A facile and efficient method for the synthesis of crystalline tetrahydro-β-carbolines via the Pictet-Spengler reaction in water

A facile and efficient synthesis of tetrahydro-β-carbolines (tryptolines) in one step from tryptamine and aldehydes, in an environmentally friendly water solvent, has been investigated. This convenient and clean synthesis of various tryptolines was facilitated by l-tartaric acid, a natural compound, to obtain the desired products as clear crystals. Among the four crystalline products, the most substituted tryptoline 2 showed the best inhibitory activity against EJ cells and the least cytotoxicity, with an LC50 value of 1.49 mg/mL, against brine shrimp larvae.


Results
We examined the substrate scope of the Pictet-Spengler reaction with four different aldehydes, as shown in Fig. 1. l-Tartaric acid facilitated the formation of colorless-crystalline tryptolines, as shown in Fig. 2. The optimum amount of l-tartaric acid was determined by carrying out the experiment with 0.1-1 equiv. of l-tartaric acid. We  found that 0.5 equiv. of l-tartaric acid was sufficient to give the highest yield, as the yield did not improve when using more than 0.5 equiv. of l-tartaric acid. The reaction was carried out in water, which is a cheap and safe solvent. The reaction mixture was set still until the crystals formed, and the products were filtered without any additional workup. Tryptolines 1-4 were obtained in crystalline form and in fair yields (25-45%), and required no chromatography. The most substituted β-carboline 4 showed the best inhibitory activity against EJ cells.

1-(4-Methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]-indole (3).
Yield: 40% (crystalline). The greater the substitution on the benzyl ring of the product, the better its potency to inhibit the growth of the EJ cells. Therefore, the most substituted tryptoline 2 showed the highest inhibitory activity against the EJ cells, as shown in Fig. 3. We speculated that tryptolines may need a bulky substituent for better binding affinity to the hydrophobic pocket of the target protein. This assumption is consistent with the fact that the clinically used anticancer drugs vinblastine and vincristine are dimers of tryptolines and are thus bulkier than monomeric tryptolines.
The cytotoxicity results for tryptolines 1-4 against brine shrimp larvae are shown in Fig. 4. Tryptolines 1, 3, and 4 showed weak cytotoxicity, with LC 50 values of 0.26 mg/mL, 0.30 mg/mL, and 0.43 mg/mL, respectively. However, 2, which showed the highest inhibitory activity against the EJ cells, exhibited the least cytotoxicity with an LC 50 value of 1.49 mg/mL. Tryptoline 2 showed no significant cytotoxicity (LC 50 > 1.0 mg/mL) against brine shrimp larvae, as opposed to the positive control (K 2 Cr 2 O 7 ), which showed significant cytotoxicity with an LC 100 value of 0.1 mg/mL, over incubation period of 24 h. Interestingly, 2 showed the highest potency against the EJ cells but the least cytotoxicity as compared to 1, 3, and 4.

Discussion
In conclusion, a facile method for the synthesis of biologically active tryptolines using l-tartaric acid, a simple and natural compound, is developed. l-tartaric acid facilitated aqueous Pictet-Spengler reactions with four different aldehydes. Moreover, the colorless crystalline tryptolines formed could be directly filtered to avoid further isolation steps such as extractions, column chromatography or HPLC. All of the major crystalline products showed (S)-conformer as a dominant species, based on the optical rotations we measured, although these reactions were not highly enantioselective. With respect to the anti-cancer activity of the crystalline products 1~4, compound 2 showed the best cytotoxicity against EJ cell line. We believe that this is one of the safest and easiest methods to prepare the key intermediates in the first step of the synthesis of biologically active vinca alkaloids, especially at the industrial-scale. (2020) 10:1057 | https://doi.org/10.1038/s41598-020-57911-0 www.nature.com/scientificreports www.nature.com/scientificreports/

Methods
General procedure for the synthesis of tryptolines. l-Tartaric acid (0.5 equiv., 0.5 mmol) was added to a mixture of tryptamine hydrochloride (1.0 equiv., 1.0 mmol) and aldehydes (1.0 equiv., 1.0 mmol) in a 15 mL falcon tube. The reaction volume was adjusted to 4.0 mL with water, and the tube was sealed. The tube was placed in a hot water bath at about 60 °C and set still for 1-2 days until crystals were formed. The crystals were then filtered, followed by rinsing with cold water and ether.
High-resolution UPLC/QTOF/MS analysis. Ultra-performance LC/MS analysis was performed on an ultra-high-resolution Q-TOF LC-MS/MS system (Micro QTOF III, Bruker Co.) using a C18 column, with a particle size of 1.7 mm, dimensions of 2.1 × 100 mm, and flow rate of 0.6 mL min −1 , and an electrospray ionization (ESI) source.
MTT assay. EJ cells (3 × 10 5 cells/well) were seeded in 6-well plates, followed by treatment with the indicated concentrations of the compounds. After 24 h, the used medium was removed, and a fresh medium containing 10 μL of MTT solution (5 mg/mL) was added to the wells, which were then allowed to stand for 1 h. Subsequently, the medium was removed, and the cells were treated with 0.1 mL of dimethyl sulfoxide (DMSO). The absorbance was measured at a wavelength of 540 nm using a microplate reader. The cell viability was expressed as the percentage of the absorbance value determined for the control cultures.