Discovery of Potent and Selective Periphery-Restricted Quinazoline

2 days ago - We disclose the discovery and X-ray co-crystal data of potent, selective quinazoline inhibitors of PDE1. Inhibitor (S)-3 readily attains ...
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Discovery of Potent and Selective Periphery-Restricted Quinazoline Inhibitors of the Cyclic Nucleotide Phosphodiesterase PDE1 John M. Humphrey, Matthew Movsesian, Christopher W. am Ende, Stacey L Becker, Thomas A. Chappie, Stephen Jenkinson, Jennifer L Liras, Spiros Liras, Christine C Orozco, Jayvardhan Pandit, Felix F. Vajdos, Fabrice Vandeput, Eddie Yang, and Frank S Menniti J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00374 • Publication Date (Web): 02 May 2018 Downloaded from http://pubs.acs.org on May 4, 2018

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Journal of Medicinal Chemistry

Discovery of Potent and Selective Periphery-Restricted Quinazoline Inhibitors of the Cyclic Nucleotide Phosphodiesterase PDE1 John M. Humphrey,*a‡ Matthew Movsesian,b‡ Christopher W. am Ende,a Stacey L. Becker,a Thomas A. Chappie,c Stephen Jenkinson,d Jennifer L. Liras,c Spiros Liras,c Christine Orozco,a Jayvardhan Pandit, a Felix F. Vajdos,a Fabrice Vandeput,b† Eddie Yang,a and Frank S. Menniti.*e ‡ a

Pfizer World Wide Research and Development, Eastern Point Road, Groton, CT 06340, USA. Division of Cardiovascular Medicine, University of Utah School of Medicine, 30 N 1900 E, Room: 4A-100, Salt Lake City, UT 84132, USA
 c Pfizer Worldwide Research and Development, 1 Portland Street, Cambridge, MA 02139, USA b

d

Pfizer World Wide Research and Development, La Jolla, CA 92121, USA
 MindImmune Therapeutics, Inc., and the George & Anne Ryan Institute for Neuroscience, University of Rhode Island, 7 Greenhouse Road, Kingston, RI 02881, USA
 e

ABSTRACT: We disclose the discovery and X-ray co-crystal data of potent, selective quinazoline inhibitors of PDE1. Inhibitor (S)-3 readily attains free plasma concentrations above PDE1 IC50 values and has restricted brain access. The racemic compound 3 inhibits >75% of PDE hydrolytic activity in soluble samples of human myocardium, consistent with heightened PDE1 activity in this tissue. These compounds represent promising new tools to probe the value of PDE1 inhibition in the treatment of cardiovascular disease.

Introduction Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes that shape the spatial and temporal aspects of second messenger signaling throughout the body. Enzymes in the PDE1 family hydrolyze both cGMP and cAMP in a mutually competitive manner. PDE1 catalytic activity is uniquely stimulated by binding to Ca2+/calmodulin,1 giving this class a defining role in the integration of Ca2+- and cyclic nucleotide-mediated signaling. The PDE1 family includes three isoforms, PDE1A, PDE1B and PDE1C, which differ with respect to their relative affinities for cGMP and cAMP. These isoforms are differentially expressed throughout the body, including the central nervous (CNS) and cardiovascular systems.2-3 While PDE1 is among the most studied of the PDE families, full elucidation of the scope of PDE1-regulated signaling has been hampered, until recently, by a lack of potent and selective inhibitors. We recently disclosed the invention of small molecule, brain-penetrant PDE1 inhibitors containing bis- and tri-methoxy-quinazoline scaffolds (compounds 1 and 2, Figure 1) along with the X-ray structure of 1 bound to the PDE1B catalytic domain.4 Others have recently reported on the discovery and X-ray co-crystal structures of selective, brain-penetrant tetracyclic pryimidone-based PDE1 inhibitors.5-6 These compounds may serve as probes to assess the role of PDE1 in brain, and the potential value of such com-

