All air or moisture sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Anhydrous solvents such as tetrahydrofuran (THF), toluene, dichloromethane, N,N
-dimethylformamide (DMF), acetonitrile, methanol and triethylamine were purchased from Sigma-Aldrich. Purifications of the compounds were performed on Waters UPLC or Biotage SP systems. Samples were analyzed for purity on an Agilent 1200 series LC/MS equipped with a Zorbax™ Eclipse XDB-C18 reverse phase (5 micron, 4.6×150 mm) column having a flow rate of 0.8 ml/min. The mobile phase was a mixture of acetonitrile and H2
O each containing 0.05% trifluoroacetic acid. Gradient of 4% to 100% acetonitrile (0.05% TFA) over 7 minutes with flow rate of 0.8 ml/min using Luna C18 3 micron 3x75 mm column. All of the analogues for assay have purity greater than 95%. High resolution mass spectrometry was recorded on Agilent 6210 Time-of-Flight LC/MS system. Note: all of the final analogues are the mixture of diastereomers which are inseparable on preparative HPLC. Purified oligonucleotides for the fluorescence-based RNase H were purchased from TriLink Biotechnologies (San Diego, CA). His-p66/His-p51 HIV-1 RT, derived from the HIV-1HXB2
, was expressed and purified as previously reported. This enzyme is identical in sequence with respect to the residues discussed in modeling studies.
5,7-Dihydroxy-2-(1-hydroxy-1-methyl-2-piperidin-1-yl-ethyl)-9-methyl-1,2,3,4-tetrahydro benzocyclohepten-6-one (1). To a solution of manicol epoxide (16) (7 mg, 0.027 mmol) in acetonitrile (0.5 ml) was added piperidine (18.2 mg, 0.213 mmol, 8.0 eq.) and lithium perchlorate (5.7 mg, 0.053 mmol, 2 eq.). The mixture was refluxed for 1 h. After cooling to room temperature, another 1.5 ml acetonitrile was added and the mixture was directly subject to preparative HPLC purification to give the desired product 1 as a brownish solid (4 mg, 54%). 1H NMR (400 MHz, DMSO-d6) δ 8.83-8.58 (br.s., 1H), 7.37 (s, 1H), 5.75 (s, 1H), 5.55-5.23 (br.s., 1H), 3.62-3.40 (m, 2H), 3.29-3.17 (m, 2H), 3.13-2.96 (m, 3H), 2.92-2.67 (m, 2H), 2.59-2.50 (m, 3H), 2.41 (s, 3H), 2.05-1.85 (m, 1H), 1.85-1.68 (m, 4H), 1.67-1.57 (m, 1H), 1.52-1.37 (m, 1H), 1.29 and 1.25 (s, 3H); LC/MS: Retention time: 3.449 min; HRMS: m/z (M+H+) = 348.2176 (Calculated for C20H30NO4S = 348.2175).
5,7-Dihydroxy-2-(1-hydroxy-2-imadazol-1-yl-1-methyl-ethyl)-9-methyl-1,2,3,4-tetrahydro benzocyclohepten-6-one (2) was prepared in the same manner as 1 except by using imidazole as a nucleophile. 1H NMR (400 MHz, DMSO-d6) δ 9.03 and 9.01 (s, 1H), 7.72-7.64 (m, 2H), 7.38 and 7.36 (s, 1H), 5.25-5.03 (br.s., 1H), 4.36-4.21 (m, 2H), 3.80-3.30 (br.s., 2H), 3.20-3.00 (m, 1H), 3.00-2.60 (m, 2H), 2.44 and 2.40 (s, 3H), 2.62-2.47 (m, 1H), 2.25-1.92 (m, 1H), 1.75-1.48 (m, 1H), 1.45-1.25 (m, 1H), 1.01 and 1.00 (s, 3H) ;LC/MS: Retention time: 3.337 min; HRMS: m/z (M+H+) = 331.1651 (Calculated for C18H23N2O4 = 331.1658).
