Design and synthesis of tetrahydropyridothieno[2,3-d]pyrimidine scaffold based epidermal growth factor receptor (EGFR) kinase inhibitors: The role of side chain chirality and michael acceptor group for maximal potency

Article abstract of DOI:10.1021/jm100607r

HTS hit 7 was modified through hybrid design strategy to introduce a chiral side chain followed by introduction of Michael acceptor group to obtain potent EGFR kinase inhibitors 11 and 19. Both 11 and 19 showed over 3 orders of magnitude enhanced HCC827 a

Full text of DOI:10.1021/jm100607r

7316 J. Med. Chem. 2010, 53, 7316–7326
DOI: 10.1021/jm100607r
Design and Synthesis of Tetrahydropyridothieno[2,3-d]pyrimidine Scaffold Based Epidermal Growth
Factor Receptor (EGFR) Kinase Inhibitors: The Role of Side Chain Chirality and Michael Acceptor
Group for Maximal Potency
Chia-Hsien Wu,†,§,# Mohane Selvaraj Coumar,†,# Chang-Ying Chu,† Wen-Hsing Lin,*,† Yi-Rong Chen,‡ Chiung-Tong Chen,†
Hui-Yi Shiao,† Shaik Rafi,† Sing-Yi Wang,† Hui Hsu,† Chun-Hwa Chen,† Chun-Yu Chang,† Teng-Yuan Chang,†
Tzu-Wen Lien,† Ming-Yu Fang,† Kai-Chia Yeh,† Ching-Ping Chen,† Teng-Kuang Yeh,† Su-Huei Hsieh,† John T.-A. Hsu,†,
Chun-Chen Liao,§,^ Yu-Sheng Chao,† and Hsing-Pang Hsieh*,†
‡
†Institute of Biotechnology and Pharmaceutical Research, and Division of Molecular & Genomic Medicine, National Health Research Institutes,
35 Keyan Road, Zhunan Town, Miaoli County 350, Taiwan, ROC, §Department of Chemistry, National Tsing Hua University, 101 Section 2,
Kuang Fu Road, Hsinchu 300, Taiwan, ROC, Department of Biological Science and Technology, National Chiao Tung University, 1001 University
Road, Hsinchu 300, Taiwan, ROC, and ^Department of Chemistry, Chung Yuan Christian University, 200 Chung Pei Road, Chung Li 320,
Taiwan, ROC. #These authors contributed equally to this work.
Received May 18, 2010
HTS hit 7 was modified through hybrid design strategy to introduce a chiral side chain followed by
introduction of Michael acceptor group to obtain potent EGFR kinase inhibitors 11 and 19. Both 11
and 19 showed over 3 orders of magnitude enhanced HCC827 antiproliferative activity compared to
HTS hit 7 and also inhibited gefitinib-resistant double mutant (DM, T790M/L858R) EGFR kinase at
nanomolar concentration. Moreover, treatment with 19 shrinked tumor in nude mice xenograft model.
Introduction
Lung cancer is the most common human cancer, with
85% being nonsmall cell lung cancer (NSCLCa). In ∼50% of
NSCLC patients, epidermal growth factor receptor (EGFR) is
overexpressed with concomitant dysregulation in their down-
stream signaling pathway, and this overexpression is correlated
with poor prognosis.1 EGFR is a transmembrane protein with
an external ligand binding receptor domain and an intracellular
tyrosine kinase activity domain. Once a native ligand (eg.,
EGF) binds to the extracellular domain, it dimerizes with
another EGFR family member (eg, EGFR, Her2), resulting
in activation of the intracellular tyrosine kinase (TK) activity.
This results in autoposphorylation of EGFR and further acti-
vation of downstream signaling pathway affects cell survival
and proliferation.2
Gefitinib (1) and erlotinib (2) were approved by US FDA in
2002 and 2004, respectively, for the treatment of NSCLC
(Figure 1).2 Gefitinib or erlotinib competitively binds to the
adenosine triphosphate (ATP) binding pocket of intracellular
EGFR TK domain and inhibit its activity, thereby blocking
Figure 1. Reversible and irreversible EGFR tyrosine kinase inhi-
bitors.
the aberrant EGFR downstream signaling essential for tumor
survival and proliferation. Although the two drugs showed
high response rates in specific subsets of NSCLC patients, for
drug resistance often occurs as a result of a secondary muta-
tion such as the Thr790 f Met790 (T790M) mutation.1,3,4
Therefore, identification of second-generation EGFR tyro-
sine kinase inhibitors that overcome the acquired T790M
most patients, who respond initially to gefitinib or erlotinib,
mutation (such as the EGFRL858R/T790M double mutation)
*To whom correspondence should be addressed. For W.-H.L.:
phone, (þ) 886-37-246166 ext 35771; fax, (þ) 886-37-586-456; E-mail,
can be beneficial for the treatment of gefinitib- or erlotinib-
resistant NSCLC.
We have recently used an EGFR-transfected 32D cell-
twstar@nhri.org.tw. For H.-P.H.: phone, (þ) 886-37-246166 ext 35708;
fax, (þ) 886-37-586-456; E-mail, hphsieh@nhri.org.tw.
aAbbreviations: ATP, adenosine triphosphate; DM, double mutant
(L858R/T790M); EGFR, epidermal growth factor receptor; HTS, high-
based high-throughput screening (HTS) assay to identify
compounds that inhibit EGFR activation and/or EGFR-
throughput screening; LCMS, liquid chromatography coupled mass
spectrometry; NSCLC, nonsmall cell lung cancer; PK, pharmacokinetic;
TK, tyrosine kinase; WT, wild type.
mediated downstream signaling pathway.5 This cell-based
r
pubs.acs.org/jmc
Published on Web 09/23/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7317
Figure 2. Design of novel and potent tetrahydropyridothieno[2,3-d]pyrimidine scaffold EGFR tyrosine kinase inhibitors, through knowledge-
based design strategy.
Table 1. Inhibition of EGFR WT Kinase by 7 and 7a-d Analoguesa
screening protocol allowed us to identify several leads which
disrupt cell proliferation mediated by aberrant EGFR signal-
ing, among which 7, a novel tricyclic tetrahydrobenzothieno-
[2,3-d]pyrimidine core compound, was identified as an initial
hit with an enzyme inhibition IC50=2.6 μM (Figure 2). In this
brief communication, we describe the development path of
hit 7, a weak EGFR kinase inhibitor, to that of potent EGFR
kinase inhibitor 11 (IC50=8 nM) and 19 (IC50=6 nM), which
can also inhibit gefitinib resistant EGFRL858R/T790M double
EGFR-WT kinase
% inhibition at 20 μM
mutant kinase. Further, in vivo activity for 19 is demonstrated
compd
n
R
in HCC827 (NSCLC cell line with Del722-726 mutation)
xenograft nude mice model, proving the potential of 19 for
cancer treatment.
7
1
1
1
1
0
-CH2Ph
76.9
44.3
1.1
7a
7b
7c
7d
-CH2CH2Ph
-CH2(4-OCH3-Ph)
-CH2(4-F-Ph)
-CH2Ph
52.7
69.1
Results and Discussion
a WT: wild type kinase.
Initial attempts to improve the activity of 7 through in-
creasing the side chain length (7a), substitution in the phenyl
ring (7b,c), or converting the fused cyclohexyl ring to cyclo-
pentyl ring (7d) led to decreased EGFR kinase activity
(Table 1). Meanwhile, through a concurrent program for
the development of aurora kinase inhibitors, we have synthe-
sized a set of kinase-targeted compound library based on
furanopyrimidine scaffold. This library was counter-screened
for EGFR kinase inhibition, leading to the identification
of an EGFR kinase selective furanopyrimidine inhibitor 8
(Figure 2) with an EGFR kinase IC50=223 nM (aurora A
kinase IC50 >10 μM).6
Table 2. Inhibition of EGFR-WT Kinase and HCC827 Cell Line by 9
and 9a-d Analoguesa
Scrutinizing both the structures of HTS hit 7 and kinase
library hit 8 revealed that even though both have different core
structures (scaffolds), they have very similar side chain. The
kinase library hit 8 possessed a chiral S-2-phenyl-2-aminoetha-
nol side chain, which is very similar to that of the HTS hit
7 except for the presence of methanol branching with an
S-configuration. This novel chiral phenylaminoethanol side
chain is not present in any of the EGFR kinase inhibitors
reported so far. As a majority of EGFR kinase inhibitors in
market or in development bear anilino type side chains linked
to quinazoline/quinoline cores (1, 2, 4-6) (Figure 1), we con-
templated the development of new EGFR kinase inhibitors
with this novel chiral phenylaminoethanol side chain, both to
explore the importance of chirality for EGFR inhibition and to
develop novel proprietary molecules. It should also be noted
that a closely related R-1-phenylethylamino side chain has
been reported a few times in EGFR kinase inhibitors,7,8 and
compound 3 (AEE788)9 bearing R-1-phenylethylamino side
EGFR-WT kinase
inhibition
HCC827
IC50 (μM)b
compd
R1
R2
IC50b (μM)
9
CH2OH
CH3
Ph
Ph
H
0.720
3.240
>20c
>20c
1.090
0.198
9a
9b
9c
9d
CH2OH
Ph
CH2OH
CH2OH
Ph
0.324
a WT: wild type kinase. b Values are expressed as the mean of at least
two independent experiments and are mostly within 15% error margins.
c<50% enzyme inhibition at 20 μM.
chain is reported to be in clinical testing; however, the im-
portance of chirality for EGFR inhibition has not been
explored in much detail through structure-activity relation-
ship (SAR) studies in those reports.
With this agenda, we synthesized the hybrid compound 9
(Figure 2) possessing the tricyclic core of hit 7 and the chiral

7318 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Wu et al.
Table 3. Inhibition of EGFR Kinase and HCC827 Cell Line by 7, 9-27d
a Values are expressed as the mean of at least two independent experiments and are mostly within 15% error margins. b<50% enzyme inhibition at
20 μM. c<50% enzyme inhibition at 10 μM. d WT: wild-type EGFR kinase. DM: double mutant EGFR kinase.
phenylaminoethanol side chain of hit 8. Subsequently, the
hybrid compound 9 showed submicromolar EGFR kinase
inhibition (IC50 = 720 nM) and further exhibited potent anti-
proliferative activity in HCC827 cell lines (IC50 = 198 nM),
which is over 50-fold better than that of the original HTS hit 7.
On the basis of these findings, new lead 9 was selected for
further structural modification to deeply investigate SAR in
order to optimize both the kinase and cell-based activity.
