Nitrophenyl-group-containing Heterocycles. 3. New Isoquinolines, as antiprolifative agents against MCF7and  HEGP2  Cell  lines. Synthesis, characterization and biological Evaluation.

doi:10.21203/rs.3.rs-5011049/v1

In this study, 7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8- (3-nitrophenyl or 4-nitrophenyl)-5,6,7,8-tetrahydrosoquinoline-3(2H)-thiones 2a-b were synthesized and used as starting materials. Thus, compounds 2a-b were reacted with methyl iodide, ethyl chloroacetate, by heating in ethanol in the presence of sodium acetate trihydrate to give 3-substituted methylthio-5,6,7,8-tetrahydroisoquinoline-4-carbonitriles 3, 4, respectively. In a similar manner, the reaction of compounds 2a-b with N-arylchloroacetamides5a-c afforded the corresponding N-aryl-(5,6,7,8-tetrahydroiso-quinolin-3-ylthio) acetamides 6a-c in excellent yields. In contrast, the reaction of 3b with N-(benzthiazol-2-yl)-2-chloroacetamide (12)under the same (above) conditions yielded 1-amino-N-(benzthiazol-2-yl)-6,7,8,9-tetrahydrothieno[2,3-c]isoquinoline-2-carboxamide13.Cyclization of compounds 6a-c into their 7a-cwas performed by heating in ethanol containing a catalytic amount of sodium ethoxide. The structures of all newly synthesized compounds were characterized by elemental and spectral analyses. Also, most of the synthesized compounds were evaluated for their anticancer activity in MCF7 andHEGP2 cell lines.The most potent compound against theMCF7 cell lines was compound 9b, and the most potent against HEGP2 cell lines was compound 3. Then the effects of compound 3 on the proliferation of HEPG2 cell lines was investigated using an apoptotic Annexin V-FITC test and flow cytometry. Compound 3 induced a 59-fold increase in HEPG2 cell line apoptosis and cell cycle arrested at the G0-G1, G2/M phases.

Anticancer

Apoptosis

Cell cycle

HEPG2 cell line

MCF7 cell line

5

6

7

8-tetrahydrosoquinoline

Cancer is a type of hereditary disease in which cells divide uncontrollably due to mutations in genetic expression, resulting in unbalanced cell proliferation [1, 2]. It is the leading cause of morbidity and mortality[3]. Whereas benign tumors remain fixed in one spot, separated from the rest of the body by a sac, making them easy to remove, malignant tumors are highly unstable and can metastasize to numerous body sections [4].

Liver cancer is the most common cause of cancer deaths worldwide and the fifth most common in the United States [5]. It is the only one of the top five deadliest malignancies with an annual percentage increase in prevalence [6, 7]. Developed countries have Developing countries have a higher incidence of liver disorders [8]. Risk factors include hepatitis B and C viruses [9], fatty liver disease, alcohol-related cirrhosis [7], smoking, obesity, diabetes, iron overload, and different dietary exposures [10]. The prognosis for liver cancer is dismal. The treatment options for more advanced stages include the following: (a) Transarterial chemoembolization (TACE), which improves 2-year survival by 23% compared to conservative therapy for intermediate-stage HCC patients (b) Oral dose with sorafenib, a kinase inhibitor and the most commonly used treatment for late-stage patients. However, less than one-third of patients benefit from the treatment, and medication resistance emerges within six months after starting the program [11]. Long-term usage of chemotherapeutic medicines, such as sorafenib, can lead to toxicity and/or pharmacological inefficacy. As a result, neither existing ablation therapies nor chemotherapy are significantly useful in improving the outcomes of this deadly disease. More research into better strategies for treating liver cancer is required. Cancer prevention, development, progression, and treatment are all linked to a patient's nutrition. A European study found that eating more fruits and vegetables lowers the risk of developing cancer [12–15].

Some chemicals exhibit selectivity in cytotoxicity to cancer cells while leaving non-cancerous cells unharmed [16]. Piperine, for example, inhibits enzymes required for drug metabolism, implying that co-administration with existing or planned chemotherapeutic medicines may be used to raise plasma concentrations [17]. Also Polysaccharides from Lentinus edodes and Tricholoma matsutake. Enhance the inhibitory action of 5-fluorouracil (5-FU) [18].

Breast cancer is the most common type of cancer in women worldwide, accounting for 2.26 million cases each year, 11.7% of all cancer cases, and 24.5% of female cancer cases [19-20]. The use of cytotoxic chemotherapy medicines is the currently accepted treatment option for breast cancer, either alone or in combination with other medical procedures such as surgery and radiation therapy. There are several FDA-approved chemotherapeutic medicines available for cancer treatment, including 5-fluorouracil (5-FU), capecitabine, docetaxel, doxorubicin, epirubicin, gemcitabine, methotrexate, paclitaxel, tamoxifen citrate, and nucleosides [21]. However, they are still considered hazardous substances and have been linked to long-term negative effects [21, 22]. A continuous research effort is underway to generate powerful new medicines or improve chemotherapeutics. A new chalcone derivative based on tetrahydro-[1,2,4]triazolo[3,4-a]isoquinolin-3-yl)-3-arylprop-2-en-1-one was produced and evaluated on mouse (Luc-4T1) and human (MDA-MB-231) breast cancer cell lines [19]. Also, a cycloplatinated (II) complex based on isoquinoline alkaloid induces ferritinophagy-dependent ferroptosis in triple-negative breast cancer cells [23].

