Synthesis and biological activity of pyrido[3′,2′:4,5]furo[3,2- d]pyrimidine derivatives as novel and potent phosphodiesterase type 4 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.07.054

A series of pyrido[3′,2′:4,5]furo[3,2-d]pyrimidines (PFP) were synthesized and tested for phosphodiesterase type 4 (PDE4) inhibitory activity, with the potential to treat asthma and chronic obstructive pulmonary disease (COPD). Structure-activity relation

Full text of DOI:10.1016/j.ejmech.2011.07.054

European Journal of Medicinal Chemistry 46 (2011) 4946e4956
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological activity of pyrido[30,20:4,5]furo[3,2-d]pyrimidine
derivatives as novel and potent phosphodiesterase type 4 inhibitors
Joan Taltavull a, Jordi Serrat a, Jordi Gràcia a, Amadeu Gavaldà b, Mònica Córdoba c, Elena Calama d,
José Luis Montero b, Míriam Andrés e, Montserrat Miralpeix d, Dolors Vilella e, Begoña Hernández f,
,
Jorge Beleta g, Hamish Ryder h, Lluís Pagès a
*
a Department of Medicinal Chemistry, Almirall S.A., R&D Centre, Laureà Miró 408-410, 08980 Sant Feliu de Llobregat, Catalonia, Spain
b Department of Integrative Pharmacology, Almirall S.A., R&D Centre, Laureà Miró 408-410, 08980 Sant Feliu de Llobregat, Catalonia, Spain
c Department of ADME & Metabolism, Almirall S.A., R&D Centre, Laureà Miró 408-410, 08980 Sant Feliu de Llobregat, Catalonia, Spain
d Respiratory Therapeutic Area Discoveryn, Almirall S.A., R&D Centre, Laureà Miró 408-410, 08980 Sant Feliu de Llobregat, Catalonia, Spain
e Biological Reagents Assay Development Screening, Almirall S.A., R&D Centre, Laureà Miró 408-410, 08980 Sant Feliu de Llobregat, Catalonia, Spain
f Structural and Computational Biology & Chemistry, Almirall S.A., R&D Centre, Laureà Miró 408-410, 08980 Sant Feliu de Llobregat, Catalonia, Spain
g Discovery Strategy and Alliances, Almirall S.A., R&D Centre, Laureà Miró 408-410, 08980 Sant Feliu de Llobregat, Catalonia, Spain
h Director of Drug Discovery, Cancer Research Technology, Angel Building, 407 St John Street, London EC1V 4AD, UK
a r t i c l e i n f o
a b s t r a c t
A series of pyrido[30,20:4,5]furo[3,2-d]pyrimidines (PFP) were synthesized and tested for phosphodies-
terase type 4 (PDE4) inhibitory activity, with the potential to treat asthma and chronic obstructive
pulmonary disease (COPD). Structureeactivity relationships within this series, leading to an increase of
potency on the enzyme, is presented.
Both gem-dimethylcyclohexyl moieties fused to the pyridine ring and the substitution at the 5 position
of the PFP scaffold, proved to be key elements in order to get a high affinity in the enzyme.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Article history:
Received 24 February 2011
Received in revised form
20 July 2011
Accepted 29 July 2011
Available online 5 August 2011
1. Introduction
bronchodilatory profile of PDE4 inhibitors could allow the
discovery of new agents, steroid-sparing compounds with utility in
Inhaled corticosteroids alone or in combination with other
agents (e.g. beta-2 adrenergic agonists and anticholinergic agents)
are widely used for the treatment of asthma and COPD [1,2].
However, there is still a significant medical unmet need. In
asthma, there are concerns related to the potential local and
systemic dose-related adverse effects, whereas in COPD there is
a need for a non-steroidal anti-inflammatory agent, because
inhaled corticosteroids have limited effectiveness [3].
diseases associated with chronic airway inflammation, particularly
in the management of asthma and COPD.
PDE4 has proved to be a chemically tractable drug target with
a wide diversity of chemotypes reported as inhibitors [8], we report
herein some derivatives of pyrido[30,20:4,5]furo[3,2-d]pyrimidines
(PFP), which were designed using a traditional medicinal chemistry
strategy, aided by the structural analysis of PDE4 inhibitor-protein
structures by X-ray crystallography.
In recent years, much attention has focused on the cyclic
nucleotide phosphodiesterase type 4 (PDE4) as a suitable target for
anti-inflammatory therapy in respiratory diseases [3,4]. Several
clinical entities that target phosphodiesterase enzymes have been
approved (PDE3 (Cilostazol) [5], PDE4 (Roflumilast) [6] and PDE5
(Sildenafil, Tadalafil and Vardenafil) [7] demonstrating the feasi-
bility of development of selective PDE inhibitors for the treatment
of various clinical disorders. The mixed anti-inflammatory and
PDE4 activity across a panel of human recombinant PDE4
subtypes (PDE4B1, PDE4A4 and PDE4D3) was determined in order
to assess potency. The compounds were also tested in a functional
assay to determine their potential to inhibit the production of pro-
inflammatory cytokines such as TNFa.
2. Chemistry
Here we describe the synthesis of a variety of new PFP deriva-
tives, in order to study the potential of this scaffold to deliver the
desired activity profile. These compounds were synthesized as
outlined in Scheme 1.
Abbreviations: PFP, pyrido[30,20:4,5]furo[3,2-d]pyrimidine; COPD, chronic
obstructive pulmonary disease; PDE4, phosphodiesterase type 4.
* Corresponding author. Tel.: þ34 932 913587; fax: þ34 932913420.
E-mail address: lluis.pages@almirall.es (L. Pagès).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.054

J. Taltavull et al. / European Journal of Medicinal Chemistry 46 (2011) 4946e4956
4947
O
H3C
CH3
H3C CH3
O
CH3 CH3
O
O
HN
O
H3C CH3
N
N
O
N
N
MeO
OMe
N
N
N
CH3
CH3
CH3
CH3
a
b
O
b
a
O
N
H3CO
N
OH
N
SH
S
S
NH2
1
MeO
O
8
O
2
3
10
9
H3C CH3
H3C CH3
H3C CH3
O
H3C CH3
O
OEt
O
Br
Br
N
NH2
O
N
O
OEt
NH2
O
c
d
d
c
NH
O
N
N
N
H3CO
N
O
H3CO
N
O
N
O
OH
O
O
OEt
OEt
11
4
5
12
H3C CH3
H3C CH3
H2N
H3C CH3
H3C CH3
O
H2N
O
N
N
N
N
N
N
O
N
e
f
N
NH
f
N
e
H3CO
N
O
HO
N
O
N
N
O
N
N
O
HN
HN
N
O
O
HN
14
13
6
7
N
N
N
H3C CH3
Scheme 1. Synthesis of pyrido[30,20:4,5]furo[3,2-d]pyrimidines. Reagents and condi-
tions: (a) CS2, MeOH, TEA, r.t.; (b) EtOH, reflux; (c) 2-Bromoethanol, NaOH, MeOH; (d)
i) K2CO3, acetone, reflux, ii) Cs2CO3, DMF, 120 ꢀC; (e) i) HC(OEt)3, reflux, ii) NH3, EtOH,
reflux; (f) i) POCl3, reflux, ii) EtOH, reflux.
HN
O
N
N
N
O
g
O
HN
N
15
Commercially available 2,2-dimethyldihydro-2H-pyran-4(3H)-
1 (or the equivalent 3,3-dimethylcyclohexanone) was
condensed with malononitrile in the presence of carbon disulfide
to yield the heterocycle 2 [9].
one
Scheme 2. Synthesis of pyrido[30,20:4,5]furo[3,2-d]pyrimidines. Reagents and condi-
tions: (a) NaH, THF, reflux; (b) NH4OAc, HOAc, toluene/MeOH, reflux; (c) i) K2CO3,
acetone, reflux, ii) Cs2CO3, DMF, 120 ꢀC; (d) i) HC(OEt)3, reflux, ii) NH3, EtOH, reflux; (e)
i) POCl3, reflux, ii) EtOH, reflux; f) HBr sol.48%, reflux; (g) (i) NaH, Tf2NPh, THF, (ii) r.t.
Reaction of compound 2 with morpholine (and other secondary
amines) yielded the pyridine derivative 3 [10]. Compound 3 was
converted to the corresponding hydroxypyridine 4 by heating with
2-bromoethanol in basic conditions. Subsequent reaction of
compound 4 with ethyl 2-bromoacetate, followed by basic treat-
ment, afforded the furopyridine 5.
The pyridofuropyrimidine 6 was synthesized by treatment of
intermediate 5 with triethylorthoformate followed by cyclization
with ammonia, as described for similar compounds [11]. The cor-
responding chloropyrimidine derivative of 6 was prepared using
phosphorous oxychloride as solvent, and then reacted with pyr-
idin-3-ylmethanamine (and other amines) to give the desired final
compound 7 [12].
other amine), to give derivative 13. Compound 13 was demethy-
lated by heating it in 48% hydrobromic acid, and the resulting
hydroxypyridine 14 was converted to the desired final compound
15 through the triflate intermediate, which was displaced by the
corresponding amine (morpholine in this case). Unfortunately, this
synthetic route was not feasible starting with 2,2-dimethyldihydro-
2H-pyran-4(3H)-one 1, so we were unable to prepare the analogous
final products.
