Discovery of potent, selective, and orally bioavailable inhibitors

Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC

Brief Article

Discovery of potent, selective, and orally bioavailable inhibitors against phosphodiesterase-9, a novel target for the treatment of vascular dementia Yinuo Wu, Qian Zhou, Tianhua Zhang, Zhe Li, Yiping Chen, Pei Zhang, Yanfa Yu, Haiju Geng, Yi-Jing Tian, Chen Zhang, Yu Wang, Jian-Wen Chen, Yan Chen, and Hai-Bin Luo J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01041 • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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.

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

Journal of Medicinal Chemistry

Discovery of potent, selective, and orally bioavailable inhibitors against phosphodiesterase-9, a novel target for the treatment of vascular dementia Yinuo Wua, #, Qian Zhoua, #, Tianhua Zhanga, #, Zhe Lia, Yi-Ping Chena, Pei Zhanga, Yan-Fa Yua, Haiju Genga, Yi-Jing Tiana, Chen Zhanga, Yu Wangb, Jian-Wen Chena,*, Yan Chenc,*, and Hai-Bin Luoa,* School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, P. R. China Infinitus (China) Co. Ltd, Guangzhou 510663, P. R. China c College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou 310014, P. R. China a

b

ABSTRACT: To identify phosphodiesterase-9 (PDE9) as a novel target for the treatment of vascular dementia (VaD), a series of pyrazolopyrimidinone analogues were discovered based on a hit 1. Hit-to-lead optimization resulted in a potent inhibitor 2 with excellent selectivity and physicochemical properties to enable in vivo studies. Oral administration of 2 (5.0 mg/kg) caused notable therapeutic effects in the VaD mouse model, providing a promising lead or chemical probe for investigating the biological functions of PDE9 inhibition.

Introduction Vascular dementia (VaD) is an age-related neurodegenerative disease that is characterized by cognitive disorders caused by cerebrovascular diseases and ischemic or hemorrhagic brain injury.1 Currently, VaD has become the second common type of dementia among the aged people after Alzheimer’s disease (AD), which occurs in approximately 20% of all dementia patients, and that occurrence will triple by 2050.2 No specific drug was approved for VaD yet, although it is the only type of dementia that can be prevented by early treatment. Thus, discovering new anti-VaD agents remains a great challenge.3,4 Phosphodiesterases (PDEs) are an enzyme superfamily in hydrolyzing the second messengers cAMP and cGMP, and they are divided into 11 subfamilies according to differences in structure and distribution.5 Up to 11 PDE inhibitors (such as cilostazol, roflumilast, sildenafil, and tadalafil) have already been approved on the market for the treatment of erectile dysfunction, pulmonary hypertension, chronic obstructive pulmonary disease, and other purposes.6-9 For the treatment of VaD, PDE3 inhibitor cilostazol and PDE5 inhibitor tadalafil exhibited excellent treatment effects for cognitive impairment in vivo and have progressed to clinical research studies.10-13 PDE9 is another cGMP-specific subfamily similar to PDE5 with the Km of 70 nM for cGMP and 230 μM for cAMP.14 PDE9 is highly expressed in the brain, kidney, spleen, and small intestine, and thus its inhibitors have worked efficiently in the preclinical and clinical trials of AD.15-18 However, no research of PDE9 inhibitors against VaD has been reported yet. Recently our preliminary animal study of VaD demonstrated that PDE9 inhibitor 1 (Table 1, IC50 = 49 nM and rat liver microsomal stability t1/2 = 8.03 min) could effectively recover learning and memory function. Herein, one of the aims of this work is to perform hit-to-lead optimization to improve binding affinity and metabolic stability, and the second aim is to identify PDE9 inhibition as a novel drug target for the treatment of VaD. As a result, lead 2 had an IC50 of 8.7 nM against PDE9 with

excellent selectivity across other PDE families and physicochemical properties to enable in vivo studies. In the animal model of VaD, lead 2 significantly improved memory impairment, providing a promising lead or chemical probe for investigating the biological functions of PDE9 inhibition. O

MeO

O

Me HN

H N O

N H

N N

Structure-based design

Me HN F

N

Metabolic stability

N O

N

2

3r IC50 = 0.6 nM F = 9.8 % T1/2 = 1.67 h

N N

N H

IC50 = 8.7 nM F = 41 % T1/2 = 2.96 h

Figure 1. Structure-based design of novel PDE9 inhibitors. Result and Discussion Rational design of novel PDE9 inhibitors to improve binding affinity and metabolic stability. We used to develop a potent PDE9 inhibitors 3r (Figure 1, IC50 = 0.6 nM) in 2014.19 Despite of its high IC50 values, the bulky N-(4-methoxyphenyl) acetamide substituents of 3r significantly reduced its metabolic stability and physicochemical properties (oral bioavailability = 9.8 %), limiting its clinical usage in treating CNS diseases. Replacement of 4-methoxyphenyl on 3r to a morpholine group led to compound 1 (Table 1 and Scheme 1), with lower IC50 against PDE9A but longer T1/2 of about 2 h in rat liver microsome. In our preliminary animal study of VaD, compound 1 was used as a tool medicine and could effectively recover learning and memory function. In order to obtain PDE9 inhibitors with better binding affinity and metabolic stability to identify PDE9 inhibition as a novel drug target for the treatment of VaD, hit-to-lead optimization on compound 1 was performed in this study. According to the X-ray crystal structure of the PDE9-3r complex (Figure S1),19,20 the pyrazolopyrimidinone ring of 3r forms two hydrogen bonds with Gln453 and π-π stacking against Phe456, which are important interactions for the high

