Bioactive Benzocycloheptene Analogues From Himachalenes and its Applications

Abstract

Functionalized benzocycloheptenes are one of the most important classes of bicyclic framework that have been investi-gated in different areas of biological activities. The claimed invention provides preparations of benzocycloheptene analogues, inhibitors of PI3K and MK2, pharmaceutical compositions containing them and their use in therapy. The compounds of Formula I, II, III, IV, V and VI may be used as anti type 2 diabetes, antipyretic, anti-inflammatory, antiepileptic, anticancer, antiulcer, CNS-stimulant, and CNS-depressant. These benzocycloheptene derivatives are useful in treatment of PI3K and MK2 related disorders.

Claims

11. A compound of formula (I), (II), (III), (IV), (V), or (VI): ##STR00014## or a pharmaceutical acceptable salt or enantiomer thereof, wherein, in each of formulas (I), (II), (III), (IV), (V), and (VI): Y is selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; Z is selected from the group consisting of —H, C1-C6 alkyl, C1-C6 aryl, halogen, oxygen, nitrogen, and sulfur; R.sup.1 is selected from the group consisting of —H, —OH, N-substituted benzylamine, N-substituted aniline, S-substituted thiophenes, carboxylic acids, and halogen; R.sup.2 is selected from the group consisting of —H, C1-C6 alkyl, C1-C6 aryl, halogen, C1-C6 amine, and C1-C6 carbonyl groups; R.sup.3 is selected from the group consisting of —H, C1-C6 alkyl, C1-C6 aryl, and thiophenes; X is selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; R.sup.4 is selected from the group consisting of —H, C1-C6 alkyl, C1-C6 aryl, halogen, and aldehyde-substituted benzenesulfonamides; R.sup.5 is selected from the group consisting of —H, C1-C6 alkyl, C1-C6 aryl, halogen, and C1-C6 carbonyl group; and R.sup.6 is selected from the group consisting of —H, C1-C6 alkyl, C1-C6 aryl, and C1-C6 amine group.

12. The compound of claim 11, wherein the compound has formula (I), where: R.sup.1 is selected from the group consisting of —H, —OH, N-substituted benzylamine, N-substituted anilines, carboxylic acids, and halogens, R.sup.2 is selected from the group consisting of —H, C1-C6 alkyl, C1-C6 aryl, halogen, C1-C6 amine, and C1-C6 carbonyl group; and R.sup.3, R.sup.4, and R.sup.5 are each independently selected from the group consisting of —H, C1-C6 alkyl, C1-C6 aryl, halogen, and C1-C6 carbonyl group.

13. The compound of claim 11, wherein the compound has formula (II), where: R.sup.1 is selected from the group consisting of —H, OH, N-substituted benzylamine, N-substituted aniline, carboxylic acids, and halogen, R.sup.2 is selected from the group consisting of —H, C1-C6 alkyl, C1-C6 aryl, halogen, C1-C6 amine, and C1-C6 carbonyl; and Y is selected from the group consisting of carbon, oxygen, nitrogen, and sulfur.

14. The compound of claim 11, wherein the compound has formula (III), where: R.sup.1 is selected from the group consisting of —H, —OH, benzylamine, aniline, carboxylic acids, and halogen; R.sup.2 is selected from the group consisting of —H, C1-C6 alkyl, C1-C6 aryl, halide, C1-C6 amine, and C1-C6 carbonyl; and Z is selected from the group consisting of —H, C1-C6 alkyl, C1-C6 aryl, halogen, oxygen, nitrogen, and sulfur.

15. The compound of claim 11, wherein the compound has formula (IV), where: R.sup.1 is selected from the group consisting of —H, —OH, N-substituted benzylamine, N-substituted aniline, carboxylic acids, and halogen, R.sup.2 is selected from the group consisting of —H, C1-C6 alkyl, C1-C6 aryl, halogen, C1-C6 amine, and C1-C6 carbonyl; and n is 5 or 6.

16. The compound of claim 11, wherein the compound has the formula (V), where: R.sup.1 is selected from the group consisting of —H, —OH, N-substituted benzylamine, N-substituted aniline, carboxylic acids, and halogen; R.sup.2 is selected from the group consisting of —H, C1-C6 alkyl, C1-C6 aryl, halogen, C1-C6 amine, and C1-C6 carbonyl group; n is from 5 to 6; and R.sup.3 is selected from the group consisting of —H, C1-C6 alkyl, C1-C6 aryl, and halogen.

17. The compound of claim 11, wherein the compound has formula (VI), where: R.sup.1 is selected from the group consisting of —H, —OH, N-substituted benzylamine, N-substituted aniline, carboxylic acids, and halogen; R.sup.2 is selected from the group consisting of —H, C1-C6 alkyl, C1-C6 aryl, halogen, C1-C6 amine, and C1-C6 carbonyl; R.sup.5 is selected from the group consisting of hydrogen, C1-C6 alkyl, halogen, sulfur, C1-C6 amine, and C1-C6 aryl; and R.sup.6 is selected from the group consisting of —H, C1-C6 alkyl, C1-C6 aryl, and C1-C6 amine.

18. A pharmaceutical formulation comprising the compound according to claim 11 and a pharmaceutically acceptable adjuvant, diluent, or carrier.

19. A method for treating type-2 diabetes, fever, inflammation, cancer, ulcers, or for stimulating or depressing the central nervous system in a subject, the method comprising administering the compound according to claim 11 to the subject.

20. A method of inhibiting PI3K and MK2 mediated activity in a subject, the method comprising administering the compound according to claim 11 to the subject.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0046] FIG. 1: Image depicts the on-rate and off-rate of binding of evaluated compounds of general formula II to PI3K (120y). Plotting association rate constant ka against dissociation rate constant kd (here on logarithmic scales) creates a plot where the affinity is represented by diagonal lines. Compounds on the same diagonal have the same affinity but differ in kinetics.

[0047] FIG. 2: Graphical representation of the on-rate and off-rate of binding of evaluated compounds of general formula II to PI3K (120y).

[0048] FIG. 3: Graphs represent the dose dependent inhibition of the PI3K (120y) by compounds of general formula II (5, 7, 8, 9, 12 and 16).

