4-Amino-7,8-dihydro-1,6-naphthyridin-5(6 H)-ones as Inhaled Phosphodiesterase Type 4 (PDE4) Inhibitors: Structural Biology and Structure-Activity Relationships

Article abstract of DOI:10.1021/acs.jmedchem.7b01751

Rational design of a novel template of naphthyridinones rapidly led to PDE4 inhibitors with subnanomolar enzymatic potencies. X-ray crystallography confirmed the binding mode of this novel template. We achieved compounds with double-digit picomolar enzymatic potencies through further structure-based design by targeting both the PDE4 enzyme metal-binding pocket and occupying the solvent-filled pocket. A strategy for lung retention and long duration of action based on low aqueous solubility was followed. In vivo efficacies were measured in a rat lung neutrophilia model by suspension microspray and dry powder administration. Suspension microspray of potent compounds showed in vivo efficacy with a clear dose-response. Despite sustained lung levels, dry powder administration performed much less well and without proper dose-response, highlighting clear differences between the two formulations. This indicates a deficiency in the low aqueous solubility strategy for long duration lung efficacy.

Full text of DOI:10.1021/acs.jmedchem.7b01751

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4-Amino-7,8-dihydro-1,6-naphthyridin-5(6H)-ones as
inhaled phosphodiesterase type 4 (PDE4) inhibitors:
structural biology and structure-activity relationships (SAR)
Richard S. Roberts, Sara Sevilla, Manel Ferrer, Joan Taltavull, Begoña Hernández, Victor
Segarra, Jordi Gracia, Martin D. Lehner, Amadeu Gavaldà, Míriam Andrés, Judit Cabedo,
Dolors Vilella, Peter Eichhorn, Elena Calama, Carla Carcasona, and Montserrat Miralpeix
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01751 • Publication Date (Web): 05 Mar 2018
Downloaded from http://pubs.acs.org on March 5, 2018
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ꢀAminoꢀ7,8ꢀdihydroꢀ1,6ꢀnaphthyridinꢀ5(6H)ꢀones
as inhaled phosphodiesterase type 4 (PDE4)
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inhibitors: structural biology and structureꢀactivity
relationships (SAR)
,
#
§
§
§
§
Richard S. Roberts,* Sara Sevilla, Manel Ferrer, Joan Taltavull, Begoña Hernández, Victor
#
#
≠
#
∂
Segarra, Jordi Gràcia, Martin D. Lehner, Amadeu Gavaldà, Miriam Andrés, Judit
#
§
∏
∂
∂
Cabedo, Dolors Vilella, Peter Eichhorn, Elena Calama, Carla Carcasona, Montserrat
§
Miralpeix
#
Almirall S.A., Centro de Investigación y Desarrollo, Medicinal Chemistry & Screening,
Crta. Laureà Miró 408ꢀ410, Sant Feliu de Llobregat, 08980 Barcelona, Spain.
∏
Almirall S.A., Centro de Investigación y Desarrollo, Pharmacokinetics & Metabolism,
Crta. Laureà Miró 408ꢀ410, Sant Feliu de Llobregat, 08980 Barcelona, Spain.
∂
Almirall S.A., Centro de Investigación y Desarrollo, Experimental Dermatology, Crta.
Laureà Miró 408ꢀ410, Sant Feliu de Llobregat, 08980 Barcelona, Spain.
≠
Bionorica SE, Kerschensteinerstraße 11ꢀ15, 92318 Neumarkt, Germany.
§
Former employee of Almirall S.A.
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ABSTRACT
Rational design of a novel template of naphthyridinones rapidly led to PDE4 inhibitors with
subꢀnanomolar enzymatic potencies. Xꢀray crystallography confirmed the binding mode of this
novel template. We achieved compounds with doubleꢀdigit picomolar enzymatic potencies
through further structureꢀbased design by targeting both the PDE4 enzyme metalꢀbinding pocket
and occupying the solventꢀfilled pocket. A strategy for lung retention and long duration of action
based on low aqueous solubility was followed. In vivo efficacies were measured in a rat lung
neutrophilia model by suspension microspray and dry powder administration. Suspension
microspray of potent compounds showed in vivo efficacy with a clear doseꢀresponse. Despite
sustained lung levels, dry powder administration performed much less well and without proper
doseꢀresponse, highlighting clear differences between the two formulations. This indicates a
deficiency in the low aqueous solubility strategy for long duration lung efficacy.
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INTRODUCTION
Cyclic nucleotide adenosine monophosphate (cAMP) is a ubiquitous secondary messenger
involved in a range of cellular responses to biological agents brought about through activation of
adenylyl cyclase. Intracellular levels of cAMP are tightly controlled by a family of cyclic
nucleotide degrading enzymes, the phosphodiesterases (PDEs). The PDEs are therefore
1
,2,3,4
implicated in the control of many biological processes.
We were particularly interested in
the inflammatory responses modulated by PDE4, especially with respect to chronic obstructive
pulmonary disease (COPD). Research into PDE4 inhibitors is decades old, and the recent
successful launches of the inhibitors roflumilast (Daliresp 1) for the oral treatment of COPD and
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apremilast (Otezla 2) for the oral treatment of psoriasis have been longꢀawaited in the field
(Figure 1). PDE4 is therefore still an active target for research and several reviews on the
5
,6,7,8,9
development of PDE4 inhibitor clinical candidates are available.
