Synthesis, antitumor screening and cell cycle analysis of novel benzothieno[3,2-b]pyran derivatives

Article abstract of DOI:10.1080/14756366.2016.1222582

Three series of benzothiophene derivatives were designed and synthesized as cytotoxic agents. The compounds were subjected to in vitro antitumor screening at the National Cancer Institute (NCI), Bethesda, MD. The results of the single dose screening indicated that only the benzothieno[3,2-b]pyran series 3a–f exhibited potent and broad spectrum cytotoxic activity and was subjected to five dose cytotoxic screening. The most active compound in this study was 2-amino-6-bromo-4-(4-nitrophenyl)-4H-[1]benzothieno[3,2-b]pyran-3-carbonitrile (3e) with MG-MID GI₅₀, TGI, and LC₅₀ values of 0.11, 7.94 and 42.66 μM, respectively. Compound 3e exhibited broad spectrum anticancer activity against a panel of 59 cell lines. To elucidate the underlying mechanism of compound 3e cytotoxic activity, we examined its effect on cell cycle progression and its ability to induce apoptosis using human colon adenocarcinoma cell line (HCT-116). The effect of compound 3e on the cell cycle progression indicated that exposure of HCT-116 cells to compound 3e for 24 and 48 h, induced a significant disruption in the cell cycle profile including time dependent decrease in cell population at G1 phase with concomitant increase in pre-G and G2/M cell population. Moreover, compound 3e induced time dependent increase in the percentage of early and late apoptotic and necrotic cell population. In conclusion, we were able to successfully design a new series of benzothieno[3,2-b]pyran derivatives with potent cytotoxic activity and their mechanism of cytotoxicity was examined.

Full text of DOI:10.1080/14756366.2016.1222582

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis, antitumor screening and cell cycle
analysis of novel benzothieno[3,2-b]pyran
derivatives
Ashraf F. Zaher, Suzan M. Abuel-Maaty, Hala B. El-Nassan, Samir A.S. Amer &
Tamer M. Abdelghany
To cite this article: Ashraf F. Zaher, Suzan M. Abuel-Maaty, Hala B. El-Nassan, Samir A.S. Amer
& Tamer M. Abdelghany (2016): Synthesis, antitumor screening and cell cycle analysis of novel
benzothieno[3,2-b]pyran derivatives, Journal of Enzyme Inhibition and Medicinal Chemistry,
DOI: 10.1080/14756366.2016.1222582
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–10
2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14756366.2016.1222582
!
RESEARCH ARTICLE
Synthesis, antitumor screening and cell cycle analysis of novel
benzothieno[3,2-b]pyran derivatives
Ashraf F. Zaher1, Suzan M. Abuel-Maaty1, Hala B. El-Nassan1, Samir A.S. Amer2, and Tamer M. Abdelghany3
2
1Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt, Department of Pathology, Molecular
Pathology Unit, National Cancer Institute, Cairo University, Cairo, Egypt, and 3Department of Pharmacology and Toxicology, Faculty of Pharmacy,
Al-Azhar University, Cairo, Egypt
Abstract
Keywords
Three series of benzothiophene derivatives were designed and synthesized as cytotoxic agents.
The compounds were subjected to in vitro antitumor screening at the National Cancer Institute
(NCI), Bethesda, MD. The results of the single dose screening indicated that only the
benzothieno[3,2-b]pyran series 3a–f exhibited potent and broad spectrum cytotoxic activity
and was subjected to five dose cytotoxic screening. The most active compound in this study
was 2-amino-6-bromo-4-(4-nitrophenyl)-4H-[1]benzothieno[3,2-b]pyran-3-carbonitrile (3e) with
MG-MID GI50, TGI, and LC50 values of 0.11, 7.94 and 42.66 mM, respectively. Compound 3e
exhibited broad spectrum anticancer activity against a panel of 59 cell lines. To elucidate the
underlying mechanism of compound 3e cytotoxic activity, we examined its effect on cell cycle
progression and its ability to induce apoptosis using human colon adenocarcinoma cell line
(HCT-116). The effect of compound 3e on the cell cycle progression indicated that exposure of
HCT-116 cells to compound 3e for 24 and 48 h, induced a significant disruption in the cell cycle
profile including time dependent decrease in cell population at G1 phase with concomitant
increase in pre-G and G2/M cell population. Moreover, compound 3e induced time dependent
increase in the percentage of early and late apoptotic and necrotic cell population. In
conclusion, we were able to successfully design a new series of benzothieno[3,2-b]pyran
derivatives with potent cytotoxic activity and their mechanism of cytotoxicity was examined.
Antitumor activity, apoptosis,
benzothieno[3,2-b]pyran, cell cycle analysis
History
Received 19 April 2016
Revised 23 July 2016
Accepted 25 July 2016
Published online 26 August 2016
Introduction
are apoptosis inducers. Taxanes and vinca alkaloids which act as
tubulin polymerization inhibitors are examples of chemothera-
peutics that function by induction of apoptosis1,3,4,9–13. Therefore,
the development of new apoptotic inducers seems to be a
promising strategy in cancer treatment.
Apoptosis or programed cell death (PCD) is a genetically
controlled process that occurs in normal cells as a part of the
normal development and maturation cycle. Apoptosis may occur
naturally as a defense mechanism in immune reactions or as a
response to cell damage by physiological or pathological
stimuli1–4. Apoptosis functions as a self-regulatory mechanism
inside the cells to prevent abnormal proliferation. It can be
initiated by intrinsic (mitochondrial) pathway or extrinsic (death
receptor) pathway. The initiation signal is followed by the
activation of caspase enzymes3,5,6. Many characteristic changes
occur within the cells during apoptosis such as: cell size
shrinkage, DNA fragmentation, chromatin condensation and
formation of membrane-bound apoptotic bodies7.
