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

Abstract

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.

Keywords:

• Antitumor activity
• apoptosis
• benzothieno[3,2-b]pyran
• cell cycle analysis

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

Introduction

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 are apoptosis inducers. Taxanes and vinca alkaloids which act as tubulin polymerization inhibitors are examples of chemotherapeutics 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.

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.

Figure 1. Design of the target compounds 2a–f and 3a–f.

The aim of the present work was to prepare 2-arylidene-7-bromo-1-benzothiophen-3(2H)-ones 2a–f as bioisosteres to benzofuranone 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 possible anticancer activity in the National Cancer Institute (NCI), Bethesda, MD. However, their cytotoxic activity was poor and their mean growth percentage compared to control on nine cancer subpanels ranged between 79.57% and 95.73%.

Molecular hybridization strategy was followed in order to improve the cytotoxic activity of these benzothiophene derivatives. Searching the literature revealed the outstanding activity of 4-aryl-4H-chromenes as apoptotic inducers and cytotoxic agents. Thus, 4-aryl-4H-chromenes (like compounds IV and V) were reported as potent apoptosis inducer in human breast cancer cells T47D18–20. Their SAR study indicated that both the 2-amino and the 3-cyano groups were essential for the antitumor activity. Replacement of the cyano with ester group or replacement of the amino with amide or urea groups decreased the antitumor activity19,20.

Therefore, structural modification of compounds 2a–f was done by molecular hybridization of the benzothiophene and pyrans rings to give benzothieno[3,2-b]pyrans 3a–f. Indeed, this modification afforded highly potent and broad spectrum cytotoxic agents. Encouraged by these results, further cyclization of compounds 3a–f was done to afford benzothieno[2′,3′:5,6]pyrano[2,3-d]pyrimidin-4-amines 4a–e.

Unfortunately, this modification led to dramatic decrease in the cytotoxic activity.

Since no previous work has been published on the mechanism of action of benzothieno[3,2-b]pyran derivatives as cytotoxic agents, we thought of examining their effect on the cell cycle. Therefore, the most active compound in the benzothieno[3,2-b]pyran series (compound 3e) was subjected to further testing to determine its effect on the cell cycle and its ability to induce apoptosis.

Experimental part

General

Melting points were determined using a Griffin apparatus (Fisher Scientific, Leicestershire, UK) and were uncorrected. IR spectra were recorded on Shimadzu IR 435 spectrophotometer (Kyoto, Japan) and values were represented in cm⁻¹. ¹H NMR and ¹³C NMR spectra were carried out on Varian Gemini 300 (Palo Alto, CA) and 75 MHz spectrophotometer (Cairo University, Cairo, Egypt), or Bruker 400 and 100 MHz spectrophotometer (Bruker BioSpin AG, Fällanden, Switzerland), respectively (Faculty of Pharmacy, Cairo University, Cairo, Egypt). TMS was used as an internal standard and chemical shifts were recorded in ppm on δ scale. Elemental analyses were carried out at the regional center for mycology and biotechnology, Al-Azhar University, Cairo, Egypt. The electron impact (EI) mass spectra were recorded on Hewlett Packard 5988 spectrometer Microanalytical center (Cairo 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 of products. All reagents and solvents were purified and dried by standard techniques. 7-Bromo-1-benzothiophen-3(2H)-one (1) was prepared according to the published method21,22.

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.

7-Bromo-2-(4-bromobenzylidene)-1-benzothiophen-3(2H)-one (2a)

Yield: 80%; m.p.: 178–180 °C; IR (cm⁻¹): 1689 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.35–8.03 (m, 8H, Ar-H+ =CH); 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.

7-Bromo-2-(4-chlorobenzylidene)-1-benzothiophen-3(2H)-one (2b)

Yield: 72%; m.p.: 110–111 °C; IR (cm⁻¹): 1681 (C=O); ¹H NMR (300 MHz, DMSO-d₆) δ ppm 7.35–7.99 (m, 8H, Ar-H + =CH); Anal. Calcd for C₁₅H₈BrClOS: C, 51.23; H, 2.29. Found: C, 51.41; H, 2.26.

