Structure-based design, synthesis and biological evaluation of novel anthra[1,2-d]imidazole-6,11-dione homologues as potential antitumor agents

European Journal of Medicinal Chemistry 69 (2013) 278e293

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European Journal of Medicinal Chemistry

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Original article

Structure-based design, synthesis and biological evaluation of novel

anthra[1,2-d]imidazole-6,11-dione homologues as potential antitumor

agents

,

Tsung-Chih Chen ^a, Dah-Shyong Yu ^{b c}, Kuo-Feng Huang ^d, Yung-Chien Fu ^e,

Chia-Chung Lee ^a, Chun-Liang Chen ^a, Fong-Chun Huang ^f, Hsi-Hsien Hsieh ^g,

,

g

,

,e,h,i,

*

Jing-Jer Lin ^f¹, Hsu-Shan Huang ^a

^aGraduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan

^bGraduate Institute of Medical Sciences, National Defense Medical Center, Taipei 114, Taiwan

^cUro-Oncology Laboratory, Division of Urology, Department of Surgery, Tri-Service General Hospital, Taipei 114, Taiwan

^dChi-Mei Medical Center, Tainan 710, Taiwan

^eSchool of Pharmacy, National Defense Medical Center, Taipei 114, Taiwan

^fInstitute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei 100, Taiwan

^gInstitute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan

^hDepartment of Pharmacy Practice, Tri-Service General Hospital, Taipei 114, Taiwan

ⁱSchool of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan

a r t i c l e i n f o

a b s t r a c t

Article history:

By using fragment-based design strategies, a series of 2-thio-substituted anthra[1,2-d]imidazole-6,11-

diones were synthesized and evaluated for hTERT repressing activities, cell proliferations, and NCI 60-

cell panel assay. Compounds 2, 3, 4, 11, 15 and 35 were selected by the NCI and 3, 4, 11 and 15 repre-

sent the GI₅₀, TGI and LC₅₀, respectively. Among them, all were moderate selectivity toward leukemia

cancer except for 4 exhibited distinctive selectivity of CNS and renal cancer with 7.403 and 6.475. The

overall of test compounds exhibited different cytostatic and cytotoxic activities for further developing

potential application as anticancer drugs.

Received 7 April 2013

Received in revised form

16 June 2013

Accepted 29 June 2013

Available online 9 August 2013

Keywords:

Anthra[1,2-d]imidazole-6,11-dione

Selectivity ratio

NCI 60-cell panel assay

Cytostatic and cytotoxic activities

1. Introduction

strong antiproliferative (or cytostatic) properties. They are clinically

used to treat many cancers for many decades, including leukemia,

Historically, natural and synthetic anthraquinones have a wide

spectrum of antitumor activities which can be mediated through

different mechanisms such as carcinogen metabolism, induction

of DNA damage, immune responses, suppression of cell cycle

progression, and/or inducing apoptosis of various human cancers

[1e4]. The anthracycline antibiotics (e.g. doxorubicin, daunoru-

bicin, mitoxantrone and ametantrone) have been shown to provide

lymphoma, breast, uterine, ovarian, and lung cancers [5]. In addi-

tion, kalafungin (kalamycin), a member of the category known as

pyranonaphthoquinone (PNQ) antibiotics, was isolated from the

zymotic broth of Streptomyces tanashiensis strain Kala in 1968 [6]. It

also exhibits activity against Gram-positive and Gram-negative

bacteria, pathogenic fungi, protozoa, yeasts and possess antiviral

activity as well as cytotoxic activity [7e9]. Moreover, recent studies

illustrated related quinone-containing molecules halawanones A

and B incorporate naphthoquinone ring systems, while halawa-

nones C and D incorporate anthraquinone moieties (Scheme 1) [10].

In the past, several studies also had found that the imidazole de-

rivatives have emerged as potent topoisomerase I (topo I) inhibitors

of the parasitic pathogen Leishmania donovani, a unicellular kinet-

oplastid protozoan parasite [11e13].

* Corresponding author. Graduate Institute of Life Sciences, National Defense

Medical Center, Taipei 114, Taiwan. Tel.: þ886 2 87923311x18869; fax: þ886 2

87923169.

E-mail addresses: jingjerlin@ntu.edu.tw (J.-J. Lin), huanghs@ndmctsgh.edu.tw,

huanghs@mail.ndmctsgh.edu.tw (H.-S. Huang).

1

Tel.: þ886 2 23123456x88208; fax: þ886 2 23915295.

http://dx.doi.org/10.1016/j.ejmech.2013.06.058

T.-C. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 278e293

279

Scheme 1. Representative investigational structural sets used in the pharmacophore studies and anthracycline antibiotics anticancer agents.

In our preliminary results, we found that some of the

anthraquinone-linked heterocyclic derivatives and synthetic tetra-

cycline scaffold might be interesting for cytotoxicity toward cancer

cells or cancer polypharmacology [14e18]. There are numerous

other reports regarding anthraquinone-derived homologs are novel

structures as compared to the known thiopurine classes of azathi-

oprine (AZA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG),

respectively (Scheme 1) [19e24]. Toward supporting the afore-

mentioned hypothesis and exploration of structureeactivity re-

lationships (SARs), we recently disclosed evidence as well as an in-

depth investigation of their fragment-based design. However,

continuous efforts toward synthesis of analogs of thio-substituted

moiety in anthra[1,2-d]imidazole-6,11-dione scaffold led to

discovery of new anticancer drugs for the polypharmacology. With

this motivation, we herein describe efﬁcient synthetic procedures

for the preparation of a series of non-nucleoside fused tetracyclic

compounds comprising imidazole moiety and anthraquinone

backbone. We attempted to identify potent enzyme inhibitors and

work towards some structural modiﬁcations in order to develop

some potential anticancer structures leads which could be

compared to parent molecules. The investigation covered advances

made and provided preliminary results on our designed small

molecules which under various headings cover the SAR studies and

expeditious structure optimization. Despite the simple and conve-

nient synthesis, studies of their biological activities have been very

limited. The encouraging results suggest this class of structures and

280

T.-C. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 278e293

their screening on a diverse panel of human tumor cell lines, which

can provide valuable information regarding their cytotoxic, cyto-

static and selectivity towards cell lines. To the best of our knowl-

edge, this is the ﬁrst report where thio-substituted anthra[1,2-d]

imidazole-6,11-dione derivatives have been evaluated for their

anticancer activity and polypharmacology.

isomers are subject to equilibration. The reaction of appropriate

alkyl halide and 2 in DMF at room temperature afforded the cor-

responding 3e25 (41e70%), respectively. Regioselective acylation

of 26e34 (29e53%) and 35 (39%) could be also obtained from

2, which could react with apposite acyl chlorides and 1,2-

dichloroethane in the presence of anhydrous DMF, respectively.

Interestingly, it was worthy to note that the thiazolidine moiety of

35 could be achieved as a one-step cyclization by using the reactant

of 1,2-dichloroethane. Compound 2 with 1,4-dichlorobutane then

gave 4-chlorobutylthio group on the imidazole ring 10 exclusively

instead of 1,3-thiazepine ring. The synthetic strategy for prepara-

tion of 3e25 was accomplished via the alkylation of 2 with various

basic catalysts in DMF treatment of alkyl halides, respectively. The

quantity of the by-products which were isolated was dependent on

substrate and puriﬁcation of the reaction mixtures which required

tedious recrystallization or chromatography. In addition, the basic

reagent affected the yield of the substitutions and simpliﬁed core

structure retains a great fraction of the cytotoxicity associated with

2. Chemistry

According to our previous studies [14,17], the structural

complexity of anthra[1,2-d]imidazole-6,11-dione, arising from the

presence of tetracyclic ring has restricted most of the SAR studies

by derivatization of the parent lead structure rather than by de novo

chemical synthesis. Such approach, as shown in Scheme 2, 1 could

react with CS₂in the presence of triethylamine (TEA) and the re-

action was reﬂuxed in DMF for 10 h to obtain the key intermediate 2

(80%) [17]. Tautomeric intermediate of this cyclization has been

found to vary with the exact conditions used as initially formed

S

O

N

O

35

iii

SH

N

S

O

NH₂

O

HN

NH

NH₂

i

1 (NSC39934)

2 (NSC747443)

ii

S R

N

O

HN

3-34

^aReagents and conditions: (i) DMF, TEA, CS₂, reflux, 10 h; (ii) (3-16) DMF, alkyl

halide, KOH, r.t., 10 h; (17-25) DMF, alkyl halide, KOH, r.t., 12 h; (26-34) DMF, acyl

chloride, TEA, r.t., 12 h; (iii) DMF, 1,2-dichloroethane, KOH, r.t., 10 h.

Scheme 2. Synthesis of 2-thiosubstituted anthra[1,2-d]imidazole-6,11-dione derivatives 2e35.

T.-C. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 278e293

281

various fragment. No matter how laborious, total synthesis efforts

can be accessible via short synthetic sequences from simple start-

ing materials which choosing the 1.0 equiv. KOH as basic catalyst

was better than TEA and K₂CO₃. All of the target compounds were

determined by ¹H NMR, ¹³C NMR and HRMS spectra and the results

are presented in the experimental part.

for further their cytostatic and cytotoxic activities against the 60

cell panel by using ﬁve dose studies (0.01, 0.1, 1, 10 and 100 M),

m

respectively. Based on the result of NCI’s analysis and the earlier

proof-of-concept, it is hypothesized that these drugs might have

the potential to be candidates for the role of anticancer drugs.

As shown in Table 2, 2 had no signiﬁcant activities for all the

cancer cell lines. Unlike 2, the SARs surrounding 3e35 were

introduced different thio substitutes onto the 2 revealed that

fragments may specialize in their steric and conformational re-

quirements to increase the inhibitory activities. Compound 3 was

effective against renal cancer cell strain CAKI-1, and could achieve

an inhibitory effect on the growth of said cancer cell up to ꢁ8.01%.

Compound 4 was effective against CNS cancer cell line SNB-75, up

to ꢁ9.55%. Compound 11 could achieve inhibiting effects against

leukemia cell lines CCRF-CEM and RPMI-8226, up to ꢁ15.39% and

ꢁ60.17%, respectively. Compound 15 was effective against leukemia

cell line K-562, MOLT-4, RPMI-8226, up to ꢁ31.59%, ꢁ2.90% and

ꢁ13.69%, and effective also against renal cancer cell line CAKI-1, up

to ꢁ14.89%, respectively. Ultimately, this signiﬁcance of 4, 11 and 15

exhibited inhibitory activities by further evaluation of human

multiple drug-resistant ovarian cancer cell against Adriamycin

(NCI/ADR-RES). Understanding the cell death pathways for selec-

tive killing of drug-resistance against ADR would lead to more

effective strategies. It seemed apparent from these results that

clinical applications and limitations of chemotherapy still have a

long way to go because of chemo-resistance.

3. Results and discussion

3.1. In vitro antiproliferative activities and hTERT repression

activities

Understanding of the mode of action of anthraquinone-based

pharmacophore as an anticancer drug has increased in recent

years, but this kind of structures has not yet been fully achieved

despite the efforts in hit-to-lead progresses. As reported in previous

sections, all of the synthetic compounds were evaluated for their

effects on cell viability in H1299 cells. In our studies, we found that

most of the target compounds showed cytotoxicity toward H1299

cells at micromolar range. Despite these promising data which

showed less cytotoxic effects toward H1299 cells, it was still impor-

tant to remain mindful of the latent liability of core structure,

possessed by these proof-of-concept compounds, to undergo

possible polypharmacology. Among them, 3, 13, 14, 16, 19, 28, and 35

showed the difference between cell viability and SEAP activity

(Table 1). Compound 3 exhibited cell viability with 73% and SEAP

activity as 17% at 100

mM; 14 and 16 also showed higher SEAP

Further and detailed evaluation of 3, 4, 11 and 15 showed cell

inhibitory effects and less cytotoxicity. Of the series of tetracyclic-

system compounds analyzed and brief inferences of SAR studies,

we found that the features for optimum antiproliferative activities

can be classiﬁed into subgroups containing [1]: the side chain at 2nd

position with a carbonyl group should be decreased in the cell

viability (11 > 23; 13 ꢀ 19) [2]; carbothioate at 2nd position, which is

crucial in decreasing their potency (antiproliferative activities:

24 ꢀ 23 > 32; 20 ꢀ 19 > 27; 22 ꢀ 21 > 34) [3]; introduced tertiary N

atom into the end of the aliphatic group, which is crucial inwhich the

potency increased (11 > 3). Although the challenge of fragment-

based design in such major structural changes was recognized with

regard to potency and good pharmacological properties, it was

deemed necessary in order to ensure the fragment characteristics for

a drug design.

growth inhibition at ﬁve concentrations (0.01, 0.1, 1, 10 and 100 mM)

with GI₅₀, TGI and LC₅₀, respectively. The doseeresponse curve is

originated from the log 10 of the corresponding drug concentration

against each cell line by subpanel groups. Compound 3, the average

concentration required to inhibit GI₅₀was 9.33

2.01 M (breast cancer: MDA-MB-468) to >100

SW-620; melanoma: UACC-257; CNS cancer: SNB-19). With 4, the

average concentration required to inhibit GI₅₀was 14.5 M with a

range of 1.94 M (non-small cell lung cancer: HOP-62) to >100 mM

mM with a range of

m

mM (colon cancer:

m

(Leukemia: CCRF-CEMMOLT-4, and RPMI-8226; non-small cell lung

cancer: NCI-H322M; colon cancer: COLO 205, HCT-15, HT29, KM-12

and SW-620; melanoma: M-14, MDA-MB-435, SK-MEL-28, SK-

MEL-5 and UACC-257; prostate cancer: DU-145). The average GI₅₀

concentration of 11 was 6.46

cancer: IGROV1) to 19.3 M (non-small cell lung cancer: NCI-H226).

