Novel thieno[2,3-d]pyrimidines: their design, synthesis, crystal structure analysis and pharmacological evaluation.

Abstract

Novel thieno[2,3-d]pyrimidines containing a cyclohexane ring fused with a six- or five-membered heterocyclic moiety along with a benzylic nitrile were designed as potential inhibitors of PDE4. Expeditious synthesis of these compounds was carried out via a multi-step sequence consisting of a few key steps such as Gewald reaction, Dieckmann type cyclisation and Krapcho decarboxylation. This newly developed strategy involved construction of the thienopyrimidine ring followed by the cyclohexanone moiety and subsequently the fused heterocyclic ring. A number of thieno[2,3-d]pyrimidine based derivatives were synthesized using this method some of which showed promising PDE4B inhibitory properties. One of them was tested for PDE4D inhibition in vitro and dose dependent inhibition of TNF-α. A few selected molecules were docked into the PE4B protein the results of which showed good overall correlations to their observed PDE4B inhibitory properties in vitro. The crystal structure analysis of representative compounds along with hydrogen bonding patterns and molecular arrangement present within the molecule is described.

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Organic &
Biomolecular
Chemistry
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PAPER
Novel thieno[2,3-d]pyrimidines: their design, synthesis, crystal structure
analysis and pharmacological evaluation†
Raju Adepu,aD. Rambabu,a,bBagineni Prasad,aChandana Lakshmi T. Meda,aAjit Kandale,a
G. Rama Krishna,cC. Malla Reddy,cLakshmi N. Chennuru,dKishore V. L. Parsa*aand Manojit Pal*a
Received 27th February 2012, Accepted 30th May 2012
DOI: 10.1039/c2ob25420d
Novel thieno[2,3-d]pyrimidines containing a cyclohexane ring fused with a six- or five-membered
heterocyclic moiety along with a benzylic nitrile were designed as potential inhibitors of PDE4.
Expeditious synthesis of these compounds was carried out via a multi-step sequence consisting of a few
key steps such as Gewald reaction, Dieckmann type cyclisation and Krapcho decarboxylation. This newly
developed strategy involved construction of the thienopyrimidine ring followed by the cyclohexanone
moiety and subsequently the fused heterocyclic ring. A number of thieno[2,3-d]pyrimidine based
derivatives were synthesized using this method some of which showed promising PDE4B inhibitory
properties. One of them was tested for PDE4D inhibition in vitro and dose dependent inhibition of
TNF-α. A few selected molecules were docked into the PE4B protein the results of which showed good
overall correlations to their observed PDE4B inhibitory properties in vitro. The crystal structure analysis
of representative compounds along with hydrogen bonding patterns and molecular arrangement present
within the molecule is described.
Introduction
A pyrimidine nucleus fused with another heterocycle has found
wide applications in the design and discovery of novel bioactive
molecules and drugs.1For example, thieno[2,3-d]pyrimidine
derivatives such as A and B (Fig. 1) exhibited remarkable
affinity and selectivity for the 5-HT3 receptor.2,3In continuation
of our research under the new drug discovery program, we
became interested in evaluating a library of small-molecules
based on thieno[2,3-d]pyrimidine that were designed as potential
inhibitors of PDE4 (phosphodiesterase 4). PDEs are a diverse
family of enzymes that hydrolyse cyclic nucleotides and thus
play a key role in regulating intracellular levels of the second
messenger cAMP and cGMP, and hence cell function.4PDE4 is
a cAMP-specific PDE and predominant isoenzyme in the
majority of inflammatory cells, with the exception of platelets,
implicated in inflammatory airways disease. Elevated levels of
cAMP play a major role in relaxation of vascular smooth
muscle, which is beneficial in treating inflammatory diseases
especially pulmonary diseases. Thus, inhibition of PDE4 is
beneficial for the treatment of respiratory diseases including
asthma and chronic obstructive pulmonary disease (COPD).5
The use of first-generation PDE4 inhibitor rolipram6(C, Fig. 2)
however was associated with dose-limiting side effects e.g.
nausea and vomiting. While these side effects were reduced
by second-generation inhibitors like cilomilast7(Ariflo) and
roflumilast (Daxas®, Nycomed), their therapeutic index has
delayed market launch so far. Recent studies have indicated that
the PDE4B subtype is linked to inflammatory cell regulation8
whereas the PDE4D subtype is implied in the emetic response.9
However, none of the PDE4 inhibitors under development are
PDE4B selective.10aRecently it has been demonstrated that inhi-
bition of PDE4D by allosteric inhibitors (maximum inhibition,
Fig. 1
derivatives.
Examplesofbiologically activethieno[2,3-d]pyrimidine
†Electronic
studies, copies of NMR spectra for all new compounds. CCDC 864129
and 864130. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c2ob25420d
supplementaryinformation(ESI)available:Docking
aInstitute of Life Sciences, University of Hyderabad Campus,
Gachibowli, Hyderabad, 500046, India.
E-mail: manojitpal@rediffmail.com
bDepartment of Chemistry, K. L. University, Vaddeswaram, Guntur,
522 502 Andhra Pradesh, India
cDepartment of Chemical Sciences, Indian Institute of Science
Education and Research, Kolkata, West Bengal, 741252, India
dDaicel Chiral Technologies (India) Pvt Ltd, IKP Knowledge Park,
Hyderabad, India
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Imax 80–90%) did not cause emetic side effects raising a
possibility that PDE4B inhibitors with partial but not complete
inhibition of PDE4D (Imaxof ∼60–80%) could be developed to
treat COPD and asthma without causing emetic side effects.10b
To identify novel and orally active PDE4 inhibitors with
decreased potential for side effects the design and synthesis of
4-cyano cyclohexane-1-carboxylic acid derivatives was under-
taken which resulted in significant improvement in reducing the
side effects of C.11Thus, cilomilast (D, Fig. 2) that belongs to
this class was discovered and finally entered into phase 3 clinical
trials. However, to address the issue of configurational isomerism
of D around the CO2H group new cyclohexane derivatives
(E, Fig. 2) were designed maintaining the benzylic nitrile as one
of the pharmacophores12and the cyclohexane ring fused with a
six- or five-membered heterocyclic moiety. Recently, cyclo-
hexane derivatives (F, Fig. 2) containing a tricyclic fused aryl
ring have been reported to possess PDE4 inhibitory properties.13
Based on these observations we designed novel cyclohexane
derivatives (F, Fig. 2) containing the thieno[2,3-d]pyrimidine
moiety along with benzylic nitrile as potential and new inhibitors
of PDE4. The cyclic rings ‘X’ and ‘Y’ were chosen to introduce
diversity into the basic scaffold for the generation of library of
small molecules. To the best of our knowledge template G has
not been previously explored for the discovery of PDE4
inhibitors.
Results and discussions
Chemistry
The retro-synthetic analysis of the target compound G revealed
that construction of a cyclohexane ring at C-4 of the thieno-
[2,3-d]pyrimidine moiety could be a key step. Overall, we envi-
sioned that sequential construction of (i) the thienopyrimidine
ring followed by (ii) the cyclohexanone moiety and subsequently
(iii) the fused heterocyclic ring could provide us with the target
compounds based on G. While introduction of an aryl or
heteroaryl or alkyne moiety14,15at C-4 of the thieno[2,3-d]-
pyrimidine moiety is known a similar method to introduce a
cyclohexyl moiety at the same position was unprecedented.
Nevertheless, the 4-chloro-thieno[2,3-d]pyrimidines (4) appeared
to be appropriate starting materials for our synthesis and were
prepared following a 3-step method (step a–c, Scheme 1) as
reported earlier.14,15The use of 4 for the preparation of sub-
sequent intermediates is shown in Scheme 1. Thus, the reaction16
of ethyl cyanoacetate with chloro derivative 4 followed by in situ
decarboxylation of the resulting ester afforded the cyano deriva-
tive 5 which on double Michael reactions with methyl acrylate
furnished the diester 6. A Dieckmann type cyclisation17of 6 fol-
lowed by Krapcho decarboxylation18of the resulting β-ketoester
7 provided the cyclohexanone derivative 8 which on reaction
with N,N-dimethylformamide dimethyl acetal (DMF-DMA)
furnished the required 2-((dimethylamino)methylene)cyclohexa-
none derivative 9. The intermediates 7 and 9 were used for the
Fig. 2
tors (C–F).
Design of novel inhibitors (G) of PDE4 based on known inhibi-
Scheme 1
line, sulphur, ethanol, 90 °C, 3–8 h; (b) for 5a–c: formamide, 190 °C,
2–4 h; for 5d: formimidine acetate, DMF, 130 °C, 16 h; (c) for 5a–c:
POCl3, 110 °C, 1–1.5 h; for 5d: POCl3, Et3N, 60 °C, 2 h; (d) for 5a–b:
ethyl cyanoacetate, K2CO3, 130 °C, 1–2 h; for 5c–d: (i) ethyl cyano-
acetate, K2CO3, DMSO, 120 °C, 1–1.5 h; (ii) NaCl, H2O, DMSO,
150 °C, 5–10 h; (e) methyl acrylate, triton-B, CH3CN, 85 °C, 3–6 h;
(f) for 5a–c: NaH, DME, 85 °C, 2–5 h; for 5d: NaH, THF, 60 °C, 1 h;
(g) NaCl, H2O, DMSO, 150 °C, 4–7 h; (h) for 9a–b: DMF-DMA, Et3N,
DMF, 80 °C, 2–4 h; for 9c–d: DMF-DMA, toluene, 95 °C, 16 h.
Reagents and conditions: (a) ethyl cyanoacetate, morpho-
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preparation of the target compounds. All the intermediates syn-
thesized were well characterized by spectral (NMR, MS and IR)
data. Additionally, the molecular structure of intermediate
7a (methyl-5-cyano-5-(5,6,7,8-tetrahydrobenzo[b]thieno[2,3-d]-
pyrimidin-4-yl)-2-oxocyclohexanecarboxylate) was established
unambiguously by single crystal X-ray diffraction (Fig. 3) the
details of which are presented in the following section. The
X-ray diffraction study indicated that 7a existed in the enol tauto-
meric form predominantly stabilized by the 6-membered ring
formed due to an intramolecular H-bond. The existence of the
enol form in solution was also supported by1H NMR data as the
enolic hydroxyl group appeared at ∼ 12.0–12.3 δ.
Compound 7a crystallizes in the triclinic P1ˉspace group with
two symmetry molecules in the asymmetric unit (Z = 4, Z′ = 2)
(Fig. 3). The two molecules in the asymmetric unit contain free
hydroxyl and ester functional groups. These molecules have the
capability to form supramolecular synthons and are confor-
mationally different (Fig. 4). The inversion related molecules of
conformer-i, and conformer-ii are both forming the same type of
intramolecular O–H⋯O synthon in between the substituted
hydroxyl group with the ortho oriented methyl ester group and
the C–H⋯O, C–H⋯S intermolecular hydrogen bonding. Conse-
quently, the two groups OH and ester come closer and interact
with the O–H⋯O synthon. These interactions propagate 3D
network packing along the ac axis (see ESI†).
