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Advances in the Development of Phosphodiesterase‑4 Inhibitors

Ting Peng,# Baowen Qi,# Jun He,# Hengming Ke,* and Jianyou Shi*

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ABSTRACT: Cyclic nucleotide phosphodiesterase 4 (PDE4) specifically hydrolyzes cyclic adenosine monophosphate (cAMP) and
plays vital roles in biological processes such as cancer development. To date, PDE4 inhibitors have been widely studied as
therapeutics for the treatment of various diseases such as chronic obstructive pulmonary disease, and many of them have progressed
to clinical trials or have been approved as drugs. Herein, we review the advances in the development of PDE4 inhibitors in the past
decade and will focus on their pharmacophores, PDE4 subfamily selectivity, and therapeutic potential. Hopefully, this analysis will
lead to a strategy for development of novel therapeutics targeting PDE4.

1. INTRODUCTION mRNA splicing, which are grouped into 11 families based on

Cyclic nucleotide phosphodiesterases (PDEs) are a family of
enzymes that uniquely hydrolyze cyclic adenosine mono-
phosphate (cAMP) and cyclic guanosine monophosphate
(cGMP) into 5′-AMP and 5′-GMP. The cAMP and cGMP
signaling systems regulate the functions of many physiological
processes, such as central nervous, cardiovascular, and immune
systems.1−4 Under normal physiological conditions, the intra-
cellular levels of cAMP and cGMP are determined by the rates of
cyclic nucleotide production and decomposition and main-
tained in a dynamic equilibrium between synthesis and
hydrolysis. There are two ways to increase intracellular
cAMP/cGMP concentrations: (a) increasing cAMP/cGMP
synthesis by stimulating adenylyl/guanylyl cyclases through
external stimuli such as hormones and neurotransmitters and
(b) preventing cAMP/cGMP hydrolysis by inhibiting PDEs.
Since many diseases are associated with low intracellular levels of
cAMP or cGMP, inhibition of PDEs would be a preferred
strategy for drug development. PDEs are involved in the
regulation of many physiological and metabolic processes,
including inflammation, ion channel signaling, cell differ-
entiation and apoptosis, muscle contraction, lipogenesis,
glycogen synthesis, and gluconeogenesis.1−4 To date, 21 PDE
genes have been identified in the human genome. These genes
express more than 100 PDE isoforms in human tissues through
their sequence similarity and biological properties. PDE4, -7,
and -8 are cAMP-specific, while PDE5, -6, and -9 specifically
hydrolyze cGMP. PDE1, -2, -3, -10, and -11 show dual activity
on hydrolysis of both cAMP and cGMP.5
2. OVERVIEW OF PDE4
PDE4, the most diverse family of PDEs, is abundantly expressed
in most cells6 and prefers to hydrolyze cAMP with the
micromolar Km values. PDE4 molecules are involved in a variety
of physiological processes, including brain function, monocyte
and macrophage activation, neutrophil infiltration, vascular
smooth muscle proliferation, and myocardial contractility
(Figure 1). PDE4 has been reported to be a target for various
inflammatory diseases, such as asthma, chronic obstructive
pulmonary disease (COPD), and rheumatoid arthritis. In
addition, PDE4 plays important roles in the development of
cardiovascular diseases, autoimmune diseases, and cancers.7−9
Received: December 28, 2019
Published: April 7, 2020

© XXXX American Chemical Society https://dx.doi.org/10.1021/acs.jmedchem.9b02170

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Figure 1. Schematic presentation of PDE4 and cAMP signaling pathway. The following abbreviations are used: β-AR, β-arrestin; GPCRs, G-protein-
coupled receptors; Gs, heterotrimeric guanine nucleotide binding protein; AC, adenylyl cyclase; AKAPs, A-kinase-anchoring proteins; PKA, protein
kinase A; EPAC, exchange protein activated by cAMP; CREB, cAMP response element binding protein; C3G, cyanidin-3-glucoside; Rap1, Ras-
proximate-1; MEK1/2, mitogen-activated protein kinase; ERK1/2, extracellular signal-regulated kinase 1/2; NF-κB, nuclear factor κ-light-chain-
enhancer of activated B-cells; MAPK, mitogen-activated protein kinase; mTORC1, mammalian target of rapamycin complex 1.

In recent years, involvement of PDE4 in regulation of the central

nervous system has received substantial attention.10
PDE4 comprises four subtypes: PDE4A, PDE4B, PDE4C,
and PDE4D, which are located on chromosomes 19p13.2, 1p31,
19p13.11, and 5q12, respectively.11 Each subtype of PDE4 genes
can express 3−11 proteins, resulting in at least 25 different PDE4
isoforms. These PDE4 proteins are located in different cell
compartments and regulated by kinases, playing critical roles in
cell functions (Figure 1). PDE4 molecules exist in long, short,
and supershort forms according to their molecular sizes (Figure
2). The long form of PDE4 has two upstream conserved regions
(UCR1 and UCR2) in the N-terminal region, while the short
form contains only UCR2 and the supershort form has a
truncated UCR2. UCR1 consists of approximate 60 amino acids,
while UCR2 has approximate 80 amino acids. All PDE4 Figure 2. Domain organization of PDE4. UCR: Upstream conserved
molecules have a highly conserved catalytic domain at the C- region.
terminus, which contains 300−350 amino acids.11
X-ray structures of PDE4 molecules have shown that the PDE4s make the discovery of PDE4 subtype-selective inhibitors
active site can be divided into three subpockets (Figure 3): a quite challenging.
divalent metal pocket that interacts with the phosphate moiety The UCRs are the unique motifs of the PDE4 family and play
of cAMP; two Q pockets that form hydrogen bonds and an important role in the regulation of PDE4 activity.14 The
hydrophobic interactions with inhibitors; and a solvated pocket phosphorylation of the UCRs by protein kinase A (PKA) and
(S pocket).12,13 PDE4 inhibitors occupy this active site with a extracellular signal-regulated kinase (ERK) regulates PDE4
variety of interactions, including hydrophobic interactions with dimerization and catalytic activities. UCR1 contains a conserved
the conserved phenylalanine and isoleucine, and hydrogen PKA phosphorylation site in PDE4 subfamilies. The activity of
bonds with the invariant glutamine. The highly conserved PDE4 is regulated by long-term transcriptional regulation or
sequences and structural homology of the catalytic domains of short-term posttranslational modifications. In long-term regu-
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Figure 3. Active site of PDE4D2. Dotted lines represent hydrogen
bonds. Three inhibitors are shown: compound 1 (green), compound
14 (yellow), and compound 15 (salmon).
lation, an increase of cAMP by PDE inhibition conjugates with
activation of adenylyl cyclase (AC) by hormone stimulation.
Studies have shown that this regulation is highly specific and is a
result of increased gene expression. In short-term regulation,
increase of cAMP levels activates PKA, which in turn
phosphorylates specific serine residues in UCR1 of the long
forms of PDE4. As a result, the activity of PDE4 increases
rapidly, and the hydrolysis of cAMP causes the cAMP level to
return to equilibrium. In addition, PDE4 activity is regulated by
several proteins, including arrestin, Src family tyrosine protein
kinases, A-kinase anchoring proteins (AKAPs), and receptor for
activated C kinase 1 (RACK1).14−16
Figure 4. Chemical structures of PDE4 inhibitors in clinic trials or in market.
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3. PDE4 AS A THERAPEUTIC TARGET FOR
TREATMENT OF HUMAN DISEASES
Members of four subfamilies, PDE4A, PDE4B, PDE4C, and
PDE4D are expressed in various tissues and cellular compart-
ments and involved in many physiological processes, including
airway immunity, asthma, signaling of CNS, cardiovascular
function, inflammatory processes, cell adhesion, and metabolic
processes.7,10,17−19 As the key enzyme hydrolyzing cAMP in
immune cells, PDE4 is one of the main targets of anti-
inflammatory drugs.6 PDE4 is involved in the regulation of the
activation and recruitment of inflammatory cells and the release
of excessive or unregulated cytokines in many allergic and
autoimmune diseases. For example, lipopolysaccharide (LPS)
selectively induces PDE4B2 mRNA expression in human
circulating monocytes.20 PDE4 inhibitors inhibit LPS-mediated
secretion of tumor necrosis factor α (TNF-α) in peripheral
monocytes,21 indicating their important roles in anti-inflamma-
tion. In addition, PDE4B and PDE4D, but not PDE4A, play
important regulatory roles in neutrophil function, such as
inhibiting the release of a variety of proinflammatory
mediators22 and delaying neutrophil apoptosis.23 In addition,
PDE4 inhibitors caused airway smooth muscle relaxation,
suggesting their potential values for the treatment of airway
diseases.
At present, four PDE4 inhibitors have been approved for the
treatment of human diseases (Figure 4 and Table 1): roflumilast
(1) for COPD,24 crisaborole (2) for atopic dermatitis,25
apremilast (3) for psoriatic arthritis,26 and ibudilast (4) for
the rare child disease Krabbe by USA and for bronchial asthma
by Japanese authority.27 In addition, the dual selective PDE3/4
inhibitor RPL554 (5) is in phase II clinical trials for the
treatment of COPD, and the PDE4 inhibitor BPN14770 (6) is
in phase II clinical trials for the treatment of Alzheimer’s disease
and fragile X syndrome (Table 1).
In recent years, studies on the medical applications of PDE4
inhibitors have shifted to their uses in treatment of cognitive
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Table 1. PDE4 Inhibitors in Clinic Trials or in Market

