Discovery of novel thieno[2,3-d]pyrimidin-4-yl hydrazone-based inhibitors of Cyclin D1-CDK4: Synthesis, biological evaluation and structure-activity relationships. Part 2

Article abstract of DOI:10.1016/j.bmc.2009.10.039

The design, synthesis and evaluation of novel thieno[2,3-d]pyrimidin-4-yl hydrazone analogues as cyclin-dependent kinase 4 (CDK4) inhibitor are described. Focusing on the optimization of the heteroaryl moiety at the hydrazone with substituted phenyl group

Full text of DOI:10.1016/j.bmc.2009.10.039

Bioorganic & Medicinal Chemistry 17 (2009) 7850–7860
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel thieno[2,3-d]pyrimidin-4-yl hydrazone-based inhibitors
of Cyclin D1-CDK4: Synthesis, biological evaluation and structure–activity
relationships. Part 2
b
b
c
a
Takao Horiuchi a, , Motoko Nagata , Mayumi Kitagawa , Kouichi Akahane , Kouichi Uoto
*
a Medicinal Chemistry Research Laboratories II, Daiichi Sankyo Co. Ltd, 16-13, Kita-Kasai 1-Chome, Edogawa-ku, Tokyo 134-8630, Japan
b Biological Research Laboratories IV, Daiichi Sankyo Co. Ltd, 16-13, Kita-Kasai 1-Chome, Edogawa-ku, Tokyo 134-8630, Japan
c R&D Division, Daiichi Sankyo Co. Ltd, 2-58, Hiromachi 1-Chome, Shinagawa-ku, Tokyo 140-8710, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, synthesis and evaluation of novel thieno[2,3-d]pyrimidin-4-yl hydrazone analogues as cyclin-
dependent kinase 4 (CDK4) inhibitor are described. Focusing on the optimization of the heteroaryl moiety
at the hydrazone with substituted phenyl groups, 4-[(methylamino)methyl]benzaldehyde (22) and 5-iso-
indolinecarbaldehyde (24) (6-tert-butylthieno[2,3-d]pyrimidin-4-yl)hydrazone derivatives have been
identified. In this paper, the potency, selectivity profile and structure–activity relationships of our syn-
thetic compounds are discussed.
Received 20 August 2009
Revised 20 October 2009
Accepted 21 October 2009
Available online 24 October 2009
Keywords:
Cyclin D1/CDK4 inhibitor
Thieno[2,3-d]pyrimidin-4-yl hydrazone
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Recently, several research groups have identified CDK inhibi-
tors.16–22 Above all, Flavopiridol,19 UCN-01,20 and R-roscovitine21
Cyclin-dependent kinases (CDKs) are a family of serine/threo-
nine kinases which play a key role in the growth, development,
proliferation, and death of eukaryotic cells.1–3 CDKs are responsible
for coordinating the events by which cells progress through the cell
cycle and they become active at specific phases: G1, S, G2 and M.4–
have been developed and have advanced to phase II/III clinical tri-
als as multi-CDK inhibitors. As well, PD033299122 is known to be a
selective CDK4 inhibitor under active development.
In our previous paper, to improve the selectivity for CDK4 of
HTS hit compound (1), we synthesized some thieno[2,3-d]pyrimi-
din-4-yl hydrazone analogues which modified both the C-2 and
6
In the G1 phase, the cyclin D1 increases to trigger the activation
of CDK4/6 in early G1 and then cyclin E/CDK2 activates. Notably,
CDK4 restricts the passage only through the G1 phase, whereas
CDK2 controls the passage through not only the G1 but also the
S phase with cyclin A.7–9 CDK4/6 activities are negatively regulated
by the tumor suppressor p16, a cyclin-dependent kinase inhibitor
of the INK4 family. But many tumors have been reported to contain
mutations, deletions or silencing of the p16 or the retinoblastoma
tumor suppressor protein (Rb) gene.10–12 Moreover, mutations in
CDKs and abnormal expressions of their regulators have been
found in a large percentage of melanoma patients.13 In addition,
Malumbres et al. have reported that knockdown of CDK4 in mam-
mary tumor cells prevents tumor formation.14 In contrast, McCor-
mick and Tetsu have found that cancer cells proliferate despite
CDK2 inhibition.15 These results suggest that selective inhibition
of CDK4 may restore normal cell activity and could be a more valu-
able approach to cancer therapy than CDK2, especially for those
who have lost the INK4 family, such as p16.
C-4 positions. As
(IC50 = 0.056 g/mL) inhibitor (2) (Fig. 1), which had good selectiv-
ity for CDK2 (IC50 = 1.40 g/mL) with moderate in vitro cytotoxic
activity and limited aqueous solubility (44 g/mL in pH 6.8 buffer
a result, we discovered the potent CDK4
l
l
l
solution).23 To further explore the potential of the thieno[2,3-
d]pyrimidine compounds, we introduced various substituted phe-
nyl rings at the end of hydrazone. Herein, we report the synthesis
and biological activity of several thieno[2,3-d]pyrimidin-4-yl
hydrazone derivatives. In addition, we also describe a docking
study of compound 2 with our homology model of CDK4.
2. Results and discussion
2.1. Molecular modeling
It has already been reported that many small molecule CDKs
inhibitors are competitive with ATP for binding.16 With this back-
ground, our early docking study with HTS hit compound (1) sup-
ported the hypothesis that the thieno[2,3-d]pyrimidin-4-yl
hydrazone analogues are also competitive with ATP and probably
* Corresponding author. Tel.: +81 3 3680 0151; fax: +81 3 5696 8772.
E-mail address: horiuchi.takao.pi@daiichisankyo.co.jp (T. Horiuchi).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.039

T. Horiuchi et al. / Bioorg. Med. Chem. 17 (2009) 7850–7860
7851
tive conformations and the space of their ATP binding sites ap-
peared to be smaller than that of our CDK4 homology model to
insert molecule (2).
N
S
N
N
N
N
N
HN
HN
N
2.2. Synthesis
Preparation of all compounds 9–29 was accomplished using a
general synthetic route as shown in Scheme 1. The thiophene
intermediate (4) was synthesized from 3,3-dimethylbutanal (3)
with methyl cyanoacetate in the presence of sulfur by the method
of Tinney et al.27 Cyclization of 4 with formamide gave 6-tert-bu-
tyl-4-oxothieno[2,3-d]pyrimidine (5). The hydrazine (7) was pre-
pared from chlorination of the carbonyl group at the C-4 position
of 5 with phosphorus oxychloride, followed by treatment with
hydrazine monohydrate according to a reported procedure.28 Fi-
nally, the hydrazone analogues (9–29) were produced by a conden-
sation reaction with 7 and appropriate aldehydes (8a–p) in
benzene, followed by deprotection in case of the compounds (14,
20, 22, 24, 28 and 29).
S
S
N
1
2
Figure 1. Lead compound
compound 1.
2
from in-house high-throughput screening hit
bind in the enzyme’s ATP binding pocket. We first carried out an in
silico docking study of 2 with the ATP binding pocket of our CDK4
homology model because no X-ray crystallographic information
was available regarding the structure of CDK4 with active confor-
mation. We have made these models using the known crystal
structures of CDK224 and CDK625 as templates.
The analysis with our model suggested several features. The
molecule (2) was buried toward the gatekeeper residue Phe93 in
the ATP binding pocket. At the hinge region, NH of hydrazone
and the nitrogen atom in the pyrimidine ring formed two backbone
hydrogen bonds with Val 96 and CH–O interaction of the pyrimi-
dine ring formed with the backbone carbonyl of Glu94. The other
key hydrogen bond was formed between the ionizable nitrogen
atom of the 6-(dimethylamino)methyl group and the side chain
carbonyl of Asp99 at the solvent accessible region (Fig. 2). This
interaction could explain the increase in potency of 2 relative to
our reported pyridine analogues,23 which lacked the substituents
capable of forming this hydrogen bond. Moreover, it appeared that
the nitrogen atom in the pyridine ring rarely contributes to in-
crease CDK4 inhibitory activity in this case. To verify this assump-
tion, we introduced various substituted phenyl rings at the end of
hydrazone.
The aldehydes (8e, f) were prepared as shown in Scheme 2. Pro-
tection of the hydroxyl group of compound 31 with tert-butyldi-
phenylsilyl chloride followed by reduction of methyl ester using
lithium aluminum hydride (LAH) and oxidation of the resulting
alcohol with MnO2 afforded 8e. Compound 33 was converted to
sulfonamide (34) with dimethylamine. Conversion of 34 to the
aldehyde (8f) was carried out by the procedure utilized for the
preparation of 8e from compound 32.
The aldehydes (8g–i, k) were synthesized as shown in Scheme
3. Amination of bromide (35, 37) with dimethylamine produced
the methyl esters (36, 38) followed by the procedure described
for compound 8f gave 8g and 8h, respectively. Similarly, the alde-
hyde (8i) was obtained from compound 37 with N-methylethanol-
amine. Reductive amination of compound 39 with methylamine
and NaBH4 followed by standard Boc protection afforded the
methyl ester (40). Finally, compound 40 was converted to 8k in a
similar manner.
Recently, two research groups26 have determined the crystal
structure of CDK4/cyclin D. However, their structures are non-ac-
O
N
CO2Me
NH2
O
a
b
c
NH
H
S
S
3
4
5
R1
NH2
N
N
N
Cl
N
HN
N
HN
N
d
e, f
N
S
S
S
9-29
6
7
Scheme 1. Reagents and conditions: (a) methyl cyanoacetate, S8, Et3N, DMF, 96%;
(b) HCONH2, 210 °C, 88%; (c) POCl3, 110 °C, 65%; (d) NH2NH2ÁH2O, EtOH, reflux, 55%;
(e) aldehydes 8a–p, benzene, reflux, 15–100%; (f) deprotection in case of 14, 20, 22,
24, 28 and 29, 81–97%.
OR2
OTBDPS
b, c
MeOOC
OHC
N
31: R2 = H
8e
a
32: R2 = TBDPS
O
S
O
S
O
O
S
O
O
d
b, c
Cl
N
HOOC
HOOC
OHC
Figure 2. Predicted binding mode of compound 2 in the ATP binding site of our
CDK4 homology model. The inhibitor is color-coded by atom type, where carbon is
yellow, oxygen is red, sulfur is orange, and nitrogen is blue. The enzyme is color-
coded by atom type in a similar fashion except that carbon is gray. Key enzyme
residues are labeled and hydrogen bonds are shown as cyan dotted lines.
33
34
8f
Scheme 2. Synthesis of aldehydes 8e and 8f. Reagents and conditions: (a) TBDPSCl,
imidazole, THF; (b) LAH, THF, 0 °C; (c) MnO2, CH2Cl2, reflux, three steps 81% (8e
from 31), two steps 58% (8f from 34); (d) Me2NH, THF, 57%.