Furthermore, the average concentration of 15 required to inhibit

GI₅₀was 2.57 M, with a range of <0.01 M (CNS cancer: SNB-75;

breast cancer: MDA-MB-468) to 19.8 M (melanoma, UACC-257).

mM, with a range of 0.56 mM (ovarian

m

3.2. NCI60 human tumor cell line anticancer drug screen

m

All the details of the methodology are described in our previous

reports [14e17]. The anticancer activity and toxicity of all the test

compounds were evaluated by using the National Cancer Institute

(NCI) Drug Screen Program of 60 human tumor cell lines. The full

NCI’s 60 panel of human tumor cell lines is derived from nine

cancer cell types: NSCLC, colon cancer, breast cancer, ovarian can-

cer, leukemia, renal cancer, melanoma, prostate cancer, and CNS

cancer [25]. There is also additional evidence that being dose-

dependent inhibited proliferations in all 60 cancer cell lines, and

cytotoxicity was derived from experiments shown in this study.

There are two stages of the screening process that starts with the

evaluation of all the test compounds against the 60 human tumor

m

Compounds 3, 4, 11 and 15 exhibited dose-dependent inhibition

of proliferation in all 60 cancer cell lines. Compound 11 is active

against most of the cancer cell lines with GI₅₀values <5 mM for 50%

of the cell lines. With initial assessment at relative doses, 15 is more

potent than 11. Compound 15 exhibited highly inhibitory effect

against most of the cancer cell lines with GI₅₀values <5

mM for 62%

and <1 M for 16% of the cell lines. The GI₅₀, LC₅₀and TGI values of

m

the active compounds within each series are given in Table 3. The

log mean values of parameter for GI₅₀, TGI and LC₅₀related to the

log delta values (the maximum sensitivity in excess of the mean)

and log range values were provided in Table 4 [25,26]. These pa-

rameters which highlighted the selectivity and potency of anti-

tumor agents were shown in Table 4. Higher values of these deltas

and ranges indicate high selectivity against some cancers over

others. The lower median log GI₅₀value (ꢁ5.59) for 15 showed it to

be the most potent compound for all cell lines with overall potency

cell lines at a single dose of 10 mM. In this stage, 2 (NSC747443), 3

(NSC747444), 4 (NSC747853), 11 (NSC747515), 15 (NSC747854) and

35 (NSC747514) were chosen by NCI and tested against a panel of 60

human tumor cell lines (Table 2). The result from the single dose

screen was reported as a mean graph (given in the Supporting

information). In addition, only compounds which showed more

than 60% growth inhibition in at least 8 tumor cell lines were

selected by NCI for further testing [26]. Furthermore, among six

compounds, with the exception of 3, 4, 11 and 15 were active drugs

in terms of GI₅₀and LC₅₀values ranging from <0.01 to 19.8

mM and

4.57 to 69.6 M; the inhibition accounts for its high range log GI₅₀

m

and log LC₅₀values with 3.30 and 1.17. The ﬁve dose graph and dose

response curves of 15 for all 60 cell lines were illustrated in Fig. 1.

282

T.-C. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 278e293

Table 1

Effects of 2-thiosubstituted anthra[1,2-d]imidazole-6,11-dione homologues on cytotoxicity by MTT assay and repressing hTERT expression activity by SEAP assay.

S R

O

HN

N

Compounds

No.

Cell type/H1299

MTT assay (viability % ꢂ SD)^a

SEAP activity (% ꢂ SD)^a

eR

1

10

100

1

10

100

1

2

105 ꢂ 10

101 ꢂ 4

95 ꢂ 4

39 ꢂ 9

98 ꢂ 11

97 ꢂ 4

77 ꢂ 12

100 ꢂ 5

18 ꢂ 4

31 ꢂ 8

99 ꢂ 2

54 ꢂ 5

3

109 ꢂ 18

127 ꢂ 18

73 ꢂ 13

100 ꢂ 9

90 ꢂ 9

17 ꢂ 5

4

5

6

7

91 ꢂ 9

82 ꢂ 11

100 ꢂ 5

94 ꢂ 7

78 ꢂ 4

135 ꢂ 27

96 ꢂ 9

12 ꢂ 2

77 ꢂ 15

79 ꢂ 5

75 ꢂ 9

103 ꢂ 13

111 ꢂ 8

97 ꢂ 8

94 ꢂ 16

105 ꢂ 10

91 ꢂ 6

33 ꢂ 25

53 ꢂ 6

69 ꢂ 5

52 ꢂ 3

96 ꢂ 7

94 ꢂ 2

85 ꢂ 3

8

91 ꢂ 8

84 ꢂ 4

83 ꢂ 4

97 ꢂ 5

82 ꢂ 7

85 ꢂ 7

83 ꢂ 11

37 ꢂ 9

88 ꢂ 6

100 ꢂ 11

92 ꢂ 12

105 ꢂ 9

96 ꢂ 5

58 ꢂ 4

98 ꢂ 8

54 ꢂ 5

41 ꢂ 8

53 ꢂ 2

9

10

11

12

90 ꢂ 11

86 ꢂ 8

89 ꢂ 6

21 ꢂ 7

102 ꢂ 8

111 ꢂ 9

98 ꢂ 5

36 ꢂ 5

14 ꢂ 6

79 ꢂ 14

30 ꢂ 16

100 ꢂ 13

13

106 ꢂ 24

127 ꢂ 15

73 ꢂ 6

119 ꢂ 6

105 ꢂ 9

36 ꢂ 3

14

15

71 ꢂ 9

70 ꢂ 9

79 ꢂ 5

38 ꢂ 11

22 ꢂ 11

85 ꢂ 6

50 ꢂ 8

86 ꢂ 9

8 ꢂ 6

108 ꢂ 7

100 ꢂ 3

15 ꢂ 4

16

17

88 ꢂ 10

83 ꢂ 8

81 ꢂ 8

83 ꢂ 8

76 ꢂ 7

57 ꢂ 8

88 ꢂ 4

88 ꢂ 3

83 ꢂ 5

83 ꢂ 7

29 ꢂ 5

61 ꢂ 3

T.-C. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 278e293

283

Table 1 (continued )

Compounds

Cell type/H1299

MTT assay (viability % ꢂ SD)^a

SEAP activity (% ꢂ SD)^a

No.

eR

1

10

100

1

10

100

18

98 ꢂ 11

91 ꢂ 7

75 ꢂ 5

72 ꢂ 5

88 ꢂ 4

87 ꢂ 4

64 ꢂ 6

40 ꢂ 3

19

20

88 ꢂ 4

70 ꢂ 4

50 ꢂ 7

103 ꢂ 8

105 ꢂ 3

87 ꢂ 8

97 ꢂ 5

99 ꢂ 8

102 ꢂ 6

45 ꢂ 5

21

22

97 ꢂ 3

94 ꢂ 6

90 ꢂ 6

77 ꢂ 6

54 ꢂ 4

44 ꢂ 2

96 ꢂ 3

97 ꢂ 3

93 ꢂ 6

49 ꢂ 2

64 ꢂ 5

98 ꢂ 10

23

24

89 ꢂ 5

102 ꢂ 5

62 ꢂ 3

57 ꢂ 7

100 ꢂ 4

87 ꢂ 13

100 ꢂ 6

90 ꢂ 8

64 ꢂ 5

107 ꢂ 13

95 ꢂ 4

110 ꢂ 15

67 ꢂ 11

25

26

86 ꢂ 10

78 ꢂ 5

93 ꢂ 8

76 ꢂ 6

94 ꢂ 10

98 ꢂ 2

91 ꢂ 11

92 ꢂ 7

85 ꢂ 13

81 ꢂ 2

80 ꢂ 4

27

28

29

89 ꢂ 9

102 ꢂ 11

95 ꢂ 12

94 ꢂ 7

120 ꢂ 8

93 ꢂ 8

90 ꢂ 5

93 ꢂ 7

89 ꢂ 5

97 ꢂ 9

87 ꢂ 3

99 ꢂ 9

95 ꢂ 12

71 ꢂ 6

38 ꢂ 7

81 ꢂ 9

71 ꢂ 10

69 ꢂ 13

30

95 ꢂ 9

100 ꢂ 9

79 ꢂ 6

102 ꢂ 11

96 ꢂ 12

75 ꢂ 9

(continued on next page)

284

T.-C. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 278e293

Table 1 (continued )

Compounds

Cell type/H1299

MTT assay (viability % ꢂ SD)^a

SEAP activity (% ꢂ SD)^a

No.

eR

1

10

100

1

10

100

31

95 ꢂ 10

91 ꢂ 6

95 ꢂ 10

104 ꢂ 5

100 ꢂ 18

89 ꢂ 6

32

33

34

110 ꢂ 4

97 ꢂ 4

117 ꢂ 4

116 ꢂ 6

119 ꢂ 10

124 ꢂ 10

116 ꢂ 9

116 ꢂ 6

96 ꢂ 11

98 ꢂ 12

106 ꢂ 9

105 ꢂ 4

104 ꢂ 6

95 ꢂ 9

81 ꢂ 9

88 ꢂ 8

118 ꢂ 4

35

115 ꢂ 10

128 ꢂ 5

99 ꢂ 13

107 ꢂ 7

53 ꢂ 4

48 ꢂ 3

e

Mitoxantrone

Doxorubicin

24 ꢂ 8

17 ꢂ 5

0 ꢂ 1

0 ꢂ 2

0 ꢂ 1

0 ꢂ 4

21 ꢂ 2

34 ꢂ 3

10 ꢂ 5

14 ꢂ 4

7 ꢂ 2

5 ꢂ 4

a

SD: standard derivatives, all experiments were independently performed at least three times.

The criterion for selectivity of compounds depends upon the

ratio obtained by dividing the full panel MID [27]. Ratios between 3

and 6 refer to moderate selectivity; ratios greater than 6 indicate

high selectivity toward the corresponding cell line, while com-

pounds not meeting either of these criteria rated non-selective

[28,29]. All of these parameters provide insight into selectivity

and the potency of antitumor activity. Based upon these parame-

ters, 3, 11 and 15 were found to be moderately selective for growth

inhibition toward leukemia cancer subpanel only with selectivity

ratios of 3.445, 3.855 and 3.301, whereas it was found to be less

selective against remaining cell panels (Table 5). It seemed

apparent from these results that 4 exhibited the unique selectivity

against CNS cancer and renal cancer with selectivity ratios of 7.403

and 6.475. For each antitumor activity parameter, the average value

of mean graph midpoint (MG-MID) was calculated over all cell

lines.

spectrum of antiproliferative activity. With regard to the relative

cellular potency of 4, it may be postulated that the n-propyl

group as a side chain showed completely diverse sensitivity by

comparison 3, 11 and 15 do not suffer unknown mechanism and

retains good cellular activity. Compounds 26e34 containing a car-

bothionate group were found the apparent debasement of the

antiproliferative activity. Opposite, 11e15 having various tertiary

amines as side chains were the most active within other com-

pounds. Finally, of the remaining most potent and stable de-

rivatives within this group and target molecules, these were

chemically novel and biologically unique in its mechanism of action

and in vitro pattern of activity.