ClinicalTrials.gov
drug, owner condition or disease status identifiersa
Roflumilast (1, Daxas, Daliresp), Severe chronic obstructive pulmonary disease Approved by EU, June 2010, and USA, March 2011
AstraZeneca Asthma Phase I December 2019 to March 2021 NCT04108377
Asthma Phase II, August 2018 to April 2020 NCT03532490
Bronchiectasis Phase II, July 2019 to December 2020 NCT03988816
Lymphoma, B-cell Phase I, May 2018 to December 2020 NCT03458546
Bronchiectasis Early Phase I October 1, 2019 to November 2020 NCT04090294
Crisaborole (2), Pfizer Atopic dermatitis ointment Approved by USA Dec 14, 2016
Morphea Phase II, September 2018 to December 2019 NCT03351114
Seborrheic dermatitis Phase IV September 1, 2018 to September 2020 NCT03567980
Atopic dermatitis eczema Phase IV September 2019 to July 2020 NCT04023084
Apremilast (3, CC-10004, Otezla), Celgene Psoriatic arthritis Approved by USA, 2014
Acne conglobata Phase II October 9, 2019 to September 2020 NCT04161456
Dermatomyositis, adult type Phase II June 12, 2018 to April 2020 NCT03529955
Lichen planus of vulva female genital disease Phase II August 27, 2019 to December 1, 2020 NCT03656666
Behcet syndrome Phase III, December 2014 to March 2021 NCT02307513
Nummular and dermatitis eczema Phase II, July 2017 to March 2020 NCT03160248
Psoriasis, psoriatic nail and vulgaris Phase IV January 2017 to December 2019 NCT03022617
Alcohol use disorder Phase II, November 2017 to December 2019 NCT03175549
Recurrent aphthous stomatitis Phase I, October 2018 to December 2019 NCT03690544
Dermatomyositis, adult type Phase II, June 2018 to April 2020 NCT03529955
Lichen planus of vulva female genital disease Phase II, January 2019 to December 2020 NCT03656666
Ibudilast (4, MN-166), MediciNova Krabbe disease Approved by USA, June 2016
poststroke complications and bronchial Approved by Japan and Korea, 1989
asthma
Methamphetamine dependence Phase II, May 2019 to January 2022 NCT03341078
Alcohol use disorder Phase II, July 2018 to June 2020 NCT03489850
Alcohol use disorder Phase II, October 2018 to September 2021 NCT03594435
Glioblastoma Phase I/II, December 2018 to December 2020 NCT03782415
RPL554 (5), Verona Pharma COPD Phase II, May 2019 to October 2019 NCT03937479
BPN14770 (6), Tetra Discovery Partners Alzheimer’s disease Phase II, April 2019 to June 2020 NCT03817684
Fragile X syndrome Phase II, June 2018 to December 2019 NCT03569631
a
Data collected from https://clinicaltrials.gov [Last accessed December 1, 2019].

Figure 5. PDE4 inhibitors in the nervous system.

