5-Hydroxy-1,4-naphthalenedione for treating cancer

Abstract

The present invention discloses compounds for inhibiting the proliferation of uncontrolled cells, particularly cancer stem cells. In particular, the present invention relates to compounds of formulas I to IV for treating cancer.

Description

Field of the Invention

[0001] The present invention relates to compounds for inhibiting uncontrolled cell proliferation, particularly cancer cells. Background Art

[0002] Newer anti-cancer drugs act directly on abnormal proteins in cancer cells; this is known as targeted therapy. Most chemotherapeutic drugs can be classified as alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other anti-tumor agents. Although molecular targeted therapies can be used at high cost to treat cancer, most people in the world rely on standard chemotherapy.

[0003] Standard anti-cancer regimens target most dividing cancer cells, rather than quiescent or slowly dividing cancer stem cells (CSCs). Although CSCs were discovered not long ago, scientists worldwide are still searching for CSC-targeting reagents. Unfortunately, until today, there are no reagents specifically targeting CSCs on the market.

[0004] Therefore, it is important to develop CSC-specific therapies that can effectively inhibit CSCs and work alone or in combination with standard therapies to provide effective treatment options for cancer patients. Summary of the Invention

[0006] The present invention relates to compounds of formula I for treating various conditions, particularly for inhibiting uncontrolled cell proliferation or uncontrolled cell growth. In particular, these compounds are effective against cancer cells. These compounds are also effective against cancer stem cells. The structure of formula I is as follows:

[0007]

[0008] Wherein,

[0009] n is 1 - 10;

[0010] Q is O, S, -NY’, where Y’ is selected from -H, alkyl;

[0011] R1, R2, R3, and R4 are each independently selected from -H, alkoxy, alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aryl having a fused ring formed by heterocycloalkyl, -NH2, -NO2, -NHCOCH3, -CN, -O-, halogen, -OCF3, heterocycloalkyl, -O-(CH2) n -heterocycloalkyl;

[0012] R is selected from substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted cycloalkyl, -NR 10 R 11 、-NR 10 R 11 .HCl or acid salts, -OR10 R 11 、 -CONR 10 R 11 、 -NR 10 R 11 CONR 10 R 11 、 -NR 10 R 11 SOONR 10 R 11 、 -COOH,

[0013] wherein R 10 and R 11 are each independently selected from -H, alkyl, substituted or unsubstituted aryl, heteroaryl, alkylamine, substituted arylamine, substituted or unsubstituted cycloalkyl, -CH2-CH2-O-alkyl, or R 10 and R 11 together form a substituted or unsubstituted cycloalkyl or heterocycloalkyl, or R 10 and R 11 together form a substituted or unsubstituted cycloalkyl or a substituted or unsubstituted heterocycloalkyl ring having -N included in the ring;

[0014] R 10 is

[0015] wherein, R 13 is selected from -OH, -NH2, -NHCOCH3, alkyl, acetyl, C3-C8 acyl, X selected from F, Cl, Br;

[0016] R 14 is selected from alkoxy, -OMe, -OH, NH2, -NHCOCH3, alkyl, acetyl, C3-C8 acyl, X selected from F, Cl, Br;

[0017] R 15 is selected from alkoxy, -OMe, -OH, -H, Br, NH2, alkyl, acetyl, C3-C8 acyl, X selected from F, Cl, Br;

[0018] R 16 is selected from -H, -CH2OH, -OH, alkyl, alkoxy;

[0019] R6 is selected from the group R as defined above, -H,

[0020] R5 is located at any position and exists as a single or multiple groups and is selected from -CH2-O-CH2, -COOH, alkyl, alkoxy, NHCOCH3, -H, -OR, -NR, -X selected from F, Cl, Br or R5 forms a fused ring having an -O-CH2-O- group.

[0021] In one aspect of the present invention, compounds of formula II represented by the following structure are covered.

[0022]

[0023] In one aspect, compounds of formula III represented by the following structure are covered.

[0024]

[0025] In one aspect of the present invention, compounds of formula IV represented by the following structure are covered.

[0026]

[0027] One aspect of the present invention relates to a pharmaceutical composition comprising the above compounds, at least one pharmaceutically acceptable excipient and optionally at least one active agent.

[0028] One aspect of the present invention relates to compounds of formula I to IV, which are used for treating or inhibiting uncontrolled cell growth such as cancer, including for targeting cancer cells such as cancer stem cells.

[0029] Another aspect of the present invention discloses a method for treating or inhibiting uncontrolled cell growth. The method includes administering to a patient an effective amount of a compound of formula I to IV or a pharmaceutical composition of formula I to IV or any of the above compounds. Description of the Drawings

[0030] Figure 1 Illustrates the sphere assay of the MDAMB231 cell line in the presence of the compound of formula 1 and cisplatin.

[0031] Figure 2 Illustrates the sphere assay of the PC3 cell line in the presence of the compound of formula 1 and cisplatin.

[0032] Figure 3 Illustrates the sphere assay of the MDAMB231 cell line in the presence of the compound of formula 2 and cisplatin.

[0033] Figure 4 Illustrates the sphere assay of the PC3 cell line in the presence of the compound of formula 2 and cisplatin.

[0034] Figure 5 Illustrates the sphere assay of the MDAMB231 cell line in the presence of the compound of formula 7 and cisplatin.

[0035] Figure 6 Illustrated the sphere assay of PC3 cell line in the presence of Compound of Formula 7 and cisplatin.

[0036] Figure 7 Illustrated the sphere assay of MDAMB231 cell line in the presence of Compound of Formula 37 and cisplatin.

[0037] Figure 8 Illustrated the sphere assay of PC3 cell line in the presence of Compound of Formula 37 and cisplatin.

[0038] Figure 9 Illustrated the sphere assay of MDAMB231 cell line in the presence of Compound of Formula 40 and cisplatin.

[0039] Figure 10 Illustrated the sphere assay of PC3 cell line in the presence of Compound of Formula 40 and cisplatin.

[0040] Figure 11 Illustrated the sphere assay of MDAMB231 cell line in the presence of Compound of Formula 41 and cisplatin.

[0041] Figure 12 Illustrated the sphere assay of PC3 cell line in the presence of Compound of Formula 41 and cisplatin.

[0042] Figure 13 Illustrated the sphere assay of MDAMB231 cell line in the presence of Compound of Formula 43 and cisplatin.

[0043] Figure 14 Illustrated the sphere assay of PC3 cell line in the presence of Compound of Formula 43 and cisplatin.

[0044] Figure 15 Illustrated the sphere assay of MDAMB231 cell line in the presence of Compound of Formula 46 and cisplatin.

[0045] Figure 16 Illustrated the sphere assay of PC3 cell line in the presence of Compound of Formula 46 and cisplatin.

[0046] Figure 17 Illustrated the sphere assay of MDAMB231 cell line in the presence of Compound of Formula 47 and cisplatin.

[0047] Figure 18 Illustrated the sphere assay of PC3 cell line in the presence of Compound of Formula 47 and cisplatin.

[0048] Figure 19 Illustrated the sphere assay of MDAMB231 cell line in the presence of Compound of Formula 52 and cisplatin.

[0049] Figure 20 Illustrated the sphere analysis of the PC3 cell line in the presence of the compound of formula 52 and cisplatin.

[0050] Figure 21 Illustrated the sphere analysis of the MDAMB231 cell line in the presence of the compound of formula 67 and cisplatin.

[0051] Figure 22 Illustrated the sphere analysis of the PC3 cell line in the presence of the compound of formula 67 and cisplatin.

[0052] Figure 23 Illustrated the sphere analysis of the MDAMB231 cell line in the presence of the compound of formula 68 and cisplatin.

[0053] Figure 24 Illustrated the sphere analysis of the PC3 cell line in the presence of the compound of formula 68 and cisplatin.

[0054] Figure 25 Illustrated the sphere analysis of the MDAMB231 cell line in the presence of the compound of formula 69 and cisplatin.

[0055] Figure 26 Illustrated the sphere analysis of the PC3 cell line in the presence of the compound of formula 69 and cisplatin.

[0056] Figure 27 Illustrated the sphere analysis of the MDAMB231 cell line in the presence of the compound of formula 70 and cisplatin.

[0057] Figure 28 Illustrated the sphere analysis of the PC3 cell line in the presence of the compound of formula 70 and cisplatin.

[0058] Figure 29 Illustrated the sphere analysis of the MDAMB231 cell line in the presence of the compound of formula 71 and cisplatin.

[0059] Figure 30 Illustrated the sphere analysis of the PC3 cell line in the presence of the compound of formula 71 and cisplatin.

[0060] Figure 31 Illustrated the sphere analysis of the MDAMB231 cell line in the presence of the compound of formula 72 and cisplatin.

[0061] Figure 32 Illustrated the sphere analysis of the PC3 cell line in the presence of the compound of formula 72 and cisplatin.

[0062] Figure 33 Illustrated the sphere analysis of the MDAMB231 cell line in the presence of the compound of formula 73 and cisplatin.

[0063] Figure 34Illustrated the sphere analysis of PC3 cell line in the presence of compound of formula 73 and cisplatin.

[0064] Figure 35 Illustrated the sphere analysis of MDAMB231 cell line in the presence of compound of formula 74 and cisplatin.

[0065] Figure 36 Illustrated the sphere analysis of PC3 cell line in the presence of compound of formula 74 and cisplatin.

[0066] Figure 37 Illustrated the sphere analysis of MDAMB231 cell line in the presence of compound of formula 75 and cisplatin.

[0067] Figure 38 Illustrated the sphere analysis of PC3 cell line in the presence of compound of formula 75 and cisplatin.

[0068] Figure 39 Illustrated the sphere analysis of MDAMB231 cell line in the presence of compound of formula 76 and cisplatin.

[0069] Figure 40 Illustrated the sphere analysis of PC3 cell line in the presence of compound of formula 76 and cisplatin.

[0070] Figure 41 Illustrated the sphere analysis of MDAMB231 cell line in the presence of compound of formula 77 and cisplatin.