ACS Paragon Plus Environment

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

affinity of PDE inhibitors. Thus, the scaffold is retained in our structure-based design procedure. Different and less bulky aliphatic amines were docked into the binding pocket of PDE9 to replace the morpholine group in 1 in order to discover new compounds with reasonable binding affinity and better metabolic stability. This rational design and molecular docking led to the discovery of the basic fragment shown in Table 1, which significantly reduced the load of chemical synthesis and subsequent bioassay. It has been reported that the low selectivity of PDE9 inhibitors over PDE1 may induce side effects as anti-AD agents.21 Thus, in the current study, a suitable candidate selected from our designed PDE9 inhibitors should have an IC50 value less than 10 nM and more than 100-fold selectivity over PDE1 to avoid possible side effects. To remove possible false hits, a pan-assay interfering compound substructures (PAINS) screening was performed by using the online program PAINS-Remover (http://cbligand.org/PAINS).22 All the designed compounds passed this test. Scheme 1. The synthetic route for the designed compounds 1a-1k, 2, and 3.a

Page 2 of 8

1b. Compounds 1c and 1d with dimethylamino and methylethyl groups yielded the worst IC50 (1c: 594 nM, 1d: 227 nM) values among all the designed compounds, indicating that conformationally restricted substituents at R position might be more preferred. Table 1. The inhibitory activity of synthesized compounds against PDE9 and PDE1B.a

Cmp. 1 1a 1b 1c 1d 2

R N O

N N S Me

N Me Et N Me

N

PDE9A IC50 (nM) 49 ± 5 112 ± 5

PDE1B IC50 (μM)b ＞ 10 (>204) 1.4 ± 0.2 (12)

LLEc LBE 6.66 6.06

20.3 20.2

13 ± 2

1.2 ± 0.2 (92)

6.33

21.8

594 ± 18

1.4 ± 0.4 ( 2)

5.70

19.6

227 ± 9

1.7 ± 0.4 (7)

5.58

20.0

8.7 ± 0.3

1.3 ± 0.1 (153) 7.18

22.2

20 ± 5

1.8 ± 0.1 (92)

6.82

21.2

88 ± 9

2.2 ± 0.9 (24)

6.11

18.2

15 ± 3

10 ± 0.3 (667)

6.87

20.2

100 ± 9

7.8 ± 0.9 (78)

5.83

18.4

103 ± 8

2.4 ± 0.3 (24)

6.23

18.7

91 ± 7

0.7 ± 0.1 (8)

5.64

19.6

19 ± 3

1.2 ± 0.5 (6)

6.15

21.5

24 ± 4

2.0 ± 0.2 (83)

7.29

21.1

F

3

N F

1e

N

1f

N

1g

N

1h

N

NMe2

NMe2

F F OMe

1i

N Me

1j 1k aReagents

and conditions: (a) Triethylamine, iso-propanol, 80 °C, 16 h; (b) NaOH, methanol-water 3:1, 80 °C, 3 h; (c) HATU, triethylamine, DMF, room temperature, 2 h. Chemistry. The synthetic route for our targeted compounds is outlined in Scheme 1. The starting compound M-1 was obtained using the same method from our previous reports.19 In the presence of triethylamine, compound M-1 reacted with Dalanine ethyl ester to produce compound M-2 in 78% yield, which was then hydrolyzed to compound M-3 with sodium hydroxide as the base and a mixture of methanol-water as the solvent. Different amines reacted with compound M-3 with the assistance of HATU,23 which led to production of compounds 1a-1k, 2, and 3 in good yields.

Structure-Activity Relationships (SARs). Most of our newly designed compounds exhibited remarkable IC50 values below 200 nM toward PDE9 in the bioassay in vitro (Table 1). This result correlated with previous reports by our group and others that the interactions formed by this scaffold with Gln453 and Phe456 are crucial for the high affinity of PDE9 inhibitors.19-21 Compounds 1a and 1b with a pyrrolidine group and a thiazolidine group at the R position had IC50 values of 112 nM and 13 nM, respectively. From the predicted binding modes of compounds 1a and 1b (Figure S2), compound 1a formed two H-bonds with PDE9 while 1b involved three H-bonds, which might contribute to the better inhibitory potentcy of compound

N H N H

O

a Bay73-6691, the positive control with an IC

50 of 50 nM.

b Number

in the parentheses is the folds of selectivity against PDE1B; c LLE = pIC50-clogP and LBE = pIC50/MW*1000. Compounds 3r and 28s19 gave the LLE or LBE values of 5.64 or 23.3 and 2.92 or 17.5, respectively.

The introduction of substituents on the pyrrolidine ring of 1a formed 2, 3, and 1e-1i, which gave the IC50 value at the range of 8.7-103 nM. Compounds 2 and 3 are two diastereomers with chiral 3-fluoropyrrolidine groups at the R position. Relative to 3 with the configuration of (R, R), 2 with the configuration of (R, S) gave a slightly higher IC50 value of 8.7 nM, which is comparable to that (8.3 nM) for Pfizer II clinical trial inhibitor PF-04447943.21 However, for the other two diastereomers, 1e and 1f, with dimethylamino substituents, the (R, R)-isomer 1f was 6 times more potent than that of the (R, S)-isomer 1e. From the docking modes of these compounds, substituents on the pyrrolidine switched into different positions due to the chirality. The pyrazolopyrimidinone ring of compounds 2 and 3 adopted a very similar binding mode and both formed three H-bonds with PDE9. Binding free energies of these diastereomers with PDE9 were predicted by the molecular dynamics simulations20 to explain the potency difference of 2 and 3, of which 2 gave a more negative value than that of 3 (-36.80 vs -31.69 kcal/mol, Table S1). For compounds 1e and 1f, the bulky dimethylamino group in compound 1e is too close to the residue Phe441 (< 3.0 Å), which possibly affected its configuration and made it only


