Design and synthesis of 4,5,6,7‐tetrahydro‐1H‐1,2‐diazepin‐7‐one derivatives as a new series of Phosphodiesterase 4 (PDE4) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.11.040

Phosphodiesterase 4 (PDE4) inhibitors have attractive therapeutic potential in respiratory, inflammatory, metabolic and CNS disorders. The present work details the design, chemical exploration and biological profile of a novel PDE4 inhibitor chemotype. A

Full text of DOI:10.1016/j.bmcl.2016.11.040

Accepted Manuscript
Design and synthesis of 4,5,6,7-tetrahydro-1H-1,2-diazepin-7-one derivatives
as a new series of Phosphodiesterase 4 (PDE4) inhibitors
Sara Guariento, Anna Karawajczyk, James A. Bull, Gessica Marchini, Martyna
Bielska, Xenia Iwanowa, Olga Bruno, Paola Fossa, Fabrizio Giordanetto
PII:
S0960-894X(16)31193-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.040
BMCL 24435
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 October 2016
14 November 2016
15 November 2016
Please cite this article as: Guariento, S., Karawajczyk, A., Bull, J.A., Marchini, G., Bielska, M., Iwanowa, X., Bruno,
O., Fossa, P., Giordanetto, F., Design and synthesis of 4,5,6,7-tetrahydro-1H-1,2-diazepin-7-one derivatives as a
new series of Phosphodiesterase 4 (PDE4) inhibitors, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://
dx.doi.org/10.1016/j.bmcl.2016.11.040
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Design and synthesis of
4,5,6,7 tetrahydro 1H 1,2 diazepin 7 one derivatives as
a new series of Phosphodiesterase 4 (PDE4) inhibitors.
Sara Guarientoa, Anna Karawajczykb, James A. Bullb, Gessica Marchinic, Martyna
Bielskab, Xenia Iwanowab, Olga Brunoa, Paola Fossaa, Fabrizio Giordanettob,§,*
aDepartment of Pharmacy, University of Genoa, Viale Benedetto XV n. 3, 16132, Gen-
oa, Italy
bMedicinal Chemistry, Taros Chemicals GmbH & Co. KG, Emil-Figge-Str. 76a, 44227
Dortmund, Germany
cPharmacology Toxicology Department, Chiesi Farmaceutici S.p.A., Nuovo Centro
Ricerche, Largo Belloli 11/a, 43122 Parma, Italy
§Present address: DE Shaw Research, 120W 45th Street, New York, 10036, United
States of America
*Corresponding Author:
Tel: +1 (0)212 4780 822; e-mail: fabrizio.giordanetto@deshawresearch.com
Keywords
Phosphodiesterase 4, PDE4 inhibitors, diazepinone derivatives, compound library

Abstract
Phosphodiesterase 4 (PDE4) inhibitors have attractive therapeutic potential in
respiratory, inflammatory, metabolic and CNS disorders. The present work details the
design, chemical exploration and biological profile of a novel PDE4 inhibitor
chemotype. A diazepinone ring was identified as an under-represented heterocyclic
system fulfilling a set of PDE4 structure-based design hypotheses. Rapid exploration of
the structure activity relationships for the series was enabled by robust and scalable
two/three-steps parallel chemistry protocols. The resulting compounds demonstrated
PDE4 inhibitory activity in cell free and cell-based assays comparable to the
Zardaverine control used, suggesting potential avenues for their further development.

Phosphodiesterase 4 (PDE4) belong to the superfamily of 3’,5’-cyclic-nucleotide
Phosphodiesterase and catalyse the hydrolysis of cyclic adenosine 3’,5’monophosphate
(cAMP) to its inactive 5’-monophosphate metabolite, thus blocking the associated
signal transduction systems.1
The PDE4 family consists of 4 isoforms (named A, B, C and D) and 25 splice
variants,2,3 which display a highly conserved catalytic site (78% of similarity).4 Distinct
promoters and mechanisms control the mRNA stability of the various PDE4 variants
therefore allowing a fine-tuned regulation and compartmentalisation of the formed
isoforms and c-AMP intracellular levels.5-7
In particular, PDE4s are mainly found in inflammatory cells, brain, pulmonary and
vascular smooth muscle cells and cardiovascular tissue both at cytosolic and plasmatic
membrane level.8-9 In those tissues, the intracellular concentration of cAMP modulates
inflammatory processes such as cellular trafficking and adhesion, cytokine and
chemokine release, the production of reactive oxygen species, the secretion of mucus in
the respiratory tract and the airway smooth muscle contraction.8,10 In the central nervous
system (CNS), cAMP is involved in regulating synaptic plasticity and memory
formation, by promoting the activation of cAMP response element-binding protein
(CREB)11 and the expression of neurotrophic factor, which enhance progenitor cell
proliferation in hippocampus and axonal growth.12-14 An excessive degradation of
cAMP promotes inflammatory diseases and CNS disorders, where behavioural, learning
and memory processes are affected.7
Therefore, increasing intracellular cAMP via PDE4 inhibition has been pursued for dec-
ades as an appealing strategy for the treatment of inflammation-related respiratory dis-
eases such as Chronic Obstructive Pulmonary Disease (COPD), asthma, allergic rhinitis