[0071] Figure 42 Illustrated the sphere analysis of PC3 cell line in the presence of compound of formula 77 and cisplatin.

[0072] Figure 43 Illustrated the sphere analysis of MDAMB231 cell line in the presence of compound of formula 78 and cisplatin.

[0073] Figure 44 Illustrated the sphere analysis of PC3 cell line in the presence of compound of formula 78 and cisplatin.

[0074] Figure 45 Illustrated the sphere analysis of MDAMB231 cell line in the presence of compound of formula 79 and cisplatin.

[0075] Figure 46 Illustrated the sphere analysis of PC3 cell line in the presence of compound of formula 79 and cisplatin.

[0076] Figure 47 Illustrated the sphere analysis of MDAMB231 cell line in the presence of compound of formula 80 and cisplatin.

[0077] Figure 48 Illustrated the sphere analysis of PC3 cell line in the presence of compound of formula 80 and cisplatin.

[0078] Figure 49 Illustrated the sphere assay of the MDAMB231 cell line in the presence of the compound of formula 81 and cisplatin.

[0079] Figure 50 Illustrated the sphere assay of the PC3 cell line in the presence of the compound of formula 81 and cisplatin.

[0080] Figure 51 Illustrated the activity of the compounds of formula 2, 40, 41, 43, 52, 67, 68, 71, 72, 73 and cisplatin against the breast cancer MDAMB231 cell line in the soft agar assay.

[0081] Figure 52 Illustrated the activity of the compounds of formula 2, 40, 41, 43, 52, 67, 68, 71, 72, 73 and cisplatin against the prostate cancer PC3 cell line in the soft agar assay.

[0082] Figure 53 Illustrated the activity of the compounds of formula 1, 2, 40, 41, 43, 52, 67, 68, 69, 70, 71, 72, 73 against lymphocytes.

[0083] Figure 54 Illustrated the wound healing effect of the compounds of formula 2, 52, 40, 43 and cisplatin on breast cancer and prostate cancer.

[0084] Figure 55 Illustrated the inhibitory effect of the compounds of formula 2, 52, 40 and cisplatin on aldehyde dehydrogenase (ALDH), a cancer stem cell (CSC) marker. Detailed Description of the Invention

[0086] The present invention relates to compounds of formula I for the treatment of various diseases, particularly for inhibiting uncontrolled cell growth or proliferation or unregulated cell growth. In particular, these compounds are effective against cancer stem cells. The structure of the compounds of formula I is:

[0087]

[0088] Wherein,

[0089] n is 1 - 10;

[0090] Q is O, S, -NY', where Y' is selected from -H, alkyl;

[0091] R1, R2, R3, and R4 are each independently selected from -H, alkoxy, alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aryl having a fused ring formed by heterocycloalkyl, -NH2, -NO2, -NHCOCH3, -CN, -O-, halogen, -OCF3, heterocycloalkyl, -O-(CH2) n -heterocycloalkyl;

[0092] R is selected from substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted cycloalkyl, -NR 10 R 11 、-NR 10 R 11 .HCl or acid salt, -OR 10 R 11 、-CONR 10 R 11 、-NR 10 R 11 CONR 10 R 11 、-NR 10 R 11 SOONR 10 R 11 、-COOH,

[0093] wherein R 10 and R 11 are each independently selected from -H, alkyl, substituted or unsubstituted aryl, heteroaryl, alkylamine, substituted arylamine, substituted or unsubstituted cycloalkyl, -CH2-CH2-O-alkyl, or R 10 and R 11 together form a substituted or unsubstituted cycloalkyl or heterocycloalkyl, or R 10 and R 11 together form a substituted or unsubstituted cycloalkyl or a substituted or unsubstituted heterocycloalkyl ring having -N included in the ring;

[0094] R 10 is

[0095] wherein, R 13 is selected from -OH, -NH2, -NHCOCH3, alkyl, acetyl, C3-C8 acyl, X selected from F, Cl, Br;

[0096] R 14 is selected from alkoxy, -OMe, -OH, NH2, -NHCOCH3, alkyl, acetyl, C3-C8 acyl, X selected from F, Cl, Br;

[0097] R 15X selected from alkoxy, -OMe, -OH, -H, Br, NH2, alkyl, acetyl, C3-C8 acyl, X selected from F, Cl, Br;

[0098] R 16 selected from -H, -CH2OH, -OH, alkyl, alkoxy;

[0099] R6 is selected from the group R as defined above, -H,

[0100] R5 is in any position and exists as a single or multiple groups and is selected from -CH2-O-CH2, -COOH, alkyl, alkoxy, NHCOCH3, -H, -OR, -NR, -X selected from F, Cl, Br or R5 forms a fused ring having an -O-CH2-O- group.

[0101] One embodiment of the present invention discloses a compound of formula II, which is represented as:

[0102]

[0103] In one embodiment of the present invention, the compound of formula III is represented as:

[0104]

[0105] In one embodiment of the present invention, the compound of formula IV is represented as:

[0106]

[0107] In one embodiment of the present invention, the compound includes:

[0108] R1, R2, R3 are each independently selected from -H;

[0109] R4 is selected from -H, alkoxy, alkyl, substituted or unsubstituted aryl, -NH2, -NO2, -NHCOCH3, -CN, -O-, halogen, -OCF3,

[0110] R6 is selected from -H,

[0111] Q is selected from -O, -NH;

[0112] R is selected from -COOH,

[0113] n is 1-6; and

[0114] * represents a connection point.

[0115] In one embodiment of the present invention, the compound comprises the following:

[0116] Group -Q-(CH2) n -R does not exist,

[0117] R1, R2, R3 are each independently selected from -H, R6 is -H,

[0118] R4 is selected from

[0119] * represents a connection point.

[0120] The compounds covered by Formulas I to IV are as follows:

[0121]

[0122]

[0123]

[0124]

[0125] The present invention also includes a pharmaceutical composition comprising a compound of Formulas I to IV or any of the above compounds, at least one pharmaceutically acceptable excipient, and optionally at least one active agent.

[0126] The active agent is selected from, but not limited to, imatinib, nilotinib, gefitinib, sunitinib, carfilzomib, salinosporamide A, retinoic acid, cisplatin, carboplatin, oxaliplatin, methyl diethyloxamate, cyclophosphamide, chlorambucil, ifosfamide, azathioprine, mercaptopurine, doxorubicin, fluorouracil, gemcitabine, methotrexate, thioguanine, vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, etoposide, teniposide, tavrolide, paclitaxel, docetaxel, irinotecan, topotecan, amsacrine, actinomycin, doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, plicamycin, mitomycin, mitoxantrone, melphalan, busulfan, capecitabine, pemetrexed, epothilone, 13-cis retinoic acid, 2-Cd-A, 2-chlorodeoxyadenosine, 5-azacytidine, 5-fluorouracil, 5-FU, 6-mercaptopurine, 6-MP, 6-TG, 6-thioguanine, albumin-bound paclitaxel (Abraxane), actinomycin-D, Ala- Aldesleukin, Alemtuzumab, Pemetrexed, Tretinoin, Alkaban- All-trans retinoic acid, Interferon alfa, Altretamine, Methotrexate, Amifostine, Aminoglutethimide, Anagrelide, Anastrozole, Cytarabine, Ara-C, Arsenic trioxide, Arzerra TM , Asparaginase, ATRA, Azacitidine, BCG, BCNU, Bendamustine, Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Bleomycin, Bortezomib, Busulfan, C225, Calcium folinate, Irinotecan, Capecitabine, Carac TM , Carboplatin, Carmustine, Carmustine wafer, CC-5013, CCI-779, CCNU, CDDP, CeeNU, Cetuximab, Chlorambucil, Citrovorum factor, Cladribine, Cortisone, CPT-11, Cytosar- Dacarbazine, Dactinomycin, Doxorubicin, Pegaspargase, Dasatinib, Daunorubicin, Daunorubicin hydrochloride, Daunorubicin liposome, Dexamethasone, Decitabine, Delta- Denileukin, Diftitox, DepoCyt TM , Dexamethasone, Dexamethasone acetate, Dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxorubicin, Doxorubicin liposome, Droxia TM , DTIC, DTIC- Eligard TM , Ellence TM , Eloxatin TM , Epirubicin, Epoetin alfa, Cetuximab, Erlotinib, Erwinia L-asparaginase, Estramustine, Ethyol, Etoposide, Etoposide phosphate, Everolimus, Exemestane, Filgrastim, Floxuridine, Fludarabine, Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic acid, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar Gleevec TM , Wafer, GM-CSF, Goserelin, Granulocyte-colony stimulating factor, Granulocyte-macrophage colony stimulating factor, Hexadrol, Altretamine, HMM, Hydrocort Hydrocortisone, Hydrocortisone sodium phosphate, Hydrocortisone sodium succinate, Hydrocortisone phosphate, Hydroxyurea, Ibritumomab tiuxetan, Ibritumomab tiuxetan, Tiuxetan, Idarubicin IFN-alpha, Ifosfamide, IL-11, IL-2, Imatinib mesylate, Temozolomide, Interferon α, Interferon α-2b (PEG conjugate), Interleukin-2, Interleukin-11, Interferon A (Interferon α-2b), Irinotecan, Isotretinoin, Ixabepilone, Ixempra TM , Lapatinib, L-Asparaginase, LCR, Lenalidomide, Letrozole, Leucovorin, Leukeran, Leukine TM , Leuprolide, Vincristine, Leustatin TM , Liposomal Ara-C, Liquid Lomustine, L-PAM, Melphalan, Mercaptopurine, Mesna, Mesnex Lupron Maxidex, Methyldioxymorphinan, Mechlorethamine hydrochloride, Megestrol acetate, Medroxyprogesterone acetate, Melphalan, Mercaptopurine, Mesna, Mesnex TM , Methotrexate, Methotrexate sodium, Methylprednisolone, Mitomycin, Mitomycin-C, Mitoxantrone, M- MTC, MTX, Mustine, Myloceli, Nelarabine, Neulasta TM , Nilotinib, Nilutamide, Mechlorethamine, Romiplostim, Octreotide, Octreotide Acetate, Ofatumumab, Onxal combination, Oprelvekin, Oxaliplatin, Paclitaxel, Albumin Bound Paclitaxel, Pamidronate Disodium, Panitumumab, Pazopanib, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON TM , PEG-L-asparaginase, Pemetrexed, Pentostatin, Melphalan, Platinol- Prednisolone, Prednisone, Procarbazine Prolifeprospan 20 with Carmustine Implant, Raloxifene, Rituximab, Roferon- (Interferon α-2a), Romiplostim, Daunorubicin Hydrochloride, Sandostatin Sargramostim, Solu- Solu- Sorafenib, SPRYCEL TM , STI-571, Streptozocin, SU11248, Sunitinib, Tamoxifen, Temozolomide, Sirolimus Ester, Teniposide, TESPA, Thalidomide, Thioguanine, Thioguanine Thiophosphoamide, Thiotepa, Topotecan, Toremifene, Tositumomab, Trastuzumab, Tretinoin, Trexall TM , TSPA, VCR, Vectibix TM , Viadur TM , Vinblastine, Vinblastine Sulfate, Vincasar Vincristine, Vinorelbine, Vinorelbine Tartrate, VLB, VM-26, Vorinostat, Pazopanib, VP-16, ZevalinTM , Zoledronic acid, vorinostat, or a combination of any of the above drugs.