This synthetic route was, nevertheless, limited by the following
issues: only secondary amines gave the desired product in step
b [10], and their introduction was at the very beginning of the
synthesis, thus we lacked a common, advanced intermediate that
allowed a shorter route to the final compounds. Both drawbacks
were overcome by the synthetic route outlined in Scheme 2.
Commercially available 3,3-dimethylcyclohexanone 8, was
reacted with dimethyl carbonate in the presence of sodium hydride
to yield compound 9 [13].
Reaction of compound 9 with malononitrile yielded the pyridine
derivative 10 [14]. Subsequent alkylation of compound 10 with
ethyl 2-bromoacetate in the presence of potassium carbonate, fol-
lowed by cyclization with a base, afforded the furopyridine 11. The
pyridofuropyrimidine 12 was synthesized by cyclization of inter-
mediate 11 with triethylorthoformate and ammonia.
3. Results and discussion
In a related series of pyrido[30,20:4,5]thieno[3,2-d]pyrimidines
(PTP) [12] where the cyclohexyl ring fused to the pyridine was
throughly studied, we had already observed that the substitution of
both gem-dimethyls by hydrogens, as well as the reduction of the
ring size, resulted in a significant loss of potency at the enzyme, so
in these PFP derivatives we only focused our efforts in the
dimethyl-substituted six-membered ring derivatives. The replace-
ment of the thiophene ring by a furane, leading to the present PFP
derivatives, was intended to improve the physico-chemical prop-
erties (permeability) of the PTP series.
3.1. In vitro binding studies
The corresponding chloroimine derivative of 12 was prepared
using phosphorous oxychloride as solvent, and the resulting
intermediate was reacted with pyridin-3-ylmethanamine (or any
Table 1 shows the in vitro results for the set of compounds 7 and
15e19.

4948
J. Taltavull et al. / European Journal of Medicinal Chemistry 46 (2011) 4946e4956
Table 1
SAR on the pyridine fused ring, R1, R2, and R3 substituents and selectivity profile.
H3C
CH3
X
N
N
N
O
N
O
NR1R2
Cpnd. nr.
15
X
R1
H
R2
PDE4B1 IC50 (nM)
PDE4A4 IC50 (nM)
20
PDE4D3 IC50 (nM)
13
HWB TNF
a IC50 (nM)
-
-
CH2
CH2
CH2
6
433
N
7
O
H
235
n.d.
n.d.
n.d.
N
-
16
17
18
CH2
O
H
8.7
337
0.8
16.3
n.d.
4.8
9.4
n.d.
2.3
n.d.
n.d.
112
N
N
N
(CH2)2
(CH2)2
(CH2)2
O
-
-
H
O
O
CH2
HO(CH2)2ꢁ
-
19
O
HO(CH2)2ꢁ
79
n.d.
n.d.
n.d.
N
(CH2)2
O
As illustrated in Table 1, we initially investigated the influence of
including an oxygen in the saturated ring of the PFP scaffold
compared to the carbocyclic derivatives. The results show that in
each case, the compounds with X ¼ CH2 (15, 16 and 18) are, at least,
one order of magnitude more potent than the corresponding
analogues with X ¼ O (7, 17 and 19) in PDE4B1, unfortunately no
selectivity over other PDE4 isoforms was observed. Besides, in the
compounds described in that previous paper [12] the substitution
in position 5 had more influence in achieving significant enzymatic
activity than the substitution at the position 8, an effect that is also
studied in the present paper.
inhibiting the enzyme (IC50 ¼ 0.1 nM in PDE4B1) and blocking the
production of the pro-inflammatory cytokine TNF
(IC50 ¼ 12 nM in
HWB TNF ). Compared to Roflumilast, compound 26 has a superior
enzymatic profile, but it is less active in the neutrophilia rat LPS
in vivo model and shows emesis in ferret at lower doses (see
Table 3).
Compound 26 had acceptable metabolic stability and perme-
ability (CaCo) but other parameters were inferior to Roflumilast:
lower blood levels and shorter half-life, as depicted in Fig. 1
(Table 4).
a
a
Table 2 shows the in vitro results for the set of compounds
20e29.
4. Structural analysis of PDE4D complexes by X-ray
crystallography
In all these PFP derivatives (see compounds from 20e29),
activity below 25 nM PDE4B1 inhibition can be maintained with
different substituents at R2, such as pyridines (27e29), basic amines
(21e23) or neutral residues (24e26), thus giving a set of potent
compounds with very diverse physico-chemical properties.
Next, we investigated different amines at the R1 position
keeping the R2 substituent fixed. If the morpholine at this position
(see, for example, compound 15) is replaced by an aminoalcohol
residue, the potency decreases (see compound 20). The most
potent compounds were obtained when the isopropylamine was
introduced at R1, whereas tertiary amines led to products one order
of magnitude less potent (for instance, compare the pairs of
compounds 21,22, 24,25 and 27,28).
It is already known [15] that Roflumilast’s cyclopropyl methoxy
group occupies the large lipophilic pocket of the active site,
whereas the difluoromethoxy group binds to a smaller pocket,
where it causes a shift of the Gln369 residue [16]; this catechol ring
is placed at the hydrophobic clamp and the 3,5-dichloropyridine
group extends to the dimetal ion site and forms one hydrogen
bond to a water molecule that is coordinated to Mg2þ. The two
chlorine atoms also form additional hydrophobic interactions with
residues in the metal binding pocket.
Compound 26 was co-crystallized with PDE4D and the structure
of the complex was resolved by X-ray crystallography (see Fig. 2).
1-(3-aminopropyl)pyrrolidin-2-one moiety in 26 is directed
towards the solvent exposed region, where no relevant interactions
are detected, which could explain the variety of substituents
From the whole set of compounds that have been analyzed
(from 20e29), compound 26 emerges as the most potent, at both

J. Taltavull et al. / European Journal of Medicinal Chemistry 46 (2011) 4946e4956
4949
Table 2
SAR on the R1 and R2 substituents and selectivity profile.
H3C
CH3
N
N
R1
N
O
NHR2
Cpnd. nr.
20
R1
R2
PDE4B1 IC50 (nM)
24
PDE4A4 IC50 (nM)
PDE4D3 IC50 (nM)
13.5
HWB TNF
a IC50 (nM)
HO
-
CH2
36
819
N
H
N
-
-
21
22
2.1
19
5
2.7
14
268
n.d.
H3C
H3C
N
H
N
N
(CH2)2
(CH2)2
O
O
N
28
CH3
CH3
-
23
0.2
0.5
0.4
268
N
(CH2)2
O
H3C
H3C
H3C
N
H
O
24
25
26
1.7
25
5
1.5
17
n.d.
n.d.
12
N
H
-
N
(CH2)3
(CH2)3
(CH2)3
O
N
62
-
-
N
CH3
CH3
O
0.1
0.3
0.1
H3C
H3C
N
H
N
-
N
CH2
CH2
CH2
27
9.7
22
240
n.d.
CH3
N
-
-
28
29
4.3
0.2
6.2
1.3
0.5
n.d.
201
H3C
H3C
N
H
N
N
CH3
0.9
N
H
accepted at this position (see R2 values in Tables 1 and 2), whereas
the gem-dimethylcyclohexyl ring occupies the large hydrophobic
pocket of the enzyme active site, as does the roflumilast’s catechol
ring, explaining why this area of the molecule is so sensitive to
small changes, as we had previously observed [12]. Neither with
the set of amines tested in Table 2 nor with the morpholine, the
compounds interacted with any of the water molecules in the
coordination sphere of the magnesium ion as the nitrogen of
Roflumilast’s pyridine does.
Table 3
As seen in Fig. 2, the most remarkable observation in the case of
26 is that the nitrogen at R1, which bears the isopropyl chain,
coincides exactly in the same position of the Roflumilast’s 4-
pyridine nitrogen. After this observation, the introduction of the
4-amino-3,5-dichloropyridine at the R1 position of the PFP was
targeted in order to achieve a similar interaction in our molecules.
Compound 30 (see Fig. 3) was synthesized with the purpose to
confirm this hypothesis and resulted to be the most potent PDE4
inhibitor in this series, with an IC50 ¼ 0.02 nM, one order of
magnitude more active than 29.
In vitro and in vivo profile of compound 26 compared with Roflumilast.
Compound Roflumilast
26
PDE4A4 IC50 (nM)
PDE4B1 IC50 (nM)
PDE4D3 IC50 (nM)
HWB TNF IC50 (nM)
PBMNC TNF IC50 (nM)
0.25
0.15
0.11
12
0.5
0.59
0.69
0.27
22
2.6
94
0.3
LPS neutrophilia rat (% inhibition at 0.3 mg/kg, p.o.) 65
Emesis ferret (max. non-emetic dose, mg/Kg, p.o.) 0.1

4950
J. Taltavull et al. / European Journal of Medicinal Chemistry 46 (2011) 4946e4956
Compound 26 PK Ferret 0.1 mg/Kg p.o.