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

Journal of Medicinal Chemistry form two H-bonds with PDE9, whereas 1f formed three Hbonds (Figure S2). Table 2. Metabolic Stability of 2 and 1b in Rat Liver Microsomes Compound k T1/2 (min) Clint (mL/min/mg) Clapp (mL/min/kg) 1b 0.0773 8.97 0.1545 278.15 2 0.0309 22.45 0.0617 111.15 sildenafil 0.1291 5.37 0.2582 462.69 The inhibitory potencies of compounds against PDE1B were tested for the calculation of selectivity, which gave the IC50 values at the range of 0.7-2.4 μM excepted compounds 1f and 1g. Thus, only derivatives 2 and 1f gave the selectivity over 100 folds. LBE and LLE values of targeted compounds were outlined in Table 1. The results showed that most of the

Clh (mL/min/kg) 45.92 36.79 49.32

Eh (%) 83% 67% 89%

the same level but its selectivity over PDE1C is significantly better than that of PF-04447943, which suggested that 2 was suitable for further physicochemical assessment studies.

compounds gave much improved LLE values than compound 3r and 28s (5.64 and 2.92).

Better metabolic stability of compound 2 than 1b and Sildenafil. Before further investigation on lead compounds, the two most potent compounds 1b and 2 were subjected to preliminary metabolic stability evaluation by rat liver microsomal experiments in vitro. As shown in Table 2, compounds 1b and 2 had T1/2 (half time) values of 8.97 min and 22.45 min as well as Eh (hepatic extraction ratio) values of 83% and 67%, respectively, which indicated that 2 was significantly more stable than 1b, 1 (T1/2 = 8.03 min), and the positive control sildenafil (T1/2 = 5.37 min). Thus, compound 2 was subjected to further studies. Tight binding of compound 2 to PDE9. The co-crystal structure of the PDE9-2 complex was achieved to explore their binding pattern (Figure 2). Compound 2 adopted a similar binding pattern as that of 3r19 as expected, but the tails of 3r and 2 switched to different positions in the binding pocket. Compound 2 formed H-bonds (2.78 Å and 3.05 Å) with the invariant Gln453 and π-π stacking against Phe456, which are the main reasons for its high affinity of PDE9. Specifically, 2 formed an extra H-bond of 2.92 Å with residue Ala452, which may enhance its potent affinity. Additionally, 3fluoropyrrolidine group in 2 involved strong hydrophobic interactions with Phe441. Inhibitor 3r may use the amide nitrogen to involve a hydrogen bond with residue Tyr424,19 but the phenomenon was not observed in the PDE9-2 complex despite that 2 also owns an amide group. Thus, the stronger IC50 of 3r replacement of the 3-fluoropyrrolidine group with the 4-methoxyphenyl group significantly improves the binding affinity of compound 3r, which possibly resulted in 0.6 nM compared with 8.7 nM for 2. Excellent selectivity of compound 2 across PDE families. Besides PDE1B, the selectivity index of compound 2 across other PDE families is outlined in Table 3. Compound 2 had weak inhibition (IC50 > 10 M) toward PDE1C, PDE2A, PDE3A, PDE4D2, and PDE8A1, and thus, it showed more than 1149-fold selectivity over these PDE subfamilies. For other families, such as PDE1B, PDE5A1, PDE7A1, and PDE10A1, compound 2 gave 153-fold, 461-fold, 555-fold, and 840-fold selectivity relative to PDE9, respectively. In common, low selectivity of PDE9 inhibitors over PDE1C may induce side effects in cardiac tissue.21 Compared with Pfizer II clinical trial inhibitor PF-04447943 (IC50: 8.3 nM of PDE9 and selectivity over PDE1C: 167-fold), its inhibition toward PDE9 is almost at

Figure 2. Tight binding of 2 to PDE9 in the crystal structure (PDB code: 6A3N). (A) Surface presentation of the 2 binding to the active site pocket of PDE9. Atoms of carbon, nitrogen, and oxygen of PDE9 are presented in colors of yellow, blue, and red, respectively. Inhibitor 2 is shown as yellow sticks. Dotted lines represent hydrogen bonds. (B) Ribbon model of the binding for the PDE9-2 complex. The magenta mesh is the electron density of the difference (2Fo − Fc) map that was calculated from the structure and contoured at 1σ. Table 3. Selectivity of Compound 2 across PDE Families PDEs IC50 (nM) Selectivity fold PDE9A2 (181-506) 8.7 ± 0.3 PDE1B (10-487) 1 330 ± 120 153 PDE1C (2-634) >10 000 >1 149 PDE2A (580-919) >10 000 >1 149 PDE3A (679-1087) >10 000 >1 149 PDE4D2 (86-413) >10 000 >1 149 PDE5A1 (535-860) 4 010 ± 530 461 PDE7A1 (130-482) 4 830 ± 250 555 PDE8A1 (480-820) >10 000 >1 149 PDE10A (449-770) 7 310 ± 690 840 Reasonable physicochemical properties to enable in vivo studies. To further characterize the physicochemical properties of compound 2, its pharmacokinetic properties, water solubility, plasma protein binding rate, cytochrome inhibition, hERG inhibition, and acute safety were evaluated. The pharmacokinetic properties of compound 2 in rats are shown in Table 4. After an oral dose of 5 mg/kg to rats, the properties included Cmax of 294 ng/mL, t1/2 of 2.96 h, and oral bioavailability of 41% (Table 4), respectively. These parameters show significant improvements over the parameters from our previously reported PDE9 inhibitors 3r (Cmax of 217 ng/mL, t1/2 of 1.67 h, and oral bioavailability of 9.8%).19 Its oral bioavailability of 41% is 4-fold higher than those of 3r, suggesting that it has practical usability and the potential to be used as a candidate.



