2-benzoylaminobenzamide derivatives as Bcl-3 inhibitors

Abstract

The invention relates to a compound of general formula (I): ##STR00001##
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, Q, m and n are as defined herein. The compounds are inhibitors of Bcl-3 and are useful for the treatment of cancer, particularly metastatic cancer.

Claims

1. A method for the treatment of cancer, the method comprising administering to a patient in need of such treatment an effective amount of a compound of general formula (Ia): ##STR00021## or a salt thereof, wherein: each R.sup.1 is independently halo, nitro, cyano, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.1-6 haloalkyl, OR.sup.5 or N(R.sup.5R.sup.6), wherein at least one R.sup.1 is fluoro; each of R.sup.5 and R.sup.6 is independently hydrogen, C.sub.1-6 alkyl or C.sub.1-6 haloalkyl; m is 0, 1 or 2; Q is (CH.sub.2).sub.p; p is 1, 2, 3 or 4; R.sup.3 is morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, pyridyl, pyrrolyl, pyrimidinyl, imidazolyl or triazolyl; each R.sup.4 is independently nitro, cyano, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.1-6 haloalkyl, OR.sup.9 or N(R.sup.9R.sup.10); each of R.sup.9 and R.sup.10 is independently hydrogen, C.sub.1-6 alkyl or C.sub.1-6 haloalkyl; and n is 0, 1 or 2.

2. A method according to claim 1, wherein R.sup.1 is halo, nitro, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl or OR.sup.5.

3. A method according to claim 1, wherein R.sup.5 is hydrogen, or methyl or ethyl, either of which may optionally be substituted by one or more halo substituents.

4. A method according to claim 1, wherein R.sup.1 is halo, nitro, methyl, trifluoromethyl, OH, methoxy or trifluoromethoxy.

5. A method according to claim 1, wherein m is 1 or 2.

6. A method according to claim 1, wherein R.sup.1 is 2-fluoro.

7. A method according to claim 1, wherein at least one R.sup.1 is OR.sup.5.

8. A method according to claim 7, wherein R.sup.5 is C.sub.1-6 alkyl.

9. A method according to claim 8, wherein R.sup.5 is methyl.

10. A method according to claim 1, wherein R.sup.3 is morpholinyl.

11. A method according to claim 1, wherein p is 2 or 3.

12. A method according to claim 1, wherein R.sup.4 is C.sub.1-4 alkyl or OR.sup.9.

13. A method according to claim 12, wherein R.sup.4 is methyl or methoxy.

14. A method according to claim 1, wherein n is 0 or 1.

15. A method according to claim 1, comprising administering to a patient an effective amount of a compound selected from: 2-[(3-fluorobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide; 2-[(4-fluorobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide; 2-[(2-methoxybenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide; 2-[(3-methoxybenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide; 2-[(4-methoxybenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide; 2-[(2-nitrobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide; 2-[(3-nitrobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide; 2-[(4-nitrobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide; 2-[(2-methylbenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide; 2-benzamido-N-(2-morpholin-4-ylethyl)benzamide; 3,4-dimethoxy-N-(2-[(2-morpholin-4-ylethyl)carbamoyl]phenyl)benzamide; 3,5-dimethoxy-N-(2-[(2-morpholin-4-ylethyl)carbamoyl]phenyl) benzamide; 3,5-difluoro-N-(2-[(2-morpholin-4-ylethyl)carbamoyl]phenyl)benzamide; 2,6-difluoro-N-(2-[(2-morpholin-4-ylethyl) carbamoyl]phenyl)benzamide; 2,4-difluoro-N-(2-[(2-morpholin-4-ylethyl)carbamoyl]phenyl)benzamide; 2-[(2-fluorobenzoyl)amino]-N-(2-morpholin-4-ylpropyl)benzamide; 2-[(4-fluorobenzoyl)amino]-N-(pyridin-3-ylmethyl)benzamide; 2-[(4-fluorobenzoyl)amino]-N-(pyrrolidin-3-ylmethyl)benzamide; 2-[(4-fluorobenzoyl)amino]-N-(piperidin-3-ylmethyl)benzamide; 2-[(4-fluorobenzoyl)amino]-N-(piperazin-3-ylmethyl)benzamide; N-(2-aminoethyl)-2-(2-fluorobenzamido) benzamide; 2-[(2-fluorobenzoyl)amino]-3-methyl-N-(2-morpholin-4-ylethyl)benzamide; 2-[(2-fluorobenzoyl)amino]-3-methoxy-N-(2-morpholin-4-ylethyl)benzamide; 2-fluoro-N-(2-((2-morpholinoethyl)carbamoyl)phenyl)benzamide; and 2-[(2-fluorobenzoyl) amino]-N-(2-morpholin-4-ylethyl) benzamide; or pharmaceutically or veterinarily acceptable salts thereof.

16. The method of claim 1, wherein the cancer is leukaemia or lymphoma.

17. The method of claim 1, wherein the cancer is anaplastic large cell lymphoma (ALCLs), classic Hodgkin lymphoma (cHL), non-Hodgkin's lymphoma or solid tumour cancer.

18. The method of claim 17, wherein the cancer is a solid tumour cancer selected from the group consisting of: breast cancer, melanoma, lung cancer, pancreatic cancer, oesophageal cancer, colorectal cancer, nasopharyngeal carcinoma and hepatocarcinoma.

19. The method according to claim 1, wherein the treatment comprises the treatment or prevention of metastasis in cancers.

20. The method according to claim 18, wherein the cancer is breast cancer.

21. The method according to claim 20, wherein the breast cancer is triple negative breast cancer or HER2 enriched breast cancer.

22. The method according to claim 1, wherein a compound of general formula (Ia) is administered in combination with one or more additional active agents which are useful in the treatment of cancer.

23. The method according to claim 22, wherein the one or more additional active agent is selected from: the anti-HER2 agents such as trastuzumab and pertuzurnab; the standard adjuvant therapy regimens 5-fluorouracil, doxorubicin, and cyclophosphamide (FAC); 5-fluorouracil, epirubicin, and cyclophosphamide (FEC); and doxorubicin and cyclophosphamide (AC); cyclophosphamide, methotrexate, and 5-fluorouracil (CMF); and docetaxel, doxorubicin, cyclophosphamide (TAC); and the anti-angiogenic/antimetastatic agent bevacizumab (Avastin).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The Invention will now be described by way of example only with reference to the

(2) Examples below and to the following Figures wherein:

(3) FIG. 1. Cell toxicity of Compound 1a. MCF-10A [A], MDA-MB-231 [B] and SKBR3 [C] breast cancer cells were cultivated with Compound 1a over a range of molarities in adherent growth conditions. Cell viability was determined after 24 hrs by the Cell Titre Blue viability assay and resulting fluorescence was normalised against fluorescence of control cells treated with DMSO in relevant concentration. Data represent average of six wells and error bars represent ±SEM. Dose response curves were generated using GraphPad software. The IC.sub.50 for each cell line is shown to the left of each graph.

(4) FIG. 2. Establishing the ability of Compound 1a to inhibit Bcl3 binding to its cognate protein partner NFKB1 (p50) by Indirect Sandwich ELISA assay. HEK-293 cells over-expressing FLAG-tagged Bcl-3 were cultivated with compound 1a or DMSO in a range of molarities in normal adherent growth conditions for 24 hrs. Cell lysates were prepared under non-denaturing conditions. [A] Indirect sandwich ELISA assay was performed on anti-FLAG coated ELISA plates using p50 antibody. Absorbance was measured at 405 nm and normalised to that of DMSO control. Error bars represent ±SEM of three independent wells. The dose response curve was generated using GraphPad software. The IC.sub.50 for Compound 1a is shown inset. [B] and [C] Indirect ELISA assay was performed on FLAG coated ELISA plates using Bcl-3 antibody. Absorbance was measured at 405 nm. Error bars represent ±SEM of three independent wells.

(5) FIG. 3. Establishing the ability of Compound 1a to inhibit NF-κB signalling by NF-κB promoter-reporter (luciferase) assay. MDA-MB-231 overexpressing Bcl-3 [A], HEK-293 cells overexpressing Bcl-3 [B] and HEK-293 overexpressing Bcl-3 and p52 [C] were cultivated with Compound 1a or DMSO control in a range of molarities for 24hrs before being transfected with NF-κB luciferase reporter for 48 hrs together with controls. NF-κB activity is represented as a % of DMSO control. Error bars represent ±SEM of three independent experiments. The dose response curve was generated using GraphPad software. IC.sub.50s for each cell line are shown inset.

(6) FIG. 4. Establishing the effect of Compound 1a on cell motility by Boyden Chamber assay. [A] MDA-MB-231 cells overexpressing Bcl-3 were cultivated with compound 1a (10 μM, 1 μM, 100 nM, 10 nM) or DMSO in corresponding concentration in normal adherent growth conditions for 24 hrs before being seeded onto Boyden motility chambers for 24 hrs in parallel with cells overexpressing the Bcl-3 binding mutant ANK M123. Migrated cells were counted from three fields of view of each of three replicate Boyden chambers. Error bars represent ±SEM. The dose response curve was generated using GraphPad software. The IC.sub.50 is shown inset. [B] Representative images of migrated cells for MDA-MB-231 cells overexpressing Bcl-3 and treated with either compound 1a or DMSO. Scale bars represent 200 μm.

(7) FIG. 5. Cell toxicity of series 3 compounds. MDA-MB-231 cells [A to C] and HEK-293 cells [D to F] were cultivated with compounds from series 1, 2 or 3 (10 nM, 100 nM, 1 μM and 10 μM) in adherent growth conditions. Cell viability was determined after 24 hrs by the Cell Titre Blue viability assay and resulting fluorescence was normalised against that of respective cells treated with DMSO under the same conditions. Data represent average of six wells and error bars represent ±SEM.

(8) FIG. 6. NF-κB assay in MDA-MB-231 cells with series 1 to 3 compounds. MDA-MB-231 cells overexpressing Bcl-3 were cultivated with compounds from series 1 [A], 2 [B] and 3 [C] at 1 μM concentration or DMSO control for 24 hrs before being transfected with NF-κB luciferase reporter for 48 hrs together with controls. NF-κB activity is plotted on a log scale as relative light units and normalised to the NF-κB activity of MDA-MB-231 cells not overexpressing Bcl-3. Error bars represent ±SEM of three independent transfections. (T-test, *=p<0.05 as compared to MDA-MB-231 Bcl-3 WT).

(9) FIG. 7. Establishing the effect of selected analogues on NF-κB signalling by luciferase-reporter assay in MDA-MB-231 cells. MDA-MB-231 cells overexpressing Bcl-3 were cultivated with analogues 1f [A], 2a [B], 2c [C] and 3a [D] or DMSO control in a range of molarities for 24 hrs before being transfected with NF-κB luciferase reporter for 48 hrs together with controls. NF-κB activity is represented as a % of DMSO control. Error bars represent ±SEM of three independent transfections. The dose response curve was generated using GraphPad software. The IC.sub.50 for each of the analogues are shown on the left of each graph.

(10) FIG. 8. Establishing the effect of selected analogues on Bcl-3 binding to p50 by Indirect Sandwich ELISA Assay. HEK-293 cells overexpressing FLAG-tagged Bcl-3 were cultivated with compound 1f [A], 2a [B], 2c [C] and 3a [D] or DMSO in a range of molarities in normal adherent growth conditions for 24 hrs. Cell lysates were prepared under non-denaturing conditions. Indirect sandwich ELISA assay was performed on FLAG coated ELISA plates using p50 antibody. Absorbance was measured at 405 nm and normalised to that of DMSO control. Error bars represent ±SEM of three independent wells. The dose response curve was generated using GraphPad software. IC.sub.50s are shown for each analogue to the left of each graph. [E-H]. Indirect ELISA assay was performed on FLAG coated ELISA plates using Bcl-3 antibody. Absorbance was measured at 405 nm. Error bars represent ±SEM of three independent wells.

(11) FIG. 9. Establishing IC.sub.50 the effect of selected analogues on cell motility by Boyden chamber assay. MDA-MB-231 cells overexpressing Bcl-3 were cultivated with compounds 1f [A], 2a [B], 2c [C] and 3a [D] or DMSO in a range of molarities in normal adherent growth conditions for 24 hrs before being seeded onto Boyden motility chambers for 24 hrs in parallel with MDA-MB-231 cells expressing the Bcl-3 binding mutant ANK M123. Migrated cells were counted from three fields of view of each of three replicate Boyden chambers. Error bars represent ±SEM. The dose response curve was generated using GraphPad software. The IC.sub.50s for each analogue are shown to the left of each graph.

(12) FIG. 10. Establishing the effect of analogue 1f on cell viability. MDA-MB-231 cells were cultivated with the analogue 1f over a range of molarities in adherent growth conditions. Cell viability was determined after 24 hrs by the Cell Titre Blue viability assay and resulting fluorescence was normalised against fluorescence of control cells treated with DMSO in relevant concentration. Data represent average of six wells and error bars represent ±SEM. Dose response curves were generated using GraphPad software. The IC.sub.50 is shown to the left of the graph.

(13) FIG. 11. Biological evaluation of di- and tri-substituted analogues [A] and [B]. MDA-MB-231 cells were cultivated with di- and tri-substituted compounds from series 1 (1l-1q) over a range of molarities in adherent growth conditions. Cell viability was determined after 24 hrs by the Cell Titre Blue viability assay and resulting fluorescence was normalised against that of respective cells treated with DMSO under the same conditions. Data represent average of six wells and error bars represent ±SEM. [C] MDA-MB-231 cells overexpressing Bcl-3 were cultivated with compounds from series 1 (1l-1q) at 1 μM concentration or DMSO control for 24 hrs before being transfected with NF-κB luciferase reporter for 48 hrs together with controls. NF-κB activity is plotted on a log scale as relative light units and normalised to the NF-κB activity of MDA-MB-231 cells. Error bars represent ±SEM of three independent transfections. (T-test, *=p<0.05 as compared to MDA-MB-231).

(14) FIG. 12. Establishing the effect of selected di- and tri-substituted analogues on NF-κB activity by luciferase-reporter assay in MDA-MB-231 cells. MDA-MB-231 cells overexpressing Bcl-3 were cultivated with analogues 10 [A], 1p [B], 1q [c] or DMSO control at a range of molarities for 24 hrs before being transfected with NF-κB luciferase reporter for 48 hrs together with controls. NF-κB activity is represented as a % of DMSO control. Error bars represent ±SEM of three independent transfections. The dose response curve was generated using GraphPad software. IC.sub.50s are shown to the left of each graph.

DETAILED DESCRIPTION

EXAMPLES

Synthesis of Compounds

(15) The compounds of the invention were synthesised according to the general method shown in Scheme 1.

(16) ##STR00012##

(17) Scheme 1 illustrates the cyclocondensation reaction of the anthranilic acid derivative (IV) and compound (V) which gives rise to the Intermediate (II). In the second step the intermediate (II) was reacted with the amine (III) to give the final product (I).

(18) All chemicals used in this investigation were obtained from commercial suppliers (Sigma Aldrich) and were used without further purification. All glassware were washed and dried before each experiment. Solvents were evaporated using the Buchi Rotavapor. Melting points were measured on a Griffin apparatus using a capillary method.

(19) The .sup.1H, .sup.13C NMR spectra were recorded on a Bruker AVANCE 500 spectrometer at 500 and 126 MHz respectively, at 25° C. Chemical shifts (δ) are reported in parts per million (ppm). J values are reported in Hertz (Hz). Dimethyl sulfoxide (DMSO) was used as a solvent. Used abbreviations include s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), dd (doublet of doublets), dt (doublet of triplets), td (triplet of doublets).

