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Novel Phosphodiesterase Inhibitors for Cognitive Improvement in Alzheimer’s Disease Miniperspective Yinuo Wu, Zhe Li, Yi-You Huang, Deyan Wu, and Hai-Bin Luo* School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, P. R. China

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S Supporting Information *

ABSTRACT: Alzheimer’s disease (AD) is one of the greatest public health challenges. Phosphodiesterases (PDEs) are a superenzyme family responsible for the hydrolysis of two second messengers: cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Since several PDE subfamilies are highly expressed in the human brain, the inhibition of PDEs is involved in neurodegenerative processes by regulating the concentration of cAMP and/or cGMP. Currently, PDEs are considered as promising targets for the treatment of AD since many PDE inhibitors have exhibited remarkable cognitive improvement effects in preclinical studies and over 15 of them have been subjected to clinical trials. The aim of this review is to summarize the outstanding progress that has been made by PDE inhibitors as anti-AD agents with encouraging results in preclinical studies and clinical trials. The binding affinity, pharmacokinetics, underlying mechanisms, and limitations of these PDE inhibitors in the treatment of AD are also reviewed and discussed. biometals, have been proposed to facilitate drug development.4 Recently, the Aβ pathological hypothesis has attracted great interest from many studies, as Aβ-plaques and neurofibrillary tangles are two major hallmarks in the brains of AD patients. However, several promising candidates targeting Aβ, such as bapineuzumab and solanuzumab, have failed in phase III clinical trials.5 Although limited information was obtained for the failure, one important possible reason is that treatment of symptomatic dementia such as β-amyloid (Aβ) plaques was too late to delay the progression of the AD.6 Furthermore, approximately 20−25% of patients have been observed with the assistance of positron emission tomography imaging to be without Aβ burden.7 Thus, the discovery of anti-AD agents, including non-Aβ-directed therapies, still has a long way to go. Phosphodiesterases (PDEs) are a superenzyme family that is responsible for the hydrolysis of two second messengers: cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP).8 The PDE superfamily is coded by 21 identified genes and can be divided into 11 subtypes (PDE1−11). Among these subtypes, PDEs 4, 7, and 8 specifically hydrolyze cAMP, whereas PDEs 5, 6, and 9 specifically hydrolyze cGMP.

1. INTRODUCTION Alzheimer’s disease (AD) is the most common type of dementia, which is characterized by progressive memory loss, a decline in language skills, and some other neurodegenerative disorders.1 According to the data provided by the Alzheimer’s Association (U.S.), approximately 46.8 million people worldwide are suffering from AD. The morbidity and mortality rates of patients with AD are high, especially for elderly individuals over the age of 60. The global societal economic cost for AD patients was estimated at $818 billion in 2015 and is expected to rise to $1 trillion in 2018 and $2 trillion in 2030.2 Thus, AD has become one of the greatest public health challenges across the world and severely impacts individuals and their families. Currently, the clinically available anti-AD drugs are three acetylcholine inhibitors (donepezil, rivastigmine, and galantamine) and one N-methyl-D-aspartate receptor (NMDA) antagonist (memantine),3 and these drugs improve only cognition and the degree of dementia in AD patients but do not reverse the progression of AD. Thus, the need to develop novel anti-AD drugs is extremely compelling. While the pathogenic mechanism of AD is highly complicated and still not fully understood, several hypotheses about the pathophysiology of AD, including low levels of acetylcholine, deposition of β-amyloid (Aβ) plaques, aggregation of Tau protein, oxidative stress, and the accumulation of © 2018 American Chemical Society

Received: September 15, 2017 Published: January 24, 2018 5467

DOI: 10.1021/acs.jmedchem.7b01370 J. Med. Chem. 2018, 61, 5467−5483

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Table 1. PDEs Inhibitors Approved on the Market drug name

target

clinical use

vinpocetine

compd

Calan, Cavinton

PDE1

cilostazol amrinone lactate ilrinone lactate roflumilast apremilast crisaborole sildenafil citrate tadalafil vardenafil hydrochloride avanafil

Pletal, Pletaal Inocor Primacor Daxas, Daliresp Otezla Eucrisa Viagra Cialis Vivanza Zepeed, Stendra

PDE3 PDE3 PDE3 PDE4 PDE4 PDE4 PDE5 PDE5 PDE5 PDE5

Parkinson’s disease Alzheimer disease antiplatelet, intermittent claudication heart failure congestive heart failure chronic obstructive pulmonary disease psoriasis and psoriatic arthritis allergic dermatitis erectile dysfunction, pulmonary arterial hypertension erectile dysfunction, pulmonary arterial hypertension erectile dysfunction erectile dysfunction

cAMP-responsive element binding protein (CREB), which has been considered a molecular switch required for learning and memory. Several preclinical and clinical studies have demonstrated that impaired CREB phosphorylation plays an important role in neurodegenerative disorders, especially AD.12 Decreasing the level of cAMP may cause a decreased concentration of CREB, which affects the transcription of genes related to synaptic plasticity and survival, such as brain-derived neurotrophic factor (BDNF), and results in the loss of synaptic plasticity and in memory decline in AD.13 Similarly, cGMP, which is derived from GTP by guanylyl cyclase (GC), could be activated by nitric oxide (NO) via the NO/cGMP pathway, which activates protein kinase G (PKG) and subsequent CREB phosphorylation, thus increasing the level of the antiapoptotic protein Bcl-2.14,15 Since the first preclinical AD study on the PDE4 inhibitor rolipram, which indicated that the inhibition of PDEs might have promising effects on the improvement of the memory deficits in AD,15 several PDE inhibitors have exhibited remarkable effects in animal models related to AD, including the Morris water maze, passive avoidance, and object recognition tasks (Table 2). In view of the encouraging results obtained in preclinical studies, several PDE inhibitors have been subjected to clinical trials for the treatment of cognitive disorders in AD patients (Table 3). Most of these inhibitors exhibited outstanding safety and tolerability profiles in phase I studies. Furthermore, over 10 PDE inhibitors have been subjected to phase II/III/IV clinical trials. Thus, this review mainly focuses on the outstanding progress that has been made in the application of PDE inhibitors for memory enhancement effects for anti-AD purposes in preclinical or clinical trials.

The remaining PDE families are capable of degrading both cGMP and cAMP.9 As cAMP and cGMP are essential in cellular signaling and function, PDE inhibitors are used to regulate many biological processes by increasing the cellular levels of cAMP and cGMP. Thus far, several PDE inhibitors have been approved as therapeutics for the treatment of erectile dysfunction, pulmonary hypertension, chronic obstructive pulmonary disease, and heart failure (Table 1).10 The most successful example of this drug class is sildenafil, a PDE5 inhibitor that is used for the treatment of male erectile dysfunction (Viagra) and pulmonary hypertension (Revatio). PDEs are highly expressed in the human brain, and their inhibitors regulate neurodegenerative processes by increasing the concentrations of cAMP and cGMP in brain tissue (Figure 1).11

2. PDE INHIBITORS STUDIED FOR THE TREATMENT OF AD 2.1. PDE1 Inhibitors. PDE1 is a Ca2+/calmodulin-dependent PDE family with three isoforms (PDE1A, -1B, and -1C) that hydrolyze both cGMP and cAMP.17 PDE1B and PDE1C are the two major isoforms existing in humans, while PDE1A has relatively low expression. Each isoform is also expressed in a tissuespecific manner. In the caudate nucleus and the nucleus accumbens, for example, PDE1B is the most abundant isoform among all PDE species, and the level of PDE1B is 10- and 100-fold higher than that of PDE1C and PDE1A, respectively. In the cortex and hippocampus, the levels of PDE1B and PDE1C are almost the same. In the periphery, PDE1C is the major isoform of PDE1 and mainly exists in the heart, bladder, and lungs (Figure S1 in Supporting Information).18 Its specific distribution in the brain and its distinctive regulation by intracellular calcium and calmodulin make PDE1 an attractive target to improve cognitive impairments in

Figure 1. Relation of PDE inhibition and the cAMP or cGMP signaling pathway in the brain: PDE, phosphodiesterase; AC, adenyl cyclase; GC, guanylyl cyclase; ATP, adenosine triphosphate; GTP, guanosine triphosphate; PKA, protein kinase A; PKG, protein kinase G; P, phosphorylated; CREB, cAMP response element binding protein.