[0049] FIG. 4: Image depicts the on-rate and off-rate of binding of evaluated compounds of general formula II to MK2. Plotting association rate constant ka against dissociation rate constant kd (here on logarithmic scales) creates a plot where the affinity is represented by diagonal lines. Compounds on the same diagonal have the same affinity but differ in kinetics.

[0050] FIG. 5: Graphical representation of the on-rate and off-rate of binding of evaluated compounds of general formula II to MK2

[0051] FIG. 6: Graphs represent the dose dependent inhibition of the MK2 by compounds of general formula II (1, 3, 6, 8, 12, 13, 14, 15 and 17 along with marketed MK2-inhibitor PF 3644022.

DETAILED DESCRIPTION OF THE INVENTION

[0052] The present invention is directed towards substituted benzocycloheptene analogues, of general formula I, II, III, IV, V or VI

##STR00002##

wherein [0053] in Formula I-VI, [0054] R.sup.1 is selected from the group consisting of H, OH, N substituted group selected from benzylamine or aniline, S-containing group selected from thiophene, carboxylic acid and its derivatives selected from propanoic acid, and halogen; [0055] R.sup.2 is selected from the group consisting of H, C1-C6 alkyl, C1-C6 aryl, halide, C1-C6 amine and C1-C6 carbonyl group; [0056] in Formula I, R.sup.3—R.sup.5 are each independently selected from the group consisting of H, C1-C6 alkyl, C1-C6 aryl, halogen and C1-C6 carbonyl group; [0057] in Formula II, Y is selected from the group consisting of C, O, N and S; [0058] in Formula III, Z is selected from the group consisting of H, C1-C6 alkyl, C1-C6 aryl, halogen, oxygen, nitrogen and sulfur; [0059] in Formula IV, n varies from 5 to 6; [0060] in Formula V, n varies from 5 to 6 and R.sup.3 is selected from the group consisting of H, OH, N substituted group selected from pyridine, carboxylic acid and its derivatives selected from propanoic acid and halogen; [0061] in Formula VI, R.sup.5 is selected from the group consisting of H, halide, oxygen, nitrogen containing group selected from the group consisting of enaminone, cyclohexane 1,3-dione, C1-C6 alkyl and C1-C6 aryl group; and R.sup.6 is selected from the group consisting of C1-C6 alkyl, C1-C6 aryl, hydrogen and hetero atoms selected from thiophene.

[0062] The present invention provides a compound of general formula I, II, III, IV, V or VI:

##STR00003##

and pharmaceutical acceptable salts and enantiomers thereof, [0063] wherein [0064] Y is selected from the group consisting of C, O, N and S; [0065] Z is selected from the group consisting of H, C1-C6 alkyl, C1-C6 aryl, halogen, oxygen, nitrogen and sulfur; [0066] R.sup.1 is selected from the group consisting of hydrogen, hydroxyl, nitrogen substituted group selected from benzylamine or aniline, S-containing groups selected from thiophene, carboxylic acid and its derivatives selected from propanoic acid, and halogen; [0067] R.sup.2 is selected from the group consisting of H, C1-C6 alkyl, C1-C6 aryl, halide, C1-C6 amine and C1-C6 carbonyl groups; [0068] R.sup.3 is selected from the group consisting of H, C1-C6 alkyl, C1-C6 aryl and sulfur containing group selected from thiophene; [0069] X is selected from the group consisting of carbon, nitrogen, oxygen and sulfur; [0070] R.sup.4 is selected from the group consisting of H, C1-C6 alkyl, C1-C6 aryl, halogen and carbonyl group selected from aldehyde substituted benzenesulphonamide; [0071] R.sup.5 is selected from the group consisting of H, C1-C6 alkyl, C1-C6 aryl, halogen and C1-C6 carbonyl group; and [0072] R.sup.6 is selected from the group consisting of H, C1-C6 alkyl, C1-C6 aryl and C1-C6 amine group. [0073] In an embodiment of the present invention there is provided a compound of the formula (I), [0074] wherein R.sup.1 is selected from the group consisting of H, OH, N substituted group selected from benzylamine or aniline, carboxylic acid and its derivatives selected from propanoic acid and halogens, [0075] R.sup.2 is selected from the group consisting of H, C1-C6 alkyl, C1-C6 aryl, halide, C1-C6 amine and C1-C6 carbonyl group; and [0076] R.sup.3-R.sup.5 are each independently selected from the group consisting of H, C1-C6 alkyl, C1-C6 aryl, halogen and C1-C6 carbonyl group. [0077] In another embodiment of the present invention there is provided a compound of the formula (II), [0078] wherein R.sup.1 is selected from the group consisting of H, OH, N substituted group selected from benzylamine, or aniline, carboxylic acid and its derivatives selected from propanoic acid, and halogen, [0079] R.sup.2 is selected from the group consisting of H, C1-C6 alkyl, C1-C6 aryl, halide, C1-C6 amine and C1-C6 carbonyl group; and [0080] and Y is selected from the group consisting of C, O, N and S. [0081] In yet another embodiment of the present invention there is provided a compound of the formula (III), [0082] wherein R.sup.1 is selected from the group consisting of H, OH, N substituted group selected from benzylamine or aniline, carboxylic acid and its derivatives selected from propanoic acid, and halogen, [0083] R.sup.2 is selected from the group consisting of H, C1-C6 alkyl, C1-C6 aryl, halide, C1-C6 amine and C1-C6 carbonyl group; and [0084] Z is selected from the group consisting of H, C1-C6 alkyl, C1-C6 aryl, halogen, oxygen, nitrogen and sulfur group. [0085] In still another embodiment of the present invention there is provided a compound of the formula (IV), [0086] wherein R.sup.1 is selected from the group consisting of H, OH, N substituted group selected from benzylamine, or aniline, carboxylic acid and its derivatives selected from propanoic acid and halogen, [0087] R.sup.2 is selected from the group consisting of H, C1-C6 alkyl, C1-C6 aryl, halide, C1-C6 amine, and C1-C6 carbonyl group; and and n varies from 5 to 6. [0088] In another embodiment of the present invention there is provided a compound of the formula (V), [0089] wherein R.sup.1 is selected from the group consisting of H, OH, N substituted group selected from benzylamine or aniline, carboxylic acid and its derivative selected from propanoic acid and halogen, [0090] R.sup.2 is selected from the group consisting of H, C1-C6 alkyl, C1-C6 aryl, halide, C1-C6 amine, and C1-C6 carbonyl group; [0091] n varies from 5 to 6; and [0092] R.sup.3 is selected from the group consisting of H, CJ-C6 alkyl, CJ-C6 aryl and halogen group. [0093] In yet another embodiment of the present invention there is provided a compound of the formula (VI), [0094] wherein R.sup.1 is selected from the group consisting of H, OH, N substituted group selected from benzylamine, or aniline, carboxylic acid and its derivatives selected from propanoic acid and halogen; [0095] R.sup.2 is selected from the group consisting of H, CJ-C6 alkyl, CJ-C6 aryl, halide, CJ-C6 amine and CJ-C6 carbonyl group; [0096] R.sup.5 is selected from the group consisting of hydrogen, CJ-C6 alkyl, halide, sulphur, C1-C6 amine and CJ-C6 aryl group; and [0097] R.sup.6 is selected from the group consisting of H, C1-C6 alkyl, C1-C6 aryl and C1-C6 amine group.