10
9
8
7
6
5
4
3
2
1
0
0
2
4
6
8
10
Time (h)
12
14
16
18
20
22
24
Fig. 1. Oral pharmacokinetic profile of compound 26 in ferret at 0.1 mg/kg p.o.
This result stimulated the design of a whole set of inter-
esting new compounds bearing the 3,5-dichloropyridin-4-amine
fragment, which will be explored and disclosed in future
communications.
5. Conclusion
A new and promising PDE4 inhibitor scaffold has been identi-
fied. In a classical SAR exercise, a series of pyrido[30,20:4,5]furo[3,2-
d]pyrimidines were synthesized and evaluated as PDE4 inhibitors.
The biological data reported in Table 1 confirm the importance of
the gem-dimethylcyclohexyl group (Table 1) and of the 4-amino-
3,5-dichloropyridine at the 5 position (Fig. 3) of the PFP scaffold to
get high PDE4 inhibition activity (<0.1 nM), as suggested by the
structural analysis of PDE4D complexes.
Fig. 2. Roflumilast (yellow) and compound 26 (green) overlay. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version
of this article.)
This scaffold showed that good enzymatic activity is achieved
and potency could be optimised through modification of the R1
substituent, where secondary alkyl amines are preferred to tertiary
ones (see compound 26). Unfortunately, compound 26 is more
emetic than Roflumilast at lower doses.
Compound 30 showed the highest potency at the enzyme, thus
confirming the X-ray co-crystal structural analysis as a useful tool in
designing new pharmacologically active molecules. With emesis
and metabolism as the main issues in this series, compound 30
seems a promising starting point for further optimization.
tetrahydropyran-4-one (1) was provided by Matrix Scientific (cat.
nr. 021441) or by Aldrich (cat. nr. 198242). concentration refers to
evaporation under vacuum using a Büchi rotatory evaporator.
reaction products were purified, when necessary, by flash chroma-
tography on silica gel (40e63 mm) with the solvent system indicated.
1H NMR spectra were performed in a Varian Gemini 2000 spec-
trometer operating at a frequency of 200 or 300 MHz. samples were
dissolved in deuterated chloroform (CDCl3) or deuterated dime-
thylsulfoxide (DMSO-d6). Tetramethylsilane (TMS) was used as
reference. the following abbreviations were used to assign spectra:
s) singlet, d) doublet, dd) double doublet, ddd) double double
doublet, t) triplet, dt) double triplet, td) triple doublet, m) multiplet,
br.s) broad signal. HPLC-UV-MS chromatograms were acquired in
a Waters Alliance 2695 chromatographer equipped with a Waters
2996 diode-array detector and a Waters ZQ mass spectrometer
detector. HPLC analysis was conducted according to the described
method, with the retention time (tR) expressed in min. UV
6. Experimental section
6.1. Chemistry: general
Reagents, starting materials, and solvents were purchased
from commercial suppliers and used as received. 3,3-Dimethyl-
cyclohexanone (8) was purchased from Aldrich (cat. no. 674893);
methyl 4,4-dimethyl-2-oxo-cyclohexanecarboxylate (9) was
synthesized as described at the literature [13]. 2,2-Dimethyl-
H3C
CH3
Table 4
PK p.o. parameters of compound 26 compared with Roflumilast.
Compound 26
Roflumilast
N
Rat metabolism (%)
Human metabolism (%)
Caco2 (cm/seg*10-6)
Cmax in ng/ml (at max non-emetic
dose in mg/Kg)
t1/2 (h)
tmax (h)
AUC0et (ng h/ml)
67
73
N
53
24
7.2 (0.1)
17
39
56 (0.3)
HN
N
Cl
N
Cl
O
HN
Compound 30
PDE4B1 IC50
0.5
0.4
13.9
4.2
4.4
95
N
= 0.02
nM
Fig. 3. Chemical structure and enzymatic inhibition of compound 30.

J. Taltavull et al. / European Journal of Medicinal Chemistry 46 (2011) 4946e4956
4951
chromatograms were processed at 210 nm with blank substraction.
HPLC method: chromatography performed on a Symmetry C18
m). the mobile phase, at a flow of
0.4 ml/min, wasa 20 min binarygradientof water(containing 0.01 M
ammonic formate at pH 3.0) and a mixture acetonitrile-methanol
50:50 (containing 0.01 M ammonic formate) (0e95%). the total
run time was 26 min.
Concerning the structural analysis of the 26 PDE4D complex
by X-ray crystallography, crystals of PDE4D were soaked with
a 150 mM solution of the compound inhibitor 26 in DMSO (addition
6.2.4. Ethyl 1-amino-8,8-dimethyl-5-morpholin-4-yl-8,9-dihydro-
6H-furo[2,3-b]pyrano[4,3-d]pyridine-2-carboxylate (5)
3,3-Dimethyl-8-morpholin-4-yl-6-oxo-3,4,6,7-tetrahydro-1H-
pyrano[3,4-c]pyridine-5-carbonitrile 4 (1.9 g, 6.6 mmol) was dis-
solved in acetone (35 ml). Potassium carbonate (908 mg, 6.6 mmol)
column (100 ꢂ 2.16 mm, 3.5
m
and ethyl 2-bromoacetate (726 ml, 6.6 mmol) were added and the
mixture was refluxed overnight. The reaction mixture was poured
onto 150 ml of ice water. The insoluble solid was filtrated and dried.
1.98 g of ethyl 2-(5-cyano-3,3-dimethyl-8-morpholin-4-yl-3,4-
dihydro-1H-pyrano[3,4-c]pyridin-6-yloxy)acetate were obtained.
of 0.3
mle2
ml crystallization drops) over 3 days. The structure was
Yield ¼ 80%. 1H NMR (300 MHz, CHLOROFORM-D)
d ppm 1.3 (t,
solved by Difference Fourier methods using the previously deter-
mined structure of the apo-protein as a starting model. The current
model had a 2.15e2.21 Å resolution. Water molecules were
included where stereochemically plausible and difference electron
J ¼ 7.1 Hz, 3 H) 1.3 (s, 6 H) 2.8 (s, 2 H) 3.2 (m, 4 H) 3.8 (m, 4 H) 4.2 (q,
J ¼ 7.1 Hz, 2 H) 4.5 (s, 2 H) 4.9 (s, 2 H).
Ethyl 2-(5-cyano-3,3-dimethyl-8-morpholin-4-yl-3,4-dihydro-
1H-pyrano[3,4-c]pyridin-6-yloxy)acetate (1.7 g, 4.6 mmol) was
suspended in DMF (20 ml) and cessium carbonate was added (3.0 g,
9.3 mmol). This reaction mixture was heated at 120 ꢀC for 4 h. The
solvent was evaporated under reduced pressure and the residue
was partitioned between water and ethyl acetate. The organic
phase was separated and the aqueous phase was extracted twice
with ethyl acetate. The organic phase was washed successively with
water and brine, dried over MgSO4, filtered and the solvent evap-
orated under reduced pressure. 980 mg of the final compound 5
were obtained. Yield ¼ 56%. 1H NMR (300 MHz, CHLOROFORM-D)
densities of more than 3.0
s where observed.
6.2. Preparation of pyridofuropyrimidine derivatives according to
Scheme 1
6.2.1. 6-Amino-3,3-dimethyl-8-thioxo-4,8-dihydro-1H,3H-thiopyrano
[3,4-c]pyran-5-carbonitrile (2)
2,2-Dimethyltetrahydropyran-4-one 1 (5.0 g, 32.0 mmol) was
dissolved in methanol (4.7 ml) and carbon disulfide (4.7 ml,
48.8 mmol) was added in one portion. Malononitrile (2.6 g,
39.0 mmol) was added portionwise and, finally, triethylamine
(1.95 ml) was added dropwise. The reaction mixture was stirred at
room temperature for 48 h. An orange precipitate was formed,
which was filtered (3.90 g), its 1H NMR being consistent with the
desired compound 2. From the filtrates, 0.89 g more of 6-amino-3,3-
dimethyl-8-thioxo-4,8-dihydro-1H,3H-thiopyrano[3,4-c]pyran-5-
carbonitrile were isolated by flash chromatography, eluting first
with CH2Cl2 and next with the mixture of solvents CH2Cl2:MeOH
d
ppm 1.4 (m, 9 H) 3.0 (s, 2 H) 3.2 (m, 4 H) 3.8 (m, 4 H) 4.4 (q,
J ¼ 7.1 Hz, 2 H) 4.7 (s, 2 H) 5.1 (s, 2 H).
6.2.5. 2,2-Dimethyl-5-morpholin-4-yl-1,4-dihydro-2H-pyrano
[400,300:40,50]pyrido[30,20:4,5]furo[3,2-d]pyrimidin-8(9H)-one (6)
Ethyl 1-amino-8,8-dimethyl-5-morpholin-4-yl-8,9-dihydro-6H-
furo[2,3-b]pyrano[4,3-d]pyridine-2-carboxylate
5 (980 mg, 2.6
mmol) were suspended in triethylorthoformate (10 ml) and heated
to reflux for 6 h. The solvent was evaporated under reduced pressure
and the residue was rinsed with ethanol. The insoluble solid was
filtrated, washed with ethyl ether and dried. 650 mg of ethyl
1-{[(1E)-ethoxymethylene]amino}-8,8-dimethyl-5-morpholin-4-
yl-8,9-dihydro-6H-furo[2,3-b]pyrano[4,3-d]pyridine-2-carboxylate
were obtained. Yield ¼ 58%. 1H NMR (300 MHz, CHLOROFORM-D)
98:2. Yield ¼ 48%.1H NMR (200 MHz, CDCl3)
d ppm 1.30 (s, 6 H), 2.62
(s, 2 H), 4.66 (s, 2 H), 7.91 (s, 2 H).
