Bifunctional conjugate molecules for cancer treatment and process of making the same

Abstract

The present invention relates to an immune-modulating bifunctional bioconjugate molecule of Formula I, comprising a functionalized HDAC inhibitory moiety covalently linked with a broad-spectrum chemotherapeutic agent using a linker. The broad-spectrum chemotherapeutic agent in formula I is Gemcitabine. The present invention also relates to the process for preparation of bifunctional conjugate molecules. Formula I wherein, Linker and HDACi is as defined in the specification.

Description

BIFUNCTIONAL CONJUGATE MOLECULES FOR CANCER TREATMENT AND PROCESS OF MAKING THE SAME FIELD OF THE INVENTION

[0001] The present invention relates to the field of medicinal chemistry. Particularly, the present invention relates to the immune-modulating bifunctional bioconjugate molecules for the treatment of cancer and process for preparation thereof. The bifunctional conjugate molecules of general Formula I, comprises a broad-spectrum chemotherapeutic agent, a linker, and a histone deacetylase inhibitor.

[0002] The process for preparation of bifunctional conjugate molecules is simple, safe, cost effective, and yields the final product with high purity. Process uses cost-effective reagents and mild reaction conditions, which makes the process more efficient for scale up. BACKGROUND OF THE INVENTION

[0003] Cancer is the second leading cause of death and the dominant barrier to increasing life expectancy globally. According to the World Health Organization (WHO) report, 19.3 million new cancer cases were registered, and almost 10 million deaths were due to cancer in 2020 worldwide. Despite enormous efforts towards advancements in cancer treatment therapy, the molecular complexity, and mechanisms of their resistance development as well as extensive clinical experience can outsmart new-generation chemotherapeutic agents. The effective and desirable chemotherapeutic effects are still far from satisfactory and yet challenging with monotherapy.

[0004] In epigenetics, it has been observed that modification of histone protein by several means of reversible acylation and methylation of histone and non-histone proteins is the most abundant post-translational modification in eukaryotic cells. Histone deacetylases (HDACs) are key regulators of histone modifications that act as transcriptional repressors by removing acetyl groups from histones. As per the literature reports in the last two decades, there is growing evidence of overexpression of HDACs in different types of cancer prognosis. The inhibition of HDACs can exert an antiproliferative effect by suppressing cancer cell viability, migration, invasion, angiogenesis, DNA repression, and induction of apoptosis. So far five Histone deacetylase inhibitor (HDACi) such as vorinostat (SAHA), romidepsin (FK228), belinostat (PXD101), panobinostat (LBH589), and chidamide (CS055, approved only in China), have been approved clinically for the management of hematological malignancies. The HDACi also demonstrated promising results with the combination of standard chemotherapeutic agents in preclinical and different phases of clinical studies. The development of multifunctional molecules with two different pharmacological mechanisms of action with immune modulating activity and delivery at the same site of action may overcome the shortcoming of both typical chemotherapeutic agents and epigenetic modifiers was the unmet need to get rid of cancer.

[0005] Despite, the amenable and great potential of HDACi for the treatment of cancer, its clinical use as a typical chemotherapeutic agent has been limited due to weak efficiency towards solid tumors. Often HDACi suffers from some other severe drawbacks to being ideal drug candidates such as lack of bioavailability, less cellular permeability in the compacted tumor environment, and development of resistance. Thus, the patients have to take frequent high dosing leading to severe off-target side effects.

[0006] Gemcitabine is one of the most active chemotherapeutic agents with broad- spectrum antitumor activity. The major limitation of Gemcitabine is developing resistance within weeks of chemotherapy initiation and short systemic stability in presence of indigenous cytidine deaminase. Due to the high potency of Gemcitabine, some adverse drug effects are also frequently observed during treatment period including bone marrow suppression, liver and kidney problems, nausea, fever, rash,shortness of breath, mouth sores, diarrhoea, neuropathy, and also hair loss. Use of Gemcitabine during pregnancy would most likely result in harm to the baby. Gemcitabine is primarily recommended in combination therapy to minimize the shortfalls of a single treatment of Gemcitabine. The translation of combination chemotherapeutics is very critical and expensive as both the drugs to be used in combination need to pass through the cumbersome drug approval procedure. Moreover, it is very challenging to deliver both drugs at the site of action at the same time. To overcome the adverse effects related to off-target toxicity and to understand the clear underlying mechanisms, there is a need for the development of better drug therapy options, particularly in providing combination treatment that address the prior art problems. The conjugation of the standard broad-spectrum chemotherapeutic agent such as Gemcitabine with HDAC inhibitor (HDACi) have shown a distinct effect in inducing synergism over different cancers.

[0007] The present invention satisfies the existing needs and addresses the forementioned drawbacks of prior arts. OBJECTIVES OF THE PRESENT INVENTION

[0008] The main objective of the present invention is to provide novel bifunctional conjugate molecules that comprises; a broad-spectrum chemotherapeutic agent, a linker, and a histone deacetylase inhibitor or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

[0009] Yet another objective of the present invention is to provide a process of preparation of the bifunctional conjugate molecules which are safe, cost effective, and yield the final product with high purity.

[0010] Yet another objective of the present invention is to provide bifunctional conjugate molecules or a pharmaceutically acceptable salt, hydrate, or solvate thereof that show biological activity as potent anticancer agent.

[0011] Yet another objective of the present invention is to provide bifunctional conjugate molecules which show no or less toxicity to normal cells.

[0012] Yet another objective of the present invention is to provide a pharmaceuticalcomposition comprising bifunctional conjugate molecules or a pharmaceutically acceptable salt, hydrate, or solvate thereof as an active ingredient in a single or a combination therapy with a pharmaceutical acceptable carrier, diluent, or excipient.

[0013] A further objective of the present invention is to provide a method for treatment of various cancers using a bifunctional conjugate molecule as an active ingredient. SUMMARY OF THE INVENTION

[0014] The present invention relates to bifunctional conjugate molecules of general Formula I, or a pharmaceutically acceptable salt, hydrate, or solvate thereof. The synthesized bifunctional conjugate molecules have potent inhibitory activity for pan- HDACs.wherein; Linker is selected from -CO-CH2-CH2-CO-NH-CH2- or -CO-CH2-CH2-CO-. HDACi is selected from formula 1.1 to 1.5wherein;m is 0-5, n is 0-5, Z is selected from CH2, O, S, or NH; X1, X2and X3are independently selected from CH or N. R1, R2and R3are independently selected from hydrogen, halogen, cyano, nitro, straight or branched chain C1-C6alkyl, C1-C4alkoxy, —OH, amino, —C(O)NH2, — NHC(O)—, Aryl, heteroaryl, heterocycle, wherein alkyl, alkoxy, —OH, amino, aryl, heteroaryl and heterocycle are optionally substituted with 1-3 substituents selected from hydrogen, halogen, cyano, nitro, C1-C6alkyl, C1-C4alkoxy, —OH and amino.

[0015] According to one aspect, the present invention provides bifunctional conjugate molecules comprising covalently linked functionalized HDAC inhibitory moiety or HDACi moiety with a broad-spectrum chemotherapeutic agent being Gemcitabine using a linker.

[0016] According to one aspect, the present invention provides a simple, safe and cost effective, process for preparation of bifunctional conjugate molecules, which produce the final product with high purity and yield. BRIEF DESCRIPTION OF THE FIGURES: The above and other aspects of the present invention will be more apparent and better understood when considered in conjunction with the following detailed description and accompanying drawings in which like characters represent like parts throughout the drawings, wherein Figure 1. General design bi-functional molecules. Figure 2. The % inhibition of the enzyme activity by the synthesized conjugate of 10 µM conc. against HeLa nuclear extract (pan-HDACs). Figure 3. Graphs used for the analysis of IC50values of the compounds C-7, C-15 and C-47 with human recombinant HDAC isozymes (A) HDAC1; (B) HDAC2; (C) HDAC3; (D) HDAC6; (E) HDAC8. [Determination of IC50was performed at thevaried concentration range of 0.016 µM – 1250 µM of these compounds in duplicate according to the protocol described. (II) IC50curves of the compounds C-65, C-70, C- 81, C-85 and C-96 when assayed with the human recombinant HDAC isozymes of (A) HDAC1; (B) HDAC2; (C) HDAC3; (D) HDAC6; and (E) HDAC8. [The experiment was carried out as per the Vendor's protocol given in their respective recombinant HDAC assay kits. The compounds were tested at the varied concentration range of 0.04 μM – 3125 μM in duplicate. The IC50values were estimated with the help of the nonlinear regression analysis method by Graph Pad Prism 8.0.1. Data represent mean ± SD (n = 2)]. Figure 4. IC50of the conjugates against different cancer cell lines (A) 4T1; (B) MCF- 7; (C) MDA-MB-231; (D) DU145; (E) PC3; and (F) MOC2 cell lines. Determination of IC50was performed at the varied concentration range of 0.07 µM to 80 µM for conjugate and 0.009 µM to 10 µM for free Gemcitabine. The IC50values were calculated using non-linear regression analysis method by Graph Pad Prism 8.0.1. Data represent mean ± SD (n = 2). Figure 5. IC50of the compound 15 in Gemcitabine-resistant prostate cancer cell lines. Determination of IC50was performed at the varied concentration range of 0.07 µM to 80 µM. The IC50values were calculated using the non-linear regression analysis method by Graph Pad Prism 8.0.1. Data represent mean ± SD (n = 2) Figure 6. The cytotoxicity of the conjugates against normal cells was assessed and the IC50values of the conjugates were determined using a similar MTT assay protocol (A) Normal human embryonic kidney cells (HEK-293); (B) Normal human corneal epithelial cells (HCEC); and (C)normal human breast (MCF10A) cell lines. The IC50values give their comparative cytotoxicity potential against cancer cell lines over normal cell lines. In the experiment, cells were treated with the compounds at the concentration range from 7.81 µM to 2000 µM for 72 h. Data represent mean ± SD (n=2). Figure 7. Apoptosis and cell cycle analysis experiment were performed in PC3 cells treated with vehicle (control), reference compound Gemcitabine (0.186 µM), compound 15 (0.177 µM) and compound 47 (0.505 µM) at the IC50concentration of the compounds for 72 h. The apoptosis and cell cycle analysis was carried out by Flowcytometry (BD Aria III) ®; (A) Apoptosis analysis using Annexin-V / PI assay double staining and analyzed by flow cytometry. (X and Y-axis represent the fluorescence intensities of annexin-V and propidium iodide respectively); (B) Graphical representation of % of apoptotic cells. Data represent mean ± SD, n=3. (C) Cell cycle analysis data and (D) Graphical representation of the % cell population at various phases of cell cycle of PC3 cells, i.e., G1, S and G2 / M phases. Figure 8. Analysis of nuclear morphology of PC3 cells based on DAPI and acridine orange staining following the treatments i.e. control, Gemcitabine, compound 15 and compound 47. Here, 'NF' represents nuclear fragmentations and 'CS' represents cytoplasmic staining. The stained nuclei were visualized under a fluorescence microscope (Leica microsystems, Germany) on 20x Magnification. (B) Quantification of fluorescence intensity was done using Image J software. Figure 9. Western blot of (A) HDAC1, HDAC2, HDAC3, HDAC4 and HDAC6 in the supernatant of 4T1 cells treated with control: lanes 1−2, Gemcitabine: lanes 3−4, compound 15: lanes 5−6 after 72 h of treatment. Results were normalized with respect to β-actin as a housekeeping protein in respective controls. (B) Quantification was done using ImageJ software. Data were analyzed using a two-tailed unpaired t test. Each column represents the mean ± SEM of protein expression: *p < 0.05 vs control and #p < 0.05 vs Gemcitabine. Figure 10. (A) Western blot of Caspase-3 and Caspase-7 in cell pellet of 4T1 cells treated with control: lanes 1−2, Gemcitabine: lanes 3−4, compound 15: lanes 5−6 after 72 h of treatment. Results were normalized with respect to β-actin as a housekeeping protein in respective controls. (B) Quantification was done using ImageJ software. Data were analyzed using a two-tailed unpair t-test. Each column represents the mean ± SEM of protein expression: *p < 0.05 vs control and #p < 0.05 vs Gemcitabine. Figure 11. In vitro hemolysis study plot using RBCs treated with different concentrations of compound 15 (2-12 µM / mL), and a positive control, Triton X-100 (0.1 % w / v). Figure 12. (A) In-vitro plasma stabilities of compound 15, compound 47, and Gemcitabine in rat plasma; (B) The major metabolite, 2,2′-difluorodeoxyuridine(dFdU) formation from Gemcitabine core moiety (2', 2'-difluoro 2'deoxycytidine, dFdC) in rat plasma after 24 h at 37ºC. The data points are represented as the mean ± SE (n= 3). Figure 13. Mean plasma concentration-time profiles of compound 15 and compound 47 following a single intraperitoneal (i.p.) administration to male wistar rats (n = 3) of dose, 23.51 µmol / kg, 7.87 µmol / kg and 22.45 µmol / kg, 7.48 µmol / kg respectively, and Gemcitabine: 23.51 µmol / kg. Figure 14. Assessment of therapeutic efficacy of Gemcitabine conjugate, compound 15 in PC3 tumor-bearing male Balb / c nude mice. (A) The schematic diagram of PC3 tumor-bearing model experimental protocol; (B) Graphical representation of tumor volume vs. days of treatment; (C) The change of weight of nude mice treated by Gemcitabine, compound 15 (higher dose), HDACi component, physical mixture of two components, compound 15 (lower dose); (D) Tumor weights at the end of the treatment i.e. on the 12th day from the start of dosing; (E) Images of the tumors collected from various groups of mice on the 12th day from the start of dosing; (F) Images of the tumor-bearing mice on different days during the treatment regimen. Figure 15. In vivo fluorescence imaging of PC3 tumor-bearing mice after i.v. injection with Dil-loaded PEG-PE micelles. Tumor regions are marked with blue circles. Figure 16. Immunohistochemical analysis for PC3 tumor-bearing nude mice that received different treatments. TUNEL assay of cryo-sectioned tumors from the mice. The tissue sections were stained with DAPI (left panel). The green fluorescence (middle panel) labeled the apoptotic nuclei. Scale bar, 100 μm. Figure 17. (A) ROS generation in tumor tissue. Live optical fluorescence images of mice administrated with Gemcitabine conjugate at the 5thday, 10thday and 12thday post-injection [DCFH-DA administration intra tumoral (i.t), 30 min before the imaging]; (B) Fluorescence micrograph of DCFH-DA-administered tumor sections (ex / em.488 / 520 nm). Figure 18. H&Estained images of various organs collected at the end of the experiment; Scale bar, 100 μm.Figure 19. (A) Images of the starting with 500+ mm3tumor-bearing mice during different days of compound 15 (higher dose) treatment considering the first dosing day as day 0; (B) Graphical representation of tumor volume vs. days of drug treatment considering the first dosing day as day 0; (C) In vivo fluorescence imaging of PC3 tumor-bearing mice after i.v. injection with Dil-loaded PEG-PE micelles. Tumors regions were marked with blue circle; (D) ROS generation in tumor tissue. Live optical fluorescence images of mice administrated with Gemcitabine conjugate at 5 days, and 10 days post-injection (DCFH-DA administration, i.t, 30 min before the imaging): The tumor region marked with blue circle for both (C) and (D); (E). Immunohistochemical expression of Ki67 (a proliferative biomarker) with the sectioned tissue from the PC3 induced tumor region and (F) Immunohistochemical expression of CD44 (expressed in proliferative cells) with the sectioned tissue from the tumor region in PC3 induced tumors. Figure 20. The blood biochemical indicator analysis after PBS, and compound 15 were intraperitoneally injected to the mice; data were obtained for (A) ALT, AST, ALP, and (B) BUN and SCr, hematology analyses of (C) WBC, (D) RBC and (E) HGB. Figure 21. The expression level (upregulation) of various biomarkers was determined by western blotting in tumor tissues after treatment with 23.51 µmol / kg (higher dose) of compound 15, 23.51 µmol / kg of Gemcitabine, 23.51 µmol / kg of HDACi, a physical mixture of (23.51 µmol of Gemcitabine and 23.51 µmol of HDACi) / kg, 11.75 µmol / kg (lower dose) of compound 15. Results were normalized with respect to β-Actin as house keeping protein. The data were represented as mean ± SD (n=2). The significance of the difference was assessed by ANOVA. *** indicates p < 0.001. Figure 22. The expression level (down regulation) of various anti-apoptotic biomarkers was determined by western blotting in tumor tissues after treatment with 23.51 µmol / kg (Higher dose) of compound 15, 23.51 µmol / kg of Gemcitabine, 23.51 µmol / kg of HDACi, a physical mixture of (23.51 µmol of Gemcitabine and 23.51 µmol HDACi) / kg, 11.75 µmol / kg (Lower dose) of compound 15. Results were normalized with respect to β-Actin as house keeping protein. The data were represented as mean ± SD (n=2). The significance of the difference was assessed by ANOVA. *** indicates p < 0.001.Figure 23. (A) Representative whole animal bioluminescence imaging of luciferin induced (i.p administration of luciferin–D, 100 µL, 100 mg / kg) 4T1-Luc tumor- bearing mice on days 0, 5, 10, and 15 by IVIS® Lumina III, PerkinElmer, USA; (B) Quantification of bioluminescence of the tumors; (C) Body weight changes in mice during the treatment period; (d) Representative graph of tumor volume over the treatment period; (E) Representative whole animal body image of tumor-bearing mice on the 15thday. Figure 24. (A) Quantification of bioluminescence of the cell implanted region using Living Image 4.5.4 software. Data represent mean ± SD (n=4) (B) Tumor volume curve after treatment with compound 15 with two doses. Figure 25. (A) Images representing the Ki67 stained tumor sections; and (B) Corresponding graph representing the percentage of Ki67 positive cells in different groups of mice. Figure 26. (A) The whole animal bioluminescence imaging of the control group animals after being injected with 1x1064T1-Luc cells at the right mammary pad; (B) The whole animal bioluminescence imaging of the compound 15 treated recued animals after being challenged with 1x1064T1-Luc cells at the mammary pad on the other side of the Balb / c female mice on the 66thday after the “15 days treatment” with compound 15. Luciferin–Ds, 100 µL, 100 mg / kg body weight dose was administered intraperitoneally, and images were captured at different time points after the 4T1-Luc cells implantations (at 15 min, 11 h, 24 h, 36 h, 48 h, 72 h, 5thday, 8thday, 11thday, 14thday, 17thday and 19thday) by IVIS® Lumina III, PerkinElmer, USA; (C) Quantification of bioluminescence of the cell implanted region using Living Image 4.5.4 software. Data represent mean ± SD (n=4). Figure 27. (A) Representative whole animal fluorescence imaging of MOC2 tumor- bearing mice. The blue circle indicates the tumor region; (B) fluorescence intensity after i.v. injection with Dil-loaded PEG-PE micelles on days 0, 5, 10, and 15 by IVIS® Lumina III, PerkinElmer, USA; C) The body weights of mice during the treatment period; D) Graphical representation of tumor volume vs. days during treatment. (E) Representative whole animal body image of tumor-bearing mice on the 15thday.Figure 28. (A) Images representing the Ki67 stained tumor sections; and (B) Corresponding graph representing the percentage of Ki67 positive cells in Gemcitabine, HDACi Physical mixture of Gemcitabine and HDACi and compound 15 treated mice. Figure 29. (A) Representative FACS plot and (B) its percentage population of CD45+, G-MDSC, M-MDSC, Total Macrophages and M1& M2 macrophage cells in the blood of 4T1cells tumor developed animals with Control, Gemcitabine, HDACi, Physical mixture of Gemcitabine+HDACi and compound 15 treatment respectively. *p < 0.05 vs, **p < 0.01 vs, ***p < 0.001 vs. Disease control and #p < 0.05, ##p < 0.01 vs Gemcitabine. Figure 30. (A) Representative FACS plot and its (B) percentage population of CD45+, G-MDSC, M-MDSC, Total Macrophages, and M1& M2 macrophage cells in Spleen of 4T1cells tumor developed animals with Control, Gemcitabine, HDACi, Physical mixture of Gemcitabine+HDACi and compound 15 treatment respectively. *p < 0.05 vs, **p < 0.01 vs, ***p < 0.001 vs. Disease control and #p < 0.05, ##p < 0.05 vs Gemcitabine. Figure 31. (A) Representative FACS plot and (B) its percentage population of CD45+, G-MDSC, M-MDSC, Total Macrophages, and M1& M2 macrophage cells in Tumor tissue of 4T1cells tumor developed animals with Control, Gemcitabine, HDACi, Physical mixture of Gemcitabine+HDACi and compound 15 treatment respectively. *p < 0.05 vs, **p < 0.01 vs, ***p < 0.001 vs. Disease control and #p < 0.05, ##p < 0.01 vs Gemcitabine. Figure 32. (A) The graph represents the percentage cell (H9c2 cells) viability in different concentrations of compound 15 (Mean ± SD, n =12). ****P<0.0001 vs control (one-way ANOVA with Dunnett's test). (B) The graph represents heart weight, body weight, and heart weight by tail length ratio (Mean ± SD, n= 8) after treatment with saline and compound 15 in mice. ****P<0.0001 vs control (unpaired t-test). Figure 33. (A) The image shows the electrocardiograph of a heartbeat and (B) different parameters of the heart altered in 7 days; (C) the electrocardiograph of aheartbeat and (D) different parameters of the heart altered in 15 days study period. Data were represented by Mean ±SD (n = 8), *p<0.05 vs control (unpaired t-test). Figure 34. (A)The image shows the left ventricle of the heart and motion-mode (M- mode) at 7 days and (B) The graph shows different parameters of heart altered in 7 days; (C) the left ventricle of the heart and motion-mode (M-mode) at 15 days and (D) The graph shows different parameters of heart altered in 15 Days (Band 15 days of study). The red dotted lines represent End Diastolic Diameter and the green dotted lines represent End Systolic Diameter. Data were represented by Mean ± SD (n = 8). *p<0.05 (unpaired t-test). DETAILED DESCRIPTION OF THE INVENTION