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Cl
SO Me
2
N
O
O
O
NH
OEt
N
H
N
O
Cl
OCHF2
OMe
O
1
roflumilast
oral for COPD
PDE4B1 IC50 0.7 nM
2 apremilast
oral for plaque psoriasis
PDE4B2 IC50 30 nM
1
0
Figure 1. Structures and affinities of approved oral drugs roflumilast 1 and apremilast 2.
Despite these successes, cardiac and emetic side effects are prevalent. PDE4 has four isotypes,
1
1
PDE4AꢀD. PDE4B is linked to the antiꢀinflammatory effects, while the side effects are
12,13
attributed to PDE4D.
domains of PDE4AꢀD.
Xꢀray crystallography has facilitated the understanding of the catalytic
1
4,15,16
It has also revealed that the four isoforms show little difference in
1
7,18
their active sites, and as a result few isoformꢀselective compounds exist.
Therefore, to
minimize the potential systemic side effects of PDE4, and with special relevant to respiratory
1
9
diseases, we and many others have turned to inhaled delivery to the lung.
The molecular design of compounds for inhaled delivery has advanced significantly over the
2
0
last few years. The lung is a highly perfused organ and so highly soluble and permeable
compounds ꢀ properties which desirable for oral administration ꢀ are readily absorbed
systemically and therefore of little use as compounds to target the lungs. Inhaled compounds
need to stay localized in the lungs and respiratory tract, and so strategies of reduced aqueous
2
1,22,23
solubility and/or increased tissue binding are common.
Reducing systemic exposure
through high clearance can help improve the safety profile of compounds: “softꢀdrugs”
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incorporate metabolicallyꢀlabile groups, susceptible to either plasma hydrolysis or hepatic
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oxidative phase I or conjugative phase II metabolism. A further approach is to incorporate slow
binding kinetics and to dissociate the pharmacodynamics (PD) effect from the pharmacokinetic
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(PK) profile.
Finally, since the dose of compound that can be administered to the lung is
28
limited, often to a milligram or less in humans, compounds should be as potent as possible.
Molecular properties addressing these issues can, and probably should therefore, deviate from
properties that are desirable for oral compounds. Since PDE4 is an intracellular target, molecular
properties should still be controlled to some degree as cellular permeability is necessary for
efficacy and wholeꢀcell assays should be employed to detect any such deficiencies in permeation
and target access.
RESULTS AND DISCUSSION
Design of the inhibitors. Here we describe our efforts to discover novel, highly potent (subꢀ
nanomolar), inhaled inhibitors of PDE4 for the treatment of COPD. We were drawn to the series
of quinolines from GSK, which showed extremely high potency and had physicochemical
properties aligned with inhaled delivery. Quinoline 3 (GSK256066) reached phase II clinical
2
9
trials for the inhaled treatment of respiratory disorders. In addition, the crystal structure of 3
30
bound to the catalytic site of PDE4B2B has been published (pdb code: 3gwt). From our own
extensive oral PDE4 program we also had obtained crystal structure data for a series of
3
1
pyridazinone compounds such as 4 (bound to PDE4D, pdb code: 5k1i, Figure 2).
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CO H
2
OMe
O
HN
O
N
N
O
NH O
N
O O
S
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N
NH2
GSK256066 3
PDE4 IC50 0.003 nM
PYR 4
PDE4 IC50 10 nM
Figure 2. Overlay of the Xꢀray structures of 4 (colored by element, pdb code: 5kli) and inhaled
clinical candidates GSK256066 3 (in blue, pdb code: 3gwt). Only glutamineꢀ369 and the two
metal ions (magnesium in green, zinc in grey) of the PDE4D enzyme are shown.
So, once we overlaid the crystal structure of 4 with that published for the quinoline 3 we
could design a new chemical family, the naphthyridinones (NAPs) 5. We envisaged rapid entry
to a new series, taking advantage of the precedent for inhaled delivery of 3 and the known SAR
from both families but in an unexploited chemical space with fast synthetic access to compounds
with a wide spread of physicochemical properties as desired (Figure 3).
[
6] solvent-
[
4] large
exposed
pocket
N
lipophilic
pocket
R
O
[
1] pi-clamp
Gln-369
[
5] metal binding zone
solvation spheres
O S
O
6
2
HO C
Ac
1
1
H
2
N
O
2
3
4
5
N
N
N
[2] glutamine
switch
N
2
R
4
8
N
H
3
N
Mg
Zn
MeO
N
H
O
7
H
O
5
N
[3] small lipophilic
pocket
6
O
NH2
H
3
4
5
GSK256066
Pyridazinone
Naphthyridinone
Asn-321
Figure 3. Conceptual design and interactions of the naphthyridinone (NAP) series. Active site
regions are defined as follows with respect to the cartoon view above: [1] The piꢀclamp region
32
(
between Pheꢀ372 and Pheꢀ340). [2] The “glutamine switch” ꢀ glutamineꢀ369. [3] The small
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lipophilic pocket. [4] The large lipophilic pocket [5]. The metal binding zone containing the zinc
and magnesium ions responsible for the catalytic activity. [6] The solvent exposed pocket ꢀ in the
full PDE4 isoenzyme, the helices of the upstream conserved region (UCR2), a negative regulator
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of cAMP hydrolysis, partially fill this area. In truncated protein sequences, coding only for the
PDE4 active site, this zone is exposed to the bulk solvent, hence the name.
