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叠氮和脱TMS

叠氮和脱TMS

Supporting Information

Wiley-VCH2012

69451Weinheim,Germany

Application of Fragment Screening and Merging to the Discovery of Inhibitors of the Mycobacterium tuberculosis Cytochrome P450

CYP121**

Sean A.Hudson,Kirsty J.McLean,Sachin Surade,Yong-Qing Yang,David Leys,Alessio Ciulli, Andrew W.Munro,and Chris Abell*

anie_201202544_sm_miscellaneous_information.pdf

Table of Contents

Supplementary Figures and Tables (2)

Figure S1. (2)

Figure S2. (2)

Figure S3. (3)

Figure S4. (4)

Figure S5. (5)

Table S1. (6)

Experimental Methods (7)

Materials (7)

Molecular biology (7)

Expression and purification of CYP121 (7)

Thermal shift screening (8)

NMR screening (8)

X-ray crystallography (9)

Fragment hit attrition (9)

Isothermal titration calorimetry (ITC) (10)

Electronic spectroscopy (10)

Enzyme kinetics (11)

Hepatic CYP inhibition (11)

In silico calculations (12)

Statistics (12)

Organic synthesis (12)

References (26)

Supplementary Figures and Tables

叠氮和脱TMS

Figure S1.Water-suppressed 1D 1H and WaterLOGSY NMR screening spectra for a representative fragment hit (1.5 mM) in the presence and absence of CYP121 (15 μM) or CYP121 (15 μM) plus cYY (0.5 mM). Only the fragment resonances in the aromatic region are shown and arrows indicate signals from cYY. The strong positive fragment signals in the presence of CYP121 indicate protein binding, and this interaction is displaced/reduced by the addition of cYY.

叠氮和脱TMS

Figure S2.Fragments soaked into CYP121 crystals but not observed bound in the crystal structure.

叠氮和脱TMS

Figure S3.Synthetic route to merged fragments and analogs.

叠氮和脱TMS

Figure S4. a) Small molecule crystal structure of 1,5-diphenoltriazole 7 resolved to 0.77 ? atomic resolution. b) Crystal packing of 7from (a), with 4 monomers per unit cell. c) Computed conformational energy landscape of 7 for all rotations of its triazole-phenol dihedral angles (force field OPLS-2005 based methods, solvent: water). The approximate dihedrals of 7from the small molecule (a) and CYP121-bound (see Figure S5a) crystal structures are marked. Using quantum mechanics (QM method: DFT(b3lyp), QM basis: 6-31g**, gas phase), the protein-bound conformation is predicted to be 8.4 kcal.mol-1 higher in conformational strain, owing to the steric clash of its phenol ortho-hydrogens.

叠氮和脱TMS

Figure S5. The CYP121 active site complex with the weak binding merged fragment 7 and its higher affinity analog 10 (green sticks) from 2.25 and 1.50 ? ligand-CYP121 structures, respectively. CYP121 is shown in cartoon and the heme b is in purple with the heme iron as a brown sphere. The I-helix running atop the heme is highlighted in yellow. Side chains and water/sulfate molecules within 5 ? of the bound ligands are shown as thin sticks and red spheres, respectively. Direct ligand-CYP121 H-bonds clear from the structures are represented as dashed yellow lines. The Fo-Fc omit electron density map associated with each ligand is shown as a green mesh contoured at 3σ.