[0017] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure.

[0018] In the following description, embodiments of the invention are described in sufficient detail to enable those skilled in the art to practice the invention and it is understood that other embodiments may be utilized and that logical changes may be made without departing from the spirit or scope of the invention. To avoid detail not necessary to enable those skilled in the art to practice the embodiments described herein, the description may omit certain information known to those skilled in the art. The following brief description is, therefore, not to be taken in a limiting sense and the scope of the illustrative embodiments are defined only by the claims appended to the complete specification to be filed hereafter.

[0019] As used throughout this description, the word “may” is used in a permissive sense (i.e. meaning having the potential to), rather than the mandatory sense, (i.e. meaning must). Further, the words “a” or “an” mean “at least one” and the word “plurality” means “one or more” unless otherwise mentioned.

[0020] Furthermore, the terminology and phraseology used herein are solely used fordescriptive purposes and should not be construed as limiting in scope. Language such as “including,” “comprising,” “having,” “containing,” or “involving,” and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents, and additional subject matter not recited, and is not intended to exclude other additives, components, integers, or steps. Likewise, the term “comprising” is considered synonymous with the terms “including” or “containing” for applicable legal purposes. Any discussion of documents acts, materials, devices, articles, and the like are included in the specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention.

[0021] In this disclosure, whenever a composition or an element or a group of elements is preceded with the transitional phrase “comprising”, it is understood that the same composition, element, or group of elements with transitional phrases “consisting of”, “consisting”, selected from the group of consisting of, “including”, or “is” preceding the recitation of the composition, element or group of elements and vice versa.

[0022] The term “alkyl”, unless otherwise specified, refers monoradical saturated hydrocarbon.

[0023] The term “alkoxy” denotes O-alkyl and; alky is same as defined earlier.

[0024] The term “aryl” relates to aromatic monocyclic or polycyclic having 5-12 carbon atom in a ring. Examples of aryl groups include, but are not limited to, phenyl naphthyl and the like.

[0025] The term “heteroaryl” relates to aromatic monocyclic or polycyclic ring having 5-12 atoms, in which 1 to 4 carbon atoms in a ring are replaced by heteroatoms selected from the group consisting of O, N and S. Examples of heteroaryl groups include, but are not limited to, thiophenyl, furanyl, pyrrolyl, pyridyl, pyrazinyl, pyrimidinyl, imidazolyl, pyrazolyl and the like.

[0026] The term “heterocycle” relates to non-aromatic monocyclic or polycyclic ringhaving 5-12 atoms, in which 1 to 4 carbon atoms in a ring are replaced by heteroatoms selected from the group consisting of O, N and S. Examples of heterocyclic groups include, but are not limited to, piperazinyl, piperidinyl, morpholinyl, thiamorpholinyl and the like.

[0027] The present invention intends to address the afore disclosed prior art needs by designing and synthesizing bifunctional conjugate molecules being a single molecule but towards different targets. In one embodiment, the bifunctional conjugate molecules comprising a functionalized HDAC inhibitory moiety covalently linked with a broad- spectrum chemotherapeutic agent using a linker. In one embodiment, the present invention provides the novel bifunctional conjugate molecules comprising a functionalized HDAC inhibitory moiety covalently linked with a broad-spectrum chemotherapeutic agent being Gemcitabine using a linker.

[0028] In one embodiment, the present invention provides a bifunctional conjugate molecule of general Formula I.or a pharmaceutically acceptable salt, hydrate, or solvate thereof. wherein; Linker is selected from -CO-CH2-CH2-CO-NH-CH2- or -CO-CH2-CH2-CO-. HDACi is selected from formula 1.1 to 1.5wherein; m is 0-5, n is 0-5, Z is selected from CH2, O, S, or NH; X1, X2and X3are independently selected from CH or N; R1, R2and R3are independently selected from hydrogen, halogen, cyano, nitro, straight or branched chain C1-C6alkyl, C1-C4alkoxy, —OH, amino, —C(O)NH2, — NHC(O)—, Aryl, heteroaryl, heterocycle, wherein alkyl, alkoxy, —OH, amino, aryl, heteroaryl and heterocycle are optionally substituted with 1-3 substituents selected from hydrogen, halogen, cyano, nitro, C1-C6alkyl, C1-C4alkoxy, —OH and amino.

[0029] In one embodiment, the present invention provides bifunctional conjugate molecules of general Formula 1 or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein bifunctional conjugate molecules, comprises: (a) a broad spectrum chemotherapeutic agent; (b) a linker; and (c) a histone deacetylase inhibitor (HDACi).

[0030] In one embodiment, a broad-spectrum chemotherapeutic agent is Gemcitabine.

[0031] In another embodiment of the present invention, bifunctional conjugatemolecule is selected from the compounds: N1-(4-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl) tetra-hydrofuran 2-yl)-2- oxo-1,2-di-hydropyrimidin-4-yl) amino)-4-oxobutanamido) methyl) phenyl) -N8- hydroxyoctanediamide, having formula:N1-(3-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl) tetrahydro furan-2-yl) 2- oxo-1,2 di hydropyrimidin-4yl) amino) 4-oxo butanamido) methyl)phenyl) - N8hydroxyoctanediamide, having formula:N1-(1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)-N4-(3-(3-(3-(hydroxyamino)-3oxopropoxy)propanamido)- benzyl) succinimide having formula:N1-(1-(3,3-difluoro-4-hydroxy-5-(hydroxy methyl) tetrahydrofuran-2-yl)-2-oxo-1,2-di hydro pyrimidin-4-yl)-N4-(3-(3-((3-(hydroxy amino)-3-oxopropyl) amino)- propanamido) benzyl) succinimide having formula:N1-(4-((2-amino phenyl) carbamoyl) benzyl)-N4-(1-(3,3-difluoro-4-hydroxy -5- (hydroxy methyl) tetrahydrofuran-2-yl)-2-oxo-1,2-di hydropyrimidin 4yl) succinimide, having formula:N1-(4-((2-amino-4-fluoro phenyl) carbamoyl) benzyl)-N4-(1-(3,3-difluoro-4-hydroxy- 5-(hydroxy methyl) tetra hydro furan-2-yl)-2-oxo-1,2-dihydropyrimidin-4yl) succinimide, having formula:N1-(4-((2-amino-4-fluoro phenyl) carbamoyl) benzyl)-N4-(1-(3,3-di fluoro-4-hydroxy- 5-(hydroxyethyl) tetra hydro furan-2-yl)-2-oxo-1,2-dihydro pyrimidin-4-yl) succinimide, having formula:4-((2-(4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo- 1,2-dihydropyrimidin-4-yl)amino)-4-oxobutanoyl)-1,2,3,4-tetrahydro-5H-pyrido[4,3- b]indol-5-yl)methyl)-N-hydroxybenzamide, having formula:N1-((5-((2-amino-4-phenyl)carbamoyl)pyrazin-2-yl)methyl)-N4-(1-(3,3-difluoro-4- hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4- yl)succinimide, having formula:N1-((5-((2-amino-4-fluorophenyl)carbamoyl)pyrazin-2-yl)methyl)-N4-(1-(3,3-difluoro- 4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4- yl)succinimide, having formula:N1-((6-((2-aminophenyl)carbamoyl)quinolin-2-yl)methyl)-N4-(1-(3,3-difluoro-4- hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydro-pyrimidin-4-yl)- succinamide, having formula:N1-(1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)-N4-((6-((2-hydroxyphenyl)carbamoyl)quinolin-2-yl)- methyl)succinimide, having formula:N1-(4-(((5-((2-aminophenyl)carbamoyl)pyrazin-2-yl)amino)methyl)benzyl)-N4-(1- (3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)succinimide, having formula:

[0032] In another embodiment of the present invention, the bifunctional conjugate molecules exist in different forms such as amorphous or crystalline solid.

[0033] Another embodiment of the present invention provides a process for the preparation of bifunctional conjugate molecule (Compound 7) comprises the following steps (scheme 1); (a) Reacting 4-amino benzylamine (1) with Di-tert-butyl dicarbonate to obtain tert- butyl (4-amino benzyl) carbamate (2); (b) Reacting tert-butyl (4-aminobenzyl) carbamate (2) as obtained in step (a), with methyl-8-chloro-8-oxo octanoate to obtain methyl 8-((4-(((tert-butoxycarbonyl) amino) methyl) phenyl) amino)-8-oxo octanoate (3);(c) Treating methyl 8-((4-(((tert-butoxycarbonyl) amino) methyl) phenyl) amino)- 8-oxo octanoate (3) as obtained in step (b), with trifluoroacetic acid to yield methyl 8-( (4-(amino methyl) phenyl) amino)-8-oxooctanoate (4); (d) Reacting methyl 8-( (4-(amino methyl) phenyl) amino)-8-oxooctanoate (4) as obtained in step (c), with succinic anhydride to obtain 4-((4-(8-methoxy-8-oxo- octanamido) benzyl) amino)-4-oxobutanoic acid (5); (e) Reacting 4-((4-(8-methoxy-8-oxooctanamido)benzyl)amino)-4-oxo butanoic acid (5) as obtained in step (d) with Gemcitabine hydrochloride to obtain methyl 8- ((4-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetra-hydrofuran-2-yl)-2- oxo-1,2-dihydropyrimidin-4-yl)amino)-4-oxobutanamido)methyl)-phenyl)amino)- 8-oxooctanoate (6); and (f) Reacting methyl 8-((4-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetra- hydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-4- oxobutanamido)methyl)-phenyl)amino)-8-oxooctanoate (6) as obtained in step (e) with hydroxylamine to obtain N1-(4-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl) tetra-hydrofuran 2-yl)-2-oxo-1,2-di-hydropyrimidin-4-yl) amino)-4-oxobutanamido) methyl) phenyl) -N8-hydroxyoctanediamide (Compound 7).Scheme 1. Synthesis of compound 7. Reagents and conditions: a) Di- tert- butyldicarbonate, Et3N, DMAP, Ethanol, 0ºC – RT, 1 h; b) Methyl 8-chloro-8- oxooctanoate, NEt3, DCM, RT, 2 h; c) TFA: DCM (1:1), 0ºC – RT, 2 h; d) succinic anhydride, EDCI, DMAP, pyridine : DCM, RT, overnight; e) Gemcitabine, NMM, EDCI, HOBT, DMF:DMSO (3:1), RT, overnight; f) 50% aq NH2OH, 2N NaOH, MeOH, RT, 3 h.

