Synthesis and biological activity of pyrido[3′,2′:4,5]thieno[3, 2-d ]pyrimidines as phosphodiesterase type 4 inhibitors

Article abstract of DOI:10.1021/jm100524j

A series of pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidines (PTP) has been synthesized and tested as phosphodiesterase IV inhibitors (PDE4), a target for the treatment of asthma and chronic obstructive pulmonary disease (COPD). Structure-activity relationships

Full text of DOI:10.1021/jm100524j

6912 J. Med. Chem. 2010, 53, 6912–6922
DOI: 10.1021/jm100524j
Synthesis and Biological Activity of Pyrido[30,20:4,5]thieno[3,2-d]pyrimidines as
Phosphodiesterase Type 4 Inhibitors
ꢀ
ꢀ
ꢁ
Joan Taltavull, Jordi Serrat, Jordi Gracia, Amadeu Gavalda, Mıriam Andres, Monica Cordoba,
Montserrat Miralpeix, Dolors Vilella, Jorge Beleta, Hamish Ryder, and Lluıs Pages*
ꢀ
ꢀ
ꢁ
Almirall, R&D Centre, Barcelona, Spain
Received March 18, 2010
A series of pyrido[30,20:4,5]thieno[3,2-d]pyrimidines (PTP) has been synthesized and tested as phos-
phodiesterase IV inhibitors (PDE4), a target for the treatment of asthma and chronic obstructive
pulmonary disease (COPD). Structure-activity relationships within this series, leading to an increase of
potency on the enzyme, are presented. The gem-dimethylcycloalkyl moiety fused to the pyridine ring
proved to be a key element of the scaffold in order to get a higher affinity in the enzyme.
Introduction
a lead compoundwhich was identified inanHTSexercise. The
optimization process was driven by a typical SAR method.
PDE4 activity from various human recombinant PDE4
subtypes (PDE4B1, PDE4A4, and PDE4D3) was monitored in
order to see whether selectivity was achieved. The compounds
were also tested to see if they were capable of blocking the pro-
duction of some pro-inflammatory cytokines such as TNFR.
The guidelines for the treatment of asthma and chronic
obstructive pulmonary disease (COPDa) show similarities in
terms of treatment options available, including the use of
inhaled corticosteroids alone or in combination with beta-2
adrenergic agonists and drugs with anticholinergic activity.1,2
However, the use of inhaled corticosteroids in asthma and
COPD still have some unmet medical needs. In asthma, the
concerns are related to the potential local and systemic dose-
related adverse effects, whereas in COPD, there is a need for a
nonsteroidal anti-inflammatory agent, because inhaled corti-
costeroids have limited effectiveness.3
In the past years, attention has been primarily focused on
cyclic nucleotide phosphodiesterase IV (PDE4) as a suitable
target for anti-inflammatory therapy in respiratory diseases.3,4
The mixed anti-inflammatory and bronchodilatory profile of
PDE4 inhibitors could allow the discovery of new agents,
steroid-sparing compounds with utility in diseases associated
with chronic airway inflammation, particularly in the manage-
ment of asthma and COPD.
PDE4 isoenzymes (PDE4A-D) are encoded by 4 genes and
more than 20 splice variants providing the basis for the
continued interest in developing selective PDE4 inhibitors for
a number of inflammatory diseases.5 Selective inhibition of
PDE4A and/or PDE4B in pro-inflammatory and immune cells
is believed to evoke the therapeutically desired effects of these
drugs.6 On one hand, aiming for PDE4B isoform-selective
inhibitors was suggested as a work-around to reduce the major
toxicity concerns, that is, emesis and the risk of cardiotoxicity
potentially related with PDE4D isoform inhibition.7,8
Chemically diverse classes of molecules have been reported as
PDE4 inhibitors, rolipram-related compounds, xanthine deri-
vatives, or nitraquazone analogues, among others.4 We report
herein some derivatives of pyrido[30,20:4,5]thieno[3,2-d]-
pyrimidines (PTP) with different substituents starting from
Chemistry
Here, we describe the synthesis of a variety of new PTP
derivatives, which were prepared with the objective of study-
ing the potential of this scaffold to deliver the desired activity
profile. These compounds were synthesized as outlined in
Schemes 1 and 2.
Ketone 1 is condensed with malononitrile in the presence of
carbon disulfide to yield heterocycle 2.9 Ketones 1 are com-
mercially available or prepared according to already known
methods.10
Reaction of compound 2 with a secondary amine, like
morpholine, yields the pyridine derivative 3.11 Subsequent
cyclocondensation of compound 3 with 2-chloroacetamide in
the presence of a base such as potassium carbonate affords the
thienopyridine compound 4, according to the procedure des-
cribed by C. Peinador et al.12 The pyridothienopyrimidinone
derivative 5 is synthesized by cyclization of intermediate 4
with triethylorthoformate. The corresponding chloropyrimidine
derivative from 5 is synthesized using phosphorus oxychloride
as solvent, and the resulting intermediate is reacted with mor-
pholinoethanamine to give the desired final compound 13.
This synthetic route is used to obtain products with amine
substituents at position 5 of the PTP scaffold. For alkyl or aryl
substituents at this position, an alternative route is depicted in
Scheme 2.
Regarding Scheme 2, ketone 6 reacts with dimethyl carbo-
nate in the presence of a strong base such as sodium hydride in
tetrahydrofurane to yield the diketone 7.13 Starting ketones
are commercially available or prepared according to already
known methods.10
ꢀ
ꢀ
*Corresponding author. Lluıs Pages, Almirall, R&D Centre, Laurea
ꢁ
Miro 408-410, 08980 Sant Feliu de Llobregat, Catalonia, Spain. Tel: þ34
932 913587. Fax: [þ34 932913420]. E-mail: lluis.pages@almirall.com.
a Abbreviations: PTP, pyrido[30,20:4,5]thieno[3,2-d]pyrimidines; COPD,
chronic obstructive pulmonary disease; PDE4, phosphodiesterase IV.
Reaction of compound 7 with cyanoacetamide in methanol
under refluxing conditions in the presence of potassium
r
pubs.acs.org/jmc
Published on Web 09/08/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6913
Scheme 1. Synthesis of Pyrido[30,20:4,5]thieno[3,2-d]pyrimidinesa
a Reagents and conditions: (a) MeOH, TEA, r.t.; (b) EtOH, reflux; (c) K2CO3, acetone, reflux; (d) reflux; (e) POCl3, reflux; (f) EtOH, reflux.
Scheme 2. Alternative Synthesis of Pyrido[30,20:4,5]thieno[3,2-d]pyrimidinesa
a Reagents and conditions: (a) NaH, THF, reflux; (b) KOH, MeOH, reflux; (c) POCl3, 170 °C; (d) K2CO3, Pd(PPh3)4, dioxane, reflux; (e) K2CO3,
EtOH, reflux; (f) reflux; (g) POCl3, reflux; (h) EtOH, reflux.

6914 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Table 1. SAR on the Pyridine Fused Ring and Selectivity Profile
Taltavull et al.
hydroxide yields the pyridine derivative 8.14 The same refer-
ence applies for theconversionof8 tothe 1,6-dichloropyridine
derivative 9 by reaction with phosphorus oxychloride.
9 is converted to 10 under classical Suzuki coupling condi-
tions by reaction with a boronic acid or the corresponding
boronate in the presence of potassium carbonate and tetrakis-
(triphenylphosphine)palladium(0) under reflux of dioxane, where
the boronic acids or their corresponding boronates are com-
mercially available or synthesized by common methodology.
Subsequent cyclocondensation of compound 10 with 2-mer-
captoacetamide in the presence of a base such as potassium
carbonate affords the thienopyridine compound 11.15
The pyridothienopyrimidinone derivative 12 is synthesized
by cyclization of intermediate 11 with triethyl orthoformate.16
The corresponding chloropyrimidine from 12 is synthesized
using phosphorus oxychloride as solvent and reacts with mor-
pholinoethanamine to give the desired final compound 28.