pounds to treat diseases of the CNS, including attention deficit disorder, Parkinson’s disease and various cognitive dysfunctions. Given cardiovascular expression, PDE1 inhibitors could offer therapeutic utility to treat cardiovascular disease (vide infra) with the caveat that CNS exposure may pose a liability. Here we report on the discovery and X-ray co-crystal data of a new set of quinazoline inhibitors, exemplified by quinazoline 3 (PF-04677490, Figure 1), that offer high potency and PDE1 selectivity and do not readily enter the CNS.7 We show further that 3 efficaciously inhibits cAMP- and cGMP-hydrolytic activity in preparations from human myocardium at concentrations selective for PDE1. These periphery-restricted inhibitors thus offer the potential to target PDE1 in the heart while limiting confounding effects that may result from PDE1 inhibition in the CNS.

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Table 2. Panel Screen IC50 values of (S)-3 for inhibition of cAMP hydrolysis by PDE1 isoforms and PDEs representative of each of the other ten PDE subtypes. a, b, c

Figure 1. Quinazoline PDE1 Inhibitors.

Results Lead matter for this series of quinazoline PDE1 inhibitors (compound 4, PDE1B IC50 = 52 nM; Figure 2) was derived from high-throughput screening of a compound library at Pfizer Inc. as previously disclosed.8 Consistent with SAR findings about inhibitors 1 and 2, incorporation of a methoxy substituent in the quinazoline 8-position enhanced potency (5, 27 nM), and deletion of the 6-position methoxy group afforded comparable potency in the racemic compound 3 (21 nM, Figure 1). Pyrazole modifications, including N-methylation, transposition of the methyl group, and transposition of the nitrogen atoms, significantly abated potency. These observations indicate that the pyrazole component, as it exists in 3, is essential for PDE1 affinity. After HPLC chiral resolution the (S)-enantiomer (S)-3 (PF-04827736) proved 15-65x more potent than the (R)enantiomer (R)-3 vs. the PDE1 isoforms (Figure 2 and Table 1). The quinazoline (S)-3 selectively inhibits human PDE1 over the other ten superfamily members (PDE2 – PDE11). The selectivity ratios are high, with the exception of PDE10, for which moderate selectivity exists (10-45x, Table 2). We note that PDE10A is expressed almost exclusively in brain, and evidence for PDE10A-mediated signaling in peripheral tissues is very limited.9-10 When assessed across a broad panel of receptors and enzymes (Cerep), (S)-3 exhibited no significant inhibition (>50%) at a concentration of 10 µM (see Supporting Information).

Figure 2. HTS lead compound 4 and derived inhibitors.

Table 1. IC50 values of 3, (S)-3, and (R)-3, for inhibition of cAMP hydrolysis by human PDE1 isoforms (nM).a 3

(S)-3

(R)-3

PDE1A

118 ± 2

42 ± 5

777 ± 60

PDE1B1

21 ± 2

9.1 ± 0.5

596 ± 50

PDE1C

83 ± 3

38 ± 1

579 ± 60

a

Values represent the mean ± SEM (n = 5 or more).

IC50

vs. PDE1B

vs. PDE1A

PDE1A

42 ± 5 nM

4.6x

-

PDE1B1

9.1 ± 0.5 nM

-

-

PDE1C

38 ± 1 nM

4.2x

-

PDE2A1

47.6 ± 5 µM

5,230x

1,133x

PDE3A1

133 ± 3 µM

14,615x

3,167x

PDE4D3

26.3 ± 1 µM

2,890x

626x

PDE5A1

54.3 ± 1 µM

5967x

1,293x

PDE6A

> 142 µM

> 15,604x

> 3380x

PDE7B

41.3 ± 5 µM

4,538x

983x

PDE8B

15.5 ± 1 µM

1,703x

369x

PDE9A1

> 200 µM

> 21,978

> 4761x

PDE10A1

409 ± 30 nM

45x

10x

PDE11A4

34.7 ± 1 µM

3736x

826x

a

Values represent the mean ± SEM (n = 3 or more).

b

Enzymes are human in origin except for PDE6A (bovine).

c

Assays utilized cAMP or cGMP substrates, as appropriate for each enzyme, at concentrations < 1/3 of the Km (see Supporting Information).