[0034] Another embodiment of the present invention provides a process for the preparation of bifunctional conjugate molecule (Compound 15) comprises the following steps (Scheme 2); (a) Treating 3-aminobenzonitrile (8) with 10% Pd / C to yield 3-(amino methyl) aniline (9); (b) Reacting 3-(amino methyl) aniline (9) as obtained in step (a), with Di-tert-butyl di carbonate to obtain tert-butyl (3-aminobenzyl) carbamate (10);(c) Reacting tert-butyl (3-aminobenzyl) carbamate (10) as obtained in step (b), with methyl-8-chloro-8-oxo octanoate to obtain methyl 8-((3-(((tert-butoxycarbonyl) amino) methyl) phenyl) amino)-8-oxo octanoate (11); (d) Treating methyl 8-((3-(((tert-butoxycarbonyl) amino) methyl) phenyl) amino)- 8-oxooctanoate (11) as obtained in step (c), with trifluoroacetic acid to yield methyl 8-( (3-(amino methyl) phenyl) amino)-8-oxooctanoate (12); (e) Reacting methyl 8-((3-(amino methyl) phenyl) amino)-8-oxooctanoate (12) as obtained in step (d), with succinic anhydride to obtain 4-((3-(8-methoxy-8- oxooctanamido) benzyl) amino)-4-oxobutanoic acid (13); (f) Reacting 4-((3-(8-methoxy-8-oxooctanamido)benzyl)amino)-4-oxobutanoic acid (13) as obtained in step (e), with Gemcitabine hydrochloride to obtain methyl 8-((3- ((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo- 1,2-dihydropyrimidin-4-yl) amino)-4 oxo butanamido) methyl)-phenyl) amino)-8- oxooctanoate (14); and (g) Reacting methyl 8-((3-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl) amino)-4-oxobutanamido) methyl) - phenyl) amino)-8-oxo-octanoate (14) as obtained in step (f), with hydroxylamine to obtain N1-(3-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl) tetrahydro furan-2-yl) 2-oxo-1,2 di hydropyrimidin-4yl) amino) 4-oxo butanamido) methyl)phenyl)N8hydroxyoctanediamide (Compound 15).Scheme 2. Synthesis of compound 15. Reagents and conditions: a) Hydrogen balloon, 10% (w / w) Pd on activated charcoal (Pd / C), methanol, RT, overnight; b) Di- tert-butyldicarbonate, Et3N, Ethanol, 0ºC – RT, 1 h; c) methyl 8-chloro-8- oxooctanoate, Et3N, DCM, RT, 2 h; d) TFA: DCM (1:1), RT, 2 h; e) succinic anhydride, EDCI, DMAP, pyridine : DCM, RT, overnight; f) Gemcitabine, NMM, EDCI, HOBT, DMF:DMSO ( 3:1) RT, overnight; g) 50% aq NH2OH, 2N NaOH, MeOH, RT, 3 h.

[0035] Another embodiment of the present invention provides a process for the preparation of bifunctional conjugate molecule (Compound 21) comprises the following steps (Scheme 3); (a) Reacting tert-butyl 4-amino benzyl carbamate (2) with 3-hydroxypropanoic acid to obtain tert-butyl (4-(3-hydroxy propanamido) benzyl) carbamate (16); (b) Reacting tert-butyl (4-(3-hydroxy propanamido) benzyl) carbamate (16) as obtained in step (a), with methyl acrylate to obtain methyl 3-(3-((4-(((tert- butoxy carbonyl) amino) methyl) phenyl) amino)-3-oxopropoxy) propionate carbamate (17); (c) Treating methyl 3-(3-((4-(((tert-butoxycarbonyl) amino) methyl) phenyl) amino)-3oxopropoxy) propanate carbamate (17) as obtain in step (b), with trifluoroacetic acid to yield methyl 3-(3-((4-(amino methyl) phenyl) amino)-3- oxopropoxy) propionate (18); (d) Reacting methyl 3-(3-((4-(amino methyl) phenyl) amino)-3-oxopropoxy) propanoate (18) as obtained in step (c), with succinic anhydride to obtain 4-((4-(3- (3-methoxy-3-oxopropoxy) propanamido) benzyl) amino)-4-oxobutanoic acid (19); (e) Reacting 4-((4-(3-(3-methoxy-3-oxopropoxy)propanamido)benzyl)amino)-4- oxobutanoic acid (19) as obtained in step (d), with Gemcitabine hydrochloride to obtain methyl 3-(3-((3-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxyl methyl) tetra- hydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)4 oxobutanamido)methyl)-phenyl) amino)-3-oxopropoxy) propanoate (20); and (f) Reacting methyl 3-(3-((3-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxy methyl) tetra-hydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl) amino)-4oxobutanamido) methyl)-phenyl) amino)-3 oxo propoxy) propanoate (20) as obtained in step (e),with hydroxylamine to obtain of N1-(1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-N4-(3-(3- (3-(hydroxyamino)-3oxopropoxy)propanamido)-benzyl) succinamide (Compound 21).Scheme 3. Synthesis of compound 21.Reagents and conditions: a) 3-hydroxy propionic acid, EDCI, DMAP, pyridine:DCM (1:1), RT, 5 h; b) Methyl acrylate, NaOMe, THF, RT, 12 h; c) TFA:DCM (1:2), d) succinic anhydride, EDCI, DMAP, pyridine:DCM (1:1), RT, overnight; e) Gemcitabine, NMM, EDCI, HOBT, DMF:DMSO (3:1), RT, overnight; f) 50% aq NH2OH, 2N NaOH, MeOH, RT, 3 h.

[0036] Another embodiment of the present invention provides a process for the preparation of bifunctional conjugate molecule (Compound 30) comprises the following steps (Scheme 4); (a) Reacting 3-aminopropanoic acid (22) with trifluoroacetic anhydride to obtain 3- (2,2,2-trifluoroacetamido) propionoic acid (23);(b) Reacting 3-(2,2,2-trifluoroacetamido) propionoic acid (23) as obtained in step (a), with tert-butyl 4-amino benzyl carbamate (2) to obtain tert-butyl-(4-(3-(2,2,2- tri-fluoro-acetamido)-propanamido)-benzyl)-carbamate (24); (c) Treating tert-butyl-(4-(3-(2,2,2-tri-fluoro-acetamido)-propanamido)-benzyl)- carbamate (24) as obtained in step (b), with K2CO3to yield tert-butyl (4-(3- aminopropanamido) benzyl) carbamate (25); (d) Reacting tert-butyl (4-(3-aminopropanamido) benzyl) carbamate (25) as obtained in step (c), with methyl acrylate to obtain methyl 3-((3-((4-(((tert- butoxycarbonyl) amino) methyl) phenyl) amino)-3-oxopropyl) amino) propanoate (26); (e) Treating 3-((3-((4-(((tert-butoxycarbonyl)-amino)-methyl)-phenyl)-amino)-3- oxopropyl)-amino)-propanoate (26) as obtained in step (d), with trifluoroacetic acid to yield methyl 3-((3-((4-(aminomethyl-) phenyl)-amino)-3-oxopropyl)-amino)- propanoate (27); (f) Reacting methyl 3-((3-((4-(aminomethyl-) phenyl)-amino)-3-oxopropyl)- amino)-propanoate (27) as obtained in step (e), with succinic anhydride to obtain 4- ((4-(3-((3-methoxy-3-oxopropyl) amino) propanamido) benzyl) amino)-4- oxobutanoic acid (28); (g) Reacting 4-((4-(3-((3-methoxy-3- oxopropyl)amino)propanamido)benzyl)amino)-4-oxobutanoic acid (28) as obtained in step (f), with Gemcitabine hydrochloride to obtain methyl 3-((3-((3-((4-((1-(3,3- di fluoro-4-hydroxy-5-(hydroxy methyl) tetra-hydro furan-2-yl)-2-oxo-1,2-di hydro pyrimidin-4-yl) amino)-4-oxobutanamido) methyl) phenyl) amino)-3-oxo propyl) amino) propanoate (29); and (h) Reacting methyl 3-((3-((3-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxy methyl) tetra-hydro-furan-2-yl)-2-oxo-1,2-di hydropyrimidin-4-yl) amino)-4-oxo butanamido) methyl)-phenyl) amino)-3-oxopropyl) amino) propanoate (29) as obtained in step (g), with hydroxylamine to obtain N1-(1-(3,3-difluoro-4-hydroxy-5- (hydroxy methyl) tetrahydrofuran-2-yl)-2-oxo-1,2-di hydro pyrimidin-4-yl)-N4-(3- (3-((3-(hydroxy amino)-3-oxopropyl) amino)-propanamido) benzyl) succinamide (Compound 30).Scheme 4. Synthesis of compound 30. Reagents and conditions: a) Trifluororacetic acid (TFA), 0ºC – RT, overnight; b) compound 2, EDCI, DMAP, pyridine:DCM (1:1), RT, 5 h; (c) K2CO3, MeOH : H2O, THF, overnight; (d) Methyl acrylate, NaOMe, THF, e) TFA : DCM (1:2), 0ºC – RT, 2 h; f) succinic anhydride, EDCI, DMAP, pyridine:DCM (1:1), RT, overnight; g) Gemcitabine, NMM, EDCI, HOBT, DMF: DMSO (3:1), overnight; h) 50% aq NH2OH, 2N NaOH, MeOH, RT, 3 h.

[0037] Another embodiment of the present invention provides a process for the preparation of bifunctional conjugate molecule (Compound 37) comprises thefollowing steps (Scheme 5): (a) Reacting 4-(amino methyl) benzoic acid (31) with trifluoroacetic anhydride to obtain 4-((2,2,2-trifluoroacetamido) methyl) benzoic acid (32); (b) Reacting 4-((2,2,2-tri fluoro acetamido) methyl) benzoic acid (32) as obtained in step (a), with tert-Butyl (2-aminophenyl) carbamate to obtain tert-butyl (2-(4-((2,2,2- tri-fluoro-acetamido)-methyl)-benzamido)-phenyl)-carbamate (33); (c) Treating tert-butyl (2-(4-((2,2,2-trifluoro acetamido) methyl) benzamido) phenyl) carbamate (33) as obtained in step (b), with K2CO3to yield tert-butyl (2-(4-(amino methyl) benzamido) phenyl) carbamate (34); (d) Reacting tert-butyl (2-(4-(amino methyl) benzamido) phenyl) carbamate (34) as obtained in step (c), with succinic anhydride to obtain 4-((4-((2-((tert-butoxycarbonyl) amino) phenyl) carbamoyl) benzyl) amino)-4-oxobutanoic acid (35); (e) Reacting 4-((4-((2-((tert-butoxycarbonyl) amino) phenyl) carbamoyl) benzyl) amino)-4-oxobutanoic acid (35) as obtained in step (d), with Gemcitabine hydrochloride to obtain tert-butyl (2-(4-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-4- oxobutanamido)-methyl) -benzamido)-phenyl)carbamate (36); and (f) Treating tert-butyl (2-(4-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl) amino)-4-oxobutanamido) methyl)-benzamido) phenyl)-carbamate (36) as obtained in step (e), with HCl to obtain N1-(4-((2-amino phenyl) carbamoyl) benzyl)-N4-(1-(3,3-difluoro-4-hydroxy -5- (hydroxy methyl) tetrahydrofuran-2-yl)-2-oxo-1,2-di hydropyrimidin-4-yl) succinamide (Compound 37).

[0038] Another embodiment of the present invention provides a process for the preparation of bifunctional conjugate molecule (Compound 42) comprises the following steps (Scheme 5): (a) Reacting 4-((2,2,2-trifluoro acetamido) methyl) benzoic acid (32) with tert-butyl (2-amino-5-fluoro phenyl) carbamate to obtain tert-butyl (5-fluoro-2-(4-((2,2,2-tri- fluoro-acetamido)-methyl)-benzamido)-phenyl)-carbamate (38);(b) Treating tert-butyl (5-fluoro-2-(4-((2,2,2-trifluoroacetamido)-methyl)-benzamido)- phenyl)-carbamate (38) as obtained in step (a), with K2CO3to yield tert-butyl (2-(4- (amino methyl) benzamido)-5-fluorophenyl) carbamate (39). (c) Reacting tert-butyl (2-(4-(amino methyl) benzamido)-5-fluorophenyl) carbamate (39) as obtained in step (b), with Succinic anhydride to obtain 4-((4-((2-((tert- butoxycarbonyl)-amino)-4-fluorophenyl)-carbamoyl)-benzyl)-amino)-4-oxobutanoic acid (40); (d) Reacting 4-((4-((2-((tert-butoxy carbonyl)-amino)-4-fluoro phenyl)- carbamoyl)- benzyl)- amino)- 4-oxobutanoic acid (40) as obtained in step (c), with Gemcitabine hydrochloride to obtain tert-butyl (2-(4-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-4- oxo-butanamido)-methyl)-benzamido)-5-fluorophenyl)carbamate (41); and (e) Treating tert-butyl (2-(4-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl) amino)-4-oxo-butanamido)- methyl)-benzamido)-5fluoro- phenyl)-carbamate: (41) as obtained in step (d), with HCl to yield N1-(4-((2-amino-4-fluoro phenyl) carbamoyl) benzyl)-N4-(1-(3,3- difluoro-4-hydroxy-5-(hydroxy methyl) tetra hydro furan-2-yl)-2-oxo-1,2- dihydropyrimidin-4yl) succinamide (Compound 42).Scheme 5. Synthesis of compound 37, 42 and 47. Reagents and conditions: a)Trifluoroacetic acid (TFA), 0°C – RT, 2 h; b) Substituted aniline, EDCI, DMAP, pyridine:DCM, RT, 8 h, (c) K2CO3, MeOH : H2O, THF, 4 h, (d) Succinic anhydride, EDCI, DMAP, pyridine:DCM (1:1), RT, overnight; (e) Gemcitabine, NMM, EDCI, HOBT, DMF:DMSO (3:1), RT, overnight; (f) 4M HCl in dioxane, DCM, RT, 2 h.

[0039] Another embodiment of the present invention provides a process for the preparation of bifunctional conjugate molecule (Compound 47) comprises the following steps (Scheme 5): (a) Reacting 4-((2,2,2-trifluoro acetamido) methyl) benzoic acid (32) with tert- butyl (2-amino-4-(thiophene-2-yl) phenyl) carbamate to obtain tert-butyl (4- (thiophen-2-yl)-2-(4-((2,2,2-trifluoroacetamido)-methyl) benzamido)-phenyl)- carbamate (43); (b) Treating tert-butyl (4-(thiophen-2-yl)-2-(4-((2,2,2-trifluoroacetamido)- methyl) benzamido)-phenyl)-carbamate (43) as obtained in step (a), with K2CO3to yield tert-butyl (2-(4-(amino methyl) benzamido)-4-(thiophen-2-yl) phenyl)-carbamate (44); (c) Reacting tert-butyl (2-(4-(amino methyl) benzamido)-4-(thiophen-2-yl) phenyl) carbamate (44) as obtained in step (b), with succinic anhydride to obtain 4-((4-((2-((tert-butoxycarbonyl)-amino)-5-(thiophen-2-yl)-phenyl)- carbamoyl)-benzyl)-amino)-4-oxobutanoic acid (45); (d) Reacting 4-((4-((2-((tert-butoxycarbonyl)amino)-5-(thiophen-2- yl)phenyl)carbamoyl)benzyl)amino)-4-oxobutanoic acid (45) as obtained in step (c), with Gemcitabine hydrochloride to obtain tert-butyl (2-(4-((4-((1-(3,3- di fluoro-4-hydroxy-5-(hydroxy methyl) tetra hydrofuran-2-yl)-2-oxo-1,2- dihydro pyrimidin-4-yl) amino)-4-oxobutanamido) methyl) benzamido)-4- (thiophen-3-yl) phenyl)carbamate (46); and (e) Treating tert-butyl (2-(4-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4- yl)amino)-4-oxo-butanamido)-methyl)-benzamido)-5fluoro- phenyl)- carbamate: (46) as obtained in step (d), with HCl to obtain N1-(4-((2-amino-4-fluoro phenyl) carbamoyl) benzyl)-N4-(1-(3,3-di fluoro-4-hydroxy-5- (hydroxyethyl) tetra hydro furan-2-yl)-2-oxo-1,2-dihydro pyrimidin-4-yl) succinamide (Compound 47).

