hendrickson's reagent [30]) in dry dichloromethane at room temperature, an intramolecular [4+2] cycloaddition smoothly afforded the pentacyclic compound 4 in excellent overall yield. In order to achieve the desired nucleophilic fluorine substitution, we chose the conditions that had been described in the patent literature [22,23] for the reaction of 8-fluoroquinoline derivatives with various amines. Thus, the fluoro compound 4 was heated to 100 °C with an excess of pyrrolidine in dimethylsulfoxide (DMSO) solution in the presence of potassium carbonate. As indicated by TLC, the starting material was completely consumed after 24 h and a single new spot was observed. After evaporation of volatiles and extractive work-up, the crude product was purified by medium-pressure liquid chromatography to afford the new compound in 49% yield as red-coloured crystals. 1 H-NMR-Spectroscopic characterisation surprisingly revealed the presence of two pyrrolidinyl residues in the molecule and the absence of the methylene unit that is part of ring C in the Luotonin A skeleton. On the other hand, the 13 C-NMR spectrum showed an additional carbonyl resonance (besides the quinazolinone C=O group at 161.4 ppm) at lower field (168.6 ppm). Mass spectrometry (EI-MS (electron-impact ionization) and ESI-TOF (electrospray ionization, time-of-flight) high-resolution MS) indicated a molecular formula of C26H25N5O2. Based on the combined spectroscopic evidence, it can be concluded that (a) the expected nucleophilic substitution of the fluorine atom with pyrrolidine has indeed taken place and (b) the C-ring of the pentacyclic In order to achieve the desired nucleophilic fluorine substitution, we chose the conditions that had been described in the patent literature [22,23] for the reaction of 8-fluoroquinoline derivatives with various amines. Thus, the fluoro compound 4 was heated to 100 • C with an excess of pyrrolidine in dimethylsulfoxide (DMSO) solution in the presence of potassium carbonate. As indicated by TLC, the starting material was completely consumed after 24 h and a single new spot was observed. After evaporation of volatiles and extractive work-up, the crude product was purified by medium-pressure liquid chromatography to afford the new compound in 49% yield as red-coloured crystals. 1 H-NMR-Spectroscopic characterisation surprisingly revealed the presence of two pyrrolidinyl residues in the molecule and the absence of the methylene unit that is part of ring C in the Luotonin A skeleton. On the other hand, the 13 C-NMR spectrum showed an additional carbonyl resonance (besides the quinazolinone C=O group at 161.4 ppm) at lower field (168.6 ppm). Mass spectrometry (EI-MS (electron-impact ionization) and ESI-TOF (electrospray ionization, time-of-flight) high-resolution MS) indicated a molecular formula of C 26 H 25 N 5 O 2 . Based on the combined spectroscopic evidence, it can be concluded that (a) the expected nucleophilic substitution of the fluorine atom with pyrrolidine has indeed taken place and (b) the C-ring of the pentacyclic scaffold has undergone an oxidative ring opening, involving another pyrrolidine unit that is now attached to the quinoline core via an amide function (structure 5, Scheme 2). To explore the scope of this unexpected reaction, the fluoro compound 4 was reacted with other amines under the same conditions (100 • C, DMSO as solvent, and K 2 CO 3 as a base). Interestingly, it turned out that only pyrrolidine is reactive enough to effect the fluorine substitution under these conditions, whereas piperidine, morpholine, and n-butylamine left the fluoro function untouched, but all led to the formation of ring-opened products with a (tertiary or secondary) carboxamide group (compounds 6-8, Scheme 3). An identical reaction behavior was found with the alkaloid Luotonin A itself (compound 9), affording the amides 10-12 on treatment with morpholine, n-butylamine, or N,N-dimethylethylenediamine, respectively. Also with the electron-rich 1,3-dimethoxy derivative [18] of Luotonin A (compound 13), an analogous oxidative ring C opening was observed, furnishing the morpholide 14, albeit in moderate yield. All of the isolated carboxamides share some characteristic NMR properties such as the carbonyl resonance in the 13 C-NMR spectra at 168-169 ppm (see supplementary material). The position of the secondary or tertiary amide function at C-3 of the quinoline nucleus is evidenced by HMBC (Heteronuclear Multiple Bond Correlation) crosspeaks between this carbonyl signal and the H-4 resonance as well as the NCH 2 protons in the amide part. The spatial proximity between the latter two kinds of protons is further proven by a corresponding crosspeak in the NOESY (Nuclear Overhauser Effect Spectroscopy) spectrum. Additionally based on HSQC (Heteronuclear Single-Quantum Correlation) and COSY (Correlation Spectroscopy) experiments, all signals in the 1 H-and 13 C-NMR spectra could be assigned and are in full agreement with the proposed structures. As with compound 5, the molecular formulae of all new compounds were confirmed by [M + H] + or [M + Na] + peaks in the high-resolution ESI-TOF mass spectra.