6.2.2. 6-Mercapto-3,3-dimethyl-8-morpholin-4-yl-3,4-dihydro-1H-
pyrano[3,4-c]pyridine-5-carbonitrile (3)
d
ppm 1.4 (m, 9 H) 1.5 (t, J ¼ 7.1 Hz, 3 H) 3.0 (s, 2 H) 3.2 (m, 4 H)
Compound 2 (3.9 g, 15.45 mmol) was suspended in ethanol
(17 ml) and morpholine (6.7 ml, 77.3 mmol) was added. The reac-
tion mixture was refluxed under nitrogen overnight. Then the
system was allowed to reach room temperature and the reaction
mixture was cooled in an ice bath for 2 h. The solid that formed was
filtrated and washed twice with ethanol. After drying, 3.12 g of the
final product were obtained as a dark solid, pure enough to perform
3.8 (m, 4 H) 4.3 (q, J ¼ 7.1 Hz, 2 H) 4.4 (q, J ¼ 7.0 Hz, 2 H) 4.7 (s, 2 H) 7.9
(s, 1 H).
Ethyl 1-{[(1E)-ethoxymethylene]amino}-8,8-dimethyl-5-morp-
holin-4-yl-8,9-dihydro-6H-furo[2,3-b]pyrano[4,3-d]pyridine-2-ca-
rboxylate (650 mg, 1.5 mmol) were suspended in ethanol (10 ml)
and concentrated ammonia (8 ml) was added. After refluxing for
5 h, the reaction was finished. The reaction mixture was cooled to
room temperature and then left overnight at þ5 ꢀC. The precipi-
tated solid was filtered, washed with ethanol and dried. 454 mg of
the final compound 6 were obtained. Yield ¼ 85%. 1H NMR
the next step. Yield ¼ 66%. 1H NMR (200 MHz, CDCl3)
d ppm 1.30 (s,
6 H), 2.75 (s, 2 H), 3.3 (m, 4 H), 3.75 (m, 4H), 4.5 (s, 2H).
6.2.3. 3,3-Dimethyl-8-morpholin-4-yl-6-oxo-3,4,6,7-tetrahydro-1H-
pyrano[3,4-c]pyridine-5-carbonitrile (4)
(300 MHz, DMSO-D6)
(m, 4 H) 4.7 (s, 2 H) 8.2 (s, 1 H).
d
ppm 1.3 (s, 6 H) 3.2 (m, J ¼ 8.2 Hz, 6 H) 3.8
6-Mercapto-3,3-dimethyl-8-morpholin-4-yl-3,4-dihydro-1H-
pyrano[3,4-c]pyridine-5-carbonitrile 3 (2.8 g, 7.2 mmol) was sus-
pended in a mixture of NaOH 1 N (7.2 ml) and methanol (20 ml) and
6.2.6. 2,2-Dimethyl-5-morpholin-4-yl-N-(pyridin-3-ylmethyl)-1,4-
dihydro-2H-pyrano[400,300:40,50]pyrido[30,20:4,5]furo[3,2-d]pyrimidin-
8-amine (7)
2-bromoethanol (513
ml, 7.2 mmol) were added. This reaction
mixture was stirred overnight and methanol was evaporated under
reduced pressure. The residue was suspended in a mixture of an
aqueous solution of NaOH 1 N (55 ml), ethanol (55 ml) and
methylglycol (55 ml) and heated at 135 ꢀC during 5 h. The reaction
solution was acidified to pH ¼ 2 with HCl 2 N and extracted with
dichloromethane. The organic phase was washed with water and
brine, dried over MgSO4 and evaporated under reduced pressure.
1.8 g of the final compound was obtained as a solid. Yield ¼ 88%. 1H
2,2-Dimethyl-5-morpholin-4-yl-1,4-dihydro-2H-pyrano[400,300:
40,50]pyrido[30,20:4,5]furo[3,2-d]pyrimidin-8(9H)-one
6 (454 mg,
1.3 mmol) was suspended in phosphorous oxychloride (2 ml) and
heated to reflux for 90 min. The solvent was evaporated under
reduced pressure and the residue was worked-up with water
and chloroform as usual. 460 mg of 8-chloro-2,2-dimethyl-5-
morpholin-4-yl-1,4-dihydro-2H-pyrano[400,300:40,50]pyrido[30,20:4,5]
furo[3,2-d]pyrimidine were obtained as a solid. Yield ¼ 96%. 1H NMR
NMR (300 MHz, DMSO-D6)
d
ppm 1.2 (s, 6 H) 2.5 (m, 2 H) 2.7 (s, 2 H)
(300 MHz, CHLOROFORM-D)
d ppm 1.4 (s, 6 H) 3.4 (s, 2 H) 3.4 (m, 4 H)
3.2 (m, 4 H) 3.7 (m, 4 H) 4.4 (m, 2 H).
3.9 (m, 4 H) 4.8 (s, 2 H) 8.9 (s, 1 H).

4952
J. Taltavull et al. / European Journal of Medicinal Chemistry 46 (2011) 4946e4956
8-Chloro-2,2-dimethyl-5-morpholin-4-yl-1,4-dihydro-2H-pyr-
for 6 h in triethylorthoformate. Then, the solvent was evaporated
under reduced pressure and the residue was redissolved in ethanol
(15 ml) and concentrated ammonia (12 ml) and heated under reflux
for 18 h. After the solvent had been evaporated, the residue was
partitioned between water and ethyl acetate. The organic phase was
separated and the aqueous phase was extracted twice with ethyl
acetate. The organic phase was washed with water, dried over
MgSO4, filtered and the solvent evaporated. 0.82 g of a brownish
solid were obtained, which was rinsed with dichloromethane to
yield 0.2 g of the final product 12 as a brownish solid. Yield ¼ 20%.1H
ano[400,300:40,50]pyrido[30,20:4,5]furo[3,2-d]pyrimidine (70 mg,
0.2 mmol) was suspended in ethanol (5 ml) and pyridine-3-
ylmethylamine (123
ml, 0.9 mmol) was added. The reaction
mixture was heated at 85 ꢀC for 48 h. The solvent was evaporated
under reduced pressure and the residue was passed through
a silica-gel column eluting with dichloromethane first and then,
successively, with mixtures of CH2Cl2/MeOH 99:1 and CH2Cl2/
MeOH 98:2. 50 mg of the final compound were obtained.
Yield ¼ 22%. 1H NMR (400 MHz, DMSO-D6)
d ppm 1.3 (s, 6 H) 3.2
(m, 4 H) 3.3 (s, 2 H) 3.8 (m, 4 H) 4.7 (s, 2 H) 4.8 (d, J ¼ 5.9 Hz, 2 H) 7.3
(dd, J ¼ 7.8, 4.7 Hz, 1 H) 7.8 (m, 1 H) 8.5 (m, 1 H) 8.6 (m, 2 H). HPLC:
99.9%, tR ¼ 10.0 min, MS(ESI) 447 m/z (M þ H)þ.
NMR (300 MHz, CHLOROFORM-D)
d
ppm 1.0 (s, 6H) 1.6 (t, J ¼ 6.6 Hz,
2 H) 2.6 (t, J ¼ 6.6 Hz, 2 H) 3.0 (s, 2 H) 4.0 (s, 3 H) 8.2 (s, 1H).
6.3.4. 5-Methoxy-2,2-dimethyl-N-(pyridin-3-ylmethyl)-1,2,3,4-
tetrahydropyrimido[40,50:4,5]furo[2,3-c]isoquinolin-8-amine (13)
5-Methoxy-2,2-dimethyl-1,2,3,4-tetrahydropyrimido[40,50:4,5]
furo[2,3-c]isoquinolin-8-ol 12 (9.9 g, 32.7 mmol) was suspended in
phosphorous oxychloride (40 ml) and heated to reflux for 3 h. The
solvent was evaporated under reduced pressure and the residue
was worked-up as usual with ethyl acetate and water. The product
was purified by silica-gel chromatography, eluting with CH2Cl2/
MeOH 99:1, to yield 5.2 g of 8-chloro-2,2-dimethyl-5-morpholin-4-
yl-1,2,3,4-tetrahydropyrimido[40,50:4,5]furo[2,3-c]isoquinoline.