2-(2-Diethylamino-1-hydroxy-1-methyl-ethyl)-5,7-dihydroxy-9-methyl-1,2,3,4-tetrahydrobenzocyclohepten-6-one (3) was prepared in the same manner as 1 except by using diethylamine as a nucleophile. 1H NMR (400 MHz, DMSO-d6) δ 8.56-8.33 (br.s., 2H), 7.38 and 7.37 (s, 1H), 5.50-5.28 (br.s., 1H), 3.95-3.35 (m, 4H), 3.34-3.10 (m, 4H), 3.10-2.90 (m, 1H), 2.87-2.55 (m, 1H), 2.54 and 2.52 (s, 3H), 2.25-1.85 (m, 1H), 1.75-1.58 (m, 1H), 1.35-1.15 (m, 10H); LC/MS: Retention time: 3.427 min; HRMS: m/z (M+H+) = 336.2171 (Calculated for C19H30NO4 = 336.2175).
2-[2-(2-Fluoro-benzylamino)-1-hydroxy-1-methyl-ethyl]-5,7-dihydroxy-9-methyl-1,2,3,4-tetrahydrobenzocyclohepten-6-one (4) was prepared in the same manner as 1 except by using 2-fluoroaniline as a nucleophile. 1H NMR (400 MHz, DMSO-d6) δ 8.90-8.60 (br.s., 2H), 7.70-7.61 (m, 1H), 7.54-7.45 (m, 1H), 7.36 and 7.35 (s, 1H), 7.34-7.35 (m, 2H), 5.48-5.15 (br.s., 1H), 4.25 (s, 2H), 3.20-2.90 (m, 3H), 2.90-2.61 (m, 2H), 2.38 and 2.34 (s, 3H), 2.50-2.35 (m, 2H), 2.15-1.92 (m, 1H), 1.80-1.65 (m, 1H), 1.33-1.13 (m, 1H), 1.20 and 1.18 (s, 3H); LC/MS: Retention time: 3.844 min; HRMS: m/z (M+H+) = 388.1914 (Calculated for C22H27FNO4 = 388.1924).
5,7-Dihydroxy-2-(1-hydroxy-1-methyl-2-phenylamino-ethyl)-9-methyl-1,2,3,4-tetrahydrobenzocyclohepten-6-one (5) was prepared in the same manner as 1 except by using aniline as a nucleophile. 1H NMR (400 MHz, CDCl3) δ 7.50 and 7.49 (s, 1H), 7.34-7.25 (m, 2H), 7.05-6.94 (m, 3H), 3.43-3.17 (m, 2H), 3.04-2.82 (m, 2H), 2.64-2.33 (m, 2H), 2.48 and 2.46 (s, 3H), 2.23-2.03 (m, 1H), 2.02-1.84 (m, 2H), 1.53-1.39 (m, 1H), 1.38 and 1.31 (s, 3H). LC/MS: Retention time min: 4.715 min; HRMS: m/z (M+H+) = 356.1864 (Calculated for C21H26NO4 = 356.1862).
2-(2-Ethylsulfanyl-1-hydroxy-1-methyl-ethyl)-5,7-dihydroxy-9-methyl-1,2,3,4-tetrahydrobenzocyclohepten-6-one (6) was prepared in the same manner as 5 except by using ethanethiol as a nucleophile. 1H NMR (400 MHz, CDCl3) δ 7.49 and 7.48 (s, 1H), 3.28-3.24 and 3.24-3.19 (m, 1H), 3.03-2.78 (m, 2H), 2.76-2.64 (m, 2H), 2.60-2.50 (m, 1H), 2.48 and 2.47 (s, 3H), 2.16-2.02 (m, 2H), 1.83-1.71 (m, 1H), 1.70-1.59 (m, 1H), 1.56-1.25 (m, 4H), 1.40 and 1.38 (s, 3H); LC/MS: Retention time: 5.529 min; HRMS: m/z (M+H+) = 325.1466 (Calculated for C17H25O4S = 325.1474).