[0040] Another embodiment of the present invention provides a process for the preparation of bifunctional conjugate molecule (Compound 54) comprises the following steps (Scheme 6): (a) Reacting phenyl hydrazine (48) with tert-butyl 4-oxopiperidine-1- carboxylate to obtain tert-butyl 1,3,4,5-tetrahydro-2H-pyrido[4,3-b] indole-2- carboxylate (49); (b) Reacting tert-butyl 1,3,4,5-tetra hydro-2H-pyrido [4,3-b] indole-2- carboxylate (49) as obtained in step (a), with methyl 4-(bromo methyl) benzoate to obtain tert-butyl 5-(4-(methoxy carbonyl) benzyl)-1,3,4,5- tetrahydro-2H-pyrido[4,3-b] indole-2-carboxylate (50); (c) Reacting tert-butyl 5-(4-(methoxy carbonyl) benzyl)-1,3,4,5-tetrahydro-2H- pyrido[4,3-b] indole-2-carboxylate (50) as obtained in step (b), with trifluoroacetic acid to obtain methyl 4-((1,2,3,4-tetra hydro-5H-pyrido [4,3-b] indol-5-yl) methyl) benzoate (51); (d) Reacting methyl 4-((1,2,3,4-tetrahydro-5H-pyrido [4,3-b] indol-5-yl) methyl) benzoate (51) as obtained in step (c), with succinic anhydride to obtain 4-(5-(4-(methoxy carbonyl) benzyl)-1,3,4,5-tetrahydro-2H-pyrido[4,3-b] indol- 2-yl)-4-oxobutanoic acid (52); (e) Reacting 4-(5-(4-(methoxycarbonyl)benzyl)-1,3,4,5-tetrahydro-2H- pyrido[4,3-b]indol-2-yl)-4-oxobutanoic acid (52) as obtained in step (d), with Gemcitabine hydrochloride to obtain methyl 4-((2-(4-((1-(3,3-difluoro-4- hydroxy-5-(hydroxy methyl) tetra-hydrofuran-2-yl)-2-oxo-1,2-di hydro pyrimidin-4-yl)amino)-4-oxo butanoyl)-1,2,3,4-tetra-hydro-5H-pyrido[4,3- b]indol-5-yl)methyl)benzoate (53); and (f) Reacting methyl 4-((2-(4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl) tetra-hydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl) amino)-4- oxobutanoyl)-1,2,3,4-tetra-hydro-5H-pyrido[4,3-b] indol-5-yl) methyl)benzoate (53) as obtained in step (e), with hydroxylamine to obtain 4-((2-(4- ((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo- 1,2-dihydropyrimidin-4-yl)amino)-4-oxobutanoyl)-1,2,3,4-tetrahydro-5H- pyrido[4,3-b]indol-5-yl)methyl)-N-hydroxybenzamide (54).Scheme 6. Synthesis of compound 54. Reagents and conditions: a) tert-butyl 4- oxopiperidine-1-carboxylate, Ethanol, reflux, 3 h; b) Methyl 4-(bromomethyl) benzoate, KOtBu, DMF, 80ºC, 3 h; c) TFA:DCM (1:2), 0ºC – RT, 2 h; d) succinic anhydride, EDCI, DMAP, pyridine: DCM (1:1), RT, overnight; e) Gemcitabine, NMM, EDCI, HOBT, DMF:DMSO (3:1), RT, overnight; f) 50% aq NH2OH, 2N NaOH, MeOH, RT, 3 h.

[0041] Another embodiment of the present invention provides a process for the preparation of bifunctional conjugate molecule (Compound 65) comprises the following steps (Scheme 7): (a) Reacting 5-methyl pyrazine carboxylic acid (55) with methanol and conc. sulfuric acid to obtain methyl 5-methylpyrazine-2-carboxylate (56);(b) Reacting 5-methyl pyrazine carboxylic acid (55) as obtained in step (a) with N-Bromo- succinimide to obtain methyl 5-(bromomethyl)pyrazine-2- carboxylate (57); (c) Reacting methyl 5-(bromomethyl)pyrazine-2-carboxylate (57) as obtained in step (b), with hexamethylene tetramine to obtain methyl 5- (aminomethyl)pyrazine-2-carboxylate (58); (d) Reacting methyl 5-(aminomethyl)pyrazine-2-carboxylate (58) as obtained in step (c), with trifluoroacetic anhydride to obtain 5-((2,2,2- trifluoroacetamido)methyl)pyrazine-2-carboxylate acid (59); (e) Treating 5-((2,2,2-trifluoroacetamido)methyl)pyrazine-2-carboxylate (59) as obtained in step (d), with LiOH to yield methyl 5-((2,2,2- trifluoroacetamido)methyl)pyrazine-2-carboxylic acid (60); (f) Reacting 5-((2,2,2-trifluoroacetamido)methyl)pyrazine-2-carboxylic acid (60) as obtained in step (e), with tert-butyl (2-aminophenyl) carbamate to obtain tert-butyl (2-(5-((2,2,2-trifluoroacetamido)methyl)pyrazine-2- carboxamido)phenyl)carbamate (61); (g) Treating tert-butyl (2-(5-((2,2,2-trifluoroacetamido)methyl)pyrazine-2- carboxamido)phenyl)carbamate (61) as obtained in step (e), with K2CO3to yield tert-butyl (2-(5-(aminomethyl)pyrazine-2- carboxamido)phenyl)carbamate (62); (h) Reacting tert-butyl (2-(5-(aminomethyl)pyrazine-2- carboxamido)phenyl)carbamate (62) as obtained in step (g), with succinic anhydride to obtain 4-(((5-((2-((tert-butoxycarbonyl)-amino)-4-phenyl)- carbamoyl)-pyrazin-2-yl)methyl)amino)-4-oxobutanoic acid (63); (i) Reacting 4-((4-((2-((tert-butoxycarbonyl) amino) phenyl) carbamoyl) benzyl) amino)-4-oxobutanoic acid (63) as obtained in step (h), with Gemcitabine hydrochloride to obtain tert-butyl (2-(5-((4-((1-(3,3-difluoro-4- hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)amino)-4-oxobutanamido)-methyl)pyrazine-2- carboxamido)-5-phenyl)carbamate (64); and (j) Treating tert-butyl (2-(5-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4- yl)amino)-4-oxobutanamido)-methyl)pyrazine-2-carboxamido)-5-phenyl)carbamate (64) as obtained in step (i), with HCl to obtain N1-((5-((2- amino-4-phenyl)carbamoyl)pyrazin-2-yl)methyl)-N4-(1-(3,3-difluoro-4- hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin- 4-yl)succinamide (65)Scheme 7. Synthesis of compound 65 and 70. Reagents and Conditions: a) MeOH, H2SO4, Reflux, 6 h; b) N-Bromo- succinimide, Azobisisobutyronitrile (AIBN), CCl4, 80ºC, 6 h; c) i. Hexamethylene tetramine (HMTA), Chloroform, 18 h, ii. 2N HCl, Methanol, 70 ºC, 3h; d) trifluoroacetic anhydride, RT, 2 h; e) aq. LiOH, Methanol, RT, 2 h; f) tert-Butyl (2-aminophenyl) carbamate or tert-butyl (2-amino-5-fluorophenyl)carbamate, 4-(dimethyl amino) pyridine, 1-(3- Dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride, DCM: Pyridine (1:1), RT, 12 h; g) K2CO3, MeOH : THF : H2O (1:1:0.5), RT, overnight; h) Succinic anhydride, 1-ethyl-3-dimethyl amino propyl carbodiimide (EDC), 4-dimethyl aminopyridine (DMAP), DCM : Pyridine (1:1), RT, overnight; i) Gemcitabine hydrochloride, 1-ethyl-3-dimethyl amino propyl carbodiimide (EDC), 1- Hydroxybenzotriazole, N-Methylmorpholine, DMF: DMSO (3:1), RT, overnight; j) 4M HCl in 1,4-dioxane, DCM, RT, 2 h.

[0042] Another embodiment of the present invention provides a process for the preparation of bifunctional conjugate molecule (Compound 70) comprises the following steps (Scheme 7): (a) Reacting 5-((2,2,2-trifluoroacetamido)methyl)pyrazine-2-carboxylic acid (60) with tert-butyl (2-amino-5-fluorophenyl)carbamate to obtain tert-butyl (5- fluoro-2-(5-((2,2,2-trifluoroacetamido)methyl)pyrazine-2- carboxamido)phenyl)carbamate (66); (b) Treating tert-butyl (5-fluoro-2-(5-((2,2,2-trifluoroacetamido)methyl)- pyrazine-2-carboxamido)phenyl)-carbamate (66) as obtained in step (a), with K2CO3to yield tert-butyl (2-(5-(aminomethyl)pyrazine-2- carboxamido)phenyl)-carbamate (67); (c) Reacting tert-butyl (2-(5-(aminomethyl)pyrazine-2-carboxamido)-5- fluorophenyl)carbamate (67) as obtained in step (b), with succinic anhydride to obtain 4-(((5-((2-((tert-butoxycarbonyl)amino)-4-fluorophenyl)carbamoyl)- pyrazin-2-yl)methyl)amino)-4-oxobutanoic acid (68); (d) Reacting 4-(((5-((2-((tert-butoxycarbonyl)amino)-4- fluorophenyl)carbamoyl)-pyrazin-2-yl)methyl)amino)-4-oxobutanoic acid (68) as obtained in step (c), with Gemcitabine hydrochloride to obtain tert-butyl (2- (5-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-2- oxo-1,2-dihydropyrimidin-4-yl)amino)-4-oxobutanamido)-methyl)pyrazine-2- carboxamido)-5-fluorophenyl)carbamate (69); and (e) Treating tert-butyl (2-(5-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4- yl)amino)-4-oxobutanamido)-methyl)pyrazine-2-carboxamido)-5- fluorophenyl)carbamate (69) as obtained in step (d), with HCl to yield N1-((5- ((2-amino-4-fluorophenyl)carbamoyl)pyrazin-2-yl)methyl)-N4-(1-(3,3- difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)succinamide (70).

[0043] Another embodiment of the present invention provides a process for the preparation of bifunctional conjugate molecule (Compound 81) comprises thefollowing steps (Scheme 8): (a) Reacting 2-methylquinoline-6-carboxylic acid with methanol and sulfuric acid to obtain methyl 2-methylquinoline-6-carboxylate (72); (b) Reacting methyl 2-methylquinoline-6-carboxylate (72) as obtained in step (a), with N-bromosuccinimide to obtain methyl 2-(bromomethyl) quinoline-6- carboxylate (73); (c) Reacting methyl 2-(bromomethyl)quinoline-6-carboxylate (73) as obtained in step (b), with hexamethylenetetramine to obtain methyl 2- (aminomethyl)quinoline-6-carboxylate (74); (d) Reacting methyl 2-(aminomethyl)quinoline-6-carboxylate (74) as obtained in step (c), with trifluoroacetic anhydride to obtain methyl 2-((2,2,2- trifluoroacetamido)methyl)quinoline-6-carboxylate (75); (e) Treating methyl 2-((2,2,2-trifluoroacetamido)methyl)quinoline-6- carboxylate (75) as obtained in step (d), with LiOH to yield 2-((2,2,2- trifluoroacetamido)methyl)quinoline-6-carboxylic acid (76); (f) Reacting 2-((2,2,2-trifluoroacetamido)methyl)quinoline-6-carboxylic acid (76) as obtained in step (e), with tert-butyl (2-aminophenyl) carbamate to obtain tert-butyl (2-(2-((2,2,2-trifluoroacetamido)methyl)quinoline-6- carboxamido)phenyl)carbamate (77); (g) Treating tert-butyl (5-fluoro-2-(5-((2,2,2-trifluoroacetamido)methyl)- pyrazine-2-carboxamido)phenyl)-carbamate (77) as obtained in step (f), with K2CO3to yield tert-butyl (2-(2-(aminomethyl)quinoline-6- carboxamido)phenyl)carbamate (78); (h) Reacting tert-butyl (2-(2-(aminomethyl)quinoline-6- carboxamido)phenyl)carbamate (78) as obtained in step (g), with succinic anhydride to obtain 4-(((6-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)quinolin-2-yl)methyl)amino)-4- oxobutanoic acid (79); (i) Reacting 4-(((6-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)quinolin-2-yl)methyl)amino)-4- oxobutanoic acid (79) as obtained in step (h), with Gemcitabine hydrochloride to obtain tert-butyl (2-(2-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4- yl)amino)-4 oxo-butanamido)-methyl)quinoline-6- carboxamido)phenyl)carbamate (80); and (j) Treating tert-butyl (2-(2-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4- yl)amino)-4-oxobutanamido)-methyl)quinoline-6- carboxamido)phenyl)carbamate (80) as obtained in step (i), with HCl to obtain N1-((6-((2-aminophenyl)carbamoyl)quinolin-2-yl)methyl)-N4-(1-(3,3-difluoro- 4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydro- pyrimidin-4-yl)-succinamide (81)Scheme 8. Synthesis of compound 81. Reagents and Conditions: a) MeOH, H2SO4, Reflux, 6 h; b) N-Bromo- succinimide, Azobisisobutyronitrile (AIBN), CCl4, 80ºC, 4 h; c) Hexamethylene tetramine (HMTA), Chloroform, 18 h, 2N HCl, Methanol, 70 ºC, 3 h; d) trifluoroacetic anhydride, RT, 2 h; e) aq. LiOH, Methanol, RT, 2 h; f) tert-butyl (2-aminophenyl) carbamate or tert-butyl (2- amino-5-fluorophenyl)carbamate, 4-(dimethyl amino) pyridine, 1-(3- Dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride, DCM: Pyridine (1:1), RT, 12 h; g) K2CO3, MeOH : THF : H2O (1:1:0.5), RT, overnight; h) Succinicanhydride, 1-ethyl-3-dimethyl amino propyl carbodiimide (EDC), 4-dimethyl aminopyridine (DMAP), DCM : Pyridine (1:1), RT overnight; i) Gemcitabine hydrochloride, 1-ethyl-3-dimethyl amino propyl carbo diimide (EDC), 1- Hydroxybenzotriazole, N-Methylmorpholine, DMF: DMSO (3:1); j) 4M HCl in Dioxane, DCM, RT, 2 h.

[0044] Another embodiment of the present invention provides a process for the preparation of bifunctional conjugate molecule (Compound 85) comprises the following steps (Scheme 9): (a) Reacting 2-((2,2,2-trifluoroacetamido)methyl)quinoline-6-carboxylic acid (76) with 2-amino phenol to obtain N-(2-hydroxyphenyl)-2-((2,2,2- trifluoroacetamido)methyl)quinoline-6-carboxamide (82); (b) Treating N-(2-hydroxyphenyl)-2-((2,2,2- trifluoroacetamido)methyl)quinoline-6-carboxamide (82) as obtained in step (a), with K2CO3 to yield 2-(aminomethyl)-N-(2-hydroxyphenyl)quinoline-6- carboxamide (83); (c) Reacting 2-(aminomethyl)-N-(2-hydroxyphenyl)quinoline-6-carboxamide (83) as obtained in step (b), with Succinic anhydride to obtain 4-(((6-((2- hydroxyphenyl)carbamoyl)quinolin-2-yl)methyl)amino)-4-oxobutanoic acid (84); and (d) Reacting 4-(((6-((2-hydroxyphenyl)carbamoyl)quinolin-2- yl)methyl)amino)-4-oxobutanoic acid (84) as obtained in step (c), with Gemcitabine hydrochloride to obtain N1-(1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-N4- ((6-((2-hydroxyphenyl)carbamoyl)quinolin-2-yl)-methyl)succinimide (85).Scheme 9. Synthesis of compound 85. Reagents and Conditions: a) 2-amino phenol, 4-(dimethyl amino) pyridine, 1-(3-Dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride, DCM: Pyridine (1:1), RT, 12 h; b) K2CO3, MeOH : THF : H2O (1:1:0.5), RT, overnight; c) Succinic anhydride, 1-ethyl-3-dimethyl amino propyl carbodiimide (EDC), 4-dimethyl aminopyridine (DMAP), DCM : Pyridine (1:1), RT, overnight; d) Gemcitabine hydrochloride, 1-ethyl-3-dimethyl amino propyl carbodiimide (EDC), 1-Hydroxybenzotriazole, N-Methylmorpholine, DMF: DMSO (3:1), RT, overnight.

