WO2023119334A1

WO2023119334A1 - Anti-cancer compound by combining ponatinib molecule with hdac inhibitor molecule using a variable length linker

Info

Publication number: WO2023119334A1
Application number: PCT/IN2022/051122
Authority: WO
Inventors: C Gopi MOHAN; Manzoor Koyakutty; Anu R MELGE
Original assignee: Amrita Vishwa Vidyapeetham
Priority date: 2021-12-25
Filing date: 2022-12-23
Publication date: 2023-06-29

Abstract

The present invention discloses novel dual-targeting designed molecules or compounds of Formula I and II. Formula I Formula II The molecules or compounds can be used for the treatment of Chronic Myeloid Leukemia (CML). The novel compounds design includes the combination of key pharmacophores that can target and inhibit both BCR-ABL and HDAC enzymes using a biocompatible linker. The inhibitions of multiple cells signaling mechanism lead towards apoptosis of CML cells and thereby preventing the activation of downstream cell survival pathways.

Description

ANTI-CANCER COMPOUND BY COMBINING PONATINIB MOLECULE WITH HDAC INHIBITOR MOLECULE USING A VARIABLE LENGTH LINKER

FIELD OF THE INVENTION

[0001] The present invention relates to the field of pharmaceutical sciences. The present invention relates to the designing of novel chemical compounds or molecules containing key pharmacophores having the multi-targeting potential. The compounds act by inhibiting the oncogenic pathways which are responsible for the pathogenesis of Chronic Myeloid Leukemia (CML).

BACKGROUND OF THE INVENTION

[0002] CML is a disease where there is an excessive proliferation of the mature granulocytes (neutrophils, eosinophils, and basophils) and its precursors. The main cause of CML is a reciprocal translocation of the BCR gene in chromosome 22 and abl gene in chromosome 9 leading to the formation of a smaller chromosome named Philadelphia containing the chimeric gene. This gene gives rise to an aberrant protein called the BCR- ABL protein having constitutive tyrosine kinase activity. In the last decade, one of the major breakthroughs in the area of cancer therapy was the invention of small molecule drugs that can specifically inhibit aberrant protein kinases through multiple mechanisms. These molecularly targeted tyrosine kinase inhibitors (TKIs) were greatly beneficial compared to the conventional DNA intercalating agents because of their high specificity to a particular kinase and thus remain non-toxic to healthy cells. Among many clinically approved TKIs, Imatinib targeting BCR-ABL fusion protein in CML, which is caused by Philadelphia chromosome (Ph) translocation t (9:22), was the most studied and clinically approved small molecule inhibitors. However, despite promising initial response, development of point mutations in the BCR-ABL kinase domain led to Imatinib resistance and relapse in CML. The most critical mutation was Thr315Ile (Threonine— dsoleucine at 315 position) a ‘gate-keeper’ mutation that offers the steric hindrance to drug binding at the BCR- ABL kinase. Drugs such as Dasatinib, Bosutinib and Nilotinib were found ineffective to the gatekeeper mutation, except ponatinib. Currently, if cancer patients develop resistance against these kinase inhibitors, there are no other treatment options available.

[0003] In addition to the BCR-ABL kinase, aberrant epigenetics was also found to play a significant role in the progression of CML. An HDAC inhibitor, Vorinostat, has shown remarkable anti-leukemic activity against CML in clinical trials. Vorinostat (SAHA; Zolinza™) is a strong inhibitor of HDAC with direct binding to the catalytic site, which catalyzes the deacetylation of a-acetyl lysine of NH2 terminal tail of histone cores. Vorinostat was the first FDA approved HDAC inhibitor for the treatment of patients with progressive, persistent, or recurrent cutaneous T cell lymphoma (CTCL). In addition, Vorinostat demonstrated remarkable in vitro activity and has shown improved survival and anti-tumor effects in rodent leukemia models. Vorinostat clinical trial study both as a single agent and in combination with other agents has emphasized its anti-leukemic effect with acceptable safety and tolerability profiles. Normal healthy cells where generally observed to be resistant to Vorinostat induced cell death. Several biological pathways are involved in cancer development and progression. However, exact cause of cancer is still unknown. Hence, targeting several aberrant proteins simultaneously can improve the inhibition of tumor progression as compared to single targeted drugs.

[0004] Discovery of hybrid compounds towards inhibiting multiple targets for the treatment of different diseases is patented earlier. The WIPO (PCT) publication no. W02008033749A3 describes the multi-targeting potential of hybrid compounds by conjugating the quinazoline moiety of Gefitinib or Erlotinib drugs (EGFR inhibitor) with hydroxamate functional group of Vorinostat or Belinostat drugs (HDAC inhibitor). The inventors claim that the conjugation of EGFR and HDAC inhibitor moieties showed greater potency and a better scope for the anticancer treatment. Similarly, several other dual binding inhibitors with HDAC targeting potential which include, dual phosphoinositide 3-kinase (PI3K) and HDAC inhibitors (US20200215039A1), where in the compounds were designed by combining the morpholinopyrimidine pharmacophore, a core scaffold present in the PI3K inhibitors (PI- 103, GDC-0941 and BKM-120) with the hydroxamate group in HDAC inhibitors (Vorinostat and Panobinostat). These compounds showed inhibition towards over expression of dual anti-cancer target PI3K and HDAC by inducing cell cycle arrest and apoptosis. The US patent application no. US20080234332A1 describes the invention of modified Raf inhibitors (Sorafenib and BAY 43- 9006) with zinc binding moiety targeting HDAC (Vorinostat) that are active against different types of cancers. The PCT publication no. W02009036020A1 refers to Mek inhibitors (Selumetinib, Cobimetinib and Selumetinib) containing HDAC targeting zinc binding pharmacophore (hydroxamate group of Vorinostat). These compounds have greater inhibitory potential than the individual drugs against cell proliferative diseases like cancer. The US patent application no. US20090076044A1 describes the development of compounds with VEGF inhibitor (Cediranib) pharmacophore containing zinc binding moiety targeting HDAC and thereby exhibiting dual inhibitory potential against cancers. A US patent no. US8563741B2 discloses dual targeting compounds that are derivatives of CDK inhibitor (SNS-032) with zinc binding moiety targeting HDAC. These compounds contain aliphatic linker combining the CDK inhibitor pharmacophore with hydroxamate group of vorinostat. The US patent no. US10464925B2 relates to the bifunctional compounds consisting of thalidomide drug targeting cereblon which in turn recruits the proteosomal complex covalently linked together to the target protein (HDAC, Hsp90 and MDM2) inhibitor. These compounds have dual targeting potential of inhibiting overexpressed proteins leading to proteosomal degradation and thereby inhibiting the different hematological malignancies and solid carcinomas. The Canadian patent no. CA2662580C describes the invention of TKIs targeting both BCR-ABL kinase and HDAC enzymes. These compounds are Dasatinib (BCR-ABL inhibitor) derivatives conjugated to hydroxamate group of Vorinostat (HDAC inhibitor) using aliphatic chain as linker. They show multi-kinase (BCR-ABL and Src) inhibitory activity along with HDAC inhibition which helps in the depletion of BCR-ABL, apoptosis induction and sensitization to TKI induced apoptosis. A Chinese patent no. CN105837596A discloses a hybrid compound with dual HDAC and BRD4 inhibitory potential. This compound was designed by combining an important pharmacophore of Entinostat (HDAC inhibitor) and JQ1 (BRD4 inhibitor) using a linker. Due to its dual inhibition this compound is active against several cancers and infectious diseases.

[0005] The present invention adopts the dual-targeting strategy by designing novel chemical compounds or molecules against BCR-ABL and HDAC enzymes for the treatment of CML. Ponatinib being a more potent drug than other BCR-ABL TKIs and due to its sensitivity to several single point and few double point mutations as opposed to other TKIs, its key pharmacophore (imidazopyridazine group) was considered in designing the new chemical compounds or molecules which are conjugated to the hydroxamate pharmacophore using methylene linkers.

SUMMARY OF THE INVENTION

[0006] The present invention relates to dual-targeting chemical compounds or molecules designed by using biocompatible linker to target both the BCR-ABL and HDAC enzymes. These molecules contain an imidazopyridazine group with triple bond and other attached groups that can target BCR-ABL and hydroxamate moiety (zinc binding groups) that target HDAC enzyme and have the potential scope in the treatment of CML.

