Synthesis of 1-indolyl-3,5,8-substituted γ-carbolines: one-pot solvent-free protocol and biological evaluation

1,5-Disubstituted indole-2-carboxaldehyde derivatives 1a–h and glycine alkyl esters 2a–c are shown to undergo a novel cascade imination-heterocylization in the presence of the organic base DIPEA to provide 1-indolyl-3,5,8-substituted γ-carbolines 3aa–ea in good yields. The γ-carbolines are fluorescent and exhibit anticancer activities against cervical, lung, breast, skin, and kidney cancer cells.

The classical Graebe-Ullmann synthesis [11] of γ-carbolines, one of the very early protocols in the domain, is based on the thermal decomposition of N-pyridylbenzotriazoles. Later, the reaction conditions were modified to make this reaction more versatile and operationally simple such as by the use of microwave irradiation [12]. Meanwhile, the Fischer indole synthesis was successfully extended for the synthesis of significant biologically active tetrahydro-γ-carboline derivatives [13,14]. A systematic assessment of the above Graebe-Ullmann and Fischer synthesis protocols revealed that these reactions are associated with i) low product yield, ii) limited scope including the use of a very specific set of substrates, and iii) involvement of extreme thermal conditions with the use of corrosive reagents. Much later, Larock and co-workers developed a Pd/ Cu-catalyzed imino-annulation of internal alkynes [15], which paved the way for transition-metal-catalyzed cyclizations as easy access to these scaffolds. Notably, the gold-catalyzed tandem cycloisomerization/Pictet-Spengler cyclization of 2-(4aminobut-1-yn-1-yl)aniline [16], the Ru and Rh-catalyzed [2 + 2 + 2] cycloadditions of yne-ynamides [17], and the Pd-catalyzed tandem coupling-cyclization [18] are significant works in the area (Scheme 1). However, the use of toxic and expensive metal catalysts has limited their development as environmentfriendly synthetic protocols. More recently, an acid-catalyzed cyclization of α-indol-2-ylmethyl TosMIC (tosylmethyl isocyanide) derivatives to synthesize heterocycles [19] has been thoroughly studied (Scheme 1), including the synthesis of the carboline alkaloid ingenine B [20]. The iodine-catalyzed [3 + 3] cycloaddition of indolyl alcohol to enaminones [21] and the thiourea-catalyzed iso-Pictet-Spengler reaction of isotryptamine with aldehydes [22], are some noteworthy contributions to the field.
A cascade or domino reaction is an interesting approach for the design of efficient one-step transformations for complex molecule synthesis [23,24]. Employing domino reactions to simplify cumbersome industrial processes can afford complex pharmaceutical products in an economical and environment-friendly manner [25]. Easy workup procedures and single-step purification reduce the efforts in the synthesis of complex molecular architectures. Therefore, cascade reactions are essential in synthetic organic chemistry, even with moderate yields [26]. Recently, such reactions have claimed their much deserving place in drug design and natural product synthesis [27]. In the literature, there is only a limited number of direct synthetic procedures to prepare γ-carbolines till date [28], and this gives a cutting edge advantage to our new protocol wherein a solvent and metal-free direct access to the γ-carboline core from substituted indole-2-aldehyes and glycine ester salts has been discovered.

Optimization of reaction conditions and chemical synthesis
The base-catalyzed imination of aromatic aldehydes is a valuable method in organic synthesis to synthesize a variety of heterocyclic building blocks [29]. Among all the reported iminoesters, alkyl N-arylideneglycinates have attracted much attention in recent years. For instance, the metal-catalyzed asymmetric [3 + 2] cycloaddition of ethyl N-benzylideneglycinates with electron-deficient alkenes has been reported to yield substituted pyrrolidines [30].
Recently, we reported the synthesis of substituted pyrrole-2aldehydes to 5-azaindole transformation during a base-catalyzed imination reaction [31]. However, we envisioned that our methodology might be strategically applied towards the synthesis of substituted γ-carbolines as a C-3 nucleophilic attack is more favored in indoles than in pyrroles. Herein, we report an interesting observation for conversion of substituted indole-2aldehydes 1 to 1-indolyl-3,5,8-substituted γ-carbolines 3 by a  Earlier in the literature, it was reported that 1H-indole-2carbaldehyde derivatives underwent condensation with N-arylideneglycinate to form pyrimidoindole derivatives [32]. However, when 1-methyl-1H-indole-2-carbaldehyde (1a) and glycine methyl ester hydrochloride salt (2a) were reacted in the presence of DIPEA (Hünig's base) at room temperature in a nonpolar solvent such as toluene, only marginal amounts of the corresponding imine were observed, that could not be isolated (Table 1, entry 1). When the reaction mixture was further heated to reflux for 16 h, only traces of 1-indolyl 3,5,8-substituted γ-carboline 3aa were formed that were still insufficient for complete characterization. Intending to improve the yield of 3aa, we screened various solvents, non-nucleophilic organic bases such as triethylamine and DBU, and several inorganic bases like K 2 CO 3 , Cs 2 CO 3, and NaH (Table 1, entries 2-6).
With the initial success at hand, the reaction was found to be equally effective with various glycine alkyl ester HCl salts 2a-c but failed to result in the formation of 1-indolyl-3-cyano-5methyl γ-carboline derivative 3ad when 2-aminoacetonitrile 2d was utilized as the condensation component. Then, a range of 1and 1,5-disubstituted indole-2-carboxaldehyde derivatives 1a-h was synthesized (for details see Supporting Information File 1) to evaluate the scope of the reaction further.
Indole-2-carbaldehyde derivatives with electron-donating substituents in the 1-position of the indole ring system 1a-e were transformed in moderate to good yields into their corresponding γ-carboline derivatives 3aa-ac and 3ba-ea due to an enhanced C-3 nucleophilicity of the indole nucleus (Scheme 2). The formation of the corresponding γ-carboline products was confirmed unequivocally by single-crystal X-ray diffraction analysis of 3ac ( Figure 2).
Plausible mechanism for the formation of γ-carbolines  substituted γ-carbolines 3aa-ac and 3ba-ea. We successfully isolated and characterized the 1,2-dihydro γ-carboline derivative 3ga, which again verifies the proposed mechanism. During the formation of carbolines, the substrates, 1a-e and 1g were exclusively transformed to γ-carbolines or the 1,2-dihydro-γcarboline 9a, and no traces of any β-carboline product were observed, which proves that the heterocyclization reaction is highly regiospecific.

