Synthesis of Indomorphan Pseudo‐Natural Product Inhibitors of Glucose Transporters GLUT‐1 and ‐3

Abstract Bioactive compound design based on natural product (NP) structure may be limited because of partial coverage of NP‐like chemical space and biological target space. These limitations can be overcome by combining NP‐centered strategies with fragment‐based compound design through combination of NP‐derived fragments to afford structurally unprecedented “pseudo‐natural products” (pseudo‐NPs). The design, synthesis, and biological evaluation of a collection of indomorphan pseudo‐NPs that combine biosynthetically unrelated indole‐ and morphan‐alkaloid fragments are described. Indomorphane derivative Glupin was identified as a potent inhibitor of glucose uptake by selectively targeting and upregulating glucose transporters GLUT‐1 and GLUT‐3. Glupin suppresses glycolysis, reduces the levels of glucose‐derived metabolites, and attenuates the growth of various cancer cell lines. Our findings underscore the importance of dual GLUT‐1 and GLUT‐3 inhibition to efficiently suppress tumor cell growth and the cellular rescue mechanism, which counteracts glucose scarcity.


SAR analysis of the indomorphan class
[a] The cells highlighted indicate where the modification was introduced compared to (±)-Glupin (entry 1).
[b] IC50 values determined for the inhibition of 2-DG uptake in HCT116 cells using the semiautomated hight-throughput assay. Data are mean values (more than three independent experiments).
Data are mean values ± s.d.

General Procedures:
General Procedure A: Fischer Indole Synthesis.
The corresponding ketone (1.0 equiv.) was dissolved in acetic acid (0.15M), the corresponding hydrazine hydrochloride (1.0 equiv.) was added and the solution was stirred and refluxed for 1.5h. The reaction was quenched with sodium bicarbonate (saturated aqueous solution), the mixture was extracted with CH2Cl2, dried over MgSO4, filtered and the solvents were concentrated in vacuo. The crude product was purified by flash chromatography (5 to 50% E.A./CH2Cl2) to afford the corresponding indole as the pure anti diasteroisomer as an orange solid.

General Procedure B: Indole N-alkylation (1).
The corresponding indole (1.0 equiv.), cesium carbonate (3.0 equiv.) and a 2-bromoacetate compound (3.0 equiv) were dissolved in DMF (0.17M) and allowed to stir at room temperature for 2h. The reaction was then partitioned between CH2Cl2 and BRINE/H2O (1:1). The aqueous phase was extracted with CH2Cl2 (5X), dried over MgSO4, filtered and the solvents were concentrated in vacuo. The crude product was purified by column chromatography (1 to 5% E.A./CH2Cl2) to afford the corresponding alkylated indole product as an orange oil.

General Procedure D: Cbz deprotection.
The corresponding Cbz N-protected compound (1.0 equiv.) was dissolved in ethanol (0.15M), Pd/C (50mg/mmol) was suspended in the reaction and the atmosphere was changed to hydrogen gas (1atm).
After 5h the mixture was filtered through Celite, solvents were concentrated in vacuo and the crude product was purified by column chromatography (5% MeOH/CH2Cl2) to afford the N-deprotected product typically as a white solid.
General Procedure E: Amide formation.
The corresponding amine (1.0 equiv.), triethylamine (1.2 equiv.) and the corresponding aroyl chloride (1.0 equiv.) were dissolved in CH2Cl2 (0.2M) and the reaction was allowed to stir overnight at room temperature. The mixture was washed with water and BRINE, solvents were concentrated in vacuo and the crude product was purified by chromatography by preparative HPLC (10% to 50% ACN/H2O with 0.1% TFA), unless otherwise stated, to afford the indomorphan class analogue.

General Procedure F: Cbz deprotection and amide formation one-pot
Pd on activated carbon (10% w/w), was added to a stirred solution of the carbamate (1 equiv.) and ammonium formate (5 equiv.) in EtOH (0.1 M) and the reaction mixture was heated to reflux. After 2 h, the reaction mixture was allowed to cool to rt, and filtered in vacuo through a celite pad, eluting with CH2Cl2 (4 × reaction volume) and the filtrate concentrated in vacuo. The crude was rediluted in CH2Cl2 (0.15 M) and Et3N (1.6 equiv.) and the corresponding acyl chloride (1.5 equiv.) were added and the reaction mixture was stirred at rt. After 24 h the reaction mixture was conc. in vacuo to give a crude. The crude product was purified by preparative HPLC (10% to 50% ACN/H2O with 0.1% TFA), unless otherwise stated.

General Procedure G: Cbz deprotection with HBr
To a solution of the corresponding Cbz-protected compound (1 equiv)l) in acetic acid (0.3 M), HBr in acetic acid 33% solution (0.98 equiv.) was added and the reaction was allowed to stir for 30 min. Then water was added and the aqueous phase was extracted with CH2Cl2 (5X). Evaporation of the solvents under reduced pressure afforded the corresponding HBr-amine salt as an orange sticky foamy solid.

