PDE2a - ACS Publications - American Chemical Society

Feb 6, 2017 - Laurent Gomez,* Mark Eben Massari, Troy Vickers, Graeme Freestone, William Vernier, Kiev Ly,. Rui Xu, Margaret McCarrick, Tami Marrone, ...
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Design and Synthesis of Novel and Selective Phosphodiesterase 2 (PDE2) Inhibitors for the Treatment of Memory Disorders. Laurent Gomez, Mark Eben Massari, Troy Vickers, Graeme Freestone, William F. Vernier, Kiev Ly, Rui Xu, Margaret McCarrick, Tami Marrone, Markus Metz, Yingzhou G Yan, Zachary W Yoder, Robert Lemus, Nicola J. Broadbent, Richard Barido, Noelle Warren, Kara Schmelzer, David Neul, Dong Lee, Carsten B. Andersen, Kristen Sebring, Kathleen Aertgeerts, Xianbo Zhou, Ali Tabatabaei, Marco Peters, and J. Guy Breitenbucher J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01793 • Publication Date (Web): 06 Feb 2017 Downloaded from http://pubs.acs.org on February 9, 2017

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Design and Synthesis of Novel and Selective Phosphodiesterase 2 (PDE2) Inhibitors for the Treatment of Memory Disorders. Laurent Gomez,* Mark Eben Massari, Troy Vickers, Graeme Freestone, William Vernier, Kiev Ly, Rui Xu, Margaret McCarrick, Tami Marrone, Markus Metz, Yingzhou G. Yan, Zachary W. Yoder, Robert Lemus, Nicola J. Broadbent, Richard Barido, Noelle Warren, Kara Schmelzer, David Neul, Dong Lee, Carsten B. Andersen, Kristen Sebring, Kathleen Aertgeerts, Xianbo Zhou, Ali Tabatabaei, Marco Peters, J. Guy Breitenbucher. Dart Neuroscience LLC, 12278 Scripps Summit Drive, San Diego, CA 92131, USA

ABSTRACT: A series of potent and selective [1,2,4]triazolo[1,5-a]pyrimidine PDE2 inhibitors is reported. The design and improvement of the binding properties of this series was achieved using X-ray crystal structures in conjunction with careful analysis of electronic and structural requirements for the PDE2a enzyme. One of the lead compounds, compound 27 (DNS-8254), was identified as a potent and highly selective PDE2a enzyme inhibitor with favorable rat pharmacokinetic properties. Interestingly, increased potency of compound 27 was facilitated by the formation of a halogen bond with the oxygen of Tyr827 present in the PDE2 active site. In vivo, compound 27 demonstrated significant memory enhancing effects in a rat model of novel object recognition. Taken together, these data

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suggest that compound 27 may be a useful tool to explore the pharmacology of selective PDE2 inhibition.

INTRODUCTION Phosphodiesterases (PDEs) represent a family of enzymes which metabolically inactivate the second messengers (cAMP and cGMP) through hydrolysis of the cyclic phosphate, and are therefore critical for the termination of the respective signaling cascades. cAMP and cGMP are ubiquitous second messengers that modulate a wide array of intracellular processes. The diverse biological actions controlled by these second messengers are tightly regulated by control over intracellular localization, as well as temporal residence. To date 21 separate genes encoding individual PDE isoforms have been identified in mammalian cells. These are classified into 11 families each family containing 1-4 isoforms.1-2 The subclassifications of PDEs are determined by different preferences for cAMP or cGMP as substrates, and the different regulatory domains contained within each family of enzyme. As a result of their importance in the cell signaling cascade, PDEs have become important biological targets for therapeutic intervention in a variety of disorders. To date the only approved uses of PDE inhibitors are for peripheral indications. There has also been significant interest in PDE inhibitors for the treatment of CNS disorders, due to the role of PDEs in terminating transcriptional cascades triggered by cAMP and cGMP up-regulation which may impact neuronal plasticity. For example, the second messenger cAMP, synthesized from ATP by adenylyl cylcase, activates protein kinase A (PKA) which increases cAMP re-

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sponse element-binding protein (CREB) function, thereby affecting the transcription of genes related to synaptic plasticity, including brain-derived neurotrophic factor (BDNF).3 Conversely, cGMP is derived from GTP by guanylyl cyclase (GC), which is activated by nitric oxide (NO). The NO/cGMP pathway activates protein kinase G (PKG), which is thought to also modulate signaling via CREB.4 Both the cAMP/PKA/CREB and the cGMP/PKG/CREB pathways are implicated in long-term potentiation (LTP), which is considered a neurophysiological correlate of memory.4-6 The importance of cAMP and cGMP in the propagation of signaling cascades which provoke gene expression changes as a result of neuronal activity have made PDEs popular targets for intervention in CNS disorders.7-9 Although phosphodiesterases are ubiquitously distributed in mammalian tissues, PDE2 enzyme is highly expressed in regions such as cortex, amygdala, and hippocampus, and relatively little expression is detected in midbrain, hindbrain, and cerebellum. PDE2 also shows low expression in peripheral tissues.10 This selective enrichment in forebrain structures suggests that PDE2 may modulate neuronal signaling of complex integrated functions such as learning, memory, and emotion. The existence of a PDE2 isoform was originally postulated in response to the finding that bovine heart tissue preparations had cAMP hydrolytic activity which could be affected by changing cGMP concentrations.11 This led to PDE2’s characterization as the mediator of cGMP dependent cAMP hydrolysis.12 PDE2 was later recognized as a dual specificity enzyme, capable of hydrolyzing both cGMP and cAMP. This ability to modulate activity in response to cGMP allows the enzyme to function as a source of “cross-talk” between the cGMP and cAMP signaling cascades. CNS penetrant small molecule inhibitors of PDE2

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may therefore increase cGMP and cAMP levels in the hippocampus and cortex, yielding enhanced synaptic plasticity and memory. Few selective PDE2 inhibitors have been disclosed thus far. The most well-known tool molecule for evaluating PDE2 inhibition, compound 1 (Chart 1), was first described in 2002 by Bayer.13 This compound inhibits the hydrolytic activity of purified PDE2 (IC50 = 0.002 µM) and is selective with respect to other PDE isoforms (50-fold over PDE1C, >100-fold selectivity vs other PDE isoforms). However due to the lack of good brain penetrant properties, in vivo results obtained with compound 1 should be treated with caution. More recently Pfizer reported the identification of 2 (Chart 1) as a potent and selective PDE2a inhibitor (PDE2a IC50 = 0.001 µM, 2000-fold selective over PDE10) with reasonable free brain/plasma ratio in rat (0.5).14a A first Phase I clinical trial was completed in August 201114b followed by a second Phase I trial in April 2012 to estimate the relative bioavailability of a modified-release formulation.14c Compound 2 was evaluated for schizophrenia14b and migraine14d but no further development has been reported since 2014. Recently a few comprehensive reviews have reported additional early stage PDE2 inhibitors.15

Chart 1. PDE2A Inhibitors

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RESULTS AND DISCUSSION

We recently initiated a medicinal chemistry program towards the identification of selective PDE2a inhibitors. A high-throughput screening campaign of our internal compound collection identified a series of 7-amino-[1,2,4]triazolo[1,5-a]pyrimidine derivatives such as 3 which displayed good PDE2 inhibition (IC50 of 0.17 µM), good PDE selectivity and encouraging metabolic stability properties (Table 1). Initial attempts to further improve the overall profile of this series were not promising. For instance, removal of the methyl substituent or modification of electronics in this area of the molecule led to 10-50 fold loss in PDE2 potency (analogs 4 and 5). In addition, initial SAR around the amide portion indicated that an aromatic system was required for activity however any substitutions or modifications made to the aromatic ring had no positive impact on PDE2 potency (analogs 6 and 7). Table 1. In vitro properties of key compounds

PDE2a Compound

R

Me

Rat Clint

(µg/mL)

(µL/min/mg)

Ar IC50 (µM)

3

Solubility a

0.17

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67.9

b

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4

H

1.52

47

76.2

5

CF3

5.36

29

94.1

6

Me

0.21

45

78.9

0.14

13

53.4

Cl

OMe

7

Me

a

Values were averaged from a minimum of three replicates. Standard error of the mean (SEM) b values were typically less than 40%. Intrinsic in vitro clearance determined in rat liver microsomes (RLM) in the presence of NADPH.