The reaction of 9 with guanidine or formamidine afforded the
compounds 10 or 11 (Scheme 2). Similarly, the reaction of 7a
with guanidine or formamidine afforded the compounds 12 or
13 whereas treating 7a with hydrazines afforded compounds 14
or 15 (Scheme 3). The reaction of 9a with hydrazine provided
the compound 16 (Scheme 4).12All the target compounds
synthesized were characterized by spectral (NMR, MS and IR)
data. For example, the presence of a –CN group was confirmed
by an IR absorption in the region 2240–2230 cm−1. The keto
form of compound 12 and 13 was characterized by the IR
absorption at 1650 and 1655 cm−1respectively, due to the CvO
moiety. The structure of compound 14 was assigned based on
two broad1H NMR signals at 11.3 and 9.6 δ due to the two NH
groups and an IR absorption at 1734 cm−1due to the amide
CvO moiety. The keto form of compound 15 was also indicated
by IR absorption at 1730 cm−1due to the CvO group and the
absence of any NH IR absorption beyond 3000 cm−1. Neverthe-
less, the molecular structure of a representative compound 10a
was
diffraction (Fig. 5). The X-ray diffraction study also indicated
the R-isomer of compound 10a.
Compound 10a crystallizes in the triclinic P1ˉ space group
with one molecule and one solvent molecule in the asymmetric
unit (Z = 6, Z′ = 2) (Fig. 5). The molecule in the asymmetric
unit contains pyrimidine substituted with a free amine and has
capability to form a supramolecular synthon via intermolecular
establishedunambiguously by singlecrystalX-ray
Fig. 3
50% probability level).
ORTEP representation of the 7a (thermal ellipsoids are drawn at
Fig. 4
molecule 7a via O–H⋯O synthon. (b) Showing the conformations
present in the asymmetric unit (i) conformer-i in blue, (ii) conformer-ii
in red.
(a) Showing the intramolecular hydrogen bonding present in the
Scheme 2
Preparation of 5,6,7,8-tetrahydroquinazoline derivatives.
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hydrogen bonding. The inversion related molecule in the asym-
metric unit formed the dimer synthon through pyrimidine amine
like N–H⋯N interactions (Fig. 6) and is stabilised by C–H⋯C,
C–H⋯N and C–H⋯S interactions with dimethoxy solvent mole-
cules. These interactions propagate in a 3D network packing
along bc axis (see ESI†).
While only the R-isomer of compound 10a was isolated
during generation of the single crystal via crystallization the
possibility of the presence of the S-isomer in the solution could
not be ruled out. It was therefore necessary to assess the chiral
purity of 10a and other target compounds synthesized. It is
worthy to mention that the stereocentre in compounds 10–16
was generated during the conversion of intermediate 6 to 7 and 8
to 9. This conversion was expected to afford a mixture of stereo-
isomers as the methodology used was not an enantiospecific
one. Thus, a chiral HPLC method was used to determine the
enantiomeric purity of some representative compounds e.g. 10a,
13, 14 and 16. All these compounds were found to be a
∼1:1 mixture of both the antipodes.
In vitro pharmacology
All the target compounds (10–16) synthesized were evaluated
for their PDE4 inhibitory properties in vitro. Initially, PDE4B
inhibitory potential was assessed by using PDE4B enzyme iso-
lated from Sf9 cells.19Some of the compounds showed signifi-
cant inhibition of PDE4B at 30 μM and the data generated for
most of the compounds are summarized in Table 1. As is evident
from Table 1 the change in ring size (10a vs. 10b and 10c) or
functionalization of the saturated cycloalkyl ring (10a vs. 10d)
fused with the thiophene moiety or removal of NH2group from
the pyrimidine ring (10a vs. 11a) did not show significant effect
on inhibitory activities. A similar trend regarding the effect of
fused cycloalkyl ring was observed for compounds 11. The func-
tionalization of the pyrimidine ring of 5,6,7,8-tetrahydroquinazo-
line-6-carbonitrile moiety was tolerated (11a vs. 12 and 13).
While replacing the 5,6,7,8-tetrahydroquinazoline-6-carbonitrile
moiety by 3-oxo-2,3,4,5,6,7-hexahydro-1H-indazole-5-carboni-
trile group decreased the activity significantly (11a vs. 14 and
15) the 4,5,6,7-tetrahydro-1H-indazole-5-carbonitrile moiety
restored the activity (11a vs. 16). Based on their initial PDE4B
inhibitory properties compounds 10a, 10c, 10d, 11a, 11c, 11d
and 16 were evaluated for dose dependent inhibitions (Fig. 7,
see also ESI†) and the corresponding IC50values are presented
in Table 1. A few of these compounds were also tested in a cell
based cAMP reporter assay in HEK 293 cells and their TNF-α
inhibitory activity was measured in lipopolysaccharide (LPS)
stimulated RAW 264.7 cells.19Rolipram, a well known inhibitor
of PDE4 was used as a reference compound in all these assays
which showed 100% inhibition of PDE4B at 30 μM. Since com-
pound 16 appeared as the promising inhibitor among all the
compounds tested its dose dependent inhibition of TNF-α was
Scheme 4
Preparation of 4,5,6,7-tetrahydro-1H-indazole derivative.
Scheme 3
3-oxo-2,3,4,5,6,7-hexahydro-1H-indazole derivatives.
Preparation of 4-oxo-3,4,5,6,7,8-hexahydroquinazoline and
Fig. 5
at 50% probability level).
ORTEP representation of the 10a (thermal ellipsoids are drawn
Fig. 6
molecule 10a via N–H⋯N synthon.
Showing the intermolecular hydrogen bonding present in the
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Table 1
In vitro data of compounds 10–16
Compound%PDE4B inhibition (30 μM) IC50(μM)TNF-α inhibition (30 μM)Fold elevation of cAMPa
80.04.48 ± 0.9166.03.70
66.0ndnd nd
70.4 5.64 ± 1.26nd3.09
78.93.24 ± 0.73 48.5nd
80.04.51 ± 0.8935.72.34
65.5ndndnd
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Table 1
(Contd.)
Compound%PDE4B inhibition (30 μM)IC50(μM)TNF-α inhibition (30 μM) Fold elevation of cAMPa
74.8 6.19 ± 1.30 ndnd
71.0 5.01 ± 0.5632.6 nd
68.0 ndndnd
70.7 nd50.03.17
51.4 ndnd nd
41.9 ndnd nd
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also determined (Fig. 8). Further the PDE4D inhibitory potential
of compound 16 was evaluated using the PDE4D enzyme assay
(Fig. 9). Based on the PDE4B and D inhibitory data it is evident
that compound 16 has 1.5 fold or balanced selectivity towards
PDE4B. Notably potent inhibitor cilomilast (D, Fig. 2) showed
reverse selectivity i.e. ∼10 fold selectivity towards PDE4D over
PDE4B and induced emesis at the first and/or second doses
though this effect apparently disappeared with continued treat-
ment.19cOverall, with respect to the in vitro data (PDE4 &
TNF-α) compound 16 seemed to be comparable with phase 2
clinical candidate CC-1088 (PDE4 IC50= 1.1 μM, TNF-α IC50
= 2.5 μM)19bof Celgene and was identified as a PDE4 inhibitor
of further interest.
To understand the nature of interactions of this class of mole-
cules with PDE4B a few selected molecules (only R-isomer)
were docked20into the PDE4B protein (see ESI†). The results of
this study showed good overall correlations to their observed
PDE4B inhibitory properties in vitro. For example, due to the
absence of an amine moiety on the pyrimidine ring though 11a
and 11b showed different orientation of binding at the active site
of PDE4B protein their overall interactions however were not
better than 10a or 10b (compounds 11a and 11d showed better
dock score but not better binding energy compared to 10a and
10d respectively). This is supported by the observed inhibition
of PDE4B shown by compounds 10a, 10d, 11a and 11d.
Conclusions
The thieno[2,3-d]pyrimidine based library of small molecules
containing a cyclohexane ring fused with a six- or five-mem-
bered heterocyclic moiety along with a benzylic nitrile was
designed as potential inhibitors of PDE4. These molecules were
prepared conveniently via a multi-step sequence consisting of a
few key steps such as Gewald reaction, Dieckmann type cyclisa-
tion and Krapcho decarboxylation. A number of thieno[2,3-d]-
pyrimidine based derivatives were synthesized and the molecular
structure ofarepresentative compoundwas established
Table 1
(Contd.)
Compound %PDE4B inhibition (30 μM) IC50(μM)TNF-α inhibition (30 μM)Fold elevation of cAMPa
83.62.0 ± 0.41 87.4 3.90
aIn a cell based reporter assay.
Fig. 7
Dose dependent inhibition of PDE4B by compound 16.
Fig. 8
Dose dependent inhibition of TNF-α by compound 16.
Fig. 9
Dose dependent inhibition of PDE4D by compound 16.
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unambiguously by single crystal X-ray diffraction. The crystal
structure analysis of this compound provided an insight on the
hydrogen bonding patterns and molecular arrangement present
within the molecule. Many of these compounds were evaluated
for their PDE4B inhibitory potential in vitro. Some of these
compounds showed promising inhibition of PDE4B initially at a
single dose and then subsequently in a dose dependent manner.
One of them i.e. 5-(5,6,7,8-tetrahydrobenzo[b]thieno[2,3-d]-
pyrimidin-4-yl)-4,5,6,7-tetrahydro-1H-indazole-5-carbonitrile was
tested for PDE4D inhibition in vitro and dose dependent
inhibition of TNF-α. The docking results of a few selected
molecules showed good overall correlations to their observed
PDE4B inhibitory properties in vitro. Overall, the strategy
involving the sequential construction of the thienopyrimidine
ring followed by the cyclohexanone moiety and subsequently
the fused heterocyclic ring provided a new framework that
appeared to be a promising template for the discovery of novel
inhibitors of PDE4.
Experimental section
Chemistry
General methods. Unless stated otherwise, reactions were per-
formed under nitrogen atmosphere using oven dried glassware.
Reactions were monitored by thin layer chromatography (TLC)
on silica gel plates (60 F254), visualizing with ultraviolet light
or iodine spray. Flash chromatography was performed on silica
gel (230–400 mesh) using distilled hexane, ethyl acetate, dichlor-
omethane.1H NMR and13C NMR spectra were determined in
CDCl3 or DMSO-d6 solution by using 400 or 100 MHz
spectrometers, respectively. Proton chemical shifts (δ) are rela-
tive to tetramethylsilane (TMS, δ = 0.00) as internal standard
and expressed in ppm. Spin multiplicities are given as s (singlet),
d (doublet), t (triplet) q (quartet) and m (multiplet) as well as bs
(broad). Coupling constants (J) are given in hertz. Infrared
spectra were recorded on a FT-IR spectrometer. Melting points
were determined using melting point apparatus and are un-
corrected. MS spectra were obtained on a mass spectrometer.