[0007] The present invention also provides a method for designing novel chemical compounds or molecules with dual-targeting potential for inhibiting BCR-ABL and HDAC. Specifically, the present invention provides the novel compounds having dual-targeting potential for inhibiting BCR-ABL and HDAC. The compounds are represented by the Formula I (also referred as

N-(4-(8-(hydroxyamino)-8-oxooctyl)-3-(trifluoromethyl)phenyl)-3-(imidazo[l,2-b]pyridazin-3- ylethynyl)-4-methylbenzamide [Formula I]

N-(4-( 11 -(hydroxy amino)- 11 -oxoundecyl)-3-(trifluoromethyl)phenyl)-3-(imidazo[ 1 ,2- b]pyridazin-3 -ylethynyl)-4-methylbenzamide [Formula II] or their pharmaceutically acceptable salt thereof.

[0008] The present invention claims a novel compound FP7, which is prepared by fusing the key pharmacophores of ponatinib and vorinostat drugs using a biocompatible linker for the treatment of CML disease. This inventive compound is equally potent like a ponatinib drug with better pharmacokinetic properties and less adverse effects and drug resistance. Also, ponatinib can inhibit BCR-ABL target only in comparison to the claimed inventive compound FP7 which has dual inhibition towards both BCR-ABL and HDAC targets. The computational process developed using BCR-ABL and HDAC inhibitors by supervised machine learning models to predict the inhibitory activity of the hybrid molecule towards both these targets. This is further validated using different CML cell lines in order to arrive at the claimed compounds.

[0009] In some embodiments, the compound of Formula (I) binds to BCR-ABL (wild type and Thr315Ile mutant) and HDAC with a binding affinity in the range of -30 to -40 kcal/mol. In some embodiments, the molecules interact with Glu286, Met318, Ile360, Arg362 and Asp381 in the wild type BCR-ABL, with Glu286, Met318 and Asp381 in the Thr315Ile mutant BCR-ABL, with Glyl54 and Zn²⁺ in HDAC enzyme.

[0010] In some embodiments, the compound of Formula (II) binds to BCR-ABL (wild type and Thr315Ile mutant) and HDAC with a binding affinity in the range of -30 to -43 kcal/mol. In some embodiments, the molecules interact with Glu286, Met318, Asp363, Asp381 and Ser385 in the wild type BCR-ABL, with Glu286, Met318 and Asp381 in the Thr315Ile mutant BCR-ABL, with Glyl54 and Zn²⁺ in HDAC enzyme.

[0011] This and other aspects are disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The features of this invention will be more readily evident from the following detailed description and the appended claims, together with the accompanying drawings.

[0013] FIG. 1A illustrates the 7 featured 3D-Pharmacophore model developed using BCR- ABL inhibitors (ADHHPRR.1980) along with the distances (A) between each feature.

[0014] FIG. IB illustrates the 6 featured 3D-Pharmacophore model developed using HDAC inhibitors (ADHHPRR.1952) along with the distances (A) between each feature.

[0015] FIG. 2A illustrates the analogous binding of FP7 (red), FP10 (green) to Ponatinib (pink) in the active site of wild type BCR-ABL. [0016] FIG. 2B illustrates the analogous binding of FP7 (red), FP10 (green) to Ponatinib (pink) in the active site of Thr315Ile mutant BCR-ABL.

[0017] FIG. 3A illustrates the interactions of FP7 to the active site residues of wild type BCR- ABL.

[0018] FIG. 3B illustrates the interactions of FP10 molecule to the active site residues of wild type BCR-ABL.

[0019] FIG. 3C illustrates the interactions of Ponatinib to the active site residues of wild type BCR-ABL.

[0020] FIG. 3D illustrates the interactions of FP7 to the active site residues of Thr315Ile mutant BCR-ABL.

[0021] FIG. 3E illustrates the interactions of FP10 to the active site residues of Thr315Ile mutant BCR-ABL.

[0022] FIG. 3F illustrates the interactions of Ponatinib to the active site residues of Thr315Ile mutant BCR-ABL.

[0023] FIG. 4 illustrates the analogous binding of FP7 (red) and FP10 (green) to Vorinostat (cyan) in the active site of HD AC.

[0024] FIG. 5 A illustrates the interactions of FP7 to the active site residues of HD AC.

[0025] FIG. 5B illustrates the interactions of FP10 to the active site residues of HD AC.

[0026] FIG. 5C illustrates the interactions of Vorinostat to the active site residues of HD AC.

[0027] FIG. 6A illustrates hemolytic activity of FP7 in whole blood samples.

[0028] FIG. 6B illustrates hemolytic activity of FP10 in whole blood samples.

[0029] FIG. 6C illustrates hemolytic activity of Ponatinib in whole blood samples.

[0030] FIG. 6D illustrates hemolytic activity of Vorinostat in whole blood samples.

[0031] FIG. 7 illustrates percentage cell viability of PBMCs following treatment with different concentrations of FP7, FP10, as well as Ponatinib and Vorinostat for 72 hours. (Cell viability percentage was determined in three different experiments for each of the above graphs) (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). [0032] FIG. 8 illustrates the fractional effect-CI plots for different constant concentrations ratios of Ponatinib and Vorinostat combination when treated for BCR-FP10 wild type K562 cell line.

[0033] FIG. 9 illustrates the growth inhibitory effect (dose-response curves) of Ponatinib and Vorinostat combinations in a 1: 1 concentration ratio in comparison to the effect of hybrid molecules FP7 and FP10 in the K562 CML cell line with wild type BCR-ABL for 72 hours.

[0034] FIG. 10A illustrates the growth inhibitory effect (dose-response curves) of hybrid molecules (FP7 and FP10) in comparison to Ponatinib, Imatinib and Vorinostat in the K562 CML cell line with wild type BCR-ABL for 72 hours. Results are derived from three separate experiments, and error bars represent standard errors.

[0035] FIG. 10B illustrates the growth inhibitory effect (dose-response curves) of hybrid molecules (FP7 and FP10) in comparison to Ponatinib, Imatinib and Vorinostat in the Ba/F3 cell line with Thr315Ile mutant BCR-ABL for 72 hours. Results are derived from three separate experiments, and error bars represent standard errors.

[0036] FIG. 10C illustrates the growth inhibitory effect (dose-response curves) of hybrid molecules (FP7 and FP10) in comparison to Ponatinib, Imatinib and Vorinostat in the Ba/F3 cell line with Tyr253His mutant BCR-ABL for 72 hours. Results are derived from three separate experiments, and error bars represent standard errors.

[0037] FIG. 11A illustrates the inhibitory activity with different concentrations of Ponatinib and Vorinostat individually and in combination on BCR-ABL wild type (K562) cell line. Each cell viability percentage bar in the graph is the averaged value of three separate experiments.

[0038] FIG. 11B illustrates the inhibitory activity with different concentrations of Ponatinib and Vorinostat individually and in combination on Imatinib resistant BCR-ABL (Ba/F3 Thr315Ile) cell line. Each cell viability percentage bar in the graph is the averaged value of three separate experiments.

[0039] FIG. 12A illustrates the fractional effect-CI plots for different non-constant concentrations ratios of Ponatinib and Vorinostat combination when treated to wild type BCR- ABL cell line (K562).

[0040] FIG. 12B illustrates the fractional effect-CI plots for different non-constant concentrations ratios of Ponatinib and Vorinostat combination when treated to Imatinib resistant BCR-ABL cell line (Ba/F3 Thr315Ile).

DETAILED DESCRIPTION OF THE INVENTION [0041] While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope.

[0042] In an embodiment of the invention, the present invention relates to dual-targeting chemical compounds or molecules designed by using biocompatible linker to target both the BCR-ABL and HDAC enzymes. These molecules contain an imidazopyridazine group with triple bond and other attached groups that can target BCR-ABL and hydroxamate moiety (zinc binding groups) that target HDAC enzyme and have the potential scope in the treatment of CML.

[0043] In another embodiment of the invention, the present invention also provides a method for designing novel chemical compounds or molecules with dual-targeting potential for inhibiting BCR-ABL and HDAC. Specifically, the present invention provides the novel compounds having dual-targeting potential for inhibiting BCR-ABL and HDAC. The compounds are represented by the Formula I (also referred as “FP7”) and Formula II (also

[0044] In yet another embodiment of the invention, the present invention discloses a novel compound FP7, which is prepared by fusing the key pharmacophores of ponatinib and vorinostat drugs using a biocompatible linker for the treatment of CML disease. This inventive compound is equally potent like a ponatinib drug with better pharmacokinetic properties and less adverse effects and drug resistance. Also, ponatinib can inhibit BCR-ABL target only in comparison to the claimed inventive compound FP7 which has dual inhibition towards both BCR-ABL and HDAC targets. The computational process developed using BCR-ABL and HDAC inhibitors by supervised machine learning models to predict the inhibitory activity of the hybrid molecule towards both these targets. This is further validated using different CML cell lines in order to arrive at the claimed compounds.