6.3. Preparation of pyridofuropyrimidine derivatives according to
Scheme 2
6.3.1. 3-Hydroxy-1-methoxy-6,6-dimethyl-5,6,7,8-tetrahydroiso-
quinolin-4-carbonitrile (10)
Methyl 4,4-dimethyl-2-oxocyclohexancarboxylate
9 (8.4 g,
45.5 mmol, prepared from 8 [13]) was dissolved in a 1:1 mixture of
toluene/methanol (2 ꢂ 30 ml). Malonodinitrile (3.3 g, 50.0 mmol),
ammonium acetate (0.5 g, 6.8 mmol) and acetic acid (2.8 ml) were
added. The reaction mixture was refluxed overnight and the
solvent was evaporated under reduced pressure. Aqueous 1 N
NaOH (120 ml) was added to the residue and the precipitated solid
was filtered and washed with water. Once dried, 6.9 g of the final
compound were obtained. Yield ¼ 66%. 1H NMR (300 MHz,
Yield ¼ 50%. 1H NMR (300 MHz, CHLOROFORM-D)
d ppm 1.0 (s, 6H)
1.6 (t, J ¼ 6.6 Hz, 2 H) 2.6 (t, J ¼ 6.6 Hz, 2 H) 3.0 (s, 2 H) 4.0 (s, 3 H) 8.9
(s, 1H).
8-Chloro-2,2-dimethyl-5-methoxy-1,2,3,4-tetrahydropyrimido
[40,50:4,5]furo[2,3-c]isoquinoline (60.0 mg, 0.2 mmol) was sus-
pended in ethanol (5 ml) and pyridin-3-ylmethylamine (0.1 ml,
1.1 mmol) was added. The reaction mixture was refluxed for two
days. The solvent was evaporated under reduced pressure and the
residue was passed through a silica-gel column, eluting with
CH2Cl2/MeOH 99:1. 60 mg of the desired compound 13 were
obtained as a solid. Yield ¼ 81%. 1H NMR (300 MHz, CHLOROFORM-
CHLOROFORM-D)
2.3 (t, J ¼ 6.6 Hz, 2 H) 3.7 (s, 3 H).
d
ppm 0.9 (s, 6H) 1.4 (t, J ¼ 6.6 Hz, 2 H) 2.2 (s, 2 H)
6.3.2. Ethyl 1-amino-5-methoxy-8,8-dimethyl-6,7,8,9-tetrahydrofuro
[2,3-c]isoquinolin-2-carboxylate (11)
3-Hydroxy-1-methoxy-6,6-dimethyl-5,6,7,8-
tetrahydroisoquinolin-4-carbonitrile 10 (6.9 g, 29.8 mmol) was
dissolved in acetone (180 ml) and potassium carbonate (9.9 g,
71.6 mmol) was added. After dropwise addition of ethyl bromoa-
cetate (3.3 ml, 29.8 mmol) at room temperature, this mixture was
refluxed under nitrogen for 3 h. The solvent was evaporated under
reduced pressure and the residue was redissolved in water/Et2O.
D)
d
ppm 1.1 (s, 6H) 1.7 (t, J ¼ 6.6 Hz, 2 H) 2.7 (t, J ¼ 6.6 Hz, 2 H) 3.2 (s,
2 H) 4.1 (s, 3 H) 4.90 (d, J ¼ 6.5, 2H) 5.5 (t, J ¼ 6.6 Hz, 1H) 7.3 (m,1H)
7.7 (m, 1H) 8.6 (m, 1H) 8.7 (m, 2H).
6.3.5. 2,2-Dimethyl-8-[(pyridin-3-ylmethyl)amino]-1,2,3,4-tetrahyd-
ropyrimido[40,50:4,5]furo[2,3-c]isoquinolin-5-ol (14)
After usual work-up, 8.4
g
of ethyl (4-cyano-1-methoxy-
5-Methoxy-2,2-dimethyl-N-(pyridin-3-ylmethyl)-1,2,3,4-tetra-
hydropyrimido[40,50:4,5]furo[2,3-c]isoquinolin-8-amine 13 (140 mg,
0.36 mmol) wasdissolved inbromhydric acid(5 ml, 48%wt. inwater)
and the mixture was heated to 100 ꢀC for 3 h. Once at room
temperature, the reaction mixture was neutralised with NaOH 6 N
and a solid precipitated, which was filtered and dried. 130 mg of the
final compound were obtained. Yield ¼ 96%. 1H NMR (400 MHz,
6,6-dimethyl-5,6,7,8-tetrahydroisoquinolin-3-yloxy)acetate were
obtained as an oil. Yield ¼ 89%. 1H NMR (300 MHz, CHLOROFORM-
D)
d
ppm 1.0 (s, 6H) 1.3 (t, J ¼ 6.6 Hz, 3 H) 1.5 (t, J ¼ 6.6 Hz, 2 H) 2.5
(t, J ¼ 6.6 Hz, 2 H) 2.6 (s, 2 H) 3.9 (s, 3 H) 4.2 (q, J ¼ 6.6 Hz, 2 H) 4.9
(s, 2H).
Ethyl (4-cyano-1-methoxy-6,6-dimethyl-5,6,7,8-tetrahydroiso-
quinolin-3-yloxy)acetate (17.2 g, 54 mmol) was dissolved in DMF
(350 ml) and potassium carbonate (7.5 g, 54 mmol) was added.
After 8 h of heating at 120 ꢀC, the solvent was evaporated under
reduced pressure and the residue was partitioned between chlo-
roform and saturated aqueous solution of ammonium chloride. The
organic phase was separated and the aqueous phase was extracted
twice with chloroform. The organic phase was washed with brine
and dried over MgSO4. Once the solvent is evaporated, 15.1 g of the
final compound 11 were obtained as a pale pink solid, pure enough
to perform the next synthetic step. Yield ¼ 88%. 1H NMR (300 MHz,
DMSO-D6)
d
ppm 1.0 (s, 6 H) 1.6 (t, J ¼ 6.2 Hz, 2 H) 2.6 (t, J ¼ 6.4 Hz,
2 H) 3.1 (s, 2 H)4.8 (d, J ¼ 6.2 Hz, 2 H) 7.3 (dd, J ¼ 7.9, 5.0 Hz,1 H)7.8(m,
1 H) 8.5 (dd, J ¼ 5.0, 1.7 Hz, 2 H) 8.6 (s, 1 H). LRMS: m/z 376 (Mþ1)þ.
6.3.6. 2,2-Dimethyl-5-morpholin-4-yl-N-(pyridin-3-ylmethyl)-
1,2,3,4-tetrahydropyrimido[40,50:4,5]furo[2,3-c]isoquinolin-8-amine
(15)
A
solution of 2,2-Dimethyl-8-[(pyridin-3-ylmethyl)amino]-
1,2,3,4-tetrahydropyrimido[40,50:4,5]furo[2,3-c]isoquinolin-5-ol 14
(25 mg, 0.07 mmol) in DMF (2 ml) was added dropwise to a suspen-
sion of sodium hydride (60% dispersion in mineral oil, 2.7 mg,
6.7 mmol) in DMF (2 ml). Then, N-phenylbistrifluorometan-
sulfonamide (2.4 mg, 0.07 mmol) was added and the reaction mixture
CHLOROFORM-D)
J ¼ 6.6 Hz, 2 H) 2.6 (t, J ¼ 6.6 Hz, 2 H) 2.8 (s, 2 H) 4.0 (s, 3 H) 4.3 (q,
J ¼ 6.6 Hz, 2 H) 4.9 (bs, 2H).
d
ppm 1.0 (s, 6H) 1.4 (t, J ¼ 6.6 Hz, 3 H) 1.6 (t,
was stirred during 1 h (its colour turning red). Morpholine (10 ml,
6.3.3. 5-Methoxy-2,2-dimethyl-1,2,3,4-tetrahydropyrimido[40,50:4,5]
furo[2,3-c]isoquinolin-8-ol (12)
Ethyl 1-amino-5-methoxy-8,8-dimethyl-6,7,8,9-tetrahydrofuro
[2,3-c]isoquinolin-2-carboxylate 11 (1.0 g, 3.3 mmol) was refluxed
0.13 mmol) was then added and the reaction was stirred overnight.
The solvent was evaporated under reduced pressure and the residue
was redissolved in chloroform and water. The organic phase was
separated and the aqueous phase was extracted twice with

J. Taltavull et al. / European Journal of Medicinal Chemistry 46 (2011) 4946e4956
4953
chloroform. The organic phases were washed with water and brine,
dried over MgSO4, filtered and evaporated. The residue was purified
by flash chromatography, eluting with CH2Cl2/MeOH 98:2. 20 mg of
the final compound 15 were obtained. Yield¼ 67%.1H NMR (400 MHz,
2 H) 3.1 (s, 2 H) 3.8 (s, 2 H) 3.9 (s, 2 H) 4.9 (d, J ¼ 5.8 Hz, 2 H) 5.2 (s,
1 H) 5.5 (s, 1 H) 7.3 (d, J ¼ 5.8 Hz, 1 H) 7.8 (d, J ¼ 6.0 Hz, 1 H) 8.6 (d,
J ¼ 4.1 Hz, 1 H) 8.6 (s, 1 H) 8.7 (s, 1 H). HPLC (Method B): 98.9%,
tR ¼ 9 min, MS(ESI) 419 m/z (M þ H)þ.