disorders. PDE4A, -4B, and -4D are expressed in the central relieve cognitive impairment.29 PDE4D is an important
nervous system, while PDE4C is mainly expressed in the regulatory factor in depression, learning, and memory
peripheral nervous system. PDE4A plays an important role in dysfunctions.30,31 The PDE4D gene is highly expressed in the
anxiety and emotional memory.28 PDE4B is involved in hippocampus, and PDE4D gene-deficient mice exhibit long-
dopamine and stress-related signaling and may thus potentially term potentiation that significantly increases learning ability,
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Figure 6. Active site of PDE4. Dotted lines represent hydrogen bonds. Residues are numbered using the PDE4B2B sequence. (A) Surface presentation
on binding of compound 1 (yellow sticks) to PDE4B (green sticks) and PDE4D (white sticks). P1−P3 and CORE denote four potential
pharmacophores of PDE4 inhibitors. (B) Coverage of the PDE4 active site by a UCR2 helix (yellow ribbons). (C) Interaction of compound 1 with the
UCR helix.
memory, and nerve regeneration.31,32 The potential applicability
of PDE4 inhibitors in improving learning and memory is well
supported experimentally. For example, compound 14 improves
learning and memory by inhibiting PDE4 activity and
influencing cAMP-mediated signaling pathways such as the
cAMP/PKA/CREB and EPAC/ERK signaling pathways
(EPAC, exchange protein directly activated by cAMP).33 The
intervention and regulation of PDE4 provide new therapeutic
strategies for the treatment of neurological diseases such as
Parkinson’s disease (PD), Alzheimer’s disease (AD), and
cerebral ischemia. PDE4 inhibitors relieve AD through
reduction of neuroinflammation and benefit memory consol-
idation.34 Although the etiology of PD has not been completely
revealed, involvement of PDE4 in regulation of oxidative stress
in neurodegeneration of PD is obvious.35,36
Many PDE4 inhibitors have been developed for the treatment
of neurological diseases; for example, HT0712 (7, Figure 5),
etazolate (8, Figure 5), compound 6 (Figure 4), and MK-0952
(9, Figure 5) were tested in clinical trials. Compound 7, also
known as IPL-455903, enhanced memory and promoted motor
function recovery after cerebral ischemia by stimulating the
generation of synapses in the cortex37 and showed the positive
results in the phase II clinical trial of age-associated memory
impairment (AAMI).38 Etazolate (9, EHT-0202), as a PDE4
inhibitor and a GABA-A receptor modulator, increased
expression of the α-secretase enzymes and APP alpha
secretion.39 Compound 9 was generally tolerated in clinical
trials for the treatment of the early Alzheimer’s disease but
showed dose-dependent and central nervous system-related
adverse events.40 Compound 6, a PDE4D allosteric modulator,
enhanced the long-term potential in hippocampal slices and is
currently in phase II clinic trials for treatment of the early
Alzheimer’s disease and fragile X syndrome (Table 1).41 MK-
0952 was tested in a phase II clinic trial for its effect on
improving cognitive impairment in patients with mild-to-
moderate AD.42 Compound (S)-Zl-n-91 (10, Figure 5) has
the IC50 values of 12 nM for PDE4D and 20 nM for PDE4B and
showed potent neuroprotection activities in ICR mice.43,44 In
addition, GEBR PDE4 inhibitors such as GEBR-20b (11, Figure
5) facilitated long-term potentiation and improved memory
without emetic-like behavior in rodent tests.45−48 Thus, in-
depth studies on the specific functions and regulatory
mechanisms of PDE4s and their inhibitors in the central
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nervous system may result in new platforms for the treatment of
neurological diseases.
In addition, regulation of the cellular cAMP level via PDE4
inhibition is directly related to pathological functions of tumors,
such as tumor cell migration and proliferation, as well as tumor
vascularization and development.8,49 Genetic studies showed
that PDE4D is related to the expression of the thiol purine gene
set, leading to an investigation of PDE4-based purine drugs for
the treatment of various tumors. Moreover, PDE4B was
reported to regulate glucocorticoid reactivity of B-cell
lymphoma.50 PDE4 has been shown to have the highest
cAMP hydrolysis activity in 41 out of 60 tumor cell lines,51
highlighting its potential for the treatment of cancers.
4. SUBFAMILY SELECTIVE PDE4 INHIBITORS AND
EMESIS
Although PDE4 has been identified as a therapeutic target, the
clinical development of inhibitors has been hampered by dose-
limiting side effects, most notably, nausea and vomiting, which
were observed with the first-generation PDE4 inhibitors such as
compound 14 and the second-generation inhibitors such as
compound 15. The early studies suggested that the emesis
results from the inhibition of PDE4D. Knocking out PDE4B, but
not PDE4A/PDE4D, significantly inhibited the synthesis of
TNF-α in LPS-mediated monocyte and peritoneal macro-
phages. In mouse allergic asthma models, Th2 cell function was
regulated by PDE4B rather than PDE4A/PDE4D.52 In addition,
a study on lighting reflection of rodents showed that the
inhibition of PDE4D but not PDE4B may be the cause of the
emesis observed in the study of nonsubfamily selective PDE4
inhibitors.53 This is shown by several PDE4B selective inhibitors
such as compound 38 that has potent anti-inflammatory effects
and reduced emetic effects.54 However, recent studies implied
that inhibition of PDE4D may not be a key factor to cause
vomiting.55
Structural superposition between the catalytic domains of
PDE4B and PDE4D showed that the active site residues for the
binding of PDE4 inhibitors are identical and have the same
conformation, including the invariant glutamine and phenyl-
alanine (Q443 and F446 of PDE4B2B, Figure 6A) that are two
basic residues for binding of substrates and inhibitors of PDEs.
The only minor difference between the active sites of PDE4B
and PDE4D is the conformation of Met431 of PDE4B (Figure
6A). Thus, it will be difficult to design PDE4 subfamily selective
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inhibitors against only the catalytic domain of PDE4. The crystal
structures of PDE4 plus UCR2 domain55,56 showed that an
UCR2 helix completely covered the active site of PDE4 (Figure
6). The sequence variation in UCR2 among the PDE4
subfamilies may allow the design of PDE4 subfamily selective
inhibitors.
On basis of the fact that a tyrosine in the UCR2 region of
PDE4A/B/C (Tyr274 of PDE4B3) corresponds to a phenyl-
alanine in PDE4D, a PDE4D-selective allosteric modulator was
designed and showed good therapeutic efficacy and reduced
emesis.55 This PDE4 allosteric modulator interacts with both
the active site and a helix of UCR2, implying a possibility of
development of a PDE4 subfamily selective inhibitor. The
crystal structure of PDE4D in complex with inhibitor PMNPQ
(12, Figure 7) showed that Phe196 of PDE4D3 from the UCR2
Figure 7. PDE4B/4D subfamily selective inhibitors 12 and 13 and
binding of 12 to PDE4B (cyan sticks and ribbons, PDB code 3G45) and
PDE4D (green sticks, PDB code 3G58). Tyr274 of PDE4B (a
phenylalanine in PDE4D) from UCR helix determines the subfamily
selectivity, while the residues in the active sites show great conservation
in both sequence and conformation.
helix, which corresponds to Tyr274 of PDE4B3, plays a key role
in ligand binding.57 By utilization of this difference, several
PDE4D selective inhibitors have been developed, such as
D159687 (13, Figure 7) and compound 6 (Figure 5), which
have a 100-fold selectivity between PDE4D and PDE4B.55,58−60
In particular, compound 6, the first PDE4D selective inhibitor,
entered phase II clinical trials for the treatment of Alzheimer’s
disease and fragile X syndrome.59 In addition, both PDE4B and
PDE4D negative allosteric modulators are shown to play
important roles in neurological diseases such as AD.58,60,61 On
the basis of the difference of tyrosine/phenylalanine, the PDE4B
allosteric modulators can be designed and showed the potential
to reduce neuroinflammation via inhibition of the production of
microglia from cerebral amyloid, while the PDE4D allosteric
modulator enhances memory consolidation.58−61
The UCR2-catalytic domain interactions block substrate
entry into the active site or product release, and therefore the
activity of different splice variants of PDE4 can be modulated
differently. This study opens a new avenue for the design of small
molecule inhibitors specifically targeting PDE4 specific splice
variants. Since the PDE4 allosteric modulators interact with
both UCR2 and the catalytic domain, they may preferentially
inhibit a certain form of PDE4 subfamilies and also have less
chance to interact with nontargeted proteins. As a result, they
may be less toxic than other PDE4 inhibitors, as shown by
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BPN14470 that is currently in phase II clinic trials for treatment
of Alzheimer’s disease and fragile X syndrome (Table 1).52
5. SURVEY OF PDE4 INHIBITORS
5.a. Catechol Ether-Based PDE4 Inhibitors. Catechol