Chromatographic purity by HPLC (Agilent 1200 series Chem
Station software) was determined by using area normalization
method and the conditions specified in each case: column,
mobile phase (range used), flow rate, detection wavelength, and
retention time. The enantiomeric purity of some representative
compounds e.g. 10a, 13, 14 and 16 was determined by using a
chiral HPLC method.
Preparation of 2-(5,6,7,8-tetrahydrobenzo[b]thieno[2,3-d]-
pyrimidin-4-yl)acetonitrile (5a)
To a mixture of ethyl cyanoacetate (4.2 mL, 40.05 mmol) and
K2CO3 (3.7 g, 26.70 mmol) was added compound 4a (3 g,
13.35 mmol). The mixture was initially heated to 60 °C for
30 min and then at 140 °C for 1 h under anhydrous conditions.
After completion of the reaction, the mixture was cooled to room
temperature and diluted with EtOAc (60 mL). The organic layer
was collected, washed with water (2 × 30 mL) followed by brine
solution (30 mL), dried over anhydrous Na2SO4, and con-
centrated under reduced pressure. The residue isolated was
purified by column chromatography using ethyl acetate–hexane
(1: 6) to give the desired product 5a (2.4 g, 78%) as a white
solid; mp 164–166 °C; Rf= 0.45 (25% EtOAc–n-hexane); IR
(KBr, cm−1): 2931, 2853, 2256, 1539;
CDCl3) δ: 8.95 (s, 1H), 4.27 (s, 2H), 2.99–2.92 (m, 4H),
1.97–1.96 (m, 4H);
152.0, 151.0, 140.1, 129.0, 125.5, 115.7, 26.3, 26.0, 25.9, 22.4,
22.3; MS (ES mass): m/z 229.9 (M + 1); HPLC: 99.3%, column:
ZORBAX XDB C-18 150 × 4.6 mm 5 μ, mobile phase A:
0.05% formic acid in water, mobile phase B: CH3CN, gradient
(T/%B): 0/50, 2/50, 9/95, 12/95, 15/50, 18/50; flow rate: 1.0 mL
min−1; UV 240 nm, retention time 5.34 min.
1H NMR (400 MHz,
13C-NMR (100 MHz, CDCl3) δ: 168.8,
Preparation of 2-(6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]-
pyrimidin-4-yl)acetonitrile (5b)
Compound 5b was synthesized in 55% yield from 4b following
a similar procedure as presented above; white solid; mp:
205–207 °C; Rf= 0.6 (30% EtOAc–n-hexane); IR (KBr, cm−1):
2911, 2861, 2261, 1552;1H NMR (400 MHz, CDCl3) δ: 8.95
(s, 1H), 4.18 (s, 2H), 3.18–3.09 (m, 4H), 2.64–2.56 (m, 2H);
13C-NMR (100 MHz, CDCl3) δ: 174.2, 152.1, 150.7, 145.6,
134.6, 125.9, 115.5, 30.0, 29.4, 27.6, 24.9; MS (ES mass): m/z
216.1 (M + 1).
Preparation of ethyl 2-cyano-2-(6,7,8,9-tetrahydro-5H-
cyclohepta[4,5]thieno[2,3-d]pyrimidin-4-yl)acetate (5cc)
A mixture of 4c (1 g, 0.42 mmol), ethyl cyanoacetate (0.04 mL,
0.42 mmol) and K2CO3(86 mg, 0.63 mmol) in DMSO (10 mL)
and water (1 mL) was heated at 120 °C for 1.5 h under anhy-
drous conditions. After completion of the reaction the mixture
was cooled to room temp, diluted with water (50 mL) and
extracted with ethyl acetate (3 × 30 mL). The organic layers
were collected, combined, washed with brine solution (30 mL),
dried over anhydrous Na2SO4, and concentrated under reduced
pressure. The residue isolated was purified by column chromato-
graphy using ethyl acetate–hexane (1: 6) to give the desired
product (0.9 g, 70%) as a white solid; mp: 139–141 °C; Rf= 0.5
(25% EtOAc–n-hexane); IR (KBr, cm−1): 2927, 2856, 2200,
1656;1H NMR (400 MHz, DMSO-d6) δ: 13.83 (bs, 1H), 8.41
(s, 1H), 4.23 (q, J = 4.5 Hz, 2H), 3.05–3.02 (m, 2H), 2.95–2.92
(m, 2H), 1.88–1.83 (m, 2H), 1.68–1.54 (m, 4H), 1.28 (t, J = 4.5
Hz, 3H);13C-NMR (100 MHz, CDCl3) δ: 170.5, 162.1, 152.8,
141.9, 139.1, 136.7, 121.9, 119.4, 66.7, 61.3, 32.0, 30.6, 27.9,
27.0, 14.4, 14.3; MS (ES mass): 315.5 (M + 1).
Preparation of 2-(6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno-
[2,3-d]pyrimidin-4-yl)acetonitrile (5c)
A mixture of compound 5cc (1 g, 0.32 mmol) and NaCl (1.47 g,
2.53 mmol) in DMSO (10 mL) and water (1 mL) was heated at
150 °C for 4.5 h under anhydrous conditions. After completion
of the reaction the mixture was cooled to room temp, diluted
with water (50 mL) and extracted with ethyl acetate (3 × 30 mL).
The organic layers were collected, combined, washed with brine
solution (30 mL), dried over anhydrous Na2SO4, and con-
centrated under reduced pressure. The residue isolated was
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purified by column chromatography using ethyl acetate–hexane
(1: 6) to give desired product (524 mg, 68%) as a white solid;
mp: 133–135 °C; Rf= 0.5 (10% EtOAc–DCM); IR (KBr, cm−1):
2925, 2853, 2254, 1533;1H NMR (400 MHz, CDCl3) δ: 8.93
(s, 1H), 4.31 (s, 2H), 3.10–3.08 (m, 2H), 3.01–2.99 (m, 2H),
2.00–1.99 (m, 2H), 1.85–1.78 (m, 4H);13C-NMR (100 MHz,
CDCl3) δ: 167.6, 151.5, 150.9, 144.1, 131.0, 129.7, 115.6, 31.4,
29.9, 29.3, 26.9, 26.8, 26.5; MS (ES mass): 243.5 (M + 1).
Preparation of ethyl 2-cyano-2-(7-(tert-butoxycarbonyl)-5,6,7,8-
tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4-yl)acetate
(5dd)
The compound 5dd was synthesized in 65% yield from 4d fol-
lowing a similar procedure as presented above; light brown
solid; mp: 128–130 °C; Rf= 0.3 (30% EtOAc–n-hexane); IR
(KBr, cm−1): 2975, 2202, 1710, 1661;
CDCl3) δ: 14.75 (s, 1H), 8.08 (d, J = 3.2 Hz, 1H), 4.72 (s, 2H),
4.33 (q, J = 7.2 Hz, 2H), 3.60–3.61 (m, 2H), 3.29–3.31 (m, 2H),
1.51 (s, 9H), 1.39 (t, J = 7.2 Hz, 3H);13C-NMR (100 MHz,
CDCl3) δ: 179.3, 170.7, 152.5 (3C), 140.1 (2C), 134.0, 120.9,
80.6, 61.6, 43.4, 41.9, 31.0, 28.4 (3C), 25.9, 14.3. MS
(ES mass): 401.1 (M + 1).
1H NMR (400 MHz,
Preparation of tert-butyl 4-acetonitrilo-5,6,7,8-tetrahydropyrido-
[4′,3′:4,5]thieno[2,3-d]pyrimidine-7-carboxylate (5d)
Compound 5d was synthesized in 62% yield from 5dd following
a similar procedure as presented above; white solid; mp:
161–163 °C; Rf= 0.4 (40% EtOAc–n-hexane); IR (KBr, cm−1):
2976, 2257, 2190, 1683;1H NMR (400 MHz, CDCl3) δ: 8.99
(s, 1H), 4.77 (s, 2H), 4.26 (s, 2H), 3.84 (t, J = 5.6 Hz, 2H),
3.08–3.09 (m, 2H), 1.51 (s, 9H);13C-NMR (100 MHz, CDCl3)
δ: 169.0, 154.2, 152.6 (3C), 128.3 (2C), 115.4, 80.9, 43.9, 40.0,
28.4 (3C), 25.9 (2C); MS (ES mass): 331.1 (M + 1).
Preparation of 4-cyano-4-(5,6,7,8-tetrahydrobenzo[b]thieno-
[2,3-d]pyrimidin-4-yl)-heptanedioic acid dimethyl ester (6a)
To a solution of 5a (2 g, 8.72 mmol) in acetonitrile (12.5 mL)
was added 40% solution of triton-B (1 mL) and the mixture was
heated to reflux under anhydrous conditions. To this was added
methyl acrylate (7.9 mL, 87.22 mmol) in acetonitrile (12.5 mL)
under refluxing conditions. The mixture was refluxed for 3 h.
After completion of the reaction the mixture was cooled to room
temperature and solvent as well as excess of methyl acrylate
was evaporated under reduced pressure. The residue was dis-
solved in EtOAc (40 mL). The organic layer was washed with
water (2 × 20 mL) followed by brine solution (30 mL), dried
over anhydrous Na2SO4, and concentrated under reduced
pressure. The residue isolated was purified by column chromato-
graphy using ethyl acetate–hexane (1: 8) to give desired product
6a (2.3 g, 65%) as a light yellow liquid; Rf= 0.5 (25% EtOAc–
n-hexane); IR (KBr, cm−1): 2946, 2861, 2236, 1738;1H NMR
(400 MHz, CDCl3) δ: 8.87 (s, 1H), 3.67 (s, 6H), 3.22 (bs, 2H),
2.98 (bs, 2H), 2.86–2.79 (m, 2H), 2.69–2.61 (m, 2H), 2.53–2.45
(m, 2H), 2.37–2.29 (m, 2H), 1.96 (bs, 4H);
(100 MHz, CDCl3) δ: 172.4 (2C), 170.2, 156.4, 150.2, 140.1,
13C-NMR
129.3, 126.3, 121.7, 51.9 (2C), 32.9 (2C), 29.8 (3C), 29.6, 26.6,
23.2, 22.2; MS (ES mass): 401.9 (M + 1).
Preparation of 4-cyano-4-(6,7-dihydro-5H-cyclopenta[4,5]thieno-
[2,3-d]pyrimidin-4-yl)-heptanedioic acid dimethyl ester (6b)
Compound 6b was synthesized in 65% yield from 5b following
a similar procedure as described above; light yellow liquid; Rf=
0.7 (30% EtOAc–n-hexane); IR (KBr, cm−1): 2954, 2859, 2240,
1735;1H NMR (400 MHz, CDCl3) δ: 8.89 (s, 1H), 3.65 (s, 6H),
3.39–3.35 (m, 2H), 3.10 (t, J = 7.2 Hz, 2H), 2.81–2.74 (m, 2H),
2.62–2.45 (m, 6H), 2.41–2.33 (m, 2H);13C-NMR (100 MHz,
CDCl3) δ: 175.3, 172.4 (2C), 156.3, 150.3, 146.4, 134.6, 125.7,
121.3, 51.9 (2C), 47.3, 33.5, 32.5 (2C), 30.1, 29.8 (2C), 28.1;
MS (ES mass): 387.5 (M + 1).
