Discovery of novel thieno[2,3-d]pyrimidin-4-yl hydrazone-based cyclin-dependent kinase 4 inhibitors: synthesis, biological evaluation and structure-activity relationships

Article abstract of DOI:10.1248/cpb.59.991

The design, synthesis, and evaluation of novel thieno[2,3-d]pyrimidin-4-yl hydrazone analogues as cyclin-dependent kinase 4 (CDK4) inhibitors are described. In continuing our program aim to search for potent CDK4 inhibitors, the introduction of a thiazole group at the hydrazone part has led to marked enhancement of chemical stability. Furthermore, by focusing on the optimization at the C-4′ position of the thiazole ring and the C-6 position of the thieno[2,3-d]pyrimidine moiety, compound 35 has been identified with efficacy in a xenograft model of HCT116 cells. In this paper, the potency, selectivity profile, and structure-activity relationships of our synthetic compounds are discussed.

Full text of DOI:10.1248/cpb.59.991

August 2011
Regular Article
Chem. Pharm. Bull. 59(8) 991—1002 (2011)
991
Discovery of Novel Thieno[2,3-d]pyrimidin-4-yl Hydrazone-Based
Cyclin-Dependent Kinase 4 Inhibitors: Synthesis, Biological
Evaluation and Structure–Activity Relationships
Takao HORIUCHI, ,a Yasuyuki TAKEDA,a Noriyasu HAGINOYA,a Masaki MIYAZAKI,a Motoko NAGATA,b
*
a
Mayumi KITAGAWA,b Kouichi AKAHANE,b and Kouichi UOTO
a Lead Discovery & Optimization Research Laboratories II, Daiichi Sankyo Co., Ltd.; and b Oncology Research
Laboratories, Daiichi Sankyo Co., Ltd.; 1–16–13 Kita-Kasai, Edogawa-ku, Tokyo 134–8630, Japan.
Received March 18, 2011; accepted May 19, 2011; published online May 27, 2011
The design, synthesis, and evaluation of novel thieno[2,3-d]pyrimidin-4-yl hydrazone analogues as cyclin-de-
pendent kinase 4 (CDK4) inhibitors are described. In continuing our program aim to search for potent CDK4 in-
hibitors, the introduction of a thiazole group at the hydrazone part has led to marked enhancement of chemical
stability. Furthermore, by focusing on the optimization at the C-4ꢀ position of the thiazole ring and the C-6 posi-
tion of the thieno[2,3-d]pyrimidine moiety, compound 35 has been identified with efficacy in a xenograft model of
HCT116 cells. In this paper, the potency, selectivity profile, and structure–activity relationships of our synthetic
compounds are discussed.
Key words cyclin-dependent kinase 4 inhibitor; thieno[2,3-d]pyrimidine-4-yl hydrazone
Cyclin-dependent kinases (CDKs), a family of serine/ 1, we found that thieno[2,3-d]pyrimidine hydrazones with a
threonine kinases, are responsible for coordinating the events phenyl ring had a common feature of poor chemical stability
by which cells progress through the cell cycle and become under acidic conditions. Since we aimed to investigate orally
active at specific phases: G1, S, G2, and M.1—6) In the G1 available drugs, we had to prevent the degradation of com-
phase, cyclin D1 increases to trigger the activation of pounds by gastric acid. Therefore, we tried to change the
CDK4/6 in early G1, and then cyclin E/CDK2 is activated. electronic environment around the hydrazone moiety by
CDK4/6 activities are negatively regulated by the tumor sup- introducing heteroaryl groups such as imidazoles, oxazoles,
pressor p16, a cyclin-dependent kinase inhibitor of the INK4 and thiazoles instead of the phenyl ring. We found that thia-
family. However, many tumors have been reported to contain zole compounds were well tolerated in the acidic condition
mutations, deletions, or silencing of p16.7—9) Moreover, mu- and they also possessed potent CDK4 inhibitory activity. To
tations in CDKs and abnormal expressions of their regulators further explore the potential of this class of compounds, we
have been found in a large percentage of melanoma pa- introduced various substituted thiazole at the hydrazone
tients.10) In addition, Malumbres and Barbacid have reported moiety and the alkyl group at the C-6 position of thieno[2,3-
that knockdown of CDK4 in mammary tumor cells prevents d]pyrimidine scaffold.
tumor formation,11) and McCormick and Tetsu have found
Herein, we report the chemical stabilities of compound 1,
that cancer cells proliferate despite CDK2 inhibition.12) and describe the design, synthesis of novel analogues of 1,3-
These results suggest that selective inhibition of CDK4 may thiazole-2-carbaldehyde thieno[2,3-d]pyrimidin-4-yl hydra-
restore normal cell activity and could be a more valuable ap- zone with marked enhancement of chemical stability, and
proach to cancer therapy than CDK2, especially for those their inhibitory activities for CDK4 with selectivity against
who have lost the INK4 family such as p16.
CDK2. We also report their cytotoxic potential against
In our previous paper, we reported the discovery of the human cancer cell lines.
potent CDK4 (IC50ꢀ0.038 mg/ml) inhibitor (1), which had
Chemistry and Chemical Stability Preparation of 1
moderate selectivity for CDK2 with strong cytotoxic activ- and 4a—n was accomplished using a general synthetic route
ity13,14) (Fig. 1). The cell cycle distribution analysis in as shown in Chart 1. The hydrazone analogues 4a—n were
HTC116 cells was carried out with selected compounds, produced by a condensation reaction with 2 and appropriate
same as 1, and the DNA content of the cells was assessed by aldehydes 3a—n in benzene, followed by deprotection in the
flow cytometry. After treating the cells with the compounds, case of 4d, 4e, and 4g—n.
significant increases in G0/G1 population were observed.
In our recent studies, the chemical stability of compound 1
Furthermore, apoptosis was induced in some compounds, was examined in pH 1.2 and pH 6.8 phosphate buffers at
which is indicated by the sub-G1 fraction. However, during
our studies to improve selectivity and the physical profile of
Reagents and conditions: (a) aldehydes 3a—n, benzene, reflux; (b)
deprotection with 4 N HCl in 1,4-dioxane in the case of 4d, e, g—n,
21—88% from 2.
Chart 1. Synthesis of Compounds 4a—n
Fig. 1. Structure of Compound 1
∗ To whom correspondence should be addressed. e-mail: horiuchi.takao.pi@daiichisankyo.co.jp
© 2011 Pharmaceutical Society of Japan

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Table 1. Enzyme Inhibitory Activity and Chemical Stability of (6-tert-Butylthieno[2,3-d]pyrimidine-4-yl)hydrazone Analogues
CDK4 IC50
CDK2 IC50
CDK4
Remaining rate (%)c)
in pH 1.2 buffer
Compd.
R1
(mg/ml)a)
(mg/ml)a)
selectivityb)
1
4-[(Methylamino)methyl]phenyl
1,3-Thiazol-2-yl
1,3-Thiazol-4-yl
0.038
0.041
0.89
0.68
0.20
2.2
17.9
4.9
2.5
1.1
38 (1.5 h)
79 (3 h)
75 (1 h)
4a
4b
4c
1,3-Thiazol-5-yl
0.052
0.059
86 (1.5 h)
a) Concentration (mg/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) Remaining rate of the substrate after incubation with pH 1.2 buffer at 37 °C. Num-
bers in parentheses indicate the incubation time.
Reagents and conditions: (a) NBS, AIBN, CCl4, 90 °C; MeNH2, THF, 0 °C; (Boc)2O,
Et3N, THF, 35% from 5a, 57% from 5b; (b) n-BuLi, N-formylmorpholine, THF,
Fig. 2. Plots of the Remaining Ratio of Compounds 1 and 4a versus Time
in Buffers
ꢁ78 °C, 35% from 6a, 32% from 6b, 44% from 6c; (c) LAH, THF, 0 °C; phthalimide,
PPh3, DEAD, 49% for two steps; (d) NH2NH2·1H2O, EtOH; (Boc)2O, NaHCO3,
CH2Cl2, 44% for two steps; (e) TBAF, THF; MnO2, CCl4, reflux, 20% for two steps.
Chart 2. Synthesis of Aldehydes 3d—g
37 °C, and the corresponding remaining ratios are given in
Fig. 2. Compound 1 is reasonably stable in neutral solution,
but a rapid decrease of 1 was observed in acidic conditions. tively. 6a was converted to aldehyde 3d with n-BuLi and N-
We then evaluated the acidic hydrolysis of 1 by HPLC analy- formylmorpholine. Aldehydes 3e and 3f were obtained from
sis. As a result, an inverse correlation was observed between 6b and commercial dimethylamine 6c in the same manner.
the production of hydrazine 2 and the decomposition of 1. In Aldehyde 3g was prepared from 7. Thiazole 7 was synthe-
order to overcome this problem, we tried to change the elec- sized using the reported method of Subhash et al.16) After re-
tronic environment around the hydrazone moiety by intro- duction of ethyl ester using lithium aluminum hydride
ducing heteroaryl groups (R1) as imidazoles, oxazoles, and (LAH), Mitsunobu reaction was carried out to introduce the
thiazoles instead of a benzene ring. We found that a series of amino methyl unit at the C-4 position. Deprotection of the
compounds 4a—c, bearing a non-substituted thiazole group, phthalimide group of 8 with hydrazine monohydrate gave the
was well tolerated in the acidic condition and they also pos- corresponding amine, followed by protection with the Boc
sessed potent CDK4 inhibitory activity as shown in Table 1. group. After deprotection of the tert-butyldiphenylsilyl
Among these compounds, 1,3-thiazole-2-yl compound 4a (TBDPS) group with tetrabutylammonium fluoride (TBAF),
showed excellent CDK4 inhibitory activity with some selec- oxidation of the resulting alcohol with MnO2 afforded alde-
tivity against CDK2 (4.9-fold). In contrast, 1,3-thiazole-5-yl hyde 3g.
compound 4c had poor selectivity. The plot of the remaining
The substituted thiazole aldehydes 3h—j were synthesized
ratio of compound 4a in pH 1.2 is shown in Fig. 2, with no as shown in Chart 3. As reported by Illig et al.,17) 11a was
data in pH 6.8 because 4a was insoluble under the condition. obtained from commercially available compound 9a using
From these results, we selected 4a to initiate our study to im- the reaction of thioamide 1016) and bromomethyl ketone in-
prove CDK4/CDK2 selectivity and cytotoxicity for further termediate, which was made by diazoketone and hydrogen
investigation.
bromide. After the protection of amine 13 with phthalic an-
The synthesis of aldehydes was described as follows. The hydride, 11b was synthesized from 9b in a similar manner.
thiazole aldehydes 3a—c were commercially available. The 12a, b were obtained from 11a, b by the procedure for 3g.
substituted thiazole aldehydes 3d—f were prepared using the 12c was prepared from 12a by deprotection of N-Boc and
reported method of Dority Jr. et al.15) as shown in Chart 2. methylation of resulting amine, followed by N-Boc protec-
After bromination of 5a, b and amination with methylamine, tion. Conversion of 12a—c to 3h—j was carried out by the
followed by N-Boc protection, 6a, b were produced, respec- procedure utilized for the preparation of 3g.

August 2011
993
Reagents and conditions: (a) SOCl2, cat. DMF, reflux; TMSCHN2, THF, 0 °C; (b) 47% HBr, Et2O; thioamide 10, EtOH, reflux, 67%
from 9a, 60% from 9b; (c) NH2NH2·1H2O, EtOH; (Boc)2O, NaHCO3, CH2Cl2, 89% from 11a, quant. from 11b; (d) MnO2, CCl4, reflux,
82% from 12a, 93% from 12b, 95% from 12c; (e) HCl, EtOH; TFAA, Et3N, CH2Cl2; MeI, K2CO3, DMF; 1 N NaOH, THF; (Boc)2O,
11%; (f) phthalic anhydride, 120 °C, 99%.
Chart 3. Synthesis of Aldehydes 3h—j
Reagents and conditions: (a) (COCl)2, cat. DMF, reflux; TMSCHN2, THF, 0 °C; 47% HBr, Et2O;
thioamide 10, EtOH, reflux, 26% from 14a, 44% from 14b, 70% from 14c; (b) BCl3, CH2Cl2;
(Boc)2O, Et3N, THF, 11% from 15a, 64% from 15b, 69% from 15c; (c) MnO2, CHCl3, reflux, 68%
from 16a, 83% from 16b, 60% from 16c, 81% from 19; (d) 18, Br2, AcOH; 17, DMF; MsCl, Et3N;
47% HBr, phenol; (Boc)2O, NaOH aq, CH2Cl2, 9% for five steps.
Chart 4. Synthesis of Aldehydes 3k—n
Reagents and conditions: (a) NH2NH2·1H2O, EtOH, reflux, 95% from 20, 31—58% from 23a—g; (b) PCC, CH2Cl2;
methyl cyanoacetate, S8, Et3N, DMF, 5—37% from 22a—f; (c) HCONH2, 210 °C; (d) POCl3, 110 °C; (e) p-cymene, p-
TsOH; LAH, Et2O, TBDPSCl, imidazole, THF, 38% for three steps; (f) OsO4, NaIO4, THF·H2O; methyl cyanoacetate, S8,
Et3N, DMF, 38% for two steps; (g) TBAF, THF, 70 °C; DAST, CH2Cl2; (h) aldehyde 3d or 3h, benzene, reflux; (i) TBAF,
THF, 70 °C in case of 24g; (j) 4 N HCl in 1,4-dioxane or 1 N HCl in EtOH, 2—97% from 21, 24a—h.
Chart 5. Synthesis of Compounds 29—38
The substituted thiazole aldehydes 3k—n, containing a clization with 17, dehydration was performed by treatment
cyclic alkyl group or fused ring, were synthesized as shown with methanesulfonyl chloride, followed by deprotection of
in Chart 4. 15a—c were obtained from 14a—c in a similar O-benzyl and N-Ts with HBr, and then N-Boc protection af-
manner to 11a, respectively. N-Cbz deprotection of 15a—c forded 19. Conversion to aldehyde 3n from 19 was per-
followed by N-Boc protection afforded 16a—c. Finally, formed as previously described.
16a—c were converted to 3k—m by the procedure for 3h.
The hydrazine 21 was prepared from 20 with hydrazine
Bicyclic compound 3n was synthesized from thioamide 17 monohydrate in ethanol under reflux. The thiophenes 23a—f
and pyrrolidin-3-one 18. After a-bromination of 18 and cy- were synthesized from appropriate alcohols 22a—f with