In our design, the arylꢀaminoꢀheteroaryl motif (Figure 3, in green) was conserved across the
three families. The rigid core of the NAPs would slot into the piꢀclamp region [1] with the 4ꢀ
aminopyridine motif acting as a powerful Hꢀbond acceptor for the glutamine switch [2]. The
lactam portion of the NAP would both fill the small lipophilic pocket [3] and be a Hꢀbond donor
to interact with Asnꢀ321 in the floor of the active site, as for the quinoline 3. We could also take
advantage of our own internal SAR of the pyridazinone series: firstly for the metal binding zone
[5] and secondly for the substitution in the NAP 2ꢀposition, filling the large lipophilic pocket [4]
and allowing access to the solventꢀfilled pocket [6]
Syntheis of the Inhibitors. The synthesis of the NAPs has been described previously, as
3
4
outlined in Scheme 1. Briefly, betaꢀamino acids 6 were condensed with Meldrum’s acid and the
resulting tricarbonyl compounds 7 were thermally cyclized to give Bocꢀprotected
piperidinediones 8. Condensation with carbon disulfide and trapping with methyl iodide gave the
ketene dithioacetals 9. The key sequence of the synthesis was the Michael addition of an
acetophenone enolate (9 → 10) and subsequent condensationꢀcyclisation with ammonia to form
the core of the NAP 11 and with a functionalized handle to introduce the 4ꢀamino substituent.
Yields were at best 50% in this sequence 9 → 11: the two methylthio groups of 9 were of equal
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reactivity, but only displacement of the methylthiol group cis to the piperidinedione ketone
carbonyl (9 → 10) led to correct cyclization and pyridine formation. Nevertheless, the route was
sufficiently robust to allow large quantities of sulfides 11 to be synthesized. The sulfides 11 were
oxidized to the sulfones 12 to increase the reactivity of the leaving group. Finalization of the
synthesis to the desired NAPs 5 could be carried out in one of two ways. In the first case, the
sulfones were thermally displaced directly with the appropriate amine or aniline. When the
displacement was carried out in refluxing ethanol, the Boc protecting group remained largely
intact, so this was removed under acidic conditions with a final treatment with trifluoroacetic
acid (TFA) (Scheme 1, f). Alternatively, the Boc group could be removed thermally, under
microwave irradiation at 160 ºC (Scheme 1, g). Despite liberating carbon dioxide under sealed
tube conditions, the increase in pressure was well within the tolerability of the microwave
apparatus. For poorly reactive amines, we moved directly to microwave irradiation to carry out
the amide displacement and Boc deprotection in one step (Scheme 1, h). In the second route, Boc
deprotection of sulfone 12 and displacement of the sulfone with ammonia gave versatile
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intermediate 13, which allowed introduction of a range of R ꢀsubstituents via palladiumꢀ
catalyzed Nꢀarylations (Scheme 1, iꢀk). Despite the moderateꢀtoꢀlow yields of the key cyclisation
sequence 9 → 11, the modular synthesis allowed us rapid access to a range of substituents to
systematically probe the SAR of each position in turn.
Scheme 1. General Synthetic Route to Naphthyridinones 5
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a
Reagents and Conditions: (a) DCC, DMAP, DCM, 5 ºC, 94% yield; (b) EtOAc, reflux, 83%
1
yield; (c) CS , K CO , DMF, rt then MeI, DMF, 70% yield; (d) R COMe, tBuOK, DMSO, THF,
2
2
3
rt, 3 h, then NH OAc, AcOH, 90 ºC, 6ꢀ55% yield; (e) mCPBA, DCM, 5 ºC, 77ꢀ100% yield; (f)
4
2
2
R NH , EtOH, reflux, then TFA, DCM, rt if necessary, 20ꢀ62% yield; (g) R NH , EtOH, reflux,
2
2
2
then 160 ºC (microwave), 0.5 h, 14ꢀ51% yield; (h) R NH , EtOH, 160 ºC (microwave), 0.5ꢀ18 h,
2
7
ꢀ83% yield; (i) TFA, DCM, rt; (j) NH (7N in MeOH), 140 ºC (microwave), 67ꢀ97% yield over
3
2
two steps; (k) R Hal, Cs CO , Pd (dba)3, xantphos, dioxane, 90 ºC, 28ꢀ41% yield.
2
3
2
1
A biaryl substitution pattern in the NAP R position was of particular interest. These
compounds 17 were synthesized according to Scheme 2. 3ꢀBromoꢀNAPs 14 were simply
extended via Suzuki reaction to give biphenyls 17. Thioethers were also accessible from the
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same intermediate 14. Alternatively, intermediate 15 was first coupled via Suzuki reaction and
the resulting amines 16 were Nꢀarylated as before.
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Scheme 2. General Synthetic Route to R ꢀaryl Substituted Naphthyridinones 17
a
3
Reagents and Conditions: (a) R Hal, Cs CO , PdCl (dppf)·CH Cl , dioxane, 90 ºC, 4ꢀ80%
2
3
2
2
2
yield from 14, 41ꢀ95% yield from 15 (b) thiol, Pd(PPh ) , Bu SnOMe, xylene, reflux, 64% yield;
3
4
3
2
(
c) R Hal, Cs O , Pd (dba)3, xantphos, dioxane, 90 ºC, 9ꢀ64% yield.