5. Materials and methods

5.1. Chemistry

4. Conclusion

Melting points were determined with Büchi B-545 melting point

apparatus and are uncorrected. All reactions were monitored by TLC

Results of the study extend the initial in vitro observation

described above and conﬁrm the signiﬁcance and importance of

anticancer activity. The cancer panel, leukemia, non-small cell lung

cancer, colon, prostate cancer lines were quite sensitive to 11 and

15, while the CNS, melanoma, ovarian, renal and breast panels were

less sensitive. The overall doseeresponse curves showed that the

leukemia subpanel was most sensitive for 11 and 15, with GI₅₀of six

(silica gel 60 F₂₅₄).¹H NMR: Varian GEMINI-300 (300 MHz);

d values

are in ppm relative to TMS as an internal standard (0 ppm). Multi-

plicities are recorded as s (singlet), d (doublet), t (triplet), q (quar-

tet), quin (quintuplet), sext (sextet), m (multiplet), dd (double of

doublet), and br (broadened). Mass spectra: High resolution elec-

tron impact ionization (HREI): Finnigan MAT MAT-95XL (Instru-

mentation Center, National Tsing Hua University, Taipei, Taiwan)

and High resolution electrospray ionization (HRESI): Finnigan MAT

95S (Instrumentation Center, National Taiwan University, Taipei,

Taiwan). Typical experiments illustrating the general procedures for

the preparation of the anthraquinones are described below.

leukemia cell lines ranging from 1.13 to 3.34 and 0.43 to 2.41

The colon cancer subpanel was ranked second most sensitive with

GI₅₀between 1.62e4.91 and 0.68e4.14 M. Additionally, 3 with

mM.

m

side chain eCH₂CH₃displayed relatively inhibitory activity but not

cytotoxic activity. However, it had signiﬁcant inﬂuence on the

T.-C. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 278e293

285

Table 2

Growth percentage of selected compounds in the NCI in vitro 60-cell Drug Screen Program.

Panel/cell lines

Compounds/growth percent^a

2 NSC747443

3 NSC747444

4 NSC747853

11 NSC747515

15 NSC747854

35 NSC747514

Leukemia

CCRF-CEM

HL-60(TB)

K-562

MOLT-4

RPMI-8226

SR

61.91

97.58

108.55

108.60

101.22

124.87

36.87

62.64

86.29

100.70

28.52

129.31

70.83

74.23

62.88

68.13

71.75

71.03

ꢁ15.39

37.41

26.94

8.49

12.74

ꢁ31.59

ꢁ2.90

ꢁ13.69

3.09

66.81

78.84

78.71

58.39

71.65

60.62

30.94

ꢁ60.17

20.25

Non-small cell lung cancer

A549/ATCC

EKVX

HOP-62

HOP-92

NCI-H226

NCI-H23

NCI-H322M

NCI-H460

NCI-H522

118.18

50.53

96.74

65.48

83.74

113.34

96.31

98.61

96.06

100.94

43.67

95.59

75.35

62.07

87.41

33.44

38.83

78.60

59.15

76.83

59.06

17.25

50.24

69.80

76.02

68.12

72.38

35.52

73.04

73.74

35.17

88.06

66.93

73.18

34.08

81.51

51.59

65.23

51.96

15.99

74.10

53.83

70.75

23.20

69.28

81.15

35.80

85.26

88.67

80.53

84.29

72.06

59.46

80.62

Colon cancer

COLO 205

HCC-2998

HCT-116

HCT-15

HT29

KM12

SW-620

103.96

113.65

96.31

71.84

89.42

68.78

32.29

75.18

68.26

84.53

102.68

81.92

47.69

67.65

75.10

55.17

104.18

17.13

71.50

38.81

40.36

e^b

62.01

72.67

27.58

36.27

17.84

20.36

26.35

95.22

94.76

68.43

57.15

e^b

83.78

103.79

101.59

97.56

19.62

22.75

84.88

69.29

CNS cancer

SF-268

SF-295

SF-539

SNB-19

SNB-75

U251

90.30

118.09

91.35

91.30

100.90

88.79

75.67

100.85

58.58

87.90

73.34

65.03

47.81

37.25

23.99

92.03

ꢁ9.55

62.95

42.04

76.91

62.25

60.47

81.75

69.07

53.13

76.03

57.50

79.67

50.54

74.11

56.15

78.56

74.06

74.47

95.57

72.48

Melanoma

LOX IMVI

MALME-3M

M14

MDA-MB-435

SK-MEL-2

SK-MEL-28

SK-MEL-5

UACC-257

UACC-62

84.00

87.50

36.72

63.33

67.07

68.42

81.65

97.72

63.93

111.80

47.64

62.57

88.24

89.99

77.38

79.33

92.59

78.52

86.3

11.89

112.31

70.87

28.57

88.23

77.32

72.18

80.03

102.07

62.55

114.50

75.39

65.93

87.59

78.50

83.11

99.88

109.36

65.88

80.59

73.06

101.06

114.02

134.35

118.39

86.66

66.47

106.62

100.23

78.52

92.77

80.95

105.26

84.17

51.07

Ovarian cancer

IGROV1

OVCAR-3

OVCAR-4

OVCAR-5

OVCAR-8

NCI/ADR-RES

SK-OV-3

138.25

86.94

91.57

129.22

139.75

109.60

103.17

73.41

69.27

55.05

85.38

96.82

85.72

24.72

53.72

62.71

42.16

99.67

44.68

50.22

38.92

23.20

e^b

30.89

59.33

29.72

87.88

61.89

54.32

32.36

62.31

e^b

57.23

87.49

49.38

53.74

55.57

89.29

105.31

75.40

84.08

87.43

Renal cancer

786-0

A489

95.78

94.31

96.23

91.23

141.66

87.98

111.74

97.83

85.38

75.49

37.57

ꢁ8.01

99.91

67.91

74.09

49.19

21.82

57.26

27.16

45.47

20.07

80.41

35.55

24.49

77.12

71.37

34.58

e^b

77.63

76.16

24.30

ꢁ14.89

38.40

37.66

31.68

25.50

92.44

73.67

59.35

e^b

ACHN

CAKI-1

RXF 393

SN12C

TK-10

22.32

52.94

36.86

21.22

46.50

74.93

58.78

45.79

UO-31

Prostate cancer

PC-3

DU-145

78.80

99.28

53.79

64.51

66.76

69.29

6.90

17.91

36.71

36.35

93.92

68.84

Breast cancer

MCF7

MDAMB231/ATCC

HS578-T

75.91

108.18

103.53

114.30

43.81

84.79

85.44

55.80

58.97

26.44

33.33

76.00

30.88

63.52

e^b

16.92

56.24

69.32

75.75

64.78

94.00

e^b

BT-549

78.62

87.90

(continued on next page)

286

T.-C. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 278e293

Table 2 (continued )

Panel/cell lines

Compounds/growth percent^a

2 NSC747443

3 NSC747444

4 NSC747853

11 NSC747515

15 NSC747854

35 NSC747514

T-47D

MDA-MB-468

Mean

Delta

Range

81.86

87.69

99.72

49.19

91.13

30.78

35.36

67.96

75.97

137.32

53.72

e^b

83.06

67.90

52.08

112.25

172.48

45.78

e^b

73.06

69.68

75.99

40.19

73.56

60.02

69.57

113.73

47.41

79.00

146.09

a

Data obtained from NCI in vitro 60-cell drug screen program at 10^ꢁ5M concentration.

Table 3

In vitro antitumor activity (GI₅₀in

m

M), toxicity (LC₅₀in

m

M) and TGI data of selected compounds 3, 4, 11 and 15.

4 (NSC747853) 11 (NSC747515)

Panel/cell line ( M) 3 (NSC747444)

m

15 (NSC747854)

GI₅₀

TGI

LC₅₀

GI₅₀

TGI

LC₅₀

GI₅₀

TGI

LC₅₀

GI₅₀

TGI

LC₅₀

Leukemia

CCRF-CEM

HL-60(TB)

K-562

MOLT-4

RPMI-8226

SR

3.66

5.26

13.5

4.05

2.83

2.36

>100

68.4

36.4

>100

4.63

>100

3.34

1.13

1.70

1.51

1.79

1.48

15.6

11.9

5.2

4.29

4.31

4.19

62.8

57.6

72.0

19.1

15.4

86.3

1.29

2.41

2.04

2.06

1.25

0.43

4.36

8.52

4.73

6.60

4.62

3.47

24.0

40.9

13.6

38.5

33.1

27.8

Non-small cell lung cancer

A549/ATCC

EKVX

HOP-62

HOP-92

NCIeH226

NCIeH23

NCIeH322M

NCI-460

9.67

2.13

25.9

90.6

14.2

8.6

3.24

5.49

4.07

>100

29.0

>100

6.37

64.9

1.94

15.0

4.37

48.0

>100

44.7

11.6

>100

4.27

>100

9.36

>100

3.80

11.2

18

15.0

26.5

33.2

N.T.

36.6

25.7

21.4

4.77

32.5

41.8

62.4

61.2

N.T.

69.7

56.9

47.3

29.3

66.2

3.26

4.20

5.90

N.T.

3.74

5.73

0.72

2.13

12.2

12.6

20.1

21.2

N.T.

22.1

19.7

20.6

12.8

24.9

38.9

47.9

48.9

N.T.

58.7

46.9

4.57

38.9

50.5

>100

N.T

19.3

11.6

8.21

1.68

15.9

NCIeH522

Colon cancer

COLO 205

HCC-2998

HCT-116

HCT-15

HT29

KM12

SW-620

17.4

12.6

5.59

3.52

23.1

8.43

>100

N.T.

3.06

>100

N.T.

>100

N.T.

3.07

4.91

1.64

1.62

1.81

2.32

1.94

16.7

18.5

3.66

10.3

4.66

6.16

5.09

58.6

46.0

8.15

34.7

15.2

24.4

19.7

3.41

4.14

1.85

0.68

1.04

1.96

1.99

17.1

17.7

8.75

15.0

2.61

7.24

6.29

43.7

43.4

30.9

40.0

6.53

27.4

27.0

>100

CNS cancer

SF-268

SF-295

SF-539

SNB-19

SNB-75

U251

18.1

4.17

22.9

>100

10.1

19.5

>100

47.4

>100

84.9

>100

3.71

3.19

3.07

9.35

2.64

7.05

>100

60.2

32.0

>100

6.43

>100

98.1

9.54

14.2

6.79

17.1

12.3

6.84

22.8

29.4

20.2

31.9

25.2

20.3

52.9

60.9

47.4

59.7

51.5

47.1

4.34

14.8

10.9

7.20

<0.01

10.1

17.7

28.4

22.7

20.7

<0.01

22.0

43.9

54.6

47.7

46.6

4.71

48.0

>100

Melanoma

LOX IMV1

MALME-3M

M14

MDA-MB-435

SK-MEL-2

SK-MEL-28

SK-MEL-5

UACC-257

UACC-62

6.00

3.58

5.62

76.5

5.10

43.6

33.6

>100

7.73

>100

94

>100

3.69

4.27

>100

1.43

14.0

8.24

4.18

17.1

2.20

12.9

18.0

15.8

2.99

28.8

21.2

16.2

32.9

4.47

26.6

31.9

30.1

6.25

59.4

47.9

43.9

63.5

9.08

54.8

56.8

57.4

1.36

13.9

7.05

10.4

13.3

13.5

11.1

19.8

10.8

3.00

27.0

20.7

22.2

26.3

23.1

34.1

23.0

6.63

52.4

46.0

47.1

52.0

51.3

48.1

58.7

49.3

>100

15.9

>100

5.78

Ovarian cancer

IGROV1

OVCAR-3

OVCAR-4

OVCAR-5

OVCAR-8

NCI/ADR-RES

SK-OV-3

2.38

10.2

3.17

6.25

15.1

8.04

3.80

>100

4.03

3.97

5.05

58.1

3.48

2.37

3.22

>100

37.6

>100

0.56

2.76

1.89

15.5

6.38

6.98

10.5

3.39

11.1

7.34

29.4

30.4

33.7

23.8

22.4

35.6

38.9

55.8

>100

54.0

0.329

3.73

0.031

12.7

6.12

5.11

1.72

12.8

18.2

5.04

25.9

21.5

20.7

37.5

43.9

31.1

52.9

56.5

69.6

51.7

>100

20.5

Renal cancer

786-0

A498

ACHN

CAKI-1

46.7

4.70

5.09

N.T.

>100

N.T.

>100

N.T.

2.13

13.9

2.69

3.07

4.39

>100

42.8

9.04

11.7

5.82

2.34

N.T

25.3

21.3

16.5

N.T.

54.7

49.4

41.1

N.T.

