Pharmacological identification of a guanidine-containing β-alanine analogue with low micromolar potency and selectivity for the betaine/GABA transporter 1 (BGT1)
Anas Al-Khawaja, Jette G. Petersen, Maria Damgaard, Mette H. Jensen, Stine B. Vogensen, Maria E. K. Lie, Bolette Kragholm, Hans Bräuner-Osborne, Rasmus P. Clausen, Bente Frølund* and Petrine Wellendorph*
Supplementary Material
Compound synthesis
All reagents and solvents were purchased from Sigma-Aldrich and were used without purification. 3-hydroxy-N-tert-butoxycarbonyl-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin (compound 13) was a gift from H. Lundbeck A/S. Thin layer chromatography (TLC) was carried out on Merck silica gel 60 F254 aluminium sheets. Compounds were detected as single spots on TLC plates and visualized using UV light (254 nm) and KMnO4 spraying reagent. Dry Column Vacuum Chromatography (DCVC) was performed on a Merck Silica gel (0.015-0.040 mm). Melting points were recorded using a Büchner instrument and are uncorrected. 1H-NMR spectra were recorded on a 300 MHz Varian Mercury 300BB spectrometer, and 13C-NMR spectra were recorded on a 75 MHz Varian Gemini 2000BB spectrometer. Assigned structures were in agreement with the 1H (300 MHz) and 13C (75 MHz) NMR spectra. Elemental analyses were performed by Mr. J. Theiner(Microanalytical Laboratory, Department of Physical Chemistry, University of Vienna, Austria)and, unless otherwise stated, within 0.4% of the calculated values.
The guanidine analogues, compound2 and 3 were synthesized from monoprotected proforms, compound 11 and 15, respectively, in two steps (Scheme 1). Ethyl piperidin-4-carboxylate (compound 11)and 3-isopropoxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine (compound 15) were converted into their respective guanidine analogues using the guanidinylation reagent N-N’-bis(Boc)-S-methylisothiourea followed by acidic deprotection. For the synthesis of compound4, 1H-pyrazole-1-carboxamidine was used as the guanidinylation reagent.
Scheme 1 Reagents and conditions: (a) N,N’-bis(tert-butoxycarbonyl)-S-methylisothiourea, DMAP, rt, 2 days (b) AcOH/H2O, conc. HCl, 110 °C, 3h (c) isopropylbromide, K2CO3, DMF (d) 4N TFA, EtO2 (e) 1H-pyrazole-1-carboxamidine hydrochloride.
1-Carbamimidoylpiperidine-4-carboxylic acid hydrochloride (2). A solution of compound 12 (490 mg, 1.23 mmol) in AcOH (3 mL), H2O (3 mL) and conc. HCl (6 mL) was stirred at 110°C for 3 h. Evaporation under reduced pressure and recrystallisation (EtOH, Et2O) gave compound 2 (250 mg, 98%). Mp > 210°C. 1H NMR 3.72–3.80 (m, 2H), 3.04–3.18 (m, 2H), 2.76–2.73 (m, 1H), 1.94–2.02 (m, 2H), 1.60–1.74 (m, 2H).13C NMR 14.2, 28.0, 28.2, 40.7, 60.6, 79.45, 81.8, 150.6, 154.9, 162.7, 173.8. Anal. (C7H13N3O2 HCl).
3-hydroxy-4,5-dihydroisoxazolo[5,4-c]pyridine-6(7H)-carboximidamide hydrochloride (3). Prepared as described for compound 2 using compound15 (320 mg, 0.75 mmol). Evaporation under reduced pressure and recrystallisation (EtOH, Et2O) gave compound 3 (125 mg, 76%). Mp > 210°C. 1H NMR (D2O): 4.52 (s, 2H), 3.62–3.74 (m, 2H), 2.48–2.57 (m, 2H).13C NMR(D2O):168.3, 164.1, 159.9, 103.8, 43.8, 43.7, 18.1. Anal. (C7H10N4O2HCl1.5 H2O).
1-((3-hydroxyisoxazol-5-yl)methyl)guanidine hydrochloride (4). A mixture of 3-hydroxyisoxazol hydrochloride (muscimol, HCl) (140 mg, 0.93 mmol), 1H-pyrazole-1-carboxamidine hydrochloride (137 mg, 0.94 mmol) and aq. Na2CO3 (1.0 M, 2 mL) was stirred for 12 h at rt. Evaporation under reduced pressure and recrystallisation (H2O) gave compound 4 (52 mg, 36%). Mp > 275°C. 1H NMR (D2O): 5.64 (s, 1H), 4.31 (s, 2H).13C NMR(D2O):180.5, 170.0, 163.4, 100.1, 40.5. Anal. (C5H8N4O20.2 H2O).