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T. Horiuchi et al. / Bioorg. Med. Chem. 17 (2009) 7850–7860
a
b, c
a, b
c
Br
Br
N
N
N
N
N
N
N
MeOOC
MeOOC
OHC
OHC
HO
HO
Boc
44
46
45
36
8g
8h
35
37
d
a
b, c
N
EtO2C
Boc
N
TfO
Boc
OH
MeOOC
MeOOC
OHC
47
38
8i
OH
e
f
d, b, c
CHO
N
N
EtO2C
Boc
Boc
OHC
OH
8o
48
Boc
Boc
e, f, g
MeOOC
b, c
N
N
Boc
NH
b
N
c
MeOOC
OHC
39
40
8k
HO
HO
49
51
50
Scheme 3. Synthesis of aldehydes 8g–i and 8k. Reagents and conditions: (a)
Me2NH, THF, 98% (35), 78% (38); (b) LAH, THF, 0 °C; (c) MnO2, CH2Cl2 or CCl4, reflux,
two steps 68% (8g), 86% (8h), 75% (8i), 58% (8k); (d) N-methylethanolamine, Et3N,
toluene, 70 °C, 74%; (e) MeNH2, toluene; (f) NaBH4, MeOH; (g) Boc2O, DMAP, CH2Cl2,
three steps 18%.
Boc
Boc
N
N
d
TfO
EtOOC
52
Boc
g
N
The isoindolinecarbaldehydes (8l–n) were prepared as shown
in Scheme 4. Bicyclic compound (8l) was synthesized using the re-
ported method of Middleton et al.29 Protection of dipropargyl-
amine (41), and successive reaction with propargyl alcohol and
Wilkinson’s catalyst gave the alcohol (43). Compound 43 was trea-
ted with MnO2 to afford the aldehyde (8l). Deprotection of the Boc
group of 43 with TFA followed by conversion to aldehydes (8m, n)
in two steps was performed according to the above method.
Dihydroisoquinoline intermediates (8o, p) were prepared as
shown in Scheme 5. Compounds 8o and 8p were synthesized fol-
lowing a reported procedure.29,30 Hydrogenation of isoquinolin-
7-ol (44) using Pt2O, followed by protection with (Boc)2O gave
compound 45, which was converted to the O-triflate (46) with
OHC
8p
Scheme 5. Synthesis of aldehydes 8o and 8p. Reagents and conditions: (a) PtO2,
AcOH, H2 (40 psi); (b) (Boc)2O, Et3N, THF, H2O, two steps 82% (45), 91% (50); (c)
Tf2NPh, Et3N, CH2Cl2, quant. (46), quant. (51); (d) ethyl acrylate, Pd(OAc)2, tri(o-
tolyl)phosphine, CH3CN, 61% (47), 18% (52); (e) OsO4, NMO, THF/acetone/H2O
(1:1:1), 76%; (f) NaIO4, THF/MeOH/H2O (1:1:1), 91%; (g) OsO4, NaIO4, THF, H2O, 46%.
is, in vitro inhibitory activity (CDK4 and CDK2) and cytotoxicity
against two cell lines (HCT116 human colon carcinoma and PC-6
human non-small cell lung carcinoma). The results are presented
in Table 1.
N-phenyl-bis(trifluoromethanesulfonimide). The
a,b-unsaturated
Compound 9, bearing non-substituted phenyl group at the C-4
ester (47) was derived from 46 with ethyl acrylate and Pd(OAc)2.
Oxidation of 47 with OsO4 followed by cleavage of the resulting
diol with NaIO4 provided the aldehyde (8o). The aldehyde (8p)
was synthesized from compound 49 in a similar manner.
position (R1), showed moderate CDK4 inhibitory activity (CDK2:
>20 lg/mL). 4-Carboxyphenyl (11) and 4-(hydroxymethyl)phenyl
(14) derivatives retained the same CDK4 activity as 9, but neither
compound had selectivity against CDK2. On the other hand, 4-
(methoxycarbonyl)phenyl (10) and 4-hydroxyphenyl (12) ana-
logues were inactive. Surprisingly the sulfonamide analogue (15)
2.3. Biological evaluation
exhibited superior activity to 2 against CDK2 (CDK4: >20 lg/mL).
To investigate our hypothesis that the nitrogen atom in the pyr-
idine ring at the C-4 hydrazone part rarely contributes to increase
CDK4 inhibitory activity, we tried to replace pyridine in the phenyl
ring. Biological activities of the novel thieno[2,3-d]pyrimidin-4-yl
hydrazone analogues were evaluated in two assay systems, that
Introduction of the dimethylamino group (16) at the 3-position
on the phenyl ring improved its CDK4 inhibitory activity with good
selectivity (42.3-fold vs CDK2) as predicted by our docking study
model. Moreover, we found that the para-substituted compounds,
as exemplified by compounds 17, 18, 20 and 22, showed potent
CDK4 inhibitory activity. In addition, analogue (22) showed excel-
lent activity to 2 against HCT116 and PC-6.
The biological activity data for thieno-pyrimidine analogues,
bearing a bicyclic ring at the hydrazone moiety, are shown in Table
1, We then introduced dihydroindoline (24–26) or tetrahydroiso-
quinoline (28, 29) rings. As a result, all the derivatives maintained
relatively potent CDK4 inhibitory activity with selectivity against
CDK2 (5.3–37.5-fold). Among these analogues, the compounds
24, 25, 28 and 29 had fairly good cytotoxicity against HCT116
N
a
b
N
H
O
O
41
42
c
N
N
Boc
N
Boc
HO
OHC
OHC
43
8l
and PC-6 (IC50 range: 0.046–0.111 lg/mL). These results support
d
e, c
our hypothesis and it is apparent that the modification of the
hydrazone moiety with a substituted phenyl ring increases CDK4
inhibitory activity and cytotoxicity.
For further characterization of our compounds, the cell cycle
distribution analysis in HCT116 cells was carried out with the se-
lected compounds 22, 24 and 29. Cells were collected after 16 h
treatment of the tested compounds and the DNA content of the
cells was assessed by flow cytometry analysis. After treating the
NH
R3
43
OHC
8m: R3 = Me
8n : R3 = i-Pr
Scheme 4. Synthesis of aldehydes 8l–n. Reagents and conditions: (a) Boc2O, Et3N,
CH2Cl2, 18%; (b) propargyl alcohol, Wilkinson’s catalyst, EtOH, two steps 44%; (c)
MnO2, CCl4, reflux, 66% (8l), 92% (8m), 33% (8n); (d) TFA, CH2Cl2; (e) HCHO or
acetone, NaBH(OAc)3, CH2Cl2, two steps 84% (R3 = Me), 38% (R3 = i-Pr).

T. Horiuchi et al. / Bioorg. Med. Chem. 17 (2009) 7850–7860
7853
Table 1
Enzyme inhibitory and cytotoxic activity for substituted benzaldehyde (6-tert-butylthieno[2,3-d]pyrimidine-4-yl)hydrazones
R1
N
HN
N
S
N
R1
CDK4 IC50
g/mL)
CDK2 IC50
g/mL)
CDK4
HCT-116 IC50
g/mL)
PC-6 IC50
(lg/mL)
G1 arrestd (%)
a
a
c
c
Compd
(
l
(
l
selectivityb
(
l
223
9
6-[(Dimethylamino)methyl]-2-pyridinyl
Phenyl
4-(Methoxycarbonyl)phenyl
4-Carboxypheny
0.056
0.61
>20
0.98
>20
1.40
>20
>20
25.0
>33
0.419
NTe
0.381
6.540
NTe
7.220
>10
0.556
2.120
0.598
0.107
0.124
0.214
0.044
0.071
0.050
0.134
0.069
0.049
10
11
12
14
15
16
17
18
20
22
24
25
26
28
29
NTe
0.39
11.0
0.26
1.22
1.10
0.60
0.29
1.60
0.68
0.52
1.00
3.00
0.18
0.64
0.4
0.7
9.990
0.844
0.665
1.420
0.405
0.128
0.169
0.334
0.056
0.111
0.088
0.206
0.107
0.046
4-Hydroxyphenyl
4-(Hydroxymethyl)phenyl
4-(N,N-Dimethylsulfonamide)phenyl
3-[(Dimethylamino)methyl]phenyl
4-[(Dimethylamino)methyl]phenyl
4-{[(2-Hydroxyethyl)(methyl)amino]methyl}phenyl
4-(Aminomethyl)phenyl
4-[(Methylamino)methyl]phenyl
Isoindolin-5-yl
2-Methylisoindolin-5-yl
0.40
>20
0.026
0.077
0.028
0.021
0.038
0.083
0.096
0.080
0.034
0.038
42.3
7.8
10.4
76.2
17.9
6.3
10.4
37.5
5.3
57 (60)
62 (100)
2-Isopropylisoindolin-5-yl
1,2,3,4-Tetrahydro-isoquinolin-7-yl
1,2,3,4-Tetrahydro-isoquinolin-6-yl
16.8
56 (50)
a
Concentration (lg/mL) needed to inhibit the Rb phosphorylation by 50%, as determined from the dose–response curve. Values are the means of at least two
determinations.
b
The values are calculated by using the following equation (CDK2 IC50)/(CDK4 IC50).
c
Dose–response curves were determined at ten concentrations. The IC50 values are the concentrations needed to inhibit cell growth by 50%, as determined from these
curves.
d
Population of G1 phase in HCT116 cells after 16 h treatment with compounds (DMSO as control: 30–34%). Numbers in parentheses indicate the concentrations of
compounds (ng/mL).
e
NT = not tested.
cells with 24, a significant increase in the G0/G1 population (62%)
was observed and decreases in the S and G2/M populations at a
concentration of 100 ng/mL. Compounds 22 and 29 also caused in-
creases in the G1 population at the concentrations of 60 and 56 ng/
mL, respectively (Table 1). In addition, the aqueous solubility of
22, 24 and 29 have been identified as potent, selective CDK4
inhibitors. These compounds have sufficient cytotoxic activities
against HCT116 human colon carcinoma cell line with the ability
to prevent cell progression. Moreover, compounds 22 and 24
showed sufficient efficacy in a xenograft model of the HCT116
compound 22 was markedly improved (783
l
g/mL), whereas those
cells. The aqueous solubility (783 lg/mL) of compound 22 also
of bicyclic ring compounds 24 and 29 were very low (2.8 and
revealed significant improvement from that of compound 2.
These results provide valuable information for the design of a po-
tent and water-soluble CDK4 inhibitor. Further work to improve
selectivity and physical profile of CDK4 will be reported in due
course.
8.5
(44
l
l
g/mL, respectively), in comparison to the lead compound 2
g/mL).
To evaluate the antitumor effects in vivo, HCT116 cells were
subcutaneously transplanted into nude mice and the synthetic
compounds 22, 24 and 29 were administered intravenously (iv)
and/or orally (po) (Table 2). Compound 18 exhibited a tumor
growth inhibition of 54% (iv, 300 mg/kg) and 57% (po, 350 mg/
kg), respectively. Similarly, a 61% reduction was observed after iv
administration of 24. In contrast, tetrahydroisoquinoline analogue
(29) had no effect against HCT116.