The synthesis of inhibitor 3 was accomplished via SNAr reaction of 4-chloro-7,8-dimethoxyquinazoline 64 and the amine 7 (Scheme), the latter of which was prepared through a modification of a published procedure as described in the Experimental Section.11-12 After resolution by chiral HPLC (see Experimental Section), the absolute stereochemistry of (S)-3 was determined by X-ray crystallography of the PDE1B-bound structure (see Supporting Information, Figure S1). Scheme. Synthesis of Inhibitor (S)-3.

X-ray crystallography of a PDE1B-derived polypeptide containing the catalytic domain4, 13 provided insight into the stringent SAR requirements of PDE1 binding for this series. The X-ray crystal structure of (S)-3 bound within the catalytic do-

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Journal of Medicinal Chemistry main is illustrated in Figure 3. According to this structure, the methoxy groups accept a bifurcated hydrogen bond from Gln 421, while the quinazoline N1 atom accepts a critical hydrogen bond from His 373. The quinazoline N3 atom likewise accepts a hydrogen bond from a water molecule that is in turn tied to both the protein (via Tyr 222 and Asp 370) and the catalytic metal centers (via a second water molecule). The pyrazole ring inserts into a pocket formed by His 223, Phe 392, Glu 391, Tyr 222 and Leu 388. The sidechain of the latter residue has shifted somewhat vs. our previously reported structure to accommodate the pyrazole methyl substituent.4

Table 3. Mean drug concentrations of (S)-3 following a 1 mg/kg sc dose in rat.a

Time (h)

Free Plasma (nM)

Free Brain (nM)

Free Brain/ Free Plasma

CSF (nM)

CSF/ Free Plasma

0.5

171

6

0.04

21

0.12

1

120

4

0.03

16

0.13

2

40

2

0.1

8

0.4

4

7

BLQ

-

BLQ

-

a

Figure 3. X-ray crystal structure of compound (S)-3 bound to the catalytic domain of PDE1B (PDB ID code 5W6E). Color code: carbon, green; nitrogen, blue; oxygen, red.

To assess the degree to which compound (S)-3 penetrates the brain after systemic administration, neuropharmacokinetics (n-PK) were studied in rat after a subcutaneous dose of 1 mg/kg. Plasma, brain, and CSF concentrations of compound (S)-3 were determined at 0.5, 1, 2 and 4 hours post dose (Table 3). Free plasma and free brain concentrations were then estimated by correcting for protein binding (plasma free fraction = 0.25 and brain free fraction = 0.20). Estimated mean free plasma concentrations were well above the PDE1 IC50 for the first hour, while the estimated mean free brain concentrations remained below the PDE1 IC50 at all time points.

n = 3 per time point.

