Ring fusion strategy for synthesis and lead optimization of sulfur-substituted anthra[1,2-c[[1,2,5]thiadiazole-6,11-dione derivatives as promising scaffold of antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2015.07.052

A series of sulfur-substituted anthra[1,2-c][1,2,5]thiadiazole-6,11-diones were synthesized and evaluated using the cell proliferations, apoptosis and NCI-60 cell panel assays. Also, the signaling pathways that account for their activities were investigated. Compounds 2, 3, 4a, 4d, 4f, 4i, 4k, 5b, 5c, 5d, 5f, 5g, 6b, 6c, 6d, 6e, 6g, 7a and 7g were selected by NCI. Among the tested compounds, 6g appeared to be the most active compound of this series that not only induced apoptosis in DU-145 cancer cells but also attenuated the ERK1/2 and p38 signaling pathways. All test compounds exhibited diverse cytostatic and cytotoxic activities that warrant further development as potential anticancer agents.

Full text of DOI:10.1016/j.ejmech.2015.07.052

Accepted Manuscript
Ring fusion strategy for synthesis and lead optimization of sulfur-substituted
anthra[1,2-c][1,2,5]thiadiazole-6,11-dione derivatives as promising scaffold of
antitumor agents
Yu-Ru Lee, Tsung-Chih Chen, Chia-Chung Lee, Chun-Liang Chen, Ahmed Atef
Ahmed Ali, Alexander Tikhomirov, Jih-Hwa Guh, Prof. Dr. Dah-Shyong Yu, Prof. Dr.
Hsu-Shan Huang
PII:
S0223-5234(15)30184-7
10.1016/j.ejmech.2015.07.052
EJMECH 8036
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 May 2015
Revised Date: 7 July 2015
Accepted Date: 31 July 2015
Please cite this article as: Y.-R. Lee, T.-C. Chen, C.-C. Lee, C.-L. Chen, A.A.A. Ali, A. Tikhomirov,
J.-H. Guh, D.-S. Yu, H.-S. Huang, Ring fusion strategy for synthesis and lead optimization of sulfur-
substituted anthra[1,2-c][1,2,5]thiadiazole-6,11-dione derivatives as promising scaffold of antitumor
agents, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.07.052.
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ACCEPTED MANUSCRIPT
Ring fusion strategy for synthesis and lead optimization of
sulfur-substituted anthra[1,2-c][1,2,5]thiadiazole-6,11-dione
derivatives as promising scaffold of antitumor agents
a,b
b
b
b
Yu-Ru Lee , Tsung-Chih Chen , Chia-Chung Lee , Chun-Liang Chen , Ahmed Atef
b,c
d,e
f
a,g,*
Ahmed Ali , Alexander Tikhomirov , Jih-Hwa Guh , Dah-Shyong Yu , Hsu-Shan
a,b,h,
Huang
*
*
Corresponding authors:
Prof. Dr. H. S. Huang (Taipei Medical University), Tel: +886-2-27361661 ext 7525,
Fax: +886-2-66387537, E-mail: huanghs99@tmu.edu.tw; Prof. Dr. D. S. Yu (National
Defense Medical Center), Tel: +886-2-87923100 ext 19390 Fax: +886-2-87923169,
E-mail: yuds@ms21.hinet.net
Abstract:
A series of sulfur-substituted anthra[1,2-c][1,2,5]thiadiazole-6,11-diones were
synthesized and evaluated for cell proliferations, apoptosis, signaling pathways, and
NCI-60 cell panel assay, respectively. Compounds 2, 3, 4a, 4d, 4f, 4i, 4k, 5b, 5c, 5d,
5
f, 5g, 6b, 6c, 6d, 6e, 6g, 7a and 7g were selected by NCI and 4d, 4f, 5f, 6g and 7a
represent the GI , TGI and LC , respectively. Among them, 6g appeared to be the
5
0
50
most active compound of this series that not only induced apoptosis in DU-145 cancer
cells but also attenuated the ERK1/2 and p-38 signaling pathways. All test compounds
exhibited diverse cytostatic and cytotoxic activities for further developing potential
anticancer agents.
Keywords: Thiadiazoles, anthra[1,2-c][1,2,5]thiadiazole-6,11-dione, SRB assay, NCI
6
0-cell panel assay, apoptosis.

ACCEPTED MANUSCRIPT
EJMECH 8036
Ring fusion strategy for synthesis and lead optimization of
sulfur-substituted anthra[1,2-c][1,2,5]thiadiazole-6,11-dione
derivatives as promising scaffold of antitumor agents
a,b
b
b
b
Yu-Ru Lee , Tsung-Chih Chen , Chia-Chung Lee , Chun-Liang Chen , Ahmed Atef
b,c
d,e
f
a,g,*
Ahmed Ali , Alexander Tikhomirov , Jih-Hwa Guh , Dah-Shyong Yu , Hsu-Shan
a,b,h,
Huang
*
a
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114,
Taiwan
b
Graduate Institute of Cancer Biology and Drug Discovery, College of Medical
Science and Technology, Taipei Medical University, Taipei 110, Taiwan
c
Institute of Molecular Biology, Taiwan International Graduate Program, Academia
Sinica, Taipei 105, Taiwan
d
Gause Institute of New Antibiotics, Moscow 119021, Russia
e
Mendeleyev University of Chemical Technology, 9 Miusskaya Square, Moscow
1
25190, Russia
f
School of Pharmacy, National Taiwan University, Taipei 110, Taiwan
g
Division of Urology, Department of Surgery, Tri-Service General Hospital, National
Defense Medical Center, Taipei114, Taiwan
h
School of Pharmacy, National Defense Medical Center, Taipei 114, Taiwan
*
Corresponding authors, Prof. Dr. H.S. Huang; Tel: +886-2-2736-1661 ext.7525, Fax:
886-2-6638-7537, E-mail: huanghs99@tmu.edu.tw
Corresponding authors, Prof. Dr. D.S. Yu (National Defense Medical Center), Tel:
+
*
+886-2-87923100 ext 19390 Fax: +886-2-87923169, E-mail: yuds@ms21.hinet.net
1

ACCEPTED MANUSCRIPT
Abstract:
A series of sulfur-substituted anthra[1,2-c][1,2,5]thiadiazole-6,11-diones were
synthesized and evaluated using the cell proliferations, apoptosisand NCI-60 cell
panel assays. Also, the signaling pathways that account for their activities were
investigated. Compounds 2, 3, 4a, 4d, 4f, 4i, 4k, 5b, 5c, 5d, 5f, 5g, 6b, 6c, 6d, 6e, 6g,
7
a and 7g were selected by NCI. Among the tested compounds, 6g appeared to be the
most active compound of this series that not only induced apoptosis in DU-145 cancer
cells but also attenuated the ERK1/2 and p-38 signaling pathways. All test compounds
exhibited diverse cytostatic and cytotoxic activities that warrant further development
as potential anticancer agents.
Keywords: Thiadiazoles, anthra[1,2-c][1,2,5]thiadiazole-6,11-dione, SRB assay, NCI
6
0-cell panel assay, apoptosis.
2

ACCEPTED MANUSCRIPT
1
. Introduction
Thiadiazoles are among the privileged pharmacological scaffold fragments
because of their unique chemical properties for diverse biological and clinical
applications [1], such as anticancer [2-5], anti-inflammatory [6], antihypertensive [7],
antidepressant [8,9], anticonvulsant [10], antileishmanial [11,12], and antimicrobial
activities [13-16]. Anthraquinone-containing extracts from different plant sources (e.g.
senna, cascara, aloe, frangula, and rhubarb etc.) have been found to have wide variety
of pharmacological activities such as anti-inflammatory, wound healing, analgesic,
antipyretic, anti-microbial, and antitumor activities [17,18]. To date, anthracycline
antibiotics (e.g. daunorubicin, doxorubicin) and the selective anthraquinone
derivatives (e.g. mitoxantrone, emodin) are important anthraquinone-containing drugs,
showing strong antiproliferative (or cytostatic) activities and diverse applications in
clinical practice [19]. In view of the above facts and the concept of bio-isosterism we
developed a ring fusion strategy that fused the thiadiazole and anthraquinone
structures to alter their pharmacological and physiochemical properties for varied
bioactivities. The scaffold hopping strategy aimed for the discovery of novel
anticancer drugs has been generally accepted as a means to transform pharmacophoric
templates, and subsequently match these with complementary features embedded into
small-molecule topologies [20] (Fig. 1).
Based on the similar chemical properties and drugs comparison studies, our
group illustrated that related various tricyclic and tetracyclic anthraquinone-derived
analogs exhibited anticancer activities by acting as potent telomerase and/or TOP1
inhibitors [18,21-24]. Thorough analysis of these scaffold according to the associated
biological activity showed that thiadiazole renders anthraquinone moieties
unprecedented broad-spectrum bioactivities [22,23,25-29]. In addition, the
five-membered thiadiazole ring connected to the plannar tricyclic anthraquinone
3

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moiety gives a compact and planar structure that exhibits an extremely low
aromaticity as a result of its high polarity and electron-withdrawing tendency [1].
Although a number of thiadiazole-containing drugs have advantages over other
commonly discovered therapeutic scaffolds, the most fully investigated of these broad
biological relevance in clinical currently on the market [30,31]. In this study we were
interested in developing the widely reported drugs containing 1,2,5-thiadiazole,
timolol, vedaclidine, sulfametrole, tazomeline and xanomeline taking into
consideration that anthra[1,2-c][1,2,5]thiadiazole-6,11-dione was evaluated as
bioisostere
of
core.
Based
on
our
previous
studies,
anthra[1,2-c][1,2,5]thiadiazole-6,11-dione was shown to exhibit significant antitumor
potency against several cancers in vitro and in vivo [26-28]. Moreover, the tricyclic
and tetracyclic systems, heterocyclic anthraquinones and substituents of these
heterocycles with thiadiazole-containing compounds typically lead to analogues with
improved activities because the sulfur atom imparts substantially increased binding to
their intracellular targets as well as increased liposolubility [31].
We have previously synthesized various tricyclic and tetracyclic derivatives
having different substituents at different positions which showed remarkable potent
anticancer activities. In the course of our continuing search for new antitumor agents
from the thiadiazole-containing anthraquinone compounds, we hypothesized that the
fused system containing thiadiazole ring will have advantage over benzene and
thiazole. Compared with other heterocycles, thiadiazole (benzene bioisostere) with
unparalleled properties display striking differential cytotoxicity profiles. The
anticancer activities of our new compounds were evaluated using a panel of 60 human
tumor cell lines primary screen provided by the NCI (USA). Toward supporting the
aforementioned hypothesis and exploration of SARs, we found the backbone of
anthra[1,2-c][1,2,5]thiadiazole-6,11-dione
derivatives
is
associated
with
4

