Design, synthesis and anticancer activity of 4-morpholinothieno[3,2-d]- pyrimidine derivatives bearing arylmethylene hydrazine moiety

Article abstract of DOI:10.1248/cpb.c12-00342

Three series of 4-morpholinothieno[3,2-d]pyrimidine derivatives containing arylmethylene hydrazine moiety (11a-f, 13a-k and 15a-h) were synthesized and their chemical structures as well as the relative stereochemistry were confirmed. The synthesized compounds were evaluated for their cytotoxicity against three cancer cell lines (H460, HT-29, MDA-MB-231). Most of them exhibited moderate to significant cytotoxicity and high-selectivity against one or more cell lines, especially compounds 11c, 13b, 15f and 15g possessing dramatically increased cytotoxicity as compared with the positive controls, which were further evaluated for six other cancer cell lines and one normal cell line. The most promising compound 11c, bearing 3,4-methylenedioxy phenyl group, showed remarkable cytotoxicity against H460, HT-29 and MDA-MB-231 cell lines with IC₅₀ values of 0.003 μM, 0.42 μM and 0.74 μM, which was 1.6- to 290-fold more potent than GDC-0941.

Full text of DOI:10.1248/cpb.c12-00342

August 2012
Regular Article
Chem. Pharm. Bull. 60(8) 1037–1045 (2012)
1037
Design, Synthesis and Anticancer Activity of 4-Morpholinothieno[3,2-d]-
pyrimidine Derivatives Bearing Arylmethylene Hydrazine Moiety
Wufu Zhu, Xin Zhai,* Qiangqiang Fu, Fei Guo, Mei Bai, Jianqiang Wang, Haiyan Wang, and
Ping Gong*
Key Laboratory of New Drugs Design and Discovery of Liaoning Province, School of Pharmaceutical Engineering,
Shenyang Pharmaceutical University; Shenyang 110016, P.R. China.
Received April 16, 2012; accepted May 15, 2012
Three series of 4-morpholinothieno[3,2-d]pyrimidine derivatives containing arylmethylene hydrazine
moiety (11a–f, 13a–k and 15a–h) were synthesized and their chemical structures as well as the relative ste-
reochemistry were confirmed. The synthesized compounds were evaluated for their cytotoxicity against three
cancer cell lines (H460, HT-29, MDA-MB-231). Most of them exhibited moderate to significant cytotoxicity
and high-selectivity against one or more cell lines, especially compounds 11c, 13b, 15f and 15g possessing
dramatically increased cytotoxicity as compared with the positive controls, which were further evaluated for
six other cancer cell lines and one normal cell line. The most promising compound 11c, bearing 3,4-methyl-
enedioxy phenyl group, showed remarkable cytotoxicity against H460, HT-29 and MDA-MB-231 cell lines
with IC50 values of 0.003µM, 0.42µM and 0.74µM, which was 1.6- to 290-fold more potent than GDC-0941.
Key words 4-morpholinothieno[3,2-d]pyrimidine; arylmethylene hydrazine; synthesis; anticancer activity
Cancer is a life-threatening disease and the leading cause (Fig. 1), which possessed potent cell-based, PI3K and mam-
of death worldwide. Although there have been significant malian target of rapamycin (mTOR) kinases activity.3) The
advances in treating cancer, drug resistance and adverse side structure–activity relationships (SARs) studies indicated that
effects are still serious problems. Therefore, the search for the 4-morpholinothieno[3,2-d]pyrimidine scaffold played a
highly efficient and safe chemotherapeutic agents for treating key role in their antitumor activity and the 4-(methylsulfonyl)-
cancer remains desirable.1)
Recently, considerable attentions have been devoted to the enhanced the solubility and metabolic stability.2–5)
construction of new 4-morpholinothieno[3,2-d]pyrimidines on The arylmethylene hydrazine moiety, an attractive active
piperazin-1-ylmethyl substituent at C-6 position remarkably
account of their reported anticancer activity.2,3) Among them, pharmacophore, has been successfully applied to the design of
GDC-0941 (Fig. 1) is a selective small phosphoinositide 3-ki- anticancer agents. It is noteworthy that arylmethylene hydra-
nase (PI3K) inhibitor currently undergoing Phase I clinical zinyl derivatives 1,7) CID13401328) (Fig. 1) and benzylidenehy-
trials.4,5) Zhang and her colleagues6) found that a high efficacy drazinyl derivatives reported by our research group9,10) showed
of antitumor activity was shown with GDC-0941 treatment potent antitumor activity against several cancer cell lines.
in two non-small cell lung cancer (NSCLC) cell lines, H460 Many researches demonstrate that the excellent antitumor ac-
and A549. In addition, H460 cells were relatively more sen- tivity of those kinds of arylmethylene hydrazinyl derivatives
sitive to GDC-0941 than A549 cells. Further optimization may be ascribed to the putative hydrogen bonds between the
of GDC-0941 led to a backup clinical candidate GNE-477 hydrazinyl group and receptors necessary for activity.11–14)
Fig. 1. Structures of 4-Morpholinothieno[3,2-d]pyrimidines (GDC-0941, GNE-477), Arylmethylene Hydrazines (1, CID1340132) and Target Com-
pounds (11a–f, 13a–k, 15a–h)
The authors declare no conflict of interest.
*To whom correspondence should be addressed. e-mail: gongpinggp@126.com
© 2012 The Pharmaceutical Society of Japan

1038
Vol. 60, No. 8
Reagents and conditions: (a) 5eq urea, 190°C, 2.5h; (b) POCl3, DMF (cat.), reflux, 8h; (c) 2.1eq morpholine, MeOH, 0°C, 30min, r.t., 1–2h; (d) DMF, n-BuLi, THF,
−78°C to r.t., 5h; (e) NaBH4, MeOH, r.t., 3h; (f) SOCl2, CH2Cl2, r.t., 2h; (g) 1-(methylsulfonyl)piperazine, DIPEA, isopropanol, reflux, 11h; (h) 80% NH2NH2·H2O,
reflux, 8–10h; (i) ArCHO, EtOH, reflux, 4–8h; (j) ArCH2Cl, K2CO3, DMF, 80°C; (k) DMF, n-BuLi, THF, −78°C to r.t., 6–8h.
Chart 1
To our knowledge, there are hardly any studies about in- 3-amino-2-thiophenecarboxylate was condensed with urea
troduction of arylmethylene hydrazinyl groups to the thieno- at 190°C for 2h to give thienopyrimidinedione 2. Chlorina-
pyrimidine core to date. Encouraged by these observations, tion of 2 with phosphorus oxychloride proceeded smoothly
we planned to synthesize new chemical entities incorporating to afford 3 as a pale yellow solid. Regioselective nucleophilic
these two active pharmacophores in a single molecular frame- displacement of the 4-chloride with morpholine gave rise
work. As a result, three series of 4-morpholinothieno[3,2- to 4 in 85–87% yields. Subsequently, lithiation of 9, fol-
d]pyrimidine derivatives bearing arylmethylene hydrazine lowed by addition of N,N-dimethylformamide (DMF), gave
moiety at C-2 position were designed and synthesized (11a–f, the aldehyde 5 in 75% yield. Treatment of 5 with NaBH4 in
13a–k and 15a–h, Fig. 1). In particular, 4-(methylsulfonyl)- MeOH at room temperature provided 6 as a primary alcohol,
piperazin-1-ylmethyl group was retained at the C-6 position which reacted with thionyl chloride and then substituted with
of thienopyrimidine core, with the aim of increasing solubil- 1-(methylsulfonyl)piperazine to furnish compound 8. Next,
ity and metabolic stability. Considering the high potency of substitution reaction of 8 with an excess of 80% hydrazine hy-
GDC-0941 and its analogous on lung cancer cells, four lung drate gave the key intermediate 9, which was then condensed
cancer cell lines (H460, A549, H1975 and H226), one normal with the corresponding aldehyde 10a–f, 12a–k and 14a–h in
human fetal lung fibroblast (WI-38) cell line and some other refluxing ethanol to afford the target compounds 11a–f, 13a–k
cell lines were chose to evaluate their anticancer activity.
and 15a–h, respectively.