Cancer is associated with failure of apoptosis where cells are
programed for death, however they do not complete the process
and instead proliferate rapidly3,8. Treatment of cancer focused
mainly on the induction of cancer cell death using different
methods such as radiation, hormones and chemotherapeutic
agents. Most of the anticancer agents described in the literature
Searching the literature indicated that many benzofuran
derivatives displayed potent cytotoxic activity and might act by
induction of apoptosis1,9,14,15. In 2007, the benzofuranone
derivatives I and II (Figure 1) had been reported by Huang
et al. as potent antiproliferative agents against four human solid
tumor cell lines; HCCLM-7, Hep-2, MDA-MB-435S and SW-
480. Both compounds arrested the cell cycle in G0/G1 phase and
displayed apoptosis-inducing effect on Hep-2 cells16. More
recently, 5-bromo-2-(4-nitrobenzylidene)-benzofuran-3(2H)-one
(III) showed potent cytotoxic activity against K562 cell line17.
Although, benzofuran-3-ones have been thoroughly studied as
cytotoxic agents, their bioisostere benzothiophen-3-ones received
little attention and needed further investigation.
The aim of the present work was to prepare 2-arylidene-7-
bromo-1-benzothiophen-3(2H)-ones 2a–f as bioisosteres to ben-
zofuranone derivatives and to examine their effect as anti-
proliferative agents. In doing this, the bromine atom at position 7
and the 3-one moiety were kept constant, while, various aryl groups
were introduced at position 2 [phenyl ring substituted with electron
donating group or electron withdrawing groups and 2-pyridyl ring
as a heteroaryl ring]. The compounds were examined for their
Address for correspondence: Hala B. El-Nassan, Department of
Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Cairo
University, 33 Kasr El-Aini Street, Cairo, Egypt. Tel: +00202
23632245. Fax: +00202 23635140. E-mail: hala_bakr@hotmail.com

2
A. F. Zaher et al
J Enzyme Inhib Med Chem, Early Online: 1–10
O
O
O
O
Br
O
N
O
N
N
N
F
N+
O-
O
III
I
II
F
Bioisosteric replacement
O
S
Ar
NH2
Br
2a-f
O
Molecular
hybridization
CN
R1R2N
O
NH2
CN
Ar
S
O
NH2
CN
N
Br
3a-f
V
OCH3
IV
O
O
Br
O
O
R1R2N= NH2, (CH3)2N-, C2H5NH-
Figure 1. Design of the target compounds 2a–f and 3a–f.
possible anticancer activity in the National Cancer Institute (NCI), Since no previous work has been published on the mechanism
Bethesda, MD. However, their cytotoxic activity was poor and their of action of benzothieno[3,2-b]pyran derivatives as cytotoxic
mean growth percentage compared to control on nine cancer agents, we thought of examining their effect on the cell cycle.
Therefore, the most active compound in the benzothieno[3,2-
subpanels ranged between 79.57% and 95.73%.
Molecular hybridization strategy was followed in order to b]pyran series (compound 3e) was subjected to further testing to
improve the cytotoxic activity of these benzothiophene derivatives. determine its effect on the cell cycle and its ability to induce
Searching the literature revealed the outstanding activity of 4-aryl- apoptosis.
4H-chromenes as apoptotic inducers and cytotoxic agents. Thus, 4-
aryl-4H-chromenes (like compounds IV and V) were reported as Experimental part
potent apoptosis inducer in human breast cancer cells T47D18–20
Their SAR study indicated that both the 2-amino and the 3-cyano
.
General
groups were essential for the antitumor activity. Replacement of Melting points were determined using a Griffin apparatus (Fisher
the cyano with ester group or replacement of the amino with amide Scientific, Leicestershire, UK) and were uncorrected. IR spectra
or urea groups decreased the antitumor activity19,20
.
were recorded on Shimadzu IR 435 spectrophotometer (Kyoto,
Therefore, structural modification of compounds 2a–f was Japan) and values were represented in cmꢀ1. H NMR and 13C
done by molecular hybridization of the benzothiophene and NMR spectra were carried out on Varian Gemini 300 (Palo Alto,
pyrans rings to give benzothieno[3,2-b]pyrans 3a–f. Indeed, CA) and 75 MHz spectrophotometer (Cairo University, Cairo,
this modification afforded highly potent and broad spectrum Egypt), or Bruker 400 and 100 MHz spectrophotometer (Bruker
1
ˆ
ˆ
cytotoxic agents. Encouraged by these results, further cycliza- BioSpin AG, FuEllanden, Switzerland), respectively (Faculty of
tion of compounds 3a–f was done to afford benzothie- Pharmacy, Cairo University, Cairo, Egypt). TMS was used as an
no[20,30:5,6]pyrano[2,3-d]pyrimidin-4-amines 4a–e.
internal standard and chemical shifts were recorded in ppm on ꢀ
Unfortunately, this modification led to dramatic decrease in the scale. Elemental analyses were carried out at the regional center
cytotoxic activity. for mycology and biotechnology, Al-Azhar University, Cairo,

DOI: 10.1080/14756366.2016.1222582
Benzothieno[3,2-b]pyrans as cytotoxic agents
3
Egypt. The electron impact (EI) mass spectra were recorded on reaction mixture was cooled, and the separated solid was filtered,
Hewlett Packard 5988 spectrometer Microanalytical center (Cairo dried and crystallized from ethanol.
