Discovery of a highly selective and potent pro-apoptotic acyclic phosphoramidate nucleotides for hepatocellular carcinoma

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

Description

[0001] Discovery of A highly selective and potent Pro-Apoptotic Acyclic Phosphoramidate Nucleotide for Hepatocellular carcinoma

[0002] BACKGROUND OF THE INVENTION:

[0003] Globally, hepatocellular carcinoma (HCC) is a leading cause of cancer and cancer- related deaths. The therapeutic efficacy of locoregional and systemic treatment in patients with advanced HCC remains low, which results in a poor prognosis. The development of sorafenib for the treatment of HCC has resulted in a new era of molecular targeted therapy for this disease.1

[0004] An important aspeXt of cancer therapeutics is the development of targeted therapy that makes use of chemi cal compounds designed to regulate the activity of specific molecular targets involved in critical oncogenic signaling pathways that ultimately govern the proliferation, growth, survival and distant metastatic dissemination of cancer cells. Consequently, targeted therapy has the advantage of delivering focused and powerful suppression of cancer development and progression, albeit with a lower toxicity to non-malignant cells, which is a common pitfail associated with systemic chemotherapy and radiotherapy.2

[0005] 2. BRIEF DESCRIPTION:

[0006] The invention described herein refers to compound(s) with identified potential Pro-Apoptotic compounds for hepatocellular carcinoma, promising antitumour activity and selectivity towards tumor cells that would be useful as anticancer drug.

[0007] Disclosed are compounds having a structure represented by formulas A and B and pharmaceutically acceptable salts thereof.

[0008]

[0009] 3. DETAILED DESCRIPTION OF THE INVENTION:

[0010] In one embodiment, the present invention provides compounds of formula A:

[0011] Wherein,

[0012] 7a: R1= Br; R2= H

[0013] 7b: R1= OCH3; R2= H

[0014] 7c: R1= R2= OCH3 7d: R1= Cl; R2= H

[0015] 7e: R1= CH3; R2= H

[0016] According to formula A, the resultant compounds will be as follows:

[0017] 9-(4-BromoDhenyl)-10-(2 -hydroxyethyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydro- acridine l,8(2 / f,5 / D-dione (7a)

[0018] 10-(2 -Hydroxyethyl)-9-(4-methoxyDhenyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydro acridine-l,8(2 / f.,5 / D-dione (7b)

[0019] 9-(3,4-DimethoxyDhenyl)-10-(2 -hydroxyethyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10- hexahydroacridine-l.,8(2 / f.,5 / D-dione (7c)

[0020]

[0021] 9-(4-ChloroDhenyl)-10-(2 -hydroxyethyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexa- hydroacridine-l,8(2 / f.,5 / D-dione (7d)

[0022] 10-(2z-Hydroxyethyl)-33.>6.,6-tetramethyl-9-(p-tolyl)-3.,4.,6.,7.,9J0-hexahydroacridine- l,8(2 / / ,5 / / )-dione (7e)

[0023] In second embodiment, the present invention provides compounds of formula B:

[0024]

[0025] Wherein,

[0026] 9a: R1= Br; R2= H

[0027] 9b: R1= OCH3; R2= H

[0028] 9c: R1= R2= OCH3

[0029] 9d: R1= Cl; R2= H

[0030] 9e: R1= CH3; R2= H

[0031] According to formula B, the resultant compounds will be as follows:

[0032] Isopropyl fGSV{2z-(9-(4-bromoDhenyl)-33.>6.,6-tetramethyl-l.,8-dioxo-2.,3.>4.,5.,6.,7.,8.,9- octahydroacridin-10(l / D-yl)ethoxyl(phenoxy)phosphoryll-L-alaninate (9a)

[0033]

[0034] Isopropyl [(5)-{2z-(9-(4-methoxyphenyl)-3,3,6,6-tetramethyl-l,8-dioxo-2,3,4,5,6,7,8,9- octahydroacridin-10( l / T)-yl)ethoxyl (phenoxy)phosphoryl] -L-alaninate ( 9b)

[0035] Isopropyl [(5)-{2z-(9-(3,4-dimethoxyphenyl)-3,3,6,6-tetramethyl-l,8-dioxo-2,3,4,5,6,7, 8,9- octahydroacridin-10(l / D-yl)ethoxyl(phenoxy)phosphoryl]-L-alaninate (9c)

[0036]

[0037] Isopropyl [GSV{2z-(9-(4-chlorophenyl)-3.,3.,6.,6-tetramethyl-l.,8-dioxo-2.,3.,4.,5.,6.,7.,8.,9- octahydroacridin-1 Of l / D-yl)ethoxyl f phenoxy)phosphoryl] -L-alaninate ( 9d)

[0038] Isopropyl IGSY)-!2z-(3,3,6,6-tetrametlnl-l,8-di()x()-9-( / Molyl)-2,3A5,6,7,8 / )-octalndro- acridin- 10 ( 1 / / )-yl )etho xy ! ( phenoxy)phosphoryl] -L-alaninate (9 e)

[0039]

[0040] 3.1. PHARMACEUTICAL PREPARATIONS AND METHODS OF ADMINISTRATION:

[0041] Compounds of the present application can be formulated with pharmaceutically acceptable excipients to produce pharmaceutical preparations suitable for human administration. Such preparations could be administered orally, parentally or as inhalations.