7-Bromo-2-(4-fluorobenzylidene)-1-benzothiophen-3(2H)-one (2c)

Yield: 70%; m.p.: 179–181 °C; IR (cm⁻¹): 1685 (C=O); ¹H NMR (300 MHz, DMSO-d₆) δ ppm 7.36–7.98 (m, 7H, Ar-H), 8.01 (s, 1H, =CH); Anal. Calcd for C₁₅H₈BrFOS: C, 53.75; H, 2.41. Found: C, 53.98; H, 2.49.

7-Bromo-2-(4-methoxybenzylidene)-1-benzothiophen-3(2H)-one (2d)

Yield: 74%; m.p.: 175–176 °C; IR (cm⁻¹): 2976, 2844 (CH-aliphatic), 1678 (C=O); ¹H NMR (300 MHz, DMSO-d₆) δ ppm 3.95 (s, 3H, OCH₃), 7.20–8.07 (m, 7H, Ar-H), 8.09 (s, 1H, =CH); ¹³C NMR (100 MHz, DMSO-d₆) δ 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 (cm⁻¹): 1678 (C=O), ¹H NMR (300 MHz, DMSO-d₆) δ ppm 7.36–7.98 (m, 7H, Ar-H), 8.00 (s, 1H, =CH); Anal. Calcd for C₁₅H₈BrNO₃S: C, 49.74; H, 2.23; N, 3.87. Found: C, 49.89; H, 2.21; N, 3.98.

7-Bromo-2-(pyridin-2-ylmethylidene)-1-benzothiophen-3(2H)-one (2f)

Yield: 70%; m.p.: 120–122 °C; IR (cm⁻¹): 1681 (C=O); ¹H NMR (300 MHz, DMSO-d₆) δ ppm 7.30–7.95 (m, 7H, Ar-H), 7.97 (s, 1H, =CH); Anal. Calcd for C₁₄H₈BrNOS: C, 52.85; H, 2.53; N, 4.40. Found: C, 53.12; H, 2.60; N, 4.49.

General procedure for the synthesis of 2-amino-4-aryl-6-bromo-4H-[1]benzothieno[3,2-b]pyran-3-carbonitriles 3a–f

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 reaction mixture was cooled, and the separated solid was filtered, dried and crystallized from ethanol.

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 (cm⁻¹): 3475, 3342 (NH₂), 2924, 2852 (CH-aliphatic), 2204 (CN); ¹H NMR (300 MHz, DMSO-d₆) δ ppm 5.18 (s, 1H, H-4), 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.

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 (cm⁻¹): 3464, 3323 (NH₂), 2924, 2852 (CH-aliphatic), 2196 (CN); ¹H NMR (300 MHz, DMSO-d₆) δ 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) δ 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.

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 (cm⁻¹): 3466, 3321 (NH₂), 2924, 2852 (CH-aliphatic), 2194 (CN); ¹H NMR (300 MHz, DMSO-d₆) δ 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) δ 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.

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 (cm⁻¹): 3423, 3323 (NH₂), 2954, 2868 (CH-aliphatic), 2196 (CN); ¹H NMR (300 MHz, DMSO-d₆) δ 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) δ 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.

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 (cm⁻¹): 3460, 3319 (NH₂), 2900, 2800 (CH-aliphatic), 2194 (CN); ¹H NMR (300 MHz, DMSO-d₆) δ ppm 5.09 (s, 1H, H-4), 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.

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); ¹H NMR (300 MHz, DMSO-d₆) δ ppm 5.18 (s, 1H, H-4), 7.23 (s, 2H, NH₂, D₂O exchangeable), 7.29–8.56 (m, 7H, Ar-H); MS m/z: 385 [(M + 2), 55.68%], 383 [M⁺, 45.82%], 307 [(M + 2-C₅H₄N)⁺, 99.98%], 305 [(M − C₅H₄N)⁺, 100%]; Anal. Calcd for C₁₇H₁₀BrN₃OS: C, 53.14; H, 2.62; N, 10.94. Found: C, 53.26; H, 2.65; N, 11.08.