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DNA-intercalating or topoisomerase inhibitory properties. The cytotoxicity of our
compounds against two classical human prostate cancer cell lines (including PC-3 and
DU-145) where evaluated by the SRB and MTT assays (Table 1). Moreover, 2
(NSC745885), 3 (NSC757963), 4a (NSC757964), 4d (NSC761882), 4f (NSC761884),
4
i (NSC757966), 4k (NSC761890), 5b (NSC761881), 5c (NSC761891), 5d
(NSC763955), 5f (NSC761885), 5g (NSC761883), 6b (NSC761889), 6c
(NSC761886), 6d (NSC761892), 6e (NSC761887), 6g (NSC763968), 7a
(NSC757965), and 7c (NSC763965) were selected by the NCI for one dose screening
program (Table 2). Considering their structure and side chains, 4d (NSC761882), 4f
NSC761884), 5f (NSC761885), 6g (NSC763968), and 7a (NSC757965) showed
(
potent growth inhibition activities, so they were evaluated at 5 dose concentration
levels (Table 3). For the most potent compound 6g, the effects on the cell cycle and
induction of apoptosis were also tested in the human prostate cancer cell line
(DU-145). In this study, it is generally accepted that we provide insight into the
correlation between the cytotoxicity and inhibition activities against ERK 1/2 and
p-38 phosphorylation of some of selective synthetic compounds for their anticancer
activities.
2
. Chemistry
According to our previous series of studies, the structural complexity of
anthra[1,2-c][1,2,5]thiadiazole-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 [22,23,26-29]. There has been a
continuous
interest
in
the
synthesis
of
sulfur-substituted
anthra[1,2-c][1,2,5]thiadiazole-6,11-diones and their systematic dissection largely on
account of their complementary activities. Our primary starting material anthra[1,2-c]
[
1,2,5]thiadiazole-6,11-dione (2) was synthesized from 1,2-diaminoanthraquinone by
5

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thionyl chloride and trimethylamine as catalyst. Subsequently, 3 was prepared by
potassium chlorate in hydrochloric acid in the presence of acetic acid and quenched
by ice water. Such approach, as shown in Scheme 1, reacting 2 with the appropriate
thiols and thiophenols by using ferric chloride as catalyst in DMF for several hours or
reacting 3 with the appropriate thiols and thiophenols in dry THF and
N,N-diisoproprylethylamine
(DIPEA)
afforded
the
sulfur-substituted
anthra[1,2-c][1,2,5]thiadiazole-6,11-dione derivatives in good yields (overall 35-88%).
It was found that the two above-mentioned methods led directly to the formation of
the desired final products. In the series of designed analogues, we maintained the
anthra[1,2-c][1,2,5]thiadiazole-6,11-dione as a core structure, and the lead
optimization focused on varying the electron-withdrawing or electron-releasing
substituents at the thio-position of anthra[1,2-c][1,2,5]thiadiazole-6,11-dione skeleton.
3
. Results and discussion
3
.1 In vitro antiproliferative activities
In an initial attempt to investigate the cytotoxic activities of
anthra[1,2-c][1,2,5]thiadiazole-6,11-dione, the IC values of each target compound
5
0
were measured by SRB and MTT assays against PC-3 and DU-145 cells. As
illustrated in Table 1, the values of IC for nine compounds (2, 4d, 4f, 4q, 5d, 6f, 6g,
5
0
7
b and 7d) were less than 10 µM against PC-3 and/or DU-145 cells, respectively.
Through structural variation as shown in Table 1, to explore the effect of substitution
in the linker, a few electron donating substituents (CH , OCH , OC H ) of terminal
3
3
2
5
phenyl group were predicted to have less efficacy in our assays. In addition to
examining different linkers and diverse heteroaromatic substitutions on the terminal
phenyl, the activity of electron withdrawing group on meta-position substitution was
superior to para-position and ortho-position. These data also indicated that a suitable
linker were important for enzymatic inhibition and anticancer cell proliferation. More
6

ACCEPTED MANUSCRIPT
importantly, compound 6f and 6g were chosen for further optimization based on
evaluating the SAR in greater detail. Among them, 6g is the most active derivative
with IC values of 3.69 µM and 4.53 µM against PC-3 and DU-145, respectively. So,
5
0
compound 6g was selected for further evaluation for the morphological changes and
apoptosis in DU-145 cells, as well as inhibition activity against the ERK 1/2 and p38
phosphorylation. Understanding the cell death pathways for selective killing against
cancer cells would lead to more effective strategies. It seemed from theresults for 6g
that the preclinical studies still have a long way to go because of its target and
mechanism.
3
.2 NCI60 human cell lines anticancer drug screening
The anticancer activity and toxicity of all the test compounds were evaluated
using the National Cancer Institute (NCI) Drug Screen Program of 60 human tumor
cell lines. Details of the methodology are described in our previous reports [21, 24, 25,
3
2-36]. There are two stages of the screening process that starts with the evaluation of
the test compounds against the 60 human tumor cell lines at a single dose of 10 µM.
In this initial screening, 2, 3, 4a, 4d, 4f, 4i, 4k, 5b, 5c, 5d, 5f, 5g, 6b, 6c, 6d, 6e, 6g,
7
a, and 7c were chosen by NCI and tested against a panel of 60 human tumor cell
lines. The growth inhibition percentages obtained from the single dose experiments
are shown in Table 2. Compounds 4d, 4f, 5f, 6g and 7a showed potent and significant
growth inhibition so they were evaluated further for their cytostatic and cytotoxic
activities against the 60 cell lines panel using the five dose studies (0.01, 0.1, 1, 10
and 100 µM). The dose-response curve is originated from the log 10 of the
corresponding drug concentration against each cell line from the subpanel groups. The
percentage growth at each concentration with GI , TGI and LC of 4d, 4f, 5f, 6g and
5
0
50
7
a are shown in Table 3. The in vitro results showed that compounds 4f and 6g have
potent broad spectrum anticancer activities against almost all of the human cancer cell
7

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lines with GI in nano molar to micro molar range. As shown in Table 3, 4f exhibited
5
0
remarkable anticancer activity against most of the tested cell lines representing nine
different subpanels with GI values between 0.31 to 20.1 ꢀM. With regard to the
5
0
sensitivity against some individual cell lines, 4f showed high activity against Ovarian
cancer (OVCAR-4) and Breast cancer (MDA-MB-231/ATCC) cell lines with GI50
values of 0.31 and 0.37 ꢀM, respectively. Sensitive cell lines to 4f, exhibited an LC50
with as little as 6.71 ꢀM and TGI with as little as 0.94 ꢀM for Ovarian cancer
(OVCAR-4). Results of our above study extended the initial in vitro observation
reported in the data above and confirmed the importance of anticancer activity.
More interestingly, 6g had the prominently potent activity against most of the
tested cell lines with GI values ranging from 0.18 to 13.3 µM. With regard to the
5
0
sensitivity against individual cell lines, 6g showed high activity against Leukemia
0.18-0.31 ꢀM); Non-small lung cancer (0.32-2.85 ꢀM); Colon Cancer (0.29-13.3
M); CNS Cancer (0.52-3.32 ꢀM); Melanoma (0.20-2.72 ꢀM); Ovarian Cancer
0.25-2.90ꢀM); Renal Cancer (0.56-5.68 ꢀM); Prostate Cancer (0.25-1.45 ꢀM);
(
ꢀ
(
Breast Cancer (0.29-2.19 ꢀM). It is worth mentioning that 6g had the highest
selectivity against the following cell lines with GI : Leukemia (0.18-0.31 ꢀM),
5
0
Non-small lung cancer (HOP-92: 0.32 ꢀM; NCI-H23: 0.56 ꢀM), Colon Cancer
HCT-116: 0.29 ꢀM; SW-620: 0.43 ꢀM), CNS Cancer (SNB-75: 0.52 ꢀM);
(
Melanoma (LOX IMVI: 0.34 ꢀM; MDA-MB-435: 0.20 ꢀM; SK-MEL-2: 0.99 ꢀM;
UACC-257: 0.57 ꢀM); Ovarian Cancer (IGROV1: 0.96 ꢀM; OVCAR-3: 0.25 ꢀM;
OVCAR-4: 0.36 ꢀM; OVCAR-8: 0.49 ꢀM; NCI/ADR-RES: 0.52 ꢀM); Renal Cancer
(A489: 0.56 ꢀM; CAKI-1: 0.59ꢀM; SN12C: 0.66 ꢀM); Prostate Cancer (PC-3: 0.25
ꢀ
M); Breast Cancer (MDA-MB-231/ATCC: 0.29 ꢀM; T-47D: 0.38 ꢀM;
MDA-MB-468: 0.37 ꢀM). The five dose graphs and dose response curves of 6g for all
tested cell lines are illustrated in Fig. 2.
8