The 5,6,7,8-tetrahydroisoquinoline ring system is a structural element of numerous alkaloids, ranking second only to indole alkaloids in abundance [24]. Compounds having a 5,6,7,8-tetrahydroisoquinoline fragment are utilized as intermediates in the synthesis of alkaloids [25]. Precursors to enzyme inhibitors [26–29] and viral infections [30]. 5,6,7,8-Tetrahydroisoquinoline compounds have also been demonstrated to have anticonvulsant properties [31]. Antibacterial [32], neurotropic [33], and antibacterial properties [34]. Also, 5,6,7,8-tetrahydroisoquinoline compounds have been used as anticancer agents [35-38].

On the other hand, numerous nitro-group-containing compounds have been shown to have a wide range of applications in biochemistry and medicine, including antioxidants and anticancers [39–42].

In light of the foregoing discoveries and as a continuation of our previous [43–45] work on tetrahyroisoquinolines, the purpose of this research was to synthesize and analyze the title compounds in the hope that these new compounds will find useful applications as anticancer drugs.

2.1. Synthesis part

The starting components are 7-acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(2-nitrophenyl, 3-nitrophenyl, or 4-nitrophenyl).-5,6,7,8-tetrahydrosoquinoline-3(2H)-thiones 2a-b were synthesized by cyclocondensation of 2,4-diacetyl-5-hydroxy-5-methyl-3-(2-nitrophenyl, 3-nitrophenyl, or 4-nitrophenyl).cyclohexanones 1a-b with 2-cyanothioacetamide by refluxing in ethanol using pipridine as a basic catalyst, similar to the described processes (Scheme 1).

Refluxing compounds 2a-b with halocompounds like methyl iodide, ethyl chloroacetate, and chloroacetonitrile in ethanol with slightly excess sodium acetate trihydrate for one hour resulted in the formation of 3-(un)substituted methylthio-5,6,7,8-tetra-hydroisoquinoline-4-carbonitriles 3-5 (Scheme 1).

In addition, under the same (above) circumstances, compounds 2a-b reacted with N-aryl-2-chloroacetamides 5a-c to produce the corresponding N-aryl-(5,6,7,8-tetrahydroiso-quinolin-3-ylthio)acetamides 6a-c in excellent yields. Compounds 6a-c undergo cyclization to provide the equivalent 7-acetyl-1-amino-N-aryl-5,8-dimethyl-8-hydroxy-6-(3-nitrophenyl/4-nitrophenyl)-6,7,8,9-tetrahydrothieno[2,3-c]Isoquinoline-2-carboxamides 7a-c were obtained by heating with catalytic quantities of sodium ethoxide in abs. ethanol for 5 mins. Compounds 7a-c were also synthesized via heating compounds 2a-b with the respective N-aryl-2-chloroacetamides 5a-c in abs. ethanol in the presence of slightly excess molar amounts of sodium ethoxide for 10 mins. (Scheme 1). Conversion of 6a-c into the corresponding 7a-c obeys intramolecular Thorpe-Ziegler cyclization which its mechanism is outlined before in our publication.

In a similar manner, reaction of compound 2a-c with N-(1-napthyl)-2-chloroacetamide (8) by refluxing in ethanol, in the presence of slightly excess molar amounts of sodium acetate trihydrate, for one hour gave the corresponding N-(1-naphthyl)-(5,6,7,8-tetrahydroisoquinolin-3-ylthio)acetamides 9a,b which canc cyclized to 10a-b (Scheme 2).

In contrast, reaction of 3b with N-(benzthiazol-2-yl)-2-chloroacetamide (11) under the same (above) conditions yielded 1-amino-2-[(N-(benzthiazol-2-yl)]-6,7,8,9-tetrahydro-thieno[2,3-c]isoquinoline-2-carboxamide 13 directly (Scheme 2).

2.2. Cytotoxic activity

The inhibition activity of all compounds 3-5,13, 2a, 6a, 7a, 9a, 9b and 10b was tested as one concentration spot 100 μg/ml against eight human cancer cell lines these cell lines were purchased from national cancer institute, Cairo – Egypt. The eight cell lines are: (human liver carcinoma (HEPG2 and HUH7).human breast carcinoma (MCF7),colon carcinoma human (HCT116 and CACO2). human lung carcinoma (H460 and A459).

human osteosarcoma (MG-63). normal human skin cell line (HSF).

The activity of these compounds against the normal human skin cell line HSF indicates that all synthetic chemicals have a lower percentage of habitation against the normal cell line than does doxorubicin, indicating that these compounds are safer and more targeted than doxorubicin. Table 1, Figure 1, provides further test details; Table S1 provides raw data.
Next, two cell lines, HEPG2 and MCF7, were assessed in vitro cytotoxcicity using the MTT test technique at various doses ranging from 0 to 100 g/mL. In this work, under the same experimental settings, doxorubicin was utilized as a positive control agent for comparison with drug candidates 3-5, 13, 2a, 6a, 7a, 9a, 9b, and 10b. Various ratios of these substances were examined in order to attain the concentration that, after 48 hours, may lead to 50% of the cancer cells dying.
Out of all the compounds that were evaluated, the results showed that: (i) two compounds, 3 and 2a, had the most active cytotoxic action against HEPG2, with IC₅₀ values of 75 and 82 g/ml, respectively (Fig. 2) (Table 2), when compared to the standard medication, doxorubicin (Table S3) in the accompanying information for the raw data.
(ii), and four compounds 9b, 3, 5, and 2a exhibited significant cytotoxic action against MCF7, with corresponding IC₅₀ values of 12, 33, 33, and 35 g/ml (Fig. 3). Additionally, the IC₅₀ of each synthesized compound is shown in Figure 3 and Table 2 in comparison to the standard medication doxorubicin (Table S4) in the supporting information for the raw data.

Table 1: Inhibition activity in one spot 100 µg/ml concentration of all compounds against normal skin cell line HSF in compared with Doxorubicin.