4. Experimental
4.1. CDK4 Homology modeling
Our homology model of CDK4 was developed using the homol-
ogy module of InsightII (Accelrys Software Inc.) with CDK2 (PDB
code 1jst24; containing ATP-Mg2+ and sharing moderate sequence
identity with CDK4) and CDK6 (1blx25; sharing 70% sequence iden-
tity with CDK4) as templates. Multiple sequence alignment was
produced using CLUSTALW (1.4) with P11802 of CDK4, 1jst, and
3. Conclusions
In summary, focusing on the optimization of the heteroaryl
moiety at hydrazone with substituted phenyl groups, compounds
Table 2
Antitumor effects of 22, 24, and 29 against HCT116 solid tumors
T/D/Nc
a
b
Compd
Route
Dose/day (mg/kg)
Total dose (mg/kg)
Administration schedule
IRTVmax (%) (day)
BWLmax (%) (day)
22
22
24
29
iv
po
iv
iv
300
350
260
300
1200
1400
1040
1200
qdx4
qdx4
qdx4
qdx4
54 (9)
57 (6)
61 (5)
5 (7)
9.7 (7)
16.0 (5)
<0
0/1/5
0/0/5
0/0/5
0/0/5
2.2 (1)
a
b
c
Maximum inhibition rate of tumor volume. Numbers in parentheses indicate the day after initial drug administration on which IRTVmax was reached.
Maximum rate of body weight loss. Numbers in parentheses indicate the day of BWLmax
Number of mice which died of toxicity/number of mice which died of tumor/number of mice used.
.

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T. Horiuchi et al. / Bioorg. Med. Chem. 17 (2009) 7850–7860
1blx. Docking studies were performed to generate solutions for 1
using Glide (Schrödinger, Inc.).
4.2.1.3. 6-tert-Butyl-4-chlorothieno[2,3-d]pyrimidine (6). Com-
pound 5 (7.06 g, 33.9 mmol) in phosphorous oxychloride (140 mL)
was stirred under reflux for 3.5 h. The reaction mixture was con-
centrated under reduced pressure. To the residue satd NaHCO3
aq was added and extracted with EtOAc. The aqueous layer was ex-
tracted with EtOAc and the combined organic layer was washed
with brine, and dried over Na2SO4. The solvent was removed under
reduced pressure to afford title compound 6 (5.03 g, 65%) as a
brownish oil. 1H NMR (CDCl3) d: 1.49 (9H, s), 7.11 (1H, s), 8.77
(1H, s); IR (KBr) cmÀ1: 2963, 1491, 1413, 1363, 1137, 839; MS
(EI) m/z: 226 (M+); HRMS (ESI) m/z: 227.04013 (calcd for
C10H12ClN2S: 227.04097); Anal. Calcd for C10H11ClN2SÁ0.25H2O: C,
51.94; H, 5.01; N, 12.11; Cl, 15.33; S, 13.87. Found: C, 51.81; H,
5.14; N, 11.95; Cl, 15.81; S, 7.57.
4.2. Chemistry
4.2.1. General
Unless otherwise noted, materials were obtained from commer-
cial suppliers and used without further purification. The solvent
and reagent names are abbreviated as follows: acetic acid (AcOH),
ethyl acetate (EtOAc), di-tert-butyl dicarbonate (Boc)2O, N,N-
dimethylformamide (DMF), diethyl ether (Et2O), lithium aluminum
hydride (LAH), N-methylmorpholine-N-oxide (NMO), palladium
acetate (Pd(OAc)2), sodium triacetoxyborohydride (NaBH(OAc)3),
tetrabutylammonium fluoride (TBAF), tert-butyldiphenylsilyl chlo-
ride (TBDPSCl), triethylamine (Et3N), trifluoroacetic acid (TFA), tet-
rahydrofuran (THF). Column chromatography was performed with
4.2.1.4. 6-tert-Butyl-4-hydrazinothieno[2,3-d]pyrimidine (7).
To a solution of 6 (5.03 g, 22.2 mmol) in EtOH (100 mL) was
added hydrazine monohydrate (50 mL). The mixture was stirred
under reflux for 5.5 h and cooled to room temperature. Water
was added to the reaction mixture and extracted with EtOAc. The
aqueous layer was extracted with EtOAc and the combined organic
layer was washed with brine, and dried over Na2SO4. The solvent
was removed under reduced pressure and the residue was recrys-
tallized from Et2O and n-hexane to afford title compound 7 (2.73 g,
55%) as a pale yellow solid. 1H NMR (DMSO-d6) d: 1.37 (9H, s), 4.48
(2H, br), 7.37 (1H, s), 1.07 (1H, s), 8.78 (1H, s); IR (KBr) cmÀ1: 3233,
2948,1576, 1440, 1345, 1305, 872; MS (EI) m/z: 222 (M+); HRMS
(ESI) m/z: 223.10077 (calcd for C10H15N4S: 223.10174); Anal. Calcd
for C10H14N4SÁ0.1H2O: C, 53.59; H, 6.39; N, 25.00; S, 14.31. Found:
C, 53.59; H, 6.29; N, 25.24; S, 14.30.
a
Merck Silica Gel 60 (particle size 0.060–0.200 or 0.040–
0.063 mm). Flash column chromatography was performed with
Biotage FLASH Si packed columns. Thin-layer chromatography
(TLC) was performed on Merck pre-coated TLC glass sheets with
Silica Gel 60 F254, and compound visualization was effected with
a 5% solution of phosphomolybdic acid in ethanol, UV lamp, iodine,
or Wako ninhydrin spray. 1H NMR spectra were recorded on a JEOL
JNM EX400, and chemical shifts are given in ppm (d) from tetra-
methylsilane as an internal standard. Significant 1H NMR data are
tabulated in the following order: number of protons, multiplicity
(s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad),
coupling constant(s) in hertz. Infrared (IR) spectra were recorded
on a Hitachi 270-30 spectrometer and a JASCO FT/IR-6100. Electron
spray ionization condition (ESI) mass spectra were recorded on an
Agilent 1100 and SCIEX API-150EX spectrometer. Fast atom bom-
bardment ionization condition (FAB) mass spectra were recorded
on a JEOL JMS-HX110 spectrometer. Electron impact ionization
4.2.2. Benzaldehyde (6-tert-butylthieno[2,3-d]pyrimidin-4-
yl)hydrazone (9)
A mixture of 7 (208 mg, 1.0 mmol) and benzaldehyde 8a
(112 lL, 1.1 mmol) in benzene (4 mL) was stirred under reflux
for 3 h and cooled to room temperature. The precipitate was col-
lected by filtration and washed with n-hexane to afford title com-
pound 9 (262 mg, 84%) as a yellow solid. 1H NMR (CDCl3) d: 1.50
(9H, s), 7.45 (3H, m), 7.70 (2H, m), 7.87 (1H, s), 7.88 (1H, s), 8.47
(1H, s), 8.98 (1H, br); IR (KBr) cmÀ1: 3195, 2966, 1577, 1552,
1429; MS (EI) m/z: 310 (M+); HRMS (ESI) m/z: 311.13045 (calcd
for C17H19N4S: 311.13304); Anal. Calcd for C17H18N4S: C, 65.78;
H, 5.84; N, 18.05; S, 10.33. Found: C, 65.81; H, 5.80; N, 18.06; S,
10.25.
condition (EI) mass spectra were recorded on
a JEOL JMS-
AX505W. High-resolution mass spectra were obtained on a JEOL
JMS-100LP AccuTOF LC-plus spectrometer (ESI) or a JEOL JMS-
700 mass spectrometer (EI). Elemental analysis was performed
using a PerkinElmer CHNS/O 2400II or a Yokohama Analysis
IC7000RS, and analytical results were within 0.4% of the theoret-
ical values unless otherwise noted.
4.2.1.1. 2-Amino-5-tert-butyl-3-methoxycarbonylthiophene (4).
3,3-Dimethylbutanal 3 (5.0 mL, 40 mmol) was added to a solu-
tion of methyl cyanoacetate (3.53 mL, 40 mmol), Et3N (4.29 mL,
40 mmol) and DMF (6.19 mL, 80 mmol) under a N2 atmosphere.
After 10 min stirring, S8 (1.28 g, 40 mmol) was added and stirred
at room temperature for 23 h. The reaction mixture was poured
into water and extracted with EtOAc. The aqueous layer was ex-
tracted with EtOAc and the combined organic layer was washed
with brine, and dried over Na2SO4. The solvent was removed under
reduced pressure to afford title compound 4 (8.21 g, 96%) as a yel-
low oil. 1H NMR (CDCl3) d: 1.29 (9H, s), 3.79 (3H, s), 5.76 (2H, br),
6.62 (1H, s); MS (ESI) m/z: 214 (M++H); HRMS (ESI) m/z:
214.08918 (calcd for C10H16NO2S: 214.09017).
4.2.3. Methyl 4-{[(6-tert-butylthieno[2,3-d]pyrimidin-4-
yl)hydrazono]methyl}benzoate (10)
A mixture of 7 (223 mg, 1.00 mmol) and methyl 4-formylbenzo-
ate 8b (181 mg, 1.10 mmol) in benzene (10 mL) was stirred under
reflux for 1 h and cooled to room temperature. The reaction mix-
ture was concentrated under reduced pressure and the residue
was recrystallized from Et2O to afford title compound 10
(360 mg, 98%) as a pale yellow solid. 1H NMR (CDCl3) d: 1.51 (9H,
s), 3.96 (3H, s), 7.77 (2H, d, J = 7.5 Hz), 7.85 (1H, br), 7.92 (1H,
br), 8.12 (2H, d, J = 7.5 Hz), 8.50 (1H, br); IR (KBr) cmÀ1: 3253,
3228, 1698; MS (FAB) m/z: 369 (M++H); HRMS (ESI) m/z:
369.13759 (calcd for C19H21N4O2S: 369.13852); Anal. Calcd for
C19H20N4O2S: C, 61.94; H, 5.47; N, 15.21; S, 8.70. Found: C,
62.19; H, 5.53; N, 15.29; S, 8.81.
4.2.1.2. 6-tert-Butyl-4-oxothieno[2,3-d]pyrimidine (5). A mix-
ture of 4 (8.21 g, 38.5 mmol) in formamide (50 mL) was stirred un-
der reflux for 2.5 h. After cooling, precipitate was formed, which
was filtered off and washed with n-hexane to afford title com-
pound 6 (5.03 g, 65%) as a pale yellow solid. 1H NMR (CDCl3) d:
1.43 (9H, s), 7.21 (1H, s), 8.01 (1H, s), 12.15 (1H, br); IR (ATR) cm
À1: 2957, 2866, 1650, 1582, 1368, 1270, 1168; MS (ESI) m/z: 209
(M++H); HRMS (ESI) m/z: 209.07370 (calcd for C10H13N2OS:
209.07486); Anal. Calcd for C10H12N2OS: C, 57.67; H, 5.81; N,
13.45; S, 15.40. Found: C, 57.88; H, 5.98; N, 13.46; S, 15.68.