Results and Discussion
In Table 1, a first group of compounds is presented.
They only differ from each other in the ring fused to the
PTP central core. According to the PDE4B affinities, it
is quite evident that this is a very sensitive part of the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6915
Table 2. SAR at the R1 Substituent and Selectivity Profile
inhibitor. It is remarkable that the substitution of the fused
tetrahydropyranyl oxygen by a methylene group favors
the affinity for the enzyme: 14 (PDE4B1 IC50=2.9 nM) is
1 order of magnitude more potent than 13 (PDE4B1 IC50=
26 nM). The absence of the gem-dimethyl substitution at the
fused tetrahydropyrane cycle leads to an evident loss of
PDE4B1 activity (see 15 compared to 13), and a similar
effect is obtained with a fused cyclopentyl ring instead
of the cyclohexyl one, no matter what the position of
the gem-dimethyl substitution is (18 and 19 are 2 orders of

6916 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Table 3. SAR at the R2 Substituent and Selectivity Profile
Taltavull et al.
magnitude less potent than 14). Moreover, this gem-di-
methyl substitution seems to be the optimum pattern to fill
the putative lipophilic pocket at the PDE4 active site: if one
of the methyls is substituted by an ethyl, the PDE4B1
inhibitor activity drops from 26 nM (13) to 90 nM (17,
racemic mixture). This loss is much more pronounced in the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6917
case where the oxygen of the tetrahydropyrane ring is
replaced by an N-methyl moiety, leading to the inactive
molecule 16. For our study, both 13 and 14 will be our
reference compounds.
Besides entry 13, in this last set of compounds a couple of
examples can be identified as selective molecules, namely, 34
and 35, both an order of magnitude less potent in PDE4D3
than in PDE4B1. As we have seen in previous examples, weak
values at HWB TNF-R could be explained by a high plasma
protein binding result: 98.35% for 34; 100% for 35. On the
other hand, less weak HWB TNF-R values for com-
pounds 40 (537 nM) and 42 (401 nM) are in good correlation
with their Caco-2 data (Papp A-B = 38.7 (cm/s) ꢀ 10-6 for
40 and 24.3 (cm/s) ꢀ 10-6 for 42).
The general low potency showed at inhibiting the PDE4B1
enzyme restrained us to test these compounds in other PDE4
subtypes, except for products 13 and 14. In the particular case
of 13, a selectivity of 1 order of magnitude was achieved as is
observed by comparing the PDE4B1 data (26 nM) with that
of PDE4D3 (604 nM); nevertheless, the HWB TNF-R value
(235 nM) is weak compared to the enzymatic values, which
can be explained through the high plasma protein binding
value (92.17% for 13). In the case of 14, its low Caco-2 value
(Papp A-B = 3.1 (cm/s)ꢀ 10-6) gave us another clue to
explain such a residual HWB TNF-R activity (1368 nM).
Concerning the compounds in Table 2, they all have the
gem-dimethyl substitution at the fused cycle (either cyclohexyl
or tetrahydropyran) and the 2-morpholin-4-ylethyl moiety at
the 8-nitrogen, but differ in the substituent of the 5-position of
the PTP central core. In the cyclohexyl series (X = C),
pyrrolidine (20) and methylamino (25) were rather equivalent
to the morpholine (14). The same tendency is observed in
the pyranyl series (X = O), although the analogues in this
case tend to be less potent (compare 24 or 26 and 25). At
this position, groups like N-methylpirazine (21) or N-ben-
zylmethylamine (23) are not welcome, potency at the enzyme
dropping almost 2 orders of magnitude in the tetrahydropyr-
an series, although naked furyl ring retains initial potency
(compare 29 and 30 to 14). Finally, in the cyclohexyl series,
alkyl groups like methyl (27) or isobutyl (28) are also a
good choice, the latter being a bit more potent. In conclusion,
it seems that small lypophilic groups give good potencies,
whereas the bigger ones are susceptible to be charged and give
poorer activities.
Conclusion
From biological data and SAR analysis using a diverse set
of substituents, the pyrido[30,20:4,5]thieno[3,2-d]pyrimidine
scaffold has shownpotential to produce new PDE4 inhibitors.
The biological data reported in Tables 1 and 2 confirm the
importance of the gem-dimethylcyclohexyl group, as well as
the substitution at position 5, whereas the substitution at the
position 8 (see Table 3) has less influence in achieving signi-
ficant enzymatic activity. Further investigation is on course in
order to complete the profile of a PDE4 inhibitor candidate
for development.
Experimental Section
Chemistry: General. Reagents, starting materials, and solvents
were purchased from commercial suppliers and used as received.
2,2-Dimethyltetrahydropyran-4-one (1) was provided by Matrix
Scientific (cat. no. 021441) or by Aldrich (cat. no. 198242); 3,3-
dimethylcyclohexanone (6) was purchased from Aldrich (cat.no.
674893); 3,3-dimethylcyclopentanone was synthesized according
to Paquette et al.;16 methyl-4,4-dimethyl-2-oxo-cyclohexanecar-
boxylate (7) was synthesized as described in the literature;13
1,3-dihydroxy-6,6-dimethyl-5,6,7,8-tetrahydroisoquinoline-4-
carbonitrile (8) and 1,3-dichloro-6,6-dimethyl-5,6,7,8-tetrahy-
droisoquinoline-4-carbonitrile (9) were prepared according to
the method described by Wenkert;14 2,2-dimethylcyclopentanone
was commercially available at Aldrich (cat. no. 31,147-5); 2,2,3-
trimethyl-5-morpholin-4-yl-1,2,3,4-tetrahydropyrimido[40,50:4,5]-
thieno[2,3-c]-2,7-naphthyridin-8(9H)-one and 2-ethyl-2-methyl-
5-morpholin-4-yl-1,4-dihydro-2H-pyrano[400,300:40,50]pyrido[30,20:
4,5]thieno[3,2-d]pyrimidin-8(9H)-one were commercially available.
It is noteworthy to observe that in this set of compounds
no selectivity among PDE4 subtypes is achieved compared
to product 13 data, with 28 perhaps the only exception (39 nM
in PDE4D3 in front of 7.21 nM in PDE4B1). Besides,
acceptable Caco-2 values for compounds 25 (Papp A-B =
13.7 (cm/s) ꢀ 10-6) and 26 (Papp A-B = 21.9 (cm/s) ꢀ 10-6
)
could justify moderately active HWB TNF-R data (233 nM
and 261 nM, respectively), which is not the case of compound
28 (Papp A-B = 0.4 (cm/s) ꢀ 10-6), a disappointing but ex-
pected value taking into account its activity in the HWB
TNF-R assay (1276 nM).
€
Concentration refers to evaporation under vacuum using a Buchi
rotatory evaporator. Reaction products were purified, when
necessary, by flash chromatography on silica gel (40-63 μm) with
the solvent system indicated. 1H NMR spectra were performed in
a Varian Gemini 2000 spectrometer operating at a frequency of
200 or 300 MHz. Samples were solved in deuterated chloroform
(CDCl3) or deuterated dimethylsulfoxide (DMSO-d6). Tetra-
methylsilane (TMS) was used as reference. The following abbre-
viations 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 de-
tector and a Waters ZQ mass spectrometer detector. HPLC ana-
lysis was conducted according to the described method, with the
retention time (tR) expressed in min. UV chromatograms were
processed at 210 nm with blank subtraction. HPLC method:
Chromatography performed on a Symmetry C18 column (100 ꢀ
2.16 mm, 3.5 μm). The mobile phase, at a flow of 0.4 mL/min, was a
20 min binary gradient of water (containing 0.01 M ammonic
formate at pH 3.0) and a mixture of acetonitrile-methanol 50:50
(containing 0.01 M ammonic formate) (0-95%). The total run
time was 26 min.