University, Cairo, Egypt). Analytical thin layer chromatography
(TLC) on silica gel plates containing UV indicator was employed
routinely to follow the course of reactions and to check the purity
2-Amino-6-bromo-4-(4-bromophenyl)-4H-[1]benzothieno[3,2-
b]pyran-3-carbonitrile (3a). Yield: 96%; m.p.: 208–210 ꢁC; IR
(cmꢀ1): 3475, 3342 (NH2), 2924, 2852 (CH-aliphatic), 2204
of products. All reagents and solvents were purified and dried by
1
(CN); H NMR (300 MHz, DMSO-d6) ꢀ ppm 5.18 (s, 1H, H-4),
standard techniques. 7-Bromo-1-benzothiophen-3(2H)-one (1)
was prepared according to the published method21,22
.
7.35 (s, 2H, NH2, D2O exchangeable), 7.27–7.77 (m, 7H, Ar-H);
MS m/z: 464 [(M + 4), 18.90%], 462 [(M + 2), 36.04%], 460 [M+,
23.56%], 307 [(M + 2-C6H4Br)+, 99.76%], 305 [(M ꢀ C6H4Br)+,
100%]; Anal. Calcd for C18H10Br2N2OS: C, 46.78; H, 2.18; N,
6.06. Found: C, 46.86; H, 2.19; N, 6.21.
General procedure for the synthesis of 2-arylidene-7-bromo-1-
benzothiophen-3(2H)-ones 2a–f
A mixture of 7-bromo-1-benzothiophen-3(2H)-one (1) (2.29 g,
10 mmol), the appropriate aromatic aldehyde (10 mmol) and
anhydrous sodium acetate (0.82 g, 10 mmol) in glacial acetic acid
(10 mL) was heated under reflux for 2 h. The reaction mixture was
cooled, poured onto water (100 mL) and the separated solid was
filtered, dried and crystallized from ethanol.
2-Amino-6-bromo-4-(4-chlorophenyl)-4H-[1]benzothieno[3,2-
b]pyran-3-carbonitrile (3b). Yield: 73%; m.p.: 203–205 ꢁC; IR
(cmꢀ1): 3464, 3323 (NH2), 2924, 2852 (CH-aliphatic), 2196
1
(CN); H NMR (300 MHz, DMSO-d6) ꢀ ppm 5.23 (s, 1H, H-4),
7.31 (s, 2H, NH2, D2O exchangeable), 7.42–8.11 (m, 7H, Ar-H);
13C NMR (300 MHz, DMSO-d6) ꢀ ppm 25.0, 55.6, 115.7, 118.7,
118.8, 119.7, 126.9, 128.2 (2), 128.7, 129.3, 130.0, 132.2 (2),
136.0, 138.7, 142.5, 160.2; Anal. Calcd for C18H10BrClN2OS: C,
51.76; H, 2.41; N, 6.71. Found: C, 51.95; H, 2.44; N, 6.87.
7-Bromo-2-(4-bromobenzylidene)-1-benzothiophen-3(2H)-one
(2a). Yield: 80%; m.p.: 178–180 ꢁC; IR (cmꢀ1): 1689 (C¼O);
1H NMR (400 MHz, DMSO-d6) ꢀ ppm 7.35–8.03 (m, 8H, Ar-
H+ ¼CH); MS m/z: 398 [(M + 4)+, 50.42%], 396 [(M + 2)+,
92.88%], 394 [M+, 47.61%], 317 [(M + 2-Br), 100%], 315
[(M ꢀ Br)+, 95.68%]; Anal. Calcd for C15H8Br2OS: C, 45.48;
H, 2.04. Found: C, 45.69; H, 2.07.
2-Amino-6-bromo-4-(4-fluorophenyl)-4H-[1]benzothieno[3,2-
b]pyran-3-carbonitrile (3c). Yield: 70%; m.p.: 237–238 ꢁC; IR
(cmꢀ1): 3466, 3321 (NH2), 2924, 2852 (CH-aliphatic), 2194
7-Bromo-2-(4-chlorobenzylidene)-1-benzothiophen-3(2H)-one
(2b). Yield: 72%; m.p.: 110–111 ꢁC; IR (cmꢀ1): 1681 (C¼O);
1H NMR (300 MHz, DMSO-d6) ꢀ ppm 7.35–7.99 (m, 8H, Ar-
H + ¼CH); Anal. Calcd for C15H8BrClOS: C, 51.23; H, 2.29.
Found: C, 51.41; H, 2.26.
1
(CN); H NMR (300 MHz, DMSO-d6) ꢀ ppm 5.14 (s, 1H, H-4),
7.22 (s, 2H, NH2, D2O exchangeable), 7.16–7.73 (m, 7H, Ar-H);
13C NMR (75 MHz, DMSO-d6) ꢀ ppm 25.4, 55.8, 115.3, 118.8,
119.8, 126.8, 128.2 (2), 129.3, 130.0 (2), 136.7, 138.6, 138.8,
139.8, 159.8, 160.1, 163.0; MS m/z: 402 [(M + 2), 45.03%], 400
[M+, 44.40%], 307 [(M + 2-C6H4F)+, 100%], 305 [(M ꢀ C6H4F)+,
96.95%], 95 [(C6H4F)+, 22.90%]; Anal. Calcd for
C18H10BrFN2OS: C, 53.88; H, 2.51; N, 6.98. Found: C, 53.94;
H, 2.60; N, 6.80.