994
Vol. 59, No. 8
methyl cyanoacetate in the presence of sulfur by the method ported procedure.20) For hydrazine 24h, intermediate 28 was
of Tinney et al.18) 27 was obtained from 25 and 26 via 2,2-di- fluorinated with (dimethylamino)sulfur trifluoride after re-
methyl-4-pentenal,19) followed by reduction of aldehyde, and moval of TBDPS protection. The hydrazone analogues 29—
protection of hydroxide with TBDPSCl. Thiophene 23g was 38 were produced by a condensation reaction with hy-
synthesized in the same manner as 23a—f after Lemieux- drazines 21, 24a—h, and aldehyde 3d or 3h in benzene, fol-
Johnson oxidation of 27. Cyclization of 23a—g with for- lowed by deprotection (Chart 5).
mamide gave 4-oxothieno[2,3-d]pyrimidines. The hydrazines
Biological Evaluation Biological activities of the novel
24a—h were prepared from chlorination of 4-oxothieno[2,3- thieno[2,3-d]pyrimidin-4-yl hydrazone analogues were eval-
d]pyrimidines with phosphorus oxychloride, followed by uated in two assay systems: enzyme inhibitory activity
treatment with hydrazine monohydrate according to the re- against CDK4/CDK2 and cytotoxicity against two cell lines
Table 2. Enzyme Inhibitory and Cytotoxic Activity for Substituted Thiazole-2-carbaldehyde (6-tert-Butylthieno[2,3-d]pyrimidine-4-yl)hydrazones
CDK4 IC50
CDK2 IC50
CDK4
HCT116 IC50
PC-6 IC50
Remaining
rate (%)d)
Compd.
R1
(mg/ml)a)
(mg/ml)a)
selectivityb)
(mg/ml)c)
(mg/ml)c)
4a
4d
1,3-Thiazol-2-yl
0.041
0.034
0.20
0.98
4.9
0.419
0.343
0.381
0.214
80
85
28.8
4e
4f
0.042
0.043
0.35
1.4
8.3
0.156
0.328
0.108
0.241
NTe)
80
32.6
4g
4h
0.090
0.019
0.38
0.83
4.2
0.730
0.177
0.490
0.103
89
81
43.7
4i
4j
0.058
0.037
1.4
1.1
24.1
29.7
0.211
0.320
0.107
0.171
84
85
4k
4l
0.073
0.070
1.0
1.8
13.7
25.7
0.768
0.812
0.706
0.635
81
NTe)
4m
4n
0.014
0.025
1.7
121.4
8.0
0.929
0.458
NTe)
NTe)
90
0.20
0.240
a) Concentration (mg/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) Remaining rate of the substrate after 3 h incubation with pH 1.2 buffer at 37 °C.
e) NTꢀnot tested.

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Table 3. Enzyme Inhibitory and Cytotoxic Activity for Substituted Thieno[2,3-d]pyrimidines Focused on the C-6 (R6) Position
CDK4 IC50
CDK2 IC50
CDK4
HCT116 IC50
PC-6 IC50
Remaining
rate (%)d)
Compd.
R6
(mg/ml)a)
(mg/ml)a)
selectivityb)
(mg/ml)c)
(mg/ml)c)
4d
0.034
0.98
28.8
25.9
0.343
0.214
85
29
30
31
32
33
34
0.058
0.260
0.045
0.029
0.030
0.020
1.50
ꢃ2.0
1.4
0.380
0.421
0.521
0.203
0.431
0.340
0.221
0.271
0.393
0.114
0.201
0.166
85
83
31.1
15.2
46.7
40.5
80 (4 h)
80 (4 h)
NTe)
0.44
1.40
0.81
NTe)
35
36
37
38
0.022
0.069
0.140
0.046
0.88
1.8
40.0
26.1
12.9
14.7
0.315
1.32
0.108
0.681
0.078
0.029
83
NTe)
80
1.8
0.184
0.061
0.68
81
a) Concentration (mg/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) Remaining rate of the substrate after 3 h incubation with pH 1.2 buffer at 37 °C.
e) NTꢀnot tested.
(HCT116 human colon carcinoma and PC-6 human non- toxic activities. These results showed that the presence of the
small cell lung carcinoma).
substituent alkyl amino group at the C-4ꢂ position of the thia-
Previously, we reported that the presence of an ionizable zole ring is very important and its steric factor influences
nitrogen atom at the C-4ꢂ position on the benzene plays an cytotoxic activity.
important role in the cytotoxic activity.14) Therefore, we in-
For further improvement of cytotoxic activity, our effort
troduced a series of substituent groups with alkyl amine at was then focused on determining the effect of the alkyl group
the C-4ꢂ or C-5ꢂ position on the thiazole ring. All the deriva- at the C-6 position. The biological activity and chemical sta-
tives in Table 2 maintained fairly good CDK4 inhibitory ac- bility data for 29—38 are shown in Table 3. Branched alkyl
tivity and chemical stability. 4-[(Methylamino)methyl]-1,3- derivatives 29, 31—35 maintained potent CDK4 inhibitory
thiazole-2-yl compound 4d and 5-[(methylamino)methyl]- activity with selectivity (15.2 to 46.7-fold vs. CDK2).
1,3-thiazole-2-yl compound 4e sustained good CDK4 activ- Among these analogues, compounds 32 and 35 had moder-
ity as 4a, and 4d had improved selectivity (28.8-fold vs. ately good cytotoxicity against HCT116 and PC-6 (IC50
CDK2). On the other hand, 4e was less selective. Therefore, range: 0.108 to 0.315 mg/ml) with sufficient chemical stabil-
we undertook replacements with a variety of alkyl amine ity. Meanwhile, cyclic alkyl compounds 30, 31, and 33 were
analogues at the C-4ꢂ position. Primary amine compound 4g less potent in cytotoxic activity. Next, we assessed the
diminished cytotoxic activity and selectivity against CDK2. branched alkyl derivatives containing a heteroatom at the C-6
In contrast, tertiary amine compound 4f exhibited similar position. Hydroxyl compound 36 retained CDK4 inhibitory
IC50 values for 4d. Then we noted the steric factors around a activity, but reduced cytotoxic activity. By contrast, hy-
nitrogen atom of alkyl amine. Among the branched alkyl drophobic fluorinated analogue 37 improved cytotoxic activi-
amines 4h—j, compound 4h improved its CDK4 inhibitory ties with weaker activity of CDK4 inhibition than that of 36.
activity with good selectivity (43.7-fold vs. CDK2), and 4h Moreover, 38 had potent kinase inhibitory activity, and cyto-
exhibited an IC50 value of 0.103 mg/ml against PC-6 cells. toxic activity was nearly equivalent to that of 1. These results
Conversely, we found that cyclic alkyl amino groups, as ex- suggest that modification of side chains at the C-6 position
emplified by compounds 4k—n, markedly decreased cyto- probably plays a crucial role for the improvement of pharma-

996
Vol. 59, No. 8
di-tert-butyl dicarbonate (Boc)2O, diethyl azodicarboxylate (DEAD), N,N-
dimethylformamide (DMF), dimethyl sulfoxide (DMSO), lithium aluminum
hydride (LAH), methanesulfonyl chloride (MsCl), N-methylmorpholine-N-
oxide (NMO), pyridinium chlorochromate (PCC), TBAF, trifluoroacetic acid
(TFA), trifluoroacetic anhydride (TFAA), tetrahydrofuran (THF). Column
chromatography was performed with Merck silica gel 60 (particle size
0.060—0.200 or 0.040—0.063 mm). Flash column chromatography was per-
formed with Biotage FLASH Si packed columns. Thin layer chromatogra-
phy (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
1
spray. H-NMR spectra were recorded on a JEOL JNM EX400, and chemi-
cal shifts are given in ppm (d) from tetramethylsilane as an internal stan-
dard. Significant 1H-NMR data are tabulated in the following order: number
of protons, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multi-
plet; br, broad), coupling constant(s) in hertz. Infrared (IR) spectra were
recorded on a Hitachi 270-30 spectrometer and a JASCO FT/IR-6100. Elec-
tron spray ionization condition (ESI) mass spectra were recorded on an Agi-
lent 1100 and SCIEX API-150EX spectrometer. Fast atom bombardment
ionization condition (FAB) mass spectra were recorded on a JEOL JMS-
HX110 spectrometer. Electron impact ionization 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 Yokokawa Analysis IC7000RS,
and analytical results were within ꢅ0.4% of the theoretical values unless
otherwise noted.
1,3-Thiazole-2-carbaldehyde (6-tert-Butylthieno[2,3-d]pyrimidin-4-
yl)hydrazone (4a) A mixture of 6-tert-butyl-4-hydrazinothieno[2,3-d]-
pyrimidine 2 (60 mg, 0.27 mmol) and 1,3-thiazole-2-carbaldehyde 3a (26 ml,
0.30 mmol) in benzene (2 ml) was stirred under reflux for 1 h and cooled to
room temperature. The solvent was removed under reduced pressure, and the
residue was chromatographed on silica gel eluted with CHCl3/MeOH
(50 : 1). The eluate was concentrated under reduced pressure, and the residue
was recrystallized from n-hexane, EtOAc, and MeOH to afford title com-
pound 4a (35.4 mg, 41%) as a yellow solid. 1H-NMR (CD3OD) d: 1.52 (9H,
s), 7.66 (1H, d, Jꢀ3.4 Hz), 7.87 (1H, br), 7.99 (1H, d, Jꢀ3.4 Hz), 8.35 (1H,
s), 8.42 (1H, s); IR (ATR) cmꢁ1: 2968, 1552, 1431, 1354, 1124; MS (EI)
m/z: 317 (Mꢆ); HR-MS (ESI) m/z: 318.08234 (Calcd for C14H16N5S2:
318.08471); Anal. Calcd for C14H15N5S2: C, 52.97; H, 4.76; N, 22.06; S,
Fig. 3. Tumor Growth Inhibition Curves of 35
cokinetics as well.
To evaluate the antitumor effects in vivo, HCT116 cells
were subcutaneously transplanted into nude mice. Compound
35 was selected as a candidate because it possesses potent
CDK4 inhibitory activity, good selectivity, and cytotoxic ac-
tivity against HCT116 cells. In addition, after treating the
cells with 35, a marked increase in G0/G1 population (51%)
was observed by the cell cycle distribution analysis. There-
fore, 35 was administered intravenously (i.v.) and orally (per
os (p.o.)). As a result, we found that 35 exhibited a tumor
growth inhibition of 52% (i.v., 300 mg/kg) and 45% (p.o.,
300 mg/kg), respectively (Fig. 3).
Conclusion
In summary, we achieved the conversion into thiazole
from the phenyl ring at the heteroaryl moiety of the C-4 hy-
drazone with adequate chemical stability. Next, we will focus
on the optimization of the thiazole ring at the hydrazone and 20.20. Found: C, 52.77; H, 4.83; N, 21.83; S, 20.33.
1,3-Thiazole-4-carbaldehyde (6-tert-Butylthieno[2,3-d]pyrimidin-4-
the C-6 position of the thieno[2,3-d]pyrimidine moiety. Also,
compounds 4h, 32, 35, and 38 have been identified as potent,
selective CDK4 inhibitors with marked chemical stability
and cytotoxic activity. Moreover, 35 showed efficacy in a
xenograft model of the HCT116 cells. These results provide
valuable information for the design of a potent CDK4 in-
hibitor and they also hold great promise for cancer therapy.
We plan to evaluate the medical safety and pharmacokinetics
using 35 for more optimization of this analogue, and further
work will be reported elsewhere on the improvement of cyto-
toxic activity, CDK4 selectivity, and the physical profile in
due course.
yl)hydrazone (4b) Compound 4b was obtained from 2 and 1,3-thiazole-4-
carbaldehyde 3b as a pale yellow solid (59%) by following the procedure de-
scribed for 4a. 1H-NMR (CDCl3) d: 1.50 (9H, s), 7.74 (1H, s), 7.87 (1H, s),
8.13(1H, s), 8.51 (1H, s), 8.88 (1H, s), 8.98 (1H, br); IR (ATR) cmꢁ1: 2960,
1562, 1442; MS (FAB) m/z: 318 (MꢆꢆH); HR-MS (ESI) m/z: 318.08587
(Calcd for C14H16N5S2: 318.08471); Anal. Calcd for C14H15N5S2: C, 52.97;
H, 4.76; N, 22.06; S, 20.20. Found: C, 53.06; H, 4.68; N, 21.90; S, 20.17.
1,3-Thiazole-5-carbaldehyde (6-tert-Butylthieno[2,3-d]pyrimidin-4-
yl)hydrazone (4c) Compound 4c was obtained from 2 and 1,3-thiazole-5-
carbaldehyde 3c as an orange solid (85%) by following the procedure de-
scribed for 4a. 1H-NMR (CDCl3) d: 1.52 (9H, s), 7.77 (1H, s), 8.03 (1H, s),
8.11 (1H, s), 8.50 (1H, s), 8.84 (1H, s); IR (ATR) cmꢁ1: 1550, 1427, 1109;
MS (FAB) m/z: 318 (Mꢆ); HR-MS (ESI) m/z: 318.08159 (Calcd for
C14H16N5S2: 318.08471); Anal. Calcd for C14H15N5S2: C, 52.97; H, 4.76; N,
22.06; S, 20.20. Found: C, 53.22; H, 4.67; N, 22.07; S, 20.28.
Experimental
Chemical Stability Assay For the chemical stability assay in aqueous
solution, a 1 ml aliquot of 0.5 mg/ml stock solution of substrate in MeOH
tert-Butyl Methyl(1,3-thiazol-4-ylmethyl)carbamate (6a) To a solu-
tion of 4-methyl-1,3-thiazole 5a (3.0 g, 30.25 mmol) in CCl4 (60 ml) were
was added to 50 ml of the appropriate buffer solution. The prepared solution added N-bromosuccinimide (5.6 g, 31.77 mmol) and 2,2ꢂ-azobisisobutyroni-
was incubated at 37 °C in the dark. At regular intervals, samples of the solu-
tion were analyzed by HPLC using the following conditions: a Waters Xterra
RP18, 2.1 mmꢄ10 cm, 3.5 mm column; mobile phase, 25 mM pH 6.8 phos-
phate buffer/acetonitrileꢀ65/35; flow rate 0.3 ml/min; detector wavelength,
210 nm and 340 nm. Samples were also analyzed by LC-MS using the fol-
lowing conditions: (LC) a Waters Xterra RP18, 2.1 mmꢄ5 cm, 3.5 mm col-
umn; mobile phase, 10 mM pH 6.8 sodium acetate-acetic acid buffer/acetoni-
trileꢀ60/40; flow rate 0.4 ml/min; detector wavelength, 340 nm; (MS) a
ThermoQuest LC-Q; ionization condition, Electron spray ionization (ESI);
scanning method, positive scan (M.W. 100—400).
trile (250 mg, 1.51 mmol, 5 mol%). The reaction mixture was refluxed for
30 min under an atmosphere of nitrogen and cooled to room temperature.
The resulting white precipitate was filtered. The filtrate was diluted with
THF and partially evaporated to afford crude 4-bromomethyl-1,3-thiazole as
a THF solution, which was used in the next step without purification. Methyl-
amine (2 m THF solution, 45 ml, 90.75 mmol) was added successively to the
THF (15 ml) solution of 4-(bromomethyl)thiazole. The resulting mixture
was stirred for 18 h at room temperature. After completion of the reaction,
the insoluble matter was filtered off and the filtrate was evaporated. The
residue was dissolved in THF (60 ml) and the solution was cooled to 0 °C.
Boc2O (6.9 ml, 30.25 mmol) and Et3N (4.6 ml, 33.28 mmol) were added. The
solution was allowed to warm to room temperature and stirred for 5 h. The
resulting precipitate was filtered. The filtrate was concentrated under re-
Chemistry Unless otherwise noted, materials were obtained from com-
mercial suppliers and used without further purification. The solvent and
reagent names are abbreviated as follows: acetic acid (AcOH), tert-butyl-
diphenylsilyl chloride (TBDPSCl), 1,1ꢂ-carbonylbis-1H-imidazole (CDI), duced pressure, and the residue was chromatographed on silica gel eluted

