Discovery of 2-(cyclopentylamino)thieno[3,2-D]pyrimidin-4(3 H)-one derivatives as a new series of potent phosphodiesterase 7 inhibitors

Article abstract of DOI:10.1021/jm5008215

The discovery of a new series of potent phosphodiesterase 7 (PDE7) inhibitors is described. Novel thieno[3,2-d]pyrimidin-4(3H)-one hit compounds were identified from our chemical library. Preliminary modifications of the hit compounds were performed, resulting in the discovery of a fragment-sized compound (10) with highly improved ligand efficiency. Compound design was guided by structure-activity relationships and computational modeling. The 6-substituted derivatives of the thienopyrimidinone showed diminished activity and enzyme selectivity. However, synthesis of the 7-substituted derivatives resulted in the discovery of 28e, a desirable lead compound that selectively inhibits PDE7 with single-digit nanomolar potency while displaying potent cellular efficacy.

Full text of DOI:10.1021/jm5008215

Article
pubs.acs.org/jmc
Discovery of 2‑(Cyclopentylamino)thieno[3,2‑d]pyrimidin-4(3H)‑one
Derivatives as a New Series of Potent Phosphodiesterase 7 Inhibitors
Kentaro Kawai,*,† Yusuke Endo,† Takeshi Asano, Seiji Amano, Keisuke Sawada, Noriko Ueo,
Nobuaki Takahashi, Yo Sonoda,† Mika Nagai, Noriyuki Kamei, and Naoya Nagata
Drug Research Center, Kaken Pharmaceutical Co. Ltd., 14 Shinomiya Minamigawara-cho, Yamashina, Kyoto 607-8042, Japan
S
* Supporting Information
ABSTRACT: The discovery of a new series of potent
phosphodiesterase 7 (PDE7) inhibitors is described. Novel
thieno[3,2-d]pyrimidin-4(3H)-one hit compounds were iden-
tified from our chemical library. Preliminary modifications of
the hit compounds were performed, resulting in the discovery
of a fragment-sized compound (10) with highly improved
ligand efficiency. Compound design was guided by structure−
activity relationships and computational modeling. The 6-
substituted derivatives of the thienopyrimidinone showed
diminished activity and enzyme selectivity. However, synthesis of the 7-substituted derivatives resulted in the discovery of 28e, a
desirable lead compound that selectively inhibits PDE7 with single-digit nanomolar potency while displaying potent cellular
efficacy.
Chart 1. Examples of Published PDE7 Inhibitors
INTRODUCTION
■
Phosphodiesterases (PDEs) are enzymes that hydrolyze the
second messenger molecules 3′,5′-cyclic adenosine mono-
phosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate
(cGMP). Currently, 11 families of PDE enzymes and 21 human
genes have been identified. Some PDE enzymes (PDE4, PDE7,
and PDE8) selectively degrade cAMP; others (PDE5, PDE6,
and PDE9) selectively degrade cGMP, and the remaining
enzymes degrade both cAMP and cGMP. This substrate
selectivity is regulated by the orientation of a conserved
glutamine, which is anchored by the hydrogen-bonding
network provided by the surrounding residues.1
PDE enzymes are recognized as promising targets to treat
various diseases.2,3 For example, inhibitors of PDE3, PDE4, and
PDE5 have been approved for clinical use in the treatment of
heart failure, respiratory diseases, and erectile dysfunction,
respectively. Currently, PDE7 is considered to be a possible
target for treating various diseases, including leukemias,4 central
nervous system (CNS) disorders,5 and airway diseases.6 To
date, many PDE7 inhibitors have been discovered by a number
of institutions, including CSIC,7,8 Celltech,9 Pfizer,10,11
Biocrea,12 BMS,13 and others14−16 (Chart 1). In addition, a
PDE7 inhibitor from Asubio (ASB16165) was found to be
effective in a mouse skin inflammation model, indicating that
PDE7 is a potential target for treating inflammatory diseases.16
Aimed at the development of remedies to treat the
aforementioned diseases, our objective is to find a lead
compound with potency and structural novelty that selectively
inhibits PDE7. In this paper we describe our use of a chemical
library, bioassays, computational modeling, and medicinal
chemistry efforts to discover and identify novel PDE7
inhibitors.
RESULTS AND DISCUSSION
■
Synthetic Strategy. Thienopyrimidinone hits, represented
by compounds 5−7, were discovered to be the most potent
compound series from our chemical library (Figure 1). None of
the three hits have the structures of pan assay interference
compounds.17
The thienopyrimidinone hits not only exhibited strong
affinity for PDE7 (IC50 range 160−1100 nM), but are also
structurally different from known PDE7 inhibitors. Further-
Received: May 20, 2014
© XXXX American Chemical Society
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be unique, because 5 has a linear and rigid structure and
contains a large substituent on the thienopyrimidinone scaffold.
In this model, the thienopyrimidinone core was found to have
two π−π interactions and one hydrogen bond with PDE7
residues: a face-to-face orientation with the phenyl ring of
Phe416, a T-shaped edge-to-face orientation with the phenyl
ring of Phe384, and a hydrogen bond between the 4-carbonyl
group of thienopyrimidinone and the carboxamide side chain of
Gln413. Therefore, we decided to maintain these three key
interactions in the drug design and syntheses for the discovery
of potent PDE7 inhibitors.
This model was used to devise the synthetic strategies that
would generate a series of compounds useful for the exploration
of structure−activity relationships. Our synthetic strategies are
summarized as follows: (1) reduce the molecular size to reveal
the minimal structural requirement for demonstrating PDE7
activity; (2) re-establish the SAR of the 6-position because
insufficient information was obtained around this position from
the library screening data; (3) explore the SAR of the 7-
position, where there is a large space in that region of the
binding pocket. These three strategies were applied in the
discovery of potent and selective PDE7 inhibitors.
Minimal Structural Requirements of the Thienopyr-
imidinone. As a starting point, we investigated the initial SAR
of this series to reveal the minimal structural requirements for
demonstrating activity against PDE7 (Figure 3). First,
compound 8 was designed and synthesized with the maximum
common substructure of the three hit compounds. The result
was a substantial loss in activity, but with an almost identical
ligand efficiency (8; IC50 = 6800 nM, LE = 0.34). Further
removal of a phenyl ring slightly increased the activity and
provided higher ligand efficiency than the hit compounds (9;
IC50 = 5000 nM, LE = 0.48). Compound 9, which is considered
to have two π−π interactions and one hydrogen bond, was
successfully determined as the minimal structure displaying
measurable enzymatic activity.
Figure 1. Structures of the thienopyrimidinone hits.
more, these hits exhibited good ligand efficiency18,19 values of
approximately 0.30. Ligand efficiency was proposed to be an
important metric for the discovery of better lead compounds, in
which good compounds show higher ligand efficiency values. In
this study, ligand efficiency (kcal·mol−1·HA−1) was calculated
by the formula −RT[ln(IC50)](HA)−1, where R = 1.987 × 10−3
kcal·mol−1·K−1, T = 298 K, and HA represents the heavy atom
count.
To identify the molecular determinants responsible for the
ligand binding to PDE7, computational modeling was
performed for the hit compounds 5−7. The Glide and
Maestro20 applications with a PDE7 cocrystal structure (PDB
ID 1ZKL21) were used for this purpose. A proposed model of 5
in the PDE7 catalytic site is shown in Figure 2, while the
The allylamine group of 9 was replaced with a cyclo-
alkylamino group. Pfizer’s compounds include a cycloalkyl
group on the thiadiazole scaffold, and ASB16165 also has a
cycloalkyl group on its fused ring scaffold (Chart 1). We hence
considered that the cycloalkylamino group could replace the
allylamine group of 9. The assay result was beyond our
expectation, as introduction of a cyclopentylamino group at the
2-position resulted in a substantial increase in PDE7 inhibitory
activity (10). The introduction of a cyclohexylamino group also
resulted in an increase in the inhibitory activity (11). The
bulkier cycloalkyl group was considered to have a better fit in
the hydrophobic binding subpocket, resulting in an increase in
activity. Because 10 demonstrated superior ligand efficiency
(LE = 0.52), we decided to use 10 as a starting point to
evaluate the SAR around the thienopyrimidinone scaffold.
Figure 2. Proposed binding mode of 5. A hydrogen bond is
represented by a dashed line.
binding modes of 6 and 7 are shown in the Supporting
Information, Figure S1. The binding model was determined to
Figure 3. Results of the initial SAR study.
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Design, Synthesis, and SAR at the 6-Position. As the hit
compounds have substituents at the 6-position, various 6-
substituted thienopyrimidinone derivatives were designed with
substituents ranging in size. To focus on the critical interactions
with the enzyme, small substituents such as methyl and ethyl
groups were initially introduced. The design of 20 was inspired
by the structure of ASB16165 (1); however, we did not
perform structural overlays, because without cocrystal struc-
tures, we were uncertain whether our compounds and the
known inhibitors would bind to PDE7 in the same manner.
Derivatives were synthesized in four to eight steps from the
starting materials 12a,b (Scheme 1). Amines 12a,b were treated
Both the secondary alcohol and the bromine were removed by
this reduction. The reductive amination of intermediate 17,
followed by reduction of the bromine, gave amine 19 in
moderate yield. The introduction of a carboxylate group was
achieved by treatment of 16 with LDA and CO2 (dry ice).
Finally, amidation followed by reduction of the bromine gave
amide 20.
The SAR at the 6-position of the thienopyrimidinone is
shown in Table 1. To improve the inhibition against PDE7 and
Table 1. Structure−Activity Relationship for the 6-Position
a
Scheme 1. Synthesis of 6-Substituted Compounds
a
b
IC50 values are the mean of at least two experiments. ND = not
c
determined. Ligand efficiency was calculated for PDE7A.
the selectivity profile of the compounds, the activity against
PDE4, which is also a cAMP-specific enzyme and is most
similar to PDE7, was measured at this lead discovery stage.
PDE4 is an attractive target for treating a number of
inflammatory diseases, including asthma and chronic obstruc-
tive pulmonary disease.24 However, the clinical utility of PDE4
inhibitors has been limited due to the occurrence of side effects,
including nausea and emesis.25,26 Thus, the discovery of new
inhibitors with good selectivity against PDE4 is desirable.