ether simulates the hydrophobic properties of the adenine
portion of cAMP and, thus, has been used as a scaffold for many
PDE4 inhibitors,62,63 such as the first-generation drug rolipram
(14, Figure 8) and more advanced roflumilast (1) analogs. The
crystal structures (PDB code 1XMY, Figure 9) showed that the
catechol ether-containing compounds form a hydrogen bond
with the invariant glutamine across the PDE families and also
π−π stacking against a conserved phenylalanine (Figure 3).
Compound 14 is sandwiched by the hydrophobic clamp made
up of conserved phenylalanine and isoleucine on the two sides
(Figures 3 and 9). These interactions, hydrogen bonding with
the invariant glutamine and stacking against the phenylalanine,
occurred in almost all the inhibitors-PDEs crystal structures and
thus form the basic affinities of the PDE inhibitors.62
The dialkoxyphenyl attachments of the compounds in this
category are often the methoxy and cyclopentyloxy groups, such
as in compound 14 and cilomilast (15, Figure 8). The methoxy
group oriented into the small Q1 pocket that is formed by
Tyr233, Asn395, Thr407, and Tyr403 of PDE4B. The larger
hydrophobic cyclopentyl moiety occupied the Q2 pocket formed
by Phe414, Met411, Met431, and Phe446 of PDE4B.12
Compound 14 has been extensively studied for the treatment
of various diseases, including asthma, but shows severe side
effects including dizziness, headache, nausea, and vomiting.6 It is
often used as a positive control for studies of PDE4 inhibitors.
Compound 15 (Figure 8), developed by GlaxoSmithKline, is a
selective PDE4 inhibitor with an IC50 of 95 nM. Compound 15
was tested for the treatment of COPD but caused severe adverse
reactions, including nausea, vomiting, headache, abdominal
pain, diarrhea, and indigestion.64 In the phase III clinical trial on
asthma, the FEV1 values of the patients treated with compound
15 were <0.03 L, indicating lack of clinical efficacy on the
improvement of lung function.65
Tetomilast (16, Figure 8), a PDE4 inhibitor with an IC50 value
of 74 nM, suppresses the expression of TNFα and IL-12 in LPS-
stimulated human monocytes and the production of interferon γ
and IL-10 in CD4 lymphocytes at the transcriptional level.66 In a
phase II clinical trial of 246 patients with COPD, the 12 week
treatment with compound 16 (50 mg) improved peak FEV1 to
50 mL and forced vital capacity (FVC) to 130 mL. However, this
clinical trial was terminated in 2017.67
Compound 3 (Figure 4) is an oral and selective PDE4
inhibitor with IC50 of 74 nM and was studied for the treatment of
autoimmune diseases. Compound 3 acts on many types of cells
and shows a wide range of anti-inflammatory activities such as
inhibition on production of TNF-α, IL-12, and IL-23 and
responses of natural killer cells and keratinocytes. Clinical data
show that compound 3 significantly improved psoriasis of nails,
scalp, and palmar (hands and feet). Compound 3 was approved
by the U.S. FDA in 2014 for the treatment of active psoriatic
arthritis and moderate to severe plaque psoriasis by oral
administration. Additional clinical trials on the treatment of
other diseases are underway (Table 1), including cardiovascular
diseases, parapsoriasis, palmoplantar psoriasis, psoriasis of the
scalp, and psoriasis vulgaris.68
Replacement of the phenyl ring of compound 3 with a
thiophene yields compound 17 (Figure 8) that has an IC50 value
of 26 nM.69 Expansion of the acetyamide group of compound 3
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Figure 8. Chemical structures of catechol ether-based PDE4 inhibitors.
Figure 9. X-ray crystal structure of PDE4B-compound 14 (PDB code
1XMY).
led to a series of highly selective PDE4 inhibitors (18, n = 3−9,
Figure 8) with IC50 values of 3−8 nM.70 Compound 19 (IC50 =
40 nM, Figure 8) and its analogs are benzodiazepinone-derived
catechol ether with amine-containing chains.71,72
In short, it can be summarized as follows: (1) The two
substituents on the catechol ether occupy the hydrophobic Q1
and Q2 pockets. (2) The hydrophilic group orients to the
solvation region and hence effectively enhances the affinity of
the compounds. (3) The interactions with water molecules in
metal ion pocket significantly increase the inhibitory capacity of
the compounds.
5.b. Compound 1 Analogs. Compound 1 (Figure 4), the
first marketed PDE4 inhibitor, was approved for the treatment of
severe COPD-associated chronic bronchitis in Europe in 2010
and in the USA in 2011. Clinical studies showed that compound
1 reduced inflammation and fibrosis in lung, enhanced mucosal
clearance, and remodeled the airways. Adverse reactions were
mainly diarrhea, weight loss, nausea, atrial fibrillation, and
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increased mental illness (such as insomnia, anxiety, and
depression). In addition, compound 1 was studied for the
treatment of asthma, noncystic fibrosis bronchiectasis, obesity,
and lymphoma B-cells (Table 1).
Compound 1 has a remarkable inhibitory affinity toward
PDE4B and PDE4D, with IC50 values of 0.84 nM and 0.68 nM,
respectively.12 The X-ray crystal structures (PDB code 1XMU,
Figure 11) showed that the difluoromethoxy group of
compound 1 interacts with the Q1 pocket and the cyclo-
propylmethoxy group occupies the Q2 pocket. The 3,5-
dichloropyridyl group extends into the metal binding pocket
to form a hydrogen bond with the water molecule.12
Replacement of the methyl cyclopropane of compound 1 with
longer-chain aminofuran leads to compound 20 (Figure 10).
Compound 20 showed IC50 of 5.4 and 18 nM for PDE4B1 and
PDE4D2, respectively.73 Compounds 21, 22, and 23 (Figure
10) were designed by modification of the amide linker between
the dichloropyridine and catechol ether of roflumilst and
showed subnanomolar inhibitory activities against PDE4.74
CHF6001 (23, IC50 = 26 pM, Figure 10) explored the chemical
space of the solvent-exposed region of the enzyme and inhibited
the release of the proinflammatory cytokine TNF-α from LPS-
stimulated PBMCs.75 The phase II clinical trial of compound 23
on 1130 COPD patients was completed in January 2018 and
showed no significant emesis at tested doses.76 However, the
clinical results of the phase III trial have not been revealed yet.
Piclamilast (24, Figure 10) combines the structural features of
compound 14 and compound 1 and showed IC50 values of 41
and 21 pM for PDE4B and PDE4D, respectively. The
cyclopentyl ether binds to the Q2 pocket, while the methoxy
group occupies the Q1 pocket (PDB code 1XM4, Figure 11b).
The 3,5-dichloropyridyl group forms hydrophobic interactions
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Figure 10. Chemical structures of compound 1 analogs.
Figure 11. X-ray crystal structure of PDE4. (A) Compound 1 bound to PDE4B (PDB code 1XMU). (B) Compound 24 bound to PDE4B (PDB code
1XM4).
with Met347, Leu393, and Phe414 and extends into the metal
pocket to form a hydrogen bond with the water molecule
(Figure 11).12 The replacement of the central phenyl group of
compound 24 with chromenone yields compound 25 (Figure
10) that has an IC50 of 5 nM against PDE4B.77
Compound 26 (LEO-29102, Figure 10) was designed by
modification of dimethyl catechol ether and dichloropyridine of
compound 1.78 By introduction of a soft spot in the 2′-position,
no cases of emesis were observed in the clinical study and may
thus work as a soft drug for the treatment of AD. Compound 26
was tested in a phase II clinical trial for the treatment of eczema,
atopic dermatitis and psoriasis, but no recent progress has been
reported. The introduction of catechol pyrimidine core, which is
a potential bioisostere of adenosine and also mimics the
nucleotide part of the cAMP, yielded a series of novel
compounds, such as compound 27 (Figure 10) that shows an
IC50 of 15 nM against PDE4B.79
In short, we can conclude that (1) the 3,5-dichloropyridine
moiety interacts with Mg2+-bound water; (2) the Q1 and Q2
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pockets are filled respectively with the small and large
attachments of the catechol ether; and (3) inappropriate
modification of the amide linker reduced the affinity of
inhibitors, such as in the cases of compounds 25, 26, and 27
(Figure 12).
5.c. Quinoline-Based PDE4 Inhibitors. GSK256066 (28,
Figure 13) has an IC50 value of 3.2 pM for PDE4 and high
selectivity over other PDEs and is an inhaled PDE4 inhibitor for
the treatment of asthma.80,81 A large number of studies showed
that compound 28, delivered orally or by inhalation, reduced
FVC and inhibited allergen-mediated eosinophilia, airway
hyperresponsiveness and remodeling, mucin production, and
goblet cell hyperplasia in CD rats or other rodent models.80
Compound 28 significantly inhibited early and late allergic
responses by 26.2−34.3% but limitedly decreased the FEV1 and
weighted FEV1 values by 40.9% and 57.2%, respectively. This
compound underwent phase II clinical trials for asthma, COPD,
and allergic rhinitis. Clinical trials were completed, but further
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Figure 14. X-ray crystal structure of compound 28 bound to PDE4B