General procedure for the synthesis of 5-aryl-7-bromo-5H-[1]benzothieno[2′,3′:5,6]pyrano[2,3-d]pyrimidin-4-amines 4a–e

A mixture of benzothienopyran derivatives 3a–e (2 mmol) and formamide (5 mL) was heated under reflux for 5 h. The reaction mixture was cooled, poured onto water (25 mL) and the separated solid was filtered, dried and crystallized from aqueous ethanol.

7-Bromo-5-(4-bromophenyl)-5H-[1]benzothieno[2′,3′: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 (CH-aliphatic); ¹H NMR (300 MHz, DMSO-d₆) δ ppm 4.14 (s, 1H, H-5), 7.25–7.99 (m, 7H, Ar-H), 8.39 (s, 1H, H-2), 10.11 (s, 2H, NH₂, D₂O exchangeable); Anal. Calcd for C₁₉H₁₁Br₂N₃OS: C, 46.65; H, 2.27; N, 8.59. Found: C, 46.81; H, 2.32; N, 8.72.

7-Bromo-5-(4-chlorophenyl)-5H-[1]benzothieno[2′,3′:5,6] pyrano[2,3-d]pyrimidin-4-amine (4b)

Yield: 40%; m.p.: 103–105 °C; IR (cm ^− 1): 3383, 3307 (NH₂), 2974, 2858 (CH-aliphatic); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 4.16 (s, 1H, H-5), 7.23–8.47 (m, 7H, Ar-H), 8.40 (s, 1H, H-2), 10.11 (br s, 2H, 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.

7-Bromo-5-(4-fluorophenyl)-5H-[1]benzothieno[2′,3′: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 (CH-aliphatic); ¹H NMR (400 MHz, DMSO-d₆) δ 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.

7-Bromo-5-(4-methoxyphenyl)-5H-[1]benzothieno[2′,3′: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 (CH-aliphatic); ¹H NMR (300 MHz, DMSO-d₆) δ 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.

7-Bromo-5–(4-nitrophenyl)-5H-[1]benzothieno[2′,3′: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 (CH-aliphatic) 1530, 1346 (NO₂); ¹H NMR (400 MHz, DMSO-d₆) δ 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) δ 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, 50.12; H, 2.44; N, 12.31. Found: C, 50.29; H, 2.49; N, 12.49.

Biological testing23–27

Materials and methods

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.

Measurement of potential antiproliferative activity27

The human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM l-glutamine. For a typical screening experiment, cells were inoculated into 96 well microtiter plates in 100 μL at plating densities ranging from 5000 to 40 000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37 °C, 5% CO₂, 95% air and 100% relative humidity for 24 h prior to addition of test compounds.

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 at 400-fold the desired final maximum test concentration and 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 containing 50 μg/mL gentamicin. Additional four, 10-fold or ½ log serial dilutions were made to provide a total of five drug concentrations plus control. Aliquots of 100 μL of the different test compounds dilutions were added to the appropriate microtiter wells already containing 100 μL of medium, resulting in the 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 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 μL 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 and air dried. SRB solution (100 μL) at 0.4% (w/v) in 1% acetic 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 μL of 80% TCA (final concentration, 16% TCA). Using the seven absorbance measurements [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:

[(Ti − Tz)/(C − Tz)] × 100 for concentrations for which Ti ≥ Tz.

[(Ti − Tz)/Tz] × 100 for concentrations for which Ti < Tz.

Three dose response parameters were calculated for each compound. Growth inhibition of 50% (GI₅₀) was calculated from:

[(Ti − Tz)/(C − Tz)] × 100 = 50, which is the drug concentration resulting in a 50% lower net protein increase in the treated cells (measured by SRB staining) as compared to the net protein increase seen in the control cells. The drug concentration resulting in TGI was calculated from Ti = 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 from [(Ti − Tz)/Tz] × 00= −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 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.