August 2011
997
with n-hexane/EtOAc (3 : 1) to afford title compound 6a (2.4 g, 35%) as a
room temperature for 5 h. After cooling at 0 °C, MeOH (0.2 ml), water
yellow oil. 1H-NMR (CDCl3) d: 1.46 (9H, s), 2.94 (3H, s), 4.58 (2H, s), 7.12 (0.1 ml), 15% NaOH aq. (0.1 ml), and water (0.3 ml) were added to a reac-
(1H, s), 8.74 (1H, d, Jꢀ2.2 Hz); IR (ATR) cmꢁ1: 2976, 1686, 1390, 1142;
MS (ESI) m/z: 251 (MꢆꢆNa); MS (FAB) m/z: 229 (MꢆꢆH); HR-MS (ESI)
m/z: 229.10345 (Calcd for C10H17N2O2S: 229.10107).
tert-Butyl [(2-Formyl-1,3-thiazol-4-yl)methyl]methylcarbamate (3d)
To a solution of n-butyllithium (1.52 m in n-hexane, 3.02 ml, 4.60 mmol) in
Et2O (10 ml) under an atmosphere of argon at ꢁ78 °C was added dropwise a
solution of 6a (1.0 g, 4.38 mmol) in Et2O (10 ml). The mixture was stirred at
tion mixture successively. The precipitate was filtered through Celite. The
filtrate was concentrated under reduced pressure and the residue was chro-
matographed on silica gel eluted with n-hexane/EtOAc (2 : 1—1 : 1). The
eluate was concentrated under reduced pressure and the residue was dis-
solved in THF (20 ml). To this solution were added phthalimide (154.4 mg,
1.05 mmol), triphenylphosphine (275.3 mg, 1.05 mmol), and DEAD (165 ml,
1.05 mmol) under an atmosphere of nitrogen. The mixture was stirred at
ꢁ78 °C for 1 h, and then 4-formylmorpholine (462 ml, 4.60 mmol) was room temperature for 4.5 h. The reaction mixture was concentrated under re-
added. The mixture was stirred for 1 h at ꢁ78 °C, and then at ꢁ5 °C duced pressure, and the residue was chromatographed on silica gel eluted
overnight. The reaction mixture was then 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 pressure and the residue was chromatographed
with n-hexane/EtOAc (2 : 1) to afford title compound 8 (441.9 mg, 49%) as a
colorless solid. 1H-NMR (CDCl3) d: 1.10 (9H, s), 4.91 (2H, s), 4.94 (2H, s),
7.17 (1H, s), 7.34—7.42 (6H, m), 7.64—7.78 (8H, m), 7.85—7.88 (2H, m);
MS (FAB) m/z: 513 (MꢆꢆH).
on silica gel eluted with n-hexane/EtOAc (4 : 1) to afford title compound 3d
tert-Butyl (2-Formyl-1,3-thiazol-5-yl)carbamate (3g) To a solution of
1
(392.7 mg, 35%) as a yellow oil. H-NMR (CDCl3) d: 1.47 (9H, s), 2.98 8 (441.9 mg, 0.86 mmol) in EtOH (4.4 ml) was added hydrazine monohy-
(3H, s), 4.62 (2H, s), 7.48—7.58 (1H, br), 9.97 (1H, s); IR (ATR) cmꢁ1
:
drate (0.95 ml, 1.89 mmol) at room temperature. After 2 h, Boc2O (0.65 ml,
2.84 mmol), CH2Cl2 (4.4 ml), and satd NaHCO3 aq. (4.4 ml) were added to
the reaction mixture, and stirred for a further 2 h. The solution was parti-
2975, 2931, 2847, 1683, 1390, 1365, 1141; MS (EI) m/z: 256 (Mꢆ); HR-MS
(EI) m/z: 256.08964 (Calcd for C11H16N2O3S: 256.08816).
4-[(Methylamino)methyl]-1,3-thiazole-2-carbaldehyde (6-tert-Butyl- tioned between CH2Cl2 and water. The aqueous layer was washed several
thieno[2,3-d]pyrimidin-4-yl)hydrazone (4d) A mixture of 2 (82.6 mg, times with CH2Cl2 and the combined organic fractions were dried over
0.37 mmol) and 3d (100 mg, 0.39 mmol) in benzene (2 ml) was stirred under Na2SO4. The reaction mixture was concentrated under reduced pressure and
reflux for 0.5 h and cooled to room temperature. The reaction mixture was the residue was chromatographed on silica gel eluted with n-hexane/EtOAc
concentrated under reduced pressure. The residue was chromatographed on (4 : 1) to afford tert-butyl {[2-({[tert-butyl(diphenyl)silyl]oxy}methyl)-1,3-
silica gel eluted with CHCl3/MeOH (50 : 1) to afford tert-butyl [(2-{[(6-tert- thiazol-4-yl]methyl}carbamate. To a solution of the afforded compound
butylthieno[2,3-d]pyrimidin-4-yl)hydrazono]methyl}-1,3-thiazol-4-yl)-
methyl]methylcarbamate (112.5 mg, 66%) as a pale yellow solid. To a solu-
tion of hydrazone (76.9 mg, 0.17 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 11.5 h and concentrated under reduced pressure. The residue
was recrystallized from EtOAc and n-hexane to afford title compound 4d
(62.2 mg, 85%) as a pale yellow solid. 1H-NMR (DMSO-d6) d: 1.47 (9H, s),
(181.1 mg, 0.38 mmol) in THF (1.8 ml) was added TBAF (1 M THF solution,
0.37 ml, 0.37 mmol) at 0 °C, and stirred at room temperature for 16 h. The
reaction mixture was concentrated 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 residue was dis-
solved in CCl4 (1.0 ml). To this solution was added MnO2 (160 mg) and
stirred under reflux for 4.5 h. The reaction mixture was filtered through
2.61 (3H, br), 4.28 (2H, d, Jꢀ4.4 Hz), 7.83 (1H, s), 7.92 (1H, s), 8.49 (1H, Celite. The filtrate was concentrated under reduced pressure to afford title
s), 8.54 (1H, s), 9.25 (1H, br); IR (ATR) cm-1: 2359, 1801, 1560, 1417; MS
(FAB) m/z: 361 (MꢆꢆH); HR-MS (ESI) m/z: 361.12640 (Calcd for
C16H21N6S2: 361.12691); Anal. Calcd for C16H20N6S2·2HCl·0.25H2O: C,
43.88; H, 5.18; Cl, 16.19; N, 19.19; S, 14.64. Found: C, 43.67; H, 5.05; Cl,
15.99; N, 18.95; S, 14.56.
compound 3g (41.3 mg, 20%) as a yellow oil. 1H-NMR (CDCl3) d: 1.46
(9H, s), 4.52 (2H, d, Jꢀ6.1 Hz), 5.19 (1H, br), 7.59 (1H, s), 9.96 (1H, s); MS
(EI) m/z: 242 (Mꢆ).
4-(Aminomethyl)-1,3-thiazole-2-carbaldehyde (6-tert-Butylthieno[2,3-
d]pyrimidin-4-yl)hydrazone (4g) Compound 4g was obtained from 2 and
3g as a pale yellow solid (49%) by following the procedure described for 4d.
1H-NMR (DMSO-d6) d: 1.47 (9H, s), 4.18 (2H, br), 7.87 (1H, br), 7.88 (1H,
tert-Butyl Methyl(1,3-thiazol-5-yl)carbamate (6b) Compound 6b was
obtained from 5b as a yellow oil (57%) by following the procedure de-
1
scribed for 6a. H-NMR (CDCl3) d: 1.50 (9H, s), 2.85 (3H, s), 4.5 (2H, s), s), 8.49 (3H, br), 8.53 (1H, s); IR (ATR) cmꢁ1: 2615, 1637, 1508, 1107; MS
7.73 (1H, s), 8.73 (1H, s); MS (FAB) m/z: 229 (MꢆꢆH).
(FAB) m/z: 347 (MꢆꢆH); HR-MS (ESI) m/z: 347.11089 (Calcd for
C15H19N6S2: 347.11126); Anal. Calcd for C15H18N6S2·2HCl·0.5H2O: C,
42.05; H, 4.94; Cl, 16.55; N, 19.62; S, 14.97. Found: C, 42.27; H, 4.65; Cl,
16.74; N, 19.32; S, 14.69.
tert-Butyl [(2-Formyl-1,3-thiazol-5-yl)methyl]methylcarbamate (3e)
Compound 3e was obtained from 6b as a yellow oil (32%) by following the
procedure described for 3d. 1H-NMR (CDCl3) d: 1.50 (9H, s), 2.90 (3H, s),
4.63 (2H, s), 7.93 (1H, s), 9.94 (1H, s); MS (EI) m/z: 256 (Mꢆ).
2-{1-[2-(Hydroxymethyl)-4,5-dihydro-1,3-thiazol-4-yl]ethyl}-1H-isoin-
5-[(Methylamino)methyl]-1,3-thiazole-2-carbaldehyde (6-tert-Butyl- dole-1,3(2H)-dione (11a) A mixture of N-phtahloyl-DL-alanine 9a (2.19 g,
thieno[2,3-d]pyrimidin-4-yl)hydrazone (4e) Compound 4e was obtained 10 mmol), a catalytic amount of DMF (2 drops) and thionyl chloride
from 2 and 3e as a yellow solid (81%) by following the procedure described (1.09 ml, 15 mmol), was stirred under reflux for 30 min and cooled to room
1
for 4d. H-NMR (DMSO-d6) d: 1.47 (9H, s), 2.58 (3H, br), 4.48 (2H, br),
temperature. The solvent was removed under reduced pressure. The residue
was dissolved in THF (10 ml), and (trimethylsilyl)diazomethane (2 M n-
7.82 (1H, s), 8.07 (1H, s), 8.50 (1H, br), 8.53 (1H, s), 9.44 (1H, br), 9.52
(1H, br); IR (ATR) cmꢁ1: 2665, 1637, 1591, 1101; MS (ESI) m/z: 361 hexane solution, 12.5 ml, 25 mmol) in THF (25 ml) was added at 0 °C. The
(MꢆꢆH); HR-MS (ESI) m/z: 361.12789 (Calcd for C16H21N6S2:
reaction mixture was stirred at 0 °C for 18 h. After removal of the solvent
361.12691); Anal. Calcd for C16H20N6S2·2HCl·0.5H2O: C, 43.44; H, 5.24; under reduced pressure, the residue was separated with satd NaHCO3 aq.
Cl, 16.03; N, 18.99; S, 14.50. Found: C, 43.71; H, 5.14; Cl, 15.74; N, 18.74; and Et2O. The aqueous layer was extracted with Et2O and the combined or-
S, 14.37. ganic layer was washed with brine and dried over Na2SO4. The solvent was
4-[(Dimethylamino)methyl]-1,3-thiazole-2-carbaldehyde (3f) Com- removed under reduced pressure, and the residue was chromatographed on
pound 3f was obtained from 6c as a brown solid (44%) by following the pro-
silica gel eluted with n-hexane/EtOAc (2 : 1) to afford 1-diazo-3-phthalimi-
dobutan-2-one (2.35 g, 97%) as an orange oil. To the resulting oil (0.49 g,
2.0 mmol) in Et2O (20 ml) was added hydrobromic acid (47%, 0.28 ml,
1
cedure described for 3d. H-NMR (CDCl3) d: 2.32 (6H, s), 3.69 (2H, s),
7.58 (1H, s), 10.00 (1H, s); MS (FAB) m/z: 171 (MꢆꢆH).
4-[(Dimethylamino)methyl]-1,3-thiazole-2-carbaldehyde (6-tert-Butyl- 2.4 mmol) at 0 °C, and stirred at room temperature for 50 min. The reaction
thieno[2,3-d]pyrimidin-4-yl)hydrazone (4f) Compound 4f was obtained
mixture was neutralized with NaHCO3 aq, the aqueous layer was extracted
with Et2O, and the combined organic layer was washed with brine and dried
from 2 and 3f as a yellow solid (21%) by following the procedure described
1
for 4a. H-NMR (CDCl3) d: 1.52 (9H, s), 2.33 (6H, s), 3.63 (2H, s), 7.20 over Na2SO4. The solvent was removed under reduced pressure, and the
(1H, s), 3.75 (2H, s), 7.84 (1H, s), 8.14 (1H, s), 8.52 (1H, s); IR (ATR)
cmꢁ1: 2964, 1549, 1421, 1169; MS (FAB) m/z: 375 (MꢆꢆH); HR-MS (ESI) butyl(diphenyl)silyl]oxy}ethanethioamide 10 (0.66 g, 2.0 mmol), and the re-
m/z: 375.14159 (Calcd for C17H23N6S2: 375.14256); Anal. Calcd for action mixture was stirred under reflux for 12 h. After removal of the solvent
C17H22N6S2: C, 54.52; H, 5.92; N, 22.44; S, 17.12. Found: C, 54.66; H, 5.89; under reduced pressure, the residue was separated with water and EtOAc.
residue was dissolved in EtOH (20 ml). To this solution was added 2-{[tert-
N, 22.44; S, 17.09.
2-{[2-({[tert-Butyl(diphenyl)silyl]oxy}methyl)-1,3-thiazol-4-yl]methyl}-
The aqueous layer was extracted with EtOAc and the combined organic layer
was washed with brine and dried over Na2SO4. The solvent was removed
1H-isoindole-1,3(2H)-dione (8) To a solution of ethyl 2-(tert-butyl- under reduced pressure, and the residue was chromatographed on silica gel
diphenylsilyloxymethyl)thiazole-4-carboxylate 7 (748.3 mg, 1.76 mmol) in eluted with n-hexane/EtOAc (2 : 3) to afford title compound 11a (0.4 g,
1
THF (14 ml) was added LAH (67 mg, 1.76 mmol) at 0 °C, and stirred at
69%) as an orange oil. H-NMR (CDCl3) d: 1.92 (3H, d, Jꢀ7.3 Hz), 2.60