[0098] In still another embodiment of the present invention there is provided a compound of the formula I-VI, for use as anti-type 2 diabetes, antipyretic, anti-inflammatory, anticancer, antiulcer, CNS-stimulant, and CNS-depressant.

[0099] Another embodiment of the present invention provides a pharmaceutical formulation comprising the compound of formula I-VI along with a pharmaceutically acceptable adjuvant, diluent or carrier.

[0100] In yet another embodiment of the present invention there is provided a compound of the formula I-VI, for use as an inhibitor of PI3K and MK2 mediated activity.

[0101] These substituted benzocycloheptene analogues are semi-synthesized from himachalenes extracted from Cedrus deodara oil.

[0102] The present invention discloses synthesis of benzocycloheptene compounds using easily available and low cost natural precursors i.e. Cedrus deodara oil following less steps, economic and cost-effective approach.

[0103] Under this investigation, semi-synthetic approaches have been developed to minimize number of challenging steps in organic synthesis and avoiding high cost reagents and chemicals to reduce overall cost of production.

[0104] Under this investigation, different new methodologies have been developed to overcome the existing challenges in this type of organic synthesis. High purity of the molecules (>98-99%) has been achieved through column chromatography.

[0105] The process is easy to apply for scale-up synthesis which is a very important issue to evaluate biological activities and future industrial interest.

[0106] These class of compounds are known for different biological activities therefore, facile approaches have been developed for different new classes of benzocycloheptene analogues synthesis following new process and further applied for treatment of PI3K and MK2 related disorders.

[0107] Further, the use of natural analogues also reduces the toxic effect and its specific structure enhances the chance of biological activities for therapeutic development.

[0108] Compounds constituted of complex bicyclic framework with alkyl group are difficult to introduce. Thus, the core structure is derived from natural precursor which reduces the cost of production, reagents and overall makes the process economic.

[0109] Compounds constituted from natural precursor are less toxic in nature and indicated different applications for treatment of PI3K and MK2 related disorders responsible for different diseases.

EXAMPLES

[0110] The following examples are given by way of illustration and therefore should not construed to limit the scope of the present invention.

Experimental Part

[0111] All reagents and solvents were purchased from commercial sources (Sigma-Aldrich, Merck India Ltd). Reactions were monitored by TLC plates coated with 0.2 mm silica gel 60 F.sub.254. TLC plates were visualized by UV irradiation (254 nm) and iodine spray. The products were purified by column chromatography employing silica gel of 60-120 mesh size (Merck). The .sup.1H and .sup.13C NMR spectra were recorded at 298 K with a Bruker AM-300 spectrometer; using TMS as internal reference standard in CDCl.sub.3. HRMS were conducted with UHR-QTOF (ultra-high resolution Q-time of flight). IR spectra were obtained on a Nicolet 5700 FTIR (Thermo, USA) spectrophotometer in the region 4,000-400 cm-1 using KBr disks. CEM Discover™ focused microwaves (2450 MHz, 300 W) were used. The temperature on the surface of the reaction flask was measured with an inbuilt infrared temperature probe in the microwave experiment. The coupling constants (J) are reported in hertz (Hz) and the following abbreviations are used to designate signal multiplicity: s=singlet; d=doublet; t=triplet; m=multiplet; br=broad singlet.

Example 1

[0112] General Procedure for the Synthesis of Formula I

##STR00004##

Synthesis of N-benzyl-N-((8-bromo-3,5,5-trimethyl-6,7-dihydro-5H-benzo[7] annulen-9-yl)methyl)-4-methylbenzenesulfonamide

[0113] A mixture of 8-bromo-9-(bromomethyl)-3,5,5-trimethyl-6,7-dihydro-5H-benzo[7]annulene (0.279 mmol, 1.0 equiv.), N-benzyl-4-methylbenzzenesulfonamide (0.41 mmol, 1.5 equiv.), K.sub.2CO.sub.3 (0.55 mmol, 2 equvi.) in DMF (3 ml) were placed in reaction tube (15 mL) at 90° C. for 16 h. After cooling the reaction mixture to ambient temperature, water was added and extracted with ethyl acetate (10×3 times). The combined organic layer was washed with water and dried over Na.sub.2SO.sub.4 and vacuum evaporated. The viscous liquid obtained was purified by column chromatography on silica gel (mesh 60-120) using the mixture of hexane: EtOAc (80:20) to obtain the desired product as yellow oily (45 mg, 30%).

[0114] .sup.1H NMR (CDCl.sub.3, 300 MHz, δ, ppm): 1.08 (s, 6H), 1.94-1.89 (m, 2H), 2.32-2.24 (m, 8H), 4.33 (s, 2H), 4.36 (s, 2H), 6.96-6.94 (d, J=7.8, 1H), 7.08-7.05 (d, 3H), 7.21-7.11 (m, 5H), 7.36-7.29 (m, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) δ, 21.4, 21.5, 31.6, 37.6, 38.6, 47.3, 51.8, 52.6, 126.6, 126.6, 127.2, 127.3, 128.1, 129.0, 133.3, 134.5, 136.4, 136.9, 137.1, 142.8, 146.2. ESI-MS: calcd for C.sub.29H.sub.32BrNO.sub.2S [M+H].sup.+ 538.1410, found 538.0189.