6

Table S1. Macromolecule X-ray crystallography data collection and final refinement statistics 1-CYP121 PDB ID 4G44 2-CYP121 PDB ID 4G45 3-CYP121 PDB ID 4G46 4-CYP121 PDB ID 4G47 7-CYP121 PDB ID 4G2G 10-CYP121 PDB ID 4G48 14-CYP121 PDB ID 4G1X Data collection a Space group P6522 P6522 P6522 P6522 P6522 P6522 P6522 Cell dimensions a, b, c (?) 77.64, 77.64, 264.10 77.80, 77.80, 264.30 77.57, 77.57, 262.99 77.45, 77.45, 263.60 77.37, 77.37, 263.65 77.73, 77.73, 263.38 77.41, 77.41, 263.95 α, β, γ (°) 90.0, 90.0, 120.0 90.0, 90.0, 120.0 90.0, 90.0, 120.0 90.0, 90.0, 120.0 90.0, 90.0, 120.0 90.0, 90.0, 120.0 90.0, 90.0, 120.0 Resolution (?) 47.11 - 1.24 (1.32 - 1.24) 36.87 - 1.52 (1.62 - 1.52) 46.99 - 1.52 (1.62 - 1.52) 33.27 - 1.33 (1.42 - 1.33) 67.00 - 2.25 (2.35 - 2.25) 47.07 - 1.50 (1.68 - 1.50) 38.70 - 1.30 (1.37 - 1.30) No. reflections (total) 1250135 (124644) 1448816 (175858) 1454417 (177892) 1019249 (99374) 312765 (4913) 767804 (216845) 1001945 (87419) No. reflections (unique) 128278 (18082) 72835 (11394) 70252 (10184) 102181 (13425) 21393 (2264) 136080 (38707) 115362 (16202) R merge 4.7 (21.6) 2.4 (10.5) 2.1 (8.4) 6.8 (45.3) 1.1 (9.7)b 7.5 (12.3) 5.8 (42.6) I / σI 25.5 (7.1) 46.6 (12.8) 53.7 (17.0) 22.9 (3.5) 33.9 (5.9) 24.5 (13.6) 20.5 (3.6) Completeness (%) 96.6 (85.4) 99.6 (98.5) 96.7 (88.5) 95.5 (79.2) 91.8 (81.8) 99.7 (99.8) 99.7 (98.0) Multiplicity 9.7 (6.9) 19.9 (15.4) 20.7 (17.5) 10.0 (7.4) 14.6 (2.2) 5.6 (5.6) 8.7 (5.4) Refinement Resolution (?) 1.24 1.53 1.52 1.34 2.25 1.50 1.30 No. reflections 121874 69154 66684 97114 20131 76377 109432 R work / R free 16.1 / 17.9 15.3 / 17.8 14.9 / 17.2 15.5 / 17.7 13.7 / 19.9 17.1 / 18.9 18.3 / 19.9 R.m.s. deviations Bond lengths (?) 0.033 0.030 0.030 0.030 0.018 0.006 0.030 Bond angles (°) 3.139 3.127 3.123 3.136 2.404 1.049 3.169 a Data for the highest resolution shell is shown in parentheses b R pim

Experimental Methods

Materials

All experiments with CYP121 were performed using the N-terminal His6-tagged form unless otherwise specified. All reagents and chemicals were of analytical grade and were obtained from Sigma-Aldrich Company (Dorset, UK) unless otherwise described. The empty pHAT2 vector was a gift from Dr. Marko Hyv?nen (Department of Biochemistry, University of Cambridge). The E. coli C41(DE3) expression strain was kindly provided by Dr J. E. Walker (MRC Laboratory of Molecular Biology, Cambridge, UK). The fragment library was a first-generation Rule of Three[1]compliant set purchased from Maybridge (Cornwall, UK). No known target-specific bias was placed on the fragments selected for incorporation. The vast majority of the commercial fragments contain benzene and/or heterocyclic aromatic subunits. The fragments have a calculated aqueous solubility of >1 mM according to the vendor. cYY was bought from PolyPeptide Group (Strasbourg, France). SYPRO Orange protein gel stain was purchased as a 5000x concentrate in DMSO from Invitrogen (Paisley, UK). The merged fragments and analogs were either synthesized directly (Figure S3) or ordered commercially from Ambinter (Paris, France) or ChemBridge Corporation (San Diego, USA).

Molecular biology

The Rv2276 gene encoding Mtb CYP121 was PCR amplified from the pET11a/CYP121 plasmid previously described.[2]Primers used in the reaction were as follows. Forward/upstream: 5’-TATGACCATGGCAACCGCGACCGTTCTGCTCG-3’. The underlined bases indicate the engineered restriction site for Nco I. Reverse/downstream: 5’-AAGACGGATCCTACCAGAGCACCGGAAGG-3’. Underlined bases show the engineered site for Bam HI and the TAG amber stop codon is incorporated. The CYP121 PCR product was cloned into empty pHAT2 vector to generate a pHAT2/CYP121 construct that encodes non-cleavable N-terminal His6-tagged CYP121. All molecular biology was performed using standard protocols.[3]

Expression and purification of CYP121

Recombinant untagged Mtb CYP121 was expressed and purified for crystallography from the pET11a/CYP121expression vector as previously described.[2]His6-tagged CYP121 was expressed and purified from the pHAT2/CYP121vector using the protocol previously

described for His6-tagged Mtb CYP125[4], but produced in E. coli C41(DE3) grown in 10 L batches of Terrific Broth medium. The Ni-affinity and anion exchange chromatography steps were carried out on an ?KTA FPLC (GE Healthcare, Little Chalfont, UK) with HisTrap FF 5 mL and Mono Q 10/100 GL 8 mL columns, respectively. The gel filtration step previously described was not necessary to achieve sufficient purity for screening, and thus the final buffer used for storage was50 mM Tris-HCl at pH 7.2 with 1 mM EDTA. The mass of purified His6-tagged CYP121 was confirmed on an Applied Biosystems QSTAR nanoESI Q-TOF mass spectrometer (Applied Biosystems, CA, USA): expected 44324 Da without the initial methionine; observed 44316.