998
Vol. 59, No. 8
citric acid aq. The aqueous layer was extracted with CH2Cl2 and the com-
bined organic layer was washed with satd NaHCO3 aq. and brine and dried
over Na2SO4. The solvent was removed under reduced pressure and the
residue was dissolved in DMF (20 ml). To this solution, K2CO3 (1.9 g,
13.7 mmol) and methyl iodide (0.43 ml, 6.9 mmol) were added, and the reac-
tion mixture was stirred overnight at room temperature. The reaction mix-
ture was separated with EtOAc and 10% sodium hydrosulfite aq. The aque-
ous layer was extracted with EtOAc and the combined organic layer was
washed with water and brine and dried over Na2SO4. The solvent was re-
moved under reduced pressure and the residue was dissolved in THF (10 ml)
and 1 N NaOH aq. (10 ml). After stirring at room temperature for 1 h, Boc2O
(1.5 g, 0.9 mmol) in THF (5 ml) was added to the reaction mixture and
stirred for a further 30 min. The solution was partitioned between EtOAc
and water. The aqueous layer was washed with EtOAc and the combined or-
ganic layer was dried over Na2SO4. The reaction mixture was concentrated
under reduced pressure and the residue was chromatographed on silica gel
eluted with CHCl3/MeOH (40 : 1) to afford title compound 12c (0.17 g,
11%) as a colorless oil. 1H-NMR (CDCl3) d: 1.47 (9H, s), 1.53 (3H, d,
Jꢀ7.1 Hz), 2.69 (3H, s), 2.95 (1H, br), 4.90 (2H, s), 7.00 (1H, s).
(1H, br), 4.85 (2H, s), 5.66 (1H, q, Jꢀ7.3 Hz), 7.24 (1H, s), 7.69—7.72 (2H,
m), 7.81—7.84 (2H, m).
tert-Butyl {1-[2-(Hydroxymethyl)-4,5-dihydro-1,3-thiazol-4-yl]ethyl}-
carbamate (12a) To a solution of 11a (0.4 g, 1.39 mmol) in EtOH (10 ml)
was added hydrazine monohydrate (0.2 ml, 4.17 mmol). The mixture was
stirred at room temperature for 1 h. The solvent was removed under reduced
pressure and the residue was dissolved in CHCl3. The precipitate was filtered
through Celite. The filtrate was concentrated under reduced pressure and the
residue was dissolved in THF (5 ml). To this solution was added Boc2O
(0.33 g, 1.5 mmol) and stirred for 1 h. The reaction mixture was concentrated
under reduced pressure and the residue was chromatographed on silica gel
eluted with CHCl3/MeOH (40 : 1) to afford title compound 12a (0.32 g,
1
89%) as a pale yellow oil. H-NMR (CDCl3) d: 1.44 (9H, s), 1.48 (3H, d,
Jꢀ6.9 Hz), 2.90 (1H, br), 4.92 (3H, m), 5.09 (1H, br), 7.05 (1H, s).
tert-Butyl [1-(2-Formyl-4,5-dihydro-1,3-thiazol-4-yl)ethyl]carbamate
(3h) To a solution of 12a (0.32 g, 1.24 mmol) in CCl4 (10 ml) was added
MnO2 (500 mg) and the mixture was stirred under reflux for 18.5 h. The re-
action mixture was filtered through Celite. The filtrate was concentrated
under reduced pressure and the residue was chromatographed on silica gel
eluted with CHCl3/MeOH (100 : 1) to afford title compound 3h (0.26 g,
82%) as a yellow oil. 1H-NMR (CDCl3) d: 1.44 (9H, s), 1.56 (3H, d,
Jꢀ6.8 Hz), 5.01 (1H, br), 5.09 (1H, br), 7.53 (1H, s), 9.95 (1H, d,
Jꢀ1.3 Hz).
tert-Butyl [1-(2-Formyl-4,5-dihydro-1,3-thiazol-4-yl)ethyl]methylcar-
bamate (3j) Compound 3j was obtained from 12c as a pale yellow oil
1
(95%) by following the procedure described for 3h. H-NMR (CDCl3) d:
1.48 (9H, s), 1.62 (3H, d, Jꢀ7.3 Hz), 2.75 (3H, br), 5.62 (1H, br), 7.48 (1H,
s), 9.97 (1H, d, Jꢀ1.2 Hz).
4-(1-Aminoethyl)-1,3-thiazole-2-carbaldehyde (6-tert-Butylthieno[2,3-
d]pyrimidin-4-yl)hydrazone (4h) Compound 4h was obtained from 2 and
3h as a yellow solid (47%) by following the procedure described for 4d. 1H-
NMR (DMSO-d6) d: 1.46 (9H, s), 1.57 (3H, d, Jꢀ6.8 Hz), 4.57—4.61 (1H,
m), 7.87 (2H, br), 8.52 (4H, br); IR (KBr) cmꢁ1: 3338, 1631, 1587, 1552,
1504, 1421, 1367, 1338; MS (FAB) m/z: 361 (Mꢆꢆ1); HR-MS (ESI) m/z:
361.12668 (Calcd for C16H21N6S2: 361.12691); Anal. Calcd for
C16H20N6S2·2.4HCl·1.75H2O: C, 40.08; H, 5.44; Cl, 17.74; N, 17.59; S,
13.37. Found: C, 40.34; H, 5.25; Cl, 17.92; N, 17.74; S, 13.43.
2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)butanoic Acid (9b) A mix-
ture of 2-aminobutyric acid 13 (10.3 g, 100 mmol) and phthalic anhydride
(14.8 g, 100 mmol) was heated at 120 °C for 4 h. After cooling to room tem-
perature, CHCl3 (50 ml) and 1 N HCl aq. (50 ml) were added to the reaction
mixture. The aqueous layer was extracted with CHCl3 and the combined or-
ganic layer was washed with brine and dried over Na2SO4. The solvent was
removed under reduced pressure to afford title compound 9b (23 g, 99%) as
a colorless oil. 1H-NMR (CDCl3) d: 0.95 (3H, t, Jꢀ7.4 Hz), 2.25—2.32 (2H,
m), 4.81—4.85 (1H, m), 7.73—7.75 (2H, m), 7.86—7.88 (2H, m).
4-[1-(Methylamino)ethyl]-1,3-thiazole-2-carbaldehyde (6-tert-Butyl-
thieno[2,3-d]pyrimidin-4-yl)hydrazone (4j) Compound 4j was obtained
from 2 and 3j as a yellow solid (88%) by following the procedure described
1
for 4d. H-NMR (DMSO-d6) d: 1.45 (9H, s), 1.60 (3H, d, Jꢀ6.8 Hz), 2.44
(3H, t, Jꢀ5.4 Hz), 4.52—4.57 (1H, m), 7.90 (1H, br), 7.97 (1H, s), 8.53 (1H,
s), 8.58 (1H, br), 9.25—9.26 (1H, br), 9.61 (1H, br); IR (ATR) cmꢁ1: 2965,
2468, 1735, 1641, 1592, 1546, 1508, 1469, 1428; MS (FAB) m/z: 375
(MꢆꢆH); HR-MS (ESI) m/z: 375.14256 (Calcd for C17H23N6S2: 375.1444);
Anal. Calcd for C17H22N6S2·2HCl·0.5H2O: C, 44.73; H, 5.52; Cl, 15.53; N,
18.41; S, 14.05. Found: C, 44.41; H, 5.20; Cl, 15.20; N, 18.39; S, 14.05.
Benzyl 3-[2-(Hydroxymethyl)-1,3-thiazol-4-yl]azetidine-1-carboxylate
(15a) A mixture of 1-benzyloxycarbonylazetidine-3-carboxylic acid 14a
(1.2 g, 5.0 mmol), a catalytic amount of DMF (2 drops), and oxalyl chloride
(0.63 ml, 7.5 mmol), was stirred at 0 °C for 15 min and then stirred at room
temperature for 1 h. The solvent was removed under reduced pressure. The
residue was dissolved in THF (30 ml), and (trimethylsilyl)diazomethane
(2 M n-hexane solution, 6.25 ml, 12.5 mmol) was added at 0 °C. The reaction
mixture was stirred at 0 °C for 15 h. After removal of the solvent under re-
duced pressure, the residue 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 pressure. To the resulting residue in Et2O (20 ml) was added
hydrobromic acid (47%, 0.7 ml, 6.0 mmol) at 0 °C, and stirred at 0 °C for
1 h. The reaction mixture was neutralized with NaHCO3 aq., 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 EtOH (30 ml). To this solution
was added 2-{[tert-butyl(diphenyl)silyl]oxy}ethanethioamide 10 (1.65 g,
5.0 mmol), and the reaction mixture was stirred under reflux for 38 h. After
removal of the solvent under reduced pressure, the residue was separated
with water 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 pressure, and the residue was chro-
matographed on silica gel eluted with n-hexane/EtOAc (1 : 2) to afford title
2-{1-[2-(Hydroxymethyl)-4,5-dihydro-1,3-thiazol-4-yl]propyl}-1H-
isoindole-1,3(2H)-dione (11b) Compound 11b was obtained from 9b as a
1
brownish oil (60%) by following the procedure described for 11a. H-NMR
(CDCl3) d: 1.00 (3H, t, Jꢀ7.4 Hz), 2.32—2.39 (1H, m), 2.47—2.55 (1H,
m), 2.63 (1H, br), 4.85 (2H, s), 5.44 (1H, q, Jꢀ4.2 Hz), 7.26 (1H, s), 7.70—
7.72 (2H, m), 7.82—7.84 (2H, m).
tert-Butyl {1-[2-(Hydroxymethyl)-4,5-dihydro-1,3-thiazol-4-yl]ethyl}-
carbamate (12b) Compound 12b was obtained from 11b as a brownish
oil (quant.) by following the procedure described for 12a. 1H-NMR (CDCl3)
d: 0.87 (3H, t, Jꢀ7.3 Hz), 1.47 (9H, s), 1.81—1.90 (2H, m), 2.82 (1H, br),
4.67 (1H, d, Jꢀ7.1 Hz), 4.91 (2H, s), 5.12 (1H, br), 7.05 (1H, s).
tert-Butyl [1-(2-Formyl-4,5-dihydro-1,3-thiazol-4-yl)ethyl]carbamate
(3i) Compound 3i was obtained from 12b as a yellow oil (93%) by follow-
ing the procedure described for 3h. 1H-NMR (CDCl3) d: 0.92 (3H, t,
Jꢀ7.3 Hz), 1.47 (9H, s), 1.87—1.99 (2H, m), 4.79 (1H, br), 5.13 (1H, br),
7.52 (1H, s), 9.96 (1H, d, Jꢀ1.2 Hz).
4-(1-Aminopropyl)-1,3-thiazole-2-carbaldehyde (6-tert-Butylthieno-
[2,3-d]pyrimidin-4-yl)hydrazone (4i) Compound 4i was obtained from 2
and 3i as a yellow solid (57%) by following the procedure described for 4d.
1H-NMR (DMSO-d6) d: 0.82 (3H, t, Jꢀ7.3 Hz), 1.44 (9H, s), 1.88—2.03
(2H, m), 4.36 (1H, m), 7.95 (1H, s), 8.56 (1H, s), 8.66 (4H, br); IR (ATR)
cmꢁ1: 2960, 2069, 1943, 1731, 1633, 1577, 1504, 1482, 1421; MS (FAB)
m/z: 375 (MꢆꢆH); HR-MS (ESI) m/z: 375.13947 (Calcd for C17H23N6S2:
375.14256); Anal. Calcd for C17H22N6S2·2HCl·1.75H2O: C, 43.45; H, 5.68;
Cl, 15.09; N, 17.88: S, 13.65. Found: C, 43.65; H, 5.60; Cl, 14.80; N, 18.23;
S, 13.40.
1
compound 15a (0.4 g, 26%) as a brownish oil. H-NMR (CDCl3) d: 2.58
(1H, br s), 3.92—3.95 (1H, m), 4.19 (2H, dd, Jꢀ6.1, 8.8 Hz), 4.36 (1H, t,
Jꢀ8.5 Hz), 4.94 (2H, s), 5.12 (2H, s), 7.01 (1H, s), 7.35—7.37 (5H, m).
tert-Butyl 3-[2-(Hydroxymethyl)-1,3-thiazol-4-yl]azetidine-1-carboxy-
late (16a) To the solution of 15a (0.4 g, 1.3 mmol) in CH2Cl2 (20 ml) was
added boron trichloride (1 M CH2Cl2 solution, 4.0 ml, 4.0 mmol) at 0 °C
under N2 atmosphere, and stirred at room temperature for 3 d. The reaction
mixture was neutralized with NaHCO3 aq. and the solvent was removed
under reduced pressure. To the resulting residue were added Boc2O (0.43 g,
1.97 mmol) in THF (10 ml) and 1 N NaOH aq. (10 ml), and stirred at room
temperature for 30 min. To the reaction mixture EtOAc and water were
added, and 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 chromatographed on silica gel eluted with CHCl3/MeOH (40 : 1)
to afford title compound 16a (40 mg, 11%) as a brownish oil. 1H-NMR
tert-Butyl {1-[2-(Hydroxymethyl)-4,5-dihydro-1,3-thiazol-4-yl]ethyl}-
methylcarbamate (12c) To a solution of 12a (1.5 g, 5.8 mmol) in EtOH
(5.0 ml) was added 1 N HCl in EtOH (5.0 ml) at 0 °C. The mixture was
stirred at room temperature for 17 h and concentrated under reduced pres-
sure. To a suspension of the residue in CH2Cl2 (30 ml) were added Et3N
(4.1 ml, 29.1 mmol) and TFAA (2.1 ml, 14.5 mmol) at 0 °C, and stirred at
room temperature for 1 h. The reaction mixture was then separated with 10%