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3
.3 The in vitro selectivity of the full NCI60 cell panel assay
All the NCI60 cell panel assays, representing nine tumor subpanels, were
incubated at five different concentrations of the test compounds. The sensitivity of
compounds against some individual cell lines depends on the ratio obtained by
dividing the full panel MID and particular subpanel MID [37]. Ratios between 3 and 6
refer to moderate selectivity; ratios greater than 6 indicate high selectivity toward the
corresponding cell line, while compounds not meeting either of these criteria are rated
non-selective [38]. All of these parameters provide insight into the selectivity and
potency of the antitumor activity. Based on these parameters, 4d, 4f, 5f, 6g and 7a
were found to be moderately selective for growth inhibition toward leukemia cancer
subpanel where 6g and 7a showed selectivity ratios of 6.44 and 3.66, respectively,
whereas they were found to be less selective against the remaining cell panels (Table
4
). It seemed from these results that only 6g exhibited the unique selectivity against
leukemia cancer.
.4 The cytotoxicity of 6g to SV-HUC-1 cells (human normal urothelial cells),
WMPY-1) normal human prostatic stromal myo-fibroblasts and (RWPE-1) normal
3
(
prostate epithelial cells.
In this study, we compared the cytotoxic effect of SV-HUC-1 cells (human
normal urothelial cells), WMPY-1 (normal human prostatic stromal myo-fibroblasts)
and RWPE-1 (normal prostate epithelial cells) induced by 6g, and also compared this
cytotoxicity to doxorubicin (DXR). Based on our result, compound 6g had less
cytotoxic effect than DXR. As shown in Fig. 3a, 6g and doxorubicin were compared
regarding cytotoxicity in the normal cell line (SV-HUC-1 cells), 80% survival rate of
the cells was observed with the highest concentration of 6g. As shown in Fig. 3b and
3
c, 50% and 60% survival rate was observed with the highest concentration of 6g of
WMPY-1 (normal human prostatic stromal myo-fibroblasts) and RWPE-1 (normal
9

ACCEPTED MANUSCRIPT
prostate epithelial cells), respectively. These data are of interest, as it suggests that
DXR are more toxic to WMPY-1 (normal human prostatic stromal myo-fibroblasts.
The response of the well-known anticancer drug, DXR, was more toxic at the same
concentration.
3
.5. Morphological changes and apoptosis in DU-145 cells
In the present study, we assessed the effect of 6g on human prostate cancer
DU-145 cells in vitro, and elucidated its possible mechanisms. DU-145 cells were
treated with different concentrations of 6g and cell proliferation was assessed by MTT
assay, cell apoptosis by flow cytometry, and protein levels of Bcl-2 by Western
blotting. The results showed that 6g inhibited the proliferation of DU-145 cells in a
dose-dependent manner. DU-145 cells treated with different concentrations of 6g
displayed obvious morphological changes indicating apoptosis under the fluorescence
microscope. In addition, 6g increased the proportion of early apoptotic DU-145 cells,
down-regulated the protein levels of Bcl-2. Morphology analysis confirmed the
cytotoxic effects of 6g. As shown in Fig. 4, the apoptotic morphological changes
included cell rounding and shrinkage after 24 hour incubation with 5 µM of 6g.
Following above observation, we concluded that 6g caused apoptosis in DU-145 cells.
To investigate whether apoptosis induced by 6g was involved in activating caspase
cascades, we exposed DU-145 cells to various concentrations of the compound for 48
h. The activity of caspase-3 was then determined using western blotting analysis,
which revealed the activation of caspase-3 within 48 h of 6g treatment. The PARP, a
1
13 kDa nuclear enzyme, is cleaved in fragments of 89 during apoptosis. Exposure to
6
g also decreased the total PARP, increased the levels of cleaved PARP (Fig. 5) and
decreased the levels of cyclinD1 and Bcl-2 (Fig. 6a, 6b). Interesting, there are more
cleaved PARP and cleaved caspase 3 at concentration 3 uM than at 12 uM of 6g might
be apoptosis and necrosis.
1
0

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3
.6 Inhibition activity of 6g against ERK1/2 and p38 phosphorylation
In view of the previous rationale and in continuation of our ongoing program,
ERK1/2 is an important mitogen-activated protein kinase which control cellular
activities and physiological processes [39]. Western blot analysis demonstrated that
compound 6g had significant inhibition activities against ERK 1/2 and p-38
phosphorylation in DU-145 cells. As shown in Fig. 7, compound 6g inhibited p-38
phosphorylation in DU-145 cells at 3, 12, 18 µM with obvious concentration
dependency. Interestingly, compound 6g might induce ERK ½ phosphorylation at low
concentrations but against ERK ½ phosphorylation at high concentrations. Thus, these
compounds can inhibit the proliferation of DU-145 cells through the mediated
signaling pathway of ERK1/2 and p-38.
4
. Conclusion
In this investigation, we continue to pay attention to the role of our synthesized
tetracyclic anthraquinone moiety pharmacophore and to understand the
pharmacological effects of thiadiazole-containing anthraquinone derivatives. Thus, a
novel structure of substituted anthra [1,2-c][1,2,5] thiadiazole-6,11-dione derivatives
were design and synthesized. The NCI60 cancer panel, leukemia (0.18-0.31 µM),
non-small cell lung cancer (0.32-2.85 µM), colon cancer (0.29-13.3 µM), CNS cancer
(0.52-3.32 µM), melanoma (0.20-2.72 µM), ovarian cancer (0.25-2.90 µM), renal
cancer (0.56-5.68 µM), prostate cancer (PC-3: 0.25 µM; DU-145: 1.45 µM), breast
cancer (0.29-2.19 µM) were sensitive to 6g. Also, the leukemia (0.56-0.64 µM),
non-small cell lung cancer (1.00-18.6 µM), colon cancer (0.44-20.1 µM), melanoma
(0.54-2.34 µM), ovarian cancer (0.31-3.97 µM), breast cancer (0.37-3.27 µM) were
sensitive to 4f whereas the CNS cancer, renal cancer and prostate cancer panels were
less sensitive. The average GI50 values of 6g and 4f were 1.61 and 3.68 ꢀM,
respectively. The data showed that the compounds bearing sulfur alkyl side chains
11

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displayed potent cytotoxicity in prostate cancer cells. Furthermore, response of the
well-known anticancer drug, DXR, showed more toxicity than 6g at high
concentrations in the normal human urothelial cells. Given the striking correlations
between the proliferative capacity and topoisomerase activity in tumor cells (data not
shown), we expected that analysis of the anthra[1,2-c][1,2,5]thiadiazole-6,11-dione as
a chromophore with various thiol groups side chain substitutions might produce
further insight into designing better lead compounds for anti-tumor agents.
Morphology analysis confirmed that the cytotoxic effects of 6g not only induced
apoptosis but also activated the caspase cascade and PARP as well as decreasing the
levels of cyclinD1 and Bcl-2. Additionally, the inhibition activities of 6g on the
ERK1/2 and p38 protein phosphorylation were also tested by Western blotting.
Results showed that such inhibition was concentration dependent. Thus, compound 6g
can inhibit the proliferation of DU-145 cells through the mediated signaling of
ERK1/2 and p38 pathways. Collectively, the extensive SARs studies discussed here
have helped us to design a further novel series of potent anticancer drugs to enhance
the bioavailability and in vivo activity. Comparison of our experimental results lead to
the conclusion that the in vitro anticancer activity is dependent on the length of linker
between phenyl and sulfur substituents, with one and two carbons of linker typically
giving rise to compounds with higher activity. Compounds containing
1
,2,5-thiadiazole ring was observed to have high potency, and some of them displayed
excellent activities against range of tumor cells. The ability of
a
thiadiazole-containing anthraquinone compounds to target DNA could explain their
broad activity in unique ways which could potentially be used for chemical
intervention at the gene level. Sulfur containing substituents in various heterocyclic
derivatives might have a promising effect on the biological activity, so our
above-mentioned results may contribute to substantial progress in this field.
1
2

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5
. Materials and methods
.1 Chemistry
5
®
Melting points were determined by melting point apparatus (Büchi 545). All
1
reactions were monitored by TLC (Silica Gel 60 F ). H NMR spectra were recorded
2
54
®
®
with GEMINI-300 MHz (Varian ) and AM-500 MHz (Bruker ). Chemical shift (δ)
values were in ppm relative to TMS as an internal standard. Fourier-transform IR
spectra (KBr): Perkin-Elmer 983G spectrometer. Mass spectra were obtained on
Finnigan MAT 95 XL HRMS and Finnigan/Thermo Quest MAT HRMS. Reagents and
solvents were purchased from Merck and Aldrich used without further purification.
Typical experiments illustrating the general procedures for the preparation of the
anthraquinones are described below.
5
.2.General synthetic methods A：Preparation of compound 2
1.19g (5 mmole) of 1,2-diaminoanthraquinone (compound 1) was dissolved in
THF (30ml). The thionyl chloride (SOCl ) (0.5mL) and triethylamine (TEA) (3 mL)
2
were added and the reaction mixture was stirred at room temperature for 24 hours.
The mixture was quenched by ice water (100ml) and then collected by filtering. The
crystals were recrystallized from hot EtOH to get a hot compound NSC745885
(anthra[1,2-c][1,2,5]thiadiazole-6,11-dione).
General synthetic methods B Preparation of compound 3
：
A solution of 0.50 g (2 mmole) of compound NSC745885 in 15 mL of acetic
acid at 90℃ was treated with 5 ml of concentrated hydrochloric acid. A solution of
2
.0 g of potassium chlorate was added to the boiling suspension during a 45 minutes
time interval. The mixture was then boiled for another 4 hours and the precipitate was
separated in ice bath. Finally the reaction product NSC757963 was crystallized from
acetic acid.
General synthetic methods C: preparation of compounds 4a~ 4r, 5a~ 5h, 6a~ 6g
1
3