The substituted benzyl-1H-indole-3-carbaldehydes 12a–k
were prepared by nucleophilic substitution of the various ben-
Results and Discussion
Chemistry The synthesis of title compounds is out- zyl chloride with 1H-indole-3-carbaldehyde in the presence
lined in Chart 1.15,16) The commercially available methyl of K2CO3 in DMF. The substituted 1-benzyl-1H-benzo[d]-

August 2012
1039
Fig. 2. Key NOESY of Representative Compounds 13b and 15b
imidazole-2-carbaldehydes 14a–h were synthesized via a two- cell lines, and three of them (11c, 13b, 15g) were superior to
step reaction. Reaction of benzimidazole with the correspond- the positive controls against all the tested cell lines. Generally,
ing benzyl chloride under basic condition in DMF yielded the N-benzyl indoles 13a–k and N-benzyl benzimidazoles
16a−f. Subsequently, lithiation of 16a–f, followed by addition 15a–h were more potent than the aromatic derivatives 11a–f,
of DMF, gave aldehydes 14a–h in 75–85% yield.
with the exception of compound 11c, which suggested that the
The structures and relative stereochemistry of the new introduction of arylmethylene hydrazinyl group was necessary
1
synthesized compounds were confirmed by IR, H-NMR, 2D for improving activity and a heteroarylmethylene hydrazinyl
nuclear Overhauser effect spectroscopy (NOESY) NMR and moiety (such as indolyl, benzimidazolyl group) was more fa-
MS spectra. It is interesting to note that the experimental vorable. Although compounds 11a–f showed a remarkable de-
procedure shown in Chart 1 led selectively to the E isomer, crease in the activity, compound 11c with 3,4-methylenedioxy
which can be confirmed by 2D NOESY NMR spectroscopic phenyl group possessed the strongest cytotoxicity (0.003,
approach17,18) (Fig. 2).
For the representative compound 13b, the NOESY effect GDC-0941.
0.42, 0.74µM), which was 1.6- to 290-fold more active than
was observed between the protons of N–H (10.49, s) and N=
Taken Table 1 and 2 together, the four selected compounds
CH (8.26, s) in E isomer (E-13b, Fig. 2) (see supplementary (11c, 13b, 15f, 15g) possess equal to enhanced cytotoxicity
information), which should not be observed in the putative against the tested cell lines as compared with the positive
Z isomer due to the larger intra-molecular H–H distances. control GDC-0941, especially against H460, A549 and H226
Similarly, for compound 15b, the nuclear Overhauser effect cell lines. The results indicated that these compounds exhib-
(NOE) interactions between N–H (11.40, s) and N=CH (8.28, ited excellent selectivity for lung cancer. Compounds 11c and
s) confirmed the E isomer. Moreover, the E isomers are rela- 15f displayed less toxicity against normal cell line WI-38 than
tively stable in air at room temperature during the period of compounds 13b, 15g and GDC-0941. The selective index (IC50
assay, revealing that they are not easily converted to the cor- normal cell/IC50 cancer cell) of the most promising compound
responding Z isomers. On the basis of the above analysis, the 11c for H460 and HT-29 cancer cell lines was 2467 and 23, re-
relative stereochemistry of target compounds was established spectively. These data demonstrated that the tumor cells were
unambiguously.
more sensitive than the normal cells.
Biological Evaluation All the target compounds and the
The SARs based on IC50 values (Tables 1, 2) showed that
precursor 9 were evaluated for their cytotoxicity against hu- variations in substitutions of the benzyl group had a marked
man lung cancer (H460), human colon cancer (HT-29) and impact on the cytotoxicity. Substituents at C-2 or C-6 posi-
human breast cancer (MDA-MB-231) cell lines together with tion of the benzyl group were unfavorable (13c, 13j, 13k vs.
reference compounds GDC-0941 and 1 by 3-(4,5-dimethylthi- 13i; 15e vs. 15f), which might be due to its steric hindrance.
azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. On For compounds 13a–k, substitution of the benzyl group at the
the basis of the cytotoxicity (IC50) against these three cancer C-3 position was well tolerated and 3-fluoro produced the best
cell lines, four potent compounds (11c, 13b, 15f, 15g) were potency. Furthermore, electron donating groups (EDGs) at
further evaluated for their cytotoxicity against human lung C-4 position were preferred. It was noticeable that strong elec-
cancer (A549, H1975, H226), human glioblastoma (U87MG), tron withdrawing groups (EWGs) at C-4 position weakened
human liver cancer (HepG2), human stomach cancer the activity dramatically (13d, 13h vs. 13b). Turn to com-
(SGC-7901) and normal human fetal lung fibroblast (WI-38) pounds 15a–h bearing benzimidazole moiety, interestingly,
cell lines in vitro. The results expressed as IC50 were summa- replacement of indolyl group with its bioisostere benzimid-
rized in Tables 1 and 2.
azolyl group remarkably enhanced the selectivity and activity
As illustrated in Table 1, most of the synthesized com- against HT-29 cancer cell line. A case in point is that com-
pounds showed moderate to significant activity to the tested pounds with 4-chloro (15g) or no substituent (15f) exhibited

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Vol. 60, No. 8
Table 1. Structures and Cytotoxicity of Compounds 9, 11a–f, 13a–k and 15a–h against H460, HT-29 and MDA-MB-231 Cancer Cell Lines in Vitro
IC50 (µmol/L)
IC50 (µmol/L)
Compd.
R
Compd.
R
H460
HT29
MDA-MB-231
H460
HT29
MDA-MB-231
9
—
39.81
64
46.22
51.41
13g
13h
2,4-Dichloro
4-Cyano
0.64
1
28
11a
3,4,5-Tri-
methoxy
>100
>100
38
24
>100
11b
11c
11d
11e
2,3,4-Tri-
methoxy
32
8.5
0.42
56
13i
H
0.8
0.87
>100
>100
3.4
1.8
>100
96
3,4-Methylene-
dioxy
0.003
14
0.74
13
13j
13k
15a
2,3-Dichloro >100
4-Trifluoro-
methoxy
6.8
2,6-Dichloro
3,4-Dichloro
19
0.75
2-Chloro-
4-fluoro
2.7
>100
56
8.3
11f
4-Cyano
3-Fluoro
0.79
0.28
0.79
13
0.64
1.2
16
14
15b
15c
15d
15e
15f
4-tert-Butyl
1.2
43
2.6
0.62
>100
>100
58
13a
13b
13c
13d
4-Methyl
1.9
4-tert-Butyl
2-Chloro
0.53
0.69
>100
2.12
2,3-Dichloro >100
2-Chloro
0.17
0.79
0.02
3.2
3.4
4-Trifluoro-
methyl
8.8
>100
H
1.1
1.8
13e
13f
3-Cyano
3,4-Dichloro
—
0.28
0.34
0.87
1.8
34
15g
15h
1
4-Chloro
3-Chloro
—
0.83
0.01
0.74
20.2
7.4
>100
>100
5.6
73
12
9.2
GDC-0941
0.66
73.82
Bold values show IC50 of <1µmol/L.