4.2.4. 4-{[(6-tert-Butylthieno[2,3-d]pyrimidin-4-
yl)hydrazono]methyl}benzoic acid (11)
Compound 11 was obtained from 4-formylbenzoic acid 8c as a
yellow solid (quant.) by following the procedure described for 9. 1H
NMR (DMSO-d6) d: 1.46 (9H, s), 7.82 (2H, d, J = 8.3 Hz), 7.83 (1H, s),
8.02 (2H, d, J = 8.3 Hz), 8.29 (1H, s), 8.46 (1H, s), 11.95 (1H, br); IR

T. Horiuchi et al. / Bioorg. Med. Chem. 17 (2009) 7850–7860
7855
(KBr) cmÀ1: 1701, 1558, 1358, 1273, 885; MS (FAB) m/z: 355
(M++H); HRMS (ESI) m/z: 355.12227 (calcd for C18H19N4O2S:
355.12287); Anal. Calcd for C18H18N4O2S: C, 61.00; H, 5.12; N,
15.81; S, 9.05. Found: C, 60.65; H, 5.13; N, 15.00; S, 8.78.
1577, 1556, 1512, 1431, 1360, 1344; MS (FAB) m/z: 341 (M++H).
HRMS (ESI) m/z: 341.14399 (calcd for C18H21N4OS: 341.14361);
Anal. Calcd for C18H20N4OSÁ0.1EtOAc: C, 63.28; H, 6.00; N, 16.04;
S, 9.18. Found: C, 63.61; H, 6.26; N, 15.84; S, 9.04.
4.2.5. 4-Hydroxybenzaldehyde (6-tert-butylthieno[2,3-
d]pyrimidin-4-yl)hydrazone (12)
4.2.5.4. 4-[(Dimethylamino)sulfonyl]benzoic acid (34). To a
solution of 4-chlorosulfonylbenzoic acid 33 (1.0 g, 4.53 mmol) in
THF (20 mL) was added dimethylamine (2 M THF solution,
4.76 mL, 9.52 mmol) at 0 °C. The mixture was stirred at room tem-
perature for 16 h. After EtOAc and 1 N NaOH aq were added, the
two layers were separated. The aqueous layer was acidified with
6 N HCl aq and the precipitate was collected by filtration to afford
title compound 34 (589 mg, 57%) as a colorless solid. 1H NMR
(DMSO-d6) d: 2.63 (6H, s), 7.86 (2H, d, J = 6.9 Hz), 8.16 (2H, d,
J = 7.3 Hz); MS (FAB) m/z: 230 (M++H).
Compound 12 was obtained from 4-hydroxybenzaldehyde 8d
as a colorless solid (92%) by following the procedure described
for 9. 1H NMR (DMSO-d6) d: 1.43 (9H, s), 6.84 (2H, d, J = 8.5 Hz),
7.54 (2H, d, J = 8.5 Hz), 7.82 (2H, br), 8.13 (1H, s), 8.36 (1H, s); IR
(ATR) cmÀ1: 1564, 1512, 1433, 1292, 1153; MS (FAB) m/z: 327
(M++H); HRMS (ESI) m/z: 327.12799 (calcd for C17H19N4OS:
327.12796); Anal. Calcd for C17H18N4OS: C, 62.55; H, 5.56; N,
17.16; S, 9.82. Found: C, 62.42; H, 5.64; N, 17.08; S, 9.96.
4.2.5.1. 4-({[tert-Butyl(diphenyl)silyl]oxy}methyl)benzaldehyde
(8e). To a mixture of methyl 4-(hydroxymethyl)benzoate 31
(1.0 g, 6.02 mmol) and imidazole (860 mg, 12.64 mmol) in THF
(20 mL) was added TBDPSCl (1.64 mL, 6.32 mmol). The reaction
mixture was stirred at room temperature for 7 h. After EtOAc and
brine were added, the two layers were separated. The aqueous
layer was extracted with EtOAc and the combined organic layer
was washed with brine, and dried over Na2SO4. The solvent was re-
moved under reduced pressure, and the residue was chromato-
graphed on silica gel eluted with n-hexane/EtOAc (5:1) to afford
methyl 4-({[tert-butyl(diphenyl)silyl]oxy}methyl)benzoate 32.
4.2.5.5. 4-Formyl-N,N-dimethylbenzenesulfonamide (8f). To a
suspension of LAH (192 mg, 5.06 mmol) in Et2O (6 mL) was added
34 (580 mg, 2.53 mmol) at 0 °C, and stirred at room temperature
for 1.5 h. After cooling at 0 °C, MeOH (0.55 mL), water (0.25 mL),
15% NaOH aq (0.25 mL) and water (0.75 mL) were added to a reac-
tion mixture successively. The precipitate was filtered through Cel-
ite. The filtrate was concentrated under reduced pressure and the
residue was dissolved in CHCl3 (10 mL). To this solution was added
Mn2O (1.0 g) and the mixture was stirred under reflux for 4 h. The
reaction mixture was filtered through Celite. The filtrate was con-
centrated under reduced pressure and the residue was recrystal-
lized from EtOAc and n-hexane to afford title compound 8f
(309 mg, 62%) as a pale yellow solid. 1H NMR (CDCl3) d: 2.76 (6H,
s), 7.95 (2H, d, J = 8.0 Hz), 8.06 (2H, d, J = 8.0 Hz), 10.12 (1H, s);
MS (FAB) m/z: 214 (M++H).
The residue was added to
a suspension of LAH (246 mg,
6.48 mmol) in THF (25 mL) at 0 °C, and stirred at room temperature
for 5 h. After cooling at 0 °C, MeOH (0.55 mL), water (0.25 mL), 15%
NaOH aq (0.25 mL) and water (0.75 mL) were added to a reaction
mixture successively. The precipitate was filtered through Celite.
The filtrate was concentrated under reduced pressure and the res-
idue was dissolved in CHCl3 (46 mL). To this solution was added
Mn2O (4.6 g) and the mixture was stirred under reflux for 6 h.
The reaction mixture was filtered through Celite. The filtrate was
concentrated under reduced pressure and the residue was chro-
matographed on silica gel eluted with CHCl3 to afford title com-
pound 8e (1.86 g, 81%) as a yellow oil. 1H NMR (CDCl3) d: 1.12
(9H, s), 4.84 (2H, s), 7.39–7.44 (6H, m), 7.51 (2H, d, J = 8.3 Hz),
7.68 (4H, dd, J = 1.7, 7.8 Hz), 7.85 (2H, d, J = 8.3 Hz), 10.00 (1H, s);
MS (FAB) m/z: 375 (M++H).
4.2.5.6. 4-{[(6-tert-Butylthieno[2,3-d]pyrimidin-4-yl)hydrazon-
o]methyl}-N,N-dimethylbenzenesulfonamide (15). Compound
15 was obtained from 8f as a pale yellow solid (quant.) by follow-
ing the procedure described for 9. 1H NMR (CDCl3) d: 1.51 (9H, s),
2.76 (6H, s), 7.81 (1H, s), 7.86 (4H, s), 7.99 (1H, s), 8.49 (1H, s); IR
(KBr) cmÀ1: 1560, 1550, 1435, 1342, 1157, 1128, 750; MS (FAB) m/
z: 418 (M++H); HRMS (ESI) m/z: 418.13540 (calcd for
C19H24N5O2S2: 418.13714); Anal. Calcd for C19H23N5O2S2: C,
54.65; H, 5.55; N, 16.77; S, 15.36. Found: C, 54.67; H, 5.53; N,
16.83; S, 15.42.
4.2.5.2. 4-({[tert-Butyl(diphenyl)silyl]oxy}methyl)benzaldehyde
(6-tert-butylthieno[2,3-d]pyrimidin-4-yl)hydrazone (13). Com-
pound 13 was obtained from 8e as a colorless solid (90%) by fol-
lowing the procedure described for 9. 1H NMR (CDCl3) d: 1.12
(9H, s), 1.50 (9H, s), 4.81 (2H, s), 7.37–7.46 (8H, m), 7.67–7.71
4.2.5.7. Methyl 3-[(dimethylamino)methyl]benzoate (36). To a
solution of methyl 4-(bromomethyl)benzoate 35 (2.3 g,
10.0 mmol) in THF (13 mL) was added dimethylamine (2 M THF
solution, 10 mL, 20 mmol) at 0 °C. The mixture was stirred at room
temperature for 16 h. The reaction mixture was separated with
satd NaHCO3 aq and EtOAc. The aqueous layer was extracted with
EtOAc and the combined organic layer was washed with brine, and
dried over Na2SO4. The solvent was removed under reduced pres-
sure to afford title compound 36 (1.89 g, 98%) as a yellow oil. 1H
NMR (CDCl3) d: 2.24 (6H, s), 3.46 (2H, s), 3.91 (3H, s), 7.40 (1H, t,
J = 7.6 Hz), 7.53 (1H, d, J = 7.6 Hz), 7.94 (1H, d, J = 7.6 Hz), 7.97
(1H, s); MS (EI) m/z: 193 (M+).
(6H, m), 7.89 (1H, br), 7.96 (1H, br), 8.44 (1H, br); IR (KBr) cmÀ1
:
1558, 1429, 1115, 1074, 702; MS (FAB) m/z: 579 (M++H); HRMS
(ESI) m/z: 579.25812 (calcd for C34H39N4OSSi: 579.26138); Anal.
Calcd for C34H38N4OSSi: C, 70.55; H, 6.62; N, 9.68; S, 5.54. Found:
C, 70.61; H, 6.63; N, 9.70; S, 5.81.
4.2.5.3. 4-(Hydroxymethyl)benzaldehyde (6-tert-butylthieno[2,
3-d]pyrimidin-4-yl)hydrazone (14). To a solution of 13 (345 mg,
0.60 mmol) in THF (7 mL) was added TBAF (1 M THF solution,
1.2 mL, 1.19 mmol), and the mixture was stirred at 60 °C for 4 h.
After EtOAc and brine were added to the reaction mixture, the
two layers were separated. The aqueous layer was extracted with
EtOAc and the combined organic layer was dried over Na2SO4.
The solvent was removed under reduced pressure, and the residue
was recrystallized from MeOH, EtOAc, and n-hexane to afford title
compound 14 (191 mg, 94%) as a colorless solid. 1H NMR (CDCl3) d:
1.50 (9H, s), 4.76 (2H, s), 7.45 (2H, d, J = 8.1 Hz), 7.70 (2H, d,
4.2.5.8. 3-[(Dimethylamino)methyl]benzaldehyde (8g). Com-
pound 8g was obtained from 36 as a yellow oil (68%) by following
the procedure described for 8f. 1H NMR (CDCl3) d: 2.24 (6H, s), 3.50
(2H, s), 7.49 (1H, t, J = 7.6 Hz), 7.59 (1H, d, J = 7.6 Hz), 7.78 (1H, d,
J = 7.6 Hz), 7.83 (1H, s), 10.02 (1H, s); MS (FAB) m/z: 164 (M++H).