In general, cAMP is synthesized from ATP by adenylate cyclase (AC), activates protein kinase A (PKA), and phosphorylates 5468

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Table 2. PDEs inhibitors studied in the animal models of Alzheimer’s disease compd (no., its target) Vinpocetine (1, PDE1)

subject Streptozotocin injected rats22

model Morris water maze, 10 mg/kg, icv; Passive avoidance, 10 mg/kg, icv

ITI-214 (2, PDE1) BAY 60-7550 (4, PDE2)

Rats26 Chronic stress-induced mice36

Rats and C57BL/6J mice31

Aged rats33 APP/PS1 mice

Object recognition, 0.3−3.0 mg/kg, ip Morris water maze, 3.0 mg/kg, ip; Novel object recognition and location task, 3.0 mg/kg, ip Object recognition and social memory task, 1.0 mg/kg, po Object recognition task, 0.3 mg/kg, ip

35

Object location test, 0.3 mg/kg, po; Y- maze, 0.3 mg/kg, po; Forced swim test, 0.3 mg/kg, po; Elevated zero maze, 0.3 mg/kg, po

Cilostazol (8, PDE3)

Aβ25−35-injected mice

Y-maze, 30 mg/kg, po;54 Passive avoidance, 30 mg/kg, po;54 Morris water maze, 10 mg/kg, ip55

Rolipram (9, PDE4)

APP/PS1 mice66

Morris water maze, 0.03 mg/kg, sc Radial-arm water maze, 0.03 mg/kg, sc Morris water maze, 0.5 mg/kg, ip; Passive avoidance, 0.5 and 1.25 mg/kg, ip

Aβ25−35 or Aβ40 infused rats68,71

Roflumilast (10, PDE4)

Streptozotocin injected and aged mice72

Morris water maze, 0.05 mg/kg and 0.1 mg/kg, ip

Iron-impaired aged rat73 Mice75

Object recognition, 0.01−0.1 mg/kg, ip Object location, 0.03 mg/kg, sc; Y-maze, 0.1 mg/kg, sc Xylazine/ketamine induced anesthesia. Object recognition, 0.1 mg/kg, ip

Scopolamine-impaired rats75

HT-0712 (11, PDE4)

Aged mice82

Fear conditioning, 0.1 mg/kg, ip; Morris water maze, 0.15 mg/kg, ip

MK-0952 (12, PDE4)

Rats84

FFPM (14, PDE4)

APP/PS1 mice91

Object recognition, 0.01 mg/kg, ip; Water maze delayed matching to position test, 0.3 mg/kg, ip Morris water maze, 0.5 mg/kg, po Passive avoidance, 0.5 mg/kg, po

GEBR-7b (15, PDE4)

Mice96

D159687 (17, PDE4)

Scopolamine-injected mice98 Mice98

Sildenafil (19, PDE5)

Tg2576 mice107 SAMP8 mice108 APP/PS1 mice103,104

Xylazine/ketamine induced anesthesia. Object location and recognition, 0.003 mg/kg, sc Xylazine/ketamine induced anesthesia. Y-maze, 0.001 mg/kg, iv Object recognition, 0.01 mg/kg, iv Xylazine/ketamine induced anesthesia. Morris water maze, 15 mg/kg, ip; Fear conditioning, 15 mg/kg, ip Morris water maze, 7.5 mg/kg, ip Fear conditioning, 3 mg/kg, ip Reversal Morris water maze, 3 mg/kg, ip Object recognition, 10 mg/kg, ip

SAMP8 and SAMR1 mice109

Passive avoidance, 7.5 mg/kg, ip

Tadalafil (20, PDE5)

Aged J20-mice114

Morris water maze, 15 mg/kg, po

S14 (23, PDE7)

APP/PS1 mice116

Radial arm water maze, 3 mg/kg, ip 5469

results Improve memory, Reduce oxidative−nitritive stress, Modulate cholinergic functions, Prevent neuronal damage. Improve acquisition, consolidation, and retrieval memory. Reverse cognitive impairment via neuroplasticity-related NMDAR-CaMKII-cGMP/cAMP signaling. Improve memory, Enhance LTP, Reversed MK801-induced deficits. Improve cognition and memory through enhancing the nNOS activity. Improve memory without anxiety, depressive-like behavior, or hypothalamus−pituitary−adrenal axis regulation; No changes on the level of Aβ, pCREB, BDNF or presynaptic density. Improve memory possibly due to the prevention of oxidative damage51 or by decreasing Aβ accumulation by the reduction of Aβ accumulation and Tau phosphorylation.52 Improve memory. Improve memory; Reduce oxidative−nitritive stress; Upregulate thioredoxin; Inhibit iNOS/NO pathway. Improve memory probably due to its anti-cholinesterase, anti-amyloid, anti-oxidative, and anti-inflammatory effects Improve memory. Improve memory, Increased emetic-like properties at a dose 100 times the memory-enhancing dose. No improvement on memory when given alone; Fully restored memory deficit when given in combination with donepezil (0.1 mg/kg, po). Enhance long-term memory; Facilitate expression of CREB-regulated genes in aged hippocampus. Improve long-term recognition, memory and cognition

Improve memory probably due to stimulation of the cAMP/PKA/CREB/BDNF pathway and antiinflammatory effects; Little emetic potential. Improve memory at doses without emesis-like behavior.

Improve memory at doses without emesis-like behavior.

Reverse memory deficits by regulating the Akt/GSK3b and p25/CDK5 pathways. Ameliorate age-dependent cognitive impairments, Reduce the Tau phosphorylation. Improve memory by a long-lasting reduction of Aβ levels. Restore cognitive deficits by the regulation of PKG/ pCREB signaling, anti-inflammatory response, and reduction of Aβ levels. Improve memory by reducing APP processing, Aβ levels, and Tau hyperphosphorylation. Improve memory; Reduce Tau phosphorylation but not Aβ. Improve memory. DOI: 10.1021/acs.jmedchem.7b01370 J. Med. Chem. 2018, 61, 5467−5483

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Table 2. continued compd (no., its target) BAY 73-6691 (24, PDE9)

subject

model

Scopolamine-injected rats127

T-maze, 10 mg/kg, po Passive avoidance, 10 mg/kg, po Social recognition, 0.3 mg/kg, po Object recognition, 0.1 mg/kg, po Object location, 5 mg/kg, po

Rats128 Tg2576 mice129

PF-04447943 (25, PDE9)

Aβ25−35-injected mice130 Scopolamine-injected rats131

Morris water maze, 1 mg/kg, ip Conditioned avoidance attention, 3 mg/kg, ip Object recognition,1 mg/kg, po Social recognition, 1 mg/kg, po Y-maze, 1 mg/kg, po

Mice131

results Improve long-term and short-term memory. Improve both early and late LTP. Transform early into late LTP. Improve memory; Restore Aβ induced impairment of long-term potentiation. Improve memory. Improve learning and memory.

Improve cognitive performance.

Table 3. PDEs Inhibitors in Clinical Trials for the Treatment of Alzheimer’s Disease or Related Diseasesa target

condition

current status

clinical trial registry no.