Table 2. Cytotoxicity of Four Potent Compounds against A549, H1975, H226, U87MG, HepG2, SGC-7901 Cancer Cell Lines and WI-38 Normal Cell
Line in Vitro
IC50 (µmol/L)
Compd. No.
A549
H1975
H226
U87MG
HepG2
SGC-7901
WI-38
11c
13b
1.6
2.4
2.62
2.4
2.9
9.6
40
0.84
5.4
0.9
1.1
1.0
1.4
3.5
7.4
5.3
7.77
1.9
1.1
3.6
1.3
6.9
1.7
4.4
2.1
1.6
1.8
7.4
4.6
9.6
3.2
7.3
15f
4.9
15g
4.8
GDC-0941
0.27
excellent HT-29 cytotoxicity, with the IC50 values of 0.01µM against H460, HT-29 and MDA-MB-231 cell lines with IC50
and 0.02µM, respectively. In our opinion, this dramatic boost values of 0.003µM, 0.42µM and 0.74µM, which was 1.6- to
in the HT-29 potency and selectivity can be ascribed to the 290-fold more active than GDC-0941. Furthermore, the cyto-
specificity of cancer cells. In addition, contrary to compounds toxicity and selective index (2467 and 23 for H460 and HT-29
13a–k, EDGs at C-4 position (15b, 15c) and chlorine atom at cell lines, respectively) of compound 11c demonstrated that
C-2 or C-3 position (15d, 15h) of benzene ring were not favor- these compounds exhibited excellent anticancer activity, with
able for this series of compounds.
high-selectivity for lung cancer.
The initial SARs showed that the heteroarylmethylene
hydrazinyl group was beneficial for improving activity. More-
Conclusion
In conclusion, three series of 4-morpholinothieno[3,2-d]- over, the substituents in the benzene ring had significant ef-
pyrimidine derivatives bearing arylmethylene hydrazine fects on cytotoxicity. For compounds 13a–k, substitution of
moiety were designed, synthesized and evaluated for their cy- the benzyl group at the C-3 position was well tolerated, while
totoxicity. The pharmacological results indicated that most of 4-chloro or no substituent was more preferred for 15a–h.
them exhibited moderate to excellent cytotoxicity and high-se- Studies on the mechanism of action of these compounds are in
lectivity against one or more cell lines, especially compounds progress and will be reported in the future.
11c, 13b, 15f and 15g which possessed dramatically increased
cytotoxicity as compared with the positive controls. The most
Experimental
promising compound 11c showed the strongest cytotoxicity
Reagents and General Procedures All melting points

August 2012
1041
were obtained on a Büchi Melting Point B-540 apparatus 5.1Hz), 8.29 (1H, s), 10.21 (1H, s).
(Büchi Labortechnik, Flawil, Switzerland) and were uncor-
Preparation of (2-Chloro-4-morpholinothieno[3,2-d]-
rected. The IR spectra were recorded by means of the KBr pyrimidin-6-yl)methanol (6) To a solution of 5 (25.0g,
1
pellet technique on a Bruker FTS 135 spectrometer. H-NMR 0.09mol) in 200mL dry methanol at −10°C was added So-
spectra were performed using Bruker 300MHz spectrometers dium borohydride (4.0g, 0.1mol) by batch. After stirring at
(Bruker Bioscience, Billerica, MA, U.S.A.) with TMS as an −10°C for 1h, the mixture was concentrated to 50mL under
internal standard. Mass spectra (MS) were taken in electro- reduced pressure. Then the mixture poured onto 500mL
spray ionization (ESI) mode on Agilent 1100 LC-MS (Agilent, water yielding a white precipitate. This was collected by fil-
Palo Alto, CA, U.S.A.). Elemental analysis was determined on tration and washed with 100mL water. The precipitate was
a Carlo-Erba 1106 Elemental analysis instrument (Carlo Erba, dried at 25°C for 24h to afford white power (23.2g, 92.1%).
Milan, Italy). All the materials were obtained from commer- mp 184–186°C. ESI-MS m/z: 285.8 (M+H)+, 307.9 (M+Na)+,
cial suppliers and used without purification, unless otherwise 324.0 (M+K)+.
specified. Yields were not optimized. TLC analysis was car-
ried out on silica gel plates GF254 (Qindao Haiyang Chemi- d]pyrimidin-4-yl)morpholine (7) A mixture of 6 (33.2g,
cal, China). 0.12mol) and 1.66mL DMF was added to thionyl chloride
Preparation of 4-(2-Chloro-6-(chloromethyl)thieno[3,2-
Preparation of 1H-Thieno[3,2-d]pyrimidine-2,4-dione (2) (232mL) at room temperature and the mixture was heated to
A mixture of methyl 3-amino-2-thiophenecarboxylate (100g, reflux for 1h. The reaction mixture was then concentrated to
0.64mol) and urea (191g, 3.2mol) was heated at 190°C for 60mL, and the solution poured into 500mL ice water yield-
2.5h. Upon cooling to about 120°C, the reaction mixture was ing a yellow precipitate. This was collected by filtration and
poured into sodium hydroxide (2000mL, 1N) solution and any washed with 300mL water. The precipitate was dried to
1
insoluble material removed by filtration. The mixture was then yield light yellow solid (34.4g, 97.3%). H-NMR (300MHz,
acidified (HCl, 2N) to yield 1H-thieno[3,2-d]pyrimidine-2,4- DMSO-d6) δ: 3.75 (4H, t, J1=5.4Hz, J2=4.2Hz), 3.89 (4H, t,
dione as a white precipitate, which was collected by filtra- J1=4.2Hz, J2=5.4Hz), 5.17 (2H, s), 7.48 (1H, s).
tion and dried (105g, 98%). mp >300°C. ESI-MS m/z: 167.1
[M−H]−. 1H-NMR (300MHz, DMSO-d6) δ: 6.93 (1H, d, piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpho-
J=5.2Hz), 8.04 (1H, d, J=5.2Hz), 11.19 (1H, s), 11.61 (1H, s). line (8) A mixture of 4-(2-chloro-6-(chloromethyl)thieno-
Preparation
of
4-(2-Chloro-6-((4-(methylsulfonyl)-
Preparation of 2,4-Dichloro-thieno[3,2-d]pyrimidine (3) [3,2-d]pyrimidin-4-yl)morpholine (7, 29.1g, 0.096mol),
A mixture of 1H-thieno[3,2-d]pyrimidine-2,4-dione (105g, 1-methanesulfonyl-piperazine (38.4g, 0.19mol) and DIPEA
0.63mol), phosphorous oxychloride (1000mL) and catalytic (37.1g, 0.29mol) in 200mL isopropanol was refluxed for 11h.
amount DMF (2mL) was heated at reflux and the reaction The reaction solution was then cooled, collected by filtra-
was monitored by TLC. The reaction mixture was concen- tion and washed with 300mL water, 100mL ethanol. The
trated under reduced pressure and the residue was poured onto precipitate was dried at 25°C for 24h to give 8 as an oyster
1
ice/water with vigorous stirring yielding a precipitate. The white solid (39.5g, 95.6%). H-NMR (300MHz, DMSO-d6) δ:
mixture was then filtered to yield 2,4-dichloro-thieno[3,2-d]- 2.57 (4H, s), 2.90 (3H, s), 3.14 (4H, s), 3.75 (4H, t, J1=4.5Hz,
pyrimidine as a white solid (93g, 72%). mp 142–144°C. ESI- J2=3.9Hz), 3.91–3.84 (6H, q), 7.31 (1H, s).