[0042] Accordingly, the oral pharmaceutical preparation containing the present compounds can be tablets, pellets and capsules. Pharmaceutically acceptable excipients suitable for oral preparations can be selected from, for example but not limited to lactose, Poly vinyl pyrrolidine (PVP), hydroxyl propyl methyl cellulose, sodium lauryl sulfate, silicone dioxide, magnesium stearate, calcium stearate, talc, PEG, HPMC, starch, alginates, etc.

[0043] 3.2. INDICATIONS AND CO- ADMINISTRATION:

[0044] The present compound is very useful in treatment of hepatocellular carcinoma if it is administered alone in therapeutically effective dose or in combination with other chemotherapeutic agents to promote their action against the targeted cancerous cells and reduce the severe side effects thereof.

[0045] Examples of the compounds that can administered simultaneously or sequentially with the compounds of the present invention include but not limited to: A- alkylating agents such as cyclophosphamide, mechlorethamine, chlorambucil, melpahalan, dacarbazine, nitrosoureas, and temozolomide. Anthracyclins such as daunorubicin, doxorubicin, valrubicin and idarubicin.

[0046] B- Taxanes such as paclitaxel, docetaxel, abraxane, taxorete.

[0047] C- Nucleotide analogues such as: Azacitidine, Azathioprine, Capecitabine, Cytarabine, Doxifluridine, Fluorouracil, Hydroxyurea, Mercaptopurin, methptrexate and Tioguanin.

[0048] D- Platinum based agents such as Carboplatin, Cisplatin and Oxaliplatin.

[0049] E- Vinca alkaloids such as vinblastine and vincristine

[0050] 3.3. SYNTHESIS:

[0051] 9-Aryl-10-(2-hydroxyethyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine- l,8(2H,5H)-dionesr7a-e1:

[0052] General Procedure: in a 50 mL round flask a mixture of dimedone (2.8 g, 20 mmol), different aromatic aldehydes (10 mmol), 2-aminoethanol (0.72 mL, 12 mmol) was dissolved in N,N- dimethylformamide (9ml) then p-Toluenesulfonic acid (PTS A) (1.5gm) or hydrochloric acid (HC1) 37% (1ml) was added as a catalyst. The mixture was stirred at 110 °C under reflux for 24 h. The progression of the reaction was monitored by TLC. After completion, the reaction mixture was cooled to room temperature then dropped into water (120 mL) with stirring until complete precipitation. The product was filtered off, washed with water, and dried at room temperature. The crude products were purified by recrystallizing in toluene to get pure compounds 7a-e. 9-(4-bromophenyl)-10-(2-hydroxyethyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10- hexahydroacridine-l,8(2H,5H)-dione (7a)

[0053] Pale-yellow powder; (3.3 g, 70% yield); m.p. 229-231 °C. IR (KBr) vmax (cm-1): 3488 (O- H); 2955 (=C-H, sp2); 2869 (C-H, sp3); 1629 (C=O, conjugated ketone); 1567, 1484 (0=0); 1233 (C-0 stretching) 3H-NMR (400 MHz, DMSO-d6) δ (ppm): 0.87 (s, 6 H, 2 CH3), 1.01 (s, 6 H,2 CH3), 2.02 (d, 2 H, 2JH-H = -16 Hz, CH2), 2.14 (d, 2 H, 2JH-H = -16 Hz, CH2), 2.44 (d, 2 H, 2JH-H = -16 Hz, CH2), 2.72 ( d, 2 H, 2JH-H = -16 Hz, CH2), 3.56 (t, 2 H, J = 4.0 Hz, CH2- N), 3.85 (t, 2 H, J = 4.0 Hz, CH2-O), 4.94 (s, 1 H, acridinedione-H9), 5.04 (t, 1 H, J = 4.0 Hz, D2O-exchangeable, O-H), 7.16 (d, 2 H, J = 8.0 Hz, Ar-H), 7.30 (d, 2 H, J = 8.0 Hz, Ar-H) .13C-NMR (125 MHz, DMSO-d6) δ (ppm): 195.63 (2 C=O), 152.40, 146.16, 130.93, 130.36, 118.95 (Ar-C), 113.60, 61.30, 49.93, 46.61, (40.11; under DMSO-d6), 32.48, 31.75, 29.29, 27.38. Anal. Calcd. for C25H3oBrN03: C, 63.56; H, 6.40; N, 2.96; Br, 16.91. Found: C, 63.66;

[0054] H, 6.50; N, 2.88; Br, 16.81.