ITI-214 (2) PF-0999 (PF-05180999, 6) TAK-915

PDE1 PDE2

Cilostazol (8)

PDE3

Roflumilast (10)

PDE4

HT-0712 (11)

PDE4

MK-0952 (12) Etazolate (13) (EHT0202) BPN14770 (18)

PDE4 PDE4 PDE4

Sildenafil (19)

PDE5

Tadalafil (20) PF-04447943 (25)

PDE5 PDE9

BI-409306

PDE9

PF-02545920 (31)

PDE10

TAK-063 (32) OMS643762 EVP-6308

PDE10 PDE10 PDE10

Schizophrenia Schizophrenia Healthy volunteers Healthy volunteers Healthy volunteers Alzheimer’s disease Mild cognitive impairment Dementia Memory impairment, Alzheimer’s disease Alzheimer disease Age-associated memory impairment Healthy elderly volunteers Alzheimer’s disease Alzheimer’s disease Alzheimer disease Alzheimer’s disease Alzheimer’s disease Schizophrenia Parkinson’s disease Parkinson’s disease Dementia, vascular Alzheimer’s disease Alzheimer’s disease Healthy Alzheimer’s disease Alzheimer’s disease Huntington’s disease Huntington’s disease Schizophrenia Schizophrenia Schizophrenia

Phase I, 2013, terminated Phase I, 2011, completed Phase I, 2012, completed Phase I, 2015, completed Phase I, 2015, completed Phase IV, 2011, completed Phase II, 2015, recruiting Phase II, 2011, completed Phase I,2014, completed Preclinical, 2016, recruiting Phase II, 2013, completed Phase I, 2010, terminated Phase II, 2006, terminated Phase II, 2009, completed Phase I, 2017, ongoing but not recruiting participants Phase I, 2016, completed Phase I, 2016, completed Phase IV, 2007, completed Phase IV, 2013 Phase II, 2007, terminated due to not enough subjects Phase II, 2017, recruting Phase II, 2009, completed Phase I, 2009, completed Phase I, 2010, completed Phase II, 2014, recruiting Phase II, 2014, recruiting Phase II, 2015, terminated Phase II, 2014, completed Phase II, 2015, terminated Phase II, 2013, terminated Phase I, 2014, completed

NCT01900522 NCT01429740 NCT01530529 NCT02584569 NCT02461160 NCT01409564 NCT02491268 NCT01433666 NCT02051335 NCT02835716 NCT02013310 NCT01215552 NCT00362024 NCT00880412 NCT03030105 NCT02840279 NCT02648672 NCT00455715 NCT01941732 NCT02162979 NCT02450253 NCT00930059 NCT00988598 NCT01097876 NCT02240693 NCT02337907 NCT02342548 NCT02197130 NCT02477020 NCT01952132 NCT02037074

compd

a

PDE2

Information from www.clinicaltrials.gov.

were also regulated by 1. These results suggested that its effect on memory impairment might be related to oxidative stress and cholinergic function mechanisms. Pharmacokinetics studies of patients with cerebrovascular disorders demonstrated that 1 is able to pass through the blood−brain barrier and reach the central nervous system.23 A clinical study of patients with mild to moderate organic psychosyndromes, including primary dementia, was carried out in 1991 to evaluate the efficacy and tolerance of orally administering 1. The result showed that 1 could efficiently enhance the cognitive performance of these patients.24 ITI-214 (2), a PDE1 inhibitor reported by Intra-Cellular Therapies in 2012, had an IC50 value of 0.058 nM against PDE1

neurodegenerative diseases, such as AD, schizophrenia, and Parkinson’s disease.19 Vinpocetine (1), a derivative of the alkaloid vincamine, shows weak inhibition against PDE1 (IC50 = 30 μM) and has been widely used for the treatment of cognitive dysfunction (Figure 2).20,21 In streptozotocin (STZ)-induced rat models, which closely simulate the clinical and pathologic features of sporadic AD22 (Table 2), chronic treatment with 1 significantly improved learning and memory abilities in the Morris water maze and passive avoidance tests. Furthermore, the levels of malondialdehyde (MDA) and nitrite decreased, whereas that of glutathione (GSH) increased. The concentrations of acetylcholinesterase and lactate dehydrogenase 5470

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Figure 2. Current PDE1 inhibitors for the treatment of AD.

Figure 3. Current PDE2 inhibitors for the treatment of AD.

properties have limited its clinical use. Thus, this compound has usually worked as a tool medicine to investigate the potential therapeutic utility of PDE2 inhibitors. The inhibition of PDE2A by 4 enhanced the long-term potentiation of synaptic transmission without altering basal synaptic transmission. In addition, 4 improved memory performance in social and object recognition memory tasks and rescued deficits of spontaneous alternation in the T-maze induced by the N-methyl-D-aspartate (NMDA) antagonist MK801.31 Further study of object location and recognition tasks indicated that its role in memory improvement was probably due to the enhancement of memory consolidation instead of attention.32 In memory-impaired, aged rats, 4 enhanced the impaired learning and recognition memory for objects at different ages and was accompanied by increased basal constitutive nitric oxide synthase activity in the hippocampus and striatum.33 Another study discovered that the inhibition of PDE2 in the hydrolysis of both cGMP and cAMP using 4 improved spatial object memory consolidation in both early and late stages, whereas the vardenafilinduced inhibition of PDE5 in the hydrolysis of cGMP only enhanced the early stage, and the rolipram-induced inhibition of PDE4 in the hydrolysis of cAMP enhanced only the late stage.34 In APP/PS1 mice, chronic treatment with 4 improved memory performance without causing anxiety, indicating that signaling pathways other than the NO/cAMP/CREB pathway might be responsible for its cognition-enhancing effects.35 Most recently, another study demonstrated that 4 reduced stress-induced activation of an extracellularly regulated protein kinase and attenuated the loss of stress-induced transcription factors and plasticityrelated proteins via the neuroplasticity-related NMDAR-CaMKIIcGMP/cAMP signaling pathway.36 Pharmaceutical study in rats revealed that the brain penetration of BAY 60-7550 was poor. The administration of BAY 607550 at the dose of 10 mg/kg only resulted in a brain concentration of 3.6 ng/g.37 Thus, the main reason for the good memory improvement effects obtained by BAY 60-7550 is probably due to the highest expression of PDE2 in brain, where low brain concentrations of PDE2 inhibitor might be sufficient to activate

and an excellent selectivity (>1000-fold) across other PDEs (Figure 2).25 The good pharmacokinetic properties of 2 made it suitable as a CNS drug candidate. In a novel object recognition test in rats, with a single oral dose of 0.1−10 mg/kg, 2 could significantly enhance memory performance during acquisition and consolidation without changing exploratory behavior or basal locomotor activity. Furthermore, its administration did not change or have an effect on the antipsychotic activity or pharmacokinetic profile of risperidone in the conditioned avoidance response test.26 Currently, 2 has already completed four phase I studies (Table 3), including a single rising dose study in normal healthy volunteers and a multiple rising dose study once daily.27 Its safety and tolerability in healthy volunteers and patients with schizophrenia have also been evaluated. Although this trial has been terminated due to a business decision, the obtained results proved that 2 might work as an efficient candidate for the treatment of cognitive dysfunction as seen in AD, schizophrenia, and Parkinson’s disease. Most recently, a thienotriazolopyrimidinone PDE1 inhibitor, DNS-0056 (3), with good pharmacokinetic properties and brain penetration has been developed (Figure 2).28 In a rat model of recognition memory, 3 significantly increased long-term memory without altering exploratory behavior in rats. 2.2. PDE2 Inhibitors. Similar to PDE1, PDE2 is another subfamily that hydrolyzes both cAMP and cGMP. Unlike other PDE families, in which activating cGMP signals could decrease the level of local cAMP, the inhibition of PDE2 selectively abolishes the negative effects of cGMP on cAMP.29 PDE2A is the only isoform of PDE2 that is highly expressed in most brain regions, including the caudate, nucleus accumbens, cortex, and hippocampus (Figure S2).18 In most peripheral tissues, except the spleen, the level of PDE2 is relatively low. This spatial distribution makes PDE2 inhibitors ideal as therapeutic agents against cognitive disorder diseases but with less cardiovascular and other side effects that commonly exist in other PDE inhibitors.30 BAY-60-7550 (4) is a highly selective PDE2 inhibitor developed by Bayer with an IC50 of 4.7 nM and up to 100-fold selectivity across other PDEs (Figure 3).30 Its poor pharmacokinetic 5471