Figure 2. GI₅₀ of compound 3e in μM against leukemia, non-small cell lung cancer, colon cancer and CNS cancer cell lines.

Figure 3. GI₅₀ of compound 3e in μM against melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer cell lines.

Table 1. Results of in vitro anticancer screening of compounds 2–4.

			Total MG-MID^a^,^b(μM)
Compound No.	Ar	Mean growth (%)	GI50	TGI	LC50
2a	4-BrC6H4	95.73	n.t	n.t	n.t
2b	4-ClC6H4	91.82	n.t	n.t	n.t
2c	4-FC6H4	79.57	n.t	n.t	n.t
2d	4-CH3OC6H4	88.62	n.t	n.t	n.t
2f	2-Pyridyl	93.14	n.t	n.t	n.t
3a	4-BrC6H4	20.25	0.76	6.02	32.36
3b	4-ClC6H4	15.92	0.67	5.24	33.11
3c	4-FC6H4	22.64	0.50	14.79	72.44
3d	4-CH3OC6H4	26.03	2.45	13.18	57.54
3e	4-NO2C6H4	17.13	0.11	7.94	42.66
3f	2-Pyridyl	36.88	n.t	n.t	n.t
4a	4-BrC6H4	89.26	n.t	n.t	n.t
4b	4-ClC6H4	91.28	n.t	n.t	n.t
4c	4-FC6H4	90.86	n.t	n.t	n.t
4d	4-CH3OC6H4	85.09	n.t	n.t	n.t

^aMG-MID is the calculated mean full panel or individual subpanel GI₅₀, TGI, or LC₅₀.

^b n.t: not tested.

Cell cycle analysis

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 × 10⁶ cell/T 75 flask, HCT-116 cells were exposed to compound 3e at its GI₅₀ concentration (0.05 μM) 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 Cycletest^TM Plus DNA Reagent Kit (BD Biosciences, San Jose, CA), according to the manufacturer’s instructions. Cell cycle distribution was determined using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA). The results are displayed in Figure 4.

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.

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 × 10⁶ cell/T 75 flask were exposed to compound 3e at its GI₅₀ concentration (0.05 μM) for 24 and 48 h. The cells then were collected by trypsinization and 0.5 × 10⁶ cells were washed twice with phosphate-buffered saline (PBS) and stained with 5 μl Annexin V-FITC and 5 μl PI in 1 × 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.

Figure 5. Effects of compound 3e on apoptosis of HCT-116 cells after 24 h and 48 h. The data were presented as means ± standard error of the mean for three independent replicates.

Results and discussion

Chemistry

Scheme 1 outlines the synthesis of the target compounds 2a–f, 3a–f and 4a–e.

Scheme 1. 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 C=O band at 1678–1689 cm⁻¹. ¹H NMR spectra of compounds 2a–f revealed the appearance of methine proton (=CH) at δ 7.97–8.09 ppm together with the introduced aromatic protons. Moreover, ¹³C NMR spectrum of compound 2d showed C=O carbon signal at δ 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 bromine 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 C=O band in the IR spectra of compounds 3a–f, together with the appearance of CN band at 2193–2204 cm⁻¹ and NH₂ bands at 3319–3475 cm⁻¹ 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 δ 5.03–5.23 ppm as well as an exchangeable singlet signal at δ 7.11–7.35 ppm corresponding to the NH₂ protons. Besides, ¹³C NMR spectra of compounds 3b–d indicated the disappearance of C=O carbon signal around δ 188 ppm and the appearance of C-4 and C-3 of the pyran ring at δ 24.4–25.0 ppm and δ 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 benzothieno[2′,3′: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 cm⁻¹ with no CN bands. Besides, their ¹H NMR spectra demonstrated the appearance of an exchangeable singlet signal at δ 10.05–10.11 ppm corresponding to the NH₂ protons. The pyrimidine proton (H-2) appeared as singlet signal at δ 8.39–8.40 ppm and notably the signal of the pyran proton (H-5) was shifted to lower frequency and appeared at δ 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.