August 2011
999
(CDCl3) d: 1.45 (9H, s), 3.21 (1H, br), 3.85—3.91 (1H, m), 4.07 (1H, dd, pyrrolidin-3-one 18 (1.4 g, 5.86 mmol) in AcOH (15 ml) was added bromine
Jꢀ6.4, 8.3 Hz), 4.27 (1H, t, Jꢀ8.5 Hz), 4.93 (2H, s), 7.01 (1H, s).
(0.33 ml, 6.4 mmol) in AcOH (15 ml). The mixture was stirred at 50 °C for
tert-Butyl 3-(2-Formyl-1,3-thiazol-4-yl)azetidine-1-carboxylate (3k) 10 min. The reaction mixture was separated with water and CHCl
3. The
aqueous layer was extracted with CHCl3 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 with DMF (15 ml). To this
solution was added 2-(benzyloxy)ethanethioamide 17 (1.2 g, 6.6 mmol), and
the reaction mixture was stirred at 50 °C for 7 h under N2 atmosphere. After
completion of the reaction, NaHCO3 (0.5 g, 5.86 mmol) was added, and the
solvent was removed under reduced pressure. To the residue, EtOAc and
water were added, and 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 pres-
sure, and the residue was dissolved in CH2Cl2 (50 ml). To this solution were
added Et3N (4.1 ml, 29.3 mmol) and MsCl (0.91 ml, 11.7 mmol) at 0 °C
under N2 atmosphere, and stirred at room temperature for 22 h. To the reac-
tion mixture, CHCl3 and water were added, and the two layers were sepa-
rated. The aqueous layer was extracted with CHCl3 and the combined or-
ganic 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 CHCl3/MeOH (100 : 1). The eluate was concentrated
under reduced pressure. To the resulting residue were added phenol (2.0 g)
and hydrobromic acid (47%, 3.0 ml) at 0 °C, and stirred under reflux for 1 h.
To a solution of 16a (40 mg, 0.148 mmol) in CHCl3 (10 ml) was added
MnO2 (0.12 g), and stirred under reflux for 21 h. The reaction mixture was
filtered through Celite. The filtrate was concentrated under reduced pressure
and the residue was chromatographed on silica gel eluted with n-
hexane/EtOAc (2 : 1) to afford title compound 3k (27 mg, 68%) as a yellow
1
oil. H-NMR (CDCl3) d: 1.47 (9H, s), 3.96—4.03 (1H, m), 4.14 (2H, dd,
Jꢀ6.1, 8.3 Hz), 4.34 (2H, t, Jꢀ8.5 Hz), 7.47 (1H, s), 9.98 (1H, d, Jꢀ0.8 Hz).
4-Azetidin-3-yl-1,3-thiazole-2-carbaldehyde (6-tert-Butylthieno[2,3-
d]pyrimidin-4-yl)hydrazone (4k) Compound 4k was obtained from 2 and
3k as a yellow solid (85%) by following the procedure described for 4d. 1H-
NMR (DMSO-d6) d: 1.45 (9H, s), 4.15 (2H, m), 4.25 (3H, m), 7.74 (1H, s),
7.88 (1H, br), 8.52 (1H, s), 9.05 (1H, br), 9.37 (1H, br); IR (ATR) cmꢁ1
:
3380, 2962, 1633, 1583, 1560, 1508, 1423, 1365; MS (FAB) m/z: 373
(MꢆꢆH); MS (ESI) m/z: 373 (MꢆꢆH); HR-MS (ESI) m/z: 373.12682
(Calcd for C17H21N6S2: 373.12691); Anal. Calcd for C17H20N6S2·2.25HCl·
1.5H2O: C, 42.40; H, 5.28; Cl, 16.56; N, 17.45: S, 13.32. Found: C, 42.24;
H, 5.32; Cl, 16.35; N, 17.31; S, 13.15.
Benzyl 3-[2-(Hydroxymethyl)-1,3-thiazol-4-yl]pyrrolidine-1-carboxy-
late (15b) Compound 15b was obtained from 14b as a brownish oil (44%)
1
by following the procedure described for 15a. H-NMR (CDCl3) d: 2.10—
2.17 (1H, m), 2.28—2.30 (1H, m), 2.96 (1H, br), 3.44—3.69 (4H, m), After cooling to ambient temperature, Et
2O and water were added to the re-
action mixture. The two layers were separated, and the organic layer was ex-
tracted with 1 N HCl aq. The combined aqueous layer was alkalized with
10 N NaOH aq. To the basic solution, Boc2O (0.55 g, 2.5 mmol) in THF
(20 ml) was added, and stirred at room temperature for 23 h. To the reaction
mixture, EtOAc and water were added, and the two layers were separated.
3.84—3.89 (1H, m), 4.91 (2H, s), 5.14 (2H, d, Jꢀ2.4 Hz), 6.93 (1H, d,
Jꢀ2.2 Hz), 7.32—7.36 (5H, m).
tert-Butyl 3-[2-(Hydroxymethyl)-1,3-thiazol-4-yl]pyrrolidine-1-car-
boxylate (16b) Compound 16b was obtained from 15b as a brownish oil
1
(64%) by following the procedure described for 16a. H-NMR (CDCl3) d:
1.46 (9H, s), 2.06—2.14 (1H, m), 2.25 (1H, br), 2.76 (1H, br), 3.41—3.59 The aqueous layer was extracted with EtOAc and the combined organic layer
(4H, m), 3.74—3.77 (1H, m), 4.92 (2H, s), 6.94 (1H, s).
was washed with brine and dried over Na2SO4. The solvent was removed
tert-Butyl 3-(2-Formyl-1,3-thiazol-4-yl)pyrrolidine-1-carboxylate (3l)
Compound 3l was obtained from 16b as a yellow oil (83%) by following the
procedure described for 3k. H-NMR (CDCl3) d: 1.48 (9H, s), 2.15—2.20
(1H, m), 2.33 (1H, br), 3.46—3.63 (4H, m), 3.81—3.86 (1H, m), 7.41 (1H,
s), 9.96 (1H, s).
under reduced pressure and the residue was chromatographed on silica gel
eluted with CHCl3/MeOH (50 : 1) to afford title compound 19 (0.13 g, 9%)
as a brownish solid. 1H-NMR (CDCl3) d: 1.52 (s, 9H), 2.48 (br, 1H), 4,52—
4.53 (m, 1H), 4.58 (br, 1H), 4.66—4.67 (m, 2H), 4.94 (s, 2H); MS (EI) m/z:
257 (MꢆꢆH).
1
4-Pyrrolidin-3-yl-1,3-thiazole-2-carbaldehyde (6-tert-Butylthieno[2,3-
d]pyrimidin-4-yl)hydrazone (4l) Compound 4l was obtained from 2 and
3l as a yellow solid (84%) by following the procedure described for 4d. 1H-
NMR (DMSO-d6) d: 1.45 (9H, s), 2.07—2.10 (1H, m), 2.32—2.36 (1H, m),
tert-Butyl 2-Formyl-4,6-dihydro-5H-pyrrolo[3,4-d][1,3]thiazole-5-car-
boxylate (3n) Compound 3n was obtained from 19 as a brownish solid
(81%) by following the procedure described for 3k. H-NMR (CDCl3) d:
1.53 (s, 9H), 4.64—4.66 (m, 1H), 4.69 (br, 1H), 4.77—4.88 (m, 2H), 9.93
1
3.25—3.27 (2H, m), 3.35 (1H, m), 3.58—3.64 (1H, m), 3.66—3.70 (1H, m), (s, 1H).
7.70 (1H, s), 7.87 (1H, br), 8.51 (2H, s), 9.32 (2H, br); IR (ATR) cmꢁ1
:
5,6-Dihydro-4H-pyrrolo[3,4-d][1,3]thiazole-2-carbaldehyde (6-tert-
2960, 1735, 1633, 1587, 1556, 1504, 1402; MS (FAB) m/z: 387 (MꢆꢆH); Butylthieno[2,3-d]pyrimidin-4-yl)hydrazone (4n) Compound 4n was
HR-MS (ESI) m/z: 387.14215 (Calcd for C18H23N6S2: 387.14256); Anal. obtained from 2 and 3n as a brownish solid (40%) by following the proce-
1H-NMR (DMSO-d6) d: 1.47 (9H, s), 4,43 (2H, br),
Calcd for C18H22N6S2·2.7HCl·H2O: C, 42.98; H, 5.35; Cl, 19.03; N, 16.71: dure described for 4d.
S, 12.75. Found: C, 43.14; H, 5.35; Cl, 18.83; N, 16.52; S, 12.66.
4.62 (2H, br), 7.77 (1H, s), 8.42 (1H, br), 8.52 (1H, s), 10.26 (1H, br); IR
(ATR) cmꢁ1: 2962, 1635, 1581, 1515, 1425, 1365; MS (FAB) m/z: 359
(MꢆꢆH); MS (ESI) m/z: 359 (MꢆꢆH); HR-MS (ESI) m/z: 359.11144
(Calcd for C16H19N6S2: 359.11126); Anal. Calcd for C16H18N6S2·1.9HCl·
2H2O: C, 41.35; H, 5.40; Cl, 14.49; N, 18.08; S, 13.80. Found: C, 41.27; H,
5.06; Cl, 14.68; N, 17.88; S, 13.77.
Benzyl 4-[2-(Hydroxymethyl)-1,3-thiazol-4-yl]piperidine-1-carboxy-
late (15c) Compound 15c was obtained from 14c as a brownish oil (30%)
1
by following the procedure described for 15a. H-NMR (CDCl3) d: 1.56—
1.64 (4H, m), 2.02—2.04 (1H, m), 2.53 (1H, br), 2.89—2.96 (3H, m), 4.27
(1H, br), 4.91 (2H, s), 5.14 (2H, s), 6.86 (1H, s), 7.31—7.37 (5H, m).
tert-Butyl 4-[2-(Hydroxymethyl)-1,3-thiazol-4-yl]piperidine-1-car-
boxylate (16c) Compound 16c was obtained from 15c as a yellow oil
4-Hydrazino-6-isopropylthieno[2,3-d]pyrimidine (21) To a solution
of 20 (13.8 g, 64.9 mmol) in EtOH (250 ml) was added hydrazine monohy-
drate (140 ml). The mixture was stirred under reflux for 3 h and cooled to
1
(69%) by following the procedure described for 16a. H-NMR (CDCl3) d:
1.47 (9H, s), 1.59—1.66 (2H, m), 1.99—2.03 (2H, m), 2.67 (1H, br), room temperature. The precipitate was collected by filtration and washed
2.802.92 (3H, m), 4.20 (2H, br), 4.92 (2H, s), 6.86 (1H, s).
with n-hexane to afford title compound 21 (12.9 g, 95%) as a pale yellow
tert-Butyl 4-(2-Formyl-1,3-thiazol-4-yl)piperidine-1-carboxylate (3m) solid. 1
Compound 3m was obtained from 16c as an orange oil (60%) by following
H-NMR (DMSO-d6) d: 1.32 (6H, d, Jꢀ6.8 Hz), 3.15—3.21 (1H, m),
7.35 (1H, br), 8.28 (1H, s); IR (KBr) cmꢁ1: 3195, 2957, 1576, 1504, 1345,
1306, 1142; MS (ESI) m/z: 209 (MꢆꢆH); HR-MS (ESI) m/z: 209.08350
(Calcd for C9H13N4S: 209.08609); Anal. Calcd for C9H12N4S: C, 51.90; H,
5.81; N, 26.90; S, 15.39. Found: C, 51.71; H, 5.84; N, 26.77; S, 15.41.
4-[(Methylamino)methyl]-1,3-thiazole-2-carbaldehyde (6-Isopropyl-
thieno[2,3-d]pyrimidin-4-yl)hydrazone (29) Compound 29 was obtained
from 3d and 21 as a yellow solid (45%) by following the procedure de-
scribed for 4d. 1H-NMR (DMSO-d6) d: 1.41 (6H, d, Jꢀ6.6 Hz), 2.59—2.61
(3H, br), 4.29 (2H, br), 7.80 (1H, s), 7.93 (1H, s), 8.52 (2H, s), 9.21 (2H,
br); IR (ATR) cmꢁ1: 2696, 1637, 1587; MS (ESI) m/z: 347 (MꢆꢆH); MS
(ESI) m/z: 347 (MꢆꢆH); HR-MS (ESI) m/z: 347.10962 (Calcd for
C15H19N6S2: 347.11126); Anal. Calcd for C15H18N6S2·13/7HCl·2.5H2O: C,
39.23; H, 5.46; Cl, 14.34; N, 18.30; S, 13.96; Found: C, 39.28; H, 5.20; Cl,
1
the procedure described for 3k. H-NMR (CDCl3) d: 1.48 (9H, s), 1.63—
1.73 (2H, m), 2.04—2.07 (2H, m), 2.88 (1H, br), 2.99—3.05 (1H, m), 3.49
(2H, d, Jꢀ4.6 Hz), 4.22 (1H, br), 7.34 (1H, s), 9.96 (1H, d, Jꢀ1.2 Hz).
4-Piperidin-4-yl-1,3-thiazole-2-carbaldehyde (6-tert-Butylthieno[2,3-
d]pyrimidin-4-yl)hydrazone (4m) Compound 4m was obtained from 2
and 3m as a yellow solid (48%) by following the procedure described for
1
4d. H-NMR (DMSO-d6) d: 1.46 (9H, s), 1.82—1.98 (2H, m), 2.12—2.16
(2H, m), 2.98—3.10 (3H, m), 3.32—3.35 (2H, m), 7.55 (1H, s), 7.86 (1H,
s), 8.47 (1H, br), 8.51 (1H, s), 8.71 (1H, br), 8.95 (1H, br); IR (ATR) cmꢁ1
:
2962, 1864, 1631, 1587, 1552, 1504, 1400, 1367; MS (FAB) m/z: 401
(MꢆꢆH); MS (ESI) m/z: 401 (MꢆꢆH); HR-MS (ESI) m/z: 401.15792
(Calcd
for
C19H25N6S2:
401.15821);
Anal.
Calcd
for
C19H24N6S2·2.75HCl·1.25H2O: C, 43.60; H, 5.63; Cl, 18.63; N, 16.06: S, 14.12; N, 18.00; S, 13.84.
12.25. Found: C, 43.20; H, 5.36; Cl, 19.02; N, 15.91; S, 12.17.
tert-Butyl 2-(Hydroxymethyl)-4,6-dihydro-5H-pyrrolo[3,4-d][1,3]thia-
zole-5-carboxylate (19) To a solution of 1-[(4-methylphenyl)sulfonyl]-
Methyl 2-Amino-5-cyclopropylthiophene-3-carboxylate (23a) To a
suspension of pyridinium chlorochromate (7.50 g, 34.8 mmol) in CH
2Cl2
(50 ml) was added 2-cyclopropylethanol 22a (2.0 g, 23.2 mmol), and stirred