(20) TLC was performed using Merck TLC silica gel 60 plates F254 (40-60 μM) with detection by UV light (254-366 nm).

(21) Mass spectrometry was run using electron ionisation (EI) and electrospray (ES) on a Waters GCT Premier or a Waters LCT Premier XE, respectively. The mass spectrometry was performed as a service by School of Chemistry, Cardiff University. Elemental analysis (CHN) microanalysis was performed as a service by MEDAC Ltd, Surrey. High resolution mass spectrometry was performed on LTQ Orkitrap XL by the EPSRC National Mass Spectrometry Service (Swansea, UK).

General Method for Step 1

(22) In the first step, an anthranilic acid derivative (IV) was dissolved in pyridine (5 ml) and 2.2 equivalent of a substituted benzoyl chloride (V) and stirred at r.t. The reaction was monitored by TLC and stopped after approximately an hour after complete disappearance of the anthranilic acid (V). The reaction mixture was poured into 10% solution of sodium carbonate (3 g sodium carbonate, 27 ml distilled water). The formed precipitate was collected by filtration under reduced pressure as intermediate (II).

General Method for Step 2

(23) In the second step, to a stirred solution of intermediate (II) DMF (10 ml) were added 2 equivalents of DIPEA and 2.2 equivalents of amine (III). The reaction mixture was stirred at r.t overnight. The complete disappearance of starting material (II) was monitored by TLC. The reaction mixture was dissolved in DCM and washed three times with water. The product was evaporated under reduced pressure and the obtained solid was recrystallized from ethanol. All synthesised compounds were analysed by .sup.1H, .sup.13C NMR spectra and mass spectrometry. The purity of final compounds was also confirmed by elemental analysis.

Example 1

Synthesis of Compounds of Series 1

(24) The compounds of series 1 have the general formula

(25) ##STR00013##

(26) TABLE-US-00001 Compound R.sup.1 1a 2-F 1b 3-F 1c 4-F 1d 2-OCH.sub.3 1e 3-OCH.sub.3 1f 4-OCH.sub.3 1g 2-NO.sub.2 1h 3-NO.sub.2 1i 4-NO.sub.2 1j 2-CH.sub.3 1k H 1l 3,4-OCH.sub.3 1m 3,5-OCH.sub.3 1n 3,4,5-OCH.sub.3 1o 3,5-F 1p 2,6-F 1q 2,4-F

Step 1—Synthesis of Intermediates

Synthesis of 2-(2-fluorophenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1a)

(27) Chemical Formula: C14H8FNO2, Molecular Weight: 241.22

(28) The synthetic procedure followed the general method above using anthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of 2-fluorobenzoyl chloride (0.96 ml, 8.02 mmol). Collected as a white solid, yield 85% (0.75 g), mp 97° C.

(29) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.18 (dd, J=8.0, 1.5 Hz, 1H, ArH), 8.10 (td, J=7.8, 1.8 Hz, 1H, ArH), 7.98 (td, J=9.1, 2.2 Hz, 1H, ArH), 7.74 (d, J=7.9 Hz, 1H, ArH), 7.68 (td, J=7.6, 1.2 Hz, 2H, ArH), 7.47-7.40 (m, 2H, ArH).

(30) .sup.13C NMR (126 MHz, DMSO-d6) δ 160.48 (d, J.sub.C-F=258.3 Hz, ArC—F), 158.66 (ArC═O), 154.23 (ArC), 146.03 (ArC), 136.88 (ArCH), 134.52 (d, J.sub.C-F=8.8 Hz, ArCH), 131.10 (ArCH), 129.02 (ArCH), 127.98 (ArCH), 127.03 (ArCH), 124.83 (d, J.sub.C-F=3.8 Hz, ArCH), 118.66 (d, J.sub.C-F=10.1 Hz, ArC), 117.21 (d, J.sub.C-F=21.4 Hz, ArCH), 116.92 (ArC).

(31) MS (APCI.sup.+): 242.05 [M+1].

Synthesis of 2-(3-fluorophenyl-4H-3,1-benzoxazin-4-one (Intermediate 1b)

(32) Chemical Formula: C14H8FNO2, Molecular Weight: 241.22

(33) The synthetic procedure followed the general method above using anthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of 3-fluorobenzoyl chloride (0.98 ml, 8.02 mmol). Collected as a white solid, yield 80% (0.71 g), mp 96° C.

(34) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.48 (d, J=8.3 Hz, 1H, ArH), 8.17 (d, J=7.9 Hz, 1H, ArH), 8.06-7.93 (m, 2H, ArH), 7.87 (d, J=10.4 Hz, 1H, ArH), 7.81 (d, J=10.1 Hz, 1H, ArH), 7.77-7.48 (m, 1H, ArH), 7.27 (t, J=7.7 Hz, 1H, ArH).

(35) .sup.13C NMR (126 MHz, DMSO-d6) δ 162.17 (d, J.sub.C-F=245.7 Hz, ArC—F), 158.56 (ArC═O), 145.93 (ArC), 139.71 (ArC), 136.89 (ArCH), 134.20 (ArCH), 131.28 (d, J J.sub.C-F=8.2 Hz, ArCH), 130.64 (ArCH), 128.90 (ArCH), 127.00 (ArCH), 123.69 (d, J.sub.C-F=2.8 Hz, ArCH), 121.14 (ArC), 119.35 (d, J.sub.C-F=88.2 Hz, ArCH), 117.37 (d, J.sub.C-F=85.7 Hz, ArC).

(36) MS (APCI.sup.+): 242.06 [M+1]. O N O F

Synthesis of 2-(4-fluorophenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1c)

(37) Chemical Formula: C14H8FNO2 Molecular Weight: 241.22

(38) The synthetic procedure followed the general method above using anthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of 4-fluorobenzoyl chloride (0.95 ml, 8.02 mmol). Collected as a white solid, yield 91% (0.80 g), mp 159° C.

(39) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.26 (td, J=14.1, 5.4 Hz, 2H, ArH), 8.16 (dd, J=8.0, 1.5 Hz, 1H, ArH), 7.96 (t, J=15.8 Hz, 1H, ArH), 7.72 (d, J=8.1 Hz, 1H, ArH), 7.63 (td, J=7.6, 1.2 Hz, 1H, ArH), 7.48-7.40 (m, 2H, ArH).

(40) .sup.13C NMR (126 MHz, DMSO-d6) δ 164.74 (d, J.sub.C-F=251.4 Hz, ArC—F), 158.72 (ArC═O), 155.58 (ArC), 146.18 (ArC), 136.87 (ArCH), 130.53 (d, J.sub.C-F=9.4 Hz, ArCH), 128.57 (ArCH), 128.05 (ArCH), 126.83 (d, J.sub.C-F=3.8 Hz, ArCH), 126.61 (ArC), 116.81 (ArC), 116.17 (d, J.sub.C-F=22.3 Hz, ArCH).

(41) MS (APCI.sup.+): 242.06 [M+1].

Synthesis of 2-(2-methoxyphenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1d)

(42) Chemical Formula: C15H11NO3 Molecular Weight: 253.25

(43) The synthetic procedure followed the general method above using anthranilic acid (0.50 g, 3.65 mmol) dissolved in dry pyridine (5 ml) under anhydrous conditions in nitrogen atmosphere and 2.2 equivalent of 2-methoxybenzoyl chloride (1.19 ml, 8.02 mmol). Collected as a white solid, yield 98% (0.91 g), mp 107° C.

(44) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.17 (dd, J=7.9, 1.6 Hz, 1H, ArH), 7.97 (td, 16.0, 1.1 Hz, 1H, ArH), 7.78 (dd, J=7.7, 1.8 Hz, 1H, ArH), 7.71 (d, 7.9 Hz, 1H, ArH), 7.66 (td, J=7.6, 1.2 Hz, 1H, ArH), 7.60 (td, J=8.8, 1.8 Hz, 1H, ArH), 7.24 (d, J=8.4 Hz, 1H, ArH), 7.12 (t, J=7.5 Hz, 1H, ArH), 3.88 (s, 3H, OCH3).

(45) .sup.13C NMR (126 MHz, DMSO-d6) δ 159.15 (ArC═O), 157.89 (ArC), 157.16 (ArC), 146.31 (ArC), 136.88 (ArCH), 133.24 (ArCH), 130.97 (ArCH), 128.81 (ArCH), 127.90 (ArCH), 126.89 (ArCH), 120.36 (ArCH), 120.27 (ArC), 116.53 (ArC), 112.56 (ArCH), 55.66 (OCH3).

(46) MS (APCI.sup.+): 254.07 [M+1].

Synthesis of 2-(3-methoxyphenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1e)

(47) Chemical Formula: C15H11NO3 Molecular Weight: 253.25

(48) The synthetic procedure followed the general method above using anthranilic acid (0.50 g, 3.65 mmol) dissolved in dry pyridine (5 ml) under anhydrous conditions in nitrogen atmosphere and 2.2 equivalent of 3-methoxybenzoyl chloride (1.13 ml, 8.02 mmol). Collected as a white solid, yield 97% (0.90 g), mp 103° C.

(49) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.14 (dd, J=7.9, 1.5 Hz, 1H, ArH), 7.94 (t, J=7.9 Hz, 1H, ArH), 7.76 (d, J=7.7, Hz, 1H, ArH), 7.71 (d, J=8.1 Hz, 1H, ArH), 7.66-7.58 (m, 2H, ArH), 7.50 (t, J=8.0 Hz, 1H, ArH), 7.21 (dd, J=8.4, 2.6 Hz, 1H, ArH), 3.85 (s, 3H, OCH3).

(50) .sup.13C NMR (126 MHz, DMSO-d6) δ 159.42 (ArC═O), 158.79 (ArC), 156.10 (ArC), 146.09 (ArC), 136.86 (ArCH), 131.27 (ArC), 130.16 (ArCH), 128.64 (ArCH), 128.01 (ArCH), 126.90 (ArCH), 120.15 (ArCH), 118.72 (ArCH), 116.79 (ArC), 112.36 (ArCH), 55.35 (OCH3).

(51) MS (APCI.sup.+): 254.07 [M+1].

Synthesis of 2-(4-methoxyphenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1f)

(52) Chemical Formula: Cl 5H11NO3 Molecular Weight: 253.25

(53) The synthetic procedure followed the general method above using anthranilic acid (0.50 g, 3.65 mmol) dissolved in dry pyridine (5 ml) under anhydrous conditions in nitrogen atmosphere and 2.2 equivalent of 4-methoxybenzoyl chloride (1.09 ml, 8.02 mmol). Collected as a white solid, yield 71% (0.66 g), mp 121° C.

(54) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.14 (td, J=17.8, 1.8 Hz, 3H, ArH), 7.93 (td, J=8.5, 1.6 Hz, 1H, ArH), 7.68 (d, J=7.9 Hz, 1H, ArH), 7.59 (td, J=7.4, 1.2 Hz, 1H, ArH), 7.14 (d, J=9.0 Hz, 2H, ArH), 3.88 (s, 3H, OCH3).

(55) .sup.13C NMR (126 MHz, DMSO-d6) δ 162.88 (ArC═O), 158.95 (ArC), 156.39 (ArC), 146.59 (ArC), 136.80 (ArCH), 132.66 (ArCH), 129.80 (ArCH), 126.61 (ArCH), 122.10 (ArCH), 120.18 (ArC), 116.55 (ArC), 114.45 (ArCH), 55.56 (OCH3).

(56) MS (APCI.sup.+): 254.08 [M+1].

Synthesis of 2-(2-nitrophenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1g)

(57) Chemical Formula: C14H8N2O4 Molecular Weight: 268.22

(58) The synthetic procedure followed the general method above using anthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of 2-nitrobenzoyl chloride (1.06 ml, 8.02 mmol). Collected as a yellow solid, yield 77% (0.75 g), mp 169° C.

(59) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.22 (dd, J=7.8, 1.5 Hz, 1H, ArH), 8.17 (dd, J=8.0, 1.3 Hz, 1H, ArH), 8.10 (dd, J=7.6, 1.5 Hz, 1H, ArH), 8.02 (td, J=7.7, 1.6 Hz, 1H, ArH), 7.96 (td, J=7.6, 1.3 Hz, 1H, ArH), 7.91 (td, J=7.8, 1.6 Hz, 1H, ArH), 7.76-7.69 (m, 2H, ArH).

(60) .sup.13C NMR (126 MHz, DMSO-d6) δ 158.20 (ArC═O), 154.50 (ArC), 148.15 (ArC), 145.58 (ArC), 137.26 (ArCH), 133.60 (ArCH), 132.97 (ArCH), 131.16 (ArCH), 129.61 (ArCH), 128.22 (ArCH), 127.04 (ArCH), 124.98 (ArC), 124.57 (ArCH), 116.68 (ArC).

(61) MS (APCI.sup.+): 269.05 [M+1].

Synthesis of 2-(3-nitrophenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1h)

(62) Chemical Formula: C14H8N2O4 Molecular Weight: 268.22

(63) The synthetic procedure followed the general method above using anthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of 3-nitrobenzoyl chloride (1.06 ml, 8.02 mmol). Collected as a yellow solid, yield 96% (0.94 g), mp 115° C.

(64) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.74 (t, J=2.0 Hz, 1H, ArH), 8.48 (dd, J=8.3, 1.1 Hz, 1H, ArH), 8.37 (dd, J=8.5, 1.3 Hz, 2H, ArH), 7.99 (dd, J=7.9, 1.6 Hz, 1H, ArH), 7.91 (t, J=8.0 Hz, 1H, ArH), 7.70 (td, J=8.5, 1.7 Hz, 1H, ArH), 7.30 (td, J=7.6, 1.3 Hz, 1H, ArH).

(65) .sup.13C NMR (126 MHz, DMSO-d6) δ 167.70 (ArC═O), 162.76 (ArC), 148.00 (ArC), 139.20 (ArC), 135.84 (ArC), 133.98 (ArCH), 133.23 (ArCH), 130.69 (ArCH), 126.53 (ArCH, 124.09 (ArCH), 122.02 (ArCH), 118.95 (ArC).

(66) MS (APCI.sup.+): 269.06 [M+1].

Synthesis of 2-(3-nitrophenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1i)

(67) Chemical Formula: C14H8N2O4 Molecular Weight: 268.22

(68) The synthetic procedure followed the general method above using anthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of 4-nitrobenzoyl chloride (1.49 g, 8.02 mmol). Collected as a yellow solid, yield 96% (0.94 g), mp 169° C.

(69) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.19 (t, J=7.8 Hz, 2H, ArH), 8.00 (t, J=7.8 Hz, 2H, ArH), 7.79 (d, J=8.1 Hz, 2H, ArH), 7.69 (t, J=7.6 Hz, 2H, ArH).

(70) .sup.13C NMR (126 MHz, DMSO-d6) δ 158.43 (ArC═O), 154.74 (ArC), 149.62 (ArC), 145.77 (ArC), 136.98 (ArCH), 135.81 (ArC), 129.37 (ArCH), 129.13 (ArCH), 128.13 (ArCH), 127.24 (ArCH), 124.08 (ArCH), 117.20 (ArC).

(71) MS (APCI.sup.+): 269.08 [M+1].

Synthesis of 2-(o-tolyl)-4H-3,1-benzoxazin-4-one (Intermediate 1j)

(72) Chemical Formula: C15H11NO2 Molecular Weight: 237.25

(73) The synthetic procedure followed the general method above using anthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of 2-methyl benzoyl chloride (1.05 ml, 8.02 mmol). Collected as a yellow solid, yield 99% (0.86 g), mp 102° C.