and idiopathic pulmonary fibrosis.16,17,18 More recently, PDE4 inhibition also demon-
strated positive effects against aberrant immune response diseases, such as atopic der-
matitis, rheumatoid arthritis, multiple sclerosis, psoriasis and lupus erythematosus.9,19,20
22
Further studies using archetypical PDE4 inhibitors (e.g. Resveratrol,21 Rolipram and
Roflumilast23) have also implicated these enzymes in the modulation of metabolic dis-
orders such as obesity, type 2 diabetes and metabolic syndrome, and glomerulonephritis,
as they all share an underlying inflammatory component.9,24,25 The therapeutic potential
of PDE4 inhibition in pulmonary artery hypertension, renal transplantation and renal
failure was also reported.9 Finally, the influence of PDE4s on long term potentiation of
the synaptic activity and on neuronal plasticity in general,26,27 as well as the beneficial
effect of PDE4 inhibitors on depression, tissue injury and neurodegeneration12,13 are
well documented.
Among the wide variety of compounds active against this enzyme,28 Rolipram,
Cilomilast,29 Roflumilast and Apremilast30 (Figure 1) are some of the most characterised
PDE4 inhibitors to date. It is noteworthy that Roflumilast and Apremilast were ap-
proved by the Food and Drug Administration (FDA) as oral drugs for the treatment of
COPD and psoriatic arthritis in 201123 and 2014,31 respectively.

Figure 1. Molecular structure of selected PDE4 inhibitors: Rolipram, Cilomilast,
Roflumilast, Apremilast.
Despite these successes, the development of novel PDE4 inhibitors is typically ham-
pered by the occurrence of dose-limiting side effects like nausea, emesis, sedation18,32,33
and novel chemical starting points are required to seed future drug discovery efforts.
Data mining of the scientific and patent literature indicated a large body of chemical
matter developed towards PDE4A-D inhibition, for a total of 5537 unique compounds
with measured PDE4 inhibition. Chemoinfomatics analysis of this data set served to
identify and compare various chemotypes with the aim to identify suitable starting
points for additional chemical exploration. Here, a cluster of catechol-containing cyclic
hydrazides was found to be of particular relevance due to the relatively high PDE4 inhi-
bition, adequate efficiency parameters (i.e. ligand efficiency (LE) and ligand
lipophilicity efficiency (LLE)) and available structural biology data, as shown by select-
ed examples in Figure 2. This molecular cluster included the archetypical PDE4 inhibi-
tor Zardaverine (IC50: 1.41 μM) 34, whose binding mode to the enzyme has been eluci-
dated (PDB code: 1XOR)35. As shown in Figure 3A, Zardaverine establishes productive
polar interactions with the catalytic site: a) bi-dentate hydrogen bond between the cate-
chol’s oxygen atoms and the side chain of Q369, a pivotal residue in the interaction with
c-AMP and with PDE4 inhibitors in general,35 b) coordination of the Zinc ion and hy-
drogen bond to H160 via the pyridazinone ring’s oxygen (displacing one of the six con-
served coordinated waters of the PDE4 active site)35 and nitrogen atoms, respectively.
Interestingly, the pyridazinone moiety of Zardaverine is surrounded by two side pock-
ets, the so called S pocket35 (including G206, S208, E339, F340, Q343, S355, C358)