Thermal shift screening

Samples (100 μL) comprising 10 μM CYP121 and 2.5x SYPRO Orange in 100 mM potassium phosphate buffer at pH 6.8 with 5% (v/v) DMSO, were prepared in 96-well plates (LightCycler 480 Multiwell Plate 96, Roche, Burgess Hill, UK) in the presence and absence of 5 mM fragment or 1 mM cYY. The plates were covered with the provided sealing foils, vortexed briefly and centrifuged using a Sigma 3-18K SciQuip centrifuge with Sigma 11240 rotor (DJB Labcare, Buckinghamshire, UK) at 4,500 rpm for 5 min. The steady-state fluorescence emission intensity (λex483 nm, λem568 nm) from each sample was then recorded as the temperature was increased linearly from 37 to 75 o C at a rate of 0.01 o C.s-1 using a LightCycler480 Real-Time PCR System (Roche). The melting curve for CYP121 in each well was plotted (fluorescence intensity vs. temperature) and the melting point/denaturing temperature (point of sigmoidal inflection) was rapidly identified by finding the maximum of each curve’s derivative[5].

NMR screening

Samples (200 μL) comprising 1.5 mM fragment with and without 15 μM CYP121 were prepared in 50 mM Tris-HCl buffer at pH 7.5 with 50 mM potassium chloride, 10% (v/v) D2O, 20 μM deuterated 3-trimethylsilylpropanoate(TSP-d4) and 1.5% (v/v) DMSO-d6. For displacement experiments, 0.5 mM cYY was added with an additional 1% (v/v) DMSO-d6. The samples were then pipetted into 3-mm NMR capillaries (Hilgenberg GmbH, Malsfeld, Germany) and loaded into 528-PP-8 NMR tubes (Wilmad-LabGlass, NJ, USA). STD[6] and WaterLOGSY[7] 1D 1H NMR spectra were acquired at 278 K on a Bruker DRX 700 MHz NMR spectrometer (Bruker, MA, USA) equipped with a 5 mm triple resonance inverse (TXI) cryoprobe with z-gradients and an auto sampler. STD experiments employed a 40 ms

s elective Gaussian 180° pulse at a frequency alternating between ‘on-resonance’ (0.9 ppm) and ‘off-resonance’ (40 ppm) after every scan. WaterLOGSY experiments employed a 20 ms selective Gaussian 180° shaped pulse at the water signal frequency and a NOE mix ing time of 1 s. Water signal suppression was achieved using a W5 Watergate gradient spin-echo pulse sequence.[8] The spectra were processed using TopSpin 3.0 software (Bruker UK, Coventry, UK) and WaterLOGSY spectra were scaled relative to the same 20 μM TSP peak intensity at 0 ppm.

X-ray crystallography

Crystals of recombinant untagged CYP121 were grown as previously reported by the sitting-drop vapour-diffusion method at 4 oC with a well solution consisting of 0.1 M sodium cacodylate-HCl buffer at pH 5-6.25 and 1.5-2.3 M ammonium sulfate as precipitant.[9] The drops were prepared by adding 0.5 or 1 μL mother liquor to 1 μL 12 mg.mL-1 enzyme in 10 mM Tris-HCl buffer at pH 7.5 following a final gel filtration purification step on an ?KTA FPLC (GE Healthcare, Little Chalfont, UK) with a Superdex 75 10/300 GL or Sephacryl S-200 (1.6 x 70 cm) column. Individual crystals were then soaked in a saturated solution of fragment or merged fragment (1-25 mM) in mother liquor with 10% (v/v) DMSO. The crystals were then immersed in mother liquor supplemented with 30% (v/v) ethylene glycol as a cryoprotectant and flash-cooled in liquid nitrogen. Complete diffraction data sets were collected from single crystals at 100 K either in-house using a Bruker X8 Proteum diffractometer (Bruker), at the I04-1 X-ray beamline, Diamond Light Source, Didcot, UK, or at ID14 stations, European Synchrotron Radiation Facility, Grenoble, France. The diffraction data were reduced and scaled using either PROTEUM2 (Bruker), XDS[10]or MOSFLM[11] and SCALA[12]. Structures were refined using REFMAC5[13] or PHENIX[14] with the native CYP121 structure (PDB 1N40)[15] as the starting model. Data collection and final refinement statistics are given in Table S1. Images for presentation were rendered with PyMOL academic 1.3 (Schr?dinger, Camberley, UK). Methodology for determining the small molecule crystal structure of the 1,5-diphenoltriazole 7is given in the organic synthesis section below.

Fragment hit attrition

The hit attrition rates as the fragments were progressed through the biophysical screening cascade (thermal shift, NMR then X-ray) were typical of our experiences. The screening techniques all report on different aspects of binding (thermal-shift for fold stabilisation, NMR

for magnetisation transfer etc.). The loss of hits at each screening step could be the result of any number of factors, e.g. non-specific binding, aggregation effects, variations in solubility between the screening buffers and crystallisation liquors, inability to access the crystalline active site. These factors will all vary for each individual fragment.