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the cAMP and cGMP signal, causing signal amplification for memory improvement. ND-7001 (5), developed by the Neuro3d company, showed an IC50 of 50 nM against PDE2 and good selectivity over PDE3 and PDE4 (Figure 3).38 Its safety, tolerability, and pharmacokinetics were evaluated in phase I trials, which demonstrated that it was safe and well tolerated at high doses and produced no sedation, memory disturbance, or withdrawal effects.39 However, no further development has been reported since 2010. PF-999 (6, also named as PF-05180999), developed by Pfizer, showed remarkable inhibitory activity against PDE2 (IC50 = 2.3 nM) and selectivity over other PDEs, as well as a reasonable free brain/plasma ratio in rats (Figure 3).40 PF-999 was found to increase the level of cGMP in the cerebrospinal fluid of rats, attenuate ketamine-induced memory deficits, and reverse spatial learning and memory in scopolamine-induced models.39 In 2015, a study that explored the primarily presynaptic mechanism of PDE2A inhibition was also performed by using PF-999. The results showed that the inhibition of PDE2 might be involved in short-term synaptic plasticity by modulating the hydrolysis of cAMP to accommodate changes in cGMP levels associated with presynaptic short-term plasticity.41 Currently, 6 has already completed two phase I clinical trials: one trial evaluated its safety, tolerability, and pharmacokinetics in healthy subjects, and the other evaluated the relative bioavailability of a modified-release formulation at 30 mg and 120 mg in patients with schizophrenia.42−44 However, no further information has been reported since 2012. Recently, compound 71 (7), developed by Pfizer, showed an IC50 of 2 nM against PDE2 and selectivity of up to 4000-fold over other PDEs (Figure 3).45 7 increased the level of cGMP in rodent brain regions expressing the highest levels of the PDE2A enzyme. In the radial arm maze, 7 significantly attenuated memory impairments induced by ketamine in rats. Furthermore, 7 reversed a maximum of 80% of the MK-801-induced local field potential disruption at a steady-state Cbu of 11 nM. These results indicated selective PDE2A inhibition in brain-potentiated, pharmacologically induced NMDA hypofunction in vivo and that it is suitable to be used in treating neurological and neuropsychiatric disorders associated with NMDA hypofunction, such as schizophrenia. TAK-915, developed by Takeda with no structural or other information disclosed, has already finished two phase I clinical trials of healthy participants in 2015. One trial served to examine the degree and duration of brain PDE2A enzyme occupancy/target engagement after its administration in order to provide evidence of dosing and a schedule for future clinical studies of schizophrenia.46 The other trial was conducted to characterize its safety, tolerability, and plasma pharmacokinetic properties when administered as single and multiple oral suspension doses at escalating dose levels in healthy participants, including elderly participants.47 2.3. PDE3 Inhibitors. Similar to PDE1 and PDE2, PDE3 is another subfamily responsible for hydrolyzing both cAMP and cGMP and has two isoforms: PDE3A and PDE3B. In the brain, the expression of PDE3A and PDE3B is relatively low and is mainly in the cerebellum. In the periphery, the expression of PDE3A is mainly in the heart, while the expression of PDE3B is mostly in the lungs and liver (Figure S3).18 PDE3A variants have differential expression in cardiovascular tissues. Intracellularly, PDE3B is predominantly membrane-associated and localized to endoplasmic reticulum and microsomal fractions. Furthermore, although the concentration of PDE3 is low in brain compared with other PDE subfamilies, the inhibition of PDE3 might be sufficient to active the cAMP and cGMP signal, causing signal amplification for memory improvement.8,48

AD and vascular dementia are two major types of dementia in elderly persons. A number of elderly people may suffer from both conditions. Cerebral white matter lesions have been demonstrated to be one important risk factor for transforming mild cognitive impairment to AD and are the main reason for the age-dependent coexistence of AD and cerebrovascular disease.49,50 The neurovascular dysfunction of aged AD is associated with cerebral ischemia and the accumulation of Aβ.51,52 Cilostazol (8) is a selective PDE3 inhibitor (IC50 = 0.2 μM) that has been clinically used as an antiplatelet drug for the prevention of cerebrovascular disease (Figure 4).53 Recently, therapy

Figure 4. Selective PDE3 inhibitor, cilostazol, may be used to treat cerebrovascular diseases and AD.

using 8 directed at both vascular and neurodegenerative aspects of dementia has been considered a more suitable approach for elderly patients with AD. In an Aβ25−35-injected mouse model of AD, repeated administration of 8 significantly attenuated impairment of spontaneous alternation and reversed the shortening of step-down latency induced by Aβ25−35. The increased level of MDA was completely prevented, indicating that 8 may reverse oxidative damage induced by Aβ25−35.54 Another study demonstrated that 8 significantly attenuated Aβ accumulation and improved spatial learning and memory. The level of apolipoprotein E (ApoE), a protein associated with Alzheimer’s neurofibrillary tangles and Aβ protein, was decreased, resulting in reduced Aβ aggregation.55 A clinical study of 10 patients with moderate AD proved that 8 ameliorated cognitive decline efficiently when coadministered with donepezil after an average follow-up period. However, this combinatorial therapy had no effect on patients with moderate or severe dementia.56,57 A retrospective analysis revealed that 8 could significantly improve memory in patients with mild cognitive impairment, but no significant effect was observed in patients with normal cognitive function or dementia.58 The clinical effects of monotherapy with galantamine or 8 added to combinatorial therapy in AD patients were also evaluated in a clinical study. Galantamine or 8 monotherapy increased the cognitive, affective, and activities of daily living functions in AD patients, while the combination of galantamine and 8 resulted in a better therapeutic effect than the monotherapy.59 Most recently, a clinical study was performed in a large Asian population to investigate the potential risk and benefit of using 8 to reduce the risk of dementia (9148 participants free of dementia at 40 years or older). Patients given 8 had a significantly decreased risk of incident dementia compared with patients without the drug. Notably, the use of 8 had a dose-dependent association with the reduced rate of dementia emergence. Subgroup analysis identified a decrease in dementia in 8 users with diagnosed ischemic heart disease and cerebrovascular disease.60 Two clinical trials of 8 related to AD or dementia were registered at clinicaltrials.gov. A phase IV study was performed by Seoul National University Hospital in 2011 to examine the added effect of 8 with donepezil treatment using cognitive tasks and PET imaging in mild to moderate AD patients with subcortical white matter hyperintensities (WMHI).61 A phase II trial in 5472

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Figure 5. Current PDE4 inhibitors for the treatment of AD.