As calculated from AUCs, the free partition coefficient (Kpuu) in brain exposure and the cerebrospinal fluid exposure relative to free plasma were 0.04 and 0.14 (see also Table 3), indicating that compound (S)-3 has limited access to the brain from the circulation. Although (S)-3 has a relatively high polar surface area (tPSA = 85.0) and has two hydrogen-bond donors, good brain exposure was predicted from a CNS multiparameter optimization algorithm (CNS MPO score- 5.5 out of 6, Table 4). 14 15 However, (S)-3 is subject to high efflux by Pglycoprotein (P-gp efflux ratio = 41) and breast cancer resistance protein (BCRP efflux ratio = 84), the two major efflux transporters at the blood-brain barrier. Thus, the restricted access to brain is hypothesized to result predominantly from efflux of (S)-3 from the brain mediated by these transporters. Transporter activities may also account for the observation that CSF concentrations of (S)-3 were slightly higher than free brain concentrations (Table 3). Given the potential therapeutic utility of PDE1 inhibitors in cardiovascular disease, we investigated the potency and efficacy of 3 for inhibition of cyclic nucleotide hydrolysis by human myocardium. The racemate was used for these experiments instead of (S)-3 because only the former was available at the time of experimentation. In this tissue, PDE1C1, an isoform with comparable affinity for cGMP and cAMP, is the predominant phosphodiesterase, with additional cGMPhydrolytic activity contributed by PDE3 and PDE5, and additional cAMP-hydrolytic activity contributed by PDE3. When examined for inhibitory activity against recombinant enzymes, compound 3 at a concentration of 1.0 µM inhibited ~90% of PDE1C1 activity with minimal inhibition of PDE5 and PDE3 (Figure 4). We therefore quantified the effects of compound 3 at 1.0 µM on cAMP and cGMP hydrolysis in soluble protein extracts from human left ventricle, with and without additive EGTA.16 As illustrated in Figure 5, compound 3 inhibited ~76% of the cAMP-hydrolytic activity, and ~80% of the cGMP-hydrolytic activity in these preparations. For both substrates, compound 3 inhibited ~90% of the Ca2+/calmodulinstimulated activity. These data are interpreted to indicate that compound 3 is a potent inhibitor of native cAMP- and cGMPhydrolytic activity expressed in human myocardium, consistent with PDE1 comprising the great majority of cyclic nucleotide-hydrolytic activity in similar human myocardium preparations.16

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Figure 4. Inhibition of cAMP and cGMP hydrolytic activity of recombinant enzymes by compound 3.17 All measurements were made at cAMP or cGMP concentrations of 0.1 µM.

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Figure 5. Inhibition of cAMP and of cGMP hydrolytic activity in human myocardium by compound 3 (1 uM). Assays were carried out in soluble protein extracts prepared from human myocardium, i) in the presence of CaCl2 and exogenous calmodulin (CM) (“total” activity) or ii) in the presence of the Ca2+ chelating agent EGTA, as described previously. All assays were carried out at substrate concentrations of 0.1 µM. Reported Ca2+/CM-dependent activity is the difference between total activity and activity in the presence of EGTA.16

Discussion and Conclusions We disclose a novel quinazoline that is a potent and selective PDE1 inhibitor. The SAR for this inhibitory activity is consistent with that described previously by our group with regard to quinazoline PDE1 inhibitors.4 In addition to being among the most potent analogs disclosed in the quinazoline series, the (S)-3 is distinguished by limited brain exposure, a feature that stems primarily from the compound being a substrate for blood brain barrier efflux transporter activities. Given that PDE1 isoforms are highly expressed throughout the brain, restricted brain exposure may be advantageous to the use of this compound as a probe for in vivo studies of the role of PDE1 in peripheral organ systems. In this regard, we report that systemic administration of compound (S)-3 readily delivers free plasma concentrations well in excess of the IC50 values for inhibition of PDE1 isoforms, supporting the utility of the compound as a probe for in vivo studies.

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Journal of Medicinal Chemistry Mounting evidence suggests an important role for PDE1 in cardiovascular disease. For example, inhibition of PDE1 prevents phenylephrine-induced myocyte hypertrophy in neonatal and adult rat ventricular myocytes.18 Inhibition of PDE1A reduces angiotensin-II or TGF-β-induced activation of rat cardiac fibroblasts, and attenuates isoproterenol-induced interstitial fibrosis in mice.19 Cellular senescence in vascular smooth muscle myocytes leads to elevated PDE1 expression, and PDE1 inhibition restores vasodilatory responses to sodium nitroprusside in aging mice.20 PDE1C expression in vascular smooth muscle cells in vitro increases with the transition from the contractile to the synthetic phenotype, and PDE1 inhibition attenuates proliferation and migration of vascular smooth muscle cells in culture.21 PDE1C expression is increased in mouse vascular injury models in vivo and in neointimal smooth muscle cells of human coronary arteries, and injuryinduced neointimal formation is reduced by PDE1 inhibition in coronary arteries of mice.21 Knockout of the PDE1C gene has anti-hypertrophic, anti-fibrotic and anti-apoptotic actions in mouse hearts.22 These observations suggest that PDE1 is a promising therapeutic target for cardiovascular disease. The PDE1 inhibitor 3 described here is highly efficacious for inhibition of both cAMP- and cGMP-hydrolytic activity in preparations from human myocardium, consistent with its potency and selectivity for PDE1. These characteristics combined with restricted CNS penetration ((S-3) to minimize central side effects, suggest utility for these compounds as new tools to further assess the value of PDE1 inhibition in the treatment of cardiovascular disease. Such studies are currently underway utilizing (S)-3 as a probe.