Compd.no.	Inhibition percent of HSF cell line	Compd.no.	Inhibition percent of HSF cell line
3	52	9a	59
4	50	9b	50
5	70	10b	58
13	56	Doxorubicin	60
2a	48
6a	56
7a	55

Table 2: IC₅₀of the synthesized compounds aganist MCF7 and HEPG2 cell lines.

Compd.no.	IC₅₀ FOR MCF7±S.D. μg/ml	IC₅₀ FOR HEPG2±S.D. μg/ml
3	33±0.034	75±0.063
4	47±0.055	80±0.070
5	33±0.042	97±0.053
13	>1000	>100
2A	35±0.061	82±0.054
6A	21±0.057	>100
7A	50±0.031	97±0.054
9A	50±0.019	88±0.072
9B	12±0.056	94±0.82
10B	80±0.050	87±0.61
DOX	4.58±0.081	4.13±0.054

2.3. Cell cycle arrest of HEPG2 Cells

We use flow cytometry to study the growth inhibitory cell cycle mechanism of HEPG2 cell lines after adding compound 3 in order to evaluate the growth inhibition mechanism of compound 3 in connection to cell cycle progression and regulation in HEPG2 cancer cells. DNA flow cytometry analysis was performed to evaluate the effect on cell cycle distribution. HEPG2 cells were incubated with compound 3 for 48 hours at its IC₅₀concentration of 75 µg/ml. HEPG2 cells exposed to compound 3 arrested in the G0-G1, G2/M phase of the cell cycle Figure 4, Table 3, with the G2/M phase fraction rising from 12.12% (in control cells) to 25.67%. Also compound 3 can arrested HEPG2 cells at the G0-G1% phase of the cell cycle, with the G2/M phase fraction increasing from 34.54 to 40.10.

Table 3. Cell cycle analysis of HEPG2 treated with compound 3

code	%G0-G1	%S	%G2/M
3/HEPG2	40.1	22.10	25.67
Cont.HEPG2	34.54	23.51	12.12

2.4. Apoptosis induced

According to the findings of the Annexin V-FITC/PI assay, compound 3 treatment of HEPG2 cells caused both early and late cellular apoptosis, as shown by a significant increase in the percentage of apoptotic cells in both the early and late apoptotic phases (from 0.20% to 13.45%) and (from 0.31% to 16.78%) respectively. indicating a significant increase in total apoptosis in comparison to the untreated control. Furthermore, as seen in Figures 5 and 6, Table 4. The percentage of necrotic cells rose from 1.36% to 2,96%. The previously cited findings demonstrate a 59-fold rise in HEPG2 cell death after treatment with compounds 3. So compound 3 possesses a biological mechanism that inhibits HEPG2 cell development, resulting in an anticancer cytotoxic impact.

Table 4: Apoptosis/necrosis assessment of HEPG2 cells after treatment with compounds 3.

code	Apoptosis			Necrosis
	Total	Early	Late
3/HEGP2	33.19	13.45	16.78	2.96
Cont.HEGP2	1.87	0.2	0.31	1.36

3.1. Instrumentations

Melting points were determined on a Gallan-Kamp apparatus and are uncorrected. The IR spectra were recorded on a Shimadzu 470 IR-spectrophotometer (KBr; νmax in cm-1). The 1H and 13C NMR spectra were recorded on a Varian A5 500 MHz spectrometer using DMSO-d6 or CDCl3 as a solvent and tetramethylsilane (TMS) as internal reference. Coupling constants (J values) are given in Hertz (Hz). The purity of the obtained products is checked by TLC.

3.2. Reaction of 2-acetylcyclohexanones 1a-b with cyanothioacetamide (2); Synthesis of compounds 2a-b; general method.

A mixture of compound 1a-b (10 mmol), cyanothioacetamide (2) (1.0 g, 10 mmol) and piperidine or morpholine (10 mmol) in ethanol (100 mL) was refluxed for 2 h. The yellow crystals that formed on hot were collected, washed with methanol, dried in air to give compounds 2a-b. The purity of these products is 100 % and need no any purification.

3.2.1. 7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(3-nitrophenyl)-5,6,7,8-tetra-hydroisoquinoline-3(2H)-thione (2a). Yield: 96 %; m. p: 279-280 °C. , FT-IR : 3429 cm-1 for (O-H), 3235 cm-1 for (N-H); 3139 cm-1 for (C-H, sp2); 2971 cm-1 for (C-H, sp3); 2221 cm-1 for (C≡N); 1710 cm-1 for (C=O). 1H NMR spectrum of compound 2a (400 MHz, DMSO-d6) showed signals at δ: 13.68 (s, 1H, NH); 7.95-8.05 (m, 2H, ArH); 7.51-7.58 (m, 2H, ArH); 5.05 (s, 1H, OH); 4.61-4.63 (d, J =10, 1H, C8H); 3.23-3.26 (d, J =15, 1H, C5H), 2.88-2.90 (d, J =10, 1H, C7H), 2.83-2.87 (d, J =20, 1H, C5H); 2.12 (s, 3H, COCH3); 1.86 (s, 3H, CH3);1.23 (s, 3H, CH3). Anal. calcd for C20H19N3O4S (397.11): C, 60.44; H, 4.82; N, 10.57%. Found: C, 60.67; H, 5.11; N, 10.28%.