Example 2

[0115] General Procedure for the Synthesis of Formula II

##STR00005##

Synthesis of 6,6,8-trimethyl-1,4,5,6-tetrahydro-3H-benzo[3,4]cycloheptal[1,2-c]furan-3-one

[0116] A mixture of 8-bromo-9-(bromomethyl)-3,5,5-trimethyl-6,7-dihydro-5H-benzo[7]annulene (0.279 mmol, 1.0 equiv.), oxalic acid (1.67 mmol, 6 equiv.), PdCl2 (0.013 mmol, 0.05 equiv.), dppe (0.013 mmol, 0.05 equiv.), TBACl (0.139 mmol, 0.50 equiv.) in DMF and t-amyl alcohol in 1:1 ratio (0.5:0.5 ml) was placed in a pressurized reaction tube (5 ml) under conventional heating (130° C.) for 20 hrs. After cooling to ambient temperature, water was added to the reaction mixture and extracted with ethyl acetate (3×10 ml). The combined organic layer was washed with water and dried over Na.sub.2SO.sub.4 and solvent was removed under reduced pressure. The viscous liquid obtained was purified by column chromatography on silica gel (60-120 mesh) using hexane:EtOAc (90:10) to afford the desired product as a white solid (97 mg, 40%).

[0117] .sup.1H NMR (CDCl.sub.3, 300 MHz, δ, ppm):1.35 (s, 6H), 2.15-2.11 (m, 2H), 2.37 (s, 3H), 2.51-2.47 (t, 3H), 4.63 (s, 2H), 7.11-7.08 (d, J=6.99 Hz, 1H), 7.23 (s, 1H), 7.33-7.30 (d, J=7.86 Hz, 1H); .sup.13C NMR (CDCl.sub.3, 75 MHz, δ, ppm): 20.1, 22.2, 27.5, 34.2, 36.6, 69.2, 125.1, 125.5, 125.7, 125.9, 139.1, 150.2, 151.6, 173.6. ESI-MS: calcd for C.sub.16H.sub.18O.sub.2[M+H].sup.+ 243.1380, found 243.3711.

Example 3

[0118] General Procedure for the Synthesis of Formula II

##STR00006##

Formula II (a)

Synthesis of 6,6,8-trimethyl-1,4,5,6-tetrahydrobenzo[3,4]cyclohepta[1,2-c]pyrrol-3(2H)-one

[0119] A mixture of 8-bromo-9-(bromomethyl)-3,5,5-trimethyl-6,7-dihydro-5H-benzo[7]annulene (0.279 mmol, 1.0 equiv.), ammonium carbamate (1.1172 mmol, 4 equiv.), oxalic acid (1.67 mmol, 6 equiv.), PdCl.sub.2 (0.013 mmol, 0.05 equiv.), dppe (0.013 mmol, 0.05 equiv.), TBACl (0.139 mmol, 0.50 equiv.) in DMF and t-amyl alcohol in 1:1 ratio (0.5:0.5 ml) was placed in a pressurized reaction tube (5 ml) under conventional heating (130° C.) for 20 hrs. After cooling to ambient temperature, water was added to the reaction mixture and extracted with ethyl acetate (3×10 ml). The combined organic layer was washed with water and dried over Na.sub.2SO.sub.4 and solvent was removed under reduced pressure. The viscous liquid obtained was purified by column chromatography on silica gel (60-120 mesh) using hexane:EtOAc (70:30) to afford the desired product as a yellow solid (20 mg, 30%). 1H NMR (CDCl3, 600 MHz, δ, ppm): 1.25 (s, 6H), 1.76-1.74 (t, J=6.66 Hz, 2H), 2.31 (s, 3H), 2.50-2.46 (m, 2H), 4.25 (s, 2H), 7.07-7.06 (d, J=7.92 Hz, 1H), 7.27 (s, 1H), 7.38-7.37 (d, J=7.98 Hz, 1H); 13C NMR (CDCl3, 150 MHz, δ, ppm): 21.5, 24.1, 28.9, 36.4, 38.0, 47.9, 126.7, 127.2, 128.2, 128.3, 133.4, 138.4, 146.6, 150.5, 174.1. ESI-MS: calcd for C.sub.16H.sub.19NO.sub.2 [M+H].sup.+ 242.1539, found 242.1002.

Formula II (b)

Synthesis of 2-benzyl-6,6,8-trimethyl-1,4,5,6-tetrahydrobenzo[3,4]cyclohepta[1,2-c]pyrrol-3(2H)-one

[0120] ##STR00007##

[0121] A mixture of 8-bromo-9-(bromomethyl)-3,5,5-trimethyl-6,7-dihydro-5H-benzo[7]annulene (0.279 mmol, 1.0 equiv.), phenylmethanamine (1.1172 mmol, 1.2 equiv.), oxalic acid (1.67 mmol, 6 equiv.), PdCl.sub.2 (0.013 mmol, 0.05 equiv.), dppe (0.013 mmol, 0.05 equiv.), TBACl (0.139 mmol, 0.50 equiv.) in DMF and t-amyl alcohol in 1:1 ratio (0.5:0.5 ml) was placed in a pressurized reaction tube (5 ml) under conventional heating (130° C.) for 20 hrs. After cooling to ambient temperature, water was added to the reaction mixture and extracted with ethyl acetate (3×10 ml). The combined organic layer was washed with water and dried over Na.sub.2SO.sub.4 and solvent was removed under reduced pressure. The viscous liquid obtained was purified by column chromatography on silica gel (60-120 mesh) using hexane:EtOAc (70:30) to afford the desired product as a semisolid (25%). 1H NMR (CDCl3, 600 MHz) δ 1.34 (s, 6H), 1.90-1.88 (t, J=6.6 Hz, 2H), 2.36 (s, 3H), 2.77-2.75 (t, J=6.5 Hz, 2H), 4.19 (s, 2H), 4.73 (s, 2H), 7.01-7.00 (d, J=7.3 Hz, 1H), 7.18-7.16 (d, J=7.9 Hz, 1H), 7.32-7.28 (m, 4H), 7.38-7.35 (m, 2H); 13C NMR (CDCl3, 150 MHz,) δ 22.01, 24.94, 29.38, 36.68, 38.57, 46.68, 52.70, 127.23, 127.34, 127.91, 128.04, 128.21, 128.56, 129.27, 134.08, 137.4, 137.91, 139.20, 144.77, 151.25, 172.68. ESI-MS: calcd. for C.sub.23H.sub.25NO [M+H].sup.+=332.2009, found 331.8708.