Surprisingly, the introduction of small substituents such as a
methyl or ethyl group resulted in a 43−73-fold loss in potency
and a decrease in ligand efficiency. The introduction of bulkier
substituents slightly increased the potency, but the ligand
efficiency was further decreased (19, 20). In addition, 18 and
19 both showed stronger potency against PDE4 than PDE7,
indicating an undesirable selectivity profile. We halted further
exploration at the 6-position, including investigation of the
original hits, because the assay results indicated that the
introduction of substituents at this position led to decreased
ligand efficiency and undesirable selectivity profiles.
In reviewing the SAR, the thienopyrimidinone scaffold was
found to have a 32-fold selectivity for PDE7 over PDE4 (10).
This result encouraged us to design and synthesize additional
compounds in our efforts to discover lead compounds from this
series. In consideration of the 6-position SAR, it is possible that
obstructive factors, such as steric hindrance, prevented the
formation of favorable interactions between the ligand and the
PDE7 enzyme. In addition, the PDE7 catalytic site in the
modeling structure suggested that there is an open space for
substituents at the 7-position. We thus decided to synthesize
compounds with substituents at the 7-position.
a
Reaction conditions: (a) ethyl isothiocyanate, pyridine, reflux, 76−
84%; (b) NaH, MeI, DMF, 0 °C, 98%; (c) K2CO3, MeI, CH3CN, rt,
75%; (d) m-CPBA, CH2Cl2, rt, 66−87%; (e) cyclopentylamine, THF,
reflux, 74−89%; (f) PhNMe3·Br3, CaCO3, CH2Cl2, MeOH, rt, 48%;
(g) LDA, DMF, THF, 0 °C, 71%; (h) MeMgBr, THF, 0 °C, 84%; (i)
PdCl2, Et3SiH, EtOH, rt, 54%; (j) 3-aminopyridine, toluene, reflux,
then NaBH4, MeOH, rt, 62%; (k) H2, Pd(OH)2, MeOH, rt, 47%; (l)
LDA, CO2, THF, 0 °C, 100%; (m) 1-(5-aminopyridin-2-yl)piperidin-
4-ol,22 WSC, HOBt, NMM, DMF, rt, 62%; (n) H2, Pd/C, THF, rt,
5%.
with ethyl isothiocyanate to give the fused ring products 13a,b,
respectively. Methylation of 13a,b with iodomethane, followed
by oxidation of the thio group, yielded 14a,b. The
methylsulfonyl moiety of 14a,b was then substituted by
cyclopentylamine, yielding 10 and 15. The more functionalized
6-substituted thienopyrimidinone derivatives 18−20 were
prepared in four steps from 10.
Regioselective bromination of 10 was achieved with
phenyltrimethylammonium tribromide. Treatment of 16 with
LDA and DMF gave aldehyde 17 in 71% yield. A Grignard
reaction on 17, followed by hydrogenolysis using Et3SiH and
PdCl2 in ethanol,23 gave the ethyl-substituted compound 18.
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a
Scheme 2. Synthesis of 7-Substituted Compounds
a
Reaction conditions: (a) trimethylboroxine, Pd(PPh3)4, K2CO3, H2O, 1,4-dioxane, 100 °C, 83%; (b) potassium vinyltrifluoroborate, PdCl2(dppf)·
CH2Cl2, TEA, EtOH, 80 °C, 71%; (c) H2 (3 atm), 10% Pd/C, THF, rt, 85−99%; (d) ethynyltrimethylsilane, PdCl2(PPh3)2, CuI, TEA, CH3CN, 80
°C, 19%; (e) TBAF, THF, rt, 92%; (f) CuCN, DMF, 150 °C, 24%; (g) alkyne, PdCl2(PPh3)2, CuI, CH3CN, 80 °C, 38−49%; (h) Ar−B(OH)2,
Pd(OAc)2, PPh3, saturated aq NaHCO3, DMF, EtOH, 80 °C, 77−99%; (i) ArBPin, Pd(PPh3)4, K3PO4, 1,4-dioxane, 90 °C or reflux, 28−64%; (j)
tert-butyl 3-oxopiperazine-1-carboxylate, CuI, N,N-dimethylethylenediamine, K3PO4, 1,4-dioxane, reflux, 59%.
Design, Synthesis, and SAR at the 7-Position. The 7-
substituted thienopyrimidinone derivatives were designed and
prepared starting from the key intermediate 16 (Scheme 2).
Cross-coupling reactions such as the Suzuki−Miyaura reaction
and the Sonogashira reaction were used to prepare 21−30.
Hydrogenation of 22 and 26a,b was performed with a
palladium catalyst to give 23 and 27a,b, respectively, in 85−
99% yield.
interaction diagram in Figure 4b shows that the carbonyl
oxygen of 24 forms a hydrogen bond with Gln413. The
thiophene ring has two π−π interactions, one each with the
phenyl groups of Phe384 and Phe416. In addition, the NH
group of 24 forms a hydrogen bond with a water molecule.
This water molecule is stabilized by other hydrogen bonds
formed with the side chains of Tyr211 and Asp362 (Figure 4a).
The modeled structure of 5 (green) is overlaid with the
cocrystal of 24 in Figure 4c. Although the interaction points of
the thienopyrimidinone ring (i.e., two π−π interactions and a
hydrogen bond) were correctly estimated from the modeling,
the position of the thieno region was slightly different from that
in the cocrystal structure of 24. This overlaid structure revealed
that the region predicted to be occupied by the substituent at
the 6-position was slightly different from the region predicted
by the cocrystal structure.
The cocrystal structure of 24 and the SAR results could
possibly explain the difference in activity between substitution
at the 6-position and substitution at the 7-position. When a
substituent was introduced at the 6-position, the PDE7
inhibitory activity decreased due to the steric hindrance
between the ligand molecule and the surrounding residues,
such as Leu401. In contrast to the 6-position, there is an open
space in the direction of the 7-position; therefore, introducing
substituents at the 7-position leads to increased inhibitory
activity.
The inhibitory activities of 21−30 against PDE7 and PDE4,
and the effects of these compounds on IL-2 production in
activated mouse lymphocytes, are shown in Table 2. Li et al.
reported that activation of human blood T cells is associated
with induction of PDE7.27 Although PDE4 is considered to be
more important for cytokine production in T cells than
PDE7,28 a selective PDE7 inhibitor (ASB16165) was shown to
block cytokine production in activated T cells.29 In addition,
another selective PDE7 inhibitor (PF 0332040) has also been
shown to inhibit human T cell activation.30 Therefore, our
PDE7 inhibitors were tested to see whether they could inhibit
IL-2 production in T cells. Ligand efficiency values are also
specified. First, the effect of 7-substitution on 10 with aliphatic
groups was examined (21−24). The introduction of methyl
and ethyl groups (21, 23) showed a positive effect of increasing
PDE7 inhibitory activity while maintaining high ligand
efficiency. In contrast to the 6-substituted compounds, these
compounds showed good selectivity against PDE4. The PDE7
inhibitory activities for compounds containing unsaturated
groups, such as vinyl, ethynyl, and cyano groups (22, 24, 25),
were the same as those for compounds containing the methyl
and ethyl groups. However, significantly increased affinities
were observed for much bulkier substituents (26a, 27a). The
pyridine-containing compounds 26a and 27a exhibited
especially high ligand efficiencies and single-digit nanomolar
IC50 values. These two compounds also exhibited greater than
150-fold selectivity for PDE7 over PDE4.
We think that the binding model and the X-ray structure
perform a complementary role. The binding model, which was
readily obtained at the hit identification stage, gave critical
information to establish the strategy to introduce the
substituent at the 7-position. As expected, we successfully
increased the potency and selectivity by exploring substitution
at the 7-position. On the other hand, this study revealed the
limitation of the use of the binding model; we found it difficult
to explain the SAR between the 6-position and 7-position.
However, as mentioned, this SAR could successfully explain by
The X-ray cocrystal structure of 24 bound to the PDE7
catalytic site is shown in Figure 4a (PDB ID 4PM0). The 2D
D
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potent PDE7 inhibition, with 96-fold selectivity over PDE4,
and strong cellular activity (IC50 = 290 nM). The
oxopiperadine 30 showed good potency for PDE7 and 275-
fold selectivity over PDE4.
Table 2. Structure−Activity Relationship for the 7-Position
With respect to the pyridyl derivatives, 28e exhibited single-
digit nanomolar potency, and the morpholine-substituted
compound 29b showed the strongest activity (IC50 = 1.0
nM) among the synthesized compounds given in Tables 1 and
2. In particular, 28e exhibited very potent activity as well as
good ligand efficiency. An aromatic substituent, such as a
phenyl or pyridyl ring, provided greater positive effects on
PDE7 enzyme inhibition than an aliphatic side chain. The
pyridyl compound 28e exhibited a selectivity of 190-fold for
PDE7 over PDE4. In addition, 28e showed high selectivity
against other PDE enzymes, including a selectivity of 800-fold
over PDE3 and 1650-fold over PDE5. We believe that
selectivity, especially for the three enzymes PDE3, PDE4, and
PDE5, is important to avoid side effects. Compound 28e also
has strong cellular activity (IC50 = 89 nM), indicating that this
thienopyrimidinone derivative is a suitable lead compound.
Whereas the IC50 values for PDE3, PDE4, PDE5, and PDE7
were measured internally, the full PDE selectivity profiling assay
for 30 was performed at Caliper Discovery Alliances & Services
using the Caliper LabChip 3000 to reveal the selectivity profile
of the thienopyrimidinone. The result is shown in the
Supporting Information (Figure S2). The data show that 30
has a good selectivity profile. Although the selectivity profile of
28e was not evaluated, considering structural similarity, 28e is
believed to exhibit selectivity for other PDE enzymes.
In the present set of active inhibitors, we further evaluated
the pharmacokinetic profile of 28e, 29a, and 29b. The data are
provided in Table S1 of the Supporting Information. Whereas
these compounds showed good permeability, they are
metabolically unstable in human and rat liver microsomes.
The findings from this study will be useful for further
optimization studies.