7-Bromo-2-(4-fluorobenzylidene)-1-benzothiophen-3(2H)-one
(2c). Yield: 70%; m.p.: 179–181 ꢁC; IR (cmꢀ1): 1685 (C¼O);
1H NMR (300 MHz, DMSO-d6) ꢀ ppm 7.36–7.98 (m, 7H, Ar-H),
8.01 (s, 1H, ¼CH); Anal. Calcd for C15H8BrFOS: C, 53.75; H,
2.41. Found: C, 53.98; H, 2.49.
2-Amino-6-bromo-4-(4-methoxyphenyl)-4H-[1]benzothieno[3,2-
b]pyran-3-carbonitrile (3d). Yield: 77%; m.p.: 180–182 ꢁC; IR
(cmꢀ1): 3423, 3323 (NH2), 2954, 2868 (CH-aliphatic), 2196
(CN); 1H NMR (300 MHz, DMSO-d6) ꢀ ppm 3.74 (s, 3H, CH3O),
5.03 (s, 1H, H-4), 7.11 (s, 2H, NH2, D2O exchangeable), 6.90–
8.00 (m, 7H, Ar-H); 13C NMR (75 MHz, DMSO-d6) ꢀ ppm 25.0,
55.0 (CH3O), 56.3, 114.1, 115.7, 118.7, 119.9, 126.8, 128.0 (2),
128.1, 128.5, 130.1 (2), 135.7, 136.0, 138.4, 158.6, 160.0; MS m/
z: 414 [(M + 2), 22.96%], 412 [M+, 24.57%], 307 [(M + 2-
C6H4OCH3)+, 100%], 305 [(M ꢀ C6H4OCH3)+, 99.80%]; Anal.
Calcd for C19H13BrN2O2S: C, 55.22; H, 3.17; N, 6.78. Found: C,
55.34; H, 3.08; N, 6.53.
7-Bromo-2-(4-methoxybenzylidene)-1-benzothiophen-3(2H)-one
(2d). Yield: 74%; m.p.: 175–176 ꢁC; IR (cmꢀ1): 2976, 2844
1
(CH-aliphatic), 1678 (C¼O); H NMR (300 MHz, DMSO-d6) ꢀ
ppm 3.95 (s, 3H, OCH3), 7.20–8.07 (m, 7H, Ar-H), 8.09 (s,
1H, ¼CH); 13C NMR (100 MHz, DMSO-d6) ꢀ ppm 55.5, 114.7,
118.0, 125.4, 126.7, 126.8 (2), 127.8, 133.2, 133.5 (2), 134.9,
137.3, 147.6, 161.6, 188.3; Anal. Calcd for C16H11BrO2S: C,
55.34; H, 3.19. Found: C, 55.47; H, 3.26.
7-Bromo-2-(4-nitrobenzylidene)-1-benzothiophen-3(2H)-one
(2e). Yield: 72%; m.p.: 128–130 ꢁC; IR (cmꢀ1): 1678 (C¼O),
1H NMR (300 MHz, DMSO-d6) ꢀ ppm 7.36–7.98 (m, 7H, Ar-H),
8.00 (s, 1H, ¼CH); Anal. Calcd for C15H8BrNO3S: C, 49.74; H,
2.23; N, 3.87. Found: C, 49.89; H, 2.21; N, 3.98.
2-Amino-6-bromo-4-(4-nitrophenyl)-4H-[1]benzothieno[3,2-
b]pyran-3-carbonitrile (3e). Yield: 82%; m.p.: 188–190 ꢁC; IR
(cmꢀ1): 3460, 3319 (NH2), 2900, 2800 (CH-aliphatic), 2194
1
(CN); H NMR (300 MHz, DMSO-d6) ꢀ ppm 5.09 (s, 1H, H-4),
7-Bromo-2-(pyridin-2-ylmethylidene)-1-benzothiophen-3(2H)-one
(2f). Yield: 70%; m.p.: 120–122 ꢁC; IR (cmꢀ1): 1681 (C¼O);
1H NMR (300 MHz, DMSO-d6) ꢀ ppm 7.30–7.95 (m, 7H, Ar-H),
7.97 (s, 1H, ¼CH); Anal. Calcd for C14H8BrNOS: C, 52.85; H,
2.53; N, 4.40. Found: C, 53.12; H, 2.60; N, 4.49.
7.16 (s, 2H, NH2, D2O exchangeable), 7.27–7.73 (m, 7H, Ar-H);
MS m/z: 429 [(M + 2), 0.10%], 427 [M+, 0.09%], 307 [(M + 2-
C6H4NO2)+, 100%], 305 [(M ꢀ C6H4NO2)+, 99.06%]; Anal.
Calcd for C18H10BrN3O3S: C, 50.48; H, 2.35; N, 9.81. Found:
C, 50.67; H, 2.39; N, 9.94.
General procedure for the synthesis of 2-amino-4-aryl-6-bromo-
4H-[1]benzothieno[3,2-b]pyran-3-carbonitriles 3a–f
2-Amino-6-bromo-4-(pyridin-2-yl)-4H-[1]benzothieno[3,2-
b]pyran-3-carbonitrile (3f). Yield: 74%; m.p.: 205–207 ꢁC; IR
(cm ꢀ 1): 3400, 3360 (NH2), 2924, 2852 (CH-aliphatic), 2193
(CN); H NMR (300 MHz, DMSO-d6) ꢀ ppm 5.18 (s, 1H, H-4),
7.23 (s, 2H, NH2, D2O exchangeable), 7.29–8.56 (m, 7H, Ar-H);
A mixture of 2-arylidene-benzothiophen-3-ones 2a–f (1 mmol),
malononitrile (0.07 g, 1 mmol) and piperidine (three drops) in
absolute ethanol (10 mL) was heated under reflux for 5 h. The
1

4
A. F. Zaher et al
J Enzyme Inhib Med Chem, Early Online: 1–10
MS m/z: 385 [(M + 2), 55.68%], 383 [M+, 45.82%], 307 [(M + 2- Measurement of potential antiproliferative activity27
C5H4N)+, 99.98%], 305 [(M ꢀ C5H4N)+, 100%]; Anal. Calcd for
The human tumor cell lines of the cancer screening panel were
C17H10BrN3OS: C, 53.14; H, 2.62; N, 10.94. Found: C, 53.26; H,
grown in RPMI 1640 medium containing 5% fetal bovine serum
2.65; N, 11.08.