low-dose administration of rolipram causes emesis. Currently, rolipram is widely used to investigate the underlying mechanisms of PDE4 inhibitors in AD. The neuroprotective effect of rolipram in AD models was first demonstrated on hippocampal-dependent memory tasks in 1998, which suggested that the inhibition of PDE4 might decrease the threshold for generating long-lasting long-term potentiation (LTP) and increase behavioral memory through the cAMP pathway.16 Gong and co-workers demonstrated that 9 improved cognitive deficits in both LTP and contextual learning in APP/PS1 mouse models. Notably, its effect on the improvement of LTP and basal synaptic transmission, as well as working, reference, and associative memory deficits, lasted at least 2 months after treatment.66 The loss of dendritic spines and dystrophic neurites existed in the hippocampus of APP/PS1 transgenic mice and AD patients. Gong also demonstrated that 9 reversed spine density to a normal level in APP/PS1 mice and aged mice. The changes in dendritic structure and function caused by Aβ peptides were also reversed by rolipram.67 In Aβ25−35- or Aβ1−40-injected rats, chronic administration or repeated treatment with 9 reversed memory impairment, while acute treatment with 9 did not. Furthermore, the decreased levels of Pcreb and Bcl-2 and the increased level of NFκB p65 and Bax in the hippocampus were regulated by 9 in a dose-dependent manner.68 These results indicated that the attenuation of neuronal inflammation and apoptosis mediated by cAMP/CREB signaling might be involved in the process, which is in accordance with a previous study showing that 9 reversed scopolamine-induced and time-dependent memory in object

patients with mild cognitive impairment was performed by the National Cerebral and Cardiovascular Center in 2015 to evaluate whether 8 could prevent the conversion from mild cognitive impairment to dementia.62 2.4. PDE4 Inhibitors. PDE4 is a subfamily that hydrolyzes cAMP only. The four isoforms of PDE4 (PDE4A, PDE4B, PDE4C, and PDE4D) are widely expressed in the CNS and have been found to remain present in the aged and Alzheimer’s brain.63 PDE4B is highly expressed in most areas of brain except in the dorsal root ganglia, where the levels of all the PDE4 isoforms are low. The levels of PDE4A and PDE4B are equally high in the cortex, but PDE4A is 2- to 4-fold lower in other brain regions. PDE4C has very low expression in the brain. PDE4D has relatively high expression in the frontal cortex but is 3- to 10-fold lower than PDE4B in all CNS tissues. In the periphery, PDE4B and PDE4D are two major isoforms (Figure S4).18 Currently, three PDE4 inhibitors, namely, roflumilast, crisaborole, and apremilast, have been approved on the market for the treatment of chronic obstructive pulmonary disease (COPD), atopic dermatitis, and psoriatic arthritis, respectively.64 In general, PDE4 inhibitors have been widely explored for the treatment of AD.65 However, most of the current PDE4 inhibitors may cause emetic side effects, limiting their usage in CNS diseases. Rolipram (9) is the first generation of PDE4 inhibitors with an IC50 of 230 nM (Figure 5). Compared with the approved drugs roflumilast and apremilast, rolipram has good brain penetration, or an ability to pass the blood−brain barrier. The concentration of 9 in the brain is twice as high as that in the plasma.65 However, 5473

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recognition tasks by elevating cAMP levels.69,70 Most recently, it has been proven that 9 significantly improved learning and memory ability in streptozotocin-induced and naturally aged mice.71 Its potential for treatment of memory dysfunctions can probably be attributed to its anti-cholinesterase, anti-amyloid, anti-oxidative, and anti-inflammatory actions, and other effects.72,73 Roflumilast (10, Daliresp or Daxas) was the first selective PDE4 inhibitor approved by the FDA for the treatment of COPD in 2011 (Figure 5). Compared with 9, 10 showed a significantly better inhibitory affinity against PDE4 with decreased emetic properties and may be suitable as a candidate drug for the treatment of AD.74 A preclinical study demonstrated that both 10 and 9 have positive effects on memory improvement in an object location task in male C57BL/6NCrl mice. In the Y-maze test, 10 improved spatial memory performance, while 9 did not. More importantly, 10 produced emetic-like effects at a dose 100 times that of the memory-enhancing dose, indicating that it is relatively safe in AD treatment. A single administration of donepezil or 10 did not improve memory, while combining efficacious doses of both fully reversed scopolamine-induced memory deficits in object recognition tasks.75 Its beneficial effect on memory and its nonemetic properties promoted 10 to clinical trials of AD. Currently, 10 has already finished one phase II study as a translational cognition enhancer76 and one phase I study to evaluate whether coadministration of 10 and donepezil could attenuate scopolamine-induced cognitive impairment.77 A preclinical trial is underway to evaluate its effect on preventing the onset of AD in high-risk individuals.78 Although the IC50 of 10 is almost 1000 times higher than that of 9 in vitro, the effects obtained in memory improvement experiments by roflumilast are almost the same or even less than those of rolipram. The difference has been attributed to the relatively poor ability of roflumilast to penetrate the blood−brain barrier. Recent studies demonstrated that 10 efficiently reversed cognitive impairment induced by hypertension in rodents, even at low doses.79,80 HT-0712 (11) was developed as a novel PDE4 inhibitor by Bourtchouladze and Scott (Figure 5). Both 11 and 9 ameliorated long-term memory in a mouse model of Rubinstein−Taybi syndrome.81,82 Currently, 11 has already completed a phase II trial to evaluate its efficacy in improving memory and cognitive performance in subjects with age-associated memory impairment (AAMI) and had positive results.83 MK-0952 (12), developed by Merck, shows an IC50 of 0.6 nM against PDE4 (Figure 5).84 A phase II clinical trial with 12 was performed to determine its effect on improving cognitive impairments in patients in mild to moderate stages of AD.85 Etazolate (13, also named as EHT0202) is a PDE4 inhibitor and a GABA-A receptor modulator (Figure 5).86 Its underlying mechanism of memory improvement in AD might be crosslinked.87 Currently, 13 has already finished one phase II trial to evaluate its safety and tolerability as an adjunctive therapy to acetylcholinesterase inhibitors in mild to moderate AD.88 The results showed that 13 was safe and generally well tolerated. Dosedependent numbers of early withdrawal and central nervous system-related adverse events were observed.89 FFPM (14) is a novel PDE4 inhibitor that was developed by Ke and co-workers and that has an IC50 of 26 nM and good selectivity over other PDEs (Figure 5).90 Its effects on learning and memory were investigated by Xu and co-workers using the APP/PS1 mouse model of AD.91 After 3 weeks of treatment, the learning and memory abilities of APP/PS1 transgenic mice were significantly improved, as measured by the Morris water maze test and the step-down passive avoidance task. 14 had no

significant effect on the duration of xylazine/ketamine anesthesia in mice, indicating that 14 might not cause emesis during the treatment of AD. Furthermore, 14 penetrated the blood−brain barrier quickly after oral administration, with a half-life in plasma of 1.5 h. PDE4D has been demonstrated to be the main subtype involved in the process of memory consolidation and LTP. The inhibition of PDE4 by 9 in PDE4D-deficient mice did not alter memory performance.92 Thus, selective PDE4D inhibitors may have beneficial effects on cognition enhancement. However, the inhibition of PDE4D is also the main reason for the emetic side effects of a PDE4 inhibitor, as it may mimic the pharmacological actions of α2-adrenoceptor antagonists.93,94 GEBR-7b (15) is a selective PDE4D inhibitor, showing low inhibitory activities toward PDE4A4, PDE4B2, and PDE4C2 (Figure 5).95 An in vivo study demonstrated that 15 improved spatial and object recognition memory in late-phase consolidation processes in object recognition and location tests. The level of cAMP in the hippocampus was increased without affecting the level of Aβ. The effect of 15 on memory was 3- to 10-fold more potent than that of 9. In the xylazine/ketamine test, no emetic effect was observed in mice, even at doses of 30 times higher than those in the object location test for behavioral performance improvement. The greatly reduced emetic effect of 15 is probably due to the relatively low doses required to improve memory.96 Most recently, the development of 15 led to a new molecule, 8a (16), which showed good PDE4D3 selective inhibition and the ability to cross the blood−brain barrier.97 The brain/plasma ratio of 16 is 0.8, while those of roflumilast and rolipram are approximately 1 and 2, respectively. In the object recognition task, administration of 16 at a dose of 0.003 mg/kg significantly enhanced long-term memory performance and fully reversed the scopolamine induced short-term memory deficit without causing any emetic-like behavior. These results offered another strategy for the discovery of PDE4 inhibitors with nonemesis behavior for the treatment of AD. Recent studies demonstrated that allosteric modulators of PDE4D that do not completely inhibit enzymatic activity may enhance memory with reduced emetic effects. Unlike the usual PDE4 inhibitors, these PDE4D allosteric modulators form an allosteric binding to the specific phenylalanine in primates in the UCR2 region of PDE4D, resulting in its high affinity and selectivity. In the scopolamine-impaired mouse model, the PDE4D allosteric modulator D159687 (17), with an IC50 of 27 nM against PDE4D and good selectivity, efficiently improved cognitive memory in novel object recognition and Y-maze tests (Figure 5). Compared with 9, 17 showed 100-fold less emetic in S. murinus, 3000-fold less in the beagle dog, and 500-fold less in monkey. Thus, 17 may work efficiently to improve memory with reduced emetic potential. The total brain/plasma AUC ratio for D159687 was above 1 across both rodents and primates.98 Another PDE4D allosteric modulator, BPN14770 (18), had IC50 values of 8 and 130 nM against human and mouse PDE4D, respectively (Figure 5). This modulator also enhanced the longterm potential in hippocampal slices.99 In 2016, two clinical trials of 18 were conducted for the evaluation of its safety, tolerability, and pharmacokinetic profile in different subjects.100,101 Another phase I trial to assess its effect on reversing scopolamine-induced cognitive impairment in healthy volunteers is in progress, with donepezil used as the positive control.102 2.5. PDE5 Inhibitors. Phosphodiesterase-5 (PDE5) is a subfamily that hydrolyzes cGMP and has one isoform, PDE5A. Compared with other PDE subfamilies, the expression of PDE5A in the brain is relatively low. The levels of PDE5A mRNA are 5474