MS m/z: 205.3 (M+H)+.
Preparation of 4-(2-Hydrazinyl-6-((4-(methylsulfonyl)-
Preparation of 2-Chloro-4-morpholine-4-yl-thieno[3,2- piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpho-
d]pyrimidine (4) To the mixture of 2,4-dichloro-thieno[3,2- line (9) A solution of 80% hydrazine hydrate (NH2NH2·H2O,
d]pyrimidine (93g, 0.45mol) and MeOH (1500mL), mor- 170mL, 4.9mol) and 8 (20.0g, 0.046mol) was stirred at 80°C
pholine (81mL, 2.1eq) was added drop-wise at 0°C. Subse- for 10h. The precipitate was collected by filtration and washed
quently, the reaction mixture was stirred at room temperature with water, and dried to give 9 as a yellow solid (10.3g, 52%).
1
for 1.5h. After completion of reaction as indicated by TLC, ESI-MS m/z: 428.3 (M+H)+. H-NMR (300MHz, DMSO-d6)
the mixture was then filtered, washed with water and MeOH, δ: 2.54 (4H, s), 2.89 (3H, s), 3.13 (4H, s), 3.90–3.71 (10H, m),
to yield the title compound as a white solid (114g, 98%). mp 4.34 (2H, s), 7.03 (1H, s), 7.49 (1H, s).
204–205°C. ESI-MS m/z: 255.9 (M+H)+, 277.6 (M+Na)+.
General Procedure for the Preparation of Compounds
1H-NMR (300MHz, DMSO-d6) δ: 3.76 (4H, s), 3.90 (4H, d, 10a–k 1H-Indole-3-carbaldehyde (14.5g, 0.1mol), together
J=4.2Hz), 7.41 (1H, d, J=5.4Hz), 8.31 (1H, d, J=5.4Hz). with the respective benzyl chloride (0.11mol) and anhydrous
Preparation of 2-Chloro-4-morpholin-4-yl-thieno[3,2-d]- K2CO3 (27.6g, 0.2mol) were dissolved in DMF (30mL) and
pyrimidine-6-carbaldehyde (5) To a suspension of 4 (50g, heated at 80°C for 5–8h. Upon cooling to room temperature,
0.20mol) in 300mL absolute tetrahydrofuran (THF) under the mixture poured into ice-water and then the precipitated
nitrogen atmosphere at −78°C was slowly added 2.5M solution was collected by filtration and dried. The crude product was
of n-butyllithium in hexanes (0.23mol, 94mL). After stirring purified by recrystallized from ethanol to give the solids
at −78°C for 0.5h, dry DMF (0.29mol, 22.7mL) was added 12a–k.
within 0.5h and stirring at −78°C for 1h. The mixture was
General Procedure for the Preparation of Compounds
warmed slowly to 0°C and stirring for 1h. After a further 14a–h. General Procedure for the Preparation of Com-
2h at room temperature, the mixture poured onto HCl (aq, pounds 16a–h 1H-Benzo[d]imidazole (11.8g, 0.1mol),
1000mL, 0.25mol/L)/ice (400g) yielding a yellow precipitate. together with the respective benzyl chloride (0.11mol) and
This was collected by filtration and washed with water. The anhydrous K2CO3 (27.6g, 0.2mol) were dissolved in DMF
precipitate was dried at 25°C for 24h to give light yellow (30mL) and heated at 50°C for 5–6h. Upon cooling to room
powder (41.6g, 75.0%). 1H-NMR (300MHz, DMSO-d6) δ: temperature, the mixture poured into ice-water and then the
3.77 (4H, t, J1=5.1Hz, J2=4.5Hz), 3.95 (4H, t, J1=4.5Hz, J2= precipitated was collected by filtration and dried. The crude

1042
Vol. 60, No. 8
product was purified by recrystallizing from ethanol to give
the solids 16a–h.
(E)-4-(6-((4-(Methylsulfonyl)piperazin-1-yl)methyl)-2-(2-
(4-(trifluoromethoxy)benzylidene)hydrazinyl)thieno[3,2-d]-
General Procedure for the Preparation of Compounds pyrimidin-4-yl)morpholine (11d): This compound was
14a–h To a stirred, cooled (−78°C) solution of substituted obtained as light yellow solid in 75% yield. mp 240–241°C.
1-benzyl-1H-benzo[d]imidazole 16a–h (0.03mol) in 60mL ESI-MS m/z: 599.8 (M+H)+. IR (KBr) cm−1: 3431.8, 1626.2,
THF under nitrogen atmosphere was added dropwise n-butyl- 1548.6, 1199.0, 1158.5, 1043.4, 780.4. 1H-NMR (300MHz,
lithium solution (0.04mol, 2.5M in hexanes) for half an hour. DMSO-d6) δ: 2.38 (4H, s), 2.98 (3H, s), 3.12 (4H, s), 3.82 (4H,
When addition was complete, stirring at −75°C was continued s), 4.07 (4H, s), 4.57 (2H, s), 7.68 (1H, s), 8.15 (1H, s), 8.17
for 1h. The reaction mixture was then treated with 0.1mol (4H, s), 12.65 (1H, s). Anal. Calcd for C24H28F3N7O4S2 (%):
dried DMF and stirring at −75°C was continued for 1h. The C, 48.07; H, 4.71; N, 16.35. Found (%): C, 48.02; H, 4.74; N,
mixture was then allowed to warm to ambient temperature 16.39.
and stirred for a future 2h. The reaction was quenched with
(E)-4-(2-(2-(2-Chloro-4-fluorobenzylidene)hydrazinyl)-6-
saturated NH4Cl (50mL) and extracted with ether (100mL×2). ((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]-
The organic phase was washed with brine, dried (MgSO4), pyrimidin-4-yl)morpholine (11e): This compound was ob-
filtered and concentrated. The crude product was purified tained as light yellow solid in 78% yield. mp 237–238°C.
by chromatography on silica gel using petroleum–EtOAc to ESI-MS m/z: 568.0 (M+H)+. IR (KBr) cm−1: 3433.0, 3281.4,
give substituted 1-benzyl-1H-benzo[d]imidazole-2-carbaldehyde 1547.2, 1515.6, 1345.2, 1163.0, 962.2, 776.9. 1H-NMR
14a–h.