The observed flat SAR around the amide functionality as well as the importance of the methyl substituent was quickly rationalized after examination of the crystal structure of compound 7 bound to PDE2. The compound binds to the active site of the enzyme (Figure 1). The bicyclic triazolopyrimidine ring is situated in the conserved hydrophobic π-clamp (Phe862, Phe830 and Ile826), forming parallel π - π stacking interactions with Phe862. The bicyclic core of compound 7 forms two direct hydrogen bonds with the PDE2 protein, involving the conserved glutamate residue Gln859 and the PDE2-specific glutamate Gln812. The methyl substituent occupies a small lipophilic pocket adjacent to Gln812 previously defined as “the HC1 pocket” of PDEs.16 In PDE2, the HC1 pocket is flanked by Leu809 and Ile822. The size and electronic characteristics of the HC1 pocket fits well with a small lipophilic group from the bound ligand, which explains the drastically improved PDE2 activity for compounds containing 5-methyl substitution. The naphthalene group is positioned in

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the solvent-exposed Q2 pocket adjacent to Gln85916 and is sandwiched between Met847, Phe862 and Ile866. The interactions in the Q2 pocket are mostly hydrophobic. The Q2 pocket can accommodate a diverse set of aromatic moieties, resulting in relatively flat SAR around the amide functionality. The overall shape of the protein bound compound 7 adopts a “U-shape” conformation, with an internal hydrogen bond between the amide nitrogen and triazole nitrogen. The piperidine ring forms the basis of the turn. A racemic mixture of compound 7 was used for structural determination, but only the Rconfiguration at the chiral center C3 was selectively bound in the protein structure and the amide functional group was in an axial position on the piperidine ring. Figure 1: Crystal structure of compound 7 (5TZ3) in the PDE2a catalytic domain

Utilizing the structural knowledge of this unique binding mode, we implemented a strategy to modify the existing chemical series in order to improve PDE2 potency. More specifi-

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cally, we decided to focus on the electronic properties of the triazolopyrimidine heterocycle primarily based on the observation that subtle changes to the central heterocylic system might improve PDE binding. Data from the literature have shown that similar heterocyclic systems can have different PDE potencies that cannot be solely explained on the basis of X-ray crystallographic data (due to the similar binding pattern) but rather by electronic factors (π-stacking interactions and H-bond strengths).15a, 17 Our strategy was to maintain some of the unique interactions between the core and PDE2 (i.e. glutamate residues and HC1 pocket) and focus on evaluating a number of alternative substitutions around our aromatic system in order to identify the optimal electronic requirements for PDE2 potency. After a number of structural modifications, we determined that replacing the amino-linker with a more neutral substitution such as a carbon-carbon bond led to up to 140-fold improvement of PDE2 potency (Figure 2). Figure 2: Amino-linked to carbon-linked core modification

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Confirmation that the improved PDE2 potency was primarily driven by subtle electronic changes of the heterocycle, and not from a different binding pattern, came with the X-ray structure of compound 9 bound to PDE2. The high affinity compound 9 displayed a similar overall binding profile to compound 7 (Figure 3). A racemic mixture of compound 9 was used for structural determination and the S-configuration is selectively bound in the protein structure. The two aromatic moieties, triazolopyrimidine and naphthalene, are positioned in the π-clamp and the Q2 pocket, respectively. The key interactions remain the same, although there is a notable shift of naphthalene group in the Q2 pocket, resulting in small rotation of Met847 and other sur-

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rounding residues. The main difference between the two compounds is centered on the piperidine ring, which adopts distinct torsion angles in relation to the triazolopyrimidine in the crystal structures. It should be noted that both compounds 7 and 9 adopt low energy conformations in the bound-state as determined by the torsional profile of each compound (40.4 and 282.2° respectively) depicted in Figure 4. This slight difference is likely due to the different locations of piperidine nitrogens as well as the difference in the length of amide linker between the two compounds. The amide carbonyl of compound 9 is in hydrogen bonding distance to three non-conserved protein surface waters. The significance of these hydrogen bonds to PDE2 binding potency is not clear. Figure 3: Compound 9 (5TZA; in cyan) shares a similar overall binding pose to compound 7 (5TZ3; in grey), despite much improved PDE2 inhibition.

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Figure 4: Torsional profiles of compounds 7 (dashed line) and 9 (solid). Energy conformations in the bound-state are highlighted in red.

Scheme 1. Synthetic route to carbon-linked analogs.

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a

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o

Reagents and conditions: a. CDI, THF, then acetone/LDA, - 78 C to rt; b. 3-Amino-1,2,4-triazole, AcOH, o 125 C; c. HCl, MeOH, DCM; d. DIEA, ArCOCl, DCM or DIEA, HATU, ArCO2H, DCM.

The synthesis of compounds such as 8 and 9 is depicted in Scheme 1. Condensation of the commercially available 1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid 10 with carbonyl diimidazole followed by addition of lithium prop-1-en-2-olate afforded the desired diketone intermediate 11. A subsequent cyclization reaction using intermediate 11 and aminotriazole under acidic conditions yielded the corresponding [1,2,4]triazolo[1,5a]pyrimidine 12. Deprotection of the N-Boc functionality was accomplished using HCl in DCM/MeOH to afford the unsubstituted piperidine derivative 13 as the hydrochloride salt. The target amides were prepared by either a HATU mediated coupling reaction of the intermediate 13 and acids or a direct condensation of 13 with a variety of aromatic acyl chlorides under basic conditions. 18

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Table 2. In vitro properties of key compounds

PDE2a Compound

Rat Clint

Human Clint

Solubility

PAMPA-BBB

(µg/mL)

(10 cm/s)

Ar IC50 (µM)

a

(µL/min/mg)

b

(µL/min/mg)

c

-6

8

0.007

83.9

56.9

47

3.1

9

0.001

85.6

28.6

37

9.4

14

0.003

21.7

2.2

36

0.4

15

0.08

33.5

9.5

35

3.8

16

0.002

6.3

3.1

47

1.2

0.010

30.2

17.7

36

2.4

17

OMe Me a

Values were averaged from a minimum of three replicates. Standard error of the mean (SEM) b,c values were typically less than 40%. Intrinsic in vitro clearance determined in rat liver microsomes (RLM) or human liver microsomes (HLM) in the presence of NADPH.

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The in vitro inhibitory activity against human PDE2 as well as ADME properties were evaluated for all final compounds. A representative set of amide analogs is shown in Table 2. Our initial exploration focused on evaluating different benzamide analogs in order to expand our SAR knowledge. Unfortunately, despite good PDE2 inhibitory activity of some examples, the majority of analogs tested in the series displayed poor in vitro microsomal stability and/or sub-optimal predicted BBB permeation properties. For example analog 9 displayed good PDE2a potency (hPDE2a IC50=0.001 µM) and acceptable in vitro brain permeability (PAMPA-BBB: 9.4x10-6 cm/s) but poor human and rat microsomal stability. Any structural modifications made to the aromatic ring (compounds 14-17) slightly improved microsomal stability to the detriment of permeability (PAMPA-BBB < 5x10-6 cm/s).19 We also profiled both individual enantiomers of analog 9 in order to assess the influence of chirality on microsomal stability (Table 3). It was determined that one of the enantiomers was about 60-fold more potent at PDE2a (S-enantiomer-19), but unfortunately neither of them displayed acceptable microsomal stability. Based on these results, we investigated the effect of alternative saturated heterocyclic systems (in place of piperidine) on PDE2a potency and ADME properties. After evaluating a number of different ring sizes (five to seven membered-rings) and various substitutions, we discovered that 3,3-difluoro-piperidine derivatives offered noticeable improvements in the overall profile of our PDE2a inhibitors.

Table 3. In vitro properties of pure enantiomers.

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O

O

N

H

O

N

H

N

3 (R)

Chiral N N N

SFC N

N

Me

9 racemic

N

N N N

Me

18

PDE2a Compound

(S)

+

N N

IC50 (µM)

a

(µL/min/mg)

Me

19

Rat Clint

C3-configuration

N

Human Clint b

(µL/min/mg)

c

Solubility

PAMPA-BBB

(µg/mL)

(10 cm/s)

-6

9

R, S

0.001

85.6

28.6

37

9.4

18

R

0.047

41.4

35.4

37

8.6

19

S

0.0008

124.0

24.4

37

8.4

a

Values were averaged from a minimum of three replicates. Standard error of the mean (SEM) b,c values were typically less than 40%. Intrinsic in vitro clearance determined in rat liver microsomes (RLM) or human liver microsomes (HLM) in the presence of NADPH.