CONCLUSION
■
In conclusion, we have described the discovery of a series of 2-
(cyclopentylamino)thieno[3,2-d]pyrimidin-4(3H)-one com-
pounds as novel and potent PDE7 inhibitors. Screening of
our chemical library led to the identification of hit
thienopyrimidinone compounds with substituents at the 6-
position. SAR studies around the hit compounds, and
computational modeling, contributed to the discovery of 7-
substituted derivatives as a series of novel and potent PDE7
inhibitors. As a result, we identified the lead compound 28e,
which demonstrated single-digit nanomolar potency for PDE7
with 190-fold selectivity over PDE4, as well as potent cellular
efficacy (IL-2 IC50 < 100 nM). This lead compound also
showed good selectivity against other PDE enzymes.
a
b
IC50 values are the mean of at least two experiments. IC50 values are
c
the mean of at least four experiments. Ligand efficiency was calculated
for PDE7A.
using the X-ray structure, which was obtained at the lead
discovery stage.
EXPERIMENTAL SECTION
Further modifications were carried out to study the influence
of substituents at the 7-position. Compounds with a phenyl
ring (28a−d and 29a) and compounds with a pyridyl ring (28e,
29b) were synthesized. The introduction of a phenyl group
resulted in a compound with single-digit nanomolar potency
against PDE7 (28a), with a 27-fold enhancement of activity
over that of 10. Although both meta- and para-substituted
methyl benzoate compounds (28c,d) exhibited almost identical
inhibition of PDE7 activity, 28c showed good cellular activity.
The ortho-substituted methyl benzoate 28b showed a 68-fold
loss in potency. Among the methyl benzoates, 28c showed both
■
General Methods. Reagents and solvents were purchased from
commercial sources and used without further purification unless
otherwise indicated. Moisture-sensitive reactions were carried out
under an argon atmosphere in dried glassware using anhydrous
1
solvents from commercial suppliers. H NMR spectra were recorded
on a JEOL JNM-ECA400 spectrometer (400 MHz). Chemical shifts
are reported in δ units (ppm) relative to TMS as an internal standard.
Coupling constants (J) are reported in hertz. High-resolution mass
spectrometry (HRMS) analyses were performed on a Thermo
Scientific Exactive mass spectrometer in positive or negative ionization
electrospray mode operating at 25 000 resolution. The purities of the
E
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Figure 4. Cocrystal structure of 24 bound to the PDE7A catalytic site (PDB ID 4PM0, 2.1 Å resolution). (a) Complex of 24 (orange) with binding
site residues. A hydrogen bond is represented by a dashed line, and only the important water molecule is shown. (b) 2D ligand interaction diagram
of 24 generated with Maestro 9.3. (c) Complex of 24 overlaid with the modeled structure 5 (green).
final compounds were determined using HPLC (area percentage at
254 nm). HPLC analyses were performed using a Hitachi La Chrom
Elite system. The HPLC purities were ≥95% for all of the compounds,
with the exception of compounds 18 and 19.
2-(Allylamino)-6-(4-(4-isopropylpiperazin-1-yl)phenyl)-3-
methylthieno[3,2-d]pyrimidin-4(3H)-one (5). This compound
was purchased from BioFocus DPI (purity 98%). 1H NMR
(CDCl3): δ 1.11 (6H, d, J = 6.9 Hz), 2.71 (1H, m), 3.30 (8H, m),
3,52 (3H, s), 4.16 (2H, m), 4.64 (1H, m), 5.22−5.34 (2H, m), 5.99−
6.09 (1H, m), 6.93 (2H, d, J = 8.7 Hz), 7.17 (1H, s), 7.58 (2H, d, J =
9.2 Hz). MS (ESI): m/z 424 (M + H)+.
white solid. 1H NMR (CDCl3): δ 1.22 (3H, t, J = 6.9 Hz), 4.44 (2H, q,
J = 6.9 Hz), 7.01 (1H, d, J = 5.5 Hz), 8.16 (1H, d, J = 5.5 Hz).
3-Ethyl-2-(methylthio)thieno[3,2-d]pyrimidin-4(3H)-one. To
a solution of 13a (27.2 g, 0.128 mmol) in DMF (0.43 L) were added
methyl iodide (16 mL, 0.26 mol) and sodium hydride (60% in oil, 7.6
g, 0.19 mol). The reaction mixture was stirred at 0 °C for 1 h.
Saturated aq NH4Cl solution and water were then sequentially added
to the reaction mixture. The mixture was filtered, and the precipitate
was washed with water and dried under reduced pressure to yield 28.6
1
g (98%) of a white solid. H NMR (CDCl3): δ 1.38 (3H, t, J = 6.9
Hz), 2.63 (3H, s), 4.24 (2H, q, J = 7.3 Hz), 7.20 (1H, d, J = 5.0 Hz),
3-(2-(Allylamino)-3-methyl-4-oxo-3,4-dihydrothieno[3,2-d]-
pyrimidin-6-yl)-N-(2-hydroxyethyl)benzamide (6). This com-
pound was purchased from BioFocus DPI (purity 98%). MS (ESI):
m/z 385 (M + H)+.
4-(2-(Allylamino)-3-methyl-4-oxo-3,4-dihydrothieno[3,2-d]-
pyrimidin-6-yl)-N-(3-(dimethylamino)propyl)benzamide (7).
This compound was purchased from BioFocus DPI (purity 97%).
MS (ESI): m/z 426 (M + H)+.
3-Ethyl-2-thioxo-2,3-dihydrothieno[3,2-d]pyrimidin-4(1H)-
one (13a). To a solution of 12a (9.58 g, 60.9 mmol) in pyridine (122
mL) was added ethyl isothiocyanate (10.7 mL, 122 mmol). The
reaction mixture was heated under reflux for 14 h. After the mixture
was cooled to rt, the solvent was evaporated under reduced pressure.
The residue was washed with chloroform to yield 10.8 g (84%) of a
7.70 (1H, d, J = 5.5 Hz).
3-Ethyl-2-(methylsulfonyl)thieno[3,2-d]pyrimidin-4(3H)-one
(14a). To a solution of 3-ethyl-2-(methylthio)thieno[3,2-d]pyrimidin-
4(3H)-one (28.6 g, 0.126 mol) in dichloromethane (0.63 L) was
added 70% m-CPBA (79 g, 0.32 mol). The mixture was stirred at rt for
1 h. Saturated sodium bicarbonate and saturated sodium thiosulfate
were added to the reaction mixture. The mixture was extracted with
chloroform and dried over anhydrous sodium sulfate. The solvent was
evaporated under reduced pressure. The residue was purified by flash
chromatography to yield 28.5 g (87%) of a yellow solid. H NMR
(CDCl3): δ 1.48 (3H, t, J = 6.9 Hz), 3.54 (3H, s), 4.58 (2H, q, J = 6.9
Hz), 7.34 (1H, d, J = 5.5 Hz), 7.87 (1H, d, J = 5.0 Hz).
2-(Cyclopentylamino)-3-ethylthieno[3,2-d]pyrimidin-4(3H)-
one (10). To a solution of 14a (11.0 g, 42.6 mmol) in THF (114 mL)
1
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was added cyclopentylamine (20 mL, 0.20 mol). The mixture was
heated under reflux for 18 h. After the mixture was cooled to rt, the
solvent was evaporated under reduced pressure. The residue was
purified by flash chromatography to yield 8.73 g (89%) of a white
(3H, t, J = 6.9 Hz), 1.56−1.74 (6H, m), 2.03−2.07 (2H, m), 4.11 (2H,
q, J = 6.9 Hz), 4.38−4.43 (1H, m), 7.06 (1H, d, J = 6.4 Hz), 10.08
(1H, s).
7-Bromo-2-(cyclopentylamino)-3-ethyl-6-(1-hydroxyethyl)-
thieno[3,2-d]pyrimidin-4(3H)-one. To a solution of 17 (50 mg,
0.135 mmol) in THF (2.7 mL) was added a 1.1 M solution of
methylmagnesium bromide in THF (0.184 mL, 0.203 mmol) at 0 °C.
The solution was stirred for 5 h at 0 °C. Saturated aq NH4Cl solution
was added, and the mixture was extracted with chloroform. The
organic layer was dried over anhydrous sodium sulfate, and the solvent
was evaporated under reduced pressure. The residue was purified by
flash chromatography to yield 44 mg (84%) of a white solid. 1H NMR
(CDCl3): δ 1.32 (3H, t, J = 7.2 Hz), 1.45−1.57 (2H, m), 1.59 (3H, d, J
= 6.9 Hz), 1.64−1.78 (4H, m), 2.16−2.27 (2H, m), 2.42 (1H, d, J =
6.9 Hz), 4.06 (2H, q, J = 7.2 Hz), 4.40−4.55 (2H, m), 5.28−5.35 (1H,
m).
2-(Cyclopentylamino)-3,6-diethylthieno[3,2-d]pyrimidin-
4(3H)-one (18). To a solution of 7-bromo-2-(cyclopentylamino)-3-
ethyl-6-(1-hydroxyethyl)thieno[3,2-d]pyrimidin-4(3H)-one (20 mg,
0.052 mmol) in ethanol (1.0 mL) were added palladium(II) chloride
(9.2 mg, 0.052 mmol) and triethylsilane (83 μL, 0.52 mmol), and the
mixture was stirred at rt for 6 h. The reaction solution was filtered
through a Celite pad, and the solvent was evaporated under reduced
pressure. The residue was purified by flash chromatography to yield
8.2 mg (54%) of a white solid. HPLC purity: 75.9%. 1H NMR
(CDCl3): δ 1.32 (3H, t, J = 7.3 Hz), 1,34 (3H, t, J = 7.6 Hz) 1.40−
1.51 (2H, m), 1.60−1.78 (4H, m), 2.10−2.20 (2H, m), 2.88 (2H, q, J
= 7.6 Hz), 4.06 (2H, q, J = 7.3 Hz), 4.30−4.41 (2H, m), 6.78 (1H, s).
HRMS (ESI): m/z calcd for C15H22N3OS (M + H)+ 292.1478, found
292.1473.
1
solid. HPLC purity: 97.2%. H NMR (CDCl3): δ 1.33 (3H, t, J = 7.3
Hz), 1.46−1.54 (2H, m), 1.66−1.79 (4H, m), 2.12−2.20 (2H, m),
4.09 (2H, q, J = 7.3 Hz), 4.37−4.45 (1H, m), 4.43 (1H, br s), 7.05
(1H, d, J = 5.5 Hz), 7.62 (1H, d, J = 5.5 Hz). HRMS (ESI): m/z calcd
for C13H18N3OS (M + H)+ 264.1165, found 264.1164.