According to the results shown in Table 3, substitution at
position 8 was not a very sensitive part of the scaffold, except
for piperidinethylamino (31), which loses ten times in potency
with relation to 13. The discovered tendency of the cyclohexyl
series to be more potent than the pyran one (compare 13 with
14) is no longer the case when substituting this 8-nitrogen by
pyridine-3-ylmethyl or pyridine-4ylmethyl groups (see com-
pounds from 34 to 37). Even a reversed example (2-methoxy-
benzyl) is reported (compare 38 with 39), although that pattern
is followed in the case of N-propylpyrrolidone (compare 32
with 33) and 2-(2-morpholinoethylamino)ethanol (compare 40
with 41). This part of the inhibitor may be located in a relatively
uncrowded area of the active site, thus explaining the astonish-
ing result of 42 (4.5 nM), one of the most potent compounds
even with such a bulky residue. The variability of substituents
accepted at this position (small and large residues, charged and
noncharged moieties, lipophilic and hydrophilic groups) makes
it ideal for potential modulation of the compound’s physico-
chemical properties.
6-Amino-3,3-dimethyl-8-thioxo-4,8-dihydro-1H-3H-thiopyrano-
[3,4-c]pyran-5-carbonitrile (2). 5.0 g (32.0 mmol) of compound 1
was dissolved in 4.7 mL of methanol, and 4.7 mL (48.8 mmol) of

6918 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Taltavull et al.
carbon disulfide was added in one portion. 2.6 g (39.0 mmol) of
malonodinitrile was added portionwise followed by a final addi-
tion of 1.95 mL of triethylamine. The reaction mixture was stirred
at room temperature for 48 h. An orange precipitate formed,
which was filtered and washed with diethyl ether. 3.90 g of the final
product was isolated. From the filtrates, an additional 0.89 g was
obtained after purification by flash chromatography, eluting first
with CH2Cl2 and then with a mixture of CH2Cl2/MeOH (98:2).
Yield=48%. 1H NMR (200 MHz, CDCl3) δ ppm 1.30 (s, 6 H),
2.62 (s, 2 H), 4.66 (s, 2 H), 7.91 (s, 2 H).
δ ppm 1.32 (s, 6 H), 3.20 (m, 4 H), 3.44 (s, 2 H), 3.76 (m, 4 H), 4.70
(s, 2 H), 8.33 (s, 1 H).
2,2-Dimethyl-5-isobutyl-1,2,3,4-tetrahydropyrimido[40,50:4,5]-
thieno[2,3-c]isoquinolin-8(9H)-one (12). Prepared following the
general method described for the synthesis of compound 5 but
starting from 0.31 g (0.92 mmol) of 11. Reaction time: 3 h. 0.23 g
of the final product was obtained. Yield=72%. 1H NMR (300
MHz, CDCl3) δ ppm 0.95 (d, 6H), 1.10 (s, 6 H), 1.70 (t, 2 H), 2.30
(dq, 1H), 2.80 (d, 2H), 2.90 (t, 2 H), 3.40 (s, 2H), 8.30 (s, 1H). MS
(ESI) 341 m/z (Mþ).
General Procedure for Mercaptoaminopyridinecarbonitriles
3. 6-Mercapto-3,3-dimethyl-8-morpholin-4-yl-3,4-dihydro-1H-
pyrano[3,4-c]pyridine-5-carbonitrile (3). 3.9 g (15.45 mmol) of
compound2 wassuspendedin17 mLofethanol, and6.7 mL (77.3
mmol) of morpholine was added to the resulting suspension. The
reaction mixture was refluxed under nitrogen overnight. Then, it
was allowed to reach room temperature, and the reaction mix-
ture was left in an ice bath for two hours. The solid formed
was filtered and washed twice with ethanol. After drying, 3.12 g
of the title compound was obtained as a dark solid, pure enough
General Procedure for Thienopyridines 13 and 28. 2.84 g (7.63
mmol) of 5 was suspended in phosphorus oxychloride (30 mL)
and heated to reflux for 90 min. The excess phosphorus oxychlo-
ride was evaporated under vacuum and the residue partitioned
between chloroform and a cooled 2 N aqueous solution of
NaOH. The aqueous phase was extracted twice with chloro-
form, and the organic phases were washed with water and brine,
dried over magnesium sulfate, filtered, and the solvent evapo-
rated. 2.98 g of 8-chloro-2,2-dimethyl-5-morpholin-4-yl-1,2,3,4-
tetrahydropyrimido[40,50:4,5]thieno[2,3-c]isoquinoline was ob-
tained. Yield = 100%. 1H NMR (200 MHz, CDCl3) δ ppm 1.44
(s, 6 H), 3.35 (m, 4 H), 3.57 (s, 2 H), 3.88 (m, 4 H), 4.78 (s, 2 H),
9.02 (s, 1 H).
1
to perform the next step. Yield = 66%. H NMR (200 MHz,
CDCl3) δ ppm 1.30 (s, 6 H), 2.75 (s, 2 H), 3.3 (m, 4 H), 3.75 (m,
4H), 4.5 (s, 2H).
General Procedure for Thienopyridines 4 and 11. 1-Amino-8,
8-dimethyl-5-morpholin-4-yl-8,9-dihydro-6H-pyrano[4,3-d]thieno-
[2,3-b]pyridine-2-carboxamide (4). To a suspension of 6-mercapto-
3,3-dimethyl-8-morpholin-4-yl-3,4-dihydro-1H-pyrano[3,4-c]-
pyridine-5-carbonitrile 3 (3.12 g, 10.22 mmol) in ethanol (150 mL),
potassium carbonate (3.3 g, 24.5 mmol) and 2-chloroacetamide
(1.05 g, 11.24 mmol) were added, and the reaction mixture was
then refluxed for 4 h under nitrogen. The solvent was evaporated
under vacuum and water was then added to the residue. The pre-
cipitated solid was filtered and dried, yielding 3.0 g (81%) of the
title compound. 1H NMR (200 MHz, DMSO-d6) δ ppm 1.29 (s, 6
H), 3.08 (m, 4 H), 3.20 (s, 2 H), 3.73 (m, 4H), 4.64 (s, 2 H), 6.81 (s, 2
H), 7.07 (s, 2 H).
3-Chloro-1-isobutyl-6,6-dimethyl-5,6,7,8-tetrahydroisoquino-
line-4-carbonitrile (10). 1,3-Dichloro-6,6-dimethyl-5,6,7,8-tet-
rahydroisoquinoline-4-carbonitrile 9 (0.5 g 1.96 mmol) reacted
with isobutylboronic acid (0.24 g, 2.35 mmol), potassium carbo-
nate (0.81 g, 5.88 mmol), and tetrakistriphenylphosphine palla-
dium (0.27 g, 0.19 mmol) by refluxing overnight in dioxane (10 mL).
Once the reaction was over, the solvent was evaporated under
reduced pressure and the residue worked up as usual with
dichloromethane and water. After flash chromatography eluting
with CH2Cl2/MeOH 7:3, 0.26g of the final compoundis isolated.
Yield=47%. 1H NMR (300 MHz, CDCl3) δ ppm 0.95 (d, 6H),
1.10 (s, 6 H), 1.65 (t, 2 H), 2.20 (dq, 1H), 2.65 (d, 2H), 2.75 (m, 4 H).
GC: 95%, tR = 8.1 min, MS(ESI) 276 m/z (Mþ).