(PDB code 3GWT).

was highly efficacious with ED50 = 0.1 mg/kg following oral

Figure 12. Schematic presentation of compound 1 interaction with administration.85
PDE4. The binding mode of quinoline inhibitors to PDE4 is
summarized as follows (Figure 15): (1) the methoxy and
development of compound 28 is not evident since the
compound no longer appears in the GSK product pipeline.
Compound 29 (Figure 13) has a 2-trifluoromethyl-8-
methoxyquinoline core and exhibited high PDE4 inhibitory
potency (IC50 = 0.01 nM).82 Derivatizing the amide group
afforded compound 30 (Figure 13), whose xinafoate salt
inhibited inflammatory responses by 69% at 0.02 mg kg−1
day−1 in rat model, and monkey PK studies of 30 showed no
emesis (AUC = 31 ng·h/mL at 10 mg/kg po).83,84 Optimization
of both the 4-benzylcarboxamide and 5-α-aminoethyl groups
leads to an orally active, highly selective compound with
picomolar potency against PDE4. For example, compound 31
(IC50 = 60 pM, Figure 13) is a very potent PDE4 inhibitor with
high selectivity over PDE10 (15 800-fold) and has an IC50 of 0.6
nM for the inhibition of TNF-α release in a cell-based assay.85 At
an oral dose of 10 mg/kg, compound 31 was found to have Cmax
= 960 nM and 24% bioavailability in a rat PK study. In an in vivo
rat LPS-induced pulmonary inflammation model, compound 31 Figure 15. Interactions of compounds 29−31 with PDE4.

Figure 13. Chemical structures of quinolone analogs.