3-Ethyl-6-methyl-2-thioxo-2,3-dihydrothieno[3,2-d]-
pyrimidin-4(1H)-one (13b). According to the method described for
derivative 13a, transformation of 12b (150 mg, 0.878 mmol) yielded
1
150 mg (76%) of a white solid. H NMR (CDCl3): δ 1.36 (3H, t, J =
6.9 Hz), 2.57 (3H, s), 4.56 (2H, q, J = 6.9 Hz), 6.64 (1H, s).
3-Ethyl-6-methyl-2-(methylthio)thieno[3,2-d]pyrimidin-
4(3H)-one. To a solution of 13b (150 mg, 0.663 mmol) in acetonitrile
(2 mL) were added methyl iodide (50 μL, 0.795 mmol) and potassium
carbonate (137 mg, 0.994 mmol). The reaction mixture was stirred at
rt for 2 h. Then the solvent was evaporated under reduced pressure.
Water was added to the reaction mixture. The mixture was extracted
with chloroform and dried over anhydrous sodium sulfate. The solvent
was evaporated under reduced pressure. The residue was purified by
1
flash chromatography to yield 120 mg (75%) of a yellow solid. H
NMR (CDCl3): δ 1.37 (3H, t, J = 7.3 Hz), 2.59 (3H, s), 2.61 (3H, s),
4.21 (2H, q, J = 7.3 Hz), 6.87 (1H, s).
3-Ethyl-6-methyl-2-(methylsulfonyl)thieno[3,2-d]pyrimidin-
4(3H)-one (14b). To a solution of 3-ethyl-6-methyl-2-(methylthio)-
thieno[3,2-d]pyrimidin-4(3H)-one (120 mg, 0.499 mol) in dichloro-
methane (1 mL) was added 75% m-CPBA (267 mg, 1.25 mol). The
mixture was stirred at rt for 2 h. Saturated sodium bicarbonate was
added to the reaction mixture. The mixture was extracted with
chloroform and dried over anhydrous sodium sulfate. The solvent was
evaporated under reduced pressure. The residue was purified by flash
7-Bromo-2-(cyclopentylamino)-3-ethyl-6-[(pyridin-3-
ylamino)methyl]thieno[3,2-d]pyrimidin-4(3H)-one. To a solu-
tion of 17 (40 mg, 0.11 mmol) in toluene (1.5 mL) was added 3-
aminopyridine (32 mg, 0.33 mmol), and the reaction mixture was
heated under reflux for 2 h. After the mixture was cooled to rt, the
solvent was evaporated under reduced pressure. After the residue was
dissolved in methanol (1.5 mL), sodium borohydride (12.3 mg, 0.33
mmol) was added, and the resulting solution was stirred at rt for 30
min. Saturated aq NH4Cl solution was added, and the mixture was
extracted with ethyl acetate. The organic layer was dried over
anhydrous sodium sulfate. The residue was purified by flash
1
chromatography to yield 90 mg (66%) of a yellow solid. H NMR
(CDCl3): δ 1.46 (3H, t, J = 7.2 Hz), 2.64 (3H, s), 3.51 (3H, s), 4.55
(2H, q, J = 7.2 Hz), 7.00 (1H, s).
2-(Cyclopentylamino)-3-ethyl-6-methylthieno[3,2-d]-
pyrimidin-4(3H)-one (15). To a solution of 14b (90 mg, 0.33 mmol)
in THF (1 mL) was added cyclopentylamine (563 mg, 6.61 mmol).
The mixture was heated under reflux for 4 h. After the mixture was
cooled to rt, the solvent was evaporated under reduced pressure. The
residue was purified by flash chromatography to yield 68 mg (74%) of
a white solid. 1H NMR (CDCl3): δ 1.32 (3H, t, J = 7.1 Hz), 1.42−1.54
(2H, m), 1.61−1.79 (4H, m), 2.09−2.19 (2H, m), 2.53 (3H, s), 4.06
(2H, q, J = 7.1 Hz), 4.34−4.41 (2H, m), 6.74 (1H, s). HRMS (ESI):
m/z calcd for C14H20N3OS (M + H)+ 278.1322, found 278.1319.
HPLC purity: 98.0%.
1
chromatography to yield 25 mg (62%) of a yellow solid. H NMR
(CDCl3): δ 1.30 (3H, t, J = 7.2 Hz), 1.45−1.58 (2H, m), 1.65−1.81
(4H, m), 2.17−2.28 (2H, m), 4.04 (2H, q, J = 7.2 Hz), 4.38 (1H, t, J =
6.4 Hz), 4.43−4.56 (2H, m), 4.60 (2H, d, J = 6.4 Hz), 6.91 (1H, ddd, J
= 1.5, 4.2, 8.2 Hz), 7.08 (1H, dd, J = 4.2, 8.2 Hz), 8.02 (1H, d, J = 4.2
Hz), 8.09 (1H, d, J = 1.5 Hz).
7-Bromo-2-(cyclopentylamino)-3-ethylthieno[3,2-d]-
pyrimidin-4(3H)-one (16). To a solution of 10 (8.73 g, 33.1 mmol)
in a mixture of dichloromethane (55.2 mL) and methanol (55.2 mL)
were added phenytrimethylammonium tribromide (21.5 g, 99.3
mmol) and calcium carbonate (13.2 g, 132 mmol). The reaction
mixture was stirred at rt for 14 h. The residue was filtered, and the
solvent was evaporated under reduced pressure. Then the residue was
washed with water and extracted with ethyl acetate. The residue was
purified by flash chromatography to yield 5.45 g (48%) of a yellow
2-(Cyclopentylamino)-3-ethyl-6-[(pyridin-3-ylamino)-
methyl]thieno[3,2-d]pyrimidin-4(3H)-one (19). To a solution of
7-bromo-2-(cyclopentylamino)-3-ethyl-6-[(pyridin-3-ylamino)-
methyl]thieno[3,2-d]pyrimidin-4(3H)-one (10 mg, 0.022 mmol) in
methanol (1.0 mL) was added 20% palladium hydroxide on carbon
(3.2 mg, 32%, w/w), and the mixture was stirred at rt for 12 h under a
hydrogen atmosphere. The reaction solution was filtered through a
pad of Celite, and the solvent was evaporated under reduced pressure.
The residue was purified by flash chromatography to yield 6.2 mg
(47%) of a pale yellow solid. HPLC purity: 67.7%. 1H NMR (CDCl3):
δ 1.24 (3H, t, J = 6.9 Hz), 1.43−1.53 (2H, m), 1.64−1.80 (4H, m),
2.10−2.20 (2H, m), 4.06 (2H, q, J = 6.9 Hz), 4.33−4.41 (1H, m), 4.48
(1H, d. J = 5.5 Hz), 4.65 (2H, d, J = 5.5 Hz), 6.57 (1H, s), 7.02 (1H, d,
J = 7.7 Hz), 7.39 (1H, dd, J = 5.0, 7.7 Hz), 7.91 (1H, d, J = 5.0 Hz),
8.44 (1H, s). HRMS (ESI): m/z calcd for C19H24N5OS (M + H)+
370.1696, found 370.1701.
7-Bromo-2-(cyclopentylamino)-3-ethyl-4-oxo-3,4-
dihydrothieno[3,2-d]pyrimidine-6-carboxylic Acid. To a solu-
tion of 16 (1.50 g, 4.38 mmol) in THF (14.6 mL) was added a 2.0 M
solution of LDA in THF (6.55 mL, 13.1 mmol), and the mixture was
stirred at 0 °C for 30 min. Dry ice (one piece) was added and the
resulting mixture was stirred at 0 °C for 30 min. Water was added, and
the mixture was washed with chloroform. Aqueous hydrochloride was
1
solid. H NMR (CDCl3): δ 1.33 (3H, t, J = 7.3 Hz), 1.47−1.56 (2H,
m), 1.67−1.78 (4H, m), 2.19−2.28 (2H, m), 4.10 (2H, q, J = 7.3 Hz),
4.44−4.58 (2H, m), 7.61 (1H, s).
7-Bromo-2-(cyclopentylamino)-3-ethyl-4-oxo-3,4-
dihydrothieno[3,2-d]pyrimidine-6-carbaldehyde (17). To a
solution of 16 (1.49 g, 4.35 mmol) in THF (14.5 mL) was added a
2 M solution of LDA in THF (6.55 mL, 13.1 mmol) at 0 °C under an
argon atmosphere. Anhydrous DMF (1.34 mL, 17.4 mmol) was then
added to the solution and the resulting solution was stirred for 2 h at 0
°C. Saturated aq NH4Cl solution was added, and the solution was
extracted with ethyl acetate. The organic layer was dried over
anhydrous sodium sulfate, and the solvent was evaporated under
reduced pressure. The residue was purified by flash chromatography to
1
yield 1.15 g (71%) of a yellow solid. H NMR (DMSO-d6): δ 1.15
G
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Journal of Medicinal Chemistry
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then added to weakly acidify the mixture, which was then extracted
with chloroform. The solvent was evaporated under reduced pressure
to yield 1.70 g (100%) of a yellow solid. H NMR (CDCl3): δ 1.33
(3H, t, J = 7.3 Hz), 1.45−1.58 (2H, m), 1.72−1.76 (4H, m), 2.20−
2.28 (2H, m), 4.07 (2H, q, J = 7.3 Hz), 4.45−4.50 (2H, m), 9.58 (1H,
br s).