Scheme 2. Synthetic routes to difluoropiperidine analogs.

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a

Reagents and conditions: a. CDI, THF, then acetone/LDA, - 78 °C to rt; b. 3-Amino-1,2,4-triazole, AcOH, 125 °C; c. HCl, EtOAc then TEA/DCM; d. DIEA, ArCOCl, DCM or DIEA, HATU, ArCO2H, DCM. e. LiHMDS, - 78 °C then 7-chloro-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine, 0 °C. H2O/MeOH/LiOH, 0 °C to rt.

Difluoro-piperidine analogs can be synthesized using the two alternative synthetic routes shown in Scheme 2. The first synthesis (Route A) utilized the previously described approach. The second and improved approach (Route B) takes advantage of the reactivity of chloro-triazolopyrimidine toward nucleophilic displacement. Deprotonation of 1-tertbutyl-3-methyl-5,5-difluoropiperidine-1,3-dicarboxylate 21 with LiHMDS followed by addition of 7-chloro-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine at low temperature afforded the corresponding

α-arylated

ester.

A

subsequent

base-promoted

saponifica-

tion/decarboxylation step using lithium hydroxide at rt yielded the desired product 22 in excellent yield (86%). Removal of the tert-butyloxycarbonyl protecting group was accomplished under standard acidic conditions (HCl) followed by neutralization of the resulting hydrochloride salt to produce the key intermediate 23 in good yield. Single enantiomers (26-29) were synthesized after super critical fluid (SFC) chromatography of the racemic intermediate 23 followed by amide bond formation. The activity of the di-fluoropiperidines 24-29 at PDE2a is shown in Table 4, along with in vitro microsomal stability and permeability data.

Table 4. In vitro properties of pure enantiomers.

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PDE2a Compound

C3-configuration

R, S

25

R,S

Human Clint

PAMPA-BBB

Ar IC50 (µM)

24

Rat Clint

OMe

a

(µL/min/mg)

b

(µL/min/mg)

c

-6

(10 cm/s)

0.001

78.8

25.1

44.9

0.003

67.7

16.7

7.3

Me

26

R

0.052

17.3

6.6

20.1

27

S

0.008

25.7

3.2

20.1

28

S

0.023

162

25.1

37.3

29

S

0.062

52.2

4.3

17.1

a

Values were averaged from a minimum of three replicates. Standard error of the mean (SEM) b,c values were typically less than 40%. Intrinsic in vitro clearance determined in rat liver microsomes (RLM) or human liver microsomes (HLM) in the presence of NADPH.

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Analysis of the data indicated that di-fluoro substitution of the piperidine ring had no noticeable effects on PDE2a potency or microsomal stability but seemed to improve permeability properties compared to the unsubstituted piperidine as seen by comparing 9 and 24; PAMP-BBB: 9.4 and 44.9x10-6 cm/s respectively. Confirmation of this observed SAR trend was obtained after a second di-fluoro analog was synthesized in which both potency and stability properties were maintained relative to the piperidine analog but the permeability was improved (analogs 17 and 25; PAMP-BBB: 2.4 and 7.3x10-6 cm/s respectively). Further SAR investigations of the benzamides and testing of pure enantiomers consistently revealed that the S-enantiomer was more potent than the corresponding R-enantiomer as seen by comparing PDE2a activity of 26 and 27 (0.052 µM vs. 0.008 µM). Absolute configuration of compound 27 was determined by single crystal diffraction analysis. Furthermore we discovered that replacing the ortho-bromo substituent in 27 with small alkyls such as methyl (29) or cyclopropyl (28) led to a significant loss in PDE2a potency. The first insight into a possible reason for the SAR came from the structural analysis of analog 27 bound to PDE2a.

Figure 5: Crystal structure of 27 bound to PDE2A at 2.35 Å resolution (5TZC). Key residues are labeled, including the hydrophobic clamp (Phe862, Phe830 and Ile826) and glutamate residues Gln859 and Gln812.

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As seen in Figure 5, the overall binding conformation was well maintained as demonstrated by the key interactions with both the hydrophobic π-clamp and the two glutamate residues (Gln859 and Gln812). This binding mode also placed the aromatic amide functionality in the Q2 pocket near Ph862 as observed previously. Interestingly, the 4-bromo substituent appears to be engaged in a halogen-bond interaction with the oxygen of Tyr827 with a near linear angle (C-Br…O) of 171.9 degrees and a Br···O-CTyr angle of 117.1 degrees. These geometrical features are optimal for the introduction of effective halogen bonds as described in the literature 20-21 In order to further confirm the halogen-bond hypothesis, we decided to conduct a systematic analysis aimed at correlating meta-substitution with PDE2A activity. For this purpose, additional analogs were synthesized where the bromo substituent was replaced with R = H, F, Cl and I (Table 5). The compounds were then tested against PDE2a and crystallized with

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the enzyme in order to accurately determine the appropriate parameters (halogen bond lengths and angles).

Table 5. Determined parameters of halogen bonds and in vitro inhibitory activity of the compounds against PDE2a.

PDE2a Compound

d(X…O), Å

Angle C-X…O (°)

Angle X…O-CTyr (°)

measured

measured

measured

X IC50 (µM)

a

30

H

0.679

N/A

N/A

N/A

31

F

0.987

3.4

172.2

119.0

32

Cl

0.052

3.2

174.9

117.0

33

Br

0.014

3.1

176.2

119.0

34

I

0.002

3.1

174.5

115.7

a

Values were averaged from a minimum of three replicates. Standard error of the mean (SEM) values were typically less than 40%. X represents halogen of interest; C is the carbon covalently linked to halogen; CTyr is the carbon covalently linked to Tyr827 hydroxyl oxygen. N/A: not applicable

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Racemic mixture of compounds 30-34 were used for structural determination, and the Sconfiguration is selectively bound in the protein structures as expected. All five compounds are bound in similar pose to compound 27, with no notable conformational changes to binding site residues. The distances and angles determined between the halogen atoms and the oxygen atom of Tyr827 from the crystal structures are in accordance with literature data20-21 further suggesting the presence of halogen bonds. It has been shown that a large halogen atom bound to a carbon tends to form an electropositive crown (also referred to as the σ-hole), an electroneutral ring and an electronegative belt.22-23 It is therefore not surprising that the shorter distances (3.1 Å) were observed with the larger halogens (X=Cl, Br and I) whereas the longer X…O distance (3.3 Å) was observed with X=F. Compound 31 also shows two alternative conformations: one with 3-fluoro buried near Tyr827 and the other with 3-fluoro exposed in solvent. This is presumably due to the repulsion between the two electronegative atoms instead of favorable halogen bond. This observation is also in accordance with the measured PDE2a inhibitory activities (I10

>10

>10

>10

1.7

>10

a

PDE1B, PDE2A1, PDE3A, PDE4D3 and PDE10A1 IC50 values were averaged from a minimum of b three replicates. Standard error of the mean (SEM) is typically less than 40%. PDE5A, PDE6C, PDE7A1, PDE8A1, PDE9A2 and PDE11A4 IC50 values were determined at BPS Bioscience.

Figure 6. Rat plasma concentrations of 27 following either a single 1 mg/kg PO or 1 mg/kg IV administration in 5:35:65 NMP:PEG400:H20.

Next, pharmacokinetic properties of 27 were evaluated in male Sprague Dawley rats (200250 g) following administration of a single 1 mg/kg IV or 1 mg/kg PO dose formulated in a mixture of NMP:PEG400:water (5:35:65) (Figure 6). The compound was characterized as

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having a moderate systemic clearance (Cl=32 min/mL/kg; 50% of liver blood flow) and the elimination half-life was determined to be 1 hour.

Following oral administration 27 was

rapidly absorbed with a Tmax of 0.67 hr (Figure 7). It was orally bioavailable (F = 87%) in rats and penetrates the brain with total brain concentration of 30 ng/mL and cerebrospinal fluid (CSF) of 15 nM (Figure 8). Figure 7. Rat pharmacokinetic parameters of 27 after 1 mg/kg administration. a

Route of Administration Pharmacokinetic Parameter i.v. (1 mg/kg)

p.o. (1 mg/kg)

Tmax (hr)

ND

0.67

Cmax (ng/mL)

ND

201

AUCinf (ng.hr/mL)

508

442

t ½ (hr)

1.01

ND

Vss (L/kg)

0.98

ND

CL (mL/min/kg)

32.8

ND

F (%)

87

a

Male SD rats following administration of a single dose formulated in a mixture of NMP:PEG400:water (5:35:65).