EXPERIMENTAL SECTION Reagents and starting materials were obtained from commercial sources and used without purification unless otherwise indicated. The chloroquinazoline 6 was obtained from commercial sources or prepared as described previously.4 Depending on storage conditions, 6 may undergo partial hydrolysis to the pyridone over time. In this event, either silica gel chromatography or a partition between DCM and 1M sodium hydroxide may be used to purge the pyridone impurity. Silica gel chromatography was performed with glass columns handpacked with JT Baker 40 µM flash silica gel. NMR spectra are presented as chemical shifts in ppm relative to the solvent with multiplicities reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br s (broad singlet), and br m (broad multiplet). Products were confirmed to be >95% pure by 1H NMR analysis and by HPLC (Waters Acuity instrumentation, CSH C18 1.7 µM 2.1 x 50 mm column, elution with an increasing gradient of acetonitrile in water with 0.1% formic acid as a modifier) with compound identified by UV = 215 nM and ESI positive ion mass spectrometry. (±)-7,8-Dimethoxy-N-(1-(5-methyl-1H-pyrazol-3yl)propan-2-yl)quinazolin-4-amine (3). To 4-chloro-7,8dimethoxyquinazoline (6) (1.00 g, 4.45 mmol) and 1-(5methyl-1H-pyrazol-3-yl)propan-2-amine dihydrochloride (1.06 g, 4.98 mmol) in DMF (10 mL) was added triethylamine (2.50 g, 25 mmol) and the mixture was heated at 75 °C for 2 h and an additional 10 h at 55 °C. The mixture was diluted with 10 mL water and cooled to rt with continued stirring. The mixture was stirred at rt for 1 h, and then was cooled in an ice bath and stirred for an additional 40 min. The resultant precipitate