and 2 mM L-glutamine. For a typical screening experiment, cells
were inoculated into 96 well microtiter plates in 100 mL at plating
densities ranging from 5000 to 40 000 cells/well depending on the
General procedure for the synthesis of 5-aryl-7-bromo-5H-
[1]benzothieno[20,30:5,6]pyrano[2,3-d]pyrimidin-4-amines 4a–e
doubling time of individual cell lines. After cell inoculation, the
microtiter plates were incubated at 37 ꢁC, 5% CO2, 95% air and
A mixture of benzothienopyran derivatives 3a–e (2 mmol) and
100% relative humidity for 24 h prior to addition of test
formamide (5 mL) was heated under reflux for 5 h. The reaction
compounds.
mixture was cooled, poured onto water (25 mL) and the separated
solid was filtered, dried and crystallized from aqueous ethanol.
After 24 h, two plates of each cell line were fixed in situ with
trichloroacetic acid (TCA), to represent a measurement of the cell
population for each cell line at the time of test compound addition
(Tz). The test compounds were solubilized in dimethyl sulfoxide
7-Bromo-5-(4-bromophenyl)-5H-[1]benzothie-
no[20,30:5,6]pyrano[2,3-d]pyrimidin-4-amine (4a). Yield: 45%;
m.p.: 145–147 ꢁC; IR (cm ꢀ 1): 3423, 3207 (NH2), 2924, 2854
at 400-fold the desired final maximum test concentration and
1
(CH-aliphatic); H NMR (300 MHz, DMSO-d6) ꢀ ppm 4.14 (s,
1H, H-5), 7.25–7.99 (m, 7H, Ar-H), 8.39 (s, 1H, H-2), 10.11 (s,
stored frozen prior to use. At the time of compound addition, an
aliquot of frozen concentrate was thawed and diluted to twice the
desired final maximum test concentration with complete medium
2H, NH2, D2O exchangeable); Anal. Calcd for C19H11Br2N3OS:
containing 50 mg/mL gentamicin. Additional four, 10-fold or ½ log
serial dilutions were made to provide a total of five drug
C, 46.65; H, 2.27; N, 8.59. Found: C, 46.81; H, 2.32; N, 8.72.
concentrations plus control. Aliquots of 100 mL of the different
test compounds dilutions were added to the appropriate microtiter
7-Bromo-5-(4-chlorophenyl)-5H-[1]benzothieno[20,30:5,6]
pyrano[2,3-d]pyrimidin-4-amine (4b). Yield: 40%; m.p.: 103–
wells already containing 100 mL of medium, resulting in the
105 ꢁC; IR (cm ꢀ 1): 3383, 3307 (NH2), 2974, 2858 (CH-
required final drug concentrations.
Following compound addition, the plates were incubated for an
additional 48 h at 37 ꢁC, 5% CO2, 95% air and 100% relative
H-5), 7.23–8.47 (m, 7H, Ar-H), 8.40 (s, 1H, H-2), 10.11 (br s, 2H,
1
aliphatic); H NMR (400 MHz, DMSO-d6) ꢀ ppm 4.16 (s, 1H,
NH2, D2O exchangeable); Anal. Calcd for C19H11BrClN3OS: C,
51.31; H, 2.49; N, 9.45. Found: C, 51.43; H, 2.51; N, 9.66.
humidity. For adherent cells, the assay was terminated by the
addition of cold TCA. Cells were fixed in situ by the gentle
addition of 50 mL of cold 50% (w/v) TCA (final concentration,
10% TCA) and incubated for 60 min at 4 ꢁC. The supernatant was
discarded, and the plates were washed five times with tap water
7-Bromo-5-(4-fluorophenyl)-5H-[1]benzothie-
no[20,30:5,6]pyrano[2,3-d]pyrimidin-4-amine (4c). Yield: 40%;
m.p.: 158–160 ꢁC; IR (cm ꢀ 1): 3213, 3292 (NH2), 2920, 2850
and air dried. SRB solution (100 mL) at 0.4% (w/v) in 1% acetic
1
(CH-aliphatic); H NMR (400 MHz, DMSO-d6) ꢀ ppm 4.16 (s,
1H, H-5), 7.13–8.31 (m, 7H, Ar-H), 8.40 (s, 1H, H-2), 10.10 (s,
2H, NH2, D2O exchangeable); Anal. Calcd for C19H11BrFN3OS:
C, 53.28; H, 2.59; N, 9.81. Found: C, 53.49; H, 2.64; N, 9.89.
acid was added to each well, and plates were incubated for 10 min
at room temperature. After staining, unbound dye was removed by
washing five times with 1% acetic acid and the plates were air
dried. Bound stain was subsequently solubilized with 10 mM
trizma base, and the absorbance was read on an automated plate
reader at a wavelength of 515 nm.
For suspension cells, the methodology was the same except
that the assay was terminated by fixing settled cells at the bottom
of the wells by gently adding 50 mL of 80% TCA (final
concentration, 16% TCA). Using the seven absorbance measure-
ments [time zero, (Tz), control growth, (C), and test growth in the
presence of the test compounds at the five concentration levels
(Ti)], the percentage growth was calculated at each of the test
compounds concentrations levels. Percentage growth inhibition
was calculated as:
7-Bromo-5-(4-methoxyphenyl)-5H-[1]benzothie-
no[20,30:5,6]pyrano[2,3-d]pyrimidin-4-amine (4d). Yield: 45%;
m.p.: 143–145 ꢁC; IR (cm ꢀ 1): 3400, 3209 (NH2), 2920, 2800
1
(CH-aliphatic); H NMR (300 MHz, DMSO-d6) ꢀ ppm 3.73 (s,
3H, CH3O), 4.08 (s, 1H, H-5), 6.87–7.60 (m, 7H, Ar-H), 8.40 (s,
1H, H-2), 10.05 (s, 2H, NH2, D2O exchangeable); MS m/z: 441
[(M + 2)+, 0.31%], 439 [M+, 0.23%], 334 [(M + 2-C6H4OCH3)+,
2.29%], 332 [(M ꢀ C6H4OCH3)+, 2.54%], 69 [100%]; Anal. Calcd
for C20H14BrN3O2S: C, 54.56; H, 3.20; N, 9.54. Found: C, 54.72;
H, 3.28; N, 9.73.