Preparation of 4-cyano-4-(6,7,8,9-tetrahydro-5H-cyclohepta[4,5]-
thieno[2,3-d]pyrimidin-4-yl)-heptanedioic acid dimethyl ester (6c)
Compound 6c was synthesized in 68% yield from 5c following a
similar procedure as described above; light yellow liquid; Rf=
0.55 (10% EtOAc–DCM); IR (KBr, cm−1): 2929, 2853, 2233,
1738;1H NMR (400 MHz, CDCl3) δ: 8.88 (s, 1H), 3.68 (s, 6H),
3.32–3.29 (m, 2H), 3.03–3.00 (m, 2H), 2.88–2.80 (m, 2H),
2.73–2.64 (m, 2H), 2.60–2.48 (m, 2H), 2.39–2.32 (m, 2H),
2.03–1.95 (m, 2H), 1.78–1.72 (m, 4H);13C-NMR (100 MHz,
CDCl3) δ: 172.5 (2C), 168.9, 156.3, 149.9, 144.8, 132.1, 129.1,
120.8, 51.9 (2C), 32.4 (2C), 32.3, 31.0, 30.7, 29.8 (3C), 26.8,
26.7; MS (ES mass): 415.5 (M + 1).
Preparation of 4-cyano-4-(7-(tert-butoxycarbonyl)-5,6,7,8-
tetrahydropyrido[4′,3′: 4,5]thieno[2,3-d]pyrimidin-4-yl)-
heptanedioic acid dimethyl ester (6d)
Compound 6d was synthesized in 65% yield from 5d following
a similar procedure as described above; white solid; mp:
133–135 °C; Rf= 0.5 (35% EtOAc–n-hexane); IR (KBr, cm−1):
2976, 2236, 1736, 1686;1H NMR (400 MHz, CDCl3) δ: 8.92
(s, 1H), 4.79 (s, 2H), 3.80–3.78 (m, 2H), 3.67 (s, 6H), 3.35–3.33
(m, 2H), 2.85–2.78 (m, 2H), 2.69–2.62 (m, 2H), 2.54–2.46 (m,
2H), 2.38–2.31 (m, 2H), 1.52 (s, 9H);
CDCl3) δ: 172.3 (2C), 170.4, 152.9, 150.7 (3C), 128.4 (2C),
121.5, 80.8, 51.9 (2C), 46.4, 45.1, 32.7 (2C), 32.3, 29.7 (2C),
28.4 (3C), 28.0. MS (ES mass): 503.2 (M + 1).
13C-NMR (100 MHz,
Preparation of methyl 5-cyano-5-(5,6,7,8-tetrahydrobenzo[b]-
thieno[2,3-d]pyrimidin-4-yl)-2-oxocyclohexanecarboxylate (7a)
A cold solution of compound 6a (2 g, 4.98 mmol) in dry DME
(15 mL) was added slowly to a mixture of 60% NaH (359 mg,
14.96 mmol) in dry DME (15 mL) at 0 °C under nitrogen atmos-
phere. The reaction mixture was heated at 60–70 °C for 2.5 h.
After completion of the reaction, the mixture was quenched with
ice cold 1 N hydrochloric acid (20 mL) and extracted with ethyl
acetate (2 × 30 mL). The organic layers were collected, com-
bined, washed with water (2 × 30 mL) followed by brine
(20 mL), dried over anhydrous Na2SO4, and concentrated under
reduced pressure. The residue waspurifiedbycolumn
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chromatography using ethyl acetate–hexane (1: 9) to give the
desired product 7a (1.5 g, 82%) as a white solid; mp:
171–173 °C; Rf= 0.6 (25% EtOAc–n-hexane); IR (KBr, cm−1):
3267, 2951, 2230, 1657;1H NMR (400 MHz, CDCl3) δ: 12.25
(s, OH), 8.89 (s, 1H), 3.81 (s, 3H), 3.30–3.26 (m, 3H),
3.05–3.00 (m, 3H), 2.93–2.85 (m, 1H), 2.63–2.58 (m, 2H),
2.44–2.37 (m, 1H), 1.98 (bs, 4H);13C-NMR (100 MHz, CDCl3)
δ: 171.8, 170.6, 169.7, 157.7, 150.4, 140.0, 129.1, 126.2, 121.7,
94.5, 51.8, 42.2, 32.8, 31.3, 29.2, 26.8, 26.6, 23.2, 22.3; MS (ES
mass): 369.9 (M + 1); HPLC: 98.9%, column: ZORBAX XDB
C-18 150 × 4.6 mm 5 μ, mobile phase A: 0.05% formic acid in
water, mobile phase B: CH3CN, gradient (T/%B): 0/80, 2/80,
9/98, 12/98, 15/80, 18/80; flow rate: 1.0 mL min−1; UV 245 nm,
retention time 4.37 min.
Preparation of methyl 5-cyano-5-(6,7-dihydro-5H-cyclopenta-
[4,5]thieno[2,3-d]pyrimidin-4-yl)-2-oxocyclohexanecarboxylate
(7b)
Compound 7b was synthesized in 70% yield from 6b following
a procedure similar to that of compound 7a; white solid; mp:
153–155 °C; Rf= 0.8 (30% EtOAc–n-hexane); IR (KBr, cm−1):
3535, 2955, 2235, 1656;1H NMR (400 MHz, CDCl3) δ: 12.26
(bs, 1H), 8.90 (s, 1H), 3.81 (s, 3H), 3.41–3.36 (m, 2H), 3.25
(d, J = 16.0 Hz, 1H), 3.12 (t, J = 7.2 Hz, 2H), 2.97 (d, J = 16.5
Hz, 1H), 2.92–2.84 (m, 1H), 2.64–2.53 (m, 4H), 2.44–2.36
(m, 1H);13C-NMR (100 MHz, CDCl3) δ: 174.9, 171.8, 170.6,
157.4, 150.6, 146.2, 134.8, 125.8, 121.6, 94.4, 51.8, 41.9, 33.2,
32.3, 30.4, 30.1, 28.2, 26.6; MS (ES mass): 355.4 (M + 1).
Preparation of methyl 5-cyano-5-(6,7,8,9-tetrahydro-5H-
cyclohepta[4,5]thieno[2,3-d]pyrimidin-4-yl)-2-oxocyclo-
hexanecarboxylate (7c)
Compound 7c was synthesized in 72% yield from 6c following a
procedure similar to that of compound 7a; white solid; mp:
166–168 °C; Rf= 0.7 (25% EtOAc–n-hexane); IR (KBr, cm−1):
3482, 2931, 2232, 1656;1H NMR (400 MHz, CDCl3) δ: 12.26
(bs, 1H), 8.90 (s, 1H), 3.80 (s, 3H), 3.39–3.26 (m, 3H),
3.04–2.98 (m, 3H), 2.94–2.85 (m, 1H), 2.66–2.60 (m, 2H),
2.46–2.38 (m, 1H), 2.01–1.98 (m, 2H), 1.78–1.70 (m, 4H);
13C-NMR (100 MHz, CDCl3) δ: 171.8, 170.7, 168.5, 157.6,
150.3, 144.7, 132.1, 128.9, 121.0, 94.5, 51.8, 42.0, 32.5,
32.4, 30.9(2C), 30.6, 26.9, 26.8, 26.7; MS (ES mass): 384.2
(M + 1).
Preparation of methyl 5-cyano-5-(7-(tert-butoxycarbonyl)-
5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4-yl)-
2-oxocyclohexanecarboxylate (7d)
Compound 7d was synthesized in 55% yield from 6d using dry
THF as a solvent following a procedure similar to that of com-
pound 7a; white solid; mp: 189–191 °C; Rf= 0.5 (40% EtOAc–
n-hexane); IR (KBr, cm−1): 2975, 2232, 1727, 1695, 1664;1H
NMR (400 MHz, CDCl3) δ: 12.25 (bs, 1OH), 8.94 (s, 1H), 4.81
(s, 2H), 3.81 (s, 5H), 3.42–3.35 (m, 2H), 3.26 (d, J = 16.0 Hz,
1H), 2.98 (d, J = 16.0 Hz, 1H), 2.92–2.85 (m, 1H), 2.65–2.58
(m, 2H), 2.47–2.39 (m, 1H), 1.52 (s, 9H).13C-NMR (100 MHz,
CDCl3) δ: 171.7, 170.6, 169.9, 158.3, 154.2, 151.0 (2C), 128.2
(2C), 121.5, 94.3, 80.7, 51.9, 42.1 (2C), 37.0, 32.7, 30.9, 29.6,
28.4 (3C), 26.7. MS (ES mass): 471.2 (M + 1). HPLC: 95.7%,
column: ZORBAX XDB C-18 150 × 4.6 mm 5 μ, mobile phase
A: 5 mM ammonium acetate in water, mobile phase B: CH3CN,
gradient (T/%B): 0/70, 2/70, 9/95, 13/95, 15/70, 18/70; flow
rate: 1.0 mL min−1; UV 240 nm, retention time 5.29 min.
Preparation of 1-(5,6,7,8-tetrahydrobenzo[b]thieno[2,3-d]-
pyrimidin-4-yl)-4-oxocyclohexanecarbonitrile (8a)
A mixture of 7a (0.5 g, 1.35 mmol) and NaCl (628 mg,
10.84 mmol) in DMSO (5 mL) and water (0.5 mL) was heated
at 150 °C for 5 h under anhydrous conditions. After completion
of the reaction, the mixture was cooled to room temp, diluted
with water (25 mL) and extracted with ethyl acetate (3 × 20 mL).
The organic layers were collected, combined, washed with brine
solution (15 mL), dried over anhydrous Na2SO4, and concen-
trated under reduced pressure. The isolated residue was purified
by column chromatography using ethyl acetate–hexane (1: 6) to
give desired product 8a (240 mg, 58%) as a white solid; mp:
167–169 °C; Rf= 0.5 (30% EtOAc–hexane); IR (KBr, cm−1):
2947, 2883, 2233, 1713;1H NMR (400 MHz, CDCl3) δ: 8.89
(s,1H), 3.28–3.26 (m, 2H), 3.01–2.99 (m, 2H), 2.96–2.87 (m, 2H),
2.87–2.76 (m, 2H), 2.67–2.56 (m, 4H), 1.99 (bs, 4H);13C-NMR
(100 MHz, CDCl3) δ: 206.8, 169.8, 157.2, 150.4, 140.4, 129.1,
125.9, 121.5, 43.3, 37.8 (2C), 35.5 (2C), 29.3, 26.6, 23.3, 22.3;
MS (ES mass): 311.9 (M + 1); HPLC: 98.4%, column:
ZORBAX XDB C-18 150 × 4.6 mm 5 μ, mobile phase A:
0.05% formic acid in water, mobile phase B: CH3CN, gradient
(T/%B): 0/50, 2/50, 9/90, 14/90, 16/50, 20/50; flow rate: 0.8 mL
min−1; UV 245 nm, retention time 7.74 min.