[0055] 10-(2-hydroxyethyl)-9-(4-methoxyphenyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10- hexahydroacridine-l,8(2H,5H)-dione (7b)

[0056] Pale-yellow powder, (3.05 g, 72% yield); m.p.: 199-200 °C. IR (KBr) vmax (cm1): 3472 (O- H); 2957 (=C-H, sp2),- 2869, 2843 (C-H, sp3),- 1615 (C=O, conjugated ketone); 1561, 1465 (C=C); 1236 (C-0 stretching). 'H-NMR (400 MHz, DMSO-tf6) δ (ppm): 0.87 (s, 6 H, 2 CH3),

[0057] I.02 (s, 6 H, 2 CH3), 2.01 (d, 2 H,2JH-H = -16 Hz, CH2), 2.14 (d, 2 H,2JH-H = -16 Hz, CH2), 2.44 (d, 2 H,2JH-H = -16 Hz, CH2), 2.71 (d, 2 H,2JH-H = -16 Hz, CH2), 3.55 (t, 2 H, J= 4.0 Hz, CH2- N), 3.66 (s, 3 H, OCH3), 3.83 (t, 2 H, J= 4.0 Hz, CH2-O), 4.90 (s, 1 H, acridinedione-H9), 5.08 (t, 1 H, J= 4.0 Hz, O-H), 6.68 (d, 2 H, J= 8.0 Hz, Ar-H), 7.11 (d, 2 H, J= 8.0 Hz, Ar-H).13C- NMR (125 MHz, DMSO-tffc) δ (ppm): 195.69 (2 C=O), 157.53, 151.82, 139.20, 129.05, 125.81 (Ar-C), 114.19, 113.40, 61.30, 55.25 (OCH3), 49.94, 46.54, 39.81 (under DMSO-O, 32.50, 30.98, 29.43, 27.34. Anal. Calcd. for C26H33NO4: C, 73.73; H, 7.85; N, 3.31. Found: C, 73.59; H, 7.90; N, 3.22. 9-(3,4-dimethoxyphenyr)-10-(2-hydroxyethyr)-3,3,6,6-tetramethyl-3,4,6,7,9,10- hexahydroacridine-l,8(2H,5H)-dione (7c)

[0058] Pale-yellow powder, (3.63 g, 80% yield); m.p.: 206-209 °C. IR (KBr) Vmax Ccm'1): 3311 (O- H); 2990 (=C-H, sp2); 2867 (C-H, sp3); 1644 (C=O, conjugated ketone); 1563, 1460 (C=C);

[0059] 1237 (C-0 stretching). 'H-NMR (400 MHz, DMSO-tf6) δ (ppm): 0.86 (s, 6 H, 2 CH3), 1.01 (s, 6 H, 2 CH3), 2.01 (d, 2 H,2JH-H = -16 Hz, CH2), 2.15 (d, 2 H,2JH-H = -16 Hz, CH2), 2.44 (d, 2 H,2JH-H = -16 Hz, CH2), 2.71 (d, 2 H,2JH-H = -16 Hz, CH2), 3.55 (t, 2 H, J= 4.0 Hz, CH2-N), 3.63 (s, 3 H, OCH3), 3.64 (s, 3 H, OCH3), 3.91 (t, 2 H, J = 4.0 Hz, CH2-O), 4.90 (s, 1 H, acridinedione-H9), 5.16 (t, 1 H, J = 4.0 Hz, O-H), 6.70-6.75 (m, 3 H, Ar-H).13C-NMR (125 MHz, DMSO-tf6) d (ppm): 196.25 (2 C=O), 152.12, 148.45, 147.03, 139.20, 120.20, 111.49, 111.34 (Ar-C), 113.83, 61.35, 55.72 (OCH3), 55.53 (OCH3), 49.84, 46.49, 39.51 (under DMSO- J6), 32.39, 31.26, 29.62, 26.94. Anal. Calcd. for C27H35NO5: C, 71.50; H, 7.78; N, 3.09. Found: C, 71.39; H, 7.69; N, 2.99.