General Procedure H: Carboxylic acid chlorination and amide formation in situ
To a solution of nicotinic acid (1.2 equiv.) (13.5 mg, 0.110 mmol) in toluene (0.067 M) thionyl chloride (1.2 equiv) freshly distilled and a drop of DMF were added. The solution was stirred at reflux for 1h, then solvents were evaporated and the crude was redissolved in CH2Cl2 (0.55M). The solution was canulated to another solution of compound 12a (1.0 equiv), triethylamine (1.2 equiv.) in CH2Cl2 (0.37 M). The mixture was allowed to stir at room temperature overnight. Then the organic phase was washed with water and BRINE, concentrated in vacuo to afford the crude product.

General Procedure J: Synthesis of 2-Substituted nicotinic acid and amide bond formation
2-chloronicotinic acid (1.0 equiv.) and sodium thiometoxide (2.5 equiv.) were suspended in dioxane (0.4 M) and water (1.0 M) in a sealed tube. The reaction was stir overnight at 120°. The reaction was then acidify with citric acid (20% aqueous solution) to pH 3-4, extracted with ethyl acetate and concentrated in vacuo to afford the crude product.
LiCl (2 × 10 mL), dried over MgSO4, filtered and conc. in vacuo to give a crude mixture. Purification by flash chromatography eluting with 20-50% EtOAc in Petrol afforded the product 13 as a yellow amorphous solid (350 mg, 80% yield) with spectroscopic data matching those reported in literature.
NaHCO3 solution and extracted with CH2Cl2 (4 × 10 mL). The combined organic layers were dried over MgSO4, filtered and conc. in vacuo. The crude was filtered through a silica pad eluting with 1:1 Petrol-EtOAc (50 mL) and conc. in vacuo to give a crude. The crude was rediluted in Toluene (1 M) and Et3N (3 equiv.) and TBDMS chloride as a 1.5 M solution in toluene (3 equiv.) was added and the resulting mixture was stirred at rt. After 24 h, the reaction mixture was conc. in vacuo and filtered through a silica pad eluting with 9:1 CH2Cl2-EtOAc (50 mL) and conc. in vacuo to give a crude which was directly submitted to General procedure C using ethyl bromoacetate. Filtration through a silica pad eluting with 7:3 Petrol-EtOAc (50 mL) and conc. in vacuo afforded a crude product which required no further purification as judged by spectroscopic data 1H NMR (CDCl3, 500MHz): δ 0.20 (s, 6H), 0.80 (s, 9H),

Cheminformatic Analysis
Supporting Figure S2: Chemoinformatic analysis of the indomorphan pseudo-NP collection. a) NP-score distribution. This class of pseudo-NPs displays a narrow NP-score distribution (black line) in a part of the graph that is poorly occupied by NPs (red line) yet densely covered by compounds in the DrugBank collection (blue line). b) PMI plot of the compounds in this collection. The majority of compounds reside away from the rod-like to disc-like vertex exhibiting higher three-dimensional character. Most synthetic molecules tend to congest along the rod-disk axis (for comparison see Sauer and Schwarz) [5] . In contrast, the distribution of these pseudo-NPs in the PMI plot moves away from this axis and is comparable to the distribution displayed by diverse NPs and bioactive compounds which incorporate the indole and morphan fragments. c) The ALogP vs MW plot, demonstrates that the majority of the prepared indomorphans fall within Lipinski Ro5 space.

Determination of the absolute configuration of the enantiomers of the indomorphan class.
In order to determine the stereochemistry of the three chiral carbons of the morphan ring of (+)-Glupin, acetylated (S)-mandelate acid was introduced on R1 position at compound 1 (see Supporting Scheme S7).

3H-2,6-methanoazocino[5,4-b]indole-3-carboxylate (25):
Compound 25 (0.022 mg, 19%) was synthesized from 24 following General Procedure B, and purified by preparative HPLC from a complicated mixture of products and characterized only by HRMS (ESI) before proceeding with the synthesis.  Compound 25' (0.022 mg, 19%) was synthesized from 24' following General Procedure B, and purified by preparative HPLC from a complicated mixture of products and characterized only by HRMS (ESI) before proceeding with the synthesis.