Preparation of 1-(6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]-
pyrimidin-4-yl)-4-oxocyclohexanecarbonitrile (8b)
Compound 8b was synthesized in 62% yield from 7b following
a procedure similar to that of compound 8a; white solid; mp:
152–154 °C; Rf= 0.5 (30% EtOAc–n-hexane); IR (KBr, cm−1):
2960, 2909, 2234, 1710;1H NMR (400 MHz, CDCl3) δ: 8.90
(s, 1H), 3.42 (t, J = 7.2 Hz, 2H), 3.12 (t, J = 7.2 Hz, 2H),
2.95–2.87 (m, 2H), 2.78–2.72 (m, 2H), 2.69–2.63 (m, 2H),
2.61–2.53 (m, 4H);
174.9, 156.8, 150.6, 146.5, 134.6, 125.8, 121.3, 42.9, 37.8 (2C),
34.9 (2C), 33.2, 30.1, 28.2; MS (ES mass): 297.5 (M + 1).
13C-NMR (100 MHz, CDCl3) δ: 206.8,
Preparation of 1-(6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno-
[2,3-d]pyrimidin-4-yl)-4-oxocyclohexanecarbonitrile (8c)
Compound 8c was synthesized in 65% yield from 7c following a
procedure similar to that of compound 8a; white solid; mp:
144–146 °C; Rf= 0.5 (25% EtOAc–n-hexane); IR (KBr, cm−1):
2916, 2856, 2232, 1720;
(s, 1H), 3.37–3.34 (m, 2H), 3.05–3.02 (m, 2H), 2.96–2.87
(m, 2H), 2.80–2.76 (m, 2H), 2.67–2.55 (m, 4H), 2.04–1.98
(m, 2H), 1.81–1.74 (m, 4H);
δ: 206.8, 168.6, 157.0, 150.2, 144.9, 131.8, 128.9, 120.7, 43.1,
1H NMR (400 Hz, CDCl3) δ: 8.89
13C-NMR (100 MHz, CDCl3)
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37.8 (2C), 35.1 (2C), 32.3, 30.9, 30.6, 26.9, 26.7; MS (ES
mass): 326.2 (M + 1).
Preparation of 1-(7-(tert-butoxycarbonyl)-5,6,7,8-
tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4-yl)-4-
oxocyclohexanecarbonitrile (8d)
Compound 8d was synthesized in 58% yield from 7d following
a procedure similar to that of compound 8a; white solid; mp:
259–261 °C; Rf= 0.4 (40% EtOAc–n-hexane); IR (KBr, cm−1):
2973, 2935, 2235, 1699;1H NMR (400 MHz, CDCl3) δ: 8.87
(s, 1H), 4.75 (s, 2H), 3.76 (t, J = 4.8 Hz, 2H), 3.33 (bs, 2H),
2.88–2.80 (m, 2H), 2.71–2.67 (m, 2H), 2.61–2.49 (m, 4H), 1.45
(s, 9H);13C-NMR (100 MHz, CDCl3) δ: 206.6, 170.1, 150.9,
150.1, 132.1, 129.8, 128.7, 126.9, 121.2, 80.9, 43.7, 43.2, 37.7
(2C), 35.2 (2C), 29.6, 28.4 (3C), 28.3. MS (ES mass): 413.2
(M + 1). HPLC: 97.6%, column: ZORBAX XDB C-18
150 × 4.6 mm 5 μ, mobile phase A: 5 mM ammonium acetate in
water, mobile phase B: CH3CN, gradient (T/%B): 0/50, 2/50,
9/95, 13/95, 15/50, 18/50; flow rate: 1.0 mL min−1; UV 240 nm,
retention time 6.72 min.
Preparation of 3-[(dimethylamino)methylene]-1-(5,6,7,8-
tetrahydrobenzo[b]thieno[2,3-d]pyrimidin-4-yl)-4-oxocyclo-
hexanecarbonitrile (9a)
A mixture of 8a (0.5 g, 1.60 mmol), DMF-DMA (0.8 mL,
6.43 mmol) and Et3N (0.7 mL, 4.82 mmol) in dry DMF (5 mL)
was heated to 110 °C for 4 h under a nitrogen atmosphere. After
completion of the reaction the mixture was cooled to room
temp, diluted with water (25 mL) and extracted with ethyl
acetate (3 × 20 mL). The organic layers were collected, com-
bined, washed with brine solution (20 mL), dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The residue
was purified by column chromatography using ethyl acetate–
hexane (4 :1) to give desired product 9a (0.4 g, 64%) as a brown
solid; mp: 214–216 °C; Rf = 0.2 (100% EtOAc); IR (KBr,
cm−1): 2947, 2230, 1735, 1647;1H NMR (400 MHz, CDCl3)
δ: 8.88 (s, 1H), 7.69 (s, 1H), 3.61 (s, 2H), 3.41–3.37 (m, 1H),
3.17 (s, 6H), 3.13–3.08 (m, 1H), 2.99 (s, 2H), 2.87–2.77
(m, 1H), 2.72–2.62 (m, 1H), 2.60–2.53 (m, 1H), 2.36–2.28
(m, 1H), 1.97 (bs, 4H);13C-NMR (100 MHz, CDCl3) δ: 193.7,
169.7, 158.4, 152.4, 150.4, 139.9, 129.1, 126.2, 122.2, 98.9,
43.7, 43.7, 43.2, 35.6, 34.6, 32.9, 29.1, 26.5, 23.2, 22.3; MS
(ES mass): 367.0 (M + 1).
Preparation of 3-[(dimethylamino)methylene]-1-(6,7-dihydro-
5H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-4-yl)-4-oxocyclo-
hexanecarbonitrile (9b)
Compound 9b was synthesized in 58% yield from 8b following
a procedure similar to that of compound 9a; light brown solid;
mp: 227–229 °C; Rf = 0.1 (100% EtOAc); IR (KBr, cm−1):
2947, 2232, 1646, 1541;
(s, 1H), 7.70 (s, 1H), 3.57 (s, 2H), 3.51–3.43 (m, 1H), 3.34–3.25
(m, 1H), 3.17 (s, 6H), 3.11 (t, J = 7.2 Hz, 2H), 2.88–2.80
(m, 1H), 2.67–2.52 (m, 4H), 2.38–2.30 (m, 1H);
1H NMR (400 Hz, CDCl3) δ: 8.89
13C-NMR
(100 MHz, CDCl3) δ: 193.6, 174.8, 158.1, 152.7, 150.6, 146.1,
134.9, 125.8, 122.0, 98.8, 43.8, 42.9, 34.8, 34.5, 33.1, 32.4,
30.1 (2C), 28.2; MS (ES mass): 353.2 (M + 1).
Preparation of 3-[(dimethylamino)methylene]-1-(6,7,8,9-
tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidin-4-yl)-4-
oxocyclohexanecarbonitrile (9c)
A mixture of 8c (0.2 g, 0.615 mmol) and DMF-DMA (0.16 mL,
1.23 mmol) in toluene (5 mL) was heated to 95 °C for 16 h
under anhydrous conditions. After completion of the reaction,
the mixture was cooled to room temp and the solvent was
removed under reduced pressure. The residue was diluted with
water (25 mL) and extracted with ethyl acetate (3 × 10 mL). The
organic layers were collected, combined, washed with brine
solution (10 mL), dried over anhydrous Na2SO4, and concen-
trated under reduced pressure. The residue was purified by
column chromatography using ethyl acetate–hexane (4: 1) to
give desired product 9c (105 mg, 45%) as a white solid; mp:
185–187 °C; Rf= 0.1 (100% EtOAc); IR (KBr, cm−1): 2920,
2851, 2228, 1646;1H NMR (400 MHz, CDCl3) δ: 8.89 (s, 1H),
7.71 (s, 1H), 3.61 (s, 2H), 3.33 (t, J = 5.6 Hz, 2H), 3.17 (s, 6H),
3.03–3.01 (m, 2H), 2.88–2.78 (m, 1H), 2.68–2.56 (m, 2H),
2.39–2.30 (m, 1H), 2.04–1.96 (m, 2H), 1.78–1.74 (m, 4H);
13C-NMR (100 MHz, CDCl3) δ: 193.7, 168.4, 158.3, 152.7,
150.2, 144.5, 132.2, 128.9, 121.5, 99.0, 43.8, 43.2, 35.6, 34.6,
32.5 (2C), 32.4, 30.8, 30.6, 27.1, 26.7; MS (ES mass): 381.2
(M + 1).
Preparation of 3-[(dimethylamino)methylene]-1-(7-(tert-
butoxycarbonyl)-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno-
[2,3-d]pyrimidin-4-yl)-4-oxocyclohexanecarbonitrile (9d)
Compound 9d was synthesized in 45% yield from 8d following
a procedure similar to that of compound 9c; brown solid; mp:
113–115 °C; Rf= 0.1 (100% EtOAc); IR (KBr, cm−1): 2976,
2237, 1729, 1696;1H NMR (400 MHz, CDCl3) δ: 8.90 (s, 1H),
8.67 (s, 1H), 4.80 (s, 2H), 3.83 (bs, 2H), 3.60 (bs, 3H),
3.39–3.29 (m, 2H), 3.08 (s, 3H), 3.03 (s, 3H), 2.71–2.63
(m, 3H), 1.52 (s, 9H).13C-NMR (100 MHz, CDCl3) δ: 193.5,
169.9, 159.0, 152.7 (2C), 150.9 (2C), 128.2, 122.1, 121.2, 98.7,
80.8, 43.8, 43.2, 42.3, 35.4, 34.6, 32.8, 31.0, 29.2, 28.4 (3C),
28.3. MS (ES mass): 468.2 (M + 1).
Preparation of 2-amino-6-(5,6,7,8-tetrahydrobenzo[b]thieno-
[2,3-d]pyrimidin-4-yl)-5,6,7,8-tetrahydroquinazoline-6-
carbonitrile (10a)
A mixture of 9a (0.1 g, 0.27 mmol), guanidine HCl (24.1 mg,
0.41 mmol) and NaOMe (22 mg, 0.41 mmol) in methanol
(8 mL) was stirred at 80 °C for 1 h under nitrogen. After com-
pletion of the reaction the excess of sodium methoxide was
quenched with ice cold water and methanol was removed under
reduced pressure. The residue was diluted with water (25 mL)
and extracted with ethyl acetate (3 × 10 mL). The organic layers
were collected, combined, washed with brine solution (10 mL),
dried over anhydrous Na2SO4, and concentrated under reduced
pressure. The residue was purified by column chromatography
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using ethyl acetate–hexane (4: 1) to give desired product 10a
(80 mg, 78%) as a light brown solid; mp: 153–155 °C; Rf= 0.35
(100% EtOAc); IR (KBr, cm−1): 3320, 3172, 2937, 2235;1H
NMR (400 MHz, CDCl3) δ: 8.89 (s, 1H), 8.17 (s, 1H), 5.15 (s,
2H), 3.67 (d, J = 16.1 Hz, 1H), 3.43–3.36 (m, 2H), 3.26–3.16
(m, 2H), 3.01–2.91 (m, 3H), 2.79–2.75 (m, 1H), 2.45–2.37
(m, 1H), 1.99 (bs, 4H);13C-NMR (100 MHz, CDCl3) δ: 169.8,
163.9, 161.9, 158.6, 157.5, 150.4, 140.3, 129.1, 126.1, 121.5,
115.7, 42.3, 35.9, 32.5, 29.3, 29.2, 26.6, 23.3, 22.3; MS
(ES mass): 362.9 (M +1); HPLC: 99.3%, column: ZORBAX
XDB C-18 150 × 4.6 mm 5 μ, mobile phase A: 0.05% formic
acid in water, mobile phase B: CH3CN (isocratic) (A :B) 40: 60;
flow rate: 0.8 mL min−1; UV 245 nm, retention time 2.9 min;
chiral HPLC: column: chiral pak IC (250 × 4.6 mm) 5 μm,
mobile phase: A: MeOH: B: 0.1% DEA, flow: 1.0 mL min−1,
wavelength: 245 nm, retention time (area %): 16.9 min (50%)
and 19.4 min (50%).