1k 3 2
Deepꢀreaching NAPs, to study the binding interactions with the floor of the active site, were
synthesized as shown in Scheme 3. NAP compounds 5 were converted to their corresponding
thiolactams 18 with phosphorous pentasulfide. SꢀAlkylation with methyl iodide (19) and
displacement with hydroxylamine or cyanamide gave access to the desired amidoximes and
cyanoamidates 20 respectively.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 3. General Synthetic Route to CarbonylꢀVariant Naphthyridinones 20
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and Conditions: (a) P S , iPrNEt , dioxane, 90 ºC, 18 h, 58ꢀ99% yield; (b) NaH,
4
10
2
MeI, DMF, 60 ºC, 3 h, 81ꢀ82% yield; (c) NH OH·HCl, K CO , EtOH, 70 ºC, 18 h, 23% yield;
2
2
3
(
d) NH CN, EtOH or THF, 70 ºC, 1ꢀ2 h, 26ꢀ51% yield.
2
Xꢀray binding mode of Naphthyridinones. Following the synthetic routes outlined above,
the first simple NAP 21 was synthesized and was highly potent with an IC of 1.6 nM against
50
35
PDE4B1, corresponding to a high ligand efficiency (LE) of 0.46.
The proposed binding mode of NAP 21, as outlined in Figure 3, was confirmed by Xꢀray
crystallography (pdb code: 5k32, Figure 4). In addition to the expected interactions, the 3ꢀ
methoxyaniline substituent was held in place by an intramolecular hydrogen bond between the
aniline NH and the lactam carbonyl. The NAP lactam NꢀH formed a hydrogen bond directly to
the side chain of Asnꢀ321 (at 2.7 Å) in the floor of the binding pocket. The lactam also took part
in a hydrogenꢀbonding network with the floor of the binding pocket via a water molecule at 2.5
Å from the lactam carbonyl. In the metalꢀbinding zone, the 3ꢀmethoxy group hydrogen bonded
(at 3.1 Å) with one of the water molecules in the outer coordination shell of the magnesium ion.
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
MeO
N
H
O
N
H
2
1
PDE4B IC50 1.6 nM
LE 0.46
Figure 4. Xꢀray structure of first synthesized NAP 21 bound to the active site of PDE4D
(magnesium, green sphere. zinc, grey. Pdb code: 5k32)
PDE4 inhibitory activity and StructureꢀActivity Relationships. With the molecular design
of the NAPs validated, we expanded the SAR in both the metal binding zone and the large
lipophilic / solventꢀexposed pocket (Tables 1ꢀ7). Compounds were typically tested in two assays.
In the first assay, we measured enzyme binding potency through the inhibition of radiolabelled
3
[
H]ꢀcAMP hydrolysis from the human PDE4B1 construct. As PDE4 is an intracellular target,
for many compounds we also measured functional inhibition in the production of tumour
necrosis factor alpha (TNFα) from peripheral blood mononucleated cell (PBMCs).
1
We first varied the monocyclic ring in the R position in the large lipophilic pocket with simple
substitutions available from acetophenone building blocks (Table 1). In general, little difference
was observed in the potency but with two exceptions: the 2ꢀmethoxy group (24) lost over 10ꢀfold
potency, undoubtedly due to a greater twist in the biaryl bond. Pyridyl (25) was wellꢀtolerated,
however the Nꢀoxide 26 (a synthetic byꢀproduct formed during the sulfide to sulfone oxidation
step) was not. Clearly, the large lipophilic pocket craves lipophilic groups. Cyclopropyl (27) was
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1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
also tolerated but other aliphatic R groups were not followed up. Since the R group was
introduced from a methyl ketone (see Scheme 1, step (d)), enolization of aliphatic methyl
ketones would occur in both directions and mixtures of two regioisomers would occur. Both
compounds 21 and 25 displayed similar potencies in the enzymatic assay and the functional
cellular assay, indicative of an effective cell permeability. The permeability of compound 21 was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ
6
measured at 2×10 cm/s by parallel artificial membrane permeability assay (PAMPA).
1
Table 1. R Ring Substituent SAR in the Large Lipophilic Pocket
PDE4B
TNFα
1
No
1
R
IC (nM) IC (nM)
5
0
50
roflumilast
0.70
1.6
4.0
2
2
2
2
2
1
2
3
4
5
5.4
n.d.
n.d.
n.d.
6.5
8.2
8.4
32
3.6
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2
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7
51
n.d.
n.d.
8.5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
n.d. not determined. All experimental data n≥2. pIC SD ± <0.2. Data shown to two significant
5
0
figures.
1
We also extended the R monocycle to a series of biphenyl groups (Table 2). Here the
intention was often twoꢀfold – access the solvent filled pocket with hydrophilic groups and use
these groups as metabolic handles for glucuronidation. We derived the metaꢀsubstitution pattern
3
1
from known SAR of our internal PYR series. Biarylꢀsubstituted NAP 28 had a potency of 0.35
nM, further validating our design hypothesis and confirming a close analogy between the PYR
and the NAP series.
Biaryl substituted compound, acid 28 was subꢀnanomolar in the enzymatic assay. However,
this compound showed a 16ꢀfold loss of potency in the cellular assay. Compound 28 showed an
ꢀ6
acceptable permeability of 3.5×10 cm/s as measured by the PAMPA assay. Another acid
compound, 30 also displayed this loss of potency between the enzymatic and cellular assays.