[0045] In still another embodiment of the invention, the compound of Formula (I) binds to BCR-ABL (wild type and Thr315Ile mutant) and HDAC with a binding affinity in the range of - 30 to -40 kcal/mol. In some embodiments, the molecules interact with Glu286, Met318, Ile360, Arg362 and Asp381 in the wild type BCR-ABL, with Glu286, Met318 and Asp381 in the Thr315Ile mutant BCR-ABL, with Glyl54 and Zn²⁺ in HDAC enzyme.

[0046] In yet another embodiment of the invention, the compound of Formula (II) binds to BCR-ABL (wild type and Thr315Ile mutant) and HDAC with a binding affinity in the range of - 30 to -43 kcal/mol. In some embodiments, the molecules interact with Glu286, Met318, Asp363, Asp381 and Ser385 in the wild type BCR-ABL, with Glu286, Met318 and Asp381 in the Thr315Ile mutant BCR-ABL, with Glyl54 and Zn²⁺ in HDAC enzyme.

[0047] In still another embodiment of the invention, the cells are granulocytes and their precursors including common myeloid progenator cells, myeloblast, monoblast, monocyte, eosinophil promyelocyte, neutrophil promyelocyte, basophil promyelocyte, proerythroblast, megakaryoblast, myeloid dendritic cell, macrophage, eosinophil, neutrophil, basophil, mast cell, erythrocytes, and thrombocytes.

[0048] In yet another embodiment of the invention, the oral absorption of N-(4-(8- (hydroxyamino)-8-oxooctyl)-3-(trifluoromethyl)phenyl)-3-(imidazo[l,2-b]pyridazin-3-ylethynyl )-4-methylbenz- amide denoted as FP7 and represented by Formula (I) (C31H30F3N5O3) is approximately 70 %, Log P is 7.05, non-mutagenic with optimum plasma protein binding potential, blood brain barrier penetration, gastrointestinal penetration, non-hepatotoxic and lower cardiotoxicity than Ponatinib and Imatinib FDA approved drugs.

[0049] In still another embodiment of the invention, the oral absorption of N-(4-(l l- (hy droxy amino)- 1 l-oxoundecyl)-3-(trifluoromethyl)phenyl)-3-(imidazo[l,2-b]pyridazin-3-yleth- ynyl)-4-methylbenzamidedenoted as FP10 and represented by Formula (II) (C34H36F3N5O3)is approximately 65 %, Log P is 8, non-mutagenic with optimum plasma protein binding potential, blood brain barrier penetration, gastrointestinal penetration, non-hepatotoxic and lower cardiotoxicity than Ponatinib and Imatinib drugs. [0050] In yet another embodiment of the invention, the molecule with Formula (I) also denoted as FP7 binds to wild type BCR-ABL with a binding affinity of -33.18 kcal/mol, Thr315Ile mutant BCR-ABL with -33.30 kcal/mol and HDAC enzyme with -38.45 kcal/mol.

[0051] In still another embodiment of the invention, the molecule with Formula (II) also denoted as FP10 binds to wild type BCR-ABL with a binding affinity of -40.29 kcal/mol, Thr315Ile mutant BCR-ABL with -42.21 kcal/mol and HDAC enzyme with -30.91 kcal/mol.

[0052] In another embodiment of the invention, a compound of general Formula A is provided:

Formula A or their pharmaceutically acceptable salt thereof;

Characterized in that R is selected from the following:

[0053] In still another embodiment of the invention, the compound is (N-(4-(8-(hydroxyamino)- 8-oxooctyl)-3-(trifluoromethyl)phenyl)-3-(imidazo[l,2-b]pyridazin-3-ylethynyl)-4- methylbenzamide) (Formula I):

[Formula I] [Compound FP7] or their pharmaceutically acceptable salt thereof.

[0054] In yet another embodiment of the invention, compound is (N-(4-(l l-(hydroxyamino)-l l- oxoundecyl)-3-(trifluoromethyl)phenyl)-3-(imidazo[l,2-b]pyridazin-3-ylethynyl)-4-methylbenz- amide) (Formula II):

[Formula II] [Compound FP10] or their pharmaceutically acceptable salt thereof.

[0055] In still another embodiment of the invention, the oral absorption of compounds is from 55 to 80 %, preferably 65 to 75 %.

[0056] In yet another embodiment of the invention, Log P of compounds is from 7 to 8.

[0057] In still another embodiment of the invention, a method of preparing compound of Formula I is provided, wherein the method comprising the following steps: a) Degassing and purging of a mixture of compound 1, compound 1A, DIEA, Pd(dba)2, PPhs and Cui in 1,4-dioxane with N2, followed by stirring the mixture to obtain a residue of compound 2.

b) Adding [acetoxy(phenyl)-iodanyl] acetate and TEMPO to a solution of compound 2 obtained from step a) in MeCN and H2O and stirring to obtain compound 3.

d) Adding Pd/C to a solution of compound 4 in MeOH and stirring under H2 to obtain a compound 5

e) Adding the compound 5A, HATU, DIPEA to a solution of compound 5 in DMF and stirring to obtain compound 6

f) Adding the NaOH to a solution of compound 6 in MeOH and H2O to a solution of compound 6 in MeOH and H2O and stirring to obtain the compound 7;

g) Adding HATU and TEA to a solution of compound 7 and O-(tetrahydro-2H-pyran-2- yl)hydroxylamine (25.0 mg, 213 pmol) in DMF and stirring to obtain compound 8;

h) Adding TsOH.PEO to a solution of compound 8 in MeOH and stirring to obtain compound FP7

[0058] In still another embodiment of the invention, the stirring in step a) is conducted at 20 °C to 35 °C, preferably at 28 °C, stirring in step b), step e), step f), step g) and step h) is conducted at 15 °C to 35 °C, preferably at 25 °C, stirring in step c) is conducted at 70°C to 90°C, preferably at 80 °C and stirring in step d) is conducted at 20 °C to 40 °C, preferably at 30 °C..

[0059] In yet another embodiment of the invention, the stirring in step a) is conducted for 1 to 2 hours, preferably for 1.5 hours, stirring in step b) and step d) is conducted for 10 to 22 hours, preferably for 16 hours, stirring in step c), step f) and step h) is conducted for 20 to 40 minutes, preferably for 30 minutes, stirring in step e) is conducted for 2 to 6 hours, preferably for 4 hours and stirring in step g) is conducted for 8 to 16 hours, preferably for 12 hours.

[0060] In yet another embodiment of the invention, a method of preparing compound of Formula II is provided, wherein the method comprising the following steps: a) Degassing and purging a mixture of undec- 10-yn-l-ol, l-bromo-4-nitro-2- (trifluoromethyl)benzene, Cui, Pd(dba)2, PPh3 and DIEA in dioxane with N2 and stirring to obtain compound 2;

b) Adding iodobenzene diacetate to a solution of compound 2 and TEMPO in CH3CN and water and stirring to obtain compound 3;

c) Adding O-(tetrahydro-2H-pyran-2-yl)hydroxylamine to a solution of compound 3,

HATU and DIPEA in DMF to obtain compound 4;

d) Adding Pd/C to a solution of compound 4 in MeOH, followed by degassing and purging with H2 and stirring the mixture to obtain compound 5;

e) Stirring the solution of Compound 5, Compound 2A, HATU and DIPEA to obtain

Compound 6;

f) Stirring a solution of compound 6 and HCl/EtOAc in EtOAc to obtain FP10

[0061] In still another embodiment of the invention, the stirring in step a), step b), step c) and step d) is conducted at 15 °C to 25 °C, preferably at 20 °C and stirring in step e) and step f) is conducted at 20 °C to 30 °C, preferably at 25 °C.

[0062] In yet another embodiment of the invention, the stirring in step a), step e) and step f) is conducted for 12 to 20 hours, preferably for 16 hours, stirring in step b) is conducted for 4 to 8 hours, preferably for 6 hours, stirring in step c) is conducted for 8 to 16 hours, preferably for 12 hours and stirring in step d) is conducted for 2 to 6 hours, preferably for 4 hours.

[0063] In still another embodiment of the invention, imidazopyridazine group with triple bond and other attached groups target BCR-ABL and hydroxamate moiety (zinc binding groups).

[0064] In yet another embodiment of the invention, the compound binds to BCR-ABL (wild type and Thr315Ile mutant) and HD AC with a binding affinity in the range selected from -30 to -40 kcal/mol.

[0065] In still another embodiment of the invention, the compound interacts with Glu286, Met318, Ile360, Arg362 and Asp381 in the wild type BCR-ABL, with Glu286, Met318 and Asp381 in the Thr315Ile mutant BCR-ABL, with Glyl54 and Zn²⁺ in HD AC enzyme.