7-Bromo-2-(cyclopentylamino)-3-ethyl-N-[6-(4-hydroxypi-
peridin-1-yl)pyridin-3-yl]-4-oxo-3,4-dihydrothieno[3,2-d]-
pyrimidine-6-carboxamide. To a solution of 7-bromo-2-(cyclo-
pentylamino)-3-ethyl-4-oxo-3,4-dihydrothieno[3,2-d]pyrimidine-6-car-
boxylic acid (151 mg, 0.391 mmol) in DMF (1.96 mL) were added
NMM (154 μL, 1.17 mmol), HOBt (68.6 mg, 0.508 mmol), and WSC
(97.5 mg, 0.508 mmol). 1-(5-Aminopyridin-2-yl)piperidin-4-ol21 (90.6
mg, 0.469 mmol) was then added, and the reaction was stirred at rt for
14 h. The residue was filtered and purified by flash chromatography to
yield 132.3 mg (62%) of a yellow amorphous powder. 1H NMR
(CDCl3): δ 1.35 (3H, t, J = 7.3 Hz), 1.48−1.64 (4H, m), 1.68−1.80
(4H, m), 1.98−2.02 (2H, m), 2.22−2.28 (2H, m), 3.14−3.21 (2H, m),
3.90−3.97 (1H, m), 4.04−4.15 (4H, m), 4.47 (1H, sext, J = 6.4 Hz),
4.61 (1H, d, J = 6.0 Hz), 6.72 (1H, d, J = 9.2 Hz), 8.02 (1H, dd, J =
2.7, 9.2 Hz), 8.29 (1H, d, J = 2.7 Hz), 8.91 (1H, s).
mL) was added 10% Pd/C (30 mg, 54.5%, w/w). The reaction
mixture was stirred at rt for 18 h under a hydrogen atmosphere. The
reaction solution was filtered through a Celite pad, and the solvent was
evaporated under reduced pressure to yield 55 mg (99%) of a white
1
1
solid. HPLC purity: 98.3%. H NMR (CDCl3): δ 1.29 (3H, t, J = 7.3
Hz), 1.33 (3H, t, J = 7.3 Hz), 1.49−1.55 (2H, m), 1.60−1.80 (4H, m),
2.15−2.21 (2H, m), 2.73 (2H, dq, J = 1.4, 7.3 Hz), 4.08 (2H, q, J = 7.3
Hz), 4.36−4.41 (2H, m), 7.27 (1H, t, J = 1.4 Hz). HRMS (ESI): m/z
calcd for C15H22N3OS (M + H)+ 292.1478, found 292.1476.
2-(Cyclopentylamino)-3-ethyl-7-[(trimethylsilyl)ethynyl]-
thieno[3,2-d]pyrimidin-4(3H)-one. To a solution of 16 (300 mg,
0.88 mmol) in acetonitrile (9 mL) were added triethylamine (3 mL,
21.5 mmol) and ethynyltrimethylsilane (0.3 mL, 2.12 mmol). Under
an argon atmosphere, bis(triphenylphosphine)palladium(II) dichloride
(60 mg, 0.085 mmol) and copper(I) iodide (20 mg, 0.11 mmol) were
added, and the mixture was stirred at 80 °C for 18 h. After being
cooled to rt, the solution was filtered through a Celite pad, and the
solvent was evaporated under reduced pressure. The residue was
purified by flash chromatography to yield 60 mg (19%) of a white
1
solid. H NMR (CDCl3): δ 0.27 (9H, s), 1.31 (3H, t, J = 7.3 Hz),
1.47−1.56 (2H, m), 1.65−1.79 (4H, m), 2.20−2.28 (2H, m), 4.07
(2H, q, J = 7.3 Hz), 4.40−4.48 (1H, m), 4.52 (1H, d, J = 5.5 Hz), 7.75
(1H, s).
2-(Cyclopentylamino)-3-ethyl-N-[6-(4-hydroxypiperidin-1-
yl)pyridin-3-yl]-4-oxo-3,4-dihydrothieno[3,2-d]pyrimidine-6-
carboxamide (20). To a solution of 10% Pd/C (11.7 mg) in THF
(1.07 mL) was added 7-bromo-2-(cyclopentylamino)-3-ethyl-N-[6-(4-
hydroxypiperidin-1-yl)pyridin-3-yl]-4-oxo-3,4-dihydrothieno[3,2-d]-
pyrimidine-6-carboxamide (117 mg, 0.214 mmol) under argon. The
reaction mixture was stirred at rt for 48 h under a hydrogen
atmosphere. The mixture was filtered through a Celite pad, and the
solvent was evaporated under reduced pressure. The residue was
purified by flash chromatography to yield 5.2 mg (5%) of a yellow
2-(Cyclopentylamino)-3-ethyl-7-ethynylthieno[3,2-d]-
pyrimidin-4(3H)-one (24). To a solution of 2-(cyclopentylamino)-3-
ethyl-7-[(trimethylsilyl)ethynyl]thieno-[3,2-d]pyrimidin-4(3H)-one
(55 mg, 0.15 mmol) in THF (1 mL) was added a 1 M solution of
TBAF in THF (0.3 mL, 0.3 mmol), and the mixture was stirred at rt
for 1 h. The solvent was evaporated under reduced pressure. The
residue was purified by flash chromatography to yield 40 mg (92%) of
a white solid. HPLC purity: 97.2%. 1H NMR (CDCl3): δ 1.33 (3H, t, J
= 7.3 Hz), 1.47−1.54 (2H, m), 1.65−1.78 (4H, m), 2.17−2.23 (2H,
m), 3.23 (1H, s), 4.08 (2H, q, J = 7.3 Hz), 4.45−4.51 (2H, m), 7.81
(1H, s). HRMS (ESI): m/z calcd for C15H18N3OS (M + H)+
288.1165, found 288.1163.
2-(Cyclopentylamino)-3-ethyl-4-oxo-3,4-dihydrothieno[3,2-
d]pyrimidine-7-carbonitrile (25). To a solution of 16 (100 mg,
0.29 mmol) in DMF (2 mL) was added copper(I) cyanide (130 mg,
1.45 mmol). The reaction mixture was stirred at 150 °C for 6 h. After
the mixture was cooled to rt, aqueous ammonia was added. The
residue was filtered and purified by flash chromatography to yield 20
mg (24%) of a white solid. HPLC purity: 98.2%. 1H NMR (CDCl3): δ
1.34 (3H, t, J = 7.3 Hz), 1.47−1.52 (2H, m), 1.70−1.80 (4H, m),
2.18−2.24 (2H, m), 4.07 (2H, q, J = 7.3 Hz), 4.48−4.53 (1H, m), 4.60
(1H, br s), 8.16 (1H, s). HRMS (ESI): m/z calcd for C14H17N4OS (M
+ H)+ 289.1118, found 289.1111.
1
solid. HPLC purity: 95.1%. H NMR (CDCl3): δ 1.34 (3H, t, J = 7.3
Hz), 1.46−1.80 (8H, m), 1.96−2.02 (2H, m), 2.10−2.20 (2H, m),
3.13−3.20 (2H, m), 3.88−3.95 (1H, m), 4.50 (1H, d, J = 6.4 Hz), 6.71
(1H, d, J = 9.2 Hz), 7.54 (1H, s), 7.73 (1H, s), 7.94−7.98 (1H, m),
8.21 (1H, d, J = 2.7 Hz). HRMS (ESI): m/z calcd for C24H31N6O3S
(M + H)+ 483.2173, found 483.2172.
2-(Cyclopentylamino)-3-ethyl-7-methylthieno[3,2-d]-
pyrimidin-4(3H)-one (21). To a solution of 16 (100 mg, 0.29 mmol)
in a mixture of 1,4-dioxane (0.9 mL) and water (0.1 mL) were added
trimethylboroxine (0.1 mL, 0.72 mmol), potassium carbonate (120
mg, 0.87 mmol), and tetrakis(triphenylphosphine)palladium (35 mg,
0.03 mmol) under an argon atmosphere, and the mixture was stirred at
100 °C for 18 h. After the mixture was cooled to rt, water was added,
and the mixture was extracted with ethyl acetate. The organic layer was
dried over anhydrous sodium sulfate. The residue was purified by flash
chromatography to yield 67 mg (83%) of a white solid. HPLC purity:
99.4%. 1H NMR (CDCl3): δ 1.32 (3H, t, J = 7.3 Hz), 1.49−1.53 (2H,
m), 1.65−1.80 (4H, m), 2.16−2.21 (2H, m), 2.28 (3H, d, J = 1.4 Hz),
4.08 (2H, q, J = 7.3 Hz), 4.40−4.47 (2H, m), 7.26−7.27 (1H, m).
HRMS (ESI): m/z calcd for C14H20N3OS (M + H)+ 278.1322, found
278.1320.
2-(Cyclopentylamino)-3-ethyl-7-vinylthieno[3,2-d]-
pyrimidin-4(3H)-one (22). To a solution of 16 (100 mg, 0.29 mmol)
in a mixture of potassium vinyltrifluoroborate (80 mg, 0.60 mmol) and
triethylamine (0.1 mL, 0.72 mmol) in ethanol (3 mL) was added
PdCl2(dppf)·CH2Cl2 (25 mg, 0.031 mmol) under an argon
atmosphere. The reaction mixture was stirred at 80 °C for 18 h.
After the mixture was cooled to rt, water was added, and the mixture
was extracted with ethyl acetate. The organic layer was dried over
anhydrous sodium sulfate. The solvent was evaporated under reduced
pressure. The residue was purified by flash chromatography to yield 60
mg (71%) of a white solid. HPLC purity: 96.7%. 1H NMR (CDCl3): δ
1.33 (3H, t, J = 7.3 Hz), 1.52−1.55 (2H, m), 1.66−1.79 (4H, m),
2.14−2.20 (2H, m), 4.09 (2H, q, J = 7.3 Hz), 4.37−4.47 (2H, m), 5.34
(1H, dd, J = 1.8, 11.5 Hz), 6.32 (1H, dd, J = 1.8, 17.9 Hz), 6.85 (1H,
dd, J = 11.5, 17.9 Hz), 7.54 (1H, s). HRMS (ESI): m/z calcd for
C15H20N3OS (M + H)+ 290.1322, found 290.1323.
2-(Cyclopentylamino)-3-ethyl-7-(pyridin-3-ylethynyl)thieno-
[3,2-d]pyrimidin-4(3H)-one (26a). To a mixture of 16 (100 mg,
0.29 mmol), 3-ethynylpyridine (60 mg, 0.58 mmol), and triethylamine
(1.0 mL, 7.15 mmol) in acetonitrile (3 mL) were added PdCl2(dppf)·
CH2Cl2 (20 mg, 0.028 mmol) and copper(I) iodide (5.0 mg, 0.027
mmol) under an argon atmosphere. The reaction mixture was stirred
at 80 °C for 18 h. After being cooled to rt, the reaction solution was
filtered through a Celite pad. The solvent was evaporated under
reduced pressure. The residue was purified by flash chromatography to
1
yield 40 mg (38%) of a white solid. HPLC purity: 99.0%. H NMR
(CDCl3): δ 1.34 (3H, t, J = 7.3 Hz), 1.50−1.56 (2H, m), 1.67−1.81
(4H, m), 2.21−2.27 (2H,m), 4.09 (2H, q, J = 7.3 Hz), 4.45−4.52 (1H,
m), 4.55 (1H, br s), 7.30 (1H, ddd, J = 0.9, 4.6, 7.8 Hz), 7.82 (1H,
ddd, J = 1.8, 1.8, 7.8 Hz), 7.84 (1H, s), 8.56 (1H, dd, J = 1.8, 4.6 Hz),
8.79 (1H, dd, J = 0.9, 1.8 Hz). HRMS (ESI): m/z calcd for
C20H21N4OS (M + H)+ 365.1431, found 365.1428.