5,7-Dihydroxy-2-(1-hydroxy-1-methyl-2-phenylsulfanyl-ethyl)-9-methyl-1,2,3,4-tetrahydrobenzocyclohepten-6-one (7). To a solution of 16 (15 mg, 0.057 mmol) in THF (1 ml) was added thiophenol (0.20 ml, 1.94 mmol, 34 eq.) and Et3N (0.08 ml, 0.57 mmol, 10 eq.) and the solution was refluxed overnight. After cooling to room temperature, the mixture was directly purified by HPLC to give the desired product 7 as a light yellow solid (10 mg, 47%). 1H NMR (400 MHz, CDCl3) δ 7.50 and 7.49 (s, 1H), 7.46-7.39 (m, 2H), 7.33-7.18 (m, 3H), 3.37-3.10 (m, 3H), 3.05-2.91 (m, 1H), 2.91-2.76 (m, 1H), 2.67-2.50 (m, 2H), 2.48 and 2.42 (s, 3H), 1.98-1.85 (m, 1H), 1.49-1.33 (m, 1H), 1.32 and 1.26 (s, 3H); LC/MS: Retention time: 6.101 min; HRMS: m/z (M+H+) = 373.1472 (Calculated for C21H25O4S = 373.1474).
2-(2-Benzylsulfanyl-1-hydroxy-1-methyl-ethyl)-5,7-dihydroxy-9-methyl-1,2,3,4-tetrahydrobenzocyclohepten-6-one (8). To a solution of BnSH (26 mg, 0.21 mmol) in MeOH (1 ml) was added NaH (4.6 mg, 0.19 mmol) and the mixture was stirred for 10 min. 16 (5 mg, 0.019 mmol) was added and the mixture was stirred overnight at room temperature and directly purified by preparative HPLC to give the desired product 8 (3 mg, 58%). 1H NMR (400 MHz, DMSO-d6) δ 7.35 and 7.34 (s, 1H), 7.33-7.28 (m, 4H), 7.25-7.20 (m, 1H), 3.79-3.76 (m, 2H), 3.06-2.94 (m, 1H), 2.82-2.60 (m, 5H), 2.48-2.36 (m, 1H), 2.34 and 2.33 (s, 3H), 1.30-1.16 (m, 2H), 1.15 and 1.13 (s, 3H); Retention time: 6.157 min; HRMS: m/z (M+H+) = 387.1620 (Calculated for C22H27O4S = 387.1630).
2-(2-Ethanesulfonyl-1-hydroxy-1-methyl-ethyl)-5,7-dihydroxy-9-methyl-1,2,3,4-tetrahydrobenzocyclohepten-6-one (9) was prepared through oxidation of corresponding sulfide 6 with m-CPBA in CH2Cl2 at room temperature. 1H NMR (400 MHz, DMSO-d6) δ 7.36 and 7.35 (s, 1H), 3.42-3.26 (m, 2H), 3.11-2.98 (m, 1H), 2.89-2.67 (m, 2H), 2.60-2.45 (m, 4H), 2.40 and 2.39 (s, 3H), 2.02-1.79 (m, 1H), 1.77-1.65 (m, 1H), 1.45-1.12 (m, 3H), 1.07 and 1.05 (s, 3H); LC/MS: Retention time: 4.323; HRMS: m/z (M+H+) = 357.1370 (Calculated for C17H25O6S = 357.1372).
2-(2-Benzylsulfanyl-1-hydroxy-1-methyl-ethyl)-5,7-dihydroxy-9-methyl-1,2,3,4-tetrahydrobenzocyclohepten-6-one (10) was prepared by oxidation of corresponding sulfide with m-CPBA at −78°C, while 2-(2-benzenesulfinyl-1-hydroxy-1-methyl-ethyl)-5,7-dihydroxy-9-methyl-1,2,3,4-tetrahydrobenzocyclohepten-6-one (11) was prepared by oxidation using m-CPBA at room temperature. 10: 1H NMR (400 MHz, DMSO-d6) δ 7.72-7.66 (m, 2H), 7.63-7.52 (m, 3H), 7.37 and 7.36 (s, 1H), 3.23-2.80 (m, 4H), 2.62-2.50 (m, 2H), 2.42 and 2.41 (s, 3H), 2.15-1.90 (m, 3H), 1.40-1.15 (m, 4H); LC/MS: Retention time: 4.767 min; HRMS: m/z (M+H+) = 389.1422 (Calculated for C21H25O5S = 389.1423). 11: 1H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 9.98 (s, 1H), 7.96-7.87 (m, 2H), 7.74-7.66 (m, 1H), 7.75-7.58 (m, 1H), 7.45-7.38 (m, 2H), 4.10-3.50 (m, 3H), 2.64-2.54 (m, 2H), 2.53 and 2.52 (s, 3H), 2.34-2.18 (m, 2H), 2.17-1.94 (m, 1H), 1.94-1.76 (m, 1H), 1.43-1.28 (m, 1H), 1.24 and 1.23 (s, 3H). LC/MS Retention time: 5.123 min; HRMS: m/z (M+H+) = 405.1354 (Calculated for C21H25O6S = 405.1360).
