Lead Diversification at the Nanomole Scale Using Liver Microsomes

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Lead Diversification at the Nanomole Scale Using Liver Microsomes and Quantitative Nuclear Magnetic Resonance Spectroscopy: Application to Phosphodiesterase 2 Inhibitors R. Scott Obach, Gregory S Walker, Raman Sharma, Stephen Jenkinson, Tuan P. Tran, and Antonia F. Stepan J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00116 • Publication Date (Web): 30 Mar 2018 Downloaded from http://pubs.acs.org on March 30, 2018

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Journal of Medicinal Chemistry

Lead Diversification at the Nanomole Scale Using Liver Microsomes and Quantitative Nuclear Magnetic Resonance Spectroscopy: Application to Phosphodiesterase 2 Inhibitors

R. Scott Obach*,† Gregory S. Walker,† Raman Sharma,† Stephen Jenkinson,‡ Tuan P. Tran,† and Antonia F. Stepan§ †Pfizer Worldwide Research and Development, Eastern Point Road, Groton, Connecticut, 06340, United States ‡Pfizer Worldwide Research and Development, 10770 Science Center Drive, La Jolla, California, 92121, United States

§Pfizer Worldwide Research and Development, 610 Main Street, Cambridge, Massachusetts, 02139, United States

1 ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT In this report, we describe a method whereby lead molecules can be converted into several new analogues each using liver microsomes. Less than 1 micromole of substrate is incubated with liver microsomes (mouse, rat, hamster, guinea pig, rabbit, dog, monkey, or human) to produce multiple products which are isolated and analyzed by quantitative cryomicroprobe NMR (qNMR) spectroscopy. The solutions from qNMR analysis were then used as stocks that were diluted into biochemical assays. Nine human phosphodiesterase-2 (PDE2) inhibitors yielded 36 new analogues. Products were tested for PDE2 inhibition, intrinsic clearance in human hepatocytes, and membrane permeability. Two of the products (2c and 4b) were 3-10X more potent than their respective parent compounds and also had improved metabolic stability. Others offered insights into structure activity relationships. Overall, this process of using liver microsomes at a submicromole scale of substrate is a useful approach to rapid and cost-effective late-stage lead diversification.

KEYWORDS Phosphodiesterase 2, liver microsomes, quantitative NMR spectroscopy, lead diversification, intrinsic clearance, membrane permeability


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INTRODUCTION Late stage diversification offers medicinal chemists an option to rapidly generate multiple analogues from a favored lead molecule to enable understanding of structure-activity relationships, offers insights into the potential for new design strategies without investing in lengthy syntheses, and in some instances offers a compound that is superior to the lead molecule to a sufficient extent that it replaces that lead molecule. Highly desired are C-H functionalization reactions that can be gentle enough so as to not alter other functional groups on the lead molecule and to not require protection/deprotection steps. Methods have included chemical and chemo-enzymatic procedures.1,2 Cytochrome P450 enzymes can be used as reagents for lead diversification.3,4 These enzymes can catalyze a variety of oxidative reactions, but most important for lead diversification are aliphatic and aromatic hydroxylations of carbon atoms. Previously we have reported the use of P450 enzymes and liver microsomes to carry out drug metabolite biosynthesis and lead diversification.5,6 In our approach, we couple this biosynthetic technology with the use of quantitative cryomicroprobe NMR spectroscopy which permits the use of sub-milligram quantities of substrate. We have extended this approach to add subsequent fluorination of the initial hydroxyl products at the nanomole scale in an effort to introduce more metabolically stable analogues where the fluorine is now present at the very site of metabolic lability.7 Liver microsomes are mixtures of P450 enzymes, and as such are likely to generate more than one product than when using single enzymes or reactions. Quantitative cryomicroprobe NMR spectroscopy permits gathering spectral information down to concentrations of 100 nM.8 Overall, this approach is consistent with principles of green chemistry in that minimal amounts of compound are used, reactions and product isolation



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procedures employ aqueous conditions, with organic solvent usage limited to relatively small amounts of acetonitrile or methanol. Phosphodiesterase 2 (PDE2) is an enzyme that is part of the large phosphodiesterase family, all of which hydrolyze cyclic nucleotide messenger molecules.9

Inhibition of PDE2 in

endothelial cells could be important in the treatment of conditions associated with dysregulation of vascular permeability, such as stroke.10 Recently we described the generation of a new lead candidate for a phosphodiesterase-2 inhibitor using monkey liver microsomes.6 In that example, a lead molecule that was potent at PDE2 but suffered from high in vitro intrinsic clearance was converted into two monohydroxy and one dihydroxy product, all of which showed much lower turnover by cytochrome P450 enzymes. One of these retained the target potency of the parent molecule and was then of greater interest. Investment was thus justified to develop a synthetic procedure and prepare a larger quantity of this new lead for further characterization. In the present work, we have applied liver microsomes and quantitative cryomicroprobe NMR spectroscopy to carry out lead diversification for several PDE2 inhibitors in an effort to develop structure-activity relationships for target potency, metabolic lability, and membrane permeability. Nine substrates of three structural classes (Figure 1) were diversified to thirty-six new analogues using liver microsomes from various species. Results of this investigation are reported herein.


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RESULTS General Approach. Nine lead compounds, seven of which were prepared as shown in Schemes 1-4, were selected for diversification using liver microsomes. This began with an initial screen of metabolite profiles generated by liver microsome preparations from eight different species. The results were used to determine adequate generation of products from a selected lead, and if so, which species’ microsome preparation would be best for scale up. An example of the screen data for compound 1 are shown in Figure 2. What is sought is a product profile that offers a variety of products and effective consumption of the parent compound. Generally, reactions wherein the substrate has lost a substantial moiety or is broken into two or more major portions are undesired. For Compound 1, rabbit liver microsomes appeared to offer the most promising profile of products (Figure 2). This incubation was scaled and fractionated by HPLC (Figure 3). Fractions in the region where products of interest eluted were checked for identity and purity by UHPLC-UV-MS and pooled as appropriate for analysis by qNMR. This procedure was carried out for all nine lead compounds and it yielded a total of 36 new analogues, using less than 1 mg of each lead compound. Concentrations of solutions in d6-DMSO returned from qNMR ranged from 0.12 to 5.28 mM. These materials were tested for inhibition of PDE2, intrinsic clearance in human hepatocytes, and membrane permeability in RRCK cells. Compounds 1-6. Compounds 1-6 all possessed a common core structure of an imidazotriazinone with a 4-pyrazole substituent at the 7-position. These were used as lead substrates to generate analogues that would offer structure-activity information and even potentially new leads. They mostly underwent various hydroxylation reactions at methyl substituents and demethylation reactions and provided two to six new compounds per substrate. Target potency values of the lead substrates ranged from 0.06 to 11 nM.



Compound 1 was converted into six new analogues using rabbit liver microsomes (Table 1). Among these, one compound (1e), wherein a hydroxyl group was added to the 5-methyl substituent of the imidazotriazinone had equivalent target potency and demonstrated increased metabolic stability. Hydroxylation of the 3-methyl substituent of the pyrazole or the 3-methyl substituent of the phenyl seemed to diminish target potency (compounds 1a-1d). Oxidation of the 3-methyl of the pyrazole up to the carboxylic acid caused a marked decrease in potency. Addition of hydroxyl groups had a consistent effect of decreasing metabolic lability in human hepatocytes and decreasing membrane permeability. Compound 1e retained suitable permeability through membranes (flux = 13 x 10-6 cm/sec). Compound 2 underwent O- and N-demethylation, hydroxylation, and N-oxidation reactions (Table 2). O-Demethylation (2c) led to a 10-fold improvement in potency, increased metabolic stability and small decrease in membrane permeability. Further N-demethylation of 2c to 2a yielded a small decrease in target potency. Compound 2 also yielded an N-oxide metabolite at the bridging nitrogen of the imidazotriazine ring (compound 2d) and while target activity only decreased a little, it was more rapidly metabolized in human hepatocytes. Compound 3 yielded just two new analogues (hydroxyl 3a and N-desmethyl 3b; Table 3). Relative to the parent compound, potency and permeability decreased for both, while metabolic lability was unchanged. In the case of compound 4, hydroxylation occurred at two positions, one where potency decreased (4a) and the second where potency increased three fold (4b; Table 4). Compound 4b was also about twice as stable in human hepatocytes as the parent, while retaining adequate permeability and as such a clearly better candidate than 4. Compounds 5 and 6 possess pyridine in place of the phenyl rings of compound 1-4 and were less potent at PDE2. They were subject to lead diversification in an attempt to increase


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potency to levels measured for compounds 1-4. Compound 5 underwent hydroxylation (to 5d) and N-demethylation (to 5c) and secondary metabolism to 5a and 5b (Table 5). While hydroxylation yielded a small decrease in target potency, other transformation products had considerably less target potency. Products generally were metabolically stable, but also had low membrane permeability. Compound 6 underwent hydroxylation reactions to three products (6a, 6b, and 6d) and also generated an isolable aldehyde metabolite (6c) (Table 6). Compound 6d retained target potency, but all others had markedly decreased potency. Permeability was low for all the products. All were metabolically stable, except for the aldehyde product. Compound 7. Compound 7 represented a second prototype structure which possessed target potency of only 792 nM (Table 7). The intent in attempting lead diversification for this structure was to determine whether introduction of a hydroxyl group would improve the potency. Human liver microsomes were used to generate two hydroxyl products, 7a and 7b and NMR data indicated that the substitution for both was on the 2-position of the cyclopentane ring indicating diastereomers, although for 7a, it cannot be completely ruled out that the site of hydroxylation is not on the 3-position. Nevertheless, neither of these two compounds yielded an improvement in PDE2 inhibition potency (Table 7) and as such determination of their exact configuration was not addressed. Compounds 8 and 9. Compounds 8 and 9 are diastereomers of an alternate chemical series that is more lipophilic than compound 1-6 and compound 7. Compound 8 and 9 yielded seven and five new analogues, respectively (Tables 8 and 9). Products arose via O-demethylation of the dimethoxyphenyl substituent, hydroxylation of the 2-hydroxyhexane, and secondary oxidation of one of the hydroxyl metabolites to a ketone. For compound 8, potency at PDE2 inhibition was 0.03 nM and the potency of the next most potent among the products was 80-fold lower (8d). It



should be noted that absolute configuration of all of the products wherein the benzylic position of the hexanol portion was hydroxylated (i.e. compounds 8a-8e) was not determined. Furthermore, the specific sites of hydroxylation in products 8a and 8f could not be distinguished among two possibilities. However as these products had 300-3000 fold lower potency they are not of interest. Compound 8 had high metabolic lability and the products all showed decreases in lability, but markedly decreased membrane permeability was observed for all products. Similar observations were made from the products from compound 9; potency, metabolic lability, and membrane permeability all decreased.