3.2..2. 7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(4-nitrophenyl)-5,6,7,8-tetra-hydroisoquinoline-3(2H)-thione (2b). Yield: 93 %; m. p 290-291°C. FT-IR : 3482 cm-1 for (O-H); 3235 cm-1 for (NH); 3106 cm-1 for (C-H, sp2); 2971, 2872 cm-1 for (C-H, sp3); 2220 cm-1 for (C≡N); 1708 cm-1 for (C=O). 1H NMR spectrum of compound 2b in (400 MHz, DMSO-d6) showed signals at δ: δ13.83(S, H, NH), 7.84-7.86 (d, J =10, H, ArH); 7.62-7.64 (d, J =10, H, ArH); 7.51-7.53 (d, J =10, H, ArH); 7.33-7.34 (d, J =5, H, Ar), 5.04 (s, 1H, OH); 4.97-4.99 (d, J =10, 1H, C8H); 3.33-3.16 (d, J =20, 1H, C5H), 3.16-3.10 (d, J =5, 1H, C7H), 2.86-2.90 (d, J =20, 1H, C5H); 2.02 (s, 3H, COCH3); 1.93 (s, 3H, CH3); 1.29 (s, 3H, CH3). Anal. calcd for C20H19N3O4S (397.11): C, 60.44; H, 4.82; N, 10.57%. Found: C, 60.32; H, 5.04; N, 10.33%.

3.3. Reaction of compounds 2a-b with methyl iodide, ethyl chloroacetate, chloroacetonitrile or its N-aryl-2-chloroacetamides 5a-c; Synthesis of compounds 4-5, 6a-c general method.

A mixture of 2a-b (10 mmol), appropirate halocompound (10 mmol) and sodium acetate trihydrate (1.50 g, 11 mmol) in ethanol (100 mL) was refluxed for one hour. The solid that formed after cooling was collected and then recrystallized from ethanol to give white crystals of compounds 3-5, 6a-c.

3.3.1. 7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-3-methylthio-8-(3-nitrophenyl)-5,6,7,8-tetrahydroisoquinoline (3): Yield: 94 %; 148-150 m.p.: °C. IR: 3500 cm-1 for (O-H); 3077 cm-1 for (N-H); 2971 cm-1 for (C-H, sp2); -2931 cm-1 for (C-H, sp3); 2213 cm-1 for (C≡N); 1701 cm-1 for (C=O, acetyl). 1H NMR spectrum of compound 3 in (400 MHz, DMSO-d6) showed signals at δ 7.95-8.09 (m, J = 32 Hz, 2H, ArH); 7.55-7.95 (m, J = 28 Hz, 2H, ArH); 4.96-4.98 ( d, J = 28 Hz , 1H,OH) , 4.77- 4.79(d, J =38.8 Hz, 1H, C8H), 3.13-3.15 (t, J =12.6, 6.8 Hz , 4H, CH2, C5H, C7H), 2.87-2.97 (dd, J =17.2, 5.5 Hz , 4H,CH3, C5H);2.18(s,3H, COCH3 ), 1.96 (s, 3H, , CH3);1.27-1.29 (t, 3H, CH3).

13C NMR of compound 3 (126 MHz, dmso) δ 208.96, 160.66, 158.33, 150.10, 147.96, 146.09, 135.17, 130.18 128.28, 122.69 , 121.73, 115.18, 104.50, 67.40, 65.96 ,43,.27, 42.9, 31.04 , 27.54, 24.90 , 23.79 , 14.49.Anal. calcd for C22H23N3O4S( 425.1): C, 62.10; H, 5.45; N, 9.88; O, 15.04; S, 7.54, found C, 62.18; H, 5.44; N, 10.00.

3.3.2. Ethyl 2-[(7-acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(3-nitrophenyl)-5,6,7,8-tetra-hydroisoquinolin-3-yl)thio]acetate (4): : Yield:93 %; m.p.: 177- 180°C.

3495 cm-1 (O-H); 3079 cm-1 (N-H); 2984 cm-1 for (C-H, sp2); -2933 cm-1 (C-H, sp3); 2217 cm-1 (C≡N); 1723, 1700 cm-1 (C=O, acetyl). 1H NMR spectrum of compound 4 in (400 MHz, DMSO-d6) showed signals at δ: 8.12-8.14 (dd, J = 8.2, 1.0 Hz 1H, ArH); 7.8 (s, J = 8.2 Hz, 1H, ArH); 7.50-7.54(t, J = 7.9 Hz, 1H, ArH); 7.35-7.36(d, J = 7.7 Hz, 1H, ArH); 4.5-4.52 ( d, J = 10.7 Hz 1H,OH) , 4.11-4.14 (J = 6.9, 5.7 Hz, dd, 2H, CH2 acetate), 3.91 (t, J =12.6, 6.8 Hz , 2H, C8 H, C5H ), 2.91-3.18 (m, J =17.2, 5.5 Hz , 4H, CH2, C7H,C5H);1.85-1.88 (d, J = 16.6 Hz 6H,CH3, COCH3 ), 1.38 (s, 3H,CH3);1.2 (s, 3H, CH3). Anal. calcd for C24H25N3O6S( 483.1): C, 59.61; H, 5.21; N, 8.69; O, 19.85; S, 6.635, found C, 59.59; H, 5.20; N, 8.75.

3.3.3. 2-[(7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(4-nitrophenyl)-5,6,7,8-tetra-hydroisoquinolin-3-yl)thio]acetoonitriile (5): It is synthesized by reaction of 2b with chloroacetonitrile. Yield:89 %; m.p.: 154-155°C. IR: 3508 (O-H) cm-1; 3108 (N-H); 2972 cm-1 for (C-H, sp2), 2931 cm-1 (C-H, sp3); 2250, 2216 cm-1 (C≡N); 1701 cm-1 (C=O, acetyl). 1H NMR spectrum of compound 5 in (400 MHz, DMSO-d6) showed signals at δ 8.15( d, J = 8.5 Hz, 98.3 Hz, 2H,2 Ar-H),7.38 ( d, J = 8.5 Hz, 58.3 Hz, 2H, 2Ar-H), 4.98 (d, J =38.8 Hz, 1H, OH), 4.81(sd, J = 10.3 Hz, 1H, C8H), 4.32( d, J = 8.1 Hz, 2H,CH2), 3.34 (d , d, J = 17.2 Hz 1H, C5H), 2,96 (t, J =17.,15,13.2 Hz , 2H, C7H, C5H); 2.19 ( d, 3H, COCH3), 2.05 (d, 3H, CH3);1.31 (s, 3H, CH3). 13C NMR spectrum of compound 5 (126 MHz, DMSO-d6) showed 20 environments of carbon as expected at δ: 209.08, 161.57, 155.28, 152.02, 151.10, 146.70, 130.07, 124.33,118.02, 115.02,106.8, 67.9, 66.18, 65.52, 43.80, 31.65, 27.97, 25.04, 18.98, 15.82. Anal. calcd for C23H20N4O5S(464.1): C, 59.47; H, 4.34; N, 12.06. Found C, 59.55; H, 4.30; N, 12.1 %.