It was found that either removal of the secondary hydroxyl
group (9a) or phenyl group (9b) from the side chain of 9 led to
decreased EGFR kinase inhibition, particularly removal of
the phenyl group led to complete loss of activity, showing the
indispensible nature of the phenyl ring in maintaining activ-
ity (Table 2). Moreover, the orientation of the phenyl ring
is critical, as the R-enantiomer (9c) of 9 was found to be
completely inactive. Also, converting the six-membered car-
bocyclic ring fused to the thienopyrimidine to five-membered
ring (9d) did not improve the activity. Although compounds
9 and 9d showed nanomolar level inhibition in wild-type
enzyme and cell-based assay, both were not capable of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7319
inhibiting double mutant EGFR kinase (<50% inhibition at
10 μM), which could overcome gefitinib resistance.
Recent research reveals that a possible way to overcome
gefitinib-resistant T790M mutant EGFR kinase is through
irreversible inhibition of the enzyme by covalent modification of
Cys733 residue in the ATP binding domain of the kinase.1,4,10,11
This has been achieved with three clinical trial compounds 4
(HKI-272),12,13 5 (EKB-569),14,15 and 6 (BIBW2992)16 for the
treatment of gefitinib-resistant NSCLC. These clinical trial
compounds possess a Michael acceptor group in common,
which plays a crucial role in inhibiting gefitinib resistant T790M
mutant kinase irreversibly through covalent modification of
cysteine residue.11,16 This knowledge stimulated us to intro-
duce a Michael acceptor group in our series of compounds to
incorporate activity toward gefitinib-resistant T790M mutant
kinase. One possibility to introduce a Michael acceptor group
into the tricyclic core of 9 is through the introduction of
additional diversity point by replacing one methylene unit
with a nitrogen in the six-membered carbocyclic ring to
form 10. Then the newly designed tricyclic tetrahydro-
pyridothieno[2,3-d]pyrimidine core was used to construct 11
incorporating an acrylamide Michael acceptor group at the
diversity point (Figure 2).
First, the hybrid compound 11 with the acrylamide Michael
acceptor group was examined for both wild-type and double
mutant EGFR kinase inhibition (Table 3). It not only dis-
played an excellent wild-type enzyme inhibition but also dis-
played double mutant kinase inhibition at nanomolar level.
Compared to 9, 11 displayed 90-fold enhanced EGFR wild-
type kinase inhibition. Most interestingly, 11 displayed sig-
nificantly enhanced double mutant kinase inhibition, which is
over 25-fold better than gefitinib. In addition to showing
potent EGFR kinase inhibition, 11 also showed potent anti-
proliferative activity in HCC827 cell line, with growth inhibi-
tion in the single-digit nanomolar range (IC50 = 3 nM). It
should be noted that compound 10, a close analogue of 11
lacking the Michael acceptor acrylamide function shows
complete loss of activity. Thus incorporation of acrylamide
Michael acceptor group had resulted in both enhancing the
wild-type enzyme inhibition as well as imparting potent activ-
ity toward gefitinib-resistant double mutant kinase. Further,
the importance of the acrylamide Michael acceptor group in
imparting EGFR activity was shown by complete inactivity of
12, wherein the terminal double bond of 11 is reduced to give a
simple propanamide functional group. Next, we attempted to
replace the acrylamide group with butynamide (13) or with
vinyl sulfonamide (14) as different Michael acceptor groups,
both of which led to loss of activity. The butynamide analogue
13 showed poor activity in both wild and double mutant
kinase, while the vinyl sulfonamide analogue 14 inhibited
the double mutant kinase in a range very similar to that of
gefitinib, suggesting the superiority of acrylamide 11 as an
EGFR inhibitor.
Figure 3. Western blot analysis using anti-phospho-EGFR Y1068
and anti-EGFR immunoblots of HCC827 cell lysates treated with 1
or 11 for 2 h. A representative anti-tubulin immunoblot is shown as
a loading control.
detected inappreciableamounts in plasma within 10min of IV
injection (data not shown). The poor PK properties precluded
testing of 11 for in vivo efficacy, and hence further efforts were
aimed at identifying potent compounds with improved PK
profile compared to that of 11. For this purpose, we first
turned our attention to the chiral phenylaminoethanol side
chain linked to the pyrimidine core. When the S-enatiomer 11
was converted to R-enatiomer 15, the compound lost EGFR
wild and double mutant kinase activity over 200-fold com-
pared to that of 11. However, either the removal of hydroxyl
group (16) or converting it to methyl group (17) led to loss of
3-6-fold potency for wild-type and 8-22-fold loss of potency
for double mutant kinase compared to that of 11. These
results suggest that presence of hydroxyl group plays an
important role in maintaining potent activity for both wild-
type and double mutant EGFR kinase. Hence, by retaining
the chiral phenylaminoethanol side chain linked to the py-
rimidine core, we attempted modification in the Michael
acceptor region of 11 in order to identify potent compounds.
Introduction of a hydrophobic phenyl ring at the terminal
double bond (18) led to complete loss of potency for both
wild-type and double mutant kinase. On the basis of this
observation, a hydrophilic N,N-dimethyl group was intro-
duced through a methylene linker to the terminal double bond
of 11 to get 19, which retained the wild-type kinase activity
and led to only a 3-fold loss of activity for double mutant
kinase compared to that of 11. When the size of the hydro-
philic moiety was increased by changing the N,N-dimethyl
group (19) to N,N-diethyl (20), morpholine (21), or N-methyl
piperazine (22), both the wild-type as well as double mutant
kinase activity decreased; the loss of potency was more
prominent in the case of double mutant kinase activity,
particularly N-methyl piperazine analogue 22 was completely
inactive.
Second, the full potential of 11 was probed by evaluating its
ability to interfere with EGFR signaling inside the cell by
carrying out Western blotting analysis. For this purpose,
HCC827 cells were treated with 11 in dose-dependent manner
and then analyzed for EGFR autophosphorylation (pEGFR
at residue Tyr1068) (Figure 3). When compared to gefitinib,
11 showed better inhibition of EGFR autoposphorylation,
further demonstrating the potential of 11 as an EGFR kinase
inhibitor.
As compound 19 is found to be better than related ana-
logues 20-22 by retaining the N,N-dimethyl side chain on the
Michael acceptor side of 19, we further attempted to explore
the SAR in the chiral phenylaminoethanol side chain linked to
the pyrimidine core. It was found that the R-enatiomer (23)
again lost the activity for both wild and double mutant kinase
completely. Removal of hydroxyl group (24) only led to 2-fold
loss of wild-type and double mutant kinase activity, while
converting it to methyl group (25) led to loss of double mutant
kinase activity (IC50>5 μM) without much affecting the wild-
type kinase activity compared to that of 19. These findings in
Third, wecarriedoutinvivopharmacokinetic (PK) studyin
rat for 11, which showed that the compound could not be

7320 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Wu et al.
the chiral phenylaminoethanol side chain are very similar to
the SAR observation in 11 containing the acrylamide Michael
acceptor group. This demonstrates that apart from the pres-
ence of Michael acceptor functionality, the presence of the
hydroxyl group and the orientation of the phenyl group in the
side chain has played a critical role determining the activity in
this series, in particular for double mutant kinase activity. To
further explore the role of the phenyl group, it was moved one
carbon away from the chiral center or completely removed
from the side chain, which resulted in 26 and 27, respectively.
Both moving the phenyl group through chain extension or
removing the phenyl group had resulted in drastic loss of
activity for both wild-type and double mutant kinase, with
compound 27 being completely inactive toward both enzymes
and 26 still maintaining nanomolar level wild-type kinase
inhibition. These studies further demonstrate that the pres-
ence aswell as orientationofthe phenyl group in the sidechain
is essential for potent EGFR kinase activity in this series of
compounds.
Having identified 19, which has similar EGFR kinase
inhibition profile as that of 11, we carried out PK evaluation
of 19 in rats and found that 19 showed better IV PK profile
(Table 4) compared to that of 11, suggesting that 19 is a more
suitable candidate for in vivo efficacy evaluation. The in vivo
efficacy of 19 was evaluated in nude mice bearing HCC827
tumor xenografts. Treatment mice received 19 as IV injec-
tion through the tail veins for 5 days/week for 2 consecutive
weeks (days 1-5 and 8-12). At the dosage of 5 and 15 mg/kg,
19 shrank tumor growth significantly (p<0.05), indicating
potent in vivo anticancer activity for 19 (Figure 4). Adminis-
tration of reference compound gefitinib (1) 100 mg/kg by
perioral route using the same dosing schedule also shrinks the
tumor significantly (p < 0.05).
Table 4. Intravenous (iv) Pharmacokinetics Profile of 19 in Rat
parameter
unit
value (SE)
N
dose
3
mg/kg
hr
5
t1/2
clearance
Vss
1.4 (0.1)
248.7 (7.6)
27.3 (3.7)
337 (7)
mL/min/kg
L/kg
ng/mL h
AUC (0-¥)
3
Synthesis. Compounds 7, 7a-d, 9, and 9a-d were synthe-
sized as shown in Scheme 1. Appropriate cyclic ketones 28a,b
were condensed with ethyl cyanoacetate under basic condi-
tion and then cyclized in the presence of sulfur to construct
the thiophene cores 29a,b through Gewald reaction.17 The
thieno[2,3-d]pyrimidine ring system was then constructed
using a modified Niementowski quinazoline synthesis by
condensation of 29a,b with formamide to afford 30a,b.
Finally, chlorination of pyrimidone 30a,b with phosphrous
oxychloride gave the intermediate 31a,b, which underwent
nucleophilic reaction with appropriate amines to give the
desired compounds 7, 7a-d, 9, and 9a-d.
Figure 4. In vivo antitumor effect of 19 in human nonsmall
cell lung cancer (HCC827) xenograft nude mice model. The growth
of HCC827 tumor xenograft is inhibited by 19 (5 mg/kg, iv or
15 mg/kg, iv) and gefitinib (100 mg/kg, po) with p < 0.05. Drug
treatment on days 1-5 and 8-12.
For the preparation of thieonpyrimidines 11-27 with the
nitrogen bridge in the alicyclic ring, the key intermediate 31c
was constructed starting from 4-oxo-piperidine-1-carboxylic
acid tert-butyl ester (28c) using a similar sequence of reaction
Scheme 1a
a Reagents and conditions: (a) NCCH2CO2Et, S8, EtOH, Et3N, reflux or rt, 16 h; (b) formamide, reflux, 2 h or formamidine acetate, DMF, 100 ꢀC,
16 h; (c) POCl3, 0 f 60 ꢀC, 3 h; (d) primary amine (R1R2CHNH2), n-BuOH, reflux, 8 h.