8-Chloro-2,2-dimethyl-5-morpholin-4-yl-1,2,3,4-tetrahydro-
pyrimido[40,50:4,5]thieno[2,3-c]isoquinoline (1.00 g, 2.56 mmol)
is suspended in ethanol (60 mL) and (2-morpholin-4-ylethyl)-
amine (1.68 mL, 12.8 mmol) is added. The mixture is refluxed
overnight and then allowed to cool to room temperature. At
þ5 °C, a precipitate is formed, which is filtered and washed with
ethanol and ethyl ether. Once dried, it weighs 0.70 g and its 1H
NMR is consistent with the desired final compound 13. Yield =
56%. 1H NMR (300 MHz, DMSO-d6) δ ppm 1.32 (s, 6 H), 2.46
(d, J = 4.27 Hz, 3 H), 2.48-2.53 (m, 3 H), 2.56 (t, J = 6.87 Hz, 2
H), 3.11-3.24 (m, 4 H), 3.35 (s, 2 H), 3.54-3.60 (m, 4 H),
3.71-3.85 (m, 4 H), 4.70 (s, 2 H), 7.71 (t, J = 5.65 Hz, 1 H), 8.58
(s, 1 H). HPLC: 99.0%, tR = 9 min, MS(ESI) 485 m/z (Mþ1)þ.
2,2-Dimethyl-5-morpholin-4-yl-N-(2-morpholin-4-ylethyl)-1,2,
3,4-tetrahydropyrimido[40,50:4,5]thieno[2,3-c]isoquinolin-8-amine
(14). Obtained (62%) from compound 6 and (2-morpholin-4-
ylethyl)amine following the experimental procedure described
1
for compound 13. mp 210.2-210.9 °C. H NMR (300 MHz,
DMSO-d6) δ ppm 1.07 (s, 6 H), 1.55 (t, J = 6.10 Hz, 2 H), 1.55 (t,
J = 6.10 Hz, 2 H), 2.36 (s, 1 H), 2.45 (m, 2 H), 2.56 (t, J = 6.87 Hz,
2H),2.75(t, J=6.10Hz,2H),3.20(m, 4H),3.43(m,1H), 3.57(m,
2 H), 3.63 (m, 2 H), 3.78 (m, 4 H), 7.64 (t, J = 5.49 Hz, 1 H), 8.59 (s,
1 H). HPLC: 93.20%, tR = 12 min, MS(ESI) 483 m/z (Mþ1)þ.
5-Morpholin-4-yl-N-(2-morpholin-4-ylethyl)-1,4-dihydro-2H-
pyrano[400,300:40,50]pyrido[30,20:4,5]thieno[3,2-d]pyrimidin-8-amine
(15). Obtained (43%) from tetrahydropyran-4-one and (2-mor-
pholin-4-ylethyl)amine following the experimental procedure de-
1-Amino-5-isobutyl-8,8-dimethyl-6,7,8,9-tetrahydrothieno[2,3-c]-
isoquinoline-2-carboxamide (11). 3-Chloro-1-isobutyl-6,6-dimeth-
yl-5,6,7,8-tetrahydroisoquinoline-4-carbonitrile (10) (0.26 g, 0.92
mmol) is dissolved in ethanol (5 mL). To this solution, potassium
carbonate (0.31 g, 2.22 mmol) and thioacetamide (1.01 mL, 1.11
mmol) are added and the mixture is heated overnight to 100 °C.
After the usual workup, 0.31 g (yield = 99%) of the desired final
1
scribed for compound 13. mp 184.5-185.3 °C. H NMR (300
MHz, CD3OD) δ ppm 2.69 (t, J = 6.87 Hz, 2 H), 3.22 (m, 4 H),
3.76 (m, 16 H), 4.10 (t, J = 6.10 Hz, 2 H), 4.79 (m, 2 H), 8.52 (s,
1 H). HPLC: 93.6%, tR = 9 min, MS(ESI) 457 m/z (Mþ1)þ.
2,2,3-Trimethyl-5-morpholin-4-yl-N-(2-morpholin-4-ylethyl)-
1,2,3,4-tetrahydropyrimido[40,50:4,5]thieno[2,3-c]-2,7-naphthyri-
din-8-amine (16). Obtained (53%) from commercially available
8-oxo-2,2,3-trimethyl-5-morpholin-4-yl-1,2,3,4-tetrahydropyri-
mido[40,50:4,5]thieno[2,3-c]-2,7-naphthyridine and (2-morpho-
lin-4-ylethyl)amine following the experimental procedure de-
1
compound is obtained as a yellow solid. H NMR (300 MHz,
CDCl3) δ ppm 0.95 (d, 6H), 1.10 (s, 6 H), 1.65 (t, 2 H), 2.20 (dq,
1H), 2.65 (d, 2H), 2.80 (t, 2 H), 3.05 (s, 2H), 5.30 (br.s, 2H), 6.50
(br.s, 2H).
1
General Procedure for Thienopyridines 5 and 12. 2,2-Dimethyl-
5-morpholin-4-yl-1,4-dihydro-2H-pyrano[400,300:40,50]pyrido[30,20:
4,5]thieno[3,2-d]pyrimidin-8(9H)-one (5). 3.0 g (8.3 mmol) of 4
was suspended in triethyl orthoformate (50 mL) and p-toluensul-
fonic acid hydrate (0.16 g, 0.83 mmol) was added. This mixture
was heated under reflux overnight. The reaction mixture was
then allowed to reach room temperature and it was left in an ice
bath for two hours. The precipitated that formed was filtered
and washed with ethyl ether. After drying, 2.8 g of the final pro-
duct was obtained. Yield = 92%. 1H NMR (200 MHz, DMSO-d6)
scribed for compound 13. mp 103.2-105.1 °C. H NMR (300
MHz, DMSO-d6) δ ppm 1.13 (s, 6 H), 2.31 (m, 2 H), 2.50 (m,
4 H), 2.57 (m, 1 H), 3.20 (s, 4 H), 3.33 (d, J = 7.02 Hz, 5 H), 3.57
(m, 4 H), 3.63 (s, 4 H), 3.77 (d, J = 5.19 Hz, 4 H), 8.59 (s, 1 H).
HPLC: 95.1%, tR = 6 min, MS(ESI) 498 m/z (Mþ1)þ.
2-Ethyl-2-methyl-5-morpholin-4-yl-N-(2-morpholin-4-ylethyl)-
1,4-dihydro-2H-pyrano[400,300:40,50]pyrido[30,20:4,5]thieno[3,2-d]-
pyrimidin-8-amine (17). Obtained (49%) from commercially avai-
lable 8-oxo-2-ethyl-2-methyl-5-morpholin-4-yl-1,4-dihydro-2H-
pyrano[400,300:400,50]pyrido[30,20:4,5]thieno[3,2-d]pyrimidine and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6919
(2-morpholin-4-ylethyl)amine following the experimental proce-
dure described for compound 13. mp 92.5-93.8 °C. H NMR
(300 MHz, DMSO-d6) δ ppm 0.92 (t, J = 7.48 Hz, 3 H), 2.45 (d,
J = 4.27 Hz, 2 H), 2.51 (m, 4 H), 2.56 (t, J = 7.02 Hz, 2 H), 3.18
(m, 4 H), 3.33 (d, J = 7.02 Hz, 3 H), 3.50 (d, J = 3.66 Hz, 2 H),
3.57 (m, 4 H), 3.67 (m, 2 H), 3.76 (s, 4 H), 4.67 (d, J = 7.32 Hz,
2 H), 7.73 (m, 1 H), 8.58 (s, 1 H). HPLC (Method B): 96.3%,
4.59 (s, 3 H), 5.55 (br.s, 1 H), 8.67 (s, 1 H). HPLC: 97.9%, tR
8 min, MS(ESI) 429 m/z (Mþ1)þ.
=
1
N5,2,2-Trimethyl-N8-(2-morpholin-4-ylethyl)-1,2,3,4- tetra-
hydropyrimido[40,50:4,5]thieno[2,3-c]isoquinolin-5,8-diamine (25).
