Lead Diversification at the Nanomole Scale Using Liver Microsomes

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Article Cite This: J. Med. Chem. 2018, 61, 3626−3640

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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§,∥ †

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 ‡

S Supporting Information *

ABSTRACT: In this report, we describe a method whereby lead molecules can be converted into several new analogues each using liver microsomes. Less than one 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− 10× 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.

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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 μM.8 Overall, this approach is consistent with principles of green chemistry in that minimal amounts of compound are used, reactions and product isolation 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 moderate 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

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 submilligram 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 © 2018 American Chemical Society

Received: January 22, 2018 Published: March 30, 2018 3626

DOI: 10.1021/acs.jmedchem.8b00116 J. Med. Chem. 2018, 61, 3626−3640

Journal of Medicinal Chemistry

Article

Figure 1.

Scheme 1. Synthesis of Compounds 1, 4, and 5

potency of the parent molecule and was then of greater interest than the parent compound. 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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Scheme 2. Synthesis of Compounds 2 and 3 from Intermediate 14

RESULTS

General Approach. Nine lead compounds, seven of which were prepared as shown in Schemes 1, 2, 3, and 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 3627

DOI: 10.1021/acs.jmedchem.8b00116 J. Med. Chem. 2018, 61, 3626−3640

Journal of Medicinal Chemistry

Article

Scheme 3. Synthesis of Compound 6

Scheme 4. Synthesis of Compound 7

Figure 2. HPLC-UV chromatograms of extracts of liver microsomal incubations of 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 3628

DOI: 10.1021/acs.jmedchem.8b00116 J. Med. Chem. 2018, 61, 3626−3640

Journal of Medicinal Chemistry

Article

Figure 3. HPLC fractionation of biosynthesis of products from compound 1. (Top) UV data (200−400 nm). (Bottom) Combined Extracted Ion Chromatogram of m/z 417 (compound 1), 431, 433, and 449. Products 1a−1f are denoted with arrows. Other products that were not of adequate purity or concentration are denoted with dashed arrows.

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

compound

R1

R2

R4

c log P

TPSA (Å2)

PDE2 IC50 (nM)a

hepatocyte intrinsic clearance; CLint (μL/min/million cells)b

RRCK permeability; Papp (10−6 cm/sec)c

lipE

1 1a 1b 1c 1d 1e 1f

−CH3 −CH3 −CH2OH −CH3 −CH2OH −CH3 −COOH

−CH3 −CH2OH −CH3 −CH3 −CH2OH −CH2OH −CH3

−CH3 −CH2OH −CH2OH −CH2OH −CH3 −CH3 −CH3

2.2 −0.7 −0.7 0.6 −0.4 0.9 1.4

80.9 121.3 121.3 101.1 121.3 101.1 118.2

0.16 19.7 186 13.4 5.94 0.20 28.6

19 stable stable 3.9 stable stabled stabled

28 2000 69.2 70.1 18.6

stable stable stable 1.9 stable

no data 99%. 5-(Methoxymethyl)-2-methyl-7-(3-methyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)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%. 7-(1,3-Dimethyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-5(methoxymethyl)-2-methyl-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)-1H-pyrazol-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-4-yl)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)-2-methylimidazo[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+). 1 H 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-pyrazol4-yl)-2,5-dimethylimidazo[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%.

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 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 LC−MS [M + H]+ m/z = 404. Synthesis of Substrate 7. tert-Butyl 3-(4-methoxy-7methylimidazo[5,1-f ][1,2,4]triazin-5-yl)-6,7-dihydropyrazolo[1,5-a]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 and then added to a degassing and preheated (50 °C) solution of bromide 23 (1.0 g, 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, and then heated to 80 °C under nitrogen atmosphere. 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 LC− MS [M + H]+ m/z = 386. 5-(5-(Cyclopentylmethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-3-yl)-7-methylimidazo[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 aminotriazinone hydrochloride salt as a yellow solid. The resulting solid was taken up in 1,2-dichloroethane (10 mL) and 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 in succession. 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 (30 mL), washed with saturated sodium bicarbonate and water (15 mL each). The organic layer was dried over magnesium sulfate, filtered, and filtrateconcentrated. 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 LC−MS [M + H]+ m/z = 354.2033. Spectral Properties of Biosynthesized Analogues. 5-(Hydroxymethyl)-7-(5-(3-(hydroxymethyl)-4-(trifluoromethyl)phenyl)-1,3-dimethyl-1H-pyrazol-4-yl)-2-methylimidazo[5,1-f ][1,2,4]triazin4(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-1H-pyrazol-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 (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)-2,5-dimethylimidazo[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%. 3637