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Figure 6. Current PDE5 inhibitors for the treatment of AD.

highest in the periphery, bladder, and lungs (Figure S5).18 However, several studies have demonstrated that PDE5 inhibitors have a potential therapeutic effect on the treatment of AD through stimulation of nitric oxide (NO)/cGMP signaling by elevating the level of cGMP. Currently, several PDE5 inhibitors, such as sildenafil and tadalafil, have been approved by the FDA for the treatment of erectile dysfunction and pulmonary arterial hypertension.103 Sildenafil (19) has shown an IC50 of 2.2 nM against PDE5A and moderate selectivity across other PDEs (Figure 6).104 Its pharmaceutical properties, such as easily crossing the blood−brain barrier and lower toxicity, indicate that 19 is suitable as a candidate drug for the investigation of the effect and mechanism of PDE5 inhibitors in neurodegenerative processes.104−109 19 produced an immediate and long-lasting improvement of synaptic function, CREB phosphorylation, and memory in an APP/PS1 mouse model.105 In the same study, PDE5 inhibitor tadalafil and a highly selective PDE1 inhibitor IC354 (IC50 = 80 nM) were also tested. Tadalafil (1 mg/kg, ip) did not improve either contextual fear conditioning or spatial working memory in APP/PS1 mice, and IC354 had no effect on the LTP amplitude in hippocampal slices. Further study proved that 19 regulates the level of Aβ possibly by modifying their production, metabolism, or clearance. The increased CREB phosphorylation might be due to the anti-inflammatory effect of 19 via the cGMP/PKG/pCREB signaling pathway.106 Currently, 19 has already finished one phase IV trial for schizophrenia and one phase IV trial for Parkinson’s disease.110,111 However, the clinical trial for AD has not been performed yet. It is worth mentioning that the selectivity of 19 for PDE1 and PDE6 is 180 and 12, respectively, and may cause mild vasodilatory effects and transiently disturb vision. As is mentioned before, there are direct links between AD pathology with cerebrovascular disease. Compared to healthy controls, patients with AD usually have reduced cerebral blood flow (CBF), increased cerebrovascular resistance, and reduced cerebral metabolic rate. A clinical study on AD patients was performed in 2017, demonstrating that 19 could significantly improve the cerebral blood flow and oxygen consumption at a single dose of 50 mg. The cerebrovascular reactivity was decreased. This result indicated that the memory effect of PDE5 inhibitors may possibly be due to the increase in cerebral blood flow mediated by PDE5 expressed in endothelial brain tissue.112 Tadalafil (20) is an efficient PDE5 inhibitor developed by Eli Lilly (Figure 6). Compared with 19, 20 has shown better selectivity against PDE6 and a longer half-life.113 These properties suggest that 20 might be more suitable than 19 as an anti-AD drug. However, the administration of 20 (1 mg/kg, ip) did not

improve either contextual fear conditioning or spatial working memory in APP/PS1 mice, while 19 did.105 The main reason for this result has been attributed to the fact that 19 can cross the BBB, while 20 cannot. A further study in 2013 proved that 12% of tadalafil found in the bloodstream crosses the BBB and reaches the brain. Chronic treatment with 20 leads its accumulation in the brain of the J20 transgenic mouse model of AD, which reverses memory deficits. 20 is even more effective than 19 in the improvement of memory performance. The level of hyperphosphorylated Tau protein decreased via activation of the pAkt/GSK3 pathway. Neither 20 nor 19 decreased the level of Aβ plaques, indicating that cognition enhancement by PDE5 inhibitors may occur without gross improvement of Aβ pathology.114 Currently, a phase II clinical trial of tadalafil is underway in patients with cerebral small vessel disease, to identify whether tadalafil improves blood flow in deep brain tissue and potentially improves cognitive function115 A quinoline compound, 7a (21), exhibited a higher inhibitory affinity (IC50 = 0.27 nM) and better selectivity than 19, vardenafil, and 20 (Figure 6). In the APP/PS1 mouse model of AD, 21 crossed the blood−brain barrier readily and increased the level of cGMP in the mouse hippocampus, thus restoring synaptic plasticity and memory damage caused by the elevation of Aβ42.116 The pathogenic mechanism of AD is complicated. A combination of multiple targets involved in AD has been considered more appropriate than individual ones. Histone deacetylase inhibitors (HDACIs) are potential modulators of cognitive impairment in AD. Administration of both the pan-HDACI vorinostat and PDE5 inhibitor 20 in aged Tg2576 mice effectively reversed the cognitive deficits and increased the reduced dendritic spine density in hippocampal neurons. The coadministration of these two drugs gave better and longer-lasting effects than each drug alone.117 On the basis of this result, CM-414 (22), an inhibitor acting on both PDE5 and HDAC, was developed with remarkable IC50 values against PDE5 and moderate inhibition against HDAC class I (Figure 6).118 22 was able to cross the blood−brain barrier, inducing AcH3K9 acetylation and CREB phosphorylation in the hippocampus and rescuing LTP in APP/PS1 mice. Chronic treatment of Tg2576 mice with 22 decreased the level of Aβ and Tau phosphorylation in the brain and increased the inactive form of GSK3β. Both the decrease in dendritic spine density in hippocampal neurons and the cognitive deficits were reversed. Thus, 22 may act as an efficient PDE5/HDAC dual inhibitor with a good safety profile, and it may be worth exploration in the clinical trials for AD patients.119 2.6. PDE7 Inhibitors. PDE7 is a cAMP-specific subfamily composed of two isoforms, PDE7A and PDE7B. Compared with 5475