Other factors could be responsible for the loss of activity, for example active transport, not
contemplated in the PAMPA assay. This was not investigated further, but we took the shift in
potencies as a warning not to proceed with acidic compounds. This shift could be avoided by
using the esters 29 and 31 respectively. However, to avoid the complications associated with
pharmacologically active prodrugs, we dropped this line of research. A series of amides also
gave potent compounds. Amide 34 demonstrated that large groups could be introduced while
maintaining enzymatic potency, and could be applicable to the modulation of physical chemical
properties at a later date. Cyclopropylamide 33 was particularly effective and showed subꢀ
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
nanomolar cellular potency. Phenol (35) and thioether (36) could also be introduced but with no
impact on potency compared to 28.
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. R Biaryl Substituent SAR Towards the Solvent Exposed Pocket
PDE4B
TNFα
3
No
R
IC (nM) IC (nM)
5
0
50
2
2
1
8
H
1.6
5.4
0.35
7.2
5.7
1.2
140
2
3
9
0
9.6
31
5.6
0.28
2.1
4.2
2.3
3
2
33
0.43
2.8
3
4
0.23
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35
36
1.8
1.6
4.2
3.4
All experimental data n≥2. pIC SD ± <0.2. Data shown to two significant figures.
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
The Xꢀray structure of NAP 21 showed the 3ꢀmethoxyphenyl R substituent was directed
towards the metal binding zone of PDE4, making a hydrogen bond to one of the water molecules
in the outer coordination sphere of the magnesium ion. A similar interaction was observed from
the benzoic acid group of our pyridazinone 4 (pdb: 5k1i). Many enzymes, particularly the
hydrolase family, possess metal ions in their active sites. Metal binding groups, such as thiols,
hydroxamic acids or othe bidentate ligands are often used to obtain high affinity inhibitors.
However, the common mechanism of metal chelation of these compounds can also lead to a lack
of selectivity across this family of enzymes. Through our NAP ligand design, we were not
especially troubled by this possibility: firstly, the metalꢀbinding group was designed to target
either the outer or inner hydration sphere of the metals, not the metal atoms themselves.
Secondly, a large proportion of the binding affinity of our ligands came from the NAP core
structure (see zones [1]ꢀ[4], Figure 3). Nonꢀselective interactions with other metalꢀcontaining
enzymes not possessing this specific recognition pattern would therefore be much less potent.
With plenty of opportunity to vary the strength and direction of the hydrogen bonds in that
zone, we synthesized a series of analogues. The SAR was reasonably flat (Table 3), with no
significant increase of affinity over 21. An aromatic ring was at least necessary, as demonstrated
by the poor activity of truncated amine 37. Interestingly, despite the drop in potency this
compound still maintained a ligand efficiency (LE) of 0.43, little changed from the LE of 0.46
for 21. Compound 37 could therefore be considered as an efficient fragment for PDE4 and
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
validation that the NAP core provided much of the binding energy of the whole compound. For
the remainder of the changes, small substituents 38ꢀ39, alcohols 40ꢀ42, acid 43, amides 44ꢀ45
and pyridines 46ꢀ48 in general showed potencies within an order of magnitude of the first NAP
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
1.
2
Table 3. R Monocyclic Amine SAR in the Metal Binding Zone
PDE4B
TNFα
2
No
R
IC (nM) IC (nM)
5
0
50
2
1
1.6
5.4
37
H
2400
3.3
n.d.
13
3
3
8
9
27
n.d.
40
24
12
n.d.
n.d.
0.49
4
4
1
2
8.7
4
3
2.3
9.9
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4
4
1.5
4.2
45
12
42
25
12
n.d.
n.d.
n.d.
n.d.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
4
6
7
48
n.d. not determined. All experimental data n≥2. pIC SD ± <0.2. Data shown to two significant
50
figures.
We also synthesized bicyclic metal binding groups (Table 4). Plain naphthyl (49) lost
potency, showing that at least some kind of polar substituent was beneficial. The series of
quinoline analogues 50ꢀ52 showed that the location of this polarity did have some impact.
Quinoline 50 was the first compound with subꢀnanomolar potency both the enzymatic and the
cellular assay. Pyrazolopyridines 53ꢀ55, designed to maintain the same interaction as the
quinoline 50, lost potency. Finally, quinoline Nꢀoxide 56 provided a final small increase of
potency in both enzymatic and cellular assays. Ligand efficiency of 56 was still high at 0.44.
2
Table 4. R Bicyclic Amine SAR in the Metal Binding Zone
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
No
R
IC (nM) IC (nM)
50
50
2
4
1
9
1.6
5.4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
11
n.d.
50
0.96
0.92
5
5
1
2
14
2.3
5.6
n.d.
4.8
3.0
N
53
5
5
5
4
5
6
11
56
n.d.
n.d.
0.47
0.43
n.d. not determined. All experimental data n≥2. pIC SD ± <0.2. Data shown to two significant
5
0
figures.