[0127] Pharmaceutically acceptable excipients include carriers, adjuvants, vehicles or mixtures thereof.

[0128] The compounds of the present invention are used for treating or inhibiting the growth of uncontrolled cells such as cancer cells. The compounds effectively target cancer cells including cancer stem cells.

[0129] The present invention also relates to methods for inhibiting and treating the growth of uncontrolled cells such as cancer cells. It has been found that these compounds target cancer cells, including cancer stem cells. The methods include administering to a patient an effective amount of one or more compounds of Formulas I to IV.

[0130] The present invention also relates to methods for treating or inhibiting uncontrolled cell growth such as cancer by administering to a patient an effective amount of a pharmaceutical composition comprising one or more compounds of Formulas I to IV or any of the above compounds.

[0131] The compounds of the present invention can also be provided together with standard therapies useful for treating cancer.

[0132] The compounds of the present invention are used for treating or inhibiting breast cancer, prostate cancer, brain cancer, blood cancer, bone marrow cancer, liver cancer, pancreatic cancer, skin cancer, kidney cancer, colon cancer, ovarian cancer, lung cancer, testicular cancer, penile cancer, thyroid cancer, parathyroid cancer, pituitary cancer, thymic cancer, retinal cancer, uveal cancer, conjunctival cancer, spleen cancer, head cancer, neck cancer, tracheal cancer, gallbladder cancer, rectal cancer, salivary gland cancer, adrenal cancer, laryngeal cancer, esophageal cancer, lymph node cancer, sweat gland cancer, sebaceous gland cancer, muscle cancer, heart cancer and gastric cancer, particularly for treating at least one of breast cancer and prostate cancer.

[0133] Compared with their activity against cancer cells, these compounds have lower activity against normal cells (lymphocytes).

[0134] These compounds are found to have a wound healing effect in breast cancer and prostate cancer.

[0135] These compounds are found to inhibit aldehyde dehydrogenase (ALDH) - a cancer stem cell (CSC) marker.

[0136] In one embodiment, the compounds can be used for treating malaria, dengue fever.

[0137] The synthesis process of the compounds is described below. Examples

[0138] The examples described below define the present invention but do not limit the present invention.

[0139] Scheme 1:

[0140]

[0141] Reagents and conditions: a. Acetic anhydride, pyridine, RT, 12 h, b. NBS, AcOH, H2O, 65 °C, 2 h, c. 5N H2SO4, retarder, 90 °C, 2 h

[0142] Synthesis of Compound 2 (1,5 - diacetoxynaphthalene):

[0143] Add 1,5 - dihydroxynaphthalene (20 g, 0.1249 mol) in pyridine (100 ml) to a clean and dry three - necked RB, and stir the reaction mixture at room temperature (RT) for 15 min. Then, cool the temperature to 0 °C. Add the weighed acetic anhydride (57.28 g, 0.5620 mol) dropwise to the RM at 0 °C and stir the reaction mixture for 12 h and monitor using TLC. Slowly pour the reaction mixture into ice - cold water (1000 ml) and stir. Stir the reaction mixture for 45 min using a overhead stirrer. Filter the reaction mixture and dissolve the precipitate in MDC (1000 ml). Wash the organic layer with copper sulfate solution (250 ml * 5 times) and brine solution (200 ml * 3 times). Concentrate the reaction mixture under reduced pressure. The obtained crude compound is purified by simple flash column chromatography. (Hexane:Ethyl acetate - 40:60). Pure compound = 24 g. % Yield = 79%.

[0144] 1 H NMR (CDCl3, 400 MHz): δ = 7.77 (dd, J = 8.5 Hz, 2H), 7.49 (t, J = 8.0 Hz, 2H), 7.28 (d, J = 7.5 Hz, 2H), 2.44 (s, 6H).

[0145] Synthesis of Compound 3 (2 - bromo - 1,4 - dihydro - 1,4 - dioxonaphthalen - 5 - yl acetate):

[0146] Add NBS (58.07 gm, 0.3277 mol) in (500 ml) water and (500 ml) acetic acid to a clean and dry 3-neck RB, and the reaction mixture is at 45 °C for 15 minutes. Compound 1 (20 gm, 0.0819 mol) is dissolved in (500 ml) acetic acid and heated at 45 °C. The solution of Compound 1 is added dropwise to the reaction mixture of NBS at 45 °C within 30 minutes. The reaction mixture is stirred at 45 °C for 40 minutes. The temperature is raised to 65 °C and stirred for 1 hour. The reaction mixture is monitored by TLC. After completion, the reaction mixture is cooled to RT and the reaction mixture is poured into water (1500 ml) and extracted with MDC (250 ml * 6 times). The organic layer is washed with saturated sodium bicarbonate and brine solution (200 ml × 3 times). The combined organic layers are dried under vacuum and concentrated under reduced pressure. Crude compound = 33 gm.

[0147] 1 H NMR (CDCl3, 400 MHz): δ = 8.15 (dd, J = 1.2, 8.0 Hz, 1H), 7.77 (t, J = 8.0 Hz, 1H), 7.42 (dd, J = 1.2, 8.0 Hz, 1H), 7.38 (s, 1H), 2.44 (s, 3H).

[0148] Synthesis of Compound 4 (2-bromo-5-hydroxynaphthalene-1,4-dione):

[0149] Add Compound 3 dissolved in a retarder (715 ml) at 45 °C to a clean and dry 3-neck RB for 15 minutes and stir. Then, 5N sulfuric acid (396 ml) is slowly added thereto. The reaction mixture is refluxed at 90 °C for 2 hours. The reaction is monitored by TLC. The reaction material is evaporated to dryness on a rotary evaporator. The reaction mixture is poured into 1000 ml of water and extracted with MDC (250 ml * 6 times). The organic layer is washed with brine solution (200 ml * 3 times) and dried with sodium sulfate. The organic layer is concentrated under reduced pressure. The obtained crude compound is purified by simple filtration column chromatography hexane:ethyl acetate - 80:20. Pure compound 9.9 gm. Yield 35.%.

[0150] 1 H NMR (CDCl3, 400 MHz): δ = 11.81 (s, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.67 (t, J = 8.2 Hz, 1H), 7.32 (d, J = 8.2 Hz, 1H), 7.20 (s, 1H).

[0151] Scheme 2:

[0152]

[0153] Reagents and conditions: a. Substituted phenylboronic acid, Pd(PPh3)4, Na2CO3, THF, water, RT, 12 h, b. 4-(2-chloroethyl)morpholine hydrochloride, K2CO3, DMF, 100 °C, 3 h

[0154] Synthesis of compound 5a (5-hydroxy-2-(4-methoxyphenyl)naphthalene-1,4-dione):

[0155] To a solution of compound 4 (1.0 g, 39.2 mmol) and 4-methoxyphenylboronic acid (0.72 g, 47.4 mmol) in THF (108 ml) and water (12 ml). Na2CO3 (0.82 g, 78.4 mmol) was added to the reaction mixture. Pd(PPh3)4 (0.226 g, 1.97 mmol) was added under a nitrogen atmosphere and stirred at RT for 30 min. The reaction mixture was stirred at RT for 16 h and monitored using TLC. The reaction mixture was cooled to RT and the reaction mixture was poured into water (200 ml) and extracted with ethyl acetate (100 ml * 3 times). The organic layer was washed with water (100 ml). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (hexane:ethyl acetate - 90:10). Pure compound = 0.45 g. % yield = 45%.

[0156] 1 H NMR (CDCl3, 400 MHz): δ = 12.07 (s, 1H), 7.71 (d, J = 8.2 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 2.4 Hz, 1H), 7.29 (d, J = 1.2 Hz, 1H), 7.0 (m, 3H), 3.87 (s, 3H).

[0157] Synthesis of compound 5b (2-(4-fluorophenyl)-5-hydroxynaphthalene-1,4-dione):

[0158] To a solution of Compound 4 (1.0 g, 39.2 mmol) and 4-fluorophenylboronic acid (0.66 g, 47.4 mmol) in THF (108 ml) and water (12 ml). Na2CO3 (0.82 g, 78.4 mmol) was added to the reaction mixture. Pd(PPh3)4 (0.226 g, 1.97 mmol) was added under a nitrogen atmosphere and stirred at RT for 30 minutes. The reaction mixture was stirred at RT for 16 hours and monitored by TLC. The reaction mixture was cooled to RT and poured into water (200 ml) and extracted with ethyl acetate (100 ml * 3 times). The organic layer was washed with water (100 ml). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained crude compound was purified by column chromatography (hexane:ethyl acetate - 90:10). Pure compound = 0.40 g. % Yield = 41%.