[0060] 9-(4-chlorophenyl)-10-(2-hydroxyethyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine- l,8(2H,5H)-dione (7d)

[0061] Pale-yellow powder, (3.124 g, 73% yield); m.p.: 176-179 °C. IR (KBr) vmax (cm1): 3335 (O- H); 2959 (=C-H, sp2); 2886 (C-H, sp3)- 1619 (C=O, conjugated ketone); 1563, 1487 (C=C);

[0062] 1238 (C-0 stretching).XH-NMR (400 MHz, DMSO-tf6) δ (ppm): 0.86 (s, 6 H, 2 CH3), 1.02 (s, 6 H, 2 CH3), 2.02 (d, 2 H,2JH-H = -16 Hz, CH2), 2.15 (d, 2 H,2JH-H = -16 Hz, CH2), 2.45 (d, 2 H,2JH-H = -16 Hz, CH2), 2.73 (d, 2 H,2JH-H = -16 Hz, CH2), 3.56 (t, 2 H, J= 4.0 Hz, CH2-N), 3.85 (t, 2 H, J= 4.0 Hz, CH2-O), 4.95 (s, 1 H, acridinedione-H9), 5.12 (broad s, 1 H, O-H), 7.18 (d, 2 H, J= 8.0 Hz, Ar-H), 7.23 (d, 2 H, J= 8.0 Hz, Ar-H).13C-NMR (125 MHz, DMSO-tf6) J (ppm): 195.69 (2 C=O), 152.40, 145.76, 130.46, 129.95, 128.06 (Ar-C), 113.56, 61.29, 49.84, 46.54, 39.82 (under DMSO-J6), 32.52, 31.66, 29.34, 27.33. Anal. Calcd. for C25H30CINO3: C, 70.16; H, 7.07; N, 3.27; Cl, 8.28. Found: C, 70.01; H, 6.98; N, 3.16; Cl, 8.11.

[0063] 10-(2-hydroxyethyl)-3,3,6,6-tetramethyl-9-(p-tolyl)-3,4,6,7,9,10-hexahydroacridine- l,8(2H,5H)-dione (7e) Pale-yellow powder, (3.34 g, 82% yield); m.p.: 202-205 °C. IR (KBr) vmax (cm’1): 3380 (O- H); 2957 (=C-H, sp2),- 2875 (C-H, sp3),- 1623 (C=O, conjugated, ketone); 1564, 1465 (C=C); 1237 (C-0 stretching).

[0064] 'H-NMR (400 MHz, DMSO-tf6) δ (ppm): 0.87 (s, 6 H, 2 CH3), 1.02 (s, 6 H, 2 CH3), 2.01 (d, 2 H,2JH-H = -16 HZ, CH2), 2.14 (d, 2 H,2JH-H = -16 HZ, CH2), 2.18 (s, 3 H, CH3), 2.44 (d, 2

[0065] H,2JH-H = -20 Hz, CH2), 2.71 (d, 2 H,2JH-H = -20 Hz, CH2), 3.57 (t, 2 H, J= 4.0 Hz, CH2-N), 3.83 (t, 2 H, J= 4.0 Hz, CH2-O), 4.92 (s, 1 H, acridinedione-H9), 5.07 (t, 1 H, J = 4.0 Hz, O- H), 6.92 (d, 2 H, J = 8.0 Hz, Ar-H), 7.07 (d, 2 H, J = 8.0 Hz, Ar-H).13C-NMR (125 MHz, DMSO-tf6) δ (ppm): 195.69 (2 C=O), 151.94, 143.93, 134.75, 128.71, 127.98 (Ar-C), 114.04, 61.28, 49.93, 46.55, 39.82 (under DMSO-J6), 32.49, 31.42, 29.44, 27.30, 21.03 (CH3). Anal. Calcd. for C26H33NO3: C, 76.62; H, 8.16; N, 3.44. Found: C, 76.53; H, 8.22; N, 3.56.

[0066] Isopropylf(5,)-{2-(9-aryl-3,3,6,6-tetramethyl-l,8-dioxo-2,3,4,5,6,7,8,9-octahydroacridin-10- (l / O-yl)ethoxy) (phenoxy)phosphoryll-L-alaninate (9a-e):

[0067] General Procedure: In a 50 mL round flask compounds 7a-e (5.0 mmol) were dissolved in N,N- dimethylformamide (20 mL), then isopropyl [( / ?)-(perfluorophenoxy)(phenoxy)phosphoryl)]-L- alaninate (2.27 g, 5.0 mmol) and l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.75 mL, 5.0 mmol) were added. The reaction mixture was stirred at room temperature for 4 h. The progression of the reaction was monitored by TLC. After completion, the reaction mixture was dropped into water (100 mL) with stirring untill complete precipitation. The product was filtered off, washed with water, and dried at room temperature. The crude products were purified by using silica column chromatography with ethyl acetate / w-hexane (1:1 v / v) as eluent to get the corresponding pure compounds 9a-e.