Isothermal titration calorimetry (ITC)

ITC binding isotherms were recorded on a MicroCal iTC200microcalorimeter (GE Healthcare) by injecting 2 μL aliquots of 10-20 mM fragment in protein buffer with 10% (v/v) DMSO (or merged fragments at their maximum solubility) into 50-100 μM CYP121 in 50 mM Tris-HCl buffer at pH 7.5 with 1 mM EDTA and also 10% (v/v) DMSO. For cYY and lead aminoquinoline 14, the binding isotherm was too steep at their maximum solubility and the ligand concentration was lowered to 2.5 and 1 mM, respectively. Binding isotherms were integrated to give the change in enthalpy for each injection and a standard one-set-of-sites binding model was fitted using the instrument software to estimate the K D with stoichiometry N = 1. For all ligand to protein titrations, the background heat of ligand dilution was subtracted by recording an identical titration series for ligand into buffer with 10% (v/v) DMSO only. Excessively large heats of dilution were observed for the quinoline fragment 2, cyclohexanone fragment 3and 1,2,3-triazolylquinoline merged fragment 17. Their K D values were instead determined by competition ITC experiment, injecting 2.5 mM cYY into 50 μM CYP121 solution pre-incubated with 0.5-5 mM ligand. The apparent K D of cYY in the presence of the competing ligand was then determined as described above, and the K D of the competitor was back-calculated using the equations described by Zhang and Zhang.[16]

Electronic spectroscopy

Heme absorbance shift assays to determine the K D of the merged fragment aminoquinolines 14-19 were performed as previously described for azole antifungals binding to CYP121.[2, 17] Absorption spectra were recorded at room temperature on a Cary 400 UV-Vis spectrophotometer (Varian, CA, USA). The individual samples (200 μL) comprised 5 μM CYP121 in 50 mM Tris-HCl buffer at pH 7.5 with 1 mM EDTA, 5% (v/v) DMSO and varying concentrations of aminoquinoline (1-1000 μM) as required to achieve the maximal heme absorbance change consistent with ligand saturation. Curve fitting was done with GraphPad Prism 5.01 (GraphPad Software, San Diego, USA). Owing to the strong inherent UV absorptivity of the aminoquinolines obscuring the ferric heme iron Soret band below 400

nm, if concentrations greater than 300 μM were required the heme absorbance change was quantified using only the absorption maxima in the series of the difference spectra.

Enzyme kinetics

To first determine the kinetics of cYY transformation by CYP121 in the absence of inhibitor, steady-state measurements comprising 50 nM untagged CYP121, 250 nM spinach ferredoxin, 50 nM spinach ferredoxin-NADP+ reductase and varying concentrations of cYY (0-1000 μM) in 50 mM potassium phosphate buffer at pH 7.2 with 100 mM potassium chloride at 37 o C were made. Assays were performed in a Cary 300 UV-Vis dual beam spectrophotometer (Agilent Technologies, Winnersh, UK) to take account of any non enzyme-mediated spectral changes. The reference cuvette contained the same mix as the sample cuvette, but with the requisite volume of buffer replacing CYP121. The enzymatic mixtures were pre-incubated for 3 minutes and the reaction was then initiated by adding 200 μM NADPH (Melford, UK) to both cuvettes. The initial rate of cYY transformation was recorded in triplicate by monitoring NADPH oxidation at 340 nm (ε340nm = 6210 M-1cm-1) over 4 minutes. NADPH oxidation rates (expressed as moles of NADPH oxidized per minute per mole of P450) were plotted versus the relevant cYY concentration. Data were fitted using the Michaelis-Menten equation to establish the k cat and K m (79 ± 18 μM) parameters. The same procedure was then repeated for identical samples with a single concentration of 200 μM cYY but with varying concentrations of lead aminoquinoline 14(0-2000 μM). A one-phase exponential decay equation was fitted to a plot of the initial NADPH oxidation rates vs. 14concentrations to determine an IC50. The K i for 14 was then calculated using the equations described by Cheng and Prusoff.[18]

Hepatic CYP inhibition

Human hepatic CYP inhibition was performed by Cyprotex Discovery Ltd. (Macclesfield, UK). Test compound (0.1-100 μM) was incubated with human liver microsomes (0.1-1 mg.mL-1) and NADPH (1 mM) with 0.25-0.3% (v/v) DMSO in the presence of a hepatic CYP isoform-specific probe substrate [mephenytoin (25 μM) for CYP2C19; dextromethorphan (5 μM) for CYP2D6; midazolam (2.5 μM) or testosterone (50 μM) (two probe substrates) for CYP3A4] for 5-60 min at 37 °C. The reactions were then terminated by adding methanol, the samples centrifuged and 0.1% (v/v) formic acid in deionised water containing internal standard was added to the supernatant. The supernatant was then analysed by LC-MS/MS to quantify the formation of each isoform-specific

metabolite 4-hydroxymephenytoin, dextrorphan, 1-hydroxymidazolam and 6?-hydroxytestosterone. Cyprotex generic LC-MS/MS conditions were used. The IC50 for each hepatic CYP was determined from the inhibition of formation of specific metabolite in the presence of test compound (compared to vehicle control). Known selective CYP inhibitors [tranylcypromine for CYP2C19; quinidine for CYP2D6; ketoconazole for CYP3A4] were also assayed alongside the lead aminoquinoline 14 as positive controls.