was isolated via filtration, rinsed with water, and dried under a stream of air to afford a white solid in the amount of 1.21 g (83%): mp 230-232 °C; 1H NMR (400 MHz, CD3OD) δ 8.37 (s, 1H), 7.93 (d, J = 9.3 Hz, 1H)), 7.34 (d, J = 9.3 Hz, 1H), 5.90 (s, 1H), 4.69 (m, 1H), 3.99 (s, 3H), 3.90 (s, 3H), 2.92 (m, 2H), 2.17 (br s, 3H) 1.29 (d, J = 7.5 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) 159.2, 155.3, 154.5, 144.9, 142.1, 119.3, 112.9, 110.7, 103.6, 61.4, 56.8, 46.7, 34.8, 20.3; HRMS m/z: [M + H]+ calcd for C17H21N5O2, 328.1768; found, 328.1764. (S)-7,8-Dimethoxy-N-(1-(5-methyl-1H-pyrazol-3yl)propan-2-yl)quinazolin-4-amine ((S)-3) and (R)-7,8Dimethoxy-N-(1-(5-methyl-1H-pyrazol-3-yl)propan-2yl)quinazolin-4-amine ((R)-3). Chiral resolution was accomplished by SFC-HPLC on a Lux Amylose-1 250 mm x 30 mm 5 u chiral column employing isocratic elution with 70% CO2, 30% ethanol + 0.2% (7 N ammonia in MeOH) at a flow rate of 80.0 mL/min at a backpressure of 120 Bar with 210 nM detection. A starting mass of 2.37 g of racemate provided 1.22 g of (R)-3 (the first to elute, 5.96 min) followed by 1.26 g of (S)-3 (the second to elute, 7.19 min). Recrystallization from acetonitrile afforded pure (R)-3 (940 mg, 79% of the theoretical yield) and pure (S)-3 (946 mg, 80% of the theoretical yield). NMR and MS data are consistent with reported values for the racemate 3. For the more potent enantiomer (S)-3: mp 170-171 °C; HRMS m/z: [M + H]+ calcd for C17H21N5O2, 328.1768; found, 328.1764. Analysis calcd for C17H21N5O2: C, 62.37; H, 6.47; N, 21.39. Found: C, 62.00; H, 6.53; N, 21.05; [α]20D +131.1 (c 0.745, CH3OH). For (R)-3: mp 170-171 °C; HRMS m/z: [M + H]+ calcd for C17H21N5O2, 328.1768; found, 328.1765; Analysis calcd for C17H21N5O2: C, 62.37; H, 6.47; N, 21.39. Found: C, 62.20; H, 6.47; N, 21.39; [α]20D -128.2 (c 1.095, CH3OH). A sample of (S)-3 was converted into the highly water soluble bis-hydrochloride salt as follows: to a stirred solution of (S)-3; 98 mg, 0.30 mmol) in methanol (4 mL) was added concentrated aqueous HCl (3 drops from a Pasteur pipet, ~0.82 mmol). The solution was concentrated under vacuum to afford a colorless film. This was dissolved in 0.25 mL of methanol, and diluted with 4 mL of ethyl ether to afford a white cloudy mixture. This was concentrated under vacuum to constant mass to afford the bis-HCl salt as a white foam in the amount of 118 mg (98%). 1H NMR (400 MHz, D2O) δ 8.51 (s, 1H), 7.97 (d, 1H, J = 9.4 Hz), 7.48 (d, 1H, J = 9.4 Hz), 6.28 (s, 1 H), 4.97 (m, 1 H), 4.04 (s, 3H), 3.93 (s, 3H), 3.17 (dd, J = 5.8, 14.8 Hz, 1H), 3.11 (dd, J = 7.8, 14.8 Hz, 1H), 2.24 (s, 3 H), 1.41 (d, 3H, J = 7.0). (±) 6,7-Dimethoxy-N-(1-(3-methyl-1H-pyrazol-5yl)propan-2-yl)quinazolin-4-amine (4). To 1-(3-methyl-1Hpyrazol-5-yl)propan-2-amine (220 mg, 1.58 mmol) and 4chloro-6,7-dimethoxyquinazoline (323 mg, 1.44 mmol) in 2propanol was added NaHCO3 (242 mg, 2.88 mmol). The reaction mixture was heated at reflux for 24 h and was then cooled, filtered through celite, concentrated and chromatographed (5:95 MeOH/EtOAc) to provide 320 mg (68%) of the title compound as a white solid: mp 192-195 °C (dec); 1H NMR (400 MHz, CD3OD) δ 8.30 (s, 1 H), 7.53 (s, 1 H), 7.03 (s, 1 H), 5.90 (s, 1 H), 4.61 (m, 1 H), 3.94 (s, 6 H), 3.27 - 3.40 (m, 1 H), 2.92 - 3.05 (m, 1 H), 2.77 - 2.92 (m, 1 H), 2.18 (s, 3 H), 1.30 (d, J = 6.64 Hz, 3 H); 13C NMR (100 MHz, DMSOd6) δ 157.6, 153.7, 153.6, 148.2, 146.1, 108.5, 107.0, 103.1,