2-(1,2-Dihydroxy-1-methyl-ethyl)-5,7-dihydroxy-9-methyl-1,2,3,4-tetrahydrobenzocyclohepten-6-one (12). To a solution of diacetate 15 (5 mg, 0.014 mmol) in acetone/H2O (0.19 ml/0.02 ml) was added 2.5wt% tert-BuOH OsO4 solution (7.7 µl, 0.757 µmol) and N-methylmorpholine oxide (3.4 mg, 0.029 mmol) and the mixture was stirred for 3 h. EtOAc (10 ml) was added and the solution was washed with 10% aqueous Na2SO3 solution and brine. The organic layer was dried over MgSO4. After the removal of EtOAc, the crude product was redissolved in MeOH (2 ml) and the refluxed for 5 h. The solution was concentrated to 2 ml and directly subject to preparative HPLC purification to give the desired diol 12 (3 mg, 71%). 1H NMR (400 MHz, DMSO-d6) δ 7.36 and 7.35 (s, 1H), 3.42-3.27 (m, 2H), 3.11-2.97 (m, 1H), 2.89-2.65 (m, 2H), 2.58-2.44 (m, 1H), 2.39 and 2.38 (s, 3H), 2.00-1.81 (m, 1H), 1.76-1.64 (m, 1H), 1.36-1.20 (m, 2H), 1.06 and 1.05 (s, 3H); LC/MS: Retention time: 3.795 min; HRMS: m/z (M+H+) = 281.1386 (Calculated for C15H21O5 = 281.1389).
5,7-Dihydroxy-2-(1-hydroxy-ethyl)-9-methyl-1,2,3,4-tetrahydrobenzocyclohepten-6-one (13). To a solution of 15 (22 mg, 0.07 mmol) in tert-BuOH/H2O (1.8 ml/0.36 ml) was added 2.5wt% tert-BuOH OsO4 solution (0.42 ml, 0.042 mmol), NaIO4 (123 mg, 0.58 mmol) and NaHCO3 (81 mg, 0.96 mmol) and the mixture was stirred 2 h at room temperature. 10% Na2SO3 (1.5 ml) was added and the mixture was stirred for 0.5 h and extracted with CH2Cl2 (3x10 ml) and the combined organic layers were washed with brine and dried over Na2SO4. After the removal of organic solvent, the residue was purified by column chromatography (EtOAc/Hexane 1/1) to give the desired ketone 17 (20 mg, 90%). 17 was then dissolved in MeOH (2 ml) and NaBH4 (6 mg, 0.16 mmol) was added and stirred at room temperature. for 0.5 h. After the complete reduction of the ketone monitored by LC/MS, the mixture was further refluxed for 5 h and directly purified by preparative HPLC to give 13. 1H NMR (400 MHz, CDCl3) δ 7.51 and 7.50 (s, 1H), 3.84-3.77 and 3.76-3.63 (m, 1H), 3.29-3.18 (m, 1H), 3.05-2.94 (m, 2H), 2.84 and 2.79 (d, J= 3.9 Hz, 1H), 2.70-2.51 (m, 1H), 2.51 and 2.48 (s, 3H), 2.16-2.06 and 2.03-1.93 (m, 1H), 1.78-1.66 (m, 1H), 1.49-1.34 (m, 1H), 1.31 (d, J= 6.3 Hz, 3H); LC/MS: Retention time: 4.315 min; HRMS: m/z (M+H+) = 251.1280 (Calculated for C14H19O4 = 251.1283).