[0068] Isopropylf(S)-{2-(9-(4-bromophenyl)-3,3,6,6-tetramethyl-l,8-dioxo-2,3,4,5,6,7,8,9- octahydroacridin-10(l / O-yl)ethoxyl(phenoxy)phosphoryll-L-alaninate (9a)

[0069] Off-white powder, (2.89 g, 78 %yield); m.p.: 114-117 °C. IR (KBr) Vmaxfcm1): 3435 (N-H); 2959 (=C-H, sp2); 2875 (C-H, sp3),- 1736 (C=O, ester); 1634 (C=O, conjugated ketone); 1574, 1488 (C=C); 1151, 1211, 1244 (C-O, P=O stretching). 'H-NMR (400 MHz, DMSO-tf6) J (ppm): 0.87 (s, 6 H, 2 CH3), 1.00 (s, 6 H, 2 CH3), 1.13-1.19 (m, 9 H, 3 CH3), 2.00, 2.04 (dd, 2 H, Ji = 8.0 Hz, J2= 8.0 Hz, CH2), 2.14, 2.16 (dd, 2 H, Jx= 8.0 Hz, J2= 8.0 Hz, CH2), 2.46-2.50 (m, 2 H, CH2), 2.69-2.76 (m, 2 H, CH2), 3.77-3.79 (m, 1 H, CH-CH3), 4.11^.21 (m, 4 H, CH2- CH2), 4.79-4.87 [m, 1 H, CH(CH3)2], 4.95 (s, 1 H, acridinedione-H9), 6.09-6.18 (m, 1 H, D2O- exchangeable, N-H), 7.13-7.20 (m, 5 H, Ar-H), 7.30 (d, 2 H, J= 8.0 Hz, Ar-H), 7.34 (d, 2 H, J= 8.0 Hz, Ar-H).13C-NMR (125 MHz, DMSO-tf6) δ (ppm): 195.68 (2 C=O), 173.10 (C=O, ester), 151.99, 151.01, 145.82, 131.11, 130.29, 130.14, 125.10, 120.33, 119.05 (Ar-C), 113.90, 68.61, 66.01, 50.38, 49.79, 44.45, 40.00 (under DMSO-J6), 32.52, 31.70, 29.14, 27.52, 21.86, 20.20. Anal. Calcd. for C37H46BrN2O7P: C, 59.92; H, 6.25; N, 3.78; Br, 10.77; P, 4.18. Found: C, 60.00; H, 6.30; N, 3.82; Br, 10.70; P, 4.28.

[0070] Isopropyl[(S)-{2-(9-(4-methoxyphenyl)-3,3,6,6-tetramethyl-l,8-dioxo-2,3,4,5,6,7,8,9- octahydroacridin-10( l / D-yl)ethoxy) (phenoxy)phosphoryl] -L-alaninate ( 9b)

[0071] Off-white powder, (2.667 g, 77% yield); m.p.: 92-94 °C. IR (KBr) Vmax ^nr1): 3468 (N-H); 2957 (=C-H, sp2); 2835 (C-H, .sp3); 1737 (C=O, ester); 1631 (C=O, conjugated ketone); 1572, 1464 (C=C); 1151, 1210, 1242 (C-O, P=O stretching).XH-NMR (400 MHz, DMSO-tf6) J (ppm): 0.89 (s, 6 H, 2 CH3), 1.00 (s, 6 H, 2 CH3), 1.10-1.19 (m, 9 H, 3 CH3), 1.99, 2.03 (dd, 2 H, Ji = 8.0 Hz, J2= 8.0 Hz, CH2), 2.12, 2.16 (dd, 2 H, Jx= 8.0 Hz, J2= 8.0 Hz, CH2), 2.44-2.49 (m, 2 H, CH2), 2.68, 2.72 (dd, 2 H, Jx= 8.0 Hz, J2= 8.0 Hz, CH2), 3.59 (s, 3 H, OCH3), 3.74- 3.81 (m, 1 H, CH-CH3), 4.09-4.12 (m, 4 H, CH2-CH2), 4.80-4.87 [m, 1 H, CH(CH3)2], 4.92 (s, 1 H, acridinedione-H9), 6.10 (broad t, 1 H, J= 12.0 Hz, N-H), 6.67 (d, 2 H, J= 8.0 Hz, Ar-H), 7.08 (d, 2 H, J= 8.0 Hz, Ar-H), 7.14-7.19 (m, 3 H, Ar-H), 7.32 (t, 2 H, J= 8.0 Hz, Ar-H).13C- NMR (125 MHz, DMSO-tf6) δ (ppm): 195.78 (2 C=O), 173.13 (C=O, ester), 157.54, 151.49, 151.02, 138.82, 130.15, 128.96, 125.09, 120.33, 114.58 (Ar-C), 113.50, 68.64, 65.86, 55.15, 50.37, 49.89, 44.39, 39.81 (under DMSO-J6), 32.50, 30.95, 29.25, 27.45, 21.83, 20.21. Anal. Calcd. for C38H49N2O8P: C, 65.88; H, 7.13; N, 4.04; P, 4.47. Found: C, 65.80; H, 7.02; N, 3.99; P, 4.31.