[0066] In yet another embodiment of the invention, the molecule of compounds binds to wild type BCR ABL with a binding affinity is from -33.18 kcal/mol to -40.29 kcal/mol.

[0067] In yet another embodiment of the invention, the molecule with compound binds to Thr315Ile mutant BCR-ABL with from -33.30 kcal/mol to -42.21 kcal/mol.

[0068] In still another embodiment of the invention, the molecule with compound binds to HD AC enzyme with from -38.45 kcal/mol to -30.91 kcal/mol.

[0069] While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material the teachings of the invention without departing from its scope.

EXAMPLES

[0070] The scope of the present invention is illustrated by the following examples as disclosed herein which are not meant to restrict the scope of the invention in any manner whatsoever.

[0071 ] Example 1: Designing of novel compounds or molecules based on knowledge-based strategy

[0072] Novel compounds or molecules were designed by combining the imidazopyridazine group with triple bond moiety: BCR-ABL and hydroxamate group: HDAC using different types and lengths of biocompatible and non-toxic linkers, which include methylene and triazole linked methylene. [0073] The designed molecules were virtually screened based on their inhibitory activities, good pharmacokinetic/pharmacodynamic properties and binding affinity towards both BCR-ABL and HD AC enzymes. Thus, for predicting the inhibitory activities of these molecules, 2D-QSAR and 3D-Pharmacophore models for BCR-ABL and HDAC inhibition was developed. These in silico models were built using a dataset of 44 BCR-ABL inhibitors and 62 HDAC inhibitors. The models were validated using test set and the statistical parameters associated with the training and test set of models explain the robustness of the model (Tables 1& 2).

[0074] The 2D-QSAR model for BCR-ABL (Eq. la) inhibition includes electronic, spatial, electro-topological, and physiochemical type of 2D and 3D descriptors. pICso (BCR-ABL) = 7.8029 + 0.019797 * Dipole_X + 0.38035 * ES_Count_aasN + 0.51523 *ES_Sum_aaaC - 0.39855 * LogD + 0.00036542 * PMI_Y [la]

[0075] The descriptors identified for successful BCR-ABL inhibition using 2D-QSAR model include Dipole_X, ES_Count_aasN, ES_Sum_aaaC, LogD and PMI_Y. Dipole_X(3D descriptor) describes the polarity of the molecule while ES_Count_aasN (2D descriptor) is the count of nitrogen atoms with two aromatic and a single bond. ES_Sum_aaaC(2D descriptor) explains the sum of electrotopological states of carbon with triple aromatic bond. LogD(2D descriptor) defines the hydrophobic nature of the molecule and PMI_Y (3D descriptor) is a spacial descriptor explaining the orientation and conformational rigidity along the Y axis. Among these descriptors Dipole_X, ES_Count_aasN, ES_Sum_aaaC and PMI_Y descriptors are positively correlated to BCR-ABL inhibitory activity while LogD descriptor is negatively correlated.

[0076] The 2D-QSAR model for HDAC (Eq. lb) inhibition includes electronic, spatial, electrotopological, and physiochemical type of 2D and 3D descriptors. pICso (HDAC) =10.197 - 0.40515 * ALogP + 0.35464 * ES_Count_sCH3 - 1.6633 * QED HBD - 11.117 * Molecular FractionalPolarSASA + 0.015986 * Jurs_DPSA_3 [lb]

[0077] The descriptors required for HDAC inhibition include AlogP, ES_Count_sCH3, QED_HBD, Molecular_FractionalPolarSASA and Jurs_DPSA_3. AlogP(2D descriptor) represents the lipophilic nature of the molecule. ES_Count_sCH3 (2D descriptor) is the count of the electrotopological state count for the singly bonded methyl group. QED_HBD (2D descriptor) describes the quantitative estimate of drug likeliness score for the contribution of hydrogen bond donor property. Molecular Fractional Polar SASA(2D descriptor) explains the fraction of the polar solvent accessible surface area in nature. Jurs_DPSA_3 (3D descriptor) is a surface atomic charge descriptor. ES_Count_sCH3 and Jurs_DPSA_3 descriptors are positively correlated to HDAC activity while QED_HBD, Molecular_Fractional Polar SASA and A log P descriptors are negatively correlated.

[0078] Table 1 : Statistical parameters of the training set and test set of the best 2D-QSAR model generated using BCR-ABL and HD AC inhibitors.

[0079] The 3D-Pharmacophore model for BCR-ABL inhibition identified 7 pharmacophoric features including an acceptor, a donor, 2 hydrophobic groups, a positive ion group and 2 rings arranged in an appropriate conformational in 3D space for proper target inhibition (FIG. 1A). Similarly, 3D-Pharmacophore model for HDAC inhibition was developed which contained 6 chemical features including an acceptor, a donor, a positive ion group and 3 ring features. The HDAC 3D-Pharmacophore model is shown in FIG. IB that illustrates the correct conformational orientation in 3D space for the inhibitor to inhibit the HDAC target.

[0080] Table 2: Training and test set statistical parameters of the developed 3D -Pharmacophore models.

[0081] Using these models, the activity of the designed novel compounds or molecules were predicted and compared with the activity of the existing drugs Ponatinib and Vorinostat. Activity of 0.5 pICso units less than the existing drugs were filtered, while the remaining molecules were subjected to ADMET based filtering (Table 3).

[0082] Table 3: BCR-ABL and HDAC inhibitory activity values of the generated designed molecules predicted using 2D-QSAR and 3D-Pharmacophore models.

[0083] The ADMET properties of the designed compounds FP7 and FP10 showed no hepatotoxicity, lower cardiotoxicity, good gastrointestinal absorption and blood brain barrier penetration, optimum plasma protein binding in comparison to Ponatinib and Imatinib drugs which was known to be hepatotoxic and more cardiotoxic (Table 4). The binding affinity studies of designed molecules FP7 and FP10 revealed an analogous binding conformation at the BCR- ABL binding sites with respect to that of the Ponatinib drug (FIG. 2 A, B). Different molecular interactions shown by Ponatinib BCR-ABL complex crystal structure was similar to that of our docking model of FP7 and FP10 molecules (FIG. 3A-F). In FP7 and FP10, the N1 of imidazo[l,2-b]pyridazin group forms hydrogen bonds with Met318 residue, 01 and N4 of amide group bond with Asp381 and Glu286 residues respectively similar to Ponatinib, while its N5 and 03 atoms of hydroxamate moiety in FP7 interacts with Ile360 and Arg362 residues, while the 02 and 03 atoms in FP10 interacts with Ser385 and Asp36 in the wild type BCR-ABL protein. Other vdW interactions between FP7 and FP10 designed molecules and residues include Val256, Ala269, Lys271, Val299, Met290, Asp381, Phe317, Phe382, Leu248 and Leu370 that stabilizes the molecule within the BCR-ABL active binding site (FIG. 3A, B). FP7 binding to mutant BCR-ABL (Thr315Ile) also has similar conformation and interactions as seen in the crystal structure of its Ponatinib complex where the phenyl ring accommodates itself in the presence of bulky Ile315 residue without any steric interactions. The Nl, 01 and N4 atoms of FP7 and FP10 designed molecules form hydrogen bonds with Met318, Asp381 and Glu286 residues respectively similar to Ponatinib drug and thus can hold the BCR-ABL kinase in DFG-out conformation even in the presence of its Thr315Ile mutation (FIG. 3C, D).

[0084] The binding conformation of the designed molecules FP7 and FP10 were slightly similar to Vorinostat (FIG. 4). In the HDAC binding, hydroxamate region of FP7and FP10 molecules, the 03 atom established only a single Zn²⁺ co-ordination bond. Moreover, the capping group interaction seen in Vorinostat where the N2 atom forms hydrogen bonding with Asp 104 was also absent (FIG. 5A-C). In the present docking model, a single hydrogen bonding interaction was observed between the N5 atom of the hydroxamate group in the novel molecules with Glyl54 in the catalytic site unlike Vorinostat where in 3 hydrogen bonding interactions were seen with Aspl04, Hisl45 and Tyr308 residues. This might be the reason for the lower binding affinity of FP7 and FP10 against HDAC in comparison to the Vorinostat drug (Table 5).

[0085] Table 5: Binding affinity of the best screened novel molecules and its fragments towards wild type BCR-ABL, Thr315Ile mutant BCR-ABL and HDAC proteins.