4.2.5.9. 3-[(Dimethylamino)methyl]benzaldehyde (6-tert-butyl-
thieno[2,3-d]pyrimidin-4-yl)hydrazone (16). Compound 16 was
obtained from 8g as a colorless solid (67%) by following the proce-
dure described for 9. 1H NMR (CDCl3) d: 1.52 (9H, s), 2.27 (6H, s),
J = 8.1 Hz), 7.87 (1H, s), 7.90 (1H, s), 8.46 (1H, s); IR (KBr) cmÀ1
:

7856
T. Horiuchi et al. / Bioorg. Med. Chem. 17 (2009) 7850–7860
3.48 (2H, s), 7.35–7.42 (2H, m), 7.57 (1H, d, J = 7.1 Hz), 7.73 (1H, s),
7.87 (1H, s), 7.92 (1H, s), 8.48 (1H, s); IR (KBr) cmÀ1: 2958, 2775,
1558, 1434, 1346, 1157, 1120, 773; MS (FAB) m/z: 368 (M++H).
HRMS (ESI) m/z: 368.19011 (calcd for C20H26N5S; 368.19089); Anal.
Calcd for C20H25N5SÁ0.25H2O: C, 65.36; H, 6.86; N, 19.06; S, 8.37.
Found: C, 64.90; H, 6.84; N, 18.96.
(ESI) m/z: 398.20146 (calcd for C21H28N5OS: 398.20235); Anal. Calcd
for C21H27N5OS: C, 63.45; H, 6.85; N, 17.62; S, 8.07. Found: C, 63.97;
H, 6.79; N, 17.52; S, 8.01.
4.2.5.15. tert-Butyl (4-{[(6-tert-butylthieno[2,3-d]pyrimidin-4-
yl)hydrazono]methyl}benzyl)carbamate (19). Compound 19
was obtained from tert-butyl (4-formylbenzyl)carbamate 8j as a
pale yellow solid (90%) by following the procedure described for
9. 1H NMR (CDCl3) d: 1.48 (9H, s), 1.50 (9H, s), 4.37 (2H, d,
J = 5.6 Hz), 4.90 (1H, br), 7.36 (1H, s), 7.37 (2H, d, J = 8.1 Hz), 7.68
(2H, d, J = 8.1 Hz), 7.86 (1H, s), 7.87 (1H, s), 8.48 (1H, s); IR (ATR)
cmÀ1: 2972, 1712, 1558, 1157; MS (FAB) m/z: 440 (M++H); HRMS
(ESI) m/z: 440.21149 (calcd for C23H30N5O2S: 440.21202); Anal.
Calcd for C23H29N5O2S: C, 62.84; H, 6.65; N, 15.93; S, 7.29. Found:
C, 63.02; H, 6.53; N, 15.75; S, 7.44.
4.2.5.10. Methyl 4-[(dimethylamino)methyl]benzoate (38). Com-
pound 38 was obtained from methyl 4-(bromomethyl)benzoate 37
as a yellow oil (78%) by following the procedure described for 36. 1H
NMR (CDCl3) d: 2.25 (6H, s), 3.47 (2H, s), 3.91 (3H, s), 7.38 (2H, d,
J = 8.3 Hz), 7.99 (2H, d, J = 8.3 Hz); MS (FAB) m/z: 194 (M++H).
4.2.5.11. 4-[(Dimethylamino)methyl]benzaldehyde (8h). Com-
pound 8h was obtained from 38 as a yellow oil (86%) by following
the procedure described for 8f. 1H NMR (CDCl3) d: 2.26 (6H, s), 3.50
(2H, s), 7.49 (2H, d, J = 8.1 Hz), 7.84 (2H, d, J = 8.1 Hz), 10.00 (1H, s);
MS (FAB) m/z: 164 (M++H).
4.2.5.16. 4-(Aminomethyl)benzaldehyde (6-tert-butylthieno-
[2,3-d]pyrimidin-4-yl)hydrazone (20). To
a solution of 19
(100 mg, 0.23 mmol) in 1,4-dioxane (1.0 mL) was added 4 N
HCl in 1,4-dioxane (1.0 mL) at 0 °C. The mixture was stirred at
room temperature for 10.5 h and concentrated under reduced
pressure. The residue was recrystallized from EtOAc and n-hex-
ane to afford title compound 20 (82 mg, 87%) as a pale yellow so-
lid. 1H NMR (DMSO-d6) d: 1.46 (9H, s), 3.68 (2H, br), 4.08 (2H, s),
7.60 (2H, d, J = 8.3 Hz), 7.83 (2H, d, J = 8.3 Hz), 7.95 (1H, s), 8.46
(1H, s), 8.48 (1H, s); IR (ATR) cmÀ1: 1641, 1587, 1086; MS
(FAB) m/z: 340 (M++H); HRMS (ESI) m/z: 340.15959 (calcd for
C18H22N5S: 340.15952); Anal. Calcd for C18H21N5SÁ2HCl: C,
52.43; H, 5.62; Cl, 17.19; N, 16.98; S, 7.78. Found: C, 52.09; H,
5.74; Cl, 17.13; N, 16.65; S, 7.82.
4.2.5.12. 4-[(Dimethylamino)methyl]benzaldehyde(6-tert-butyl-
thieno[2,3-d]pyrimidin-4-yl)hydrazone (17). Compound 17 was
obtainedfrom 8h as a colorlesssolid (54%) byfollowingthe procedure
described for 9. 1H NMR (CDCl3) d: 1.50 (9H, s), 2.30 (6H, s), 3.50 (2H,
s), 7.41(2H, d, J = 8.1 Hz), 7.68(2H, d, J = 8.1 Hz), 7.87(1H, s), 7.89 (1H,
s), 10.00 (1H, s); IR (KBr) cmÀ1: 1558, 1427, 1348, 773; MS (FAB) m/z:
368 (M++H). HRMS (ESI) m/z: 368.19245 (calcd for C20H26N5S:
368.19089); Anal. Calcd for C20H25N5S: C, 65.36; H, 6.86; N, 19.06; S,
8.73. Found: C, 65.14; H, 6.86; N, 19.00; S, 8.60.
4.2.5.13. 4-{[(2-Hydroxyethyl)(methyl)amino]methyl}benzalde-
hyde (8i). To a solution of methyl 4-(bromomethyl)benzoate 37
(1.0 g, 4.37 mmol) in toluene (10 mL) was added N-methyl etha-
4.2.5.17. Methyl 4-{[(tert-butoxycarbonyl)(methyl)amino]methyl}-
benzoate (40). To a solution of methyl 4-formylbenzoate 39 (2.0 g,
12.18 mmol) in toluene (20 mL) was added methylamine (2 M THF
solution, 6.09 mL, 12.18 mmol). The solution was stirred at room tem-
perature for 30 min and concentrated under reduced pressure. The res-
idue was diluted with MeOH (200 mL) and NaBH4 (510 mg,
13.40 mmol) was added and stirred at room temperature for 2 h. After
being acidified with 1 N HCl aq, the mixture was concentrated under
reduced pressure. The residue was diluted with CHCl3 and washed
with 10% NaOH aq. The two layers were separated. The aqueous layer
was extracted with EtOAc, and the combined organic layer was
washed with brine, and dried over Na2SO4. The solvent was removed
under reduced pressure. To a solution of the residue in CH2Cl2 (50 mL)
were added Boc2O (2.8 mL, 12.2 mmol) and a catalytic amount of
DMAP. The reaction mixture was stirred for 4 h at room temperature.
After evaporation of the solvent, the residue was chromatographed on
silica gel eluted with n-hexane/EtOAc (10:1) to afford title compound
40 (600 mg, 18%) as a colorless oil. 1H NMR (CDCl3) d: 1.45–1.49 (9H,
br), 2.81–2.88 (3H, br), 3.91 (3H, s), 4.47 (2H, s), 7.30 (2H, d, J = 8.1 Hz),
8.00 (2H, d, J = 8.1 Hz); MS (FAB) m/z: 280 (M++H).
nolamine (420 lL, 5.24 mmol). After stirring at room temperature
for 1 h, Et3N (3.0 mL, 21.8 mmol) was added to the reaction mix-
ture, and stirred at 70 °C for 19.5 h. The mixture was separated
with EtOAc and water. The aqueous layer was extracted with
EtOAc and the combined organic layer was washed with brine,
and dried over Na2SO4. The solvent was removed under reduced
pressure and the residue was dissolved in THF (7 mL) and was
added to a suspension of LAH (122 mg, 3.21 mmol) in THF (7 mL)
at 0 °C. The reaction mixture was stirred at room temperature for
5 h. After cooling at 0 °C, MeOH (0.29 mL), water (0.13 mL), 15%
NaOH aq (0.13 mL) and water (0.39 mL) were added to a reaction
mixture successively. The precipitate was filtered through Celite.
The filtrate was concentrated under reduced pressure and the res-
idue was dissolved in CHCl3 (10 mL). To this solution was added
Mn2O (1.4 g) and the mixture was stirred under reflux for 3.5 h.
The reaction mixture was filtered through Celite. The filtrate was
concentrated under reduced pressure and the residue was chro-
matographed on silica gel eluted with CHCl3/MeOH (10:1) to afford
title compound 8i (468 mg, 55%) as a pale yellow oil. 1H NMR
(CDCl3) d: 2.26 (3H, s), 2.63 (2H, t, J = 5.4 Hz), 3.65 (4H, s), 7.49
(2H, d, J = 8.1 Hz), 7.85 (2H, dd, J = 1.7, 8.1 Hz), 10.00 (1H, s); MS
(FAB) m/z: 194 (M++H); HRMS (ESI) m/z: 194.11582 (calcd for
C11H16NO2: 194.11810).
4.2.5.18. tert-Butyl4-(formylbenzyl)methylcarbamate(8k). Com-
pound 8k was obtained from 40 as a colorless oil (58%) by following
the procedure described for 8f. 1H NMR (CDCl3) d: 1.45–1.50 (9H, br),
2.83–2.89 (3H, br), 4.50 (2H, br), 7.38 (2H, d, J = 8.1 Hz), 7.86 (2H, d,
J = 8.1 Hz), 10.01 (1H, s); MS (ESI) m/z: 272 (M++Na); HRMS (ESI) m/
z: 272.12570 (calcd for C14H19NNaO3: 272.12626).
4.2.5.14. 4-{[(2-Hydroxyethyl)(methyl)amino]methyl}benzalde-
hyde (6-tert-butylthieno[2,3-d]pyrimidin-4-yl)hydrazone (18).