Figure 8. CNS penetration parameters of 27. Parameter Brain concentration (nM)

Value a

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CSF concentration (nM)

a

Plasma concentration (nM)

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15 a

6

204

PAMPA-BBB (10- cm/s)

20.1

Plasma free fraction (%)

8

Brain free fraction (%)

4

a

Male SD rats following administration of a single dose 1 mg/kg PO formulated in a mixture of NMP:PEG400:water (5:35:65).

Based on these data, compound 27 was evaluated in a test of novel object recognition (NOR) in rat (Figure 9). NOR is a benchmark task to assess visual recognition memory, and is based on an animals innate preference for novelty. In this task, rats are presented with two identical objects to explore in a training phase. In the testing phase, rats are presented with a trained object and a novel object. Rats with memory for the previously presented trained object will preferentially explore the novel object. Rats that receive submaximal training in conjunction with a memory enhancing drug are expected to exhibit improved memory performance similar that of untreated animals that have received stronger training (i.e., more exposure to objects during the training phase). Studies in non-human primates and rodents suggest that the hippocampus and perirhinal cortex mediate NOR performance.24 PDE2 is highly expressed in both rat, non-human primate and human hippocampus25-26, and PDE2 inhibitors have been shown to enhance recognition memory using the NOR task.27-31

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Journal of Medicinal Chemistry

Rats were dosed orally with compound 27 or vehicle 60 minutes prior to training and then given 3 mins (submaximal memory training) or 20 mins (strong memory training) to explore the arena and objects (Figure 9A). Rats treated with compound 27 (1 or 3 mg/kg) exhibited enhanced long-term object recognition memory compared to sub-maximally trained vehicle-treated rats at the 24 hr retention test (p’s 0.4) and these groups also exhibited a significant preference for the object at test (one sample t-test, p 99% ee) was assigned the (S)stereoconfiguration. The second eluted compound (18; 36.1 mg, 71%, 97.7% ee) was assigned the (R)-stereoconfiguration. The purified enantiomers were analyzed using a Shimadzu LC-20AB instrument with a Chiralpak® AS-H, 150×4.6 mm I.D. 5 µm column and eluting with 40% ethanol in hexanes. Assignments of absolute configuration were made from crystal structures obtained with analoguous compounds.

(R)-(3-(5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)piperidin-1-yl)(naphthalen-2yl)methanone (18) 1H NMR (400 MHz, CD3OD) δ 8.40 - 8.60 (m, 1H), 7.80 - 8.10 (m, 4H), 7.40 - 7.60 (m, 3H), 7.05 - 7.25 (m, 1H), 4.60 - 5.00 (m, 1H), 4.20 - 4.35 (m, 1H), 3.70 3.90 (m, 1H), 3.30 - 3.50 (m, 1H), 3.10 - 3.25 (m, 1H), 2.55 - 2.70 (m, 3H), 2.25 - 2.40 (m, 1H), 1.70 - 2.20 (m, 3H); LCMS found 372.1 [M+H]+. (S)-(3-(5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)piperidin-1-yl)(naphthalen-2yl)methanone (19) 1H NMR (400 MHz, CD3OD) δ 8.40 - 8.60 (m, 1H), 7.80 - 8.10 (m,

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4H), 7.40 - 7.60 (m, 3H), 7.05 - 7.25 (m, 1H), 4.60 - 5.00 (m, 1H), 4.20 - 4.35 (m, 1H), 3.70 3.90 (m, 1H), 3.30 - 3.50 (m, 1H), 3.10 - 3.25 (m, 1H), 2.55 - 2.70 (m, 3H), 2.25 - 2.40 (m, 1H), 1.70 - 2.20 (m, 3H); LCMS found 372.1 [M+H]+.

Preparation of 7-(5,5-Difluoropiperidin-3-yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine Synthetic route A: tert-Butyl 3,3-difluoro-5-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)piperidine-1carboxylate (22). Step 1: To a solution of diisopropylamine (76 g, 754 mmol) in THF (400 mL) was added n-butyllithium (301.6 mL, 754 mmol) dropwise at -70 °C. The mixture was stirred for 1 h at -70 °C. Acetone (43.7 g, 754 mmol) was added dropwise at -70 °C and the mixture was stirred for 1 h at -70 °C.

In a separate flask, to a solution of 1-(tert-

butoxycarbonyl)-5,5-difluoropiperidine-3-carboxylic acid (20; 100 g, 377 mmol) in THF (800 mL) was added CDI (61.13 g, 377 mmol) in portions at 0 °C. The mixture was stirred for 5 h at rt and then added dropwise to the enolate solution at -60 °C under a nitrogen atmosphere. The reaction mixture was stirred for 1 h at -60 °C, and TLC (PE/EtOAc, 3:1) showed the starting material was consumed completely. The mixture was warmed to rt and EtOAc (1 L) was added. The pH value of the mixture was adjusted to 6 by with sat. aq. critic acid. The organic layer was separated, and the aqueous layer was extracted with EtOAc (2 x 500 mL). The combined organic layer was washed with water (500 mL) and brine (500 mL), dried (Na2SO4), filtered, and concentrated under reduce pressure. Purification (FCC, SiO2, PE/EtOAc, 50:1 to 3:1) afforded tert-Butyl 3,3-difluoro-5-(3-

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oxobutanoyl)piperidine-1-carboxylate (60 g, 52%) as a yellow oil.

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1

H NMR (CDCl3, 400

MHz) δ 15.22 (br s, 1H), 5.52 (s, 1H), 4.17-4.32 (m, 2H), 2.76–2.92 (m, 3H), 2.40-2.42 (m, 1H), 1.97 (s, 3H), 1.40 (s, 9H). Step 2: To a solution of tert-butyl 3,3-difluoro-5-(3-oxobutanoyl)piperidine-1-carboxylate (80 g, 262 mmol) in AcOH (400 mL) was added 4H-1,2,4-triazol-3-amine (22 g, 262 mmol) at rt. Then the reaction mixture was heated to 125 °C for 2 h. The mixture was cooled to rt and poured into water (1 L), and was extracted with EtOAc (3 x 500 mL). The combined organic layer was washed with sat. aq. NaHCO3 (1 L) and brine (500 mL), dried (Na2SO4), filtered, and concentrated under reduce pressure to give the crude product (80 g) as brown oil, which was used in the next step without further purification. 1H NMR (CDCl3, 400 MHz) δ 8.44 (s, 1H), 6.85 (s, 1H), 4.26-4.44 (m, 3H), 3.81 (bs, 1H), 3.32-3.39 (m, 2H), 2.70 (s, 3H), 1.49 (s, 9H).

7-(5,5-Difluoropiperidin-3-yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (23). solution

of

tert-butyl

To a

3,3-difluoro-5-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-

yl)piperidine-1-carboxylate (115 g, 325.8 mmol) in EtOAc (200 mL) was added a solution of 4N hydrochloric acid in EtOAc (600 mL) at 0 °C. Then the reaction mixture was stirred for 3 h at rt. The resulting white precipitate was filtered, and the filter cake was collected. The crude material was mixed with DCM (500 mL) and TEA (200 mL) at 0 °C. After stirring for 3 h the reaction mixture became homogeneous. Water (300 mL) was added and the organic layer separated. The aqueous layer was extracted with DCM (3 x 100 mL). The combined organic layer was washed with brine (300 mL), dried (Na2SO4), filtered, and concentrated

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Journal of Medicinal Chemistry

under reduce pressure to afford the title compound (92 g) as a white solid.

1

H NMR

(DMSO-d6, 400 MHz) δ 8.58 (s, 1H), 7.25 (s, 1H), 3.77 (t, J = 11.2 Hz, 1H), 3.15-3.23 (m, 1H), 3.09-3.15 (m, 1H), 2.84-2.92 (m, 2H), 2.66-2.72 (m, 1H), 2.59 (s, 3H), 2.32-2.36 (m, 2H); LCMS found 254.1 [M+H]+.

Synthetic route B: tert-Butyl 3,3-difluoro-5-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)piperidine-1carboxylate (22).