(74) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.17 (d, J=8.0 Hz, 1H, ArH), 7.99-7.90 (m, 2H, ArH), 7.71 (d, J=8.1 Hz, 1H, ArH), 7.65 (t, J=7.6 Hz, 1H, ArH), 7.51 (t, J=7.5 Hz, 1H, ArH), 7.40 (t, J=6.9 Hz, 2H, ArH), 2.66 (s, 3H, CH3).

(75) .sup.13C NMR (126 MHz, DMSO-d6) δ 159.07 (ArC═O), 157.59 (ArC), 146.17 (ArC), 138.32 (ArC), 136.74 (ArCH), 131.69 (ArCH), 131.48 (ArCH), 129.88 (ArC), 129.79 (ArCH), 128.69 (ArCH), 127.84 (ArCH), 126.93 (ArCH), 126.07 (ArCH), 116.68 (ArC), 21.31 (CH3).

(76) MS (APCI.sup.+): 238.07 [M+1].

Synthesis of 2-phenyl-4H-3,1-benzoxazin-4-one (Intermediate 1k)

(77) Chemical Formula: C14H9NO2 Molecular Weight: 223.23

(78) The synthetic procedure followed the general method above using anthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of benzoyl chloride (0.93 ml, 8.02 mmol). Collected as a yellow solid, yield 95% (0.77 g), mp 91° C.

(79) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.22 (dd, J=7.3, 1.7 Hz, 1H, ArH), 8.03 (dd, J=7.9, 1.6 Hz, 1H, ArH), 7.98 (dd, 8.1, 1.6 Hz, 2H, ArH), 7.72-7.64 (m, 2H, ArH), 7.62 (dd, J=8.1, 6.6 Hz, 3H, ArH).

(80) .sup.13C NMR (126 MHz, DMSO-d6) δ 168.04 (ArC═O), 164.76 (ArC), 140.25 (ArC), 136.87 (ArCH), 134.33 (ArCH), 132.19 (ArCH), 130.69 (ArCH), 129.00 (ArCH), 127.03 (ArCH), 123.34 (ArCH), 120.75 (ArCH), 116.99 (ArC).

(81) MS (APCI.sup.+): 224.08 [M+1].

Synthesis of 2-(3,4-dimethoxyphenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1l)

(82) Chemical Formula: C16H13NO4 Molecular Weight: 283.28

(83) The synthetic procedure followed the general method above using anthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of 3,4-dimethoxybenzoyl chloride (1.61 g, 8.02 mmol). Collected as a white solid, yield 83% (0.86 g), mp 166° C.

(84) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.15 (dd, J=7.8, 1.6 Hz, 1H, ArH), 7.94 (td, J=8.4, 1.6 Hz, 1H, ArH), 7.83 (dd, J=8.5, 2.1 Hz, 1H, ArH) 7.71 (dd, J=6.8, 1.7 Hz, 2H, ArH), 7.60 (td, J=8.3, 1.1 Hz, 1H, ArH), 7.18 (d, J=8.6 Hz, 1H, ArH), 3.89 (d, J=4.7 Hz, 6H, OCH3).

(85) .sup.13C NMR (126 MHz, DMSO-d6) δ 159.35 (ArC═O), 158.95 (ArC), 152.82 (ArC), 148.79 (ArC), 146.56 (ArC), 136.84 (ArCH), 128.09 (ArCH), 126.65 (ArCH), 122.07 (ArC), 121.86 (ArCH), 116.65 (ArC), 111.56 (ArCH), 110.19 (ArCH), 55.77 (OCH3), 55.63 (OCH3).

(86) MS (EI.sup.+): 283.1.

Synthesis of 2-(3,5-dimethoxyphenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1m)

(87) Chemical Formula: C16H13NO4 Molecular Weight: 283.28

(88) The synthetic procedure followed the general method above using anthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of 3,5-dimethoxybenzoyl chloride (1.61 g, 8.02 mmol). Collected as a white solid, yield 93% (0.97 g), mp 165° C.

(89) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.71 (dd, J=7.8, 1.6 Hz, 1H, ArH), 7.97 (t, J=7.6 Hz, 1H, ArH), 7.75 (dd, J=8.0, 2.1 Hz, 1H, ArH), 7.65 (t, J=7.6 Hz, 1H, ArH), 7.31 (d, J=2.5 Hz, 2H, ArH), 6.81 (d, J=2.3 Hz, 1H, ArH), 3.86 (s, 6H, OCH.sub.3)

(90) .sup.13C NMR (126 MHz, DMSO-d6) δ 160.72 (ArC═O), 158.76 (ArC), 152.82 (ArC), 146.11 (ArC), 145.80 (ArC), 136.84 (ArCH), 132.01 (ArC), 131.55 (ArCH), 128.73 (ArCH), 128.08 (ArCH), 126.99 (ArCH), 116.96 (ArC), 105.49 (ArCH), 104.83 (ArCH), 55.59 (OCH.sub.3).

(91) MS (EI.sup.+): 283.1.

Synthesis of 2-(3,4,5-trimethoxyphenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1n)

(92) Chemical Formula: C17H15NO5 Molecular Weight: 313.30

(93) The synthetic procedure followed the general method above using anthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of 3,4,5-dimethoxybenzoyl chloride (1.85 g, 8.02 mmol). Collected as a white solid, yield 91% (1.04 g), mp 165° C.

(94) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.17 (dd, J=9.3, 1.8 Hz, 1H, ArH), 7.98-7.94 (m, 1H, ArH), 7.75 (d, J=2.9 Hz, 1H, ArH), 7.63 (td, J=15.7, 1.2 Hz, 1H, ArH), 7.49 (s, 2H, ArH), 3.92 (s, 6H, OCH3), 3.80 (s, 3H, OCH.sub.3).

(95) .sup.13C NMR (126 MHz, Chloroform) δ 159.61 (ArC═O), 156.82 (ArC), 153.34 (ArC), 147.07 (ArC), 142.21 (ArC), 136.58 (ArCH), 128.64 (ArCH), 128.09 (ArCH), 127.09 (ArCH), 125.23 (ArC), 116.78 (ArC), 105.60 (ArCH), 61.01 (OCH.sub.3), 56.42 (OCH.sub.3).

(96) MS (EI.sup.+): 313.1.

(97) Syntheiss of 2-(3,5-difluorophenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1o)

(98) Chemical Formula: C14H7F2NO2 Molecular Weight: 259.21

(99) The synthetic procedure followed the general method above using anthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of 3,5-difluorobenzoyl chloride (0.95 ml, 8.02 mmol). Collected as a white solid, yield 94% (0.89 g), mp 122° C.

(100) .sup.1H NMR (500 MHz, Chloroform) δ 8.28 (dd, J=9.4, 1.6 Hz, 1H, ArH), 7.88 (m, 3H, ArH), 7.74 (dd, 8.1, 0.6 Hz, 1H, ArH), 7.60 (tt, 15.3, 1.4 Hz, 1H, ArH), 7.05 (m, 1H, ArH).

(101) .sup.13C NMR (126 MHz, Chloroform) δ 164.11 (d, J.sub.C-F=10.1 Hz, ArC—F), 162.12 (d, J.sub.C-F=18.1 Hz, ArC—F), 158.78 (ArC═O), 146.35 (ArC), 136.78 (ArCH), 133.75 (ArC), 128.98 (ArCH), 128.78 (ArCH), 127.49 (ArCH), 117.14 (ArC), 111.40 (d, J.sub.C-F=7.7 Hz, ArCH), 111.23 (d, J.sub.C-F=7.2 Hz, ArCH), 107.93 (ArCH), 105.93 (ArC).

(102) MS (EI.sup.+): 259.1.

Synthesis of 2-(2,6-difluorophenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1p)

(103) Chemical Formula: C14H7F2NO2 Molecular Weight: 259.21

(104) The synthetic procedure followed the general method above using anthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of 2,6-difluorobenzoyl chloride (1.01 ml, 8.02 mmol). Collected as a white solid, yield 97% (0.92 g), mp 119° C.

(105) .sup.1H NMR (500 MHz, Chloroform-d) δ 8.31 (dd, J=7.8, 2.0 Hz, 1H, ArH), 7.92-7.88 (m, 1H, ArH), 7.76 (dd, J=8.1, 1.7 Hz, 1H, ArH), 7.64 (td, J=7.3, 1.2 Hz, 1H, ArH), 7.52 (tt, J=8.5, 6.2 Hz, 1H, ArH), 7.08 (tt, J=7.1, 4.5 Hz, 2H, ArH).

(106) .sup.13C NMR (126 MHz, DMSO-d6) δ 161.85 (d, J.sub.C-F=6.2 Hz, ArC—F), 159.75 (d, J.sub.C-F=7.4 Hz, ArC—F), 158.91 (C═O), 150.95 (ArC), 146.19 (ArC), 136.68 (ArCH), 133.55 (ArC), 133.03 (t, J.sub.C-F=21.1 Hz, ArCH), 129.38 (ArCH), 128.68 (ArCH), 127.49 (ArCH), 117.26 (ArC), 112.25 (d, J.sub.C-F=4.2 Hz, ArCH), 112.07 (d, J.sub.C-F=4.4 Hz, ArCH).

(107) MS (EI.sup.+): 259.1.

Synthesis of 2-(2,4-difluorophenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1q)

(108) Chemical Formula: C14H7F2NO2 Molecular Weight: 259.21

(109) The synthetic procedure followed the general method above using anthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of 2,4-difluorobenzoyl chloride (0.99 ml, 8.02 mmol). Collected as a white solid, yield 81% (0.76 g), mp 102° C.

(110) .sup.1H NMR (500 MHz, Chloroform-d) δ 8.28 (dd, J=7.9, 0.6 Hz, 1H, ArH), 8.20 (td, J=8.6, 6.4 Hz, 1H, ArH), 7.91-7.84 (m, 1H, ArH), 7.73 (dd, J=8.2, 0.6 Hz, 1Hz, ArH), 7.59 (td, J=7.8, 1.2 Hz, 1H, ArH), 7.08 (m, 2H, ArH).

(111) .sup.13C NMR (126 MHz, DMSO-d6) δ 164.50 (d, J.sub.C-F=252.9 Hz, ArC—F), 162.20 (d, J.sub.C-F=289.0 Hz, ArC—F), 158.99 (ArC═O), 146.63 (ArC), 136.68 (ArCH), 132.85 (ArCH), 128.78 (ArCH), 128.62 (ArCH), 127.48 (ArCH), 116.97 (ArC), 113.95 (ArC), 112.00 (d, J.sub.C-F=21.9 Hz), 107.25 (ArC), 105.63 (ArCH).

(112) MS (EI.sup.+): 259.0.

Step 2—Synthesis of Example Compounds

Synthesis of 2-[(2-fluorobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide (Compound 1a)

(113) Chemical Formula: C20H22FN3O3 Molecular Weight: 371.41

(114) The synthetic procedure followed the general method for step 2 above using Intermediate 1a (1.00 g, 4.15 mmol) in DMF (10 ml), 2 equivalents of DIPEA (1.19 ml, 8.29 mmol) and 2.2 equivalents of 2-morpholinoethanamine (1.44 ml, 9.12 mmol). The product was recrystallized from ethanol as a white solid. Yield 47% (0.73 g), mp 131° C.

(115) .sup.1H NMR (500 MHz, DMSO-d6) δ 12.00 (s, 1H, NH), 8.72 (s, 1H, NH), 8.57 (d, J=8.3 Hz, 1H, ArH), 7.89 (td, J=7.7, 1.9 Hz, 1H, ArH), 7.76 (dd, J=8.0, 1.5 Hz, 1H, ArH), 7.68-7.64 (m, 1H, ArH), 7.57 (td, J=8.6, 1.5 Hz, 1H, ArH), 7.46-7.35 (m, 2H, ArH), 7.24 (td, J=7.6, 1.2 Hz, 1H, ArH), 3.53 (t, J=4.6 Hz, 4H, CH.sub.2), 3.40 (q, J=6.4 Hz, 2H, CH.sub.2), 2.51 (t, J=6.7 Hz, 2H, CH.sub.2), 2.47 (t, J=4.7 Hz, 4H, CH.sub.2).

(116) .sup.13C NMR (126 MHz, DMSO-d6) δ 168.03 (C═O), 160.86 (d, J.sub.C-F=146.2 Hz, ArC—F), 158.28 (C═O), 138.32 (ArC), 133.77 (d, J.sub.C-F=8.8 Hz, ArCH), 131.83 (ArCH), 130.62 is (d, J.sub.C-F=1.3 Hz, ArCH), 128.02 (ArCH), 125.04 (d, J.sub.C-F=3.8 Hz, ArCH), 123.30 (ArCH), 122.94 (d, J.sub.C-F=13.9 Hz, ArC) 121.73 (ArC), 120.96 (ArCH), 116.57 (d, J.sub.C-F=22.7 Hz, ArCH), 66.15 (CH.sub.2), 56.98 (CH.sub.2), 53.18 (CH.sub.2), 36.43 (CH.sub.2).

(117) MS (ES+): 372.19 [M+1].

Synthesis of Hydrochloride Salt of Compound 1a

(118) Compound 1a (0.31 g, 0.81 mmol) was dissolved in 150 ml of methanol. Hydrogen chloride in methanol (1.2 ml, 1.25M) was added and the mixture was stirred at r.t. for an hour. Methanol was evaporated from the mixture, followed by addition and evaporation of hexane. DCM (2 ml) was added to the mixture followed by addition of hexane and the formed white precipitate was filtered under reduced pressure. Yield 79%, (0.26g), mp 145° C.

(119) .sup.1H NMR (500 MHz, Chloroform) δ 11.61 (s, 1H, NH), 9.00 (s, 1H, NH), 8.65 (d, J=8.2 Hz, 1H, ArH), 8.00 (td, J=7.6, 2.0 Hz, 2H, ArH), 7.50-7.43 (m, 2H, ArH), 7.25 (t, J=7.5 Hz, 1H, ArH), 7.15 (t, J=5.3 Hz, 1H, ArH), 7.12 (d, J=7.0 Hz, 1H, ArH), 4.06 (t, J=12.2 Hz, 2H, CH.sub.2), 3.93-3.78 (m, 4H, CH.sub.2) 3.56 (d, J=11.8 Hz, 2H, CH.sub.2), 3.20 (s, 2H, CH.sub.2), 3.01 (s, 1H, NH.sup.+), 2.87 (t, J=8.3, 7.6 Hz, 2H, CH.sub.2).

(120) .sup.13C NMR (126 MHz, DMSO-d6) δ 169.51 (C═O), 162.15 (ArC—F), 162.16 (ArC), 161.23 (ArC), 159.24 (ArC), 139.50 (ArC), 133.25 (d, J.sub.C-F=8.8 Hz, ArC), 132.85 (ArCH), 131.70 (d, J.sub.C-F=2.2 Hz, ArCH), 128.40 (ArCH), 124.69 (d, J.sub.C-F=3.4 Hz, ArCH), 123.73 (ArCH), 122.04 (ArCH), 120.17 (ArC), 116.35 (d, J.sub.C-F=23.9 Hz, ArCH), 63.53 (CH.sub.2), 58.72 (CH.sub.2), 53.11 (CH.sub.2), 34.00 (CH.sub.2).

(121) Calculated analysis for C20H22FN3O3 (371.41): C, 64.68; H, 5.97; N, 11.31. Found C, 64.49; H, 6.08; N, 11.29.

(122) MS (EI.sup.+): 372.2

Synthesis of 2-[(4-fluorobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide (Compound 1c)

(123) Chemical Formula: C20H22FN3O3 Molecular Weight: 371.41

(124) The synthetic procedure followed the general method for step 2 above using Intermediate 1c (0.50 g, 2.06 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.72 ml, 4.12 mmol) and 2.2 equivalents of 2-morpholinoethanamine (0.60 ml, 4.54 mmol). The product was recrystallized from ethanol as a white solid. Yield 8% (0.05 g), mp 129° C.