3.3.4. 2-[(7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(3-nitrophenyl)-5,6,7,8-tetra-hydroisoquinolin-3-yl)thio]-N-(4-acetylphenyl)acetamide (6a): It was synthesized by reaction of 2a with N-(4-acetylphenyl)-2-chloroacetamide . Yield: 93 %; m.p.: 231- 235 °C. IR: 3420 cm-1 (O-H); 3343 cm-1 (N-H); 2924 cm-1 (C-H, sp2); 2217 cm-1 (C≡N); 1703,1674 cm-1(C=O, acetyl).1H NMR spectrum of compound 6a in (400 MHz, DMSO-d6) showed signals at δ: 10.57 (s, 1H, NH), 8.06-8.11 (d, 2H, ArH), 7.84-7.86 (d, 2H, ArH), 7.62-7.65 (d, 2H, ArH), 7.28-7.31 (d, 2H, ArH), 5.02 (s, 1H, OH), 4.76-4.78 (d, 1H, C8H), 4.36-4.38 (d, 1H, C5H), 4.11-4.13 (dd, 2H, SCH2), 2.88-2.91 (m, 2H, C7H and C5H), 2.12 (s, 3H,COCH3), 1.80 (s, 3H, COCH3), 1.23 (s, 3H, CH3 attached to pyridine ring), 1.03 (s, 3H, CH3). Anal. Calcd. for C30H28N4O6S (572.17): C, 62.92; H, 4.93; N, 9.78%. Found: C, 62.95; H, 5.09; N, 9.75 %.

3.3.5. 2-[(7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(4-nitrophenyl)-5,6,7,8-tetra-hydroisoquinolin-3-yl)thio]-N-(4-acetylphenyl)acetamide (6b): It was synthesized by reaction of 2b with N-(4-acetylphenyl)-2-chloroacetamide . Yield: 90 %; m.p.: 213-215 °C. IR: 3540 cm-1 for (O-H); 3337 cm-1 for (N-H); 3109 cm-1 for (C-H, sp2); 2968 cm-1 for (C-H, sp3); 2220 cm-1 for (C≡N); 1683 cm-1 for (3 C=O); 1595 cm-1 for (C=N).1H NMR spectrum of compound 6b in (400 MHz, DMSO-d6) showed signals at δ: 10.57 (s, 1H, NH), 8.06-8.11 (d, 2H, ArH), 7.84-7.86 (d, 2H, ArH), 7.62-7.65 (d, 2H, ArH), 7.28-7.31 (d, 2H, ArH), 5.02 (s, 1H, OH), 4.76-4. 78 (d, 1H, C8H), 4.36-4.38 (d, 1H, C5H), 4.11-4.13 (dd, 2H, SCH2), 2.88-2.91 (m, 2H, C7H and C5H), 2.12 (s, 3H, COCH3), 1.80 (s, 3H, COCH3), 1.23 (s, 3H, CH3 attached to pyridine ring), 1.03 (s, 3H, CH3).

Anal. Calcd. for C30H28N4O6S (572.17): C, 62.92; H, 4.93; N, 9.78%. Found: C, 63.05; H, 5.00; N, 9.65 %.

3.3.6. 2-[(7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(4-nitrophenyl)-5,6,7,8-tetra-hydroisoquinolin-3-yl)thio]-N-(4-chlorophenyl)acetamide (6c): Yield: 94%; m.p.: 144-145 °C. IR: 3563 cm-1 for (O-H), 3344 cm-1 for (N-H); 3134 cm-1 for (C-H, sp2); 2971, 2937 cm-1 for (C-H, sp3); 2221 cm-1 for (C≡N); 1705 cm-1 for (C=O, acetyl); 1681 cm-1 for (C=O, amide). 1H NMR of compound 6c in (400 MHz, DMSO-d6) showed signals at δ: 10.35 (s, 1H, NH), 8.08-8.11 (m, 2H, ArH), 7.60-7.62 (d, 2H, ArH), 7.29-7.54 (m, 4H, ArH), 4.98 (s, 1H, OH), 4.71-4.73 (d, J = 10, 1H, CH at C-8), 4.06-4.14 (dd, J =15.0, 2H, SCH2), 3.42-3.44 (d, J = 7.0, 1H, C5H), 2.90-2.92 (m, 2H: C7H and C5H), 2.15(s, 3H, COCH3), 1.85 (s, 3H, CH3), 1.27 (s, 3H, CH3).13C NMR spectrum of compound 6c (126 MHz-, DMSO-d6) showed 28 environments of carbon as expected at δ: 208.53, 166.23, 164.75, 160.47, 157.63, 151.75, 150.04, 146.07, 137.85, 129.52, 128.73, 128.60, 126.83, 123.77, 120.90, 120.53, 114.98, 103.98, 67.39, 65.71, 55.99, 43.21, 42.65, 34.72, 31.02, 27.46, 24.45, 18.50.