Scheme 2a
a Reagents and conditions: (a) amine (R1R2CHNH2), EtOH, reflux, 8 h; (b) TFA, CH2Cl2, 0 ꢀC f rt, 2 h; (c) acid (RCO2H), EDCI, CH2Cl2, rt, 2 h
(d) Cl(CH2)2SO2Cl, CH2Cl2, rt, 4 h; (e) (i) (E)-BrCH2CHdCHCO2H, EDCI, CH2Cl2, rt, 2 h; (ii) secondary amine (R2NH), THF, 0 ꢀC, 2 h.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7321
as that for obtaining 31a,b (Scheme 1). Nucleophilic reaction
of 31c with appropriate primary amines gave 32a-f, which
were then subjected to Boc deprotection using TFA resulting
in the intermediates 33a-f. The desired compounds 11-13
and 15-18 were obtained by amide coupling reaction of
appropriate secondary amine (33) and the acid (RCO2H)
using EDCI as a coupling agent. For the preparation of
compounds 19-27, a two-step reaction sequence was uti-
lized to get the final products; an initial amide coupling of
4-bromo crotonoic acid with appropriate intermediate 33,
followed by SN2 reaction with different secondary amines
(R2NH), was carried out. For the sulphonamide derivative
14, acylation of 33a with sulphonyl chloride was carried out
(Scheme 2).
A-acetonitrile and mobile phase B-water containing 0.1% for-
mic acid þ 10 mmol NH4OAc. Elution condition, at 0 min phase
A 10% þ phase B 90%; at 45 min phase A 90% þ phase B 10%;
at 50 min phase A 10% þ phase B 90%; at 60 min phase A
10% þ phase B 90%. The flow-rate was 0.5 mL/min, and the
injection volume was 5 μL. The system operated at 25 ꢀC. Peaks
were detected at 210 nm. Purity of all the tested compounds were
found to be g95% unless otherwise stated.
Ethyl 2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxyl-
ate (29a). To a mixture of cyclohexanone (28a) (9.80 g, 100.0
mmol), ethyl cyanoacetate (11.32 g, 100.0 mmol), and sulfur
(3.20 g, 100.0 mmol) in absolute ethanol (200 mL) was added
triethylamine (20 mL) and refluxed for 16 h; the reaction mixture
was concentrated and the residue was partitioned between water
and ethyl acetate. The organic layer was separated, dried over
MgSO4, and concentrated, and the crude product was purified by
silica gel column chromatography using a mixture of hexanes:
ethyl acetate (4:1), to give 29a (18.6 g, 82%). 1H NMR (400 MHz,
CDCl3) δ 1.31 (t, 3H, J = 7.2 Hz), 1.68-1.77 (m, 4H), 2.45-2.49
(m, 2H), 2.66-2.69 (m, 2H), 4.23 (q, 2H, J = 7.2 Hz), 5.90 (brs,
2H). LCMS (ESI): m/z 226.0 [M þ H]þ.
Conclusion
In summary, HTS hit 7 identified from cell-based screening
strategy was modified through a knowledge-based design
approach to obtain potent and novel tetrahydropyridothieno-
[2,3-d ]pyrimidine scaffold compounds as EGFR kinase inhi-
bitors. Designing-in an unusual S-chiral 2-phenyl-2-amino-
ethanol side chain identified from counter-screening approach,
and then introduction of Michael acceptor group into the
tricyclic core through a diversity point “NH” in HTS hit 7, led
to the development of 11 and 19. Both 11 and 19 showed >
300-fold enhanced EGFR wild-type kinase inhibition
and >3000-fold enhanced HCC827 antiproliferative activity
compared to HTS hit 7 and also inhibited gefitinib-resistant
double mutant (DM, T790M/L858R) EGFR kinase at nano-
molar concentration. Detailed SAR study in this series reveals
that, in the presence of the side chain, the nature of chirality
and the presence of the phenyl group are very critical
for maintaining activity, while the presence of secondary
hydroxyl function provides maximum potency. The presence
of S-chiral 2-phenyl-2-aminoethanol side chain along with the
acrylamide-type Michael acceptor group imparts maximal
activity toward gefitinib-resistant double mutant EGFR
kinase. Six of the compounds (11, 19-21, 24, 25) possess
excellent antiproliferative activity against HCC827 lung can-
cer cell line in single-digit nanomolar range, with compound
19 showing in vivo efficacy in nude mice bearing HCC827
tumor xenografts. Further testing could identify the full
potential of this novel series for the treatment of cancers.
Ethyl 2-Amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxyl-
ate (29b). Compound 29b was synthesized in 78% yield from
cyclopentanone (28b), in a manner similar to 29a. 1H NMR (300
MHz, CDCl3): δ 1.30 (t, 3H, J = 7.2 Hz), 2.28 (dddd, 2H, J =
7.2, 7.2, 7.2, 7.2 Hz), 2.66-2.71 (m, 2H), 2.77-2.81 (m, 2H), 4.21
(q, 2H, 7.2 Hz), 5.59 (brs, 2H). LCMS (ESI): m/z 212.0 [M þ H]þ.
6-(tert-Butyl) 3-Ethyl 2-Amino-4,5,6,7-tetrahydrothieno[2,3-c]-
pyridine-3,6-dicarboxylate (29c). To a mixture of 4-oxo-piperi-
dine-1-carboxylic acid tert-butyl ester (28c) (12.90 g, 64.7 mmol),
ethyl cyanoacetate (7.31 g, 64.7 mmol), and sulfur (2.07 g, 64.7
mmol) in absolute ethanol (50 mL) was added triethylamine (9
mL). After stirring for 16 h at room temperature, the precipitate
was collected by filtration and washed with ethanol to give 29c
(18.7 g, 88%). 1H NMR (300 MHz, CDCl3) δ 1.30 (t, 3H, J = 7.2
Hz), 1.44 (s, 9H), 2.72-2.80 (brs, 2H), 3.58 (t, 2H, J = 6.0 Hz),
4.23 (q, 2H, J = 7.2 Hz), 4.31 (s, 2H), 6.02 (s, 2H). LCMS (ESI):
m/z 327.0 [M þ H]þ.
3,4,5,6,7,8-Hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-one
(30a). A mixture of 29a (0.90 g, 4.0 mmol) and formamide (10 mL)
was refluxed for 2 h. The reaction mixture was cooled, water was
added, and the precipitate formed was collected by filtration, and
washed thoroughly with water to give 30a (0.8 g, 77%). 1H NMR
(400 MHz, CDCl3): δ 1.83-2.02 (m, 4H), 2.76-2.79 (m, 2H),
2.99-3.02(m, 2H), 7.91(s, 1H). LCMS(ESI):m/z207.0 [M þ H]þ.
3,5,6,7-Tetrahydro-4H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-
4-one (30b). Compound 30b was synthesized in 83% yield from
29b, in a manner similar to 30a. 1H NMR (400 MHz, CDCl3): δ
2.47 (dddd, 2H, J= 7.2, 7.2, 7.2, 7.2 Hz), 2.95-2.98 (m, 2H), 3.05-
3.09 (m, 2H), 8.03 (s, 1H). LCMS (ESI): m/z 193.0 [M þ H]þ.
tert-Butyl 4-Oxo-3,4,5,6,7,8-hexahydropyrido[40,30:4,5]thieno-
[2,3-d]pyrimidine-7-carboxylate (30c). A mixture of 29c (18.50 g,
56.7 mmol) and formamidine acetate (8.85 g, 85.0 mmol) in
DMF (100 mL) were heated at 100 ꢀC for 16 h. The reaction
mixture was cooled, DMF removed under vacuum, and the solid
obtained was washed thoroughly with water to give 30c (15.8 g,
Experimental Section
General Methods. All commercial chemicals and solvents are
reagent grade and were used without further treatment unless
otherwise noted. All reactions were carried out under an atmo-
sphere of dry nitrogen. Reactions were monitored by TLC using
Merck 60 F254 silica gel glass backed plates (5 cm ꢀ 10 cm); zones
were detected visually under ultraviolet irradiation (254 nm) or
by spraying with phosphomolybdic acid reagent (Aldrich),
followed by heating at 80 ꢀC. Flash column chromatography
was done using silica gel (Merck Kieselgel 60, no. 9385, 230-400
1
90%). H NMR (400 MHz, CDCl3) δ 1.42 (s, 9H), 3.02-3.08
(brs, 2H), 3.62-3.70 (brs, 2H), 4.56-4.62 (brs, 2H), 7.88 (s, 1H).
LCMS (ESI): m/z 308.1 [M þ H]þ.
1
4-Chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine
(31a). Amixture of30a (12.0 g, 58.2 mmol) and POCl3 (10mL) was
heated at 55-65 ꢀC for 3 h. The reaction mixture was cooled in an
ice bath and then carefully neutralized by the addition of aqueous
sodium bicarbonate solution. The resulting mixture was extracted
with ethyl acetate, the organic layer was separated, dried over
MgSO4, and concentrated, and the crude product was purified by
silica gel column chromatography using a mixture of hexanes:ethyl
mesh ASTM). H NMR spectra were obtained with a Varian
Mercury-300 spectrometer operating at 300 MHz. Chemical
shifts were recorded in parts per million (ppm, δ) and were
reported relative to the solvent peak or TMS. High-resolution
mass spectra (HRMS) were measured with a Finnigan (MAT-
95XL) electron impact (EI) or by using Finnigan/Thermo Quest
MAT 95XL FAB mass spectrometer. LCMS data were mea-
sured on an Agilent MSD-1100 ESI-MS/MS System. Purity of
the final compounds were determined with an Hitachi 2000
series HPLC system using C-18 column (Agilent ZORBAX
Eclipse XDB-C18 5 μm. 4.6 mm ꢀ 150 mm) using mobile phase
1
acetate (20:1), to give 31a (9.6 g, 73%). H NMR (300 MHz,
CDCl3): δ 1.89-1.92 (m, 4H), 2.88-2.86 (m, 2H), 3.10-3.07
(m, 2H), 8.69 (s, 1H). LCMS (ESI): m/z 225.3 [M þ H]þ.

7322 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Wu et al.
4-Chloro-6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimi-
dine (31b). Compound 31b was synthesized in 88% yield from
30b in a manner similar to 31a. 1H NMR (400 MHz, CDCl3): δ
2.48-2.56 (m, 2H), 3.04-3.08 (m, 2H), 3.13-3.17 (m, 2H), 8.70
(s, 1H). LCMS (ESI): m/z 210.9 [M þ H]þ.
(m, 4H), 2.74 (m, 2H), 2.88-2.93 (m, 2H), 3.95-3.99 (m, 2H),
5.36(dd, 1H, J =5.6, 9.6 Hz), 6.14 (d, 1H, J =6.0Hz), 7.26-7.34
(m, 5H), 8.20 (s, 1H). 13C NMR (75 MHz, CDCl3): δ 22.3 (CH2),
22.4 (CH2), 25.3 (CH2), 26.1 (CH2), 57.0 (CH), 67.1 (CH2), 116.2
(C), 125.3 (C), 126.4 (CH), 127.7 (CH). 128.8 (CH), 133.6 (C),
139.7 (C), 152.4 (CH), 156.8 (C), 165.0 (C). HRMS (EI): calcd for
C18H19N3OS (Mþ) 325.1249, found 325.1247.