Obtained (51%) from compound 6 and methylamine following the
experimental procedure described for compound 13. mp 1H NMR
(300 MHz, CDCl3) δ ppm 1.07 (s, 6 H), 1.70 (br.s, 2 H), 2.43 (br.s,
4H),2.55(br.s,3H),2.71(br.s,2H),3.16(d,J=3.30Hz, 3H),3.37
(br.s, 2 H), 3.54-3.90 (m, 5 H), 4.67 (br.s, 1 H), 5.47 (br.s, 1 H), 8.68
(s, 1 H). HPLC: 97.6%, tR = 11 min, MS (ESI) 427 m/z (Mþ1)þ.
N5,N5,2,2-Tetramethyl-N8-(2-morpholin-4-ylethyl)-1,4-dihydro-
2H-pyrano[400,300:40,50]pyrido[30,20:4,5] thieno[3,2-d]pyrimidine-5,8-
diamine (26). Obtained (83%) from compound 1 and (2-morpho-
lin-4-ylethyl)-pyridin-2-ylmethylamine following the experimental
procedure described for compound 13. mp 195.1-195.8 °C. 1H
NMR (300 MHz, CDCl3) δ ppm 1.43 (s, 6 H), 2.55 (s, 4 H), 2.72
(t, J = 6.04 Hz, 2 H), 2.98 (s, 6 H), 3.58 (s, 2 H), 3.74 (m, 6 H), 4.80
(s, 2 H), 5.56 (m, 1 H), 8.70 (s, 1 H). HPLC: 99.8%, tR=10 min,
MS (ESI) 443 m/z (Mþ1)þ.
t
R = 10 min, MS(ESI) 499 m/z (Mþ1)þ.
2,2-Dimethyl-4-morpholin-4-yl-N-(2-morpholin-4-ylethyl)-2,3-
dihydro-1H-cyclopenta[40,50]pyrido[30,20:4,5]thieno[3,2-d]pyrimidin-
7-amine (18). Obtained (39%) from 3,3-dimethylcyclopentanone
and (2-morpholin-4-ylethyl)amine following the general experi-
1
mental procedure described for compound 13. H NMR (300
MHz, CDCl3) δ ppm1.2 (m, 6 H), 2.6 (s, 4 H), 2.7 (t, J = 5.9 Hz, 2
H), 2.8 (s, 2 H), 3.4 (s, 2 H), 3.6 (m, 4 H), 3.7 (m, 6 H), 3.9 (m, 4 H),
5.6 (m, 1 H), 8.7 (s, 1 H). HPLC: 98.9%, tR = 11 min, MS(ESI)
469 m/z (Mþ1)þ.
1,1-Dimethyl-4-morpholin-4-yl-N7-(2-morpholin-4-ylethyl)-2,3-
dihydro-1H-cyclopenta[40,50]pyrido[30,20:4,5]thieno[3,2-d]pyrimidin-
7-amina (19). Obtained (81%) from 2,2-dimethylcyclopentanone
and (2-morpholin-4-ylethyl)amine following the general experi-
2,2,5-Trimethyl-N-(2-morpholin-4-ylethyl)-1,2,3,4- tetrahydro-
pyrimido[40,50:4,5]thieno[2,3-c]isoquinolin-8-amine (27). Obtained
(50%) from compound 6 and methylboronic acid following the
experimental procedure described for compound 28. mp 1H NMR
(400 MHz, CDCl3) δ ppm 1.09 (s, 6 H), 1.63-1.77 (m, 2 H), 2.49-
2.58 (m, 4 H), 2.62 (s, 3 H), 2.68-2.74 (m, 2 H), 2.75-2.86 (m,
2 H), 3.45-3.55 (m, 2 H), 3.64-3.87 (m, 6 H), 5.44-5.67 (m, 1 H),
8.68-8.83 (m, 1 H). HPLC (Method B): 95.8%, tR = 12 min, MS
(ESI) 412 m/z (Mþ1)þ.
1
mental procedure described for compound 13. H NMR (400
MHz, DMSO-d6) δ ppm 1.68 (s, 3 H), 1.99 (t, J = 7.26 Hz, 2 H),
2.45 (d, J=4.15Hz,4H),2.53-2.61 (m, 2 H), 2.98 (t, J=7.26Hz,2
H), 3.31 (s, 3 H), 3.37-3.47 (m, 4 H), 3.54-3.59 (m, 4 H), 3.60-3.68
(m, 2 H), 3.70-3.80 (m, 4 H), 7.55 (t, J = 5.39 Hz, 1 H), 8.56 (s,
1 H). HPLC: 99.1%, tR = 12 min, MS (ESI) 469 m/z (Mþ1)þ.
2,2-Dimethyl-N-(2-morpholin-4-ylethyl)-5-pyrrolidin-1-yl-1,2,
3,4-tetrahydropyrimido[40,50:4,5]thieno[2,3-c]isoquinolin-8-amine
(20). Obtained (50%) from compound 6 and (2-morpholin-4-
ylethyl)amine following the experimental procedure described for
compound 13. mp 173.3-174.0 °C. 1H NMR (300 MHz, DMSO-
d6) δ ppm 1.06 (s, 6 H), 1.50 (t, J = 6.10 Hz, 2 H), 1.90 (m, 4 H),
2.44 (m, 4 H), 2.56 (m, 2 H), 2.77 (t, J=5.95 Hz, 2 H), 3.30 (s,
2 H), 3.60 (m, 10 H), 7.41 (t, J = 5.49 Hz, 1 H), 8.52 (s, 1 H).
HPLC: 98.6%, tR=15 min, MS(ESI) 467 m/z (Mþ1)þ.
2,2-Dimethyl-5-(4-methylpiperazin-1-yl)-N-(2-morpholin-4-
ylethyl)-1,4-dihydro-2H-pyrano[400,300:40,50]pyrido[30,20:4,5]thieno-
[3,2-d]pyrimidin-8-amine (21). Obtained (40%) from compound 5
and N-methylpyperazine following the experimental procedure
described for compound 13. 1H NMR (300 MHz, CDCl3) δ ppm
1.42 (s, 6 H) 2.40, (s, 3 H), 2.54 (br.s, 4 H), 2.64 (br.s, 4 H), 2.72 (s,
2 H), 3.32 (br.s, 4 H), 3.60 (s, 2 H), 3.78 (br.s, 6 H), 4.78 (s, 2 H),
5.61 (br.s, 1 H), 8.71 (s, 1 H). HPLC: 95.6%, tR = 6 min, MS(ESI)
498 m/z (Mþ1)þ.
5-Isobutyl-2,2-dimethyl-N-(2-morpholin-4-ylethyl)-1,2,3,4-tetra-
hydropyrimido[40,50:4,5]thieno[2,3-c]isoquinolin-8-amine (28). Ob-
tained (68%) from compound 12 and (2-morpholin-4-ylethyl)-
amine following the experimental procedure described for
compound 13. 1H NMR (400 MHz, CDCl3) δ ppm 1.12 (s, 6 H),
1.58 (s, 4 H), 1.87 (t, J = 5.67 Hz, 2 H), 2.07 (quin, J = 7.53 Hz,
2 H), 2.48 (t, J = 8.22 Hz, 3 H), 2.77 (t, J = 6.46 Hz, 2 H), 3.30 (d,
J = 4.30 Hz, 4 H), 3.37-3.50 (m, 6 H), 3.66 (q, J = 6.13 Hz, 2 H),
3.88 (d, J = 4.70 Hz, 2 H), 6.29 (t, J = 6.06 Hz, 1 H), 8.69 (s,
1 H). HPLC: 97.7%, tR = 15 min, MS (ESI) 454 m/z (Mþ1)þ.
5-(2-Furyl)-2,2-dimethyl-N-(2-morpholin-4-ylethyl)-1,2,3,4-
tetrahydropyrimido[40,50:4,5]thieno[2,3-c]isoquinolin-8-amine (29).