Two other areas were briefly explored: the 3ꢀposition of the NAP core and the small lipophilic
pocket (Table 5). Pyrimidine analogue 57 was synthesized in a variation of Scheme 1, replacing
the acetophenone enolate of step (d) with phenylamidine. Nitrogen at the 3ꢀposition of the NAP
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core lost potency, possibly because of the weaker hydrogen bonding potential of a 4ꢀ
aminopyrimidine versus a 4ꢀaminopyridine. Similar structures have been reported with potencies
3
6
also generally in the doubleꢀdigit nM range. In the small lipophilic pocket, the role of the C7ꢀ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C8 ethylene unit of the NAP core was identified. 7ꢀMethyl (58) and 8ꢀmethyl (59) analogues of
2
1 were synthesized from the appropriate betaꢀamino acids according to Scheme 1 (in 4% and
1
5% overall yield respectively). Methyl substitution of these positions resulted in a 70ꢀ and 400ꢀ
fold drop in potency respectively, indicating a close fit to the small lipophilic pocket in the initial
design. The lipophilic nature of the ethylene unit to fill the small lipophilic pocket was also
evident: open amide structures 60 and 61 lost between 30 and 150ꢀfold potency with respect to
3
7
their NAP counterparts (60 ↔ 21 and 61 ↔ 50 respectively). The methyl amide 62 essentially
lost all potency ꢀ the favored conformation of the amide would direct the methyl group into the
floor of the binding site, thus dislodging the rest of the structure from its ideal fitting position.
Hence, the initial NAP core design was optimal.
Table 5. Core Variations
PDE4B
TNFα
No
IC (nM)
IC (nM)
5
0
50
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
5
1
7
1.6
52
5.4
40
58
120
640
240
35
270
n.d.
n.d.
n.d.
n.d.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
6
9
0
61
6
2
5800
n.d. not determined. All experimental data n≥2. pIC SD ± <0.2. Data shown to two significant
50
figures.
We also extended the NAP lactam group down to the floor of the PDE4 binding site.
Displacement of energetically unfavoured water molecules from binding sites can add significant
binging affinity, although our approach was done empirically, without applying any of the
38
sophisticated methods for analyzing water molecules. The Xꢀray crystal structure of NAP 21
showed just a single water molecule between the NAP and the wall of the large lipophilic region,
indicating an already close fit (Figure 4). However this water molecule forms a stable Hꢀbonded
network between an NH of Glnꢀ369 and the backbone carbonyl of Valꢀ365. We instead targeted
the water molecule located at 2.5 Å from the NAP carbonyl (Figure 4). Our approach was to
extend the structure down from the NAP carbonyl to leave a polar atom where the water
molecule should be.
Some example lactamꢀcontaining NAPs were converted to their extended analogues following
the steps of Scheme 3 (24 → 63 and 64, 65 → 66, respectively). NAP Hydroxyamidine 63 (NꢀO
bond length typically 1.3 Å) was, as expected, a bond too short, and whether by a steric clash
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with the trapped water molecule, or by leaving a highꢀenergy gap between its hydroxyl group
and Asnꢀ321, lost three orders of magnitude of potency with respect to the parent NAP 24. The
NAP cyanoamidines 64 and 66 were much more promising (NꢀC≡N typically 2.5 Å long). While
they effectively maintained the enzymatic potency of their NAP lactam analogues, 66 showed
some cellular dropꢀoff, so these compounds were not explored further (Table 6).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 6. CarbonylꢀVariant NAPs to Displace a Deep Water Molecule
R1
R1
N
N
Scheme 3
R2
R2
N
N
H
H
H O
N
X
N
H
O
N
H
2
º
2.5 A
H O
2
24
63, 64
6
5
66
PDE4B
TNFα
1
2
No
R
R
X
IC (nM)
IC (nM)
50
50
2
6
6
4
3
4
-
8.3
2000
13
n.d.
n.d.
n.d.
OH
CN
6
6
5
6
-
0.75
0.89
1.5
17
CN
n.d. not determined. All experimental data n≥2. pIC SD ± <0.2. Data shown to two significant
5
0
figures.
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1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
3
Finally, some of the best groups of the R and R substituents were combined and the SAR was
suitably additive (Table 7). Many of the compounds showed subꢀnanomolar potencies in both
enzymatic and cellular assays. Of particular interest, quinoline 72 and its Nꢀoxide 73 combined
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
the best R and R substituents from Tables 2 and 4. Quinoline 72 was 170 pM in enzymatic and
1
4
20 pM in the cellular assay. NꢀOxide 73 was the most potent compound of the series with IC50
5 pM in the enzymatic and 75 pM in the cellular assay. This in vitro activity is comparable to
26
2
3
the best compounds of our previous PYR series, from which the selection of the R and R
groups of the NAPs were derived.
2
3
Table 7. SAR with Combinations of R and R .
PDE4B
TNFα
2
3
No
21
R
R
IC (nM) IC (nM)
5
0
50
MeO
1.6
5.4
6
6
7
8
0.89
0.11
0.50
0.52
6
9
1.2
0.66
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70
71
0.27
0.32
0.17
0.045
0.11
0.46
1.1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
7
7
2
3
4
0.12
0.075
0.36
All experimental data n≥2. pIC SD ± <0.2. Data shown to two significant figures.
5
0
Lung PK and In vivo Neutrophilia in Rats. From the series of NAPs, compound 72 was
selected for further profiling, both in vitro and in vivo, due to its excellent cellular potency in the
TNFα assay and to allow likeꢀforꢀlike comparison with other inꢀhouse compounds from the PYR
ꢀ6
series (unpublished results). Permeability of 72 was modest (PAMPA Papp 0.15 ×10 cm/s).