2-(Cyclopentylamino)-3-ethyl-7-(3-morpholinoprop-1-yn-1-
yl)thieno[3,2-d]pyrimidin-4(3H)-one (26b). To a mixture of 16
(100 mg, 0.29 mmol), 3-morpholinopropyne (200 mg, 1.60 mmol),
and triethylamine (1.0 mL, 7.15 mmol) in acetonitrile (3 mL) were
added PdCl2(dppf)·CH2Cl2 (40 mg, 0.057 mmol) and copper(I)
iodide (10 mg, 0.053 mmol) under an argon atmosphere. The reaction
mixture was stirred at 80 °C for 18 h. After being cooled to rt, the
reaction solution was filtered through a Celite pad. The solvent was
2-(Cyclopentylamino)-3,7-diethylthieno[3,2-d]pyrimidin-
4(3H)-one (23). To a solution of 22 (55 mg, 0.19 mmol) in THF (10
H
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evaporated under reduced pressure. The residue was purified by flash
chromatography to yield 55 mg (49%) of a white solid. HPLC purity:
99.3%. 1H NMR (CDCl3): δ 1.32 (3H, t, J = 7.3 Hz), 1.49−1.56 (2H,
m), 1.64−1.80 (4H, m), 2.16−2.21 (2H, m), 2.71 (4H, t, J = 5.0 Hz),
3.59 (2H, s), 3.77 (4H, t, J = 5.0 Hz), 4.07 (2H, q, J = 7.3 Hz), 4.45−
4.52 (2H, m), 7.70 (1H, s). HRMS (ESI): m/z calcd for C20H27N4O2S
(M + H)+ 387.1849, found 387.1845.
= 1.3, 7.7 Hz), 8.78 (1H, t, J = 1.3 Hz). HRMS (ESI): m/z calcd for
C21H24N3O3S (M + H)+ 398.1533, found 398.1526.
Methyl 4-[2-(Cyclopentylamino)-3-ethyl-4-oxo-3,4-
dihydrothieno[3,2-d]pyrimidin-7-yl]benzoate (28d). According
to the method described for derivative 28a, transformation of 16 (20
mg, 0.06 mmol) yielded 22 mg (95%) of a white solid. HPLC purity:
96.9%. 1H NMR (CDCl3): δ 1.36 (3H, t, J = 7.3 Hz), 1.50−1.62 (2H,
m), 1.65−1.83 (4H, m), 2.10−2.22 (2H, m), 3.95 (3H, s), 4.11 (2H, q,
J = 7.3 Hz), 4.30−4.41 (1H, m), 4.50 (1H, d, J = 6.0 Hz), 7.83 (1H, s),
8.07−8.13 (4H, m). HRMS (ESI): m/z calcd for C21H24N3O3S (M +
H)+ 398.1533, found 398.1529.
2-(Cyclopentylamino)-3-ethyl-7-pyridin-3-ylthieno[3,2-d]-
pyrimidin-4(3H)-one (28e). According to the method described for
derivative 28a, transformation of 16 (20 mg, 0.06 mmol) yielded 18
mg (90%) of a white solid. HPLC purity: 99.3%. 1H NMR (CDCl3): δ
1.36 (3H, t, J = 7.3 Hz), 1.50−1.62 (2H, m), 1.64−1.80 (4H, m),
2.10−2.21 (2H, m), 4.12 (2H, q, J = 7.3 Hz), 4.34−4.42 (1H, m), 4.51
(1H, d, J = 5.5 Hz), 7.36 (1H, dd, J = 5.0, 7.8 Hz), 7.83 (1H, s), 8.27
(1H, ddd, J = 2.0, 2.0, 7.8 Hz), 8.57 (1H, dd, J = 2.0, 5.0 Hz), 8.78
(1H, d, J = 2.0 Hz). HRMS (ESI): m/z calcd for C18H21N4OS (M +
H)+ 341.1431, found 341.1429.
2-(Cyclopentylamino)-3-ethyl-7-[4-(morpholinomethyl)-
phenyl]thieno[3,2-d]pyrimidin-4(3H)-one (29a). To a solution of
16 (300 mg, 0.88 mmol) in 1,4-dioxane (3 mL) were added 4-([4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl]morpholine (266
mg, 0.88 mmol) and tripotassium phosphate (186 mg, 0.88 mmol).
Tetrakis(triphenylphosphine)palladium (1013 mg, 0.88 mmol) was
added under an argon atmosphere and the resulting solution was
stirred at reflux for 14 h. The solution was filtered through a Celite
pad, and the solvent was evaporated under reduced pressure. The
residue was purified by flash chromatography to yield 245 mg (64%)
of a white solid. HPLC purity: 95.1%. 1H NMR (CDCl3): δ 1.33−1.37
(3H, m), 1.56−1.76 (6H, m), 2.14−2.20 (2H, m), 2.49 (4H, br s),
3.54 (2H, s), 3.73 (4H, t, J = 4.6 Hz), 4.11 (2H, q, J = 6.0 Hz), 4.38
(1H, m), 4.51 (1H, br d, J = 5.0 Hz), 7.38 (2H, d, J = 7.8 Hz), 7.76
(1H, s), 7.97 (2H, d, J = 7.8 Hz). HRMS (ESI): m/z calcd for
C24H31N4O2S (M + H)+ 439.2162, found 439.2164.
2-(Cyclopentylamino)-3-ethyl-7-(2-pyridin-3-ylethyl)thieno-
[3,2-d]pyrimidin-4(3H)-one (27a). To a solution of 26a (35 mg,
0.095 mmol) in THF (10 mL) was added 10% Pd/C (30 mg). The
reaction mixture was stirred at rt for 24 h under a hydrogen
atmosphere (3 atm). The reaction solution was filtered through a
Celite pad, and the solvent was evaporated under reduced pressure to
1
yield 30 mg (85%) of a white solid. HPLC purity: 95.0%. H NMR
(CDCl3): δ 1.34 (3H, t, J = 7.3 Hz), 1.53−1.58 (2H, m), 1.68−1.80
(4H, m), 2.12−2.21 (2H, m), 2.97−3.06 (4H, m), 4.09 (2H, q, J = 7.3
Hz), 4.39−4.43 (1H,m), 4.45 (1H, br s), 7.20 (1H, dd, J = 4.6, 7.8
Hz), 7.22 (1H, s), 7.47 (1H, ddd, J = 1.8, 1.8, 7.8 Hz), 8.45 (1H, dd, J
= 1.8, 4.6 Hz), 8.49 (1H, d, J = 1.8 Hz). HRMS (ESI): m/z calcd for
C20H25N4OS (M + H)+ 369.1744, found 369.1739.
2-(Cyclopentylamino)-3-ethyl-7-(3-morpholinopropyl)-
thieno[3,2-d]pyrimidin-4(3H)-one (27b). To a solution of 26b (50
mg, 0.13 mmol) in THF (10 mL) was added 10% Pd/C (30 mg, 60%,
w/w). The reaction mixture was stirred at rt for 24 h under a hydrogen
atmosphere (3 atm). The reaction solution was filtered through a
Celite pad, and the solvent was evaporated under reduced pressure to
1
yield 49 mg (96%) of a colorless oil. HPLC purity: 98.1%. H NMR
(CDCl3): δ 1.33 (3H, t, J = 7.3 Hz), 1.49−1.55 (2H, m), 1.64−1.78
(4H, m), 1.86−1.93 (2H, m), 2.11−2.19 (2H, m), 2.41 (2H, t, J = 7.3
Hz), 2.45 (4H, t, J = 4.6 Hz), 2.73 (2H, t, J = 7.3 Hz), 3.72 (4H, t, J =
4.6 Hz), 4.08 (2H, q, J = 7.3 Hz), 4.34−4.45 (2H, m), 7.28 (1H, s).
HRMS (ESI): m/z calcd for C20H31N4O2S (M + H)+ 391.2162, found
391.2158.
2-(Cyclopentylamino)-3-ethyl-7-phenylthieno[3,2-d]-
pyrimidin-4(3H)-one (28a). To a DMF (0.3 mL) solution of
diacetoxypalladium (3.28 mg, 0.015 mmol) and triphenylphosphine
(15.3 mg, 0.058 mmol) was added dropwise a DMF (1.5 mL) solution
of 16 (100 mg, 0.292 mmol). After the resulting solution was stirred at
rt for 5 min, an ethanol (0.3 mL) solution of phenylboronic acid (71.3
mg, 0.58 mmol) and saturated sodium bicarbonate (0.3 mL) were
added. The reaction mixture was stirred at 80 °C for 2 h. After the
mixture was cooled to rt, water was added, and the mixture was
extracted with diethyl ether. The organic layer was dried over sodium
sulfate, and the solvent was evaporated under reduced pressure. The
residue was purified by flash chromatography to yield 76 mg (77%) of
a white solid. HPLC purity: 98.9%. 1H NMR (CDCl3): δ 1.36 (3H, t, J
= 7.3 Hz), 1.50−1.61 (2H, m), 1.63−1.80 (4H, m), 2.11−2.21 (2H,
m), 4.11 (2H, q, J = 7.3 Hz), 4.33−4.41 (1H, m), 4.47 (1H, d, J = 5.5
Hz), 7.30−7.36 (1H, m), 7.40−7.46 (2H, m), 7.77 (1H, s), 7.96−8.01
(2H, m). HRMS (ESI): m/z calcd for C19H22N3OS (M + H)+
340.1478, found 340.1474.