In vitro anticancer screening

All the synthesized compounds except 2e and 4e (Ar= 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 10⁻⁵ 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 μM). 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 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-benzothieno[3,2-b]pyran-3-carbonitriles 3a–f and 5-aryl-benzothieno[2′,3′:5,6]pyrano[2,3-d]pyrimidin-4-amines 4a–d. 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 (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-benzothiophenones into benzothieno[3,2-b]pyrans greatly enhanced the cytotoxic activity, while, further cyclization into benzothieno[2′,3′:5,6]pyrano[2,3-d]pyrimidines reduced the cytotoxic 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 ring [compare 3a–e and 3f]. In addition, the presence of electron withdrawing substituent on the phenyl ring like nitro group and 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 μM, MG-MID TGI in the range of 5.24–14.79 μM and MG-MID LC₅₀ in the range of 32.36–72.44 μM. Four of the test compounds (3a–c and 3e) exhibited MG-MID GI₅₀ values less than 1 μM.

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 all the panels tested. Thus, compound 3e displayed GI₅₀ at submicromolar concentrations [GI₅₀ less than 1 μM in 83% of the tested cell lines and GI₅₀ less than 0.1 μM 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).

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

During cellular proliferation, cells pass through four distinct phases leading eventually to cell replication: G1 phase, S phase, G2 phase (collectively known as interphase) and M phase. These groups of events are known as the cell cycle. Disruption of cell cycle progression in cancer cells is considered as a milestone in cancer treatment29. Therefore the effect of compound 3e, which displayed the highest cytotoxic activity, on the cell cycle progression was characterized, in an attempt to understand its mechanism of action. Figure 4 showed that exposure of HCT-116 cells to compound 3e at the GI₅₀ concentration (0.05 μM) for 24 and 48 h, induced a significant disruption in the cell cycle profile including time dependent decrease in cell population at G1 phase (proliferation phase) with concomitant increase at pre-G and G2/M phase. This might indicate an ability of compound 3e to reduce the cellular proliferation and to induce cell cycle arrest at G2/M phase and induce DNA fragmentation, the hallmark of cell death, preventing the cells from proceeding towards replication and proliferation.

Apoptosis determination

To further study the mechanism of compound 3e-induced cell death, the ability of compound 3e to induce apoptosis was determined using an Annexin V (conjugated to FITC) apoptosis detection kit. This assay detects phosphatidylserine (PS) expressed on the surface of the apoptotic cells and fluoresces green after interacting with the labeled Annexin V. During early apoptosis, membrane asymmetry is lost, and PS translocates from the cytoplasmic side of the membrane to the external leaflet. Propidium iodide (PI), the counter stain used in this assay, has the ability to cross only compromised membranes to intercalate into the DNA. Therefore, PI is used to detect the late stages of apoptosis and necrosis by the presence of red fluorescence30. Figure 5 showed that, exposure of HCT-116 cells to compound 3e at its GI₅₀ (0.05 μM) for 24 h decreased the percentage of the survived cells and increased the percentage of Annexin-V positive cells indicating an early apoptosis (lower right quadrant). This effect further increased after 48 h of exposure with concomitant increase in PI positive cells indicating late apoptosis (upper right quadrant) compared to control cells.

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 μM, MG-MID TGI in the range of 5.24–14.79 μM and MG-MID LC₅₀ in the range of 32.36–72.44 μM. Four of the test compounds (3a–c and 3e) exhibited MG-MID GI₅₀ values less than 1 μM. Compound 3e, the most active compound in this work, displayed MG-MID GI₅₀, TGI, and LC₅₀ values of 0.11, 7.94 and 42.66 μM, respectively. The latter compound induced a significant disruption in the cell cycle profile parallel to its effect on apoptotic induction. Further studies are still needed to examine the possible anticancer effect of compound 3e in vivo as well as to determine its ADME profile.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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

Acknowledgements

The authors are grateful to NCI staff, Bethesda, MD, USA, for carrying out the antitumor testing of the synthesized compounds.