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In 2005, the first selective PDE9 inhibitor, BAY 73-6691 (24), was reported by Bayer. 24 showed an IC50 of 55 nM against PDE9A and moderate selectivity over PDE1 (Figure 8).126 A program was performed to investigate its potential treatment effect on neurological diseases, including AD, but was terminated in 2004 with no reason disclosed. Currently, 24 is often used as a tool in medicine to investigate the underlying mechanism of PDE9A inhibition in AD.127−130 24 enhanced the ability of acquisition, consolidation, and retention of LTP in social and object recognition tasks in rodents, improving the scopolamine-induced passive avoidance deficit and the MK-801-induced short-term memory deficits.127 Compared with donepezil, which only increased early LTP, 24 improved both early and late LTP, even transforming early into late LTP. These results indicated that PDE9 inhibition might have better therapeutic effects on AD patients than donepezil.128 In APP transgenic Tg2576 mice with Alzheimer’s plaque pathology, 24 restored Aβ42 oligomer-induced LTP and improved memory performance.129,130 PF-04447943 (25) is a highly selective and brain penetrant PDE9A inhibitor that was developed by Pfizer in 2011, with an IC50 of 8.3 nM against PDE9A and above 100-fold selectivity over other PDEs (Figure 8).131 Compared with other current AD drugs, which only ameliorate learning and memory function, 25 was able to reverse scopolamine-induced deficits in both the Morris water maze and novel object recognition tests.132 In the Y-maze spatial recognition memory, social recognition memory, and scopolamine-deficit novel object recognition tasks, 25 significantly improved cognitive performance.133 In the Tg2576 mouse model of amyloid precursor protein (APP) overexpression, 25 regulated the dendritic spine density of hippocampal neurons.134 A preclinical test proved that 25 has excellent tolerability, exposure, and a long half-life of 19−31 h. Currently, 25 has already completed six phase I trials and one phase II trial for AD. In the six phase I trials, its safety, tolerability, and blood level after multiple doses were evaluated in different subjects with good results. One phase I trial was performed to evaluate its safety when given in combination with donepezil in AD patients and to evaluate the absorption and distribution of both it and donepezil.135 Its efficacy, safety, and pharmacokinetics were investigated in a phase II trial in subjects with mild to moderate probable AD.136

other PDEs, the expression of PDE7 is relatively low in all tissues. The expression of PDE7B is relatively higher than that of PDE7A in the CNS, such as in the caudate, nucleus accumbens, cortical tissues, and hippocampus. In the periphery, the level of PDE7A is higher than or equal to that of PDE7B in the heart, lungs, etc. (Figure S6).18 Moreover, in situ hybridization demonstrated that the expression of PDE7B remained unchanged and that PDE7A was reduced in the hippocampal regions of advanced AD patients.120 Several PDE7 inhibitors have been recently reported to be potential new agents for the treatment of brain diseases. However, investigation of PDE7 inhibitors for the treatment of AD is still limited.121 A quinazoline compound, S14 (23), was identified by the CODES program with IC50 values of 4.7 and 8.8 μM against PDE7A and PDE7B, respectively, as well as good selectivity over the other five PDEs (Figure 7).122,123 In the APP/PS1 mouse, its

Figure 7. Current PDE7 inhibitor for the treatment of AD.

administration reduced behavioral impairment in the T-maze via the cAMP/CREB signaling pathway. Moreover, 23 reduced the accumulation of Aβ deposits and modulated brain astrocyte distribution. Significant decreases in Tau phosphorylation, cell death, and proapoptotic protein expression were also observed.124 2.7. PDE9 Inhibitors. PDE9 is a subfamily that hydrolyzes cGMP only. Among the 11 PDE subfamilies, PDE9 has the highest binding affinity for cGMP (Km = 70 nM) with only isoform PDE9A.125 The expression of PDE9A occurs in most human tissues (Figure S8). In the CNS, the highest levels of PDE9A are in the caudate nucleus and cerebellum. In the periphery, the highest level of PDE9 occurs in the bladder. PDE9 has been regarded as an important target since several efficient inhibitors are in clinical trials for the treatment of AD.

Figure 8. Current PDE9 inhibitors for the treatment of AD. 5476

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Figure 9. Current PDE10 inhibitors for the treatment of AD and other diseases.

relatively high in the nucleus accumbens. In other parts of the brain and the peripheral tissues examined, the level of PDE10 mRNA could be detected but was very low (Figure S8).18 Furthermore, the level of PDE10A decreased in the striatum of patients with Huntington’s disease and immediately preceded the onset of motor symptoms.144 Currently, PDE10 is considered a promising target for CNS diseases, especially for schizophrenia and Huntington’s disease. PF-02545920 (31, MP-10) was developed by Pfizer (Figure 9).145 After its safety, tolerability, and pharmacokinetics in different subjects were identified in five phase I trials, its efficacy in the treatment of schizophrenia was evaluated in three phase II trials. However, 31 failed to reach its primary efficacy end-point in a trial with schizophrenia patients in 2012, and thus, its condition was changed to Huntington’s disease, which was completed in 2014.146 Another phase II trial in 2015 sought to investigate its long-term safety, tolerability, and efficacy in subjects with Huntington’s disease. However, this study was terminated in 2016 due to no significant difference being observed in the primary end point between 31 and placebo.147 TAK-063 (32), developed by Takeda by using structure-based drug design (SBDD) techniques, has an IC50 of 0.30 nM against PDE10 (Figure 9). Its selectivity over other PDEs could be up to 15 000-fold. Its pharmacokinetics were promising, including high brain penetration in mice.148 In vivo studies have demonstrated that 32 produced dose-dependent, antipsychotic-like effects in METH-induced hyperactivity and prepulse inhibition in rodents.149 Furthermore, 32 attenuated both phencyclidine-induced working memory deficits in a Y-maze test in mice and MK-801-induced working memory deficits in an eight-arm radial maze task in rats.150 Recently, 32 has been evaluated in a phase II trial for the treatment of acutely exacerbated schizophrenia.151 OMS-824 (33, OMS643762), developed by Omeros Corporation, has finished one phase II trial that evaluated its safety, tolerability, and pharmacokinetics in psychiatrically stable schizophrenia patients (Figure 9).152 Another phase I trial of 33 in subjects with Huntington’s disease has been suspended with no reason disclosed.153 Another PDE10 inhibitor, EVP-6308, has finished a phase II trial in subjects with schizophrenia who are on a stable antipsychotic regimen.154

However, its administration over 2 weeks did not improve cognitive behavior or cause global changes with statistically significant differences compared with a placebo. BI-409306 was developed by Boehringer Ingelheim for the treatment of cognitive disorder diseases, such as AD and schizophrenia (IC50 = 52 nM). Currently, BI-409306 has already completed 15 phase I trials. The safety, tolerability, and pharmacokinetics of BI-409306 were investigated in different subjects, including healthy males, CYP2C19-genotyped persons, and patients with AD. The results showed that BI-409306 was safe and well tolerated in most subjects. The most commonly experienced adverse effects were headache and photopsia, with mild to moderate intensity, which might be caused by the modulation of PDE9A of some processes in the retina. Furthermore, in a study aiming to assess its exposure in cerebrospinal fluid, BI-409306 was found to cross the blood−CSF barrier and be rapidly absorbed in plasma and distributed in CSF. The level of cGMP in CSF increased in a dose-dependent manner. Currently, two phase II trials for AD are in progress.137,138 One trial aims to investigate its effect on cognitive impairment in AD patients in comparison with a placebo or donepezil. The other trial aims to explore the effect of different doses on the treatment of AD. IMR-687(26) and PF-4181366, developed by H. Lundbeck company and Pfizer, respectively were evaluated in preclinical and clinical trials of AD, but no progress has been reported. A series of PDE9 inhibitors was reported by Luo’s group with the assistance of structure-based design and computational docking (Figure 8).139 Compound 27 has an IC50 of 21 nM against PDE9A and >150-fold selectivity over other PDEs. Further structural modification of compound 27 led to compound 3r (28), with an improved IC50 of 0.6 nM. However, these two compounds were designed for the treatment of diabetes, and thus, their physicochemical properties might be beyond the range of CNS drugs.140 Compound C33 (29), with an IC50 value of 16 nM against PDE9 and moderate selectivity over other PDEs, could significantly improve memory and cognitive impairment induced by scopolamine or Aβ25−35.141 In 2015, Luo’s group used a combinatorial method to discover novel PDE9 inhibitors with new scaffolds rather than pyrazolopyrimidinones from the SPECS database.142 Fifteen hits out of 29 molecules, with five novel scaffolds, were identified as PDE9 inhibitors. Further structural modification of compound AG-690/40135604 (IC50 = 8.0 μM) led to a new compound 30, with an improved inhibitory affinity of 2.1 μM. The five novel scaffolds discovered could be used for the rational design of PDE9 inhibitors with higher affinities. 2.8. PDE10 Inhibitors. PDE10A is a dual-specificity subfamily that hydrolyzes cAMP (Km = 0.05 μM) and cGMP (Km = 3 μM).143 The highest expression of PDE10A in the brain is in the caudate nucleus, and it is also the most prevalent PDE species in this tissue, together with PDE1B. The level of PDE10A is