(125) .sup.1H NMR (500 MHz, DMSO-d6) δ 12.52 (s, 1H, NH), 8.80 (s, 1H, NH), 8.61 (d, J=8.3 Hz, 1H, ArH), 8.0 (td, J=14.1, 2.1 Hz, 2H, ArH), 7.83 (dd, J=7.9, 1.5 Hz, 1H, ArH), 7.58 (td, J=8.2, 7.8, 1.5 Hz, 1H, ArH), 7.45 (t, J=17.6 Hz, 2H, ArH), 7.23 (t, J=7.6, 1H, ArH), 3.55 (t, J=4.6 Hz, 4H, CH.sub.2), 3.44 (q, J=6.5 Hz, 2H, CH.sub.2), 2.51 (q, J=6.3 Hz, 6H, CH.sub.2).

(126) .sup.13C NMR (126 MHz, DMSO-d6) δ 168.50 (C═O), 164.29 (d, J.sub.C-F=253.3 Hz, ArC—F), 158.31 (C═O), 139.17 (ArC), 132.15 (ArCH), 131.05 (d, J.sub.C-F=3.8 Hz, ArC), 129.61 (d, J.sub.C-F=10.1 Hz, ArCH), 128.10 (ArCH), 122.94 (ArCH), 120.58 (ArC), 120.38 (ArCH), 115.98 (d, J.sub.C-F=20.2 Hz, ArCH), 66.17 (CH.sub.2), 56.96 (CH.sub.2), 53.20 (CH.sub.2), 36.55 (CH.sub.2).

(127) MS (ES+): 372.20 [M+1].

(128) Calculated analysis for C20H22FN3O3 (371.41): C, 64.62; H, 6.09; N, 11.32.

(129) Found C, 64.49; H, 6.08; N, 11.29.

Synthesis of 2-[(3-methoxybenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide (Compound 1e)

(130) Chemical Formula: C21H25N3O4 Molecular Weight: 383.44

(131) The synthetic procedure followed the general method for step 2 above using Intermediate le (0.50 g, 1.97 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.69 ml, 3.94 mmol) and 2.2 equivalents of 2-morpholinoethanamine (0.57 ml, 4.34 mmol). The product was recrystallized from ethanol as a white solid. Yield 0.33 g (44%), mp 105° C.

(132) .sup.1H NMR (500 MHz, DMSO-d6) δ 12.49 (s, 1H, NH), 8.80 (s, 1H, NH), 6 8.63 (d, J=8.5 Hz, 1H, ArH), 7.82 (dd, J=7.9, 1.6 Hz, 1H, ArH), 7.58 (td, J=17.0, 1.2 Hz, 1H, ArH), 7.52 (d, J=6.5, Hz, 2H, ArH), 7.48 (t, J=3.2 Hz, 1H, ArH), 7.25-7.16 (m, 2H, ArH), 3.86 (s, 3H, CH.sub.3), 3.54 (t, J=4.7 Hz, 4H, CH.sub.2), 3.44 (q, J=6.5 Hz, 2H, CH.sub.2), 2.50 (d, J=15.1 Hz, 2H, CH.sub.2), 2.42 (t, J=4.7 Hz, 4H, CH.sub.2). .sup.13C NMR (126 MHz, DMSO-d6) 6 168.50 (C═O), 164.09 (C═O), 159.52 (ArC), 139.19 (ArC), 136.01 (ArC), 132.14 (ArCH), 130.13 (ArCH), 128.10 (ArCH), 122.87 (ArCH), 120.55 (ArC), 120.27 (ArCH), 118.80 (ArCH), 117.63 (ArCH), 112.46 (ArCH), 66.17 (CH.sub.2), 56.99 (CH.sub.2), 55.28 (0 CH.sub.3), 53.18 (CH.sub.2), 36.53 (CH.sub.2).

(133) MS (ES+): 384.19 [M+1].

(134) Calculated analysis for C21H25N304 (383.44): C, 65.78; H, 6.57; N, 10.95. Found C, 65.71; H, 6.77; N, 10.98.

Synthesis of 2-[(4-methoxybenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide (Compound 1f)

(135) Chemical Formula: C21H25N3O4 Molecular Weight: 383.44

(136) The synthetic procedure followed the general method for step 2 above using Intermediate 1f (0.50 g, 1.97 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.69 ml, 3.94 mmol) and 2.2 equivalents of 2-morpholinoethanamine (0.57 ml, 4.34 mmol). The product was recrystallized from ethanol as a white solid. Yield 32% (0.24 g), mp 110° C.

(137) .sup.1H NMR (500 MHz, DMSO-d6) δ 12.44 (s, 1H, NH), 8.78 (s, 1H, NH), 8.65 (d, J=8.3 Hz, 1H, ArH), 7.92 (d, J=8.9 Hz, 2H, ArH), 7.81 (dd, J=8.0, 1.5 Hz, 1H, ArH), 7.56 (td, J=8.7, 1.5 Hz, 1H, ArH), 7.19 (d, J=15.5, 1.3 Hz, 3H, ArH), 3.86 (s, 3H, OCH.sub.3), 3.55 (t, J=4.6 Hz, 4H, CH.sub.2), 3.45 (q, J=6.4 Hz, 2H, CH.sub.2), 2.51 (m, 3H, CH.sub.2), 2.43 (d, J=9.1 Hz, 3H, CH.sub.2).

(138) .sup.13C NMR (126 MHz, DMSO-d6) δ 168.60 (C═O), 163.87 (C═O), 162.20 (ArC), 140.73 (ArC), 132.10 (ArCH), 128.80 (ArCH), 128.06 (ArCH), 126.63 (ArC), 122.50 (ArCH), 120.23 (ArC), 120.18 (ArCH), 114.18 (ArCH), 66.18 (CH.sub.2), 56.98 (CH.sub.2), 55.45 (OCH.sub.3), 53.21 (CH.sub.2), 36.54 (CH.sub.2).

(139) MS (ES+): 384.19 [M+1].

(140) Calculated analysis for C21H25N3O4 (383.44): C, 65.78; H, 6.57; N, 10.95. Found C, 65.63; H, 6.62; N, 10.95.

Synthesis of 2-[(2-nitrobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide (Compound 1g)

(141) Chemical Formula: C20H22N4O5 Molecular Weight: 398.16

(142) The synthetic procedure followed the general method for step 2 above using Intermediate 1g (0.50 g, 1.87 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.65 ml, 3.74 mmol) and 2.2 equivalents of 2-morpholinoethanamine (0.54 ml, 4.11 mmol). The product was recrystallized from ethanol as a white solid. Yield 49% (0.37 g), mp 139° C.

(143) .sup.1H NMR (500 MHz, DMSO-d6) δ 12.02 (s, 1H, NH), 8.75 (s, 1H, NH), 8.41 (d, J=8.2 Hz, 1H, ArH), 8.13 (d, J=8.3, 1H, ArH), 7.91 (t, J=15.2 Hz, 1H, ArH), 7.82 (m, 3H, ArH), 7.59 (t, J=8.2 Hz, 1H, ArH), 7.27 (td, J=7.6, 1.3 Hz, 1H, ArH), 3.53 (t, J=4.6 Hz, 4H, CH.sub.2), 3.37 (q, J=6.5 Hz, 2H, CH.sub.2), 2.46 (t, J=6.8 Hz, 2H, CH.sub.2), 2.39 (t, J=4.7 Hz, 4H, CH.sub.2)

(144) .sup.13C NMR (126 MHz, DMSO-d6) δ 168.01 (C═O), 163.35 (C═O), 147.02 (ArC), 138.23 (ArC), 134.11 (ArCH), 132.02 (ArC), 131.58 (ArCH), 131.68 (ArCH), 128.38 (ArCH), 128.11 (ArCH), 124.57 (ArCH), 123.67 (ArCH), 121.76 (ArC), 120.89 (ArCH), 66.14 (CH.sub.2), 56.95 (CH.sub.2), 53.19 (CH.sub.2), 36.48 (CH.sub.2). MS (ES+): 399.19 [M+1].

(145) Calculated analysis for C20H22N4O5 (398.16): C, 60.29; H, 5.57; N, 14.06. Found C, 60.34; H, 5.61; N, 13.97.

Synthesis of 2-[(4-nitrobenzoyl)amino]-N-(2-morpholin-4-ylethyl) benzamide (Compound 1i)

(146) Chemical Formula: C20H22N4O5 Molecular Weight: 398.16

(147) The synthetic procedure followed the general method for step 2 above using Intermediate 1 i (0.50 g, 1.87 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.65 ml, 3.74 mmol) and 2.2 equivalents of 2-morpholinoethanamine (0.54 ml, 4.11 mmol). The product was recrystallized from ethanol as a white solid. Yield 51% (0.38 g), mp 148° C.

(148) .sup.1H NMR (500 MHz, DMSO-d6) δ 12.70 (s, 1H, NH), 8.83 (s, 1H, NH), 8.59 (d, J=8.4 Hz, 1H, ArH), 8.44 (d, 8.9 Hz, 2H, ArH), 8.16 (d, 8.9 Hz, 2H, ArH), 7.85 (dd, J=8.0, 1.5 Hz, 1H, ArH), 7.60 (td, J=8.6, 1.5 Hz, 1H, ArH), 7.27 (td, J=7.5, 1.2 Hz, 1H, ArH), 3.54 (t, J=4.6 Hz, 4H, CH.sub.2), 3.44 (q, J=6.5 Hz, 2H, CH.sub.2), 2.44 (m, 6H, CH.sub.2).

(149) .sup.13C NMR (126 MHz, DMSO-d6) δ 68.38 (C═O), 162.69 (C═O), 149.41 (ArC), 140.10 (ArC), 138.76 (ArC), 132.21 (ArCH), 128.43 (ArCH), 128.15 (ArCH), 124.13 (ArCH), 123.47 (ArCH), 120.92 (ArC), 120.64 (ArCH), 66.17 (CH.sub.2), 56.93 (CH.sub.2), 53.20 (CH.sub.2), 36.58 (CH.sub.2).

(150) MS (ES+): 399.21 [M+1].

Calculated analysis for C20H22N4O5 (398.16): C, 60.29; H, 5.57; N, 14.06. Found C, 60.36; H, 5.54; N, 14.05.

Synthesis of 2-[(2-methylbenzoyl)amino]-N-(2-morpholin-4-ylethyl) benzamide (Compound 1j)

(151) Chemical Formula: C21H25N3O3 Molecular Weight: 367.44

(152) The synthetic procedure followed the general method for step 2 above using Intermediate 1j (0.50 g, 2.11 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.61 ml, 4.64 mmol) and 2.2 equivalents of 2-morpholinoethanamine (0.74 ml, 4.22 mmol). The product was recrystallized from ethanol as a white solid. Yield 14% (0.11 g), mp 76° C.

(153) .sup.1H NMR (500 MHz, DMSO-d6) δ 11.78 (s, 1H, NH), 8.72 (s, 1H, NH), 8.59 (d, J=8.3 Hz, 1H, ArH), 7.78 (dd, J=7.8, 1.5 Hz, 1H, ArH), 7.56 (q, J=23.6 Hz, 2H, ArH), 7.43 (td, J=7.5, 1.4 Hz, 1H, ArH), 7.34 (dt, J=7.3, 3.4 Hz, 2H, ArH), 7.22 (td, J=7.6, 1.2 Hz, 1H, ArH), 3.53 (t, J=4.6 Hz, 4H, CH.sub.2), 3.37 (q, J=6.5 Hz, 2H, CH.sub.2), 2.45 (d, J=3.9 Hz, 5H, CH.sub.2/3), 2.39 (t, J=4.7 Hz, 4H, CH.sub.2).

(154) .sup.13C NMR (126 MHz, DMSO-d6) δ 168.22 (C═O), 167.10 (C═O), 138.96 (ArC), 136.44 (ArC), 135.82 (ArC), 131.96 (ArCH), 131.14 (ArCH), 130.24 (ArCH), 128.04 (ArCH), 126.75 (ArCH), 126.07 (ArCH), 122.92 (ArCH), 121.00 (ArC), 120.32 (ArCH), 66.14 (CH.sub.2), 56.99 (CH.sub.2), 53.19 (CH.sub.2), 36.43 (CH.sub.2), 19.59 (CH.sub.3). MS (ES+): 368.22 [M+1].

(155) Calculated analysis for C21H25N3O3 (367.44): C, 68.64; H, 6.86; N, 11.44. Found C, 68.27; H, 6.71; N, 11.28.

Synthesis of 3,4-dimethoxy-N-(2-[(2-morpholin-4-ylethyl) carbamoyl]phenyl) benzamide (Compound 11)

(156) Chemical Formula: C22H27N3O5 Molecular Weight: 413.47

(157) The synthetic procedure followed the general method for step 2 above using Intermediate 11 (0.25 g, 0.88 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.31 ml, 1.77 mmol) and 2.2 equivalents of 2-morpholinoethanamine (0.26 ml, 1.94 mmol). The product was recrystallized from ethanol as a white solid. Yield 22% (0.19 g), mp 106° C.

(158) .sup.1H NMR (500 MHz, DMSO-d6) δ 12.47 (s, 1H, NH), 8.79 (s, 1H, NH), 8.64 (d, J=8.4 Hz, 1H, ArH), 7.81 (d, J=7.8 Hz, 1H, ArH), 7.60-7.50 (m, 3H, ArH), 7.23-7.13 (m, 2H, ArH), 3.86 (s, 6H, OCH.sub.3), 3.55 (t, J=4.4 Hz, 4H, CH.sub.2), 3.45 (q, J=6.5 Hz, 2H, CH.sub.2), 2.42 (t, J=4.6 Hz, 6H, CH.sub.2).

(159) .sup.13C NMR (126 MHz, DMSO-d6) δ 168.62 (C═O), 163.98 (ArC), 151.93 (ArC), 148.69 (ArC), 139.52 (ArC), 132.12 (ArCH), 128.06 (ArCH), 126.83 (ArC), 122.50 (ArCH), 120.26 (ArC), 120.07 (ArCH), 119.86 (ArCH), 111.38 (ArCH), 110.45 (ArCH), 66.17 (CH.sub.2), 56.98 (CH.sub.2), 55.70 (OCH.sub.3), 55.45 (OCH.sub.3), 53.20 (CH.sub.2), 36.52 (CH.sub.2). MS (El+): 413.2.

(160) Calculated analysis for C22H27N3O5 (413.47): C, 63.91; H, 6.58; N, 10.16. Found C, 63.54; H, 6.86; N, 10.11

Synthesis of 3,5-dimethoxy-N-(2-[(2-morpholin-4-ylethyl) carbamoyl]phenyl) benzamide (Compound 1m)

(161) Chemical Formula: C22H27N3O5 Molecular Weight: 413.47

(162) The synthetic procedure followed the general method for step 2 above using Intermediate 1m (0.25 g, 0.88 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.31 ml, 1.77 mmol) and 2.2 equivalents of 2-morpholinoethanamine (0.26 ml, 1.94 mmol). The product was recrystallized from ethanol as a white solid. Yield 58% (0.21 g), mp 120° C.