Example 4

[0122] General Procedure for the Synthesis of Formula IV

##STR00008##

Formula IV (a)

Synthesis of 3,5,5-trimethyl-5,6,7,9,10,11-hexahydro-12H-benzo[3,4]cycloheptal[1,2-b]naphthalene-12-one

[0123] A mixture of 8-bromo-9-(bromomethyl)-3,5,5-trimethyl-6,7-dihydro-5H-benzo[7]annulene (0.279 mmol, 1.0 equiv.), 3-amino-2-cyclohexen-1-one/3-aminocyclopent-2-en-1-one (0.33 mmol, 1.2 equiv.), Pd(OAc)2 (0.055 mmol, 20 mol %), Xanthphos (0.055 mmol, 20 mol %), K.sub.2CO.sub.3 (0.55 mmol, 2 equvi.) in 2-methyl THF (1.5 ml) were placed in reaction tube (15 mL) at 90° C. for 12 h. After cooling the reaction mixture to ambient temperature, water was added and extracted with ethyl acetate (10×3 times). The combined organic layer was washed with water and dried over Na.sub.2SO.sub.4 and vacuum evaporated. The viscous liquid obtained which was purified by column chromatography on silica gel (mesh 60-120) using the mixture of hexane: EtOAc (80:20) to obtained the desired product as semisolid (38 mg, 45%).

[0124] .sup.1H NMR (CDCl.sub.3, 600 MHz, δ, ppm): 1.18 (s, 6H), 2.18-2.13 (m, 4H), 2.35 (s, 3H), 2.66-2.62 (m, 2H), 2.76-2.71 (t, J=7.0 Hz, 2H), 3.12-3.08 (t, J=6.1 Hz, 2H), 7.11 (d, 1H), 7.21-7.16 (m, 2H), 8.10 (s, 1H); .sup.13C NMR (CDCl.sub.3, 150 MHz, δ, ppm): 22.07, 22.7, 29.6, 31.7, 35.5, 37.6, 38.5, 47.3, 126.7, 127.1, 127.3, 130.6, 134.3, 137.3, 138.03, 145.8, 161.5, 164.5, 198.2. ESI-MS: calcd for C.sub.21H.sub.23NO [M+H].sup.+ 306.1852, found 306.1964.

Formula IV (b)

Synthesis of 3,5,5,10-tetramethyl-5,6,7,9,10,11-hexahydro-12H-benzo[3,4]cyclohepta[1,2-b]quinolin-12-one

[0125] ##STR00009##

[0126] A mixture of 8-bromo-9-(bromomethyl)-3,5,5-trimethyl-6,7-dihydro-5H-benzo[7]annulene (0.279 mmol, 1.0 equiv.), 3-amino-2-cyclohexen-1-one/3-amino-6-methylcyclohex-2-en-1-one (0.33 mmol, 1.2 equiv.), Pd(OAc)2 (0.055 mmol, 20 mol %), Xanthphos (0.055 mmol, 20 mol %), K.sub.2CO.sub.3 (0.55 mmol, 2 equvi.) in 2-methyl THF (1.5 ml) were placed in reaction tube (15 mL) at 90° C. for 12 h. After cooling the reaction mixture to ambient temperature, water was added and extracted with ethyl acetate (10×3 times). The combined organic layer was washed with water and dried over Na.sub.2SO.sub.4 and vacuum evaporated. The viscous liquid obtained which was purified by column chromatography on silica gel (mesh 60-120) using the mixture of hexane: EtOAc (80:20) to obtained the desired product as semisolid (48%).

[0127] .sup.1H NMR (CDCl.sub.3, 600 MHz, δ, ppm): 1.23 (s, 6H), 1.24 (m, 3H), 2.27-2.24 (m, 4H), 2.45 (s, 3H), 2.91-2.79 (m, 4H), 3.27-3.24 (m, 2H), 3.12-3.08 (t, J=6.1 Hz, 2H), 7.20-7.19 (m, 1H), 7.28-7.27 (m, 1H), 8.18 (s, 1H); .sup.13C NMR (CDCl.sub.3, 150 MHz, δ, ppm): 21.3, 21.6, 29.5, 31.7, 35.6, 37.6, 40.6, 46.7, 47.3, 126.5, 126.7, 127.4, 130.6, 132.6, 134.3, 137.2, 138.0, 145.8, 161.0, 164.6, 198.3. ESI-MS: calcd for C.sub.22H.sub.25NO [M+H].sup.+ 320.2009, found 320.1964.

Example 5

[0128] General Procedure for the Synthesis of Formula VI

##STR00010##

Formula VI (a)

Synthesis of 3,5,5-Trimethyl-9-((phenylsulfonyl)methyl)-6,7-dihydro-5H-benzo[7]annulene

[0129] A mixture of 2,9,9-trimethyl-5-methylene-6,7,8,9-tetrahydro-5H-benzo[7]annulene (0.25 mmol, 1.0 equiv.), sodium benzenesulfinate (0.3 mmol, 1.2 equiv.), K.sub.2S.sub.2O.sub.8 (0.62 mmol, 2.5 equiv.), and I.sub.2 (0.3 mmol, 1.2 equiv.), in ACN:H.sub.2O (1:1) were placed in reaction tube (5 mL) at room temperature for 12 h. After completion of reaction, a saturated solution of sodium thiosulfate was added to the reaction mixture and extracted with ethyl acetate (10×3 times). The combined organic layer was washed with water and dried over Na.sub.2SO.sub.4 and vacuum evaporated. The viscous liquid obtained was purified by column chromatography on silica gel (mesh 60-120) using the mixture of hexane: EtOAc (80:20) to obtained the desired product as semisolid (71 mg, 84%).