(300MHz, DMSO-d6) δ: 2.57 (4H, s), 2.90 (3H, s), 3.15 (4H, s),
General Procedure for Preparation of Compounds 11a–f, 3.77 (4H, s), 3.82 (6H, s), 7.16 (1H, s), 7.30 (1H, s), 7.47 (1H, d,
13a–k and 15a–h A mixture of 9 (0.12g, 0.28mmol), sub- J=7.1Hz), 8.04 (1H, s), 8.44 (1H s), 11.08 (1H, s). Anal. Calcd
stitued aldehyde (10a–f, 12a–k, 14a–h, 0.28mmol) and a drop for C23H27ClFN7O3S2 (%): C, 48.63; H, 4.79; N, 17.26. Found
of glacial acetic acid in absolute ethanol (8mL) was refluxed (%): C, 48.60; H, 4.76; N, 17.31.
for 4–8h. The mixture was cooled, separated by filtration and
(E)-4-((2-(6-((4-(Methylsulfonyl)piperazin-1-yl)methyl)-4-
washed with EtOH to give off a white to light yellow solid. morpholinothieno[3,2-d]pyrimidin-2-yl)hydrazono)methyl)-
The crude product was recrystallized from ethanol or puri- benzonitrile (11f): This compound was obtained as light
fied by chromatography on silica gel using MeOH/CH2Cl2 to yellow solid in 79% yield. mp 266–267°C. ESI-MS m/z: 541.2
afford white to yellow solids 11a–f, 13a–k and 15a–h, respec- (M+H)+. IR (KBr) cm−1: 3436.3, 2850.3, 2222.9, 1513.7,
1
tively.
1345.2, 1161.0, 963.4, 779.3. H-NMR (300MHz, DMSO-d6) δ:
(E)-4-(6-((4-(Methylsulfonyl)piperazin-1-yl)methyl)-2-(2- 2.57 (4H, s), 2.90 (3H, s), 3.14 (4H, s), 3.76 (4H, s), 3.86 (6H,
(3,4,5-trimethoxybenzylidene)hydrazinyl)thieno[3,2-d]pyrimi- s), 7.17 (1H, s), 7.82 (4H, d, J=3.7Hz), 8.10 (1H, s), 11.11 (1H,
din-4-yl)morpholine (11a): This compound was obtained as s). Anal. Calcd for C24H28N8O3S2 (%): C, 53.32; H, 5.22; N,
light yellow solid in 77% yield. mp 265–266°C. ESI-MS m/z: 20.73. Found (%): C, 53.27; H, 5.24; N, 20.79.
1
606.3 (M+H)+. H-NMR (300MHz, DMSO-d6) δ: 2.58 (4H,
(E)-4-(2-(2-((1-(3-Fluorobenzyl)-1H-indol-3-yl)methylene)-
s), 2.90 (3H, s), 3.14 (4H, s), 3.96–3.56 (19H, m), 7.01 (2H, hydrazinyl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-
s), 7.19 (1H, s), 8.03 (1H, s), 11.11 (1H, s). Anal. Calcd for thieno[3,2-d]pyrimidin-4-yl)morpholine (13a): This com-
C26H35N7O6S2 (%): C, 51.55; H, 5.82; N, 16.19. Found (%): C, pound was obtained as pale yellow solid in 75% yield. mp
51.50; H, 5.82; N, 16.25.
199–201°C. ESI-MS m/z: 662.5 (M+H)+. IR (KBr) cm−1:
1
(E)-4-(6-((4-(Methylsulfonyl)piperazin-1-yl)methyl)-2-(2- 3436.8, 1629.1, 1531.4, 1198.2, 1046.2, 780.4, 747.0. H-NMR
(2,3,4-trimethoxybenzylidene)hydrazinyl)thieno[3,2-d]pyrimi- (300MHz, DMSO-d6) δ: 2.58 (4H, s), 2.90 (3H, s), 3.15 (4H,
din-4-yl)morpholine (11b): This compound was obtained as s), 3.80 (4H, d, J=4.3Hz), 3.85 (2H, s), 3.94 (4H, s), 5.46 (2H,
light yellow solid in 75% yield. mp 184–185°C. ESI-MS m/z: s), 7.24–7.03 (6H, m), 7.36 (1H, dd, J1=14.5Hz, J2=7.4Hz),
606.4 (M+H)+. IR (KBr) cm−1: 3431.1, 2931.2, 1625.5, 1594.4, 7.49 (1H, d, J=8.0Hz), 7.83 (1H, s), 8.26 (1H, s), 8.49 (1H, d,
1
1198.8, 1047.2, 783.1. H-NMR (300MHz, DMSO-d6) δ: 2.39 J=7.6Hz), 10.51 (1H, s). Anal. Calcd for C32H35FN8O3S2 (%):
(4H, s), 3.00 (3H, s), 3.21 (4H, s), 3.79 (3H, s), 3.84 (4H, d, C, 57.99; H, 5.32; N, 16.91. Found (%): C, 57.91; H, 5.34; N,
J=4.4Hz), 3.87 (3H, s), 3.89 (3H, s), 4.08 (4H, d, J=4.2Hz), 16.96.
4.67 (2H, s), 7.00 (1H, d, J=9.0Hz), 7.71 (1H, s), 7.97 (1H,
(E)-4-(2-(2-((1-(4-tert-Butylbenzyl)-1H-indol-3-yl)methyl-
d, J=8.9Hz), 8.49 (1H, s), 12.49 (1H, s). Anal. Calcd for ene)hydrazinyl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-
C26H35N7O6S2 (%): C, 51.54; H, 5.82; N, 16.19. Found (%): C, thieno[3,2-d]pyrimidin-4-yl)morpholine (13b): This compound
51.50; H, 5.81; N, 16.26.
was obtained as off-white solid in 78% yield. mp 253–255°C.
(E)-4-(2-(2-(Benzo[d][1,3]dioxol-5-ylmethylene)hydrazinyl)- ESI-MS m/z: 700.9 (M+H)+. IR (KBr) cm−1: 3431.7, 2957.7,
1
6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyri- 1553.8, 1515.3, 1372.9, 1346.3, 1161.5, 776.7, 745.4. H-NMR
midin-4-yl)morpholine (11c): This compound was obtained as (300MHz, DMSO-d6) δ: 1.22 (9H, s), 2.57 (4H, s), 2.89 (3H,
light yellow solid in 72% yield. mp 200–202°C. ESI-MS m/z: s), 3.14 (4H, s), 3.79 (4H, s), 3.84 (2H, s), 3.94 (4H, s), 5.37
560.2 (M+H)+. IR (KBr) cm−1: 3429.8, 2855.0, 1566.1, 1515.8, (2H, s), 7.26–6.96 (5H, m), 7.32 (2H, d, J=7.8Hz), 7.50 (1H,
1
1325.9, 1161.6, 779.2. H-NMR (300MHz, DMSO-d6) δ: 2.57 d, J=7.6Hz), 7.79 (1H, s), 8.26 (1H, s), 8.47 (1H, d, J=7.2Hz),
(4H, s), 2.89 (3H, s), 3.14 (4H, s), 3.76 (4H, d, J=4.3Hz), 10.49 (1H, s). Anal. Calcd for C36H44N8O3S2 (%): C, 61.69; H,
3.95–3.81 (6H, m), 6.06 (2H, s), 6.94 (1H, d, J=8.0Hz), 7.05 6.33; N, 15.99. Found (%): C, 61.69; H, 6.30; N, 15.98.