Following our strategy for low aqueous solubility, thermodynamic solubility for 72 was low (4
µg/mL at 37 ºC), but comparable to GSK compound 3 (19 µg/mL at 37 ºC) and higher than that
3
9
published for roflumilast (0.52 µg/mL at 22ºC). Compound 72 was selective against several
other phosphodiesterases and in a counterꢀscreen of 30 kinases (see Supporting Information
Table S2). Pharmacokinetic data is shown in Figure 5. Intravenous rat PK at 500 µg/kg showed a
moderateꢀtoꢀhigh volume of distribution (3.9 L/kg) and a desirably high systemic clearance of
around hepatic blood flow (79 mL/min/kg). Intraꢀtracheal PK in rats using dry powder at 1000
µg/kg (5% w/w with lactose) showed a prolonged exposure. The percentage of the total
administered dose present in the lungs reached a maximum of 36% and dropped to 15% over a
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1
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1
1
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2
2
2
2
2
2
3
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3
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6
2
4 h period, based on compound recovered from lung biopsies. Corresponding plasma levels
measured after intraꢀtracheal dosing were around or below the level of quantification (<5 ng/mL)
up to 1 h postꢀdose, and undetectable after that (Figure 5).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. PK profiles of compound 72 in rat. (a) i.v. dose: 500 µg/kg. (b) i.t. dose of dry powder:
1000 µg/kg, as a 5% w/w mixture with lactose. Percentages in the i.t. PK graph refer to the
percentage of the total dose (250 µg compound / animal) recovered from the lungs by methanol
extraction.
Compound 72 was tested in a rat lipopolysaccharide (LPS)ꢀinduced neutrophilia model,
measuring the inhibition of increase of neutrophils in the bronchoalveolar lavage (BAL) fluid.
Compounds were prepared in aqueous suspension and administered intraꢀtracheally by microꢀ
spray using the PennꢀCentury device. Animals were challenged 1 h later with nebulized LPS,
with the neutrophilia readout 4 h later, i.e. a total of 5 h after test compound administration.
Roflumilast 1 was used as a control, showing a clear doseꢀresponse and reaching 80% inhibition
at 1000 µg/kg. Compound 72 also showed a clear doseꢀresponse, however it only reached an
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inhibition of 58% at 1000 µg/kg (Figure 6a). Other compounds were also tested at single doses
but showed lower efficacies than 72 (see Supporting information Table S3).
Clinical delivery of inhaled compounds is almost always carried out using a dry powder
formulation of active ingredient mixed with a lactose carrier. We also measured inhibition of
lung neutrophilia after dry powder i.t. administration of compound 72 with roflumilast 1 as
control (Figure 6b). Both compounds were micronized before coꢀmixing with lactose to enhance
their kinetic solubility and lung deposition. Compounds were administered with a dry powder
insufflator. Animals were again challenged with nebulized LPS 1 h or 18 h later, with the
neutrophilia readout 4 h later, i.e. a total of 5 h or 22 h after test compound administration.
Roflumilast again performed well 5 h after dosing, with the inhibition dropping off 22 h post
administration. Compound 72 showed much more erratic results: a moderate but sustained
efficacy at 100 µg/kg, moderate efficacy but short duration at 1000 µg/kg and a slight
improvement of efficacy at 3000 µg/kg at the only tested time of 22 h postꢀdose. Despite
extended lung levels of compound 72, a maximum of 35% inhibition was seen across this range
of doses. The absence of clear doseꢀresponses, either for different doses or at different timeꢀ
points shows that other factors are impeding the efficacy of 72 in the dry powder formulation.
1
1
1
1
1
1
1
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1
2
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2
2
2
2
2
2
2
2
3
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3
3
4
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5
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5
5
5
5
6
0
1
2
3
4
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8
9
0
1
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4
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0
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2
3
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0
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9
0
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9
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1
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2
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2
2
2
2
2
2
3
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0
1
2
3
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9
0
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2
3
4
5
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9
0
1
2
3
4
5
6
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8
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0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. (a) Doseꢀresponses of roflumilast 1 and compound 72 in a LPSꢀinduced rat
neutrophilia model. Compounds were administered in suspension by intraꢀtracheal microꢀspray
delivery. (b) Timeꢀcourse inhibition of LPSꢀinduced rat neutrophilia. Roflumilast 1 and
compound 72 were given by i.t. administration of dry powder coꢀmixed with lactose. n.t.: not
tested.
The low lung neutrophilia efficacy of compound 72 after inhaled delivery is probably related
to its solubility (thermodynamic solubility 4 µg/mL at 37 ºC). Compound 72 was micronized
from its amorphous solid/crystalline state without detailed crystallization experiments, and so
should represent a more soluble solid form. The separation between lung and plasma levels after
i.t. administration is evident of dissolution rateꢀdriven PK (Figure 5b). Despite the apparently
long lung PK profile, the lung levels observed were possibly misleading in that they represent the
total amount of compound in the lung. A low solubility may well mean that most of the
compound remained unꢀdissolved in solid microparticles, or even phagocytosed by macrophages
within the lung, and with little of the total compound dissolved and available to inhibit the PDE4
enzymes present. Lung free fraction or exꢀvivo target engagement studies could have shed more
light on this situation.
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Nonꢀdissolving particles in the lung can often lead to irritation and a proꢀinflammatory
response. Compound 72 itself was not proꢀinflammatory – dry powder administration of 1000 or
3000 µg/kg to the lung did not result in an increase in neutrophils in BAL (0.7ꢀfold and 1.3ꢀfold
increase on basal levels observed respectively, n = 4). However, the sustained PK profile of
Figure 5b is not characteristic of an effective dissolution in the lung.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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0
Ultimately, in a preliminary safety assay, compound 72 also produced emesis in ferrets in a
doseꢀdependent manner (Table 8).