Supporting Figure S11. Influence of Glupin in GLUT-2 or GLUT-4 overexpression CHO cells. (a)
Uptake of 2DG in CHO cells that ectopically express GLUT2 as compared to cells transfected with an empty vector (mock). (b) Uptake of 2DG in CHO cells that ectopically express GLUT-3 as compared to cells transfected with an empty vector (mock). 2DG uptake was determined using the resazurin/diaphorase detection system. Data are mean values ± SD (N=3, n=3).
Supporting Figure S12: Influence of Glupin on the expression of GLUT-1 and GLUT-3. DLD-1 cells were treated with Glupin at 25 mM glucose for 48 h prior to isolation of total RNA and cDNA synthesis.
Quantitative PCR was performed to assess the expression levels of GLUT1 and GLUT3 or ATP1A1, TUBB and ACTB as reference genes. Data are mean values of n=3 ± SD.

Supporting Tables
Supporting Table S3: Influence of (±)-Glupin on metabolites in Molt16 cells. Data is displayed as fold changes of significantly altered metabolites (p = 0.05, n=2) in MOLT16 cells treated with 50 nM (±)-Glupin for 24 h compared to DMSO controls. control (which was set to 100 %) and cells without 2DG (set to 0 %) using Graph Pad Prism 5 or 6 software and IC50 values were determined using a four-parameter fit.

Metabolites
The observed difference in the obtained IC50 values for Glupin in the automated screening vs. manually performed assay is most likely attributed to the minituarization that is required for high-troughput screening and usually requires different plate format (384 vs. 96-well plates) and different cell number.
In this case, the automated assay was performed in 384-well plates (vs. 96-well plates for the manual assay) with 15,000 cells per well (vs. 40,000 cells per well in the 96-well plates).
For kinetic measurements, 15,000 cells were seeded into black CellBIND 384-well plates (Corning) and allowed to attach overnight. Then, the plates were washed 3X with KRB buffer containing 0.1%BSA and treated with different concentrations of 2DG (0-60mM) and Glupin (0-1µM) for 30s to 30min. The uptake was determined using the resazurin/diaphorase system as described above. 2DG uptake was stopped by aspirating the 2DG solution followed by quick wash with ice-cold KRB buffer (0.1 % BSA). Km and Vmax were determined by fitting the data according to Michaelis-Menten using non-linear fitting.

Metabolite profiling
For each data point approximately 4 * 10 6 MOLT-16 cells in log phase were cultured in 6 mL medium in a 6-well culture plate. Cells were either treated with 0.1% DMSO (controls) or 50 nM (±)-Glupin and 0.1% DMSO. After 24 h the cell densities were determined using counting chambers. Working on ice, cell suspensions were transferred to centrifuge tubes and centrifuged at 200 g for 10 min at 4°C. Cell pellets were washed twice with 2 mL cold PBS followed by centrifugation and removal of the supernatant.
Cell pellets were frozen and stored at -80 °C before extraction of the metabolites. Cell pellets were then thawed on ice and 400 µL extraction mix (methanol:water 9:1 + internal standards) were added and the samples were transferred into Eppendorf tubes. Two tungsten carbide beads were added to each tube and the samples were extracted with a Retsch MM301 vibration mill at 30 Hz for two minutes. After storage on ice for 45 min the tungsten beads were removed and the samples were centrifuged at 14000 rpm (18620 g) for 15 min at 4 °C. 150 µL of the supernatants were transferred to vials and concentrated under reduced pressure to complete dryness and then stored at -80 °C. Prior to GC-MS analysis the samples were derivatized. The cell pellets were thawed on ice and then dried under reduced pressure at room temperature for 20 min. 20 µL of a 15 µg/µL methoxamine solution in pyridine was added and the samples were dissolved by shaking them for 10 min and they were then allowed to stand for 20 h at room temperature. 20 µL MSTFA with 1% TMSCl was then added and after 1 h at room temperature, 20 µL heptane containing 15 ng/µL methyl stearate was added.
The samples were analyzed at the Swedish Metabolomics Center in Umeå, Sweden, using a Leco Pegasus HT time-of-flight mass spectrometer equipped with an Agilent 7890A gas chromatograph (GC) and a 30 m DB-5ms Ultra Inert GC-column with an inner diameter of 0.25 mm. Automated splitless injection of 1 μl sample was performed at an injection temperature of 270 °C. The purge time was 75 seconds with a rate of 20 mL/min. Helium was used as carrier gas (1 mL/min). The primary GC oven temperature was 70 °C for 2 minutes and then increased 20 °C/minute to 320 °C, where it was held constant for 8 minutes.
The transfer line temperature between the gas chromatograph and mass spectrometer was 250 °C. The ion source temperature was 200 °C and the electron impact ionization energy was 70 eV. Mass spectra were collected at 20 Hz in the mass range 50 to 800 m/z and the detector voltage vas 1670 V. A series of n-alkanes (C8-C40) were used as external retention index standards.
The raw GC-chromatograms were aligned using the internal standards and the GC-MS data was compared against an in-house spectral library using the in-house RDA software (Swedish Metabolomics Centre, Umeå, Sweden). The data was then curated using NIST MS search v2.0. The annotated integrated data was normalized against the cell density and the injection standard, methyl stearate, in each sample. For statistical evaluation, two-sided student's t-tests were performed in Microsoft Excel, assuming equal variance.