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102.0, 56.2, 55.6, 46.1, 19.9; MS m/z (ion) 328 (M + H); Analysis calcd for C17H21N5O2: C, 62.37; H, 6.47; N, 21.39. Found: C, 62.15; H, 6.45; N, 21.03. (±) - 6,7,8-Trimethoxy-N-(1-(5-methyl-1H-pyrazol-3yl)propan-2-yl)quinazolin-4-amine (5). Prepared similarly, from 1-(5-methyl-1H-pyrazol-3-yl)propan-2-amine and 4chloro-6,7,8-dimethoxyquinazoline to yield 45 mg (41%) of the title compound as a white solid: mp 229-231 °C; 1H NMR (400 MHz, CD3OD) δ 8.34 (s, 1H), 7.41 (s, 1H), 5.90 (s, 1H), 4.71 (m, 1H), 3.98 (s, 3H), 3.97 (s, 3H), 3.96 (s, 3H), 2.93 (br m, 2H), 2.19 (br s, 3H), 1.30 (d, J = 6.9 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 157.9, 152.6, 151.5, 147.0, 145.7, 140.2, 111.1, 103.1, 98.0, 61.7, 60.9, 56.4, 46.2, 19.9; HRMS m/z: [M + H]+ calcd for C18H23N5O3, 358.1874; found, 358.1870; Analysis calcd for C18H23N5O3: C, 60.49; H, 6.49; N, 19.59. Found: C, 60.30; H, 6.42; N, 19.47. (±)1-(5-Methyl-1H-pyrazol-3-yl)propan-2-amine dihydrochloride (7). Prepared through a modification of a reported procedure:12 To 2,6-dimethyl-4-pyrone (2.50 g, 20.1 mmol) in methanol (6 mL) in a room temperature water bath (to control a minor exothermic event) was added with stirring hydrazine hydrate (19.4 mg, 60.4 mmol). The solution was stirred at rt for 40 h, at which point acetic acid (8 mL) was added. The mixture was then hydrogenated over platinum oxide hydrate (375 mg) at 60 °C while maintaining a pressure between 50 and 100 psi. After 5 h, the mixture was cooled to rt and filtered through celite. The filter cake was rinsed with ethanol. The filtrate was concentrated to a thin oil under reduced pressure. The oil was basified to a pH of 11-12 via the slow addition of 5 N KOH. The resultant solution was then extracted with an equal volume of 2-butanol (4x) and the combined extracts were stirred with solid NaCl for 20 min to draw out water. The resultant brine layer was discarded, and the organic layer was concentrated to afford a viscous oil. This was dissolved in 100 mL toluene, and the solution was concentrated under vacuum at 60 °C to afford a viscous oil containing some white solids. The oil was dissolved in DCM and filtered through a generous wad of cotton to complete the drying process. The resultant pale yellow solution was concentrated once again to afford a constant mass of 3.79 g (89%) of the free base amine as a viscous syrup. The material was dissolved in 20 mL of methanol and cooled in an ice bath. Concentrated HCl (5 mL, ~60 mmol) was added and the solvent was removed under vacuum. The resultant oil was dissolved in 100 mL of methanol and again concentrated to an oil. This was then dissolved in 25 mL of methanol, and with stirring was diluted with 100 mL of ethyl acetate. Continued stirring afforded a white precipitate. After stirring overnight the solid was collected via filtration and rinsed with 1:9 Methanol/ethyl acetate followed by ethyl ether. Drying under a stream of air afforded 2.84 g (67%) of the title compound as a white solid: mp 187.9-189.5 °C; 1H NMR (400 MHz, CD3OD) δ 6.56 (s, 1H), 3.70 (m, 1H), 3.20 (dd, J = 5.9, 14.8 Hz, 1H), 3.04 (dd, J = 8.6, 14.8 Hz, 1H), 2.46 (s, 3H), 1.34 (d, J = 6.6 Hz, 3H); MS m/z (ion) 140 (M + H).

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AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected] and [email protected].

Present Addresses † Current address for Fabrice Vandeput: Wuxi Apptec co., Ltd, 7F, 2 Huajing Rd, Waigaoqiao Free Trade Zone, Shanghai 200131. Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. / ‡ JMH, MM, and FSM contributed equally.

Notes Compound (S)-3 (PF-04837736) has been made commercially available via Sigma Aldrich (catalog #PZ0379).