Obtained (75%) from compound 6 and 2-furanylboronic acid
following the experimental procedure described for compound
28. 1H NMR (400 MHz, CDCl3) δ ppm 1.14 (s, 6 H), 1.57 (s, 10
H), 1.72 (t, J = 6.65 Hz, 1 H), 2.56 (br.s, 2 H), 2.73 (t, J = 6.06
Hz, 2 H), 3.15 (s, 1 H), 3.76 (d, J = 5.09 Hz, 2 H), 5.62 (br.s, 1 H),
6.61 (dd, J = 3.33, 1.76 Hz, 1 H), 7.12 (d, J = 3.52 Hz, 1 H), 7.68
(d, J = 1.96 Hz, 1 H), 8.77 (s, 1 H). HPLC: 95.1%, tR = 13 min,
MS (ESI) 464 m/z (Mþ1)þ.
2,2-Dimethyl-5-(4-methylpiperazin-1-yl)-N-(2-morpholin-4-
ylethyl)-1,2,3,4-tetrahydropyrimidino[40,50:4,5]thieno[2,3-c]iso-
quinolin-8-amine (22). Obtained (50%) from compound 6 and
N-methylpiperazine following the experimental procedure
described for compound 13. mp 1H NMR (300 MHz, CDCl3) δ
ppm 1.13 (s, 6 H), 1.63 (s, 2 H), 2.39 (s, 3 H), 2.55 (br.s, 4 H), 2.62
(br.s, 4 H), 2.71 (s, 2 H), 3.34 (br.s, 4 H), 3.45 (s, 2 H), 3.74 (br.s,
8 H), 5.55 (s, 1 H), 8.72 (s, 1 H). HPLC: 100.0%, tR = 7 min,
MS(ESI) 496 m/z (Mþ1)þ.
5-(3-Furyl)-2,2-dimethyl-N-(2-morpholin-4-ylethyl)-1,2,3,4-
tetrahydropyrimido[40,50:4,5]thieno[2,3-c]isoquinolin-8-amine (30).
Obtained (79%) from compound 6 and 3-furanylboronic acid
following the experimental procedure described for compound
1
28. H NMR (400 MHz, CDCl3) δ ppm 1.13 (s, 6 H) 1.72 (t,
J=6.65 Hz, 2 H) 2.56 (d, J=4.30 Hz, 2 H) 2.73 (t, J=6.06 Hz,
2 H) 2.98 (t, J=6.65Hz, 2 H) 3.47 (t, J=7.04 Hz, 1 H) 3.57(s, 2 H)
3.65 - 3.86 (m, 7 H) 5.60 (br. s., 1 H) 7.12 (d, J=1.96 Hz, 1 H) 7.54
(d, J=1.57 Hz, 1 H) 7.97 (s, 1 H) 8.77 (s, 1 H). HPLC: 97.5%,
N5-Benzyl-N5,2,2-Trimethyl-N-(2-morpholin-4-ylethyl)-1,2,3,
4-tetrahydropyrimido[40,50:4,5]thieno[2,3-c]isoquinolin-8-amine
(23). Obtained (67%) from compound 6 and benzylmethylamine
following the experimental procedure described for compound 13.
1H NMR (300 MHz, CDCl3) δ ppm 1.12 (s, 6 H), 1.59 (t, J = 6.32
Hz, 4 H), 2.56 (s, 3 H), 2.73 (s, 2 H), 2.84 (t, J = 6.59 Hz, 2 H), 2.88
(s, 4 H), 3.45 (s, 2 H), 3.77 (s, 4 H), 4.49 (s, 2 H), 5.54 (s, 1 H),
7.27-7.48 (m, 5 H), 8.72 (s, 1 H). HPLC: 97.1%, tR = 18 min,
MS(ESI) 517 m/z (Mþ1)þ.
t
R = 13 min, MS (ESI) 464 m/z (Mþ1)þ
2,2-Dimethyl-5-morpholin-4-yl-N-(2-piperidin-1-ylethyl)-1,4-di-
hydro-2H-pyrano[400,300:40,50]pyrido[30,20:4,5]thieno[3,2-d]pyrimidin-
8-amine (31). was obtained (81%) from compound 5and (piperidin-
1-yl-ethyl)amine following the experimental procedure described
for compound 13. 1H NMR (300 MHz, DMSO-d6) δppm 1.32 (s, 6
H) 1.37 (br. s., 2 H) 1.47 (br. s., 4 H) 2.41 (br. s., 6 H) 3.19 (br. s., 4 H)
3.50 (s, 2 H) 3.62 (br. s., 2 H) 3.76 (br. s., 4 H) 4.70 (s, 2 H) 7.56 - 7.79
(m, 1 H) 8.58 (s, 1 H). HPLC: 97.3%, tR = 11 min, MS (ESI) 483
m/z (Mþ1)þ
N5,2,2-trimethyl-N8-(2-morpholin-4-ylethyl)-1,4-dihydro-2H-
pyrano[400,300:40,50]pyrido[30,20:4,5]thieno[3,2-d]pyrimidin-5,8-dia-
mine (24). Obtained (12%) from compound 5 and methylamine
following the experimental procedure described for compound
13. 1H NMR (300 MHz, CDCl3) δ ppm 1.38 (s, 6 H), 2.57 (d, J =
4.12 Hz, 4 H), 2.72 (t, J = 5.91 Hz, 2 H), 3.15 (d, J = 4.94 Hz, 3 H),
3.38-3.59 (m, 2 H), 3.64-3.95 (m, 5 H), 4.24 (d, J= 4.94 Hz, 1H),
1-{3-[(2,2-Dimethyl-5-morpholin-4-yl-1,4-dihydro-2H-pyrano-
[400,300:40,50]pyrido[30,20:4,5] thieno[3,2-d]pyrimidin-8-yl)amino]-
propyl}pyrrolidin-2-one (32). Obtained (80%) from compound

6920 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Taltavull et al.
5and 1-(3-aminopropyl)-pyrrolidin-2-one following the experimen-
tal procedure described for compound 13. mp 215.9-216.7 °C. 1H
NMR (400 MHz, DMSO-d6) δ ppm 1.3 (s, 6 H), 1.8 (m, 2 H), 1.9
(m, 2 H), 2.2 (t, J = 8.2 Hz, 2 H), 3.2 (m, 4 H), 3.3(m, 2 H), 3.4 (m,
2 H), 3.5 (m, 4 H), 3.8 (m, 4 H), 4.7 (s, 2 H), 7.7 (t, J = 5.5 Hz, 1
H), 8.6 (s, 1 H). HPLC: 98.2%, tR = 15 min, MS (ESI) 497 m/z
(Mþ1)þ.
1H NMR (300 MHz, CDCl3) δ ppm 1.13 (s, 6 H), 1.50-1.68
(m, 4 H), 2.77 (t, J = 6.46 Hz, 2 H), 3.30 (d, J = 4.67 Hz, 4 H),
3.45 (s, 2 H), 3.89 (d, J = 4.94 Hz, 5 H), 4.88 (d, J = 5.77 Hz,
2 H), 5.16-5.39 (m, 1 H), 6.93 (d, J = 7.97 Hz, 2 H), 7.21-
7.35 (m, 1 H), 7.39 (d, J=7.69 Hz, 1 H), 8.75 (s, 1 H). HPLC:
98.3%, tR=11 min, MS (ESI) 427 m/z (Mþ1)þ.