[0045] Another embodiment of the present invention provides a process for the preparation of bifunctional conjugate molecule (Compound 96) comprises the following steps (Scheme 10): (a) Reacting 4-(aminomethyl)benzonitrile (86) with trifluoroacetic anhydride to obtain N-(4-cyanobenzyl)-2,2,2-trifluoroacetamide (87); (b) Treating N-(4-cyanobenzyl)-2,2,2-trifluoroacetamide (87) as obtained in step (a), with Pd / C under hydrogen to obtain N-(4-(aminomethyl)benzyl)- 2,2,2-trifluoroacetamide (88); (c) Reacting N-(4-(aminomethyl)benzyl)-2,2,2-trifluoroacetamide (88) as obtained in step (b), with methyl 5-chloropyrazine-2-carboxylate (89) to obtainmethyl 5-((4-((2,2,2-trifluoroacetamido)methyl)benzyl)amino)pyrazine-2- carboxylate (90); (d) Treating methyl 5-((4-((2,2,2- trifluoroacetamido)methyl)benzyl)amino)pyrazine-2-carboxylate (90) as obtained in step (c), with LiOH to obtain 5-((4-((2,2,2- trifluoroacetamido)methyl)benzyl)amino)pyrazine-2-carboxylic acid (91); (e) Reacting 5-((4-((2,2,2-trifluoroacetamido)methyl)benzyl)amino)pyrazine-2- carboxylic acid (91) as obtained in step (d), with tert-butyl (2-aminophenyl) carbamate to obtained tert-butyl (2-(5-((4-((2,2,2-trifluoroacetamido)- methyl)benzyl) amino)-pyrazine-2-carboxamido)phenyl)carbamate (92); (f) Treating tert-butyl (2-(5-((4-((2,2,2-trifluoroacetamido)-methyl)benzyl) amino)-pyrazine-2-carboxamido)phenyl)carbamate (92) as obtained in step (e), with K2CO3to obtain tert-butyl (2-(5-((4- (aminomethyl)benzyl)amino)pyrazine-2-carboxamido)phenyl)carbamate) (93); (g) Reacting tert-butyl (2-(5-((4-(aminomethyl)benzyl)amino)pyrazine-2- carboxamido)phenyl)carbamate (93) as obtained in step (f), with succinic anhydride to obtain 4-((4-(((5-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)pyrazin-2- yl)amino)methyl)benzyl)amino)-4-oxobutanoic acid (94); (h) Reacting 4-((4-(((5-((2-((tert-butoxycarbonyl)-amino)-phenyl)- carbamoyl)pyrazin-2-yl)amino)-methyl)-benzyl)-amino)-4-oxobutanoic acid (94) as obtained in step (g), with Gemcitabine hydrochloride to obtain tert- butyl (2-(5-((4-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4- yl)amino)-4-oxobutanamido)methyl)benzyl)amino)pyrazine-2- carboxamido)phenyl)carbamate (95); and (i) Treating tert-butyl (2-(5-((4-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4- yl)amino)-4-oxo-butanamido)methyl)benzyl)amino)pyrazine-2- carboxamido)phenyl)carbamate (95) as obtained in step (h), with HCl to obtain N1-(4-(((5-((2-aminophenyl)carbamoyl)pyrazin-2-yl)amino)methyl)benzyl)- N4-(1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo- 1,2-dihydropyrimidin-4-yl)succinamide (96).Scheme 10. Synthesis of compound 96. Reagents and Conditions: a) trifluoroacetic anhydride, RT, 2h; b) Hydrogen balloon, 10% (w / w) Pd on activated charcoal (Pd / C), methanol, RT, overnight; c) Triethylamine, Isopropyl alcohol, 85 ºC, 8 h; d) aq. LiOH, Methanol, RT, 2 h; e) tert-Butyl (2-aminophenyl) carbamate, 4-(dimethyl amino) pyridine, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride, DCM: Pyridine (1:1), RT, 12 h; f) K2CO3, MeOH : THF : H2O (1:1:0.5), RT, overnight; g) Succinic anhydride, 1-ethyl-3-dimethyl amino propyl carbodiimide (EDC), 4-dimethyl aminopyridine (DMAP), DCM : Pyridine (1:1), RT, overnight; h) Gemcitabine hydrochloride, 1-ethyl-3-dimethyl amino propyl carbo diimide (EDC), 1- Hydroxybenzotriazole, N-Methylmorpholine, DMF: DMSO (3:1), RT, overnight; i) 4M HCl in Dioxane, DCM, RT, 2 h.

[0046] Another embodiment of the present invention provides a pharmaceuticalcomposition comprising an effective amount of a bifunctional conjugate molecule of said invention or a pharmaceutically acceptable salt, hydrate, or solvate thereof, and a pharmaceutical acceptable carrier, diluent, or excipient.

[0047] Yet another embodiment of the present invention provides use of a bifunctional conjugate molecule and aforementioned pharmaceutical composition for the treatment of cancer.

[0048] Yet another embodiment of the present invention provides use of a bifunctional conjugate molecule and aforementioned pharmaceutical composition as HDAC inhibitors.

[0049] Yet another embodiment of the present invention provides use of a bifunctional conjugate molecule for the treatment of cancer consisting of carcinoma, lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia, squamous cell cancer, lung cancer, , cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, , prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, and other lymphoproliferative disorders.

[0050] The following examples further describe and demonstrate embodiments within the scope of the present invention. The examples are given solely for the purpose of illustration and not to be construed as limitations of the present invention, as many variations are possible without departing from the spirit and scope of the invention. All embodiments apparent to a process in the art are deemed to fall within the scope of the present invention The general method of preparation of compounds described in scheme is provided below. EXAMPLES