[0072] Isopropyl [(S)-{2-(9-(3,4-dimethoxyphenyl)-3,3,6,6-tetramethyl-l,8-dioxo-2,3,4,5,6,7, 8,9- octahydroacridin-10(l / D-yl)ethoxyl(phenoxy)phosphoryl]-L-alaninate (9c) Off-white powder, (2.89 g, 80% yield); m.p.: 93-96 °C. IR (KBr) vmax (cm1): 3468 (N-H); 2957 (=C-H, sp2),- 2875 (C-H, sp3); 1737 (C=O, ester); 1632 (C=O, conjugated ketone); 1571, 1463 (C=C); 1146, 1181, 1243 (C-O, P=O stretching). 'H-NMR (400 MHz, DMSO-tf6) d (ppm): 0.88 (s, 6 H, 2 CH3), 1.01 (s, 6 H, 2 CH3), 1.12-1.20 (m, 9 H, 3 CH3), 2.00, 2.04 (dd, 2 H, Ji = 8.0 Hz, J2= 8.0 Hz, CH2), 2.14, 2.18 (dd, 2 H, Jx= 8.0 Hz, J2= 8.0 Hz, CH2), 2.46 (broad s, 2 H, CH2), 2.69-2.76 (m, 2 H, CH2), 3.60 (s, 3 H, OCH3), 3.66 (s, 3 H, OCH3), 3.75-3.78 (m,

[0073] 1 H, CH-CH3), 4.09-4.13 (m, 4 H, CH2-CH2), 4.82-4.93 [m, 1 H, CH(CH3)2], 4.93 (s, 1 H, acridinedione-H9), 6.09 (broad t, 1 H, J = 12.0 Hz, N-H), 6.68-6.72 (m, 3 H, Ar-H), 7.14-720 (m, 3 H, Ar-H), 7.32-7.36 (m, 2 H, Ar-H).13C-NMR (125 MHz, DMSO-tf6) δ (ppm): 195.86 (2 C=O), 173.13 (C=O, ester), 151.51, 150.97, 148.35, 147.13, 139.17, 130.15, 125.13, 120.37, 119.69, 114.34, 111.78, 68.64, 65.72, 55.15, 50.37, 49.89, 44.41, 39.82 (under DMSO-J6), 32.46, 31.08, 29.41, 27.22, 21.84, 20.16. Anal. Calcd. for C39H5IN2O9P: C, 64.81; H, 7.11; N, 3.88; P, 4.29. Found: C, 64.92; H, 7.01; N, 3.79; P, 4.34.

[0074] IsopropylH2-(9-(4-chlorophenyl)-3,3,6,6-tetramethyl-l,8-dioxo-2,3,4,5,6,7,8,9- octahydroacridin-10(lH)-yl)ethoxyl(phenoxy)phosphoryll-L-alaninate (9d)

[0075] Off-white powder, (2.615 g, 75% yield); m.p.: 110-113 °C. IR (KBr) Vmax (cm’1): 3448 (N- H); 2958 (=C-H, .s / r); 2873 (C-H, sp3\ 1131 (0=0, ester); 1630 (C=O, conjugated ketone); 1569, 1490 (C=C); 1149, 1210, 1241 (C-O, P=O stretching).XH-NMR (400 MHz, DMSO-tf6) δ (ppm): 0.87 (s, 6 H, 2 CH3), 1.00 (s, 6 H, 2 CH3), 1.10-1.20 (m, 9 H, 3 CH3), 2.00, 2.04 (dd,

[0076] 2 H, Ji = 8.0 Hz, J2= 8.0 Hz, CH2), 2.14, 2.18 (dd, 2 H, Jx= 8.0 Hz, J2= 8.0 Hz, CH2), 2.46- 2.50 (m, 2 H, CH2), 2.69-2.76 (m, 2 H, CH2), 3.74-3.81 (m, 1 H, CH-CH3), 4.11-4.21 (m, 4 H, CH2-CH2), 4.78-4.89 [m, 1 H, CH(CH3)2], 4.95 (s, 1 H, acridinedione-H9), 6.12 (broad t, 1 H, J= 12.0 Hz, N-H), 7.13-7.20 (m, 5 H, Ar-H), 7.30 (d, 2 H, J= 8.0 Hz, Ar-H), 7.34 (d, 2 H, J = 8.0 Hz, Ar-H).13C-NMR (125 MHz, DMSO -tf6) J (ppm): 195.68 (2 C=O), 173.15 (C=O, ester), 151.98, 151.01, 145.82, 131.92, 131.09, 130.29, 130.14, 125.10, 120.33 (Ar-C), 113.91, 68.61, 66.00, 50.38, 49.79, 44.43, 39.86 (under DMSO-J6), 32.52, 31.75, 29.14, 27.51, 21.85, 20.20.AnaL Calcd. for C37H46C1N2O7P: C, 63.74; H, 6.65; N, 4.02; Cl, 5.08; P, 4.44. Found: C, 63.66; H, 6.59; N, 3.79; Cl, 5.18; P, 4.35. Isopropyl _ r(S)-{2-(3,3,6,6-tetramethyl-l,8-dioxo-9-(p-tolyl)-2,3,4,5,6,7,8,9-octahydro- acridin- 10( lH)-yl)ethoxy 1 ( phenoxy)phosphoryll -L-alaninate ( 9e)