2-[(2,2-Dimethyl-5-morpholin-4-yl-1,4-dihydro-2H-pyrano[400,
300:40,50]pyrido[30,20:4,5]thieno[3,2-d]pyrimidin-8-yl)(2-morpholin-
4-ylethyl)amino]ethanol (40). Obtained (41%) from compound 5
and 2-(2-morpholin-4-ylethylamino)-ethanol following the ex-
perimental procedure described for compound 13. mp 111.9-
112.6 °C. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.3 (s, 6 H), 2.5
(m, 4 H), 2.6 (t, J = 6.8 Hz, 2 H), 3.2 (m, 4 H), 3.5 (s, 2 H), 3.6 (m,
4 H), 3.7 (m, 6 H), 3.9 (t, J = 6.1 Hz, 2 H), 3.9 (t, J = 6.8 Hz, 2 H),
4.7 (s, 2 H), 5.0 (t, J = 5.7 Hz, 1 H), 8.6 (s, 1 H). HPLC: 97.8%,
1-(3-{[5-Morpholin-4-yl)-2,2-dimethyl-1,2,3,4-tetrahydropyri-
mido[40,50:4,5]thieno[2,3-c]isoquinolin-8-yl]amino}propyl)pyrrolilydin-
2-one (33). Obtained (81%) from compound 6 and 1-(3-aminopro-
pyl)pyrrolidin-2-one following the experimental procedure de-
1
scribed for compound 13. H NMR (400 MHz, CDCl3) δ ppm
1.12 (s, 6 H), 1.59-1.63 (m, 2 H), 1.81-1.93 (m, 2 H), 1.99-2.14
(m, 2 H), 2.48 (t, J = 8.22 Hz, 2 H), 2.77 (t, J = 6.46 Hz, 2 H),
3.25-3.32 (m, 4 H), 3.38-3.52 (m, 6 H), 3.66 (q, J= 6.13 Hz, 2H),
3.85-3.92 (m, 4 H), 6.29 (t, J =6.06Hz, 1H), 8.69(s, 1H). HPLC:
96.9%, tR = 19 min, MS (ESI) 495 m/z (Mþ1)þ.
t
R = 9 min, MS (ESI) 529 m/z (Mþ1)þ.
2-[(2,2-Dimethyl-5-morpholin-4-yl-1,2,3,4-tetrahydropyrimido-
[40,50:4,5]thieno[2,3-c]isoquinolin-8-yl)(2-morpholin-4-ylethyl)-
amino]ethanol (41). Obtained (57%) from compound 6 and
2-(2-morpholin-4-ylethylamino)ethanol following the experi-
2,2-Dimethyl-5-morpholin-4-yl-N-(pyridin-3-ylmethyl)-1,4-di-
hydro-2H-pyrano[400,300:40,50]pyrido[30,20:4,5]thieno[3,2-d]pyri-
midin-8-amine (34). Obtained (76%) from compound 5 and
pyridin-3-ylmethylamine following the experimental procedure
described for compound 13. mp 260.6-261.7 °C. 1H NMR (300
MHz, DMSO-d6) δ ppm 1.32 (s, 6 H), 3.19 (m, 4 H), 3.50 (s, 2 H),
3.76 (m, 4 H), 4.70 (s, 2 H), 4.77 (d, J = 5.80 Hz, 2 H), 7.35 (dd,
J = 7.63, 4.58 Hz, 1 H), 7.77 (m, 1 H), 8.39 (t, J = 5.80 Hz, 1 H),
8.46 (dd, J = 4.58, 1.53 Hz, 1 H), 8.59 (s, 1 H), 8.61 (d, J = 1.53
Hz, 1 H). HPLC: 98.3%, tR = 14 min, MS (ESI) 463 m/z (Mþ1)þ.
2,2-Dimethyl-5-morpholin-4-yl-N-(pyridin-3-ylmethyl)-1,2,3,
4-tetrahydropyrimido[40,50:4,5]thieno[2,3-c]isoquinolin-8-amine
(35). Obtained (84%) from compound 6 and (pyridin-3-yl-
methyl)amine following the experimental procedure described
for compound 13. mp 248.8-249.3 °C. 1H NMR (300 MHz,
DMSO-d6) δ ppm 1.07 (s, 6 H), 1.55 (t, J = 5.80 Hz, 2 H), 2.76 (s,
2 H), 3.21 (s, 4 H), 3.36 (m, 2 H), 3.78 (s, 4 H), 4.77 (d, J = 5.80 Hz,
2 H), 7.36 (m, 1 H), 7.77 (d, J = 7.94 Hz, 1 H), 8.32 (d, J = 6.10
Hz, 1 H), 8.46 (d, J = 4.88 Hz, 1 H), 8.61 (m, 2 H). HPLC: 99.8%,
tR=18 min, MS (ESI) 461 m/z (Mþ1)þ.
1
mental procedure described for compound 13. H NMR (300
MHz, CDCl3) δ ppm 1.1 (s, 6 H), 1.6 (t, J = 6.3 Hz, 2 H), 2.6 (s,
4 H), 2.8 (t, J = 6.3 Hz, 2 H), 2.9 (m, 2 H), 3.3 (m, 4 H), 3.5 (s,
3 H), 3.7 (m, 4 H), 3.9 (m, 4 H), 4.0 (m, 6 H), 8.6 (s, 1 H). HPLC:
96.8%, tR=12 min, MS (ESI) 527 m/z (Mþ1)þ.
2,2-Dimethyl-5-morpholin-4-yl-N-(2-morpholin-4-ylethyl)-N-
(pyridin-3-ylmethyl)- 1,4-dihydro-2H-pyrano[400,300:40,50]pyrido-
[30,20:4,5]thieno[3,2-d]pyrimidin-8-amine (42). Obtained (14%)
from compound 5 and (2-morpholin-4-ylethyl)-pyridin-2-yl-
methylamine following the experimental procedure described
1
for compound 13. mp 149.8-150.3 °C. H NMR (400 MHz,
DMSO-d6) δ ppm 1.3 (s, 6 H), 2.4 (s, 6 H), 2.7 (t, J = 6.8 Hz, 2 H),
3.2 (m, 4 H), 3.5 (d, J = 8.7 Hz, 4 H), 3.8 (m, 4 H), 3.9 (t, J = 6.6
Hz, 2 H), 4.7 (s, 2 H), 5.2 (s, 2 H), 7.3 (dd, J = 7.5, 4.6 Hz, 1 H),
7.7 (d, J = 8.3 Hz, 1 H), 8.5 (m, 1 H), 8.6 (d, J = 1.7 Hz, 1 H), 8.6
(s, 1 H). HPLC: 96.6%, tR=10 min, MS (ESI) 576 m/z (Mþ1)þ.
Biology: General. PDE4 Enzyme Preparation. Yeast strains
of P. pastoris overexpressing different recombinant PDE4 sub-
types (PDE4B1, PDE4A4, and PDE4D3, Accession numbers
L20966, L20965, and L20970, respectively) were inoculated in
500 mL flasks with 100 mL YPD (1 g yeast 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 one reaching near
OD600 = 2.0. Then, cells were harvested by centrifugation at
1500 rpm for 10 min at 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 sulfate, 5 g (NH4)2SO4, 30%
galactose, 0.5% adenine sulfate, and 050 mg/mL L-lysine-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
centrifugation at 4000 rpm for 20 min at 4 °C was carried out.
Supernatants were discarded and using a spatula yeast paste were
transfered into a 10 mL syringe, and yeast “noodles” were made
and placed into small plastic beakers filled with liquid nitrogen.