ACCEPTED MANUSCRIPT
and 7a~ 7e
These compounds were synthesized from 1,2-diaminoanthraquinone. All the
chemicals and organic solvents required for synthesis were purchased from either
Sigma Aldrich Chemical Company or Merck Chemical Company. A mixture of
compound 3 (0.3 g, 1 mmole) in dry THF and N,N-Diisoproprylethylamine (DIPEA)
(0.5 mL) were heated to 65ꢀ, and then series organosulfur compounds (2 mmole) in
same solvent were added. The reaction mixtures were refluxed for 2~4 hours at 65ꢀ.
Then the crude products were purified by ethanol to afford desired compounds.
5
.2.1.
4-(Phenylthio)anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4a)
The pure compound was obtained as an orange red powder (yield 76ꢁ). Mp: 224-225
-1
℃
(EtOH). FT-IR (KBr, νmax cm ): 1508.2 (aromatic C=C), 1519.8 (CO), 1589.2
1
(
CO), 1672.2 (C=N). H NMR (300 MHz, CDCl ): 7.58-7.62 (m, 3H), 7.71-7.74 (m,
3
2
H, m), 7.76-7.86 (m, 3H), 8.16 (dd, J= 7.8 Hz, J= 1.5 Hz, 1H), 8.35 (dd, J= 7.8 Hz,
1
3
J= 1.5 Hz, 1H). C NMR (75 MHz, CDCl ): δ ppm 119.93, 121.40, 127.04, 127.44,
3
1
1
3
5
28.32, 130.67, 130.87, 132.30, 133.97, 134.03, 134.88, 135.73, 135.90, 143.57,
+
+
51.32, 155.18, 181.68, 183.52. HRMS (EI) m/z: calcd for C H N O S [M] ;
2
0
10
2
2 2
74.0184, found, 374.0184.
.2.2. 4-(2-Fluorophenylthio)anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4b)
The pure compound was obtained as a yellow orange powder (yield 88ꢁ). Mp:
-
1
2
1
7
72-273 ℃ (EtOH). FT-IR (KBr, νmax cm ): 1508.2 (aromatic C=C), 1541.0 (CO),
1
591.2 (CO), 1674.1 (C=N). H NMR (300 MHz, CDCl ): δ ppm 7.32-7.40 (m, 2H),
3
.61-7.67 (m, 1H), 7.68-7.69 (m, 1H), 7.71-7.75 (m, 1H), 7.76-7.86 (m, 2H), 8.17 (dd,
1
3
J= 7.5 Hz, J= 1.5 Hz, 1H), 8.34 (dd, J= 7.5 Hz, J= 1.5 Hz , 1H). C NMR (75 MHz,
CDCl ): δ ppm 117.21, 117.51, 119.84, 121.67, 126.04, 126.09, 127.08, 127.48,
3
1
1
27.85, 132.20, 133.61, 133.72, 133.85, 134.13, 134.95, 135.67, 137.77, 151.28,
+
55.14, 161.71, 181.74, 183.47. HRMS (EI) m/z: calcd [M] , 392.0089
1
4

ACCEPTED MANUSCRIPT
+
(
C H FN O S ); found, 392.0087.
20 9 2 2 2
5
.2.3. 4-(2-Chlorophenylthio)anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4c)
The pure compound was obtained as a yellow powder (yield 81ꢁ). Mp: 263-264 ℃
-1
(
EtOH). FT-IR (KBr, νmax cm ): 1506.3 (aromatic C=C), 1542.9 (CO), 1670.2 (C=N).
1
H NMR (300 MHz, CDCl ): δ ppm 7.47 (td, J= 7.5 Hz, J= 1.5 Hz, 1H), 7.57 (td, J=
3
7
.5 Hz, J= 1.5 Hz, 1H), 7.63 (s, 1H), 7.70 (dd, J= 8.4 Hz, J= 1.5 Hz, 1H), 7.75-7.87
1
3
(
m, 3H), 8.16-8.19 (m, 1H), 8.34-8.37 (m, 1H). C NMR(75 MHz, CDCl ): δ ppm
3
1
1
20.24, 121.78, 127.05, 127.44, 127.76, 128.59, 131.45, 132.26, 132.41, 133.93,
34.05, 134.90, 135.67, 137.98, 140.04, 140.75, 151.37, 155.24, 181.64, 183.41.
+
+
2 2
HRMS(EI) m/z: calcd [M] , 407.9794 (C H ClN O S ); found, 407.9796.
2
0
9
2
5
.2.4.
4-(2-Bromophenylthio)anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4d)
The pure compound was obtained as an orange powder (yield 85ꢁ). Mp: 210-211 ℃
-1
(
EtOH). FT-IR(KBr, νmax cm ): 1506.3 (aromatic C=C), 1539.1 (CO), 1593.1 (CO),
1
1
670.2 (C=N). H NMR (300 MHz, CDCl ): δ ppm 7.46-7.53 (m, 2H), 7.62 (s, 1H),
3
7
.74-7.82 (m, 4H), 8.16 (dd, J= 7.5 Hz, J= 1.5 Hz, 1H), 8.33 (dd, J= 7.5 Hz, J= 1.5
13
Hz, 1H). C NMR (75 MHz, CDCl ): δ ppm 120.24, 121.74, 127.09, 127.48, 129.30,
3
1
1
29.88, 130.85, 132.26,132.45, 133.92, 134.11, 134.96, 135.68, 138.00, 140.91,
+
51.39, 155.19, 181.74, 183.47. HRMS (EI) m/z: calcd [M] , 451.9289
+
(
C H BrN O S ); found, 451.9295.
1
9
9
2
2 2
5
.2.5.
4-(3-Fluorophenylthio)anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4e)
The pure compound was obtained as a yellow powder (yield 78ꢁ). Mp: 254-255 ℃
-1
(
EtOH). FT-IR (KBr, νmax cm ): 1508.2 (aromatic C=C), 1575.7 (CO), 1670.2 (C=N).
1
H NMR (300 MHz, CDCl ): δ ppm 7.28-7.34 (m, 1H), 7.41-7.45 (m, 1H), 7.49-59
3
(m, 2H), 7.78 (s, 1H), 7.80-7.88 (m, 2H), 8.18 (dd, J= 7.2 Hz, J= 1.5 Hz, 1H),
13
8
.33-8.36 (m, 1H). C NMR (75 MHz, CDCl ): δ ppm 117.83, 118.03, 119.93,
3
1
22.24, 122.46, 126.87, 127.24, 131.26, 131.29 131.70, 131.79, 131.83, 133.48,
1
5

ACCEPTED MANUSCRIPT
1
33.96, 134.79, 135.33, 141.98, 150.90, 154.72, 161.71, 164.48, 181.45, 183.09.
+
+
HRMS (EI) m/z: calcd [M] , 392.0089 (C H FN O S ); found, 392.0091.
2
0
9
2
2 2
5
.2.6.
4-(3-Chlorophenylthio)anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4f)
The pure compound was obtained as an orange powder (yield 58ꢁ). Mp: 252-253 ℃
-1
(
EtOH). FT-IR (KBr, νmax cm ): 1500.5 (aromatic C=C), 1539.1 (CO), 1668.3 (C=N).
1
H NMR (300 MHz, CDCl ): δ ppm 7.50-7.63 (m, 3H), 7.71 (t, J= 1.5 Hz, 1H), 7.79
3
(s, 1H), 7.81-7.87 (m, 2H), 8.19 (dd, J= 7.8 Hz, J= 1.8 Hz, 1H), 8.36 (dd, J= 7.5 Hz,
1
3
J= 1.8 Hz, 1H). C NMR (75 MHz, CDCl ): δ ppm 120.61, 121.87, 127.15, 127.50,
3
1
1
4
5
30.43, 131.05, 131.58, 132.26, 133.78, 133.91, 134.18, 135.00, 135.33, 135.71,
+
36.28, 142.03, 151.31, 155.22, 181.73, 183.41. HRMS (EI) m/z: calcd [M] ,
+
07.9794 (C H ClN O S ); found, 407.9794.
2
0
9
2
2 2
.2.7.
4-(3-Bromophenylthio)anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4g)
The pure compound was obtained as an orange powder (yield 74ꢁ). Mp: 197-198 ℃
-1
(
EtOH). FT-IR (KBr, νmax cm ): 1508.2 (aromatic C=C), 1558.4 (C=O), 1668.3
1
(
C=N). H NMR(400 MHz, CDCl ): δ ppm 7.40 (t, J= 7.6 Hz, 1H), 7.66 (d, J= 7.6 Hz,
3
1
H), 7.74 (d, J= 7.6 Hz, 1H), 7.78-7.81 (m, 2H), 7.83-7.87 (m, 2H), 8.19 (d, J= 7.6
1
3
Hz, 1H), 8.36 (d, J= 7.6 Hz, 1H). C NMR (75 MHz, CDCl ): δ ppm 120.04, 121.39,
3
1
1
23.87, 126.91, 127.27, 130.01, 131.65, 131.86, 133.50, 133.79, 134.01, 134.12,
34.82, 135.36, 137.95, 141.91, 150.93, 154.73, 181.50, 183.12. HRMS (EI) m/z:
+
+
calcd [M] , 451.9289 (C H BrN O S ); found, 451.9295.
20
9
2
2
5
.2.8.
4-(4-Fluorophenylthio)anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4h)
The pure compound was obtained as an orange red powder (yield 79ꢁ). Mp: 224-225
-1
℃
(EtOH). FT-IR (KBr, νmax cm ): 1508.2 (aromatic C=C), 1539.1 (CO), 1519.2
1
(
CO), 1674.1 (C=N). H NMR (300 MHz, CDCl ): δ ppm 7.31(d, J= 8.7 Hz, 2H),
3
7
.67 (s, 1H), 7.69-7.74 (m, 2H), 7.75-7.89 (m, 2H), 8.17 (d, J= 7.5 Hz, 1H), 8.34 (d,
1
3
J= 7.2 Hz, 1H). C NMR (75 MHz, CDCl ): δ ppm 117.90, 118.19, 119.74, 121.52,
3
1
6