CHLOROFORM-D)
d
ppm 1.1 (s, 6 H) 1.6 (t, J ¼ 6.2 Hz, 2 H) 2.8 (t,
J ¼ 6.4Hz,2 H) 3.2 (s, 2 H)3.3 (m, 4 H) 3.9 (m, 4 H)4.9(d, J ¼ 6.2Hz,2 H)
5.5 (t, J ¼ 6.0 Hz, 1 H) 7.3 (dd, J ¼ 7.9, 5.0 Hz, 1 H) 7.7 (m, 1 H) 8.6 (dd,
J ¼ 5.0, 1.7 Hz, 1 H) 8.7 (s, 1 H) 8.7 (d, J ¼ 2.1 Hz, 1 H). HPLC: 97.4%,
tR ¼ 15.0 min, MS(ESI) 445 m/z (M þ H)þ.
6.3.12. N5-Ethyl-2,2-dimethyl-N8-(2-morpholin-4-ylethyl)-1,2,3,4-
tetrahydropyrimido[40,50:4,5]furo[2,3-c]isoquinoline-5,8-diamine
(21)
Obtained (56%) according to Scheme 2 from compound 12 and
morpholinoethanamine following the same experimental proce-
dure as for compound 13, subsequent demethylation as described
for compound 14, and final reaction with ethylamine following the
experimental procedure described for 15. 1H NMR (300 MHz,
6.3.7. 2,2-Dimethyl-5-morpholin-4-yl-N-(2-morpholin-4-ylethyl)-
1,2,3,4-tetrahydropyrimido[40,50:4,5]furo[2,3-c]isoquinolin-8-amine
(16)
Obtained (34%) from compound 14 and 2-morpholinoethanamine
CHLOROFORM-D)
d
ppm 1.1 (s, 6 H) 1.4 (t, J ¼ 7.1 Hz, 3 H) 1.7 (t,
following the experimental procedure described for 15. 1H NMR
J ¼ 6.6 Hz, 2 H) 2.4 (t, J ¼ 6.3 Hz, 2 H) 2.5 (s, 4 H) 2.7 (t, J ¼ 5.5 Hz,
2 H) 3.1 (s, 2 H) 3.6 (m, 2 H) 3.8 (m, 6 H) 4.6 (t, J ¼ 5.2 Hz, 1 H) 5.8 (t,
J ¼ 4.0 Hz, 1 H) 8.6 (s, 1 H). HPLC: 94.5%, tR ¼ 9 min, MS(ESI) 425 m/z
(M þ H)þ.
(300 MHz, CHLOROFORM-D) d ppm 1.1 (s, 6 H) 1.6 (m, 2 H) 2.5 (s, 4 H)
2.7 (m, 2 H) 2.8 (t, J ¼ 6.2 Hz, 2 H) 3.2 (s, 2 H) 3.3 (m, 4 H) 3.8 (m, 6 H)
3.9 (m, 4 H) 5.9 (t, J ¼ 4.8 Hz,1 H)8.6 (s,1 H). HPLC: 91.8%, tR ¼ 11.0 min,
MS(ESI) 467 m/z (M þ H)þ.
6.3.13. N5-Ethyl-N5,2,2-trimethyl-N8-(2-morpholin-4-ylethyl)-
1,2,3,4-tetrahydropyrimido[40,50:4,5]furo[2,3-c]isoquinoline-5,8-
diamine (22)
Obtained (41%) from compound 12 and morpholinoethanamine
following the same experimental procedure as for compound 13,
then subsequent demethylation as described for compound 14 and
final reaction with ethyl(methyl)amine following the experimental
procedure described for 15. 1H NMR (300 MHz, CHLOROFORM-D)
6.3.8. 2,2-Dimethyl-5-morpholin-4-yl-N-(2-morpholin-4-ylethyl)-
1,4-dihydro-2H-pyrano[400,300:40,50]pyrido[30,20:4,5]furo[3,2-d]
pyrimidin-8-amine (17)
Obtained (57%) from compound 6 and 2-morpholinoethanamine
following the experimental procedure described for 7. 1H NMR
(400 MHz, DMSO-D6)
d
ppm 1.3 (s, 6 H) 2.4 (m, 4 H) 2.5 (d, J ¼ 2.0 Hz,
3 H) 2.6 (t, J ¼ 6.8 Hz, 2 H) 3.2 (m, 4 H) 3.3 (s, 2 H) 3.6 (m, 4 H) 3.8 (m,
4 H) 4.7 (s, 2 H) 8.4 (s,1 H). HPLC: 92.3%, tR ¼ 8.0 min, MS(ESI) 469 m/z
(M þ H)þ.
d
ppm 1.1 (s, 6 H) 1.2 (t, J ¼ 7.1 Hz, 3 H) 1.6 (t, 2 H) 2.5 (m, 4 H)
2.7 (m, 4 H) 3.0 (s, 3 H) 3.2 (s, 2 H) 3.3 (q, J ¼ 6.9 Hz, 2 H) 3.7 (m, 6 H)
5.9 (t, J ¼ 4.8 Hz,1 H) 8.6 (s, 1 H). HPLC: 97.0%, tR ¼ 12.0 min, MS(ESI)
439 m/z (M þ H)þ.
6.3.9. 2-[(2,2-Dimethyl-5-morpholin-4-yl-1,2,3,4-tetrahydropyrimido
[40,50:4,5]furo[2,3-c]isoquinolin-8-yl)(2-morpholin-4-ylethyl)amino]
ethanol (18)
6.3.14. N5-Isopropyl-2,2-dimethyl-N8-(2-morpholin-4-ylethyl)-
1,2,3,4-tetrahydropyrimido[40,50:4,5]furo[2,3-c]isoquinoline-5,8-
diamine (23)
8-Chloro-2,2-dimethyl-5-morpholin-4-yl-1,2,3,4-tetrahydro-
pyrimido[40,50:4,5]furo[2,3-c]isoquinoline (90 mg, 0.2 mmol,
prepared according to Scheme
thylcyclohexanone 8 instead of 2,2-dimethyltetrahydropyran-4-
one 1) was suspended in ethanol (5 ml) and 2-(2-morpholin-4-
1
starting from 3,3-dime-
Obtained (18%) from compound 12 and morpholinoethanamine
following the same experimental procedure as for compound 13,
subsequent demethylation as described for compound 14 and final
reaction with isopropylamine following the experimental proce-
ylethylamino)ethanol (169 ml, 1.2 mmol) was added. The reaction
mixture was heated to 85 ꢀC for 48 h. The solvent was evaporated
under reduced pressure and the residue was passed through a silica-
gel column eluting first with dichloromethane and then successively
with the mixtures CH2Cl2/MeOH 99:1 and CH2Cl2/MeOH 98:2. 40 mg
of the final compound were obtained. Yield ¼ 16%. 1H NMR
dure described for 15. 1H NMR (300 MHz, CHLOROFORM-D)
d ppm
1.1 (s, 6 H) 1.3 (d, J ¼ 6.0 Hz, 6 H) 1.7 (t, J ¼ 6.7 Hz, 2 H) 2.4 (t,
J ¼ 6.6 Hz, 2 H) 2.5 (m, 4 H) 2.7 (m, 2 H) 3.1 (s, 2 H) 3.7 (m, 7 H) 4.5
(m, 1 H) 5.8 (t, J ¼ 4.8 Hz,1 H) 8.6 (s, 1 H). HPLC: 94.6%, tR ¼ 11.0 min,
MS(ESI) 439 m/z (M þ H)þ.
(400 MHz, CHLOROFORM-D)
d
ppm 1.1 (s, 6 H) 1.65 (t, J ¼ 7.5 Hz, 2 H)
2.6 (m, 6 H) 2.7 (m, 2 H) 2.9 (m, 2 H) 3.3 (s, 2 H) 3.35 (m, 4 H) 3.5 (m,
2 H) 3.7 (m, 5 H) 3.9 (m, 2 H) 4.0 (m, 4 H) 8.4 (s, 1 H).). HPLC: 97.4%,
tR ¼ 11.0 min LRMS: m/z 511 (Mþ1)þ.
6.3.15. 1-(3-{[5-(Ethylamino)-2,2-dimethyl-1,2,3,4-tetrahydropyri-
mido[40,50:4,5]furo[2,3-c]isoquinolin-8-yl]amino}propyl)pyrrolidin-
2-one (24)
Obtained (72%) from compound 12 and 1-(3-aminopropyl)pyr-
rolidin-2-one, following the same experimental procedure as for
compound 13, subsequent demethylation as described for
compound 14 and final reaction with ethylamine following the
experimental procedure described for 15. 1H NMR (300 MHz,
6.3.10. 2-[(2,2-Dimethyl-5-morpholin-4-yl-1,4-dihydro-2H-pyrano
[400,300:40,50]pyrido[30,20:4,5]furo[3,2-d]pyrimidin-8-yl)(2-morpholin-
4-ylethyl)amino]ethanol (19)
Obtained (57%) from the title compound (6) and 2-(2-
morpholin-4-ylethylamino)ethanol, following the experimental
procedure described for compound 7. 1H NMR (400 MHz, DMSO-
CHLOROFORM-D)
d
ppm 1.1 (s, 6 H) 1.3 (t, J ¼ 7.3 Hz, 3 H) 1.7 (t,
J ¼ 6.5 Hz, 2 H) 1.9 (m, 2 H) 2.1 (m, 2 H) 2.4 (m, 4 H) 3.1 (s, 2 H) 3.4
(m, 4 H) 3.6 (m, 4 H) 4.6 (t, J ¼ 4.3 Hz, 1 H) 5.8 (s, 1 H) 8.6 (s, 1 H).