In silico calculations

The relative conformational energy of 1,5-diphenoltriazole7 for full 360o rotations of its triazole-phenol dihedral angles was computed rapidly in 5o increments using a MacroModel coordinate scan (force field: OPLS-2005, solvent: water, default settings) in Schr?dinger Maestro 9.0 (Schr?dinger, Camberley, UK) and with the triazole of 7 constrained planar (dihedral C6-N1-C5-C13 at 0o). The energy landscape was plotted as a 3D map using Graphis 2.9.33 (Kylebank Software, Ayr, UK). The difference in gas phase energy between 7 in its conformation from the small molecule and CYP121-bound crystal structures was calculated quantum mechanically using a Jaguar relaxed coordinate scan in Schr?dinger Maestro 9.0 (QM method: DFT(b3lyp), QM basis: 6-31g**, default settings) and with the triazole of 7 constrained planar (dihedral C6-N1-C5-C13 at 0o).

Statistics

All repeated measurements are given as the mean ± s.e.m. and the sample size, n, is indicated in the text.

Organic synthesis

General synthesis method A

A mixture of bromoquinoline and azole in 1,4-dioxane (2-10 mL) was refluxed at 120 o C for 1-2 d, cooled to room temperature and then partitioned between CHCl3 (30 mL) and saturated sodium bicarbonate(30 mL). The organic layer was separated, washed with brine (2 x 30 mL), dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was chromatographed using a Biotage Isolera One flash chromatography system with a Biotage SNAP cartridge (10 g silica, Biotage) and linear DCM/MeOH gradient (0-20% MeOH) as eluant, to afford the azolequinoline product.

General synthesis method B

Palladium (as 10% on charcoal, 10 mol %) was added to a solution of nitroquinoline in EtOH (1 mL) and HOAc (10 μL), and the mixture was stirred at room temperature for 2 h under an atmosphere of hydrogen. The solution was filtered and the solvent removed in vacuo to yield the amine product, which was either of sufficient purity or chromatographed as indicated using a Biotage Isolera One flash chromatography system with a Biotage SNAP cartridge (10 g silica, Biotage) and linear DCM/MeOH gradient (0-20% MeOH) as eluant.

4-bromoquinoline[19]

N

B r

PBr3 (210 mg, 0.760 mmol) was added to a suspension of 4-hydroxyquinoline (100 mg, 0.690 mmol) in DMF (3 mL) and the mixture was stirred at room temperature under nitrogen for 30 min. The reaction mixture was then poured onto ice and following complete hydrolysis of phosphorous tribromide,was neutralized by addition of solid sodium acetate and extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with water (2 x 10 mL), dried over anhydrous MgSO4, and the solvent was removed in vacuo to yield the bromoquinoline(110 mg, 0.520 mmol, 76%) as a light yellow oil. TLC (Hexane:EtOAc, 1:1 v/v): R f = 0.55; 1H NMR (400 MHz, CDCl3): δ 8.68 (d, J = 4.7 Hz, 1H), 8.20 (dd, J = 8.4, 1.4 Hz, 1H), 8.13 (dd, J = 8.4, 1.4 Hz, 1H), 7.78 (ddd, J = 8.4, 7.0, 1.4 Hz, 1H), 7.72 (d, J = 4.7 Hz, 1H), 7.66 (ddd, J = 8.4, 7.0, 1.4 Hz, 1H); MS (m/z): [M + H]+ 207.9 (79Br, 100%), 209.9 (81Br, 100%).

4-(1H-1,2,4-triazol-1-yl)quinoline[20] (12)

N N N N

Synthesis by method A using 4-bromoquinoline (38.6 mg, 0.185 mmol) and 1,2,4-triazole (138 mg, 2.00 mmol). The triazolylquinoline (22.9 mg, 0.117 mmol, 63 %) was purified as a white powder. TLC (DCM:MeOH, 9:1 v/v): R f = 0.45; 1H NMR (400 MHz, DMSO-d6): δ9.25 (s, 1H), 9.08 (d, J = 4.6 Hz, 1H), 8.46 (s, 1H), 8.19 (dd, J = 8.4, 1.4 Hz, 1H), 8.11 (dd, J

= 8.4, 1.4 Hz, 1H), 7.90 (ddd, J = 8.4, 7.0, 1.4 Hz, 1H), 7.81 (d, J = 4.6 Hz, 1H), 7.73 (ddd, J = 8.4, 7.0, 1.4 Hz, 1H); MS (m/z): [M + H]+ 197.2 (100%).