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DISCUSSION AND CONCLUSIONS The use of liver microsomes as a lead diversification tool has appeal in that incubations can be carried out with very small amounts of reactant, are conducted under mild conditions in aqueous solution, can catalyze reactions at unactivated C-H bonds, and can yield multiple products from a single incubation. Liver microsomes contain a mixture of cytochrome P450 enzymes, each of which has the potential to generate more than one product. Screening liver microsomes from several species offers an even greater potential to generate multiple products since the P450 enzymes across species also have differences in active site architecture, even among the same subfamilies. In the present work, as many as seven new analogues were generated from one lead. In the procedure described in this report, a new lead compound can be screened, scaled-up, products isolated, and analyzed by quantitative NMR spectroscopy within a few days. Less than a micromole of lead compound is needed to carry this out. The new analogues are obtained as stock solutions of known concentration (ranging between 0.1 and 10 mM) in hexadeuterated DMSO that can be diluted for use in testing target potency, metabolic lability, membrane permeability, and other in vitro assays. At present, the specific products that will be generated for any lead from any liver microsome sample cannot be predicted; only that the types of reactions that will occur include hydroxylations, heteroatom dealkylations, heteroatom oxygenations, desaturations, among others. Mammalian drug-metabolizing cytochrome P450 enzymes have broad substrate specificities and product profiles generated will depend on the binding orientations into individual enzymes, and not merely on oxidation states of various substituents in the substrate. Selection of the source species of liver microsomes to use in biosynthesis varies with the substrate. In our experience for these PDE2 inhibitors as well as other drug design programs



there is no single species that consistently outperformed the others. Thus, the generation of products which provide useful structure-activity relationships or even better compounds than the lead itself requires serendipity. In some instances, the exact site of modification and/or stereochemical configuration cannot be known from spectral data alone, yet these materials still have usefulness in the approach. (And in some cases, the product may not be pure, although the stock concentration will still be known from qNMR.) If such a product is shown to have desired activity, then further pursuit of that compound(s) can be done using organic synthesis methods. For these PDE2 inhibitors, lead diversification offered several SAR insights, and in a couple of instances a substantially superior compound was generated. Enough material was generated to measure three fundamental parameters: target intrinsic potency, intrinsic clearance, and membrane permeability. These are the three most important fundamental parameters on which to optimize in most drug design programs. These are generally related to lipophilicity, with higher lipophilicity favoring target potency and membrane permeability and lower lipophilicity favoring low intrinsic clearance; thus a balance is needed. Metabolic lability was carried out in human hepatocytes because many of the new leads now possessed substituents that could be metabolized by phase 2 drug metabolizing enzymes (e.g. glucuronosyl transferases, sulfotransferases). Three chemical series were evaluated. In one, lead diversification was attempted in order to test whether a weak lead could be made more potent (compound 7), but this failed for this example. In a second, an attempt was made to use lead diversification to reduce lipophilicity of leads while maintaining target potency and improving metabolic stability (compounds 8 and 9). LipE scores for these compounds did not show substantial improvement, because lipophilicity decreases had a negative impact on target potency. Furthermore, membrane permeability was seriously impacted such that almost all new analogues had flux


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values below the cutoff of 5 x 10-6 cm/sec. Success was obtained for some of the compounds in the chemical series represented by compounds 1-6. In almost all cases, hydroxylation on the 5methyl substituent of the imidazotriazinone yielded a new analogue of similar target potency but improved metabolic stability (compounds 1 vs 1e, 4 vs 4b, 5 vs 5d, and 6 vs 6d). The corresponding product that arose via O-demethylation of compound 2 (i.e. compound 2c) yielded a new lead that was 10 times more potent at PDE2 inhibition and was permeable and metabolically stable. Thus, in some instances like this, a superior compound can come from lead diversification using liver microsomes.6 This represents an unconventional and different approach to drug design. Instead of preparing milligram quantities of a pure and highly characterized single product using chemical reactions that are optimized to provide high yields, in the lead diversification approach described here nanomole quantities of multiple products are prepared simultaneously as solutions of known concentration with overall yields that can be as low as 1% for any given product. Structures of products are elucidated using high resolution mass spectrometry and 1D and 2D NMR, aided by the known spectral characteristics of the lead compound. Quantities generated, typically ranging from 5 nmoles to hundreds of nmoles, yield proton NMR spectra, but are too low to yield direct observed C-13 NMR data. Consequently, homonuclear and heteronuclear 2D NMR spectral data (i.e. COSY, TOCSY, HSQC and HMBC experiments) are instead utilized to elucidate structures (Examples are shown in the Supplemental Data.) Additionally, when isolates are below the 50 nmole level, complete proton accountability may not be possible, but when NMR data and MS are used together, reasonable structures can be proposed. Multiple new SAR data points are obtainable, in some cases from products that would require large investments in developing multistep synthetic procedures. In a few instances, the exact structure of a product may remain



unknown, however even these materials are of value in that if they lack activity they are no longer of interest and if they possess activity they can be further pursued using conventional organic synthesis methods. Individual products that are potentially superior to the leads from which they were generated (based on initial potency, metabolic stability, and permeability properties) will merit investment in the development of conventional synthetic methods that will yield larger quantities for further characterization (i.e. confirmation of initial results, additional in vitro and in vivo assays). We have utilized this approach to help support other drug design projects. In almost all cases, useful SAR is learned from products generated using this approach, in a few instances superior leads have been generated, and a few others have failed to generate analogues of interest. Future research efforts include the investigation of alternate enzyme systems and coupling these nanoscale enzymatic lead diversification reactions to other chemistries to broaden the array of possible new analogues.


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EXPERIMENTAL SECTION Materials. Compound 8 was from Cayman Chemical (Ann Arbor, MI) and compound 9 was prepared as previously described (Hendrick, et al., 2015)11. Liver microsomes were from the following sources: female mouse, male dog, female cynomolgus monkey, and pooled male and female human (Corning, Woburn, MA); male guinea pig and male rabbit (Xenotech, Lenexa, KS); male gerbil and dexamethasone-induced male rat (prepared in-house at Pfizer). Cryopreserved pooled human hepatocytes (from ten donors) were prepared under contract by Celsis IVT (Baltimore, MD). Williams E medium was from Gibco (Gaithersburg, MD). NADPH was from Sigma-Aldrich (St. Louis, MO). Metabolite Profile in Liver Microsomes. In order to determine a liver microsome source that would yield acceptable conversion to a variety of products, the metabolite profiles were determined. Substrate (20 µM) was incubated with liver microsomes (2 mg/mL) from mouse, rat (dexamethasone induced), hamster, guinea pig, rabbit, dog, monkey, and human in a volume of 0.2 mL potassium phosphate buffer (100 mM, pH 7.5) containing MgCl2 (3.3 mM) and NADPH (1.3 mM). Incubations were commenced with the addition of NADPH and carried out in a shaking water bath maintained at 37oC for 45 min. Incubations were terminated with the addition of CH3CN (0.6 mL), the precipitated protein was removed by centrifugation (1700 g, 5 min) and the supernatant was removed by vacuum centrifugation. The residues were reconstituted in 0.05 mL 1% formic acid in water. (Note that human and monkey liver microsomes are considered potentially biohazardous materials.) Reconstituted samples were analyzed by UHPLC-UV-HRMS. The system consisted of a Thermo Orbitrap Elite high resolution ion trap mass spectrometer in line with a Thermo Accela UHPLC and diode array UV/VIS detector, along with a CTC Analytics Leap autoinjector