9.73

12.1

1.74

0.822

23.8

24.8

15.0

11.0

57.1

51.0

38.7

36.0

>100

T.-C. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 278e293

287

Table 3 (continued )

Panel/cell line (

m

M)

3 (NSC747444)

4 (NSC747853)

11 (NSC747515)

15 (NSC747854)

GI₅₀

TGI

LC₅₀

GI₅₀

TGI

LC₅₀

GI₅₀

TGI

LC₅₀

GI₅₀

TGI

LC₅₀

RXF 393

SN12C

TK-10

5.17

10.3

7.40

3.31

>100

2.65

14.0

3.25

2.53

8.10

>100

21.6

70.2

>100

3.08

5.96

4.22

2.08

16.3

19.6

19.1

15.8

48.5

46.1

46.8

44.9

1.89

3.26

1.37

0.564

12.8

16.5

13.5

11.7

42.3

40.6

38.3

36.5

UO-31

Prostate cancer

PC-3

DU-145

65.0

7.02

>100

11.3

>100

2.57

3.42

11.0

14.9

43.4

39.6

N.T.

2.38

N.T.

12.9

N.T.

35.9

Breast cancer

MCF7

MDA-MB-231/ATCC

HS 578T

BT-549

T-47D

4.84

20.7

5.12

9.34

4.21

2.01

>100

59.6

>100

57.0

5.14

3.45

33.4

2.66

6.38

>100

83.0

>100

1.74

4.46

6.24

12.5

17.5

1.33

7.52

16.2

25.8

26.4

40.3

3.06

45.6

43.9

79.9

55.8

92.8

7.05

0.202

4.96

4.84

9.13

8.73

4.21

19.3

22.9

21.8

24.8

0.439

28.4

45.0

65.0

48.7

61.7

21.8

MDA-MB-468

<0.01

5.2. General synthetic methods

5.2.4. General procedure D: preparation of compound 35

Compound 2 (1.40 g, 5 mmol) was dissolved in DMF (30 mL) and

1,2-dichloroethane (0.74 g, 7.5 mmol) and KOH solution (1 M,

10 mL, 10 mmol) was added at room temperature for 10 h (TLC

monitoring). After ﬁnished, the mixture was poured into ice water.

The resulting precipitate was collected by ﬁltration, washed with

distilled water and puriﬁed by crystallization from ethanol to afford

desired compound as a yellowebrown solid.

These compounds were synthesized from 1,2-diaminoanthra

quinone (1). All the chemicals and organic solvents required for

synthesis were purchased from either Sigma Aldrich Chemical

Company or Merck Chemical Company.

5.2.1. General procedure A: preparation of compound 2

Compound 1 (1.19 g, 5 mmol) was dissolved in DMF (30 mL) and

CS₂(0.4 g, 5 mmol) and TEA (3 mL) was added. The reaction mixture

was continued under reﬂuxing condition for 10 h. The reaction

mixture was cooled to room temperature and treated with crushed

ice and the resulting precipitate was collected by ﬁltration, washed

with diethyl ether and puriﬁed by crystallization from ethanol to

afford desired compound as a brown solid.

5.3. Synthesis of target compounds 2e35

5.3.1. 2-Thioxo-2,3-dihydro-1H-anthra[1,2-d]imidazole-6,11-dione

or 2-mercapto-1(3)H-anthra[1,2-d] imidazole-6,11-dion (2)

The pure compound was obtained as a brown solid (yield 80%).

(R_f¼ 0.8 at ethyl acetate:dichloromethane ¼ 1:4). Mp 407e409 ^ꢃ

C

(EtOH) [17]. FT-IR (KBr;

n

cm^ꢁ1): 3221 (NH), 3192 (NH), 1665 (CO).

5.2.2. General procedure B: preparation of compounds 3e25

Compound 2 (1.40 g, 5 mmol), alkyl halide (6 mmol) and KOH

(10 mmol) were dissolved in DMF (30 mL) stirred at room tem-

perature for 10e12 h. Water (300 mL) was added to precipitate out

the crude product. The resulting precipitate was collected by

ﬁltration, washed with water and puriﬁed by crystallization from

ethanol to afford desired compounds.

¹H NMR (300 MHz, DMSO-d₆):

(d, J ¼ 8.1 Hz, 1H), 7.91e7.94 (m, 2H), 8.18e8.22 (m, 2H), 12.73 (s,

1H), 13.29 (s, 1H). ¹³C NMR (75 MHz, DMSO-d₆):

(ppm) 113.89,

d

(ppm) 7.54 (d, J ¼ 8.1 Hz, 1H), 8.02

d

115.27, 122.41, 126.26, 126.76, 126.88, 130.95, 132.89, 133.06, 134.25,

134.47, 138.19, 172.89, 181.79 (CO), 182.46 (CO). HRMS (ESI-TOF) m/z

calcd for C₁₅H₈N₂O₂S [M]^þ: 280.0306, found [M þ H]^þ: 281.0389.

5.3.2. 2-(Ethylthio)-1H-anthra[1,2-d]imidazole-6,11-dione (3)

Product 3 was prepared from 2 and iodoethane. The yellow solid

material was isolated in 65% yield (R_f¼ 0.33 at ethyl acetate:n-

hexane ¼ 1:1). Mp 239e240 ^ꢃC (EtOH). ¹H NMR (300 MHz, CDCl₃):

5.2.3. General procedure C: preparation of compounds 26e34

To a solution of 2 (1.40 g, 5 mmol) in DMF (30 mL) was added

acyl chloride (6 mmol) dropwise with stirring at room temperature.

After 30 min, TEA (6 mmol) was added into the mixture and stirred

for 12 h. Water (200 mL) was added to precipitate out the crude

product. The resulting precipitate was collected by ﬁltration,

washed with water and puriﬁed by crystallization from ethanol to

afford desired compounds.

d

(ppm) 1.53 (t, J ¼ 7.5 Hz, 3H, eCH₃), 3.44 (q, J ¼ 7.5 Hz, 2H, eCH₂e

), 7.78e7.80 (m, 2H, AreH), 7.94 (d, J ¼ 8.4 Hz, 1H, AreH), 8.17 (d,

J ¼ 8.4 Hz, 1H, AreH₃), 8.24e8.27 (m, 1H, AreH), 8.31e8.34 (m, 1H,

AreH), 10.83 (br, 1H, eNH). ¹³C NMR (75 MHz, CDCl₃):

d (ppm)

13.55 (eCH₃), 34.44 (eSCH₂CH₃), 117.10, 121.94, 123.78, 126.78,

127.72, 127.77, 133.48, 133.91, 134.10, 134.30, 134.57, 149.64, 158.22,

182.90, 185.22. HRMS (EI) m/z calcd for

308.0619, found, 308.0609.

C

₁₇H₁₂N₂O₂S [M]^þ:

Table 4

Cytostatic (GI₅₀and TGI) and cytotoxic (LC₅₀) parameters of active compounds 3, 4,

11, and 15.

Compounds

Log GI₅₀

3

4

11

15

5.3.3. 2-(Propylthio)-1H-anthra[1,2-d]imidazole-6,11-dione (4)

Product 4 was prepared from 2 and 1-chloropropane. The yellow

solid material was isolated in 55% yield (R_f¼ 0.45 at ethyl acetate:n-

hexane ¼ 1:1). Mp 212e213 ^ꢃC (EtOH). ¹H NMR (300 MHz, CDCl₃):

Median

Delta

Range

ꢁ5.03

0.67

ꢁ4.84

0.87

ꢁ5.19

1.21

1.68

ꢁ5.59

2.41

3.30

1.70

1.71

Log TGI

Median

Delta

Range

ꢁ4.02

0.52

ꢁ4.14

1.23

ꢁ4.71

0.78

1.09

ꢁ4.93

3.07

3.53

d

(ppm) 1.10 (t, J ¼ 7.5 Hz, 3H, eCH₃), 1.88 (sext, J ¼ 7.5 Hz, 2H, e

CH₂e), 3.41 (q, J ¼ 7.5 Hz, 2H, eSCH₂e), 7.75e7.82 (m, 2H, AreH),

7.93 (d, J ¼ 8.4 Hz, 1H, AreH), 8.16 (d, J ¼ 8.4 Hz, 1H, AreH), 8.24e

8.27 (m, 1H, AreH), 8.31e8.34 (m, 1H, AreH), 10.85 (br, 1H, eNH).

0.54

1.37

Log LC₅₀

Median

Delta

Range

ꢁ4.0

ꢁ4.04

1.00

ꢁ4.29

0.36

0.65

ꢁ4.42

0.91

1.17

¹³C NMR (75 MHz, CDCl₃):

d

(ppm) 13.55 (eCH₃), 23.05 (eCH₂e),

0

1.04

34.44 (eSCH₂e), 117.10, 121.94, 123.76, 126.78, 127.72, 127.77,

288

T.-C. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 278e293

Fig. 1. Drug response curve from ﬁve dose study against compound 15.

133.48, 133.91, 134.10, 134.30, 134.57, 149.64, 158.22, 182.90, 185.22.

HRMS (EI) m/z calcd for C₁₈H₁₄N₂O₂S [M]^þ: 322.0776, found,

322.0762.

(300 MHz, CDCl₃):

d

(ppm) 0.96 (t, J ¼ 7.5 Hz, 3H, eCH₃), 1.51 (sext,

J ¼ 7.5 Hz, 2H, eCH₂e), 1.81 (quin, J ¼ 7.5 Hz, 2H, eCH₂e), 3.41 (t,

J ¼ 7.5 Hz, 2H, eSCH₂e), 7.74e7.78 (m, 2H, AreH), 7.90 (d,

J ¼ 8.4 Hz, 1H, AreH), 8.13 (d, J ¼ 8.4 Hz, 1H, AreH), 8.20e8.23 (m,

1H, AreH), 8.28e8.30 (m, 1H, AreH), 10.87 (br, 1H, eNH). ¹³C NMR

(75 MHz, CDCl₃):

CH₂e), 32.23 (eSCH₂e), 117.04, 121.89, 123.69, 126.74, 127.65,

5.3.4. 2-(Butylthio)-1H-anthra[1,2-d]imidazole-6,11-dione (5)

Product 5 was prepared from 2 and 1-chlorobutane. The yel-

lowebrown solid material was isolated in 54% yield (R_f¼ 0.51 at

ethyl acetate:n-hexane ¼ 1:1). Mp 227e228 ^ꢃC (EtOH). ¹H NMR

d (ppm) 13.82 (eCH₃), 22.11 (eCH₂e), 31.56 (e

127.71, 133.40, 133.85, 134.05, 134.23, 134.53, 149.61, 158.31, 182.84,

Table 5

Median growth inhibitory concentration (GI₅₀, mM) and GI₅₀selectivity ratios of selected compounds 3, 4, 11 and 15 in the NCI in vitro 60-cell Drug Screen.

Subpanel cell lines

Compounds

3 (NSC747444)

Subpanel MID^bSelectivity ratio Subpanel MID^bSelectivity ratio Subpanel MID^bSelectivity ratio Subpanel MID^bSelectivity ratio

4 (NSC747853)

11 (NSC747515)

15 (NSC747854)

Leukemia

Non-small cell lung cancer 18.211

5.277

3.445

0.998

0.746

0.624

0.581

2.600

1.539

0.505

2.360

68.238

32.987

83.843

4.835

58.849

11.460

5.528

55.650

18.005

35.793

0.525

1.085

0.427

7.403

0.608

3.123

6.475

0.643

1.988

1.825

11.211

2.473

11.128

10.428

6.367

5.029

2.995

7.295

7.036

3.855

0.628

2.845

0.632

0.675

1.105

1.399

2.349

0.964

1.580

4.735

2.153

7.892

11.246

4.249

3.935

2.380

4.645

5.215

3.244

1.101

2.422

0.661

0.464

1.227

1.325

2.191

1.123

Colon cancer

CNS cancer

Melanoma

Ovarian cancer

Renal cancer

Prostate cancer

Breast cancer

MID^a

24.377

29.128

31.303

6.991

11.810

36.010

7.703

18.179

MID^a¼ average sensitivity of all cell line in

mM.

MID^b¼ average sensitivity of all cell line of a particular subpanel in

Selectivity ratio ¼ MID^a:MID^b.

mM.