(163) .sup.1H NMR (500 MHz, DMSO-d6) δ 12.46 (s, 1H, NH), 8.79 (s, NH), 8.61 (dd, J=8.4, 1.2 Hz, 1H, ArH), 7.81 (dd, J=7.9 Hz, 1H, ArH), 7.57 (t, J=7.6 Hz, 1H, ArH), 7.22 (td, J=7.5, 1.3 Hz, 1H, ArH), 7.07 (d, J=2.2 Hz, 2H, ArH), 6.77 (t, J=2.2 Hz, 1H, ArH), 3.84 (d, J=1.5 Hz, 6H, OCH.sub.3), 3.54 (t, J=4.6 Hz, 4H, CH.sub.2), 3.44 (q, J=6.4 Hz, 2H, CH.sub.2), 2.49 (t, J=6.8 Hz, 2H, CH.sub.2), 2.42 (t, J=4.7 Hz, 4H, CH.sub.2)

(164) .sup.13C NMR (126 MHz, DMSO-d6) δ 168.49 (C═O), 163.95 (ArC), 160.70 (ArC), 139.12 (ArC), 136.72 (ArC), 132.13 (ArCH), 128.09 (ArCH), 122.89 (ArCH), 120.59 (ArC), 120.22 (ArCH), 104.99 (ArCH), 103.43 (ArCH), 66.16 (CH.sub.2), 56.99 (CH.sub.2), 55.44 (OCH.sub.3), 53.21 (CH.sub.2), 36.52 (CH.sub.2).

(165) MS (EI+): 413.2.

(166) Calculated analysis for C22H27N3O5 (413.47): C, 63.91; H, 6.58; N, 10.16. Found C, 63.66; H, 6.43; N, 10.03.

Synthesis of 3,4,5-trimethoxy-N-(2-[(2-morpholin-4-ylethyl) carbamoyl]phenyl) benzamide (Compound 1n)

(167) Chemical Formula: C23H29N3O6 Molecular Weight: 443.21

(168) The synthetic procedure followed the general method for step 2 above using Intermediate 1n (0.25 g, 0.80 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.28 ml, 1.60 mmol) and 2.2 equivalents of 2-morpholinoethanamine (0.23 ml, 1.76 mmol). The product was recrystallized from ethanol as a white solid. Yield 52% (0.18 g), mp 117° C.

(169) .sup.1H NMR (500 MHz, DMSO-d6) δ 11.78 12.49 (s, 1H, NH), 8.80 (s, 1H, NH), 8.59 (dd, J=8.3, 1.3 Hz, 1H, ArH), 7.81 (dd, J=7.9, 1.5 Hz, 1H, ArH), 7.57 (td, J=8.8, 2.0 Hz, 1H, ArH), 7.26 (s, 2H, ArH), 7.21 (td, J=8.8, 1.4 Hz, 1H, ArH), 3.89 (s, 6H, OCH.sub.3), 3.77 (s, 3H, OCH.sub.3), 3.54 (t, J=4.7 Hz, 4H, CH.sub.2), 3.43 (q, J=6.5 Hz, 2H, CH.sub.2), 2.49 (d, J=6.8 Hz, 2H, CH.sub.2), 2.41 (t, J=7.3 Hz, 4H, CH.sub.2).

(170) .sup.13C NMR (126 MHz, DMSO-d6) 6 168.54 (C=0), 163.92 (ArC), 152.90 (ArC), 140.63 (ArC), 139.25 (ArC), 132.12 (ArCH), 129.91 (ArC), 128.08 (ArCH), 122.77 (ArCH), 120.62 (ArC), 120.14 (ArCH), 104.56 (ArCH), 66.15 (CH.sub.2), 60.13 (OCH.sub.3), 56.99 (CH.sub.2), 55.94 (OCH.sub.3), 53.20 (CH.sub.2), 36.49 (CH.sub.2).

(171) MS (EI+): 443.2.

(172) Calculated analysis for C23H29N3O6 (443.21): C, 62.29; H, 6.59; N, 9.47. Found C, 62.29; H, 6.46; N, 9.49.

Synthesis of 3,5-difluoro -N-(2-[(2-morpholin-4-ylethyl) carbamoyl]phenyl) benzamide (Compound 1o)

(173) Chemical Formula: C20H21F2N3O3 Molecular Weight: 389.40

(174) The synthetic procedure followed the general method for step 2 above using Intermediate 1o (0.25 g, 0.96 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.33 ml, 1.92 mmol) and 2.2 equivalents of 2-morpholinoethanamine (0.28 ml, 2.11 mmol).

(175) The product was recrystallized from ethanol as a white solid. Yield 40% (0.15 g), mp 116° C.

(176) .sup.1H NMR (500 MHz, DMSO-d6) δ 12.54 (s, 1H, NH), 8.81 (s, 1H, NH), 8.52 (dd, J=8.4, 1.2 Hz, 1H, ArH), 7.83 (dd, J=7.9, 1.5 Hz, 1H, ArH), 7.61-7.59 (m, 1H, ArH), 7.59-7.55 (m, 3H, ArH), 7.26 (td, J=7.6, 1.3 Hz, 2H, ArH), 3.55 (t, J=4.6 Hz, 4H, CH.sub.2), 3.44 (q, J=6.1 Hz, 2H, CH.sub.2), 2.49 (d, J=6.8 Hz, 1H, CH.sub.2), 2.42 (t, J=6.5 Hz, 4H, CH.sub.2).

(177) .sup.13C NMR (126 MHz, DMSO-d6) δ 168.33 (C═O), 163.50 (d, J.sub.C-F=13.1 Hz, ArC—F), 161.50 (d, J.sub.C-F=11.2 Hz, ArC—F), 150.10 (ArC), 143.55 (ArC), 138.5 (d, J.sub.C-F=37.7 Hz, ArC), 132.15 (ArCH), 128.16 ArCH), 123.47 (ArCH), 121.16 (ArC), 120.69 (ArCH), 110.50 (d, J.sub.C-F=7.04 Hz, ArCH), 110.29 (d, J.sub.C-F=6.7 Hz, ArCH), 107.50 (ArCH), 66.15 (CH.sub.2), 56.98 (CH.sub.2), 53.21 (CH.sub.2), 36.56 (CH.sub.2).

(178) MS (EI+): 389.2.

(179) Calculated analysis for C20H21F2N3O3 (389.40): C, 61.69; H, 5.44; N, 10.79.

(180) Found C, 61.56; H, 5.27; N, 10.68.

Synthesis of 2,6-difluoro -N-(2-[(2-morpholin-4-ylethyl) carbamoyl]phenyl) benzamide (Compound 1p)

(181) Chemical Formula: C20H21F2N3O3 Molecular Weight: 389.40

(182) The synthetic procedure followed the general method for step 2 above using Intermediate 1 p (0.25 g, 0.96 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.33 ml, 1.92 mmol) and 2.2 equivalents of 2-morpholinoethanamine (0.28 ml, 2.11 mmol). The product was recrystallized from ethanol as a white solid. Yield 61% (0.25 g), mp 136° C.

(183) .sup.1H NMR (500 MHz, DMSO-d6) δ 12.03 (s, 1H, NH), 8.76 (s, 1H, NH), 8.52 (dd, J=8.3, 1.2 Hz, 1H, ArH), 7.79 (dd, J=8.0, 1.6 Hz, 1H, ArH), 7.66-7.62 (m, 1H, ArH), 7.62-7.57 (m, 1H, ArH), 7.28 (td, J=7.9, 6.2 Hz, 3H, ArH), 3.53 (t, J=4.7 Hz, 4H, CH.sub.2), 3.37 (q, J=6.4 Hz, 2H, CH.sub.2), 2.46 (t, J=6.8 Hz, 2H, CH.sub.2), 2.39 (t, J=5.0 Hz, 4H, CH.sub.2).

(184) .sup.13C NMR (126 MHz, DMSO-d6) δ 167.96 (C═O), 159.76 (d, J.sub.C-F=7.1 Hz, ArC—F), 157.72 (d, J.sub.C-F=5.5 Hz, ArC—F), 137.98 (ArC), 132.79 (t, J.sub.C-F=20.4 Hz, ArCH), 132.13 (ArCH), 128.14 (ArCH), 127.75 (ArC), 123.77 (ArCH), 121.32 (ArC), 120.54 (ArCH), 112.52 (d, J.sub.C-F=3.8 Hz, ArCH), 112.34 (d, J.sub.C-F=4.2 Hz, ArCH), 111.50 (ArC), 66.13 (CH.sub.2), 56.92 (CH.sub.2), 53.18 (CH.sub.2), 36.45 (CH.sub.2).

(185) MS (EI+): 389.2.

(186) Calculated analysis for C20H21F2N3O3 (389.40): C, 61.69; H, 5.44; N, 10.79.

(187) Found C, 61.68; H, 5.31; N, 10.82.

Synthesis of 2,4-difluoro-N-(2-[(2-morpholin-4-ylethyl) carbamoyl]phenyl) benzamide (Compound 1q)

(188) Chemical Formula: C20H21F2N3O3 Molecular Weight: 389.40

(189) The synthetic procedure followed the general method for step 2 above using Intermediate 1q (0.25 g, 0.96 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.33 ml, 1.92 mmol) and 2.2 equivalents of 2-morpholinoethanamine (0.28 ml, 2.11 mmol). The product was recrystallized from ethanol as a white solid. Yield 55% (0.21 g), mp 121° C.

(190) .sup.1H NMR (500 MHz, DMSO-d6) δ 12.00 (s, 1H, NH), 8.72 (s, 1H, NH), 8.55 (d, J=8.3 Hz, 1H, ArH), 7.98 (tt, J=8.8, 6.6 Hz, 1H, ArH), 7.76 (dd, J=7.9, 1.6 Hz, 1H, ArH), 7.57 (td, J=8.5, 1.5 Hz, 1H, ArH), 7.49 (td, J=11.6, 2.5 Hz, 1H, ArH), 7.30 (td, J=8.4, 3.8 Hz, 1H, ArH), 7.24 (td, J=7.6, 1.2 Hz, 1H, ArH), 3.53 (t, J=5.6 Hz, 4H, CH.sub.2), 3.39 (q, J=6.3 Hz, 2H, CH.sub.2), 2.48 (t, J=6.7 Hz, 2H, CH.sub.2), 2.41 (t, J=4.6 Hz, 4H, CH.sub.2).

(191) .sup.13C NMR (126 MHz, DMSO-d6) δ 168.02 (C═O), 163.20 (d, J.sub.C-F=5.3 Hz, ArC—F), 158.10 (d, J.sub.C-F=10.5 Hz, ArC—F), 138.24 (d, J.sub.C-F=5.4 Hz, ArC), 132.69 (d, J.sub.C-F 14.9 Hz, ArC), 131.83 (ArCH), 128.02 (ArCH), 123.38 (ArCH), 121.80 (ArC), 121.04 (ArCH), 119.75 (ArC), 112.57 (d, J.sub.C-F=3.3 Hz, ArCH), 112.41 (d, J.sub.C-F=3.7 Hz, ArCH), 104.99 (ArCH), 66.16 (CH.sub.2), 56.98 (CH.sub.2), 53.18 (CH.sub.2), 36.44 (CH.sub.2). MS (EI+): 389.2.

(192) Calculated analysis for C20H21F2N3O3 (389.40): C, 61.69; H, 5.44; N, 10.79.

(193) Found C, 61.59; H, 5.24; N, 10.70.

Example 2

Synthesis of Compounds of Series 2

(194) The compounds of series 2 have the general formula

(195) ##STR00014##

(196) TABLE-US-00002 Compound R.sup.1 2a 2b 2c 2d 2e 2f —NH.sub.2

Step 1—Synthesis of Example Compounds

(197) Synthesis of 2-[(2-fluorobenzoyl)amino]-N-2-morpholin-4-ylpropyl)benzamide (Compound 2a)

(198) Chemical Formula: C21H24FN3O3 Molecular Weight: 385.43

(199) The synthetic procedure followed the general method for step 2 above using Intermediate 1a (0.50 g, 2.07 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.72 ml, 4.14 mmol) and 2.2 equivalents of 3-morpholinopropan-1-amine (0.67 ml, 4.56 mmol). The product was recrystallized from ethanol as a white solid. Yield 17% (0.14 g), mp 108° C.

(200) .sup.1H NMR (500 MHz, DMSO-d6) δ 12.06 (s, 1H, NH), 8.82 (s, 1H, NH), 8.58 (d, J=8.3 Hz, 1H, ArH), 7.89 (td, J=7.7, 1.9 Hz, 1H, ArH), 7.77 (dd, J=7.9, 1.5 Hz, 1H, ArH), 7.69-7.61 (m, 1H, ArH), 7.57 (td, J=8.6, 1.5 Hz, 1H, ArH), 7.44-7.36 (m, 2H, ArH), 7.23 (td, J=7.6, 1.2 Hz, 1H, ArH), 3.55 (t, J=4.6 Hz, 4H, CH.sub.2), 3.30 (dd, J=7.0 Hz, 2H, CH.sub.2), 2.32 (t, J=6.6 Hz, 6H, CH.sub.2), 1.69 (p, J=7.0 Hz, 2H, CH.sub.2).

(201) .sup.13C NMR (126 MHz, DMSO-d6) δ 168.01 (C═O), 160.86 (d, J.sub.C-F=146.2 Hz, ArC—F), 158.29 (C═O), 138.37 (ArC), 133.76 (d, J.sub.C-F=8.8 Hz, ArCH), 131.78 (ArCH), 130.64 (d, J.sub.C-F=1.3 Hz, ArCH), 128.01 (ArCH), 125.01 (d, J.sub.C-F=3.8 Hz, ArCH), 123.24 (ArCH), 122.91 (ArC), 121.70 (ArC), 120.94 (ArCH), 116.64 (d, J.sub.C-F=22.9 Hz, ArCH), 66.16 (CH.sub.2), 55.99 (CH.sub.2), 53.30 (CH.sub.2), 37.75 (CH.sub.2), 25.59 (CH.sub.2).

(202) MS (ES+): 386.18 [M+1].

(203) Calculated analysis for C21H24FN3O3 (385.43): C, 65.44; H, 6.28; N, 10.09. Found C, 65.44; H, 6.39; N, 10.94.

Synthesis of 2-[(4-fluorobenzoyl)amino]-N-(pyridin-3-ylmethyl)benzamide (Compound 2b)

(204) Chemical Formula: C21H18FN3O2 Molecular Weight: 363.38

(205) The synthetic procedure followed the general method for step 2 above using Intermediate 1a (0.50 g, 2.07 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.72 ml, 4.14 mmol) and 2.2 equivalents of 2-(pyridin-2-yl) ethanamine (0.55 ml, 4.56 mmol). The product was recrystallized from ethanol as a brown solid. Yield 44% (0.34 g), mp 88° C.

(206) .sup.1H NMR (500 MHz, DMSO-d6) δ 11.99 (s, 1H, NH), 8.89 (s, 1H, NH), 8.57 (d, J=8.4 Hz, 1H, ArH), 8.48 (d, J=6.6 Hz, 1H, ArH), 7.88 (td, J=7.8, 1.8 Hz, 1H, ArH), 7.72 (dd, J=7.8, 1.6 Hz, 1H, ArH), 7.68 (m, 2H, ArH), 7.56 (td, J=17.4, 1.3 Hz, 1H, ArH), 7.41 (m, 2H, ArH), 7.27 (d, J=7.7 Hz, 1H, ArH), 7.22 (td, J=7.6, 1.3 Hz, 1H, ArH), 7.16 (td, J=7.5, 1.2 Hz, 1H, ArH), 3.63 (m, 2H, CH.sub.2), 3.00 (t, J=7.2 Hz, 2H, CH.sub.2).

(207) .sup.13C NMR (126 MHz, DMSO-d6) δ 168.03 (C═O), 160.88 (d, J.sub.C-F=144.9 Hz, ArC—F), 158.94 (ArC), 158.30 (ArC), 148.97 (ArCH), 138.37 (ArC), 136.33 (ArCH), 133.76 (d, J.sub.C-F=8.8 Hz, ArCH), 131.81 (ArCH), 130.60 (d, J.sub.C-F=1.3 Hz, ArCH), 127.99 (ArCH), 125.02 (d, J.sub.C-F=2.6 Hz, ArCH), 123.23 (ArCH), 123.12 (ArCH), 121.60 (ArC), 121.42 (ArCH), 120.89 (ArCH), 116.49 (d, J.sub.C-F=22.7 Hz, ArCH), 36.98 (CH.sub.2).