Preparation of 2-amino-6-(6,7-dihydro-5H-cyclopenta-
[4,5]thieno[2,3-d]pyrimidin-4-yl)-5,6,7,8-tetrahydroquinazoline-
6-carbonitrile (10b)
Compound 10b was synthesized in 70% yield from 9b following
a procedure similar to that of compound 10a; white solid; mp:
200–202 °C; Rf= 0.3 (100% EtOAc); IR (KBr, cm−1): 3318,
3161, 2950, 2242;1H NMR (400 MHz, CDCl3) δ: 8.89 (s, 1H),
8.16 (s, 1H), 5.10 (bs, 2H), 3.62 (d, J = 16.0 Hz, 1H), 3.52–3.45
(m, 1H), 3.40–3.36 (m, 1H), 3.34–3.17 (m, 2H), 3.15–3.11
(m, 2H), 2.96–2.89 (m, 1H), 2.78–2.72 (m, 1H), 2.62–2.54
(m, 2H), 2.46–2.38 (m, 1H);
δ: 169.3, 163.9, 161.7, 158.6, 157.0, 150.6, 146.5, 134.7, 125.8,
121.4, 115.5, 41.9, 34.7, 33.2, 31.9, 30.1, 29.7, 28.9; MS
(ES mass): 349.1 (M + 1); HPLC: 90.7%, column: X Bridge
C-18 150 × 4.6 mm 5 μ, mobile phase A: 0.05% formic acid in
water, mobile phase B: CH3CN, gradient (T/%B): 0/30, 2/30,
9/95, 12/95, 15/30, 18/30; flow rate: 0.8 mL min−1; UV 241 nm,
retention time 7.0 min.
13C-NMR (100 MHz, CDCl3)
Preparation of 2-amino-6-(6,7,8,9-tetrahydro-5H-cyclohepta[4,5]-
thieno[2,3-d]pyrimidin-4-yl)-5,6,7,8-tetrahydroquinazoline-6-
carbonitrile (10c)
Compound 10c was synthesized in 68% yield from 9c following
a procedure similar to that of compound 10a; white solid; mp:
234–236 °C; Rf= 0.2 (100% EtOAc); IR (KBr, cm−1): 3456,
3314, 2930, 2232;1H NMR (400 MHz, CDCl3) δ: 8.90 (s, 1H),
8.15 (s, 1H), 5.17 (s, 2H), 3.64 (d, J = 16.4 Hz, 1H), 3.44–3.17
(m, 4H), 3.04 (t, J = 5.4 Hz, 2H), 2.99–2.92 (m, 1H), 2.81–2.76
(m, 1H), 2.45–2.37 (m, 1H), 2.05–1.97 (m, 2H), 1.79–1.76
(m, 4H);13C-NMR (100 MHz, CDCl3) δ: 168.6, 164.1, 161.9,
158.6, 157.3, 150.3, 144.9, 131.9, 128.9, 120.8, 115.7, 42.1,
35.7, 32.4, 32.1, 30.9, 30.6, 29.1, 26.9, 26.7; MS (ES mass):
377.1 (M + 1); HPLC: 99.1%, column: X Bridge C-18 150 ×
4.6 mm 5 μ, mobile phase A: 0.1% formic acid in water, mobile
phase B: CH3CN, gradient (T/%B): 0/50, 2/50, 9/95, 12/95,
15/50, 18/50; flow rate: 0.8 mL min−1; UV 245 nm, retention
time 4.5 min.
Preparation of 2-amino-6-(7-(tert-butoxycarbonyl)-5,6,7,8-
tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4-yl)-5,6,7,8-
tetrahydroquinazoline-6-carbonitrile (10d)
Compound 10d was synthesized in 55% yield from 9d following
a procedure similar to that of compound 10a; light yellow solid;
mp: 141–143 °C; Rf= 0.3 (100% EtOAc); IR (KBr, cm−1):
3327, 3194, 2971, 2235, 1696;
δ: 8.92 (s, 1H), 8.21 (s, 1H), 5.82 (s, 2H), 4.82 (s, 2H),
3.89–3.77 (m, 2H), 3.72 (d, J = 16.1 Hz, 1H), 3.57–3.46
(m, 1H), 3.40 (d, J = 16.3 Hz, 1H), 3.31–3.19 (m, 2H),
3.03–2.91 (m, 1H), 2.81–2.71 (m, 1H), 2.49–2.39 (m, 1H),
1.52 (s, 9H).
163.8, 161.2 (2C), 158.3, 154.2, 151.0 (2C), 128.2, 121.3,
115.4, 80.9, 42.1, 35.4, 32.1, 31.5, 28.7, 28.4 (3C), 28.3, 26.9.
MS (ES mass): 464.2 (M + 1). HPLC: 97.9%, column:
ZORBAX XDB C-18 150 × 4.6 mm 5 μ, mobile phase
A: 5 mM ammonium acetate in water, mobile phase B: CH3CN,
gradient (T/%B): 0/20, 2/20, 9/95, 13/95, 15/20, 18/20; flow
rate: 1.0 mL min−1; UV 240 nm, retention time 8.6 min.
1H NMR (400 MHz, CDCl3)
13C-NMR (100 MHz, CDCl3) δ: 175.3, 170.1,
Preparation of 6-(5,6,7,8-tetrahydrobenzo[b]thieno[2,3-d]-
pyrimidin-4-yl)-5,6,7,8-tetrahydroquinazoline-6-carbonitrile
(11a)
Compound 11a was synthesized in 45% yield from 9a and
formimidine acetate (1.5 mmol) following a procedure similar to
that of compound 10a; brown solid; mp: 182–184 °C; Rf= 0.45
(100% EtOAc); IR (KBr, cm−1): 2941, 2868, 2234, 1557;1H
NMR (400 MHz, CDCl3) δ: 9.05 (s, 1H), 8.86 (s, 1H), 8.61
(s, 1H), 3.99 (d, J = 16.9 Hz, 1H), 3.54 (d, J = 16.9 Hz, 1H),
3.46–3.34 (m, 2H), 3.15–3.02 (m, 4H), 2.86–2.82 (m, 1H),
2.51–2.43 (m, 1H), 2.05–1.99 (m, 4H);13C-NMR (100 MHz,
CDCl3) δ: 169.9, 162.9, 157.1, 157.0, 156.7, 150.4, 140.6,
129.0, 126.7, 125.9, 121.4, 41.7, 36.3, 32.3, 29.3, 29.1, 26.6,
23.2, 22.2; MS (ES mass): 347.9 (M + 1); HPLC: 98.5%,
column: ZORBAX XDB C-18 150 × 4.6 mm 5 μ, mobile phase
A: 0.05% formic acid in water, mobile phase B: CH3CN
(Isocratic) (A:B) 40: 60; flow rate: 0.8 mL min−1; UV 245 nm,
retention time 4.2 min.
Preparation of 6-(6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]-
pyrimidin-4-yl)-5,6,7,8-tetrahydroquinazoline-6-carbonitrile
(11b)
Compound 11b was synthesized in 78% yield from 9b and
formimidine acetate (1.5 mmol) following a procedure similar to
that of compound 10a; white solid; mp: 248–250 °C; Rf= 0.5
(100% EtOAc); IR (KBr, cm−1): 2954, 2863, 2243, 1535;1H
NMR (400 MHz, CDCl3) δ: 9.05 (s, 1H), 8.86 (s, 1H), 8.60
(s, 1H), 3.91 (d, J = 16.8 Hz, 1H), 3.57–3.49 (m, 2H), 3.41–3.25
(m, 2H), 3.13 (t, J = 7.2 Hz, 2H), 3.08–3.01 (m, 1H), 2.84–2.77
(m, 1H), 2.65–2.54 (m, 2H), 2.51–2.44 (m, 1H);
(100 MHz, CDCl3) δ: 175.0, 162.9, 157.2, 157.1, 156.3, 150.5,
146.8, 134.6, 126.5, 125.7, 121.2, 41.3, 34.9, 33.2, 31.9, 30.2,
28.9, 28.2; MS (ES mass): 334.1 (M + 1); HPLC: 97.9%.
column: X Bridge C-18 150 × 4.6 mm 5 μ, mobile phase A:
0.05% formic acid in water, mobile phase B: CH3CN, gradient
13C-NMR
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(T/%B): 0/30, 2/30, 9/95, 12/95, 15/30, 18/30; flow rate: 0.8 mL
min−1; UV 241 nm, retention time 7.9 min.
Preparation of 6-(6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno-
[2,3-d]pyrimidin-4-yl)-5,6,7,8-tetrahydroquinazoline-6-
carbonitrile (11c)
Compound 11c was synthesized in 67% yield from 9c and formi-
midine acetate (1.5 mmol) following a procedure similar to that
of compound 10a; white solid; mp: 157–159 °C; Rf= 0.4 (100%
EtOAc); IR (KBr, cm−1): 2934, 2853, 2230, 1551;
(400 MHz, CDCl3) δ: 9.06 (s, 1H), 8.88 (s, 1H), 8.61 (s, 1H),
3.97 (d, J = 16.8 Hz, 1H), 3.55 (d, J = 16.8 Hz, 1H), 3.43–3.28
(m, 3H), 3.13–3.00 (m, 3H), 2.87–2.84 (m, 1H), 2.50–2.42
(m, 1H), 2.10–1.95 (m, 2H), 1.80–1.77 (m, 4H);
(100 MHz, CDCl3) δ: 168.7, 163.0, 157.2, 157.1, 156.6, 150.2,
145.2, 131.8, 128.9, 126.7, 120.6, 41.6, 36.1, 32.4, 31.9, 30.9,
30.6, 29.1, 26.9, 26.7; MS (ES mass): 362.1 (M + 1); HPLC:
97.3%. column: X Bridge C-18 150 × 4.6 mm 5 μ, mobile phase
A: 0.1% formic acid in water, mobile phase B: CH3CN, gradient
(T/%B): 0/50, 2/50, 9/95, 12/95, 15/50, 18/50; flow rate: 0.8 mL
min−1; UV 245 nm, retention time 6.4 min.