4-Hydroxy-6-nitroquinoline[21]

OH

N

NO2

A solution of 4-hydroxyquinoline (1.00 g, 6.90 mmol) in H2SO4 (5.2 mL) was cooled on ice, and a mixture of H2SO4 (0.5 mL) and HNO3 (0.5 mL) was added dropwise, maintaining the temperature between 0–5 o C. After the addition was complete, the mixture was allowed to warm to room temperature and stirred for an additional 2 h. The solution was then poured onto ice and the resulting precipitate was filtered and dried in vacuo to yield the nitroquinolone (830 mg, 4.40 mmol, 63%) as a bright yellow solid. TLC (DCM:MeOH, 9:1 v/v): R f = 0.3; 1H NMR (400 MHz, DMSO-d6): δ 8.85 (d, J = 2.7 Hz, 1H), 8.43 (dd, J = 9.2, 2.7 Hz, 1H), 8.05 (d, J = 7.5 Hz, 1H), 7.74 (d, J = 9.2 Hz, 1H), 6.20 (d, J = 7.5 Hz, 1H); MS (m/z): [M + H]+ 190.9 (100%).

4-Bromo-6-nitroquinoline

B r

N

NO2

A mixture of 4-hydroxy-6-nitroquinoline (510 mg, 2.70 mmol) and POBr3(6.10 g, 21.0 mmol) in toluene (13 mL) was refluxed at 125 o C for 5 h under nitrogen, cooled to room temperature and then poured onto ice. After complete hydrolysis of phosphoryl bromide, the mixture was neutralized with addition of solid sodium acetate and then extracted with CHCl3 (3 x 125 mL). The organic layer was washed with water (3 x 100 mL), dried over anhydrous MgSO4, and the solvent was removed in vacuo to yield the bromoquinoline (530 mg, 2.10 mmol, 78%) as a light yellow powder. TLC (Hexane:EtOAc, 1:1 v/v): R f= 0.3; 1H NMR (400 MHz, DMSO-d6): δ 8.98 (d, J = 2.5 Hz, 1H), 8.96 (d, J = 4.7 Hz, 1H), 8.57 (dd, J = 9.2, 2.5 Hz, 1H), 8.33 (d, J = 9.2 Hz, 1H), 8.19 (d, J = 4.7 Hz, 1H); 13C NMR (125 MHz, CDCl3): δ 152.9 (CH), 150.6 (C), 146.5 (C), 136.1 (C), 132.0 (CH), 127.4 (C), 126.8 (CH), 124.0 (CHx2); IR: 3099, 3055, 3015, 1618, 1582, 1563, 1525, 1484, 1337 cm-1; MS (m/z): [M +

H]+253.1 (79Br, 100%), 255.1 (81Br, 100%); HRMS (m/z): [M + H]+calcd. for C9H679BrN2O2, 252.9613; found, 252.9630.

6-Nitro-4-(1H-1,2,4-triazol-1-yl)quinoline (13)

N N N

N

NO2

Synthesis by method A using 4-bromo-6-nitroquinoline (54.4 mg, 0.215 mmol) and 1,2,4-triazole (138 mg, 2.00 mmol). The triazolylquinoline(33.5 mg, 0.139 mmol, 65%) was purified as a light yellow powder. TLC (DCM:MeOH, 9:1 v/v): R f= 0.35; 1H NMR (500 MHz, CDCl3): δ 9.28 (d, J = 2.5 Hz, 1H), 9.20 (d, J = 4.6 Hz, 1H), 8.65 (s, 1H), 8.58 (dd, J = 9.2, 2.5 Hz, 1H), 8.38 (d, J = 9.2 Hz, 1H), 8.36 (s, 1H), 7.61 (d, J = 4.6 Hz, 1H); 13C NMR (125 MHz, CDCl3): δ 154.2 (CH), 153.5 (CH), 151.5 (C), 146.5 (C), 144.3 (CH), 142.3 (C), 132.0 (CH), 124.2 (CH), 121.2 (C), 121.1 (CH), 116.3 (CH); IR: 3105, 1603, 1512, 1501, 1342, 1323 cm-1; MS (m/z): [M + H]+242.3 (100%); HRMS (m/z): [M + H]+calcd. for C11H8N5O2, 242.0678; found, 242.0670.