The frozen yeast “noodles” can be stored at -80 °C until the day
of extract preparation. Yeast “noodles” were poured into a cooled
porcelain mortar full of liquid nitrogen and crushed under liquid
nitrogen until yeast showed a powdery smooth consistency. Then,
the yeast/liquid nitrogen suspension was poured into a plastic
beaker and the remaining yeast powder scraped into a 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 supplemented with DTT and
2,2-Dimethyl-5-morpholin-4-yl-N-(pyridin-4-ylmethyl)-1,4-di-
hydro-2H-pyrano[400,300:40,50]pyrido[30,20:4,5]thieno[3,2-d]pyri-
midin-8-amine (36). Obtained (34%) from compound 5 and
pyridin-4-ylmethylamine following the experimental procedure
described for compound 13. mp 238.0-239.7 °C. 1H NMR (300
MHz, DMSO-d6) δ ppm 1.32 (s, 6 H), 3.20 (m, 4 H), 3.50 (s, 2 H),
3.77 (m, 4 H), 4.71 (s, 2 H), 4.77 (d, J = 5.80 Hz, 2 H), 7.33 (d, J =
6.10 Hz, 2 H), 8.44 (t, J = 6.10 Hz, 1 H), 8.49 (m, 2 H), 8.56 (s,
1 H). HPLC: 97.8%, tR=12 min, MS (ESI) 463 m/z (Mþ1)þ.
2,2-Dimethyl-5-morpholin-4-yl-N-(pyridin-4-ylmethyl)-1,2,3,
4-tetrahydropyrimido[40,50:4,5]thieno[2,3-c]isoquinolin-8-amine
(37). Obtained (45%) from compound 6 and (pyridin-4-yl-
methyl)amine following the experimental procedure described
1
for compound 13. mp 239.9-240.8 °C. H NMR (300 MHz,
DMSO-d6) δppm 1.06(s, 6 H), 1.54 (t, J = 5.49Hz, 2 H), 2.76 (m,
2 H), 3.22 (s, 4 H), 3.38 (m, 2H), 3.78 (s, 4 H), 4.76 (d, J = 5.49 Hz,
2 H), 7.33 (d, J = 4.88 Hz, 2 H), 8.37 (t, J = 5.49 Hz, 1 H), 8.49 (d,
J = 5.49 Hz, 2 H), 8.56 (s, 1 H). HPLC: 98.4%, tR=16 min, MS
(ESI) 461 m/z (Mþ1)þ.
N-(2-Methoxybenzyl)-2,2-dimethyl-5-morpholin-4-yl-1,4-di-
hydro-2H-pyrano[400,300:40,50]thieno[3,2-d]pyrimidin-8-amine (38).
Obtained (74%) from compound 5 and 2-methoxybenzylamine
following the experimental procedure described for compound
13. 1H NMR (300 MHz, DMSO-d6) δ ppm 1.32 (s, 6 H), 3.20 (d,
J = 4.58 Hz, 4 H), 3.76 (d, J = 4.58 Hz, 4 H), 3.85 (s, 4 H), 4.61-
4.80 (m, 5 H), 6.87 (t, J= 7.48Hz, 1 H), 7.01(d, J = 7.63 Hz, 1H),
7.15 (d, J = 7.32 Hz, 1 H), 7.23 (t, J = 7.78 Hz, 1 H), 8.20 (t, J =
5.65 Hz, 1 H), 8.54 (s, 1 H). HPLC: 99.3%, tR = 21 min, MS (ESI)
490 m/z (Mþ1)þ.
N-(2-Methoxybenzyl)-2,2-dimethyl-5-morpholin-4-yl-1,2,3,4-
tetrahydropyrimido[40,50:4,5]thieno[2,3-c]isoquinolin-8-amine (39).
Obtained (90%) from compound 6 and 2-methoxybenzylamine
following the experimental procedure described for compound 13.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6921
protease inhibitors) was then added to resuspended yeast powder
and then centrifuged at 25 000 rpm for 2 h at 4 °C. The lipidic layer
was removed; supernatants were recovered and transferred to
dialysis tubing and dialyzed overnight against 500 mL YDB.
After dialysis, supernatants were transferred to microtubes in
small aliquots (100 μL) 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.
PDE4 Activity Determination. 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 extracts (∼4 μg
of protein) were incubated in “low binding” plates (Costar 3604)
for 60 min at room temperature. The assay mixture (80 μL)
contains 15 nM [3H]-cAMP (1 μCi/mL) in the assay buffer
(50 mM Tris pH 7.5, 8.3 mM MgCl2, 1.7 mM EGTA) and 10 μL
of testcompound. These compounds wereresuspendedin DMSO
(the final DMSO concentration 5% (v/v)) at a stock concentra-
tion of 1 mM. The compounds were tested at different concen-
trations varying from 10 μM to 10 pM to calculate an IC50. These
dilutions were done in 96-well plates. In some cases, plates con-
taining diluted compounds were frozen before being assayed. In
these cases, the plates were thawed at room temperature and agi-
tated for 15 min.
Detection of bound hTNF-R was carried out with 100 μL of
substrate solution (H2O2 and tetramethylbenzidine) followed by
measurement at 450 nm in a SPECTRA max Plus (Molecular
Devices).
These experiments were performed 2-3 times using the same
experimental design. Duplicates from each series of experiments
were averaged and expressed as hTNF-R levels in pg/mL.
In Vitro Rat, Mouse, and Human Plasma Protein Binding.
Plasma protein binding is typically determined using the
ultrafiltration technique. The NCE solution is first prepared
by adding an aliquot of concentrated NCE (typically 5 μL of
100 μg/mL in DMSO) to 1 mL of plasma to achieve the desired
final concentration of 2.5 μg/mL and 1% organic solvent. The
plasma is mixed and incubated for 2 h at 37 °C in a shaker water
bath (85 U/min).
Each solution is then loaded onto the Centrifree filter (YM-
30, Millipore) and centrifuged at 2000 g for 15 min at 37 °C. An
aliquot of the ultrafiltrate is diluted (1:1) with acetonitrile/water
and analyzed by HPLC-MS. A blank incubation without plas-
ma is performed in parallel to account for the unspecific binding.
Samples are quantified using 25 and 250 ng/mL standard points.
Study of Transport through Caco-2 Cell Barrier (Active or
Passive Transport). 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 (1 μm pore size)
with a layer of fibrillar collagen are also used for the experi-
ments. Briefly, Caco-2 cells are seeded at 500 000 cell/well in
Mitoþ Serum Extender supplemented medium incubated
for 56 h. Then, medium is changed to Mitoþ supplemented
Entero-STIM Differentiation Media (ESMþ) and incubated for
another 48 h. Then, ESMþ is renewed by fresh ESMþ and
incubated for 24 h extra. Six days postseeding cells are used to
perform transport experiments. The NCE (solution in HBSS
supplemented buffer and maximum of 1% DMSO) or supple-
mented HBSS buffer (10 mM Hepes and 25 mM glucose at pH
7.4) will be placed in the apical or basolateral side as indicated in
the table below and according to the AB or BA transport study.
After a 3 h incubation period, samples are 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 absorp-
tion, material balance after 3 h, and stability at 37 °C after 3 h.
Hydrolysis of [3H]-cAMP was initiated by adding 10 μL of a
solution containing PDE4 enzyme, and the plate was then
incubated under agitation at room temperature. The reaction
was stopped after 60 min (with ∼10-20% substrate conversion)
by addition of 50 μL 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 of the beads, although the signal was
quite stable, and samples may be counted from 1 to 48 h after
bead addition.
LPS Induced TNF-r in Human Whole Blood (HWB-TNF-r).
Human whole blood of healthy donors was collected in 50 mL
Falcon tubes with heparin (5000 units/mL, Heparin Mayne 5%,
MAYNE PHARMA). LPS (lipopolysaccharidefromEscherichia
coli, Sigma, St. Louis, MO) dissolved in PBS (Dulbecco’s phos-
phate buffered saline, without calcium and magnesium chloride
Sigma, St. Louis, MO) was added to the tubes to give a final
concentration in the assay of 1 μg/mL and preincubated at 37 °C
for 10 min with rocking. Increasing concentrations of different
inhibitors (2 μL), dissolved in 100% DMSO, were added to the
96-well plates and 200 μL of blood containing LPS (except
for controls) then distributed into wells, plates were shaken for
1-2 min, sealed with aluminum foil lid (Beckman Coulter), and
then incubated for 24 h at 37 °C under agitation in KelvitronT
(Heraeus Instruments).
Acknowledgment. We acknowledge the significant techni-
cal support provided by Silvia Petit and Carmen Cabello. This
work was funded by Almirall, Spain.
After the 24 h period, plates were placed on ice, 50 μL 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.
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