[0051] Example 1: Experimental details 1.1 Chemistry section of materials and methods for the preparation of bifunctional conjugateReagents and conditions: a) Di -tert-butyl di carbonate, Et3N, EtOH, 0ºC – RT, 1h. 1. Preparation of tert-butyl (4-amino benzyl) carbamate (2) 4-amino benzylamine (1) (1.00g; 8.19mmol) was dissolved in 10 mL of ethanol. To the reaction mixture triethylamine (1.7 mL) was added at 0 °C. Di-tert-butyl dicarbonate (2.142g; 9.81mmol) was dissolved in ethanol in a separate round bottom flask. The ethanolic solution of di-tert-butyl di carbonate was slowly transferred into the stirring reaction mixture with a syringe, the reaction was stirred for another 30 min at room temperature. The reaction was monitored by thin layer chromatography (TLC). After completion of the reaction, the mixture was dissolved in water and extracted with ethyl acetate. The organic layer was collected, dried with sodium sulfate, and concentrated in a rotary evaporator under reduced pressure. The crude mixture was purified by column chromatography ethyl acetate hexane; 40:60) to afford 1.09g (yield: 60%) of (2).1H NMR (400 MHz, DMSO-d6) δ : 7.18 (d, J = 5.8 Hz, 1H), 6.95 (d, J = 8.4 Hz, 2H), 6.55 (d, J = 8.4 Hz, 2H), 4.96 (s, 2H), 3.99 (d, J = 6.0 Hz, 2H), 1.43 (s, 9H). C12H18N2O2[M]: 222.14; MS (ESI) m / z: [M+H]+: 223.08.Reagents and conditions: a) methyl-8-chloro-8-oxo octanoate, Et3N, DCM, RT, 2 h. 2. Preparation of methyl 8-((4-(((tert-butoxycarbonyl) amino) methyl) phenyl) amino)-8-oxo octanoate (3) Here, tert-butyl (4-aminobenzyl) carbamate (2) (0.1 g; 0.449 mmol) was dissolved in dichloromethane. To this solution triethylamine (0.313 mL) was added at 0°C, the reaction was stirred for 5 min and to the stirring reaction mixture 70 µL of methyl-8- chloro-8-oxo octanoate (0.102 g; 0.494 mmol) was added dropwise, reaction wasperformed in nitrogen atmosphere and continuously stirred for 2 h at room temperature. The reaction was monitored by thin layer chromatography (TLC). After completion of the reaction, 10 mL dichloromethane (DCM) was added in to the reaction mixture, washed with brine solution and extracted 3 times with DCM. The organic fractions were dried with sodium sulphate, concentrated using rotavap under reduced pressure and purified by column chromatography (ethylacetate: hexane (40:60)) to afford 0.119g (yield: 48%) pure compound 3.1H NMR (400 MHz, DMSO- d6) δ : 9.87 (s, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.38 (t, J = 6.0 Hz, 1H), 7.20 (d, J = 8.8 Hz, 2H), 4.11 (d, J = 6.0 Hz, 2H), 3.65 (s, 3H), 2.36 (m, 4H), 1.60 (m, 4H), 1.41 (s, 9H), 1.34 (m, 4H). C21H32N2O5[M]: 392.23; MS (ESI) m / z: [M+H]+: 393.08.Reagents and conditions: a) Trifluoroacetic acid:DCM (1:1), 0ºC – RT, 2 h. 3. Preparation of methyl 8-( (4-(amino methyl) phenyl) amino)-8-oxooctanoate (4) Methyl 8-((4-(((tert-butoxycarbonyl) amino) methyl) phenyl) amino)-8-oxo octanoate (3) (0.1 g; 0.255 mmol) was dissolved in dichloromethane and was cooled in an ice bath. In another round bottom flask, 5 mL trifluoroacetic acid (TFA) was added to 5 mL of dichloromethane. This solution was slowly transferred to compound (3) solution at 0 °C, and the reaction was stirred for 3 h at room temperature. The completion of the reaction was monitored by thin-layer chromatography (TLC). After completion of the reaction, a saturated solution of sodium bicarbonate was added and extracted with ethyl acetate three times. The organic layers were collected, dried with sodium sulfate, concentrated under reduced pressure using rotavap, and purified by column chromatography (chloroform: methanol (80:20)) to afford 0.0743 g (yield: 99.83%) of pure compound 4.1H NMR (400 MHz, DMSO-d6) δ : 9.90 (s, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.30 (d, J = 8.8 Hz, 2H), 3.79 (s, 2H), 3.58 (s, 3H), 2.31 (m, 4H), 1.56 (m, 4H), 1.29 (m, 4H), 1.25 (t, J = 7.2 Hz, 2H). C16H24N2O3[M] : 292.18; MS (ESI) m / z : [M+H]+: 293.54. Scheme 14:Reagents and conditions: a) Succinic anhydride, 1-ethyl-3- dimethylaminopropylcarbodiimide (EDCI), 4-di methyl amino pyridine (DMAP), DCM: Pyridine (1:1), RT, overnight. 4. Preparation of 4-((4-(8-methoxy-8-oxo-octanamido) benzyl) amino)-4- oxobutanoic acid (5) Methyl 8-( (4-(amino methyl) phenyl) amino)-8-oxooctanoate (4) (0.325 g; 1.111 mmol) was dissolved in dichloromethane (DCM): pyridine (1:1) mixture. To the solution, a catalytic amount of 4-di methyl amino pyridine (DMAP) and 1-ethyl-3- dimethylamino propyl carbodiimide (EDC) (0.310 g; 1.999 mmol) were added. The reaction mixture was stirred for 20 min at room temperature. After 20 min succinic anhydride (0.444 g; 4.45 mmol) was added and stirred for 5 h at room temperature. The reaction was monitored by thin layer chromatography (TLC). After completion, the reaction mixture was evaporated, and 10 mL water was added. The reaction mixture was extracted up to three times with ethyl acetate, collected organic layers and the combined organic solution was washed with saturated brine solution. The organic layer was then dried over sodium sulfate, concentrated under reduced pressure in rotavapor, and crude was purified by column chromatography. (methanol: ethyl acetate: acetic acid (5:94:1)) to afford 0.412g (yield: 92%) of pure compound 5.1H NMR (400 MHz, DMSO-d6) δ : 9.82 (s, 1H), 8.32 (t, J = 6.0 Hz, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.4 Hz, 2H), 4.19 (d, J = 6.0 Hz, 2H), 3.58 (s, 3H), 2.46 (t, J = 6.4 Hz, 2H), 2.39 (t, J = 7.6 Hz, 2H), 2.29 (m, 4H), 1.58 (m, 4H), 1.29 (m, 4H). C20H28N2O6[M] : 392.19; MS (ESI) m / z : [M+H]+: 393.30. M-H]+: 391.15.Reagents and conditions: a) Gemcitabine HCl, 1-ethyl-3- dimethylaminopropylcarbodiimide (EDC), 1-hydroxybenzotriazole, N-methyl morpholine, DMF: DMSO (1:3), RT, overnight. 5. Preparation of methyl 8-((4-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)tetra-hydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)- 4-oxobutanamido)methyl)-phenyl)amino)-8-oxooctanoate (6) Gemcitabine hydrochloride (0.1 g; 0.380 mmol) and a 15 mL mixture of DMF: DMSO (3:1) were added into a 50 mL flask. The mixture was stirred and 1-(3-dimethyl aminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) (0.070 g; 0.456 mmol), N- methyl morpholine (0.038 g; 0.380 mmol), 1-hydroxy benzotriazole monohydrate (0.051 g; 0.380 mmol), and 4-((4-(8-methoxy-8-oxooctanamido)benzyl)amino)-4-oxo butanoic acid (5) (0.163 g; 0.418 mmol) were added into it. The reaction mixture was protected by a nitrogen environment and stirred at room temperature overnight; the reaction was monitored by thin-layer chromatography (TLC). After the completion of the reaction, 100 mL of 10% (w / v) NaCl solution and 50 mL of water were added slowly into the reaction solution to quench the reaction. The resulting yellow suspension was extracted with 3x50 mL of ethyl acetate, combined ethyl acetate layer was washed with 2x20 mL of lithium chloride solution and 50 mL of saturated sodium bicarbonate solution followed by saturated brine solution. The ethyl acetate layer was evaporated at vacuum conditions to afford 0.159 g (yield 60%) of (6).1H NMR (400 MHz, DMSO-d6) δ : 10.67 (s, 1H), 9.46 (s, 1H), 7.97 (t, J = 5.6 Hz, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.14 (d, J = 8.4 Hz, 2H), 6.89 (d, J = 7.6 Hz, 1H), 6.78 (d, J = 8.8 Hz, 1H), 5.95 (d, J = 6.4 Hz, 1H), 5.81 (t, J = 7.2 Hz, 1H), 4.96 (t, J = 5.2 Hz, 1H), 3.84 (d, J = 5.6 Hz, 2H), 3.54 – 3.51 (td, J = 3.2 Hz, 3.2 Hz, 1H), 3.46 (dd, J = 3.2 Hz, 3.2 Hz, 2H), 3.28 (td, J = 5.6 Hz, 5.6 Hz, 1H), 3.21 (s, 3H), 2.29 (t, J = 6.8 Hz, 2H), 2.08 (t, J = 6.8 Hz, 2H), 1.93 (m, 4H), 1.72 (s, 1H), 1.16 (m, 4H), 0.92 (m, 4H). C29H37F2N5O9[M] : 637.64; MS (ESI) m / z : [M+H]+: 638.25, [M-H]-: 636.20. Scheme 16:Reagents and conditions: a) 50%, aq. NH2OH, 2N NaOH, MeOH, RT, 3 h. 6. Preparation of N1-(4-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl) tetra- hydrofuran 2-yl)-2-oxo-1,2-di-hydropyrimidin-4-yl) amino)-4-oxobutanamido) methyl) phenyl) -N8-hydroxyoctanediamide (7) Methyl 8-((4-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetra-hydrofuran-2-yl)- 2-oxo-1,2-dihydropyrimidin-4-yl)amino)-4-oxobutanamido)methyl)-phenyl)amino)-8- oxooctanoate (6) (0.1g; 0.156mmol) was dissolved in methanol. Then aq. solution of hydroxylamine (0.095g; 2.893 mmol) was added and stirred for 30min at room temperature. After 30min 2N NaOH solution was added to the reaction mixture and stirring was continued for 3h at room temperature. The completion of the reaction was monitored by thin layer chromatography (TLC). After completion, water was added to the reaction mixture and neutralized with 1N HCl, and precipitate was formed. The reaction mixture was centrifuged at 5000 rpm for 5min and after discarding the supernatant, the residue was collected as a product. The washing of the solid with pentane afforded 0.040g. (yield: 40%) of (7).1H NMR (400 MHz, DMSO-d6) δ : 10.19 (s, 1H), 7.76 (s, 1H), 7.74 (d, J = 7.6 Hz, 2H), 7.55 (s, 2H), 7.35 (s, 1H), 7.31 (d, J = 7.9 Hz, 1H), 7.18 (d, J = 7.6 Hz, 1H), 6.15 (t, J = 8.4 Hz, 1H), 5.86 (d, J = 7.2 Hz, 1H), 4.17 (td, J = 4.4 Hz, 4.4 Hz, 1H), 3.93 (s, 2H), 3.79 (td, J = 3.2 Hz, 3.2 Hz, 1H), 3.64 (d, J = 3.6 Hz, 1H), 3.62 (d, J = 3.6 Hz, 1H), 2.52 (m, 4H), 2.32 (t, J = 7.2 Hz, 2H), 1.96 (t, J = 7.6 Hz, 2H), 1.58 (t, J = 9.6 Hz, 4H), 1.28 (m, 4H).13C NMR (101 MHz, DMSO-d6) δ : 171.98, 170.05, 166.11, 155.40, 141.60, 140.24, 135.67, 129.32, 123.71, 119.97, 119.46, 95.16, 83.96, 80.92, 69.03, 59.37, 42.91, 36.94, 32.67, 28.72,25.36. HRMS (APESI) m / z calculated for HRMS (APESI) m / z calculated for C28H36F2N6O9[M+H]+ :639.2590; found: 639.2644.Reagents and conditions: a) Hydrogen balloon, 10% (w / w) Pd on activated charcoal (Pd / C), methanol, RT, overnight. 7. Preparation of 3-(amino methyl) aniline (9) 3-aminobenzonitrile (8) (1.00 g; 8.46 mmol) was dissolved in 50 mL of methanol and stirred overnight at room temperature in presence of 10% Pd / C (100 mg) under hydrogen balloon. The reaction completion was monitored by thin layer chromatography (TLC). After completion of the reaction, Pd / C was removed by filtration through a pad of celite, and resulting filtrate was evaporated under reduced pressure to give 1.2 g (yield: 98%) of (9).1H NMR (400 MHz, DMSO-d6) δ : 6.99 (t, J = 7.7 Hz, 1H), 6.58 (s, 1H), 6.53 (m, 1H), 6.47 (m, 1H), 5.01 (s, 2H), 3.64 (s, 2H), 1.29 (t, J = 7.2 Hz, 2H). C7H10N2[M] : 122.14; MS (ESI) m / z: [M+H]+: 123.06.Reagents and conditions: a) Di -tert-butyl di-carbonate, Et3N, EtOH, 0ºC – RT, 1 h. 8. Preparation of tert-butyl (3-aminobenzyl) carbamate (10). 3-(amino methyl) benzylamine (9) (1.00 g; 8.19 mmol) was dissolved in 10 mL of ethanol. To the reaction mixture triethylamine (1.7 mL) was added at 0 °C. Di-tert- butyl di carbonate (2.142 g; 9.81 mmol) was dissolved in ethanol in a separate conical flask. The Boc anhydride solution was slowly transferred to the reaction mixture by syringe over a period of 10 min, reaction was stirred for another 30 min at room temperature. After completion of the reaction, the mixture was dissolved in water andextracted with ethylacetate. The organic layer was collected, dried with sodium sulphate and concentrated under reduced pressure in rotavap. The crude mixture was purified by column chromatography (ethyl acetate : hexane; 40:60) to get 1.28g. (yield : 70%) of (10).1H NMR (400 MHz, CDCl3) δ : 7.03 (t, J = 7.7 Hz, 1H), 6.59 (d, J = 7.5 Hz, 1H), 6.57 – 6.40 (m, 2H), 4.15 (d, J = 5.4 Hz, 2H), 1.39 (s, 9H). C12H18N2O2[M]: 222.18; MS (ESI) m / z: [M+H]+: 223.08.Reagents and conditions: a) methyl-8-chloro-8-oxo octanoate, Et3N, DCM, RT, 2 h. 9. Preparation of methyl 8-((3-(((tert-butoxycarbonyl) amino) methyl) phenyl) amino)-8-oxo octanoate (11) A tert-butyl (3-aminobenzyl) carbamate (10) (0.350 g; 1.57 mmol) was dissolved in 5 mL dichloromethane. Triethylamine (0.313 mL) was added to this solution at 0 °C. The reaction was stirred for 5 min, and to the stirring reaction mixture, methyl-8- chloro-8-oxo octanoate (0.355 g; 1.73 mmol) was added dropwise, and the reaction was performed in a nitrogen atmosphere. The reaction was continuously stirred for 2 h at room temperature. The completion of the reaction was monitored by thin-layer chromatography (TLC). After the completion of the reaction, the mixture was dissolved in dichloromethane and washed with brine solution. The organic layer was separated, dried with sodium sulfate, and concentrated under reduced pressure. The crude mixture was purified by column chromatography (ethylacetate: hexane (40:60)) to get pure 0.537 g (yield: 86%) of (11).1H NMR (400 MHz, DMSO-d6) δ : 9.89 (s, 1H), 7.52 (d, J = 7.7 Hz, 2H), 7.41 (t, J = 6.0 Hz, 1H), 7.26 (t, J = 7.9 Hz, 1H), 6.95 (d, J = 7.6 Hz, 1H), 4.13 (d, J = 6.1 Hz, 2H), 3.63 (s, 3H), 2.34 (dd, J = 14.1, 6.9 Hz, 4H), 1.60 (dt, J = 16.9, 7.2 Hz, 4H), 1.45 (s, 9H), 1.34 (dd, J = 7.0, 3.6 Hz, 4H). C21H32N2O5[M]: 392.23; MS (ESI) m / z: [M+H]+: 393.08.Reagents and conditions: a) Trifluoroacetic acid: DCM (1:1), 0ºC – RT, 2 h. 10. Preparation of methyl 8-( (3-(amino methyl) phenyl) amino)-8-oxooctanoate (12) Methyl 8-((3-(((tert-butoxycarbonyl) amino) methyl) phenyl) amino)-8-oxooctanoate (11) (0.500 g; 1.27 mmol) was dissolved in 5 mL dichloromethane and cooled in ice bath. In another round bottom flask 3 mL trifluoroacetic acid was dissolved in 3 mL of dichloromethane. This solution was slowly transferred to compound (11) solution at 0 °C and reaction was stirred for 1 h at room temperature. The reaction completion was monitored by TLC. After completion of the reaction, a saturated solution of sodium bicarbonate was added and extracted with ethyl acetate, the separated organic layer was dried over sodium sulfate and concentrated under reduced pressure. The crude mixture was purified by column chromatography (chloroform: methanol (80:20)) afforded 0.342 g (yield: 99.83%) of (12).1H NMR (400 MHz, DMSO-d6) δ: 9.89 (s, 1H), 7.51 (s, 2H) 7.41 (t, J = 6.0 Hz, 1H), 7.26 (t, J = 7.7 Hz, 1H), 6.93 (d, J = 7.6 Hz, 1H), 4.13 (d, J = 5.6 Hz, 2H), 3.63 (s, 3H), 2.40 – 2.31 (m, 4H), 1.66 – 1.52 (m, 4H), 1.39 – 1.30 (m, 4H). C16H24N2O3[M]: 292.18; MS (ESI) m / z : [M+H]+: 293.54.Reagents and conditions: a) Succinic anhydride, 1-ethyl-3- dimethylaminopropylcarbodiimide (EDC), 4-di methyl amino pyridine (DMAP), DCM: Pyridine (1:1), RT, overnight. 11. Preparation of 4-((3-(8-methoxy-8-oxooctanamido) benzyl) amino)-4- oxobutanoic acid (13) Compound methyl 8-((3-(amino methyl) phenyl) amino)-8-oxooctanoate (12) (0.200 g; 0.684 mmol) was dissolved in 5 mL dichloromethane: pyridine (1:1) mixture. To the solution, catalytic amount of 4-di methyl amino pyridine and 1-ethyl-3-dimethyl amino propyl carbo-di-imide (0.191 g; 1.23 mmol) was added. The reaction mixture was stirred for 20 min at room temperature. After 20 min, succinic anhydride (0.070 g; 0.704 mmol) was added and stirred for 5 h at room temperature. Reaction wasmonitored by TLC. After completion, the reaction mixture was evaporated. To the reaction mixture, water was added and extracted with ethyl acetate, and the organic layer was washed with saturated brine solution, dried over sodium sulfate, and concentrated under reduced pressure in rotavap. The crude mixture was purified by column chromatography (Methanol: DCM: acetic acid (5:94:1)) which afforded 0.233 g. (yield : 86.79%) of (13).1H NMR (400 MHz DMSO-d6) δ :12.06 (s, 1H), 9.75 (s, 1H), 8.27 (t, J = 5.8 Hz, 1H), 7.46 – 7.34 (m, 2H), 7.14 (t, J = 7.8 Hz, 1H), 6.84 (d, J = 7.6 Hz, 1H), 4.14 (d, J = 5.9 Hz, 2H), 3.51 (s, 3H), 2.45 – 2.38 (m, 2H), 2.33 – 2.29 (m, 2H), 2.21 (dd, J = 7.3, 7.6 Hz, 4H), 1.53 – 1.41 (m, 4H), 1.26 – 1.19 (m, 4H). C20H28N2O6[M]: 392.446; MS (ESI) m / z : [M-H]+: 391.25Reagents and conditions: a) Gemcitabine HCl, 1-ethyl-3-dimethyl amino propyl carbodiimide (EDC), 1-Hydroxybenzotriazole, N-methyl morpholine, DMF: DMSO, RT, overnight. 