A Novel Structural Modification Strategy to Incorporate LAT-1 Transporter Inhibitory Activity

Research Article

J Drug Discov Develop and Deliv. 2020; 6(1): 1033.

A Novel Structural Modification Strategy to Incorporate LAT-1 Transporter Inhibitory Activity

Li L1, Li Y2, Gong M2 and Zhang B1*

¹School of Chemical Engineering and Technology, Tianjin University, China

²Tianjin Medical University General Hospital, China

*Corresponding author: Bao Zhang, School of Chemical Engineering and Technology, Tianjin University, 135 Yaguan Road, Jinnan District, Tianjin, China

Received: February 14, 2020; Accepted: April 23, 2020; Published: April 30, 2020


L-type Amino Acid Transporter 1 (LAT-1), over expressed on the membrane of various tumor cells, was considered as a potential target for solid tumor therapy. It was hypnotized that conjugating a LAT-1 affinity molecule upon cell toxic agent could enhance the tumor targeting. In this study, quercetin was modified by overhanging a LAT-1 affinity group, with the aim of developing an effective LAT-1-mediated chemotherapeutic agent. On the other hand, the poor water solubility of quercetin limited its clinical utility in fact; this obstacle was ameliorated by conjugating the LAT-1 affinity group.

The anti-tumor assays in this study as certained that the novel compound possessed LAT-1 inhibitory activity in high affinity which lead to progress of antitumor activity of quercetin, and increase on water-solubility of quercetin which enhanced the bioavailability of quercetin. All those data suggested that chemconjugating a LAT-1 affinity group with traditional cellular toxic agent might be a powerful approach aiming to re-utilize and ameliorate its original anti-tumor characterizations.

Keywords: Quercetin; LAT-1 transporter; Solubility


The amino acid transporters on the plasma membrane is crucial for mediating the amino acid transportation in and out of cells and organelles which is essential for cellular physiological function [1]. The L-type Amino Acid Transporter-1 (LAT-1), a Na+-independent neutral amino acid transporter [2,3] which has been identified as potential as a target for tumor therapy since LAT-1 transporters is not only over expressed in various tumor cells but also in organ barrier system including blood brain barrier, testis barrier and placental barrier. The existence of these barriers extremely decreased the drug bioavailability following the lesion in drug efficacy [4-6].

Quercetin (3,3’,4’,5,7-pentahydroxyflavone), a natural flavonoid found in various vegetables and fruits, was reported to possess anticarcinogenic properties against a wide range of human cancer [7,8]. It was ascertained that this compound plays its effects by inhibiting tyrosine kinases involved in cell signaling pathways, including HMGB1, JAK-STAT, and TSLP [9-11]. Following its comprehensive characterization, quercetin has been approved by FDA A as a therapeutic medicine for the treatment of prostatic cancer.

However, the poor water solubility of quercetin limited the efficacy in clinical utility of quercetin, the maximal solubility of quercetin of 0.1mg/ml even in 100% DMSO solution.

In this study, quercetin was conjugated with various amino acids possessing high affinity to the LAT-1 transporter containing a hydrolysis linker, with a view to enhancing affinity to LAT-1 over expressed on tumor cells and water solubility relative to parent quercetin. The increased water solubility induced by the introduced amino acid led to improved bioavailability. Moreover, as expected, the derivatives synthesized in this study possessed higher LAT-1 affinity, which facilitated increased quercetin transport into tumor cells.

Materials and Methods

Reaction Scheme for synthesis of compound 2

Step 1: A suspension of starting material (0.9g, 1.36mmol, 1eq), 2-bromoacetic acid (940mg, 6.79mmol, 5eq), K2CO3 (1.5g, 10.86mmol, 8eq), and NaI (40mg, 0.27, 0.2eq) in EtOH (50mL) was refluxed overnight. The solvent was removed under vacuum and the residue re-dissolved in a mixture of ethyl acetate/water. HCl (2N) was added to neutralize the mixture until pH reached 2-3. The desired product was extracted twice with EA. After drying and concentration, the residue was purified using silicagel column chromatography (Pure PE/EA = 2/1 to DCM/MeOH = 20/1) to obtain target compound as a yellow solid (0.9g, 92%).

Step 2: The starting material (4.2g, 17mmol, 1eq) was dissolved in H2O/CH3CN (50/50mL).Na2CO3 (1.81g, 17.05mmol, 1eq) was added and stirred to dissolve into the mixture. TrocCl was added (3.97, 18.76, 1.1eq) within 15 min and further stirred for 4 h at room temperature. LC-MS analysis revealed the formation of an intermediate, which was reacted with BnBr (3.21g, 18.76, 1.1eq). The mixture was warmed to 50oC and stirred for an additional 4 h. EA was added to the reaction mixture, the organic phase separated, and the aqueous layer extracted once with EA. The combined organic phase was dried and concentrated. The residue obtained was purified directly via silicagel column chromatography (PE/EA = 8/1 to 5/1) to obtain the target compound as a colorless oil (4.88g, 56%).

Step 3: The starting material (4.88g, 9.53mmol, 1eq) was dissolved in a solution of EtOH/AcOH (60/2mL). Zn powder (6.23g, 95.3mmol, 10eq) was added and the mixture stirred for 0.5 h at rt. The reaction suspension was filtered and washed with MeOH. The filtrate was concentrated under vacuum and re-dissolved in a mixture of DCM/water. After separation of the DCM phase, the aqueous phase was extracted once with DCM. The combined organic layer was dried and concentrated, and the residue further purified using a silicagel column [DCM/MeOH = 20/1 to 10/1 to 5/1(3% NH3))] to obtain target material as a yellow oil (1.4g, 46%).

Step 4: The starting acid material (502mg, 0.7mmol, 1eq), EDC (336mg, 2.09mmol, 3eq), and HOBt (292mg, 2.09mmol, 3eq) were dissolved in dry DCM (30ml) and stirred for 5 min, followed by the addition of DIPEA (280mg, 2.09mmol, 3eq) and amino compound (291mg, 0.84mmol, 1.2eq). The mixture was stirred for 16 h at rt and the reaction mixture concentrated. The residue was re-dissolved in EA and washed once with HCl, once with Na2CO3, and once with brine. After drying and removal of solvent, the residue was purified using column chromatography (PE/EA = 2/1 to 3/2) to obtain TM as a yellow oil (600mg, 73%).

Step 5: The starting material (600mg, 0.58mmol) was dissolved in DCM (10mL). After addition of TFA (3mL), the mixture was stirred for 30 min at rt. Next, the solvent and TFA were removed, and the residue re-dissolved in DCM (20mL). A Na2CO3 aqueous solution (20mL) was added and the mixture stirred for 15 min. The DCM layer was separated, dried, and concentrated. The residue was purified using silica-gel column chromatography (PE/acetone = 2/1 to DCM/ MeOH = 15/1, 0.5% NH3) and TM obtained as a yellow oil (488mg, 90%).

Step 6: SM (150mg) was dissolved in THF and Pd/C (10%, 200mg) added under a hydrogen atmosphere, and stirred for 6 h. Then the residue was dissolved into DMSO and filtered to obtain a DMSO solution of the desired compound. The solution was purified directly using reverse C18 column chromatography (CH3CN/H2O=90/1 to 80/20, 3% NH3HCO3) to obtain TM as a yellow solid (65mg, 83%).

The Mass spectrum and NMR spectrum of this compound was showed in supplementary Figure 1.