ACCEPTED MANUSCRIPT
1
1
3
5
23.47, 127.10, 127.48, 132.28, 133.94, 134.12, 134.97, 135.70, 138.15, 138.26,
+
43.33, 151.32, 155.82, 162.95, 181.69, 183.50. HRMS (EI) m/z: calcd [M] ,
+
92.0089 (C H FN O S ); found, 392.0087.
2
0
9
2
2 2
.2.9. 4-(4-Chlorophenylthio)anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4i)
The pure compound was obtained as a brown powder (yield 63ꢁ). Mp: 260-261 ℃
-1
(
EtOH). FT-IR (KBr, νmax cm ): 1506.3 (aromatic C=C), 1519.4 (CO), 1582.2 (CO),
1
1
664.4 (C=N). H NMR (300 MHz, CDCl ): δ ppm 7.56 (d, J= 8.4 Hz, 2H), 7.65 (d,
3
J= 8.7 Hz, 2H), 7.76 (s, 1H), 7.78-7.85 (m, 2H), 8.19 (dd, J= 7.2 Hz, J= 1.2 Hz, 1H),
1
3
8
1
1
4
5
.35 (dd, J= 7.8 Hz, J= 1.5 Hz, 1H). C NMR(75 MHz, CDCl ): δ ppm 120.20,
3
21.76, 126.95, 127.13, 127.50, 130.97, 132.30, 133.97, 134.15, 135.00, 135.73,
+
37.02, 137.50, 142.54, 151.35, 155.22, 181.68, 183.48. HRMS (EI) m/z calcd [M] ,
+
07.9794 (C H ClN O S ); found, 407.9791.
2
0
9
2
2 2
.2.10. 4-(4-Bromophenylthio)anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4j)
The pure compound obtained as an orange powder (yield 68ꢁ). Mp: 246-247 ℃
-1
(
EtOH). FT-IR (KBr, νmax cm ): 1506.3 (aromatic C=C), 1522.4 (CO), 1591.2 (CO),
1
1
2
668.3 (C=N). H NMR (300 MHz, CDCl ): δ ppm 7.13 (dd, J= 6.6 Hz, J= 1.8 Hz,
3
H), 7.51 (dd, J= 6.6 Hz, J= 1.8 Hz, 2H), 7.75 (s, 1H), 7.77-7.86 (m, 2H), 8.19 (dd,
1
3
J= 7.2 Hz, J= 1.2 Hz, 1H), 8.34 (dd, J= 7.8 Hz, J= 1.5 Hz, 1H). C NMR (75 MHz,
CDCl ): δ ppm 120.27, 121.78, 125.64, 127.14, 127.50, 127.60, 127.83, 132.29,
3
1
33.95, 134.17, 135.00, 135.72, 137.16, 142.32, 151.33, 155.22, 181.69, 183.48.
+
+
HRMS (EI) m/z: calcd [M] , 451.9289 (C H BrN O S ); found, 451.9296.
2
0
9
2
2 2
5
.2.11. 4-(o-Tolylthio)anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4k)
The pure compound was obtained as a dark red powder (yield 78ꢁ). Mp: 251-252 ℃
-1
(
EtOH). FT-IR (KBr, νmax cm ): 1502.4 (aromatic C=C), 1541.0 (CO), 1593.1 (CO),
1
1
7
670.2(C=N). H NMR (300 MHz, CDCl ): δ ppm 2.47 (s, 3H), 7.37-7.42 (m, 1H),
3
.50 (s, 1H), 7.52-7.55 (m, 2H), 7.69 (d, J= 7.8 Hz, 1H), 7.76 (td, J= 7.2 Hz, J= 1.2
1
7

ACCEPTED MANUSCRIPT
Hz, 1H), 7,83 (td, J= 7.5 Hz, J= 1.5 Hz, 1H), 8.15 (dd, J= 7.5 Hz, J= 1.5 Hz, 1H),
1
3
8
1
1
.35 (dd, J= 7.5 Hz, J= 1.5 Hz, 1H). C NMR (75 MHz, CDCl ): δ ppm 20.62,
3
19.17, 121.22, 127.03, 127.25, 127.43, 128.11, 131.54, 131.99, 132.32, 134.01,
34.48, 134.88, 135.79, 137.22, 142.92, 143.51, 151.43, 155.29, 181.71, 183.56.
+
+
2 2
HRMS (EI) m/z: calcd [M] , 388.0340 (C H N O S ); found, 388.0343.
2
1
12
2
5
.2.12. 4-(m-Tolylthio)anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4l)
The pure compound was obtained as an orange red powder (yield 76ꢁ). Mp: 261-262
-1
℃
(EtOH). FT-IR (KBr, νmax cm ): 1508.2 (aromatic C=C), 1541.0 (CO), 1593.1
1
(
CO), 1670.2 (C=N). H NMR (300 MHz, CDCl ): δ ppm 2.46 (s, 3H), 7.42 (t, J= 8.4
3
Hz, 1H), 7.47-7.53 (m, 3H), 7.73 (s, 1H), 7.76 (td, J= 7.5 Hz, J= 1.5 Hz, 1H), 7.83 (td,
J= 7.5 Hz, J= 1.5 Hz, 1H), 8.15 (dd, J = 7.5 Hz, J = 1.5 Hz, 1H), 8.33 (dd, J= 7.5 Hz,
1
3
J= 1.5 Hz, 1H). C NMR (75 MHz, CDCl ): δ ppm 21.38, 119.93, 121.29, 127.02,
3
1
1
3
5
27.43, 127.90, 130.44, 131.71, 132.32, 132.88, 134.00, 134.87, 135.75, 136.32,
+
40.75, 143.86, 151.31, 155.18, 181.70, 183.58. HRMS (EI) m/z: calcd [M] ,
+
88.0340 (C H N O S ); found, 388.0344.
2
1
12
2
2 2
.2.13. 4-(p-Tolylthio)anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4m)
The pure compound was obtained as a light orange powder (yield 80ꢁ). Mp: 294-295
-1
℃
(EtOH). FT-IR (KBr, νmax cm ): 1502.4 (aromatic C=C), 1591.2 (CO), 1666.4
1
(
C=N). H NMR (300 MHz, CDCl ): δ ppm 2.50 (s, 3H), 7.40 (d, J= 7.8 Hz, 2H),
3
7
1
1
1
1
.73 (d, J= 8.1 Hz, 2H), 7.71 (s, 1H), 7.76 (td, J= 1.5 Hz, J= 7.2 Hz, 1H), 7.83 (td, J=
.5 Hz, J= 7.5 Hz, 1H), 8.16 (dd, J= 7.2 Hz, J= 1.2 Hz, 1H), 8.35 (dd, J= 7.2 Hz, J=
1
3
.2 Hz, 1H). C NMR (75 MHz, CDCl ): δ ppm 21.52, 119.67, 121.26, 124.55,
3
27.03, 127.45, 131.50, 132.35, 134.01, 134.89, 135.78, 135.88, 141.38, 144.25,
+
51.35, 155.18, 181.73, 183.68. HRMS (EI) m/z: calcd [M] , 388.0340
+
(
C H N O S ); found, 388.0341.
2
1
12
2
2 2
5
.2.14. 4-(2-Isopropylphenylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4n)
1
8

ACCEPTED MANUSCRIPT
The pure compound was obtained as a light brown powder (yield 68ꢁ). Mp: 266-267
-1
1
℃
(EtOH). FT-IR (KBr, νmax cm ): 1506.3 (aromatic C=C), 1672.2 (C=N). H NMR
(
300 MHz, CDCl ): δ ppm 1.23 (d, J = 6.9 Hz, 6H), 3.47-3.52 (m, 1H), 7.35-7.41 (m,
3
1
1
1
1
1
4
5
H), 7.54 (s, 1H), 7.58-7.62 (m, 2H), 7.67 (d, J = 7.2 Hz, 1H), 7.75 (td, J= 7.5 Hz, J=
.5 Hz, 1H), 7.82 (td, J= 7.5 Hz, J= 1.2 Hz, 1H), 8.13-8.16 (m, 1H), 8.32-8.35 (m,
1
3
H). C NMR (75 MHz, CDCl ): δ ppm 23.99, 31.48, 119.52, 121.15, 126.05, 127.02,
3
27.41, 127.69, 127.95, 131.98, 132.30, 133.98, 134.86, 134.87, 135.73, 137.41,
+
44.01, 151.36, 153.90, 155.14, 181.70, 183.53. HRMS (EI) m/z: calcd [M] ,
+
16.0653 (C H N O S ); found, 416.0651.
2
3
16
2
2 2
.2.15. 4-(4-Isopropylphenylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4o)
The pure compound was obtained as a brown powder (yield 57ꢁ). Mp: 232-233 ℃
-1
(
EtOH). FT-IR (KBr, νmax cm ): 1508.2 (aromatic C=C), 1539.1 (CO), 1593.1 (CO),
1
1
666.4 (C=N). H NMR(300 MHz, CDCl
3
): δ ppm 1.36 (d, J= 7.2 Hz, 6H), 3.03-3.07
(m, 1H), 7.45 (d, J= 7.8 Hz, 2H), 7.63 (d, J= 7.8 Hz, 2H), 7.74 (s, 1H), 7.77 (d, J= 7.5
Hz, 1H), 7.81 (d, J= 7.2 Hz, 1H), 8.15 (d, J= 7.5 Hz , 1H), 8.33 (d, J= 7.8 Hz, 1H).
1
3
C NMR (75 MHz, CDCl
3
): δ ppm 23.86, 34.21, 119.73, 121.27, 124.84, 127.04,
1
1
27.45, 128.87, 132.37, 134.00, 134.87, 135.81, 135.93, 144.17, 151.35, 152.20,
+
+
16 2 2 2
55.21, 181.75, 183.65. HRMS (EI) m/z: calcd [M] , 416.0653 (C23H N O S );
found, 416.0657.
.2.16. 4-(2-Ethylphenylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4p)
The pure compound was obtained as a brown powder (yield 57ꢁ). Mp: 232-233℃
5
-1
(
EtOH). FT-IR (KBr, νmax cm ): 1508.2 (aromatic C=C), 1542.9 (CO), 1593.1 (CO),
1
1
668.3 (C=N). H NMR (300 MHz, CDCl ): δ ppm 1.21 (t, J = 7.5 Hz, 3H), 2.84 (q,
3
J= 12.9 Hz, J= 7.5 Hz, 2H), 7.39 (td, J= 1.8 Hz, J= 7.4 Hz, 1H), 7.51 (s, 1H), 7.54 (d,
J= 5.1 Hz, 1H), 7.58 (td, J= 7.8 Hz, J= 1.5 Hz, 1H), 7.67 (dd, J= 7.8 Hz, J= 1.2 Hz,
1
H), 7.75 (td, J= 7.5 Hz, J= 1.8 Hz, 1H), 7.81 (td, J= 7.5 Hz, J= 1.2 Hz, 1H), 8.13 (dd,
1
9