Anal. Calcd. for C28H25ClN4O5S (564.12): C, 59.52; H, 4.46; N, 9.92%. Found: C, 59.20; H, 4.67; N, 10.07%.

3.4. Synthesis of 7-Acetyl-1-amino-2-(N-arylcarbamoyl)-5,8-dimethyl-8-hydroxy-6-(2-nitrophenyl, 3-nitrophenyl or 4-nitrophenyl)-6,7,8,9-tetrahydrothieno[2,3-c]isoquinolines 11a,d,e,f,g; general methods.

5.4.1. Method A)

To a suspension of 6a-c (10 mmol) in abs. ethanol (60 mL), a methanolic sodium methoxide was added (prepared by dissolving 0.25 g of sodium in 30 ml of methanol). The reaction mixture was stirred for one hour at room temperature. The yellow solid that formed after dilution with water (60 ml) was collected, washed with water, dried in air and then recrystallized from dioxane to give 7a-c, repectively.

3.4.1. 7-Acetyl-N-(4-acetylophenyl)-1-amino-5,8-dimethyl-8-hydroxy-6-(3-nitro-phenyl)-6,7,8,9-tetrahydrothieno[2,3-c]isoquinoline-2-carboxamide (7a).

It was obtained by cyclization of compound. Yield: %; m.p.:277-280 °C. IR: 3416, 3316 cm-1 for (O-H, NH2, NH); 2987 for (C-H, sp2);, 2917 cm-1 for (C-H, sp3);1701 cm-1 for (C=O. 1H NMR spectrum of compound 7a in (400 MHz, DMSO-d6) showed signals at δ: 9.73 (s, 1H, NH), 8.06 (s, 1H, Ar-H), 7.96 – 7.80 (m, 5H,NH2 , 3Ar-H), 7.54 (dd, J = 19.1, 7.6 Hz, 2H, Ar-H), 7.20 (s, 2H, Ar-H), 4.86 (s, 2H, OH, C7H), 3.66 (d, J = 17.4 Hz, 1H, C6H), 2.94 (d, J = 10.6 Hz, 1H, C6H), 2.55 (s, 3H COCH3), 2.20 (s, 3H,CO CH3), 2.03 (s, 3H, CH3), 1.33 (s, 3H, CH3).

13C NMR spectrum of compound 7a (126 MHz-, DMSO-d6) showed 24 environments of carbon as expected at δ: 13C NMR (126 MHz, dmso) δ: 209.42, 164.35, 158.33, 156.65, 149.62, 147.92, 147.04, 142.94, 135.07, 130.10 ,128.27, 128.32, 126.96, 122.95, 122.6, 122.4, 121.51, 96.8, 67.14, 65.88, 42.89, 41.99, 31.17, 27.94, 24.74. Anal. Calcd. for C30H28N4O6S (572.17): C, 62.92; H, 4.93; N, 9.78%. Found: C, 62.87; H, 5.29; N, 9.88 %.

3.4.2. 7-Acetyl-N-(4-acetylophenyl)-1-amino-5,8-dimethyl-8-hydroxy-6-(4-nitro-phenyl)-6,7,8,9-tetrahydrothieno[2,3-c]isoquinoline-2-carboxamide (7b). It was obtained by cyclization of compound 6b. Yield:89 %; m.p.: 301-302 °C. IR: 3424 cm-1 for (O-H); 3320 cm-1 for (N-H); Anal. Calcd. for: C32H34N4O4S: (570.23): C, 67.35; H, 6.00; N, 9.82%. Found: C, 67.51; H, 6.09; N, 9.74%.

3.4.3. 7-Acetyl-1-amino-N-(4-chlorophenyl)-5,8-dimethyl-8-hydroxy-6-(3-nitrophenyl)-6,7,8,9-tetrahydrothieno[2,3-c]isoquinoline-2-carboxamide (7c).

It was obtained by cyclization of compound 7c . Yield: 94 %; m.p.: 293-294 °C.

The FT-IR spectrum of compound 7c: 3417, 3383, 3314 cm-1 for (O-H, NH2, N-H); 3095 cm-1 for (C-H, sp2); 2967 for (C-H, sp2), 2916 cm-1 for (C-H, sp3); 1706 cm-1 for (C=O, acetyl); 1622 cm-1 for (C=O, amide) .1H NMR spectrum of compound 7c in (400 MHz, DMSO-d6) showed signals at δ: 9.56 (s, 1H, NH); 7.36-8.08 (m, 8H, ArH); 7.13 (s, 2H, NH2); 4.86-4.88 (d, J =10,1H, C6H); 4.85 (s, 1H, OH); 3.64-3.67 (d, J =15, 1H, C9H), 3.40-3.44 (d, J =20, 1H, C7H); 2.93-2.95 (d, J =10,1H, C9H); 2.21 (s, 3H, COCH3); 2.04 (s, 3H, CH3); 1.33 (s, 3H, CH3) (Figure 119). 13C NMR spectrum of compound 7c (126 MHz, DMSO-d6) showed 25 environments of carbon as expected at δ: 209.42, 164.35, 158.33, 156.65, 149.62, 147.92 , 147.04, 142.94, 135.07, 130.10 , 128.27, 128.23, 126.96, 122.95, 122.65, 122.41, 121.51, 96.81, 67.14, 65.88, 42.8, 41.99, 31.17, 27.94, 24.74. Anal. Calcd. for C28H25ClN4O5S (564.12): C, 59.52; H, 4.46; N, 9.92%. Found: C, 59.3; H, 5.01; N, 9.90%.