and a smaller one (including L319, S320, N321), as highlighted in Figure 3. We rea-
soned both of them could be exploited to engage additional interactions with the en-
zyme and optimize the functional potency of the inhibitors. Indeed, analogous cyclic
hydrazides such as tetrahydrophtalazinone 236 and 4,4-dimethylpyrazolone 3,37 showed
favourable PDE4 inhibition results (Figure 2) suggesting opportunities for further chem-
ical
diversification
in
the
area.
We
thus
designed
a
4,5,6,7‐tetrahydro‐1H‐1,2‐diazepin‐7‐one ring as a likely scaffold satisfying this struc-
ture-based framework as summarized in Figure 2.
O
O
O
F
N
O
O
O
N
R
3
F
O
O
O
O
O
N
4
H
N
N
N
O
N
R
2
N
N
R
3
O
NH
N
H
N
O
O
O
O
O
O
2
3
Zardaverine (1)
N
IC : 4.0 nM
50
IC : 32 nM
50
IC :1.41
50
M
N
R
LE: 0.41
LE: 0.31
LE: 0.42
3
LLE: 4.63
LLE: 6.45
LLE: 4.86
O
5
PRIOR ART
THIS WORK
Figure 2. Design process underpinning the present work. IC50 values for Zardaverine
(this work), 234 and 335 are indicated. Ligand efficiency (LE) was calculated as 1,37*-
log10(PDE4 IC50)/Heavy Atom Count (HAC) and Ligand lipophilicity efficiency (LLE)
was calculated as -log10(PDE4 IC50)- clogP. clogP was calculated with ChemDraw®.

Figure 3. (A) Experimental binding modes of Zardaverine (PDB code:1XOR). (B) Pre-
dicted binding mode of 4. Amino acid residues from the S pocket are colored in green,
while residues from the smaller side pocket are colored in blue. The Zinc and Magnesi-
um ions are represented as grey and green spheres, respectively. Water molecules are
shown as red spheres. H-bonds between the compounds and the active site amino acids
are highlighted as red solid lines.
Importantly, the seven-membered ring nature of the intended scaffold served multiple
purposes: a) it would allow for a pragmatic, lipophilicity efficient steric extension to-
ward the smaller side pocket b) it could be rapidly assembled and diversified using ro-
bust synthetic chemistry protocols and c) it offered an opportunity to explore an innova-
tive, underrepresented heterocyclic chemical space as only 25 tetrahydro-[1,2]-
diazepinone derivatives have been described to date,38 none of which in the context of
PDE4 inhibition. Based on these premises, the dimethoxyphenyl moiety substituting C3
of the 4,5,6,7‐tetrahydro‐1H‐1,2‐diazepin‐7‐one ring was maintained due to its PDE4
warhead character. Two additional diversification elements at the C5 and N1 position of
the designed scaffold were considered (Figure 2). Here, C5 included a methylene and a
geminal-dimethyl group to further extend towards the end of the smaller side pocket. On

N1, a wider range of alkyl substituents could be introduced following robust N-
alkylation protocols in a parallel chemistry format to verify structure activity relation-
ships (SAR) at the S pocket. Selection of appropriate side chains was informed by a
combination of chemical diversity and physicochemical properties considerations.39,40
In particular, docking calculations were performed to assess binding of the
3‐(3,4‐dimethoxyphenyl)‐4,5,6,7‐tetrahydro‐1H‐1,2‐diazepin‐7‐one
4
and
3‐(3,4‐dimethoxyphenyl)‐5,5‐dimethyl‐4,5,6,7‐tetrahydro‐1H‐1,2‐diazepin‐7‐one
5
within PDE4 active site (Figure 3). The aforementioned crystal structure of PDE4 active
site in complex with Zardaverine (PDB code: 1XOR) was selected for the simulations. 4
and 5 displayed a comparable binding mode to Zardaverine (with compound 4 showing
the best docking score, Table 1) maintaining key hydrogen bonds to the side chains of
H160 and Q369. (Figure 3B).
We therefore synthesized 3-(3,4-dimethoxyphenyl)-5,6-dihydro-1H-1,2-diazepin-7(4H)-
one 4 and 3-(3,4-dimethoxyphenyl)-5,5-dimethyl-5,6-dihydro-1H-1,2-diazepin-7(4H)-
one 5 to establish comparison baselines to Zardaverine and verify any PDE4 inhibition
difference across scaffolds. Compounds 6, where the catechol moiety of Zardaverine
was replaced by 4-methoxyphenyl served as a negative control for the structure-based
hypothesis (Figure 3). A one-step Friedel-Crafts acylation between phenyl derivatives 7,
8 and anhydrides 9, 10 yielded the acid intermediates 11-13 in 41% to 92% yields
(Scheme 1). Interestingly, the reactivity of the two anhydrides was different. Using 10
the reaction proceeded to completion in 1h and longer reaction time using 8 as starting
material led to the de-methylation of one of the two phenolic oxygens. The same side
reaction occurred with 9 only after 12 h. Presence of diphenyl-containing side product
was detected, which was easily removed by an acid-base extraction (ethyl acetate/10%