ACCEPTED MANUSCRIPT
1
3
J= 7.5 Hz, J= 1.2 Hz, 1H), 8.32 (dd, J= 7.5 Hz, J= 1.2 Hz, 1H). C NMR (75 MHz,
CDCl ): δ ppm 15.54, 27.47, 119.28, 121.07, 126.44, 126.99, 127.40, 128.12, 130.49,
3
1
31.78, 132.20, 133.87, 134.02, 134.89, 135.68, 137.49, 143.74, 149.35, 151.29,
+
+
1
55.12, 181.73, 183.55. HRMS (EI) m/z: calcd [M] , 402.0497 (C H N O S );
22 14 2 2 2
found, 402.0499.
5
.2.17. 4-(4-Ethylphenylthio)anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4q)
The pure compound was obtained as an orange powder (yield 80ꢁ). Mp: 232-233 ℃
-1
(
EtOH). FT-IR (KBr, νmax cm ): 1502.4 (aromatic C=C), 1591.2 (CO), 1668.3 (C=N).
1
H NMR (300 MHz, CDCl ): δ ppm 1.35 (t, J= 7.5 Hz, 3H), 2.80 (q, J= 7.5 Hz, 2H),
3
7
7
.42 (d, J= 8.1 Hz, 2H), 7.62 (d, J= 8.1 Hz, 2H), 7.71 (s, 1H), 7.80 (t, J= 7.8 Hz, 1H),
1
3
.79-7.84 (m, 1H), 8.15 (dd, J= 7.5 Hz, J= 1.2 Hz, 1H), 8.33 (d, J= 7.5 Hz, 1H). C
NMR (75 MHz, CDCl ): 15.20, 28.85, 119.56, 121.32, 124.55, 127.01, 127.43,
3
1
30.27, 132.25, 133.91, 134.02, 134.89, 135.71, 135.95, 144.22, 147.58, 151.27,
+
+
1
55.07, 181.74, 183.47. HRMS (EI) m/z: calcd [M] , 402.0497 (C H N O S );
22 14 2 2 2
found, 402.0500.
5
.2.18. 4-(4-Tert-butylphenylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (4r)
The pure compound was obtained as a yellow powder (yield 77ꢁ). Mp: 298-299℃
-1
1
(
EtOH). FT-IR (KBr, νmax cm ): 1508.2 (aromatic C=C), 1670.2 (C=N). H NMR
(
300 MHz, CDCl ): δ ppm 1.43 (s, 9H), 7.58-7.65 (m, 4H), 7.75 (s, 1H), 7.76 (td, J=
3
7
1
3
1
.8 Hz, J= 1.5 Hz, 1H), 7.83 (td, J= 7.8 Hz, J= 1.5 Hz, 1H), 8.15 (dd, J= 7.5 Hz, J=
1
3
.2 Hz, 1H), 8.32 (dd, J= 7.8 Hz, J= 1.2 Hz, 1H). C NMR (75 MHz, CDCl ): δ ppm
3
1.34, 35.13, 119.74, 121.25, 124.60, 127.01, 127.42, 127.73, 127.74, 132.33, 133.98,
34.84, 135.59, 135.79, 144.04, 151.32, 154.52, 160.69, 181.71, 183.61. HRMS (EI)
+
+
m/z: calcd [M] , 430.0810 (C H N O S ); found, 430.0813.
2
4
18
2
2 2
5
.2.19. 4-(2,5-Dimethylphenylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (5a)
The pure compound was obtained as a dark brown powder (yield 47ꢁ). Mp: 243-244
2
0

ACCEPTED MANUSCRIPT
-1
℃
(EtOH). FT-IR (KBr, νmax cm ): 1500.5 (aromatic C=C), 1522.8 (CO), 1591.2
1
(
CO), 1674.2 (C=N). H NMR (300 MHz, CDCl ): δ ppm 2.40 (6H, s, -CH ), 7.33 (d,
3
3
J= 7.8 Hz, 1H), 7.39 (d, J= 7.5 Hz, 1H), 7.51-7.53 (m, 2H), 7.76 (t, J= 7.5 Hz, 1H),
1
3
7
.83 (t, J= 7.8 Hz, 1H), 8.15 (d, J= 7.5 Hz, 1H), 8.34 (d, J= 7.5 Hz, 1H). C NMR
(
75 MHz, CDCl ): δ ppm 20.08, 20.82, 119.24, 121.23, 125.36, 126.79, 127.02,
3
1
1
27.45, 131.79, 132.41, 134.00, 134.01, 134.87, 135.84, 137.54, 137.93, 140.25,
+
43.17, 151.45, 155.30, 181.74, 183.68. HRMS (EI) m/z: calcd [M] , 402.0497
+
(
C H N O S ); found, 402.0506.
2
2
14
2
2 2
5
.2.20. 4-(2,4-Dimethylphenylthio)
anthra[1,2-c][1,2,5]thiadiazole-6,11-dione
(5b)
The pure compound was obtained as an orange powder (yield 39ꢁ). Mp: 250-251 ℃
-1
(
EtOH). FT-IR (KBr, νmax cm ): 1502.4 (aromatic C=C), 1522.4 (CO), 1591.2 (CO),
1
1
668.3 (C=N). H NMR (300 MHz, CDCl ): δ ppm 2.42 (s, 3H), 2.46 (s, 3H), 7.20 (d,
3
J= 7.8 Hz, 1H), 7.32 (s, 1H), 7.52 (s, 1H), 7.56 (d, J= 8.1 Hz, 1H), 7.76 (td, J= 7.5 Hz,
J= 1.5 Hz, 1H), 7.83 (td, J= 7.2 Hz, J= 1.5 Hz, 1H), 8.16 (dd, J= 7.8 Hz, J= 1.2 Hz,
1
3
1
2
1
H), 8.35 (dd, J= 7.5 Hz, J= 1.2 Hz, 1H). C NMR(75 MHz, CDCl ): δ ppm 20.51,
3
1.43, 119.03, 121.12, 123.55, 126.98, 127.42, 128.96, 132.32, 132.84, 133.97,
34.87, 135.78, 137.12, 141.96, 143.26, 143.53, 151.45, 155.31, 181.75, 183.76.
+
+
HRMS (EI) m/z: calcd [M] , 402.0497 (C H N O S ); found, 402.0497.
2
2
14
2
2 2
5
.2.21. 4-(3,5-Dimethylphenylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (5c)
The pure compound was obtained as an orange red powder (yield 41ꢁ). Mp: 283-284
-1
℃
(EtOH). FT-IR (KBr, νmax cm ): 1502.4(aromatic C=C), 1539.1(CO), 1595.0(CO),
1
1
2
1
672.2 (C=N). H NMR (300 MHz, CDCl ): δ ppm 2.41 (s, 6H), 7.22 (s, 1H), 7.32 (s,
3
H), 7.73 (s, 1H), 7.78 (td, J= 7.5 Hz, J= 1.2 Hz, 1H), 7.85 (td, J= 7.5 Hz, J= 1.5 Hz,
1
3
H), 8.16 (dd, J= 7.5 Hz, J= 1.5 Hz, 1H), 8.35 (dd, J= 7.8 Hz, J= 1.5 Hz, 1H). C
NMR(75 MHz, CDCl ): δ ppm 21.29, 119.87, 121.13, 127.00, 127.42, 132.29, 132.72,
3
2
1

ACCEPTED MANUSCRIPT
1
1
5
33.35, 133.94, 134.00, 134.87, 135.75, 140.47, 144.53, 151.27, 155.13, 181.73,
+
+
83.66. HRMS (EI) m/z: calcd [M] , 402.0497 (C H N O S ); found, 402.0491.
22
14
2
2 2
.2.22. 4-(2,6-Dimethylphenylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (5d)
The pure compound was obtained as a dark brown powder (yield 35ꢁ). Mp: 213-214
-1
1
℃
(EtOH). FT-IR(KBr, νmax cm ): 1508.2 (aromatic C=C), 1668.3 (C=N). H NMR
(
300 MHz, CDCl ): δ ppm 2.48 (s, 6H), 7.30 (d, J= 7.8 Hz, 2H), 7.40 (s, 1H),
3
7
1
.41-7.46 (m, 1H), 7.76 (td, J= 7.5 Hz, J= 1.5 Hz, 1H), 7.82 (td, J= 7.5 Hz, J= 1.5 Hz,
1
3
H), 8.12 (dd, J= 7.8 Hz, J= 1.5 Hz, 1H), 8.34 (dd, J= 7.5 Hz, J= 1.2 Hz, 1H). C
NMR(75 MHz, CDCl ): δ ppm 21.54, 29.77, 118.29, 121.22, 126.65, 127.01, 127.43,
3
1
1
28.30, 129.38, 129.54, 131.22, 132.35, 134.00, 135.88, 142.34, 143.75, 144.31,
+
51.54, 158.87, 181.73, 183.66. HRMS (EI) m/z: calcd [M] , 402.0497
+
(
C H N O S ); found, 402.0496.
2
2
14
2
2 2
5
.2.23. 4-(3,4-Dimethylphenylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (5e)
The pure compound was obtained as an orange powder (yield 67ꢁ). Mp: 221-222 ℃
-1
(
EtOH). FT-IR (KBr, νmax cm ): 1506.3 (aromatic C=C), 1541.0 (CO), 1668.3 (C=N).
1
H NMR(300 MHz, CDCl ): δ ppm 2.35 (s, 3H), 2.40 (s, 3H), 7.33 (d, J= 7.8 Hz, 1H),
3
7
.42-7.46 (m, 2H), 7.73 (s, 1H), 7.76 (dd, J= 7.5 Hz, J= 1.5 Hz, 1H), 7.83 (dd, J= 7.5
Hz, J= 1.5 Hz, 1H), 8.15 (dd, J= 7.2 Hz, J= 1.5 Hz, 1H), 8.35 (dd, J= 7.5 Hz, J= 1.5
1
3
Hz, 1H). C NMR(75 MHz, CDCl ): δ ppm 19.85, 19.88, 119.22, 120.68, 123.84,
3
1
1
4
5
26.76, 127.21, 131.69, 131.91, 133.20, 133.56, 133.84, 134.71, 135.41, 136.54,
+
39.16, 139.90, 144.27, 150.93, 154.67, 181.52, 183.44. HRMS (EI) m/z: calcd [M] ,
+
02.0497 (C H N O S ); found, 402.0489.
2
2
14
2
2 2
.2.24. 4-(2,4-Difluorophenylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (5f)
The pure compound was obtained as a brown powder (yield 78ꢁ). Mp: 217-218 ℃
-1
(
EtOH). FT-IR (KBr, νmax cm ): 1510.2 (aromatic C=C), 1589.2 (CO), 1666.4 (C=N).
1
H NMR (400 MHz, CDCl ): δ ppm 7.09-7.15 (m, 2H), 7.65 (s, 1H), 7.71-7.74 (m,
3
2
2