(208) MS (ES+): 362.13 [M+1]. High resolution MS: 364.1456 [M−1]—Composition to C21H19FN3O2 (delta ppm 0.1) or C18H21F5O2 (delta ppm −0.1).

Synthesis of 2-[(4-fluorobenzoyl)amino]-N-(pyrrolid in-3-ylmethyl)benzamide (Compound 2c)

(209) Chemical Formula: C20H22FN3O2 Molecular Weight: 355.17

(210) The synthetic procedure followed the general method for step 2 above using

(211) Intermediate 1a (0.50 g, 2.07 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.72 ml, 4.14 mmol) and 2.2 equivalents of 2-(pyrrolidin-1-yl) ethanamine (0.58 ml, 4.56 mmol). The product was recrystallized from ethanol as a yellow/brown solid. Yield 41% (0.30 g), mp 89° C.

(212) .sup.1H NMR (500 MHz, DMSO-d6) δ 12.04 (s, 1H, NH), 8.74 (s, 1H, NH), 8.58 (d, J=8.4 Hz, 1H, ArH), 7.88 (td, J=7.7, 1.9 Hz, 1H, ArH), 7.77 (dd, J=7.9, 1.5 Hz, 1H, ArH), 7.66 (m, 1 H, ArH), 7.57 (td, J=8.6, 1.5 Hz, 1H, ArH), 7.41 (m, 2H, ArH), 7.23 (td, J=7.6, 1.2 Hz, 1H, ArH), 3.38 (q, J=6.5 Hz, 2H, CH.sub.2), 2.57 (t, J=6.8 Hz, 2H, CH.sub.2), 2.47 (t, J=6.6, Hz, 4H, CH.sub.2), 1.71-1.60 (m, 4H, CH.sub.2).

(213) .sup.13C NMR (126 MHz, DMSO-d6) δ 167.98 (C═O), 161.44 (d, J.sub.C-F=144.9 Hz, ArC—F), 158.29 (C═O), 138.38 (ArC), 133.77 (d, J.sub.C-F=8.6 Hz, ArCH), 131.83 (ArCH), 130.60 (ArCH), 128.04 (ArCH), 125.03 (d, J.sub.C-F=3.8 Hz, ArCH), 123.27 (ArCH), 122.91 (d, J.sub.C-F=12.6 Hz, ArC), 121.59 (ArCH), 120.93 (ArCH), 116.58 (d, J.sub.C-F=22.7 Hz, ArC), 54.49 (CH.sub.2), 53.56 (CH.sub.2), 38.58 (CH.sub.2), 23.12 (CH.sub.2).

(214) MS (ES+): 356.15 [M+1].

(215) Calculated analysis for C20H22FN3O2 (355.17): C, 67.59; H, 6.24; N, 11.82. Found C, 67.66; H, 6.19; N, 11.67.

Synthesis of 2-[(4-fluorobenzoyl)amino]-N-(piperidin-3-ylmethyl)benzamide (Compound 2d)

(216) Chemical Formula: C21H24FN3O2 Molecular Weight: 369.43

(217) The synthetic procedure followed the general method for step 2 above using Intermediate 1a (0.50 g, 2.07 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.72 ml, 4.14 mmol) and 2.2 equivalents of 2-(piperidin-1-yl) ethanamine 16d (0.65 ml, 4.56 mmol). The product was recrystallized from ethanol as a white solid. Yield 51% (0.39 g), mp 94° C.

(218) .sup.1H NMR (500 MHz, DMSO-d6) δ 11.99 (s, 1H, NH), 8.69 (s, 1H, NH), 8.57 (d, J=8.3 Hz, 1H, ArH), 7.88 (td, J=17.1, 1.7 Hz, 1H, ArH), 7.76 (d, J=9.1 Hz, 1H, ArH), 7.67-7.62 (m, 1H, ArH), 7.56 (t, J=16.4 Hz, 1H, ArH), 7.42-7.37 (m, 1H, ArH), 7.23 (t, J=15.4 Hz, 2H, ArH), 3.36 (q, J=19.6 Hz, 2H, CH.sub.2), 2.43 (t, J=14.1 Hz, 2H, CH.sub.2), 2.35 (t, J=18.6 Hz, 4H, CH.sub.2), 1.44 (q, J=22.6 Hz, 4H, CH.sub.2), 1.34 (d, J=15.1 Hz, 2H, CH.sub.2).

(219) .sup.13C NMR (126 MHz, DMSO-d6) δ 167.99 (C═O), 160.89 (d, J.sub.C-F=155.0 Hz, ArC—F), 158.31 (C═O), 154.28 (ArC), 138.39 (ArC), 133.76 (d, J.sub.C-F=8.8 Hz, ArCH), 131.77 (ArCH), 130.60 (d, J.sub.C-F=1.8 Hz, ArCH), 128.01 (ArCH), 125.00 (d, J.sub.C-F=3.7 Hz, ArCH), 123.26 (ArCH), 121.81 (ArC), 120.97 (ArCH), 116.56 (d, J.sub.C-F=22.8 Hz, ArCH), 57.31 (CH.sub.2), 53.96 (CH.sub.2), 36.87 (CH.sub.2), 25.56 (CH.sub.2), 23.99 (CH.sub.2).

(220) MS (EI+): 369.19.

(221) Calculated analysis for C21H24FN3O2 (369.43): C, 68.27; H, 6.55; N, 11.37. Found C, 67.95; H, 6.82; N, 11.44.

Synthesis of N-(2-aminoethyl)-2-(2-fluorobenzamido) benzamide (Compound 2f)

(222) Chemical Formula: C16H16FN3O2 Molecular Weight: 301.32

(223) The synthetic procedure followed the general method for step 2 above using Intermediate 1a (0.50 g, 2.07 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.72 ml, 4.14 mmol) and 2.2 equivalents of ethane-1,2-diamine (0.31 ml, 4.56 mmol). The product was recrystallized from ethanol as a yellow solid. Yield 43% (0.27 g), mp 96° C.

(224) .sup.1H NMR (500 MHz, DMSO-d6) δ 12.51 (s, 1H, NH), 11.62 (s, 2H, NH.sub.2), 8.80 (s, 1H, NH), 8.60 (dd, J=9.4, 1.1 Hz, 1H, ArH), 8.03 (dd, J=9.4, 1.6 Hz, 1H, ArH), 7.98 (dd, J=9.7, 1.6 Hz, 2H, ArH), 7.72-7.64 (m, 2H, ArH), 7.62 (td, J=16.6, 1.9 Hz, 1H, ArH), 7.26 (td, J=16.4, 1.1 Hz, 1H, ArH), 3.91 (s, 4H, CH.sub.2).

(225) .sup.13C NMR (126 MHz, DMSO-d6) δ 168.24 (C═O), 160.87 (d, J.sub.C-F=151.5 Hz, ArC—F), 158.29 (C═O), 154.31 (ArC), 138.41 (ArC), 133.74 (d, J.sub.C-F=9.5 Hz, ArCH), 131.80 (ArCH), 130.61 (d, J.sub.C-F=1.8 Hz, ArCH), 128.21 (ArCH), 125.02 (d, J.sub.C-F=3.6 Hz, ArCH), 123.22 (ArCH), 121.65 (ArC), 120.86 (ArCH), 116.57 (d, J.sub.C-F=22.5 Hz, ArCH), 42.79 (CH.sub.2), 40.88 (CH.sub.2).

(226) MS (EI+): 301.12. High resolution MS: 302.1301 [M−1].

(227) Composition: C21H18O2 (delta ppm −0.1), C13H19O2F5 (delta ppm 0.4), C16H17O2N3F1 (delta ppm 0.6).

Example 3

Synthesis of Compounds of Series 3

(228) The compounds of series 3 have the general formula

(229) ##STR00020##

(230) TABLE-US-00003 Compound R.sup.4 3a 3-OCH.sub.3 3b 3-CH.sub.3 3c 5-I 3d 6-Cl

Step 1—Synthesis of Intermediates

Synthesis of 2-(2-fluorophenyl)-8-methoxy-3,1-benzoxazin-4-one (Intermediate 3a)

(231) Chemical Formula: C15H10FNO3 Molecular Weight: 271.24

(232) The synthetic procedure followed the general method set out above for step 1 using 2-amino-3-methoxybenzoic acid (0.50 g, 2.99 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of 2-fluorobenzoyl chloride (0.79 ml, 6.58 mmol). Collected as a white solid, yield 92% (0.75 g), mp 131° C.

(233) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.08 (td, J=7.7, 1.8 Hz, 1H, ArH), 7.72 (d, J=1.6 Hz, 1H, ArH), 7.71 (d, J=1.6 Hz, 1H, ArH), 7.63-7.54 (m, 2H, ArH), 7.42 (m, 2H, ArH), 3.97 (s, 3H, OCH.sub.3).

(234) 13C NMR (126 MHz, DMSO-d6) δ 160.31 (d, J.sub.C-F=257.0 Hz, ArC—F), 158.73 (ArC═O), 154.15 (ArC), 152.92 (ArC), 135.83 (ArC), 134.31 (d, J.sub.C-F=8.8 Hz, ArCH), 131.14 (ArCH), 129.48 (ArCH), 124.79 (d, J.sub.C-F=3.8 Hz, ArCH), 118.90 (d, J.sub.C-F=10.1 Hz, ArC), 118.78 (ArCH), 118.32 (ArCH), 117.22 (d, J.sub.C-F=21.4 Hz, ArCH), 56.34 (OCH3).

(235) MS (APCI+): 272.07 [M+1].

Synthesis of 2-(2-fluorophenyl)-8-methoxy-3,1-benzoxazin-4-one (Intermediate 3b)

(236) Chemical Formula: C15H10FNO2 Molecular Weight: 255.21

(237) The synthetic procedure followed the general method set out above for step 1 using 2-amino-3-methylbenzoic acid (0.50 g, 3.31 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of 2-fluorobenzoyl chloride (0.87 ml, 7.28 mmol). Collected as a white solid, yield 97% (0.81 g), mp 106° C.

(238) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.13 (td, J=7.7, 1.9 Hz, 1H, ArH), 8.0 (d, J=8.8Hz, 1H, ArH), 7.84 (d, J=8.8, 1H, ArH), 7.75-7.67 (m, 1H, ArH), 7.55 (t, J=7.7 Hz, 1H, ArH), 7.47-7.40 (m, 2H, ArH), 2.58 (s, 3H, CH.sub.3).

(239) .sup.13C NMR (126 MHz, DMSO-d6) δ 160.51 (d, J.sub.C-F=259.6 Hz, ArC—F), 159.01 (ArC═O), 153.06 (ArC), 144.29 (ArC), 137.40 (ArCH), 135.61 (ArC), 134.44 (d, J.sub.C-F=8.82 Hz, ArCH), 131.07 (ArCH), 128.42 (ArCH), 125.54 (ArCH), 124.82 (d, J.sub.C-F=3.8 Hz, ArCH), 118.93 (d, J.sub.C-F=1.3 Hz, ArC), 117.32 (d, J.sub.C-F=21.4 Hz, ArC), 116.78 (ArCH), 16.50 (CH.sub.3).

(240) MS (APCI+): 256.07 [M+1].

Synthesis of 2-(2-fluorophenyl)-8-methoxy-3,1-benzoxazin-4-one (Intermediate 3c)

(241) Chemical Formula: C14H7FINO2 Molecular Weight: 367.11

(242) The synthetic procedure followed the general method set out above for step 1 using 2-amino-5-iodobenzoic acid (0.50 g, 1.90 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of 2-fluorobenzoyl chloride (0.50 ml, 4.18 mmol). Collected as a white solid, yield 90% (0.63 g), mp 159° C.

(243) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.41 (d, J=2.0 Hz, 1H, ArH), 8.26 (dd, J=8.4, 2.0 Hz, 1H, ArH), 8.09 (td, J=7.8, 1.8 Hz, 1H, ArH), 7.75-7.69 (m, 1H, ArH), 7.50 (d, J=8.4 Hz, 1H, ArH), 7.44 (m, 2H, ArH).

(244) .sup.13C NMR (126 MHz, DMSO-d6) δ 160.48 (d, J.sub.C-F=258.3 Hz, ArC—F), 157.35 (ArC═O), 154.59 (ArC), 145.39 (ArC), 145.12 (ArCH), 135.85 (ArCH), 134.74 (d, J.sub.C-F=8.8 Hz, ArCH), 131.10 (ArCH), 128.95 (ArCH), 124.86 (d, J.sub.C-F=3.8 Hz, ArCH), 118.82 (ArC), 118.45 (d, J.sub.C-F=8.8 Hz, ArC), 117.28 (d, J.sub.C-F=21.4 Hz, ArCH), 93.91 (ArC—I).

Synthesis of 5-chloro-2-(2-fluorophenyl)-3,1-benzoxazin-4-one (Intermediate 3d)

(245) Chemical Formula: C14H7CIFNO2 Molecular Weight: 275.66

(246) The synthetic procedure followed the general method set out above for step 1 using 2-amino-6-chlorobenzoic acid (0.50 g, 2.91 mmol) dissolved in pyridine (5 ml) and 2.2 equivalent of 2-fluorobenzoyl chloride (0.76 ml, 6.41 mmol). Collected as a white solid, yield 89% (0.71 g), mp 104° C.

(247) 1H NMR (500 MHz, DMSO-d6) δ 8.01 (td, J=17.6, 1.8 Hz, 1H, ArH), 7.90 (t, J=16.1 Hz, 1H, ArH), 7.71 (dd, J=9.1, 1.2 Hz, 2H, ArH), 7.67 (dd, J=9.1, 1.2 Hz, 1H, ArH), 7.46-7.42 (m, 2H, ArH),

(248) .sup.13C NMR (126 MHz, DMSO-d6) δ 160.53 (d, J.sub.C-F=256.9 Hz, ArC—F), 155.43 (ArC═O), 148.53 (ArC), 144.39 (ArC), 136.6 (ArCH), 134.81 (d, J.sub.C-F=10.1 Hz, ArCH), 134.01 (ArC), 131.09 (ArCH), 130.93 (ArCH), 126.44 (ArCH), 124.86 (d, J.sub.C-F=3.9 Hz, ArCH), 118.18 (d, J.sub.C-F=10.3 Hz, ArC), 117.28 (d, J.sub.C-F=22.3 Hz, ArCH), 114.77 (ArC—Cl).

(249) MS (EI+): 275.01.

Step 2—Synthesis of Example Compounds

Synthesis of 2-[(2-fluorobenzoyl)amino]-3-methoxy-N-(2-morpholin-4-ylethyl) benzamide (Compound 3a)

(250) Chemical Formula: C21H24FN3O4 Molecular Weight: 401.43

(251) The synthetic procedure followed the general method for step 2 using Intermediate 3a (0.50 g, 1.84 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.64 ml, 3.69 mmol) and 2.2 equivalents of 2-morpholinoethanamine (0.53 ml, 4.06 mmol). The product was recrystallized from ethanol as a white solid. Yield 14% (0.27 g), mp 131° C.

(252) .sup.1H NMR (500 MHz, DMSO-d6) δ 9.53 (s, 1H, NH), 8.02 (s, 1H, NH), 7.79 (t, J=15.1 Hz, 1H, ArH), 7.60 (q, J=7.0 Hz, 1H, ArH), 7.38-7.30 (m, 3H, ArH), 7.21 (d, J=8.2 Hz, 1H, ArH), 7.12 (d, J=7.7 Hz, 1H, ArH), 3.81 (s, 3H, OCH.sub.3), 3.31 (t, J=10.1 Hz, 6H, CH.sub.2), 2.33 (m, 6H, CH.sub.2).