[0086] The key pharmacophore of Ponatinib fragment and Vorinostat fragment and its two different linker units (7 and 10) containing molecules binding affinity towards BCR-ABL and HDAC active sites were also studied using molecular docking method (Table 5). The binding conformation of these fragments and its linker molecules at the active sites of enzymes showed broadly similar mode of interaction as that of its parent molecule. However, the binding affinity of these molecules (fragments and with linkers) varies considerably and was very week in comparison to its parent molecule and FP7 and FP10 (Table 5).

[0087] Example 2: Hemocompatibility analysis of novel compounds

The hemolytic toxicities of the novel compounds (FP7 and FP10) were tested and compared with those of the existing drugs (Ponatinib and Vorinostat).

[0088] Hemolytic potential of hybrid molecules (Methodology): Whole blood samples from the healthy donors were collected in EDTA-coated vacutainers. Anticoagulated human peripheral blood was diluted in 1: 1 ratio. The treated concentrations of hybrid molecules (FP7 and FP10) were compared with the concentrations of individual drugs (Ponatinib and Vorinostat) for their hemolytic activity. For this, lOO I of each test sample at different concentrations were added separately to the diluted blood sample. Samples were incubated for 3 hours at 37°C with mild mixing every 15 minutes. DMSO solvent served as positive control while 1% Triton-X 100 was the negative control. After incubation, the samples were centrifuged at 800 g for 15 minutes at 20°C. Following centrifugation, 100 pl plasma from every sample was transferred to a 96-well plate, and its absorbance was measured at 540 nm using Epoch 2 spectrophotometer, BioTek. The hemolytic potential of the hybrid molecules and individual drugs were calculated based on the degree of hemoglobin leakage into the plasma.

[0089] PBMC cytotoxicity study of the hybrid molecules (Methodology): Peripheral blood samples from healthy donors were collected in EDTA-coated vacutainers with informed consent and approval by the Institute Ethical Committee (IEC) at Amrita Institute of Medical Sciences and Research Centre, Kochi Kerala, India. The isolated PBMC’s were washed initially with 3x and then with 2x concentration of basal RPMI media to remove any contaminants. PBMCs were seeded in a 96 well plate at 3xl0⁴ cells/well concentration. Individual drugs (Ponatinib and Vorinostat) and hybrid molecules (FP7 and FP10) were treated at different concentrations on PBMCs for 72 hours. Each well was then treated with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) reagent and incubated for 4 hours. After incubation with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent, the plates were read at 570 nm and 660 nm wavelength on an automated microtiter plate reader (Epoch 2, BioTek) to determine PBMC cytotoxicity of the hybrid molecules (FP7 and FP10) with respect to individual drugs.

[0090] Hemolytic toxicity determines the percentage of hemolysis caused by the agent in whole blood samples. This study is important for CML drug discovery with less adverse effect to normal human blood cells. Hemolytic potential of FP7 and FP10 showed negligible toxicities (FIG. 6A-D). Further, compatibility of the FP7with healthy mononuclear cells was also investigated compared to the individual drugs (Ponatinib and Vorinostat) for 72 hours inhuman PBMCs. Experimental analysis revealed that FP7and FP10 showed cytotoxicity in PBMCs at20pM, which is much higher than the IC50 value of this designed molecules (FP7 ICso=O.OOO2pM; FP1OIC5O=O.25 pM) (FIG. 7). The hemocompatibility study revealed that the incorporation of methylene linker with 7 and 10 units has served to reduce the toxicity with respect to the methyl piperazine moiety in Ponatinib thus demonstrating the superiority of the designed molecules over individual drugs. As these molecules had negligible hemolytic potential and PBMC cytotoxicity, we continued its studies in BCR-ABL positive cell lines.

[0091 ] Example 3: Growth inhibitory effects of designed compounds in comparison to drug combinations. Ponatinib and Vorinostat drug combination was studied in terms of dose-response curves in wild type K562 CML cell line to compare its activity with the designed molecules FP7 and FP10. The synergistic effect of Ponatinib, Vorinostat and designed drugs were also determined using Compusyn software.

[0092] Combination Index study of Ponatinib and Vorinostat. Combination index (CI) calculation provides a quantitative value for the drug interactions based on the synergism and antagonism for a particular drug treatment concentration effect. CI is based on the mass -action law derived from enzyme kinetic models and receptor binding theory. The CompuSyn (version 1.0) (ComboSyn, NJ, USA) software calculates the CI using IC50 values of cell lines treated with test sample. A CI of 1 explains drugs additivity, less than 1 indicates drugs synergism and greater than 1 indicates drugs antagonism.

[0093] Interestingly, FP7 showed IC5o=O.OOO16 pM close to Ponatinib and Vorinostat drug combination of 0.0004 pM, while FP10 had a lower inhibitory activity than FP7 (ICso=O.16 pM). FP7, being a single agent could produce a synergistic effect similar to the combination of drugs in K562 cell line. A significant number of data points were below 1 explaining synergism (FIG. 8, 9). This again highlights the significance of the 7 unit length of methylene linker which showed more favorable inhibitory activity against BCR-ABL positive cell line. Thus, 7 units of methylene linker were required for favorable BCR-ABL inhibitory activity than 10 units of methylene linker.

[0094] Example 4: Inhibitory activities of designed molecule FP7 in BCR-ABL expressing cell lines.

Inhibitory activities of the designed molecules FP7 and FP10 were assessed using the colorimetric cell proliferation assay with MTT reagent.

[0095] In vitro cell culture and cell proliferation assay (Methodology): CML cell line, K562 was purchased from NCCS, Pune and Ba/F3 cell lines stably expressing BCR-ABL mutants (Thr315Ile, Tyr253His) were kindly gifted by Deininger/O’Hare Lab, Huntsman Cancer Institute, USA. These cell lines were regularly cultured in RPMI media supplemented with 10% FBS and 1% and penicillin/streptomycin. Cell lines were maintained in ESCO CelCulture® CO2 incubator with 5% CO2 in air at 37 °C temperature and equipped with an air jacket which provides isolation against ambient temperature fluctuations. Cell viability assay was performed using MTT reagent which forms blue solid crystals when in contact with an enzyme in living cells. For this study, cells (K562 (wild type BCR-ABL), Ba/F3 Thr315Ile and Ba/F3 Tyr253His) were seeded at 8 x 10³ cells/well in 96-well plate. The cells were treated with varying concentrations of synthesized hybrid molecules (FP7 and FP10) and control drugs (Ponatinib, Imatinib and Vorinostat). The plate was incubated for 72 hours following treatment. MTT reagent was added to each well with a 4 hours’ incubation period and the insoluble crystals were dissolved to measure the absorbance of the plate at 570 nm and 660 nm using an automated microtiter plate reader (Epoch 2, BioTek). The IC50 values were determined from the growth inhibition curves plotted for the studied molecules.

[0096] The inhibitory activities of the designed molecules were compared with the control drugs (Imatinib, Ponatinib and Vorinostat) by plotting dose -response curves (Table 6). The cell viability assay exhibited an interesting observation with respect to K562 cell line, where FP7 (ICso= 0.00016 pM) showed an inhibition curve similar to that of Ponatinib (IC5o=O.OOO79 pM) while FP10 showed lower inhibitory activity (IC5o=O.16 pM). FP7 showed around 1000 fold higher inhibitory activity than Vorinostat and Imatinib in K562 cell line with wild type BCR- ABE while FP10 had a similar inhibitory curve as that of Vorinostat (FIG. 10A). The inhibitory activity of the designed molecules was also tested in Ba/F3 Imatinib resistant single point mutant cell lines containing the gatekeeper (Thr315Ile) and Tyr253His mutations. FP7 had -100 fold greater inhibitory activity than Vorinostat in Ba/F3 Thr315Ile cell lines while -10 fold greater activity in Ba/F3 Tyr253 His cell lines and more than 1000 fold greater inhibitory activity than Imatinib in these cell lines (FIG. 10B, C). FP10 however had lower activity than Ponatinib and Vorinostat but greater than Imatinib. FP7 had an inhibitory action similar to Ponatinib in K562 CME cell line and also in BCR- ABE positive Ba/F3 Thr315Ile and Tyr253His mutant cell lines.

[0097] Table 6. Growth Inhibitory (Dose-Response Relationships) Activities (IC50) of the Hybrid Molecules (FP7 and FP10) and Ponatinib, Imatinib, and Vorinostat towards K562 (Drug Sensitive) and Ba/F3 (Imatinib Resistant) Cell Lines³

aICso values are representative of three separate cell proliferation assays with respect to each treatment molecule bImatinib resistant cell lines

[0098] In spite of the elaborate description containing many specifications, these should not be thought to limit the scope of the invention but instead as demonstrating various examples and aspects of the invention. It should be appreciated that the scope of the invention includes other embodiments not discussed herein. Various other modifications, changes and variations which will be apparent to those skilled in the art may be made in the arrangement, operation and details of the system and method of the present invention disclosed herein without departing from the spirit and scope of the invention as described here. While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material the teachings of the invention without departing from its scope.