HPLC: 97.5%, tR ¼ 14 min, MS(ESI) 437 m/z (M þ H)þ.
D6)
d ppm 1.3 (s, 6 H) 2.6 (m, 2 H) 3.2 (s, 4 H) 3.3 (s, 2 H) 3.5 (s,
4 H) 3.7 (m, 9 H) 4.0 (s, 4 H) 4.7 (s, 2 H) 4.9 (m, 2 H) 8.4 (s, 1 H).
HPLC: 98.0%, tR ¼ 8.0 min, MS(ESI) 513 m/z (M þ H)þ.
6.3.16. 1-[3-({5-[Ethyl(methyl)amino]-2,2-dimethyl-1,2,3,4-
tetrahydropyrimido[40,50:4,5]furo[2,3-c]isoquinolin-8-yl}amino)
propyl]pyrrolidin-2-one (25)
Obtained (66%) from compound 12 and 1-(3-aminopropyl)pyr-
rolidin-2-one, following the same experimental procedure as
for compound 13, subsequent demethylation as described for
compound 14 and final reaction with ethyl(methyl)amine following
6.3.11. 2-({2,2-Dimethyl-8-[(pyridin-3-ylmethyl)amino]-1,2,3,4-
tetrahydropyrimido[40,50:4,5]furo[2,3-c]isoquinolin-5-yl}amino)
ethanol (20)
Obtained (44%) from compound 14 and 2-aminoethanol
following the experimental procedure described for 15. 1H NMR
(300 MHz, CHLOROFORM-D)
d ppm 1.1 (s, 6 H) 1.7 (s, 2 H) 2.5 (s,

4954
J. Taltavull et al. / European Journal of Medicinal Chemistry 46 (2011) 4946e4956
the experimental procedure described for 15. 1H NMR (300 MHz,
CHLOROFORM-D)
J ¼ 6.5 Hz, 2 H) 1.9 (m, 2 H) 2.1 (m, 2 H) 2.4 (t, J ¼ 8.1 Hz, 2 H) 2.7 (t,
J ¼ 6.3 Hz, 2 H) 3.0 (s, 3 H) 3.2 (s, 2 H) 3.3 (q, J ¼ 7.1 Hz, 2 H) 3.4 (m,
4 H) 3.7 (q, J ¼ 6.4 Hz, 2 H) 6.0 (s, 1 H) 8.6 (s, 1 H). HPLC: 97.5%,
tR ¼ 18 min, MS(ESI) 451 m/z (M þ H)þ.
extract, 2 g peptone, 95 ml H2O, and 5 ml 40% glucose) and grown
at 30 ꢀC under orbital shaking (150 rpm) for 48 h to ensure to
reach one near OD600 ¼ 2.0. Then cells were harvested by
centrifugation at 1500 rpm for 10 min, 20 ꢀC. All supernatants
were removed and pellets resuspended in the appropriate volume
of SD (1.7 g yeast nitrogen base w/o amino acids and sulphate, 5 g
(NH4)2SO4, 30% galactose, 0.5% adenine sulphate and 050 mg/ml
d
ppm 1.1 (s, 6 H) 1.2 (t, J ¼ 7.1 Hz, 3 H) 1.6 (t,
6.3.17. 1-(3-{[5-(Isopropylamino)-2,2-dimethyl-1,2,3,4-tetrahydro-
pyrimido[40,50:4,5]furo[2,3-c]isoquinolin-8-yl]amino}propyl)
pyrrolidin-2-one (26)
Obtained (56%) according to Scheme 2 from compound 12 and
1-(3-aminopropyl)pyrrolidin-2-one, following the same experi-
mental procedure as for compound 13, subsequent demethylation
as described for compound 14 and final reaction with isopropyl-
amine following the experimental procedure described for 15. 1H
L-lysin-HCl) to obtain an OD600 between 0.4 and 0.6 and then
incubated with shaking at 30 ꢀC until an OD600 of 2.0 was
reached (usually 48 h).
Then cells were centrifuged at 4000 rpm for 20 min at 4 ꢀC and
supernatants discarded. Pellets were resuspended in 100 ml of ice-
cold sterile water and centrifuged at 4000 rpm for 20 min at 4 ꢀC.
Supernatants were discarded and the wet weight of pellets was
measured and 1.3 volume/weight of Homogenization Buffer, YHB
(2 M KCl, 0.5 M EDTA, 1 M HEPES) supplemented with DTT and
protease inhibitors added and then resuspended. Another centri-
fugation at 4000 rpm for 20 min at 4 ꢀC was carried out. Super-
natants were discarded and using a spatula yeast paste was transfer
into 10-ml syringe and yeast “noodles” were made and placed into
small plastic beaker filled with liquid nitrogen. The frozen yeast
“noodles” can be stored at ꢁ80 ꢀC until the day of extract prepa-
ration. Yeast “noodles” were poured into cooled porcelain mortar
full with liquid nitrogen and crushed under liquid nitrogen until
yeast showed a powdery smooth consistency. Then yeast/liquid
NMR (300 MHz, CHLOROFORM-D) d ppm 1.1 (s, 6 H) 1.3 (s, 6 H) 1.7
(m, 2 H) 1.9 (m, 2 H) 2.1 (m, 2 H) 2.4 (m, 4 H) 3.1 (s, 2 H) 3.4 (m, 4 H)
3.7 (m, 2 H) 4.4 (m, 2 H) 5.8 (s, 1 H) 8.6 (s, 1 H). HPLC: 95.1%,
tR ¼ 15 min, MS(ESI) 451 m/z (M þ H)þ.
6.3.18. N5-Ethyl-N5,2,2-trimethyl-N8-(pyridin-3-ylmethyl)-1,2,3,4-
tetrahydropyrimido[40,50:4,5]furo[2,3-c]isoquinoline-5,8-diamine (27)
Obtained (34%) from compound 14 and ethyl(methyl)amine
following the experimental procedure described for 15.
LRMS: m/z 417 (M þ 1)þ.
nitrogen suspension was poured into
a plastic beaker and
6.3.19. N5-Ethyl-2,2-dimethyl-N8-(pyridin-3-ylmethyl)-1,2,3,4-
tetrahydropyrimido[40,50:4,5]furo[2,3-c]isoquinoline-5,8-diamine
(28)
remaining yeast powder scraped into beaker with spatula. Dialysis
buffer (1.4 volume/weight), YDB (2 M KCl, 0.5 M EDTA, 1 M HEPES,
100% glycerol, 1 M DTT, 0.25 M PMSF, 1 mg/ml leupeptin and sup-
plemented with DTT and protease inhibitors) was then added to
resuspended yeast powder and then centrifuged at 25 000 rpm for
2 h at 4 ꢀC. Lipidic layer was removed, supernatants recovered and
transferred to dialysis tubing and dialyze overnight against 500 ml
YDB.
Obtained from compound 14 and ethylamine, following the
experimental procedure described for 15. Yield ¼ 33%. 1H NMR
(300 MHz, CHLOROFORM-D)
d
ppm 1.1 (s, 6 H) 1.3 (t, J ¼ 7.3 Hz, 3 H)
1.7 (t, J ¼ 6.5 Hz, 2 H) 2.4 (t, J ¼ 6.2 Hz, 2 H) 3.1 (s, 2 H) 3.6 (m, 2 H)
4.7 (t, J ¼ 4.5 Hz, 1 H) 4.9 (d, J ¼ 6.0 Hz, 2 H) 5.5 (t, J ¼ 5.6 Hz, 1 H) 7.3
(m, 1 H) 7.7 (d, J ¼ 8.0 Hz, 1 H) 8.6 (d, J ¼ 4.7 Hz, 1 H) 8.6 (s, 1 H) 8.7
(s, 1 H). HPLC: 98.6%, tR ¼ 12 min, MS(ESI) 403 m/z (M þ H)þ.
After dialysis supernatants were transferred to microtubes in
small aliquots (100
m
l) and quick-frozen and stored at ꢁ80 ꢀC; also
protein concentration was determined by the method of Bradford
with the Bio-Rad protein assay kit and using bovine serum albumin
(BSA) as a standard.