2-(1-Benzylamino-ethyl)-5,7-Dihydroxy-9-methyl-1,2,3,4-tetrahydrobenzocyclohepten-6-one (14). To a solution of 17 (5 mg, 0.015 mmol) in 1,2-dichloroethane (0.1 ml) was added benzylamine (1.6 µl, 0.016 mmol) and sodium triacetoxyborohydride (4.5 mg, 0.021 mmol) and the mixture was stirred overnight at room temperature. LC/MS indicated the formation of de-acetylated product 14. The mixture was directly purified by preparative HPLC to give desired product 14 (3 mg, 59%). 1H NMR (400 MHz, DMSO-d6) δ 9.03-8.85 (br.s., 1H), 8.73-8.56 (br.s., 1H), 7.63-7.51 (m, 2H), 7.51-7.40 (m, 3H), 7.37 (s, 1H), 4.37-4.14 (m, 2H), 3.45-3.27 (m, 1H), 3.20-3.00 (m, 1H), 2.97-2.63 (m, 2H), 2.58-2.44 (m, 2H), 2.39 and 2.37 (s, 3H), 2.25-2.05 (m, 1H), 2.03-1.93 and 1.92-1.83 (m, 1H), 1.42-1.28 (m, 4H). LC/MS: Retention time: 3.913 min; HRMS: m/z (M+H+) = 340.1908 (Calculated for C21H26NO3 = 340.1913).
HIV-1 RT Expression and Purification for Biochemical Analysis.
-tagged p66/p51 HIV-1HXB2
RT and the RNase H-deficient mutants were expressed from recombinant Escherichia coli
and purified by a combination of immobilized metal affinity and ion exchange chromatography as previously described37
. Purified enzymes were stored at −20°C in a buffer of 50 mM Tris/HCl, pH 7.0, 25 mM NaCl, 1 mM EDTA, 1 mM dithiothereitol and 50% (v/v) glycerol.
RNase H Inhibitor Analysis.
values were determined as previously reported6
, using an 18-nucleotide 3’-fluourescein-labeled RNA annealed to a complementary 18-nucleotide 5’-dabsyl-labeled DNA. Cleavage of the HIV-1 polypurine tract (PPT) primer was performed with a 29 nt Cy5-labeled RNA (5’-Cy5-UUU UAA AAG AAA AGG GGG G*AC UGG AAG GG
-3’, where *represents the PPT 3’ terminus) hybridized to a 40 nt DNA (5’-ATT AGC CCT TCC AGT CCC CCC TTT TCT TTT AAA AAG TGG C-3’). The reaction was initiated by adding 1 µL of 100 mM MgCl2
to 9 µL of mixture containing 4 ng enzyme, 200 nM substrate, 20 µM α-hydroxytropolones in 50 mM Tris, pH 8.0, 80 mM KCl, 2 mM DTT, and 10% DMSO at 37°C and quenched with 10 µL of a gel-loading buffer after 10 min. Hydrolysis products were fractionated by denaturing polyacrylamide gel electrophoresis and visualized by fluorescent imaging (Typhoon Trio+, GE Healthcare).
DNA Polymerase Assay. DNA-dependent DNA synthesis was measured on a fluorescently-labeled duplex DNA prepared by annealing a 33-nt template, 5’-CAC TGC TCA AGA AGT TCC AAT CCT AAA TAC ATA -3’, to the 5’-Cy5 -labeled primer 5’-ATG TAT GGG TAT GTA TTT AGG-3’. Polymerization was initiated by adding 1 µL of 2 mM dNTPs to 9 µL of mixture containing 4 ng enzyme, 200 nM substrate, 20 µM α-hydroxytropolones in 10 mM Tris, pH 7.8, 80 mM KCl, 1 mM DTT, 10 mM MgCl2, and 10% DMSO at 37°C. DNA synthesis was quenched with 10 µL of a gel-loading buffer after 10 min and reaction products were analyzed by denaturing polyacrylamide gel electrophoresis and fluorescent imaging.
HIV-1 Cytopathicity assay.
This assay was conducted as previously reported38
. Samples were dissolved in DMSO at 10 mM and diluted to a final high concentration of 50 µM in the 96-well assay plate, with 2-fold dilutions made to a low concentration of 0.78 µM. All samples were tested in duplicate. The HIV-1 virus strain RF was used to infect CEM-SS cells. Compound cytotoxicity was measured in the same assay plate using uninfected cells. Regression analysis was used to estimate the effective concentration (EC50
) as well as the cytotoxic concentration (CC50
Expression and Purification of HIV-1 RT for structural studies.