T.-C. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 278e293

289

185.13. HRMS (EI) m/z calcd for C₁₉H₁₆N₂O₂S [M]^þ: 336.0932,

found, 336.0926.

eSCH₂e), 3.54 (t, J ¼ 6.3 Hz, 2H, eCH₂Cl), 7.76e7.78 (m, 2H, AreH),

7.90 (d, J ¼ 8.4 Hz, 1H, AreH), 8.14 (d, J ¼ 8.4 Hz, 1H, AreH), 8.22 (d,

J ¼ 6.0 Hz, 1H, AreH), 8.29 (d, J ¼ 6.0 Hz, 1H, AreH), 10.87 (br, 1H, e

5.3.5. 2-(Allylthio)-1H-anthra[1,2-d]imidazole-6,11-dione (6)

Product 6 was prepared from 2 and 3-bromoprop-1-ene. The

brown solid material was isolated in 41% yield (R_f¼ 0.55 at ethyl

NH). ¹³C NMR (75 MHz, CDCl₃):

d (ppm) 26.60, 28.17, 29.43, 32.33,

32.60 (eSCH₂e), 45.15 (eCH₂Cl), 117.13, 121.97, 123.79, 126.79,

127.53, 127.79, 133.47, 133.94, 134.12, 134.29, 134.63, 149.61, 158.04,

182.92, 185.24. HRMS (EI) m/z calcd for C₂₁H₁₉N₂O₂SCl^þ[M]^þ:

398.0856, found, 398.0855.

acetate:n-hexane

(300 MHz, CDCl₃):

¼

1:1). Mp 302e304 ^ꢃC (EtOH). ¹H NMR

(ppm) 4.06 (d, J ¼ 6.6 Hz, 2H, eSCH₂e), 5.28 (d,

d

J ¼ 10.2 Hz,1H, trans-CH₂), 5.47 (d, J ¼ 16.5 Hz,1H, cis-CH₂), 6.06 (m,

1H, eCHe), 7.78e7.80 (m, 2H, AreH), 7.95 (d, J ¼ 8.4 Hz, 1H, AreH),

8.17 (d, J ¼ 8.1 Hz, 1H, AreH), 8.25e8.33 (m, 2H, AreH), 10.93 (br,

5.3.10. 2-((2-(Pyrrolidin-1-yl)ethyl)thio)-1H-anthra[1,2-d]

imidazole-6,11-dione (11)

Product 11 was prepared from 2 and 1-(2-chloroethyl)pyrroli-

dine. The yellow solid material was isolated in 36% yield (R_f¼ 0.42

at dichloromethane). Mp 157e159 ^ꢃC (EtOH). ¹H NMR (300 MHz,

1H, eNH). ¹³C NMR (75 MHz, CDCl₃):

d

(ppm) 37.12 (eSCH₂e),

118.18, 118.25, 121.99, 123.89, 126.37, 126.80, 127.79, 127.96, 133.49,

133.85, 134.13, 134.29, 134.63, 149.59, 157.69, 182.89, 185.18. HRMS

(EI) m/z calcd for C₁₈H₁₂N₂O₂S^þ[M]^þ: 320.0619, found, 320.0623.

CDCl₃):

d

(ppm) 1.98 (quin, J ¼ 3.3 Hz, 4H, eCH₂e), 2.87 (t, 2H,

J ¼ 5.7 Hz, 4H, eNCH₂e), 3.11 (t, J ¼ 5.4 Hz, 2H, eNCH₂e), 3.38 (t,

J ¼ 5.4 Hz, 2H, eSCH₂e), 7.75e7.79 (m, 2H, AreH), 7.91 (d,

J ¼ 8.4 Hz, 1H, AreH), 8.16 (d, J ¼ 8.4 Hz, 1H, AreH), 8.20e8.23 (m,

1H, AreH), 8.30e8.32 (m, 1H, AreH). ¹³C NMR (75 MHz, CDCl₃):

5.3.6. 2-(Prop-2-ynylthio)-1H-anthra[1,2-d]imidazole-6,11-dione

(7)

Product 7 was prepared from 2 and 3-bromoprop-1-yne. The

yellow solid material was isolated in 41% yield (R_f¼ 0.44 at ethyl

d

(ppm) 24.14 (eCH₂e), 31.91 (eSCH₂e), 55.11 (eNCH₂e), 58.83 (e

acetate:n-hexane

(300 MHz, CDCl₃):

¼

1:1). Mp 132e133 ^ꢃC (EtOH). ¹H NMR

(ppm) 2.47 (s, 1H, eCH), 4.17 (s, 2H, eSCH₂e),

NCH₂CH₂Se), 117.74, 121.77, 123.71, 126.64, 127.57, 127.67, 133.80,

134.14, 134.23, 134.29,134.40, 150.36, 159.06, 183.21, 184.41. EI-MS

m/z calcd for (C₂₁H₁₉N₃O₂S^þ) [M]^þ: 377.1, found, 377.1.

d

7.78e7.83 (m, 2H, AreH), 7.99 (d, J ¼ 10.5 Hz, 1H, AreH), 8.20 (d,

J ¼ 10.5 Hz, 1H, AreH), 8.25e8.29 (m, 1H, AreH), 8.32e8.36 (m, 1H,

AreH), 11.07 (br, 1H, eNH). ¹³C NMR (75 MHz, CDCl₃):

d

(ppm) 27.12

5.3.11. 2-((2-(Piperidin-1-yl)ethyl)thio)-1H-anthra[1,2-d]

imidazole-6,11-dione (12)

Product 12 was prepared from 2 and 1-(2-chloroethyl)piperi-

dine. The yellow solid material was isolated in 56% yield (R_f¼ 0.45

at dichloromethane). Mp 160e162 ^ꢃC (EtOH). ¹H NMR (300 MHz,

(eSCH₂e), 67.45 (eCCH), 79.45 (eCCH), 117.18, 121.99, 123.82,

126.42, 126.80, 127.79, 127.93, 133.44, 133.95, 134.12, 134.26, 149.51,

157.63, 182.89, 185.19. HRMS (EI) m/z calcd for C₁₈H₁₀N₂O₂S^þ[M]^þ:

318.0463, found, 318.0462.

CDCl₃):

d

(ppm) 1.50 (m, 2H, eCH₂e), 1.63 (quin, J ¼ 5.1 Hz, 4H, e

5.3.7. 2-(Chlorobutylthio)-1H-anthra[1,2-d]imidazole-6,11-dione

(8)

CH₂e), 2.61 (t, J ¼ 5.1 Hz, 4H, eNCH₂e), 2.86 (t, J ¼ 6.3 Hz, 2H, e

CH₂Ne), 3.49 (t, J ¼ 6.3 Hz, 2H, eSCH₂e), 7.75e7.79 (m, 2H, AreH),

7.90 (d, J ¼ 8.4 Hz, 1H, AreH), 8.15 (d, J ¼ 8.4 Hz, 1H, AreH), 8.23e

8.26 (m, 1H, AreH), 8.30e8.32 (m, 1H, AreH). ¹³C NMR (75 MHz,

Product 8 was prepared from 2 to 1,4-dichlorobutane. The

brown solid material was isolated in 52% yield (R_f¼ 0.4 at ethyl

acetate:dichloromethane ¼ 1:1). Mp 177e179 ^ꢃC (EtOH). ¹H NMR

CDCl₃):

d (ppm) 24.56 (eCH₂e), 25.22 (eCH₂e), 30.66 (eSCH₂e),

(300 MHz, CDCl₃):

d

(ppm) 1.98e1.99 (m, 4H, eCH₂CH₂e), 3.44 (t,

54.49 (eNCH₂e), 58.13 (eSCH₂e), 117.42, 121.90, 123.70, 126.75,

127.68, 127.73, 133.53, 133.63, 133.88, 134.21, 134.46, 149.86, 158.68,

183.04, 184.82. ESI-MS m/z calcd for (C₂₂H₂₁N₃O₂S^þ) [M]^þ:

391.1354, found [M þ H]^þ: 392.1.

J ¼ 6.3 Hz, 2H, eSCH₂e), 3.59 (t, J ¼ 6.3 Hz, 2H, eCH₂Cl), 7.74e7.77

(m, 2H, AreH), 7.90 (d, J ¼ 8.4 Hz, 1H, AreH), 8.14 (d, J ¼ 8.4 Hz, 1H,

AreH), 8.20e8.24 (m, 1H, AreH), 8.28e8.31 (m, 1H, AreH), 10.85

(br, 1H, eNH). ¹³C NMR (75 MHz, CDCl₃):

d (ppm) 27.02, 30.28, 31.67

(eSCH₂e), 44.45 (eCCl), 117.18, 121.99, 123.82, 126.80, 127.79,

127.93, 133.44, 133.95, 134.12, 134.26, 134.63, 149.51, 157.63, 182.89,

185.19. HRMS (EI) m/z calcd for C₁₉H₁₅N₂O₂SCl [M]^þ: 370.0543,

found, 370.0546.

5.3.12. 2-((2-Morpholinoethyl)thio)-1H-anthra[1,2-d]imidazole-

6,11-dione (13)

Product 13 was prepared from 2 and 3-bromoprop-1-yne. The

yellow solid material was isolated in 54% yield (R_f¼ 0.55 at

dichloromethane). Mp 177e179 ^ꢃC (EtOH). ¹H NMR (300 MHz,

5.3.8. 2-(5-Bromopentylthio)-1H-anthra[1,2-d]imidazole-6,11-

dione (9)

CDCl₃):

d

(ppm) 2.60 (t, J ¼ 4.5 Hz, 4H, eNCH₂e), 2.86 (t, J ¼ 6.6 Hz,

2H, eCH₂Ne), 3.56 (t, J ¼ 6.6 Hz, eSCH₂e), 3.76 (t, J ¼ 4.5 Hz, 4H, e

CH₂Oe), 7.77e7.80 (m, 2H, AreH), 7.92 (d, J ¼ 8.1 Hz, 1H, AreH),

8.17 (d, J ¼ 8.4 Hz, 1H, AreH), 8.24e8.27 (m, 1H, AreH), 8.30e8.34

Product 9 was prepared from 2 to 1,5-dibromopentane. The

brown solid material was isolated in 44% yield (R_f¼ 0.37 at ethyl

acetate:n-hexane

(300 MHz, CDCl₃):

¼

1:1). Mp 222e223 ^ꢃC (EtOH). ¹H NMR

(ppm) 1.63e1.68 (m, 2H, eCH₂e), 1.87e1.97

(m, 1H, AreH). ¹³C NMR (75 MHz, CDCl₃):

d (ppm) 30.08 (eSCH₂e),

d

53.08 (eNCH₂e), 58.02 (eCH₂Ne), 66.87 (eCH₂Oe), 117.27, 121.96,

123.79,126.78,127.73,133.42, 133.94,134.06,134.20,134.40,134.56,

149.61, 158.11, 182.90, 184.97. HRMS (ESI) m/z calcd for

(m, 4H), 3.41e3.46 (m, 4H, eSCH₂, eCH₂Br), 7.78e7.81 (m, 2H, Are

H), 7.94 (d, J ¼ 8.1 Hz, 1H, AreH), 8.17 (d, J ¼ 8.4 Hz, 1H, AreH),

8.24e8.34 (m, 2H, AreH). ¹³C NMR (75 MHz, CDCl₃):

d

(ppm) 27.43,

C

₂₁H₁₉N₃O₃S^þ[M]^þ: 393.1147, found [M þ H]^þ: 394.1253.

28.81, 32.33, 33.58, 117.15, 121.97, 123.79, 126.79, 127.77, 128.25,

131.59, 133.45, 133.92, 134.59, 135.01, 140.14, 149.55, 159.79, 182.87,

185.18. HRMS (EI) m/z calcd for C₂₀H₁₇N₂O₂SBr^þ[M]^þ: 428.0194,

found, 428.0164.

5.3.13. 2-((3-(Piperidin-1-yl)propyl)thio)-1H-anthra[1,2-d]

imidazole-6,11-dione (14)

Product 14 was prepared from 2 and 1-(3-chloropropyl)piperi-

dine. The red solid material was isolated in 41% yield (R_f¼ 0.52 at

dichloromethane). Mp 159e162 ^ꢃC (EtOH). ¹H NMR (300 MHz,

5.3.9. 2-((6-Chlorohexyl)thio)-1H-anthra[1,2-d]imidazole-6,11-

dione (10)

CDCl₃):

d

(ppm) 1.42e1.48 (m, 2H, eCH₂e), 1.60 (quin, J ¼ 5.4 Hz,

Product 10 was prepared from 2 to 1,6-dichlorohexane. The

brown solid material was isolated in 56% yield (R_f¼ 0.43 at ethyl

4H, eNCCH₂e), 2.05 (quin, J ¼ 5.4 Hz, 2H, eSCCH₂e), 2.40e2.45 (m,

4H, eNCH₂e), 2.51 (t, J ¼ 6.9 Hz, 2H, eCH₂Ne), 3.46 (t, J ¼ 6.9 Hz,

2H, eSCH₂e), 7.77e7.81 (m, 2H, AreH), 7.94 (d, J ¼ 8.4 Hz, 1H, Are

H), 8.18 (d, J ¼ 8.4 Hz, 1H, AreH), 8.24e8.27 (m, 1H, AreH), 8.31e

acetate:n-hexane

(300 MHz, CDCl₃):

¼

1:1). Mp 159e160 ^ꢃC (EtOH). ¹H NMR

(ppm) 1.77e1.86 (m, 8H), 3.41 (t, J ¼ 3.9 Hz, 2H,

d

290

T.-C. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 278e293

8.34 (m, 1H, AreH), 10.04 (br, 1H, eNH). ¹³C NMR (75 MHz, CDCl₃):

(ppm) 24.58 (eCH₂e), 25.95 (eCH₂e), 26.85 (eCH₂e), 30.67 (e

SCH₂e), 54.74 (eCH₂Ne), 57.70 (eNCH₂e), 117.13, 121.90, 123.66,

126.43,126.75,127.70,133.44,133.86,134.09,134.23,134.50,149.60,

158.31, 182.86, 185.04. HRMS (ESI) m/z calcd for C₂₃H₂₃N₃O₂S^þ

[M]^þ: 405.1511, found [M þ H]^þ: 406.1589; [M ꢁ H]^ꢁ: 404.1452.

