Synthesis and cytotoxic activity of novel 2,6-disubstituted-4-mor- pholinothieno[3,2-d]pyrimidines as potent anti-tumor agents

Article abstract of DOI:10.1016/j.cclet.2012.04.012

A series of 2,6-disubstituted-4-morpholinothieno[3,2-d]pyrimidine derivatives were synthesized and their cytotoxic activity against H460, HT-29, MDA-MB-231, U87MG and H1975 cancer cell lines were evaluated in vitro. Most of the target compounds exhibited moderate to excellent activity to the tested cell lines. The most promising compound 23 (0.84 μmol/L, 0.23 μmol/L, 2.52 μmol/L, 1.80 μmol/L) was 1.0, 2.9, 29.3 and 4.3 times more active than GDC-0941 (0.87 μmol/L, 0.66 μmol/L, 73.8 μmol/L, 7.77 μmol/L) against H460, HT-29, MDA-MB-231 and U87MG cell lines, respectively.

Full text of DOI:10.1016/j.cclet.2012.04.012

Available online at www.sciencedirect.com
Chinese Chemical Letters 23 (2012) 703–706
www.elsevier.com/locate/cclet
Synthesis and cytotoxic activity of novel 2,6-disubstituted-4-mor-
pholinothieno[3,2-d]pyrimidines as potent anti-tumor agents
*
Wu Fu Zhu, Xin Zhai, Sai Li, Yun Yun Cao, Ping Gong, Ya Jing Liu
Key Laboratary of Original New Drugs Design and Discovery of Ministry of Education, School of Pharmaceutical Engineering,
Shenyang Pharmaceutical University, Shenyang 110016, China
Received 7 March 2012
Available online 11 May 2012
Abstract
A series of 2,6-disubstituted-4-morpholinothieno[3,2-d]pyrimidine derivatives were synthesized and their cytotoxic activity
against H460, HT-29, MDA-MB-231, U87MG and H1975 cancer cell lines were evaluated in vitro. Most of the target compounds
exhibited moderate to excellent activity to the tested cell lines. The most promising compound 23 (0.84 mmol/L, 0.23 mmol/L,
2.52 mmol/L, 1.80 mmol/L) was 1.0, 2.9, 29.3 and 4.3 times more active than GDC-0941 (0.87 mmol/L, 0.66 mmol/L, 73.8 mmol/L,
7.77 mmol/L) against H460, HT-29, MDA-MB-231 and U87MG cell lines, respectively.
# 2012 Ya Jing Liu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: 4-Morpholinothieno[3,2-d]pyrimidine; Synthesis; Cytotoxic activity
The derivatives of thienopyrimidine have attracted great attentions over many years due to their broad bioactivities,
including anti-tumor [1–3], antimicrobial [4], anti-inflammatory [5], etc. Among them, 4-morpho-linothieno[3,2-
d]pyrimidines (e.g. 1, GDC-0941 and GNE-477, Fig. 1) have been reported to show marked anti-tumor activities
[2,3,6]. For example, GDC-0941, which is a potent, selective, orally bioavailable small inhibitor of PI3K, exerted
antiproliferative effects against an array of human tumor cell lines and is currently under going Phase I clinical trials
[2,7]. Further studies of GDC-0941 led to a backup clinical candidate GNE-477, which possessed potent kinase and
cytotoxic activity [3]. The SARs studies revealed that the 4-morpholinothieno[3,2-d]pyrimidine scaffold played a key
role in their anti-tumor activities. Furthermore, the introduction of 4-(methylsulfonyl)-piperazin-1-ylmethyl
substituent at C6-position remarkably enhanced the solubility and metabolic stability, which may attribute to the
hydrogen bonds formed between sulfonylpiperazine moiety and the residues located near the mouth of the ATP-
binding pocket [2,3].
The benzylidenehydrazinyl group or its mimics are key pharmacophores in the design of different potential
anticancer agents [8–10]. In the past few years, many of the benzylidenehydrazine derivatives are identified as potent
anticancer agents, such as, PAC-1 [8] and compound 5 [9] (Fig. 1). In addition, we have reported a series of 2-(2-
benzylidenehydrazinyl)pyrido[2,3-b]pyrazin-3(4H)-ones derivatives and most of them showed potent in vitro
antitumor activities against several cell lines [10,11]. Encouraged by these observations, we planned to synthesize new
* Corresponding author.