(Thermo-Fisher, Palo Alto, CA). Samples were injected (0.01 mL) onto a Waters HSS T3 C18 column (2.1 x 100 mm; 1.8 µ) in a mobile phase of 0.1% formic acid in water (A) and CH3CN (B) at a flow rate of 0.4 mL/min, maintained at 45oC in a column heater. The initial composition of 95%A/5%B was held for 0.5 min followed by linear gradients to 60%A/40%B at 6 min and 20%A/80%B at 8 min. The column was then washed with 5%A/95%B for 1 min and reequilibrated to initial conditions for 1.5 min before the next injection. The mass spectrometer was operated in the positive ion mode with source potentials and temperatures optimized to maximize signal for the substrates. MS2 spectra were obtained in HCD mode at an energy setting of 55. These HPLC conditions were also used to estimate purity of isolated products using the diode array full scan data of 200-400 nm (see below) and purity of all products was ≥95% unless otherwise noted. General Procedure for Biosynthesis of Analogues Using Liver Microsomes. Biosynthesis and isolation was carried out using methods similar to those previously described for drug metabolites (Walker, et al., 2014). Substrates (20 µM; 800 nmoles) were incubated with liver microsomes (2 mg/mL) in a volume of 40 mL potassium phosphate (0.1 M, pH 7.5) containing MgCl2 (3.3 mM) and NADPH (1.3 mM). Incubations were carried out in a 500 mL Erlenmeyer flask in a shaking water bath maintained at 37oC. Other specifics regarding incubation conditions for each substrate are in Table 10. Incubations were stopped by the addition of 40 mL CH3CN and the precipitate was removed by centrifugation (1700 g; 5 min). The supernatant was transferred to 50 mL polypropylene tubes and subject to evaporation in a vacuum centrifuge (Genevac, Valley Cottage, NY) for 1-2 hrs to yield a volume of ~20 mL. To this mixture was added formic acid (0.5 mL), CH3CN (0.5 mL) and water to a final volume of 50 mL. This mixture was spun in a centrifuge at 40000 g for 30 min. The clarified supernatant was applied to


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a Polaris C18 column (4.6 x 250 mm; 5 µ; Agilent, Palo Alto, CA) at 0.8 mL/min through an HPLC pump (Jasco, Easton, MD) followed by 7 min of pumping 0.1% formic acid in water to ensure the entire mixture was applied to the column. The column was moved to a Thermo LTQ Velos mass spectrometer in line with a Waters Acquity UHPLC system comprised of a quaternary pump, autosampler, and photodiode array UV/VIS detector. A gradient was applied to separate products of interest (for conditions used for each substrate, refer to Table 10). After passing through the PDA detector, the eluent was split at a ratio of approximately 15:1 with the larger portion going to a fraction collector (Collect PAL, Leap Technologies, Carrboro, NC) and the smaller portion going to the mass spectrometer. Fractions were collected every 20 sec. Fractions containing peaks of interest were analyzed by UHPLC-UV-HRMS using the system described above to ascertain identity and purity. Based on that analysis, fractions were pooled and the solvent was removed by vacuum centrifugation. The dried tubes were analyzed by NMR spectroscopy. NMR Spectroscopy. All samples were dissolved DMSO-d6 “100%” and placed in a 1.7 mm NMR tube (0.04 ml isolated materials) or a 3 mm NMR tube (0.150 ml parent compound) under a dry argon atmosphere. Proton and 13C spectra were referenced using residual DMSO-d6 (1H δ=2.50 ppm relative to TMS, δ=0.00, 13C δ=39.50 ppm relative to TMS, δ=0.00). NMR spectra were recorded on a Bruker Avance 600 MHz (Bruker BioSpin Corporation, Billerica, MA) controlled by Topspin V3.2 and equipped with a 1.7 mm TCI Cryo probe (isolated materials) or a 5 mm BBFO cryo probe (parent compound). 1D Spectra were recorded using an approximate sweep width of 8400 Hz and a total recycle time of approximately 7 s. 2D data were recorded using the standard pulse sequences provided by Bruker. Post-acquisition data processing was performed with either Topspin V3.2 or MestReNova V9.1 Quantitation of NMR isolates was



performed by external calibration against the 1H NMR spectrum of a 5 mM benzoic acid standard using the ERETIC2 function within Topspin V3.2. Synthesis of Substrates 1, 4, and 5. 5-Chloro-1,3-dimethyl-N-(1-(3-methyl-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)ethyl)-1Hpyrazole-4-carboxamide (12). To a solution of acid 11 (0.37 g, 2.10 mmol) in dichloromethane (25 mL) was added thionyl chloride (2.5 mL, 35 mmol) and the mixture was heated to reflux for 1 h. The reaction mixture was concentrated under vacuo, the resulting residue was taken up in dichloromethane (5mL), then added dropwise to a cooled (0 °C) solution of amine 10 (0.33 g, 2.10 mmol) and triethylamine (0.60 mL, 4.26 mmol) in dichloromethane (25 mL). After 15min, the reaction mixture was taken up in dichloromethane, washed with sat’d sodium bicarbonate solution and water. The organic layer was dried over magnesium sulfate, filtered and filtrate concentrated. Purification via flash column chromatography eluding with 0 to 5% methanol in dichloromethane afforded amide 12 as a white solid (461mg, 70%). 1H NMR (400 MHz, DMSO-d6): δ 13.54 (br s, 1 H), 7.90 (d, J= 8.0 Hz, 1 H), 5.10 (t, J= 8.0 Hz, 1 H), 3.71 (s, 3 H), 2.25 (s, 3 H), 2.24 (s, 3 H), 1.34 (d, J= 8.0 Hz, 3 H). Mass calculated for [M+H]+ (C12H16ClN6O2) is m/z = 311, found LCMS [M+H]+ m/z = 311. 7-(5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)-2,5-dimethylimidazo[5,1-f][1,2,4]triazin-4(3H)one (13). A mixture of amide 12 (0.55 g, 1.77 mmol) in propylphosphonic anhydride solution (50% in ethyl acetate, 5.60 g, 8.84 mmol) was heated to 120 °C. After 16 h, the reaction mixture was cooled to room temp, slowly quenched with saturated sodium bicarbonate. The mixture was taken up in dichloromethane, washed with saturated sodium bicarbonate and water. The organic layer was dried over magnesium sulfate, filtered, and filtrate concentrated under vacuo. Trituration from ethyl acetate and heptane, filtered and dried under vacuum afforded imidazo-


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triazinone 13 as a white solid (0.32 g, 62%). 1H NMR (400 MHz, DMSO-d6): δ 11.62 (br s, 1 H), 3.77 (s, 3 H), 2.48 (s, 3 H), 2.13 (d, J= 4.0 Hz, 3 H). Mass calculated for [M+H]+ (C12H14ClN6O) is m/z = 293, found LCMS [M+H]+ m/z = 293. Library synthesis of compounds 1, 4, and 5. Library synthesis of compounds 1, 4, and 5. To degassed mixtures containing triazinone 12 (100 umol), boronate (100 umol), diisopropyl ethylamine (200 umol), in ethanol and water (6:1 mixture) was added tetrakis(triphenylphosphine)palladium (5 umol). The mixture was evacuated with vacuum and flushed with nitrogen, repeated twice more, then heated to 100 °C under nitrogen atmosphere for 18 h. Standard work-up and automated HPLC purification afforded: 7-(1,3-dimethyl-5-(3-methyl-4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-2,5dimethylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (1). 1H NMR (400 MHz, CDCl3): δ 10.28 (br s, 1 H), 7.56 (d, J= 8.8 Hz, 1 H), 7.28 (s, 1 H), 7.16 (d, J= 8.0 Hz), 3.82 (s, 3 H), 2.61 (s, 3 H), 2.46 (s, 3 H), 2.36 (s, 3 H), 2.06 (s, 3 H).

13

C NMR (100 MHz, CDCl3): δ 187.58, 156.83,

148.44, 146.37, 142.54, 141.37, 137.41, 136.80, 133.82, 132.83, 128.93 (q, J= 30 Hz), 125.80 (q, J= 5.5 Hz), 124.24 (q, J= 272 Hz), 113.65, 107.95, 37.33, 19.30, 18.23, 14.70, 12.82. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for (C20H20F3N6O) 417.1645, found 417.1637. 7-(5-(4-chlorophenyl)-1,3-dimethyl-1H-pyrazol-4-yl)-2,5-dimethylimidazo[5,1f][1,2,4]triazin-4(3H)-one (4). 1H NMR (400 MHz, CDCl3): δ 9.56 (br s, 1 H), 734-7.31 (m, 2 H), 7.23-7.20 (m, 2 H), 3.81 (s, 3 H), 2.61 (s, 3 H), 2.35 (s, 3 H), 2.07 (s, 3 H). Mass calculated for [M+H]+ (C18H18ClN6O) is m/z = 369.1225, found LCMS [M+H]+ m/z = 369.1222 7-(5-(4-methoxyphenyl)-1,3-dimethyl-1H-pyrazol-4-yl)-2,5-dimethylimidazo[5,1f][1,2,4]triazin-4(3H)-one (5). 1H NMR (400 MHz, CDCl3): δ 9.88 (br s, 1 H), 8.10 (d, J= 2.0 Hz, 1 H), 7.50 (dd, J= 8.8, 2.4 Hz, 1 H), 6.73 (d, J= 8.4 Hz, 1 H), 3.93 (s, 3 H), 3.81 (s, 3 H),



2.60 (s, 3 H), 2.34 (s, 3 H), 2.12 (s, 3 H). Mass calculated for [M+H]+ (C18H20N7O2) is m/z = 366.1673, found LCMS [M+H]+ m/z = 366.1674. Synthesis of Substrates 2 and 3. 7-(1,3-Dimethyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-5-(methoxymethyl)-2methylimidazol[5,1-f][1,2,4]triazin-4-(3H)-one (2). To a solution of the alcohol 146 (55 mg, 0.12 mmol) in tetrahydrofuran (2 mL) at 0 °C was added sodium hydride (60% in mineral oil, 14 mg, 0.35 mmol). After 30 min, iodomethane (34 mg, 0.24 mmol) was added dropwise. The reaction was warmed to room temp for 3 h, then saturated ammonium chloride solution was added. The mixture was extracted with ethyl acetate, the organic layer was dried over magnesium sulfate, filtered and filtrate concentrated under vacuo. The resulting crude residue was taken up in dioxane (1 mL) and 1 N hydrochloric acid (0.1 mL) and heated to 80 °C. After 16 h, the reaction mixture was cooled to room temp, concentrated under vacuo, and purification via flash column chromatography eluting with 0 to 5% methanol in ethyl acetate to furnish triazinone 2 as a white solid (27 mg, 53%). 1H NMR (400 MHz, CDCl3): δ 7.61 (d, J= 8.0 Hz, 2 H), 7.43 (d, J= 8.0 Hz, 2 H), 4.76 (s, 2 H), 3.84 (s, 3 H), 3.42 (s, 3 H), 2.38 (s, 3 H), 2.06 (s, 3 H).