Ethyl 1-(N’,N’’-di-tert-butoxycarbonyl)carbamimidoylpiperidine-4-carboxylate (12). To a solution of N,N’-bis(tert-butoxycarbonyl)-S-methylisothiourea (3.47 mmol, 1.01 g) in DMF (10 mL) was added compound 11 (1.07 mL, 6.95 mmol) and DMAP (42 mg, 0.34 mmol), and the reaction mixture was stirred at rt for 2 days. The solvent was evaporated under reduced pressure, and the residue was dissolved in EtOAc (30 mL). The solution was washed successively with 10% aq citric acid (3 x 25 mL), aq sat. NaHCO3 solution (3 x 25 mL) and H2O (25 mL). The organic phase was then dried (MgSO4) and evaporated under reduced pressure to dryness. DCVC (Tol:EtOAc, 2:1) gave compound 12 (656 mg, 48%) as white crystals. Mp 131.0–131.5 C. 1H NMR 4.13 (q, 2H, J = 7.2 Hz), 4.05 (br s, 1H), 3.04–3.13 (m, 2H), 2.47–2.58 (m, 1H), 1.91–2.01 (m, 2H), 1.75–1.89 (m, 2H), 1.50 (br s, 18 H), 1.25 (t, 3H, J = 6.9 Hz). 13C NMR 14.3, 28.0, 28.2, 40.8, 60.6, 79.4, 81.8, 150.6, 154.9, 162.7, 173.8. Anal. (C19H33N3O6).
3-Isopropoxy-N-tert-butoxycarbonyl-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin (14). A solution of 3-hydroxy-N-tert-butoxycarbonyl-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin (compound 13)(3.0 g, 12.5 mmol) and K2CO3 (1.9 g, 13.7 mmol) in dry DMF (20 mL) was stirred under a nitrogen atmosphere for 30 min at 60 C. Isopropyl bromide (1.8 mL, 19.2 mmol) was added, and stirring was continued overnight at 60 C. Water (30 mL) was added, and the mixture was extracted with EtOAc (3x50 mL). The combined organic phase was dried with MgSO4, filtered, and evaporated. Purification by DCVC (Toluene/EtOAc, 1:2) resulted in compound 14 (2.65 g, 75%) as yellow oil. 1H-NMR (300 MHz, CDCl3): δ 4.64 (septet, J = 6.1 Hz, 1H), 4.36 (br s, 2H), 3.62 (br s, 2H), 2.21 (br s, 2H), 1.51 (s, 9H), 1.34 (d, J = 6.1 Hz, 6H). 13C-NMR (75 MHz, CDCl3): δ 168.6, 164.8, 154.3, 102.5, 80.5, 42.1, 41.5, 28.3, 21.9, 18.7.
3-isopropoxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin, trifluoromethylacetate (15). A mixture of compound14 (2.34 g, 8.27 mmol) and 4N TFA in ether (4.7 mL/16 mL) was stirred at room temperature for 2 h.Bromide (1.8 mL, 19.2 mmol) was added, and stirring was continued overnight at 60 C. Evaporation under reduced pressure and recrystallisation (EtOH, EtO2) gave compound 15 (1.25g, 51%). Mp 147.5–148.0C.1H-NMR (300 MHz, CDCl3): δ 4.91 (septet, J = 6.1 Hz, 1H), 4.38 (br s, 2H), 3.50 (t,J = 6 Hz, 2H), 2.79 (m, 2H), 1.41 (d, J = 6,1 Hz, 6H). 13C-NMR (75 MHz, CDCl3): δ 170.4, 162.2, 104.0, 76.0, 43.5, 42.4, 23.0, 17.9.Anal. (C9H14N2O2CF3COOH).