[0077] Off-white powder, (2.875 g, 85% yield); m.p.: 95-98 °C. IR (KBr) Vmax ^m1): 3450 (N-H); 2959 (=C-H, sp2); 2873 (C-H, sp3); 1738 (C=O, ester); 1638 (C=O, conjugated ketone); 1572, 1465 (C=C); 1142, 1161, 1200, 1242 (C-O, P=O stretching). 'H-XMR (400 MHz, DMSO-tf6) δ (ppm): 0.86 (s, 6 H, 2 CH3), 1.00 (s, 6 H, 2 CH3), 1.10-1.17 (m, 9 H, 3 CH3), 1.97, 2.01 (dd, 2 H, Ji = 8.0 Hz, J2= 8.0 Hz, CH2), 2.13 (s, 3 H, CH3), 2.15-2.19 (m, 2 H, CH2), 2.43-2.48 (m,

[0078] 2 H, CH2), 2.65-2.71 (m, 2 H, CH2), 3.74-3.81 (m, 1 H, CH-CH3), 4.08-4.19 (m, 4 H, CH2- CH2), 4.81-4.86 [m, 1 H, CH(CH3)2], 4.92 (s, 1 H, acridine- dione-H9), 6.04 (broad t, 1 H, J= 12.0 Hz, N-H), 6.91 (d, 2 H, J= 8.0 Hz, Ar-H), 7.04 (d, 2 H, J= 8.0 Hz, Ar-H), 7.12-7.19 (m,

[0079] 3 H, Ar-H), 7.31 (t, 2 H, J= 8.0 Hz, Ar-H).13C-NMR (125 MHz, DMSO-tf6) δ (ppm): 196.05 (2 C=O), 173.13 (C=O, ester), 151.18, 150.94, 143.47, 134.83, 130.16, 128.95, 128.85, 128.13, 125.16, 120.28 (Ar-C), 114.71, 68.76, 65.92, 50.37, 49.82, 44.30, 39.60 (under DMSO-J6), 32.43, 31.33, 29.22, 27.35, 21.81, 21.00, 20.13. Anal. Calcd. for C38H49N2O7P: C, 67.44; H, 7.30; N, 4.14; P, 4.58. Found: C, 67.19; H, 7.19; N, 3.89; P, 4.49.

[0080] 3.4. Biological evaluation:

[0081] 3.4.1 In-vitro Cytotoxicity assay

[0082] The in vitro anti-proliferative activity of the novel synthesized compounds was evaluated against different cancer cells including A549 (human lung adenocarcinoma), Caco-2 (human colon adenocarcinoma), Huh-7 (human hepatocellular carcinoma), and HepG-2 (human hepatocellular carcinoma) cell lines as well as to normal cell line WISH (derived from normal amnion) was used as a control cell.3

[0083] The results revealed that some compounds exhibited moderate to potent antitumor activities against four different cancer cell lines with IC5o values ranged between 12.82 and 77.85 pM (Table 1). Interestingly, this antitumor activity was remarkably potentiated upon insertion of phosphoramidate prodrug-forming group indicating not only enhancement of pharmacokinetic profiles of these compounds but also a synergistic antitumor activity. The notable improvement of the antitumor activities of phosphoramidate derivatives 9a-e compared to their nucleoside precursor’s 7a-e, is owing to the nature of phosphoramidate coupling group attached to the acridinedione core.

[0084] Table 1. The half maximal inhibitory concentration (IC50in pM) of the synthesized compounds 7a-e and 9a-e as compared to 5-FU (standard chemotherapy drug) on the normal (WISH) cells, Lung tumor (A549) cells, Colon tumor (Caco-2) cells, Liver tumor (Huh-7 cells) cells and Liver tumor (HepG-2) cells. All values are expressed as mean ± standard deviation.

[0085] 3.4.2. Tumor Selectivity Index (TSI)

[0086] Tumor Selectivity Index (TSI) value was determined by dividing the half maximal inhibitory concentration IC5o of normal cells by the IC5o of tumor cells.4

[0087] TSI = IC50 on normal cells / IC50 on Cancer cells

[0088] Thus, obtained TSI values seem to reflect the in vivo antitumor activity, even though these cells were different types of cells. The higher the TSI ratio, the theoretically more effective and safer a drug would be during in vivo treatment for a given tumor. A SI value > 10 was assumed to belong of a selected potential compound that can be further investigated. Ideally the drug can kill the cancer cells, but it should not affect the normal cells.5Among the tested compounds 9a exhibited the highest degree of cytotoxic tumor selectivity (TSI = 16.91, IC50 = 12.82 pM) against Liver tumor cells (Huh-7) (Table 2).