13

C NMR (100 MHz, CDCl3): δ 166.47, 155.99, 148.63, 146.63, 142.57, 140.21, 138.13,

134.01, 130.65 (q, J= 32 Hz), 129.94, 125.25, 122.45, 115.00, 107.92, 66.91, 58.38, 37.35, 18.26, 12.89. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for (C20H20F3N6O2) 433.1594, found 433.1588. 5-(Difluoromethyl)-7-(1,3-dimethyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-2methyl-4-(pyrrolidin-1-yl)imidazo[5,1-f][1,2,4]triazine (15). To a solution of alcohol 14 (0.17 g, 0.36 mmol)6 in chloroform (5 mL) was added manganese dioxide (0.16 g, 1.80 mmol). The mixture was heated to 70 °C. After 7 h, the reaction mixture was cooled to 10 °C, and filtered.


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The filtrate was concentrated under vacuo. The resulting residue was immediately taken up in dichloromethane (4 mL), cooled to 0 °C, and diethylaminosulfur trifluoride (0.15 g, 0.94 mmol) was added. The reaction mixture was gradually warmed to room temp as the ice bath expired. After 16 h, saturated sodium bicarbonate was added dropwise. The mixture was taken up in dichloromethane and washed with saturated sodium bicarbonate and water. The organic layer was dried over magnesium sulfate, filtered and filtrate concentrated to afford crude 15 (0.11 g). Mass calculated for [M+H]+ (C23H23F5N7) is m/z = 492, found LCMS [M+H]+ m/z = 492. 5-(Difluoromethyl)-7-(1,3-dimethyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-2methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (3). The reaction was carried out same as above for compound 2 to afford triazinone 3 as a white solid (27 mg, 17% over 3 steps). 1H NMR (400 MHz, CDCl3): δ 9.79 (br s, 1 H), 7.64 (d, J= 8.4 Hz, 2 H), 7.46 (d, J= 8.0 Hz, 2 H), 7.09 (t, J= 53.6 Hz, 1 H), 3.85 (s, 3 H), 2.39 (s, 3 H), 2.09 (s, 3 H).

13

C NMR (100 MHz, CDCl3): δ 156.68,

153.18, 148.28, 146.11, 141.57, 140.23, 137.18, 133.33, 125.25 (q, J= 33 Hz), 123.93, 123.36 (q, J= 271 Hz), 113.85, 108.56, 38.55, 18.22, 14.72, 12.75. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for (C19H16F5N6O) 439.1300, found 439.1299.

Synthesis of Substrate 6. Methyl 5-bromo-1,3-dimethyl-1H-pyrazole-4-carboxylate (17). To a stirred solution of the tert-butylnitrite (8.30 g, 72.7 mmol) and copper (II) bromide (11.0g, 49.2 mmol) in acetonitrile (100 mL) at -78 °C was added compound 16 (5.00g, 29.6 mmol) slowly as a solution in acetonitrile (20 mL). The reaction mixture was slowly warmed to room temp as the ice bath expired, then heated to 65 °C. After 18 h, the reaction mixture was cooled to room temp, filtered through celite, washed with acetonitrile, and the combined filtrate was concentrated. The



resulting residue was taken up in ethyl acetate and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and filtrate concentrated under vacuo. Purification via flash column chromatography eluding with 10% ethyl acetate in hexanes afforded bromide 17 (6.03 g, 87.5%). 1H NMR (400 MHz, DMSO-d6): δ 3.79 (s, 3 H), 3.76 (s, 3 H), 2.33 (s, 3 H). Mass calculated for [M+H]+ (C7H10BrN2O2) is m/z = 233 (100%); 235 (97%), found LCMS [M+H]+ m/z = 233, 235. 5-Bromo-1,3-dimethyl-1H-pyrazole-4-carboxylic acid (18). To a solution of the ester 17 (6.50 g, 27.9 mmol) in tetrahydrofuran (15 mL) and methanol (60 mL) was added lithium hydroxide (1.47 g, 61.4 mmol). After stirring at room temp for 4 h, the reaction mixture was concentrated under vacuo. The resulting residue was taken up in 2 N HCl (75 mL) and extracted twice with ethyl acetate. The organic layer were combined, dried over sodium sulfate, filtered, and filtrate concentrated to give acid 18 (5.87 g, 96.1%). 1H NMR (400 MHz, DMSO-d6): δ. Mass calculated for [M+H]+ (C6H8BrN2O2) is m/z = 219 (100%); 221 (97%), found LCMS [M+H]+ m/z = 219, 221. 5-Bromo-1,3-dimethyl-N-(1-(3-methyl-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)ethyl)-1Hpyrazole-4-carboxamide (19). To a solution of amine 10 (3.84 g, 24.9 mmol) and acid 18 (6.0 g, 27.4 mmol) in N,N-dimethylformamide (100 mL) were added 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (7.69 g, 40.3 mmol), 1-hydroxybenzotriazole (5.43 g, 40.3 mmol) and diisopropyl ethylamine (23.2 mL, 134.2 mmol). After stirring at room temp for 16 h, the reaction mixture was concentrated under vacuo. The resulting residue was recrystallized from acetone to afford amide 19 (5.56 g, 62.9%). 1H NMR (400 MHz, DMSO-d6): δ 13.61 (br s, 1 H), 7.94 (d, J= 7.6 Hz, 1 H), 5.13 (appar quint., J= 7.1 Hz, 1 H), 3.76 (s, 3 H), 2.28 (s, 3 H),


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2.27 (s, 3 H), 1.37 d, J= 6.9 Hz, 3 H). Mass calculated for [M+H]+ (C12H16BrN6O2) is m/z = 355 (100%); 357 (97%), found LCMS [M+H]+ m/z = 355, 357. 7-(5-Bromo-1,3-dimethyl-1H-pyrazol-4-yl)-2,5-dimethylimidazo[5,1-f][1,2,4]triazin-4(3H)one (20). To a solution of amide 19 (8.0 g, 22.5 mmol) in acetonitrile (100 mL) at 0 °C was added phosphorous oxychloride (20.5 mL, 225 mmol). After 10min, the reaction mixture was heated to 120 °C for 1 h. The reaction mixture was cooled to room temp and concentrated under vacuo. The resulting residue was taken up in ethyl acetate and washed with sat’d sodium bicarbonate and water. The organic layer was dried over sodium sulfate, filtered and filtrate concentrated. The resulting residue was purified via flash column chromatography eluding with 0 to 5% methanol in dichloromethane to furnish product 20 (4.10 g, 54.0%).

1

H NMR (400

MHz, DMSO-d6): δ 11.65 (s, 1 H), 3.83 (s, 3 H), 2.50 (d, J= 1.8 Hz, 3 H), 2.16 (s, 3 H), 2.14 (s, 3 H). Mass calculated for [M+H]+ (C12H14BrN6O) is m/z = 337 (100%); 339 (97%), found LCMS [M+H]+ m/z = 337, 339. 7-(5-Bromo-1,3-dimethyl-1H-pyrazol-4-yl)-2,5-dimethyl-4-(pyrrolidin-1-yl)imidazo[5,1f][1,2,4]triazin (21). To a solution of triazinone 20 (0.50 g, 1.48 mmol) in toluene (10 mL) was added phosphorous oxychloride (0.34 mL, 3.71 mmol). N,N-Diisopropyl ethylamine (1.32 mL, 7.41 mmol) was added dropwise, then the reaction mixture was heated to reflux. After 3 h, the reaction mixture was cooled to room temp, concentrated under vacuo. The resulting residue was taken up in dichloromethane (10 mL), cooled to 0 °C, then pyrrolidine (0.31 g, 4.45 mmol) was added. The reaction mixture was gradually warmed to room temp as the ice bath expired. After 16 h, the reaction mixture was taken up with more dicloromethane, washed with sat’d sodium bicarbonate and water. The organic layer was dried over magnesium sulfate, filtered and filtrate concentrated. Purification via flash column chromatography eluding with 0 to 5% methanol in