sodium hydroxide aqueous solution followed by ethyl acetate/1N hydrochloric acid
aqueous solution).
The cyclization reaction of the formed 5-oxopentanoic acids 11-13 was executed with
hydrazine hydrate at 150 °C (Scheme 2). By using a Dean-Stark apparatus, water con-
tent in the reaction mixture was minimized, firstly promoting the formation of the hy-
drazone and then avoiding the hydrolysis of the final hydrazide.41 This cyclization was
overall low-yielding (13 85%) and represented the limiting step of the synthetic route.
Its success rate was greatly influenced by the choice of the R2 substituents (Scheme 2):
the insertion of the geminal-CH3 in 5 (5) underwent the cyclisation with the best yield,
following a typical Thorpe-Ingold effect.42
Scheme 1. Synthesis of intermediates 11-13.
Reagents and conditions: (a) AlCl3, dry DCM, rt, 1 10 h.
Scheme 2. Synthesis of final compounds 4-6.

1
1
2
2
1
1
1
3
3
2
2
2
1
1
1
3
3
2
2
2
gem
gem
3
3
2
2
2
2
Reagents and conditions: (a) NH2NH2 hydrate, xylene, reflux, 5 h.
As shown in Table 1, 4 and 5 showed comparable PDE4B2 inhibition to Zardaverine,
lending support to the chosen design approach (Table 1). The dramatic loss of potency
of 6 (PDE4B2 IC50 > 30 μM) highlighted the importance of the bi-dentate H bond be-
tween the two catechol oxygen atoms and Q369 as a necessary interaction with the en-
zyme. Interestingly, the addition of the geminal-CH3 in 5 of the diazepinone did not
result in a potency gain consistent with the increased size and lipophilicity (cf. 5 and 4).
Having confirmed the 4,5,6,7‐tetrahydro‐1H‐1,2‐diazepin‐7‐one scaffold as an effective
PDE4 inhibitor starting point, we proceeded to the functionalization of its N1 position
to verify how different substituents could affect PDE4 inhibition. Three
5,5‐dimethyl‐4,5,6,7‐tetrahydro‐1H‐1,2‐diazepin‐7‐one substituted examples were also
synthesized as a further validation for the 5-geminal-dimethyl substitution.
Table 1. PDE4B2 inhibition potency and ligand efficiency parameters for compounds
4-6.

Docking
Scored
PDE4B2 IC50 (μM)a
LEb
LLEc
Entry
R1
R2
Zardaverine
-
-
1.41
4.17
3.53
>30
0.42
0.41
0.37
-
4.86
4.32
3.36
-
-24.5415
-29.6783
-24.9914
-
4
5
6
OCH3
OCH3
H
CH2
gem-CH3
CH2
a
Results are mean of at least two experiments, assay variability within 25% of reported value... b Ligand
c
efficiency (LE) was calculated as 1,37*-log10(PDE4B2 IC50)/Heavy Atom Count (HAC). Ligand
lipophilicity efficiency (LLE) was calculated as -log10(PDE4B2 IC50)- clogP. clogP was calculated with
d
ChemDraw®. Docking scores were calculated with FlexX as implemented in LeadIT® (Supporting
Material).
The alkylation of the diazepinone ring (Scheme 3) was performed in a parallel format
with a 24 position Mettler-Toledo Miniblock® apparatus, by using the relevant alkyl
bromide or iodide and a 60% dispersion of sodium hydride in mineral oil at room tem-
perature.43This set-up allowed the production of fifteen final compounds in adequate
quantity (at least 2 mg) and purity (liquid chromatography-mass spectrometry (LC-MS
>85%)) for further biochemical profiling (Table 2 and 3).
Scheme 3. Synthesis of final compounds 14-28.

Reagents and conditions: (a) R3X, NaH 60% in mineral oil, DMF, rt, 2 h.
Table 2. PDE4B2 inhibition potency and efficiency for compounds 14-25.
PDE4B2 IC50 (μM)a
LEb
0.42
0.41
0.43
LLEc
4.86
4.32
4.37
Entry
R3
-
Zardaverine
1.41
4.17
0.26
4
H
14
15
0.34
0.40
3.86
16
17
0.32
1.13
0.39
0.39
3.80
4.05
2
18
7.55
0.32
4.75
1.62
2.27
0.32
0.34
3.05
3.76
19
20

21
22
1.22
0.17
0.37
0.36
5.01
3.23
23
1.29
0.28
4.15
24
25
1.41
0.29
0.32
0.36
3.95
4.64
a
Results are mean of at least two experiments, assay variability within 25% of reported value. b.Ligand
c
efficiency (LE) was calculated as 1,37*-log10(PDE4B2 IC50)/Heavy Atom Count (HAC). Ligand
lipophilicity efficiency (LLE) was calculated as -log10(PDE4B2 IC50)- clogP. clogP was calculated with
ChemDraw®.
Table 3. PDE4B2 inhibition potency and efficiency for compounds 26-28
PDE4B2 IC50 (μM)a
LEb
0.42
0.37
0.37
LLEc
4.86
3.36
5.01
Entry
R1
-
R2
-
Zardaverine
1.41
3.53
1.22
13
19
gem-CH3
CH2
H
22
CH2
0.17
0.36
3.23