12. Preparation of methyl 8-((3-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl) amino)- 4 oxo butanamido) methyl)-phenyl) amino)-8-oxooctanoate (14) Gemcitabine hydrochloride (0.1 g; 0.380 mmol) and a 15 mL mixture of DMF: DMSO (3:1) were added into a 50 mL round bottom flask. The reaction mixture was stirred and 1-(3-dimethylamino propyl)-3-ethylcarbodiimidehydrochloride (EDC) (0.070 g; 0.456 mmol), N-methylmorpholine (0.038 g; 0.380 mmol), 1-hydroxybenzotriazole monohydrate (0.051 g; 0.380 mmol), and 4-((3-(8-methoxy-8-oxooctanamido)benzyl)amino)-4-oxobutanoic acid (13) (0.163 g; 0.418 mmol) were added. The reaction mixture was kept protected by nitrogen from moisture and stirred at room temperature overnight. Reaction was monitored by TLC. After completion of the reaction, 50 mL of 10% (w / v) aq. NaCl solution and 50 mL of water added slowly to the reaction solution to quench the reaction. The resulting yellow suspension was extracted with 3x 50 mL of ethylacetate. The combined ethyl acetate layer was washed with 2x 60 mL of lithium chloride solution and 50 mL of saturated sodium bicarbonate solution, and saturated brine solution. The ethylacetate layer was evaporated at vacuum condition to afford 0.053g (yield: 32.4%) of (14).1H NMR (400 MHz, DMSO-d6) δ : 11.04 (s, 4H), 9.83 (s, 5H), 8.33 (t, J = 7.6 Hz, 1H), 8.25 (d, J = 7.6 Hz, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.27 (d, J = 7.6 Hz, 1H), 7.14 (d, J = 8.8 Hz, 2H), 6.32 (d, J = 6.4Hz, 1H), 6.20 (t, J = 7.2 Hz, 1H), 5.31 (t, J = 5.2 Hz, 1H), 4.19 (d, J = 5.6 Hz, 2H), 3.91 (dt J = 2.8, 2.8 Hz, 1H), 3.74 (dd, J = 2.0, 2.0 Hz, 2H), 3.68 (dt, J = 3.2, 3.2 Hz, 1H )3.58 (s, 3H), 2.69 (t, J = 6.8 Hz, 2H), 2.45 (t, J = 6.8 Hz, 2H), 2.30 (m, 4H), 2.09 (m, 4H), 1.55 (m, 4H), 1.30 (m, 4H), 1.24 (s, 1H). C29H37F2N5O9 [M]: 637.34; MS (ESI) m / z: [M+H]+:638.03, M-H]-: 636.16.Reagents and conditions: a) 50% aq. NH2OH, 2N NaOH, MeOH, RT, 2 h. 13. Preparation of N1-(3-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl) tetrahydro furan-2-yl) 2-oxo-1,2 di hydropyrimidin-4yl) amino) 4-oxo butanamido) methyl)phenyl)N8hydroxyoctanediamide (15) Compound methyl 8-((3-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl) amino)-4-oxobutanamido)methyl) - phenyl) amino)-8-oxo-octanoate (14) (0.1 g; 0.15 mmol) was dissolved in methanol. Then aq. solution of hydroxylamine (0.095 g; 2.893 mmol) was added and stirred for 30 min at room temperature. After 30 min 2N NaOH solution was added into the reaction mixture and stirred at room temperature for 3 h. The completion of the reaction was monitored by TLC. After completion, water was added to the reaction mixture and neutralized with 1N HCl and precipitate was formed. The reaction mixture was centrifuged at 5000 rpm for 5 min and after discarding the supernatant the solid residue was collected as crude product. The crude compound was washed with pentane to afford 0.046 g. (yield: 45.93%) of (17).1H NMR (400 MHz, DMSO-d6) δ : 8.58 (s, 1H), 8.51 (t, J = 7.2 Hz, 1H), 8.12 (s, 1H), 7.99 (d, J = 7.6 Hz, 2H), 7.59 (t, J = 8.4 Hz, 2H), 7.33 (m, 1H), 7.01 (d, J = 8.0 Hz, 1H), 6.45 (s, 1H), 6.20 (t, J = 7.6 Hz,1H), 6.10 (d, J = 8.0 Hz, 1H), 4.31 (s, 2H), (3.93 (td, J = 3.2, 3.2 Hz, ), 3.89 (s, 1H), 3.85 (s, 1H), 3.74 (d, J = 3.6 Hz, 1H), 3.71 (d, J = 3.2 Hz, 1H), 2.52 (m, 4H), 2.39 (t, J = 7.2 Hz, 2H), 2.04 (t, J = 7.2 Hz, 2H), 1.62 (t, J = 7.2 Hz, 4H), 1.37 (m, 4H).13C NMR (101 MHz, DMSO – d6) δ: 171.77, 169.88, 166.34, 155.35, 141.21, 139.91, 129.62, 126.09, 124.12, 123.84, 123.54, 120.02, 119.82, 95.22, 80.88, 69.43, 59.36, 43.03, 36.72, 32.82, 28.77, 25.46. HRMS (APESI) m / z calculated for HRMS (APESI) m / z calculated for C28H36F2N6O9[M+H]+ :639.2590; found: 639.2644.Reagents and conditions: a) 3-hydroxypropanoic acid, 1-ethyl-3-dimethyl amino propyl carbo diimide (EDC), 4-di methyl amino pyridine (DMAP), DCM : Pyridine (1:1), RT, 5 h. 14. Preparation of tert-butyl (4-(3-hydroxy propanamido) benzyl) carbamate (16) Compound tert-butyl 4-amino benzyl carbamate (2) (0.500 g; 2.25 mmol) was dissolved in 5 mL dichloromethane: pyridine (1:1) mixture. To this solution catalytic amount of 4-di methyl amino pyridine and 1-ethyl-3- dimethylaminopropylcarbodiimide (0.628 g; 4.053 mmol) was added. Reaction mixture was stirred for 20 min at room temperature. After 20 min 3-hydroxypropanoicacid (0.202 g; 2.25 mmol) was added stirred for 5 h at room temperature. Reaction was monitored by TLC. After completion the reaction mixture was evaporated. To the reaction mixture water was added and extracted with ethylacetate, separated the organic layer and washed with saturated brine solution. The organic portion was dried over sodium sulphate, concentrated under reduced pressure. The crude mixture was purified by column chromatography. (methanol: DCM: acetic acid (5:94:1)) afforded 0.362 g. (yield: 54.68%) of (16).1H NMR (400 MHz, CDCl3) δ : 7.38 (d, J = 7.6 Hz, 2H), 7.13 (d, J = 7.2 Hz, 2H), 4.18 (s, 2H), 3.89 (t, J = 3.6 Hz, 2H), 2.54 (t, J = 4.4 Hz, 2H), 1.61 (s, 1H), 1.39 (s, 9H).C15H22N2O4[M] : 294.35; MS (ESI) m / z : [M+H]+: 295.18.Reagents and conditions: a) Methyl acrylate, sodium methoxide, THF, 12 h. 15. Preparation of methyl 3-(3-((4-(((tert- butoxy carbonyl) amino) methyl) phenyl) amino)-3-oxopropoxy) propionate carbamate (17) tert-butyl (4-(3-hydroxy propanamido) benzyl) carbamate (16) (0.300 g; 1.02mmol) was dissolved in a solution of sodium methoxide (0.148 g; 2.75 mmol) in 10 mL THF and methyl acrylate (0.097 g; 1.12 mmol) in THF (5 mL) was added dropwise into the above solution. The mixture was stirred at room temperature for overnight. The solvent was removed under reduced pressure. The crude mixture was purified by chromatography on silica gel chromatography (ethylacetate: hexane; 20:80) afforded 0.225g. (yield: 58%) of (17).1H NMR (400 MHz, DMSO-d6) δ : 9.87 (s, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.32 (s, 1H), 7.14 (d, J = 8.0 Hz, 2H), 4.05 (d, J = 6.0 Hz, 2H), 3.64 (m, 4H), 3.52 (s, 6H), 2.50 (m, 4H), 1.39 (s, 9H).C19H28N2O6[M] : 380.44; MS (ESI) m / z : [M+H]+: 381.25.Reagents and conditions:a) trifluoroacetic acid (50%v / v), DCM, RT, 2 h. 16. Preparation of methyl 3-(3-((4-(amino methyl) phenyl) amino)-3-oxopropoxy) propionate (18) Methyl3-(3-((4-(((tert-butoxycarbonyl) amino) methyl) phenyl) amino)-3oxopropoxy) propanate carbamate (17) (0.300 g; 0.788 mmol) was dissolved in 5 mL dichloromethane cooled in ice bath. In another round bottom flask 5 mL trifluoroacetic acid (TFA) was dissolved in 5 mL of dichloromethane. The TFA solution was slowly transferred to compound (17) solution at 0 °C and reaction was stirred for 2 h at room temperature. The reaction completion was monitored by TLC. After completion of the reaction, saturated solution of sodium bicarbonate was added in to it and extracted with ethylacetate. Separated organic layer was dried over sodium sulphate and concentrated under reduced pressure. The crude mixture was purified by column chromatography. (DCM: methanol (90:10)) afforded 0.158 g (yield: 71.48%) of (18). 1H NMR (400 MHz, DMSO-d6) δ :10.03 (s, 1H), 8.14 (s, 2H), 7.61 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 3.97 (s, 4H), 3.63 (m, 4H), 3.56 (s, 3H), 2.52 (t, J = 6.8 Hz, 4H). C14H20N2O6[M]: 280.32; MS (ESI) m / z: [M+H]+: 281.20.Reagents and conditions: a) Succinic anhydride, 1-ethyl-3- dimethylaminopropylcarbodiimide (EDC), 4-di methyl amino pyridine (DMAP), DCM: pyridine (1:1), RT, overnight. 17. Preparation of 4-((4-(3-(3-methoxy-3-oxopropoxy) propanamido) benzyl) amino)-4-oxobutanoic acid (19) Compound methyl 3-(3-((4-(amino methyl) phenyl) amino)-3-oxopropoxy) propanoate (18) (0.100 g; 0.356 mmol) was dissolved in 5 mL dichloromethane: pyridine (1:1). To the solution catalytic amount of 4-di methyl amino pyridine and 1-ethyl-3- dimethylaminopropylcarbo-di-imide (0.19 g; 1.23 mmol) were added. Reaction mixture was stirred for 20 min at room temperature. After 20 min succinic anhydride (0.036 g; 0.356 mmol) was added stirred for overnight at room temperature. Reactionwas monitored by TLC. After completion of the reaction, pyridine solution was evaporated. To the reaction mixture water was added and extracted with ethylacetate, and the organic layer was washed with saturated brine solution and was dried over sodium sulphate, concentrated under reduced pressure. The crude mixture was purified by column chromatography. (Methanol: DCM: acetic acid (10:90:1)) that afforded 0.117 g. (yield: 86.22%) of (19).1H NMR (400 MHz, DMSO-d6) δ : 12.48 (s, 1H), 10.18 (s, 1H), 8.68 (s, 1H), 7.79 (d, J = 8.4 Hz 2H), 7.56 (d, J = 7.6Hz, 1H), 7.25 (d, J = 7.6 Hz, 1H), 4.55 (s, 2H), 3.96 (s, 3H), 2.86 (t, J = 1.6 Hz, 4H), 2.63 (t, J = 7.2 Hz, 4H), 1.90 (t, J = 2.0 Hz, 4H). C18H24N2O7[M]: 380.32; MS (ESI) m / z: [M-H]+: 379.20.Reagents and conditions: a) Gemcitabine hydrochloride, 1-ethyl-3-dimethyl amino propyl carbo diimide (EDC), 1-Hydroxybenzotriazole, N-methylmorpholine, DMF : DMSO, RT, overnight. 18. Preparation of methyl 3-(3-((3-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxyl methyl) tetra-hydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)4 oxobutanamido)methyl)-phenyl) amino)-3-oxopropoxy) propanoate (20). Gemcitabine hydrochloride (0.1 g; 0.380 mmol) and 15 mL mixture of DMF:DMSO (3:1) was added into a 50 mL flask. The reaction mixture was stirred and 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (0.070 g; 0.456 mmol),N-methylmorpholine (0.038 g; 0.380 mmol), 1-hydroxybenzotriazole monohydrate (0.051 g; 0.380 mmol), and 4-((4-(3-(3-methoxy-3- oxopropoxy)propanamido)benzyl)amino)-4-oxobutanoic acid (19) (0.158 g; 0.418 mmol) were added. The reaction mixture was protected with nitrogen ballon, and stirred at room temperature for overnight. Reaction was monitored by TLC. After thecompletion of the reaction 100 mL of 10% (w / v) NaCl solution and 50mL of water were added slowly to the reaction solution to quench the reaction. The resulting yellow suspension was extracted with 3x 50 mL of ethylacetate. Combined ethyl acetate layers were washed with 2x 20 mL of lithium chloride solution and 50 mL of saturated sodium bicarbonate solution and saturated brine solution. The ethyl acetate mixture was evaporated at vacuum condition to afford 0.126 g. (yield: 53%) of (20).1H NMR (400 MHz, DMSO-d6) δ : 11.04 (s, 1H), 9.83 (s, 1H), 8.32 (t, J = 5.6 Hz, 1H), 8.23 (d, J = 7.6 Hz, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.27 (d, J = 7.6 Hz, 1H), 7.14 (d, J = 8.8 Hz, 1H), 6.32 (d, J = 6.4 Hz, 1H), 6.18 (t, J = 7.2 Hz, 1H), 5.33 (s, 1H), 4.19 (d, J = 5.7 Hz, 2H), 3.91 (td, 3.2 Hz,3.2 Hz, 1H), 3.80 (dd, J = 2.4 Hz, 2.4 Hz, 2H), 3.66 (td, J = 3.6 Hz, 3.6 Hz, 1H), 3.58 (s, 3H), 2.66 (t, J = 7.2 Hz, 2H), 2.45 (t, J = 6.8 Hz, 2H), 2.09 (s, 2H), 1.55 (m, 4H), 1.29 (m, 4H). C27H33F2N5O10[M] : 625.22; MS (ESI) m / z : [M+H]+: 626.25.Reagents and conditions: a) 50% aq. NH2OH, 2N NaOH, MeOH, 3 h. 19. Preparation of N1-(1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-N4-(3- (3-(3-(hydroxyamino)-3oxopropoxy)propanamido)-benzyl) succinamide (21) Compound methyl 3-(3-((3-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxy methyl) tetra- hydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl) amino)-4oxobutanamido) methyl)- phenyl) amino)-3 oxo propoxy) propanoate (20) (0.08 g; 0.128 mmol) was dissolved in methanol. Then aq. solution of hydroxylamine (0.076 g; 2.304 mmol) was added and stirred for 30 min at room temperature. After 30 min 2N NaOH solution wasadded into the reaction mixture and stir at room temperature for 3 h. The completion of the reaction was monitored by TLC. After completion, water was added to the reaction mixture and neutralized with 1N HCl and precipitate was formed. The reaction mixture was centrifuged at 5000 rpm for 5 min and the supernatant was discarded to collect the residue as crude product, washed with pentane to afford 0.039 g (yield: 48.6%) of (21).1H NMR (400 MHz, DMSO-d6) δ : 10.15 (s, 1H), 7.76 (s, 1H), 7.70 (d, J = 7.2 Hz, 2H), 7.53 (d, J =8.4 Hz, 1H), 7.50 (s, 1H), 7.34 (s, 1H), 7.30 (d, J = 7.6 Hz, 1H), 7.17 (d, J = 7.6 Hz, 1H), 6.13 (t, J = 8.0 Hz, 1H), 5.83 (d, J = 6.5 Hz, 1H), 4.16 (dt, J = 4.0 Hz, 1H), 3.93 (s, 2H), 3.79 (dt, J = 3.2 Hz, 1H), 3.75 (s, 2H), 3.64 (dd, J = 3.2, 3.6 Hz, 2H), 2.32 (t, J = 7.6 Hz, 2H), 1.95 (t, J = 7.2 Hz, 2H), 1.57 (t, J = 6.8 Hz, 2H), 1.48 (t, J = 7.2 Hz, 2H), 1.28 (t, J = 2.8 Hz, 4H).13C NMR (101 MHz, DMSO-d6) δ : 171.94,166.12,169.28155.39, 141.45, 140.34, 136.06, 129.59, 126.03, 123.71, 123.65, 121.21, 119.98, 119.46, 116.25, 95.11, 80.81, 69.06, 59.68, 43.05, 32.96 – 32.76, 28.84, 25.58. HRMS (APESI) m / z calculated for HRMS (APESI) m / z calculated for C26H33F2N7O9 [M+H]+ :627.2226; found: 627.2293.Reagents and conditions: a) Trifluoroacetic anhydride, overnight. 20. Preparation of 3-(2,2,2-trifluoroacetamido) propionoic acid (23) The solution of 3-aminopropanoic acid (22) (1.00 g; 11.22 mmol) in 10 mL of tri- fluoro-acetic anhydride was stirred at room temperature under a nitrogen atmosphere for overnight. Reaction completion was monitored by TLC. After completion, the reaction mixture was evaporated under reduced pressure in rotavap. The obtained solid was washed with di-ethyl-ether to afford 1.640 g (yield: 79%) of (23).1H NMR (400 MHz, DMSO-d6) δ : 12.33 (s, 1H), 9.45 (s, 1H), 3.40 (q, J = 3.6 Hz, 2H), 2.51 ( t, J = 6.8 Hz, 2H). C5H6F3NO3[M] :185.10; MS (ESI) m / z: [M-H]+: 184.20. Scheme 31:Reagents and conditions: a) 3-(2,2,2-trifluoroacetamido) propanoic acid (23), 1-ethyl- 3-dimethylaminopropyl carbodiimide (EDC), 4-di methyl amino pyridine (DMAP), DCM: pyridine (1:1), RT, 5 h. 21. Preparation of tert-butyl-(4-(3-(2,2,2-tri-fluoro-acetamido)-propanamido)- benzyl)-carbamate (24) Compound methyl tert-butyl 4-amino benzyl carbamate (2) (0.500 g; 2.25 mmol) was dissolved in 5 mL dichloromethane: pyridine (1:1). To this solution catalytic amount of 4-di methyl amino pyridine and 1-ethyl-3-dimethylaminopropylcarbodiimide (0.191 g; 1.23 mmol) was added. Reaction mixture was stirred for 20 min at room temperature. After 20 min, 3-(2,2,2-trifluoroacetamido) propanoic acid (23) (0.416g; 2.25 mmol) was added and stirred for 5 h at room temperature. Reaction was monitored by TLC. After completion of the reaction, pyridine solution was evaporated. To the reaction mixture, water was added and extracted with ethylacetate, organic layer was separated and washed with saturated brine solution, dried over sodium sulphate, concentrated under reduced pressure. The crude mixture was purified by column chromatography in ethylacetate: hexane (40:60) to afford 0.475 g. (yield: 54.23%) of (24).1H NMR (400 MHz, DMSO-d6) δ: 10.01 (s, 1H), 9.59 (s, 1H), 7.56 d, J = 8.0 Hz, 2H), 7.39 (s, 1H), 7.22 (d, J = 8.0 Hz, 2H), 4.13 (d, J = 5.6 Hz, 2H), 3.53 (q, J = 6.0 Hz, 2H), 2.67 (t, J = 11.2 Hz, 2H), 1.45 (s, 9H). C17H22F3N3O4[M] : 389.37; MS (ESI) m / z : [M+H]+: 389.16.Reagents and conditions: a) K2CO3, MeOH : THF : H2O (1:1:0.5), RT, overnight.22. Preparation of tert-butyl (4-(3-aminopropanamido) benzyl) carbamate (25) Compound tert-butyl-(4-(3-(2,2,2-tri-fluoro-acetamido)-propanamido)-benzyl)- carbamate (24) (0.500 g; 1.28 mmol) was dissolved in 25 mL mixture of methanol: tetrahydrofuran: water (1:1:0.5) followed by aq. solution of K2CO3was added and stirred at room temperature overnight. After reaction completion, the solvent was evaporated, and ethyl acetate was added. The ethylacetate layer was washed with saturated brine solution, and ethylacetate was dried over sodium sulfate, concentrated under reduced pressure, afforded 0.335 g (yield: 88.93%) of (25).1H NMR (400 MHz, DMSO-d6) δ : 7.54 (d, J = 8.0 Hz, 2H), 7.34 (s, 1H), 7.14 (d, J = 8.4 Hz, 2H), 4.05 (s, 2H), 2.80 (t, J = 6.4 Hz, 2H), 2.42 (t, J = 6.8 Hz, 2H), 1.65 (s, 3H), 1.38 (s, 9H). C15H23N3O3[M]: 293.3; MS (ESI) m / z : [M+H]+: 293.16.Reagents and conditions: a) Methyl acrylate, chloroform, 40 ºC 12 h. 23. Preparation of methyl 3-((3-((4-(((tert-butoxycarbonyl) amino) methyl) phenyl) amino)-3-oxopropyl) amino) propanoate (26) Compound tert-butyl (4-(3-aminopropanamido) benzyl) carbamate (25) (0.300 g; 1.02 mmol) and methyl acrylate (0.097g; 1.12mmol) were dissolved in 10 mL chloroform. The mixture was stirred at 40 ºC for 12 h. Then 20 mL water was added into the reaction mixture and extracted with ethyl acetate; ethylacetate layer was washed with saturated brine solution and dried over sodium sulfate concentrated under reduced pressure. The crude mixture was purified by silica gel chromatography with methanol: DCM; 10:90 to afford 0.362 g (yield: 67.52%) of (26).1H NMR (400 MHz, MeOD-d4) δ : 7.40 (d, J = 8.4 Hz 2H), 7.13 (d, J = 8.8 Hz, 2H), 4.08 (s, 2H), 3.61 (s, 3H), 3.04 (m, 4H), 2.58 (q, J = 6.4 Hz, 4H), 1.82 (s, 2H), 1.35 (s, 9H). C19H29N3O5[M]: 379.46; MS (ESI) m / z : [M+H]+: 380.16.Reagents and conditions: a) Trifluoroacetic acid (50 %v / v), DCM, RT, 2 h. 24. Preparation of methyl 3-((3-((4-(aminomethyl-) phenyl)-amino)-3-oxopropyl)- amino)-propanoate (27) Methyl 3-((3-((4-(((tert-butoxycarbonyl)-amino)-methyl)-phenyl)-amino)-3- oxopropyl)-amino)-propanoate (26) (0.250g; 0.658mmol) was dissolved in 5 mL dichloromethane and was cooled in ice bath. In another round bottom flask, 5 mL of trifluoroacetic acid was mixed with 5 mL of dichloromethane. This solution was slowly transferred to the solution of compound (26) at 0 °C and the reaction was stirred for 2 h at room temperature. The completion of the reaction was monitored by TLC. After completion, a saturated solution of sodium bicarbonate was added and extracted with ethyl acetate. Separated organic layer was dried over sodium sulphate and concentrated under reduced pressure. The crude mixture was purified by column chromatography (DCM: methanol (90:10)) to afford 0.174 g (yield: 94.55%) of (27). 