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Figure 16. Chemical structures of PDE4 inhibitors with a fused bicyclic ring.
difluoromethyl groups form hydrophobic interactions with the
Q1 and Q2 pockets of PDE4, respectively; (2) the quinoline ring
stacks against the phenylalanine via π−π interactions; (3) R2
interacts with the S pocket, and thus its size influences the
activity.
NVP-ABE-171 (32, Figure 13) has the IC50 values of 602, 34,
1230, and 1.5 nM for four subtypes of PDE4 (A, B, C, D) and
was tested for treatment of asthma and chronic obstructive
pneumonia.86,87 Compound 32 inhibited the activation of
airway neutrophils with an ED50 value of 3 mg/kg in BALB/c
mice and of 0.2 mg/kg in the Norway rat model. It also inhibited
ovalbumin-induced airway eosinophil activation with an ED50 of
0.1 mg·kg−1. However, the relatively poor pharmacokinetic
parameters (especially solubility) of compound 32 hinder its
further development. Replacement of the benzoxadiazole of 32
with fluorobenzene leads to compound 33 (Figure 13), which
exhibited 400-fold improvement of water solubility, good
pharmacokinetics, and no significant nausea and emesis.
YM976 (34,Figure 13) is a pyrimidine derivative and
structurally different from the other PDE4 inhibitors such as
compound 14.87 YM976 inhibited PDE4 with an IC50 value of
2.2 nM, inhibited cell infiltration into the pleural cavity with an
oral ED30 of 9.1 mg/kg in a rat carrageenan-induced pleurisy
model, and produced no emesis at doses of up to 10 mg/kg.88
5.d. PDE4 Inhibitors with a Scaffold of Fused Bicyclic
Rings. Several compounds with a scaffold of the fused bicyclic
ring show excellent PDE4 inhibitory activities. Ronomilast
(ELB353) (35, Figure 16) is a potent oral PDE4 inhibitor with
an IC50 of 3 nM and good pharmacokinetic properties in Lewis
rat or ferrets.89 In the preclinical and phase I trials, compound 35
demonstrated efficacy for the treatment of inflammation-related
diseases and no severe adverse events in humans. AWD12-281
(36, GSK842470, Figure 16) is an inhaled PDE4 inhibitor with
an IC50 of 9.7 nM and an EC50 of 110 nM for TNF-α release. It
was tested in phase II clinical trials for the treatment of COPD,
asthma, and allergic rhinitis.90
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With the combination of dimethoxybenzene, indole, and
dihydropyridine, compound 37 (Figure 16) was found to be a
potent PDE4 inhibitor with IC50 values of 0.54 and 0.65 nM for
PDE4B and PDE4D, respectively.91 The docking of 37 to
PDE4B/PDE4D showed that its indole moiety has two possible
orientations in PDE4D and forms hydrogen bonds with Glu396
or Glu505, whereas 37 exhibits a completely different
orientation in PDE4B and forms a hydrogen bond with
Ser442. The dimethoxy groups of 37 interact with the metal
ions in PDE4B, and the indole moiety forms hydrogen bonds to
Gln443.
GSK356278 (38, Figure 16), a selective and brain-penetrable
PDE4B inhibitor with the pIC50 value 8.8, showed a therapeutic
index superior to both compound 14 and compound 1 and had
beneficial effects on anxiety and cognition in primates without
emesis or other dose-limiting side effects.92 However,
compound 38 was apparently discontinued after phase I safety
and tolerability studies in 2012.
High throughput screening led to the discovery of
pyrazolopyridine-containing compound 39 (Figure 16), a
potent and selective PDE4 inhibitor. Compound 39 inhibits
LPS-induced TNF-α production and shows an encouraging PK
profile in rat, indicating its suitability for oral administration.93
The structure of PDE4B-39 showed that the pyrazolo[3,4-
b]pyridine moiety forms a hydrogen bond with Gln443, and the
N-ethyl substituent occupies the lipophilic pocket, similar to the
pattern of compound 14 binding (PDB code 3D3P, Figure 17).
Exploration of the SAR on the amide fragment of 39 led to the
discovery of compound 40 (Figure 16), which has a few tens of
picomolar affinity with PDE4 and subnanomolar inhibition on
LPS-induced TNFα production in human peripheral blood
mononuclear cells.94
Modification of the pyrazolopyridine scaffold led to
preparation of compound 41 (IC50 = 26 nM, Figure 16).95
Molecular docking of compound 41 to the crystal structure of
PDE4B showed that interactions of the amide, 3-methoxyaryl,
and sulfone groups with PDE4B completely coincide with those
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Figure 17. X-ray crystal structure of 39 bound to PDE4B (PDB code
3D3P).
of compound 28 (PDB code 3GWT, Figure 14). A nitrogen in
the heterocyclic ring and the amide form hydrogen bonds with
Gln443 through a water molecule, in addition to a hydrogen
bond between the amide of 39 and Asn395.95 Cyclization of the
primary amide at the 5-position of the pyrazolo[1,5-a]-
pyrimidine yielded compound 42 (Figure 16) and improved
the affinity by 200-fold (IC50 = 0.03 nM). The amide of 42
contacted Asn395 and displaced a water molecule.95
We can summarize the above discussion as follows (Figure
18). (1) The pyridine core inserts into the hydrophobic clamp
Figure 18. Interactions of compounds 38−42.
and forms a hydrogen bond with Gln443 of PDE4B to increase
the binding strength. (2) The amide branch forms a hydrogen
bond with Asn321 of PDE4D2 in the active site while interacting
with the small hydrophobic pocket (Q1). The inhibitory activity
could be significantly increased if the amide branch is a lactam.
(3) The substituent at the 2- or 3-position of pyridine fills the
large hydrophobic pocket (Q2). (4) The aryl fragment at the 4-
position of pyridine interacts with ion pocket to increase its
inhibitory activity.
5.e. Pyrimidine-Based PDE4 Inhibitors. Compound 43
(Figure 19) was identified as an orally active PDE4B-selective
inhibitor and had IC50 values of 5.5 nM and 440 nM for PDE4B
and PDE4D, respectively.96 Its therapeutic index, which was
calculated based on its inhibitory effect on LPS inhalation-
induced neutrophilia and gastric emptying, was similar to that of
compound 1 in mice. Replacements of the sulfide with a sulfone
and of the fluorine with chlorine of 43 afforded highly selective
PDE4B inhibitor 44 (Figure 19), which had an IC50 of 3 nM for
PDE4B and 433-fold selectivity over PDE4D.97 Changing the
sulfonyl moiety to a lactam and the carboxylic acid to an N-
neopentylacetamide afforded compound 45 (Figure 19), which
has an IC50 of 7.3 nM against PDE4B and potently inhibited
LPS-induced TNF-α production in mouse splenocytes (IC50 =
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0.21 nM) and LPS-induced pulmonary neutrophilia in vivo in
mice by 41% at a dose of 1.0 mg/kg (i.t.).98 The crystal structure
of PDE4B-45 showed that its pyrimidine ring forms π−π
stacking against Phe446 of PDE4B, while the neopentyl amide
moiety occupies a lipophilic Q1 pocket.
Compound 46 (Figure 19), with a 2-arylpyrimidine core,
showed anti-inflammatory therapeutic potential and a wider
safety margin than compound 15.99 The central pyrimidine ring
of 46 is sandwiched by Phe618 and Ile582 of PDE4B and forms a
hydrogen bond with Gln615. The aryl group extends into the
metal pocket and forms a hydrogen bond with the water
molecule bound to Mg2+, while the ethyl and methyl
substituents fit the Q1 pocket. In the mouse model, 46 showed
a strong antidepressant effect in mouse models of forced swim
and tail suspension.99
Compound 47 (Figure 19) had an IC50 of 7 nM for PDE4 and
an EC50 of 0.01 mg/kg for TNF-α inhibition in a mouse sepsis
model and reduced ear thickness by 40% in a psoriasis model.100
Introduction of a pyrido[3,2-d]pyrimidine substituent resulted
in compounds such as 48 (Figure 19) with IC50 = 0.4 nM against
PDE4.101
5.f. Pyridazinone-Based PDE4 Inhibitors. Zardaverine
(49 Figure 20) interacts with the catalytic site via hydrogen
bonds between (1) the catechol oxygen and the side chain of
invariant Gln369 of PDE4D2 and (2) the pyridazinone oxygen
and the nitrogen atom of His160. The pyridazinone moiety of
compound 49 is surrounded by residues Leu319, Ser320, and
Asn321 (PDB code 1XOR, Figure 21).12
Tetrahydrophthalazinone derivatives such as compound 50
(IC50 = 4 nM, Figure 20) can be considered as an analog of
compound 49, but molecular modeling revealed that the cis-
fused cyclohexane ring occupies a different region from that of
compound 49. Therefore, it was assumed that the steric
interactions of these rings with the residues in the binding site of
PDE4 play an important role in enzymatic inhibition. On the
other hand, substitution of the pyridazinones with pyrazoles/
1,2-diazepin-7-ones or N-substitution on compound 49 led to a
series of new derivatives such as 51 (Figure 20) that has pIC50 of
9.45.102
Optimization of the pyridazinone branch resulted in
compound 52 (Figure 20).103 The X-ray crystal structure of
human PDE4D in complex with 52 demonstrated that the
pyridazinone core sits in the hydrophobic clamp and forms a
hydrogen bond with Glu369. The ethyl branch at the 6-position
of the benzene ring of 52 fits in the lipophilic pockets, while the
benzoate extends into the outer solvated space. Replacement of
the acetyl carbonyl group with a urea link between 3,5-
dichloropyridine and pyridazinone led to compounds 53 and 54
(Figure 20), which were shown to be potent, long-acting, and
potentially safe inhaled PDE4 inhibitors.103,104 The combina-
torial assembly of functional linkers led to compounds such as
55, in which a phenyl ring is connected to the C4 pyridazine
atom.105
5.g. Methoxy Heteroaromatic-Based PDE4 Inhibitors.
Compound 4 (AV-411 or MN-166, 4, Figure 4), an anti-
inflammatory drug approved in Japan and Korea, inhibited
human PDE4A-D with IC50 values of 54, 65, 239, and 166 nM,
respectively, as well as weakly inhibiting other PDE families.106
Compound 4 was shown to be a bronchodilator and vasodilator,
had neuroprotective effects, and was used for oral treatment of
asthma, stroke, and topical treatment of allergic conjunctivi-
tis.107 In addition, phase II clinical trials are underway for
treatments of progressive multiple sclerosis, neuroinflammation,
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Figure 19. Chemical structures of pyrimidine-based PDE4 inhibitors.
Figure 20. Chemical structures of pyridazinone-based PDE4 inhibitors.
amyotrophic lateral sclerosis as an adjunct to riluzole, chronic
migraine, and alcohol use disorder (Table 1). Compound 4 was
approved as an orphan drug in the U.S. for the treatment of
progressive Krabbe disease in 2015 and for the treatment of
poststroke complications and bronchial asthma in Japan and
Korea in 1989.
Installation of a methoxyl substituent and 3,5-dichloropyr-
idine onto the heterocycle of compound 4 leads to compound
56 (Figure 22) that showed dramatic improvement in PDE4
inhibition (IC50 of 0.27 nM). The docking of compound 56 to
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the PDE4 catalytic domain suggested that the amide linker is
largely responsible for the improved activity.108,109 The nitrogen
at position 1 of the pyrazolopyridine interacted with the
invariant glutamine, and the pyridine ring extended into the
metal binding pocket to form hydrogen bonds with water.
Lotamilast (E6005, RVT-501, 57, Figure 22) is a selective
PDE4 inhibitor with an IC50 of 2.8 nM and suppresses the
production of various cytokines in human lymphocytes and
monocytes.110 Topical application of 0.03% compound 57 to
the rostral back significantly inhibited the activity of the
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Figure 21. X-ray crystal structure of PDE4D2 (PDB code 1XOR) in complex with compound 49.