(1H, d, J=8.1Hz), 7.26 (1H, s), 7.13 (1H, s), 8.01 (1H, d,
(E)-4-(2-(2-((1-(2-Chlorobenzyl)-1H-indol-3-yl)methylene)-
J=9.0Hz), 10.65 (s, 1H). Anal. Calcd for C24H29N7O5S2 (%): hydrazinyl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-
C, 51.51; H, 5.22; N, 17.52. Found (%): C, 51.44; H, 5.25; N, thieno[3,2-d]pyrimidin-4-yl)morpholine (13c): This compound
17.56.
was obtained as white solid in 82% yield. mp 272–273°C.

August 2012
1043
ESI-MS m/z: 679.4 (M+H)+. IR (KBr) cm−1: 3430.6, 3306.4, (KBr) cm−1: 3451.3, 2846.4, 2223.7, 1557.1, 1514.0, 1346.1,
1
1
2845.0, 1555.7, 1513.0, 1375.7, 1158.9, 778.3, 748.9. H-NMR 1159.1, 961.1, 778.0. H-NMR (300MHz, DMSO-d6) δ: 2.57
(300MHz, DMSO-d6) δ: 2.57 (4H, s), 2.90 (3H, s), 3.15 (4H, s), (4H, s), 2.89 (3H, s), 3.14 (4H, s), 4.10–3.65 (10H, m), 5.68
3.82 (6H, d, J=13.1Hz), 3.94 (4H, s), 5.53 (2H, s), 6.76 (1H, d, (2H, s), 6.96 (1H, s), 7.19–7.11 (3H, m), 7.47 (2H, s), 7.60 (1H,
J=6.7Hz), 7.36–7.08 (5H, m), 7.41 (1H, d, J=7.7Hz), 7.52 (1H, s), 7.76 (1H, s), 7.92 (1H, s), 8.26 (1H, s), 8.54 (1H, s), 10.55
d, J=7.5Hz), 7.72 (1H, s), 8.25 (1H, s), 8.52 (1H, d, J=6.9Hz), (1H, s). Anal. Calcd for C33H35N9O3S2(%): C, 59.17; H, 5.27; N,
10.53 (1H, s). Anal. Calcd for C32H35ClN8O3S2 (%): C, 56.58; 18.82. Found (%): C, 59.13; H, 5.29; N, 18.86.
H, 5.19; N, 16.50. Found (%): C, 56.52; H, 5.17; N, 16.55.
(E)-4-(2-(2-((1-Benzyl-1H-indol-3-yl)methylene)hydra-
(E)-4-(6-((4-(Methylsulfonyl)piperazin-1-yl)methyl)-2-(2- zinyl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-
((1-(4-(trifluoromethyl)benzyl)-1H-indol-3-yl)methylene)hydra- d]pyrimidin-4-yl)morpholine (13i): This compound was ob-
zinyl)thieno[3,2-d]pyrimidin-4-yl)morpholine (13d): This tained as off-white solid in 70% yield. mp 267–268°C. ESI-MS
compound was obtained as white solid in 72% yield. mp m/z: 645.6 (M+H)+. IR (KBr) cm−1: 3450.7, 2853.6, 1553.3,
1
283–284°C. ESI-MS m/z: 713.1 (M+H)+. IR (KBr) cm−1: 1515.2, 1344.3, 1161.1, 777.7, 746.8. H-NMR (300MHz, DM-
1
3447.1, 2853.1, 1554.1, 1515.3, 1327.9, 1161.9, 1118.9, 778.1. H- SO-d6) δ: 2.58 (4H, s), 2.90 (3H, s), 3.15 (4H, s), 3.79 (4H, s),
NMR (300MHz, DMSO-d6) δ: 2.58 (4H, s), 2.90 (3H, s), 3.15 3.85 (2H, s), 3.94 (4H, s), 5.43 (2H, s), 7.21 (8H, m), 7.48 (1H,
(4H, s), 3.80 (6H, s), 3.95 (4H, s), 5.56 (2H, s), 7.18–7.14 (3H, d, J=8.2Hz), 7.81 (1H, s), 8.26 (1H, s), 8.48 (1H, d, J=7.6Hz),
m), 7.60–7.50 (5H, m), 7.86 (1H, s), 8.27 (1H, s), 8.52 (1H, s), 10.52 (1H, s). Anal. Calcd for C32H36N8O3S2(%): C, 59.61; H,
10.53 (1H, s). Anal. Calcd for C33H35F3N8O3S2 (%): C, 55.60; 5.63; N, 17.38. Found (%): C, 59.56; H, 5.60; N, 17.45.
H, 4.95; N, 15.72. Found (%): C, 55.56; H, 4.94; N, 15.79.
(E)-4-(2-(2-((1-(2,3-Dichlorobenzyl)-1H-indol-3-yl)methyl-
(E)-3-((3-((2-(6-((4-(Methylsulfonyl)piperazin-1-yl)- ene)hydrazinyl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-
methyl)-4-morpholinothieno[3,2-d]pyrimidin-2-yl)hydrazono)- thieno[3,2-d]pyrimidin-4-yl)morpholine (13j): This compound
methyl)-1H-indol-1-yl)methyl)benzonitrile (13e): This com- was obtained as off-white solid in 76% yield. mp 287–288°C.
pound was obtained as light yellow solid in 74% yield. mp ESI-MS m/z: 713.6 (Cl=35), 715.6 (Cl=37) (M+H)+. IR
266–268°C. ESI-MS m/z: 670.4 (M+H)+. IR (KBr) cm−1: (KBr) cm−1: 3443.4, 1553.4, 1515.4, 1344.6, 1159.2, 960.8,
3429.8, 3310.3, 2845.1, 2229.0, 1558.2, 1513.4, 1157.9, 778.9. 777.4, 742.5. 1H-NMR (300MHz, DMSO-d6) δ: 2.58 (4H,
1H-NMR (300MHz, DMSO-d6) δ: 2.57 (4H, s), 2.90 (3H, s), s), 2.90 (3H, s), 3.15 (4H, s), 3.89–3.76 (6H, m), 3.95 (4H, s),
3.15 (4H, s), 3.80 (6H, s), 3.94 (4H, s), 5.50 (2H, s), 7.18–7.14 5.58 (2H, s), 6.60 (1H, d, J=7.7Hz), 7.35–7.03 (4H, m), 7.42
(3H, m), 7.53 (3H, s), 7.76 (2H, s), 7.85 (1H, s), 8.27 (1H, s), (1H, d, J=7.4Hz), 7.58 (1H, d, J=8.1Hz), 7.75 (1H, s), 8.26
8.51 (1H, s), 10.54 (1H, s). Anal. Calcd for C33H35N9O3S2 (%): (1H, s), 8.53 (1H, d, J=7.1Hz), 10.63 (1H, s). Anal. Calcd for
C, 59.17; H, 5.27; N, 18.82. Found (%): C, 59.14; H, 5.29; N, C32H34Cl2N8O3S2(%): C, 53.85; H, 4.80; N, 15.70. Found (%):
18.86.
(E)-4-(2-(2-((1-(3,4-Dichlorobenzyl)-1H-indol-3-yl)methyl-
C, 53.86; H, 4.82; N, 15.68.