ACCEPTED MANUSCRIPT
1
H), 7.75-7.79 (m, 1H), 7.81-7.85 (m, 1H), 8.16 (d, J= 7.6 Hz, 1H), 8.33 (d, J= 7.6
1
3
Hz, 1H). C NMR (75 MHz, CDCl ): δ ppm 105.63, 105.89, 106.15, 110.39, 110.43,
3
110.58 113.40, 113.44, 113.62, 113.66, 119.20, 119.262, 121.38, 126.82, 127.22,
1
1
1
5
27.29, 131.77, 133.42, 133.95, 134.79, 134.83, 135.25, 138.69, 138.79, 140.42,
50.89, 154.62, 162.23, 163.81, 163.92, 164.75, 164.88, 166.35, 166.47, 181.36,
+
+
83.40. HRMS (EI) m/z: calcd [M] , 409.9995 (C H F N O S ); found, 409.9992.
20
8
2
2
2 2
.2.25. 4-(2,5-Dichlorophenylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (5g)
The pure compound was obtained as a yellow powder (yield 77ꢁ). M.p: 248-249 ℃
-1
(
EtOH). FT-IR (KBr, νmax cm ): 1508.2 (aromatic C=C), 1539.1 (CO), 1670.2 (C=N).
1
H NMR (300 MHz, CDCl ): δ ppm 7.52 (dd, J= 8.4 Hz, J= 2.7 Hz, 1H), 7.62 (d, J=
3
7
7
7
1
1
4
5
.2 Hz, 1H), 7.72 (s, 1H), 7.77 (d, J= 2.7 Hz, 1H), 7.81 (td, J= 7.5 Hz, J=1.5 Hz, 1H),
.85 (td, J= 7.5 Hz, J= 1.8 Hz, 1H), 8.20 (dd, J= 7.2 Hz, J= 1.8 Hz, 1H), 8.36 (dd, J=
1
3
.5 Hz, J= 1.5 Hz, 1H). C NMR (75 MHz, CDCl ): δ ppm 121.16, 122.33, 127.16,
3
27.52, 129.91, 132.18, 132.27, 133.92, 134.10, 134.19, 135.02, 135.66, 136.94,
+
38.02, 139.12, 151.40, 155.35, 181.66, 183.31. HRMS (EI) m/z: calcd [M] ,
+
41.9404 (C H Cl N O S ); found, 441.9399.
2
0
8
2
2
2 2
.2.26. 4-(2,4,5-Trichlorophenylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione
5h)
The pure compound was obtained as a yellow powder (yield 75ꢁ). Mp: 261-262 ℃
(
-1
(
EtOH). FT-IR (KBr, νmax cm ): 1506.3 (aromatic C=C), 1542.9 (CO), 1593.1 (CO),
1
1
670.2 (C=N). H NMR (300 MHz, CDCl ): δ ppm 7.76 (s, 1H), 7.79 (s, 1H), 7.81 (d,
3
J= 7.5 Hz, 1H), 7.83 (t, J= 1.5 Hz, 1H), 7.86 (s, 1H), 8.20-8.23 (m, 1H), 8.35-8.38 (m,
1
3
1
1
1
H). C NMR (75 MHz, CDCl ): δ ppm 120.61, 122.02, 126.95, 127.31, 127.44,
3
31.81, 132.20, 132.48, 133.46, 134.10, 134.91, 135.27, 136.20, 137.87, 138.18,
+
38.35, 151.00, 154.81, 181.43, 183.02. HRMS (EI) m/z: calcd [M] , 475.9015
+
(
C H Cl N O S ); found, 475.9013.
2
0
7
3
2
2 2
2
3

ACCEPTED MANUSCRIPT
5
.2.27. 4-(2-Methoxyphenylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (6a)
The pure compound was obtained as a yellow powder (yield 83ꢁ). Mp: 227-228 ℃
-1
1
(
EtOH). FT-IR(KBr, νmax cm ): 1506.3 (aromatic C=C), 1670.2 (C=N). H NMR (300
MHz, CDCl ): δ ppm 3.85 (s, 3H), 7.15-4.17 (m, 2H), 7.60-7.63 (m, 2H), 7.62 (s, 1H),
3
7
7
5
1
1
5
.76 (td, J= 7.5 Hz, J= 1.5 Hz, 1H), 7.80 (td, J= 7.5 Hz, J= 1.5 Hz, 1H), 8.14 (dd, J=
1
3
.2 Hz, J= 1.2 Hz, 1H), 8.32 (d, J= 7.2 Hz, 1H). C NMR (75 MHz, CDCl ): δ ppm
3
6.25, 112.61, 115.84, 119.61, 121.15, 122.30, 126.97, 127.42, 132.38, 133.14,
33.91, 134.06, 134.82, 135.72, 137.69, 142.52, 151.39, 155.35, 160.68, 181.71,
+
+
83.76. HRMS (EI) m/z: calcd [M] , 404.0289 (C H N O S ); found, 404.0295.
21
12
2
3 2
.2.28. 4-(3-Methoxyphenylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (6b)
The pure compound was obtained as a yellowish powder (yield 81ꢁ). Mp: 237-238
-1
℃
(EtOH). FT-IR(KBr, νmax cm ): 1506.3 (aromatic C=C), 1544.9 (CO), 1589.2
1
(
CO), 1668.3 (C=N). H NMR (300 MHz, CDCl ): δ ppm 3.87 (s, 3H), 7.14 (dd, J=
3
8
.4 Hz, J= 2.7 Hz, 1H), 7.25 (d, J= 7.8 Hz, 1H), 7.30 (d, J= 7.8 Hz, 1H), 7.51 (t, J=
.8 Hz, 1H), 7.74-7.86 (m, 3H), 8.15 (dd, J= 7.5 Hz, J= 1.5 Hz, 1H), 8.35 (dd, J= 7.5
7
1
3
Hz, J= 1.5 Hz, 1H). C NMR (75 MHz, CDCl ): δ ppm 55.71, 116.94, 120.14,
3
1
1
4
5
20.90, 121.47, 127.08, 127.47, 127.91, 129.25, 131.43, 132.37, 134.04, 134.91,
+
35.81, 143.40, 151.35, 155.22, 161.31, 181.72, 183.56. HRMS (EI) m/z: calcd [M] ,
+
04.0289 (C H N O S ); found, 404.0281.
2
1
12
2
3 2
.2.29. 4-(4-Methoxyphenylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (6c)
The pure compound was obtained as an orange powder (yield 85ꢁ). Mp: 293-294 ℃
-1
(
EtOH). FT-IR(KBr, νmax cm ): 1506.3 (aromatic C=C), 1593.1 (CO), 1664.5 (C=N).
1
H NMR (300 MHz, CDCl ): δ ppm 3.94 (s, 3H), 7.11 (d, J= 8.4 Hz, 2H), 7.63 (d, J=
3
8
.7 Hz, 2H), 7.69 (s, 1H), 7.77-7.83 (m, 2H), 8.17 (d, J= 7.2 Hz, 1H), 8.35 (d, J= 7.5
1
3
Hz, 1H). C NMR (75 MHz, CDCl ): δ ppm 55.50, 116.07, 117.66, 119.30, 126.80,
3
1
27.21, 129.35, 131.94, 133.59, 133.84, 134.71, 135.41, 137.47, 144.60, 150.98,
2
4

ACCEPTED MANUSCRIPT
+
1
54.62, 161.65, 181.53, 183.41. HRMS (EI) m/z: calcd [M] , 404.0289
+
(
C H N O S ); found, 404.0283.
2
1
12
2
3 2
5
.2.30. 4-(3-Ethoxyphenylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (6d)
The pure compound was obtained as an orange powder (yield 84ꢁ). Mp: 249-250 ℃
-1
(
EtOH). FT-IR(KBr, νmax cm ): 1506.3 (aromatic C=C), 1589.2 (CO), 1670.2 (C=N).
1
H NMR (300 MHz, CDCl ): δ ppm 1.44 (t, J= 6.9 Hz, 3H), 4.09 (q, J= 6.9 Hz, 2H),
3
7
7
8
.10-7.14 (m, 1H), 7.23 (t, J= 2.4 Hz, 1H), 7.27-7.30 (m, 1H), 7.49 (t, J= 8.0 Hz, 1H),
.77 (dd, J= 7.5 Hz, J= 1.5 Hz, 1H), 7.79 (s, 1H), 7.83 (dd, J= 7.5 Hz, J= 1.5 Hz, 1H),
1
3
.17 (dd, J= 7.5 Hz, J= 1.5 Hz, 1H), 8.35 (dd, J= 7.8 Hz, J= 1.2 Hz, 1H). C NMR
(
75 MHz, CDCl ): δ ppm 14.75, 64.11, 117.54, 120.12, 121.47, 127.08, 127.65,
3
1
1
27.75, 129.12, 131.40, 132.37, 134.04, 134.90, 135.83, 136.80, 143.53, 151.35,
+
55.22, 160.66, 181.74, 183.58. HRMS (EI) m/z: calcd [M] , 418.0446
+
(
C H N O S ); found, 418.0445.
2
2
14
2
3 2
5
.2.31. 4-(4-(Methylthio)phenylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (6e)
The pure compound was obtained as a dark brown powder (yield 70ꢁ). Mp: 214-215
-1
℃
(EtOH). FT-IR (KBr, νmax cm ): 1508.2 (aromatic C=C), 1558.4 (CO), 1668.3
1
(
C=N). H NMR (300 MHz, CDCl ): δ ppm 2.58 (s, 3H), 7.41 (d, J= 8.4 Hz, 2H),
3
7
.60 (d, J= 8.4 Hz, 2H), 7.76 (s, 1H), 7.80 (td, J= 7.5 Hz, J= 1.5 Hz, 1H), 7.83 (td, J=
.5 Hz, J= 1.5 Hz, 1H), 8.17 (dd, J= 7.5 Hz, J= 1.5 Hz, 1H), 8.35 (dd, J= 7.5 Hz, J=
7
13
1
.5 Hz, 1H). C NMR(75 MHz, CDCl ): 15.27, 119.69, 123.43, 127.07, 127.47,
3
1
27.75, 132.27, 133.92, 134.10, 134.95, 135.72, 136.13, 143.21, 143.77, 151.31,
+
+
1
53.79, 181.76, 183.63. HRMS (EI) m/z: calcd [M] , 420.0061 (C H N O S );
21 12 2 2 3
found, 420.0069.
5
.2.32. 4-(Propylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (6f)
The pure compound was obtained as a dark brown powder (yield 85ꢁ). Mp: 194-195
-1
℃
(EtOH). FT-IR (KBr, νmax cm ): 1508.2 (aromatic C=C), 1591.2 (CO), 1668.3
2
5