ethyl acetate afforded triazine 21 as a beige foam (0.55 g, 95%). 1H NMR (400 MHz, DMSOd6): δ. Mass calculated for [M+H]+ (C16H21BrN7) is m/z = 390 (100%); 392 (97%), found LCMS [M+H]+ m/z = 390, 392. 7-(1,3-Dimethyl-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrazol-4-yl)-2,5-dimethyl-4(pyrroldin-1-yl)imidazo[5,1-f][1,2,4]triazine (22). To a dried vial under nitrogen atmosphere was added bromide 21 (0.10 g, 0.26 mmol) and 2-methyltetrahydrofuran (2 mL). The solution was cooled to -10 °C, then isopropylmagnesium chloride lithium chloride complex (1.3 M in tetrahydrofuran, 0.46 mL, 0.60 mmol) was added over 3 min. After stirring for 30 min, zinc chloride (1.9 M solution in 2-methyltetrahydrofuran, 0.15 mL, 0.28 mmol) was added. The mixture was stirred for 5 min, then warmed to room temp. After 1h, this solution was added to a dried flask containing 2-bromo-5-(trifluoromethyl)pyridine (0.086 g, 0.38 mmol), tris(dibenzylideneacetone)dipalladium (2.80 mg, 3.0 µmol), and Xantphos (8.80 mg, 1.5 µmol). The mixture was heated to 80 °C for 16 h, then cooled to room temp, diluted with 10% citric acid, and stirred rigorously. The mixture was filtered over celite, rinsed with tetrahydrofuran, and the combined filtrate was concentrated to afford crude 22 (0.11 g), which was used without further purification. Mass calculated for [M+H]+ (C22H24F3N8) is m/z = 457, found LCMS [M+H]+ m/z = 457. 7-(1,3-Dimethyl-5-(5-(trifluoromethyl)pyridine-2-yl)-1H-pyrazol-4-yl)-2,5dimethylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (6). To a solution of the crude triazine 22 (0.11 g) in dioxane (3 mL) was added 1 N hydrochloric acid (1 mL). The mixture was heated to 90 °C. After heating for 18 h, the reaction mixture was cooled to room temp and concentrated. The resulting residue was purified via reverse phase, automated HPLC system to afford triazinone product 6 as a white solid (38 mg, 37% over 2 steps). 1H NMR (400 MHz, CDCl3): δ 22 ACS Paragon Plus Environment

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9.27 (br s, 1 H), 8.97 (s, 1 H), 7.80 (appar d, J= 8.0 Hz, 1 H), 7.28 (d, J= 8.0 Hz, 1 H), 4.08 (s, 3 H), 2.65 (s, 3 H), 2.38 (s, 3 H), 1.98 (s, 3 H). Mass calculated for [M+H]+ (C18H17F3N7O) is m/z = 404, found LCMS [M+H]+ m/z = 404. Synthesis of Substrate 7. tert-Butyl 3-(4-methoxy-7-methylimidazo[5,1-f][1,2,4]triazin-5-yl)-6,7-dihydropyrazolo[1,5a]pyrazine-5(4H)-carboxylate (25). A solution of CatacXium A (0.31 g, 0.82 mmol) and palladium acetate (0.097 g, 0.41 mmol) in toluene (10 mL) was stirred for 5 min, then added to a degassing and preheated (50 °C) solution of bromide 23 (1.0g, 4.11 mmol),12 bromide 24 (1.49 g, 4.94 mmol), cesium fluoride (3.12 g, 20.6 mmol), and bis(pinacolato)diboron (1.57 g, 6.17 mmol) in methanol (15 mL). The mixture was evacuated with vacuum, flushed with nitrogen, repeated twice more, then heated to 80 °C under nitrogen atmosphereosphere. After 18 h, the reaction mixture was cooled to room temperature, filtered through celite, and rinsed with ethyl acetate. The combined filtrate was washed with water and brine. The organic layer was dried over magnesium sulfate, filtered and filtrate concentrated under vacuo. Purification via flash column chromatography eluting with 40 to 100% ethyl acetate in heptane afforded 25 as a yellow solid (937 mg, 59%). Mass calculated for [M+H]+ (C18H24N7 O3) is m/z = 386, found LCMS [M+H]+ m/z = 386. 5-(5-(Cyclopentylmethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-3-yl)-7methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (7). To a solution of the carbamate 25 (0.90 g, 2.34 mmol) in methanol (10 mL) was added hydrochloric acid (4 N in ether, 3 mL). After 4 h, the reaction mixture was concentrated under vacuo to afford the resulting amino-triazinone hydrochloride salt as a yellow solid. This solid was taken up in 1,2-dichloroethane (10 mL) then cyclopentane carboxaldehyde (0.47 g, 4.82 mmol), acetic acid (1.38 mL, 24.1 mmol) and sodium



triacetoxyborohydride (1.61 g, 7.23 mmol) were added. The reaction mixture was heated to 40 °C for 3 h, cooled to room temp, and concentrated under vacuo. The resulting residue was taken up in dichloromethane, washed with saturated sodium bicarbonate and water. The organic layer was dried over magnesium sulfate, filtered and filtrate concentrated. Purification via flash column chromatography eluting with 0 to 8% methanol in ethyl acetate gave product 7 as a white solid (0.34 g, 40%). 1H NMR (600 MHz, DMSO-d6) δ 11.60 (s, 1H), 8.39 (s, 1H), 7.83 (s, 1H), 4.12 (t, J = 5.5 Hz, 2H), 3.98 (s, 2H), 2.92 (dd, J = 4.7, 6.3 Hz, 2H), 2.47 (d, J = 7.6 Hz, 2H), 2.16 (hept, J = 7.6 Hz, 1H), 1.82 – 1.67 (m, 3H), 1.62 – 1.54 (m, 2H), 1.54 – 1.44 (m, 2H), 1.30 – 1.16 (m, 3H). Mass calculated for [M+H]+ (C18H24N7 O) is m/z = 354.2036, found LCMS [M+H]+ m/z = 354.2033.

Spectral Properties of Biosynthesized Analogues. 5-(hydroxymethyl)-7-(5-(3-(hydroxymethyl)-4-(trifluoromethyl)phenyl)-1,3-dimethyl-1Hpyrazol-4-yl)-2-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (1a). Yield: 8.5 nmol, 1.1% from (1). HRMS: m/z 449.1543 (-0.1 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 7.80 (s, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 5.50 (t, J = 5.8 Hz, 1H), 5.07 (t, J = 5.8 Hz, 1H), 4.65 (m, 4H), 3.81 (s, 3H), 2.21 (s, 3H), 1.95 (s, 3H). Purity by HPLC-UV (200-400 nm) >99%.

7-(3-(hydroxymethyl)-5-(3-(hydroxymethyl)-4-(trifluoromethyl)phenyl)-1-methyl-1Hpyrazol-4-yl)-2,5-dimethylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (1b). Yield: 20 nmol, 2.5% from (1). HRMS: m/z 449.1538 (-1.2 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 7.74 (s, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 5.51 (s, 1H), 5.24 (t, J = 5.5 Hz, 1H), 4.65


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(s, 2H), 4.49 (d, J = 5.5 Hz, 2H), 3.84 (s, 3H), 2.46 (s, 3H), 1.81 (s, 3H). Purity by HPLC-UV (200-400 nm) >99%.

7-(5-(3-(hydroxymethyl)-4-(trifluoromethyl)phenyl)-1,3-dimethyl-1H-pyrazol-4-yl)-2,5dimethylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (1c). Yield: 18.3 nmol, 2.3% from (1). HRMS: m/z 433.1590 (-1.0 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 7.75 (s, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 5.48 (s, 1H), 4.63 (s, 2H), 3.79 (s, 3H), 2.44 (s, 3H), 2.19 (s, 3H), 1.89 (s, 3H). Purity by HPLC-UV (200-400 nm) >99%.

5-(hydroxymethyl)-7-(3-(hydroxymethyl)-1-methyl-5-(3-methyl-4-(trifluoromethyl)phenyl)1H-pyrazol-4-yl)-2-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (1d). Yield: 15 nmol, 1.9% from (1). HRMS: m/z 449.1544 (0.1 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 7.67 (d, J = 8.1 Hz, 1H), 7.57 (s, 1H), 7.29 (d, J = 8.1 Hz, 1H), 5.29 (s, 1H), 4.65 (d, J = 4.7 Hz, 2H), 4.48 (d, J = 5.4 Hz, 2H), 3.84 (s, 3H), 2.45 (s, 3H), 1.83 (s, 3H). Purity by HPLC-UV (200-400 nm) >99%.

7-(1,3-dimethyl-5-(3-methyl-4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-5(hydroxymethyl)-2-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (1e). Yield: 11 nmol, 1.4% from (1). HRMS: m/z 433.1593 (-0.3 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 7.65 (d, J = 8.2 Hz, 1H), 7.58 (s, 1H), 7.29 (d, J = 8.2 Hz, 1H), 4.65 (d, J = 4.3 Hz, 2H), 3.81 (s, 3H), 2.44 (s, 3H), 2.20 (s, 3H), 1.94 (s, 3H). Purity by HPLC-UV (200-400 nm) >99%.



4-(2,5-dimethyl-4-oxo-3,4-dihydroimidazo[5,1-f][1,2,4]triazin-7-yl)-1-methyl-5-(3-methyl-4(trifluoromethyl)phenyl)-1H-pyrazole-3-carboxylic acid (1f). Yield: 6.5 nmol, 0.8% from (1). HRMS: m/z 447.1388 (0.2 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 7.65 (d, J = 8.1 Hz, 1H), 7.56 (s, 1H), 7.31 (d, J = 8.1 Hz, 1H), 3.83 (s, 3H), 2.41 (s, 3H), 2.39 (s, 3H), 2.04 (s, 3H). Purity by HPLC-UV (200-400 nm) >99%.

5-(hydroxymethyl)-2-methyl-7-(3-methyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4yl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one (2a). Yield: 26 nmol, 3.2% from (2). HRMS: m/z 405.1270 (-2.8 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 7.60-7.66 (m, 4H), 4.72 (d, J=5.7 Hz, 2H), 1.99 (s, 3H). Purity by HPLC-UV (200-400 nm) 98%.