26
gem-CH3
gem-CH3
gem-CH3
13.08
2.69
1.22
0.28
0.32
0.29
2.64
3.63
1.35
27
28
a.Results are mean of at least two experiments, assay variability within 25% of reported value. b.Ligand
c
efficiency (LE) was calculated as 1,37*-log10(PDE4B2 IC50)/Heavy Atom Count (HAC). Ligand
lipophilicity efficiency (LLE) was calculated as -log10(PDE4B2 IC50)- clogP. clogP was calculated with
ChemDraw®.
Small apolar groups (compounds 14- 17) appeared to be tolerated overall, with n-propyl
affording the best inhibitory profile (IC50: 0.26 μM) when normalized by size and
lipophilicity (cf. LE and LLE, Table 2). Interestingly, branched and cyclized analogues
(15, 16) did not increase potency over the linear counterparts. The introduction of
polarity on the N1 side chain, as from amine (18) and ether (19) linkages or amide (20)
and alcohol (21) functionalities did not have the sought effect on potency, probably due
to the lack of rewarding interactions with the PDE4 enzyme.
Incorporation of aryl moieties at N1 via a methylene bridge was more successful from a
PDE4 inhibition perspective. Here, 22 and 25 afforded two of the most potent PDE4
inhibitors of this study (IC50: 0.17 and 0.29 μM, respectively). Still, none of the aromatic
appendages evaluated were as efficient as the smaller and less lipophilic alkylic
substituents (cf. 14 and 15, Table 2). Interestingly, the 3-pyridylmethyl substituent
increased potency ten-fold compared to its “para” regioisomer (cf. 24 and 25), thus
hinting at potential directionality effects of substitution for heterocyclic derivatives.

Finally matched pair analysis indicated a general deterioration of PDE4 inhibition for
the 5,5‐dimethyl‐4,5,6,7‐tetrahydro‐1H‐1,2‐diazepin‐7‐one scaffold (Table 3), confirm-
ing that the insertion of steric bulk at 5 position did not provide any advantage.
The newly designed compounds were able to inhibit the enzymatic activity of PDE4. In
particular, 14 offered a 10-fold potency gain and comparable ligand efficiency over
Zardaverine. We therefore evaluated the ability of the best compounds so far identified
to inhibit the Lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α)
release in human peripheral blood mononuclear cells (PBMC), a relevant PDE4-based
biomarker of functional utility in a clinical setting.44Gratifyingly, both 14 and 15
showed significant TNF-α release inhibition (IC50: 0.21 and 0.11 µM, respectively, Ta-
ble 4), with virtually no drop-off effects from their cell-free IC50 values, indicating op-
portunities for further optimization.
Table 4. Inhibition of LPS-induced TNF-αrelease in human PBMCs for Zardaverine
and compounds 14 and 15
PBMC IC50 (μM)a
LEb
LLEc
Comp.
Zardaverine
0.515 (0.098-2.700)
0.214 (0.072-0.635)
0.106 (0.052-0.215)
0.45
0.44
0.43
5.30
4.46
4.36
14
15
a.Results are mean of n=1 independent experiment performed in quadruplicate. 95% confidence intervals
of the mean are displayed within brackets. b.Ligand efficiency (LE) was calculated as 1,37*-log10(PBMC
IC50)/Heavy Atom Count (HAC). c Ligand lipophilicity efficiency (LLE) was calculated as -log10(PBMC
IC50)- clogP. clogP was calculated with ChemDraw®.

In summary, starting from chemoinformatics analysis of the PDE4 inhibitor prior art, a
novel heterocyclic scaffold was designed based on structure-based considerations, di-
versification opportunities and developability potential. By focussing on the exploration
of underrepresented chemical space, a parallel synthetic chemistry protocol was devel-
oped using robust and scalable transformations. This resulted in the identification of a
series of novel, potent PDE4 inhibitors with promising cellular activity. This prelimi-
nary proof of concept coupled with the compact nature of the chemotype formed the
basis for its future development and profile optimization.

Author Contributions
The manuscript was written through contributions of all authors.
Notes
The authors declare no competing financial interest.
Acknowledgements
We thank E. Armani for her support in the biological profiling of the compounds.

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