[(Ti ꢀ Tz)/(C ꢀ Tz)] ꢂ 100 for concentrations for which
Ti ꢃ Tz.
7-Bromo-5–(4-nitrophenyl)-5H-[1]benzothie-
no[20,30:5,6]pyrano[2,3-d]pyrimidin-4-amine (4e). Yield: 50%;
m.p.: 110–112 ꢁC; IR (cm ꢀ 1): 3400, 3319 (NH2), 2918, 2848
[(Ti ꢀ Tz)/Tz] ꢂ 100 for concentrations for which Ti5Tz.
1
(CH-aliphatic) 1530, 1346 (NO2); H NMR (400 MHz, DMSO-
Three dose response parameters were calculated for each
compound. Growth inhibition of 50% (GI50) was calculated from:
[(Ti ꢀ Tz)/(C ꢀ Tz)] ꢂ 100 ¼ 50, which is the drug concentra-
tion resulting in a 50% lower net protein increase in the treated
cells (measured by SRB staining) as compared to the net protein
d6) ꢀ ppm 4.17 (s, 1H, H-5), 7.32–8.29 (m, 7H, Ar-H), 8.40 (s, 1H,
H-2), 10.10 (s, 2H, NH2, D2O exchangeable); 13C NMR
(100 MHz, DMSO-d6) ꢀ ppm 29.4, 116.3, 119.9, 123.2, 123.5,
128.1 (2), 129.7, 134.1 (2), 134.2, 134.8, 141.5, 142.5, 153.5,
155.2, 155.7, 160.6, 163.4; Anal. Calcd for C19H11BrN4O3S: C, increase seen in the control cells. The drug concentration resulting
50.12; H, 2.44; N, 12.31. Found: C, 50.29; H, 2.49; N, 12.49.
in TGI was calculated from Ti ¼ Tz. The LC50 (concentration of
drug resulting in a 50% reduction in the measured protein at the
end of the drug treatment as compared to that at the beginning)
indicating a net loss of cells following treatment was calculated
Biological testing23–27
Materials and methods
from [(Ti ꢀ Tz)/Tz] ꢂ 00¼ ꢀ50. Values were calculated for each
of these three parameters if the level of activity is reached;
however, if the effect was not reached or was exceeded, the value
for that parameter was expressed as greater or less than the
The operation of this screen utilized 59 different human tumor
cell lines, representing leukemia, melanoma and cancers of the
lung, colon, brain, ovary, breast, prostate and kidney.

DOI: 10.1080/14756366.2016.1222582
Benzothieno[3,2-b]pyrans as cytotoxic agents
5
Table 1. Results of in vitro anticancer screening of compounds 2–4.
NH2
N
O
O
N
O
CN
NH2
Ar
S
S
S
Ar
Ar
Br
Br
Br
2a-f
3a-f
4a-d
Total MG-MIDa,b(mM)
Compound No.
Ar
Mean growth (%)
GI50
TGI
LC50
2a
2b
2c
2d
2f
3a
3b
3c
3d
3e
3f
4-BrC6H4
4-ClC6H4
4-FC6H4
95.73
91.82
79.57
88.62
93.14
20.25
15.92
22.64
26.03
17.13
36.88
89.26
91.28
90.86
85.09
n.t
n.t
n.t
n.t
n.t
n.t
n.t
n.t
n.t
n.t
n.t
n.t
4-CH3OC6H4
2-Pyridyl
n.t
n.t
n.t
4-BrC6H4
4-ClC6H4
4-FC6H4
0.76
0.67
0.50
2.45
0.11
n.t
n.t
n.t
n.t
n.t
6.02
5.24
14.79
13.18
7.94
n.t
n.t
n.t
n.t
n.t
32.36
33.11
72.44
57.54
42.66
n.t
n.t
n.t
n.t
n.t
4-CH3OC6H4
4-NO2C6H4
2-Pyridyl
4a
4b
4c
4d
4-BrC6H4
4-ClC6H4
4-FC6H4
4-CH3OC6H4
aMG-MID is the calculated mean full panel or individual subpanel GI50, TGI, or LC50
.
in 1 ꢂ binding buffer for 15 minutes at room temperature in the
dark. Analyses were performed using FACS Calibur flow
cytometer (BD Biosciences, San Jose, CA). The results are
displayed in Figure 5.
maximum or minimum concentration tested. The log GI50, log
TGI, log LC50 were then determined, defined as the mean of the
log’s of the individual GI50, TGI, LC50 values respectively. The
lowest values are obtained with the most sensitive cell lines. The
data are presented in Table 1 and Figures 2 and 3 as well as
supplementary data.
Results and discussion
Cell cycle analysis
Chemistry
To elucidate the mechanism of the cytotoxic activity of the
newly synthesized compounds, the effects of compound 3e on
the cell cycle progression were examined against HCT-116 cells.