[0159] 1 H NMR (CDCl3, 400 MHz): δ = 11.99 (s, 1H), 7.72 (d, J = 6.0 Hz, 1H), 7.68 (d, J = 6.0 Hz, 1H), 7.60 (m, 2H), 7.31 (d, J = 6.8 Hz, 1H), 7.19 (m, 2H), 7.02 (s, 1H).

[0160] Synthesis of Compound 5c (2-(benzo[d][1,3]dioxol-6-yl)-5-hydroxynaphthalene-1,4-dione):

[0161] To a solution of Compound 4 (1.0 g, 39.2 mmol) and 3,4-(methylenedioxy)phenylboronic acid (0.65 g, 39.2 mmol) in THF (90 ml) and water (10 ml). Na2CO3 (0.83 g, 78.4 mmol) was added to the reaction mixture. Pd(PPh3)4 (0.226 g, 1.96 mmol) was added under a nitrogen atmosphere and stirred at RT for 30 minutes. The reaction mixture was stirred at RT for 16 hours and monitored by TLC. The reaction mixture was cooled to RT and poured into water (200 ml) and extracted with ethyl acetate (100 ml * 3 times). The organic layer was washed with water (100 ml). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained crude compound was purified by column chromatography (hexane:ethyl acetate - 90:10). Pure compound = 0.72 g. % Yield = 64%.

[0162] 11H NMR (CDCl3, 400 MHz): δ = 12.03 (s, 1H), 7.71 (d, J = 8.2 Hz, 2H), 7.69 (d, J = 8.2 Hz, 1H), 7.30 (d, J = 8.2 Hz, 1H), 7.14 (m, 2H), 6.98 (s, 1H), 6.91 (d, J = 8.0 Hz, 1H), 6.04 (s, 2H).

[0163] Synthesis of Compound of Formula 7 (5-(2-Morpholinoethoxy)-2-(4-fluorophenyl)naphthalene-1,4-dione):

[0164] A two-necked RBF (100 mL) was charged with Compound 5b (0.25 gm, 9.36 mmol) and DMF (20 ml). K2CO3 (0.26 g, 18.7 mmol) and KI (0.015 gm, 0.93 mmol) were added to the reaction mixture and stirred at RT for 15 minutes. 4-(2-Chloroethyl)morpholine hydrochloride (0.209 gm, 11.23 mmol) was added to the reaction mixture. The reaction mixture was heated at 100 °C for 4 hours. The reaction was monitored by TLC. After completion, the reaction mixture was cooled to RT and the reaction mixture was poured into water (100 ml) and extracted with ethyl acetate (100 ml * 3 times). The organic layer was washed with water (100 ml * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (ethyl acetate: methanol - 95:5). Pure compound = 206 mg. % Yield = 58%.

[0165] 1 1H NMR (CDCl3, 400 MHz): δ = 7.84 (d, J = 6.0 Hz, 1H), 7.71 (d, J = 6.0 Hz, 1H), 7.58 (m, 2H), 7.33 (d, J = 6.8 Hz, 1H), 7.17 (m, 2H), 6.93 (s, 1H), 4.31 (t, J = 4.4 Hz, 2H), 3.75 (m, 4H), 2.98 (t, J = 4.8 Hz, 2H), 2.72 (m, 4H).

[0166] Synthesis of Compound of Formula 1 (5-(2-Morpholinoethoxy)-2-(benzo[d][1,3]dioxol-6-yl)naphthalene-1,4-dione):

[0167] A two-necked RBF (100 mL) was charged with compound 5c (0.2 g, 8.40 mmol) and DMF (20 mL). K2CO3 (0.23 g, 16.8 mmol) and KI (0.013 g, 0.84 mmol) were added to the reaction mixture and stirred at RT for 15 minutes. 4-(2-Chloroethyl)morpholine hydrochloride (0.187 g, 10.08 mmol) was added to the reaction mixture. The reaction mixture was heated at 100 °C for 4 hours. The reaction was monitored by TLC. After completion, the reaction mixture was cooled to RT and the reaction mixture was poured into water (100 mL) and extracted with ethyl acetate (100 mL * 3 times). The organic layer was washed with water (100 mL * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (ethyl acetate: methanol - 95:5). Pure compound = 40 mg. % Yield = 16%.

[0168] 1 1H NMR (CDCl3, 400 MHz): δ = 7.83 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.11 (m, 2H), 6.90 (m, 2H), 6.02 (s, 2H), 4.32 (t, J = 4.4 Hz, 2H), 3.78 (m, 4H), 3.04 (t, J = 4.8 Hz, 2H), 2.82 (m, 4H).

[0169] Scheme 3:

[0170]

[0171] Reagents and conditions: a. K2CO3, DMF, RT, 4 hrs, b. 4-(2-Chloroethyl)morpholine hydrochloride, K2CO3, DMF, 100 °C, 4 hrs

[0172] Synthesis of compound 7a (2-(4-Methoxyphenoxy)-5-hydroxynaphthalene-1,4-dione)

[0173] A two-necked RBF (250 mL) was charged with 4-methoxyphenol (0.972 g, 78.4 mmol) and DMF (50 mL). K2CO3 (1.08 g, 78.4 mmol) was added to the reaction mixture and stirred at RT for 15 minutes. Compound 4 (2.0 g, 78.4 mmol) was added to the reaction mixture. The reaction mixture was stirred at RT for 3 hours. The reaction was monitored by TLC. After completion, the reaction mixture was poured into water (200 mL) and extracted with ethyl acetate (100 mL * 3 times). The organic layer was washed with water (100 mL * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (petroleum ether: ethyl acetate - 95:5). Pure compound = 0.538 g. % Yield = 24%.

[0174] 1 H NMR (CDCl3, 400 MHz): δ = 12.07 (s, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 0.8 Hz, 1H), 7.65 (d, J = 0.8 Hz, 1H), 7.20 (d, J = 9.2 Hz, 2H), 7.07 (d, J = 9.2 Hz, 2H), 5.60 (s, 1H).

[0175] Synthesis of compound 7b (2-(4-fluorophenoxy)-5-hydroxynaphthalene-1,4-dione):

[0176] A two-necked RBF (100 mL) was charged with 4-fluorophenol (0.443 g, 39.5 mmol) and DMF (50 mL). K2CO3 (0.54 g, 39.5 mmol) was added to the reaction mixture and stirred at RT for 15 minutes. Compound 4 (1.0 g, 39.5 mmol) was added to the reaction mixture. The reaction mixture was stirred at RT for 3 hours. The reaction was monitored by TLC. After completion, the reaction mixture was poured into water (200 mL) and extracted with ethyl acetate (100 mL * 3 times). The organic layer was washed with water (100 mL * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (petroleum ether: ethyl acetate - 95:5). Pure compound = 1.05 g. % Yield = 78%.

[0177] 1 H NMR (CDCl3, 400 MHz): δ = 12.07 (s, 1H), 7.74 (d, J = 1.2 Hz, 1H), 7.62 (m, 1H), 7.31 (dd, J = 1.2 Hz & 7.6 Hz, 1H), 7.17 (m, 4H), 5.87 (s, 1H).

[0178] Synthetic Compound 7c (2-(Benzo[d][1,3]dioxol-5-yloxy)-5-hydroxynaphthalene-1,4-dione):

[0179] A two-necked RBF (100 mL) was charged with sesamol (1.08 g, 78.4 mmol) and DMF (50 mL). K2CO3 (0.54 g, 78.4 mmol) was added to the reaction mixture and stirred at RT for 15 minutes. Compound 4 (2.0 g, 78.4 mmol) was added to the reaction mixture. The reaction mixture was stirred at RT for 4 hours. The reaction was monitored by TLC. After completion, the reaction mixture was poured into water (200 mL) and extracted with ethyl acetate (100 mL * 3 times). The organic layer was washed with water (100 mL * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (petroleum ether:ethyl acetate - 95:5). Pure compound = 0.676 g. % yield = 25%.

[0180] 1 1H NMR (CDCl3, 400 MHz): δ = 12.11 (s, 1H), 7.74 (d, J = 0.8 Hz, 1H), 7.72 (dd, J = 1.2 Hz, 8.4 Hz, 1H), 7.30 (d, J = 0.8 Hz, 1H), 6.85 (d, J = 8 Hz, 1H), 6.62 (dd, J = 2.4 Hz, 7.6 Hz, 1H), 6.58 (d, J = 2.4 Hz, 1H), 6.05 (s, 2H).

[0181] Synthesis of Compound 43 (5-(2-Morpholinoethoxy)-2-(4-methoxyphenoxy)naphthalene-1,4-dione)

[0182] A two-necked RBF (100 mL) was charged with Compound 7a (0.32 g, 10.94 mmol) and DMF (20 mL). K2CO3 (0.30 g, 21.9 mmol) was added to the reaction mixture and stirred at RT for 15 minutes. 4-(2-Chloroethyl)morpholine hydrochloride (0.41 g, 21.9 mmol) was added to the reaction mixture. The reaction mixture was heated at 100 °C for 6 hours. The reaction was monitored by TLC. After completion, the reaction mixture was cooled to RT and poured into water (100 mL) and extracted with ethyl acetate (100 mL * 3 times). The organic layer was washed with water (100 mL * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (ethyl acetate:methanol - 95:5). Pure compound = 69 mg. % yield = 21%.

[0183] 11H NMR (CDCl3, 400 MHz): δ = 7.88 (d, J = 6.8 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.17 (m, 4H), 5.82 (s, 1H), 4.27 (t, J = 5.6 Hz, 2H), 3.83 (s, 3H), 3.72 (m, 4H), 2.92 (t, J = 5.6 Hz, 2H), 2.68 (m, 4H).

[0184] Synthesis of Compound of Formula 75 (5-(2-Morpholinoethoxy)-2-(4-fluorophenoxy)naphthalene-1,4-dione)

[0185] A two-necked RBF (100 mL) was charged with Compound 7b (0.32 g, 10.94 mmol) and DMF (20 mL). K2CO3 (0.30 g, 21.9 mmol) was added to the reaction mixture and stirred at RT for 15 minutes. 4-(2-Chloroethyl)morpholine hydrochloride (0.41 g, 21.9 mmol) was added to the reaction mixture. The reaction mixture was heated at 100 °C for 6 hours. The reaction was monitored by TLC. After completion, the reaction mixture was cooled to RT and the reaction mixture was poured into water (100 mL) and extracted with ethyl acetate (100 mL * 3 times). The organic layer was washed with water (100 mL * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (ethyl acetate: methanol - 95:5). Pure compound = 69 mg. % Yield = 21%.