3.4.4. 2-[(7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(3-nitrophenyl)-5,6,7,8-tetrahydro-isoquinolin-3-yl)thio]-N-(naphthalen-1-yl)acetamide (9a)

It was obtained by reaction of compound 2a with N-(1-naphthyl)-2-chloroacetamide Yield: 86 %; m.p.: 237-238 °C. The FT-IR spectrum of compound 9a: 3527 cm-1 for (O-H); 3401 cm-1 for (N-H); 3063 cm-1 for (C-H, sp2); 2970, 2928 cm-1 for (C-H, sp3); 2214 cm-1 for (C≡N); 1702 cm-1 for (C=O, acetyl); 1665 cm-1 for (C=O, amide).1597 cm-1 for (C=N). 1H NMR spectrum of compound 9a in (400 MHz, DMSO-d6) showed signals at δ: 10.19 (s, 1H, NH); 7.30-8.01 (m, 11H, ArH); 5.01 (s, 1H, OH); 4.76-4.78 (d, J =10, 1H, C8H); 4.22-4.30 (dd, J =15, 2H, SCH2); 3.25-3.27 (d, J =10, 1H, C5H), 2.87-2.95 (m, 2H: C7H and C5H ), 2.15 (s, 3H, COCH3); 1.97 (s, 3H, CH3); 1.27 (s, 3H, CH3). Anal. Calcd. for C32H28N4O5S (580.18): C, 66.19; H, 4.86; N, 9.65%. Found: C, 65.88; H, 4.75; N, 9.41%.

3.4.5. 2-[(7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(4-nitrophenyl)-5,6,7,8-tetrahydro-isoquinolin-3-yl)thio]-N-(naphthalen-1-yl)acetamide (9b)

It was synthesized by reaction of 2b with N-(1-naphthyl)-2-chloroacetamide. Yield:93 %; m.p.: 230-232 °C. Yield: %; m.p.: 230 °C. IR: 3600-3489 cm-1 for (O-H); 3256 cm-1 for (N-H); 2970-2928 cm-1 for (C-H, sp2); 2217 cm-1 for (C≡N); 1703-1689 cm-1 for (C=O, acetyl). 1H NMR of compound 9b δ: (500 MHz, ) δ 10.23 (s, 1H, NH), 7.79 (t, J = 4.2 Hz, 5H, Ar-H), 7.56 (dd, J = 11.3, 5.5 Hz, 8H, Ar-H), 4.79 (d, J = 10.4 Hz, 1H, OH), 4.36 – 4.27 (m, 2H, SCH2), 3.46 (dd, J = 14.1, 7.0 Hz, 1H, C8H), 3.33 (d, J = 17.1 Hz, 1H, C5H), 3.02, – 2.93 (m, 2H, C7H and C5H), 2.17 (d, J = 3.3 Hz, 3H, CH3), 2.00 (d, J = 3.3 Hz, 3H, CH3), 1.31 (d, J = 3.3 Hz, 3H, CH3).13C NMR spectrum of compound 9b (126 MHz-, DMSO-d6) showed 28 environments of carbon as expected at δ 208.5, 166. 87, 165.62, 160.63, 157.87 ,151.84, ,150. 08, 146.09, 133.69, 131.8, 128.16, 126.12, 126.00, 122.53, 122.52, 121.88, 121.88, 115.1, 104.19, 67.4, 65.7, 61.96, 56.06, 43.35, 31.17, 27.51, 24.63, 18.53. Anal. Calcd. for C32H28N4O5S (580.18): C, 66.19; H, 4.86; N, 9.65%. Found: C, 66.33; H, 4.92; N, 9.68%.

3.4.6. 7-Acetyl-1-amino-N-(naphthalen-1-yl)-5,8-dimethyl-8-hydroxy-6-(3-Nitro phenyl)-6,7,8,9-tetrahydrothieno[2,3-c]isoquinoline-2-carboxamide (10a).

It was obtained by cyclization of compound 9a Yield: 96 %; m.p.: 290-293 °C. The FT-IR spectrum of compound 10a:, 3389 cm-1 for (O-H, NH2, NH); 2928 cm-1 for (C-H, sp2); 2855 cm-1 for (C-H, sp3); 1705 cm-1 for (C=O, acetyl). Anal. Calcd for: C33H30N4O4S( 578.69): C, 68.49; H, 5.23; N, 9.68%. Found: C, 69.1; H, 5.18; N, 9.52%.

3.4.7. 7-Acetyl-1-amino-N-(naphthalen-1-yl)-5,8-dimethyl-8-hydroxy-6-(4-Nitro phenyl)-6,7,8,9-tetrahydrothieno[2,3-c]isoquinoline-2-carboxamide (10b).

It was obtained by cyclization of compound 9b Yield: 89 %; m.p.: 286-289 °C. The FT-IR spectrum of compound 10b:

3479 cm-1 for (O-H, NH2, NH); 2928 cm-1 for (C-H, sp2); 2855 cm-1 for (C-H, sp3); 1715 cm-1 for (C=O, acetyl). Anal. Calcd for: C33H30N4O4S( 578.69): C, 68.49; H, 5.23; N, 9.68%. Found: C, 69.33; H, 5.25; N, 9.59%.

3.5. Synthesis of 7-Acetyl-1-amino-N-(benzthiazol-2-yl)-5,8-dimethyl-8-hydroxy-6-(3-nitrophenyl)-6,7,8,9-tetrahydrothieno[2,3-c]isoquinoline-2-carboxamide (13). It was obtained by cyclization of compound 12. A mixture of 2a (10 mmol), N-(benzthiazol-2-yl)-2-chloroacetamide (11) ( 10 mmol), and sodium acetate trihydrate (1.50 g, 11 mmol) in ethanol (100 mL) was refluxed for one hour. The solid that formed after cooling was collected and then recrystallized from ethanol to give white crystals of compound 13 directlly.

