# Polynitrogen Bicyclic and Tricyclic Compounds as PDE4 Inhibitors
## 2 Materials and Methods  
### Chemistry  
Reagents and starting materials were obtained from commercial sources (Sigma Aldrich (Merck Spa), Via Monte Rosa, 93-20149 Milan, Italy; Fluorochem Limited, Unit 14, Graphite Way, Rossington Park, Hadfield, Derbyshire SK13 IQH, UK). Extracts were dried over Na\({}_{2}\)SO\({}_{4}\), and the solvents were removed under reduced pressure. All melting points were determined on a Buchi apparatus (New Castle, DE, USA) and are uncorrected. All reactions were monitored by thin layer chromatography (TLC) using commercial plates pre-coated with Merck silica gel 60 F-254. Visualization was performed by UV fluorescence (\(\Lambda\)max = 254 nm) or by staining with iodine or potassium permanganate. Chromatographic separations were performed on a silica gel column by gravity (Kieselgel 40, 0.063-0.200 mm; Merck, Rahway, NJ, USA) or flash chromatography (Kieselgel 40, 0.040-0.063 mm; Merck). Yields refer to chromatographically and spectroscopically pure compounds, unless otherwise stated. When reactions were performed in anhydrous conditions, the mixtures were maintained under a nitrogen atmosphere. Compounds were named following IUPAC rules, as applied by Beilstein-Institut AutoNom 2000 (4.01.305) or CA Index Name. The identity and purity of intermediates and final compounds were ascertained through TLC chromatography and NMR. \({}^{1}\)H NMR and \({}^{13}\)C NMR spectra were recorded with Avance 400 or Bruker Avance 400 Neo (Billerica, MA, USA) instruments (Bruker Biospin Version 002 with SGU). Chemical shifts (\(\delta\)) are reported in ppm to the nearest 0.01 ppm, using the solvent as an internal standard. Coupling constants (\(J\) values) are given in Hz and were calculated using 'TopSpin 1.3' software, rounded to the nearest 0.1 Hz. Data are reported as follows: chemical shift, multiplicity [exch, exchange; br, broad; s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; sext, sextet; sept, septet; m, multiplet; or as a combination of these (e.g., dd, dt etc. )], integration, assignment and coupling constant (s). High resolution mass spectrometry (HRMS) analysis was performed with a Thermo Finnigan (San Jose, CA, USA) LTQ Orbitrap mass spectrometer equipped with an electrospray ionization source (ESI). Analyses were carried out in positive ion mode, monitoring protonated molecules, [M+H]\({}^{+}\) species, and a proper dwell time acquisition was used to achieve 30,000 units of resolution at Full Width at Half Maximum (FWHM). Elemental composition of compounds was evaluated on the basis of their measured accurate masses, accepting only results with an attribution error less than 2 ppm and a non-integer RDB (double bond/ring equivalents) value [24]. All new compounds possess a purity > 95%; microanalyses indicated by the symbols of the elements were performed with a Perkin-Elmer 260 (Waltham, MA, USA) elemental analyzer for C, H, and N, and they were within \(\pm\) 0.4% of the theoretical values. **General procedure for 2a-g.** A total of 0.98 mmol of commercial starting material **1** was dissolved in an excess of SOCI\({}_{2}\) (3 mL), and a catalytic amount of anhydrous DMF (0.15 mL) was added. The mixture was stirred at reflux for 4-5 h. After cooling, the excess of SOCI\({}_{2}\) was removed in vacuo, and the residual oil was dissolved in anhydrous tetrah  
Figure 4: General structure of new pyrazolo [1’.5’:1.6]pyrimido [4,5-\(d\)]pyridazines from our in-house compound library. drofuran (2.5 mL) and added to a mixture previously prepared consisting of 1.18 mmol of suitable amine and 1.96 mmol of triethylamine in anhydrous tetrahydrofuran (5 mL) at 0 \({}^{\circ}\)C. The mixture was stirred for 2 h at 0 \({}^{\circ}\)C, and then at room temperature overnight. The mixture was filtered to remove the solid, and the filtrate was recovered. Evaporation of the solvent resulted in a residue that was purified by flash column chromatography using dichloromethane/methanol 9:1 (for **2a,2d**) or 9:5:05 (for **2b,2c,2e,2f,2g**) as eluents.