[0099] Results and Discussions (in-vitro studies)

[001001 In-vitro study of hybrid molecules FP7 and FP10

Hemocompatibility analysis of hybrid molecules.

The hemolytic toxicities of the hybrid molecules (FP7 and FP10) were tested and compared with those of the existing drugs (Ponatinib and Vorinostat). Hemolytic toxicity determines the percentage of hemolysis caused by the agent in whole blood samples. This study is important for CML drug discovery with less adverse effect on normal human blood cells. The hemolytic potential of FP7 and FP10 showed negligible toxicities, which were similar to Ponatinib and Vorinostat (FIG. 6A-D). Further, hemocompatibility of the FP7 and FP10 with healthy mononuclear cells were also investigated compared to the individual drugs (Ponatinib and Vorinostat) for 72 hours in human PBMCs. Experimental analysis revealed that FP7 and FP10 showed cytotoxicity in PBMCs at 20p M, which was considerably higher than the IC50 value of these molecules (FP7 ICso=5O pM; FP10 ICso=8O pM) (FIG.7). The hemocompatibility study revealed that the presence of methylene linker with 7 and 10 units has reduced the toxicity with respect to the methyl piperazine moiety in Ponatinib thus demonstrating the superiority of the hybrid molecules over individual drugs. As these hybrid molecules had negligible hemolytic potential and PBMC cytotoxicity, the inventors continued the studies in BCR-ABL positive cell lines.

[001011 Growth inhibitory effects of hybrid molecules in comparison to drug combinations.

The inhibitory activities of hybrid molecules were studied in comparison to the existing drugs by plotting dose-response curves. The synergistic effect of Ponatinib and Vorinostat drug combination was explored by Okabe et.al in Imatinib resistant Ba/F3 Thr315Ile cell line. This combination was studied in wild type K562 CML cell line (FIG.11A) and on the Ba/F3 Thr315Ile mutant cell line (FIG.11B). The growth inhibitory curve of FP7 and FP10 hybrid molecules were compared to Ponatinib and Vorinostat combination inhibition in K562 cell lines at a 1: 1 ratio of drug concentrations. Interestingly, FP7 showed ICso= 0.00016 pM similar to Ponatinib and Vorinostat combination ICso= 0.0004 pM, while FP10 showed around 1000 fold lower inhibitory activity in K562 cell line, thus highlighting the potency of FP7 with respect to FP10 (FIG. 9). FP7, being a single agent, could produce a synergistic effect similar to the combination of drugs in K562 cell line. As discussed above, these results clearly suggest that FP7 can act as a better alternative to inhibit CML than combination drugs. The inhibitory activity of Ponatinib to Vorinostat in constant and non-constant concentration ratios was studied in K562 cell line while in Ba/F3 Thr315Ile mutant cell line, only non-constant concentration ratios were studied. The combination of Ponatinib and Vorinostat drugs showed enhanced inhibitory activity in comparison to individual drugs on both BCR-ABL wild type and mutant cell lines as observed in the study by Okabe et al. To further evaluate the synergy between Ponatinib and Vorinostat drugs, CI was calculated depending on their inhibitory effects at different concentrations. The CI values at different concentrations of drug combinations in constant (1: 1) (FIG.8) and nonconstant concentration ratios (FIG.12A, B) were plotted with respect to its fractional effect to determine whether the combination drugs yield a synergy or antagonism at each treatment concentration. The majority of the values were below 1, clearly indicating synergistic effect in the BCR-ABL wild type and mutant cell lines. Lower drug combination concentrations showed poor synergy, while higher concentrations showed good synergy. FP7 maintained its Ponatinib pharmacophore based inhibitory activity towards BCR-ABL with similar inhibitory action in both wild type and mutant BCR-ABL positive cell lines (K562, Ba/F3 Thr315Ile and Ba/F3 Tyr253His respectively) while FP10 with a greater number of methylene units than FP7 could not produce an inhibitory effect close to drug combination (Ponatinib and Vorinostat) or Ponatinib alone. Thus, the inventors identified 7 units of methylene linker is optimally required for favorable BCR-ABL inhibitory activity. 001021 Inhibitory activities of hybrid molecules FP7 and FP10 in BCR-ABL expressing cell lines. Inhibitory activity of the hybrid molecules FP7 and FP10 were assessed using the colorimetric cell proliferation assay with MTT reagent. The activities of the hybrid molecules were compared with the control drugs (Imatinib, Ponatinib and Vorinostat) (Table 6). The cell viability assay exhibited an interesting observation with respect to K562 cell line, where FP7 (ICso= 0.00016 p M) showed an inhibition curve similar to that of Ponatinib (IC5o=O.OOO79 p M) while FP10 (ICso= 0.16 pM) showed similar curve to that of Vorinostat (ICso= 0.32 pM). FP7 showed around 1000 fold higher inhibitory activity than Vorinostat, Imatinib and FP10 in the K562 cell line. FP10 showed approximately 10 fold lower inhibitory activity to Imatinib and -1000 fold lower inhibitory activity to FP7 and Ponatinib (FIG. 10A). FP7 and FP10 inhibitory activity were also tested in Ba/F3 Imatinib resistant single point mutant cell lines containing the gatekeeper (Thr315Ile) and Tyr253His mutations. FP7 had -1000 fold more inhibitory activity than FP10 in Ba/F3 Thr315Ile cell line while -100 fold greater inhibitory activity in Ba/F3 Tyr253His cell line, -100 fold greater than Vorinostat in Ba/F3 Thr315Ile cell line while -10 fold greater inhibitory activity in Ba/F3 Tyr253His cell line and almost 10000 fold more inhibitory activity than Imatinib in Ba/F3 Thr315Ile cell line while -1000 fold greater inhibitory activity in Ba/F3 Tyr253His cell line. FP10 molecule, on the other hand, showed -10 fold higher inhibitory activity to Imatinib in Ba/F3 single point mutant cell lines, while its inhibitory activity was nearing 10 fold lower to Vorinostat, -100 fold lower to Ponatinib (FIG. 10B, C). FP7 with 7 units of methylene linker was more potent than FP10 with 10 units of methylene linker. FP7 had inhibitory activity similar to Ponatinib in K562 CML cell line and also in BCR-ABL positive Ba/F3 Thr315Ile and Tyr253His mutant cell lines (FIG. 10A-C). Thus, the hybrid molecule FP7 is a better alternative than FP10 in both the wild type CML cell line and against the multi-drug resistant gate keeper mutation containing Ba/F3 Thr315Ile cell line. 001031 In summary, the hybrid molecules (FP7 and FP10), which were finally selected following a detailed in silico screening, were validated in vitro based on their hemocompatibility and inhibitory activities in different types of wild type and drug resistant CML cell lines.

[001041 Example 5: synthesis of the compounds

General procedure for preparation of compound 2

A mixture of compound 1 (1.54 g, 12.2 mmol), compound 1A (3 g, 11.1 mmol), DIEA (5.74 g, 44.4 mmol), Pd(dba)2 (1.28 g, 2.22 mmol), PPh3 (1.17 g, 4.44 mmol) and Cui (423 mg, 2.22 mmol) in 1,4-dioxane (40 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 28 °C for 1.5 hr under N2 atmosphere. LCMS (EW8484-3-Plc) showed the starting material (R2) was consumed. The reaction mixture was concentrated to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=10: l to 3: 1; TLC: R2:Rf=0.95;Pl:Rf=0.12) to compound 2 (2.5 g, 71.36% yield) as a yellow oil.

LC-MS: EW8484-3-P1C

General procedure for preparation of compound 3

To a solution of compound 2 (500 mg, 1.59 mmol) in MeCN (15 mL) and H2O (15mL) was added [acetoxy(phenyl)-iodanyl] acetate (1.53 g, 4.76 mmol) and TEMPO (74.8 mg, 475.76 umol),the mixture was stirred at 25 °C for 16 h. TLC (Petroleum ether/Ethyl acetate=l:l, material Rf=0.5, product Rf=0.1) indicated Reactant 1 was consumed completely and one new spot formed. The reaction was diluted with H2O (50mL) and extracted with Ethyl acetate (35mL*3). The combined organic phase was dried over Na2SO4 and concentrated to give compound 3 (0.8 g, crude) as a yellow oil.