(253) .sup.13C NMR (126 MHz, DMSO-d6) δ 166.71 (C═O), 159.56 (d, J.sub.C-F=249.5 Hz, ArC—F), 154.59 (C═O), 144.51 (ArC), 138.35 (ArC), 134.90 (d, J.sub.C-F=1.3 Hz, ArC), 133.09 (d, J.sub.C-F=1.3 Hz, ArCH), 130.62 (ArCH), 127.27 (ArCH), 124.50 (ArCH), 119.74 (ArCH), 118.6 (ArC), 116.34 (d, J.sub.C-F=22.7 Hz, ArCH), 113.45 (ArCH), 66.05 (CH.sub.2), 57.03 (CH.sub.2), 56.04 (OCH.sub.3), 53.14 (CH.sub.2), 36.23 (CH.sub.2).

(254) MS (ES+): 402.21 [M+1].

(255) Calculated analysis for C21H24FN3O4 (401.43): C, 62.83; H, 6.03; N, 10.47. Found C, 62.76; H, 6.17; N, 10.53.

Synthesis of 2-[(2-fluorobenzoyl)amino]-3-methyl-N-(2-morpholin-4-ylethyl) benzamide (Compound 3b)

(256) Chemical Formula: C21H24FN3O3, Molecular Weight: 385.43

(257) The synthetic procedure followed the general method for Step 2 using 2-(2-fluorophenyl)-8-methyl-3,1-benzoxazin-4-one (0.50 g, 1.96 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.68 ml, 3.91 mmol) and 2.2 equivalents of 2-morpholinoethanamine (0.57 ml, 4.31 mmol). The product was recrystallized from ethanol as a white solid. Yield 42% (0.32 g), mp 165° C.

(258) .sup.1H NMR (500 MHz, DMSO-d6) δ 9.96 (s, 1H, NH), 8.12 (s, 1H, NH), 7.80 (td, J=7.6, 1.9 Hz, 1H, ArH), 7.61 (td, J=7.4, 1.9 Hz, 1H, ArH), 7.37 (m, 4H, ArH), 7.30 (t, J=7.6 Hz, 1H, ArH), 3.32 (d, J=5.4 Hz, 4H, CH.sub.2), 2.39 (t, J=6.8 Hz, 2H, CH.sub.2), 2.32 (m, 6H, CH.sub.2), 2.27 (s, 3H, CH.sub.3).

(259) .sup.13C NMR (126 MHz, DMSO-d6) δ 167.43 (C═O), 162.08 (d, J.sub.C-F=206.6 Hz, ArC—F), 158.45 (C═O), 135.89 (ArC), 133.90 (ArC), 133.42 (ArC), 133.04 (d, J.sub.C-F=8.8 Hz, ArCH), 131.88 (ArCH), 130.36 (d, J.sub.C-F=1.3 Hz, ArCH), 126.37 (ArCH), 125.59 (ArCH), 125.10 (ArCH), 124.62 (d, J.sub.C-F=2.5 Hz, ArC), 116.30 (d, J.sub.C-F=22.7 Hz, ArCH), 66.08 (CH.sub.2), 57.04 (CH.sub.2), 53.16 (CH.sub.2), 36.30 (CH.sub.2), 18.22 (CH.sub.3).

(260) MS (ES+): 386.22 [M+1].

(261) Calculated analysis for C21H24FN3O3 (385.43): C, 65.44; H, 6.28; N, 10.90. Found C, 65.66; H, 6.27; N, 10.96.

Synthesis of 2-[(2-fluorobenzoyl)amino]-2-chloro-N-(2-morpholin-4-ylethyl) benzamide (Compound 3d)

(262) Chemical Formula: C20H21FCIN3O3 Molecular Weight: 405.85

(263) The synthetic procedure followed the general method for Step 2 using 2-(2-fluorophenyl)-5-chloro-3,1-benzoxazin-4-one (0.50 g, 1.83 mmol) in DMF (8 ml), 2 equivalents of DIPEA (0.64 ml, 3.65 mmol) and 2.2 equivalents of 2-morpholinoethanamine (0.53 ml, 4.02 mmol). The product was recrystallized from ethanol as a yellow solid. Yield 33% (0.25 g), mp 121° C.

(264) .sup.1H NMR (500 MHz, DMSO-d6) δ 9.60 (s, 1H, NH), 8.60 (s, 1H, NH), 7.97 (dd, J=8.6

(265) Hz, 1H, ArH), 7.87 (td, J=15.6, 1.8 Hz, 1H, ArH), 7.67-7.63 (m, 1H, ArH), 7.48 (t, 16.5 Hz, 1H, ArH), 7.42-7.36 (m, 3H, ArH), 3.48 (t, J=9.3 Hz, 4H, CH.sub.2), 3.38 (q, J=19.5 Hz, 2H, CH.sub.2), 2.43 (t, J=13.7 Hz, 2H, CH.sub.2), 2.33 (s, 4H, CH.sub.2).

(266) .sup.13C NMR (126 MHz, DMSO-d6) δ 164.21 (C═O), 162.52 (d, J.sub.C-F=114.8 Hz, ArC—F), 158.45 (C═O), 155.98 (ArC), 142.8 (ArC), 136.0 (ArC), 133.97 (d, J.sub.C-F=9.4 Hz, ArCH), 130.87 (ArCH), 130.24 (ArCH, 130.20 (d, J.sub.C-F=19.9 Hz, ArC), 125.94 (ArCH), 124.99 (d, J.sub.C-F=3.4 Hz, ArCH), 122.60 (ArCH), 116.44 (d, J.sub.C-F=23.0 Hz, ArCH), 66.08 (CH.sub.2), 56.74 (CH.sub.2), 53.16 (CH.sub.2), 36.47 (CH.sub.2).

(267) MS (EI+): 405.12.

(268) Calculated analysis for C20H21FCIN3O3 (405.85): C, 59.19; H, 5.22; N, 10.35.

(269) Found C, 59.40; H, 5.21; N, 10.38.

Biological Examples

Materials and Methods

Cloning Procedures

NF-κB Luciferase Reporter Plasmid

(270) NF-κB luciferase assays were carried out using the 3×κB luciferase reporter plasmid, which was a kind gift from Professor Ron Hay (University of St. Andrews). A pcDNA3.1 plasmid containing the LacZ sequence was used as a control for transfection efficiency (gift from Professor Trevor Dale, School of Biosciences, Cardiff University). For positive and negative controls pGL3 luciferase reporter vectors (Promega) were used, pGL3control and pGL3basic, respectively.

Cell Culture Maintenance and Storage

Experimental Cell Lines

(271) The human embryonic kidney cells (HEK-293) were a gift from Prof. Vladimir Buchman (School of Biosciences, Cardiff University). The human breast cancer cell lines, MDA-MB-231, SKBR3 and ZR-7S-1 were a gift from Dr. Julia Gee (Department of Pharmacy and Pharmaceutical Sciences, Cardiff University). The human normal breast cancer cell line MCF-10A was a gift from Dr. Torsten Stein (Division of Cancer Sciences and Molecular Pathology, University of Glasgow). Descriptions of the main cell lines used are outlined below:

(272) HEK-293 is a non-tumorigenic cell line derived from human embryonic kidney cells. HEK-293 cells are convenient for our investigation, because they are easily transfected and have undetectable basal level of Bcl-3 protein.

(273) MDA-MB-231 is a highly metastatic, human basal epithelial cell line isolated from the pleural effusion of an adenocarcinoma. The cells are ‘triple negative’ as they lack estrogen, progesterone and ERBB2 receptor and they strongly over-express EGFR. The expression of receptors in this line has been confirmed by the host laboratory.

(274) SKBR3 cell line is a poorly metastatic human luminal epithelial cell line derived from a pleural effusion. SKBR3 cells are estrogen and progesterone receptor negative, over-express the ERBB2 receptor and have very low levels of the EGFR receptor.

(275) The ZR-7S-1 cell line is a moderately metastatic human luminal epithelial cell line derived from a malignant ascitic effusion with infiltrating ductal carcinoma. ZR-7S-1 cells are oestrogen and progesterone receptor positive. They express very low levels of the ErbB2 receptor and over-express EGFR.

(276) MCF-10A is a mammary epithelial cell line and is considered as a model of non-tumorigenic mammary cells. MCF-10 cells were derived from a mammary tissue from a 36-year-old woman in a good health and the immortalized MCF-10A line can grow in culture and has a stable, near-diploid karyotype with modest genetic modifications typical of culture-adapted breast epithelial cells.

Maintenance of Cell Lines

(277) The HEK-293 cell line was maintained in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% v/v foetal bovine serum (FBS, Sigma, Dorset, UK), penicillin (50 u/ml, Invitrogen), streptomycin (50 u/ml, Invitrogen) and L-glutamine (2 mM, Invitrogen). The MDA-MB-231, ZR-75-1 and SKBR3 cell lines were maintained in RPMI medium (Invitrogen) supplemented with 10% v/v FBS, penicillin (50 u/ml Invitrogen) streptomycin (50 u/ml, Invitrogen) and L-glutamine (2 mM, Invitrogen). MCF-10A cell line was maintained in Dulbecco's modified Eagle's medium nutrient mixture F-12 (DMEM/F-12, Invitrogen) supplemented with 5% v/v horse serum (Sigma, Dorset, UK), penicillin (50 u/ml, Invitrogen), streptomycin (50 u/ml, Invitrogen), epidermal growth factor (EGF, 20 ng/ml, Sigma), hydrocortisone (0.5 mg/ml, Sigma), cholera toxin (100 mg/ml, Sigma) and insulin (10 μg/ml, Sigma).

(278) All cell lines were incubated at 37° C. and 5% CO.sub.2 in T25 or T80 tissue culture flasks (Nunc, Leics, UK) and were routinely passaged every 3-8 days at a split ratio of 1:4-1:12, when they became 80-90% confluent.

Cell Based Assays

Cell Titre Blue Viability Assay

(279) The viability of cells at experimental endpoints for particular assays was determined using the Cell Titre Blue reagent (Promega, Southampton, UK). This reagent measures the cellular metabolic activity using resazurin as an indicator dye. Viable cells, therefore metabolically active, will reduce resazurin into highly fluorescent resofurin. The resulting fluorescence levels are measured and indicate cell viability.

(280) Cells were plated at low confluency into 96 well plates in 100 μl of complete growth media in triplicates and were incubated at 37° C. in 5% CO.sub.2 for the desired test exposure period. For each 100 μl of media in 96 well plates, 20 μl Cell Titre Blue reagent was added followed by incubation for an hour at 37° C. in 5% CO.sub.2. Fluorescence was then measured by setting excitation/emission wavelengths to 560/590 nm on a Flurostar Optima plate reader (BMG tabtech, Bucks, UK).

Cell Count

(281) To establish cell viability over time period of three days, respective cells were seeded at low confluency into 96 well plates in 100 μl of complete growth media in triplicates and were incubated at 37° C. in 5% CO.sub.2. After 24 hrs, cells from triplicate wells for the first time point were detached using 0.25% Trypsin/EDTA (Invitrogen) and resuspended in complete growth media and individually counted. The same was done for each cell line at 48 hrs and 72 hrs post-seeding.

Determination of NF-κB Activity in Cells

(282) For NF-κB luciferase assays, cells were seeded into clear bottom black 96-well plates (Corning Inc., Lowell, US) in antibiotic free culture media in appropriate density. After 24 hrs, cells were transfected with 10 ng of 3×κB luciferase plasmid and 10 ng of pcDNA3.1-LacI plasmid per well. Empty pcDNA3.1 plasmid was also included to normalize the total weight of DNA transfected to 100 ng. For positive and negative controls respectively, 10 ng of pGL3control or pGL3basic were transfected in place of 3×κB luciferase plasmid. Transfection was carried out using Lipofectamine LTX reagents (Invitrogen, Paisley, UK).

(283) After 48 hrs post-transfection with luciferase reporter plasmid, the media was aspirated and cells were lyzed using 50 μl/well of Glo-lysis buffer (Promega, Southampton, UK). The plate was left on a rocker for 20 min to facilitate complete cell lysis. Then, 20 μl of lysate from each well was removed and transferred into a new clear bottom black well plate for measuring LacZ activity as a transfection efficiency control and followed by addition of 20 ul/well of Beta-Glo substrate (Promega, Southampton, UK) and cultivation at room temperature for at least 20 min. Subsequently, 30 μl/well of Bright-Glo luciferase substrate (Promega, Southampton, UK) was added to the original plate and assess immediately for luminescence activity. The luminescence produced from either reaction was read using a Flurostar Optima plate reader (BMG tabtech, Bucks, UK). The resulting luciferase activity was then normalized against IacZ activity obtained from Beta-glo measurement and is displayed as relative light units (R.t.U).

Boyden Chamber Migration Assay

(284) The migratory or invasive capacity of human mammary cancer cell lines was assessed by the Boyden chamber assay. Cells were seeded in low serum media in a chamber with porous membrane (transparent polyethylene terephthalate (PET) membranes with 8 μm pores) as a solid support for motility assays or with a porous membrane coated with Matrigel Basement Membrane Matrix (BD BioCoat Growth factor reduced invasion chambers) for invasion assay. The cell insert was placed into a well with complete growth media, therefore cells are stimulated by a serum gradient to migrate or invade across the membrane through pores.

(285) Total of 750 μl of complete growth media containing 10% of serum was added to appropriate wells of a 24 well cell culture insert companion plate (BD Biosciences, Oxford, UK). A cell culture insert (BD Biosciences, Oxford, UK) was then carefully placed into each well of the insert companion plate using tweezers. Cells were is detached from tissue culture plates using 0.25% w/v Trypsin/EDTA (Invitrogen) and centrifuged at 13000 rpm for 5 min. Cells were washed twice in serum free media by resuspension and centrifugation. The appropriate number of cells (2×10.sup.5 cells/ml for MDA-MB-231 cells) was resuspended in normal growth media containing only 0.1% serum. 350 μl of the cell suspension was added to the appropriate upper chambers of the cell culture inserts and plates were incubated for 24 hrs at 37° C. and 5% CO.sub.2.

(286) After incubation, cells on membranes were fixed by replacing the media in the top and bottom sections of the chamber with 70% ice-cold ethanol (Fisher Scientific). Plates were incubated at −20° C. for at least an hour. After fixation, inserts were immersed in a tap water using tweezers to ensure all ethanol was removed. A moistened cotton wool bud was then used to mechanically remove all cells fixed on the upper side of the membranes. Cells were stained by individually immersing the inserts into filtered Harris' Haematoxylin (Sigma, Dorset, UK) for 1 min. Following this, inserts were washed in a beaker of tap water to remove the dye and immersed in 0.5% filtered Eosin (Sigma, Dorset, UK) for 2 min. Stained inserts were then washed again in a tap water.

(287) Glycerol Gelatin (Sigma, Dorset, UK) was heated in a beaker of boiling water and once liquefied, a drop was placed onto an appropriately labelled microscope slide (R. A. Lamb, Loughborough, UK). Membranes were cut out of the insert and transferred onto the corresponding slide with tweezers. Glycerol Gelatin was added to the top of the membranes and a cover slip was placed on the slide under firm pressure. Mounted slides were left to air dry before being analysed.

Protein Analysis

Protein Extraction From Cells

(288) Proteins were extracted from cells in order to be analyzed by ELISA assay. The media from tissue culture flask was removed and cells were rinsed with ice cold PBS (Sigma, Dorset, UK). Appropriate volume of PBS (5 ml for T25, 10 ml for T80) was added into the flask and cells were removed with a cell scraper (Nunc, Leics, UK). The cell suspension was then transferred to 15 ml tubes and centrifuged at 11000 rpm for 5 min at room temperature. Resulting pellet was used for protein extraction immediately or stored at −20° C. prior use.