Figure 22. Chemical structures of methoxy heteroaromatics PDE4 inhibitors.

cutaneous nerves and increased the cAMP concentration in

mice with chronic dermatitis.111 Lotamilast completed phase II
clinical trials sponsored by Dermavant Sciences Inc. for the
treatment of atopic dermatitis in July, 2017.
Oglemilast (58, Figure 22) is an oral PDE4 inhibitor for
asthma and rheumatoid arthritis and exhibited an IC50 value of
1.4 nM for PDE4 and showed high selectivity over other PDEs.
Compound 58 inhibited LPS-induced TNF-α production (IC50
= 190 nM) and blocked LPS-induced pulmonary neutrophilia in
rats (ID50 = 1.45 mg/kg) without obvious side effects.53
However, the phase IIb clinical data were discouraging and
showed no evidence of clinical effects.112 Revamilast (59, Figure Figure 23. Chemical structures of triazine-based PDE4 inhibitors.
22) was launched by Glenmark as a PDE4 inhibitor after the
failure of compound 58 and showed an IC50 of 2.7 nM for PDE4
and high selectivity over other PDEs. In 2011, Glenmark
globally announced the initiation of a phase IIb trial of
compound 59 on chronic inflammatory disorders such as
asthma and rheumatoid arthritis. However, the phase IIb data
were discouraging and the trial was discontinued.
5.h. Triazine-Based PDE4 Inhibitors. Compound 60, a
1,3,5-substituted triazine derivative (Figure 23), was identified
by screening a compound library and inhibited PDE4A with an
IC50 value of 245 nM. Structure-based design leads to
compound 61 (PDB code 4KP6, Figure 23), a potent and
orally active PDE4 inhibitor with an IC50 value of 1 nM against
PDE4A, -4B, and -4D.113 The crystal structure (Figure 24)
showed that the triazine core was located in the hydrophobic
clamp and that the substituents occupied the Q1 and Q2 pockets.
The triazine ring forms hydrogen bonds directly with Gln443
Figure 24. Diagram of the interactions between PDE4B and 61 (PDB
and indirectly with Asn395, Tyr233, and Asp392 via a water
code 4KP6).
molecule, in addition to π−π stacking interactions with Phe446
and Phe414 of PDE4B.
5.i. Cyclohexanediamide-Based PDE4 Inhibitors. A hexanediamide, such as compound 62 (Figure 25), have been
class of pyridopyrimidinedione derivatives containing cyclo- reported.114,115 Compound 62 inhibited PDE4B2 with a pIC50
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Figure 25. Chemical structures of cyclohexanediamide-based PDE4 inhibitors.
of 9.9. UK-500,001 (63, Figure 25), developed by Pfizer for
inhaled delivery, has IC50 values of 0.38−1.9 nM for PDE4A, -B,
and -D and strongly inhibited TNF-α production (IC50 = 0.13
nM). However, the phase II clinical data of its use against asthma
showed that this compound does not effectively improve
allergen-induced FEV1 but shows the typical side effects of
PDE4 inhibitors. Therefore, Pfizer ceased further development
of compound 63. AstraZeneca obtained several patents for 2-
phenoxypyridin-3-ylamide derivatives, such as compound 64
(pIC50 = 11, Figure 25).116 These compounds contain a
cyclohexanediamide motif similar to that of compound 63 and
show pIC50 values between 10 and 11.
In summary, for the conservation of the binding patterns,
PDE4 inhibitors can be divided into four pharmacophores for
design: P1−P3 and CORE (Figures 6A and 26). The CORE
Figure 26. Superposition of PDE4 inhibitors, which are colored with
green (compound 1), yellow (GEBR20b, 11), cyan (compound 24),
salmon (coumarin), orange (compound 46), and pink (naph).
contributes the basic affinity for all inhibitors and fits to the
hydrophobic clamp of the PDE4 active site. Pharmacophores
P1−P3 respectively oriented to subpockets of Q1 and Q2 and the
solvent region.
6. NATURAL PRODUCTS AS PDE4 INHIBITORS
Natural products have served as original sources of current drugs
and attracted a great deal of attention for their benefits for
human health in the past years. While functions of natural
products may be achieved via interactions with multiple targets,
regulation on the second messenger cAMP has been recognized
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as an important mechanism.117 As the key regulator of the cAMP
signaling pathways in physiological processes, PDE4 probably
serves as a cellular receptor for many natural products and
traditional herbal medicines to exert their beneficial effects on
human health.117 Nonselective inhibition of PDEs may be a
general pattern for functions of many natural products and
traditional herbal medicines. A well-known example of such
natural products is theophylline (65, Figure 27), which is a
nonselective inhibitor of PDEs with IC50 values of 50−100 μM
that is a first generation respiratory drug for the treatment of
asthma and COPD.118,119
Another example is the superfamily of flavonoids that are
abundant and key bioactive components of our daily foods and
have been thought to exert their nutritional effects via the
nonselective inhibition of PDEs.120 Many flavonoids with a
chromone scaffold (quercetin (66), genistein, luteolin, daidzein,
hesperidin, prunetin, diosmetin, biochanin A, apigenin, and
myricetin), which are nutrition supplements involved in
regulation of various biological processes, are nonselective
inhibitors of PDEs with affinity in the tens of micromolar
range.121 A recent study showed that the cocktail of antileukemia
drug dasatinib and natural product compound 66 (Figure 27)
prolongs the lifespan of mice by 36%.122 The mechanism for the
cocktail to increase lifespan is not clear, but perhaps the PDE4-
cAMP signaling pathway plays a critical role since compound 66
is a well-known PDE4 inhibitor.121
The catechol ether, a characteristic motif of PDE4 inhibitors,
also appears in some of the natural products. For example,
mesembrenone (67, Figure 27), which is an alkaloid isolated
from the South African plant Sceletium tortuosum and has
antianxiety and antidepressant effects, is an inhibitor of PDE4
with an IC50 value of 0.47 μM but not other PDEs.123
Compound 67 is also a selective inhibitor of the serotonin
transporter (SERT) with Ki = 27 nM to inhibit reuptake of
neurotransmitter serotonin.124
While most natural products nonselectively inhibit PDEs or
even simultaneously target other proteins, some of them showed
moderate or high selectivity among PDE families. The most
potent component 68 (Figure 27) out of 13 compounds that
were isolated from Gaultheria yunnanensis has an IC50 of 245 nM
for PDE4D and moderate selectivity over other PDEs.125
Compounds, which were extracted from the Chinese herb
“Sang-Bai-Pi” (Morus alba L.) and have been used for the
treatment of coughing and inflammatory diseases, also show
relatively good selectivity for PDE4 over other PDEs.126
Moracin M (69, Figure 27) has IC50 values of 2.9, 4.5, >40,
and >100 μM for PDE4D2, PDE4B2, PDE5A1, and PDE9A2,
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Figure 27. Chemical structures of natural products that inhibit PDE4.
respectively. Muracin A (70, Figure 27), a prenylated flavonoid,
shows the highest affinity for PDE4 (IC50 of 5.4 nM for
PDE4D2) and outstanding selectivity over other PDEs.127 Thus,
PDE4 may be the receptor mediating the functions of the
moracin compounds.
The natural product selaginpulvilin K (71) is a highly selective
inhibitor of PDE4D with an IC50 of 11 nM (Figure 27).128 The
cocrystal structure of PDE4D-71 (PDB code 5WQA) revealed
that 71 shallowly binds to the active site pocket and forms a
hydrogen bond with the side chain of Gln369, while a benzene
ring of 71 contacts Phe372 with π−π stacking (Figure 28).128
Figure 28. Diagram of the binding of compound 71 (yellow sticks)
PDE4D (PDB code 5WQA). The dotted line represents hydrogen
bond. Compound 1 is shown as green sticks. The binding residues of
PDE4D2 are shown as cyan and white sticks, respectively, for the
structures of PDE4D-compound 71 and PDE4D-compound 1.
These interactions are comparable with those of synthetic PDE4
inhibitors. However, compound 71 shows more extensive
interactions with some PDE4 residues that have rarely been
reported in the literature, such as Ser208, Pro356, and Ile376
(Figure 28).
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In short, although natural products may target multiple
proteins, PDE4 must be an important receptor for their
functions. For their different skeletons and potentially different
binding patterns, natural products may be an abundant resource
for the development of PDE4 selective inhibitors.
7. DUAL PDE4 INHIBITORS
For the important roles of the cAMP and cGMP signaling in
various physiological processes1−4 and the existence of multiple
PDEs in certain tissues such as seven PDE families in the
heart,129 simultaneous inhibition of multiple PDE families may
produce synergistic effects of physiological processes. In
addition, simultaneous regulation on the cAMP/cGMP signal-
ing pathways and the key proteins in the physiological processes
such as cancer development might produce additive or even
synergistic effect. On the basis of these considerations, dual
selective PDE4 inhibitors that target PDE4 and other PDEs or
that conjugate with other functional proteins have been
developed in recent decades.
7.a. Dual Inhibitors of PDE4 and Other PDEs. Dual
inhibition of PDE3/PDE4 and PDE4/PDE7 may synergistically
suppress inflammatory diseases such as COPD. However,
because of the conservation of the active site of the PDE
isozymes, the PDE4 dual inhibitors may also inhibit other PDEs,
resulting in unpredictable beneficial or adverse effects.
A survey of nonselective PDE inhibitors containing
pyrazolopyridine-pyridazinone motifs led to the development
of dual PDE3/4 selective inhibitors for the treatment of
respiratory diseases. These dual PDE3/4 inhibitors often show
both bronchodilatory and anti-inflammatory activities, thus
having advantages over the singly selective inhibitors. For
example, KCA-1490 (72, Figure 29) displayed IC50 values of
360 and 42 nM against PDE3 and PDE4, respectively.137
Compounds 73 and 74 (Figure 29) are orally active and have
well-balanced dual PDE3/4-inhibitory activities. However, most
of these agents caused adverse reactions in the gastrointestinal
system.138,139 In the past, at least five dual PDE3/4 inhibitors,
compound 49 (Figure 20), pumafentrine (75, Figure 29),
benzafentrine (76, Figure 29), tolafentrine (77, Figure 29), and
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Figure 29. Chemical structures of dual PDE inhibitors 72−83.
compound 5 (Figure 4, Table 1), entered clinical trials, but most
of them, except compound 5, have been discontinued.140
Compound 5 is a long-acting dual PDE3/PDE4 inhibitor
developed by Verona Pharma as a bronchodilator and anti-
inflammatory agent for the treatment of asthma and COPD.
Compound 5 triggers significant bronchiectasis in asthmatics,
which is even detectable 7 h after administration, and induces
bronchial protection against methacholine. For patients with
allergic rhinitis, a single dose of compound 5 (18 μg/kg)
reduced inflammatory cells (eosinophils and neutrophils) 7 and
24 h after the allergen challenge. In the phase II clinical trial, a
single dose of nebulized compound 5 (18 μg/kg) showed a rapid
bronchodilator response and a >15% increase in FEV1 in COPD
patients and was tolerated without significant side effects. In
addition, a phase II clinical trial on compound 5 for the
treatment of cystic fibrosis is in progress.
Compound 78 (Figure 29), which contains a compound 49
fragment, was claimed as a dual PDE4/PDE7 inhibitor with an
IC50 of 12 nM for PDE4, but its exact number for PDE7 was not
reported.141 Compound 79 (Figure 29) is a moderate dual
inhibitor with IC50 values of 2.9 μM and 1.4 μM against PDE4
and PDE7.142 In a series of spiroisoxazoline-containing dual
inhibitors, compound 80 (PDE4 IC50 = 5 nM, PDE7 IC50 = 55
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nM, Figure 29) binds to PDE4 through its pyrazolopyridine
group in a pattern similar to that of compound 28.143
Compound 81 (Figure 29) was identified by yeast-based
screening and displayed dual PDE4/PDE7 inhibition at the
micromolar level: 64−90% inhibition of PDE4 and 98%
inhibition of PDE7 at 2 μM concentration of the compound.144
A novel butanehydrazide derivative of purine-2,6-dione
(compound 82,Figure 29) appeared to be a dual PDE4/7