7-(3-(hydroxymethyl)-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-5-(methoxymethyl)2-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (2b). Yield: 10 nmol, 1.3% from (2). HRMS: m/z 435.1381 (-1.3 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 7.78 (d, J=7.5 Hz, 2H), 7.59 (d, J=7.5 Hz, 2H), 4.67 (d, J=4.7 Hz, 2H), 4.51 (d, J=5.7 Hz, 2H), 3.85 (s, 3H). Purity by HPLC-UV (200-400 nm) >99%.

5-(methoxymethyl)-2-methyl-7-(3-methyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4yl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one (2c). Yield: 250 nmol, 31% from (2). HRMS: m/z 419.1425 (-3.1 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 7.76 (d, J=8.1 Hz, 2H), 7.57 (d, J=8.1 Hz, 2H), 5.14 (s, 1H), 4.66 (s, 2H), 3.82 (s, 3H), 2.22 (s, 3H), 1.91 (s, 3H). Purity by HPLC-UV (200-400 nm) >99%.


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7-(1,3-dimethyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-5-(methoxymethyl)-2methyl-4-oxo-3,4-dihydroimidazo[5,1-f][1,2,4]triazine 1-oxide (2d). Yield: 14 nmol, 1.8% from (2). HRMS: m/z 449.1532 (-2.6 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 7.76 (d, J=8.2 Hz, 2H), 7.56 (d, J=8.2 Hz, 2H), 4.59 (s, 2H), 3.80 (s, 3H), 3.20 (s, 3H), 2.24 (s, 3H), 2.06 (s, 3H). Purity by HPLC-UV (200-400 nm) >99%.

5-(difluoromethyl)-7-(3-(hydroxymethyl)-1-methyl-5-(4-(trifluoromethyl)phenyl)-1Hpyrazol-4-yl)-2-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (3a). Yield: 264 nmol, 33% from (3). HRMS: m/z 455.1244 (-1.2 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6) δ 12.13 (s, 1H), 7.79 (d, J = 8.1 Hz, 2H), 7.62 (d, J = 8.1 Hz, 2H), 7.21 (t, J = 53.6 Hz, 1H), 4.95 (t, J = 6.0 Hz, 1H), 4.55 (d, J = 6.0 Hz, 2H), 3.85 (s, 3H), 1.97 (s, 3H). Purity by HPLC-UV (200-400 nm) 94%.

5-(difluoromethyl)-2-methyl-7-(3-methyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4yl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one (3b). Yield: 100 nmol, 13% from (3). HRMS: m/z 425.1138 (-1.3 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6) δ 13.34 (s, 1H), 7.66 (d, J = 8.1 Hz, 2H), 7.59 (d, J = 8.1 Hz, 2H), 7.28 (t, J = 53.8 Hz, 1H), 2.26 (s, 3H), 2.02 (s, 3H). Purity by HPLC-UV (200-400 nm) >99%. Purity by HPLC-UV (200-400 nm) >99%.

7-(5-(4-chlorophenyl)-1,3-dimethyl-1H-pyrazol-4-yl)-5-(hydroxymethyl)-2methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (4a). Yield: 17 nmol, 2.1% from (4). HRMS: m/z 385.1171 (-0.9 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 7.46 (d, J=8.3 Hz, 2H),



7.37 (d, J=8.3 Hz, 2H), 4.64 (d, J=5.2 Hz, 2H), 3.77 (s, 3H), 2.18 (s, 3H), 1.98 (s, 3H) Purity by HPLC-UV (200-400 nm) 95%.

7-(5-(4-chlorophenyl)-3-(hydroxymethyl)-1-methyl-1H-pyrazol-4-yl)-2,5dimethylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (4b). Yield: 13 nmol, 1.6% from (4). HRMS: m/z 385.1173 (-0.3 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 7.48 (d, J=8.48 Hz, 2H), 7.36 (d, J=8.48 Hz, 2H), 4.47 (d, J=5.20 Hz, 2H), 3.80 (s, 3H), 2.46 (s, 3H), 1.89 (s, 3H). Purity by HPLC-UV (200-400 nm) 94%.

7-(5-(6-hydroxypyridin-3-yl)-3-methyl-1H-pyrazol-4-yl)-2,5-dimethylimidazo[5,1f][1,2,4]triazin-4(3H)-one (5a). Yield: 45 nmol, 5.6% from (5). HRMS: m/z 338.1361 (0.3 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 11.59 (bs, 1H), 8.17 (s, 1H), 7.58 (s, 1H), 6.31 (d, J = 9.5 Hz, 1H), 2.17 (s, 3H), 2.08 (s, 3H). Purity by HPLC-UV (200-400 nm) >99%.

5-(hydroxymethyl)-7-(5-(6-methoxypyridin-3-yl)-3-methyl-1H-pyrazol-4-yl)-2methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (5b). Yield: 33 nmol, 4.1% from (5). HRMS: m/z 368.1468 (0.6 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 11.8 (s, 1H), 8.15 (s, 1H), 7.75 (dd, J = 2.5, 8.7 Hz, 1H), 6.77 (d, J = 8.7 Hz, 1H), 5.11 (t, J = 5.9 Hz, 1H), 4.72 (d, J = 5.9 Hz, 2H), 3.83 (s, 3H), 2.21 (s, 3H), 2.06 (s, 3H). Purity by HPLC-UV (200-400 nm) >99%.

7-(5-(6-methoxypyridin-3-yl)-3-methyl-1H-pyrazol-4-yl)-2,5-dimethylimidazo[5,1f][1,2,4]triazin-4(3H)-one (5c). Yield: 113 nmol, 14% from (5). HRMS: m/z 352.1517 (0.1 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 13.07 (s, 1H), 11.56 (s, 1H), 8.18 (s, 1H), 7.72


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(dd, J = 2.5, 8.6 Hz, 1H), 6.78 (d, J = 8.6 Hz, 1H), 3.83 (s, 3H), 2.19 (s, 3H), 2.02 (s, 3H). Purity by HPLC-UV (200-400 nm) >99%.

5-(hydroxymethyl)-7-(5-(6-methoxypyridin-3-yl)-1,3-dimethyl-1H-pyrazol-4-yl)-2methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (5d). Yield: 210 nmol, 26% from (5). HRMS: m/z 382.1623 (0.2 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 11.68 (s, 1H), 8.15 (d, J = 2.4 Hz, 1H), 7.71 (dd, J = 2.4, 8.6 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 5.06 (t, J = 5.9 Hz, 1H), 4.65 (d, J = 5.7 Hz, 2H), 3.86 (s, 3H), 3.77 (s, 3H), 2.19 (s, 3H), 2.02 (s, 3H). Purity by HPLCUV (200-400 nm) >99%.

5-(hydroxymethyl)-7-(3-(hydroxymethyl)-1-methyl-5-(5-(trifluoromethyl)pyridin-2-yl)-1Hpyrazol-4-yl)-2-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (6a). Yield: 11.5 nmol, 1.4% from (6). HRMS: m/z 436.1337 (-0.6 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 11.70 (s, 1H), 9.08 (d, J = 2.2 Hz, 1H), 8.21 (dd, J = 2.2, 8.4 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 5.34 (t, J = 6.3 Hz, 1H), 5.17 (t, J = 5.9 Hz, 1H), 4.70 (d, J = 5.8 Hz, 2H), 4.54 (d, J = 6.2 Hz, 2H), 4.01 (s, 3H), 1.72 (s, 3H). Purity by HPLC-UV (200-400 nm) 65%.

7-(1,3-dimethyl-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrazol-4-yl)-5-(hydroxymethyl)-2methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (6b). Yield: 196 nmol, 25% from (6). HRMS: m/z 420.1383 (-1.7 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 9.06 (d, J = 2.3 Hz, 1H), 8.19 (dd, J = 2.3, 8.3 Hz, 1H), 7.46 (d, J = 8.3 Hz, 1H), 5.14 (s, 1H), 4.69 (s, 2H), 3.99 (s, 3H), 2.28 (s, 3H), 1.80 (s, 3H). Purity by HPLC-UV (200-400 nm) >99%.



7-(3-(hydroxymethyl)-1-methyl-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrazol-4-yl)-2,5dimethylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (6c). Yield: 241 nmol, 30% from (6). HRMS: m/z 420.1384 (-1.5 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 11.51 (s, 1H), 9.08 (s, 1H), 8.21 (dd, J = 2.4, 8.4 Hz, 1H), 7.50 (d, J = 8.3 Hz, 1H), 5.34 (t, J = 6.2 Hz, 1H), 4.53 (d, J = 6.2 Hz, 2H), 4.00 (s, 3H), 2.51 (s, 3H), 1.69 (s, 3H). Purity by HPLC-UV (200-400 nm) >99%.

4-(2,5-dimethyl-4-oxo-3,4-dihydroimidazo[5,1-f][1,2,4]triazin-7-yl)-1-methyl-5-(5(trifluoromethyl)pyridin-2-yl)-1H-pyrazole-3-carbaldehyde (6d). Yield: 10.5 nmol, 1.3% from (6). HRMS: m/z 418.1232 (-0.4 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 11.64 (s, 1H), 9.12 (d, J = 2.3 Hz, 1H), 8.27 (dd, J = 2.3, 8.4 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 4.18 (s, 3H), 2.48 (s, 3H), 1.92 (s, 3H).

Purity by HPLC-UV (200-400 nm) 86%.