Table 4. Pharmacokinetic Profile of Compound 2 in SD rats t1/2 tmax Cmax AUC(0-t) (h) (h) (ng/mL) (h·ng /mL) i.v. 0.32  0.00 0.083  0.001 1261  25 596  38 p.o. 2.96  1.92 0.33  0.14 294  91 486  118 Table 5. Physicochemical Properties of Compound 2 Content Value Water solubility (pH: 7.3) 126 μg/mL Rat liver microsome stability (T1/2) 22.45 min Pharmacokinetic properties (p.o.) t1/2 2.96 h F% 41% Human plasma binding rate 76 % Mice plasma binding rate 78 % Unbound brain concentration* 8.6 ng/g Cytochrome P450 inhibition (IC50) >10 μM CYP1A2, 2B6, 2C9, 2D6, and 3A4 hERG inhibition (IC50) > 30 μM Acute toxicity >1.5 g/kg * After an oral dose of 10 mg/kg at 15 min in mice By using the same approach as pharmacokinetic studies, we examined the unbound brain concentration of compound 2 in the brain homogenates after an oral dose of 10 mg/kg (Table S2). For mice, the unbound brain concentrations of compound 2 were 8.6 and 5.5 ng/g after 15 min and 30 min, respectively, while for rats, its concentrations were 10.6 and 7.3 ng/g. Even after 30 min at an intraperitoneal dose of 10 mg/kg, its unbound brain concentration was 24.8 ng/g for mice, which was over four-fold higher than that at the oral dose. It was approximately calculated that the unbound brain concentrations of compound 2 at an oral dose of 10 mg/kg for mice were about 24 nM at 15 min and 15 nM at 30 min, respectively, which demonstrated that both of them were higher than the IC50 value (8.7 nM) of 2. Besides the high expression of PDE9A in brain, the considerable unbound brain penetration of compound 2 after the oral dose and the potent inhibition (IC50) of 2 potentially made it to activate the cGMP signaling and cause signal amplification for memory improvement. Similar phenomenon was observed for PDE2 inhibitor Bay 60-7550.24 The human plasma protein binding rate of 2 (76%) was lower than the value (98%) for the positive control propranolol (Table 5). The inhibitory IC50 values of 2 against five human hepatic CYP P450 enzymes (CYP1A2, 2B6, 2C9, 2D6, and 3A4) were >10 μM, which indicates that 2 may not exhibit significant pharmacokinetic interactions with drugs that are metabolized by the five major CYP isoforms. Additionally, the IC50 of 2 against hERG was above 30 μM, which suggests that 2 exhibited a weak inhibitory effect on hERG. Finally, the acute toxicity of 2 was evaluated in mice and it was well tolerated up to a dose of 1.5 g/kg with no acute toxicity. Based on the good physicochemical properties, inhibitor 2 is suitable for further pharmacodynamic experiments. Notable therapeutic effects in vivo to identify PDE9 as a novel drug target for VaD. To determine the in vivo memory improvement effect of the most potent PDE9 inhibitor 2 in VaD,

AUC(0-∞) (h·ng /mL) 604  39 544  72

Page 4 of 8

MRT(0-t) (h) 0.35  0.02 1.93  0.56

F (%) 41  5

Morris water maze (MWM) test in a unilateral common carotid artery occlusion (UCCAO) mouse model was adopted. The UCCAO mouse model relies on the induction of chronic cerebral hypoperfusion to produce a vascular cognitive impairment (VCI)-related pathology, such as white matter damage, neuronal degeneration, cognitive deficits, and inflammation.25 Escape latency time (ELT) of the animal to locate the hidden platform, frequency of the platform site crossings in spatial probe trial and mean time spent by the animal in the target quadrant for searching the hidden platform were recorded for the evaluation of learning and memory ability for each groups.26 Compared with those of the control group, the ELT in the model group was significantly increased while the frequency of platform area crossings and times in the target area were decreased after the platform was removed. This result in Figure 3 demonstrated that UCCAO mouse model is successfully built. Oral administration of compound 2 (daily 2.5 mg/kg and 5.0 mg/kg for 21 days) to UCCAO-treated mice significantly reduced the day 6 ELT and increased the frequency of platform area crossings compared to those of UCCAO-treated mice, which indicated there was an improvement in the learning capabilities of the animals (Figure 3A-3B). The duration of time spent in the target quadrant was considerably longer for the UCCAO/2-treated mice (Figure 3C) than that for the model mice, which also suggested that 2 effectively recovered learning and memory function. Statistically, there was no significant difference in the day 6 ELT between the high and low dose groups (2H and 2L, Figure 3A). But qualitatively, the high dose group possibly improved the ELT of mice, from 34.55 s of 2L to 22.04 s of 2H, which demonstrated that compound 2 effectively recovered learning and memory function. The same phenomenon also occurs in frequency of platform area crossings (Figure 3B). In addition, oral administration of compound 2 at the dose of 10.0 mg/kg gave a slightly better result on the ELT and the duration of time spent in the target quadrant than that at the dose of 5.0 mg/kg (Figure S3), in which the high and low doses of 2 did not statistically show dose response in their therapeutic effects. Similar phenomenon was observed when using PDE9 inhibitor PF-04447943 was applied in the AD preclinical experiments, which suggested that lower doses were more likely to be efficacious, and though the reason for this remains to be clarified in their manuscript.27 Donepezil (0.7 mg/kg, i.g.) served as the positive control, which nearly provided a comparable memory improvement effect to 2.28 Representative swimming trajectories of each group in the spatial probe trial are presented in Figure 3D, which showed that both compound 2 and donepezil increased the number of platform crossings compared with the model group. All these results indicated that 2 exhibited notable therapeutic effects and efficiently improved cognitive impairment in the UCCAO mouse model of VaD, which identified PDE9 inhibition as a novel target for the treatment of VaD.