Yield: 97 %; m.p.:300-305 °C. : 3431, 3319 cm-1 for (O-H, NH2, NH); 2973cm-1 for (C-H, sp2); 2918 cm-1 for (C-H, sp3); 1707 cm-1 for (C=O, acetyl);

1H NMR (400 MHz, DMSO-d6) showed signals at δ 7.54 (dd, J = 19.1, 7.6 Hz, 11H,Ar-H), 4.86 (s, 1H, OH ), 3.66 (d, J = 17.4 Hz, 2H, C6H, C9H), 3.65 (d, J = 10.6 Hz, 1H, , C7H), 2.93 (s, 1H, , C9H ), 2.21 (s, 3H, COCH3), 2.05 (s, 3H, CH3), 1.34 (s, 3H, CH3).

13C NMR of compound 13 (126 MHz, dmso) δ 209.47, 158.18, 157.84, 147.91, 147.12, 142.94, 135.08, 130.10, 127.91.67, 123.27, 123.1, 122.4, 121.50, 67.16, 65.93, 42.90, 41.97, 31.17, 27.98, 24.74, 18.53.

Anal. Calcd. For C29H25N5O5S2 (587.67)%: C, 59.27; H, 4.29; N, 11.92; O, 13.61; S, 10.91., Found C, 58.07; H, 4.35; N, 12.00.

5. 1 Cytotoxicity against human cancer cell lines

In this work, all synthesized compounds were tested as anticancer activity in IC50 against two cell lines HEPG2 and MCF7 cells were tested using the sulphorhodamine-B (SRB) . Cells were seeded at a density of 3 *103 cells/well in 96-well microtiterplates [46]. They were allowed to connect for 24 hours before incubating with our compounds. Cells were treated with five doses of our synthesized compounds (0, 12.5, 25, 50, and 100 µg/mL). For each concentration, three wells were employed, and the incubation period lasted 48 hours. DMSO was employed as the control vehicle (1% v/v). Finally, they incubation, the cells were fixed with 20% trichloroacetic acid and stained with 0.4% SRB dye. The optical density (O.D.) of each well was measured spectrophotometrically at 570 nm with an ELISA microplate reader. The proportion of cell survival was determined as follows: Survival fraction = O.D. (treated cells) / O.D. (control cells). Sigmoidal dose-response curve-fitting models (GraphPad Prizm software, version 5) were used to obtain the IC50 (concentration that inhibits cell growth by 50%) for each chemical.

5.2. Cell cycle analysis:

The cell cycle arrests of compound 3 against HEGP2 at their IC50 concentration was carried out according to Abcam method (code ab139418), (www.abcam.co.jp) . The HEPG2 cells were collected using ice-cold ethanol and kept at -20 °C for 1 hour following treatment with an IC50 concentration dose of compounds 3. Then the cells were centrifuged and washed with ice-cold PBS, and incubated at 4°C for 20 minutes. The cell cycle was assessed using a flow cytometry kit (Propidium Iodide [ab13941]). Finally, use the Cell Quest software to undertake statistical analysis of the cell fractions in sub-G0/G1, S, and G2/M phases [45, 47,48].

5.3. Annexin-V FITC apoptosis assay

The Annexin-V FITC apoptosis assay of compounds 3 against HEPG2 at their IC50 values was performed according to (BioVision Research Products (code k101-25). (www.biovision.com).Thus, HEPG2 cell line were treated with the IC5O conc of the compound 3 for 24 h then collected by trypsin, centrifuged then rinsed with PBS and suspended in binding buffer, then dual-stained with Annexin V-FITC (5 μL) and propidium iodide (5 μL) in the dark for 15 min at romm temp. using flow cytometry to measure the cells with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. The final results were analyzed with the Cell quest software [45, 49, 50].

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

All data generated or analyzed during this study are in this published article and supplementary information.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This research received no funds.

Authors' contributions

Etify A. Bakhite: Conceptualization, Formal analysis, Supervision, Investigation. Reda Hassanien: Investigation, Methodology. Writing–review & editing. Nasser Farhan:Investigation, Writing–original draft, Writing–review & editing. Eman M. Sayed: Investigation, Methodology, Writing–original draft, Visualization, Software, Validation Marwa Sharaky: Conceptualization, Formal analysis, Supervision, Investigation, Methodology, Writing-original draft, Writing- review & editing.

AUTHOR INFORMATION

Corresponding Author

Eman M. Sayed- Chemistry Department, Faculty of Science, New Valley University, 72511 El-Kharja , Egypt. Email [email protected]

Authors

Etify A. Bakhite^_ Department of Chemistry, Faculty of science, Assiut University, Assiut 71516, Egypt; Orcid.org/0000-0003-3994-5629; Phone: +201006670292; Email: [email protected]

Reda Hassanien- Chemistry Department, Faculty of Science, New Valley University, 72511 El-Kharja, Egypt. Email: [email protected]

Nasser Farhan- Chemistry Department, Faculty of Science, New Valley University, 72511 El-Kharja, Egypt. Email: [email protected]

Eman M. Sayed- Chemistry Department, Faculty of Science, New Valley University, 72511 El-Kharja , Egypt. Email: [email protected]

Marwa Sharaky- Cancer biology department, national cancer institute, Cairo University, Egypt, Email: [email protected].

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No competing interests reported.

Schemes 1-2 are available in the supplementary files section.

Nitrophenyl-group-containing Heterocycles. 3. New Isoquinolines, as antiprolifative agents against MCF7and HEGP2 Cell lines. Synthesis, characterization and biological Evaluation.

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Abstract

Figures

1. INTRODUCTION

2. RESULTS AND DISCUSSION

3. EXPERIMENTAL

Declarations

References

Additional Declarations

Supplementary Files

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Version 1