134.24, 134.69, 134.76, 140.02, 141.92, 155.89, 182.98, 183.04,

184.03. HRMS (EI) m/z calcd for C₂₀H₁₄N₂O₄S^þ[M]^þ: 378.0674,

found, 378.0683.

d

5.3.18. 2-((2-Morpholino-2-oxoethyl)thio)-1H-anthra[1,2-d]

imidazole-6,11-dione (19)

Product 19 was prepared from 2 and 2-chloro-1-morpholi

noethanone. The solid material was isolated in 53% yield

(R_f¼ 0.82 at ethyl acetate:n-hexane ¼ 3:1). Mp 279e281 ^ꢃC (EtOH).

5.3.14. 2-((3-(Dimethylamino)propyl)thio)-1H-anthra[1,2-d]

imidazole-6,11-dione (15)

Product 15 was prepared from 2 and 3-chloro-N,N-dimethyl-

propan-1-amine. The yellow solid material was isolated in 55%

yield (R_f¼ 0.35 at dichloromethane). Mp 154e156 ^ꢃC (EtOH). ¹H

¹H NMR (300 MHz, CDCl₃):

d (ppm) 3.60e3.67 (m, 4H, eN(CH₂)₂e),

3.70e3.79 (m, 4H, e(CH₂)₂Oe), 4.18 (s, 2H, eSCH₂e), 7.78e7.82 (m,

2H, AreH), 7.94 (d, J ¼ 8.4 Hz, 1H, AreH), 8.19 (d, J ¼ 8.4 Hz, 1H, Are

H), 8.31e8.36 (m, 2H, AreH), 12.39 (br, 1H, eNH). ¹³C NMR (75 MHz,

NMR (300 MHz, CDCl₃):

d

(ppm) 2.03 (quin, J ¼ 6.3 Hz, 2H, eCH₂e),

2.37 (s, 6H, eN(CH₃)₂), 2.62 (t, J ¼ 6.3 Hz, 2H, eCH₂Ne), 3.43 (t,

J ¼ 6.3 Hz, 2H, eSCH₂), 7.73e7.77 (m, 2H, AreH), 7.87 (d, J ¼ 8.4 Hz,

1H, AreH), 8.13 (d, J ¼ 8.4 Hz, 1H, AreH), 8.21e8.24 (m, 1H, AreH),

CDCl₃): d (ppm) 34.27 (eSCH₂e), 47.25, 47.64, 65.05, 65.64, 118.02,

121.91, 123.99, 127.12, 127.52, 128.03, 133.59, 133.74, 133.91, 134.17,

134.35, 149.61, 157.03, 167.65, 183.19, 184.55. HRMS (EI) m/z calcd

for C₂₁H₁₇N₃O₄S^þ[M]^þ: 407.0940, found, 407.0944.

8.27e8.30 (m, 1H, AreH). ¹³C NMR (75 MHz, CDCl₃):

d (ppm) 25.25,

30.56, (eSCH₂e) 44.50 (eN(CH₃)₂), 58.23, 117.22, 121.89, 123.63,

126.75, 127.68, 127.73, 133.53, 133.83,134.22, 134.29, 134.46, 149.86,

158.63, 183.02, 184.22. HRMS (ESI) m/z calcd for C₂₀H₁₉N₃O₂S^þ

[M]^þ: 365.1198, found [M þ H]^þ: 366.1295.

5.3.19. 2-((3-Morpholino-3-oxopropyl)thio)-1H-anthra[1,2-d]

imidazole-6,11-dione (20)

Product 20 was prepared from 2 and 3-chloro-1-morpholino

propan-1-one. The brown solid material was isolated in 56% yield

(R_f¼ 0.6 at ethyl acetate:n-hexane ¼ 3:1). Mp 242e244 ^ꢃC (EtOH).

5.3.15. 2-(2-Methylbenzylthio)-1H-anthra[1,2-d]imidazole-6,11-

dione (16)

¹H NMR (300 MHz, CDCl₃):

3.57 (m, 2H, eSCH₂e), 3.70 (s, 8H, morpholineeCH₂e), 7.78e7.82

(m, 2H, AreH), 7.92 (d, J ¼ 8.1 Hz, 1H, AreH), 8.18 (d, J ¼ 8.1 Hz, 1H,

AreH), 8.26e8.35 (m, 2H, AreH), 11.21 (br, 1H, eNH). ¹³C NMR

d

(ppm) 2.98 (t, J ¼ 6.3 Hz, 2H, eCH₂e),

Product 16 was prepared from 2 and 1-(chloromethyl)-2-

methylbenzene. The brown solid material was isolated in 60%

yield (R_f¼ 0.51 at ethyl acetate:n-hexane ¼ 1:1). Mp 218e219 ^ꢃC

(EtOH). ¹H NMR (300 MHz, CDCl₃):

d

(ppm) 2.46 (s, 3H, eCH₃), 4.69

(75 MHz, CDCl₃): d (ppm) 29.27, 34.22 (eSCH₂e), 47.24, 47.62,

(s, 2H, eSCH₂e), 7.14e7.23 (m, 3H), 7.43 (d, J ¼ 6.9 Hz, 1H), 7.74e

7.80 (m, 2H, AreH), 7.97 (d, J ¼ 8.4 Hz, 1H, AreH), 8.18 (d, J ¼ 8.4 Hz,

1H, AreH), 8.22e8.26 (m, 1H, AreH), 8.31e8.37 (m, 1H, AreH),

65.04, 65.62, 118.02, 121.91, 123.99, 127.02, 127.52, 128.03, 133.59,

133.74, 133.91, 134.07, 134.35, 149.60, 157.03, 168.05, 183.19, 184.55.

HRMS (ESI) m/z calcd for C₂₂H₁₉N₃O₄S^þ[M]^þ: 421.1096, found

[M þ H]^þ: 422.1182; found [M ꢁ H]^ꢁ: 420.1037.

10.85 (br, 1H, eNH). ¹³C NMR (75 MHz, CDCl₃):

d (ppm) 19.52 (e

CH₃), 35.17 (eSCH₂e), 117.25, 122.00, 123.91, 126.67, 126.80, 127.72,

127.90,128.63,130.46,130.98,133.42,133.69,133.94,134.06,134.24,

134.61, 137.39, 149.49, 157.64, 182.89, 185.13. HRMS (EI) m/z calcd

for C₂₃H₁₆N₂O₂S^þ[M]^þ: 384.0932, found, 384.0931.

5.3.20. 2-((6,11-Dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-

yl)thio)-N,N-diethyl-acetamide (21)

Product 21 was prepared from 2 and 2-chloro-N,N-dieth-

ylacetamide. The brown solid material was isolated in 66% yield

(R_f¼ 0.78 at ethyl acetate:n-hexane ¼ 3:1). Mp 219e220 ^ꢃC (EtOH).

5.3.16. Methyl-(6,11-dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazole-

2-ylthio) butanoate (17)

¹H NMR (300 MHz, CDCl₃):

d

(ppm) 1.22 (t, J ¼ 7.2 Hz, 4H), 1.30 (t,

Product 17 was prepared from 2 and methyl 4-chlorobutanoate.

The brown solid material was isolated in 65% yield (R_f¼ 0.18 at

ethyl acetate:dichloromethane ¼ 1:1). Mp 193e195 ^ꢃC (EtOH). ¹H

J ¼ 7.2 Hz, 4H), 3.48 (q, J ¼ 7.2 Hz, 2H, eNCH₂e), 3.56 (q, J ¼ 7.2 Hz,

2H, eNCH₂e), 4.09 (s, 1H, eSCH₂e), 7.75e7.81 (m, 2H, AreH), 7.92

(d, J ¼ 8.4 Hz, 1H, AreH), 8.18 (d, J ¼ 8.4 Hz, 1H, AreH), 8.30e8.36

(m, 2H, AreH), 12.91 (br, 1H, eNH). ¹³C NMR (75 MHz, CDCl₃):

NMR (300 MHz, CDCl₃):

d

(ppm) 2.20 (quin, J ¼ 6.9 Hz, 2H, eCH₂e),

2.54 (t, J ¼ 6.9 Hz, 2H, eCH₂COe), 3.47 (t, J ¼ 6.9 Hz, 2H, eSCH₂e),

3.70 (s, 3H, eOCH₃), 7.76e7.80 (m, 2H, AreH), 8.03 (d, J ¼ 8.4 Hz,1H,

AreH), 8.18 (d, J ¼ 8.4 Hz, 1H, AreH), 8.23e8.25 (m, 1H, AreH),

8.35e8.37 (m, 1H, AreH), 10.83 (br, 1H, eNH). ¹³C NMR (75 MHz,

d (ppm) 13.13 (eCH₃), 14.80 (eCH₃), 33.52 (eSCH₂e), 41.43 (e

NCH₂e), 43.37 (eNCH₂e), 117.97, 122.00, 123.83, 127.09, 127.62,

128.09, 133.56, 133.66, 133.92, 134.15, 134.40, 149.28, 157.15, 168.42,

183.12, 184.58. HRMS (EI) m/z calcd for C₂₁H₁₉N₃O₃S^þ[M]^þ:

393.1147, found, 393.1145.

CDCl₃):

d (ppm) 24.98 (eCH₂e), 31.63 (eCH₂COe), 32.75 (eSCH₂),

52.01 (eOCH₃), 117.04, 121.98, 123.87, 126.81, 127.78, 127.83, 133.45,

133.95, 134.12, 134.26, 134.63, 149.52, 157.44, 173.41 (eCOOe),

182.92(CO), 185.18(CO). HRMS (EI) m/z calcd for C₂₀H₁₆N₂O₂S^þ

[M]^þ: 380.0831, found, 380.0841.

5.3.21. 3-((6,11-Dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-

yl)thio)-N,N-diethyl-propanamide (22)

Product 22 was prepared from 2 and 3-chloro-N,N-dieth-

ylpropanamide. The brown solid material was isolated in 61% yield

(R_f¼ 0.8 at ethyl acetate:n-hexane ¼ 3:1). Mp 387e388 ^ꢃC (EtOH).

5.3.17. (E)-Methyl 4-((6,11-dioxo-6,11-dihydro-1H-anthra[1,2-d]

imidazol-2-yl)thio)but-2-enoate (18)

¹H NMR (300 MHz, CDCl₃):

d

(ppm) 1.12 (t, J ¼ 5.1 Hz, 3H, eCH₃),

Product 18 was prepared from 2 and (E)-methyl 4-chlorobut-2-

enoate. The yellow solid material was isolated in 70% yield

(R_f¼ 0.2 at ethyl acetate:n-hexane ¼ 1:1). Mp 220e222 ^ꢃC (EtOH).

1.17 (t, J ¼ 5.1 Hz, 3H, eCH₃), 2.97 (t, J ¼ 6.6 Hz, 2H, eCH₂COe), 3.32

(q, J ¼ 7.2 Hz, 2H, eNCH₂e), 3.44 (q, J ¼ 7.2 Hz, 2H, eNCH₂e), 3.70 (t,

J ¼ 6.6 Hz, 2H, eSCH₂e), 7.74e7.80 (m, 2H, AreH), 7.89 (d, J ¼ 8.1 Hz,

1H, AreH), 8.15 (d, J ¼ 8.4 Hz, 1H, AreH), 8.24e8.33 (m, 2H, AreH),

¹H NMR (300 MHz, CDCl₃):

d (ppm) 3.72 (s, 3H, eOCH₃), 4.19 (d,

J ¼ 5.7 Hz, 2H, eSCH₂e), 6.15 (d, J ¼ 14.7 Hz, 1H, eC]CHCOOe),

7.07e7.12 (m, 1H, eCH]Ce), 7.79e7.81 (m, 2H, AreH), 7.95 (d,

J ¼ 8.1 Hz, 1H, AreH), 8.19 (d, J ¼ 8.1 Hz, 1H, AreH₃), 8.26e8.27 (m,

1H, AreH), 8.34e8.35 (m, 1H, AreH), 10.87 (br, 1H, eNH). ¹³C NMR

11.36 (br, 1H, eNH). ¹³C NMR (75 MHz, CDCl₃):

d (ppm) 13.35,

14.46, 28.24 (eSCH₂e), 33.77, 40.65, 42.12, 117.30, 121.84, 123.58,

126.93, 127.65, 127.71, 133.45, 133.89, 134.11, 134.46, 134.76,

149.54, 158.41, 169.39, 182.92, 184.82. HRMS (ESI) m/z calcd for

(75 MHz, CDCl₃):

d

(ppm) 32.92 (eSCH₂e), 51.99 (eOCH₃), 118.71,

C

₂₂H₂₁N₃O₃S^þ[M]^þ: 407.1304, found [M þ H]^þ: 408.1388; found

122.06, 124.16, 124.23, 124.57, 126.83, 127.83, 133.93, 134.02,

[M ꢁ H]^ꢁ: 406.1246.