Methyl 2-[2-(Cyclopentylamino)-3-ethyl-4-oxo-3,4-
dihydrothieno[3,2-d]pyrimidin-7-yl]benzoate (28b). According
to the method described for derivative 28a, transformation of 16 (20
mg, 0.06 mmol) yielded 23 mg (99%) of a white solid. HPLC purity:
98.5%. 1H NMR (CDCl3): δ 1.31 (3H, t, J = 7.3 Hz), 1.29−1.42 (2H,
m), 1.55−1.71 (4H, m), 1.95−2.06 (2H, m), 3.56 (3H, s), 4.07 (2H, q,
J = 7.3 Hz), 4.20−4.30 (1H, m), 4.37 (1H, d, J = 6.4 Hz), 7.41−7.47
(2H,m), 7.53−7.58 (1H, m), 7.61 (1H, s), 7.93−7.98 (1H, m). HRMS
(ESI): m/z calcd for C21H24N3O3S (M + H)+ 398.1533, found
398.1531.
2-(Cyclopentylamino)-3-ethyl-7-(6-morpholinopyridin-3-yl)-
thieno[3,2-d]pyrimidin-4(3H)-one (29b). To a solution of 16 (63
mg, 0.184 mmol) in 1,4-dioxane (0.61 mL) were added 4-[5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)pyridinyl]morpholine (64.1 mg,
0.221 mmol) and tripotassium phosphate (78.1 mg, 0.368 mmol).
Tetrakis(triphenylphosphine)palladium (10.6 mg, 0.0072 mmol) was
added under an argon atmosphere and the resulting solution was
stirred at 90 °C for 14 h. The solution was filtered through a Celite
pad, and the solvent was evaporated under reduced pressure. The
residue was purified by flash chromatography to yield 21.9 mg (28%)
1
of a white solid. HPLC purity: 97.8%. H NMR (DMSO-d6): δ 1.15
(3H, t, J = 6.9 Hz), 1.56−1.73 (6H, m), 1.96−2.00 (2H, m), 3.49 (4H,
t, J = 5.0 Hz), 3.71 (4H, t, J = 5.0 Hz), 4.14 (2H, q, J = 6.9 Hz), 4.30
(1H, m), 6.75 (1H, d, J = 6.0 Hz), 6.90 (1H, d, J = 8.7 Hz), 8.28 (1H,
dd, J = 2.3, 8.7 Hz), 8.85 (1H, d, J = 2.3 Hz). HRMS (ESI): m/z calcd
for C22H28N5O2S (M + H)+ 426.1958, found 426.1959.
tert-Butyl 4-(2-(Cyclopentylamino)-3-ethyl-4-oxo-3,4-
dihydrothieno[3,2-d]pyrimidin-7-yl)-3-oxopiperazine-1-car-
boxylate (30). To a solution of 16 (995 mg, 2.91 mmol) in 1,4-
dioxane (7.28 mL) were added tripotassium phosphate (1.85 g, 8.73
mmol), N,N-dimethylethylenediamine (0.25 mL, 2.33 mmol), and tert-
butyl 3-oxopiperazine-1-carboxylate (640 mg, 3.20 mmol). Copper(I)
iodide (333 mg, 1.75 mmol) was added under an argon atmosphere
and the resulting solution was refluxed for 14 h. The solution was
filtered through a Celite pad, and the solvent was evaporated under
reduced pressure. The residue was purified by flash chromatography to
Methyl 3-[2-(Cyclopentylamino)-3-ethyl-4-oxo-3,4-
dihydrothieno[3,2-d]pyrimidin-7-yl]benzoate (28c). According
to the method described for derivative 28a, transformation of 16 (20
mg, 0.06 mmol) yielded 23 mg (99%) of a white solid. HPLC purity:
95.6%. 1H NMR (CDCl3): δ 1.36 (3H, t, J = 7.3 Hz), 1.48−1.61 (2H,
m), 1.63−1.80 (4H, m), 2.11−2.25 (2H, m), 3.93 (3H, s), 4.12 (2H, q,
J = 7.3 Hz), 4.37−4.47 (1H, m), 4.51 (1H, d, J = 5.9 Hz), 7.50 (1H, t,
J = 7.7 Hz), 7.84 (1H, s), 8.01 (1H, dt, J = 1.3, 7.7 Hz), 8.15 (1H, dt, J
1
yield 788 mg (59%) of a yellow solid. HPLC purity: 98.8%. H NMR
(CDCl3): 1.32 (3H, t, J = 7.3 Hz), 1.50 (9H, s), 1.47−1.56 (2H, m),
1.63−1.77 (4H, m), 2.05−2.12 (2H, m), 3.80−3.83 (2H, m), 3.94
(2H, br s), 4.06 (2H, q, J = 7.3 Hz), 4.23−4.31 (1H, m), 4.28 (2H, s),
4.49 (1H, d, J = 5.5 Hz), 7.69 (1H, s). HRMS (ESI): m/z calcd for
C22H32N5O4S (M + H)+ 462.2097, found 462.2162.
I
dx.doi.org/10.1021/jm5008215 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Modeling of the Phosphodiesterase 7 Protein−5 Structure.
The substructure of 5 (2-amino-3-methylthieno[3,2-d]pyrimidin-
4(3H)-one) was docked into the PDE7A catalytic site using the
Glide SP docking protocol. An appropriate model with spaces for the
substituents was obtained. The substituents were then manually added
to the model structure. The torsion angles were adjusted by visual
inspection to place the substituents in a reasonable orientation.
PDE Activity Assay. Human PDE7A1 and PDE4B1 were
overexpressed in insect cells. The enzymatic activity of PDE7A1 was
monitored by measuring the hydrolysis of [3H]cAMP to [3H]AMP.
The reaction mixture (100 μL final volume) contained 10 nM
[3H]cAMP and 40 nM cAMP in the assay buffer (20 mM Tris, pH 7.5,
10 mM MgCl2, 0.1 mM EDTA). In the case of PDE4B1, 20 nM
[3H]cAMP and 480 nM cAMP were added to the assay buffer.
The enzymatic reactions were initiated by adding a solution
containing the phosphodiesterase protein. The mixtures were
incubated for 30 min at room temperature. The reactions were
stopped by the addition of 50 μL of 25 mM ZnCl2, followed by 50 μL
of 10 mM Ba(OH)2. The precipitates were filtered through GF-B glass
fiber filters (Millipore), and the filters were washed several times with
tap water. The filters were dried and counted in TopCount NTX
(PerkinElmer).
IL-2 Assay. Freshly isolated mouse lymphocytes were resuspended
in culture media composed of RPMI1640 medium supplemented with
10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 0.5 mM
monothioglycerol, 100 U/mL penicillin, and 100 μg/mL streptomycin.
The cells were stimulated with anti-CD3 and anti-CD28 antibodies
and incubated for 3 days at 37 °C and 5% CO2. The supernatants were
collected, and the IL-2 concentration was measured by ELISA.
Crystallography. The PDE7A1 coding region of amino acids
130−482 was subcloned into vector pET32a. The plasmid pET32-
PDE7A1-130−482 was transformed into Escherichia coli strain BL21
(codonplus) for overexpression. E. coli cells were grown in LB medium
at 37 °C to absorption A600 = 0.6, and then 0.1 mM isopropyl β-D-
thiogalactopyranoside was added for further growth at 15 °C for 24 h.
The catalytic domain of PDE7A1 was purified using three chromato-
graphic columns of nickel−nitrilotriacetic acid, Q-Sepharose, and
Superdex 200. The PDE7A1−24 complex was crystallized by the
hanging drop method at 4 °C. The protein drops contained 2 μL of
PDE7A1−24 complex and 2 μL of well buffer containing 0.7−0.9 M
(NH4)2SO4, 3 mM β-mercaptoethanol, 10 mM EDTA, 10 mM MgCl2,
and 0.1 M Tris·HCl at pH 7.5. The well buffer plus 20% glycerol was
used as the cryosolvent for freezing the crystals in liquid nitrogen. Data
were collected at 100 K using a Micromax 007 generator and an R-
AXIS VII image plate detector (Rigaku MSC). The data were
integrated and reduced with HKL2000. Structures were determined by
molecular replacement using the structure of the PDE7A1 catalytic
domain (PDB ID 1ZKL21) as a starting model for rigid body
refinement, with REFMAC as implemented in CCP4. The model was
rebuilt manually with Coot and completed using iterative rounds of
refinement and rebuilding. The coordinates and structural factors have
been deposited into the Protein Data Bank with the accession code
4PM0.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Masanao Shimano, Hiroshi Shin, Yuuta Onodera,
and Hirohide Ishige for their helpful discussions, Rie Suzuki
and Sayuri Kataoka for analytical assistance, and Hironobu
Ogura for manuscript preparation.
ABBREVIATIONS USED
■
dppf, 1,1′-bis(diphenylphosphino)ferrocene; FBS, fetal bovine
serum; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid; HOBt, 1-hydroxybenzotriazole; IL-2, interleukin 2; LE,
ligand efficiency; NMM, N-methylmorpholine; Pin, pinacolate;
PDE, phosphodiesterase; SAR, structure−activity relationship;
WSC, water-soluble carbodiimide
REFERENCES
■
(1) Zhang, K. Y.; Card, G. L.; Suzuki, Y.; Artis, D. R.; Fong, D.;
Gillette, S.; Hsieh, D.; Neiman, J.; West, B. L.; Zhang, C.; Milburn, M.
V.; Kim, S. H.; Schlessinger, J.; Bollag, G. A glutamine switch
mechanism for nucleotide selectivity by phosphodiesterases. Mol. Cell
2004, 15, 279−286.
(2) Manallack, D. T.; Hughes, R. A.; Thompson, P. E. The next
generation of phosphodiesterase inhibitors: structural clues to ligand
and substrate selectivity of phosphodiesterases. J. Med. Chem. 2005, 48,
3449−3462.
(3) Verhoest, P. R.; Chapin, D. S.; Corman, M.; Fonseca, K.; Harms,
J. F.; Hou, X.; Marr, E. S.; Menniti, F. S.; Nelson, F.; O’Connor, R.;
Pandit, J.; Proulx-Lafrance, C.; Schmidt, A. W.; Schmidt, C. J.; Suiciak,
J. A.; Liras, S. Discovery of a novel class of phosphodiesterase 10A
inhibitors and identification of clinical candidate 2-[4-(1-methyl-4-
pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]-quinoline (PF-
2545920) for the treatment of schizophrenia. J. Med. Chem. 2009,
52, 5188−5196.
(4) Zhang, L.; Murray, F.; Zahno, A.; Kanter, J. R.; Chou, D.; Suda,
R.; Fenlon, M.; Rassenti, L.; Cottam, H.; Kipps, T. J.; Insel, P. A.