Compound 18 was obtained from 8i as a pale yellow solid (80%)
by following the procedure described for 9. 1H NMR (DMSO-d6) d:
1.46 (9H, s), 2.17 (3H, s), 2.50 (2H, d, J = 1.2 Hz), 3.54 (4H, br), 4.41
(1H, t, J = 5.4 Hz), 7.42 (2H, d, J = 7.8 Hz), 7.67 (2H, d, J = 7.8 Hz),
7.86 (1H, br), 8.23 (1H, s), 8.42 (1H, s), 11.77 (1H, br); IR (KBr) cm
À1: 1581, 1558, 1433, 1356; MS (FAB) m/z: 398 (M++H); HRMS
4.2.5.19. tert-Butyl (4-{[(6-tert-butylthieno[2,3-d]pyrimidin-4-
yl)hydrazono]methyl}benzyl)methylcarbamate (21). A mixture
of 7 (81 mg, 0.37 mmol) and 8k (100 mg, 0.40 mmol) in benzene
(3 mL) was stirred under reflux for 3 h and cooled to room temper-
ature. The precipitate was collected by filtration and recrystallized
with EtOAc and n-hexane to afford title compound 21 (61 mg, 37%)

T. Horiuchi et al. / Bioorg. Med. Chem. 17 (2009) 7850–7860
7857
as a pale yellow solid. 1H NMR (CDCl3) d: 1.51 (18H, s), 2.88 (3H,
br), 4.47 (2H, s), 7.30 (1H, s), 7.32 (1H, s), 7.68 (2H, d, J = 8.3 Hz),
7.88 (2H, s), 8.49 (1H, s), 9.12 (1H, br); IR (KBr) cmÀ1: 2962,
1703, 1680, 1556, 1437, 1126; MS (FAB) m/z: 454 (M++H); HRMS
(ESI) m/z: 454.22882 (calcd for C24H32N5O2S: 454.22767); Anal.
Calcd for C24H31N5O2S: C, 63.55; H, 6.89; N, 15.44; S, 7.37. Found:
C, 63.26; H, 6.81; N, 15.35; S, 7.01.
(M++H); HRMS (ESI) m/z: 452.21125 (calcd for C24H30N5O2S:
452.21202); Anal. Calcd for C24H29N5O2S: C, 63.83; H, 6.47; N,
15.51; S, 7.10. Found: C, 63.91; H, 6.44; N, 15.48; S, 7.36.
4.2.5.25. 5-Isoindolinecarbaldehyde (6-tert-butylthieno[2,3-d]-
pyrimidin-4-yl)hydrazone (24). Compound 24 was obtained
from 23 as a colorless solid (97%) by following the procedure de-
scribed for 20. 1H NMR (DMSO-d6) d: 1.44 (9H, s), 4.55 (4H, br),
7.53 (1H, d, J = 7.8 Hz), 7.85 (1H, br), 7.90 (1H, s), 8.49 (1H, s),
9.80 (2H, br); IR (KBr) cmÀ1: 3388, 2698, 2582, 1643; MS (FAB)
m/z: 352 (M++H); HRMS (ESI) m/z: 352.15884 (calcd for
C19H22N5S: 352.15959); Anal. Calcd for C19H21N5SÁ2HClÁ0.75H2O:
C, 52.11; H, 5.64; N, 15.99; Cl, 16.19; S, 7.32. Found: C, 52.28; H,
5.55; N, 15.88; Cl, 16.45; S, 7.27.
4.2.5.20. 4-[(Methylamino)methyl]benzaldehyde (6-tert-butyl-
thieno[2,3-d]pyrimidin-4-yl)hydrazone (22). Compound 22 was
obtained from 21 as a pale yellow solid (81%) by following the pro-
cedure described for 20. 1H NMR (DMSO-d6) d: 1.46 (9H, s), 2.55–
2.57 (3H, m), 4.18 (2H, m), 7.64 (1H, s), 7.66 (1H, s), 7.98 (2H, br),
8.53 (1H, br), 9.19 (2H, br); IR (ATR) cmÀ1: 3396, 2738, 2667, 1651,
1595; MS (FAB) m/z: 354 (M++H); HRMS (ESI) m/z: 354.17533
(calcd
for
C19H23N5S:
354.17524);
Anal.
Calcd
for
4.2.5.26. 2-Methylisoindoline-5-carbaldehyde (8m). To a solu-
tion of 43 (200 mg, 0.80 mmol) in CH2Cl2 (0.4 mL) was added TFA
(1 mL) and the mixture was stirred for 2 min at room temperature.
After evaporation of the solvent, CH2Cl2 (2.4 mL) and Et3N
C19H23N5SÁ2HClÁH2O: C, 51.35; H, 6.12; Cl, 15.95; N, 15.76; S,
7.22. Found: C, 51.63; H, 6.04; Cl, 15.96; N, 15.73; S, 7.28.
4.2.5.21. N-tert-Butoxycarbonyl-dipropargylamine (42). To
a
(223
ture was stirred at room temperature for 15 min followed by the
addition of AcOH (115 L, 2.00 mmol), 35% aq formaldehyde
(138 L, 1.60 mmol), and NaBH(OAc)3 (272 mg, 1.28 mmol). The
lL, 1.60 mmol) were added to the residue. The reaction mix-
mixture of dipropargylamine 41 (5.45 g, 58.5 mmol) and Et3N
(10 mL, 72 mmol) in CH2Cl2 (60 mL) was added Boc2O (15 mL,
65 mmol). The reaction mixture was stirred overnight at room
temperature. After evaporation of the solvent, the residue was par-
titioned between EtOAc and water. The organic layer was washed
with brine and dried over Na2SO4. The solvent was removed under
reduced pressure to afford title compound 42 (11.5 g, quant.) as a
brown oil. 1H NMR (CDCl3) d: 1.48 (9H, s), 2.22 (2H, t, J = 2.5 Hz),
4.17 (4H, br).
l
l
mixture was stirred at room temperature for 15 h. After EtOAc
and water were added to the reaction mixture, the two layers
were separated. The aqueous layer was extracted with EtOAc,
and the combined organic layer was washed with brine, and dried
over Na2SO4. The solvent was removed under reduced pressure,
and the residue was diluted with CCl4 (2 mL). To this solution
was added Mn2O (220 mg) and the mixture was stirred under
reflux for 7 h. The reaction mixture was filtered through Celite.
The filtrate was concentrated under reduced pressure to afford
title compound 8m (100 mg, 92%) as a pale yellow solid. 1H
NMR (CDCl3) d: 2.60 (3H, s), 3.91 (4H, d, J = 2.9 Hz), 7.16 (2H, d,
J = 1.7 Hz), 7.71 (1H, s), 9.97 (1H, s); MS (FAB) m/z: 162
(M++H).
4.2.5.22. tert-Butyl 5-(hydroxymethyl)-1,3-dihydro-2H-isoin-
dole-2-carboxylate (43). To a solution of N-tert-butoxycarbonyl-
dipropargylamine 42 (7.35 g, 38 mmol) in EtOH (160 mL) was
dropwised propargylalcohol (9.0 mL, 0.155 mol) followed by the
addition of chlorotris(triphenylphosphine)rhodium(I) (1.0 g,
1.1 mmol) at 0 °C. The reaction mixture was stirred at room tem-
perature for 24 h, and concentrated under reduced pressure. The
residue was chromatographed on silica gel eluted with n-hexane/
EtOAc (2:1). The eluate was concentrated under reduced pressure,
and the residue was recrystallized from Et2O and n-hexane to af-
ford title compound 43 (4.17 g, 44%) as a colorless solid. 1H NMR
(CDCl3) d: 1.52 (9H, s), 4.62 (4H, br), 4.69 (2H, br), 7.21–7.27 (3H,
m); IR (KBr) cmÀ1: 3421, 2863, 1673; MS (FAB) m/z: 250 (M++H);
Anal. Calcd for C14H19NO3: C, 67.45; H, 7.68; N, 5.62. Found: C,
67.53; H, 7.58; N, 5.58.
4.2.5.27. 2-Methylisoindoline-5-carbaldehyde (6-tert-butylthie-
no[2,3-d]pyrimidin-4-yl)hydrazone (25). A mixture of
7
(125 mg, 0.56 mmol) and 8m (100 mg, 0.62 mmol) in benzene
(4.5 mL) was stirred under reflux for 2.5 h and cooled to room tem-
perature. The reaction mixture was concentrated under reduced
pressure. The residue was chromatographed on silica gel eluted
with CHCl3/MeOH (10:1) to afford title compound 25 (31 mg,
15%) as a yellow solid. 1H NMR (CDCl3) d: 1.50 (9H, s), 2.64 (3H,
s), 3.97 (4H, s), 7.28 (1H, s), 7.50 (1H, d, J = 7.1 Hz), 7.61 (1H, s),
7.86 (1H, s), 7.88 (1H, s), 8.48 (1H, s); IR (KBr) cmÀ1: 2954, 1562,
1429, 1354, 1344; MS (FAB) m/z: 366 (M++H); HRMS (ESI) m/z:
366.17483 (calcd for C20H24N5S: 366.17524); Anal. Calcd for
C20H23N5S: C, 65.72; H, 6.34; N, 19.16; S, 8.77. Found: C, 65.34;
H, 6.40; N, 19.02; S, 8.91.
4.2.5.23. tert-Butyl 5-formyl-1,3-dihydro-2H-isoindole-2-car-
boxylate (8l). To a solution of 43 (500 mg, 2.00 mmol) in CCl4
(15 mL) was added MnO2 (2.0 g) and the mixture was stirred for
30 min at room temperature. The reaction mixture was filtered
through Celite and washed with CHCl3. The filtrate was concen-
trated under reduced pressure and the residue was chromato-
graphed on silica gel eluted with n-hexane/EtOAc (4:1) to afford
title compound 8l (325 mg, 66%) as a colorless solid. 1H NMR
(CDCl3) d: 1.53 (9H, s), 4.72 (2H, br), 4.76 (2H, br), 7.38–7.45 (1H,
m), 7.75–7.81 (2H, m), 10.00 (1H, s); IR (KBr) cmÀ1: 3050, 1693;
MS (EI) m/z: 247 (M+); Anal. Calcd for C14H17NO3Á0.1H2O: C,
67.70; H, 6.98; N, 5.64. Found: C, 67.51; H, 6.98; N, 5.45.
4.2.5.28. 2-Isopropylisoindoline-5-carbaldehyde (8n). Compound
8n was obtained from 43 as a yellow oil (33%) by following the pro-
cedure described for 8m with acetone instead of formaldehyde. 1H
NMR (CDCl3) d: 1.21 (6H, d, J = 6.3 Hz), 2.75–2.80 (1H, m), 3.95 (2H,
d, J = 8.1 Hz), 4.02 (2H, s), 7.16 (1H, s), 7.36 (1H, d, J = 8.1 Hz), 7.72
(1H, d, J = 3.4 Hz), 9.98 (1H, s); MS (FAB) m/z: 190 (M++H); HRMS
(ESI) m/z: 190.12308 (calcd for C12H16NO: 190.12319).