Optical properties of γ-carbolines
Interestingly, the γ-carboline derivatives were found to be highly fluorescent under UV light irradiation. A systematic literature survey revealed that the structural core of carbolines had  The fluorescence studies carried out for 3ac in four different solvents revealed that the emission maxima shifted bathochromically by almost 40 nm upon changing the solvent polarity, for instance, from non-polar hexane to moderately polar dichloromethane and then highly polar DMSO (Table 2, Figure 3). The fluorescence quenching of 3ac in methanol is attributed to the partial protonation of the carboline unit's nitrogen atoms facilitated by polar-protic solvents [33]. The fluorescence lifetimes were measured by time-correlated single-photon counting (TCSPC) experiments. The average fluorescence lifetime of compound 3ac was found to be 8.35 ns and 4.73 ns in DMSO and DCM, respectively (Table 2, Figure 4).

Biological evaluation of γ-carbolines as anticancer agents
A panel of carboline derivatives 3ac, 3bc, 3ca, and 3ga, along with a standard drug, doxorubicin, were screened for their cytotoxicity against various cancer lines ( Figure 5, Table 3 and Figure S2, in Supporting Information File 1) such as MCF-7 (breast cancer), HeLa (cervical cancer), HEK293 (human   At last, to evaluate cell uptake of the novel γ-carboline for fluorescence imaging, live-cell imaging experiments were performed. In brief, HeLa cells were incubated with carboline 3ac (10 μM and 100 nM), and the cellular uptake was examined using confocal microscopy (λ ex = 405 nm; λ em = 420-470 nm). Compound 3ac showed excellent cytosolic uptake in cancer cells when incubated at a 10 μM concentration (Figure 7), whereas only little uptake was observed at a concentration of 100 nM ( Figure S3, Supporting Information File 1).

Conclusion
In summary, we have developed an operationally simple onepot synthetic protocol for the synthesis of highly substituted γ-carboline derivatives. The metal-and solvent-free method provides direct access to complex molecular structures in good yield from inexpensive substrates. The optical and biological evaluations carried out for representative γ-carbolines revealed promising photophysical and anticancer properties of the core framework for developing novel theranostic applications to diagnose and treat cancer in the future.

Experimental General methods and materials
All reactions were carried out in oven-dried glassware with magnetic stirring. Starting materials and other reagents were obtained from a commercial supplier and used without further purification. NMR spectra were recorded on an Avance III 400 Ascend Bruker spectrometer. CDCl 3 and D 2 O were used as NMR solvents. Chemical shifts (δ) were reported as part per   In vitro cytotoxicity studies Cytotoxicity analysis in cancer and macrophage cells Cancer (MCF7, A431, A549, HEK293 or HeLa cell lines) or RAW264.7 cells were seeded in a 96-well plate (4,200 cells/ well) and allowed to form a monolayer for a period of 48 h. Old medium was replaced with fresh medium (0.2 mL) containing an increasing concentration of γ-carboline derivatives 3ac, 3bc, 3ca, 3ga and doxorubicin (0.1 μM, 0.25 μM, 0.5 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM) and incubated for 48 h or 3 h, respectively. The medium in each well was discarded, and the cells were rinsed with PBS (3 × 0.2 mL) followed by treatment with 0.5% crystal violet (0.05 mL) for 20 minutes at room temperature. The cells were rinsed with PBS (3 × 0.2 mL) and methanol (0.20 mL) was added to each well followed by incubation for 20 minutes. The absorbance from each well proportional to the live cell was measured using a Synergy H1 multimode plate reader (BioTek Instruments, Inc., Winooski, VT, USA) at an excitation and emission wavelength of 530 nm and 590 nm, respectively.
Dose-response curves were obtained from a plot of the semilogarithmic [conc] vs the intensity of the fluorescence emission, and the IC 50 (concentration at which 50% of the enzymatic activity is inhibited) was calculated for the carboline derivatives or doxorubicin using GraphPad Prism, version 7.02 for Windows (GraphPad Software, San Diego, CA).

HeLa cell uptake study of γ-carboline 3ac
A live-cell imaging experiment was performed with HeLa cells. The HeLa cells were placed in a 4-well confocal dish (cell count ≈ 100 cells per well) and incubated for 48 h at 37 °C under 5% CO 2 . After 3 h of incubation with carboline derivative 3ac (10 nM, 100 nM, 1 μM, 10 μM, and 100 μM), the cellular uptake and distribution was monitored by using confocal microscopy (λ ex = 405 nm; λ em range = 420-470 nm).