inhibitor (PDE4 IC50 = 1.4 μM, PDE7 IC50 = 3.2 μM).145
In addition, a number of pyrazolopyridine derivatives showed
inhibitory activities on PDE4/PDE10/JNK (Jun N-terminal
kinase) with binding affinity in the micromolar range. For
example, compound 83 (Figure 29) showed IC50 values of 1.2,
7.4, 0.3, and 1.2 μM against PDE4, PDE10, JNK2, and JNK3,
respectively. These compounds were considered to be useful for
treating neurological diseases such as Parkinson’s disease and
neuropathic pain, and some were tested in a chronic constriction
injury model in rats.146
7.b. Dual PDE4 Inhibitors in Conjugation with Other
Proteins. Successful examples of the dual PDE4 inhibitors are
conjugated with β2 agonists, M3 antagonists, and selective
serotonin reuptake inhibitors (SSRIs). These inhibitors are
designed by covalently grafting two pharmacophores together
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Figure 30. Chemical structures of dual PDE4 inhibitors 84−93.
with a flexible linker, and each pharmacophore targets its own
protein in the anticipation of a synergistic effect.
Compound 85 (Figure 30), which combines compound 28
(Figure 13) and carmoterol (84,Figure 30), showed activities of
both β2 agonist and PDE4 inhibitor with IC50 values of 8 and 5
nM, respectively.130 The combination of the β2 agonist
formoterol (86,Figure 30) and the PDE4 inhibitor 55 (Figure
20) yielded compound 87 (Figure 30) that possessed moderate
inhibitory activity against PDE4B2 (IC50 = 278 nM) and
excellent relaxant effects on tracheal tissue precontracted by
histamine (pEC50 = 9.3).131 By exploration of the composition
and length of the linker, a new series of hybrids that combined
formoterol (86) and the PDE4 inhibitor phthalazinone (50,
Figure 20) were investigated as potent agents for the treatment
of asthma and COPD. The most potent one in this class is
compound 88 (Figure 30), which exhibited both a β agonist
effect (EC50 = 1.05 nM, pEC50 = 9.0) and PDE4B2 inhibition
(IC50 = 92 nM).132
Compound 89 (Figure 30), a dual PDE4/SSRI inhibitor,
grafted the PDE4 inhibitor compound 50 to the 2,5-
Q https://dx.doi.org/10.1021/acs.jmedchem.9b02170
pubs.acs.org/jmc Perspective
disubstituted tetrahydrofuran of an SSRI inhibitor and inhibited
serotonin reuptake with an IC50 value of 127 nM and PDE4D3
with Ki of 2.0 nM. This combination showed synergistic
antidepressant effects.133
The combination of pyrazolopyridine of PDE4 inhibitor 39
with M3 antagonists yielded a series of compounds with the
capacity of dual PDE4 inhibition and M3 antagonism, which
have IC50 values of 0.1−2511 nM for PDE4B and 10 nM to 33
μM for M3 antagonist. The best compound 90 (Figure 30)
shows potency for the treatment of respiratory and allergic
diseases, such as COPD, asthma, rhinitis, and inflammatory
diseases.134 Compound 91 (Figure 30), a carbamate derivative,
showed both activities of PDE4 inhibition (IC50 ≈ 100 nM) and
muscarinic M3 receptor antagonism (IC50 ≈ 100 nM), but its
therapeutic effects need further study.135
In addition, a series of orally bioavailable dual inhibitors of
epoxide hydrolase (sEH)/PDE4 were developed for the
treatment of inflammatory pain.136 Unlike other dual inhibitors
that were prepared by simple grafting, compound 92 (Figure 30)
was designed by linking the benzyl amide moiety of the sEH
J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
inhibitor to a methoxybenzene fragment (compound 14
simulation) and showed highly potent and dual inhibition of
PDE4 and sEH with IC50 values of 8.1 nM and 2.1 nM,
respectively. Compound 93 (Figure 30) is a triple p38α MAPK/
JNK3/PDE4 inhibitor with IC50 values of 500, 700, and 200 nM,
respectively, and demonstrated desirable pharmacokinetic
properties and low plasma protein binding in vitro and in vivo
even at the nanomolar doses.
8. PERSPECTIVE
While PDE4 inhibitors have been extensively investigated as
therapeutics for treatment of human diseases such as COPD,
only a few of them have been approved for practical use, due to
side effects. It was thought that the side effects of nausea and
emesis are caused by the poor selectivity of the PDE4 inhibitors
between subfamilies of PDE4B and PDE4D. However, this
argument is not supported by the recent studies of PDE4D
inhibitors (see the discussion in section 4).
A statistical survey shows that the four approved PDE4
inhibitors for the treatment of human diseases in market have
only nanomolar affinity with PDE4, but the compounds that
have picomolar affinity never progressed beyond phase II clinical
trials. For example, Merck’s compounds 29 (IC50 = 10 pM
against PDE4B)147 and 31 (IC50 = 60 pM against PDE4B) and
compound 42 (IC50 of 30 pM for PDE4B)148 have never been
reported to enter clinical trials. The tightest-bound compound
28 (IC50 = 3.2 pM against PDE4) was tested in a dozen phase II
clinical trials on treatment of various disease but does not seem
to advance to further trials. The failure of these very tight
binding PDE4 inhibitors might be associated with their side
effects. A possible reason to explain the failure might be
inhibition of nontargeted cellular proteins, which causes
disturbance on normal physiological functions of cAMP
signaling. Besides, we believe that the cellular cAMP level
needs to be maintained in an equilibrium for cellular functions.
Deep inhibition of the PDE4 activity may cause redundant
accumulation of cAMP and thus disturb normal physiological
processes. This hypothesis of cellular balance of cAMP levels is
supported by the case of the first generation PDE4 inhibitor
rolipram (compound 14) that was shown to have two binding
sites with high and low binding affinity states (HARBS/LARBS)
against PDE4.149 The binding of compound 14 to the high
affinity site showed antidepressant and anti-inflammatory
activities but was also correlated with side effects such as
vomiting and gastric acid secretion.150,151
At present, several dual selective PDE4 inhibitors have been
reported to show synergistic effects on human health, implying
that simultaneous inhibition on multiple PDEs produces
synergistic therapeutic effects. This argument is supported in
theory by the facts that both cAMP and cGMP signaling
pathways play critical roles in physiological processes and
disease development.152−158 Inhibition of PDE4 and other
PDEs may increase levels of cAMP, cGMP, or both in cells and
thus boost physiological processes. This assumption of potential
synergistic effects from simultaneous inhibition of multiple
PDEs is also supported by several experimental observations.
First, theophylline (compound 65), the first generation
antiasthma drug, nonselectively inhibits most PDEs with a few
tens micromolar affinity and presumably functions via inhibition
of multiple PDEs. Second, many natural products that are
nutritional supplements to benefit human health have been
shown to be nonselective PDE inhibitors. For example,
resveratrol, a natural phenol that shows several beneficial effects
R https://dx.doi.org/10.1021/acs.jmedchem.9b02170
pubs.acs.org/jmc Perspective
on human diseases, modulates the PDE4/3-cAMP signaling
pathway for part of its function.159 Another example is the recent
report that the cocktail of the antileukemia drug dasatinib and
the natural product quercetin prolongs the lifespan of mice by
36%,122 which may be achieved via the regulation of the PDE4-
cAMP signaling pathway since compound 66 is a well-known
PDE4 inhibitor.121 Thus, simultaneous inhibition of multiple
PDE families would be a promising direction for discovery of
PDE inhibitors and is worthy of further exploration.
■ AUTHOR INFORMATION
Corresponding Authors
Hengming Ke − Department of Biochemistry and Biophysics, and
Lineberger Comprehensive Cancer Center, University of North
Carolina, Chapel Hill, North Carolina 27599, United States;
Email: hengming_ke@med.unc.edu
Jianyou Shi − Personalized Drug Therapy Key Laboratory of
Sichuan Province, Sichuan Academy of Medical Science &
Sichuan Provincial People’s Hospital, School of Medicine of
University of Electronic Science and Technology of China,
Chengdu 610072, China; orcid.org/0000-0002-1026-0282;
Phone: 86-18908001802; Email: shijianyoude@126.com
Authors
Ting Peng − Personalized Drug Therapy Key Laboratory of
Sichuan Province, Sichuan Academy of Medical Science &
Sichuan Provincial People’s Hospital, School of Medicine of
University of Electronic Science and Technology of China,
Chengdu 610072, China
Baowen Qi − Center for Nanomedicine and Department of
Anesthesiology, Brigham and Women’s Hospital, Harvard
Medical School, Boston, Massachusetts 02115, United States
Jun He − Cancer Center, Collaborative Innovation Center for
Biotherapy, West China Hospital, Sichuan University, Sichuan
610041, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.9b02170
Author Contributions
#
T.P., B.Q., and J.H. contributed equally to this manuscript.
Notes
The authors declare no competing financial interest.
Biographies
Ting Peng obtained her Bachelor’s degree in Pharmacy at Zhengzhou
University, China, in 2016. She is now a graduate student at the
University of Electronic Science and Technology of China. Her
research interests involve the design and synthesis of small molecules
for the treatment of tumor and neurological and inflammatory diseases.
Baowen Qi received his Ph.D. from the University of Montreal in
Pharmaceutical Science in 2016 and is now a Postdoctoral Fellow at
Brigham and Women Hospital, Harvard Medical School. His research
focuses on the development and formulation of novel therapeutic
agents for the treatment of various diseases.
Jun He, a Research Associate, received his Ph.D. in Pharmacy from
Sichuan University in 2011 and now works at the State Key Laboratory
of Biotherapy of Sichuan University in China. His research focuses on
small molecule drug discovery and computer-aided drug design for the
treatment of diseases.
Hengming Ke received his Ph.D. in Biophysics from Harvard
University in 1989 and is now a Professor at the University of North
Carolina at Chapel Hill. His research focuses on the crystal structures of
J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
medically important proteins and rational drug design for the treatment
of diseases.
Jianyou Shi received his Ph.D. in Medicinal Chemistry from Sichuan
University in 2010. After 3 years of postdoctoral training, he joined a
group at the University of North Carolina at Chapel Hill in 2016, and he
worked in this group for 2 years. He is currently working at the
University of Electronic Science and Technology as a professor. His
major scientific interests focus on the discovery of small molecules
targeting oncogenesis, neurological, and inflammatory diseases.
■ ACKNOWLEDGMENTS
The authors are thankful for the financial support from the
Sichuan Science and Technology Department Project (Grant
2017JQ0038) and the Sichuan Provincial People’s Hospital
Clinical Research and Transformation Fund Key Project (Grant
2016LZ03).
■
ABBREVIATIONS USED
PDE, phosphodiesterase; cAMP, cyclic adenosine mono-
phosphate; cGMP, cyclic guanosine monophosphate; COPD,
chronic obstructive pulmonary disease; β-AR, β-arrestin; GPCR,
G-protein-coupled receptor; Gs, heterotrimeric guanine nucleo-
tide binding protein; AC, adenylyl cyclase; AKAP, A-kinase-
anchoring protein; PKA, protein kinase A; EPAC, exchange
protein activated by cAMP; CREB, cAMP response element
binding protein; C3G, cyanidin-3-glucoside; Rap1, Ras-
proximate-1; MEK1/2, mitogen-activated protein kinase;
ERK1/2, extracellular signal-regulated kinase 1/2; NF-κB,
nuclear factor κ-light-chain-enhancer of activated B-cells;
MAPK, mitogen-activated protein kinase; mTORC1, mamma-
lian target of rapamycin complex 1; UCR, upstream conserved
regions; RACK1, receptor for activated C kinase 1; LPS,
lipopolysaccharide; TNFα, tumor necrosis factor α; PD,
Parkinson’s disease; AD, Alzheimer’s disease; AAMI, age-
associated memory impairment; IL, interleukin; CD4, cluster
of differentiation 4; FEV1, forced expiratory volume; FVC,
forced vital capacity; FST, forced swim test; TST, tailsus-
pension test; NAH, N-acylhydrazone; NK, natural killer cell;
SSRI, selective serotonin reuptake inhibitor; sEH, epoxide
hydrolase; BALF, bronchoalveolar lavage fluid; JNK, Jun N-
terminal kinase.
■
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