(E)-4-(2-(2-((1-(2,6-Dichlorobenzyl)-1H-indol-3-yl)methyl-
ene)hydrazinyl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)- ene)hydrazinyl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-
thieno[3,2-d]pyrimidin-4-yl)morpholine (13f): This compound thieno[3,2-d]pyrimidin-4-yl)morpholine (13k): This compound
was obtained as white solid in 82% yield. mp 279–281°C. was obtained as off-white solid in 75% yield. mp 275–276°C.
ESI-MS m/z: 713.1 (M+H)+. IR (KBr) cm−1: 3438.0, 3296.5, ESI-MS m/z: 713.0 (Cl=35), 715.0 (Cl=37) (M+H)+. IR (KBr)
1556.4, 1515.9, 1344.7, 1159.3, 960.0, 778.4. 1H-NMR cm−1: 3423.2, 3006.0, 1628.0, 1549.0, 1198.3, 1158.2, 937.3,
(300MHz, DMSO-d6) δ: 2.57 (4H, s), 2.89 (3H, s), 3.15 (4H, 780.1. 1H-NMR (300MHz, DMSO-d6) δ: 2.57 (4H, s), 2.89
s), 4.05–3.66 (10H, m), 5.45 (2H, s), 7.36–7.07 (4H, m), 7.49 (3H, s), 3.15 (4H, s), 3.80 (4H, d, J=4.4Hz), 3.84 (2H, s), 3.93
(1H, d, J=8.0Hz), 7.69–7.53 (2H, m), 7.83 (1H, s), 8.27 (1H, (4H, s), 5.58 (2H, s), 7.36–6.95 (4H, m), 7.55–7.41 (1H, m),
s), 8.50 (1H, d, J=7.6Hz), 10.52 (1H, s). Anal. Calcd for 7.60 (3H, dd, J1=8.0Hz, J2=3.1Hz), 8.16 (1H, s), 8.49 (1H, d,
C32H34Cl2N8O3S2 (%): C, 53.85; H, 4.80; N, 15.70. Found (%): J=7.8Hz), 10.48 (1H, s). Anal. Calcd for C32H34Cl2N8O3S2 (%):
C, 53.84; H, 4.82; N, 15.69.
C, 53.85; H, 4.80; N, 15.70. Found (%): C, 53.80; H, 4.82; N,
(E)-4-(2-(2-((1-(2,4-Dichlorobenzyl)-1H-indol-3-yl)methyl- 15.75.
ene)hydrazinyl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-
(E)-4-(2-(2-((1-(3,4-Dichlorobenzyl)-1H-benzo[d]imida-
thieno[3,2-d]pyrimidin-4-yl)morpholine (13g): This compound zol-2-yl)methylene)hydrazinyl)-6-((4-(methylsulfonyl)pipera-
was obtained as white solid in 79% yield. mp 245–246°C. zin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine (15a):
ESI-MS m/z: 712.8 (Cl=35), 714.8 (Cl=37) (M+H)+. IR (KBr) This compound was obtained as white solid in 76% yield.
1
1
cm−1: 3438.5, 1553.9, 1515.8, 1344.7, 1159.3, 960.0, 777.4. H- ESI-MS m/z: 713.1 (Cl=35), 715.1 (Cl=37) (M+H)+. H-NMR
NMR (300MHz, DMSO-d6) δ: 2.54 (4H, s), 2.86 (3H, s), 3.11 (300MHz, DMSO-d6) δ: 2.57 (4H, s), 2.90 (3H, s), 3.14 (4H, s),
(4H, s), 3.76 (4H, s), 3.81 (2H, s), 3.91 (4H, s), 5.48 (2H, s), 6.72 3.62 (4H, s), 3.73 (4H, s), 3.86 (2H, s), 6.14 (2H, s), 7.15 (1H,
(1H, d, J=8.3Hz), 7.07 (1H, s), 7.23–7.09 (2H, m), 7.31 (1H, d, s), 7.27 (3H, d, J=6.8Hz), 7.55 (2H, d, J=8.9Hz), 7.66 (1H, d,
J=8.4Hz), 7.37 (1H, d, J=7.8Hz), 7.67 (2H, d, J=5.4Hz), 8.21 J=6.8Hz), 7.89 (1H, s), 8.26 (1H, s), 11.41 (1H, s). Anal. Calcd
(1H, s), 8.48 (1H, d, J=7.6Hz), 10.47 (1H, s). Anal. Calcd for for C31H33Cl2N9O3S2 (%): C, 52.10; H, 4.65; N, 17.64. Found
C32H34Cl2N8O3S2 (%): C, 53.85; H, 4.80; N, 15.70. Found (%): (%): C, 52.06; H, 4.63; N, 17.69.
C, 53.80; H, 4.82; N, 15.76.
(E)-4-(2-(2-((1-(4-tert-Butylbenzyl)-1H-benzo[d]imidazol-2-
(E)-3-((3-((2-(6-((4-(Methylsulfonyl)piperazin-1-yl)- yl)methylene)hydrazinyl)-6-((4-(methylsulfonyl)piperazin-1-
methyl)-4-morpholinothieno[3,2-d]pyrimidin-2-yl)hydrazono)- yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine (15b): This
methyl)-1H-indol-1-yl)methyl)benzonitrile (13h): This com- compound was obtained as off-white solid in 78% yield. mp
pound was obtained as white solid in 82% yield. mp 252–254°C. ESI-MS m/z: 702.4 (M+H)+. IR (KBr) cm−1:
264–265°C. ESI-MS m/z: 670.2 (M+H)+, 692.1 (M+Na)+. IR 3437.1, 1553.7, 1516.9, 1345.9, 1159.9, 777.9, 743.9. 1H-NMR

1044
Vol. 60, No. 8
(300MHz, DMSO-d6) δ: 1.20 (9H, s), 2.57 (4H, s), 2.89 (3H, 3.59 (4H, s), 3.70 (4H, s), 3.85 (2H, s), 6.16 (2H, s), 7.13 (1H, s),
s), 3.14 (4H, s), 3.52 (4H, s), 3.68 (4H, s), 3.85 (2H, s), 6.14 (2H, 7.22–7.18 (2H, m), 7.38 (4H, dd, J1=16.9Hz, J2=8.5Hz), 7.49
s), 7.13 (1H, s), 7.21 (2H, d, J=8.6Hz), 7.30 (4H, s), 7.50 (1H, (1H, d, J=7.2Hz), 7.64 (1H, d, J=6.5Hz), 8.27 (1H, s), 11.40
d, J=8.4Hz), 7.63 (1H, d, J1=6.2Hz), 8.28 (1H, s), 11.40 (1H, (1H, s). Anal. Calcd for C31H34ClN9O3S2 (%): C, 54.74; H, 5.04;
s). Anal. Calcd for C35H43N9O3S2 (%): C, 59.89; H, 6.17; N, N, 18.53. Found (%): C, 54.67; H, 5.05; N, 18.57.