T.-C. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 278e293

291

5.3.22. 2-((2-Oxo-2-(pyrrolidin-1-yl)ethyl)thio)-1H-anthra[1,2-d]

imidazole-6,11-dione (23)

(yield 43%): Mp 239e241 ^ꢃC (EtOH) (R_f¼ 0.4 at dichloromethane).

¹H NMR (300 MHz, CDCl₃):

d (ppm) 3.62e3.83 (m, 8H, eCH₂e),

Product 23 was prepared from 2 and 2-chloro-1-(pyrrolidin-1-

yl)ethanone. The brown solid material was isolated in 56% yield

(R_f¼ 0.8 at ethyl acetate:n-hexane ¼ 3:1). Mp 345e347 ^ꢃC (EtOH).

7.78e7.83 (m, 2H, AreH), 8.03 (d, J ¼ 8.7 Hz, 1H, AreH), 8.23 (d,

J ¼ 6.9 Hz,1H, AreH), 8.29e8.36 (m, 2H, AreH), 12.92 (br, 1H, eNH).

¹³C NMR (75 MHz, CDCl₃):

d (ppm) 47.24 (eOCH₂e), 47.64 (eOCH₂e

¹H NMR (300 MHz, CDCl₃):

d

(ppm) 1.93e1.99 (t, J ¼ 6.6 Hz, 2H),

), 65.04 (eNCH₂e), 65.64 (eNCH₂e), 118.04, 121.94, 123.95, 127.04,

127.42, 128.03, 133.55, 133.74, 133.95, 134.06, 134.34, 149.60, 157.03,

168.05, 183.19, 184.55. HRMS (ESI) m/z calcd for C₂₀H₁₅N₃O₄S^þ

[M]^þ: 393.0783, found [M þ H]^þ: 394.0862; found [M ꢁ H]^ꢁ:

392.0725.

2.05e2.10 (t, J ¼ 6.6 Hz, 2H), 3.61e3.69 (m, 2H, eNCH₂e), 3.99 (s,

2H, eSCH₂), 7.76e7.78 (m, 2H, AreH), 7.91 (d, J ¼ 8.1 Hz, 1H, AreH),

8.16 (d, J ¼ 8.1 Hz,1H, AreH), 8.28e8.36 (m, 2H, AreH),13.18 (br,1H,

eNH). ¹³C NMR (75 MHz, CDCl₃):

d (ppm) 24.66, 26.37, 34.07 (e

SCH₂e), 47.05, 47.64, 118.00, 121.91, 123.99, 127.12, 127.59, 128.03,

133.58, 133.74, 134.91, 134.17, 134.35, 149.61, 157.03, 167.61, 183.19,

184.55. HRMS (ESI) m/z calcd for C₂₁H₁₇N₃O₃S^þ[M]^þ391.0991,

found [M þ H]^þ: 392.1071; found [M ꢁ H]^ꢁ: 390.0928.

5.3.27. S-(6,11-Dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-yl)

4-methylpiperazine-1-carbothioate (28)

Compound 28 was prepared from 2 and 4-methylpiperazine-1-

carbonyl chloride for 12 h. The pure product was obtained as gray

powder (yield 46%): Mp 233e235 ^ꢃC (EtOH) (R_f¼ 0.66 at ethyl

acetate:dichloromethane ¼ 1:1). ¹H NMR (300 MHz, CDCl₃):

5.3.23. 2-((3-Oxo-3-(pyrrolidin-1-yl)propyl)thio)-1H-anthra[1,2-d]

imidazole-6,11-dione (24)

Product 24 was prepared from 2 and 3-chloro-1-(pyrrolidin-1-yl)

propan-1-one. The brown solid material was isolated in 54% yield

(R_f¼ 0.8 at ethyl acetate:n-hexane ¼ 3:1). Mp 244e245 ^ꢃC (EtOH).¹H

d (ppm) 2.36 (s, 3H, eCH₃), 2.49e2.53 (m, 4H, eCH₂e), 3.59e3.66

(m, 2H, eNCH₂e), 3.76e3.84 (m, 2H, eNCH₂e), 3.35 (t, J ¼ 7.5 Hz,

2H, eNCH₂e), 7.76e7.82 (m, 2H, AreH), 8.00 (d, J ¼ 8.4 Hz, 1H, Are

H), 8.20 (d, J ¼ 8.4 Hz, 1H, AreH), 8.26e8.34 (m, 2H, AreH), 12.94

NMR (300 MHz, CDCl₃):

d

(ppm) 1.84e1.98 (m, 4H), 2.92 (t, J ¼ 6.3 Hz,

2H, eCH₂COe), 3.42 (t, J ¼ 6.6 Hz, 2H, eNCH₂e), 3.55 (t, J ¼ 6.6 Hz,

2H, eNCH₂e), 3.70 (t, J ¼ 6.3 Hz, 2H, eSCH₂e), 7.77e7.80 (m, 2H, Are

H), 7.91 (d, J ¼ 8.4 Hz,1H, AreH), 8.16 (d, J ¼ 8.4 Hz,1H, AreH), 8.26e

8.34 (m, 2H, AreH), 11.50 (br, 1H, eNH). ¹³C NMR (75 MHz, CDCl₃):

(br, 1H, eNH). ¹³C NMR (75 MHz, CDCl₃):

d (ppm) 40.07 (eCH₃),

47.04 (eNCH₂e), 47.64 (eNCH₂e), 56.04 (eNCH₂e), 56.64 (e

NCH₂e), 118.06, 121.95, 123.95, 127.05, 127.46, 128.03, 133.55,

133.70, 133.91, 134.06, 134.34, 149.60, 157.03, 168.05, 183.19, 184.50.

HRMS (EI) m/z calcd for C₂₁H₁₈N₄O₃S^þ[M]^þ: 406.1100, found,

406.1106.

d

(ppm) 24.60, 26.23, 27.80 (eCH₂COe), 35.28 (eSCH₂e), 46.31 (e

NCH₂), 46.80 (eNCH₂), 117.39, 121.87, 123.12, 123.70, 126.87, 127.68,

130.15, 133.51, 133.92, 134.21, 134.49, 149.64, 158.42, 169.52, 183.02,

184.90. HRMS (ESI) m/z calcd for C₂₂H₁₉N₃O₃S^þ[M]^þ: 405.1147,

found [M þ H]^þ: 406.1232; found [M ꢁ H]^ꢁ: 404.1087.

5.3.28. S-(6,11-Dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-yl)

3-phenylpropanethioate (29)

Compound 29 was prepared from 2 and 3-phenylpropanoyl

chloride for 11 h. The pure product was obtained as gray powder

(yield 41%): Mp 237e240 ^ꢃC (EtOH) (R_f¼ 0.82 at dichloromethane).

5.3.24. 2-((6,11-Dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-

yl)thio)-N,N-dipropyl-acetamide (25)

Product 25 was prepared from 2 and 2-chloro-N,N-dipropyla-

cetamide. The brown solid material was isolated in 55% yield

(R_f¼ 0.83 at ethyl acetate:n-hexane ¼ 3:1). Mp 193e195 ^ꢃC (EtOH).

¹H NMR (300 MHz, CDCl₃):

d

(ppm) 3.18 (t, J ¼ 7.5 Hz, 3H, eCH₂e),

3.98 (t, J ¼ 7.5 Hz, 2H, eCH₂e), 7.31 (m, 5H, phenyl-H), 7.82e7.86

(m, 2H, AreH), 8.17 (d, J ¼ 8.4 Hz, 1H, AreH), 8.27e8.35 (m, 2H, Are

H), 8.45 (d, J ¼ 8.4 Hz, 1H, AreH), 11.19 (br, 1H, eNH). ¹³C NMR

¹H NMR (300 MHz, CDCl₃):

d

(ppm) 0.92 (t, J ¼ 7.2 Hz, 3H, eCH₃),

1.00 (t, J ¼ 7.2 Hz, 3H, eCH₃), 1.61e1.76 (m, 4H, eCH₂e), 3.35 (t,

J ¼ 7.5 Hz, 2H, eNCH₂e), 3.46 (t, J ¼ 7.5 Hz, 2H, eNCH₂e), 4.11 (s, 2H,

eSCH₂), 7.76e7.79 (m, 2H, AreH), 7.91 (d, J ¼ 8.4 Hz, 1H, AreH), 8.17

(d, J ¼ 8.4 Hz, 1H, AreH), 8.29e8.35 (m, 2H, AreH), 12.87 (br, 1H, e

(75 MHz, CDCl₃): d (ppm) 30.81 (eCH₂e), 44.96 (eCOCH₂e), 118.13,

124.28, 125.21, 126.20, 127.24, 128.05, 130.50, 131.37, 133.25, 133.80,

134.39, 134.60, 134.81, 135.21, 135.94, 150.60, 156.67, 181.82, 184.14,

189.94. ESI-MS m/z calcd for C₂₄H₁₆N₂O₃S^þ[M]^þ: 412.1, found,

413.0.

NH). ¹³C NMR (75 MHz, CDCl₃):

d (ppm) 11.50 (eCH₃), 20.96, 22.70,

24.70, 33.91 (eSCH₂e), 48.57 (eNCH₂e), 50.77 (eNCH₂e), 117.82,

121.85, 123.83, 126.99, 127.56, 127.93, 133.50, 133.71, 133.88, 134.09,

134.34, 149.52, 157.26, 168.88, 184.07, 184.49. HRMS (ESI) m/z calcd

for C₂₃H₂₃N₃O₃S^þ[M]^þ: 421.1460, found [M þ H]^þ: 422.1552.

5.3.29. S-(6,11-Dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-yl)

2,5-bis(triﬂuoromethyl)benzothioate (30)

Compound 30 was prepared from 2 and 2,5-bis(triﬂuoromethyl)

benzoyl chloride for 13 h. The pure product was obtained from

silica column and eluted with ethyl acetate:dichloromethane ¼ 1:3

to get the gray powder (yield 30%): Mp 344e345 ^ꢃC (R_f¼ 0.77 at

5.3.25. S-(6,11-Dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-yl)

2-nitrobenzothioate (26)

Compound 26 was prepared from 2 and 2-nitrobenzoyl chloride.

The pure product was obtained as gray powder (yield 39%): Mp

279e281 ^ꢃC (EtOH) (R_f¼ 0.42 at dichloromethane). ¹H NMR

dichloromethane). ¹H NMR (300 MHz, CDCl₃):

d (ppm) 7.81 (s, 1H,

Ar-H), 7.84e7.88 (m, 2H, AreH), 7.91 (br, 1H, AreH), 8.27 (d,

J ¼ 8.4 Hz, 1H, AreH), 8.28e8.38 (m, 2H, AreH), 8.51 (d, J ¼ 8.4 Hz,

(300 MHz, CDCl₃):

d

(ppm) 7.61 (d, J ¼ 7.2 Hz, 1H, AreH₄₀), 7.65 (t,

1H, AreH), 11.09 (br, 1H, eNH). ¹³C NMR (75 MHz, CDCl₃):

d (ppm)

J ¼ 7.2 Hz, 1H, AreH), 7.74e7.85 (m, 3H, AreH), 8.31e8.40 (m, 2H,

AreH), 8.46 (d, J ¼ 8.7 Hz, 1H, AreH), 8.67 (d, J ¼ 8.4 Hz, 1H, AreH),

114.88, 119.96, 124.28, 126.11, 127.24, 128.05, 130.43, 130.98, 131.37,

133.25, 133.66, 133.79, 134.38, 134.60, 134.81, 134.99, 135.21, 135.94,

136.82, 166.60, 173.07, 181.82, 184.14. HRMS (ESI) m/z calcd

for C₂₄H₁₀N₂O₃SF^þ₆[M]^þ: 520.0316, found [M þ H]^þ: 521.0387;

[M ꢁ H]^ꢁ: 519.0249.