N-[(1R)-1-Phenylethyl]-N-(5,6,7,8-tetrahydrobenzo[4,5]thieno-
[2,3-d]pyrimidin-4-yl)amine (9a). Compound 9a was synthesized
in94%yieldfrom31a and R-(þ)-methylbenzylamine ina manner
similar to 9. 1H NMR (300 MHz, CDCl3): δ 1.60 (d, 3H, J = 6.6
Hz), 1.85-1.92 (m, 4H), 2.76-2.81 (m, 2H), 2.89-2.91 (m, 2H),
5.51-5.52 (m, 1H), 7.25-7.39 (m, 5H), 8.33 (s, 1H). 13C NMR
(75 MHz, CDCl3): δ 22.4 (CH2), 22.5 (CH2), 22.6 (CH3), 25.4
(CH2), 26.3 (CH2), 49.7 (CH), 115.9 (C), 125.2 (C), 126.0 (CH),
127.3 (CH), 128.3 (CH), 133.2 (C), 143.7 (C), 153.0 (CH), 156.5
(C), 165.3 (C). HRMS (EI): calcd for C18H19N3S (Mþ) 309.1300,
found 309.1296.
tert-Butyl 4-Chloro-5,6,7,8-tetrahydropyrido[40,30:4,5]thieno-
[2,3-d]pyrimidine-7-carboxylate (31c). To a mixture of POCl3
(3 mL) and triethylamine (3 mL) at 0 ꢀC was added 30c (2.3 g,
7.5 mmol). The reaction mixture was heated at 55-60 ꢀC for 3 h,
then the reaction mixture was cooled, excess of POCl3 removed
under vacuum, and the remaining residue neutralized carefully
by adding saturated aqueous sodium bicarbonate solution.
The resulting mixture was extracted with dichloromethane, the
organic layer separated, dried over MgSO4, concentrated, and
the crude product obtained was purified by silica gel column
chromatography using a mixture of hexane:ethyl acetate (10:1)
to give 31c (1.85 g, 75%). 1H NMR (400 MHz, CDCl3) δ 1.46 (s,
9H), 3.14-3.18 (brs, 2H), 3.74 (t, 2H, J = 5.6 Hz), 4.69 (s, 2H),
8.70 (s, 1H). LCMS (ESI): m/z 326.0 [M þ H]þ.
2-(5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d ]pyrimidin-4-ylamino)-
1-ethanol (9b). Compound 9b was synthesized in 88% yield from
31a and ethanolamine in a manner similar to 9. H NMR (400
N-Benzyl-N-(5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-
4-yl)amine (7). A mixture of 31a (45 mg, 0.2 mmol) and benzyl-
amine (86 mg, 4.0 equiv, 0.8 mmol) in n-butanol (2 mL) was
refluxed for 8 h. The reaction mixture was concentrated, and the
residue was partitioned between water and dichloromethane. The
organic layer was washed with brine, dried over MgSO4, and
concentrated. The crude product was purifiedbysilicagel column
chromatography using a mixture of hexane:ethyl acetate (6:1) to
give 7 (42 mg, 72%). 1H NMR (400 MHz, CDCl3): δ 1.84-1.88
(m, 4H), 2.75-2.78 (m, 2H), 2.82-2.84 (m, 2H), 4.77 (d, 2H, J =
5.6 Hz), 5.54 (brs, 1H), 7.24-7.34 (m, 5H), 8.38 (s, 1H). HRMS
(EI): calcd for C17H17N3S (Mþ) 295.1143, found 295.1129.
N-Phenethyl-N-(5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrim-
idin-4-yl)amine (7a). Compound 7a was synthesized in 52%
yield from 31a and phenylethylamine, in a manner similar to 7.
1H NMR (400 MHz, CDCl3): δ 1.75-1.80 (m, 4H), 2.49-2.52
(m, 2H), 2.70-2.72 (m, 2H), 2.94(t, 2H, J = 6.4Hz), 3.80 (td, 2H,
6.4, 6.4 Hz), 5.19 (m, 1H), 7.19-7.23 (m, 3H), 7.28-7.31 (m, 2H),
8.35 (s, 1H). LCMS (ESI): m/z 310.0 [M þ H]þ. HPLC purity
90.63%.
1
MHz, CDCl3): δ 1.78-1.84 (m, 4H), 2.70-2.72 (m, 2H), 2.82-
2.84 (m, 2H), 3.66 (dt, 2H, J = 5.2, 5.2 Hz), 3.82 (t, 2H, J = 5.2
Hz), 5.80 (t, 1H, J = 5.2 Hz), 8.24 (s,1H). 13C NMR (100 MHz,
CDCl3): δ 22.3 (CH2), 22.3 (CH2), 25.2 (CH2), 26.1 (CH2), 43.9
(CH2), 62.0 (CH2), 116.1 (C), 125.5 (C), 133.3 (C), 152.2 (CH),
157.4 (C), 164.7 (C). HRMS (EI): calcd for C12H15N3OS (Mþ)
249.0936, found 249.0958.
(2R)-2-Phenyl-2-(5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]-
pyrimidin-4-ylamino)ethan-1-ol (9c). Compound 9c was synthe-
sizedin59%yieldfrom31a and R-(-)-2-amino-2-phenyl-ethanol
in a manner similar to 9. 1H NMR (400 MHz, CDCl3): δ 1.82-
1.88 (m, 4H), 2.73-2.76 (m, 2 H), 2.88-2.93 (m, 2 H), 3.93-3.99
(m, 2 H), 5.35-5.38 (m, 1 H), 6.11 (d, 1 H, J = 6.4 Hz), 7.25-
7.34 (m, 5 H), 8.20 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 22.3
(CH2), 22.4 (CH2), 25.3 (CH2), 26.1 (CH2). 57.0 (CH), 67.1
(CH2), 116.2 (C), 125.3 (C), 126.4 (CH), 127.7 (CH), 128.8
(CH), 133.6 (C), 139.7 (C), 152.4 (CH), 156.8 (C), 165.0 (C).
HRMS (EI): calcd for C18H19N3OS (Mþ) 325.1249, found
325.1254.
N-(4-Methoxybenzyl)-N-(5,6,7,8-tetrahydrobenzo[4,5]thieno-
[2,3-d]pyrimidin-4-yl)amine (7b). Compound 7b was synthesized
in 65% yield from 31a and 4-methoxybenzylamine, in a manner
similar to 7. 1H NMR (400 MHz, CDCl3): δ 1.80-1.85 (m, 4H),
2.75-2.78 (m, 2H), 2.80-2.82 (m, 2H), 3.78 (s, 3H), 4.68 (d, 2H,
J = 5.2 Hz), 5.45 (m, 1H), 6.85-6.88(m, 2H), 7.26-7.28 (m, 2H),
8.39 (s, 1H). LCMS (ESI): m/z 326.0 [M þ H]þ.
(2S)-2-(6,7-Dihydro-5H-cyclopenta[4,5]thieno[2,3-d ]pyrimidin-
4-ylamino)-2-phenylethan-1-ol (9d). Compound 9d was synthe-
sized in 94% yield from 31b and S-(þ)-2-amino-2-phenyl-ethanol
in a manner similar to 9. 1H NMR (400 MHz, CDCl3): δ 2.48-
2.55 (m, 2H), 2.93-3.06 (m, 4H), 3.83 (brs, 1H), 4.00 (dd, 2H,
J = 5.2, 5.2 Hz), 5.34 (ddd, 1H, J = 5.2, 5.2, 5.2 Hz), 5.80 (d, 1H,
J = 6.0Hz), 7.29-7.40 (m, 5H), 8.31 (s, 1H). 13C NMR (75MHz,
CDCl3): δ 27.9 (CH2), 28.9 (CH2), 29.4 (CH2), 57.1 (CH), 67.2
(CH2), 113.4 (C), 126.4 (CH), 127.8 (CH), 128.9 (CH), 134.3 (C),
139.1 (C), 139.6 (C), 152.5 (CH), 156.3 (C), 165.0 (C). HRMS
(EI): calcd for C17H17N3OS (Mþ) 311.1092, found 311.1096.
tert-Butyl 4-[(1S)-2-Hydroxy-1-phenylethyl]amino-5,6,7,8-tetra-
hydropyrido[40,30:4,5]thieno[2,3-d]pyrimidine-7-carboxylate (32a).
A mixture of 31c (0.25 g, 0.7 mmol) and S-(þ)-2-amino-2-phenyl-
ethanol (0.13 g, 1.1 mmol) in ethanol (1 mL) was refluxed for 8 h.
The reaction mixturewas concentrated, andthe residue was parti-
tioned between water and dichloromethane; the organic layer
separated, dried over MgSO4, and concentrated, and the crude
product was purified by silica gel column chromatography using
a mixture of dichloromethane:methanol (20:1) to give 32a (0.31 g,
95%). 1H NMR (300 MHz, CDCl3) δ 1.46 (s, 9H), 2.90-3.08 (m,
2H), 3.72-3.81 (m, 2H), 3.97-4.02 (m, 2H), 4.62 (s, 2H), 5.36-
5.43(m, 1H), 6.04 (d, 1H, J = 6.6 Hz), 7.26-7.38 (m, 5H), 8.25(s,
1H). LCMS (ESI): m/z 427.0 [M þ H]þ.
N-(4-Fluorobenzyl)-N-(5,6,7,8-tetrahydrobenzo[4,5]thieno[2,
3-d]pyrimidin-4-yl)amine (7c). Compound 7c was synthesized in
76% yield from 31a and 4-fluorobenzylamine, in a manner simi-
lar to7. 1H NMR (400 MHz, CDCl3): δ 1.86-1.91 (m, 4H), 2.80-
2.82 (m, 2H), 2.85-2.88 (m, 2H), 4.77 (d, 2H, J = 6.0 Hz), 5.53
(m, 1H), 7.01-7.06 (m, 2H), 7.32-7.35 (m, 2H), 8.41 (s, 1H).
LCMS (ESI): m/z 314.0 [M þ H]þ.
N-Benzyl-N-(6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d ]pyrim-
idin-4-yl)amine (7d). Compound 7d was synthesized in 79% yield
from 31b and benzylamine in a manner similar to 7. H NMR
1
(400 MHz, CDCl3): δ 2.45-2.53 (m, 2H), 2.94-2.98 (m, 4H),
4.80 (d, 2H, J = 5.6 Hz), 5.37 (m, 1H), 7.24-7.35 (m, 5H), 8.40
(s, 1H). HRMS (EI): calcd for C16H15N3S (Mþ) 281.0987, found
281.1009.
(2S)-2-Phenyl-2-(5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]-
pyrimidin-4-ylamino)ethan-1-ol (9). A mixture of 31a (0.05 g,
0.2 mmol) and S-(þ)-2-amino-2-phenyl-ethanol (0.04 g, 1.5
equiv, 0.3 mmol) in n-butanol (2 mL) was refluxed for 8 h. The
reaction mixture was concentrated, and the residue was parti-
tioned between water and dichloromethane. The organic layer
was washed with brine, dried over MgSO4, and concentrated. The
crude product was purified by silica gel column chromatography
using a mixture of dichloromethane:methanol (30:1), to give 9
(0.048 g, 75%). 1H NMR (400 MHz, CDCl3): δ 1.83-1.88
tert-Butyl 4-[(1R)-2-Hydroxy-1-phenylethyl]amino-5,6,7,8-tetra-
hydropyrido[40,30:4,5]thieno[2,3-d]pyrimidine-7-carboxylate (32b).