[0186] 1 1H NMR (CDCl3, 400 MHz): δ = 7.78 (d, J = 1.2 Hz, 1H), 7.62 (m, 1H), 7.31 (dd, J = 1.2 Hz & 7.6 Hz, 1H), 7.17 (m, 4H), 5.82 (s, 1H), 4.27 (t, J = 5.6 Hz, 2H), 3.72 (m, 4H), 2.92 (t, J = 5.6 Hz, 2H), 2.68 (m, 4H).

[0187] Synthesis of Compound of Formula 46 (5-(2-Morpholinoethoxy)-2-(benzo[d][1,3]dioxol-5-yloxy)naphthalene-1,4-dione):

[0188] A two-necked RBF (100 mL) was charged with compound 7c (0.5 gm, 17.66 mmol) and DMF (20 ml). K2CO3 (0.49 g, 35.3 mmol) was added to the reaction mixture and stirred at RT for 15 minutes. 4-(2-Chloroethyl)morpholine hydrochloride (0.395 gm, 21.2 mmol) was added to the reaction mixture. The reaction mixture was heated at 100 °C for 2 hours. The reaction was monitored by TLC. After completion, the reaction mixture was cooled to RT and poured into water (100 ml) and extracted with ethyl acetate (100 ml * 3 times). The organic layer was washed with water (100 ml * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (ethyl acetate: methanol - 95:5). Pure compound = 294 mg. % Yield = 42%.

[0189] 1 H NMR (CDCl3, 400 MHz): δ = 7.74 (d, J = 0.8 Hz, 1H), 7.72 (dd, J = 1.2 Hz, 8.4 Hz, 1H), 7.30 (d, J = 0.8 Hz, 1H), 6.85 (d, J = 8 Hz, 1H), 6.62 (dd, J = 2.4 Hz, 7.6 Hz, 1H), 6.58 (d, J = 2.4 Hz, 1H), 6.05 (s, 2H), 5.91 (s, 1H), 4.27 (t, J = 5.6 Hz, 2H), 3.72 (m, 4H), 2.92 (t, J = 5.6 Hz, 2H), 2.68 (m, 4H).

[0190] Scheme 4:

[0191]

[0192] Reagents and conditions: a. K2CO3, DMF, RT, 4 h, b. 4-(2-Chloroethyl)morpholine hydrochloride, K2CO3, DMF, 100 °C, 4 h

[0193] Synthesis of compound 9a (2-(4-Methoxyphenylamino)-5-hydroxynaphthalene-1,4-dione):

[0194] A two-necked RBF (100 mL) was charged with 4-methoxyaniline (0.145 g, 1.17 mmol) and DMF (20 mL). K2CO3 (0.217 g, 1.56 mmol) was added to the reaction mixture and stirred at RT for 15 minutes. Compound 4 (0.2 g, 0.784 mmol) was added to the reaction mixture. The reaction mixture was stirred at RT for 3 hours. The reaction was monitored by TLC. After completion, the reaction mixture was poured into water (100 mL) and extracted with ethyl acetate (100 mL * 3 times). The organic layer was washed with water (100 mL * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (petroleum ether: ethyl acetate - 70:30). Pure compound = 0.078 g. % Yield = 34%.

[0195] 1 1H NMR (CDCl3, 400 MHz): δ = 12.93 (s, 1H), 7.67 (d, J = 0.8 Hz, 1H), 7.65 (m, 1H), 7.59 (d, J = 8 Hz, 1H), 7.28 (d, J = 8.8 Hz, 2H), 6.97 (d, J = 8.8 Hz, 2H), 6.11 (s, 1H), 3.84 (s, 3H).

[0196] Synthesis of compound 9b (2-(3,4-dimethoxyphenylamino)-5-hydroxynaphthalene-1,4-dione):

[0197] A two-necked RBF (250 mL) was charged with 3,4-dimethoxyaniline (0.72 g, 4.7 mmol) and DMF (50 mL). K2CO3 (1.08 g, 7.82 mmol) was added to the reaction mixture and stirred at RT for 15 minutes. Compound 4 (1.0 g, 3.92 mmol) was added to the reaction mixture. The reaction mixture was stirred at RT for 12 hours. The reaction was monitored by TLC. After completion, the reaction mixture was poured into water (200 mL) and extracted with ethyl acetate (100 mL * 3 times). The organic layer was washed with water (100 mL * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (petroleum ether: ethyl acetate - 80:20). Pure compound = 0.53 g. % Yield = 41%.

[0198] 11H NMR (CDCl3, 400 MHz): δ = 12.92 (s, 1H), 7.67 (d, J = 0.8 Hz, 1H), 7.65 (m, 1H), 7.59 (d, J = 8 Hz, 1H), 7.28 (d, J = 0.8 Hz, 1H), 6.91 (d, J = 8.8 Hz, 1H), 6.76 (d, J = 2.4 Hz, 1H), 6.11 (s, 1H), 3.91 (s, 6H).

[0199] Synthesis of Compound of Formula 37 (5-(2-Morpholinoethoxy)-2-(4-methoxyphenylamino)naphthalene-1,4-dione:

[0200] A two-necked RBF (100 mL) was charged with Compound 9a (0.3 g, 11.76 mmol) and DMF (30 mL). K2CO3 (0.324 g, 23.5 mmol) was added to the reaction mixture and stirred at RT for 15 minutes. 4-(2-Chloroethyl)morpholine hydrochloride (0.437 g, 23.5 mmol) was added to the reaction mixture. The reaction mixture was heated at 100 °C for 4 hours. The reaction was monitored by TLC. After completion, the reaction mixture was cooled to RT and the reaction mixture was poured into water (100 mL) and extracted with ethyl acetate (100 mL * 3 times). The organic layer was washed with water (100 mL * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (ethyl acetate: methanol - 95:5). Pure compound = 98 mg. % Yield = 21%.

[0201] 1 1H NMR (CDCl3, 400 MHz): δ = 12.74 (s, 1H), 7.45 (m, 2H), 7.21 (m, 3H), 7.05 (m, 2H), 5.74 (s, 1H), 4.06 (t, J = 6.8 Hz, 2H), 3.91 (s, 3H), 3.64 (m, 4H), 2.69 (m, 2H), 2.49 (m, 4H).

[0202] Synthesis of Compound of Formula 40 (5-(2-Morpholinoethoxy)-2-(3,4-dimethoxyphenylamino)naphthalene-1,4-dione:

[0203] A two-necked RBF (100 mL) was charged with compound 9b (0.3 g, 9.14 mmol) and DMF (30 mL). K2CO3 (0.251 g, 18.3 mmol) was added to the reaction mixture and stirred at RT for 15 minutes. 4-(2-Chloroethyl)morpholine hydrochloride (0.34 g, 18.2 mmol) was added to the reaction mixture. The reaction mixture was heated at 100 °C for 4 hours. The reaction was monitored by TLC. After completion, the reaction mixture was cooled to RT and the reaction mixture was poured into water (100 mL) and extracted with ethyl acetate (100 mL * 3 times). The organic layer was washed with water (100 mL * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (ethyl acetate: methanol - 95:5). Pure compound = 35 mg. % Yield = 8%.

[0204] 1 H NMR (CDCl3, 400 MHz): δ = 12.74 (s, 1H), 7.48 (m, 2H), 7.21 (d, J = 2 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.73 (m, 2H), 5.52 (s, 1H), 4.06 (d, J = 6.8 Hz, 2H), 3.91 (s, 6H), 3.62 (m, 4H), 2.68 (d, J = 7.2 Hz, 2H), 2.48 (m, 4H).

[0205] Scheme 5:

[0206]

[0207] Reagents and conditions: a. 1,4-Dibromobutane, TBAB, NaOH, H2O, 60 °C, 4 h, b. Morpholine, K2CO3, DMF, RT 12 h

[0208] Synthesis of compound 11c (5-(4-bromobutoxy)-2-(benzo[d][1,3]dioxol-6-yl)naphthalene-1,4-dione):

[0209] Dibromobutane (2.2 gm, 103.5 mmol) was added dropwise to a solution of compound 5c (0.35 gm, 10.35 mmol), NaOH (0.082 gm, 20.7 mmol), TBAB (33 mg, 1.35 mmol) and water (30 ml). The reaction mixture was stirred at 60 °C for 4 h. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was poured into ice-cold water and extracted with ethyl acetate (100 ml * 3 times). The organic layer was washed with water (100 ml). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (hexane:ethyl acetate - 80:20). Pure compound = 96 mg. % Yield = 19%.

[0210] Synthesis of compound of formula 2 (5-(4-morpholinobutoxy)-2-(benzo[d][1,3]dioxol-6-yl)naphthalene-1,4-dione)

[0211] Morpholine (0.159 gm, 18.2 mmol) was dissolved in DMF (20 ml) and K2CO3 (0.505 gm, 36.4 mmol) at RT. Compound 11c (0.087 gm, 18.0 mmol) was added to the reaction mixture at RT. The reaction mixture was stirred at RT for 16 h. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was poured into ice-cold water and extracted with ethyl acetate (100 ml * 3 times). The organic layer was washed with water (100 ml * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (ethyl acetate:methanol - 95:5). Pure compound = 45 mg. % Yield = 51%.

[0212] 1 1H NMR (CDCl3, 400 MHz): δ = 7.83 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.11 (m, 2H), 6.90 (m, 2H), 6.02 (s, 2H), 4.32 (t, J = 4.4 Hz, 2H), 3.78 (m, 4H), 3.04 (t, J = 4.8 Hz, 2H), 2.82 (m, 4H), 1.94 (m, 2H), 1.82 (m, 2H).