4.2.5.24. tert-Butyl 5-{[(6-tert-butylthieno[2,3-d]pyrimidin-4-
yl)hydrazono]methyl}-2H-isoindole-2-carboxylate (23). Com-
pound 23 was obtained from 8l as a pale yellow solid (96%) by fol-
lowing the procedure described for 9. 1H NMR (CDCl3) d: 1.50 (9H,
s), 1.54 (9H, s), 4.70 (2H, br), 4.74 (2H, br), 7.31–7.36 (1H, m), 7.56
(1H, s), 7.70 (1H, s), 7.85 (1H, s), 7.94 (1H, s), 8.46 (1H, s); IR (KBr)
cmÀ1: 2968, 1689, 1564, 1433, 1396, 1107; MS (FAB) m/z: 452
4.2.5.29. 2-Isopropylisoindoline-5-carbaldehyde (6-tert-butyl-
thieno[2,3-d]pyrimidin-4-yl)hydrazone (26). A mixture of
7
(47 mg, 0.21 mmol) and 8n (60 mg, 0.32 mmol) in benzene
(2 mL) was stirred under reflux for 19.5 h and cooled to room tem-
perature. The reaction mixture was concentrated under reduced
pressure and the residue was separated with water and EtOAc.

7858
T. Horiuchi et al. / Bioorg. Med. Chem. 17 (2009) 7850–7860
The aqueous layer was extracted with EtOAc and the combined or-
ganic layer was washed with brine, and dried over Na2SO4. The sol-
vent was removed under reduced pressure, and the residue was
chromatographed on silica gel eluted with CHCl3/MeOH (20:1).
The eluate was concentrated under reduced pressure, and the res-
idue was recrystallized from n-hexane to afford title compound 26
(18 mg, 21%) as a yellow solid. 1H NMR (CDCl3) d: 1.22 (6H, d,
J = 6.3 Hz), 1.50 (9H, s), 2.81 (1H, m), 4.02 (4H, s), 7.29 (1H, d,
J = 7.8 Hz), 7.52 (1H, d, J = 7.8 Hz), 7.60 (2H, s), 7.86 (1H, s), 7.88
(1H, s), 8.47 (1H, s); IR (KBr) cmÀ1: 2964, 1556, 1427, 1354,
1107; MS (FAB) m/z: 394 (M++H). HRMS (ESI) m/z: 394.20572
J = 8.1 Hz), 7.27–7.42 (2H, m), 7.65 (1H, d, J = 4.3, 16.1 Hz); MS
(ESI) m/z: 354 (M++Na), 276 (M+ÀBoc).
4.2.5.33. tert-Butyl 7-(3-ethoxy-1,2-dihydroxy-3-oxopropyl)-
3,4-dihydroisoquinoline-2(1H)-carboxylate (48). To a solution
of 47 (1.50 g, 4.52 mmol) in a mixture of THF (10 mL), acetone
(10 mL), and H2O (10 mL) were added NMO (1.06 g, 9.0 mmol)
and OsO4 (115 mg, 10 mol %), and the mixture was stirred at room
temperature for 7 h. After EtOAc and satd Na2S2O3 aq were added
to the reaction mixture, the two layers were separated. The aque-
ous layer was extracted with EtOAc, and the combined organic
layer was washed with satd NaHCO3 aq, brine, and dried over
Na2SO4. The solvent was removed under reduced pressure and
the residue was chromatographed on silica gel eluted with n-hex-
ane/EtOAc (2:1) to afford title compound 48 (1.26 g, 76%) as a col-
orless oil. 1H NMR (CDCl3) d: 1.30 (3H, t, J = 7.0 Hz), 1.49 (9H, s),
2.71 (1H, d, J = 7.3 Hz), 2.82 (2H, t, J = 5.5 Hz), 3.11 (1H, d,
J = 5.5 Hz), 3.63 (2H, t, J = 5.5 Hz), 4.29 (2H, q, J = 7.0 Hz), 4.34
(1H, dd, J = 5.5, 3.0 Hz), 4.58 (2H, s), 4.99 (1H, dd, J = 7.3, 3.0 Hz),
7.13–7.22 (3H, m); MS (ESI) m/z: 388 (M++Na).
(calcd
for
C22H28N5S:
394.20654);
Anal.
Calcd
for
C22H27N5SÁ0.5H2O: C, 65.64; H, 7.01; N, 17.40; S, 7.97. Found: C,
65.68; H, 6.80; N, 17.00; S, 8.25.
4.2.5.30. tert-Butyl 7-hydroxy-3,4-dihydroisoquinoline-2(1H)-
carboxylate (45). Isoquinolin-7-ol 44 (1.50 g, 10.3 mmol) was
hydrogenated over PtO2 (100 mg) in AcOH (15 mL) at 40 psi for
overnight. The catalyst was filtered off and the filtrate was concen-
trated under reduced pressure. The residue was diluted with the
mixture of THF (15 mL) and water (5 mL). To the solution were
added Et3N (1.67 mL, 12 mmol) and Boc2O (2.50 mL, 10.9 mmol).
The reaction mixture was stirred for 2 h at room temperature.
The mixture was partitioned between EtOAc and water. The organ-
ic layer was washed with satd NH4Cl aq and brine, and dried over
Na2SO4. The solvent was removed under reduced pressure, and the
residue was chromatographed on silica gel eluted with n-hexane/
EtOAc (4:1) to afford title compound 45 (2.05 g, 82%) as a colorless
solid. 1H NMR (CDCl3) d: 1.50 (9H, s), 2.75 (2H, t, J = 6.0 Hz), 3.62
(2H, t, J = 6.0 Hz), 4.53 (2H, s), 6.74–6.63 (2H, m), 6.98 (2H, t,
J = 6.0 Hz); IR (ATR) cmÀ1: 3337, 1654, 1435, 1159; MS (FAB) m/
z: 250 (M++H); HRMS (ESI) m/z: 272.12681 (calcd for
C14H19NNaO3: 272.12626); Anal. Calcd for C14H19NO3Á0.1H2O: C,
66.96; H, 7.71; N, 5.58. Found: C, 66.85; H, 7.58; N, 5.46.
4.2.5.34. tert-Butyl 7-formyl-3,4-dihydroisoquinoline-2(1H)-
carboxylate (8o). To a solution of 48 (1.20 g, 3.28 mmol) in a mix-
ture of THF (12 mL), MeOH (12 mL), and H2O (12 mL) were added
NaIO4 (1.40 g, 6.55 mmol), and the mixture was stirred at room
temperature for 1 h. After EtOAc and water were added to the reac-
tion mixture, the two layers were separated. The aqueous layer
was extracted with EtOAc, and the combined organic layer was
washed with brine, and dried over Na2SO4. The solvent was re-
moved under reduced pressure, and the residue was chromato-
graphed on silica gel eluted with n-hexane/EtOAc (4:1) to afford
title compound 8o (777 mg, 91%) as a colorless oil. 1H NMR (CDCl3)
d: 1.50 (9H, s), 2.92 (2H, t, J = 5.5 Hz), 3.68 (2H, t, J = 5.5 Hz), 4.65
(2H, s), 7.30 (1H, d, J = 7.5 Hz), 7.63 (1H, s), 7.68 (1H, d,
J = 7.5 Hz), 9.97 (1H, s); IR (KBr) cmÀ1: 3366, 2976, 1690, 1415,
1365, 1159; MS (FAB) m/z: 284 (M++Na).
4.2.5.31. tert-Butyl 7-(trifluoromethanesulfonyl)oxy-3,4-dihy-
droisoquinoline-2(1H)-carboxylate (46). To a solution of 45
(2.00 g, 8.00 mmol) was added N-phenyl-bis(trifluoromethanesulf-
onimide) (3.15 g, 8.8 mmol) was added in CH2Cl2 (100 mL) at 0 °C.
The reaction mixture was stirred at room temperature for 24 h and
concentrated. The residue was chromatographed on silica gel
eluted with n-hexane/EtOAc (9:1). The eluate was concentrated
under reduced pressure, and the residue was recrystallized from
n-hexane to afford title compound 46 (3.05 g, quant.) as a colorless
solid. 1H NMR (CDCl3) d: 1.49 (9H, s), 2.83 (2H, t, J = 6.0 Hz), 3.65
(2H, t, J = 6.0 Hz), 4.58 (2H, s), 7.02 (1H, d, J = 2.4 Hz), 7.06 (1H,
dd, J = 2.4, 8.5 Hz), 7.19 (1H, d, J = 8.5 Hz); IR (ATR) cmÀ1: 2987,
1670, 1423, 1208; MS (ESI) m/z: 404 (M++Na); HRMS (ESI) m/z:
404.07421 (calcd for C15H18F3NNaO5S: 404.07555).
4.2.5.35. tert-Butyl 7-{[2-(6-tert-butylthieno[2,3-d]pyrimidin-4-
yl)hydrazono]methyl}-3,4-dihydroisoquinoline-2(1H)-carboxyl-
ate (27). Compound 27 was obtained from 8o as a colorless solid
(77%) by following the procedure described for 9. 1H NMR (CDCl3)
d: 1.51 (18H, s), 2.88 (2H, br), 3.68 (2H, br), 4.63 (2H, s), 7.21 (1H, d,
J = 7.8 Hz), 7.46–7.50 (2H, m), 7.87 (2H, s), 8.45 (1H, s); IR (KBr) cm
À1: 2962, 1695, 1562, 1425, 1163; MS (FAB) m/z: 466 (M++H).
HRMS (ESI) m/z: 466.22667 (calcd for C25H32N5O2S: 466.22767);
Anal. Calcd for C25H31N5O2S: C, 64.49; H, 6.71; N, 15.04; S, 6.89.
Found: C, 63.74; H, 6.66; N, 14.38; S, 6.93.
4.2.5.36. 1,2,3,4-Tetrahydro-7-isoquinolinecarbaldehyde (6-tert-
butylthieno[2,3-d]pyrimidin-4-yl)hydrazone (28). Compound 28
was obtained from 27 as a colorless solid (97%) by following the pro-
cedure described for 20. 1H NMR (CD3OD) d: 1.52 (9H, s), 3.21 (2H, t,
J = 6.3 Hz), 3.56 (2H, t, J = 6.3 Hz), 4.46 (2H, s), 7.41 (1H, d, J = 7.8 Hz),
7.80 (1H, s), 7.88 (1H, s), 7.91 (2H, d, J = 7.8 Hz), 8.55 (1H, s), 8.59
(1H, s); IR (KBr) cmÀ1: 2627, 1641, 1587; MS (FAB) m/z: 366
(M++H). HRMS (ESI) m/z: 366.17704 (calcd for C20H24N5S:
366.17524); Anal. Calcd for C20H23N5SÁ2HClÁ0.75H2O: C, 53.15; H,
5.91; N, 15.50; Cl, 15.69; S, 7.10. Found: C, 53.71; H, 5.85; N,
14.69; Cl, 14.93; S, 7.16.
4.2.5.32. tert-Butyl 7-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-3,4-
dihydroisoquinoline-2(1H)-carboxylate (47). To a solution of 46
(3.00 g, 7.86 mmol) in CH3CN (30 mL) were added ethyl acrylate
(1.1 mL, 10 mmol), Pd(OAc)2 (176 mg, 10 mol%), tri(o-tolyl)phos-
phine (530 mg, 1.74 mmol), and Et3N (2.2 mL, 15.8 mmol). The
reaction mixture was stirred under reflux for 18 h. After EtOAc
was added to the reaction mixture, the two layers were separated.