To a solution of 1-tert-butyl 3-methyl 5,5-difluoropiperidine-1,3-

dicarboxylate (21; 58.0 g, 207.6 mmol) in THF (1038 mL, 0.2M) cooled to -78 °C was added LiHMDS (207.6 mL, 207.6 mmol, 1M in THF) dropewise over 16 min. The reaction mixture was stirred at -78 °C for 45 min. before 7-chloro-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (35.0 g, 207.61 mmol) was added. The reaction mixture temperature was raised to 0 °C and stirred for 25 min. Water (32 mL), MeOH (67 mL) and LiOH (4260 mg, 1245.7 mmol) were added and the reaction mixture was stirred at 0 - 25 °C for 18 h. The reaction mixture was diluted with DCM (500 mL) and quenched with H2O (500 mL). The aqueous layer was extracted with dichloromethane (2 x 500 mL) and the combined organic layers were dried (Na2SO4), filtered, and concentrated under reduced pressure.

Purification (LC,

EtOAc/MeOH/Hexane 45:5:50) afforded the title compound (62.8 g, 86%) as a yellow foam. 1H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H), 7.27 (s, 1H), 4.37 (d, J = 13.3 Hz, 1H), 4.18 (br s, 1H), 3.69 - 3.80 (m, 1H), 3.37 (d, J = 19.6 Hz, 1H), 3.20 (br s, 1H), 2.50 - 2.66 (m, 5H); LCMS found 354.4 [M+H]+.

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7-(5,5-Difluoropiperidin-3-yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (23). To a solution

of

tert-butyl

3,3-difluoro-5-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-

yl)piperidine-1-carboxylate (62.8 g, 177.7 mmol) in MeOH (444 mL, 0.4 M) cooled to 0 °C was slowly added 4N HCl in dioxane (600 mL). The mixture was stirred at 0 - 25 °C for 4 h and concentrated under reduced pressure to afford the title compound (58.1 g, 100%) as a beige solid (bis HCl salt). 1H NMR (400 MHz, DMSO-d6) δ ppm 11.12 (br s, 1H), 9.87 (br s, 1H), 8.65 (s, 1H), 7.31 (s, 1H), 4.08 (tt, J = 12.4, 3.6 Hz, 1H), 3.76 - 3.87 (m, 1H), 3.46 - 3.66 (m, 2H), 3.31 - 3.42 (m, 1H), 2.54 - 2.76 (m, 5H); LCMS found 254.4 [M+H]+. The beige solid was diluted with DCM (200 mL) and TEA (100 mL) at 0 °C. After stirring for 4 h the reaction mixture became homogeneous. Water (200 mL) was added and the organic layer separated. The aqueous layer was extracted with DCM (3 x 100 mL). The combined organic layer was washed with brine (200 mL), dried (Na2SO4), filtered, and concentrated under reduce pressure to afford the title compound as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 8.58 (s, 1H), 7.25 (s, 1H), 3.77 (t, J = 11.2 Hz, 1H), 3.15-3.23 (m, 1H), 3.09-3.15 (m, 1H), 2.84-2.92 (m, 2H), 2.66-2.72 (m, 1H), 2.59 (s, 3H), 2.32-2.36 (m, 2H); LCMS found 254.4 [M+H]+. 3,3-Difluoro-5-{5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl}-1-[(naphthalen-2yl)carbonyl]piperidine (24). The title compound was prepared using General Method B using 7-(5,5-Difluoropiperidin-3-yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine

23 as the

starting material. 1H NMR (400MHz, DMSO-d6) δ 8.22 - 7.86 (m, 4H), 7.77 - 7.44 (m, 3H), 7.40 - 7.18 (m, 1H), 5.07 - 4.74 (m, 1H), 4.70 - 3.33 (m, 4H), 2.83 - 2.53 (m, 5H); LCMS found 408.3 [M+H]+.

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Journal of Medicinal Chemistry

3,3-Difluoro-1-(4-methoxy-3-methylbenzoyl)-5-{5-methyl-[1,2,4]triazolo[1,5a]pyrimidin-7-yl}piperidine (25). The title compound was prepared using General Method B using 7-(5,5-Difluoropiperidin-3-yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine 23 as the starting material. 1H NMR (400MHz, CDCl3) δ 8.43 (s, 1H), 7.36 - 7.30 (m, 2H), 6.91 - 6.81 (m, 2H), 4.88 - 4.29 (m, 2H), 3.95 (d, J = 5.1 Hz, 1H), 3.87 (s, 3H), 3.63 - 3.32 (m, 2H), 2.76 - 2.56 (m, 5H), 2.57 - 2.55 (m, 1H), 2.25 (s, 3H); LCMS found 402.6 [M+H]+. Chiral separation: The racemic intermediate 23, (3.97g, 15.3 mmol) was resolved to give the pure enantiomers using a preparative SFC instrument with a Chiralpak® OZ-H (2 x 25 cm) column and eluting with 25% 1:1 MeOH:ACN(0.1% NH4OH)/CO2, 100 bar. Obtained were the two pure enantiomers: The first eluted compound (0.51 mg, >99% purity, >99% ee) was determined to be the (R)-stereoconfiguration. The second eluted compound (0.5 g, >99% purity, >99% ee) was determined to be the (S)-stereoconfiguration. The purified enantiomers were analyzed using a Chiralpak® OZ-H (25 x 0.46 cm) column and eluting with 30% methanol/CO2, 100 bar. R and S configurations were assigned after synthesis and full characterization of compound 27 (including single crystal X-ray diffraction shown in supporting information).

(5R)-1-[(3-Bromo-4-fluorophenyl)carbonyl]-3,3-difluoro-5-{5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-yl}piperidine (26). The title compound was prepared using

General

Method

B

using

(R)-7-(5,5-difluoropiperidin-3-yl)-5-methyl-

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[1,2,4]triazolo[1,5-a]pyrimidine. 1H NMR (400MHz, DMSO-d6) δ 8.75 - 8.44 (m, 1H), 7.92 7.71 (m, 1H), 7.63 - 7.46 (m, 2H), 7.36 - 7.05 (m, 1H), 5.04 - 3.74 (m, 4H), 2.79 - 2.58 (m, 6H); LCMS found 456.2 [M+H]+.

(5S)-1-[(3-Bromo-4-fluorophenyl)carbonyl]-3,3-difluoro-5-{5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-yl}piperidine (27). The title compound was prepared using

General

Method

B

using

(S)-7-(5,5-difluoropiperidin-3-yl)-5-methyl-

[1,2,4]triazolo[1,5-a]pyrimidine. 1H NMR (400MHz, DMSO-d6) δ 8.69 - 8.48 (m, 1H), 7.85 7.76 (m, 1H), 7.48 (s, 2H), 7.37 - 7.18 (m, 1H), 5.04 - 4.62 (m, 1H), 4.31 - 3.64 (m, 3H), 2.60 (br s, 6H); LCMS found 456.2 [M+H]+.

(5S)-1-(3-Cyclopropyl-4-fluorobenzoyl)-3,3-difluoro-5-{5-methyl-[1,2,4]triazolo[1,5a]pyrimidin-7-yl}piperidine (28). The title compound was prepared using General Method A using O-(benzotriazol-1-yl)N,N,N′,N′-tetramethyluronium hexafluorophosphate in place of 2-(3H-[1,2,3]triazolo[4,5b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate. 1H NMR (400MHz, DMSO-d6) δ 8.57 (s, 1H), 7.40 - 6.85 (m, 4H), 4.82 - 3.83 (m, 5H), 2.60 (s, 5H), 2.09 - 2.00 (m, 1H), 0.98 (d, J = 8.6 Hz, 2H), 0.76 (dd, J = 5.3, 18.6 Hz, 2H); LCMS found 416.4 [M+H]+.

(5S)-1-(3,4-Difluoro-5-methylbenzoyl)-3,3-difluoro-5-{5-methyl-[1,2,4]triazolo[1,5a]pyrimidin-7-yl}piperidine (29). The title compound was prepared using General

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Method A using O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate in place of 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate. 1

H NMR (400MHz, DMSO-d6) δ 8.66 - 8.40 (m, 1H), 8.30 - 8.00 (m, 1H), 7.38 (ddd, J = 2.0,

7.5, 10.1 Hz, 1H), 7.24 (d, J = 5.9 Hz, 2H), 3.88 (br s, 3H), 3.56 - 3.31 (m, 2H), 2.67 - 2.57 (m, 5H); LCMS found 408.2 [M+H]+.