At a density of 4 ꢂ 106 cell/T 75 flask, HCT-116 cells were
exposed to compound 3e at its GI50 concentration (0.05 mM) for
24 and 48 h. The cells then were collected by trypsinization,
washed with phosphate buffered saline (PBS), and fixed in ice-
cold absolute alcohol. Thereafter, the cells were stained, using
CycletestTM Plus DNA Reagent Kit (BD Biosciences, San Jose,
CA), according to the manufacturer’s instructions. Cell cycle
Scheme 1 outlines the synthesis of the target compounds 2a–f,
3a–f and 4a–e. Reacting 7-bromo-1-benzothiophen-3(2H)-one
(1)21,22 with different aromatic aldehydes in glacial acetic and
sodium acetate afforded 2-arylidene-7-bromo-1-benzothiophen-
3(2H)-ones 2a–f. Their structures were confirmed by IR spectra
that revealed the appearance of the conjugated C¼O band at
1
1678–1689 cmꢀ1. H NMR spectra of compounds 2a–f revealed
the appearance of methine proton (¼CH) at ꢀ 7.97–8.09 ppm
together with the introduced aromatic protons. Moreover,
13C NMR spectrum of compound 2d showed C¼O carbon
signal at ꢀ 188.3 ppm. Furthermore, the mass spectrum of
compound 2a showed molecular ion peaks at m/z 394, 396 and
398 corresponding to M, M + 2 and M + 4 peaks in the ratio of
100:195:105 as expected from compounds containing two brom-
ine atoms28.
Cyclization of 2-arylidene-7-bromo-1-benzothiophen-3(2H)-
ones 2a–f with malononitrile in presence of piperidine resulted in
the formation of 2-amino-4-aryl-6-bromo-4H-[1]benzothieno[3,2-
b]pyran-3-carbonitriles 3a–f.
The disappearance of the C¼O band in the IR spectra of
compounds 3a–f, together with the appearance of CN band at
2193–2204 cmꢀ1 and NH2 bands at 3319–3475 cmꢀ1 provided
proofs for the formation of compounds 3a–f.
1H NMR spectra of compounds 3a–f revealed the appearance of
the H-4 characteristic signal of the pyran ring at ꢀ 5.03–5.23 ppm as
distribution was determined using
a FACS Calibur flow
cytometer (BD Biosciences, San Jose, CA). The results are
displayed in Figure 4.
Measurement of apoptosis using Annexin-V-FITC apoptosis
detection kit
Apoptosis was determined by staining the cells with Annexin V-
fluorescein isothiocyanate (FITC) and counterstaining with PI
using the Annexin V-FITC/PI apoptosis detection kit (BD
Biosciences, San Diego, CA) according to the manufacturer’s
instructions. Briefly, 4 ꢂ 106 cell/T 75 flask were exposed to
compound 3e at its GI50 concentration (0.05 mM) for 24 and
48 h. The cells then were collected by trypsinization and
0.5 ꢂ 106 cells were washed twice with phosphate-buffered
saline (PBS) and stained with 5 ml Annexin V-FITC and 5 ml PI

6
A. F. Zaher et al
J Enzyme Inhib Med Chem, Early Online: 1–10
Figure 2. GI50 of compound 3e in mM against
leukemia, non-small cell lung cancer, colon
cancer and CNS cancer cell lines.
Figure 3. GI50 of compound 3e in mM against
melanoma, ovarian cancer, renal cancer,
prostate cancer and breast cancer cell lines.
well as an exchangeable singlet signal at ꢀ 7.11–7.35 ppm In vitro anticancer screening
corresponding to the NH2 protons. Besides, 13C NMR spectra of
All the synthesized compounds except 2e and 4e (Ar¼ 4-
nitrophenyl) were subjected to in vitro antitumor screening at
the NCI, Bethesda, MD23–27. The compounds were screened using
sulforhodamine B (SRB) assay26. The screening was done against
59 cell lines from nine subpanels: leukemia, lung, colon,
melanoma, renal, CNS, breast, prostate and ovarian cancer
panels. The compounds were first screened at single dose primary
anticancer assay (concentration 10ꢀ5 M) and the mean growth
percent of the treated cells compared to the untreated control cells
was calculated for each compound. Compounds that showed
promising results at this stage were screened further at five doses
(concentrations 0.01, 0.1, 1, 10 and 100 mM). The results were
expressed in terms of three response parameters, median growth
inhibition (GI50, the compound’s concentration that cause 50%
decrease in net cell growth), total growth inhibition (TGI, the
compound’s concentration leading to total inhibition of cell
growth) and median lethal concentration (LC50, the compound’s
concentration causing a net 50% loss of initial cells at the end of
the incubation period). Mean graph midpoints (MG-MID) were
compounds 3b–d indicated the disappearance of C¼O carbon
signal around ꢀ 188 ppm and the appearance of C-4 and C-3 of the
pyran ring at ꢀ 24.4–25.0 ppm and ꢀ 55.6–56.3 ppm, respectively.
The mass spectra of compounds 3a, 3c, 3d and 3f showed their
corresponding molecular ion peaks and base peaks due to loss of
the aryl group at position 4.
Finally, 2-amino-benzothieno[3,2-b]pyran-3-carbonitriles 3a–e
were reacted with formamide to give benzothie-
no[20,30:5,6]pyrano[2,3-d]pyrimidin-4-amines 4a–e.
The IR spectra of compounds 4a–e showed NH2 forked band at
3207–3423 cmꢀ1 with no CN bands. Besides, their 1H NMR
spectra demonstrated the appearance of an exchangeable singlet
signal at ꢀ 10.05–10.11 ppm corresponding to the NH2 protons.
The pyrimidine proton (H-2) appeared as singlet signal at ꢀ 8.39–
8.40 ppm and notably the signal of the pyran proton (H-5) was
shifted to lower frequency and appeared at ꢀ 4.08–4.17 ppm
probably due to the shielding effect of the amino group.
Furthermore, the mass spectrum of compound 4d displayed two
molecular ion peaks at m/z 439 and 441.