E-mail address: lyjpharm@126.com (Y.J. Liu).
1001-8417/$ – see front matter # 2012 Ya Jing Liu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2012.04.012

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W.F. Zhu et al. / Chinese Chemical Letters 23 (2012) 703–706
O
O
N
O
O
N
O
N
O
N
O
N
S
S
2MeSO3H
N
N
S
N
S
N
N
S
N
NH
N
N
O
N
OH
N
O
N
O
N
H3C
S
N
NH2
1
2 GDC-0941
3 GNE-477
N
S
O
N
N
N
R
N
N
H
O
N
O
18-27
OH
O
N
N
N
N
N
N
N
H
N
N
OH
H
O
4 PAC-1
5
Fig. 1. Structures of some reported thieno[3,2-d]pyrimidines (1, GDC-0941, GNE-477), hydrazones (PAC-1, 5) and target compounds.
chemical entities incorporating the two active pharmacophores, 4-morpholinothieno[3,2-d]pyrimidine and
(hetero)arylmethylenehydrazinyl group in a single molecular framework. Therefore, a series of 2,6-disubstituted-
4-morpholinothieno[3,2-d]pyrimidine derivatives, containing arylmethylene- or heteroarylmethylene-hydrazinyl
group at C2-position and 4-(methylsulfonyl)piperazin-1-ylmethyl substituent at C6-position (Fig. 1), were designed
and synthesized. Their cytotoxic activity against H460 (human lung cancer), HT-29 (human colorectal cancer), MDA-
MB-231 (human breast cancer), U87MG (human glioblastoma) and H1975 (human lung cancer) cell lines were
evaluated in vitro in attempt to find novel and potent anti-tumor agents.
The synthesis of title compounds is illustrated in Scheme 1 [2]. The commercially available methyl 3-amino-2-
thiophenecarboxylate 6 was condensed with urea at 190 8C for 2 h to give thienopyrimidinedione 7, which was
chlorinated with phosphorus oxychloride to afford 8 as a pale yellow solid. Regioselective nucleophilic displacement of
the 4-chloride with morpholine gave rise to 9 in 85–87% yields. Subsequently, lithiation of 9, followed by the addition of
DMF, gave the aldehyde 10 in 75% yield. Treatment of 10 with NaBH4 in MeOH at room temperature provided 11 as a
primary alcohol, which reacted with thionyl chloride and then substituted with 1-(methylsulfonyl)piperazine to furnish
O
N
O
N
O
N
O
O
Cl
d
e
a
b
c
S
S
S
S
S
S
OMe
NH
NH
N
N
HO
N
S
N
OHC
N
H
7
O
N
Cl
N
Cl
2
N
Cl
N
Cl
8
9
10
6
11
O
O
O
N
O
N
O
O
O
O
O
O
S
S
N
N
i
N
N
h
g
N
f
N
N
Cl
S
N
N
S
N
S
N
S
N
N
17a-17j
NH
N
N
Cl
N
R
2
N
N
Cl
N
H
H
12
13
OH
14
18-27
R ,R =H,CH
3
17a 3-allyl-4-hydroxybenzaldehyde
17b 2-hydroxy-3-(2-methylallyl)benzaldehyde
17c 3-allyl-2-hydroxy-5-methylbenzaldehyde
17d 5-tert-butyl-2-hydroxybenzaldehyde
17e 3,5-di-tert-butyl-2-hydroxybenzaldehyde
1
2
OH
O
17f 1H-indazole-3-carbaldehyde
17g 1H-indole-3-carbaldehyde
17h 4-chloro-2-hydroxybenzaldehyde
17i 3,5-dibromo-4-hydroxybenzaldehyde
17j 2-hydroxy-1-naphthaldehyde
j
k
R
2
R
CHO
15a-15c
CHO
1
R
R
CHO
1
1
R
2
16a-16c
17a-17c
Scheme 1. Reagents and conditions: (a) 5 equiv. urea, 190 8C, 2.5 h; (b) POCl3, DMF (cat.), reflux, 8 h; (c) 2.1 equiv. morpholine, MeOH, 0 8C,
30 min, r.t., 1–2 h; (d) DMF, n-BuLi, THF, À78 8C to r.t., 5 h; (e) NaBH4, MeOH, r.t., 3 h; (f) SOCl2, CH2Cl2, r.t., 2 h; (g) 1-(methylsulfonyl)piper-
azine, DIPEA, isopropanol, r.f., 11 h; (h) 80% NH2NH2ÁH2O, r.f., 6–8 h; (i) R-CHO, EtOH, reflux, 4–8 h; (j) allyl bromide, CH3CN, K2CO3, 60 8C,
5–8 h; (k) 180 8C, 6–8 h.