The aqueous layer was extracted with EtOAc, and the combined or-
ganic layer was washed with satd NH4Cl aq, brine, and dried over
Na2SO4. The solvent was removed under reduced pressure and
the residue was chromatographed on silica gel eluted with n-hex-
ane/EtOAc (10:1–3:1) to afford title compound 47 (1.59 g, 61%) as a
pale yellow oil. 1H NMR (CDCl3) d: 1.34 (3H, t, J = 7.2 Hz), 1.49 (9H,
s), 2.85 (2H, t, J = 5.9 Hz), 3.65 (2H, t, J = 5.9 Hz), 4.26 (2H, q,
J = 7.2 Hz), 4.58 (2H, s), 6.40 (1H, d, J = 16.1 Hz), 7.15 (1H, q,
4.2.5.37. tert-Butyl 6-hydroxy-3,4-dihydroisoquinoline-2(1H)-
carboxylate (50). To a mixture of 6-hydroxy-1,2,3,4-tetrahydro-
isoquinoline 49 (500 mg, 2.17 mmol) and Et3N (0.40 mL,
2.85 mmol) in THF (7.5 mL) and water (2.5 mL) was added Boc2O
(569 mg, 2.61 mmol). The reaction mixture was stirred overnight
at room temperature, and concentrated under reduced pressure.

T. Horiuchi et al. / Bioorg. Med. Chem. 17 (2009) 7850–7860
7859
The residue was partitioned between EtOAc and H2O. The organic
4.3. Kinase inhibition assays
layer was washed with satd NH4Cl aq, brine and dried over Na2SO4
and concentrated. The residue was chromatographed on silica gel
eluted with n-hexane/EtOAc (1:1) to afford title compound 50
(493 mg, 91%) as a yellow oil. 1H NMR (CDCl3) d: 1.49 (9H, s),
2.77 (2H, t, J = 5.9 Hz), 3.61 (2H, t, J = 5.9 Hz), 4.49 (2H, s), 4.95
(1H, s), 6.62 (1H, s), 6.67 (1H, t, J = 8.3 Hz), 6.96 (1H, t, J = 8.3 Hz);
MS (ESI) m/z: 272 (M++Na).
CDK4, CDK2, cyclin D, and cyclin E proteins were purified from
baculovirus infected Sf-9 insect cells. Glutathione S-transferase Rb
(GST-Rb) protein was expressed and purified from bacteria using
glutathione-sepharose beads by the standard procedure. Enzyme
assays to determine the concentration of compounds that cause
50% of kinase inhibition (IC50) were performed in 96-well filter
plates (Whatmann, GF/C filter). The assay mixture (30
10 mM ATP contained 0.2 mCi [33P]ATP, 30
L of enzymes (Cdk4/
D1 or Cdk2/E), 30 L of 25% GST-Rb with glutathione sepharose
beads in kinase assay buffer (50 mM Hepes pH 7.4, 10 mM MgCl2,
1 mM DTT, 2.5 mM EGTA, 5 mg/mL AMSF, 5 mg/mL aprotinine,
0.1 mM NaF, 10 mL b-glycerophosphate, and 0.1 mM sodium-o-
lL of
l
4.2.5.38. tert-Butyl 6-(trifluoromethanesulfonyl)oxy-3,4-dihy-
droisoquinoline-2(1H)-carboxylate (51). Compound 51 was ob-
tained from 50 as a colorless oil (quant.) by following the
procedure described for 46. 1H NMR (CDCl3) d: 1.49 (9H, s), 2.85
(2H, t, J = 6.0 Hz), 3.64 (2H, t, J = 6.0 Hz), 4.57 (2H, s), 7.05 (1H, d,
J = 2.4 Hz), 7.08 (1H, dd, J = 2.4, 8.5 Hz), 7.15 (1H, d, J = 8.5 Hz); IR
(ATR) cmÀ1: 2979, 1693, 1663, 1418, 1205, 1130; MS (ESI) m/z:
404 (M++Na), 326 (M+ÀtBu); HRMS (ESI) m/z: 404.07534 (calcd
for C15H18F3NNaO5S: 404.07555).
l
vanadate) and 10
lL of the tested compound in a final volume of
100 L) was incubated on the plate for 30 min at 30 °C. The plate
l
was washed for four times, and the kinase activities were mea-
sured. The IC50 values were determined by analyzing the dose–re-
sponse inhibition curves.
4.2.5.39. tert-Butyl 6-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-3,4-
dihydroisoquinoline-2(1H)-carboxylate (52). Compound 52 was
obtained from 51 as a yellowish oil (18%) by following the proce-
dure described for 47. 1H NMR (CDCl3) d: 1.34 (3H, t, J = 7.3 Hz),
1.49 (9H, s), 2.85 (2H, m), 3.65 (2H, m), 4.26 (2H, q, J = 7.3 Hz),
4.58 (2H, s), 6.41 (1H, d, J = 16.1 Hz), 7.12 (1H, d, J = 8.1 Hz), 7.29
(1H, s), 7.35 (1H, d, J = 8.1 Hz), 7.64 (1H, d, J = 16.1 Hz); MS (FAB)
m/z: 332 (M++H).
4.4. Cytotoxicity assay
An MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetra-
zolium bromide] assay was performed against HCT116 and PC-6
cell lines to examine the growth inhibitory effects of our com-
pounds. HCT116 cell line was purchased from American Type Cul-
ture Collection (ATCC, USA). PC-6 cell line was obtained from
Immuno-Biological Laboratories (Gunma, Japan). The cells were
maintained in RPMI 1640 medium supplemented with 10% fetal
bovine serum. The cells were plated in 96-well micro plates on
day 0, and stock solutions serially diluted in DMSO were added
to each well on day 1. After 3 days of culture (day 4), the number
of viable cells was determined by the MTT assay. The concentration
of compounds that cause 50% of growth inhibition was calculated
by the following equation 100 Â [(T À T0)/(CÀT0)] = 50 (C: control
optical density; T: test optical density; T0: optical density at time
zero).
4.2.5.40. tert-Butyl 6-formyl-3,4-dihydroisoquinoline-2(1H)-car-
boxylate (8p). To a solution of 52 (309 mg, 0.93 mmol) in a mix-
ture of THF (4 mL) and H2O (2 mL) were added OsO4 (2 mg,
1 mol %) and NaIO4 (1.40 g, 6.55 mmol) slowly, and the mixture
was stirred at 50 °C for 3.5 h. After EtOAc and satd Na2S2O3 aq
were added to the reaction mixture, the two layers were sepa-
rated. The aqueous layer was extracted with EtOAc, and the com-
bined organic layer was washed with brine, and dried over
Na2SO4. The solvent was removed under reduced pressure and
the residue was chromatographed on silica gel eluted with n-hex-
ane/EtOAc (4:1) to afford title compound 8p (112 mg, 46%) as a
colorless solid. 1H NMR (CDCl3) d: 1.50 (9H, s), 2.92 (2H, t,
J = 5.8 Hz), 3.68 (2H, t, J = 5.8 Hz), 4.65 (2H, s), 7.28 (1H, d,
J = 7.3 Hz), 7.66 (1H, s), 7.69 (1H, d, J = 7.3 Hz), 9.96 (1H, s); MS
(FAB) m/z: 262 (M++H).
4.5. Cell cycle analysis
HCT116 cells were treated with test compounds for 16 h and
were stained with propidium iodide using a Cycle Test Kit (Becton
Dickinson). Then the cells were analyzed using a FACscan flow
cytometer with CellQuest software (Becton Dickinson) in accor-
dance with the manufacturer’s recommendations. The percentage
of cells in the G0/G1, S, and G2/M phases of the cell cycle were
quantified using Modifit software (Verity Software House, Inc.).
4.2.5.41. 1,2,3,4-Tetrahydroisoquinoline-6-carbaldehyde (6-tert-
butylthieno[2,3-d]pyrimidin-4-yl)hydrazone (29). A mixture of 7
(86 mg, 0.39 mmol) and 8p (112 mg, 0.43 mmol) in benzene (2 mL)
was stirred under reflux for 2 h and cooled to room temperature.
The reaction mixture was concentrated under reduced pressure
and the residue was recrystallized from EtOAc and n-hexane to af-
ford tert-butyl 6-{[(6-tert-butylthieno[2,3-d]pyrimidin-4-yl)hydraz-
4.6. Antitumor activity assay
For in vivo studies, HCT116 tumor cells were subcutaneously
transplanted into the right hind limb of male BALB/c-nu/nu mice
(Japan SLC, Inc.) aged 5 or 6 weeks. After the subcutaneous tumors
averaged 80–100 mm3 in size, the tested compounds dissolved in
20% CaptisolÒ (b-cyclodextrin sulfobutylether sodium salt, CyDex,
Inc.) solution were administered intravenously or orally once a
day for 4 days at the doses indicated in Table 2. The tumor volume
was measured with a caliper periodically.
ono]methyl}-3,4-dihydroisoquinoline-2(1H)-carboxylate
(87 mg,
49%). To this solution isoquinolinecarboxylate in 1,4-dioxane
(1.0 mL) was added 4 N HCl/1,4-dioxane (0.5 mL) at 0 °C. The mix-
ture was stirred at room temperature for 12 h and concentrated
under reduced pressure. The residue was recrystallized from EtOAc
and n-hexane to afford title compound 29 (65 mg, 72%) as an or-
ange solid. 1H NMR (CD3OD) d: 1.53 (9H, s), 3.24 (2H, t,
J = 6.3 Hz), 3.57 (2H, t, J = 6.3 Hz), 4.45 (2H, s), 7.38 (1H, d,
J = 7.8 Hz), 7.84 (1H, s), 7.88 (1H, d, J = 7.8 Hz), 7.89 (1H, s), 8.54
(1H, s), 8.60 (1H, br); IR (ATR) cmÀ1: 2790, 1635, 1587, 1091;
MS (FAB) m/z: 366 (M++H); HRMS (ESI) m/z: 366.17526 (calcd for
C20H24N5S: 366.17524); Anal. Calcd for C20H23N5SÁ2.25HClÁ2H2O:
C, 49.68; H, 6.10; Cl, 16.50; N, 14.48; S, 6.63. Found: C, 49.89; H,
5.79; Cl, 16.68; N, 14.40; S, 6.66.
Acknowledgments
We would like to thank the analysis group of Daiichi Sankyo
R&D Associe Co., Ltd for their analytical and spectral determina-
tions. We would also like to thank Takashi Shimada of Exploratory
Research Laboratories I for his helpful suggestions on silico studies.

7860
T. Horiuchi et al. / Bioorg. Med. Chem. 17 (2009) 7850–7860
224, 771; (b) Havlicek, L.; Hanus, J.; Vesely, J.; Leclerc, S.; Meijer, L.; Shaw, G.;
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