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

Journal of Medicinal Chemistry

Figure 3. Compound 2 improved the spatial learning and memory in unilateral common carotid artery occlusion (UCCAO) mice after 21 days treatments. 2L and 2H refer to the oral doses of 2.5 mg/kg and 5.0 mg/kg for compound 2, respectively. (A) Escape latency time (ELT) of mice in spatial probe trial (s). (B) Frequency of the platform site crossings in spatial probe trial (min-1). (C) Swimming time in the target area in the spatial probe trial (s). (D) Representative swimming trajectories of each group in the spatial probe trial. The location of the original platform and the effective region (2-fold diameter of the platform) were represented as blue and bright green circles, respectively. Donepezil (0.7 mg/kg, i.g.) was used as the positive control. Mice in each group, n = 6~10. #, p < 0.05 vs. control; ###, p < 0.001 vs. control; *, p < 0.05 vs. model; **, p < 0.01 vs. model, respectively. Conclusion This study is the first report to identify PDE9 as a novel target for VaD by using potent, selective, and oral bioavailable inhibitors. In this study, structural optimization resulted in lead 2, which exhibited potent inhibition against PDE9, excellent selectivity across other PDE subfamilies, and reasonable physicochemical properties. Further animal studies identified the therapeutic potential of PDE9 inhibition for the treatment of VaD, in where 2 significantly improved memory impairment in the VaD model. Additionally, the binding pattern of PDE9 with 2 was revealed by the X-ray structure, which may provide evidence for rational design of selective PDE9 inhibitors. Experimental section General. All the reagents were purchased from commercial suppliers (Sigma-Aldrich, Adamas, Energy), which are used directly without further purification. Chemical HG/T2354-92 silica gel (200-300 mesh, Haiyang®) was used for chromatography. Silica gel plates with fluorescence F254 (0.25 mm, Huanghai®) were used for thin-layer chromatography (TLC) analysis. 1H NMR and 13C NMR spectra were recorded at room temperature on a Bruker AVANCE III 400 instrument or Bruker Ascend TM 500 instrument with tetramethylsilane (TMS) as an internal standard. The following abbreviations are used: s (singlet), d (doublet), dd (two doublets), t (triplet), q (quartet), and m (multiplet). Coupling constants were reported in Hz. High-resolution mass spectra was recorded on a Shimadzu LCMS-IT-TOF mass spectrometer. The purity of compounds was determined by reverse-phase highperformance liquid chromatography (HPLC) analysis confirming to be over 95%. HPLC instrument: SHIMADZU LC-20AT (column: Hypersil BDS C18, 5.0 μm, 4.6 × 150 mm (Elite); Detector: SPD-20A UV/VIS detector, UV detection at 254 nm; Elution, MeOH in water (50% or 70%, v/v); T = 25°C; and flow rate = 1.0 mL/min. Ethyl (1-cyclopentyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4d]pyrimidin-6-yl)-D-alaninate (M-2). To the solution of 6-

chloro-1-cyclopentyl-1, 5-dihydro-4H-pyrazolo[3,4-d] pyrimidin -4-one (M-1) (4.76 g, 20.0 mmol) and ethyl Dalaninate (2.80 g, 24.0 mmol) in iso-propanol (80 mL), triethylamine (2.69 g, 24.0 mmol) was added. The mixture was stirred at 80OC for 24 h. The solvent was evaporated and water was added to the residue. The resulting solution was extracted with ethyl acetate (3×100 mL) and washed with brine (3×100 mL). The combined organic extracts were dried over anhydrous sodium sulfate, and concentrated to give a crude product, which was purified by silica gel column chromatography (petroleum ether/EtOAc, 1:1) to get the pure M-2 (5.19 g, 80%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.96 (s, 1H), 7.02 (d, J = 6.8 Hz, 1H), 5.07- 4.92 (m, 1H), 4.71 (d, J = 6.9 Hz, 1H), 4.34 - 4.20 (m, 2H), 2.10 - 2.06 (m, 4H), 1.96-1.94 (m, 2H), 1.711.69 (m, 2H), 1.56 (d, J = 6.8 Hz, 3H), 1.28 (td, J = 7.1, 1.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 173.25, 160.20, 154.06, 151.99, 134.57, 100.23, 61.47, 57.60, 49.87, 32.00, 31.88, 24.78, 18.30, 14.22. HRMS (ESI) m/z calcd C15H21N5O3 [M+H]+ 320.1717, found 320.1694. 1-cyclopentyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4d]pyrimidin-6-yl)-D-alanine (M-3). M-2 (3.19 g, 10.0 mmol) was dissolved in the mixture of methanol (20 mL) and water (20 mL). 1M NaOH (11 mL) was added. The mixture was stirred at 70OC for 2h. After the reaction was finished, methanol in the solvent was evaporated and 1M HCl (15 mL) was added the mixture slowly. The precipitate was filtered and washed with water, giving the pure M-3 (2.72 g, 93%) as a white solid. 1H NMR (400 MHz, CD3OD) δ 7.81 (d, J = 1.4 Hz, 1H), 4.98 (m, 1H), 4.55 (m, 1H), 2.07-2.03 (m, 6H), 1.72-1.70 (m, 2H), 1.52 (dd, J = 7.2, 1.5 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ 175.11, 159.72, 153.85, 152.46, 133.92, 99.90, 57.93, 49.55, 31.32, 31.24, 24.27, 24.25, 16.64. HRMS (ESI-TOF) m/z [M+H]+ calcd for C13H17N5O3 292.1404, found 292.1395. 1-cyclopentyl-6-(((R)-1-((S)-3-fluoropyrrolidin-1-yl)-1oxopropan-2-yl)amino)-1,5-dihydro-4H-pyrazolo[3,4d]pyrimidin-4-one (2). M-3 (0.15 g, 0.50 mmol) and (R)-3fluoropyrrolidine (53 mg 0.60 mmol) were dissolved in DMF.