Cyclic nucleotide phosphodiesterase profiling reveals increased
expression of phosphodiesterase 7B in chronic lymphocytic leukemia.
Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 19532−19537.
́ ́ ̀
(5) Miro, X.; Perez-Torres, S.; Palacios, J. M.; Puigdomenech, P.;
Mengod, G. Differential distribution of cAMP-specific phosphodies-
terase 7A mRNA in rat brain and peripheral organs. Synapse 2001, 40,
201−214.
(6) Fan Chung, K. Phosphodiesterase inhibitors in airways disease.
Eur. J. Pharmacol. 2006, 533, 110−117.
(7) Castano, T.; Wang, H.; Campillo, N. E.; Ballester, S.; Gonzal
́
ez-
̃
García, C.; Hernan
́
dez, J.; Per
́
ez, C.; Cuenca, J.; Per
́
ez-Castillo, A.;
Martínez, A.; Huertas, O.; Gelpí, J. L.; Luque, F. J.; Ke, H.; Gil, C.
Synthesis, structural analysis, and biological evaluation of thioxoqui-
nazoline derivatives as phosphodiesterase 7 inhibitors. ChemMedChem
2009, 4, 866−876.
(8) Redondo, M.; Brea, J.; Perez, D. I.; Soteras, I.; Val, C.; Perez, C.;
Morales-García, J. A.; Alonso-Gil, S.; Paul-Fernandez, N.; Martin-
Alvarez, R.; Cadavid, M. I.; Loza, M. I.; Perez-Castillo, A.; Mengod, G.;
Campillo, N. E.; Martinez, A.; Gil, C. Effect of phosphodiesterase 7
(PDE7) inhibitors in experimental autoimmune encephalomyelitis
mice. Discovery of a new chemically diverse family of compounds. J.
Med. Chem. 2012, 55, 3274−3284.
ASSOCIATED CONTENT
■
S
* Supporting Information
Proposed binding models of 6 and 7, full PDE selectivity profile
of 30, and physicochemical and pharmacokinetic profiles. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(9) Barnes, M. J.; Cooper, N.; Davenport, R. J.; Dyke, H. J.; Galleway,
F. P.; Galvin, F. C.; Gowers, L.; Haughan, A. F.; Lowe, C.; Meissner, J.
W.; Montana, J. G.; Morgan, T.; Picken, C. L.; Watson, R. J. Synthesis
and structure−activity relationships of guanine analogues as
phosphodiesterase 7 (PDE7) inhibitors. Bioorg. Med. Chem. Lett.
2001, 11, 1081−1083.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +81-75-594-0787. Fax: +81-75-594-0790. E-mail:
kawai_kentaro@kaken.co.jp.
Author Contributions
(10) Lorthiois, E.; Bernardelli, P.; Vergne, F.; Oliveira, C.; Mafroud,
A. K.; Proust, E.; Heuze, L.; Moreau, F.; Idrissi, M.; Tertre, A.; Bertin,
†K.K., Y.E., and Y.S. contributed equally to this work.
J
dx.doi.org/10.1021/jm5008215 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
B.; Coupe, M.; Wrigglesworth, R.; Descours, A.; Soulard, P.; Berna, P.
Spiroquinazolinones as novel, potent, and selective PDE7 inhibitors.
Part 1. Bioorg. Med. Chem. Lett. 2004, 14, 4623−4626.
(11) Vergne, F.; Bernardelli, P.; Lorthiois, E.; Pham, N.; Proust, E.;
Oliveira, C.; Mafroud, A. K.; Royer, F.; Wrigglesworth, R.; Schellhaas,
J.; Barvian, M.; Moreau, F.; Idrissi, M.; Tertre, A.; Bertin, B.; Coupe,
M.; Berna, P.; Soulard, P. Discovery of thiadiazoles as a novel
structural class of potent and selective PDE7 inhibitors. Part 1: design,
synthesis and structure−activity relationship studies. Bioorg. Med.
Chem. Lett. 2004, 14, 4607−4613.
(12) Gewald, R.; Rueger, C.; Grunwald, C.; Egerland, U.; Hoefgen,
N. Synthesis and structure−activity relationship studies of dihydro-
naphthyridinediones as a novel structural class of potent and selective
PDE7 inhibitors. Bioorg. Med. Chem. Lett. 2011, 21, 6652−6656.
(13) Pitts, W. J.; Vaccaro, W.; Huynh, T.; Leftheris, K.; Roberge, J.
Y.; Barbosa, J.; Guo, J.; Brown, B.; Watson, A.; Donaldson, K.; Starling,
G. C.; Kiener, P. A.; Poss, M. A.; Dodd, J. H.; Barrish, J. C.
Identification of purine inhibitors of phosphodiesterase 7 (PDE7).
Bioorg. Med. Chem. Lett. 2004, 14, 2955−2958.
(27) Li, L.; Yee, C.; Beavo, J. A. CD3- and CD28-dependent
induction of PDE7 required for T cell activation. Science 1999, 283,
848−851.
(28) Yamamoto, S.; Sugahara, S.; Naito, R.; Ichikawa, A.; Ikeda, K.;
Yamada, T.; Shimizu, Y. The effects of a novel phosphodiesterase 7A
and -4 dual inhibitor, YM-393059, on T-cell-related cytokine
production in vitro and in vivo. Eur. J. Pharmacol. 2006, 541, 106−114.
(29) Kadoshima-Yamaoka, K.; Murakawa, M.; Goto, M.; Tanaka, Y.;
Inoue, H.; Murafuji, H.; Nagahira, A.; Hayashi, Y.; Nagahira, K.; Miura,
K.; Nakatsuka, T.; Chamoto, K.; Fukuda, Y.; Nishimura, T. ASB16165,
a novel inhibitor for phosphodiesterase 7A (PDE7A), suppresses IL-
12-induced IFN-γ production by mouse activated T lymphocytes.
Immunol. Lett. 2009, 122, 193−197.
(30) Jones, N. A.; Leport, M.; Holand, T.; Vos, T.; Morgan, M.; Fink,
M.; Pruniaux, M. P.; Berthelier, C.; O’Connor, B. J.; Bertrand, C.;
Page, C. P. Phosphodiesterase (PDE) 7 in inflammatory cells from
patients with asthma and COPD. Pulm. Pharmacol. Ther. 2007, 20,
60−68.
(14) Banerjee, A.; Patil, S.; Pawar, M. Y.; Gullapalli, S.; Gupta, P. K.;
Gandhi, M. N.; Bhateja, D. K.; Bajpai, M.; Sangana, R. R.; Gudi, G. S.;
Khairatkar-Joshi, N.; Gharat, L. A. Imidazopyridazinones as novel
PDE7 inhibitors: SAR and in vivo studies in Parkinson’s disease model.
Bioorg. Med. Chem. Lett. 2012, 22, 6286−6291.
(15) Martínez, A.; Castro, A.; Gil, C.; Miralpeix, M.; Segarra, V.;
Domen
́
ech, T.; Beleta, J.; Palacios, J. M.; Ryder, H.; Miro,
́
X.; Bonet,
C.; Casacuberta, J. M.; Azorín, F.; Pina, B.; Puigdomen
́
ech, P. Benzyl
̃
derivatives of 2,1,3-benzo- and benzothieno[3,2-a]thiadiazine 2,2-
dioxides: first phosphodiesterase 7 inhibitors. J. Med. Chem. 2000, 43,
683−689.
(16) Goto, M.; Kadoshima-Yamaoka, K.; Murakawa, M.; Yoshioka,
R.; Tanaka, Y.; Inoue, H.; Murafuji, H.; Kanki, S.; Hayashi, Y.;
Nagahira, K.; Ogata, A.; Nakatsuka, T.; Fukuda, Y. Phosphodiesterase
7A inhibitor ASB16165 impairs proliferation of keratinocytes in vitro
and in vivo. Eur. J. Pharmacol. 2010, 633, 93−97.
(17) Baell, J. B.; halloway, G. A. New substructure filters for removal
of pan assay interference compounds (PAINS) from screening libraries
and for their exclusion in bioassays. J. Med. Chem. 2010, 53, 2719−
2740.
(18) Abad-Zapatero, C.; Metz, J. T. Ligand efficiency indices as
guideposts for drug discovery. Drug Discovery Today 2005, 10, 464−
469.
(19) Reynolds, C. H.; Tounge, B. A.; Bembenek, S. D. Ligand
binding efficiency: trends, physical basis, and implications. J. Med.
Chem. 2008, 51, 2432−2438.
(20) Schrodinger Suite 2009; Glide, version 5.5; Maestro, version 9.0;
̈
Schrodinger, LLC: New York, 2009.
̈
(21) Wang, H.; Liu, Y.; Chen, Y.; Robinson, H.; Ke, H. Multiple
elements jointly determine inhibitor selectivity of cyclic nucleotide
phosphodiesterases 4 and 7. J. Biol. Chem. 2005, 280, 30949−30955.
(22) Inoue, H.; Murafuji, H.; Hayashi, Y. Thienopyrazole derivative
having PDE7 inhibitory activity. U.S. Patent 7,932,250, Apr 26, 2011.
(23) Mirza-Aghayan, M.; Boukherroub, R.; Rahimifard, M. A simple
and efficient hydrogenation of benzyl alcohols to methylene
compounds using triethylsilane and a palladium catalyst. Tetrahedron
Lett. 2009, 50, 5930−5932.
̀ ̀
(24) Pages, L.; Gavalda, A.; Lehner, M. D. PDE4 inhibitors: a review
of current developments (2005 - 2009). Expert Opin. Ther. Pat. 2009,
19, 1501−1519.
(25) Calverley, P. M.; Sanchez-Toril, F.; McIvor, A.; Teichmann, P.;
Bredenbroeker, D.; Fabbri, L. M. Effect of 1-year treatment with
roflumilast in severe chronic obstructive pulmonary disease. Am. J.
Respir. Crit. Care Med. 2007, 176, 154−61.
(26) Rennard, S. I.; Schachter, N.; Strek, M.; Rickard, K.; Amit, O.
Cilomilast for COPD: results of a 6-month, placebo-controlled study
of a potent, selective inhibitor of phosphodiesterase 4. Chest 2006,
129, 56−66.
K
dx.doi.org/10.1021/jm5008215 | J. Med. Chem. XXXX, XXX, XXX−XXX