[0130] .sup.1H NMR (CDCl.sub.3, 600 MHz) δ 1.34 (s, 6H), 1.96-1.99 (t, J=7.2 Hz, 2H), 2.10-2.12 (t, J=6.9 Hz, 2H), 2.34 (s, 3H), 4.22 (s, 2H), 6.24-6.26 (t, J=6.4 Hz, 1H), 6.95 (d, J=7.8 Hz, 1H), 7.20 (d, J=8.1 Hz, 2H), 7.51-7.53 (t, J=7.8 Hz, 2H), 7.61-7.64 (m, 1H), 7.90 (d, J=7.5 Hz, 2H); .sup.13C NMR (CDCl.sub.3, 150 MHz) δ 21.3, 27.0, 30.8, 38.0, 46.5, 63.7, 126.4, 126.5, 128.1, 128.3, 128.4, 129.0, 133.4, 134.4, 136.6, 137.0, 139.7, 148.0. ESI-MS: calcd. for C.sub.21H.sub.24O.sub.2S [M+H].sup.+ 341.1575, found 341.1565.

Formula VI(b)

Synthesis of 3,5,5-trimethyl-9-(tosylmethyl)-6,7-dihydro-5H-benzo[7]annulene

[0131] ##STR00011##

[0132] A mixture of 2,9,9-trimethyl-5-methylene-6,7,8,9-tetrahydro-5H-benzo[7]annulene (0.25 mmol, 1.0 equiv.), sodium 4-methylbenzenesulfinate (0.3 mmol, 1.2 equiv.), K.sub.2S.sub.2O.sub.8 (0.62 mmol, 2.5 equiv.), and I.sub.2 (0.3 mmol, 1.2 equiv.), in ACN:H.sub.2O (1:1) were placed in reaction tube (5 mL) at room temperature for 12 h. After completion of reaction, a saturated solution of sodium thiosulfate was added to the reaction mixture and extracted with ethyl acetate (10×3 times). The combined organic layer was washed with water and dried over Na.sub.2SO.sub.4 and vacuum evaporated. The viscous liquid obtained was purified by column chromatography on silica gel (mesh 60-120) using the mixture of hexane: EtOAc (80:20) to obtained the desired product as semisolid (64 mg, 72%).

[0133] 1H NMR (CDCl.sub.3, 600 MHz) δ 1.35 (s, 6H), 1.97-2.00 (t, J=7.2 Hz, 2H), 2.11-2.14 (m, 2H), 2.35 (s, 3H), 2.45 (s, 3H), 4.21 (s, 2H), 6.24-6.27 (t, J=6.5 Hz, 1H), 6.96-6.97 (d, J=7.3 Hz, 1H), 7.21-7.24 (t, J=8.5 Hz, 2H), 7.31-7.32 (d, J=8.1 Hz, 2H), 7.78-7.79 (d, J=8.2 Hz, 2H); 13C NMR (CDCl.sub.3, 150 MHz) δ 21.49, 21.65, 27.12, 30.92, 38.09, 46.69, 63.88, 126.46, 126.62, 128.38, 128.43, 128.60, 129.71, 134.63, 136.57, 136.79, 136.85, 144.50, 148.08. ESI-MS: calcd. For C.sub.22H.sub.26O.sub.2S [M+H].sup.+ 355.1732, found 355.1720.

Formula VI (c)

Synthesis of 3,5,5-Trimethyl-9-((methylsulfonyl)methyl)-6,7-dihydro-5H-benzo[7]annulene

[0134] ##STR00012##

[0135] A mixture of 2,9,9-trimethyl-5-methylene-6,7,8,9-tetrahydro-5H-benzo[7]annulene (0.25 mmol, 1.0 equiv.), sodium methanesulfinate (0.3 mmol, 1.2 equiv.), K.sub.2S.sub.2O.sub.8 (0.62 mmol, 2.5 equiv.), and 12 (0.3 mmol, 1.2 equiv.), in ACN:H.sub.2O (1:1) were placed in reaction tube (5 mL) at room temperature for 12 h. After completion of reaction, a saturated solution of sodium thiosulfate was added to the reaction mixture and extracted with ethyl acetate (10×3 times). The combined organic layer was washed with water and dried over Na.sub.2SO.sub.4 and vacuum evaporated. The viscous liquid obtained was purified by column chromatography on silica gel (mesh 60-120) using the mixture of hexane: EtOAc (60:40) to obtained the desired product as semisolid (42 mg, 60%).

[0136] .sup.1H NMR (CDCl.sub.3, 600 MHz) δ 1.40 (s, 6H), 2.00-2.02 (t, J=7.0 Hz, 2H), 2.20-2.23 (m, 2H), 2.37 (s, 3H), 2.71 (s, 3H), 4.19 (s, 2H), 6.40-6.42 (t, J=6.2 Hz, 1H), 7.09 (d, J=7.8 Hz, 1H), 7.27 (s, 1H), 7.32 (d, J=7.8 Hz, 1H); .sup.13C NMR (CDCl.sub.3, 150 MHz) δ 21.39, 27.18, 30.58, 38.09, 41.10, 45.98, 62.80, 126.74, 127.04, 127.96, 128.12, 133.68, 137.12, 137.40, 148.54. ESI-MS: calcd. for C.sub.16H.sub.22O.sub.2S [M+H].sup.+ 279.1419, found 279.1410.

Example 6

General Procedure for the Synthesis of Formula V

[0137] ##STR00013##

Synthesis of 3,5,5,10-tetramethyl-6,7-dihydro-5H-benzo[3,4]cyclohepta[1,2-b]quinolin-12-ol

[0138] A mixture of 3,5,5,10-tetramethyl-5,6,7,9,10,11-hexahydro-12H-benzo[3,4]cyclohepta[1,2-b]quinolin-12-one (1.0 equiv.), oxidant (0.3 mmol, 2 equiv.) in solvent (1:1) were placed in reaction tube (5 mL) for 12 h. After cooling the reaction mixture to ambient temperature, water was added and extracted with ethyl acetate (10×3 times). The combined organic layer was washed with water and dried over Na.sub.2SO.sub.4 and vacuum evaporated. The viscous liquid obtained which was purified by column chromatography on silica gel (mesh 60-120) using the mixture of hexane: EtOAc (70:30) to obtained the desired product.