ACKNOWLEDGMENT We thank Artem Evdokimov, Mahmoud Mansour, and Samuel P. Simons for assistance in expression, purification, and crystallization of PDE1B for this study. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DEAC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). Matthew Movsesian was supported by Medical Research Funds from the United States Department of Veterans Affairs and a Univa grant from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (R37HD014939).

ABBREVIATIONS USED AUC, area under the curve; BLQ, below limit of quantitation; cAMP, 3’,5’-cyclic adenosine monophosphate; cGMP, 3’,5’cyclic guanosine monophosphate; CM, calmodulin; CSF, cerebrospinal fluid; DCM, dichloromethane; DMF, N,Ndimethylformamide; DMSO, dimethyl sulfoxide; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid; ESI, electrospray ionization; HRMS, high resolution mass spectrometry; Kpuu, free partition coefficient; m/z, mass / charge number; MPO, multi-parameter optimization; mp, melting point; MS, mass spectrometry; PDB, Protein Data Bank; PDE, phosphodiesterase; SAR, structure-activity relationship; SNAr, nucleophilic aromatic substitution; sc, subcutaneous; SEM, Standard error of the mean; SFC, supercritical fluid chromatography; TGF-β, transforming growth factor beta; tPSA, topological polar surface area.

ASSOCIATED CONTENT Supporting Information : Methods and statistics for PDE assays. Methods for determination of compound concentrations in plasma, brain and cerebrospinal fluid of rat. Methods for crystal structure determination. Electron density image for PDE1B-bound (S)3. Proton NMR spectrum of the bis-hydrochloride salt of (S)-3. Molecular formula strings. This material is available free of charge via the Internet at http://pubs.acs.org. The PDB ID code for Figure 4 is 5W6E. Authors will release the atomic coordinates and experimental data upon article publication.

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18. Miller, C. L.; Oikawa, M.; Cai, Y.; Wojtovich, A. P.; Nagel, D. J.; Xu, X.; Xu, H.; Florio, V.; Rybalkin, S. D.; Beavo, J. A.; Chen, Y.-F.; Li, J.-D.; Blaxall, B. C.; Abe, J.-i.; Yan, C. Role of Ca2+/calmodulinstimulated cyclic nucleotide phosphodiesterase 1 in mediating cardiomyocyte hypertrophy. Circ. Res. 2009, 105, 956-964. 19. Miller, C. L.; Cai, Y.; Oikawa, M.; Thomas, T.; Dostmann, W. R.; Zaccolo, M.; Fujiwara, K.; Yan, C. Cyclic nucleotide phosphodiesterase 1A: a key regulator of cardiac fibroblast activation and extracellular matrix remodeling in the heart. Basic Res. Cardiol. 2011, 106, 1023-1039. 20. Bautista, N. P. K.; Durik, M.; Danser, A. H. J.; de, V. R.; Musterd-Bhaggoe, U. M.; Roks, A. J. M.; Meima, M. E.; Kavousi, M.; Ghanbari, M.; Franco, O. H.; Dehghan, A.; Hoeijmakers, J. H.; O'Donnell, C. J.; Franceschini, N.; Janssen, G. M. J.; De, M. J. G. R.; Liu, Y.; Shanahan, C. M. Phosphodiesterase 1 regulation is a key mechanism in vascular aging. Clin Sci (Lond) 2015, 129, 1061-1075. 21. Cai, Y.; Nagel, D. J.; Zhou, Q.; Cygnar, K. D.; Zhao, H.; Li, F.; Pi, X.; Knight, P. A.; Yan, C. Role of cAMP-phosphodiesterase 1C signaling in regulating growth factor receptor stability, vascular smooth muscle cell growth, migration, and neointimal hyperplasia. Circ. Res. 2015, 116, 1120-1132. 22. Knight, W. E.; Chen, S.; Zhang, Y.; Oikawa, M.; Wu, M.; Zhou, Q.; Miller, C. L.; Cai, Y.; Mickelsen, D. M.; Moravec, C.; Small, E. M.; Abe, J.; Yan, C. PDE1C deficiency antagonizes pathological cardiac remodeling and dysfunction. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, E7116-E7125.

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