[0213] Scheme 6:

[0214]

[0215] Reagents and conditions: a. dibromobutane, TBAB, NaOH, H2O, 60 °C, 4 h, b. morpholine, K2CO3, DMF, RT 12 h

[0216] Synthetic compound 13a (5-(4-bromobutoxy)-2-(4-methoxyphenoxy)naphthalene-1,4-dione)

[0217] Dibromobutane (14.53 gm, 67.5 mmol) was added dropwise to a solution of compound 7a (2.0 gm, 6.75 mmol), NaOH (0.54 gm, 13.5 mmol), TBAB (218 mg, 0.67 mmol) and water (50 ml). The reaction mixture was stirred at 60 °C for 3 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was poured into ice-cold water and extracted with ethyl acetate (100 ml * 4 times). The organic layer was washed with water (100 ml). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (hexane:ethyl acetate - 80:20). Pure compound = 1.79 gm. % Yield = 61%.

[0218] Synthetic compound 13c (5-(4-bromobutoxy)-2-(benzo[d][1,3]dioxol-5-yloxy)naphthalene-1,4-dione

[0219] Dibromobutane (0.5 gm, 16.1 mmol) was added dropwise to a solution of compound 7c (0.35 gm, 10.35 mmol), NaOH (0.129 gm, 32.2 mmol), TBAB (52 mg, 1.61 mmol) and water (20 ml). The reaction mixture was stirred at 60 °C for 3 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was poured into ice-cold water and extracted with ethyl acetate (100 ml * 3 times). The organic layer was washed with water (100 ml). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (hexane:ethyl acetate - 80:20). Pure compound = 0.4 gm. % Yield = 55%.

[0220] Synthetic compound of formula 44 (2-(4-methoxyphenoxy)-5-(4-morpholinobutoxy)naphthalene-1,4-dione)

[0221] At RT, morpholine (0.121 gm, 1.16 mmol) was dissolved in DMF (20 ml) and K2CO3 (0.320 gm, 2.32 mmol). At RT, compound 13a (0.5 gm, 1.16 mmol) was added to the reaction mixture. The reaction mixture was stirred at RT for 16 h. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was poured into ice-cold water and extracted with ethyl acetate (100 ml * 3 times). The organic layer was washed with water (100 ml * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (ethyl acetate: methanol - 95:5). Pure compound = 235 mg. % Yield = 48%.

[0222] Synthesis of compound of formula 47 (5-(4-morpholinobutoxy)-2-(benzo[d][1,3]dioxol-5-yloxy)naphthalene-1,4-dione)

[0223] At RT, morpholine (0.082 gm, 9.43 mmol) was dissolved in DMF (20 ml) and K2CO3 (0.217 gm, 15.7 mmol). At RT, compound 13c (0.35 gm, 7.86 mmol) was added to the reaction mixture. The reaction mixture was stirred at RT for 16 h. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was poured into ice-cold water and extracted with ethyl acetate (100 ml * 3 times). The organic layer was washed with water (100 ml * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (ethyl acetate: methanol - 95:5). Pure compound = 25 mg. % Yield = 7%.

[0224] 1 H NMR (CDCl3, 400 MHz): δ = 7.85 (d, J = 0.8 Hz, 1H), 7.66 (dd, J = 1.2 Hz, 8.4 Hz, 1H), 7.32 (d, J = 0.8 Hz, 1H), 6.83 (d, J = 8 Hz, 1H), 6.61 (dd, J = 2.4 Hz, 7.6 Hz, 2H), 6.03 (s, 2H), 5.88 (s, 1H), 4.16 (t, J = 5.6 Hz, 2H), 3.71 (m, 4H), 2.45 (m, 6H), 1.91 (m, 2H), 1.85 (m, 2H)

[0225] Scheme 7:

[0226]

[0227] Reagents and conditions: a. CuCl, CAN, O2, RT, 10 h, b. bromine, AcOH, RT, 30 min

[0228] Synthesis of 5-hydroxy-1,4-naphthoquinone (15):

[0229] In a 450 ml autoclave reactor, acetonitrile (30 ml) and CuCl (0.78 g, 39.4 mmol) were added batchwise at RT. A solution of 1,5-dihydroxynaphthalene (1 g, 31.3 mmol) in acetonitrile (200 ml) was added to the reaction mixture at RT. A 3 kg / cm 2 oxygen pressure was applied to the reaction mixture. An oxygen atmosphere was maintained with vigorous stirring. The reaction mixture was stirred at RT for 10 h. The solution was concentrated in vacuo and the crude product was purified by column chromatography (hexane:EtOAc, 80:20). Pure compound = 0.45 g. % yield = 37%, M.p. 157 °C; 1 1H NMR (300 MHz, CDCl3): δ = 11.91 (s, 1H), 7.69–7.60 (m, 2H), 7.29 (dd, J = 7.3, 2.5 Hz, 1H), 6.96 ppm (s, 2H).

[0230] Synthesis of 3-bromo-5-hydroxy-(1,4)naphthoquinone (16):

[0231] 5-Hydroxy-1,4-naphthoquinone (15) (1 g, 57.1 mmol) was suspended in 15 ml of glacial acetic acid. At RT and in the dark, bromine (1.00 eq. 0.3 ml, 57.1 mmol) was added in RM. The reaction mass was stirred in the dark for 20 min and then poured into ice (100 g). The mixture was stirred vigorously for 30 min and the precipitate was filtered in vacuo as an orange solid and washed with a small amount of ice water. The mixture was immediately placed in a single-neck RBF and ethanol (8 ml) was added thereto. The reaction mixture was stirred under reflux with a preheated oil bath for 10 min. The crude product obtained as a red solid from the reaction solution was filtered in vacuo and washed with 5 ml of cold ethanol. The crude product was purified by column chromatography (hexane:EtOAc, 80:20). Pure compound = 0.7 g. % yield = 47%.

[0232] M.p. 168 °C; 1 1H NMR (300 MHz, CDCl3): δ = 11.73 (s, 1H), 7.68 (t, J = 7.4 Hz, 1H), 7.64 (dd, J = 7.4, 2.0 Hz, 1H), 7.50 (s, 1H), 7.31 ppm (dd, J = 7.5, 2.0 Hz, 1H)

[0233] Scheme 8:

[0234]

[0235] Reagents and conditions: a. K2CO3, DMF, RT, 4 h, b. 4-(2-chloroethyl)morpholine hydrochloride, K2CO3, DMF, 100 °C, 4 h

[0236] Synthesis of compound 17a (2-(4-methoxyphenoxy)-8-hydroxynaphthalene-1,4-dione)

[0237] A two-necked RBF (250 mL) was charged with 4-Methoxy phenol (0.972 gm, 78.4 mmol) and DMF (50 ml). K2CO3 (1.08 g, 78.4 mmol) was added to the reaction mixture and stirred at RT for 15 min. Compound 16 (2.0 gm, 78.4 mmol) was added to the reaction mixture. The reaction mixture was stirred at RT for 3 h. The reaction was monitored by TLC. After completion, the reaction mixture was poured into water (200 ml) and extracted with ethyl acetate (100 ml * 3 times). The organic layer was washed with water (100 ml * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained crude compound was purified by column chromatography (petroleum ether: ethyl acetate - 95:5). Pure compound = 1.0 gm. % Yield = 43%.

[0238] Synthesis of compound 17b (2-(4-fluorophenoxy)-8-hydroxynaphthalene-1,4-dione)

[0239] A two-necked RBF (100 mL) was charged with 4-fluorophenol (0.879 gm, 78.4 mmol) and DMF (50 ml). K2CO3 (1.08 g, 78.4 mmol) was added to the reaction mixture and stirred at RT for 15 min. Compound 16 (2.0 gm, 78.4 mmol) was added to the reaction mixture. The reaction mixture was stirred at RT for 3 h. The reaction was monitored by TLC. After completion, the reaction mixture was poured into water (200 ml) and extracted with ethyl acetate (100 ml * 3 times). The organic layer was washed with water (100 ml * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained crude compound was purified by column chromatography (petroleum ether: ethyl acetate - 95:5). Pure compound = 1.13 gm. % Yield = 51%.

[0240] Synthesis of compound 17c (2-(benzo[d][1,3]dioxol-6-yloxy)-8-hydroxynaphthalene-1,4-dione)

[0241] A two-necked RBF (100 mL) was charged with sesamol (1.08 gm, 78.4 mmol) and DMF (50 ml). K2CO3 (0.54 g, 78.4 mmol) was added to the reaction mixture and stirred at RT for 15 minutes. Compound 4 (2.0 gm, 78.4 mmol) was added to the reaction mixture. The reaction mixture was stirred at RT for 4 hours. The reaction was monitored by TLC. After completion, the reaction mixture was poured into water (200 ml) and extracted with ethyl acetate (100 ml * 3 times). The organic layer was washed with water (100 ml * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained crude compound was purified by column chromatography (petroleum ether: ethyl acetate - 95:5). Pure compound = 0.676 gm. % Yield = 25%.

[0242] Synthesis of Compound 80 (8-(2-Morpholinoethoxy)-2-(4-fluorophenoxy)naphthalene-1,4-dione)

[0243] A two-necked RBF (100 mL) was charged with Compound 17b (0.5 gm, 17.66 mmol) and DMF (20 ml). K2CO3 (0.488 g, 35.33 mmol) was added to the reaction mixture and stirred at RT for 15 minutes. 4-(2-Chloroethyl)morpholine hydrochloride (0.394 gm, 21.2 mmol) was added to the reaction mixture. The reaction mixture was heated at 100 °C for 3 hours. The reaction was monitored by TLC. After completion, the reaction mixture was cooled to RT and the reaction mixture was poured into water (100 ml) and extracted with ethyl acetate (100 ml * 3 times). The organic layer was washed with water (100 ml * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained crude compound was purified by column chromatography (ethyl acetate: methanol - 95:5). Pure compound = 35 mg. % Yield = 33%.