1000
Vol. 59, No. 8
(1H, s), 8.54 (1H, s), 8.61 (1H, br), 9.33 (2H, br); IR (ATR) cmꢁ1: 2663,
1630, 1589, 1101; MS (FAB) m/z: 359 (MꢆꢆH); MS (ESI) m/z: 359
(MꢆꢆH); HR-MS (ESI) m/z: 359.10991 (Calcd for C16H19N6S2:
359.11126); Anal. Calcd for C16H18N6S2·2HCl·0.5H2O: C, 43.63; H, 4.81;
Cl, 16.10; N, 19.08; S, 14.56; Found: C, 43.54; H, 4.65; Cl, 16.07; N, 19.13;
S, 14.56.
Methyl 2-Amino-5-(1-methylpropyl)thiophene-3-carboxylate (23c)
Compound 23c was obtained from 22c as a pale yellow oil (36%) by follow-
ing the procedure described for 23a. 1H-NMR (CDCl3) d: 0.88 (3H, t,
Jꢀ7.0 Hz), 1.22 (3H, d, Jꢀ7.0 Hz), 1.50—1.60 (2H, m), 2.62—2.69 (1H,
m), 3.79 (3H, s), 5.79 (2H, br), 6.62 (1H, s); MS (ESI) m/z: 254
(MꢆꢆMeCN).
6-sec-Butyl-4-hydrazinothieno[2,3-d]pyrimidine (24c) Compound
24c was obtained from 23c as a pale yellow oil (56%) by following the pro-
cedure described for 24a. 1H-NMR (CDCl3) d: 0.91 (3H, t, Jꢀ7.0 Hz), 1.36
(3H, d, Jꢀ7.0 Hz), 1.64—1.72 (2H, m), 2.91—2.99 (1H, m), 6.57 (1H, br),
6.89 (1H, s), 8.50 (1H, s); IR (KBr) cmꢁ1: 3270, 2954, 1583; MS (ESI) m/z:
223 (MꢆꢆH); HR-MS (ESI) m/z: 223.10034 (Calcd for C10H15N4S:
223.10174); Anal. Calcd for C10H14N4S: C, 54.03; H, 6.35; N, 25.20; S,
14.42. Found: C, 53.99; H, 6.39; N, 25.52; S, 14.56.
4-[(Methylamino)methyl]-1,3-thiazole-2-carbaldehyde (6-sec-Butyl-
thieno[2,3-d]pyrimidin-4-yl)hydrazone (32) Compound 32 was obtained
from 3d and 24c as a yellow solid (45%) by following the procedure de-
scribed for 4d. 1H-NMR (DMSO-d6) d: 0.92 (3H, t, Jꢀ7.3 Hz), 1.38 (3H, d,
Jꢀ6.8 Hz), 1.68—1.76 (2H, m), 2.58—2.61 (3H, m), 3.06—3.13 (1H, m),
4.29 (2H, m), 7.83 (1H, s), 7.95 (1H, s), 8.53 (1H, s), 8.59 (1H, br), 9.29
(2H, br); IR (ATR) cmꢁ1: 2692, 1639, 1099; MS (FAB) m/z: 361 (MꢆꢆH);
HR-MS (ESI) m/z: 361.12685 (Calcd for C16H21N6S2: 361.12691); Anal.
Calcd for C16H20N6S2·2HCl·0.5H2O: C, 43.44; H, 5.24; Cl, 16.03; N, 18.99;
S, 14.50. Found: C, 43.70; H, 5.07; Cl, 16.04; N, 19.18; S, 14.53.
Methyl 2-Amino-5-(1-methylbutyl)thiophene-3-carboxylate (23d)
Compound 23d was obtained from 22d as a yellow oil (24%) by following
the procedure described for 23a. 1H-NMR (CDCl3) d: 0.89 (3H, t,
Jꢀ7.3 Hz), 1.21 (3H, d, Jꢀ7.1 Hz), 1.26—1.35 (2H, m), 1.41—1.53 (2H,
m), 2.75 (1H, td, Jꢀ13.9, 7.1 Hz), 3.78 (3H, s), 5.77 (2H, br), 6.61 (1H, s);
IR (ATR) cmꢁ1: 3332, 2954, 1664, 1584, 1500, 1439, 1263; MS (ESI) m/z:
228 (MꢆꢆH); HR-MS (ESI) m/z: 228.10266 (Calcd for C11H18NO2S:
228.10582).
4-Hydrazino-6-(1-methylbutyl)thieno[2,3-d]pyrimidine (24d) Com-
pound 24d was obtained from 23d as a colorless solid (52%) by following
the procedure described for 24a. 1H-NMR (CDCl3) d: 0.91 (3H, t,
Jꢀ7.3 Hz), 1.27—1.36 (2H, m), 1.36 (3H, d, Jꢀ6.8 Hz), 1.56—1.69 (2H,
m), 2.50 (1H, m), 3.06 (1H, td, Jꢀ13.9, 6.8 Hz), 6.48 (1H, br), 6.88 (1H, s),
8.50 (1H, s); IR (ATR) cmꢁ1: 3260, 2958, 2925, 1579, 1516, 1355; MS
(ESI) m/z: 237 (MꢆꢆH); HR-MS (ESI) m/z: 237.11384 (Calcd for
C11H17N4S: 237.11739); Anal. Calcd for C11H16N4S: C, 55.90; H, 6.82; N,
23.71; S, 13.57. Found: C, 55.96; H, 6.79; N, 23.72; S, 13.41.
vigorously at room temperature under N2 atmosphere. After 1.5 h of stirring,
Et2O (180 ml) was added and stirred for 30 min. The reaction mixture passed
through a Florisil® column to afford cyclopropylacetaldehyde (1.59 g) as a
colorless oil. The resulting aldehyde was added to a solution of methyl
cyanoacetate (1.50 ml, 17 mmol), Et3N (2.40 ml, 17 mmol), and DMF (2.65
ml, 34 mmol) under N2 atmosphere. After 10 min of stirring, S8 (545 mg,
17 mmol) was added and stirred at room temperature for 40 h. The reaction
mixture was poured into water 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 pres-
sure, and the residue was chromatographed on silica gel eluted with n-
hexane/EtOAc (9 : 1) and recrystallized from n-pentane to afford title com-
pound 23a (580 mg, 13%) as a pale yellow solid. 1H-NMR (CDCl3) d:
0.60—0.67 (2H, m), 0.80—0.86 (2H, m), 1.77—1.83 (1H, m), 3.78 (3H, s),
5.78 (2H, br), 6.59 (1H, s); IR (KBr) cmꢁ1: 3423, 3311, 1662, 1602; MS
(EI) m/z: 197 (Mꢆ); Anal. Calcd for C9H11NO2S: C, 54.80; H, 5.62; N, 7.10;
S, 16.26. Found: C, 55.10; H, 5.62; N, 7.02; S, 16.18.
6-Cyclopropyl-4-hydrazinothieno[2,3-d]pyrimidine (24a) A mixture
of 23a (550 mg, 2.79 mmol) in formamide (5.5 ml) was stirred at 210 °C for
2.5 h. After cooling, precipitate formed, which was separated from the solu-
tion by filtration and washed with water. The crude product was chro-
matographed on silica gel eluted with n-hexane/EtOAc (2 : 1) and recrystal-
lized from EtOAc and n-hexane to afford 6-cyclopropylthieno[2,3-d]pyri-
midin-4(3H)-one (285 mg, 53%) as a colorless solid. The resulting com-
pound (255 mg, 1.32 mmol) in phosphorous oxychloride (3.0 ml) was stirred
at 110 °C for 6 h. The reaction mixture was concentrated under reduced pres-
sure. The residue was poured into ice water, and sat. NaHCO3 aq. was added
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 chro-
matographed on silica gel eluted with n-hexane/EtOAc (9 : 1) to afford 4-
chloro-6-cyclopropylthieno[2,3-d]pyrimidine (275 mg, 99%) as a colorless
solid. To a solution of the resulting compound (250 mg, 1.18 mmol) in EtOH
(5.0 ml) was added hydrazine monohydrate (2.5 ml). The mixture was stirred
under reflux for 1.5 h and cooled to room temperature. The reaction mixture
was concentrated under reduced pressure. After EtOAc and water 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 removed under reduced pressure and the
residue was recrystallized from EtOAc and n-hexane to afford title com-
1
pound 24a (190 mg, 78%) as a colorless solid. H-NMR (CDCl3) d: 0.81—
0.86 (2H, m), 1.08—1.12 (2H, m), 2.11—2.18 (1H, m), 6.39 (1H, br), 6.85
(1H, d, Jꢀ1.0 Hz), 8.48 (1H, s); IR (KBr) cmꢁ1: 3243, 3197, 1585; EI m/z:
206 (Mꢆ). MS (ESI) m/z: 207 (MꢆꢆH); HR-MS (ESI) m/z: 207.06637
(Calcd for C9H11N4S: 207.07044); Anal. Calcd for C9H10N4S: C, 52.41; H,
4.89; N, 27.16; S, 15.54. Found: C, 52.32; H, 4.85; N, 27.17; S, 15.67.
4-[(Methylamino)methyl]-1,3-thiazole-2-carbaldehyde (6-Cyclopropyl-
thieno[2,3-d]pyrimidin-4-yl)hydrazone (30) Compound 30 was obtained
from 3d and 24a as a yellow solid (49%) by following the procedure de-
scribed for 4d. 1H-NMR (DMSO-d6) d: 0.84—0.88 (2H, m), 1.15—1.23
(2H, m), 4.29 (2H, t, Jꢀ5.4 Hz), 7.77 (1H, br), 7.98 (1H, s), 8.53 (1H, s),
8.66 (1H, br), 9.83 (2H, br); IR (ATR) cmꢁ1: 2657, 1630, 1585, 1099; MS
(FAB) m/z: 345 (MꢆꢆH); MS (ESI) m/z: 345 (MꢆꢆH); HR-MS (ESI) m/z:
345.09333 (Calcd for C15H17N6S2: 345.09561); Anal. Calcd for
C15H16N6S2·1.9HCl·0.75H2O: C, 42.17; H, 4.58; Cl, 15.77; N, 19.67; S,
15.01. Found: C, 42.31; H, 4.44; Cl, 16.17; N, 19.43; S, 14.66.
4-[(Methylamino)methyl]-1,3-thiazole-2-carbaldehyde [6-(1-Methyl-
butyl)thieno[2,3-d]pyrimidin-4-yl]hydrazone (33) Compound 33 was
obtained from 3d and 24d as a colorless solid (79%) by following the proce-
dure described for 4d. 1H-NMR (DMSO-d6) d: 0.91 (3H, t, Jꢀ7.3 Hz),
1.30—1.36 (2H, m), 1.38 (3H, d, Jꢀ6.8 Hz), 1.63—1.71 (2H, m), 2.59 (3H,
t, Jꢀ5.3 Hz), 3.17 (1H, q, Jꢀ6.6 Hz), 4.28 (2H, t, Jꢀ5.4 Hz), 7.81 (1H, s),
7.95 (1H, s), 8.52 (1H, s), 8.56 (1H, br), 9.27 (2H, br); IR (ATR) cmꢁ1
:
1643, 1596, 1550, 1511, 1463, 1423, 1382, 1242, 1101; MS (FAB) m/z: 375
(MꢆꢆH); MS (ESI) m/z: 375 (MꢆꢆH); HR-MS (ESI) m/z: 375.14012
(Calcd for C17H23N6S2: 375.14256); Anal. Calcd for C17H22N6S2·1.8HCl: C,
46.39; H, 5.45; Cl, 14.50; N, 19.09; S, 14.57. Found: C, 46.40; H, 5.34; Cl,
14.24; N, 19.12; S, 14.51.
Methyl 2-Amino-5-cyclobutylthiophene-3-carboxylate (23b) Com-
pound 23b was obtained from 22b as a pale yellow oil (37%) by following
1
the procedure described for 23a. H-NMR (CDCl3) d: 1.79—2.34 (6H, m),
3.40—3.49 (1H, m), 3.79 (3H, s), 5.79 (2H, br), 6.62 (1H, s).
Methyl 2-Amino-5-cyclohexylthiophene-3-carboxylate (23e) Com-
pound 23e was obtained from 22e as a yellow oil (5%) by following the pro-
6-Cyclobutyl-4-hydrazinothieno[2,3-d]pyrimidine (24b) Compound
24b was obtained from 23b as a pale yellow oil (41%) by following the pro-
1
cedure described for 23a. H-NMR (CDCl3) d: 1.16—1.25 (1H, m), 1.28—
1
cedure described for 24a. H-NMR (CDCl3) d: 1.90—2.12 (2H, m), 2.19—
1.37 (4H, m), 1.67—1.71 (1H, m), 1.77—1.81 (2H, m), 1.93—1.98 (2H, m),
2.51—2.56 (1H, m), 3.78 (3H, s), 5.78 (2H, br), 6.61 (1H, d, Jꢀ1.1 Hz); IR
(ATR) cmꢁ1: 3299, 2918, 2851, 1657, 1500, 1439, 1260; MS (ESI) m/z: 240
(MꢆꢆH); HR-MS (ESI) m/z: 240.10716 (Calcd for C12H18NO2S:
240.10582).
2.29 (2H, m), 2.42—2.50 (2H, m), 3.71—3.80 (1H, m), 6.43 (1H, br), 6.86
(1H, d, Jꢀ1.0 Hz), 8.50 (1H, s); IR (KBr) cmꢁ1: 3234, 3193, 1585; MS
(ESI) m/z: 221 (MꢆꢆH); HR-MS (ESI) m/z: 221.08765 (Calcd for
C10H12N4S2: 221.08609); Anal. Calcd for C10H12N4S: C, 52.52; H, 5.49; N,
25.43; S, 14.55. Found: C, 52.26; H, 5.41; N, 25.58; S, 14.68.
6-Cyclohexyl-4-hydrazinothieno[2,3-d]pyrimidine (24e) Compound
24e was obtained from 23e as a colorless solid (31%) by following the pro-
cedure described for 24a. 1H-NMR (DMSO-d6) d: 1.21—1.24 (1H, m),
1.35—1.43 (4H, m), 1.67—1.70 (1H, m), 1.76—1.80 (2H, m), 2.00—2.04
(2H, m), 2.81—2.86 (1H, m), 4.49 (2H, br), 7.34 (1H, br), 8.28 (1H, s), 8.81
(1H, br); IR (ATR) cmꢁ1: 3286, 2917, 2849, 1581, 1510; MS (ESI) m/z: 249
(MꢆꢆH); HR-MS (ESI) m/z: 249.11937 (Calcd for C12H17N4S: 249.11739);
4-[(Methylamino)methyl]-1,3-thiazole-2-carbaldehyde (6-Cyclobutyl-
thieno[2,3-d]pyrimidin-4-yl)hydrazone (31) Compound 31 was obtained
from 3d and 24b as a yellow solid (55%) by following the procedure de-
scribed for 4d. 1H-NMR (DMSO-d6) d: 1.89—1.97 (1H, m), 2.01—2.12
(1H, m), 2.19—2.29 (2H, m), 2.45—2.49 (2H, m), 2.58—2.61 (3H, t,
Jꢀ5.8 Hz), 3.86 (3H, td, Jꢀ8.5, 16.8 Hz), 4.29 (2H, m), 7.83 (1H, s), 7.96