[0089] 4. Conclusion

[0090] A novel series of 9-Aryl-10-(2'-hydroxyethyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10- hexahydroacri dine- 1,8 (2H,5H)-diones 7a-e and their phosphoramidate derivatives 9a-e were successfully prepared. Phosphoramidate 9a exhibited the highest degree of tumor selectivity of 16.91 with IC50 determined to be 12.82 pM against liver tumor (Huh-7) cells with high safety against normal cells. These encouraging results suggest phosphoramidate compound 9a as a prospective candidate for further development as a new anticancer agent especially for liver carcinoma. In the mechanistic studies, Cell cycle analysis and gene expression were carried out and it was found that compound 9a inhibit the cell proliferation in both G0 / G1 and G2 / M phases of the cell cycle and the primary cause of death of the cells was found to be through apoptosis. Moreover, the most active compound 9a showed a significant effect on intrinsic apoptotic protein upregulated expression levels of Bax and downregulated Bcl-2 gene expression through RT-PCR assay.

[0091] References

[0092] 1. Chow, A. K. M., Yau, S. W. L., & Ng, L. (2020). Novel molecular targets in hepatocellular carcinoma. World Journal of Clinical Oncology, 11(8), 589.

[0093] 2. Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A., & Jemal, A. (2018). Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians, 68(6), 394-424.

[0094] 3. Setyowati, E. P., Pratiwi, S. U. T., & Purwantini, I. (2018). In-vitro cytotoxicity and apoptosis mechanism of ethyl acetate extract from Trichoderma reesei strain TV221 associated with marine sponge: Stylissa flabelliformis. Journal of Applied Pharmaceutical Science, 8(9), 151-157.

[0095] 4. Pena-Moran, O. A., Villarreal, M. L., Alvarez-Berber, L., Meneses-Acosta, A., & Rodriguez-Lopez, V. (2016). Cytotoxicity, post-treatment recovery, and selectivity analysis of naturally occurring podophyllotoxins from Bursera fagaroides var. fagaroides on breast cancer cell lines. Molecules, 21(8), 1013. Lopez-Lazaro, M. (2015). How many times should we screen a chemical library to discover an anticancer drug?. Drug Discovery Today, 2(20), 167-169. Ye, X. Y., Wang, H. X., Liu, F., & Ng, T. B. (2000). Ribonuclease, cell-free translation- inhibitory and superoxide radical scavenging activities of the iron-binding protein lactoferrin from bovine milk. The international journal of biochemistry & cell biology, 32(2), 235-241. Vilar, S., Cozza, G., & Moro, S. (2008). Medicinal chemistry and the molecular operating environment (MOE): application of QSAR and molecular docking to drug discovery. Current topics in medicinal chemistry, 8(18), 1555-1572.

Claims

Claims

1. A non-cyclic phosphoramidate nucleotide analog represented by the structures 9a-e or a pharmaceutically acceptable drug thereof, wherein said non-cyclic phosphoramidate nucleotide analog 9a exhibits potent anti-cancer activity against hepatocellular carcinoma cells, specifically human liver tumor cells Huh-7 and HepG2, with half- inhibitory concentration (IC5o) values of 12.82 and 23.37 micromolar, respectively, and high tumor selectivity ratios of 16.91 and 9.28, respectively. Claim 2] The non-cyclic phosphoramidate nucleotide analog of claim 1, wherein compound 9a demonstrates a high safety profile for normal cells, indicating a reduced cytotoxic effect on non-tumor cells. Claim 3] The non-cyclic phosphoramidate nucleotide analog of claim 1, wherein the synthesis method involves a simple two-step process, allowing for efficient and cost-effective production of compound 9a using readily available starting materials. Claim 4] The non-cyclic phosphoramidate nucleotide analog of claim 1, wherein compound 9a exhibits selectivity towards liver tumor cells, providing a targeted treatment option for hepatocellular carcinoma. Claim 5] The non-cyclic phosphoramidate nucleotide analog of claim 1, wherein compound 9a exhibits selectivity towards liver tumor cells, providing a targeted treatment option for colorectal cancer. Claim 6] The non-cyclic phosphoramidate nucleotide analog of claim 1, wherein compound 9a exhibits selectivity towards liver tumor cells, providing a targeted treatment option for Lung cancer. Claim 7] The non-cyclic phosphoramidate nucleotide analog of claim 1, wherein the synthesis process is scalable to an industrial level, enabling large-scale production of compound 9a for pharmaceutical applications.