[0244] 1 H NMR (CDCl3, 400 MHz): δ = 7.71 (d, J = 1.2 Hz, 1H), 7.68 (m, 1H), 7.31 (dd, J = 1.2 Hz & 7.6 Hz, 1H), 7.17 (m, 4H), 5.84 (s, 1H), 4.31 (t, J = 5.6 Hz, 2H), 3.76 (m, 4H), 2.98 (t, J = 5.6 Hz, 2H), 2.72 (m, 4H).

[0245] Synthesis of Compound 79 (8-(2-Morpholinoethoxy)-2-(benzo[d][1,3]dioxol-6-yloxy)naphthalene-1,4-dione)

[0246] A two-necked RBF (100 mL) was charged with compound 7c (0.5 gm, 17.66 mmol) and DMF (20 ml). K2CO3 (0.49 g, 35.3 mmol) was added to the reaction mixture and stirred at RT for 15 minutes. 4-(2-Chloroethyl)morpholine hydrochloride (0.395 gm, 21.2 mmol) was added to the reaction mixture. The reaction mixture was heated at 100 °C for 2 hours. The reaction was monitored by TLC. After completion, the reaction mixture was cooled to RT and the reaction mixture was poured into water (100 ml) and extracted with ethyl acetate (100 ml * 3 times). The organic layer was washed with water (100 ml * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound obtained was purified by column chromatography (ethyl acetate: methanol - 95:5). Pure compound = 294 mg. % Yield = 42%.

[0247] 1 H NMR (CDCl3, 400 MHz): δ = 7.74 (d, J = 0.8 Hz, 1H), 7.68 (dd, J = 1.2 Hz, 8.4 Hz, 1H), 7.29 (d, J = 0.8 Hz, 1H), 6.83 (d, J = 8 Hz, 1H), 6.60 (dd, J = 2.4 Hz, 7.6 Hz, 1H), 6.56 (d, J = 2.4 Hz, 1H), 6.03 (s, 2H), 5.91 (s, 1H), 4.30 (t, J = 5.6 Hz, 2H), 3.76 (m, 4H), 2.98 (t, J = 5.6 Hz, 2H), 2.72 (m, 4H).

[0248] Test data

[0249] The following tests were used to determine the effectiveness and non-toxicity of the compounds.

[0250] Cancer cell assay

[0251] 1. In vitro anti-proliferation assay (MTT assay)

[0252] The MTT assay is a simple and sensitive assay that can measure the metabolic reduction activity of cells. The increase in activity over time is used as a parameter for cell growth. If the increase in activity is disrupted by drug treatment, it is either the result of growth inhibition, cell killing, or both. The compounds of the present invention and standard cytotoxic drugs (such as cisplatin) were tested using breast cancer and prostate cancer cell lines at different concentrations (1, 0.1, 0.01, 0.001 mM). All cell lines were cultured in a 5% CO2 environment, 37 °C incubator. The compounds were dissolved in DMSO at a concentration of 0.1 M (stock solution). The cells were seeded into 96-well plates at an appropriate plating efficiency (seeding rate).

[0253] The following plate efficiencies for the MTT assay were standardized:

[0254] Table 1

[0255]

[0256] During the MTT assay, cells were seeded in 96-well plates at a predetermined plate efficiency (Table 1). The plates were placed in a 5% CO2 gas environment and incubated at 37 °C for 24 hours. Subsequently, an appropriate concentration of the drug was added to the plates and further incubated for 48 hours (37 °C, 5% CO2 gas environment). Then, the assay plates were centrifuged twice at 3000 rpm for 3 minutes, and the supernatant was discarded. Then, 100 μL of MTT solution (0.5 mg / ml) was added to each well of the plates and further incubated for 4 hours (37 °C, 5% CO2 gas environment). After 4 hours, the plates were centrifuged twice, and the supernatant was carefully aspirated. Then, 200 μL of DMSO was added to each well to dissolve. The MTT crystals were evenly mixed by shaking the plates. Then, an XY plot of the logarithm of the survival percentage versus the logarithm of the drug concentration was plotted. The IC50 (the drug concentration that inhibits 50% of the cell population) was calculated by regression analysis.

[0257] MTT assay results of the compounds against breast cancer (MDAMB231 cell line) and prostate cancer (PC3 cell line).

[0258] Table 2

[0259]

[0260]

[0261] The above table shows that these compounds exhibited very high potency against breast and prostate cancer cell lines in the MTT assay compared to the standard therapeutic drug cisplatin.

[0262] Figures 1 to 50 The activities of Compounds of Formula 1, 2, 7, 37, 40, 41, 43, 46, 47, 52, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 and 81 against breast and prostate cancer cell lines were compared with cisplatin. It was found that these compounds exhibited higher anti-cancer activities compared to cisplatin.

[0263] 2. Soft agar assay

[0264] The soft agar colony formation assay is an anchorage-independent growth assay in soft agar and is one of the most stringent assays for detecting cell malignant transformation. For this assay, malignant cells and appropriate controls are cultured in soft agar medium for 1 - 2 weeks. After this culture period, the colonies formed can be morphologically analyzed using cell staining and the number of colonies formed can be quantified. The results of this assay are comparable to those obtained after injecting tumorigenic cells into nude mice, which is regarded as the "gold standard" for testing cell tumorigenicity (one of the important characteristics of cancer stem cells (CSCs)) in vitro.

[0265] Briefly, for the soft agar assay, a mixture of 50 μl of 2X medium (appropriately taken according to the cell line) and 50 μl of 1.2% Bacto agar is inoculated onto each well of a 96-well microtiter assay plate. 10 μl of cells (with a specific plating efficiency pre-standardized for the corresponding cell line) are mixed with 20 μl of 2X medium, 30 μl of 0.8% Bacto agar, and 1.6 μl of drug (at an appropriate concentration) in a vial and transferred to the solidified pre-layer of the assay plate. Then the cells are allowed to grow at 37 °C and 5% CO2 for one week to form colonies. After a 3-day experimental setup, intermittent feeding is carried out with 50 μl of appropriate 2X medium. Then 16 μl of Alamar Blue (1.5 mg / ml) is added to all wells to quantify the developed colonies. The plate is incubated at 37 °C for 24 hours. Then the absorbance is measured at 630 nm. Then an XY plot of the percentage of logarithmic survival rate versus logarithmic drug concentration is drawn. The IC50 (drug concentration that inhibits 50% of the cell population) is calculated by regression analysis.

[0266] The following plating efficiencies for the soft agar assay were standardized:

[0267] Table 3

[0268] Cell Line Cell Line Name Plating Efficiency (Cells per Well) Breast MDAMB231 7500 Prostate PC3 5000

[0269] Results of the soft agar assay of the compounds on breast cancer (MDAMB231 cell line) and prostate cancer (PC3 cell line).

[0270] Table 4

[0271]

[0272]

[0273] The above table shows that these compounds exhibit very high potency against breast cancer and prostate cancer cell lines in the soft agar assay compared to the standard therapeutic drug cisplatin.

[0274] Figure 51Show the activities of the compounds of formula 2, 40, 41, 43, 52, 67, 68, 71, 72 and 73 against breast cancer cell lines respectively as compared with cisplatin. It was found that these compounds exhibited higher anti-cancer activities as compared with cisplatin.

[0275] Figure 52 Show the activities of the compounds of formula 2, 40, 41, 43, 52, 67, 68, 71, 72 and 73 against prostate cancer cell lines respectively as compared with cisplatin. It was found that these compounds exhibited higher anti-cancer activities as compared with cisplatin.

[0276] 3. Stem cell assay

[0277] In vitro sphere formation assay: The sphere assay measures the ability of cancer stem cells (CSCs) to form spheres in a specially designed serum-free medium. This assay is used to measure the killing efficiency of test compounds compared to the standard chemotherapy drug cisplatin.

[0278] Materials and reagents: 50X B27 Supplement (Life Technologies, Invitrogen, catalog number: 17502-044), fibroblast growth factor (FGF) (Sigma-Aldrich, catalog number: F029125), epidermal growth factor (EGF) (Sigma-Aldrich, catalog number: E9644), insulin (Sigma, catalog number: 19278), Dulbecco's Modified Eagle Medium / F12 (HiMedia catalog number: AL139-6), Dulbecco's Phosphate Buffered Saline (HiMedia catalog number: TL1006), Trypan Blue (TC193), Prostate Epithelial Medium (LONZA, catalog number: CC-3166), MEGM (LONZA, catalog number: CC-3051), heparin (Sigma, catalog number: H3393), Penstrep (HiMedia, catalog number: A002)

[0279] Preparation of mammosphere medium (100 ml): Add 1 g of autoclaved methylcellulose and 100 ml of normal medium (MEBM) with a magnetic stirrer and dissolve under magnetic stirring. After complete dissolution, add 80 μl of FGF, 40 μl of EGF, 1 ml of Penstrep, and 400 μl of heparin.

[0280] Preparation of prostate sphere medium (100 ml): Under magnetic stirring, add 1 g of autoclaved methylcellulose and 100 ml of normal medium (prostate epithelial basal medium) and dissolve. After complete dissolution, add 40 μl of insulin, 2 ml of B27, 80 μl of EGF, and 1 ml of Penstrep.

[0281] Procedure - Cells are trypsinized and made into a single-cell suspension through cell filters (100 μl and 40 μl respectively). The cells are diluted and suspended at a concentration of 2000 cells / 100 μl in mammosphere medium (for mammary cell lines) or prostate medium (for prostate cell lines). Add 100 μl of this suspension to each well of a 96-well suspension plate and culture at 37 °C, 5% CO2 for 24 hours. Add an appropriate concentration of drug (2 μl) to each well with 100 μl of stem cell medium. Incubate the plate at 37 °C, 5% CO2 for 72 hours. After incubation, add 2.5 μl of each drug concentration and 50 μl of stem cell medium to each well, and further culture the plate at 37 °C, 5% CO2 for 72 hours. After incubation, add 3 μl of each drug concentration together with 50 μl of stem cell medium again, and culture the plate at 37 °C, 5% CO2 for 72 hours. Calculate the number of primary spheres formed at each concentration. Calculate the percentage of survival of spheres transformed into spheres compared to untreated (growth control using DMSO, GCD). Plot a comparison graph of the percentage of sphere survival against drug concentration and compare it with the standard therapeutic drug cisplatin.