General procedure for preparation of compound 4

To a solution of compound 3 (700 mg, 2.13 mmol) in EtOH (15 mL) was added drop-wise SOCh (4.92 g, 41.35 mmol), the mixture was stirred at 80°C for 0.5h. TLC (Petroleum ether/Ethyl acetate=l:l, material Rf=0.1, product Rf=0.8) indicated Reactant 1 was consumed completely and one new spot formed. The reaction was concentrated. The residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate=50/l to 10:1) to give compound 4 (0.8 g, crude) as a light yellow oil, which was confirmed by LCMS (EW8484-6- P1A).

LC-MS: EW8484-6-PlA,MS (ESI) m/z = 358.1 [M+H]⁺

General procedure for preparation of compound 5

To a solution of compound 4 (0.8 g, 2.24 mmol) in MeOH (30 mL) was added Pd/C (0.4 g, 10% purity) , the mixture was stirred at 30°C for 16h under H2 (15 psi). TLC (Petroleum ether/Ethyl acetate=5:l, material Rf=0.5, product Rf=0.3) indicated Reactant 1 was consumed completely and one new spot formed. The reaction was filtered via a celite pad, the filtrate was concentrated. The residue was purified by prep-TLC (SiO2, Petroleum ether/Ethyl acetate=5 : 1 ) to give compound 5 (0.2 g, crude) as a light yellow oil, which was confirmed by LCMS (EW8484-7-P1C).

LC-MS: EW8484-7-PlC,MS (ESI) m/z = 332.1 [M+H]⁺

General procedure for preparation of compound 6

To a solution of compound 5 (100 mg, 302 pmol) in DMF (2 mL) was added compound 5A (83.7 mg, 302 pmol) and HATU (138 mg, 362 pmol), DIEA (58.50 mg, 453 pmol). After the additional, the mixture was stirred at 25°C for 4h. LCMS (EW8484-8-P1A) showed -29% desired MS was detected. The reaction was diluted with H2O (5 mL) and extracted with EA (5 mL*4). The combined organic phase was dried over Na2SC>4 and concentrated. The residue was purified by prep-TLC (SiCh, Petroleum ether/Ethyl acetate=l: l, Rf = 0.26) to give compound 6 (0.09 g, crude) as a light-yellow solid, which was confirmed by LCMS (EW8484-8-P1A2).

LC-MS: EW8484-8-P1A2, MS (ESI) m/z = 591.2 [M+H]⁺

General procedure for preparation of compound 6

To a solution of compound 6 (0.08 g, 135 pmol) in MeOH (5 mL) and H2O (1 mL) was added NaOH (1 M,406 pL).The mixture was stirred at 25 °C for 30 min. LCMS (EW8484-10-P1A) showed -88% desired MS detected. The reaction was concentrated; the residue was adjusted pH to 4 with sat. citric acid. The solid was filtered and dried under reduce pressure to give compound 7 (80 mg, crude) as a light-yellow solid.

LC-MS: EW8484-1O-P1A,MS (ESI) m/z = 563.2 [M+H]⁺

General procedure for preparation of compound 6

To a solution of compound 7 (60 mg, 107 pmol) and O-(tetrahydro-2H-pyran-2- yl)hydroxylamine (25.0 mg, 213 pmol) in DMF (1 mL) was added HATU (81.1 mg, 213.3 pmol) and TEA (54.0 mg, 533 pmol). After the addition, the mixture was stirred at 25 °C for 12 hr. LCMS (EW8484-11-P1A) showed the desired MS was detected. The reaction was diluted with water (lOmL) and extracted with EA(10 mL*3). The combined organic phase was dried over Na2SC>4 and concentrated to give compound 8 (60 mg, crude) as a light yellow solid.

General procedure for preparation of FP7

FP7

To a solution of compound 8 (0.07 g, 106 pmol) in MeOH (3 mL) was added TsOH.H2O (8.05 mg, 42.3 pmol). The mixture was stirred at 25 °C for 0.5 hr. LCMS (EW8484-12-P1A) showed the reaction was completed. The reaction was diluted with H2O (10 mL) and extracted with EA (10 mL*3). The combined organic phase was dried over Na2SC>4 and concentrated. The residue was purified by prep-TLC (SiCL, EtOAc: MeOH=20: l) to give 30 mg the crude product, the crude product was washed with MeOH (5 mL), filtered and dried under reduce pressure to give Mol-1 (14 mg, 22.91% yield) as a white solid, which was confirmed by HNMR (EW88055+43- P1A), HPLC (EW8055-43-P1C) and LCMS (EW8055-43-P1C). 'H NMR:EW8055-43-PlA 400 MHz CDC13

5 10.52 (s, 1H), 10.33 (s, 1H), 8.74 (dd, J = 1.5, 4.4 Hz, 1H), 8.73 (s, 1H) 8.29 - 8.16 (m, 4H), 8.00 - 7.96 (m, 1H), 8.05 - 7.93 (m, 1H), 7.98 - 7.92 (m, 1H), 7.56 (d, J = 8.2 Hz, 1H), 7.48 (d, J = 8.7 Hz, 1H), 7.43 - 7.38 (m, 1H), 2.72 - 2.68 (m, 2H), 2.62 (s, 3H), 1.93 (t, J = 7.3 Hz, 2H), 1.57 (br s, 2H), 1.57 - 1.47 (m, 4H), 1.32 - 1.24 (m, 6H)

LC-MS: EW8055-43-PlC,MS (ESI) m/z = 578.4 [M+H]⁺

HPLC: EW8055-43-P1C, purity = 95.5%

[001071 Synthesis of FP10

General procedure for preparation of compound 2

A mixture of undec- 10-yn-l-ol (1.03 g, 6.11 mmol), l-bromo-4-nitro-2-(trifluoromethyl)benzene (1.5 g, 5.56 mmol), Cui (212 mg, 1.11 mmol), Pd(dba)2 (639 mg, 1.11 mmol), PPhs (583 mg, 2.22 mmol) and DIEA (2.87 g, 22.2 mmol) in dioxane (30 m ) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 20 °C for 16 hr under N2 atmosphere. TLC (Petroleum ether : Ethyl acetate = 3 : 1) showed reactant 1 was and a new main (Rf = 0.2) spot was formed. The mixture was diluted with water (50 mL) and extracted with ethyl acetate (40 mb * 3). The combined organic layer was washed with brine (30 mL), dried over Na2SC>4, filtered and concentrated under reduced pressure to give the crude product. The residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate=20/lto8:l) to give Compound 2 (1.5 g, crude) as a brown oil, which was confirmed by HNMR (EW8923-24-P1C).

'H NMR:EW8923-24-PlC 400 MHz CDCI3

General procedure for preparation of compound 3

To a solution of Compound 2 (1.5 g, 4.20 mmol) and TEMPO (198 mg, 1.26 mmol) in CH3CN (15 mL) and Water (15 mL) was added iodobenzene diacetate (4.06 g, 12.6 mmol), and then stirred at 20 °C for 6 hr. TLC (Petroleum ether : Ethyl acetate = 2:1) showed reactant 1 was consumed completely and a new main spot (Rf = 0.2) was formed. The mixture was diluted with water (50 mL) and extracted with ethyl acetate (40 mL * 3). The combined organic layer was washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (Petroleum ether: Ethyl acetate = 10: 1 to 3:1) to give Compound 3 (0.9 g, crude) was obtained as a light-yellow solid, which was confirmed by HNMR (EW8923-25-P1A)

'H NMR:EW8923-25-PlA 400 MHz DMSO-d6

5 11.89 (br s, 1H), 8.42 - 8.33 (m, 2H), 7.83 (d, J = 8.3 Hz, 1H), 2.49 (t, J = 6.8 Hz, 2H), 2.11 (t, J= 7.4 Hz, 2H), 1.54 - 1.46 (m, 2H), 1.45 - 1.30 (m, 4H), 1.21 (br s, 6H)

General procedure for preparation of compound 4

To a solution of Compound 3 (0.5 g, 1.35 mmol), HATU (614 mg, 1.62 mmol) and DIPEA (261 mg, 2.02 mmol) in DMF (5 mL) was added O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (157 mg, 1.35 mmol) at 20 °C. After the additional, the mixture was stirred at 20 °C for 12 hr. LCMS (EW8923-26-P1A) showed the desired MS (RT = 1.124 min) was detected. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (20 mL * 3). The combined organic layer was washed with brine (20 mL), dried over Na2SC>4, filtered and concentrated under reduced pressure to give Compound 4 (0.35 g, crude) as a yellow oil, which was confirmed by HNMR (EW8923-26-P1A).