An HIV-1 RT variant designated RT52A was used for X-ray diffraction studies. In this variant, which was optimized for crystallization of RT with nucleoside or non-nucleoside RT inhibitors (NRTIs and NNRTIs, respectively), the p66 subunit was truncated at residue 555. The p51 subunit contained a HRV14 3C protease cleavable N-terminal hexahistidine tag and was truncated at residue 428. Both subunits contained the mutation C280S. The p66 subunit also contained the mutations K172A and K173A39
. RT52A was expressed and purified as previously described39
. Briefly, 1 mM IPTG was used to induce BL21-CodonPlus®-RIL (Stratagene) containing a plasmid encoding both subunits of RT69A at an OD600 of 0.9 and the culture was incubated for three hours at 37°C. The cells were pelleted and lysed by sonication. Protein was purified by Ni-NTA according to manufacturers’ recommendations (Qiagen) with the following modifications: each buffer contained 600 mM NaCl, no lysozyme was added, and a 1.2 M NaCl wash was added. Eluted protein was incubated with HRV14 3C protease overnight at 4°C. A Mono Q purification step was performed as described40
. The protein was concentrated to 20 mg/ml in "RT storage buffer" (10 mM Tris pH 8.0, 75 mM NaCl) and stored at −80°C.
Crystallization and Data Collection.
HIV-1 RT52A was co-crystallized with manicol and 18
at 4°C by vapor diffusion in microseeded hanging drops containing 1.2 µl each of 20 mg/ml protein (in a solution of 9.2 mM Tris pH 8.0, 68.7 mM NaCl, 3.6 mM manganese sulfate, 0.7 mM tris(2-carboxyethyl) phosphine (TCEP), 0.27% (w/v) β-ocytl glucopyranoside, 7% (v/v) DMSO, 0.9 mM manicol, and 0.7 mM 18
, pre-incubated for 30 minutes on ice) and a reservoir solution containing 50 mM HEPES pH 7.5, 100 mM ammonium sulfate, 15 mM manganese sulfate, 10 mM spermine, 5 mM TCEP, and 11% (w/w) PEG 8000. The chosen crystal was soaked for 120 seconds in a solution containing 50 mM HEPES pH 7.5, 50 mM NaCl, 100 mM ammonium sulfate, 15 mM manganese sulfate, 10 mM spermine, 15% (w/w) PEG 8000, 5% (w/w) PEG 400, 10% (v/v) DMSO, 11% (v/v) ethylene glycol, 6.5% (w/v) trimethylamine-N
-oxide (TMAO), 0.69 mM manicol, and 0.34 mM 18
. The crystal was subsequently flash-cooled and stored in liquid N2
. X-ray data were collected at 100K and a wavelength of 1.1 Å at the National Synchrotron Light Source at Brookhaven National Laboratories, Beamline X25. The data were processed using the HKL-DENZO/SCALEPACK software suite41, 42
Structure Determination and Refinement.
Phases for the diffraction data were determined by molecular replacement with the CCP4 program PHASER43
, using an RT/18
structure (PDB accession number 2ZD1)44
as an initial search model. Stepwise model building and refinement were conducted using the "O" graphics package45
, the Coot graphics package46
, and CNS47
with a bulk solvent correction (). Water molecules were built in both manually in Coot and using the program Refmac/ARP/wARP in the CCP4 software suite48–52
. The geometry of the inhibitor and refinement of the RNH active site were improved by energy minimization using the Impact and PrimeX facilities of the Schrödinger software package (Schrödinger, LLC).
Molecular Modeling. A model of the HIV-1 RNase H domain in complex with an inhibitor and two Mn2+ cations was constructed from the HIV-1 RT-manicol co-crystal structure using Discovery Studio 2.0 (Accelrys, San Diego, CA). To create the starting structure for energy minimization, RT was truncated to include only the RNase H domain, the inhibitors were constructed directly from manicol using various “build” functions, and the CHARMm forcefield was applied to the entire structure via the “Simulation” tool. The modified structure was then subjected to two rounds of energy minimization (500 iterations each). Inhibitor atoms outside of the tropolone and cyclohexane rings, as well as the entire “His-loop” and side chains of α-helix E’, were permitted full flexibility according to the dictates of the minimization algorithm. The positions of other atoms were held fixed in order to minimize perturbation of the overall complex. Minimized structures were saved as PDB files and imported into PyMOL (Delano Scientific, LLC, San Francisco, CA) to generate the final image.