Compound 32b was synthesized in 99% yield from 31c and
R-(-)-2-amino-2-phenyl-ethanol in a manner similar to 32a. 1H
NMR (400 MHz, CDCl3): δ 1.46 (s, 9H), 2.98-3.02 (m, 2H),
3.73-3.76 (m, 2H), 3.96-3.99 (m, 2H), 4.61 (s, 2H), 5.37-5.38

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7323
(m, 1H), 6.04 (d, 1H, J = 6.0 Hz), 7.26-7.34 (m, 5H), 8.23 (s,
1H). 13C NMR (75 MHz, CDCl3): δ 25.8 (CH2), 27.7 (CH2), 28.1
(CH3), 39.8 (CH2), 40.9 (CH2), 42.6 (CH2), 43.4 (CH2), 56.0
(CH), 65.7 (CH2), 80.3 (C), 115.3 (C), 124.4 (C), 126.3 (CH),
127.2 (CH), 128.4 (CH), 129.2 (C), 139.6 (C), 152.7 (CH), 154.0
(C), 156.5 (C), 164.9 (C). LCMS (ESI): m/z 427.1 [M þ H]þ.
tert-Butyl 4-[(1R)-1-Phenylethyl]amino-5,6,7,8-tetrahydropyrido-
[40,30:4,5]thieno[2,3-d]pyrimidine-7-carboxylate (32c). Compound
32c was synthesized in 90% yield from 31c and R-(þ)-methylben-
zylamine in a manner similar to 32a. 1H NMR (300 MHz, CDCl3):
δ 1.47 (s, 9H), 1.57 (d, 3H, J = 9.6 Hz), 2.95-3.03 (m, 2H),
3.73-3.79 (m, 2H), 4.62 (brs, 2H), 5.35-5.37 (m, 1H), 5.49-5.54
(m, 1H), 7.24-7.38 (m, 5H), 8.37 (s, 1H). LCMS (ESI): m/z 411.2
[M þ H]þ.
(m, 1H), 5.49-5.53 (m, 1H), 7.24-7.38 (m, 5H), 8.35 (s, 1H).
LCMS (ESI): m/z 311.1 [M þ H]þ.
N-[(1R)-1-Phenylpropyl]-N-(5,6,7,8-tetrahydropyrido[40,30:4,5]-
thieno[2,3-d]pyrimidin-4-yl)amine (33d). Compound 33d was
synthesized in 82% yield from 32d in a manner similar to 33a.
1H NMR (400 MHz, CDCl3): δ 0.93 (t, 3H, J = 7.6 Hz), 1.89-
1.97 (m, 2H), 2.90-3.00 (m, 2H), 3.24 (t, 2H, 5.6 Hz), 4.04
(s, 2H), 5.30 (td, 1H, 7.2, 7.2 Hz), 5.46 (d, 1H, 7.2 Hz), 7.22-
7.33 (m, 5H), 8.32 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 10.6
(CH3), 27.2 (CH2), 29.6 (CH2), 42.9 (CH2), 45.0 (CH2), 55.6
(CH), 115.7 (C), 123.9 (C), 126.4 (CH), 127.2 (CH), 128.6
(CH), 132.2 (C), 142.4 (C), 153.3 (CH), 156.9 (C), 165.4 (C).
LCMS (ESI): m/z 325.0 [M þ H]þ.
(2S)-3-Phenyl-2-(5,6,7,8-tetrahydropyrido[40,30:4,5]thieno[2,3-d]-
pyrimidin-4-ylamino)propan-1-ol (33e). Compound 33e was synthe-
sized in 96% yield from 32e in a manner similar to 33a. 1H NMR
(400 MHz, CDCl3): δ 2.38-2.42 (m, 1H), 2.67-2.71 (m, 1H),
2.89-2.95 (m, 1H), 3.03-3.19 (m, 3H), 3.73 (dd, 1H, J = 5.6, 11.2
Hz), 3.76 (dd, 1H, J =2.8, 11.2 Hz), 4.05 (s, 2H), 4.44-4.47 (m,
1H), 5.37 (d, 1H, J=6.0 Hz), 7.22-7.31 (m, 5H), 8.32 (s, 1H).
LCMS (ESI): m/z 341.0 [M þ H]þ.
tert-Butyl 4-[(1R)-1-phenylpropyl]amino-5,6,7,8-tetrahydropyrido-
[40,30:4,5]thieno[2,3-d]pyrimidine-7-carboxylate (32d). Compound
32d was synthesized in 95% yield from 31c and R-(þ)-1-phenyl-
1
propylamine in a manner similar to 32a. H NMR (300 MHz,
CDCl3) δ 0.93 (t, 3H, J = 7.2 Hz), 1.42 (s, 9H), 1.87-2.00 (m, 2H),
2.98-3.00 (m, 2H), 3.77-3.78 (m, 2H), 4.62 (s, 2H), 5.29 (td, 1H,
J = 7.2, 7.2 Hz), 5.41 (d, 1H, J = 7.2 Hz), 7.22-7.33 (m, 5H), 8.33
(s, 1H). LCMS (ESI): m/z 425.1 [M þ H]þ.
2-(5,6,7,8-Tetrahydropyrido[40,30:4,5]thieno[2,3-d ]pyrimidin-4-
ylamino)-1-ethanol (33f). Compound 33f was synthesized in 49%
yield from 32f, in a manner similar to 33a. 1H NMR (400 MHz,
CDCl3): δ 2.96 (t, 2H, J = 5.6 Hz), 3.18 (t, 2H, J = 5.6 Hz), 3.65
(t, 2H, J = 5.6 Hz), 3.76 (t, 2H, J = 5.6 Hz), 4.00 (s, 2H), 8.18 (s,
1H). 13C NMR (100 MHz, CDCl3): δ 26.6 (CH2), 43.1 (CH2),
44.1 (CH2), 44.9 (CH2), 61.3 (CH2), 116.9 (C), 126.4 (C), 131.1
(C), 153.8 (CH), 158.6 (C), 165.6 (C). LCMS (ESI): m/z 251.0
[M þ H]þ.
tert-Butyl 4-[(1S)-1-Benzyl-2-hydroxyethyl]amino-5,6,7,8-tetra-
hydropyrido[40,30:4,5]thieno[2,3-d]pyrimidine-7-carboxylate (32e).
Compound 32e was synthesized in 87% yield from 31c and S-(-)-2-
amino-3-phenyl-1-propanol in a manner similar to 32a. 1H NMR
(300 MHz, CDCl3): δ 1.46 (s, 9H), 2.37-2.44 (m, 1H), 2.66-2.71
(m, 1H), 2.91 (dd, 1H, J = 8.4, 13.8 Hz), 3.06 (dd, 1H, J = 6.6, 13.8
Hz), 3.60 (m, 2H), 3.72 (dd, 1H, J = 5.4, 10.8 Hz), 3.84 (dd, 1H,
J = 2.4, 10.8 Hz), 4.43-4.48 (m, 1H), 4.56 (s, 2H), 5.37 (d, 1H, J =
5.7 Hz), 7.20-7.32 (m, 5H), 8.32 (s, 1H). 13C NMR (75 MHz,
CDCl3): δ 25.8 (CH2), 28.3 (CH3), 37.2 (CH2), 39.9 (CH2), 41.0
(CH2), 42.9 (CH2), 43.6 (CH2), 54.7 (CH), 65.1 (CH2), 80.6 (C),
115.5 (C), 124.5 (C), 127.0 (CH), 128.8 (CH), 129.3 (CH), 137.2
(C), 152.7 (CH), 154.2 (C), 157.0 (C), 165.8 (C). LCMS (ESI): m/z
441.1 [M þ H]þ.
1-(4-(((1S)-2-Hydroxy-1-phenylethyl)amino)-5,6,7,8-tetrahy-
dropyrido[40,30:4,5]thieno[2,3-d]pyrimidin-7-yl)-2-propen-1-one
(11). A mixture of acrylic acid (12 mg, 0.16 mmol) and EDCI (32
mg, 0.16 mmol) in anhydrous dicloromethane was stirred for 2 h
and 33a/10 (50 mg, 0.15 mmol) was then added. The resulting
mixture was stirred for 15 min, then partitioned between water
and ethyl acetate; the organic layer was dried over MgSO4 and
concentrated and the residue was purified by silica gel column
chromatography using a mixture of dichloromethane:methanol
tert-Butyl 4-[(2-Hydroxyethyl)amino]-5,6,7,8-tetrahydropyrido-
[40,30:4,5]thieno[2,3-d]pyrimidine-7-carboxylate (32f). Compound
32f was synthesized in 91% yield from 31c and ethanolamine in a
1
1
manner similar to 32a. H NMR (300 MHz, CDCl3): δ 1.46 (s,
(20:1) to give 11 (35 mg, 60%). H NMR (400 MHz, CDCl3)
9H), 2.97-3.00 (m, 2H), 3.71-3.77 (m, 4H), 3.84-3.88 (m, 2H),
4.62 (s, 2H), 5.81-5.83 (m, 1H), 8.33 (s, 1H). 13C NMR (75 MHz,
CDCl3): δ 25.9 (CH2), 28.4 (CH2), 39.9 (CH2), 41.0 (CH2), 43.5
(CH2), 61.3 (CH2), 80.6 (C), 115.3 (C), 124.5 (C), 129.3 (C), 152.6
(CH), 154.3 (C), 157.3 (C), 165.0 (C). LCMS (ESI): m/z 351.0
[M þ H]þ.
δ 3.06-3.12 (m, 2H), 3.94-3.99 (m, 4H), 4.76-4.83 (m, 2H),
5.39 (m, 1H), 5.75 (dd, 1H, J = 10.4, 0.8 Hz), 5.96-6.11 (m, 1H),
6.27-6.39 (m, 1H), 6.52-6.64 (m,1H), 7.25-7.36 (m, 5H), 8.26
(s, 1H). 13C NMR (100 MHz, CDCl3) δ 25.8 (CH2), 27.0 (CH2),
39.3 (CH2), 42.0 (CH2), 42.9 (CH2), 45.3 (CH2), 56.6 (CH), 66.8
(CH2), 115.4 (C), 123.6 (C), 125.4 (C), 126.4 (CH), 126.9 (CH),
127.3 (CH), 127.8 (CH), 128.5 (C), 128.9 (CH), 130.0 (C), 139.4
(C), 153.2 (CH), 156.8 (C), 165.7 (C). HRMS (EI) calcd for
C20H20N4O2S (Mþ) 380.1307, found 380.1310.