W.F. Zhu et al. / Chinese Chemical Letters 23 (2012) 703–706
705
Table 1
The structures of compounds 18–27.
Compd.
R
Compd.
R
Compd.
R
Compd.
R
Compd.
R
Br
18
20
22
24
26
HO
HO
OH
N
OH
H
Br
HO
HO
19
21
23
25
27
HO
HO
N
Cl
N
H
compound 13. Next, substitution reaction of 13 with an excess of 80% hydrazine hydrate in ethanol gave the key
intermediate 14, which was then condensed with the corresponding (hetero)aromatic aldehyde 17a–17j in refluxing
ethanol to yield the target compounds 18–27 as white to yellow solids. The substituted aromatic aldehyde 17a–17c were
prepared via a two-step reaction. Reaction of substituted hydroxybenzaldehyde 15a–15c with alkyl bromide in the
presence of K2CO3 in CH3CN gave 16a–16c, which were then heated at 190 8C to afford 17a–17c via Claisen
rearrangement. The structures of target compounds were listed in Table 1. All of them were confirmed by IR, 1H NMR
and MS spectra [12].
All the target compounds and their precursor 14, along with the reference compounds GDC-0941 and PAC-1, were
screened for their in vitro cytotoxic activity against H460, HT-29, MDA-MB-231, U87MG and H1975, using standard
MTT assay. The results, expressed as IC50 values, were summarized in Table 2.
As illustrated in Table 2, most of the synthesized compounds showed potent cytotoxic activities against one or more
cancer cell lines. Several compounds (19, 21–23) exhibited enhanced cytotoxic activities than PAC-1 against H460
cancer cell line. The most promising compound 23 (0.84 mmol/L, 0.23 mmol/L, 2.52 mmol/L, 1.80 mmol/L) was 1.0,
2.9, 29,3 and 4.3 times more active than positive control GDC-0941 (0.87 mmol/L, 0.66 mmol/L, 73.8 mmol/L,
7.77 mmol/L) against H460, HT-29, MDA-MB-231 and U87MG cell lines, respectively.
The pharmacological data indicated that the introduction of substituted arylmethylene group was favorable for
increasing the cytotoxic activity, as all the target compounds showed greater cytotoxic effects than the precursor 14
against all tested cell lines. Furthermore, the substituents in benzene ring had an important influence on antitumor
activity. In most cases, the presence of electron-donating groups (such as tert-butyl, allyl, hydroxyl in this study) in the
benzene ring (19–22) with the exception of hydroxyl group at 4-position (18, 26), enhanced the cytotoxic activity. By
contrast, the introduction of electron-withdrawing groups (such as chloro, bromo) caused a dramatic decrease in
biological activity (25, 26). In addition, hydrogen atom at 5-position of the benzene ring was more favorable, as
compound 19 was more potent than compounds 20–22 against all the tested cancer cell lines.
On the other hand, replacement of arylmethylene with indolyl methylene (24) or 2-hydroxynaphthalen-1-
ylmethylene (27) group resulted in a considerable loss in potency. Interestingly, the alternative indazol-3-ylmethylene
group (23) showed significantly improved potency. Therefore, the nature of the (hetero)arylmethylene group appears
Table 2
Cytotoxic activities of compounds against H460, HT-29, MDA-MB-231, U87MG and H1975 cancer cell lines in vitro.
Compd.