HATU (0.23 g, 0.60 mmol) and N, N-diisopropylethylamine (96 mg, 0.75 mmol) were added successively. The mixture was stirred at room temperature for 2 h. The mixture was diluted with water and extracted with ethyl acetate three times. The combined organic layers were dried over anhydrous sodium sulfate, and concentrated to give a crude product, which was purified by silica gel column chromatography (CH2Cl2/MeOH, 20:1) to get the compounds 2 as a white solid. Yield: 65%. Purity: 98%.1H NMR (500 MHz, CDCl3) δ 10.29 (dd, J = 58.7, 19.6 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.18-7.15 (m, 1H), 5.45 - 5.33 (m, 1H), 5.01 - 4.89 (m, 2H), 4.10 - 4.08 (m, 1H), 3.96 3.81 (m, 3H), 2.46 - 2.43 (m, 1H), 2.19 - 2.13 (m, 1H), 2.08 (m, 4H), 1.96 (m, 2H), 1.71 (m, 2H), 1.49 (dd, J = 12.4, 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl ) δ 172.76, 159.14, 153.71, 151.90, 3 134.59, 100.59, 91.71 (d, JF = 52.5 Hz, 1C), 57.33, 53.44, 48.19, 44.79, 32.12 (d, JF = 24.2 Hz, 1C), 32.15, 32.10, 24.86, 18.28. HRMS (ESI-TOF) m/z [M+H]+ calcd for C17H23N6O2F 363.1939, found 363.1933. In Vitro Enzymatic Activity Assay. The catalytic domains of PDE1B (10-487), PDE2A (580-919), PDE3A (679-1087), PDE4D (86-413), PDE7A (130-482), PDE8A (480-820), PDE9 (181-506), and PDE10A (449-770) were purified using a similar protocol in our previously published protocol.19-20 PDE1C (2-634) was bought from BPS Bioscience. 3H-cGMP was the substrate for the biological test against PDE9A, PDE1B, PDE1C, and PDE2A while 3H-cAMP for those of PDE3A, PDE4D, PDE7A, PDE8A, and PDE10A. Firstly, 3HcGMP or 3H-cAMP was diluted with the assay buffer (20-50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, and 1 mM DTT) to 20 000-30 000 cpm per assay. The mixture was then performed at 25 °C for 15 min and terminated by the addition of 0.2 M ZnSO4. 0.2 N Ba(OH)2 was added with a precipitate formed. The unreacted 3H-cGMP was left in the supernatant. The radioactivity in the supernatant was measured in 2.5 mL of Ultima Gold liquid scintillation cocktail (PerkinElmer) using a PerkinElmer 2910 liquid scintillation counter. For the measurement of IC50 of compounds, eight different concentrations were used, and each measurement was at least repeated three times.

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website. Characterization of target compounds, Procedures for crystallization trials, physicochemical properties of compound 2, Procedure for the UCCAO mice model of vascular dementia and the Morris water maze test, Procedure for the molecular docking and dynamics stimulations. 1H NMR, 13C NMR, Highresolution mass spectra (HRMS) data, HPLC spectra data for tested compounds and The unbound concentrations of compound 2 in the brain homogenates. Molecular formula strings and some data (CSV). Accession Codes The atomic coordinates and structure factors have been deposited into the RCSB Protein Data Bank with accession number 6A3N. Authors will release the atomic coordinates and experimental data upon article publication.

AUTHOR INFORMATION Corresponding Author

*To whom correspondence should be addressed. Fax: +86-2039943000. E-mail: [email protected] (H. -B. L.), [email protected] (Y. C.), and [email protected] (J. C.). ORCID Hai-Bin Luo: 0000-0002-2163-0509 Author Contributions # These authors contributed equally to this work.

ABBREVIATIONS AUC, area under the curve; AD, Alzheimer’s disease; cGMP, cyclic guanosine monophosphate; Clapp, apparent clearane; Clh, hepatic clearane; Clint, intrinsic clearane; Cmax, peak concentration; CYP, cytochrome P450s; DCM, dichloromethane; DIPEA, ethyldiisopropylamine; Eh, hepatic extraction ratio; ELT, escape latency time; HATU, O-(7-azabenzotriazol-1-yl)uronium hexafluoro-phosphate; hERG, the human Ether-a-go-go-Related Gene; MD, molecular dynamics; MRT, mean residence time; PAINS, pan-assay interfering compound substructures; PDB, protein data bank; PDE, phosphodiesterase; PDE9, phosphodiesterase-9; PO, oral administration; IV, intravenous administration; SAR, structure-activity relationship; t1/2, half time; tmax, peak time; UCCAO: unilateral common carotid artery occlusion; VaD, Vascular dementia.