5-(5-((2-hydroxycyclopentyl)methyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-3-yl)-7methylimidazo[5,1-f][1,2,4]triazin-4(3H) (7a and 7b). Diastereomer 1 (7a): Yield: 53 nmol, 6.6% from (7). HRMS: m/z 370.1981 (-1.3 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 11.58 (bs, 1H), 8.39 (s, 1H), 7.83 (s, 1H),4.11 (m, 3H), 3.98 (s, 2H), 2.91 (m, 2H), there are multiple resonances with chemicals shifts between δ 2.2 and 1.2 that are consistent with a single oxidation on the cyclopentyl. 2.2– 2.07 (m, 2H), 1.95 (dt, J = 7.2, 14.2 Hz, 1H), 1.86 (dd, J = 6.7, 13.4 Hz, 1H), 1.80-1.65 (m, 0H), 1.72 – 1.60 (m, 1H), 1.55 – 1.42 (m, 1H), 1.35 (ddd, J = 5.6, 8.0, 13.5 Hz, 1H), 1.22 (m, 1H). Purity by HPLC-UV (200-400 nm) 99%. Diastereomer 2 (7b): Yield: 28 nmol, 3.4% from (7). HRMS: m/z 370.1982 (-1.1 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 11.57 (s, 1H), 8.39 (s, 1H), 7.83 (s, 1H), 4.51 (d, J = 4.3 Hz, 1H), 4.12 (t, J =


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5.7 Hz, 2H), 3.99 (s, 2H), 3.77 (d, J = 6.1 Hz, 1H), 2.93 (m, 2H), 2.65 – 2.57 (m, 1H), 2.42 – 2.28 (m, 1H), 1.97 (m, 1H), 1.89 – 1.80 (m, 1H), 1.75 (m, 1H), 1.63 (m, 1H), 1.52 (m, 1H), 1.50 – 1.41 (m, 1H), 1.24 (m, 1H). Purity by HPLC-UV (200-400 nm) >99%.

2-(3,4-dimethoxybenzyl)-5-methyl-7-((2R,3R)-2,5,6-trihydroxy-6-phenylhexan-3yl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one (8a). Yield: 11 nmol, 1.4% from (8). HRMS: m/z 509.2386 (-1.7 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 11.59 (s, 1H), 6.98 (2 1H), 6.91 (d, J = 8.1Hz, 1H), 6.86 (d, J = 8.1 Hz, 1H), 6.78 – 6.69 (m, 2H), 5.96 (t, J = 10.1 Hz, 2H), 4.76 (d, J = 5.9 Hz, 1H), 3.80 (q, J = 6.6 Hz, 1H), 3.73 (s, 3H), 3.72 (s, 3H), 3.68 (s, 2H), 3.16 – 3.08 (m, 1H), 2.42 (s, 3H), 1.91 (m, 1H), 1.74 (m, 1H), 1.59 (m, 1H), 1.48 (m, 1H), 0.81 (d, J = 6.1 Hz, 3H). Purity by HPLC-UV (200-400 nm) 98%.

7-((2R,3R)-2,6-dihydroxy-6-phenylhexan-3-yl)-2-(4-hydroxy-3-methoxybenzyl)-5methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one and 7-((2R,3R)-2,6-dihydroxy-6-phenylhexan3-yl)-2-(3-hydroxy-4-methoxybenzyl)-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (structures uncertain). Isomer 1 (8b): Yield: 12.5 nmol, 1.5% from (8). HRMS: m/z 479.2279 (-2.1 ppm; M+H+).

1

H NMR (600 MHz, DMSO-d6): δ 11.55 (s, 1H), 8.91 (s, 1H), 7.26 (M,

2H), 7.19 (d, J = 7.3 Hz, 2H), 6.92 (s, 1H), 6.75 – 6.67 (m, 2H), 5.05 (d, J = 4.6 Hz, 1H), 4.75 (d, J = 5.8 Hz, 1H), 4.37 (q, J = 5.8 Hz, 1H), 3.81 (q, J = 6.7 Hz, 1H), 3.71 (s, 3H), 3.21 – 3.12 (m, 1H), 2.41 (s, 3H), 2.11 (m, 1H), 1.67 (m, 1H), 1.44 (m, 1H), 1.31 – 1.14 (m, 2H), 0.81 (d, J = 6.1 Hz, 3H). Purity by HPLC-UV (200-400 nm) 79%. Isomer 2 (8c): Yield: 10.5 nmol, 1.3% from (8). HRMS: m/z 479.2283 (-1.2 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 11.56 (s, 1H), 8.91 (s, 1H), 7.26 (m, 2H), 7.18 (m, 3H), 6.92 (d, J = 1.9 Hz, 1H), 6.76– 6.63 (m, 2H), 5.05



(d, J = 4.3 Hz, 1H), 4.74 (d, J = 5.8 Hz, 1H), 4.41 (q, J = 5.8 Hz, 1H), 3.82 (q, J = 6.8 Hz, 1H), 3.70 (s, 3H), 3.18 (ddd, J = 3.7, 8.7, 11.9 Hz, 1H), 2.43 (s, 3H), 1.93 (m, 1H), 1.82 (m, 1H), 1.29 (m, 2H), 0.81 (d, J = 6.2 Hz, 3H). Purity by HPLC-UV (200-400 nm) 93%.

7-((2R,3R)-2,6-dihydroxy-6-phenylhexan-3-yl)-2-(3,4-dimethoxybenzyl)-5methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (8d and 8e). Diastereomer 1 (8d): Yield: 52 nmol, 6.4% from (8). HRMS: m/z 493.2435 (-2.3 ppm; M+H+). 1H NMR (600 MHz, DMSOd6): δ 8.34 (s, 1H), 7.26 (m, 2H), 7.19 (d, J = 7.2 Hz, 3H), 6.96 (s, 1H), 6.85 (s, 2H), 5.05 (s, 1H), 4.74 (s, 1H), 4.36 (t, J = 6.6 Hz, 1H), 3.81 (t, J = 7.8 Hz, 1H), 3.73 (s, 3H), 3.69 (s, 3H), 3.15 (ddd, J = 3.9, 8.7, 11.9 Hz, 1H), 2.41 (s, 3H), 2.09 (m, 1H), 1.67 (m, 1H), 1.43 (m, 1H), 1.20 (m, 1H), 0.81 (d, J = 6.2 Hz, 3H). Purity by HPLC-UV (200-400 nm) 94%. Diastereomer 2 (8e): Yield: 81 nmol, 10% from (8). HRMS: m/z 493.2433 (-2.5 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 7.26 (m, 2H), 7.18 (m, 3H), 6.96 (d, J = 1.8 Hz, 1H), 6.82 (m, 2H), 5.06 (s, 1H), 4.75 (s, 1H), 4.41 (t, J = 6.6 Hz, 1H), 3.82 (t, J = 7.8 Hz, 1H), 3.71 (s, 3H), 3.70 (s, 3H), 3.18 (ddd, J = 3.8, 8.6, 12.0 Hz, 1H), 2.43 (s, 3H), 1.95 (dtd, J = 5.3, 10.5, 19.6 Hz, 1H), 1.81 (tt, J = 5.6, 11.0 Hz, 1H), 1.29 (m, 1H), 0.81 (d, J = 6.1 Hz, 3H). Purity by HPLC-UV (200-400 nm) 93%.

7-((2R,3R)-2,4 (or 2,5)-dihydroxy-6-phenylhexan-3-yl)-2-(3,4-dimethoxybenzyl)-5methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (8f). Yield: 6 nmol, 0.8% from (8). HRMS: m/z 493.2435 (-2.3 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 11.59 (s, 1H), 9.08 (s, 1H), 6.97 (s, 1H), 6.85 – 6.79 (m, 4H), 6.61 (d, J = 8.0 Hz, 2H), 4.77 (d, J = 5.7 Hz, 1H), 3.73 (s, 3H),


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3.69 (s, 3H), 2.43 (s, 3H), 2.01 (m, 1H), 1.75 (m, 1H), 1.50 (m, 2H), 0.82 (d, J = 6.4 Hz, 3H). Purity by HPLC-UV (200-400 nm) >99%.

2-(3,4-dimethoxybenzyl)-7-((2R,3R)-2-hydroxy-6-oxo-6-phenylhexan-3-yl)-5methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (8g). Yield: 29 nmol, 3.6% from (8). HRMS: m/z 491.2280 (-1.8 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 11.56 (s, 1H), 7.78 (d, J = 7.7 Hz, 2H), 7.59 (m, 2H), 7.45 (t, J = 7.6 Hz, 3H), 6.97 (s, 1H), 6.83 (d, J = 2.9 Hz, 2H), 4.87 (d, J = 5.7 Hz, 1H), 3.92 (q, J = 6.7 Hz, 1H), 3.71 (s, 3H), 3.70 (s, 3H), 3.62 (d, J = 6.4 Hz, 2H), 3.29 – 3.24 (m, 1H), 2.85 (m, 1H), 2.80 – 2.70 (m, 1H), 2.40 (s, 3H), 2.36 (m, 1H), 2.16 – 2.06 (m, 1H), 0.88 (d, J = 6.2 Hz, 3H). Purity by HPLC-UV (200-400 nm) 97%.

2-(3,4-dimethoxybenzyl)-5-methyl-7-((2S,3S)-2,4,6-trihydroxy-6-phenylhexan-3yl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one (9a). Yield: 21 nmol, 2.6% from (9). HRMS: m/z 509.2387 (-1.5 ppm; M+H+). The sample concentration and purity of this isolate prevented the explicit assignments of the majority of individual 1H resonances. The concentration was determined using the aromatic resonances. The structure was determined by comparing the 2D HSQC data of the parent with that of the isolated material. The proposed structure was also supported by HRMS and MS/MS data. Purity by HPLC-UV (200-400 nm) 90%.