ACCEPTED MANUSCRIPT
1
(
C=N). H NMR (300 MHz, CDCl ): δ ppm 1.20 (t, J= 7.2 Hz, 3H), 1.94 (s, J= 7.2 Hz,
3
2
H), 3.30 (t, J= 7.2 Hz, 2H), 7.80-7.88 (m, 2H), 8.14 (s, 1H), 8.28 (dd, J= 7.2 Hz, J
1
3
=
1.2 Hz, 1H), 8.36 (dd, J= 7.2 Hz, J= 1.2 Hz, 1H). C NMR(75 MHz, CDCl ): δ ppm
3
1
1
3
5
3.63, 21.78, 33.56, 118.47, 121.02, 127.16, 127.49, 127.60, 132.37, 134.01, 134.98,
+
35.64, 143.04, 151.22, 155.94, 181.73, 183.96. HRMS (EI) m/z: calcd [M] ,
+
40.0340 (C H N O S ); found, 340.0338.
1
7
12
2
2 2
.2.33. 4-(Isopropylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (6g)
The pure compound was obtained as a yellow powder (yield 88ꢁ). Mp: 193-194 ℃
-1
(
EtOH). FT-IR(KBr, νmax cm ): 1502.4 (aromatic C=C), 1539.1 (CO), 1591.2 (CO),
1
1
668.3 (C=N). H NMR (300 MHz, CDCl ): δ ppm 1.58 (d, J= 6.6 Hz, 6H), 4.05 (m,
3
1
3
1
H), 7.81-7.89 (m, 2H), 8.23 (s, 1H), 8.27-8.30 (m, 1H), 8.36-8.39 (m, 1H). C NMR
(
75 MHz, CDCl ): δ ppm 22.81, 36.08, 119.67, 121.17, 127.17, 127.51, 127.82,
3
1
32.40, 134.03, 134.98, 135.64, 142.33, 151.36, 156.24, 181.77, 183.95. HRMS (EI)
+
+
m/z: calcd [M] , 340.0340 (C H N O S ); found, 340.0345.
1
7
12
2
2 2
5
.2.34. 4-(Benzylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (7a)
The pure compound was obtained as a pale brown powder (yield 88ꢁ). Mp: 243-244
-1
ꢀ
(EtOH). FT-IR (KBr, νmax cm ): 1498.6 (aromatic C=C), 1585.4 (C=O), 1666.4
1
(
C=N). H NMR (400 MHz, CDCl ): δ ppm 4.56 (s, 2H), 7.31 (t, J= 7.2 Hz, 1H), 7.38
3
(t, J= 7.2 Hz, 2H), 7.55 (d, J= 7.2 Hz, 2H), 7.81 (td, J= 7.2 Hz, J= 1.6 Hz, 1H), 7.86
(dd, J= 7.2 Hz, J= 1.6 Hz, 1H), 8.26-8.28 (m, 2H), 8.36 (dd, J= 7.2 Hz, J= 1.6 Hz,
1
3
1
1
1
H). C NMR (100 MHz, CDCl ): δ ppm 36.24, 118.76, 120.01, 120.90, 126.94,
3
27.25, 128.02, 128.93, 129.07, 131.90, 133.57, 133.88, 134.67, 134.81, 135.24,
41.72, 150.79, 181.53, 183.48. HRMS (EI) m/z: calcd [M]+, 388.0340
+
(
C H N O S ); found, 388.0340.
2
1
12
2
2 2
5
.2.35. 4-(2-Chlorobenzylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (7b)
The pure compound was obtained as a dark yellowish orange powder (yield 81ꢁ).
2
6

ACCEPTED MANUSCRIPT
-1
Mp: 254-255 ℃ (EtOH). FT-IR (KBr, νmax cm ): 1508.2 (aromatic C=C), 1542.9
1
(
CO), 1674.1 (C=N). H NMR (300 MHz, CDCl ): δ ppm 4.67 (s, 2H), 7.25-7.28 (m,
3
2
H), 7.44-7.47 (m, 1H), 7.56-7.59 (m, 1H), 7.80-7.86 (m, 2H), 8.26 (dd, J= 7.35 Hz,
1
3
J= 1.5 Hz, 1H), 8.27 (s, 1H), 8.35 (dd, J= 7.35 Hz, J= 1.5 Hz, 1H). C NMR (75
MHz, CDCl ): 33.92, 118.59, 121.00, 126.92, 127.21, 127.26, 129.52, 130.05, 130.90,
3
1
1
4
5
31.85, 132.49, 133.52, 133.86, 134.55, 134.77, 135.23, 141.42, 150.75, 155.28,
+
+
81.741, 183.34. HRMS (EI) m/z: calcd [M] , 421.9950 (C H ClN O S ); found,
2
1
11
2
2 2
21.9954.
.2.36. 4-(4-Chlorobenzylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dion (7c)
The pure compound was obtained as a light yellow powder (yield 82ꢁ). Mp: 261-262
-1
1
℃
(EtOH). FT-IR (KBr, νmax cm ): 1510.2 (aromatic C=C), 1670.2 (C=N). H NMR
(
400 MHz, CDCl ): δ ppm 4.52 (s, 2H), 7.34 (d, J= 8.0 Hz , 2H), 7.48 (d, J= 7.6 Hz,
3
2
H), 7.80-7.88 (m, 2H), 8.24 (s, 1H), 8.27 (d, J= 7.6 Hz, 1H), 8.36 (d, J= 7.2 Hz, 1H).
1
3
C NMR (100 MHz, CDCl ): δ ppm 35.53, 119.00, 126.97, 127.28, 129.12, 130.36,
3
1
1
5
31.88, 133.35, 133.58, 133.93, 134.85, 135.19, 140.99, 150.82, 155.33, 181.48,
+
+
83.40. HRMS (EI) m/z: calcd [M] , 421.9950 (C H ClN O S ); found, 421.9942.
21
11
2
2 2
.2.37. 4-(4-Tert-butylbenzylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dion (7d)
The pure compound was obtained as an orange yellow powder (yield 62ꢁ). Mp:
-
1
2
1
56-257 ℃ (EtOH). FT-IR(KBr, νmax cm ): 1506.3 (aromatic C=C), 1544.9 (CO),
1
670.2 (C=N). H NMR (300 MHz, CDCl ): δ ppm 1.31 (s, 9H), 4.53 (s, 2H), 7.39 (d,
3
J= 8.4 Hz, 2H), 7.35 (d, J= 8.4 Hz, 2H), 7.83 (td, J= 1.8 Hz, J= 7.5 Hz, 2H),
1
3
8
3
1
.27-8.30 (m, 2H), 8.36 (d, J= 7.2 Hz, 1H). C NMR(75 MHz, CDCl ): δ ppm 31.29,
3
4.60, 35.89, 35.89, 118.61, 120.84, 125.89, 126.96, 127.27, 128.83, 131.52, 131.94,
33.62, 133.88, 134.81, 135.28, 142.09, 151.09, 181.56, 183.55. HRMS (EI) m/z;
+
+
calcd [M] , 444.0966 (C H N O S ); found, 444.0970.
25
20
2
2 2
5
.2.38. 4-(Phenethylthio) anthra[1,2-c][1,2,5]thiadiazole-6,11-dione (7e)
2
7

ACCEPTED MANUSCRIPT
The pure compound was obtained as a yellow powder (yield 62ꢁ). Mp: 119-120 ℃
-1
(
EtOH). FT-IR (KBr, νmax cm ): 1506.3 (aromatic C=C), 1542.9 (C=O), 1668.3
1
(
C=N). H NMR (300 MHz, CDCl ): δ ppm 3.19 (t, J= 7.5 Hz, 2H), 3.58 (t, J= 7.5 Hz,
3
2
H), 7.23-7.30 (m, 1H), 7.33-7.37 (m, 4H), 7.79-7.88 (m, 2H), 8.22 (s, 1H), 8.29 (dd,
1
3
J= 7.8 Hz, J= 1.5 Hz, 1H), 8.37 (dd, J= 7.8 Hz, J= 1.5 Hz, 1H). C NMR (75 MHz,
CDCl ): δ ppm 33.07, 34.85, 118.73, 121.16, 125.79, 127.17, 127.20, 127.41, 127.49,
3
1
1
27.68, 128.88, 129.07, 132.32, 134.03, 134.48, 134.98, 135.19, 135.58, 139.43,
+
42.28, 151.21, 155.92, 181.67, 183.80. HRMS (EI) m/z: calcd [M] , 402.0497
+
(
C H N O S ); found, 402.0491.
2
2
14
2
2 2
5
.3. Cell Cultures and Sulforhodamine B (SRB) assay
Human hormone-refractory prostate cancer cell lines (PC-3) were from American
Type Culture Collection (Rockville, MD). The cells were cultured in RPMI1640
medium with 10% FBS (v/v) and penicillin (100 units/ml)/streptomycin (100 ꢀg/ml).
o
Cultures were maintained in a humidified incubator at 37 C in 5% CO . Cells were
2
seeded in 96-well plates in medium with 5% FBS. After 24 h, cells were fixed with
1
0% trichloroacetic acid (TCA) to represent cell population at the time of compound
addition (T ). After additional incubation of vehicle (0.1% DMSO) or the indicated
0
compound for 48 h, cells were fixed with 10% TCA and SRB at 0.4% (w/v) in 1%
acetic acid was added to stain cells. Unbound SRB was washed out by 1% acetic acid
and SRB bound cells were solubilized with 10 mM Trizma base. The absorbance was
read at a wavelength of 515 nm. Using the following absorbance measurements, such
as time zero (T ), control growth (C), and cell growth in the presence of compound
0
(TX), the percentage growth was calculated at each of the compound concentrations
levels. Percentage growth inhibition was calculated as: 100 - [(T -T )/(C-T )] × 100
X
0
0
for concentrations for which T ≥ T . Growth inhibition of 50% (IC ) was determined
X
0
50
at the drug concentration which resulted in 50% reduction of total protein increased in
2
8