3,3-Difluoro-1-[(4-fluorophenyl)carbonyl]-5-{5-methyl-[1,2,4]triazolo[1,5a]pyrimidin-7-yl}piperidine (30). The title compound was prepared using General Method B. 1H NMR (400MHz, DMSO-d6) δ 8.59 (br s, 1H), 7.67 - 7.42 (m, 2H), 7.31 (t, J = 8.8 Hz, 3H), 5.02 - 4.51 (m, 1H), 3.93 - 3.78 (m, 2H), 3.40 - 3.32 (m, 1H), 2.60 (s, 6H); LCMS found 376.2 [M+H]+.

1-[(3,4-Difluorophenyl)carbonyl]-3,3-difluoro-5-{5-methyl-[1,2,4]triazolo[1,5a]pyrimidin-7-yl}piperidine (31). The title compound was prepared using General Method B. 1H NMR (400MHz, DMSO-d6) δ 8.71 - 8.26 (m, 1H), 7.67 - 7.55 (m, 2H), 7.39 - 7.33 (m, 1H), 7.31 - 7.16 (m, 1H), 4.94 - 4.58 (m, 1H), 3.98 - 3.73 (m, 2H), 3.39 - 3.32 (m, 1H), 2.75 - 2.56 (m, 6H); LCMS found 394.2 [M+H]+.

1-[(3-Chloro-4-fluorophenyl)carbonyl]-3,3-difluoro-5-{5-methyl-[1,2,4]triazolo[1,5a]pyrimidin-7-yl}piperidine (32). The title compound was prepared using General

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Method B. 1H NMR (400MHz, DMSO-d6) δ 8.67 - 8.47 (m, 1H), 7.74 - 7.71 (m, 1H), 7.55 7.50 (m, 2H), 7.36 - 7.15 (m, 1H), 4.93 - 4.65 (m, 1H), 4.23 - 3.68 (m, 3H), 2.75 - 2.53 (m, 6H); LCMS found 410.2 [M+H]+.

1-[(3-Bromo-4-fluorophenyl)carbonyl]-3,3-difluoro-5-{5-methyl-[1,2,4]triazolo[1,5a]pyrimidin-7-yl}piperidine (33). The title compound was prepared using General Method B. 1H NMR (400 MHz, DMSO-d6) δ 8.57 (br s, 1H), 7.82 (dd, J = 6.7, 2.0 Hz, 1H), 7.43 - 7.61 (m, 2H), 7.27 (br s, 1H), 4.77 (br s, 1H), 4.02 - 4.26 (m, 1H), 3.59 - 3.97 (m, 2H), 3.32 - 3.52 (m, 1H), 2.53 - 2.81 (m, 5H); LCMS found 454.2 [M+H]+.

3,3-Difluoro-1-[(4-fluoro-3-iodophenyl)carbonyl]-5-{5-methyl-[1,2,4]triazolo[1,5a]pyrimidin-7-yl}piperidine (34). The title compound was prepared using General Method B. 1H NMR (400 MHz, CDCl3) δ 8.56 (s, 1H), 7.93 (d, J = 4.30 Hz, 1H), 7.34 - 7.60 (m, 1H),, 7.12 - 7.26 (m, 1H), 6.92 (br s, 1H), 3.93 (br s, 1H), 3.38 (br s, 5H), 2.64 - 2.76 (m, 4H); LCMS found 502.1 [M+H]+.

PDE2 crystallography. Crystal structures of PDE2 co-complexes were determined similar to previously described.32 The catalytic domain of PDE2a (residues 579-918) was overexpressed in baculovirus insect cells and purified through affinity and size-exclusion chromatography. Crystals were obtained in the presence of 2mM 3-isobutyl-1-methylxanthine (IBMX) in well solution of 17-19% PEG3350, 0.2M MgCl2, 0.1M Tris, pH 8.4. Crystals were

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then soaked in reservoir buffer containing 1-2.5mM target compound overnight and flash frozen in liquid nitrogen using additional 25% glycerol as cryoprotectant. The diffraction data sets were collected at the Advanced Light Source beamlines 5.0.1 and 5.0.2. The structures were solved by molecular replacement and refined in PHENIX.33 Data collection and refinement statistics are summarized in the supplemental information. PDE Inhibition. PDE2a inhibition IC50 values were determined by an IMAP assay measuring inhibition of FAM-cAMP hydrolysis by full length PDE2a enzymes. Specifically, in 1536 well white plates, 250 pg per well GST tagged PDE2a was dispensed in 2.5 µL IMAP assay buffer consisting of 10 mM Tris pH 7.2, 10 mM MgCl2, 1 mM DTT, 0.1 % fatty acid free BSA, with 10 U/ml calmodulin, and 2.5 mM CaCl2. 30 nl compound was then added from 1 mM stock in DMSO using the Kalypsys 1536 pintool. Plates were incubated for 5 minutes at rt before dispensing 1.5 µL of 533 nM FAM-cAMP for a final concentration of 200 nM. The plates were incubated 30 minutes at rt after a brief centrifugation. The assay was terminated by adding 5 µL IMAP binding reagent Tb complex to each well, prepared according to manufacturer’s recommendations. Plates were incubated 1 hour at rt and read on a Viewlux multimode plate reader (Perkin Elmer). PDE2A, PDE3A, PDE4D3 and PDE10A1 values were determined using analogous procedures. PDE5A, PDE6C, PDE7A, PDE8A1, PDE9A and PDE11A values were determined at BPS Bioscience. Metabolic stability in the presence of liver microsomes. Test compound was incubated at a final concentration of 1 µM in a reaction mixture containing human or rat liver microsomes of 0.5 mg protein/mL in tris buffer at 37 oC. The reaction was started by addition of NADPH (2 mM). Aliquots of the incubation mixture were removed and quenched with

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three volumes of cold ACN containing internal standard, indomethacin (0.5 µM) at 0, 5, 15 and 30 min for RLM and at 0, 15, 30 and 60 min for HLM. The samples were vortex mixed and centrifuged at 4000 rpm for 10 min at 4 °C; and the supernatant was transferred to a 384-well plate followed by dilution with same volume of distilled water. Five µL of the samples were analyzed using a Shimadzu HPLC system (Kyoto, Japan) equipped with a reverse-phase column (Thermo Hypersil GOLD 3um Drop-In guard cartridges, 10 x 2.1 mm). Mobile phase was consisted of 0.1% formic acid in water and of 0.1% formic acid in ACN with a flow rate of 0.7 mL/min. Eluent was directed to an API4000 triple quadruple mass spectrometer (AB Sciex, Framingham, MA) equipped with a turbo electrospray interface. Multiple Reaction Monitoring (MRM) transition in positive ion mode was used. Integration of the sample peaks was performed using AB Sciex Analyst and DiscoveryQuant software; peak area ratio of the analyte to the internal standard was determined accordingly. In vitro intrinsic clearance (CLint) was calculated by dividing the first-order degradation rate constant (Kdeg) by the concentration of microsomal protein used in the incubation. Kdeg was the slope using least-squares fit to the curve of percent test compound remaining vs. time (0 to 30 min for RLM and 0 to 60 min for HLM).

BBB PAMPA permeability assay. The determination of permeability was performed using Evolution instrument (Pion Inc., Billerica, MA) combined with Tecan Freedom Evo Workstation (Tecan Group Ltd. Mannedorf, Switzerland) robotic liquid handling system. The liquid handling draws 8 µl of 10 mM DMSO stock of new chemical entities and mixes it into 800 µl of an aqueous universal PRIZMA buffer solution pH 7.4 (Pion Inc., Billerica,

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MA) so that the final sample concentration is 100 µM in buffer solutions. The DMSO concentration was kept at 1.0% (v/v) in the final buffer solutions. 200µL of the 100µM NCE was dispensed 96-well microtitre plate “sandwich” (pION, PN 110212, pre-loaded with magnetic stirrers) donor side and 200µL of Brain Sink Buffer (Pion Inc., Billerica, MA) to the receiver solutions contained a surfactant mixture (“lipophilic sink”) to mimic tissue binding. Vigorous stirring was employed in the assay, with stirring speed set to produce an ABL thickness of about 60 μm, to minimize the ABL contribution to the measured permeability. The PAMPA sandwich was assembled and allowed to incubate for 60 min in a controlled-environment chamber (pION Gut-Box™, PN 110205) with a built-in magnetic stirring mechanism. Both the donor and receiver wells were assayed for the amount of material present, by comparison with the UV spectrum (210–500 nm) obtained from a reference standard. Permeability values were corrected for membrane retention. Rat Cortical Neuron cGMP Assay Primary rat cortical neurons were harvested from rat E18 pups and plated onto Poly-DLysine coated 96 well plates (BD Biocoat, Cat# 356691) at 4.0x104 cells per well in 100 µL Neuronal Media (BME media supplemented with 20 mM glucose, 1 mM sodium pyruvate, 2 mM GlutaMAX, 1x pen-strep, 1% horse serum, and B27 supplement). Media was replaced 24 hours later with 200 µL fresh Neuronal Media and cultured for 6 days in a 37 °C 5% CO2 incubator. On day 7 of culture, growth media was aspirated and replaced with 25 µL of 2X compound stock made in Hank’s Balanced Salt Solution (HBSS) containing 1% DMSO. Cells were incubated for 30 min in a 37 °C, 5% CO2 incubator. Next, 25 µL of 2X cGMP activator Bay 41-8543 (300 nM) in HBSS was added and incubated for an additional 30 min.