'H NMR:EW8923-26-PlA 400 MHz DMSO-d6

510.88 (s, 1H), 8.50 - 8.40 (m, 2H), 7.90 (d, J = 8.4 Hz, 1H), 4.79 (br s, 1H), 3.96 - 3.85 (m, 1H), 3.53 - 3.41 (m, 1H), 2.56 (t, J = 6.8 Hz, 2H), 1.99 - 1.94 (m, 2H), 1.69 - 1.62 (m, 2H), 1.61 - 1.37 (m, 10H), 1.27 (br s, 6H)

General procedure for preparation of compound 5

To a solution of Compound 4 (0.25 g, 531 pmol) in MeOH (5 mL) was added Pd/C (0.1 g, 10% purity) under N2 atmosphere. The suspension was degassed and purged with H2 for 3times. The mixture was stirred under H2 (15 Psi) at 20 °C for 4 hr. LCMS (EW8923-27-P1A) showed the desired MS (RT = 1.128 min) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give Compound 5 (0.1 g, 42.34% yield) as light-yellow oil, which was confirmed by HNMR (EW8923-27-P1B).

'H NMR:EW8923-27-PlB 400 MHz CDC13

5 8.45 (br s, 1H), 7.01 (d, J = 8.2 Hz, 1H), 6.83 (d, J = 2.4 Hz, 1H), 6.69 (dd, J = 2.2, 8.2 Hz, 1H), 4.86 (br s, 1H), 3.86 (br d, J = 8.3 Hz, 1H), 3.59 - 3.52 (m, 1H), 2.61 - 2.47 (m, 2H), 2.11 - 1.99 (m, 2H), 1.73 (br s, 2H), 1.62 - 1.42 (m, 8H), 1.28 - 1.15 (m, 14H)

LC-MS: EW8923-27-P1A, MS (ESI) m/z = 443.4 [M-H]⁺

General procedure for preparation of compound 6

A solution of Compound 5 (0.09 g, 202 pmol), Compound 2A (56.14 mg, 202 pmol), HATU (92 mg, 243 pmol) and DIPEA (39 mg, 304 pmol) was stirred at 25 °C for 16 hr. LCMS (EW8923- 29-P1B1) showed the desired MS (RT = 1.095 min) was detected. The reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (20 mL * 3). The combine organic layer was washed with brine (20 mL), dried over Na2SC>4, filtered and concentrated under reduced pressure to give Compound 6 (0.09 g, crude) as a yellow oil.

LC-MS: EW8923-29-PlBl,MS (ESI) m/z = 704.5 [M+H]⁺

General procedure for preparation of FP10

A solution of compound 6 (90 mg, 127 pmol,) and HCl/EtOAc (4 M, 2 mL) in EtOAc (4 mL) was stirred at 25 °C for 16 hr. LCMS (EW8923-30-P1B) showed the desired MS (RT = 1.023 min) was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The crude product was purified by pre-HPLC (TFA condition) to give FP10 (7.04 mg 8.76% yield, 98.6% purity) was obtained as a yellow solid, which was confirmed by HNMR (EW8923-30-P1B), HPLC (EW8923-30-P1A) and LCMS (EW8923-30-P1D).

'H NMR:EW8923-3O-P1B 400 MHz CDC13

5 10.52 (s, 1H), 10.31 (s, 1H), 8.74 (dd, J = 1.5, 4.4 Hz, 1H), 8.29 - 8.16 (m, 4H), 8.00 - 7.96 (m, 1H), 8.05 - 7.93 (m, 1H), 7.98 - 7.92 (m, 1H), 7.56 (d, J = 8.2 Hz, 1H), 7.48 (d, J = 8.7 Hz, 1H), 7.43 - 7.38 (m, 1H), 2.71 (br d, J = 8.3 Hz, 2H), 2.62 (s, 3H), 1.93 (t, J = 7.3 Hz, 2H), 1.57 (br s, 2H), 1.48 (br s, 2H), 1.38 - 1.22 (m, 14H)

LC-MS: EW8923-30-Pld, MS (ESI) m/z = 620.5 [M+H]

HPLC: EW8923-30-P1A, purity = 98.6% ABBREVIATIONS:

1. DIEA = N, N-Diisopropylethylamine

2. PPhs = Triphenylphosphine

3. Cui = Copper(I) iodide

4. TEMPO = 2,2,6,6-Tetramethylpiperidin-l-yl)oxyl or (2,2,6,6-tetramethylpiperidin- l-yl)oxidanyl

5. MeCN = methyl cyanide

6. EtOH = Ethanol

7. SOCh = Thionyl chloride

8. MeOH = Methanol

9. HATU = l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5- b]pyridinium 3 -oxide hexafluorophosphate

10. DIPEA = N, N-Diisopropylethylamine

11. DMF = Dimethylformamide

12. NaOH = Sodium hydroxide

13. TEA = Triethylamine

14. TSOH.H2O = p-Toluenesulfonic acid monohydrate or tosylic acid

15. Pd(dba)2 = Tris(dibenzylideneacetone)dipalladium(0)

16. CH3CN = Acetinitrile

17. EtOAc = Ethyl acetate

Claims

The Claims :

1. A compound of general Formula A;

Formula A or their pharmaceutically acceptable salt thereof;

Characterized in that R is selected from the following:

2. The compound as claimed in claim 1, wherein the compound is (N-(4-(8- (hydroxyamino)-8-oxooctyl)-3-(trifluoromethyl)phenyl)-3-(imidazo[l,2-b]pyridazin-3- ylethynyl)-4-methylbenzamide) (Formula I):

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[Formula I] [Compound FP7] or their pharmaceutically acceptable salt thereof. The compound as claimed in claim 1, wherein compound is (N-(4-(l l-(hydroxyamino)- l l-oxoundecyl)-3-(trifluoromethyl)phenyl)-3-(imidazo[l,2-b]pyridazin-3-ylethynyl)-4- methylbenz- amide) (Formula II):

[Formula II] [Compound FP10] or their pharmaceutically acceptable salt thereof. The compound as claimed in claim 1 , wherein the oral absorption of compounds is from 55 to 80 %, preferably 65 to 75 %. The compound as claimed in claim 1, wherein Fog P of compound is from 7 to 8. A method of preparing compound of Formula I as claimed in claim 2, wherein the method comprising the following steps: a) Degassing and purging of a mixture of compound 1, compound 1A, DIEA, Pd(dba)2, PPhs and Cui in 1,4-dioxane with N2, followed by stirring the mixture to obtain a residue of compound 2.

c) Adding SOCh to a solution of compound 3 obtained from step b) in EtOH and stirring to obtain compound 4.

h) Adding TSOH.H2O to a solution of compound 8 in MeOH and stirring to obtain compound FP7

The method as claimed in claim 6, wherein the stirring in step a) is conducted at 20 °C to 35 °C, preferably at 28 °C, stirring in step b), step e), step f), step g) and step h) is conducted at 15 °C to 35 °C, preferably at 25 °C, stirring in step c) is conducted at 70°C to 90°C, preferably at 80 °C and stirring in step d) is conducted at 20 °C to 40 °C, preferably at 30 °C. The method as claimed in claim 6, wherein the stirring in step a) is conducted for 1 to 2 hours, preferably for 1.5 hours, stirring in step b) and step d) is conducted for 10 to 22 hours, preferably for 16 hours, stirring in step c), step f) and step h) is conducted for 20 to 40 minutes, preferably for 30 minutes, stirring in step e) is conducted for 2 to 6 hours, preferably for 4 hours and stirring in step g) is conducted for 8 to 16 hours, preferably for 12 hours. A method of preparing compound of Formula II as claimed in claim 3, wherein the method comprising the following steps: a) Degassing and purging a mixture of undec- 10-yn-l-ol, l-bromo-4-nitro-2- (trifluoromethyl)benzene, Cui, Pd(dba)2, PPh3 and DIEA in dioxane with N2 and stirring to obtain compound 2;

HATU and DIPEA in DMF to obtain compound 4;

e) Stirring the solution of Compound 5, Compound 2A, HATU and DIPEA to obtain Compound 6;

f) Stirring a solution of compound 6 and HCl/EtOAc in EtOAc to obtain FP10

The method as claimed in claim 9, wherein the stirring in step a), step b), step c) and step d) is conducted at 15 °C to 25 °C, preferably at 20 °C and stirring in step e) and step f) is conducted at 20 °C to 30 °C, preferably at 25 °C. The method as claimed in claim 9, wherein the stirring in step a), step e) and step f) is conducted for 12 to 20 hours, preferably for 16 hours, stirring in step b) is conducted for 4 to 8 hours, preferably for 6 hours, stirring in step c) is conducted for 8 to 16 hours, preferably for 12 hours and stirring in step d) is conducted for 2 to 6 hours, preferably for 4 hours. The compound as claimed in claim 1, wherein imidazopyridazine group with triple bond and other attached groups target BCR-ABL and hydroxamate moiety (zinc binding groups).

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