17.96. Found (%): C, 59.82; H, 6.19; N, 17.99.
(E)-4-(2-(2-((1-(3-Chlorobenzyl)-1H-benzo[d]imidazol-2-
(E)-4-(2-(2-((1-(4-Methylbenzyl)-1H-benzo[d]imidazol-2- yl)methylene)hydrazinyl)-6-((4-(methylsulfonyl)piperazin-1-
yl)methylene)hydrazinyl)-6-((4-(methylsulfonyl)piperazin-1- yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine (15h): This
yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine (15c): This compound was obtained as white solid in 81% yield. mp
compound was obtained as pale yellow solid in 76% yield. 253–255°C. ESI-MS m/z: 680.2 (M+H)+. IR (KBr) cm−1:
1
mp 281–282°C. ESI-MS m/z: 660.2 (M+H)+. IR (KBr) cm−1: 3426.1, 2852.9, 1556.8, 1516.0, 1158.5, 780.1, 743.4. H-NMR
3439.2, 2855.7, 1545.7, 1516.3, 1342.1, 1326.1, 1158.4, 781.8. (300MHz, DMSO-d6) δ: 2.56 (4H, s), 2.89 (3H, s), 3.14 (4H,
1H-NMR (300MHz, DMSO-d6) δ: 2.21 (3H, s), 2.57 (4H, s), s), 3.60 (4H, s), 3.71 (4H, s), 3.85 (2H, s), 6.17 (2H, s), 7.14
2.89 (3H, s), 3.14 (4H, s), 3.56 (4H, s), 3.71 (4H, s), 3.85 (2H, (1H, s), 7.28 (5H, d, J=11.5Hz), 7.56 (2H, d, J=10.6Hz), 7.65
s), 6.13 (2H, s), 7.07 (2H, d, J=7.9Hz), 7.13 (1H, s), 7.32–7.17 (1H, d, J=6.7Hz), 8.27 (1H, s), 11.41 (1H, s). Anal. Calcd for
(4H, m), 7.46 (1H, d, J=6.1Hz), 7.72–7.55 (1H, m), 8.27 (1H, C31H34ClN9O3S2 (%): C, 54.74; H, 5.04; N, 18.53. Found (%):
s), 11.38 (1H, s). Anal. Calcd for C32H37N9O3S2(%): C, 58.25; C, 54.69; H, 5.03; N, 18.58.
H, 5.65; N, 19.11. Found (%): C, 58.17; H, 5.69; N, 19.14.
Cytotoxicity Assay in Vitro19) The cytotoxicity of target
(E)-4-(2-(2-((1-(2,3-Dichlorobenzyl)-1H-benzo[d]imida- compounds (11a–f, 13a–k, 15a–h) were evaluated with H460,
zol-2-yl)methylene)hydrazinyl)-6-((4-(methylsulfonyl)pipera- HT-29 and MDA-MB-231 cell lines by the standard MTT as-
zin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine (15d): say in vitro, with GDC-0941 and compound 1 as the positive
This compound was obtained as white solid in 74% yield. controls. Four potent compounds (11c, 13b, 15f, 15g) were
mp 279–281°C. ESI-MS m/z: 714.0 (Cl=35), 716.1 (Cl=37) further evaluated against six cancer cell lines (A549, H1975,
(M+H)+. IR (KBr) cm−1: 3432.1, 2853.5, 1521.2, 1345.9, 1327.1, H226, U87MG, HepG2 and SGC-7901) and WI-38 normal cell
1
1161.9, 779.3. H-NMR (300MHz, DMSO-d6) δ: 2.57 (4H, s), line. The cancer or normal cell lines were cultured in mini-
2.89 (3H, s), 3.14 (4H, s), 3.63 (4H, s), 3.69 (4H, s), 3.85 (2H, mum essential medium (MEM) supplement with 10% fetal
s), 6.27 (2H, s), 6.45 (1H, d, J=7.8Hz), 7.11 (1H, s), 7.34–7.14 bovine serum (FBS). Approximately 4×103 cells, suspended
(4H, m), 7.54 (1H, d, J=7.9Hz), 7.69 (1H, d, J=6.4Hz), 8.28 in MEM medium, were plated onto each well of a 96-well
(1H, s), 11.32 (1H, s). Anal. Calcd for C31H33Cl2N9O3S2 (%): C, plate and incubated in 5% CO2 at 37°C for 24h. The test
59.89; H, 6.17; N, 17.96. Found (%): C, 59.85; H, 6.19; N, 17.81. compounds at indicated final concentrations were added to the
(E)-4-(2-(2-((1-(2-Chlorobenzyl)-1H-benzo[d]imidazol-2- culture medium and the cell cultures were continued for 72h.
yl)methylene)hydrazinyl)-6-((4-(methylsulfonyl)piperazin-1- Fresh MTT was added to each well at a terminal concentra-
yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine (15e): This tion of 5µg/mL and incubated with cells at 37°C for 4h. The
compound was obtained as light yellow solid in 76% yield. formazan crystals were dissolved in 100µL dimethyl sulfoxide
mp 255–257°C. ESI-MS m/z: 701.8 (M+Na)+. IR (KBr) cm−1: (DMSO) each well, and the absorbency at 492nm (for absor-
1
3430.7, 1552.2, 1516.6, 1345.5, 1160.1, 777.9, 746.5. H-NMR bance of MTT formazan) and 630nm (for the reference wave-
(300MHz, DMSO-d6) δ: 2.56 (4H, s), 2.89 (3H, s), 3.14 (4H, length) was measured with the enzyme-linked immunosorbent
s), 3.60 (4H, s), 3.68 (4H, d, J=4.2Hz), 3.85 (2H, s), 6.28 (2H, assay (ELISA) reader. All of the compounds were tested three
s), 6.64 (1H, d, J=7.7Hz), 7.10 (1H, s), 7.34–7.13 (5H, m), 7.55 times in each of the cell lines. The results expressed as IC50
(1H, d, J=7.9Hz), 7.67 (1H, d, J=5.8Hz), 8.30 (1H, s), 11.36 (inhibitory concentration 50%) were the averages of two de-
(1H, s). Anal. Calcd for C31H34ClN9O3S2 (%): C, 54.74; H, 5.04; terminations and calculated by using the Bacus Laboratories
N, 18.53. Found (%): C, 54.70; H, 5.02; N, 18.58.
Inc. Slide Scanner (Bliss) software.
(E)-4-(2-(2-((1-Benzyl-1H-benzo[d]imidazol-2-yl)methyl-
ene)hydrazinyl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-
Acknowledgments This work was supported by a Grant
thieno[3,2-d]pyrimidin-4-yl)morpholine (15f): This compound from National S and T Major Project (No. 2009ZX09301–012)
was obtained as white solid in 79% yield. mp 254–256°C. and National Natural Science Foundation of China (NSFC No.
ESI-MS m/z: 646.6 (M+H)+. IR (KBr) cm−1: 3430.4, 2853.6, 21002065).
1
1554.5, 1515.5, 1346.1, 1159.3, 961.5, 778.2, 744.5. H-NMR
(300MHz, DMSO-d6) δ: 2.57 (4H, s), 2.89 (3H, s), 3.14 (4H,
s), 3.55 (4H, s), 3.69 (4H, s), 3.85 (2H, s), 6.18 (2H, s), 7.13
(1H, s), 7.28–7.22 (5H, m), 7.37 (2H, d, J=6.4Hz), 7.46 (1H,
s), 7.63 (1H, s), 8.28 (1H, s), 11.39 (1H, s). Anal. Calcd for
C31H35N9O3S2 (%): C, 57.65; H, 5.46; N, 19.52. Found (%): C,
57.58; H, 5.44; N, 19.58.
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