6.3.20. N5-Isopropyl-2,2-dimethyl-N8-(pyridin-3-ylmethyl)-1,2,3,4-
tetrahydropyrimido[40,50:4,5]furo[2,3-c]isoquinoline-5,8-diamine (29)
Obtained (36%) from compound 14 and isopropylamine,
following the experimental procedure described for 15. 1H NMR
6.4.2. PDE4 activity determination
(300 MHz, CHLOROFORM-D)
d
ppm 1.1 (s, 6 H) 1.3 (d, J ¼ 6.6 Hz, 6 H)
PDE4 activity from various human recombinant PDE4 subtypes
(PDE4B1, PDE4A4 and PDE4D3) was monitored by measuring the
hydrolysis of [3H]-cAMP to [3H]-AMP using a PDE-SPA kit from
Amersham International as previously described 17. Enzyme
1.7 (t, J ¼ 6.6 Hz, 2 H) 2.4 (t, J ¼ 6.5 Hz, 2 H) 3.1 (s, 2 H) 4.4 (m, 1 H)
4.5 (m, 1 H) 4.9 (d, J ¼ 6.0 Hz, 2 H) 5.4 (t, J ¼ 5.9 Hz, 1 H) 7.3 (dd,
J ¼ 4.5, 3.4 Hz,1 H) 7.7 (d, J ¼ 7.7 Hz,1 H) 8.6 (d, J ¼ 3.8 Hz,1 H) 8.6 (s,
1 H) 8.7 (s,1 H). HPLC: 92.7%, tR ¼ 13 min, MS(ESI) 417 m/z (M þ H)þ.
extracts (w4
(Costar 3604) for 60 min at room temperature. The assay mixture
(80 l) contains 15 nM [3H]-cAMP (1 Ci/ml) in the assay buffer
(50 mM Tris pH 7.5, 8.3 mM MgCl2, 1.7 mM EGTA) and 10 l of test
mg of protein) were incubated in “low binding” plates
6.3.21. N5-(3,5-dichloropyridin-4-yl)-2,2-dimethyl-N8-(pyridin-3-
ylmethyl)-1,2,3,4-tetrahydropyrimido[40,50:4,5]furo[2,3-c]isoquino-
line-5,8-diamine (30)
m
m
m
compound. These compounds were resuspended in DMSO (the
final DMSO concentration 5% (v/v)) at a stock concentration of
1 mM. The compounds were tested at different concentrations
Obtained (2%) from compound 14 and 4-amino-3,5-
dichloropyridine, following the experimental procedure described
for 15. 1H NMR (300 MHz, CHLOROFORM-D)
d
ppm 1.15 (s, 6 H)
varying from 10 mM to 10 pM to calculate an IC50. These dilutions
1.47e1.94 (m, 4 H) 2.77 (t, J ¼ 6.25 Hz, 2 H) 3.27 (s, 2 H) 4.86 (d,
J ¼ 5.86 Hz, 2H) 5.34e5.73 (m, J ¼ 5.86 Hz, 1 H) 6.49 (s, 1 H) 7.71 (d,
J ¼ 7.81 Hz, 1 H) 8.52 (s, 2 H) 8.66 (s, 2 H). HPLC: 99.7%,
tR ¼ 15.01 min, MS(ESI) 521 m/z (M þ H)þ.
were done in 96-well plates. In some cases, plates containing
diluted compounds were frozen before being assayed. In these
cases, the plates were thawed at room temperature and agitated for
15 min.
Hydrolysis of [3H]-cAMP was initiated by the adding 10 ml of
6.4. Biology: general
a solution containing PDE4 enzyme, and the plate was then incu-
bated under agitation at room temperature. The reaction was
stopped after 60 min (with w10e20% substrate conversion) by
6.4.1. PDE4 enzyme preparation
Yeast strains of Pichia pastoris over expressing different
recombinant PDE4 subtypes (PDE4B1, PDE4A4 and PDE4D3,
Accession numbers L20966, L20965 and L20970, respectively)
were inoculate in 500 ml flasks with 100 ml YPD (1 g yeast
addition of 50 ml Phosphodiesterase Scintillation Proximity Assay
(SPA) Beads. All reactions were carried out in duplicate. [3H]-AMP,
captured by the SPA beads, was determined by counting the plates
in a Wallac-Microbeta Trilux scintillation counter 1 h after addition

J. Taltavull et al. / European Journal of Medicinal Chemistry 46 (2011) 4946e4956
4955
of the beads, although the signal was quite stable, and samples may
be counted from 1 to 48 h after bead addition.
collected, diluted with methanol (1:1) and analyzed by HPLC-UV.
[3H]Mannitol at 115 nM is used as a positive control of the
membrane integrity. Outcome of the assay obtained include
apparent permeability for directions (PappAB and PappBA),
percentage of absorption, material balance after 3 h and stability at
37 ꢀC after 3 h.
6.4.3. LPS inducing TNF-a on human whole blood
Human whole blood of healthy donors was collected in 50 ml
Falcon tubes with heparin (5000 units/ml, Heparin Mayne 5%,
MAYNE PHARMA). LPS (Lipopolysaccharide from Escherichia coli,
Sigma, St. Louis, MO) dissolved in PBS (Dulbbecco’s Phosphate
Buffered Saline, without calcium and magnesium chloride Sigma,
St. Louis, MO) was added to the tubes to give a final concentration in
6.5. Emesis in ferret
Male castrated adult ferrets (1e2 kg) provided by Marshall
Farms (North Rose, NY, USA) were used. They were housed in
humidity and temperature controlled environment and fed on
carnivore diet (standard ferret food) with water “ad libitum”.
Ferrets were fasted overnight. On the day of the experiment, ferrets
were placed into individual observation cages. Following the oral
administration of the drug or of its vehicle (5% w/v of methylcel-
lulose with 0.1% v/v tween-80 in distilled water) they were
observed continuously for a period of 3 h during which, the number
of emetic episodes (i.e. rhythmic contraction of the abdomen with
expulsion or attempt to expel solid/liquid matter from the gastro-
intestinal tract) were recorded in a timely manner. Oral adminis-
tration of drug or its vehicle was performed using a rubber feeding
tube, size 12 Ch (Rüsch AG, Kernen, Germany).
the assay of 1
rocking. Increasing concentrations of different inhibitors (2
dissolved in 100% DMSO, were added to the 96-well plates and
200 l of blood containing LPS (except for controls) were then
m
g/ml and preincubated at 37 ꢀC for 10 min with
l),
m
m
distributed into wells, plates were shaking for 1e2 min, sealed with
aluminium foil lid (Beckman Coulter) and then incubated for 24 h at
37 ꢀC under agitation in KelvitronÒT (Heraeus Instruments).
After the 24-h period, plates were placed on ice, 50 ml of PBS
added and the reaction was stopped by centrifugation of plates at
2000 rpm (800g) at 4 ꢀC for 15 min. Serum obtained was then
subjected to ELISA or kept at ꢁ80 ꢀC until use.
6.4.4. TNF-
a determination
The quantification of TNF-
a
in human serum was performed
using commercial ELISA kit (DuoSet) obtained from R&D Systems,
Inc. and following the manufacturer’s instructions.
6.6. Metabolic stability in human microsomes
Plate preparation e First, R&D DuoSet ELISA 96-well microplates
were coated with 4.0 mg/ml mouse anti-human TNF-a diluted in
This assay is conducted at 1 mg/mL of microsomal protein in
a phosphate buffer (50 mM potassium phosphate, 1 mM EDTA and
PBS, overnight at room temperature. After washing, plates were
then blocking with PBS containing 1% BSA for a minimum of 1 h at
room temperature and then washed.
3 mM magnesium chloride, pH 7.4) containing the NCE at 5 mM, 0.8
or 1% of ACN and at 37 ꢀC. The reaction is initiated by the addition of
the NADPH generating system (1 mM NADPþ; 5 mM glucose-6-
phosphate; 1.5 UI/mL glucose-6-phosphate dehydrogenase). Incu-
Assay procedure e 100
incubated at 2 h at room temperature. After washing (ELX406
Select, BIO-TEK), biotinylated anti-hTNF- antibody was added and
incubated at room temperature for 2 h, followed by incubation with
streptavidin-peroxidase for 20 min. Detection of bound hTNF- was
carried out with 100 l of substrate solution (H2O2 and tetrame-
ml of samples or standard were added and
bation time is 30 min and the volume of incubation is 250 ml. The
a
reaction is stopped by the addition of trifluoroacetic acid 0.2% in
acetonitrile, on ice. Samples are centrifuged at 4000 rpm, at room
temperature for 10 min and analysed by HPLC-UV detection using
an on-line solid phase system (SPE, Prospekt, Spark Holland)
assisted by a sampling injector (AspecXL, Gilson) or analyzed by
HPLC-MS after protein precipitation. Incubation with testosterone
a
m
thylbenzidine) followed by measurement at 450 nm in a SPECTRA
max Plus (Molecular Devices).
These experiments were performed 2e3 times using the same
experimental design. Duplicates from each series of experiments
were averaged and expressed as hTNF-a levels in pg/ml.
(100 mM) is used as a positive control. The percentage of metabo-
lism is obtained from the relative comparison to controls where the
NADPH generated system is not added.
6.4.5. Study of transport through Caco-2 cell barrier (active or
passive transport)
Acknowledgement
To study the transport characteristics of a new NCE and account
for passive or active transport, the Caco-2 cells are used to study
permeability from the apical to basolateral side (AB transport) as
well as basolateral to apical side (BA transport). Passage numbers
from 20 to 40 are used for transport studies. Experiments are
performed using monolayers grown with the Biocoat HTS-Caco-2
Assay System (Becton & Dickinson) according to the protocol of
the kit. 24-well plate format from BIOCAT with PET membranes
We acknowledge the significant technical support provided by
Silvia Petit, Carmen Cabello, Lluis Boix, Laia Benavent, Dolores
Marin, Manuel De Luca and Laura Estrella. This work was funded by
Almirall, Spain.
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