Experimental Procedures

[0139] I. Determination of affinity and kinetic constants for PI3K (120y) [0140] II. Determination of affinity of PI3K (120y) inhibitors (KD/IC50) through inhibition in solution assay of kinase PI3K (120y) by various compounds of general formula I-VI [0141] III. Determination of affinity and kinetic constants for MAPKAPK2 [0142] IV. Determination of affinity of MK2 inhibitors (IC50) through inhibition in solution assay of kinase MK2 by various compounds of general formula I-VI

Determination of Affinity and Kinetic Constants for PI3K (120y)

[0143] Immobilization buffer scouting was performed for PI3K (120y) to find a suitable buffer for immobilization. It was found that 10 mM sodium acetate pH 5.5 buffer is best suitable for the immobilization. Immobilization of the PI3K (120y) kinase was performed at 25° C. temperature using concentration of Kinase 5 μg/ml with a flow rate of 10 μl/min and 1000 sec of contact time. Successful immobilization of 9191.2 RU of kinase PI3K (120y) over flow cell 4 of sensor surface CM5 by amine coupling was achieved.

[0144] After immobilization, kinetics screening for binding of compounds with PI3K (120y) was performed with the protocol recommended as per Biacore Assay Handbook (protocol: kinetic screening using a single concentration of compounds, concentration used: 50 μMin 1×PBS with 5% DMSO, running buffer: 1×PBS with 5% DMSO, temperature: 25° C., buffer blanks: 1×PBS with 5% DMSO wizard used: Kinetics/Affinity). Compound K were prepared in 50 μM concentration in 1× PBS with 5% DMSO and placed in sample compartment at 25° C. These were passed over the sensor through flow cell 1 (reference-blank) and 2 (active-immobilized with kinase) in flow rate of 30 μl/min at 25° C. Binding responses were seen in real time for Fc 2 (active), Fc1 (reference) and Fc 2-1 (reference subtracted). Solvent correction was done for DMSO (5% in PBS) Data was analyzed using Biacore T200 evaluation software v 3.1 and fitting was done using 1:1 binding model. Also, detailed kinetics characterization was performed for compounds of general formula I.

[0145] Compounds of general formula II showed promising binding and kinetics profiles as shown in FIG. 1, FIG. 2, and Table 1 FIG. 1 depicts the on-rate and off-rate of binding of evaluated compounds of general formula II to PI3K (120y). Plotting association rate constant ka against dissociation rate constant kd (here on logarithmic scales) creates a plot where the affinity is represented by diagonal lines. Compounds on the same diagonal have the same affinity but differ in kinetics. FIG. 2 is the Graphical representation of the on-rate and off-rate of binding of evaluated compounds of general formula II to PI3K (120y).

[0146] Table 1 represents the numerical values of binding (on-rate, off-rate and affinity of binding) of evaluated compounds of general formula I to PI3K (120y). KD represents the equilibrium constant of binding in Molar value.

TABLE-US-00001 TABLE 1 Sample Ka (1/Ms) Kd (1/s) KD (M) Sample 5 9.75E+03 2.38E−03 2.44E−07 Sample 7 1.70E+04 2.46E−01 1.45E−05 Sample 8 9.88E+03 2.41E−01 2.44E−05 Sample 9 1.08E+04 2.31E−01 2.14E−05 Sample 11 1.44E+04 1.51E−01 1.05E−05 Sample 12 4.46E+04 9.48E−03 2.13E−07 Sample 16 3.16E+04 5.54E−02 1.75E−06

[0147] Detailed kinetics characterization of selected compounds (Compounds having general formula II that is pyrolone fused benzocycloheptene) for binding to PI3K (120y) were performed using the protocol recommended as per Biacore Assay Handbook (protocol: kinetic analysis using single cycle kinetics method, concentrations used: 10, 5, 2.5, 1.25, 0.625 μM in 1×PBS with 5% DMSO, running buffer: 1×PBS with 5% DMSO, temperature: 25° C. Buffer blanks: 1×PBS with 5% DMSO, wizard used: Kinetics/Affinity). Compound (Compounds having general formula II that is pyrolone fused benzocycloheptene) were prepared in 10 μM concentration in 1× PBS with 5% DMSO and serial diluted to make 5, 2.5, 1.25 and 0.625 μM concentration. They were placed in sample compartment at 25° C. These were passed over the sensor through flow cell 1 (reference-blank) and 2 (active-immobilized with kinase) in flow rate of 30 μl/min at 25° C. using single cycle kinetics injection method. Binding responses were seen in real time for Fc2 (active), Fc1 (reference) and Fc 2-1 (reference subtracted). Solvent correction was done for DMSO (5% in PBS). Data was analyzed using Biacore T200 evaluation software v 3.1. Fitting of data is done using 1:1 binding model and kinetics constants as well as equilibrium kinetics constant of dissociation (KD) was also determined.

[0148] Compounds of general formula II showed effective inhibitory potential against PI3K (120y) with IC50 ranging in between 2-2.5 μM as provided in FIG. 3, and Table 2. FIG. 3 represents the dose dependent inhibition of the PI3K (120y) by compounds of general formula II (5, 7, 8, 9, 12 and 16). Table 2: Table represents the extrapolated IC50 values of the compounds of general formula I against the PI3K(120y) from the graphs.

TABLE-US-00002 TABLE 2 S. No. Ligand (Small Molecules) IC50 1 Ligand 5  2057 nM 2 Ligand 7  1958 nM 3 Ligand 8  2173 nM 4 Ligand 9  1999 nM 6 Ligand 12 2342 nM 7 Ligand 16 2093 nM

Example 7

Determination of Affinity and Kinetic Constants for Mk2

[0149] Immobilization buffer scouting was performed for MK2 to find suitable buffer for immobilization and it was found that 10 mM Sodium acetate pH 5.5 buffer is best suitable for the immobilization. Immobilization of the MK2 kinase was performed at 25° C. temperature using concentration of Kinase 10 μg/ml with a flow rate of 10 μl/min and 1000 sec of contact time. Successful immobilization of 11119.6 RU of kinase MK2 over flow cell 4 of sensor surface CM5 by amine coupling was achieved.