1H NMR
13C-NMR
Preparation of 6-(7-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-
pyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4-yl)-5,6,7,8-tetrahydro-
quinazoline-6-carbonitrile (11d)
Compound 11d was synthesized in 50% yield from 9d and
formimidine acetate (1.5 mmol) following a procedure similar to
that of compound 10a: brown solid; mp: 191–193 °C; Rf= 0.5
(100% EtOAc); IR (KBr, cm−1): 2975, 2930, 2228, 1698;1H
NMR (400 MHz, CDCl3) δ: 9.11 (s, 1H), 8.89 (s, 1H), 8.72
(m, 1H), 4.84 (s, 2H), 4.08 (d, J = 16.0 Hz, 1H), 3.83 (s, 2H),
3.66–3.51 (m, 2H), 3.51–3.37 (m, 1H), 3.35–3.03 (m, 2H),
2.89–2.77 (m, 1H), 2.60–2.48 (m, 1H), 1.52 (s, 9H);13C-NMR
(100 MHz, CDCl3) δ: 170.3 (2C), 163.3, 156.8, 155.9, 154.2,
150.9 (3C), 128.1, 121.1, 109.9, 80.9, 41.5, 35.9, 32.0 (2C),
28.8 (2C), 28.4 (3C), 28.3; MS (ES mass): 449.1 (M + 1).
HPLC: 98.2%, column: ZORBAX XDB C-18 150 × 4.6 mm
5 μ, mobile phase A: 5 mM ammonium acetate in water, mobile
phase B: CH3CN, gradient (T/%B): 0/50, 2/50, 9/95, 13/95,
15/50, 18/50; flow rate: 0.8 mL min−1; UV 240 nm, retention
time 6.6 min.
Preparation of 2-amino-6-(5,6,7,8-tetrahydrobenzo[b]thieno-
[2,3-d]pyrimidin-4-yl)-4-oxo-3,4,5,6,7,8-hexahydroquinazoline-6-
carbonitrile (12)
A mixture of 7a (0.1 g, 0.27 mmol), guanidine HCl (48 mg,
0.81 mmol) and NaOMe (73 mg, 1.35 mmol) in methanol
(8 mL) was stirred at 80 °C for 1 h under nitrogen. After com-
pletion of the reaction the excess sodium methoxide was
quenched with ice cold water and methanol was removed under
reduced pressure. The residue was diluted with water (25 mL)
and extracted with ethyl acetate (3 × 10 mL). The organic layers
were collected, combined, washed with brine solution (10 mL),
dried over anhydrous Na2SO4, and concentrated under reduced
pressure. The residue was purified by column chromatography
using methanol–DCM (1: 19) to give desired product 12
(73 mg, 72%) as a white solid; mp: 279–281 °C; Rf= 0.5 (10%
MeOH–DCM); IR (KBr, cm−1): 3448, 3314, 3125, 2940, 2229,
1650;1H NMR (400 MHz, DMSO-d6) δ: 10.8 (bs, 1H), 9.04
(s, 1H), 6.46 (bs, 2H), 3.28–3.26 (m, 3H), 3.23–3.21 (m, 3H),
2.81–2.63 (m, 3H), 2.39–2.36 (m, 1H), 1.94 (bs, 4H);13C-NMR
(100 MHz, DMSO-d6) δ: 168.8, 162.9, 158.3, 154.1, 150.6,
145.1, 139.7, 128.4, 126.1, 122.1, 104.4, 42.2, 32.0, 31.6, 28.9,
28.5, 25.9, 22.7, 21.8; MS (ES mass): 378.9 (M + 1); HPLC:
97.4%, column: ZORBAX XDB C-18 150 × 4.6 mm 5 μ,
mobile phase A: 0.05% formic acid in water, mobile phase
B: CH3CN, gradient (T/%B): 0/20, 2/20, 9/95, 12/95, 15/20, 18/20;
flow rate: 1.0 mL min−1; UV 246 nm, retention time 6.2 min.
Preparation of 6-(5,6,7,8-tetrahydrobenzo[b]thieno[2,3-d]-
pyrimidin-4-yl)-4-oxo-3,4,5,6,7,8-hexahydroquinazoline-6-
carbonitrile (13)
Compound 13 was synthesized in 68% yield from 7a and formi-
midine acetate (3 mmol) following a procedure manner similar
to that of compound 12; white solid; mp: 202–204 °C; Rf= 0.6
(10% MeOH–DCM); IR (KBr, cm−1): 3153, 2943, 2233, 1655;
1H NMR (400 MHz, CDCl3) δ: 8.89 (s, 1H), 8.12 (s, 1H), 3.58
(d, J = 17.6 Hz, 1H), 3.46–3.34 (m, 2H), 3.26–3.18 (m, 2H),
3.01 (s, 2H), 2.95–2.89 (m, 1H), 2.78–2.74 (m, 1H), 2.55–2.47
(m, 1H), 1.99–1.98 (m, 4H);
δ: 169.8, 163.5, 160.6, 157.2, 150.4, 145.9, 140.2, 129.1, 126.1,
121.7, 119.1, 41.5, 32.4, 31.8, 29.2, 29.1, 26.6, 23.2, 22.2; MS
(ES mass): 363.9 (M + 1); HPLC: 98.6%, column: ZORBAX
XDB C-18 150 × 4.6 mm 5 μ, mobile phase A: 0.05% formic
acid in water, mobile phase B: CH3CN, gradient (T/%B): 0/20,
2/20, 9/95, 12/95, 15/20, 18/20; flow rate: 0.8 mL min−1; UV
244 nm, retention time 8.1 min; chiral HPLC: column: chiral
pak AD (250 × 4.6 mm) 3 μm, mobile phase: A: n-hexane:
B: 0.1% IPA, flow: 0.8 mL min−1, wave length: 245 nm, reten-
tion time (area %): 12.6 min (49.5%) and 15.8 min (50.5%).
13C-NMR (100 MHz, CDCl3)
Preparation of 5-(5,6,7,8-tetrahydrobenzo[b]thieno[2,3-d]-
pyrimidin-4-yl)-3-oxo-2,3,4,5,6,7-hexahydro-1H-indazole-5-
carbonitrile (14)
A mixture of 7a (0.1 g, 0.27 mmol), hydrazine (0.03 mL,
0.54 mmol) and Et3N (0.09 mL, 0.81 mmol) in methanol
(8 mL) was stirred at 80 °C for 1 h under nitrogen. After com-
pletion of the reaction methanol was removed under reduced
pressure. The residue was diluted with water (25 mL) and
extracted with ethyl acetate (3 × 10 mL). The organic layers
were collected, combined, washed with brine solution (10 mL),
dried over anhydrous Na2SO4, and concentrated under reduced
pressure. The isolated residue was purified by column chromato-
graphy using methanol–DCM (1: 49) to give desired product 14
(75 mg, 76%) as a white solid; mp: 271–273 °C; Rf= 0.5 (5%
MeOH–DCM); IR (KBr, cm−1): 3231, 2944, 2234, 1734;
NMR (400 MHz, DMSO-d6) δ: 11.31 (bs, 1H), 9.61 (bs, 1H),
9.00 (s, 1H), 3.22–3.15 (m, 4H), 3.02 (s, 2H), 2.87 (s, 2H),
2.66–2.62 (m, 1H), 2.38–2.28 (m, 1H), 1.91 (s, 4H);13C-NMR
(100 MHz, DMSO-d6) δ: 168.7, 158.5, 150.5, 150.3, 139.6,
137.9, 128.3, 126.1, 122.1, 109.5, 43.6, 32.3, 30.5, 28.5, 25.9,
1H
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22.7, 21.8, 19.4; MS (ES mass): 351.9 (M + 1); HPLC: 97.7%,
column: ZORBAX XDB C-18 150 × 4.6 mm 5 μ, mobile phase
A: 0.05% formic acid in water, mobile phase B: CH3CN, gradi-
ent (T/%B): 0/20, 2/20, 9/95, 12/95, 15/20, 18/20; flow rate:
0.8 mL min−1; UV 245 nm, retention time 7.9 min; chiral
HPLC: column: Lux Cellulose-2 (250 × 4.6 mm) 3 μm, mobile
phase: A: n-hexane: D: 0.1% TFA in EtOH, flow: 0.8 mL min−1,
wavelength: 245 nm, retention time (area %): 16.9 min (45.7%)
and 20.9 min (49.8%).
Preparation of 5-(5,6,7,8-tetrahydrobenzo[b]thieno[2,3-d]-
pyrimidin-4-yl)-3-oxo-2-phenyl-3,3a,4,5,6,7-hexahydro-2H-
indazole-5-carbonitrile (15)
Compound 15 was prepared in 65% yield from 7a and phenyl
hydrazine (2 mmol) following a procedure similar to compound
14; white solid; mp: 218–220 °C; Rf= 0.3 (70% EtOAc–n-
hexane); IR (KBr, cm−1): 3062, 2861, 2237, 1730;
(400 MHz, CDCl3) δ: 8.87 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H),
7.64 (d, J = 8.0 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.35 (t, J =
7.6 Hz, 1H), 7.22–7.13 (m, 1H), 3.41–3.33 (m, 2H), 3.27–3.17
(m, 2H), 3.09–2.84 (m, 5H), 2.76–2.65 (m, 1H), 2.40–2.33
(m, 1H), 2.01–1.94 (m, 4H);
δ: 169.8, 159.2, 157.9, 150.4, 148.1, 140.1, 129.1, 128.9, 128.9
(2C), 126.3, 125.7, 121.7, 120.3, 118.9, 42.8, 37.3, 32.1, 30.5,
29.2, 26.6, 23.2, 22.2, 20.4; MS (ES mass): 427.9 (M + 1);
HPLC: 97.9%, column: ZORBAX XDB C-18 150 × 4.6 mm
5 μ, mobile phase A: 0.05% formic acid in water, mobile phase
B: CH3CN, gradient (T/%B): 0/50, 2/50, 9/95, 12/95, 15/50, 18/50;
flow rate: 0.8 mL min−1; UV 245 nm, retention time 5.9 min.
1H NMR
13C-NMR (100 MHz, CDCl3)
Preparation of 5-(5,6,7,8-tetrahydrobenzo[b]thieno[2,3-d]-
pyrimidin-4-yl)-4,5,6,7-tetrahydro-1H-indazole-5-carbonitrile
(16)
A mixture of 9a (0.1 g, 0.27 mmol) and hydrazine (0.02 mL,
0.41 mmol) in methanol (5 mL) was stirred at 80 °C for 1 h
under nitrogen. Then, methanol was removed under reduced
pressure. The residue was diluted with water (25 mL) and
extracted with ethyl acetate (3 × 10 mL). The organic layers
were collected, combined, washed with brine solution (10 mL),
dried over Na2SO4, and concentrated under reduced pressure.