DOI: 10.1080/14756366.2016.1222582
Benzothieno[3,2-b]pyrans as cytotoxic agents
7
Figure 4. (a) Effect of compound 3e on DNA-ploidy flow cytometric analysis of HCT-116 cells after 24 h and 48 h. (b) Bar chart showing percentage of
HCT-116 cells at each phase of the cell cycle in control cells and cells treated with compound 3e after 24 h and 48 h.

8
A. F. Zaher et al
J Enzyme Inhib Med Chem, Early Online: 1–10
Figure 5. Effects of compound 3e on apop-
tosis of HCT-116 cells after 24 h and 48 h.
˘
ˆ
The data were presented as means o standard
error of the mean for three independent
replicates.
NH2
Scheme 1. Synthesis of the target compounds
2a–f, 3a–f and 4a–e.
O
O
O
CN
a
b
S
Ar
S
S
Ar
Br
Br
Br
2a-f
1
3a-f
c
N
N
O
Ar = 4-BrC H , 4-ClC H , 4-FC H , 4-CH OC H , 4-NO C H , C H N
6
4
6
4
6
4
3
6
4
2
6
4
5 4
Reagents: a) ArCHO, CH3COONa, CH3COOH, reflux 2h.;
b) CH2(CN)2, piperidine, C2H5OH, reflux 5h.;
c) HCONH2, reflux 5h.
NH2
S
Ar
Br
4a-e
halogen atoms enhanced the anti-proliferative activity more than
the electron donating groups like methoxy group [compare 3a–c,
3e and 3d].
The obtained results indicated that the test compounds in five
dose screening (3a–e) showed potent and broad spectrum
antitumor activity. The compounds tested gave MG-MID GI50
in the range of 0.11–2.45 mM, MG-MID TGI in the range of 5.24–
14.79 mM and MG-MID LC50 in the range of 32.36–72.44 mM.
Four of the test compounds (3a–c and 3e) exhibited MG-MID
GI50 values less than 1 mM.
The most active compound in this study was 2-amino-6-
bromo-4–(4-nitrophenyl)-4H-[1]benzothieno[3,2-b]pyran-3-car-
bonitrile (3e) with MG-MID GI50, TGI, and LC50 values of 0.11,
7.94 and 42.66 mM, respectively. Compound 3e exhibited broad
spectrum anticancer activity against all the panels tested. Thus,
compound 3e displayed GI50 at submicromolar concentrations
[GI50 less than 1 mM in 83% of the tested cell lines and GI50 less
than 0.1 mM in 66% of the tested cell lines]. Compound 3e also
showed TGI in submicromolar concentrations in four of the cell
lines tested, namely HL-60 (leukemia panel), NCI-H522 (non-
small cell lung cancer panel), HT29 (colon cancer panel) and
MDA-MB-435 (melanoma panel) (Figures 2 and 3 and Table 1 in
supplementary data).
calculated for each of these parameters to obtain an average
activity parameter for each compound over all the cell lines tested.
As such, the NCI antitumor drug screen is able to distinguish
between broad spectrum antitumor compounds and tumor or
subpanel-selective compounds23.
Structurally, the test compounds belonged to three series:
2-arylidene-benzothiophen-3-ones 2a–f, 2-amino-4-aryl-ben-
zothieno[3,2-b]pyran-3-carbonitriles 3a–f and 5-aryl-benzothie-
no[20,30:5,6]pyrano[2,3-d]pyrimidin-4-amines 4a–d. The results
of the single dose screening indicated that only the ben-
zothieno[3,2-b]pyran series 3a–f exhibited potent and broad
spectrum cytotoxic activity (mean growth percent between 15%
and 36%) (Table 1). While, the other two series showed weak
cytotoxic activity which appeared from their high mean growth
percent (79%–95%).
Thus, it seemed that cyclization of 2-arylidene-benzothiophe-
nones into benzothieno[3,2-b]pyrans greatly enhanced the cyto-
toxic
activity,
while,
further
cyclization
into
benzothieno[20,30:5,6]pyrano[2,3-d]pyrimidines reduced the cyto-
toxic activity significantly.
Accordingly, compounds 3a–e were further subjected to five
dose anticancer screening to determine MG-MID of GI50, TGI
and LC50 and the results are displayed in Table 1. Detailed results
of each compound on the 59 cell lines tested are provided in the
supplementary data.
SAR examination of the benzothieno[3,2-b]pyran derivatives
3a–f indicated that the presence of 4-substituted phenyl group
afforded better cytotoxic results than the presence of heterocyclic
Accordingly, compound 3e was identified as a novel lead
compound and was subjected to further studies to explore its
mechanism of action.
Cell-cycle analysis
ring [compare 3a–e and 3f]. In addition, the presence of electron During cellular proliferation, cells pass through four distinct
withdrawing substituent on the phenyl ring like nitro group and phases leading eventually to cell replication: G1 phase, S phase,

DOI: 10.1080/14756366.2016.1222582
Benzothieno[3,2-b]pyrans as cytotoxic agents
9
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Conclusion
The results reported here introduced benzothieno[3,2-b]pyran
derivatives 3a–f as novel and promising lead anticancer agents.
Derivatives belonging to this series displayed potent and broad
spectrum antitumor activity. Thus, compounds 3a–f showed MG-
MID GI50 in the range of 0.11–2.45 mM, MG-MID TGI in the
range of 5.24–14.79 mM and MG-MID LC50 in the range of
32.36–72.44 mM. Four of the test compounds (3a–c and 3e)
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The authors are grateful to NCI staff, Bethesda, MD, USA, for carrying
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Declaration of interest
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The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.

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Supplementary material available online
The supplementary data includes Tables 1 describing the GI50, TGI and LC50 values in mM of compounds 3a–e.