IC50 (mmol/L)
Compd. IC50 (mmol/L)
H460 HT29
H460 HT29 MDA-MB-231 U87MG H1975
MDA-MB-231 U87MG H1975
14
18
39.81 46.22
51.41
20.99
ND
ND
31.98
1.88
4.61
3.51
3.57
0.87
6.82
ND
ND
23
24
0.84
18.38
>100
0.23
15.65 >100
>100 >100
17.41 6.38
ND >100
0.97 6.11
2.52
1.80
ND
ND
ND
ND
ND
28.82
ND
ND
ND
ND
ND
19
20
2.90 >100
25.63
30.73
ND
>100
>100
ND
6.83
3.67
2.64
0.66
7.00
25
26
21
22
4.23
11.57
73.82
15.96
31.57
3.57
ND
ND
27
PAC-1a
GDC-0941a
7.77
0.27
ND: not determined.
a
Used as a positive control.

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W.F. Zhu et al. / Chinese Chemical Letters 23 (2012) 703–706
to have a significant impact on anti-tumor activity, presumably due to the putative hydrogen bonds between the
(hetero)aryl (e.g. indazole, 23) and the enzyme necessary for activity. Next our attention will turn to replacement of
indazolylmethylene with other heterocyclic methylene groups. Further studies in this sense are currently underway
and will be reported in the future.
Acknowledgment
This work was supported by a Grant from the Doctoral Startup Foundation of Liaoning Province (No. 20101110).
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[12] Data for new compounds. 18: Yield: 77%; mp: 185–187 8C; ESI-MS m/z: 572.1 (M+H)+; IR (KBr, cmÀ1): 3434.6, 1630.2, 1548.3, 1197.1,
1054.8, 937.7, 782.7; 1H NMR (300 MHz, DMSO-d6): d 2.40 (s, 4H), 3.00 (s, 3H), 3.19 (s, 4H), 3.83 (s, 6H), 4.07 (s, 4H), 4.65 (s, 2H), 5.07 (m,
2H), 6.02 (d, 1H, J = 6.9 Hz), 6.92 (d, 1H, J = 8.2 Hz), 7.62 (d, 2H, J = 7.3 Hz), 7.72 (s, 1H), 8.18 (s, 1H), 12.43 (s, 1H), 13.01 (s, 1H). 19:
Yield: 82%; mp: 265–265 8C; ESI-MS m/z: 586.0 (M+H)+, 609.6 (M+Na)+; IR (KBr, cmÀ1): 3440.4, 3308.2, 2920.7, 2855.3, 1554.5, 1516.3,
1158.3, 960.9; 1H NMR (300 MHz, DMSO-d6): d 1.70 (s, 3H), 2.57 (s, 4H), 2.90 (s, 3H), 3.15 (s, 4H), 3.98–3.67 (m, 12H), 4.68 (s, 1H), 4.77 (s,
1H), 6.84 (s, 1H), 7.11 (m, 3H), 8.20 (s, 1H), 11.18 (s, 1H), 12.58 (s, 1H). 20: Yield: 82%; mp: 195–196 8C; ESI-MS m/z: 586.6 (M+H)+; IR
(KBr, cmÀ1): 3432.0, 2853.5, 1558.5, 1514.7, 1326.1, 1159.7, 960.1; 1H NMR (300 MHz, DMSO-d6): d 2.35 (s, 7H), 2.57 (s, 3H), 3.20 (s, 4H),
3.83(s, 2H), 3.87 (s, 6H), 4.08 (s, 4H), 4.67 (s, 2H), 5.96–5.99 (m, 1H), 7.23 (s, 2H), 7.72 (s, 1H), 8.17 (s, 1H), 12.55 (s, 1H), 13.15 (s, 1H). 21:
Yield: 78%; mp: 273–273 8C; ESI-MS m/z: 585.6 (MÀH)À, 665.6 (M+Br)À; IR (KBr, cmÀ1): 3425.0, 3295.0, 2958.2, 2859.6, 1556.0, 1516.3,