The residue was purified by column chromatography using ethyl
acetate–n-hexane (3 :2) to give desired product 16 (65 mg, 72%)
as a light brown solid; mp: 109–111 °C; Rf= 0.3 (70% EtOAc–
n-hexane); IR (KBr, cm−1): 3647, 3248, 2230, 1513;1H NMR
(400 MHz, CDCl3) δ: 8.89 (s, 1H), 7.54 (s, 1H), 6.45 (bs, 1H),
3.61 (d, J = 16.0 Hz, 1H), 3.48 (d, J = 16.0 Hz, 1H), 3.38–3.09
(m, 4H), 3.00 (bs, 2H), 2.83–2.78 (m, 1H), 2.48–2.43 (m, 1H),
1.98 (bs, 4H);13C-NMR (100 MHz, CDCl3) δ: 169.5, 158.2,
150.2 (2C), 139.8 (2C), 129.0, 126.1 (2C), 121.8, 43.5, 33.4,
31.9, 29.1, 26.4, 23.1, 22.1, 20.1. MS (ES mass): 336.2 (M + 1);
HPLC: 99.1%, column: ZORBAX XDB C-18 150 × 4.6 mm
5 μ, mobile phase A: 0.1% formic acid in water, mobile phase B:
CH3CN, gradient (T/%B): 0/20, 2/20, 9/95, 13/95, 15/20, 18/20;
flow rate: 1.0 mL min−1; UV 245 nm, retention time 8.5 min.
Chiral HPLC: column: chiral pak IC (250 × 4.6 mm) 5 μm,
mobile phase: A: MeOH: B: 0.1% DEA, flow : 1.0 mL min−1,
wave length: 295 nm, retention time (area %): 8.8 min (50%)
and 10.9 min (50%).
Single crystal X-ray data for compound 7a and 10a
Single crystals suitable for X-ray diffraction of 7a and 10a were
grown from methanol. The crystals were carefully chosen using
a stereo zoom microscope supported by a rotatable polarizing
stage. The data was collected at room temperature on Bruker’s
KAPPA APEX II CCD Duo with graphite monochromated
Mo-Kα radiation (0.71073 Å). The crystals were glued to a thin
glass fibre using FOMBLIN immersion oil and mounted on the
diffractometer. The intensity data were processed using Bruker’s
suite of data processing programs (SAINT), and absorption cor-
rections were applied using SADABS.21The crystal structure
was solved by direct methods using SHELXS-97 and the data
was refined by full matrix least-squares refinement on F2with
anisotropic displacement parameters for non-H atoms, using
SHELXL-97.22
Crystal data of 7a: Molecular formula = C19H19N3O3S,
formula weight = 369.44, crystal system = triclinic, space group
= P1ˉ, a = 11.092 (5) Å, b = 11.448 (5) Å, c = 15.672 (7) Å, V =
1761.7 (13) Å3, T = 296 K, Z = 4, Dc = 1.401 Mg m−3,
μ(Mo-Kα) = 0.21 mm−1, 20533 reflections measured, 7395
independent reflections, 5076 observed reflections [I > 2.0σ(I)],
R1_obs = 0.081, goodness of fit = 1.003. CCDC 864130.
Crystal data of 10a: Molecular formula = C19H18N6S, formula
weight = 362.13, crystal system = triclinic, space group = P1ˉ,
a = 7.625 (4) Å, b = 10.1757 (5) Å, c = 12.243 (6) Å, V =
903.66 (8)Å3, T = 296 K, Z = 6, Dc= 1.387 Mg m−3, μ(Mo-Kα)
= 0.21 mm−1, 15612 reflections measured, 3949 independent
reflections, 3342 observed reflections [I > 2.0σ(I)], R1_obs =
0.029, goodness of fit = 0.876. CCDC 864129.
Pharmacology
Materials and methods
Cells and reagents. HEK 293 and Sf9 cells were obtained
from ATCC (Washington, DC, USA). HEK 293 cells were cul-
tured in DMEM supplemented with 10% fetal bovine serum
(Invitrogen Inc., San Diego, CA, USA). Sf9 cells were routinely
maintained in Grace’s supplemented medium (Invitrogen) with
10% FBS. RAW 264.7 cells (murine macrophage cell line) were
obtainedfrom ATCCand
1640 medium with 10% fetal bovine serum (Invitrogen Inc.).
cAMP was purchased from SISCO Research Laboratories
(Mumbai, India). PDElight HTS cAMP phosphodiesterase assay
kit was procured from Lonza (Basel, Switzerland). PDE4B1
clone was from OriGene Technologies (Rockville, MD, USA).
PDE4D2 enzyme was purchased from BPS Bioscience (San
Diego, CA, USA). Lipopolysaccharide (LPS) was from Escheri-
chia coli strain 0127:B8 obtained from Sigma (St. Louis, MO,
USA). Mouse TNF-α ELISA kit was procured from R&D
Systems (Minneapolis, MN, USA).
routinelycultured inRPMI
Evaluation of PDE4 inhibitory potential by cell based cAMP
reporter assay. One day prior to transfection, HEK 293 cells
were seeded in p60 cell culture dish (Tarsons Inc.). These were
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Page 15

transfected using Lipofectamine 2000 (as per the manufacturer’s
instructions) with 2.4 μg of PDE4B1 expression plasmid and
4.0 μg of pCRELuc plasmid. After 5 h of transfection, medium
was aspirated, cells were trypsinized and seeded in 96 well
plates at a density of 60000 cells per well. Plates were incubated
overnight in a CO2incubator set to 37 °C and 5% CO2.Twenty
four hours post transfection, cells were pre-treated with various
concentrations (0.001 to 30 μM) of compounds for 30 min,
followed by stimulation with 5 μM forskolin for 4 h. Sub-
sequently medium was removed and cells were lysed in reporter
lysis buffer (Promega Inc) for 15 min with gentle rocking at RT.
Luciferase activity in the lysates was measured by a Multilabel
Plate Reader (Perkin Elmer 1420 Multilabel Counter). Fold
elevation of cAMP is calculated using the following formula.
Foldactivation ¼ðRLUof compound ? Rluof vehiclecontrolÞ
ðRluof forskolin ? RLUof vehiclecontrolÞ
PDE4B protein production and purification. PDE4B1 cDNA
was sub-cloned into pFAST Bac HTB vector (Invitrogen) and
transformed into DH10Bac (Invitrogen) competent cells. Recom-
binant bacmids were tested for integration by PCR analysis. Sf9
cells were transfected with bacmid using Lipofectamine 2000
(Invitrogen) according to manufacturer’s instructions. Sub-
sequently, P3 viral titer was amplified, cells were infected and
48 h post infection cells were lysed in lysis buffer (50 mM Tris-
HCl pH 8.5, 10 mM 2-mercaptoethanol, 1% protease inhibitor
cocktail (Roche), 1% NP40). Recombinant His-tagged PDE4B
protein was purified as previously described elsewhere.19a
Briefly, lysate was centrifuged at 10000 rpm for 10 min at 4 °C
and supernatant was collected. Supernatant was mixed with
Ni-NTA resin (GE Life Sciences) in a ratio of 4: 1 (v/v) and
equilibrated with binding buffer (20 mM Tris-HCl pH 8.0,
500 mM KCl, 5 mM imidazole, 10 mM 2-mercaptoethanol and
10% glycerol) in a ratio of 2: 1 (v/v) and mixed gently on rotary
shaker for 1 hour at 4 °C. After incubation, lysate–Ni-NTA
mixture was centrifuged at 4500 rpm for 5 min at 4 °C and the
supernatant was collected as the flow-through fraction. Resin
was washed twice with wash buffer (20 mM Tris-HCl pH 8.5,
1 M KCl, 10 mM 2-mercaptoethanol and 10% glycerol). Protein
was eluted sequentially twice using elution buffers (Buffer I:
20 mM Tris-HCl pH 8.5, 100 mM KCl, 250 mM imidazole,
10 mM 2-mercaptoethanol, 10% glycerol, Buffer II: 20 mM
Tris-HCl pH 8.5, 100 mM KCl, 500 mM imidazole, 10 mM
2-mercaptoethanol, 10% glycerol). Eluates were collected in
four fractions and analyzed by SDS-PAGE. Eluates containing
PDE4B protein were pooled and stored at −80 °C in 50%
glycerol until further use.
PDE4 enzymatic assay. The inhibition of PDE4 enzyme was
measured using PDElight HTS cAMP phosphodiesterase assay
kit (Lonza) according to manufacturer’s recommendations.
Briefly, 10 ng of in house purified PDE4B1 or 0.5 ng commer-
cially procured PDE4D2 enzyme was pre-incubated either with
DMSO (vehicle control) or compound for 15 min before incu-
bation with the substrate cAMP (5 μM) for 1 hour. The reaction
was halted with stop solution and reaction mix was incubated
with detection reagent for 10 min in dark. Dose response studies
were performed at 13 different concentrations ranging from
200 μM to 0.001 μM. Luminescence values (RLUs) were
measured by a Multilabel Plate Reader (PerklinElmer 1420 Mul-
tilabel Counter). The percentage of inhibition was calculated
using the following formula and the IC50values were determined
by a nonlinear regression analysis from dose response curve
using Graphpad Prism software (San Diego, USA). IC50values
are presented as mean ± SD.
%inhibition ¼ðRLUof vehiclecontrol ? Rluof inhibitorÞ
RLUof vehiclecontrol
? 100
TNF-α production assay. RAW 264.7 cells were pre-incubated
either with DMSO (vehicle control) or compound for 30 min
and then stimulated with 1 μg mL−1of LPS overnight. Dose
response studies were carried out at eight different concentrations
(30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01 μM). Post-stimulation, cell
supernatants were harvested, centrifuged to clear cell debris and
the amount of TNF-α in the supernatants was measured using
mouse TNF-α DuoSet ELISA kit from R&D Systems according
to manufacturer’s recommendations. The percentage of inhi-
bition was calculated using the following formula:
%inhibition ¼ 100
?
? 100
ðLPSstimulatedcompound? unstimulatedÞ
ðLPSstimulatedDMSO? unstimulatedÞ
??
The IC50values were determined by a nonlinear regression
analysis from dose response curve using Graphpad Prism soft-
ware (San Diego, USA). IC50values are expressed as mean ±
SD.
Acknowledgements
The authors sincerely thank the management of Institute of Life
Sciences for continuous support and encouragement. M.P. and
K.P. thank DBT, New Delhi, India for financial support (Grant
No. BT/PR12829/Med/30/222/2009). R.A. and B. P. thank
CSIR, New Delhi, India for a Junior Research Fellowship.
Notes and references
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5568 | Org. Biomol. Chem., 2012, 10, 5554–5569 This journal is © The Royal Society of Chemistry 2012
Downloaded by University of Hyderabad on 29 December 2012
Published on 01 June 2012 on http://pubs.rsc.org | doi:10.1039/C2OB25420D
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