[0150] After immobilization, kinetics screening for binding of compounds with MK2 was performed with the protocol recommended as per Biacore Assay Handbook (protocol: kinetic screening using a single concentration of compounds, concentration used: 50 μM in 1×PBS with 5% DMSO, running buffer: 1×PBS with 5% DMSO, temperature: 25° C., buffer blanks: 1×PBS with 5% DMSO wizard used: Kinetics/Affinity). Compound (Compounds having general formula II that is pyrolone fused benzocycloheptene) were prepared in 50 μM concentration in 1× PBS with 5% DMSO and placed in a sample compartment at 25° C. These were passed over the sensor through flow cell 3 (reference-blank) and 4 (active-immobilized with kinase) in flow rate of 30 μl/min at 25° C. Binding responses were seen in real time for Fc 4 (active), Fc3 (reference) and Fc 4-3 (reference subtracted). Solvent correction was done for DMSO (5% in PBS). Data was analyzed using Biacore T200 evaluation software v 3.1 and fitting was done using 1:1 binding model.

[0151] Compounds of general formula II showed promising binding and kinetics profiles. Out of them, on the basis of their binding and kinetic profiles, 9 most promising lead compounds of general formula II along with one marketed MK2-inhibitor compound (PF 3644022) were selected for further in-solution inhibition assay. The results are provided in FIG. 4, FIG. 5, and Table 3. FIG. 4 depicts the on-rate and off-rate of binding of evaluated compounds of general formula II to MK2. Plotting association rate constant ka against dissociation rate constant kd (here on logarithmic scales) creates a plot where the affinity is represented by diagonal lines. Compounds on the same diagonal have the same affinity but differ in kinetics. FIG. 5 is the graphical representation of the on-rate and off-rate of binding of evaluated compounds of general formula II to MK2. Table 3 represents the numerical values of binding (on-rate, off-rate and affinity of binding) of evaluated compounds of general formula II to MK2. KD represents the equilibrium constant of binding in Molar value.

TABLE-US-00003 TABLE 3 Sample Ka (1/Ms) Kd (1/s) KD (M) Sample 1 2.11E+03 1.28E−01 6.08E−05 Sample 2 3.75E+03 9.30E−02 2.48E−05 Sample 3 2.55E+03 3.63E−02 1.42E−05 Sample 4 4.12E+03 6.61E−02 1.61E−05 Sample 5 1.11E+00 2.82E−03 2.53E−03 Sample 6 1.77E+01 5.87E−02 3.32E−03 Sample 7 3.46E+03 1.36E−01 3.94E−05 Sample 8 3.42E+03 1.24E−01 3.63E−05 Sample 9 3.57E+03 6.73E−02 1.89E−05 Sample 11 2.41E+03 1.54E−01 6.40E−05 Sample 12 2.65E+03 5.39E−02 2.04E−05 Sample 13 3.32E+03 7.88E−02 2.37E−05 Sample 14 3.26E+03 8.61E−02 2.64E−05 Sample 15 1.94E+03 1.09E−01 5.64E−05 Sample 16 1.70E+03 1.67E−01 9.81E−05

[0152] Detailed kinetics characterization of selected compounds binding to MK2 were performed using the protocol recommended as per Biacore Assay Handbook (protocol: kinetic analysis using single cycle kinetics method, concentrations used: 30, 15, 7.5, 3.75, 1.87 μMin 1×PBS with 5% DMSO, running buffer: 1×PBS with 5% DMSO, temperature: 25° C., buffer blanks: 1×PBS with 5% DMSO, wizard used: Kinetics/Affinity). Compounds were prepared in 30 μM concentration in 1× PBS with 5% DMSO and serial diluted to make 15, 7.5, 3.75 and 1.87 μM concentration. They were placed in a sample compartment at 25° C. These were passed over the sensor through flow cell 3 (reference-blank) and 4 (active-immobilized with kinase) in flow rate of 30 μl/min at 25° C. using single cycle kinetics injection method. Binding responses were observed in real time for Fc 4 (active), Fc3 (reference) and Fc 4-3 (reference subtracted). Solvent correction was done for DMSO (5% in PBS). Data was analyzed using Biacore T200 evaluation software v 3.1 and fitting of data was done using 1:1 binding model and kinetics constants as well as equilibrium kinetics constant of dissociation (KD) was also determined.

[0153] Compounds of general formula II showed effective inhibitory potential against MK2 with IC50 between ranging in between 526-709 nM. The values are in well competence with IC50 value of marketed MK2-inhibitor (585 nM) as provided in FIG. 6, and Table 4.

[0154] FIG. 6: Graphs represent the dose dependent inhibition of the MK2 by compounds of general formula II (1, 3, 6, 8, 12, 13, 14, 15 and 17 along with marketed MK2-inhibitor PF 3644022. Table 4 represents the extrapolated IC50 values of the compounds of general formula II against MK2 from the graphs.

TABLE-US-00004 TABLE 4 S. No. Ligand (Small Molecules) IC50 1 Ligand 1  650 nM 2 Ligand 3  526 nM 3 Ligand 6  550 nM 4 Ligand 8  525 nM 5 Ligand 12 709 nM 6 Ligand 13 578 nM 7 Ligand 14 545 nM 8 Ligand 15 531 nM 9 Ligand 17 596 nM 10 Marketed MK2 Inhibitor (PF 3644022) 585 nM

Advantages of the Invention

[0155] The main scaffold of benzocycloheptene comes from natural precursor. Therefore, use of natural precursor reduces the number of steps for the synthesis of the compounds. [0156] The cost of obtaining plant derived precursor is very low, as the plant is abundant in nature, hence the overall process is economic. [0157] The process is also sustainable as 60-80% of the molecules come from natural precursor and only 40-20% molecule were used through synthetic process. [0158] The compounds of general formula I-VI are less toxic. [0159] The compound of general formula I-VI are applicable for treatment of PI3K and MK2 related disorder.