Cellular thermal shift assay (CETSA)
SW480 cell lysates were prepared according to the following procedure: nearly confluent cells were washed with PBS and detached using trypsin (0.05% Trypsin, 0.02% EDTA; PAN Biotech). The trypsin was inactivated with media and the cell suspension was centrifuged at 1,200 rpm. The cell pellet was resuspended in lysis buffer (PBS, 0.4% NP40 alternative, EDTA-free Protease Inhibitor Cocktail (Roche)) and stored on ice. The cell suspension was subjected to four freeze-and-thaw cycles and short sonication (10 s). After ultracentrifugation at 4 °C (20 min, 100,000 x g) the supernatant was collected, the protein concentration was measured by means of Bradford and stored snap frozen at -80 °C. For CETSA and Thermal Proteomic Profiling (TPP) cell lysates were diluted to 2 mg/mL and divided into two reaction tubes. One fraction was treated with 10 µM Glupin, the other with 1% (v/v) DMSO and incubated for 10 min at room temperature respectively. The treated lysates were split in 10 fractions each and subjected a temperature gradient (36.9-67°C) for 3 min. The lysates were centrifuged at 100,000 x g for 20 min and 4°C and the soluble fractions were analyzed using immunoblotting (CETSA) or 10plex TMT labeling for mass spectrometry based readout. [8] For TMT-labeling the samples were reduced with TCEP, alkylated with iodoacetamide, precipitated with acetone and tryptic digested in TEAB buffer overnight. Afterwards samples were spinned down and labeled with TMT label according to the description of the manufacturer, but using just half the amount of labeling reagent. 120 µl of each labeled aliquot incubated with Glupin were combined into one sample and 120 µl of each DMSO aliquot into a second one. Both samples were evaporated to dryness. Prefractionation of samples using high pH conditions, nanoHPLC-MS/MS analysis, and data evaluation using MaxQuant software [9] (v. 1.5.3.30) and an in-house programmed excel macro to calculate melting curves were performed as described by Martin-Gago et al. [10] The mean values of the melting temperatures of GLUT1-1 and GLUT-3 of two biological replicates were calculated and a Boltzmann fit was performed. [7] 1x10 6 CHO cells were seeded 10 cm cell culture plates. After overnight incubation, cells were transfected with the different using Lipofectamine 3000 according to the supplier's instructions. Briefly, DNA-lipid complexes (100 µg plasmid DNA, DNA:lipid ratio 1:2) were prepared in OptiMEM and then added to the cells. In the case of GLUT-4-transfected cells, Insulin (100 µg/mL) was added to the transfection medium. After incubation for 48 h cells were either reseeded (40,000 cell/well) in 96-well plates for monitoring glucose uptake or cells were directly lysed and protein expression was subsequently analysed by means of immunoblotting. In the case of GLUT-4 transfected cells, Insulin (100 µg/mL) was added to the culture medium medium.

Transient overexpression of GLUT-1-4 in CHO cells and 2DG assay
The 2DG assay was performed as described above (low throughput resazurin-based assay). In the case of GLUT-4 transfected cells, Insulin (100 µg/mL) was added to the buffer.

Real-time live-cell analysis
MDA-MB-231 cells were seeded at a density of 2,500 cells/well into clear 96-well plates and allowed to adhere overnight. Increasing concentrations of compounds or vehicle control were added to the cells in fresh medium containing 0 mM, 5 mM or 25 mM glucose, respectively and analysed in real-time using an IncuCyte ZOOM Live-Cell Imaging System (Essen Bioscience, UK) at 20x or 10x magnification for 48 h. Images were acquired in phase contrast and green fluorescence mode every hour. Images were analyzed using the IncuCyte Zoom 2016/A software by automated image segmentation. For confluence measurements, data were normalized to confluence at time point t=0.

Detection of lipid droplets
MDA-MB-231 cells were seeded in black 96-well plates with clear bottom (Corning) and incubated overnight. The growth medium was then replaced with serum-reduced medium (5% FBS) containing the desired concentration of the test substance or vehicle control and oleic acid (400 μM final concentration).
After treatment for 18 h cells were fixed with 3.7% paraformaldehyde in PBS, permeabilized with 0.1% Optical density was measured at 492, 520 and 560 nm using a Deelux-LED96 plate reader (Deelux Labortechnik GmbH, Germany).

Statistics
Data from independent experiments (n) are presented as mean values ± standard deviation (SD). N is the number of technical replicates and n is the number of biological replicates. Data fitting was performed using GraphPad Prism 6.0. Statistical analysis was performed using unpaired t test with Welch's correction (GraphPad Prism 6.0).