ACKNOWLEDGMENT This work was supported by the Natural Science Foundation of China (21877134, 21572279, 81602955, and 81703341), Science Foundation of Guangdong Province (2016A030310144), Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (2016) and Guangzhou Pearl River New Star Fund Science and Technology Planning Project (201806010190). We cordially thank Prof. H. Ke at the University of North Carolina, Chapel Hill, for his help with molecular cloning, expression, purification, crystal structure, and bioassay of PDEs.

REFERENCES (1) O'Brien, J. T.; Thomas, A. Vascular Dementia. The Lancet 2015, 386, 1698-1706. (2) Gorelick, P. B.; Scuteri, A.; Black, S. E.; Decarli, C.; Greenberg, S. M.; Iadecola, C.; Launer, L. J.; Laurent, S.; Lopez, O. L.; Nyenhuis, D.; Petersen, R. C.; Schneider, J. A.; Tzourio, C.; Arnett, D. K.; Bennett, D. A.; Chui, H. C.; Higashida, R. T.; Lindquist, R.; Nilsson, P. M.; Roman, G. C.; Sellke, F. W.; Seshadri, S. Vascular Contributions to Cognitive Impairment and Dementia: A Statement for Healthcare Professionals from the American Heart Association/American Stroke Association. Stroke 2011, 42, 2672-2713. (3) Iadecola, C. The Pathobiology of Vascular Dementia. Neuron 2013, 80, 844-866. (4) Farooq, M. U.; Min, J.; Goshgarian, C.; Gorelick, P. B. Pharmacotherapy for Vascular Cognitive Impairment. CNS drugs 2017, 31, 759-776. (5) Maurice, D. H.; Ke, H.; Ahmad, F.; Wang, Y.; Chung, J.; Manganiello, V. C. Advances in Targeting Cyclic Nucleotide Phosphodiesterases. Nat. Rev. Drug Discov. 2014, 13, 290-314. (6) Hong, J. H.; Kwon, Y. S.; Kim, I. Y. Pharmacodynamics, Pharmacokinetics and Clinical Efficacy of Phosphodiesterase-5 Inhibitor. Expert Opin. Drug Meta. 2017, 13, 183-192. (7) Schudt, Christian; Hatzelmann, A.; Beume, R.; Tenor, H. Phosphodiesterase Inhibitors: History of Pharmacology. Handbook of experimental pharmacology. 2011, 204, 1-46. (8) Ahluwalia, J.; Udkoff, J.; Waldman, A.; Borok, J.; Eichenfield, L. F. Phosphodiesterase 4 Inhibitor Therapies for Atopic Dermatitis: Progress and Outlook. Drugs 2017, 77, 1389-1397.


Page 6 of 8

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

Journal of Medicinal Chemistry (9) Sakkas, Lazaros I.; Mavropoulos, Athanasios; Bogdanos, Dimitrios P. Phosphodiesterase 4 Inhibitors in Immune-Mediated Diseases: Mode of Action, Clinical Applications, Current and Future Perspectives. Curr. Med. Chem. 2017, 24, 3054-3067. (10) Kumar, A.; Kumar, A.; Jaggi, A. S.; Singh, N. Efficacy of Cilostazol a Selective Phosphodiesterase-3 Inhibitor in Rat Model of Streptozotocin Diabetes Induced Vascular Dementia. Pharmacol. Biochem. Behav. 2015, 135, 20-30. (11) Clinical trial study found in clinical trials for Cilostazol, NCT00847860,https://clinicaltrials.gov/ct2/show/NCT00847860?t erm=Cilostazol&cond=dementia&rank=2. (accessed Jun. 1, 2018). (12) Kim, K. T.; Chung, K. J.; Lee, H. S.; Ko, I. G.; Kim, C. J.; Na, Y. G.; Kim, K. H. Neuroprotective Effects of Tadalafil on Gerbil Dopaminergic Neurons Following Cerebral Ischemia. Neural Regen. Res. 2013, 8, 693-701. (13) Clinical trial study in clinical trials for Tadalafil, NCT02450253,https://clinicaltrials.gov/ct2/show/ NCT02450253?term=tadalafil&cond=vascular+dementia&rank=1 ). (accessed Jun. 1, 2018). (14) Wang, P.; Wu, P.; Egan, R. W.; Billah, M. M. Identification and Characterization of a New Human Type 9 cGMP-Specific Phodiesterase Splice Variant (PDE9A5) Differential Tissue Distribution and Subcellular Localization of PDE9A Variants. Gene 2003, 18, 15-27. (15) Wu, Y.; Li, Z.; Huang, Y.; Wu, D.; Luo, H.-B. Novel Phosphodiesterase Inhibitors for Cognitive Improvement in Alzheimer’s Disease. J. Med. Chem. 2018, 61, 5467-5483. (16) Prickaerts, J.; Heckman, P. R. A.; Heckman, P. R. A.; Blokland, A. Investigational Phosphodiesterase Inhibitors in Phase I and Phase II Clinical Trials for Alzheimer's Disease. Expert Opin. Inv. Drug 2017, 26, 1033-1048. (17) Domek-Lopacinska, K. U.; Strosznajder, J. B. Cyclic GMP and Nitric Oxide Synthase in Aging and Alzheimer’s Disease. Mol. Neurobiol. 2010, 41, 129-137. (18) Wang, Z.; Zhang, Y.; Zhang, H. T.; Li, Y. F. Phosphodiesterase: an Interface Connecting Cognitive Deficits to Neuropsychiatric and Neurodegenerative Diseases. Curr. Pharm. Design 2015, 21, 303-316. (19) Shao, Y. X.; Huang, M.; Cui, W.; Feng, L. J.; Wu, Y.; Cai, Y.; Li, Z.; Zhu, X.; Liu, P.; Wan, Y.; Ke, H.; Luo, H.-B. Discovery of a Phosphodiesterase 9A Inhibitor as a Potential Hypoglycemic Agent. J. Med. Chem., 2014, 57, 10304-10313. Meng, F.; Hou, J.; Shao, Y. X.; Wu, P. Y.; Huang, M.; Zhu, X.; Cai, Y. H.; Li, Z.; Xu, J.; Liu, P. Q.; Luo, H.-B.; Wan, Y.; Ke, H. Structure-based discovery of highly selective phosphodiesterase-9A inhibitors and implications for inhibitor design. J. Med. Chem. 2012, 55, 85498558.