(289) Non-denatured protein extract was prepared using non-denaturing lysis buffer (Table 1) and used to analyze protein-protein interaction.

(290) TABLE-US-00004 TABLE 1 Composition of buffers for whole cell protein extraction RIPA buffer pH 7.4 Non-denaturing buffer 50 mM Tris pH 8 (Sigma) 20 mM Tris pH 7.5 (Sigma) 150 mM sodium chloride (Sigma) 150 mM sodium chloride (Sigma) 1% v/v Nonidet-P40 (Roche) 1% v/v Nonidet-P40 (Roche) 0.1% w/v sodium dodecyl sulphate 1 mM EDTA pH 8.0 (Fischer (SDS, Sigma) Scientific) 0.5% w/v sodium deoxycholate 1 mM EGTA pH 8.0 (Fluka (Sigma) Biochemika)

(291) Complete mini protease inhibitor tablets (Roche, Welwyn Garden City, UK), 10 mM sodium fluoride (Fluka Biochemika), 1 mM sodium pyrophospate and 1 mM sodium orthovanadate (Sigma, Dorset, UK) were added to the buffer prior use. Cell pellets were resuspended in appropriate volume of non-denaturing buffer (50-200 μl) and cultivated on ice 5 min. Cell suspensions were transferred to microcentrifuge tubes and sonicated on ice (3 times 5 s) and centrifuged at 10000 rpm for 10 min at 4° C. The resultant supernatant was used immediately or stored at −20° C. until required.

ELISA Assay

(292) In our case, ELISA assay was used to detect either the amount of Flag-Bcl-3 protein alone or Flag-Bcl-3 in complex with p50, using indirect or sandwich ELISA respectively.

(293) For Indirect and sandwich ELISA assay, Non-denaturing cell lysate (above) was diluted with TBS/T [tris buffer saline (TBS, Calbiochem, Merck) supplemented with 0.5% v/v Tween (Sigma, Dorset, UK)] to a concentration of 0.5-1 μg/μl and 100 μl was added onto ANTI-Flag coated flat bottom ELISA plates (Sigma, Dorset, UK). Samples were added in triplicates, while TBS/T was used as a negative control. The plate was cultivated at 37° C. for an hour followed by 3×200 μl washes with TBS/T. Primary antibodies, either Bcl-3 (Santa Cruz Biotech) for indirect ELISA and p50 for sandwich ELISA (Abcam) were added in known volumes (125 μl) and concentrations to each well, cultivated covered from light for an hour at room temperature. Another three 200 μl washes with TBS/T were performed before cultivation with alkaline phospatase (AP) conjugated secondary antibody. Meanwhile, para-nitrophenylphosphate solution (pNPP, Santa Cruz Biotechnology, California, USA) was prepared according to manufacturer's instructions (5 mg of pNPP disodium salt in 5 ml of pNPP substrate buffer). The solution was mixed well and covered from light prior to use. pNPP is a substrate of choice for use with alkaline phosphatase and produces a soluble end product that is yellow in colour. Therefore colour changes can be measured and represent the amount of AP present. After cultivation with secondary antibody, the wells were washed 3×200 μl. TBS/T, pNPP solution was added (50 μl/well) and cultivated for an hour covered from light at room temperature. The reaction was stopped by addition of 3N NaOH (20 μl/well) and the colorimetric changes were measured at 405 nm using a plate reader.

Statistical Analysis

(294) The Student's T-test was used to determine statistical differences between normally distributed data sets and between data sets with sample sizes of n=3. This test was performed using Excel 2008 software.

Example 4

Characterization of Example Compound 1a

(295) We established that the solubility of Compound 1a in most commonly used solvents is very low (Table 2) which represents an issue for biological evaluation. Therefore a hydrochloric salt of Compound 1a was synthesised and the solubility was analysed. The obtained salt had improved solubility in water and methanol (Table 2), however it was not soluble (<0.1 g/100 ml) in Phosphate Buffered Saline (PBS). PBS is a water-based salt solution containing sodium chloride, sodium phosphate, potassium chloride and potassium phosphate, while the buffer's phosphate groups help to maintain a constant pH. PBS is non-toxic and is commonly used as an isotonic is buffered solution for cell-based assays and animal studies.

(296) TABLE-US-00005 TABLE 2 Solubility of the lead compound in organic solvents Solubility (g/100 ml) Compound 1a Solvent Compound 1a hydrochloride salt Water <0.01 0.95 Methanol 0.20 0.59 Ethanol 0.09 0.19 DMSO 3.71 <0.01 Ethyl acetate 0.34 0.02 Dichloromethane 2.91 0.90 Diethyl ether 0.09 <0.01

Example 5

Cell Toxicity of Compound 1a In Vitro

(297) The toxicity of Compound 1a was evaluated in vitro using human breast cancer cell lines. To compare the toxicity in tumorigenic as well as in non-tumorigenic breast cancer cells, we selected MCF-10A as a non-tumorigenic human breast cancer cell line and MDA-MB-231 and SKBR3 as cell models of tumorigenic human breast cancer cell line.

(298) MCF-10 cells were derived from a mammary tissue from a 36-year-old woman in a good health and the immortalized MCF-10A line can grow in culture and has a stable, near-diploid karyotype with modest genetic modifications typical of culture-adapted breast epithelial cells, including loss of p16 locus. The cells express normal p53 and they do not grow in immuno-compromised mice.

(299) Compound 1a was dissolved in DMSO and diluted in media in a highest concentration of 1mM (10.sup.−3M). Cell toxicity was evaluated using the Cell Titre Blue viability assay over a range of molarities for 24 hrs. The effect of Compound 1a on cell viability was always normalised against DMSO control and the dose-response curve was generated using GraphPad software (FIG. 1).

(300) IC.sub.50 values could not be established in any of the cell lines, as even the highest concentration did not cause a 50% decrease in cell viability. We could, however, see a difference in cell toxicity between non-tumorigenic and tumorigenic cell lines. At the highest concentration of 1 mM, the viability in MCF-10A cell line was 96%, 72% in MDA-MB-231 and 57% in SKBR3 compared to DMSO control (100%). This low toxicity was expected of a specific inhibitor of Bcl-3 as previous studies had shown that genetic inhibition of Bcl-3 had only a modest effect on cell viability of cancer cell lines in vitro and little or no effect on non-tumourgenic lines (11).

(301) The IC.sub.50 was calculated using GraphPad software by extrapolating the dose-response curve, giving IC.sub.50 values of 14.9 mM for MCF-10A, 2.70 mM for MDA-MB-231 and 1.37 mM for SKBR3.

Example 6

Establishing Biological Effects of Compound 1a In Vitro

A. Establishing Effect on Protein Binding by Indirect Sandwich ELISA

(302) HEK-293 cells overexpressing Bcl-3 were cultivated with Compound 1a over a range of molarities for 24 hrs before cell lysates were obtained under non-denaturing conditions. The dose response curve was generated using GraphPad software (FIG. 2A). The determined IC.sub.50 was 385.5 nM. Indirect ELISA assay was performed using Bcl-3 antibody to show an equal loading across samples treated with compound 1a (FIG. 2B) and controls (FIG. 2C).

B. Establishing the Effect on Intra-Cellular NF-κB Activity

(303) The effect of Compound 1a on NF-κB activity was determined by NF-κB luciferase assay in MDA-MB-231 and HEK-293 cells overexpressing Bcl-3 and HEK-293 cells overexpressing p52. Cells were cultivated with Compound 1a over a range of molarities for 24 hrs before being transfected with NF-κB luciferase reporter plasmid for 48 hrs and analysed for NF-κB activity. The dose response curve was generated to using GraphPad software (FIG. 3). The determined IC.sub.50 in MDA-MB-231 cells was 49.43 nM, 159.6 nM HEK-293 cells and 210.6 nM in HEK-293 p52 overexpressing cells.

C. Establishing the Effect of Compound 1a on Cell Motility

(304) It was previously determined that Compound 1a significantly suppressed migration ability in MDA-MB-231 cells at 10 μM. We therefore generated a dose response curve to determine an IC.sub.50 in this assay. MDA-MB-231 cells overexpressing Bcl-3 were cultivated with Compound 1a and DMSO control over a range of molarities for 24 hrs before being seeded onto Boyden migration chambers. The constant number of live cells present during this assay across samples was monitored by cell count. The dose response curve was generated using GraphPad software (FIG. 4). The determined IC.sub.50 was 310.4 nM.

Example 7

Biological Evaluation of Analogues From Series 1-3

A. Cell Toxicity

(305) Selected compounds were dissolved in DMSO and diluted to a highest concentration of 10 μM (0.1% DMSO). In all assays, a DMSO control was always used. Selected compounds were tested for cell toxicity in HEK-293 and MDA-MB-231 cells before being used in cell-based assays. Cell toxicity was evaluated using the Cell Titre Blue viability assay over a range of molarities for 24 hrs

(306) Results for mono-substituted compounds 1a, 1c, 1e, 1f, 1g, 1i, 1j, 2a, 2b, 2c, 2d, 2f, 3a, 3b and 3d are shown in FIG. 5. Compounds were well tolerated in both cell lines and cell viability was above 70% even at 10 μM concentration.

(307) Results for di- and tri-substituted compounds 1l, 1m, 1n, 1o, 1p and 1q are shown in FIGS. 11A & B. Compounds were well tolerated in both cell lines and cell viability was above 90% even at 10 μM concentration.

B. NF-κB Assay

(308) The effect of selected analogues on NF-κB activity was determined by NF-KB luciferase assay in MDA-MB-231 cells. MDA-MB-231 Bcl-3 over-expressing cells were cultivated with compounds from series 1-3 at 1 μM concentration for 24 hrs before being transfected with NF-κB luciferase reporter plasmid for 48 hrs together with controls and analysed for NF-κB activity.

(309) Results for mono-substituted compounds 1a, 1c, 1e, 1f, 1g, 1i, 1j, 2a, 2b, 2c, 2d, 2f, 3a, 3b and 3d are shown in FIG. 6).

(310) is It can be seen that the NF-κB activity of the mono-substituted analogues from series 1 was comparable to that of Compound 1a, with a significant decrease of in NF-KB activity observed for Compound 1a and the analogue 1f as compared to DMSO control.

(311) From series 2 analogue 2a and 2d significantly decreased NF-κB activity as compared to Bcl-3 WT DMSO control. Analogue 2a showed comparable activity with Compound 1a with other analogues having lesser activity.

(312) From series 3 analogue 3a had comparable effect on NF-κB activity with Compound 1a. Analogue 3c also showed significant decrease in NF-κB activity as compared to DMSO control, however not to the same level as Compound 1a.

(313) Based on the results for the series of analogues, we have established dose response curve for selected analogues from series 1 (10, from series 2 (2a and 2c) and from series 3 (3a) over a range of molarities. MDA-MB-231 Bcl-3 WT cells were cultivated with selected compounds from series 1-3 over a range of molarities for 24 hrs before being transfected with NF-κB luciferase reporter plasmid for 48 hrs together with controls and analysed for NF-κB activity (FIG. 7). We observed an improvement in the determined IC.sub.50 for analogue 1f (6.97 nM). Other analogues showed decreased ability to suppress NF-κB activity compared with Compound 1a. The determined IC.sub.50 for analogue 2a was 2.50 μM, 2.49 μM for analogue 2c and 1.07 μM for analogue 3a (see Table 3).

(314) Results for di- and tri-substituted compounds 1l, 1m, 1n, 1o, 1p and 1q are shown in FIG. 11C (compared with Compound 1a) and FIG. 12 as well as in Table 3.

(315) Compound 1a showed the most potent suppression of NF-κB activity as compared to Bcl-3 WT overexpressing MDA-MB-231 cells (FIG. 11C).

(316) IC.sub.50 values were established for three selected analogues (1o-q). All three tested analogues showed decreased ability to suppress NF-κB activity than Compound 1a. The determined IC.sub.50 for analogue 10 was 237.80 nM, 705.90 nM for analogue 1p and 988.87 nM for analogue 1q (FIG. 12; Table 3).

(317) TABLE-US-00006 TABLE 3 Comparison of IC.sub.50 values for test compounds ELISA assay NF-κB assay Cell motility assay Analogue (μM) (μM) (μM) 1a 0.3855 0.04543 0.3104 1f 0.0617 0.00697 0.02893 2a 15.23 2.5 1.33 2c 10.41 2.49 3.65 3a 0.01195 1.07 0.90 1o 0.2378 1p 0.7059 1q 0.98887

C. Indirect Sandwich ELISA Assay

(318) The ability to disrupt Bcl-3-p50 binding was determined by Indirect Sandwich ELISA assay for selected analogues (1f, 2a, 2c, 3a).

(319) HEK-293 Bcl-3 WT cells were cultivated with selected compounds over a range of molarities for 24 hrs before cell lysates were obtained under non-denaturing conditions. The dose response curve for selected analogues was generated using GraphPad software (FIG. 8A-D). We observed improved IC50 as compared to Compound 1a for analogues 1f and 3a, with IC50 values 60.17 nM and 119.3 nM respectively. The IC50 could not be established for analogues from series 2, 2a and 2c, and was calculated from the dose response curve using GraphPad software with IC.sub.50 of 15.23 μM for analogue 2a and 10.41 μM for analogue 2c.

(320) Indirect ELISA assay was performed using Bcl-3 antibody to show an equal loading across samples treated with compounds 1f, 2a, 2c, and 3a (FIG. 8E-H).

D. Cell Motility Assay

(321) As shown in Example 6, Compound 1a caused a significant decrease in cell motility with an IC.sub.50 value of 310.4 nM. Therefore we wanted to establish whether designed analogues have similar or improved ability to suppress cell migration. MDA-MB-231 Bcl-3 over-expressing cells were cultivated with selected analogues (1f, 2a, 2c, 3a) and DMSO control over a range of molarities for 24 hrs before being seeded onto the Boyden motility chambers. Migrated cells were visualised and counted after 24 hrs. The dose response curves were generated using GraphPad software (FIG. 9). The constant number of live cells present during this assay across samples was monitored by cell count.

(322) Consistent with results from NF-κB assay and Indirect Sandwich ELISA assay, the analogue 1f showed an improved ability to suppress cell migration, with IC50 value of 28.93 nM. Interestingly, the cell migration was suppressed below 50% even at 10 nM concentration. Other analogues showed decreased ability to suppress cell motility than Compound 1a. The determined IC50 for analogue 2a was 1.33 μM, 3.65 μM for analogue 2c and 900 nM for analogue 3a.

F. Establishing EC.SUB.50 .of Analogue 1f

(323) The activity of the analogue 1f was improved as compared to Compound 1a; therefore next we determined the toxicity of this analogue in MDA-MB-231 cell line.

(324) Compound 1a was dissolved in DMSO and diluted in media to a highest concentration of 2 mM. Cell toxicity was evaluated using the Cell Titre Blue viability assay over a range of molarities for 24 hrs. The effect of Compound 1a on cell viability was always normalised against DMSO control and the dose-response curve was generated using GraphPad software (FIG. 10). The determined EC.sub.50 for analogue 1f was 781.3 μM.

SUMMARY

(325) We have synthesised 2-[(2-fluorobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide (Compound 1a) and a number of novel analogues of this compound and we have shown that the compounds are inhibitors of Bcl3 since are capable of suppressing Bcl3-NFkB protein interactions and inhibiting NF-κB signalling. This indicates that the compounds will be useful for the treatment of cancer, particularly metastatic cancer.

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