7-((2S,3S)-2,6-dihydroxy-6-phenylhexan-3-yl)-2-(3-hydroxy-4-methoxybenzyl)-5methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (9b). Yield: 14 nmol, 1.7% from (9). HRMS: m/z 479.2285 (-0.8 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6) δ7.25 (m, 2H), 7.18 (m, 3H), 6.92 (d, J=1.8, 1H), 6.72 (dd, J=8.5, 1.8 Hz, 1H), 6.68 (d, J=8.5 Hz, 1H), 5.05 (d, J=3.6 Hz, 1H),



4.73 (d, J=6.1 Hz, 1H), 4.4 (m, 1H), 3.83 (m, 1H), 3.71 (s, 3H), 3.64 (s, 2H) 3.2 (m, 1H), 2.43 (s, 3H), 0.85 (s, 3H). Purity by HPLC-UV (200-400 nm) >99%.

7-((2S,3S)-2,6-dihydroxy-6-phenylhexan-3-yl)-2-(3,4-dimethoxybenzyl)-5methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (9c and 9d). Diastereomer 1 (9c): Yield: 50 nmol, 6.1% from (9). HRMS: m/z 493.2440 (-1.1 ppm; M+H+). 1H NMR (600 MHz, DMSOd6): δ 7.26 (m, 2H), 7.19 (d, J=7.0 Hz, 3H), 6.96 (s, 1H), 6.85 (s, 2H), 5.05 (d, J=4.6, 1H), 4.74 (d, J=5.9 Hz, 1H), 4.38 (m, 1H), 3.82 (m, 1H), 3.71 (m, 6H), 3.68 (s, 2H), 3.15 (m, 1H), 2.42 (s, 3H), 2.1 (m, 1H), 1.65 (m, 1H), 1.44 (m, 1H), 1.20 (m, 1H), 0.82 (d, J=6.2 Hz, 3H). Purity by HPLC-UV (200-400 nm) 98%. Diastereomer 2 (9d): Yield: 180 nmol, 23% from (9). HRMS: m/z 493.2440 (-1.1 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 7.26 (m, 2H), 7.19 (m, 3H), 6.96 (d, J=1.7 Hz, 1H), 6.83 (m, 2H), 4.41 (t, J=6.5 Hz, 1H), 3.82 (m, 1H), 3.71 (s, 3H), 3.69 (s, 3H), 3.68 (s, 2H), 3.18 (m, 1H), 2.43 (s, 3H), 1.87 (m, 2H), 1.30 (m, 2H), 0.81 (d, J=6.2 Hz, 3H). Purity by HPLC-UV (200-400 nm) 99%.

2-(3,4-dimethoxybenzyl)-7-((2S,3S)-2-hydroxy-6-oxo-6-phenylhexan-3-yl)-5methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (9e). Yield: 42 nmol, 5.2% from (9). HRMS: m/z 491.2283 (-1.2 ppm; M+H+). 1H NMR (600 MHz, DMSO-d6): δ 7.78 (d, J=7.5 Hz, 2H), 7.58 (m, 1H), 7.45 (m, 2H), 6.97 (s, 1H), 6.83 (m, 2H), 3.92 (m, 1H), 3.72 (m, 3H), 3.69 (m, 3H), 3.62 (m, 1H), 2.80 (m, 2H), 2.40 (s, 3H), 0.88 (d, J=6.2 Hz, 3H). Purity by HPLC-UV (200-400 nm) 95%.


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Phosphodiesterase 2A1 Assay. The human phosphodiesterase (PDE) 2A1 assay measures the conversion of 3’, 5’-[3H] cGMP to 5’-[3H] GMP. Yttrium silicate (YSi) scintillation proximity (SPA) beads bind selectively to 5’-[3H] GMP, with the magnitude of radioactive counts being directly related to PDE enzymatic activity. The assay was performed in white walled opaque bottom 384-well plates. 0.5 µL of compound in dimethyl sulfoxide was added to each well. Enzyme (15 µL) was then added to each well in buffer (in mM: Trizma, 50 (pH7.5); MgCl2, 1.3 mM) containing Brij 35 (0.01% (v/v)). Subsequently, 10 µl of 3’,5’-[3H] cGMP (125 nM) was added to each well to start the reaction and the plates were incubated for 30 minutes at 25oC. The reaction was terminated by the addition of 10 µL of PDE YSi SPA beads (Perkin Elmer). Following an additional 1 hour incubation period the plates were read on a MicroBeta radioactive plate counter (Perkin Elmer, Waltham. MA) to determine radioactive counts per well. Inhibition curves were plotted from individual experiments, and IC50 values were determined using a four parameter logistic fit. IC50 is defined as the concentration of the test article that produced a 50% inhibition of a maximal response. Measurement of Intrinsic Clearance in Pooled Human Hepatocytes. Volumes of solutions of compounds in d6-DMSO were added to a 96-well plate and subjected to vacuum centrifugation to remove the solvent (which can affect hepatocyte viability and rates of drug metabolizing enzymes). Volumes added were such that assay concentrations would be 1 µM. To the each well of the plate was added 0.05 mL Williams E medium followed by vortex mixing and sonication of the plate to reconstitute the substrate. Human hepatocytes suspended in Williams E medium were added to each well to yield a final volume of 0.2 mL and hepatocyte concentration of 680000 cells/mL. Incubations were carried out on rotating shaker inside a 37oC humidified incubator (90% humidity; 95% O2/5% CO2). At 0, 0.5, 1, 2, 3, and 4 hr, aliquots (0.025 mL)



were removed and added to 0.075 mL water/CH3CN (2:1) containing 2% formic acid and clozapine (0.5 µM; as internal standard) to terminate the incubation. The mixtures were spun in a centrifuge (1700 g; 5 min) and the supernatants were analyzed by HPLC-HRMS. Samples (0.01 mL) were injected onto a Phenomenex Kinetix C18 column (2.1 x 50 mm; 1.7 µ) equilibrated in a mobile phase consisting of 0.1% formic acid in water (95%) and CH3CN (5%) at 0.4 mL/min. This composition was held for 0.5 min followed by a linear gradient to 95% CH3CN at 2 min, held for 0.5 min, followed by re-equilibration to initial conditions for 0.5 min. The eluent was introduced into the aforementioned Thermo Orbitrap Elite mass spectrometer operated in the positive ion mode. Data were collected in MS1 full scan at a resolution setting of 30000. The m/z values for protonated molecular ions were extracted at 5 ppm to generate reconstructed ion chromatograms for each analyte. Plots of the natural logarithm of peak area ratios (normalized to those measured at t=0) vs time were used to estimate first order substrate depletion rate constants (k), which were used to calculate intrinsic clearance by dividing k by the hepatocyte concentration in the incubation. Non-specific binding was not measured in these incubations. Permeability Assay in RRCK Cells. RRCK permeability data were generated using a previously described method.13 SUPPORTING INFORMATION Supporting information include 1H spectra of compounds 1 and 1e, 1H-13C multiplicity edited HSQC spectrum of compound 1e, 1H spectra of compounds 2 and 2c, 1H spectrum of compound 4, and 1H, 1H-1H COSY, and 1H-13C multiplicity edited HSQC spectra for compound 4b.

CORRESPONDING AUTHOR INFORMATION


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Address correspondence to R. Scott Obach, Pfizer Inc., Groton, CT 06340. E-mail: [email protected]

PRESENT ADDRESS Current Address: Antonia Stepan, Boehringer Ingelheim Pharma, Biberach an der Riß, Germany

ACKNOWLEDGEMENTS The authors wish to acknowledge Jim Federico for running the permeability assay. Support and enthusiasm for this endeavor from Drs. Douglas Spracklin and Michael Brodney has been greatly appreciated.

NON-STANDARD ABBREVIATIONS USED qNMR, quantitative nuclear magnetic resonance spectroscopy; RRCK, Ralph-Russ canine kidney; TOCSY, total correlation spectroscopy



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2. Cernak, T.; Dykstra, K.D.; Tyagarajan, S.; Vachal, P.; Krska, S.W. The medicinal chemist’s toolbox for late stage functionalization of drug-like molecules. Chem. Soc. Rev. 2016, 45, 546576.

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Table 1. PDE2 Potency, Human Hepatocyte Intrinsic Clearance, and Membrane Permeability of Lead Compound 1 and its Diversified Products. R2

O HN

N N N

R1

F F N

F

N

R4 Compound 1

Compound

R1

R2

R4

clogP

TPSA (Å2)

-CH3 -CH3 -CH3 2.2 80.9 1 -CH3 -CH2OH -CH2OH -0.7 121.3 1a -CH2OH -0.7 121.3 -CH2OH -CH3 1b -CH3 -CH3 -CH2OH 0.6 101.1 1c -CH2OH -CH2OH -CH3 -0.4 121.3 1d -CH3 -CH2OH -CH3 0.9 101.1 1e -COOH -CH3 -CH3 1.4 118.2 1f a Geometric mean of three determinations b Average fit value of replicate determinations c Average of two replicates d Under the limit of quantifiable intrinsic clearance, but observable decline

PDE2 IC50 (nM)a

0.16 19.7 186 13.4 5.94 0.20 28.6


Hepatocyte RRCK Intrinsic Permeability; Clearance; CLint Papp (10-6 (µL/min/million cm/sec)c cells)b 19 28 stable 2000 69.2 70.1 18.6


Hepatocyte Intrinsic Clearance; CLint (µL/min/million cells)b stable stable stable 1.9 stable

RRCK Permeability; Papp (10-6 cm/sec)c

lipE

no data

Lead Diversification at the Nanomole Scale Using Liver Microsomes

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