To gain more insight into this remarkable transformation, we varied the solvent and observed essentially the same results when N,N-dimethylformamide (DMF) was used instead of DMSO. However, with 1-propanol as a protic solvent, a complex reaction mixture was obtained from which no defined product could be isolated. Thus, an aprotic dipolar solvent seems to be favorable in this context. As neither the amine component, nor the inorganic base, nor the solvent have oxidative properties to perform the observed oxidation of the benzylic CH 2 group of ring C into an amide function, we suspected atmospheric oxygen to play a crucial role in this process. Indeed, when Luotonin A was heated to 100 • C with morpholine/K 2 CO 3 in DMSO or DMF solution under argon atmosphere, the starting material remained essentially unchanged after several hours [31], whereas a control experiment in the presence of air oxygen showed an almost complete transformation into compound 10 after the same time span. A possible mechanism for this oxidative/nucleophilic ring opening is formulated in Scheme 4: Presumably, the benzylic carbon undergoes a two-step oxidation sequence, involving attack of the amine after the first step and hydrolytic ring cleavage after the second step (promoted by moisture in the solvent or during work-up). To the best of our knowledge, a comparable reaction has not yet been reported for Luotonin A nor for the structurally related CPT.  Although our initial aim, i.e., the synthesis of new A-ring-modified Luotonin A derivatives, was not met, the obtained quinazolinyl-substituted quinoline-3-carboxamides represent interesting and hitherto unknown molecular entities. Therefore, we briefly examined the new compounds for their in vitro growth inhibitory activity towards SW480 human colon adenocarcinoma cells. Indeed, three of the tested compounds (5, 6 and 7) showed moderate activity, slightly exceeding that of the natural lead compound, Luotonin A. Compounds 8, 11, 12 and 14 are clearly inferior and compound 10 is approximately equipotent as compared to the reference (Table 1). At present, no valid conclusions can be drawn with respect to structure-activity relationships and the mode of action. Although our initial aim, i.e., the synthesis of new A-ring-modified Luotonin A derivatives, was not met, the obtained quinazolinyl-substituted quinoline-3-carboxamides represent interesting and hitherto unknown molecular entities. Therefore, we briefly examined the new compounds for their in vitro growth inhibitory activity towards SW480 human colon adenocarcinoma cells. Indeed, three of the tested compounds (5, 6 and 7) showed moderate activity, slightly exceeding that of the natural lead compound, Luotonin A. Compounds 8, 11, 12 and 14 are clearly inferior and compound 10 is approximately equipotent as compared to the reference (Table 1). At present, no valid conclusions can be drawn with respect to structure-activity relationships and the mode of action. % Inhibition 19 ± 3 47 ± 3 54 ± 10 52 ± 7 no inhib. 22 ± 1 5 ± 4 3 ± 0 7 ± 9 3. Experimental

Biological Evaluation
Human colon adenocarcinoma cells (SW480) were seeded in a 96-well plate at a density of 5 × 10 4 cells per mL. Minimum Essential Medium Eagle (MEM) with 10% FCS (Fetal Calf Serum) was used to conduct the culture in the first 24 h at 37 • C in the presence of 5% CO 2 . Compounds were then added in defined concentrations to the cultivated cells using DMSO as solvent and diluting this solution with serum-free MEM-BSA (Minimum Essential Medium Eagle, Bovine Serum Albumin). Treated cells were incubated for further 72 h at 37 • C and an atmosphere containing 5% CO 2 . After this incubation period, the MTT viability assay [32] was performed. This test measures cell viability using the redox potential of living cells. Functional cells reduce a yellow-colored tetrazolium salt to a red formazan derivative, which can be then determined by optical density. After 3 h of incubation, the absorbance of formazan was measured at 450 nm with 620 nm as a reference wavelength.

Conclusions
In summary, we have discovered an unprecedented oxidative ring opening reaction of the central ring C in Luotonin A and two of its derivatives simply on heating with an excess amine and potassium carbonate in DMSO solution in the presence of air oxygen. The amide-type reaction products were fully characterized and all 1 H-and 13 C-NMR signals have been assigned by a combination of HSQC, HMBC, COSY, and NOESY experiments. Four of the new compounds show moderate in vitro anticancer activity towards human colon adenocarcinoma cells.