3-isopropoxy-1-(N’,N’-di-tert-butoxycarbonyl)-4,5,6,7-tetrahydroisoxazolo[5,4-c]carbamimidoylpyridin (16).Prepared as described for compound 13 using compound 15 (150 mg, 0.35 mmol). Purification by DCVC (hepthane/EtOAc 10:1) resulted incompound 16(396 mg, 55%) as colorless crystals. Mp 145–146°C (decomp.). 1H NMR 10.23 (br s, 1H), 4.91 (sep, 1H, J = 6.3 Hz), 4.54 (br s, 2H), 2.59 (br s, 2H), 1.50 (m, 1H), 1.91–2.01 (m, 2H), 1.75–1.89 (m, 2H), 1.50 (br s, 18 H), 1.25 (t, 3H, J = 6.9 Hz). 13C NMR 168.6, 163.9, 162.0, 155.6, 144.0, 103.3, 82.4, 79.9, 73.2, 28.1, 22.0, 18.5. Anal. (C20H32N4O6).
Confirmation of integration of the hGATs into the Flp-InTM-CHO cells by RT-PCR
Genomic DNA was prepared by solubilizing the cell lines stably expressing hGAT1, hGAT2or hGAT3 in a buffer containing 50 mM KCl, 10 mM Tris-HCl, 2 mM MgCl2, 0.1 mg/mL gelatin, 0.45% Nonidet P40, 0.45% TWEEN 20, pH 8.3. Proteinase K was added, and the mixture was incubated at 56 °C for 2 hrs. Next, the mixture was placed at 95 °C for 10 min and subsequently stored at 4 °C. Genomic DNA was amplified by PCR using forward and reverse primers, respectively, for the three transporters: hGAT1: 5’-gacatggcgaccaacggcag-3’ and 5’-ctagatgtaggcctccttgc-3’, hGAT2: 5’-gggatggatagcagggtctc-3’ and 5’-ctagcagtgagactctagctc-3’, hGAT3: 5’-tggagttttgggagcaccgggtcctggccatctctgacgg-3’ and 5’-tcagaagtgcgtctccttctctg-3’. All primers were designed along separate exons to avoid amplification of genomic DNA.
PCR was performed using Advantage polymerase as described by the manufacturer (Clontech, Mountain View, CA, USA). The reactions were heated to 94 °C for 1 min and then cycled 35 times at 94 °C for 30 s and 68 °C for 2 min. The final elongation step was performed at 68 °C for 10 min. The reactions were run on a 1% agarose gel containing SYBR SafeTM(Life Technologies) to visualize the PCR products.
The bands corresponded to the predicted size of 1803, 1812, and 1289 nucleotides, for hGAT1, hGAT2 and hGAT3, respectively. No visible PCR products were amplified from genomic DNA from untransfected Flp-InTM-CHO cells when using any of the primer pairs (data not shown).
Compound 9 shows a competitive inhibition mode at mBGT1
HEK-293 cells stably expressingmBGT1 were exposed to increasing concentrations of GABA and a fixed concentration of compound 9. The obtained kinetic curves are presented in Fig. S1. Values of Km and Vmaxwere estimated and are presented in Table S1.
Increasing the concentration of compound 9 resulted in a statistically significant decrease in pKm values, while Vmax was not significantly altered. A decrease in pKmalongside with a constant Vmaxindicated competitive inhibition.
Fig. S1 Effect of compound 9 on the GABA transport velocity through mBGT1. Specific transport velocity of increasing concentrations of GABA was measured in the [3H]GABA uptake assay at mBGT1 stably expressed in HEK-293 cells. The maximum concentration of [3H]GABA was 100 nM. Data are given as nmol GABA transported per minute and normalized to protein amount per well. Results are means ± SD of triplicate measurements of a single representative experiment. Two additional independent experiments gave similar results. The estimated Kmand Vmaxfor both experiments are presented in Table S1.
Table S1 Effect of compound 9 on the uptake rate of GABA at mBGT1. The experiment was performed by generating kinetic curves (Fig. S1) in the absence and presence of varying concentrations of compound 9 using the [3H]GABA uptake assay at mBGT1 stably expressed in HEK-293 cells. Three independent experiments were performed.
[Compound 9] / Km(pKm± S.E.M.) (µM) / Vmax ± S.E.M.(nmol GABA/(min×mg protein)
0 µM / 223 (3.7 ± 0.07)* / 8.7 ± 2.11
30 µM / 380 (3.4 ± 0.04)# / 8.5 ± 2.32
150 µM / 529 (3.3 ± 0.02)# / 6.4 ± 1.46
The pKm values in the presence of 30 µM and 150 µM compound 9 were significantly decreased compared to control (0 µM compound 9), but no significant difference in pKm was present between 30 µM and 150 µM compound 9. Groups with different superscript symbols are statistically different (P<0.05, One-way ANOVA followed by Tukey’s multiple comparison test).
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