9.06 (d, J ¼ 8.7 Hz, 1H, AreH). ¹³C NMR (75 MHz, CDCl₃):

d (ppm)

120.31, 124.28, 126.11, 127.24, 128.05, 130.43, 130.69, 131.37,

131.96, 133.25, 133.79, 134.17, 134.38, 134.60, 134.81, 135.21, 135.94,

166.60, 173.07, 181.82, 184.14, 187.62. HRMS (ESI) m/z calcd for

C

₂₂H₁₁N₃O₅S^þ[M]^þ: 429.0419, found [M ꢁ NO₂]^þ: 383.0501.

5.3.30. S-(6,11-Dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-yl)

2,5-dimethylfuran-3-carbothioate (31)

5.3.26. S-(6,11-Dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-yl)

morpholine-4-carbothioate (27)

Compound 27 was prepared from 2 and morpholine-4-carbonyl

chloride for 10 h. The pure product was obtained as gray powder

Compound 31 was prepared from 2 and 2-nitrobenzoyl chloride

for 9 h. The pure product was obtained from silica column and

eluted with ethyl acetate:dichloromethane ¼ 1:3 to get the

gray powder (yield 29%): Mp > 400 ^ꢃC (R_f¼ 0.71 at ethyl

292

T.-C. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 278e293

acetate:dichloromethane ¼ 1:1). ¹H NMR (300 MHz, CDCl₃):

compound as a yellowebrown solid in 39% yield (R_f¼ 0.6 at ethyl

acetate:dichloromethane ¼ 1:6). Mp 241e243 ^ꢃC (EtOH). FT-IR

d

(ppm) 2.32 (s, 3H, eCH₃), 2.68 (s, 3H, eCH₃), 7.79e7.84 (m, 2H,

AreH), 8.05 (d, J ¼ 8.4 Hz, 1H, AreH), 8.25 (d, J ¼ 8.4 Hz, 1H, AreH),

(KBr; n d (ppm) 3.94

cm^ꢁ1) 1630 (CO). ¹H NMR (300 MHz, CDCl₃):

8.33e8.38 (m, 2H, AreH), 12.86 (br, 1H, eNH). ¹³C NMR (75 MHz,

(t, J ¼ 7.2 Hz, 2H, eCH₂e), 5.16 (t, J ¼ 7.2 Hz, 2H, eCH₂e), 7.75e7.82

CDCl₃):

d (ppm) 10.81 (eCH₃), 13.96 (eCH₃), 104.88, 119.96, 124.28,

(m, 2H, AreH), 7.86 (d, J ¼ 8.4 Hz, 1H, AreH), 8.17 (d, J ¼ 8.4 Hz, 1H,

126.20, 127.24, 128.05, 130.50, 131.37, 133.25, 133.80, 134.39, 134.60,

134.81, 135.21, 135.94, 150.60, 156.67, 181.82, 184.14, 186.14. HRMS

(ESI) m/z calcd for C₂₂H₁₄N₂O₄S^þ[M]^þ: 402.0674, found [M þ H]^þ:

403.0752; found [M ꢁ H]^ꢁ: 401.0598.

AreH), 8.17e8.28 (m, 2H). ¹³C NMR (75 MHz, CDCl₃):

d (ppm) 34.21,

50.86, 118.84, 122.55, 124.40, 126.92, 127.25, 128.54, 133.45, 134.00,

134.24, 134.47, 155.67, 165.96, 166.68, 183.12 (CO), 184.05 (CO).

HRMS (ESI) m/z calcd for C₁₇H₁₀N₂O₂S^þ[M]^þ: 306.0463, found

[M þ H]^þ: 307.0531.

5.3.31. S-(6,11-Dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-yl)

pyrrolidine-1-carbothioate (32)

5.4. MTT assay for cell viability

Compound 32 was prepared from 2 and pyrrolidine-1-carbonyl

chloride for 11 h. The pure product was obtained as brown powder

A simple and regular colorimetric assay using MTT was devel-

oped to monitor mammalian cell survival and proliferation in vitro

[30,31]. Approximately 2 ꢄ 10³cells were plated onto each well of a

96-well plate and incubated in 5% CO₂at 37 ^ꢃC for 24 h. To assess

the in vitro cytotoxicity, each compound was dissolved in DMSO,

prepared immediately before the experiments and diluted into the

complete medium before being added to cell cultures. Test com-

pounds were then added to the culture medium for designated

(yield 52%): Mp 228e230 ^ꢃC (EtOH) (R_f

¼

0.67 at ethyl

acetate:dichloromethane ¼ 1:1). ¹H NMR (300 MHz, CDCl₃):

d

(ppm) 1.90e2.13 (m, 6H, eCH₂e), 3.56 (t, J ¼ 6.6 Hz, 2H, eNCH₂e),

3.71 (s, J ¼ 6.6 Hz, 2H, eNCH₂e), 7.76e7.82 (m, 2H, AreH), 7.99 (d,

J ¼ 8.4 Hz, 1H, AreH), 8.21 (d, J ¼ 8.4 Hz, 1H, AreH), 8.28e8.35 (m,

2H, AreH), 13.01 (br, 1H, eNH). ¹³C NMR (75 MHz, CDCl₃):

d (ppm)

24.46 (eCH₂e), 26.35 (eCH₂e), 47.03 (eNCH₂e), 47.64 (eNCH₂e),

118.00, 121.91, 123.99, 127.12, 127.59, 128.03, 133.54, 133.74, 133.91,

134.17, 134.35, 149.61, 157.03, 167.31, 183.19, 184.55. HRMS (ESI) m/z

calcd for C₂₀H₁₅N₃O₃S^þ[M]^þ: 377.0834, found [M þ H]^þ: 378.0921.

various concentrations. After 48 h, an amount of 25

mL of MTT was

added to each well, and the samples were incubated at 37 ^ꢃC for 4 h.

A 100 mL solution of lysis buffer containing 20% SDS and 50% DMF

was added to each well and incubated at 37 ^ꢃC for another 16 h. The

absorbency at 550 nm was measured by using an ELISA reader.

5.3.32. S-(6,11-Dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-yl)

piperidine-1-carbothioate (33)

Compound 33 was prepared from 2 and piperidine-1-carbonyl

chloride for 11 h. The pure product was obtained as brown pow-

der (yield 53%): Mp 242e244 ^ꢃC (EtOH) (R_f¼ 0.69 at ethyl

acetate:dichloromethane ¼ 1:1). ¹H NMR (300 MHz, CDCl₃):

5.5. Secreted alkaline phosphatase (SEAP) assay

Secreted alkaline phosphatase was used as the reporting system

to monitor the transcriptional activity of hTERT [32,33]. Here, about

2 ꢄ 10³cells each were grown in 96-well plates and incubated at

37 ^ꢃC for 24 h and changed with fresh media. Varying amounts of

drugs were added and cells were incubated for another 24 h. Cul-

ture media were collected and heated at 65 ^ꢃC for 10 min in order to

inactivate heat-labile phosphatases. An equal amount of SEAP

d

(ppm) 1.72 (br, 6H, eCH₂e), 3.56 (br, 2H, eNCH₂e), 3.73 (br, 2H, e

NCH₂e), 7.76e7.79 (m, 2H, AreH), 7.98 (d, J ¼ 8.4 Hz, 1H, AreH),

8.18 (d, J ¼ 8.4 Hz, 1H, AreH), 8.27e8.34 (m, 2H, AreH), 13.00 (br,

1H, eNH).¹³C NMR (75 MHz, CDCl₃):

d (ppm) 23.86 (eCH₂e), 24.98,

27.15, 45.77 (eNCH₂e), 47.49 (eNCH₂e), 118.19, 121.97, 124.49,

126.93, 127.70, 128.49, 133.63, 134.06, 134.15, 134.44, 152.97, 154.38,

168.22, 181.20, 183.89, 186.63. HRMS (EI) m/z calcd for

buffer (2 M diethanolamine, 1 mM MgCl₂, and 20 mM L-homo-

arginine) was added to the media and p-nitrophenyl phosphate was

added to a ﬁnal concentration of 12 mM. Absorptions at 405 nm

were taken, and the rate of absorption increase was determined.

C

₂₁H₁₇N₃O₃S^þ[M]^þ: 391.0991, found, 391.0995.

5.3.33. S-(6,11-Dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-yl)

diethylcarbamothioate (34)

5.6. In vitro NCI-60 panel tumor cell lines study

Compound 34 was prepared from 2 and diethylcarbamic chlo-

ride for 11 h. The pure product was obtained as brown powder

As a primary screen, six selected compounds were submitted to

the NCI cell lines screen for evaluation of their anticancer activity

[34]. From the data analysis, it follows that approximately 95% of

the active ones from the 60 cell lines screen can be identiﬁed. The

detailed methods used for the 60 cell lines panel have been

described elsewhere [35e40]. Brieﬂy, cellular protein levels were

determined after 48 h of drug exposure by SRB (Sulforhodamine B)

colorimetry. Through the use of a time zero cell control, the cell

growth can be determined for each cell line thus allowing calcu-

lations of GI₅₀, TGI, and LC₅₀. Comparison to plates not exposed to

drug permits determination of concentration and times of exposure

conferring GI₅₀, TGI, and LC₅₀. These data are then plotted as mean

bar graphs and as doseeresponse curves. All of the selected com-

pounds, which diminished the growth of any one of the cell lines

less than 32%, were sieved out for further evaluation in the full

panel of NCI’s 60 human tumor cell lines. Based on these criteria,

four compounds reported from our compounds bank were all

active and passed on for evaluation in the full panel of 60 human

tumor cell lines. The panel is organized into nine subpanels rep-

resenting diverse histology: leukemia, melanoma, and cancers of

the lung, colon, kidney, ovary, breast, prostate, and brain (central

nervous system).

(yield 53%): Mp 214e216 ^ꢃC (EtOH) (R_f

¼

0.74 at ethyl

acetate:dichloromethane ¼ 1:1). ¹H NMR (300 MHz, CDCl₃):

d

(ppm) 1.28 (t, J ¼ 6.9 Hz, 3H, eCH₃), 1.35 (t, J ¼ 6.9 Hz, 3H, eCH₃),

3.49 (q, J ¼ 6.9 Hz, 2H, eCH₂e), 3.59 (t, J ¼ 6.9 Hz, 2H, eNCH₂e),

7.76e7.82 (m, 2H, AreH), 7.99 (d, J ¼ 8.4 Hz, 1H, AreH), 8.20 (d,

J ¼ 8.4 Hz,1H, AreH), 8.29e8.35 (m, 2H, AreH), 13.04 (br, 1H, eNH).

¹³C NMR (75 MHz, CDCl₃):

d (ppm) 13.35 (eCH₃), 14.06 (eCH₃),

43.26 (eNCH₂e), 50.77 (eNCH₂e), 118.09, 121.85, 124.63, 126.87,

127.67, 128.41, 132.21, 133.58, 134.02, 134.12, 134.41, 148.13, 152.16,

162.11,183.08,184.49. HRMS (ESI) m/z calcd for C₂₀H₁₇N₃O₃S^þ[M]^þ:

379.0991, found [M þ H]^þ: 380.1066.

5.3.34. 1,2-Dihydroanthra[2⁰,1⁰:4,5]imidazo[2,1-b]thiazole-7,12-

dione (35)

Compound 2 (1.40 g, 5 mmol) was dissolved in N,N-dime-

thylformamide (30 mL) and 1,2-dichloroethane (0.74 g, 7.5 mmol)

and potassium hydroxide solution (1 M, 10 ꢄ 10 mL, 10 mmol) was

added at room temperature for 10 h (TLC monitoring). After

ﬁnished, the mixture was poured into ice water. The resulting

precipitate was collected by ﬁltration, washed with distilled water

and puriﬁed by crystallization from ethanol to afford desired

T.-C. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 278e293

293

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Acknowledgment

The authors would like to thank the NCI Developmental Ther-

apeutics Program for 60 cell line screen of compounds described in

this paper funded by National Cancer Institute, National Institutes

of Health (NIH-NCI). The present study was support by National

Science Council Grants of Taiwan (NSC100-2113-M-016 and

NSC101-2113-M-016), Chi-Mei Medical Center (CMNDMC-10101

and 10210), National Research Program for Biopharmaceuticals

(ChemBank and High-Throughput Screening Resource Center, NSC-

101-2325-B-001-029) and National Health Research Institutes

(NHRI EX100-10050SI).

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Appendix A. Supplementary data

Supplementary data related to this article can be found at

http://dx.doi.org/10.1016/j.ejmech.2013.06.058.

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Structure-based design, synthesis and biological evaluation of novel anthra[1,2-d]imidazole-6,11-dione homologues as potential antitumor agents

DOI: 10.1016/j.ejmech.2013.06.058

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