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Total cGMP levels were measured using the cGMP Kit from Cisbio (Cat# 62GM2PEC) as per the manufacturer’s instructions, except that 4 mM EDTA was added to cGMP-d2 detection reagent. Briefly, 25 µL of each detection solution was added and incubated for 1 hr at rt. Plates were read on an Envision multimode reader (Perkin Elmer). Data were analyzed and plotted in Prism 6 (GraphPad Software, Inc.). DMSO and compound 1 (5 µM) were used as the vehicle (0%) and positive control (100%), respectively. Rat Pharmacokinetics Determination. Pharmacokinetics properties in male SD rats (200-250 g) were determined following intravenous (i.v.) and oral (p.o.) administration. For i.v. dosing (N=3, 1 mg/kg), rats were catheterized in jugular and femoral vein. For p.o dosing (N=3, 1 mg/kg) rats were catheterized in femoral vein. Test article was formulated in NMP:PEG400:water (5:35:65).

Rats were not fasted during this study. Blood was sampled

at 0 (pre-dose), 0.033 , 0.083 , 0.25, 0.5, 1, 2, 4, 8, 12, 16 and 24 hours following i.v. and at 0 (pre-dose), 0.25, 0.5, 1, 2, 4, 8, 12, 16 and 24 hours following p.o. dosing. Plasma was isolated by centrifugation and all samples were frozen at -80 oC. Calibration standards were prepared by the addition of known concentrations of test article to blank rat plasma to provide a calibration range of 0.5 ng/mL-2000 ng/mL. Fifty μL plasma samples or calibration standard was added to 250 μL of internal standard solution in acetonitrile. Samples were vortex mixed and centrifuged at 12000 rpm for 5 minutes at 4 °C. Supernatant (100 μL) was transferred to labeled autosampler vials containing 300 μL of mobile phase (water/acetonitrile/formic acid, 90/10/0.2%), vortex mixed and analyzed by LC-MS/MS. A bioanalytical method was developed for the quantification of test article in rat plasma. Method development and sample analysis was conducted using a Waters Quattro Premier LC-

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MS/MS system equipped with Waters Acquity UPLC system. Five µL of the samples were analyzed using a Waters Acquity UPLC system equipped with a C 18 reverse-phase column (Phenomenex Kinetex C18, 1.7um, 2.1 x 50.0 mm). Mobile phase consisted of 0.1% formic acid in water and of 0.1% formic acid in acetonitrile with a flow rate of 0.7 mL/min. Eluent was directed to a Waters Quattro Premier mass spectrometer equipped with a turbo electrospray interface. Multiple Reaction Monitoring (MRM) transition in positive ion mode was used. Novel Object Recognition Training and Testing. Rats were habituated to handing and the empty test arena for 7 min on each of 3 consecutive days. On training day, drug was administered p.o. in a vehicle of 10:50:40 NMP:PEG400:H2O at a volume of 1 mL/kg. Sixty minutes after dosing, each rat was placed into the test arena which now contained two identical objects located centrally in the arena. Rats were given either 3 min (to induce a weak memory), or 20 min (to induce a strong memory) to explore the arena and objects. Memory retention was tested 24 h after training. Rats were placed back in the arena with one ‘familiar’ (previously trained) and one ‘novel’ object and given five minutes to explore. The spatial position of objects (left-right position) and which object was novel (ball or square) was counterbalanced across subjects. Objects and arenas were cleaned with steris solution between trials to remove rat feces and urine. To determine memory performance, an object-discrimination index (DI) was calculated as [novel exploration - familiar exploration]/ [total exploration]. Rats were excluded from the analysis if total exploration during training or test was less than 5 s. Locomotor activity and object exploration during train-

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ing was also recorded. Data were analyzed by ANOVA with least squares means planned comparisons (student’s t-test). All data are presented as the mean ± s.e.m.

ASSOCIATED CONTENT Supporting Information. Description of conformational analysis, single crystal X-ray diffraction for 27, protein crystallographic data statistics tables, cGMP levels in rat primary cortical neurons for 27 and off-target selectivity panels. PDB ID Codes. Crystal structures of PDE2a in complex with 7, 9, 27, 30-34 have been deposited in the RCSB RCSB Protein Data Bank under the accession codes of 5TZ3, 5TZA, 5TZC, 5TZH, 5TZW, 5TZX, 5TZZ AND 5U00, respectively. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Tel: 858-736-3043. ACKNOWLEDGMENT The authors thank Dr. Damian Wheeler for proof reading the manuscript. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC0205CH11231. ABBREVIATIONS

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br s, broad signal; CREB, CAMP-response element binding protein; DIEA, diisopropylethylamine; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; FAM-CAMP, FAMcAMP, 2’-(6-[fluoresceneinyl] aminohexylcarbamoyl)adenosine-3’,5’-cyclic monophosphate; HLM, human liver microsomes; IMAP; immobilized metal ion affinity-based fluorescence polarization; HOBt, 1hydroxybenzotriazole; MDCK, Madin-Darby canine kidney; NOR, novel object recognition; RLM, rat liver microsomes. REFERENCES (1) Conti, M.; Beavo,

J.

Biochemistry and physiology of cyclic nucleotide phos-

phodiesterases: essential components in cyclic nucleotide signaling. Ann. Rev. Biochem. 2007, 76, 481-511. (2) Lugnier, C. Cyclic nucleotide phosphodiesterase (PDE) superfamily: a new target for the development of specific therapeutic agents. Pharmacol. Ther. 2006, 109, 366–398 (3) Schmidt, C.J. Phosphodiesterase inhibitors as potential cognition enhancing agents. Curr. Topics Med. Chem. 2010, 10, 222-230. (4) Bitner, S.R. Cyclic AMP response element-binding protein (CREB) phosphorylation: a mechanistic marker in the development of memory enhancing Alzheimer's disease therapeutics. Biochem. Pharmacol. 2011, 83, 705-714. (5) Lu,Y.F.; Kandel, E.R.; Hawkins, R.D. Nitric oxide signaling contributes to late-phase LTP and CREB phosphorylation in the hippocampus. J. Neurosci. 1999, 19, 10250-10261. (6) Bliss, T.V.; Collingridge, G.L. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 1993, 361, 31-39.

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(7) Frey, U.; Huang, Y.Y.; Kandel, E.R. Effects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons. Science 1993, 260, 1661-1664. (8) Xu, Y.; Zhang, H-T.; O’Donnell, J.M. Phosphodiesterases in the central nervous system: implications in mood and cognitive disorders. Handb. Exp. Pharmacol. 2011, 204, 447485. (9) Reneerkens, O.A.H.; Rutten, K.; Steinbusch, H.W.M.; Blokland, A.; Prickaerts, J. Selective phosphodiesterase inhibitors: a promising target for cognition enhancement. Psychopharmacology 2009, 202, 419-443. (10) Stephenson, D.T.; Coskran, T.M.; Wilhelms, M.B.; Adamowicz, W.O.; O’Donnell, M.M.; Muravnick, K.B.; Menniti, F.S.; Kleiman, R.J.; Morton, D. Immunohistochemical localization of phosphodiesterase 2A in multiple mammalian species. J. Histochem. Cytochem. 2009, 57, 933-949. (11) Stangherlin, A.; Gesellchen, F.; Zoccarato, A.; Terrin, A.; Fields, L.A.; Berrera, M.; Surdo, N.C.; Craig, M.A.; Smith, G.; Hamilton, G.; Zaccolo, M. cGMP signals modulate cAMP levels in a compartment-specific manner to regulate catecholamine-dependent signaling in cardiac myocytes. Circ. Res. 2011, 108, 929-939. (12) a) Beavo, J.A.; Hardman, J.G.; Sutherland, E.W. Hydrolysis of cyclic guanosine and adenosine 3',5'-monophosphates by rat and bovine tissues. J. Biol. Chem. 1970, 245, 56495655; b) Erneux, C.; Couchie, D.; Dumont, J.E. Characterization of horse thyroid cyclic nucleotide phosphodiesterases. Eur. J. Biochem. 1980, 104, 297-304; c) Martins, T.J.; Mumby, M.C.; Beavo, J.A. Purification and characterization of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from bovine tissues. J. Biol. Chem. 1982, 257, 1973-1979.