4-(1H-Imidazol-1-yl)-6-nitroquinoline

N N

N

NO2

Synthesis by method A using 4-bromo-6-nitroquinoline (51.4 mg, 0.203 mmol) and imidazole (154 mg, 2.26 mmol). The imidazolylquinoline (39.1 mg, 0.162 mmol, 80%) was purified as an orange/brown powder. TLC (DCM:MeOH, 9:1 v/v): R f = 0.35; 1H NMR (500 MHz, CDCl3): δ 9.18 (d, J = 4.6 Hz, 1H), 8.81 (d, J = 2.5 Hz, 1H), 8.57 (dd, J = 9.2, 2.5 Hz, 1H), 8.38 (d, J = 9.2 Hz, 1 H), 7.91 (s, 1H), 7.52 (d, J = 4.6 Hz, 1H), 7.42 (s, 1H), 7.38 (s, 1H); 13C NMR (125 MHz, CDCl3): δ 153.9 (CH), 151.4 (C), 146.6 (C), 143.4 (C), 137.5 (CH), 132.3 (CH), 131.4 (CH), 124.1 (CH), 122.6 (C), 120.7 (CH), 119.6 (CH), 118.2 (CH); IR: 3123, 3081, 3032, 1600, 1498, 1484, 1343, 1323 cm-1; MS (m/z): [M + H]+ 241.5 (100%); HRMS (m/z): [M + H]+ calcd. for C12H9N4O2, 241.0726; found, 241.0715.

6-nitro-4-(1H-pyrazol-1-yl)quinoline

N

N N

NO2

Synthesis by method A using 4-bromo-6-nitroquinoline(250 mg, 0.990 mmol) and pyrazole (340 mg, 5.00 mmol).The pyrazolylquinoline (91.5 mg, 0.380 mmol, 38%) was purified as a light orange powder. TLC (EtOAc): R f = 0.4; 1H NMR (400 MHz, CDCl3): δ 9.52 (d, J = 2.5 Hz, 1H), 9.12 (d, J = 4.8 Hz, 1H), 8.52 (dd, J = 9.3, 2.5 Hz, 1H), 8.30 (d, J = 9.3 Hz, 1H), 8.01 (d, J = 2.5 Hz, 1H), 7.99 (d, J = 1.9 Hz, 1H), 7.56 (d, J = 4.8 Hz, 1H), 6.68 (dd, J = 2.5, 1.9 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 153.6 (CH), 151.8 (C), 146.1 (C), 145.5 (C), 143.6 (CH), 131.7 (CH), 130.8 (CH), 123.5 (CH), 122.5 (CH), 121.4 (C), 115.5 (CH), 109.1 (CH); MS (m/z): [M + H]+241.0 (100%); HRMS (m/z): [M + H]+calcd. for C12H9N4O2, 241.0721; found, 241.0733.

6-nitro-4-(1H-1,2,3-triazol-1-yl)quinoline

N N

N

N

NO2

Synthesis by method A using 4-bromo-6-nitroquinoline (39.2 mg, 0.150 mmol) and 1,2,3-triazole (51.8 mg, 0.750 mmol). The 1,2,3-triazolylquinoline(14.0 mg, 0.058 mmol, 39%) was purified as a yellow powder. TLC (DCM:MeOH, 9:1 v/v): R f= 0.65; 1H NMR (500 MHz, DMSO-d6): δ 9.33 (d, J = 4.7 Hz, 1H), 9.02 (d, J = 2.6 Hz, 1H), 8.97 (d, J = 1.2 Hz, 1H), 8.60 (dd, J = 9.3, 2.6 Hz, 1H), 8.42 (d, J = 9.3 Hz, 1H), 8.20 (d, J = 1.2 Hz, 1H), 8.04 (d, J = 4.7 Hz, 1H); 13C NMR (125 MHz, DMSO-d6): δ 154.9 (CH), 150.9 (C), 146.2 (C), 141.8 (C), 134.7 (CH), 131.8 (CH), 127.6 (CH), 124.1 (CH), 121.2 (CH), 120.6 (C), 117.8 (CH); MS (m/z): [M + H]+ 242.0 (100%); HRMS (m/z): [M + H]+ calcd. for C11H8N5O2, 242.0673; found, 242.0669.

4-(1H-1,2,4-Triazol-1-yl)quinolin-6-amine (14)

N N N

N

NH2

Synthesis by method B using 6-nitro-4-(1H-1,2,4-triazol-1-yl)quinoline (20.7 mg, 0.086 mmol). The crude product was chromatographed to yield the amine (14.6 mg, 0.069 mmol, 81%) as a light yellow powder. TLC (DCM:MeOH, 9:1 v/v): R f = 0.3; 1H NMR (400 MHz, MeOH-d4): δ 9.03 (s, 1H), 8.63 (d, J = 4.8 Hz, 1H), 8.36 (s, 1H), 7.91 (d, J = 9.1 Hz, 1H), 7.57 (d, J = 4.8 Hz, 1H), 7.40 (dd, J = 9.1, 2.4 Hz, 1H), 7.01 (d, J = 2.4 Hz, 1H); 13C NMR (125 MHz, MeOH-d4): δ 153.5 (CH), 150.4 (C), 146.6 (CH), 145.1 (CH), 144.4 (C), 140.4 (C), 130.2 (CH), 126.3 (C), 124.5 (CH), 118.3 (CH), 101.3 (CH); IR: 3381 and 3304 (br), 3175 (br), 3102, 1620, 1509 cm-1; MS (m/z): [M + H]+ 212.3 (100%); HRMS (m/z): [M + H]+ calcd. for C11H10N5, 212.0936; found, 212.0946.