Table 8. Emetic Events Observed in Ferrets for Compound 72.
a
Dose (µg/kg)
Emesis observed
30
0 / 4
1 / 4
3 / 4
1
3
00
00
a
number of animals with emesis / number of animals in study. Compound 72 was administered
interꢀtracheally to ferrets as a dryꢀpowder coꢀmixture with lactose. Observation lasted 1 h. The
first emetic event in each group occurred within 20 min.
CONCLUSIONS
We used rational design based on Xꢀray crystallography to conceive a novel chemical series of
PDE4 inhibitors, the naphthyridoinones (NAPs). The first synthesized example 21 was highly
potent at 1.6 nM. A modular chemical synthesis allowed rapid SAR expansion of all substitution
positions around the central core. Several examples with subꢀnanomolar potencies both in
enzymatic assays and inhibition of TNFꢀα production in whole cells were found. Compound 72
was advanced to the in vivo LPSꢀinduced lung neutrophilia in rats. This compound demonstrated
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1
1
1
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2
2
2
2
2
2
2
2
2
3
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3
3
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3
3
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a moderate efficacy, however physicochemical properties seemed to restrict its full potential,
especially when dosed as a dry powder formulation. In our other PDE4 series, the
3
1
pyridazinones, we observed superior in vivo results for more soluble compounds, incorporating
basic groups to facilitate a long lung duration. We conclude that the low solubility strategy of the
NAPs was not ideal: in vivo efficacy was compromised for highly potent in vitro molecules and
erratic kinetic solubility rates can lead to unpredictable or unꢀreproducible results. The NAP
series was abandoned at this stage.
0
1
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Experimental section
General Chemistry Methods. Reaction products were purified, when necessary, by flash
chromatography on silica gel (40ꢀ63 ꢁm) with the solvent system indicated. Purifications in
reverse phase were made in a Biotage SP1 automated purification system equipped with a C18
column and using a gradient of, unless otherwise stated, waterꢀacetonitrile/MeOH (1:1) (0.1%
v/v ammonium formate both phases) from 0% to 100% acetonitrile/MeOH (1:1) in 80 column
volumes. Preparative HPLCꢀMS were performed on a Waters instrument equipped with a 2767
injector/collector, a 2525 binary gradient pump, a 2996 PDA detector, a 515 pump as a makeꢀup
pump and a ZQ4000 Mass spectrometer detector. All test compounds were purified to >95%
purity by HPLC UV trace unless otherwise stated.
1
H Nuclear Magnetic Resonance Spectra were recorded on a Gemini 200 MHz spectrometer or a
Varian Mercury plus operating at a frequency of 400 MHz. Tetramethylsilane was used as
reference. Chemical shifts (δ) are given in ppm to 2 decimal places. Coupling constants (J
values) are given in ppm to 1 decimal place. The following abbreviations are used: singlet (s),
doublet (d), triplet (t), quartet (q), quintet (quin), double doublet (dd), double triplet (dt), triple
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doublet (td), multiplet (m), broad signal (br. s), apparent (app). Partial refers to spectra where a
peak or peaks are obscured under solvent peaks and cannot be assigned. Signals of protons
attached to heteroatoms may or may not be present according to solvent conditions. Mass
Spectra (m/z) were recorded on a Micromass ZMD or in a Waters ZQ mass spectrometer using
ESI ionization.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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9
0
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3
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tertꢀButyl [3ꢀ(2,2ꢀdimethylꢀ4,6ꢀdioxoꢀ1,3ꢀdioxanꢀ5ꢀyl)ꢀ3ꢀoxopropyl]carbamate (7). Bocꢀbeta
alanine (6) (83 g, 0.44 mol) was dissolved in 2.7 L dichloromethane, which had been previously
cooled to 5 ºC in the fridge. Meldrum's acid (66.6 g, 0.46 mol) was added and the mixture
shaken to dissolve. 4,4ꢀDimethylaminopyridine (75 g, 0.61 mol) was added and shaken to
dissolve. The solution was left in the fridge for 1 h to cool to approx 5 ºC.
Dicyclohexylcarbodiimide (100 g, 0.48 mol) was added, the mixture shaken to dissolve and then
left to stand in the fridge (5 ºC) for 2 d. The mixture was filtered and the solid washed twice
with cold dichloromethane. The combined filtrates were evaporated to approx 700 mL volume.
The organics were washed three times with 10% w/v potassium hydrogen sulfate solution, once
with brine and were dried over sodium sulfate. Filtration, evaporation of the filtrate and reꢀ
evaporation from ether gave 7 as a white solid (136 g, 0.41 mol, 94% yield), HPLC purity 99%.
1
H NMR (200 MHz, CDCl ) δ 1.43 (s, 9H), 1.75 (s, 6H), 3.30 (t, J = 6.4 Hz, 2H), 3.55 (q, J =
3
+
5
.9 Hz, 2H), 4.85 (br. s., 1H); MS(ESI) m/z 316 [M + H] .
tertꢀButyl 2,4ꢀdioxopiperidineꢀ1ꢀcarboxylate (8). A suspension of 7 (136 g, 0.41 mol) in 250
mL ethyl acetate and heated at reflux (bath temperature 120 ºC) for 2.5 h. The mixture was
allowed to cool and was filtered. The filtrate was evaporated, giving an orange semisolid
residue. The residue was dissolved in 200 mL diisopropyl ether. The volume of solvent was then
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