(2S)-2-Phenyl-2-(5,6,7,8-tetrahydropyrido[40,30:4,5]thieno[2,3-d ]-
pyrimidin-4-ylamino)ethan-1-ol (33a/10). To a mixture of 32a (0.6 g,
1.4 mmol) in dichloromethane (2 mL) at 0 ꢀC was added trifluo-
roacetic acid (TFA) (1 mL) and then warmed to room temperature.
The reaction mixture was stirred for 2 h, removed the solvent under
vacuum, and neutralized the residue by slow addition of sodium
bicarbonate solution. The precipitate formed was collected by
1-(4-[(1S)-2-Hydroxy-1-phenylethyl]amino-5,6,7,8-tetrahydro-
pyrido[40,30:4,5]thieno[2,3-d]pyrimidin-7-yl)-1-propanone (12).
Compound 12 was synthesized in 63% yield from 33a and
1
propionic acid in a manner similar to 11. H NMR (400 MHz,
1
filtration and washed with water to give 33a/10 (0.4 g, 90%). H
CD3OD): δ 1.12 (t, 1.2H, J = 7.6 Hz), 1.17 (t, 1.8 H, J = 7.6 Hz),
2.48 (q, 0.8 H, J = 7.6 Hz), 2.55 (q, 1.2H, J = 7.6 Hz), 3.14-3.26
(m, 2H), 3.87-3.98 (m, 4H), 4.77-4.79 (m, 2H), 5.38-5.40 (m,
1H), 7.20-7.23 (m, 1H), 7.24-7.33 (m, 2H), 7.38-7.42 (m, 2H),
8.15 (s, 1H). HRMS (EI): calcd for C20H21N4O2S (Mþ) 382.1463,
found 382.1466.
NMR (400 MHz, CDCl3) δ 2.84-3.04 (m, 2H), 3.20 (t, 2H, J =
4.4 Hz), 3.99 (d, 2H, J = 3.2 Hz), 4.01 (d, 2H, J = 7.6 Hz), 5.38
(d, 1H, J = 4.8 Hz), 6.04 (d, 1H, J = 6.4 Hz), 7.26-7.38 (m, 5H),
8.28 (s, 1H). LCMS (ESI): m/z 327.1 [M þ H]þ.
(2R)-2-Phenyl-2-(5,6,7,8-tetrahydropyrido[40,30:4,5]thieno[2,3-d]-
pyrimidin-4-ylamino)ethan-1-ol (33b). Compound 33b was synthe-
sized in 97% yield from 32b in a manner similar to 33a. 1H NMR
(400 MHz, CD3OD): δ 3.14-3.17 (m, 2H), 3.21-3.24 (m, 2H),
3.89-3.97 (m, 2H), 4.03-4.04 (m, 2H), 5.38 (dd, 1H, J = 5.6,
5.6 Hz), 7.21-7.25 (m, 1H), 7.29-7.33 (m, 2H), 7.38-7.41 (m, 2H),
8.15 (s, 1H). LCMS (ESI): m/z 327.0 [M þ H]þ.
1-(4-[(1S)-2-Hydroxy-1-phenylethyl]amino-5,6,7,8-tetrahydro-
pyrido[40,30:4,5]thieno[2,3-d]pyrimidin-7-yl)-2-butyn-1-one (13).
Compound 13 was synthesized in 68% yield from 33a and
2-butynoic acid in a manner similar to 11. 1H NMR (300 MHz,
CDCl3): δ 1.97-2.03 (m, 3H), 2.96-3.24 (m, 2H), 3.84-4.15 (m,
4H), 4.67-4.82 (m, 1H), 4.92 (s, 1H), 5.35-5.45 (m, 1H), 6.13-
N-[(1R)-1-Phenylethyl]-N-(5,6,7,8-tetrahydropyrido[40,30:4,5]-
thieno[2,3-d]pyrimidin-4-yl)amine (33c). Compound 33c was
synthesized in 90% yield from 32c in a manner similar to 33a.
1H NMR (400 MHz, CDCl3): δ 1.60 (d, 3H, J = 6.8 Hz), 2.89-
2.95 (m, 2H), 3.21-3.44 (m, 2H), 4.04 (brs, 2H), 5.26-5.41
6.16(m, 1H), 7.23-7.36 (m, 5H), 8.22 (s, 0.5H), 8.24 (s, 0.5H). 13
C
NMR (75 MHz, CDCl3): δ 4.1 (CH3), 25.7 (CH2), 26.7 (CH2),
29.6 (C), 38.4 (CH2), 41.0 (CH2), 43.7 (CH2), 46.2 (CH2), 56.5
(CH), 56.7 (CH), 66.4 (CH2), 66.5 (CH2), 72.4 (C), 72.5 (C), 90.1
(C), 91.3 (C), 115.3 (C), 115.4 (C), 124.0 (C), 124.9 (C), 126.4

7324 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Wu et al.
(CH), 126.5 (C), 127.7 (C), 127.8 (CH), 128.6 (CH), 128.8 (C),
128.8 (C), 129.0 (C), 139.3 (C), 139.4 (C), 152.8 (C), 153.2 (CH),
153.3 (CH), 156.7 (C), 156.9 (C), 164.9 (C), 165.1 (C). HRMS
(EI): calcd for C21H20N4O2S (Mþ) 392.1307, found 392.1311.
(2S)-2-Phenyl-2-[7-(vinylsulfonyl)-5,6,7,8-tetrahydropyrido[40,
30:4,5]thieno[2,3-d]pyrimidin-4-yl]aminoethan-1-ol (14). A mix-
ture of 33a (88 mg, 0.27 mmol), Et3N (0.2 mL, 5 equiv), and
2-chloroethanesulfonyl chloride (175 mg, 1.08 mmol) in dichloro-
methane (2 mL) was stirred at room temperature for 4 h. The
reaction mixture was concentrated, and the residue was parti-
tioned between water and dichloromethane; the organic layer
was washed with brine, dried over MgSO4, and concentrated
under vacuo. The crude product was purified by silica gel column
chromatography using a mixture of dichloromethane:methanol
for 2 h, and then the solvent was removed under vacuum.
The residue was reconstituted in THF (3 mL), N,N-dimethyl-
amine (0.5 mL, 28% aqueous solution) was added, and stirring
was continued for another 2 h. At the end of the reaction, the re-
sulting mixture was partitioned between water and ethyl ace-
tate; the organic layer was separated, dried over MgSO4, and
concentrated, and the residue was purified by silica gel column
chromatography using a mixture of dichloromethane:methanol
(5:1) to give 19 (88mg, 67%). 1H NMR (400 MHz, CDCl3): δ 2.24
(s, 6 H), 3.01-3.20 (m, 4H), 3.90-3.99 (m, 2H), 4.00-4.12 (m,
2H), 4.78 (s, 1H), 4.86 (s, 1H), 5.39 (s, 1H), 6.03 (d, 1H, J = 13.6
Hz), 6.38-6.57 (m, 1H), 6.80-6.98 (brs, 1H), 7.28-7.40 (m, 5H),
8.30 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 25.6 (CH2), 26.9
(CH2), 39.2 (CH2), 41.9 (CH2), 42.7 (CH2), 45.1 (CH2), 45.1
(CH3), 56.0 (CH), 60.2 (CH2), 65.8 (CH2), 115.2 (C), 121.8
(CH), 123.8 (C), 125.4 (C), 126.3 (CH), 127.3 (CH), 128.0 (C),
128.5 (CH), 129.5 (C), 139.8 (C), 142.8 (CH), 142.3 (CH), 153.1
(CH), 153.2 (CH), 156.6 (C), 156.7 (C), 165.1 (C), 165.4 (C).
HRMS (EI): calcd for C23H27N5O2S (Mþ) 437.1885, found
437.1869.
(E)-4-(Diethylamino)-1-(4-(((1R)-2-hydroxy-1-phenylethyl)-
amino)-5,6,7,8-tetrahydropyrido[40,30:4,5]thieno[2,3-d]pyrimidin-7-
yl)-2-buten-1-one (20). Compound 20 was synthesized in 59%
yield from 33a, 4-bromocrotonoic acid, and N,N-diethylamine in
a manner similar to 19. 1H NMR (300 MHz, CDCl3): δ 0.96-1.06
(m, 6H), 2.55-2.58 (m, 4H), 3.11-3.28 (m, 4H), 3.93-4.02 (m,
2H), 4.08-4.13 (m, 2H), 4.80-4.85 (m, 1H), 4.85-4.92 (m, 1H),
5.39-5.44 (m, 1H), 6.08 (s, 1H), 6.52 (dd, 1H, J = 14.7, 15.6 Hz),
6.90-6.97 (m, 1H), 7.27-7.41 (m, 5H), 8.32 (s, 1H). 13C NMR
(100 MHz, CDCl3): δ 11.6 (CH3), 25.7 (CH2), 27.0 (CH2), 39.2
(CH2), 42.0 (CH2), 42.8 (CH2), 45.1 (CH2), 47.0 (CH2), 54.1
(CH2), 56.3 (CH), 66.3(CH2), 115.3 (C), 121.4 (CH), 122.2 (CH2),
123.7 (CH2), 126.4 (CH), 127.6 (CH), 128.2 (CH2), 128.7 (CH),
129.8 (CH2), 139.7(C), 143.0(CH), 144.5 (CH), 153.2 (CH), 153.3
(CH), 156.7 (C), 165.5 (C), 165.6 (C). HRMS (EI): calcd for
C25H31N5O2S (Mþ) 465.2198, found 465.2195.
(E)-1-(4-[(1R)-2-Hydroxy-1-phenylethyl]amino-5,6,7,8-tetra-
hydropyrido[40,30:4,5]thieno[2,3-d]pyrimidin-7-yl)-4-morpholino-2-
buten-1-one (21). Compound 21 was synthesized in 61% yield
from 33a, 4-bromocrotonoic acid, and morpholine in a manner
similar to 19. 1H NMR (400 MHz, CDCl3): δ 2.42 (m, 4H), 3.10-
3.11 (m, 4H), 3.60-3.67 (m, 4H), 3.88-3.97 (m, 4H), 4.79 (m,
1H), 4.74 (m, 1H), 4.81 (m, 1H), 5.36-5.38 (m, 1H), 6.04-6.09
(m, 1H), 6.53 (t, 1H, J = 16.2 Hz), 6.84-6.88 (m, 1H), 7.22-7.33
(m, 5H), 8.23 (s, 1H). 13C NMR (75 MHz, CDCl3): δ 25.8 (CH2),
27.0 (CH2), 39.4 (CH2), 42.0 (CH2), 42.9 (CH2), 45.2 (CH2), 53.6
(CH2), 56.5 (CH), 59.9 (CH2), 66.6 (CH2), 66.7 (CH2), 115.3 (C),
121.9 (CH), 122.2 (CH), 123.6 (C), 125.5 (C), 126.4 (CH), 127.7
(CH), 128.3 (C), 128.8 (CH), 130.0 (C), 139.5 (C), 142.9 (CH),
153.2 (CH), 153.3 (CH), 156.7 (C), 165.2 (C), 165.8 (C). HRMS
(EI): calcd for C25H29N5O3S (Mþ) 479.1991, found 479.1991.