4-(1H-Imidazol-1-yl)quinolin-6-amine (16)

N N

N

NH2

Synthesis by method B using 4-(1H-imidazol-1-yl)-6-nitroquinoline (18.0 mg, 0.075 mmol) to yield the amine (13.8 mg, 0.066 mmol, 88%) as a dark brown powder. TLC (DCM:MeOH, 9:1 v/v): R f = 0.25; 1H NMR (400 MHz, MeOH-d4): δ 8.53 (d, J = 4.4 Hz, 1H), 8.05 (m, 1H), 7.83 (d, J =9.2 Hz, 1H), 7.51 (m, 1H), 7.35 (d, J =4.4 Hz, 1H), 7.29 (dd, J =9.2, 2.4 Hz, 1H), 7.26 (m, 1H), 6.73 (d, J = 2.4 Hz, 1H); 13C NMR (100 MHz, MeOH-d4): δ 148.8 (C), 144.4 (CH), 143.3 (C), 139.4 (C), 137.7 (CH), 129.2 (CH), 128.3 (CH), 125.8 (C), 122.8 (CH), 121.2 (CH), 117.5 (CH), 99.3 (CH); IR: 3334 and 3206 (br), 1621, 1511, 1493 cm-1; MS (m/z): [M + H]+ 211.2 (100%); HRMS (m/z): [M + H]+ calcd. for C12H11N4, 211.0984; found, 211.0992.

4-(1H-pyrazol-1-yl)quinolin-6-amine (15)

N

N N

NH2

Synthesis by method B using 6-nitro-4-(1H-pyrazol-1-yl)quinoline (50.4 mg, 0.210 mmol). The crude product was chromatographed to yield the amine (42.7 mg, 0.203 mmol, 97%) as an orange/brown powder. TLC (EtOAc): R f = 0.3; 1H NMR (400 MHz, MeOH-d4): δ8.44 (d, J = 4.8 Hz, 1H), 7.99 (d, J = 2.4 Hz, 1H), 7.78 (d, J = 1.6 Hz, 1H), 7.75 (d, J = 9.1 Hz, 1H), 7.28 (d, J = 4.8 Hz, 1H), 7.20 (dd, J = 9.1, 2.5 Hz, 1H), 7.00 (d, J = 2.5 Hz, 1H), 6.53 (dd, J =

2.4, 1.6 Hz, 1H); 13C NMR (100 MHz, MeOH-d4): δ148.6 (C), 144.9 (CH), 144.1 (C), 142.6

(C), 141.7 (CH), 132.2 (CH), 129.5 (CH), 125.4 (C), 123.0 (CH), 116.8 (CH), 107.6 (CH), 101.4 (CH); MS (m/z): [M + H]+ 211.4 (100%); HRMS (m/z): [M + H]+ calcd. for C12H11N4, 211.0984; found, 211.0990.

4-(1H-1,2,3-triazol-1-yl)quinolin-6-amine (17)

N N

N

N

NH2

Synthesis by method B using 6-nitro-4-(1H-1,2,3-triazol-1-yl)quinoline (14.0 mg, 0.058 mmol) to yield the amine(10.7 mg, 0.051 mmol, 88%) as a green powder. TLC (DCM:MeOH, 9:1 v/v): R f = 0.45; 1H NMR (400 MHz, MeOH-d4): δ 8.60 (d, J = 4.7 Hz, 1H), 8.46 (d, J = 1.1 Hz, 1H), 8.02 (d, J = 1.1 Hz, 1H), 7.88 (d, J = 9.1 Hz, 1H), 7.49 (d, J = 4.7 Hz, 1H), 7.33 (dd, J = 9.1, 2.4 Hz, 1H), 6.75 (d, J = 2.4 Hz, 1H); 13C NMR (100 MHz, MeOH-d4): δ 149.4 (C), 144.5 (CH), 144.0 (C), 139.2 (C), 133.8 (CH), 129.7 (CH), 127.0 (CH), 125.4 (C), 123.4 (CH), 117.6 (CH), 99.8 (CH); IR: 3394, 3297 (br), 3199 (br), 3084, 2923, 1619, 1589, 1565, 1511, 1480, 1442, 1360 cm-1; MS (m/z): [M + H]+ 212.4 (100%); HRMS (m/z): [M + H]+ calcd. for C11H10N5, 212.0931; found, 212.0929.

6-nitro-4-(pyridin-3-yl)quinoline