August 2011
1001
Anal. Calcd for C12H16N4S: C, 58.04; H, 6.49; N, 22.56; S, 12.91. Found: C, tion of methyl cyanoacetate (0.65 ml, 7.5 mmol), Et3N (1.05 ml, 7.5 mmol),
57.76; H, 6.83; N, 22.51; S, 12.68. and DMF (1.1 ml, 14 mmol) under N2 atmosphere. After 3 min of stirring, S8
4-[(Methylamino)methyl]-1,3-thiazole-2-carbaldehyde (6-Cyclohexyl- (225 mg, 7.0 mmol) was added and stirred at room temperature for 72 h. The
thieno[2,3-d]pyrimidin-4-yl)hydrazone (34) Compound 34 was obtained
from 3d and 24e as a yellow solid (58%) by following the procedure de-
reaction mixture was poured into water and extracted with EtOAc. The aque-
ous layer was extracted with EtOAc and the combined organic layer was
scribed for 4d. 1H-NMR (DMSO-d6) d: 1.23—1.34 (1H, m), 1.38—1.57 washed with brine and dried over Na2SO4. The solvent was removed under
(4H, m), 1.72 (1H, d, Jꢀ12.9 Hz), 1.83 (2H, d, Jꢀ12.9 Hz), 2.11 (2H, d, reduced pressure, and the residue was chromatographed on silica gel eluted
Jꢀ10.3 Hz), 2.60 (3H, t, Jꢀ2.9 Hz), 2.95 (1H, m), 4.29 (2H, t, Jꢀ4.6 Hz),
with n-hexane/EtOAc (9 : 1) to afford title compound 23g (2.50 g, 76%) as a
7.79 (1H, s), 7.94 (1H, s), 8.52 (1H, s), 8.55 (1H, br), 9.23 (2H, br); IR
pale yellow oil. 1H-NMR (CDCl3) d: 1.05 (9H, s), 1.29 (6H, s), 3.48 (2H, s),
(ATR) cmꢁ1: 2701, 1633, 1593, 1099; MS (FAB) m/z: 387 (MꢆꢆH); HR- 3.78 (3H, s), 5.77 (2H, br), 6.68 (1H, s), 7.33—7.61 (10H, m).
MS (ESI) m/z: 387.13763 (Calcd for C18H23N6S2: 387.14256); Anal. Calcd
for C18H22N6S2·2.1HCl·0.25H2O: C, 46.23; H, 5.30; Cl, 15.92; N, 17.97; S, d]pyrimidin-4(3H)-one (28) A solution of 23g (2.50 g, 5.34 mmol) in
13.71. Found: C, 45.83; H, 5.30; Cl, 15.92; N, 18.34; S, 13.34. formamide (12.5 ml) was heated at 210 °C for 2 h. After cooling, precipitate
Methyl 2-Amino-5-(1,2-dimethylpropyl)thiophene-3-carboxylate (23f) was formed, which was collected and washed with water. The crude solid
6-(2-{[tert-Butyl(diphenyl)silyl]oxy}-1,1-dimethylethyl)thieno[2,3-
Compound 23f was obtained from 22f as a yellow oil (29%) by following
was chromatographed on silica gel eluted with n-hexane/EtOAc (1 : 1) to af-
the procedure described for 23a. 1H-NMR (CDCl3) d: 0.88 (3H, d, ford title compound 28 (1.43 g, 58%) as a colorless solid. 1H-NMR (CDCl3)
Jꢀ6.9 Hz), 0.89 (3H, d, Jꢀ6.9 Hz), 1.19 (3H, d, Jꢀ7.2 Hz), 1.67—1.73 (1H, d: 1.04 (9H, s), 1.42 (6H, s), 3.61 (2H, s), 7.26 (1H, s), 7.32—7.59 (10H,
m), 2.56 (1H, dt, Jꢀ6.9, 14.9 Hz), 3.79 (3H, s), 5.78 (2H, s), 6.60 (1H, s); IR
m), 8.01 (1H, br); IR (KBr) cmꢁ1: 3571, 2854, 1689, 1577; MS (FAB) m/z:
(ATR) cmꢁ1: 3439, 3334, 2957, 1666, 1583, 1501, 1439, 1265; MS (ESI) 463 (MꢆꢆH); HR-MS (ESI) m/z: 463.18572 (Calcd for C26H31N2O2SSi:
m/z: 228 (MꢆꢆH); HR-MS (ESI) m/z: 228.10542 (Calcd for C11H18NO2S:
228.10582).
463.18755); Anal. Calcd for C26H30N2O2SSi: C, 67.49; H, 6.54; N, 6.05; S,
6.93. Found: C, 67.54; H, 6.82; N, 6.22; S, 6.67.
6-(1,2-Dimethylpropyl)-4-hydrazinothieno[2,3-d]pyrimidine (24f)
6-(2-{[tert-Butyl(diphenyl)silyl]oxy}-1,1-dimethylethyl)-4-hydrazino-
thieno[2,3-d]pyrimidine (24g) Compound 24g was obtained from 28 as a
Compound 24f was obtained from 23f as a colorless solid (58%) by follow-
1
ing the procedure described for 24a. H-NMR (DMSO-d6) d: 0.88 (3H, d, colorless solid (76%) by following the chlorination and hydrazinolysis pro-
1
Jꢀ6.9 Hz), 0.91 (3H, d, Jꢀ6.9 Hz), 1.27 (3H, d, Jꢀ6.9 Hz), 1.84 (1H, td,
Jꢀ13.5, 6.9 Hz), 2.85 (1H, dt, Jꢀ13.5, 6.9 Hz), 4.51 (2H, br), 7.34 (1H, s),
cedure described for 24a. H-NMR (CDCl3) d: 1.02 (9H, s), 1.42 (6H, s),
3.63 (2H, s), 6.35 (1H, br), 6.90 (1H, s), 7.32—7.55 (10H, m), 8.51 (1H, s);
8.29 (1H, s), 8.82 (1H, br s); IR (ATR) cmꢁ1: 3289, 2956, 1579, 1508, 1355; IR (KBr) cmꢁ1: 3301, 3268, 2929, 1589, 1548; MS (FAB) m/z: 477
MS (ESI) m/z: 237 (MꢆꢆH); HR-MS (ESI) m/z: 237.11924 (Calcd for (MꢆꢆH); HR-MS (ESI) m/z: 477.21018 (Calcd for C26H33N4OSSi:
C11H17N4S: 237.11739); Anal. Calcd for C11H16N4S: C, 55.90; H, 6.82; N, 477.21443).
23.71; S, 13.57. Found: C, 56.19; H, 6.99; N, 23.63; S, 13.30.
4-[(Methylamino)methyl]-1,3-thiazole-2-carbaldehyde [6-(2-Hydroxy-
4-[(Methylamino)methyl]-1,3-thiazole-2-carbaldehyde [6-(1,2-Di- 1,1-dimethylethyl)thieno[2,3-d]pyrimidin-4-yl]hydrazone (36) A mix-
methylpropyl)thieno[2,3-d]pyrimidin-4-yl]hydrazone (35) Compound
35 was obtained from 3d and 24f as a yellow solid (82%) by following the
procedure described for 4d. H-NMR (DMSO-d6) d: 0.92 and 0.93 (each
3H, d, Jꢀ6.6 Hz), 1.35 (3H, d, Jꢀ7.1 Hz), 1.91 (1H, td, Jꢀ13.2, 6.6 Hz), chromatographed on silica gel eluted with n-hexane/EtOAc (2 : 1) to afford
ture of 24g (885.6 mg, 1.86 mmol) and 3d (500 mg, 1.95 mmol) in benzene
(17 ml) was stirred under reflux for 3 h and cooled to room temperature. The
reaction mixture was concentrated under reduced pressure. The residue was
1
2.59 (3H, t, Jꢀ5.4 Hz), 2.99 (1H, q, Jꢀ6.8 Hz), 4.29 (2H, t, Jꢀ5.6 Hz), 7.89
crude hydrazone (777 mg). To a solution of the hydrazone in THF (8 ml) was
added TBAF (1 M THF solution, 1.3 ml, 1.31 mmol) at 0 °C, and stirred at
(1H, br), 7.99 (1H, s), 8.57 (1H, s), 8.68 (1H, br), 9.41 (2H, br); IR (ATR)
cmꢁ1: 1635, 1579, 1508, 1460, 1419, 1373, 1247, 1147, 1099; MS (FAB) 75 °C for 15 h. The solvent was removed under reduced pressure, and the
m/z: 375 (MꢆꢆH); HR-MS (ESI) m/z: 375.14180 (Calcd for C17H23N6S2:
375.14256); Anal. Calcd for C17H22N6S2·2.5HCl·1.25H2O: C, 41.82; H, (1 : 3) to afford crude hydroxide (35.8 mg). To a solution of the hydroxide in
residue was chromatographed on silica gel eluted with n-hexane/EtOAc
5.44; Cl, 18.29; N, 17.02; S, 12.95. Found: C, 41.68; H, 5.44; Cl, 18.29; N,
17.02; S, 12.95.
1,4-dioxane (1.5 ml) was added 4 N HCl in 1,4-dioxane (1.5 ml) at 0 °C. The
mixture was stirred at room temperature for 16 h and concentrated under re-
tert-Butyl[(2,2-dimethylpent-4-en-1-yl)oxy]diphenylsilane (27) To a duced pressure. The residue was recrystallized from EtOH, EtOAc, and n-
mixture of isobutyraldehyde 25 (25 ml, 0.275 mol) and allyl alcohol 26 hexane to afford title compound 36 (9.3 mg, 2%) as a yellow solid. 1H-NMR
(12.5 ml, 0.183 mol) in p-cymene (40 ml) was added p-TsOH (60 mg) and
stirred under reflux for 24 h. Distillation gave 2,2-dimethyl-4-pentenal
(14.8 g, 72%) as a colorless oil. To a suspension of LAH (0.8 g, 21 mmol) in
Et2O (60 ml) was added dropwise 2,2-dimethyl-4-pentenal (8.00 g, 71 mmol)
in Et2O (40 ml) at room temperature, and stirred under reflux for 3 h. After
cooling to 0 °C, MeOH (1.8 ml), water (0.8 ml), 15% NaOH aq. (0.8 ml), and
water (2.4 ml) were added to the reaction mixture successively. The precipi-
(DMSO-d6) d: 1.40 (6H, s), 2.45 (3H, s), 3.49 (2H, s), 4.00 (2H, s), 5.06
(1H, br), 7.70 (1H, s), 7.83 (1H, s), 8.42 (1H, s), 8.50 (1H, s); IR (ATR)
cmꢁ1: 1558, 1508, 1348; MS (FAB) m/z: 377 (MꢆꢆH); HR-MS (ESI) m/z:
377.11975 (Calcd for C16H21N6OS2: 377.12183); Anal. Calcd for
C16H20N6OS2·1.5HCl·0.25H2O: C, 44.11; H, 5.09; N, 19.29; S, 14.72.
Found: C, 44.46; H, 4.89; N, 18.98; S, 14.57.
6-(2-Fluoro-1,1-dimethylethyl)-4-hydrazinothieno[2,3-d]pyrimidine
tate was filtered through Celite. The filtrate was concentrated under reduced (24h) To a solution of 28 (8.60 g, 18.4 mmol) in THF (100 ml) was added
pressure and the residue was purified by vacuum distillation to afford 2,2-di-
methyl-4-pentenol (5.9 g, 73%) as a colorless oil. To a solution of pentenol
(2.28 g, 20 mmol) in THF (50 ml) were added imidazole (1.50 g, 22 mmol)
and TBDPSCl (5.7 ml, 22 mmol). The reaction mixture was stirred at room
temperature for 20 h. After EtOAc and water were added, the two layers were
separated. The organic layer was washed with brine and dried over Na2SO4.
The solvent was removed under reduced pressure, and the residue was chro-
TBAF (1 M THF solution, 36.7 ml, 36.7 mmol) at 0 °C, and stirred overnight
at 70 °C. After cooling, CHCl3 and brine were added, and the two layers
were separated. The organic layer was dried over Na2SO4. The solvent was
removed under reduced pressure, and the residue was chromatographed on
silica gel eluted with CHCl3/MeOH (100 : 3). The eluate was concentrated
under reduced pressure, and the residue was recrystallized from CHCl3 and
EtOAc to afford 6-(1,1-dimethyl-2-hydroxyethyl)thieno[2,3-d]pyrimidin-4-
matographed on silica gel eluted with n-hexane/EtOAc (50 : 1) to afford title one (2.01 g, 49%) as a brownish solid. To a solution of afforded compound
compound 27 (5.05 g, 72%) as a colorless oil. 1H-NMR (CDCl3) d: 0.85
(1.60 g, 7.14 mmol) in CH2Cl2 (240 ml) was added (dimethylamino)sulfur
(6H, s), 1.05 (9H, s), 2.05 (2H, d, Jꢀ8.0 Hz), 3.32 (2H, s), 4.95—5.02 (2H, trifluoride (3.77 ml, 28.6 mmol) at ꢁ78 °C and warmed to room temperature
m), 5.71—5.82 (1H, m), 7.35—7.77 (10H, m).
Methyl 2-Amino-5-(2-{[tert-butyl(diphenyl)silyl]oxy}-1,1-dimethyl-
over 2 h with stirring. The reaction mixture was neutralized with a solution
of sat NaHCO3, and the two layers were separated. The organic layer was
ethyl)thiophene-3-carboxylate (23g) To a solution of 27 (5.00 g, 14.2 dried over Na2SO4. The solvent was removed under reduced pressure, and
mmol) in a mixture of THF (140 ml) and H2O (70 ml) were slowly added
OsO4 (180 mg, 5 mol%) and NaIO4 (6.10 g, 28.5 mmol), and the mixture was
stirred at 50 °C for 2 h. Et2O and sat. Na2S2O3 aq. were added to the reaction
mixture, and the two layers were separated. The aqueous layer was extracted
with Et2O and the combined 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-hexane/EtOAc
(20 : 1) to afford 4-{[tert-butyl(diphenyl)silyl]oxy}-3,3-dimethylbutanal
(2.50 g, 50%) as a colorless oil. The resulting aldehyde was added to a solu-
the residue was chromatographed on silica gel eluted with CHCl3/MeOH
(100 : 1) to afford 6-(2-fluoro-1,1-dimethylethyl)thieno[2,3-d]pyrimidin-4-
one as a colorless solid. Compound 24h was obtained from the resulting
compound as a colorless solid (1.16 g, 68%) by following the procedure de-
1
scribed for 24g. H-NMR (DMSO-d6) d: 1.33 (3H, s), 1.38 (3H, s), 3.17
(2H, d, Jꢀ22.2 Hz), 4.57 (2H, br), 7.39 (1H, br), 8.31 (1H, s), 9.07 (1H, s);
IR (ATR) cmꢁ1: 335, 2985, 1580, 1513, 1317; MS (FAB) m/z: 241 (MꢆꢆH);
HR-MS (ESI) m/z: 241.09405 (Calcd for C10H14FN4S: 241.09232); Anal.
Calcd for C10H1FN4S: C, 49.98; H, 5.45; F, 7.91; N, 23.32; S, 13.34. Found:

1002
Vol. 59, No. 8
C, 49.99; H, 5.46; F, 7.91; N, 23.12; S, 13.38.
Cell Cycle Distribution Analysis HCT116 cells were treated with test
4-[(Methylamino)methyl]-1,3-thiazole-2-carbaldehyde [6-(2-Fluoro- compounds for 16 h and were stained with propidium iodide using a Cycle
1,1-dimethylethyl)thieno[2,3-d]pyrimidin-4-yl]hydrazone (37) Com-
pound 37 was obtained from 3d and 24h as a yellow solid (97%) by follow-
ing the procedure described for 4d. H-NMR (DMSO-d6) d: 1.39 (3H, s),
1.44 (3H, s), 2.59 (3H, t, Jꢀ5.4 Hz), 3.30 (2H, d, Jꢀ22.0 Hz), 4.28 (2H, t,
Test Kit (Becton Dickinson). Then the cells were analyzed using a FACscan
flow cytometer with CellQuest software (Becton Dickinson) in accordance
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
1
Jꢀ5.4 Hz), 7.89 (1H, s), 7.98 (1H, s), 8.56 (1H, s), 8.65 (1H, br), 9.40 (2H, software (Verity Software House, Inc.).
br); MS (FAB) m/z: 379 (MꢆꢆH); IR (ATR) cmꢁ1: 3350, 2696, 1631, 1589,
1372; HR-MS (ESI) m/z: 379.10884 (Calcd for C16H20FN6S2: 379.11749);
Anal. Calcd for C16H19N6FS2·2HCl: C, 42.57; H, 4.69; Cl, 15.71; F, 4.21; N,
18.62; S, 14.21. Found: C, 42.52; H, 4.74; Cl, 15.80; F, 4.27; N, 18.68; S,
14.15.
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 tu-
mors averaged 80—100 mm3 in size, the tested compounds dissolved in 20%
Captisol® (b-cyclodextrin sulfobutylether sodium salt, CyDex, Inc.) solution
4-(1-Aminoethyl)-1,3-thiazole-2-carbaldehyde [6-(2-Fluoro-1,1-di- were administered intravenously or orally once a day for 4—6 d at the doses.
methylethyl)thieno[2,3-d]pyrimidin-4-yl]hydrazone (38) Compound 38
was obtained from 3h and 24h as a yellow solid (88%) by following the pro-
cedure described for 4d. 1H-NMR (DMSO-d6) d: 1.39 (3H, s), 1.44 (3H, s),
The tumor volume was measured with a caliper periodically.
Acknowledgements We would like to thank the members of Drug Me-
1.59 (3H, d, Jꢀ6.8 Hz), 3.31 (2H, d, Jꢀ22.2 Hz), 4.52—4.64 (1H, m), 7.92 tabolism and Pharmacokinetics Research Laboratories for their chemical
(1H, s), 7.94 (1H, s), 8.54—8.90 (6H, m); MS (FAB) m/z: 379 (MꢆꢆH); IR stability assays and members of the analysis group of Daiichi Sankyo R&D
(ATR) cmꢁ1: 2872, 1633, 1581, 1552, 1424, 1211; HR-MS (ESI) m/z: Associe Co., Ltd. for their analytical and spectral determinations.
379.11307 (Calcd for C16H20FN6S2: 379.11749); Anal. Calcd for
C16H19FN6S2·2.4HCl·0.95H2O: C, 39.78; H, 4.86; Cl, 17.61; F, 3.93; N, References
17.40; S, 13.27. Found: C, 40.15; H, 4.80; Cl, 17.66; F, 3.61; N, 17.00; S,
13.01.
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