(E)-1-(4-[(1S)-2-Hydroxy-1-phenylethyl]amino-5,6,7,8-tetrahy-
dropyrido[40,30:4,5]thieno[2,3-d]pyrimidin-7-yl)-4-(4-methylpipera-
zino)-2-buten-1-one (22). Compound 22 was synthesized in 55%
yield from 33a, 4-bromocrotonoic acid, and N-methylpiperazine
in a manner similar to 19. 1H NMR (400 MHz, CDCl3): δ 2.28 (s,
3H), 2.60 (m, 8H), 3.13-3.16 (m, 4H), 3.89-4.03 (m, 4H), 4.76-
4.88 (m, 2H), 5.35-5.40 (m, 1H), 6.09 (d, 1H, J = 1.6 Hz), 6.40-
6.50 (m, 1H), 6.82-6.88 (m, 1H), 7.25-7.37 (m, 5H), 8.28 (s, 1H).
13C NMR (100 MHz, CDCl3): δ 25.8 (CH2), 27.2 (CH2), 39.4
(CH2), 42.1 (CH2), 43.0 (CH2), 45.3 (CH2), 45.8 (CH3), 52.8
(CH2), 53.1 (CH2), 54.8 (CH2), 56.4 (CH), 56.7 (CH), 59.5 (CH2),
66.6 (CH2), 115.4 (C), 122.1 (CH), 122.4 (CH), 123.6 (C), 125.5
(C), 126.4 (CH), 127.8 (CH), 128.9 (CH), 130.1 (C), 139.7 (C),
142.4 (CH), 143.4 (CH), 153.4 (CH), 156.9 (C), 165.5 (C), 166.0
(C). HRMS (EI): calcd for C26H32N6O2S (Mþ) 492.2307, found
492.2316.
1
(40:1), to give 14 (66 mg, 59%). H NMR (400 MHz, CDCl3):
δ 3.04-3.18 (m, 2H), 3.63 (t, 2H, J = 4.8 Hz), 3.96-4.23 (m,
2H), 4.47 (s, 2H), 5.41 (m, 1H), 5.99 (d, 1H, J = 6.4 Hz), 6.02 (d,
1H, J = 9.6 Hz), 6.32 (d, 1H, J = 16.4 Hz), 6.44 (dd, 1H, J =
9.6, 16.4 Hz), 7.28-7.39 (m, 5H), 8.32 (s, 1H). 13C NMR (75 MHz,
CDCl3): δ 26.2 (CH2), 42.5 (CH2), 44.6 (CH2), 56.9 (CH), 66.9
(CH2), 115.3 (C), 124.0 (C), 126.4 (CH), 128.0 (CH), 128.3 (C),
128.9 (CH), 129.0 (CH2), 133.2 (CH), 139.3 (C), 153.2 (CH),
156.9 (C), 165.7 (C). HRMS (EI): calcd for C19H20N4O3S2 (Mþ)
416.0977, found 416.0977. HPLC purity 92.63%.
1-(4-[(1R)-2-Hydroxy-1-phenylethyl]amino-5,6,7,8-tetrahydro-
pyrido[40,30:4,5]thieno [2,3-d]pyrimidin-7-yl)-2-propen-1-one (15).
Compound 15 was synthesized in 68% yield from 33b and acrylic
acid in a manner similar to 11. 1H NMR (400 MHz, CD3OD): δ
3.25-3.27 (m, 2H), 3.89 (dd, 2H, J = 6.0, 11.2 Hz), 3.94-4.04
(m, 2H), 4.90 (m, 2H), 5.39 (dd, 1H, J = 5.2, 5.2 Hz), 5.79-5.84
(m, 1H), 6.25-6.30 (m, 1H), 6.80-6.93 (m, 1H), 7.20-7.24 (m,
1H), 7.29-7.33 (m, 2H), 7.39-7.41 (m, 2H), 8.16 (s, 1H). LCMS
(ESI): m/z 381.1 [M þ H]þ. HPLC purity 93.46%.
1-(4-[(1R)-1-Phenylethyl]amino-5,6,7,8-tetrahydropyrido[40,30:
4,5]thieno[2,3-d]pyrimidin-7-yl)-2-propen-1-one (16). Compound
16 was synthesized in 74% yield from 33c and acrylic acid in
a manner similar to 11. 1H NMR (300 MHz, CD3OD): δ 1.61 (d,
3H, J = 7.2 Hz), 3.01-3.22 (m, 2H), 3.91-4.05 (m, 2H), 4.78-
5.01 (m, 2H), 5.48 (dd, 1H, J = 6.9, 13.8 Hz), 5.81 (d, 1H, J =
10.8 Hz), 6.26 (d, 1H, J = 16.5 Hz), 6.75-6.93 (m, 1H), 7.18-
7.42 (m, 5H), 8.20 (s, 1H). LCMS (ESI): m/z 365.1 [M þ H]þ.
1-(4-[(1R)-1-Phenylpropyl]amino-5,6,7,8-tetrahydropyrido[40,
30:4,5]thieno[2,3-d]pyrimidin-7-yl)-2-propen-1-one (17). Com-
pound 17 was synthesized in 60% yield from 33d and acrylic acid
in a manner similar to 11. 1H NMR (400 MHz, CDCl3): δ 0.86
(t, 3H, J = 7.6Hz), 1.81-2.01 (m, 2H), 2.97-3.11 (m, 2H), 3.81-
4.10 (m, 2H), 4.74-4.95 (m, 2H), 5.26-5.43 (m, 2H), 5.74-5.78
(m, 1H), 6.24-6.36 (m, 1H), 6.52-6.67 (m, 1H), 7.21-7.35 (m,
5H), 8.33 (s, 1H). LCMS (ESI): m/z 379.1 [M þ H]þ. HPLC
purity 92.06%.
(E)-1-(4-[(1S)-2-Hydroxy-1-phenylethyl]amino-5,6,7,8-tetrahy-
dropyrido[40,30:4,5]thieno[2,3-d]pyrimidin-7-yl)-3-phenyl-2-propen-
1-one (18). Compound 18 was synthesized in 73% yield from 33a
and trans-cinnamic acid in a manner similar to 11. 1H NMR (300
MHz, CDCl3): δ 3.08 (br, 2H), 3.92-4.04 (m, 4H), 4.75-4.92 (m,
2H), 5.41 (s, 1H), 6.16 (s, 1H), 6.91 (d, 1H, J = 15.6 Hz), 7.24-
7.27 (m, 8H), 7.47-7.52 (m, 2H), 7.67 (d, 1H, J = 15.6 Hz), 8.23
(s, 1H). 13C NMR (75 MHz, CDCl3): δ 25.8 (CH2), 27.1 (CH2),
39.4 (CH2), 42.2 (CH2), 42.9 (CH2), 45.2 (CH2), 56.7 (CH), 66.4
(CH2), 115.4 (C), 116.2 (CH), 116.6 (CH), 123.9 (C), 125.5 (C),
126.5 (CH), 127.8 (CH), 128.8 (CH), 128.8 (CH), 129.0 (C), 129.9
(CH), 134.7 (C), 139.4 (C), 143.8 (CH), 152.7 (CH), 156.8 (C),
165.0 (C), 165.8 (C). HRMS (EI): calcd for C26H24N4O2S (Mþ)
456.1620, found 456.1616. HPLC purity 92.65%.
(E)-4-(Dimethylamino)-1-(4-[(1S)-2-hydroxy-1-phenylethyl]-
amino-5,6,7,8-tetrahydropyrido[40,30:4,5]thieno[2,3-d]pyrimidin-
7-yl)-2-buten-1-one (19). A mixture of 4-bromocrotonoic acid
(49 mg, 0.33 mmol) and EDCI (63 mg, 0.33 mmol) in anhydrous
dichloromethane (3 mL) was stirred for 15 min, and 33a (0.100 g,
0.30 mmol) was then added. The resulting mixture was stirred
(E)-4-(Dimethylamino)-1-(4-[(1R)-2-hydroxy-1-phenylethyl]-
amino-5,6,7,8-tetrahydropyrido[40,30:4,5]thieno[2,3-d ]pyrimidin-7-
yl)-2-buten-1-one (23). Compound 23 was synthesized in 68%

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7325
yield from 33b, 4-bromocrotonoic acid, and N,N-dimethylamine
in a manner similar to 19. 1H NMR (400 MHz, CDCl3): δ 2.14 (s,
3H), 2.33 (s, 3H), 2.97-3.20 (m, 4H), 4.08-4.09 (m, 2H), 4.73-
4.96 (m, 4H), 5.36 (dd, 1H, J = 6.0, 10.4 Hz), 6.07 (d, 1H, J = 6.0
Hz), 6.48-6.72 (m, 1H), 6.81-6.86 (m, 1H), 7.23-7.39 (m, 5H),
8.28 (s, 1H). LCMS (ESI): m/z [M þ H]þ 438.1. HPLC purity
93.89%.
(E)-4-(Dimethylamino)-1-(4-[(1R)-1-phenylethyl]amino-5,6,7,
8-tetrahydropyrido[40,30:4,5]thieno[2,3-d]pyrimidin-7-yl)-2-buten-1-
one (24). Compound 24 was synthesized in 79% yield from 33c,
4-bromocrotonoic acid, and N,N-dimethylamine in a manner
similar to 19. 1H NMR (400 MHz, CDCl3): δ 1.62 (d, 3H, J =
6.4 Hz), 2.27 (m, 6H), 3.03-3.11 (m, 4H), 3.93-3.99 (m, 2H),
4.80-4.89 (m, 2H), 5.32-5.40 (m, 1H), 5.52-5.53 (m, 1H), 6.48
(m, 1H), 6.91 (d, 1H, J = 14.8 Hz), 7.27-7.38 (m, 5H), 8.39 (s,
1H). 13C NMR (100 MHz, CDCl3): δ 22.2 (CH3), 25.9 (CH2), 27.2
(CH2), 39.4 (CH2), 42.1 (CH2), 42.9 (CH2), 44.6 (CH2), 45.3
(CH3), 49.9 (CH), 60.1 (CH2), 115.0 (C), 122.0 (CH), 123.4 (C),
125.2 (CH), 126.0 (CH), 127.4 (CH), 128.2 (C), 128.8 (CH), 129.7
(C), 142.7 (C), 143.2 (C), 153.7 (CH), 156.5 (C), 165.5 (C), 165.9
(C). HRMS (EI): calcd for C23H27N5OS (Mþ) 421.1936, found
421.1941.