DOI: 10.1021/acs.jmedchem.8b00116 J. Med. Chem. 2018, 61, 3626−3640

Journal of Medicinal Chemistry

Article

7-(5-(6-Hydroxypyridin-3-yl)-3-methyl-1H-pyrazol-4-yl)-2,5dimethylimidazo[5,1-f ][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-1Hpyrazol-4-yl)-2-methylimidazo[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)-2,5dimethylimidazo[5,1-f ][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 (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-dimethyl1H-pyrazol-4-yl)-2-methylimidazo[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 HPLC-UV (200−400 nm) > 99%. 5-(Hydroxymethyl)-7-(3-(hydroxymethyl)-1-methyl-5-(5(trifluoromethyl)pyridin-2-yl)-1H-pyrazol-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-4yl)-5-(hydroxymethyl)-2-methylimidazo[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 HPLCUV (200−400 nm) > 99%. 7-(3-(Hydroxymethyl)-1-methyl-5-(5-(trifluoromethyl)pyridin-2yl)-1H-pyrazol-4-yl)-2,5-dimethylimidazo[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-7yl)-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)-7-methylimidazo[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, 2H), 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+). 1 H 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 = 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-6phenylhexan-3-yl)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 (s 1H), 6.91 (d, J = 8.1 Hz, 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-3methoxybenzyl)-5-methylimidazo[5,1-f ][1,2,4]triazin-4(3H)-one and 7-((2R,3R)-2,6-dihydroxy-6-phenylhexan-3-yl)-2-(3-hydroxy-4methoxybenzyl)-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+). 1H 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)-5-methylimidazo[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, DMSO-d6): 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)-5-methylimidazo[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), 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)-5-methylimidazo[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-6phenylhexan-3-yl)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 3638

DOI: 10.1021/acs.jmedchem.8b00116 J. Med. Chem. 2018, 61, 3626−3640

Journal of Medicinal Chemistry

Article

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-4methoxybenzyl)-5-methylimidazo[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)-5-methylimidazo[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, DMSO-d6): 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)-5-methylimidazo[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%. 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. Compound (0.5 μL) in dimethyl sulfoxide was added to each well. Enzyme (15 μL) was then added to each well in buffer [in mM: Trizma, 50 (pH 7.5); 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 min at 25 °C. The reaction was terminated by the addition of 10 μL of PDE YSi SPA beads (PerkinElmer). Following an additional 1 h incubation period, the plates were read on a MicroBeta radioactive plate counter (PerkinElmer, 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 37 °C humidified incubator (90% humidity; 95% O2/5% CO2). At 0, 0.5, 1, 2, 3, and 4 h, 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 (1700g; 5 min), and the supernatants were analyzed by HPLC-HRMS. Samples (0.01 mL) were injected onto a Phenomenex Kinetix C18 column (2.1 × 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 a 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) versus 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. Nonspecific binding was not measured in these incubations. Permeability Assay in RRCK Cells. RRCK permeability data were generated using a previously described method.13

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00116. 1 H 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 1 H, 1H−1H COSY, and 1H−13C multiplicity edited HSQC spectra for compound 4b (PDF) PDE2 IC50 (CSV)

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AUTHOR INFORMATION

Corresponding Author

*Address: Pfizer Inc., Groton, CT 06340. E-mail: r.scott.obach@ pfizer.com. ORCID

R. Scott Obach: 0000-0002-6604-401X Tuan P. Tran: 0000-0001-9578-639X Antonia F. Stepan: 0000-0003-2203-129X Present Address ∥

Current Address: Boehringer Ingelheim Pharma, Biberach an der Riß, Germany. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS 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 have been greatly appreciated.

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

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REFERENCES

(1) Wencel-Delord, J.; Glorius, F. C-H bond activation enables the rapid construction and late-stage diversification of functional molecules. Nat. Chem. 2013, 5, 369−375. (2) Cernak, T.; Dykstra, K. D.; Tyagarajan, S.; Vachal, P.; Krska, S. W. The medicinal chemist’s toolbox for late stage functionalization of druglike molecules. Chem. Soc. Rev. 2016, 45, 546−576. (3) Sawayama, A. M.; Chen, M. Y.; Kulanthaivel, P.; Kuo, M. S.; Hemmerle, H.; Arnold, F. H. A panel of cytochrome P450 BM3 variants

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DOI: 10.1021/acs.jmedchem.8b00116 J. Med. Chem. 2018, 61, 3626−3640

Journal of Medicinal Chemistry

Article

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NOTE ADDED AFTER ASAP PUBLICATION This paper published ASAP on 4-9-2018. The Abstract graphic was replaced and the revised version was reposted on 4-16-2018.

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DOI: 10.1021/acs.jmedchem.8b00116 J. Med. Chem. 2018, 61, 3626−3640