[0282] Results of in vitro spheroid formation assay of the compound on breast cancer (MDAMB231 cell line) and prostate cancer (PC3 cell line) at a plating efficiency of 2000 cells / well (n = 6 + S.D).

[0283] Table 5: Number of 3D spheres of MDAMB231 in mammosphere medium, plating efficiency 2000 cells / well (n = 6 ± S.D)

[0284]

[0285]

[0286]

[0287]

[0288]

[0289] The above results show that the above compound is more effective in inhibiting MDAMB231 spheres compared to cisplatin.

[0290] Figure 1 , Figure 3 , Figure 5 , Figure 7 , Figure 9 , Figure 11 , Figure 13 , Figure 15 , Figure 17 , Figure 19 , Figure 21 , Figure 23 , Figure 25 , Figure 27 , Figure 29 , Figure 31 , Figure 33 , Figure 35 , Figure 37 , Figure 39 , Figure 41 , Figure 43 , Figure 45 , Figure 47 and Figure 49 refer to the compounds of Formulas 1, 2, 7, 37, 40, 41, 43, 46, 47, 52 and 67 - 81, respectively, for the MDAMB231 cell line.

[0291] Figure 1 , Figure 3 , Figure 5 , Figure 7 , Figure 9 , Figure 11 , Figure 13 , Figure 15 , Figure 17 , Figure 19 , Figure 21 , Figure 23 , Figure 25 , Figure 27 , Figure 29 , Figure 31 , Figure 33 , Figure 35 , Figure 37 , Figure 39 , Figure 41 , Figure 43 , Figure 45 , Figure 47 and Figure 49 Illustrate the percentage viability of the spheres obtained by converting the number of spheres formed and compare with the growth control using DMSO (GCD), where GCD is considered 100% viability. The sphere count results for the individual drug concentrations shown in Table 6 have been converted to percentage viability of the spheres for graphical representation. The figures and Table 6 show that the percentage viability of MDAMB231 spheres is reduced in the presence of the compounds of Formulas 1, 2, 7, 37, 40, 41, 43, 46, 47, 52 and 67 - 81 compared to cisplatin.

[0292] Table 6: Percent viability of spheroids (MDAMB231)

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299] Table 7: 3D sphere counts of PC3 in Prostosphere medium with a plating efficiency of 2000 cells / well (n=6±SD).

[0300]

[0301]

[0302]

[0303]

[0304] The above results indicate that compounds of Formulas 1, 2, 7, 37, 40, 41, 43, 46, 47, 52, and 67-81 are more effective in inhibiting PC3 spheroids than cisplatin.

[0305] Figure 2 , Figure 4 , Figure 6 , Figure 8 , Figure 10 , Figure 12 , Figure 14 , Figure 16 , Figure 18 , Figure 20 , Figure 22 , Figure 24 , Figure 26 , Figure 28 , Figure 30 , Figure 32 , Figure 34 , Figure 36 , Figure 38 , Figure 40 , Figure 42 , Figure 44 , Figure 46 , Figure 48 and Figure 50 They refer to Formulas 1, 2, 7, 37, 40, 41, 43, 46, 47, 52 and 67-81, respectively.

[0306] Figure 2 、 Figure 4 、 Figure 6 、 Figure 8 、 Figure 10 、 Figure 12 、 Figure 14 、 Figure 16 、 Figure 18 、 Figure 20 、 Figure 22 、 Figure 24 、 Figure 26 、 Figure 28 、 Figure 30 、 Figure 32 、 Figure 34 、 Figure 36 、 Figure 38 、 Figure 40 、 Figure 42 、 Figure 44 、 Figure 46 、 Figure 48 and Figure 50 clarify the percentage viability of the spheres obtained by converting the number of spheres formed and compare it with the growth control using DMSO (GCD), where GCD is considered 100% viability. The sphere count results for the individual drug concentrations shown in Table 8 have been converted to percentage viability of the spheres for graphical representation. The figures and Table 8 show that the percentage viability of PC3 spheres is reduced in the presence of the compounds of Formulas 2, 7, 37, 40, 41, 43, 46, 47, 52 and 67 - 81 compared to cisplatin.

[0307] Table 8: Percentage viability of spheres (PC3)

[0308]

[0309]

[0310]

[0311] 4. Activity against lymphocytes

[0312] Lymphocyte assay

[0313] Human lymphocytes were isolated from peripheral blood. A pure lymphocyte population was obtained based on differential centrifugation, where the diluted defibrinated blood was layered on a solution of sodium diatrizoate and polysucrose (HiSep LSM 1077) and centrifuged at low speed for 30 minutes.

[0314] Procedure: The isolation of lymphocytes from fresh defibrinated blood was carried out by the following procedure:

[0315] 1. Dilute defibrinated fresh blood was gradually overlaid on (HiSeP LSM1077) and centrifuged at low speed for 30 minutes.

[0316] 2. The lymphocyte layer (buffy coat) was carefully removed in a new collection tube.

[0317] 3. The buffy coat was washed again with dilution buffer Dulbecco's phosphate buffered saline (D.P.B.S.).

[0318] 4. The supernatant was discarded and the pellet was resuspended in D.P.B.S.

[0319] 5. Cell viability was detected using a hemocytometer.

[0320] 6. Cells with a purity and viability above 95% were used for the experiment.

[0321] 7. The purified lymphocytes were diluted with sterile D.P.B.S. to a concentration of 700,000 / ml and the MTT procedure was performed exactly as described before.

[0322] Table 9: Lymphocyte results: IC50 in μM

[0323]

[0324]

[0325] The above table and Figure 53 showed that these compounds had higher activity against cancer cells compared to normal cells, indicating the safety of these compounds.

[0326] 5. Wound healing effect

[0327] Wound healing assay: (WHA)

[0328] The wound healing assay (WHA) determines the ability of cancer stem cells to heal wounds formed in a confluent monolayer. This assay is used to measure the ability of a test drug to inhibit the wound healing ability of cancer stem cells compared to a standard chemotherapy drug such as cisplatin.

[0329] Procedure: 0.35X10 6Cells were plated at a density of [number of cells] per well in each of six tissue culture plates. The plates were incubated at 37 °C in 5% CO₂ for 48 hours. Complete confluence of the cells was observed, and after two washes with D.P.B.S., a horizontal scratch was made at the center of each well using a sterile 100 μl tip. The width of the scratch was measured at 0 hours, immediately after making the scratch. Compounds at the IC10 concentration were added to each well. The plates were incubated at 37 °C in 5% CO₂, and the width of the scratch was measured at different time intervals (e.g., 6, 24, and 48 hours). The average of three distances was taken for each time point using an IS camera. The points of the average scratch width in micrometers were plotted against the time intervals after treatment. The anti-CSC potential was determined by calculating the percentage of inhibition of each compound after 48 hours compared to cisplatin.

[0330] Table 10: Results of wound healing assay (WHA)

[0331]

[0332] The above table and Figure 54 indicate that these anti-cancer compounds inhibit cancer cell wound healing and thus prevent cancer spread compared to the standard therapeutic drug cisplatin.

[0333] 6. Inhibitory effect of compounds on the cancer biomarker aldehyde dehydrogenase (ALDH)

[0334] Aldehyde dehydrogenase (ALDH) assay:

[0335] Aldehyde dehydrogenase (ALDH) is a class of enzymes that catalyze the metabolism of exogenous and endogenous aldehydes, preventing the accumulation of potentially reactive and toxic aldehydes and their metabolites. In addition to their role in aldehyde metabolism, ALDH enzymes also play key roles in other cellular processes such as cell proliferation, differentiation, and survival.

[0336] ALDH can also serve as a marker for certain stem cell populations, including hematopoietic stem cells and certain cancer stem cells.

[0337] ALDH concentration was determined using a (Kinesis Dx) ELISA kit according to the following protocol:

[0338] 1. Add the standard (50 μl) / sample (40 μl) to the corresponding wells (except the blank).

[0339] 2. Then add (10 μl) of the antibody-biotin conjugate to each sample well (except the blank).

[0340] 3. Then add (50 μl) of the horseradish peroxidase (HRP) conjugate to each well (except the blank).

[0341] 4. Incubate the plate in an incubator at 37 °C for 1 hour.

[0342] 5. Wash (wash 4 times with wash buffer), tap the plate forcefully on absorbent paper to wipe off the residual buffer. Wipe off any liquid at the bottom of the microplate well because any residue will interfere with the reading step.

[0343] 6. Add TMB substrate A (50 μl) and TMB substrate B (50 μl) to each well, including the blank well.

[0344] 7. Incubate in the dark at 37 °C for 10 minutes.

[0345] 8. Add (50 μl) stop solution. The wells should change from blue to yellow.

[0346] 9. Read the absorbance at 450 nm.

[0347] 10. An X-Y plot of concentration versus optical density was drawn. The ALDH concentration was calculated by substituting the optical density value into the regression analysis equation.

[0348] Table 11: Aldehyde dehydrogenase (ALDH) results

[0349]

[0350] The above table and Figure 55 show that compared with the standard therapeutic drug cisplatin, these compounds inhibit aldehyde dehydrogenase (ALDH) - a cancer stem cell (CSC) marker.

Claims

1. Compounds of formula I: Wherein, n is from 1 to 6; Q is O or the group -Q-(CH2) n -R does not exist; R1, R2 and R3 are each -H; R4 is -H, R is selected from -COOH, R6 is selected from -H, * represents the point of attachment.

2. The compound according to claim 1, wherein R6 is selected from 3. The compound according to claim 1, wherein R6 is selected from 4. The compound according to claim 1, wherein R6 is 5. The compound according to claim 1, wherein the compound is selected from any one of the following compounds:

6. A pharmaceutical composition comprising the compound according to any one of claims 1 to 5 and at least one pharmaceutically acceptable excipient, with or without one or more active agents.

7. Use of the compound according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6 in the preparation of a medicament for treating or inhibiting the growth of breast cancer stem cells or prostate cancer stem cells or breast cancer or prostate cancer.

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