(E)-4-(Dimethylamino)-1-(4-[(1S)-1-phenylpropyl]amino-5,6,7,
8-tetrahydropyrido[40,30:4,5]thieno[2,3-d]pyrimidin-7-yl)-2-buten-
1-one (25). Compound 25 was synthesized in 67% yield from 33d,
4-bromocrotonoic acid, and N,N-dimethylamine in a manner
similar to 19. 1H NMR (400 MHz, CDCl3): δ 0.95 (t, 3H, J = 7.2
Hz), 1.85-2.04 (m, 2H), 2.17 (s, 3H), 2.37 (s, 3H), 3.13-3.17 (m,
4H), 3.84-4.03 (m, 2H), 4.80-4.89 (m, 2H), 5.30-5.46 (m, 2H),
6.45-6.60 (m, 1H), 6.89-6.94 (m, 1H), 7.25-7.36 (m, 5H), 8.37
(s, 1H). HRMS (EI): calcd for C24H29N5OS (Mþ) 435.2093,
found 435.2095.
(E)-1-(4-[(1R)-1-Benzyl-2-hydroxyethyl]amino-5,6,7,8-tetrahy-
dropyrido[40,30:4,5]thieno[2,3-d]pyrimidin-7-yl)-4-(dimethylamino)-
2-buten-1-one (26). Compound 26 was synthesized in 52% yield
from 33e, 4-bromocrotonoic acid, and N,N-dimethylamine in a
manner similar to 19. 1H NMR (400 MHz, CDCl3): δ 2.27 (s, 3H),
2.33 (s, 3H), 2.49 (m, 1H), 2.77 (m, 1H), 2.92-2.95 (m, 1H), 3.07-
3.15(m, 3H), 3.60-3.93 (m, 4H), 4.46-4.49 (m, 1H), 4.76 (m, 1H),
4.85 (m, 1H), 5.37 (d, 1H, J = 11.6 Hz), 6.47 (dd, 1H, J = 16.0,
24.0 Hz), 6.87-7.00 (m, 1H), 7.27-7.36 (m, 5H), 8.36 (s, 1H).
HRMS (EI): calcd for C24H29N5O2S (Mþ) 451.2042, found
451.2048.
1 μM ATP. Following incubation, 50 μL Kinase-Glo Plus re-
agent (Promega) was added and the mixture was incubated
at 25 ꢀC for 20 min. A 70 μL aliquot of each reaction mixture was
transferred to a black microtiter plate, and the luminescence
was measured on Wallac Vector 1420 multilabel counter
(PerkinElmer).
HCC827 Antiproliferation Assay. HCC827 cell viability was
examined by the MTS assay (Promega, Madison, WI). One
thousand HCC827 cells in 100 μL of RPMI1640 with 10% FBS
medium were seeded in each well of a 96-well plate. After 96 h
incubation with the test compound, the cells were further in-
cubated with 20 μL of a MTS/PMS mixture (MTS/PMS ratio:
20:1) in each well of the 96-well plate for 2 h at 37 ꢀC in a
humidified incubator with 5% CO2 to allow viable cells to con-
vert the tetrazolium salt (MTS) into formazan. The amount/
concentration of formazan, which indicates the number of
live cells, was determined by measuring its absorbance at
490 nm using a PerkinElmer Victor2 plate reader (PerkinElmer,
Shelton, CT).
Cell Extract Preparation and Western Blot Analyses. HCC827
cells were seeded into 12-well plates at a concentration of
2.5 ꢀ 105 cells per well. After 16 h of growth in serum-containing
media, cells were incubated in serum-free media for 24 h. Com-
pounds was then added for 1 h, at which time cells were stimu-
lated for 5 min with 100 ng/mL EGF. Whole-cell lysates were
prepared by suspending 2.5 ꢀ 105 cells in 100 μL lysis buffer
(50 mM Tris, pH 8.0, 150 mM NaCl, 1% (v/v) Triton X-100,
0.5% (v/v), sodium deoxycholate, 0.1% (w/v) SDS, 500 μM
PMSF, 1 mM DTT, and 1 mM Na3VO4). The cell lysates were
kept on ice and vigorously vortexed four times at 5 min intervals.
The lysate was cleared by centrifugation at 15000g for 10 min at
4 ꢀC. After adding SDS sample buffer, the supernatants were
heated at 95 ꢀC for 5 min and cell extract samples (25 μg) were
resolved by 8% SDS-polyacrylamide gel electrophoresis and
then transferred to a nitrocellulose membrane (Sartorius Stedim
Biotech). After transfer, the membrane was first incubated in
TBST buffer (20 mM Tris, pH 7.6, 135 mM NaCl, and 0.1%
(v/v) Tween 20) with 5% (w/v) skim milk powder, then incubated
with the indicated primary antibody (dilution of antibodies was
performed according to the manufacturer’s instructions), washed,
and blotted with horseradish-peroxidase-conjugated secondary
antibody. Primary antibodies included the following: p-EGFR-
(Y1068), EGFR (Cell Signaling Technology, Danvers, MA), and
R-tubulin (Sigma, Natick, MA). The membrane was then devel-
oped using Western Lightning plus-ECL reagent (PerkinElmer,
Shelton, CT) and exposed to X-ray film.
In Vivo Pharmacokinetics Evaluation of 19 in Rats. This study
was approved by Institutional Animal Care and Use Committee
of National Health Research Institutes. A solution of test com-
pound (10 mg/mL) was prepared by dissolving appropriate
amount compound in a mixture of PEG 400/dehydrated etha-
nol/Solutol (20:30:50, v/v/v) and was diluted with two parts of
physiological saline to make the dosing solution of 3.3 mg/mL
before dosing. Male Sprague-Dawley rats, weighing 250-350 g
each (8-10 weeks old), were obtained from BioLASCO, Ilan,
Taiwan. Compound 19 was administered to three male rats each
intravenously (5 mg/kg dose) by a bolus injection to the jugular
vein. The volume of dosing solution administered was adjusted
according to the body weight recorded before dose administra-
tion. At 0 (prior to dosing), 2, 5, 15, and 30 min and at 1, 2, 4, 6, 8,
and 24 h after dosing, a blood sample (∼150 μL) was collected
from each animal via the jugular-vein cannula and stored in ice
(0-4 ꢀC). Plasma was separated from the blood by centrifuga-
tion (14000g for 15 min at 4 ꢀC in a Beckman model AllegraTM
6R centrifuge) and stored in a freezer (-60 ꢀC). All samples were
analyzed for the test compound by LC-MS/MS (ABI3000).
Data were acquired via multiple reactions monitoring. Plasma
concentration data were analyzed with standard noncompart-
mental method with WinNonLin software program (version
1.1, Pharsight Corporation, CA).
(E)-4-(Dimethylamino)-1-4-[(2-hydroxyethyl)amino]-5,6,7,8-
tetrahydropyrido[40,30:4,5]thieno[2,3-d]pyrimidin-7-yl-2-buten-1-
one (27). Compound 27 was synthesized in 42% yield from 33f,
4-bromocrotonoic acid, and N,N-dimethylamine in a manner
similar to 19. 1H NMR (300 MHz, CD3OD): δ 2.27 (s, 3H), 2.29
(s, 3H), 3.14-3.18 (m, 4H), 3.67-3.78 (m, 4H), 4.00 (dd, 2H, 6.0,
6.0 Hz), 4.83 (m, 2H), 6.63-6.88 (m, 2H), 8.25 (s, 1H). 13C NMR
(100 MHz, CD3OD): δ 26.6 (CH2), 27.8 (CH2), 40.5 (CH2), 43.0
(CH2), 44.0 (CH2), 44.2 (CH2), 45.3 (CH3), 46.1 (CH2), 61.2
(CH2), 61.3 (CH2), 116.6 (C), 124.1 (CH), 124.5 (CH), 126.5 (C),
127.3 (C), 129.3 (C), 129.9 (C), 143.3 (CH), 154.0 (CH), 158.7 (C),
166.0 (C), 167.3 (C). LCMS-ESI (m/z) 362.1 [M þ H]þ. HPLC
purity 93.35%.
EGFR WT and DM Kinase Inhibition Assay. GST-EGFR-KDWT
containing the EGFR kinase catalytic domain (residues 696-
1022) and GST-EGFR-KDL858RT/790M containing the EGFR
kinase catalytic domain (residues from 696 to 1022 and with
L858R/T790M) were expressed in Sf9 insect cells transfected
the baculovirus containing pBac-PAK8-GST-EGFR-KD plas-
mid, respectively. GST-EGFR-KDWT protein expression and
purification and kinase assay were done in a manner as reported
earlier.5 The EGFRL858R/T790M Kinase-Glo assays were car-
ried out in 96-well plates at 37 ꢀC for 60 min in a final volume
of 50 μL including the following components: 200 ng GST-
EGFR-KDL858R/T790M proteins, 25 mM HEPES, pH 7.4, 4 mM
MnCl2, 2 mM DTT, 10 mM MgCl2, 0.1 mg/mL bovine serum
albumin, 10 μM poly(Glu,Tyr) 4:1, 0.5 mM Na3VO4, and

7326 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Wu et al.
In Vivo HCC827 Xenograft Evaluation of 19 in Mice. Adult
male nude mice (Nu-Fox1nu) were purchased from BioLasco,
Taiwan Co., Ltd. Animals had access to food and water ad
libitum. Experimental procedures using animals were approved
by the Institutional Animal Care and Use Committees of The
National Health Research Institutes. Human HCC827 cells
were cultured in RPMI-1640 supplemented with 10% heat-
inactived bovine serum (FBS) and incubated at 37 ꢀC in
humidified atmosphere consisting 5% CO2. Eight-week-old
nude mice were inoculated with HCC827 cells subcutaneously
at 5 ꢀ 106 cells per mouse mixed with equal volume of Matrigel
(Becton Bickinson) in 0.1 mL via a 24-gauge needle. Tumor
volume was measured by using an electronic caliper and calcu-
lated with the formula length ꢀ width2 ꢀ 0.5, twice a week.
When the size of a growing tumor ∼200 mm3, the HCC827
tumor bearing mice were administered compound 19 (dissolved
in 10% DMSO þ 20% Cremophor EL þ70% normal saline)
IV via the tail veins for 5 days per week for 2 consecutive weeks
at 15 and 5 mg/kg dose (n = 7). Gefitinib was administered
(dissolved in 1% Tween 80 in water) as reference compound
periorally at 100 mg/kg dose (n = 8). Also a control group
received vehicle alone (10% DMSO þ 20% Cremophor EL
þ70% normal saline) as IV injection (n = 7). Tumor size and
animal body weight were measured twice a week after drug
treatments. At the end of the experiments, animals were eu-
thanized with carbon dioxide, followed by cervical dislocation.
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(12) Tsou, H. R.; Overbeek-Klumpers, E. G.; Hallett, W. A.; Reich,
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Acknowledgment. The study was financially supported by
National Health Research Institutes, National Science Coun-
cil (grant no, NSC-95-2113-M-400-001-MY3, for H.-P.H.)
and Department of Health (grant nos. DOH98-TD-G-111-
019, for Y.-S.C. and DOH98-TD-G-111-020, for H.-P.H.),
Taiwan, ROC.
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