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(13) Boess, F.-J.; Grosser, R.; Hendrix, M.; Koenig, G.; Niewoehner, U.; Schauss, D.;Schlemmer, K.-H.; Schreiber, R.; Van Der Staay F.-J. Novel substituted imidazotriazinones as PDE2-inhibitors. WO2002050078, 2002. (14) a) Helal, C. J.; Chappie, T. A.; Humphrey, J. M.; Verhoest, P. R.; Yang, E. Imidazo[5,1f ][1,2,4]triazines for the treatment of neurological disorders. US20120214791, 2012; b) ClinicalTrials.gov.

https://clinicaltrials.gov

(accessed

January

14,

2017).

https://clinicaltrials.gov/ct2/show/NCT01429740?term=PF-05180999&rank=4; c) ClinicalTrials.gov.

https://clinicaltrials.gov

(accessed

January

14,

2017).

https://clinicaltrials.gov/ct2/show/NCT01530529?term=PF-05180999&rank=1; d)

ClinicalTrials.gov.

https://clinicaltrials.gov

(accessed

January

14,

2017).

https://clinicaltrials.gov/ct2/show/study/NCT01981499?term=PF-05180999&rank=3 (15) a) Gomez, L.; Breitenbucher, J. Guy. PDE2 inhibition: potential for the treatment of cognitive disorders. Bioorg. Med. Chem. Lett. 2013, 23, 6522-6527. b) am Ende, C. W., Kormos, B. L.; Humphrey, J. M. The State of the art in selective PDE2A inhibitor design, in phosphodiesterases and their inhibitors. Methods Princ. Med. Chem. 2014, 61, 83-104. c) Trabanco, A.A.; Buijnsters, P.; Rombouts, F.J.R. Towards selective phosphodiesterase 2A (PDE2A) inhibitors: a patent review (2010 - present). Exp. Opin. Ther. Pat. 2016, 26, 933946. (16) Jansen, C.; Kooistra, A.J.; Kanev, G.K.; Leurs, R.; de Esch, I.J.P.; de Graaf, C. PDEStrIAn: A Phosphodiesterase structure and ligand interaction annotated database as a tool for structure-based drug design. J. Med. Chem. 2016, 59, 7029–7065.

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(17) Haning, H.; Niewöhner, U.; Schenke, T.; Lampe, T.; Hillisch, A.; Bischoff, E. Comparison of different heterocyclic scaffolds as substrate analog PDE5 inhibitors. Bioorg. Med. Chem. Lett. 2005, 15, 3900-3907. (18) Breitenbucher, J. Guy; Freestone, G.; Gomez, L.; Lemus, R.; Ly, K.; McCarrick, M.; Vernier, W.; Vickers, T. Dart Neuroscience LLC. Substituted [1,2,4] triazolo [1,5-a] pyrimidin-7-yl compounds as PDE2 inhibitors. WO2015164508, 2015. (19) Di, L.; Kerns, E.H.; Fan, K.; McConnell, O.J.; Carter, G.T. High throughput artificial membrane permeability assay for blood-brain barrier. Eur. J. Med. Chem. 2003,38, 223-232. (20) Cavallo, G.; Metrangolo, P.; Milani ,R.; Pilati ,T.; Priimagi ,A.; Resnati ,G.; Terraneo, G. The halogen bond. Chem. Rev. 2016, 116, 2478–2601. (21) Xu, Z.; Liu, Z.; Chen, T.; Chen, T.; Wang, Z.; Tian, G.; Shi, J.; Wang, X.; Lu, Y.; Yan, X.; Wang, G.; Jiang, H.; Chen, K.; Wang, S.; Xu, Y.; Shen, J.; Zhu, W. Utilization of halogen bond in lead optimization: a case study of rational design of potent phosphodiesterase type 5 (PDE5) inhibitors. J. Med. Chem. 2011, 54, 5607–5611. (22) Clark, T; Hennemann, M.; Murray, J.S.; Politzer, P. Halogen bonding: the sigma-hole. Proceedings of "Modeling interactions in biomolecules II", Prague, September 5th-9th, 2005. J. Mol. Model. 2007, 13, 291–296. (23) Politzer, P.; Lane, P.; Concha, M.C.; Ma, Y.G.; Murray, J.S. An overview of halogen bonding. J. Mol. Model. 2007, 13, 305–311. (24) Clark, R.E.; Squire, L.R. An animal model of recognition memory and medial temporal lobe amnesia: History and current issues. Neuropsychologia 2010, 48, 2234-2244.

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(25) Repaske, D.R.; Corbin, J.G.; Conti, M.; Goy, M.F. A cyclic GMP-stimulated cyclic nucleotide phosphodiesterase gene is highly expressed in the limbic system of the rat brain. Neuroscience 1993, 56, 673-686. (26) Stephenson, D.T.; Coskran, T.M.; Wilhelms, M.B.; Adamowicz, W.O.; O-Donnell, M.M.; Muravnick, K.B.; Menniti, F.S.; Kleiman, R.J.; Morton, D. Immunohistochemical localization of phosphodiesterase 2A in multiple mammalian species. J. Histochem. Cytochem. 2009, 57, 933-949. (27) Boess, F.G. ; Hendrix, M.; van der Staay, F.J.; Erb, C.; Schreiber, R.; an Staveren, W.; de Vente, J.; Prickaerts, J.; Blokland, A.; Koenig, G. Inhibition of phosphodiesterase 2 increases neuronal cGMP, synaptic plasticity and memory performance. Neuropharmacology 2004, 47, 1081-1092. (28) Domek-Łopacińska, K.; Strosznajder, J.B. The effect of selective inhibition of cyclic GMP hydrolyzing phosphodiesterases 2 and 5 on learning and memory processes and nitric oxide synthase activity in brain during aging. Brain Res. 2008, 1216, 68-77. (29) Rutten, K.; Prickaerts, J.; Hendrix, M.; van der Staay, F.; Sik, A.; Blockland, A. Timedependent involvement of cAMP and cGMP in consolidation of object memory: studies using selective phosphodiesterase type 2, 4 and 5 inhibitors. Eur. J. Pharmacol. 2007, 558, 107-112. (30) Bollen, E., Puzzo, D.; Rutten, K.; Privitera, L.; De Vry, J.; Vanmierlo, T.; Kenis, G.; Palmeri, A.; Balschun, D.; Steinbusch, H. W. M.; Blokland, A.; Prickaerts, J. Improved longterm memory via enhancing cGMP-PKG signaling requires cAMP-PKA signaling. Neuropharmacology 2014, 39, 2497-505.

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(31) Bollen, E.; Akkerman, S.; Puzzo, D.; Gulisano, W.; Palmeri, A.; D’Hooge, R.; Balschun, D.; Steinbusch, H.W.M.; Blokland, A.; Prickaerts, J. Object memory enhancement by combining sub-efficacious doses of specific phosphodiesterase inhibitors. Neuropharmacology 2015, 95, 361-366. (32) Zhu, J.; Yang, Q.; Dai, D.; Huang, Q. X-ray crystal structure of phosphodiesterase 2 in complex with a highly selective, nanomolar inhibitor reveals a binding-induced pocket important for selectivity. J. Am. Chem. Soc. 2013, 135, 11708-11711. (33) Afonine, P.V.; Grosse-Kunstleve, R.W.; Echols, N.; Headd, J.J.; Moriarty, N.W.; Mustyakimov, M.; Terwilliger, T.C.; Urzhumtsev, A.; Zwart, P.H.; Adams, P.D. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2012, 68, 352-367.

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