3. OTHER PDE SUBFAMILIES AND THEIR APPLICATION PDE6 is an enzyme-specific hydrolyzing cGMP that is divided into three subtypes: PDE6A, -6B, and -6C. The retina is the only human tissue with high levels of PDE6A expression. PDE6 has not been detected in the CNS.18 Currently, the purification of PDE6 enzyme is still a challenge, and no selective PDE6 inhibitor has been reported yet.155 The structural, biochemical, and pharmacologic properties of PDE6 are closely related to PDE5. PDE5 5477

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studied through effective methods. Many studies used structurebased molecular design methods to discover highly selective PDE inhibitors. In addition, the discovery of novel allosteric modulators93−97 based on the allosteric site of PDEs could significantly improve their selectivity and decrease the likelihood of side effects. Such allosteric modulators will provide a better basis for the development of future anti-AD drugs in the field of PDE inhibitors, but their role needs to be examined in further preclinical or clinical evaluations.

inhibitors, such as sildenafil, vardenafil, and tadalafil, have exhibited low selectivity toward PDE6, which may disturb the cGMP signaling pathway in the retina during treatment for other diseases and may cause side effects, including visual disturbances, blurry vision, and light sensitivity.156 PDE8 specifically hydrolyzes cAMP, which is coded by two genes: PDE8A and PDE8B. PDE8A exists throughout the brain and peripheral organs at low levels, while PDE8B is mainly expressed in the brain and thyroid.18 On the basis of its expression, PDE8 has been considered a target for the treatment of thyroid dysfunction and modulation of steroidogenesis in the testes and adrenal glands.157−159 Furthermore, the overexpression of PDE8B, as well as PDE7, has been observed in the brains of Alzheimer’s patients, indicating that the inhibition of PDE8B was able to enhance memory function by the activation of the cAMP signal pathway. However, only a few PDE8-selective inhibitors are available,160 limiting the usage of PDE8. PDE11A is the only isoform of PDE11 that hydrolyzes both cAMP and cGMP. PDE11A is expressed at relatively low levels in most tissues, especially in the cortex and the hippocampus.18 Studies of PDE11 are few, and the biological roles of PDE11 are poorly understood due to the lack of selective inhibitors.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01370. Figures showing expression of mRNA for PDEs in human tissues (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86-20-3994 3000. ORCID

4. PROSPECTIVE In light of the recent high-profile phase III failures of several Aβtargeting agents, such as bapineuzumab and solanuzumab, there has been a trend to refocus on the discovery of novel non-Aβdirected, anti-AD drugs rather than acetylcholine inhibitors and NMDA antagonists, which still have a long way to go. Over the past 20 decades, great efforts have been made to understand the roles of PDEs and to discover effective inhibitors for cognitive improvement in AD. Compared with other hot research areas related to the deposition of Aβ or aggregation of Tau protein, PDE inhibitors hydrolyze two important second messengers (cAMP/cGMP), which can be used before the formation of amyloid plaques and neurofibrillary tangles, regulating central nervous system function such as emotion-related learning, memory, and neural regeneration. Alterations in the levels of cAMP and cGMP are due to the accumulation of cyclase and the degradation of PDE regulation. The regulation of the concentration of cAMP/cGMP makes PDEs promising drug targets. Until now, there have been many reports of PDE inhibitors with outstanding and remarkable effects on memory in AD in preclinical (Table 2) or clinical (Table 3) trials. The role of PDE inhibitors in improving learning and memory is gaining more and more data, but the underlying mechanisms are still not fully understood. Sufficient evidence has demonstrated that the cAMP-mediated PKA/CREB or cGMP-mediated PKG/CREB signaling pathways are involved in this process. In addition, enhanced cAMP/cGMP concentrations by several PDE inhibitors can significantly improve both early and late LTP, even transforming early into late LTP, which demonstrates that the inhibition of PDEs might have better therapeutic effects on AD patients than acetylcholine inhibitors and suggests a possible mechanism for the use of PDE inhibitors in the treatment of AD. This finding may lead to the development of drugs for treating cognitive dysfunctions, especially in AD. As candidates for the treatment of AD, PDE inhibitors may cause several adverse reactions, which greatly limits their clinical applications. For example, most current PDE4 inhibitor including roflumilast may cause nausea, emesis, diarrhea, and headache, due to the inhibition of PDE4D subtype. PDE5 inhibitor sildenafil may cause visual abnormality as it inhibits PDE6 at the same time. Solutions for avoiding these side effects should be further

Hai-Bin Luo: 0000-0002-2163-0509 Notes

The authors declare no competing financial interest. Biographies Yinuo Wu is currently an Assistant Professor in the School of Pharmaceutical Sciences at Sun Yat-sen University (SYSU). She received her Ph.D. degree at SYSU in 2011 and subsequently went to Hong Kong Polytechnic University as a postdoctoral fellow. Her research encompasses discovery of novel PDEs inhibitors and their applications in the treatment of neurological disorders. Zhe Li is currently an Assistant Professor in the School of Pharmaceutical Sciences at Sun Yat-sen University (SYSU). He received his Ph.D. degree in Medicinal Chemistry from SYSU in 2017. During his Ph.D. study, he went to Prof. Chang-Guo Zhan’s group as an exchange student in University of Kentucky for two years. His research in medicinal chemistry focuses on the development of drug design methods and the applications of these methods in the development of novel PDEs inhibitors. Yiyou Huang is currently pursuing his Ph.D. in the School of Pharmaceutical Sciences at Sun Yat-sen University under the supervision of Prof. Hai-Bin Luo. He went to Prof. Hengming Ke’s group as a visiting scholar in the University of North Carolina at Chapel Hill from April 2017 to October 2017. His research focuses in structural biology and their applications on the design of novel PDE inhibitors. Deyan Wu is currently an Assistant Professor in the School of Pharmaceutical Sciences at Sun Yat-sen University (SYSU). He received his Ph.D. degree at East China University of Science & Technology in 2014 and subsequently worked at ChemPartner as a senior researcher. After one-year research, he joined Sun Yat-sen University as an Assistant Professor. His research is currently specialized in medicinal chemistry and total synthesis of natural products, focusing on the research and development of PDEs inhibitors for treatment of pulmonary arterial hypertension (PAH), erectile dysfunction (ED), chronic obstructive pulmonary disease (COPD), and structural modification of active natural products as novel PDEs inhibitors. Hai-Bin Luo is currently a Full Professor and the Associate Dean of the School of Pharmaceutical Sciences at Sun Yat-sen University (SYSU). He received his Bachelor’s degree from Xiamen University in 1999 and 5478

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Ph.D. degree from Hong Kong Baptist University in 2005. After oneyear postdoctoral research, he joined Sun Yat-sen University as an Associate Professor. His research currently encompasses the rational design and discovery of novel PDEs inhibitors and their applications in the treatment of several diseases.

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ACKNOWLEDGMENTS This work was supported by the National Key R&D Program of China (Grant 2017YFB0202600), Natural Science Foundation of China (Grants 21402243, 21572279, 81522041, 81373258, and 81602968), Science Foundation of Guangdong Province (Grants 2014A020210009 and 2016A030310144), Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (2016), and Foundation of Traditional Chinese Medicine, Bureau of Guangdong Province (Grant 20171049).

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DOI: 10.1021/acs.jmedchem.7b01370 J. Med. Chem. 2018, 61, 5467−5483