(21) Verhoest, P. R.; Fonseca, K. R.; Hou, X.; Proulx-Lafrance, C.; Corman, M.; Helal, C. J.; Claffey, M. M.; Tuttle, J. B.; Coffman, K. J.; Liu, S.; Nelson, F.; Kleiman, R. J.; Menniti, F. S.; Schmidt, C. J.; Vanase-Frawley, M.; Liras, S. Design and Discovery of 6-[(3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl) pyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4Hpyrazolo[3,4-d] pyrimidin-4-one (PF-04447943), a Selective Brain Penetrant PDE9 Inhibitor for the Treatment of Cognitive Disorders. J. Med. Chem. 2012, 55, 9045-9054. (22) Jonathan, B. B.; Georgina, A. H. New Substructure Filters for Removal of Pan Assay Interference Compounds (PAINS) from Screening Libraries and for Their Exclusion in Bioassays. J. Med. Chem. 2010, 53, 2719-2740. (23) Matharu, D. S.; Flaherty, D. P.; Simpson, D. S.; Schroeder, C. E.; Chung, D.; Yan, D.; Noah, J. W.; Jonsson, C. B.; White, E. L.; Aube, J.; Plemper, R. K.; Severson, W. E.; Golden, J. E. Optimization of Potent and Selective Quinazolinediones: Inhibitors of Respiratory Syncytial Virus that Block RNA-Dependent RNAPolymerase Complex Activity. J. Med. Chem. 2014, 57, 1031410328. (24) Reneerkens, O. A.; Rutten, K.; Bollen, E.; Hage, T.; Blokland, A.; Steinbusch, H. W.; Prickaerts, J. Inhibition of Phoshodiesterase Type 2 or Type 10 Reverses Object Memory Deficits Induced by Scopolamine or MK-801. Behavioural Brain Res. 2013, 236, 1622. (25) Yoshizaki, K.; Adachi, K.; Kataoka, S.; Watanabe, A.; Tabira, T.; Takahashi, K.; Wakita, H. Chronic Cerebral Hypoperfusion Induced by Right Unilateral Common Carotid Artery Occlusion Causes Delayed White Matter Lesions and Cognitive Impairment in Adult Mice. Exp. Neurol. 2008, 210, 585-591. (26) Morris, R. Developments of a Water-Maze Procedure for Studying Spatial Learning in the Rat. J. Neurosci. Methods 1984, 11 47-60. (27) Hutson, P. H.; Finger, E. N.; Magliaro, B. C.; Smith, S. M.; Converso, A.; Sanderson, P. E.; Mullins, D.; Hyde, L. A.; Eschle, B. K.; Turnbull, Z.; Sloan, H.; Guzzi, M.; Zhang, X.; Wang, A.; Rindgen, D.; Mazzola, R.; Vivian, J. A.; Eddins, D.; Uslaner, J. M.; Bednar, R.; Gambone, C.; Le-Mair, W.; Marino, M. J.; Sachs, N.; Xu, G.; Parmentier-Batteur, S. The Selective Phosphodiesterase 9 (PDE9) Inhibitor PF-04447943 (6-[(3S,4S)-4-methyl-1(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro-2H-py ran4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one) Enhances Synaptic Plasticity and Cognitive Function in Rodents. Neuropharmacology 2011, 61, 665-676. (28) Madhu, K.; Saima, Z.; Abul, K. N.; Suhel, P.; Mohd, A. Betulinic Acid, a Natural PDE Inhibitor Restores Hippocampal cAMP/cGMP and BDNF, Improve Cerebral Blood Flow and Recover Memory Deficits in Permanent BCCAO Induced Vascular Dementia in Rats. Eur. J. Pharmaco. 2018, 832, 56-66.

(20) Li, Z.; Huang, Y.-Y.; Wu, Y.; Chen, J.; Wu, D.; Zhan, C.G.; Luo, H.-B. Absolute Binding Free Energy Calculation and Design of a Subnanomolar Inhibitor of Phosphodiesterase10. J. Med. Chem. 2019, 62, 2099-2111.

Table of Contents Graphic. Crystal structure

In vivo

2 O F

Memory Improvement (VaD)

HN N O

N H

N

N

N

PDE9 IC50 = 8.7 nM PDE1 IC50 = 1330 nM F = 41%, t1/2 = 2.96 h Solubility: 0.126 mg/mL clogP = 0.88 hPPB: 76% 2.5, 5.0 hERG IC50 > 30 M CYP1A2, 2B6, 2C9, 2D6, 3A4, IC50 > 10M Acute toxicity > 1.5 g/kg


mg/kg for 2L and 2H, p.o.


Table of content graphic 135x55mm (300 x 300 DPI)


Page 8 of 8

Discovery of potent, selective, and orally bioavailable inhibitors

Recommend Documents