1160.2, 959.3; 1H NMR (300 MHz, DMSO-d6): d 1.27 (s, 9H), 2.57 (s, 4H), 2.89 (s, 3H), 3.14 (s, 4H), 3.76 (s, 4H), 3.87 (d, 6H, J = 10.1 Hz),
6.83 (d, 1H, J = 8.4 Hz), 7.11 (s, 1H), 7.39–7.18 (m, 2H), 8.20 (s, 1H), 11.16 (s, 1H), 11.92 (s, 1H). 22: Yield: 72%; mp: 248–250 8C; ESI-MS
m/z: 641.9 (MÀH)À; IR (KBr, cmÀ1): 3438.0, 2958.3, 1626.2, 1548.5, 1204.9, 1159.4, 1039.5, 782.2; 1H NMR (300 MHz, DMSO-d6): d 1.29
(s, 9H), 1.42 (s, 9H), 2.38 (s, 4H), 2.99 (s, 3H), 3.22 (s, 4H), 3.83 (s, 4H), 4.08 (s, 4H), 4.66 (s, 2H), 7.34 (s, 2H), 7.66 (s, 1H), 8.47 (s, 1H),
12.79–11.93 (m, 1H). 23: Yield: 76%; mp: 249–251 8C; ESI-MS m/z: 555.6 (M+H)+; IR (KBr, cmÀ1): 3426.9, 1625.7, 1548.2, 1327.2, 1196.1,
1055.7, 782.4; 1H NMR (300 MHz, DMSO-d6): d 2.58 (s, 4H), 2.90 (s, 3H), 3.14 (s, 4H), 3.89 (m, 10H), 7.15 (s, 1H), 7.25–7.16 (m, 1H), 7.40 (t,
1H, J = 7.5 Hz), 7.54 (d, 1H, J = 8.3 Hz), 8.39 (s, 1H), 8.50 (d, 1H, J = 8.1 Hz), 10.95 (s, 1H), 12.15 (s, 1H). 24: Yield: 68%; mp: 256–257 8C;
ESI-MS m/z: 554.3 (M+H)+; IR (KBr, cmÀ1): 3426.0, 3009.0, 1629.2, 1551.8, 1195.3, 1055.5, 782.2; 1H NMR (300 MHz, DMSO-d6): d 2.39
(s, 4H), 2.99 (s, 3H), 3.17 (s, 4H), 3.75 (s, 4H), 3.83 (s, 4H), 4.62 (s, 2H), 7.03 (s, 1H), 7.06 (s, 1H), 7.70 (s, 1H), 7.79 (d, 3H, J = 8.3 Hz), 8.18 (s,
1H), 12.42 (s, 1H), 12.97 (s, 1H). 25: Yield: 73%; mp: 251–251 8C; ESI-MS m/z: 565.5 (M+H)+; IR (KBr, cmÀ1): 3423.2, 3006.0, 1638.0,
1593.1, 1549.0, 1198.3, 937.3, 780.1; 1H NMR (300 MHz, DMSO-d6): d 2.57 (s, 4H), 2.89 (s, 3H), 3.14 (s, 4H), 4.01–3.55 (m, 10H), 5.91–6.02
(m, 1H), 6.89 (s, 1H), 6.96 (s, 1H), 7.14 (s, 1H), 8.15 (s, 1H), 11.13 (s, 1H), 12.29 (s, 1H). 26: Yield: 79%; mp: 210–211 8C; ESI-MS m/z: 690.5
(Br = 79), 692.5 (Br = 81) (M+H)+; IR (KBr, cmÀ1): 3427.8, 1626.1, 1592.1, 1548.6, 1328.4, 1199.7, 780.5; 1H NMR (300 MHz, DMSO-d6): d
2.43 (s, 4H), 3.01 (s, 3H), 3.43 (s, 4H), 3.84 (s, 4H), 4.08 (s, 4H), 4.72 (s, 2H), 7.76 (s, 1H), 8.16 (s, 3H), 12.66 (s, 1H), 13.15 (s, 1H). 27: Yield:
1
69%; mp: >300 8C; ESI-MS m/z: 580.6 (MÀH)À; IR (KBr, cmÀ1): 3433.0, 3281.4, 1547.2,1515.6, 1345.2, 1163.0, 962.2,776.9; H NMR
(300 MHz, DMSO-d6): d 2.59 (s, 4H), 2.90 (s, 3H), 3.16 (s, 4H), 4.06–3.59 (m, 10H), 7.15 (s, 1H), 7.22 (d, 1H, J = 9.0 Hz), 7.38 (t, 1H,
J = 7.4 Hz), 7.57 (t, 1H, J = 7.7 Hz), 7.95–7.76 (m, 2H), 8.08 (d, 1H, J = 8.5 Hz), 9.14 (s, 1H), 11.17 (s, 1H), 13.35 (s, 1H).