1H NMR (400 MHz, DMSO-d6) δ : 10.02 (s, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 4.40 (d, J = 5.6 Hz, 2H), 3.78 (s, 3H), 3.54 (q, J = 4.5 Hz, 4H), 2.71 (s, 1H), 1.74 (s, 2H), 1.50 (t, J = 4.5 Hz, 4H). C14H21N3O3[M]: 279.34; MS (ESI) m / z : [M+H]+: 280.16.Reagents and conditions: a) Succinic anhydride, 1-ethyl-3- dimethylaminopropylcarbodiimide (EDC), 4-di methyl amino pyridine (DMAP), DCM: pyridine (1:1), RT, overnight. 25. Preparation of 4-((4-(3-((3-methoxy-3-oxopropyl) amino) propanamido) benzyl) amino)-4-oxobutanoic acid (28)Compound methyl 3-((3-((4-(aminomethyl-) phenyl)-amino)-3-oxopropyl)-amino)- propanoate (27) (0.200g; 0.715mmol) was dissolved in 5 mL dichloromethane: pyridine (1:1). To this solution catalytic amount of 4-di methyl amino pyridine and 1- ethyl-3-dimethyl aminopropyl carbodiimide (0.200 g;1.285 mmol) was added. Reaction mixture was stirred for 20 min at room temperature. After 20 min, succinic anhydride (0.072 g; 0.723 mmol) was added and stirred for overnight at room temperature. Reaction was monitored by TLC. After completion of the reaction pyridine solution was evaporated. To the residual reaction mixture water was added and extracted with ethyl acetate and separated organic layer was washed with saturated brine solution and dried over sodium sulphate, concentrated under reduced pressure. The crude mixture was purified by column chromatography (methanol: DCM: acetic acid (10:90:1)) to afford 0.237 g. (yield: 87.2%) of (28).1H NMR (400 MHz, DMSO- d6) δ: 12.09 (s, 1H), 9.81 (s, 1H), 8.30 (s, 1H), 7.50 (d, J = 7.4 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 4.19 (s, 2H), 3.58 (s, 3H), 2.46 (t, J = 6.0 Hz, 4H), 2.29 (d, J = 7.6 Hz, 4H), 1.55 (s, 1H), 1.29 (t, J = 3.6 Hz, 4H). C18H25N3O6 [M]: 379.34; MS (ESI) m / z: [M+H]+: 380.16.Reagents and conditions: a) Gemcitabine hydrochloride, 1-ethyl 3 dimethyl amino propyl carbodiimide (EDC), 1-Hydroxybenzotriazole, N-methylmorpholine, DMF: DMSO (1:3), RT, overnight. 26. Preparation of methyl 3-((3-((3-((4-((1-(3,3-di fluoro-4-hydroxy-5-(hydroxy methyl) tetra-hydro furan-2-yl)-2-oxo-1,2-di hydro pyrimidin-4-yl) amino)-4- oxobutanamido) methyl) phenyl) amino)-3-oxo propyl) amino) propanoate (29)Gemcitabine hydrochloride (0.1 g; 0.380 mmol) and 15 mL mixture of DMF: DMSO (3:1) were added into a 50 mL RB flask. To the stirring mixture 1-(3-dimethylamino propyl)-3-ethylcarbodiimide hydrochloride (EDC) (0.070 g; 0.456 mmol), N- methylmorpholine (0.038 g; 0.380 mmol), 1-hydroxybenzotriazole monohydrate (0.051 g; 0.380 mmol), and 4-((4-(3-((3-methoxy-3- oxopropyl)amino)propanamido)benzyl)amino)-4-oxobutanoicacid (28) (0.158g; 0.418mmol) were added. The reaction mixture was protected with nitrogen balloon, and stirred at room temperature for overnight. Reaction was monitored by TLC. After completion, 100 mL of 10% aq. NaCl solution and 50 mL of water added slowly to the reaction solution to quench the reaction. The resulting yellow suspension was extracted with 3x 50 mL of ethyl acetate. The combined ethyl acetate layer was washed with 2x 50 mL of lithium chloride solution and 50 mL of saturated sodium bicarbonate solution, and saturated brine solution. The ethylacetate layer was evaporated at vacuum condition to afford 0.087 g. (yield: 33%) of (29).1H NMR (400 MHz, DMSO-d6) δ : 11.12 (s, 1H), 9.91 (s, 1H), 8.41 (t, J = 5.6 Hz, 1H), 8.31 (d, J = 7.6 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 7.6 Hz, 1H), 7.23 (d, J = 8.8 Hz, 1H), 6.40 (d, J = 6.4 Hz, 1H), 6.24 (t, J = 7.2 Hz 1H), 5.38 (t, J = 5.2 Hz, 1H), 4.27 (d, J = 5.6 Hz, 2H), 3.97 (dt, J = 3.2 Hz, 3.2 Hz 1H), 3.85 (dd, J = 2.4 Hz, 2.4 Hz, 2H), 3.74 (dt, J = 3.6 Hz, 3.6 Hz, 1H), 3.66 (s, 3H), 2.74 (t, J = 6.8 Hz, 2H), 2.53 (s, J = 6.8 Hz, 2H), 2.38 (q, J = 7.6 Hz, 4H), 1.65 (m, 4H), 1.37 (s, 2H). C27H34F2N6O9[M] : 624.58; MS (ESI) m / z : [M+H]+: 625.37.Reagents and conditions: a) aq. NH2OH, NaOH, MeOH, RT, 3 h.27. Preparation ofN1-(1-(3,3-difluoro-4-hydroxy-5-(hydroxy methyl) tetrahydrofuran-2-yl)-2-oxo-1,2-di hydro pyrimidin-4-yl)-N4-(3-(3-((3-(hydroxy amino)-3-oxopropyl) amino)-propanamido) benzyl) succinamide (30) Compound methyl 3-((3-((3-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxy methyl) tetra- hydro-furan-2-yl)-2-oxo-1,2-di hydropyrimidin-4-yl) amino)-4-oxo butanamido) methyl)-phenyl) amino)-3-oxopropyl) amino) propanoate (29) (0.08 g; 0.128 mmol). was dissolved in methanol. Then aq. solution of hydroxylamine (0.06 g; 0.192 mmol) was added and stirred for 30 min at room temperature. After 30min added 2N NaOH solution into the reaction mixture and stirred at room temperature for 3 h. The completion of the reaction was monitored by TLC. After completion, water was added to the reaction mixture and neutralized with 1N HCl resulted in formation of precipitation. The reaction mixture was centrifuged at 5000 rpm for 5min and after discarding the supernatant the residue was collected as crude product and washed with pentane to afford 0.047g (yield: 58%) of (30).1H NMR (400 MHz, DMSO-d6) δ : 10.01 (s, 1H), 7.58 (s, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.37 (d, J = 5.2 Hz, 2H), 7.17 (s, 1H), 7.13 (d, J = 8.0 Hz, 1H), 7.00 (d, J = 7.6 Hz, 1H), 5.95 (t, J =8.4 Hz, 1H), 5.68 (d, J = 7.6 Hz, 1H), 3.99 (dt, J = 4.4 Hz, 4.4 Hz 1H), 3.75 (s, 2H), 3.62 (dt, J = 3.2 Hz, 3.2 Hz 1H), 3.57 (s, 2H), 3.47 (dd, J = 3.6 Hz, 3.6 Hz 2H), 2.15 (t, J = 7.2 Hz, 2H), 1.78 (t, J = 7.6 Hz, 2H), 1.40 (t, J = 6.8 Hz, 2H), 1.31 (t, J = 6.8 Hz, 2H), 1.09 (m, 4H).13C NMR (101 MHz, DMSO - d6) δ : 176.98, 174.71, 171.11, 160.28, 146.18, 145.02, 140.75, 134.48, 131.15, 128.65, 128.59, 124.97, 124.31, 100.16, 88.70, 85.92, 74.03, 64.37, 48.07, 41.73, 37.67, 33.63, 30.28. HRMS (APESI) m / z calculated for C26H33F2N7O9[M+H]+ :626.2386; found: 626.2293.Reagents and conditions: a) Trifluoroacetic anhydride, RT, 2 h. 28. Preparation of4-((2,2,2-trifluoroacetamido) methyl) benzoic acid (32)The solution of 4-(amino methyl) benzoic acid (31) (1.00 g; 6.62 mmol) in 10 mL of trifluoroacetic anhydride was stirred at room temperature under nitrogen atmosphere for 2 h. Reaction completion was monitored by TLC. After completion, to the reaction mixture water was added and extracted with ethylacetate. The ethyl acetate layer was evaporated under reduced pressure. The obtained crude mixture was purified by column chromatography (ethyl acetate: hexane (1:1)) to afford 1.30 g (yield: 79.50%) of (32).Reagents and conditions: a) tert-Butyl (2-aminophenyl) carbamate, 4-(dimethyl amino) pyridine, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride, DCM: Pyridine (1:1), RT, 12 h. 29. Preparation of tert-butyl (2-(4-((2,2,2-tri-fluoro-acetamido)-methyl)- benzamido)-phenyl)-carbamate (33) Compound 4-((2,2,2-tri fluoro acetamido) methyl) benzoic acid (32) (0.5 g; 2.02 mmol) was dissolved in a mixture of DCM: Pyridine (1:1). 1-(3-Dimethyl amino propyl)-3-ethyl carbo di imide hydro chloride (0.563 g; 3.636 mmol) and catalytic amount of 4- (dimethyl amino) pyridine (DMAP) were added to the reaction mixture. tert-Butyl (2-aminophenyl) carbamate (0.421 g; 2.02 mmol) was added and reaction was purged with nitrogen gas. The reaction mixture was stirred for 12 h at room temperature till the completion of the reaction. The solvent was evaporated and product was fractioned between ethylacetate and saturated solution of sodium bicarbonate. The crude product was purified by column chromatography (ethylacetate: hexane; 25:75), afforded 0.630 g. (yield 71%) of compound (33).1H NMR (400 MHz, DMSO-d6) δ : 10.12 (s, 1H), 9.86 (s, 1H), 8.68 (s, 1H), 7.95 (t, J = 7.2 Hz, 2H), 7.46 (d, J = 6.4 Hz, 4H), 7.17 (m, 2H), 4.49 (s, 2H), 1.45 (s, 9H). C21H22F3N3O4[M] : 437.42; MS (ESI) m / z : [M+H]+: 438.16. Scheme 40:Reagents and conditions: a) K2CO3, MeOH: THF: H2O (1:1:0.5), RT, overnight. 30. Preparation of tert-butyl (2-(4-(amino methyl) benzamido) phenyl) carbamate (34) Compound tert-butyl (2-(4-((2,2,2-trifluoro acetamido) methyl) benzamido) phenyl) carbamate (33) (0.300g; 0.686mmol) was dissolved in 25 mL mixture of methanol: tetrahydrofuran: water (1:1:0.5), followed by aq. solution of K2CO3was added and stirred overnight at room temperature. After completion of the reaction, the mixture was evaporated, and ethyl acetate was added. The ethylacetate layer was washed with saturated brine solution and was dried over sodium sulfate, concentrated under reduced pressure that afforded 0.221g (yield: 94.93%) of (34).1H NMR (400 MHz, DMSO-d6) δ : 9.86 (s, 1H), 8.73 (s, H), 7.96 (d, J = 6.8 Hz, 2H), 7.56 (m, 4H), 7.28 (m, J = 8.8 Hz, 2H), 3.86 (s, 2H), 1.50 (s, 9H). C19H23N3O3[M]: 341.42; MS (ESI) m / z: [M+H]+: 342.16.Reagents and conditions: a) Succinic anhydride, 1-ethyl-3-dimethyl amino propyl carbodiimide (EDC), 4-dimethyl aminopyridine (DMAP), DCM: Pyridine (1:1), RT, overnight. 31. Preparation of 4-((4-((2-((tert-butoxycarbonyl) amino) phenyl) carbamoyl) benzyl) amino)-4-oxobutanoic acid (35) Compound tert-butyl (2-(4-(amino methyl) benzamido) phenyl) carbamate (34) (0.200 g; 0.585 mmol) was dissolved in 5 mL dichloromethane: pyridine (1:1). To this solution, catalytic amount of 4-dimethyl aminopyridine and 1-ethyl-3-dimethyl amino propyl carbo-di-imide (0.163 g, 1.054 mmol) were added. Reaction mixture was stirred for 20 min at room temperature. After 20 min, succinic anhydride (0.059 g;0.586 mmol) was added and stirred for overnight at room temperature. Reaction was monitored by TLC. After completion of the reaction, the pyridine mixture was evaporated. To the residue, water was added and extracted with ethyl acetate, separated the organic layer, and the organic layer was washed with saturated brine solution and was dried over sodium sulfate, concentrated under reduced pressure. The crude mixture was purified by column chromatography. (methanol: DCM: acetic acid (10:90:1)) that afforded 0.180 g (yield: 70%) of (35).1H NMR (400 MHz, DMSO-d6) δ : 9.81 (s, 1H), 8.67 (s, 1H), 8.47 (s, 1H), 8.14 (s, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.54 (t, J = 7.6 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.18 (m, J = 7.2 Hz, 2H), 4.36 (d, J = 5.6 Hz, 2H), 2.95 (t, J = 2.8 Hz, 2H), 2.43 (t, J = 2.8 Hz, 2H), 1.45 (s, 9H). C23H27N3O6[M]: 441.48; MS (ESI) m / z: [M+H]+: 342.25.Reagents and conditions: a) Gemcitabine hydrochloride, 1-ethyl-3-dimethyl amino propyl carbo diimide (EDC), 1-Hydroxybenzotriazole, N-Methylmorpholine, DMF: DMSO (3:1), RT, overnight. 32. Preparation of tert-butyl (2-(4-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)- 4-oxobutanamido)-methyl) -benzamido)-phenyl)carbamate (36) Gemcitabine hydrochloride (0.1 g; 0.380 mmol) and 8 mL mixture of DMF: DMSO (3:1) added into a 50 mL RB flask. The reaction mixture was stirred and 1-(3- dimethylamino propyl)-3-ethylcarbodiimidehydrochloride, N-methylmorpholine, 1- hydroxybenzotriazole monohydrate, and 4-((4-((2-((tert-butoxycarbonyl) amino) phenyl) carbamoyl) benzyl) amino)-4-oxobutanoic acid (35) (0.184 g; 0.418mmol) were added. The reaction mixture was protected with nitrogen balloon, and stirred at room temperature for overnight. Reaction was monitored by TLC. After completion of the reaction 100 mL of 10% NaCl solution and 50 mL of water were added slowly tothe reaction solution to quench the reaction. The resulting yellow suspension was extracted with 3x 100 mL of ethylacetate. The combined ethylacetate layer was washed with 2x 60 mL of lithium chloride solution and 100 mL of saturated sodium bicarbonate solution and saturated brine solution. The ethyl acetate layer was evaporated at vacuum condition to afford 0.121g of (36) light yellow foam.1H NMR (400 MHz, DMSO-d6) δ : 11.05 (s, 1H), 9.81 (s, 1H), 8.68 (s, 1H), 8.50 (t, J = 5.6 Hz, 1H), 8.25 (d, J = 7.6 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.54 (td, J = 6.0,1.6 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.0 Hz, 1H), 7.19 (m, 2H), 6.33 (d, J = 6.4 Hz, 1H), 6.20 (t, J = 7.2 Hz, 1H), 5.28 (t, J = 5.2 Hz, 1H), 4.35 (d, J = 5.2 Hz, 2H), 3.86 (dd, J = 3.2 Hz, 3.2 Hz, 2H), 3.83 (dt, J = 4.8, 4.8 Hz, 1H), 3.65 (dt, J = 3.6, 3.6 Hz, 1H), 2.70 (t, J = 6.2, Hz, 2H), 1.45 (s, 9H), 1.24 (t, J = 9.2, Hz, 2H). C32H36F2N6O9[M] : 686.25; MS (ESI) m / z : M+H]+: 687.20.Reagents and conditions: a) 4M HCl in Dioxane, DCM, RT, 2h. 33. Preparation of N1-(4-((2-amino phenyl) carbamoyl) benzyl)-N4-(1-(3,3- difluoro-4-hydroxy -5- (hydroxy methyl) tetrahydrofuran-2-yl)-2-oxo-1,2-di hydropyrimidin 4yl) succinamide (37) Compound tert-butyl (2-(4-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl) amino)-4-oxobutanamido) methyl)-benzamido) phenyl)-carbamate (36) (0.05g; 0.072mmol) was dissolved in 3 mL dichloromethane and cooled in ice bath and 3 mL 4M HCl in dioxane was slowly transferred to compound (36) solution at 0 °C and reaction was stirred for 1h at room temperature. After completion of the reaction, the mixture was dried and saturated solution of NaHCO3was added into it. Extracted with DCM and organic layer was separated, dried with sodium sulphate, evaporated under reduced pressure to afford0.036g. (yield: 84.29%) of (37).1H NMR (400 MHz, DMSO-d6) δ : 9.96 (s, 1H), 8.76 (s, 1H), 8.21 (d, J = 7.2 Hz, 2H), 8.10 (d, J = 7.6 Hz, 2H), 7.68 (d, J = 8.4 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.28 (t, J = 6.8 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 6.93 (t, J = 7.2 Hz, 1H), 6.58 (s, 1H), 6.42 (t, J = 8.0 Hz, 1H), 6.16 (d, J = 7.6 Hz, 1H), 4.65 (s, 2H), 4.44 (s, 2H), 4.13 (dt, J = 2.8 Hz, 2.8 Hz, 2H), 4.05 (s, 1H), 3.93 (dd, J = 3.2 Hz, 3.6 Hz, 2H), 2.80 (t, J = 2.4 Hz, 4H), 2.21 (s, 1H).13C NMR (101 MHz, DMSO-d6) δ : 167.63, 166.14, 156.84, 155.12, 142.86, 141.27, 135.35, 130.41, 130.26,127.69, 126.13, 123.57, 121.00, 117.85, 115.09, 95.11, 84.60, 80.90, 69.38, 69.05, 68.93, 59.42, 42.82. HRMS (APESI) m / z calculated for C27H28F2N6O7[M+H]+ :587.2066; found: 587.2056.Reagents and conditions: a) tert-butyl (2-amino-5-fluoro phenyl) carbamate, 4- (dimethyl amino) pyridine, 1-(3-Dimethylaminopropyl)-3- ethylcarbodiimidehydrochloride, DCM: Pyridine (1:1), RT, 12 h 34. Preparation of tert-butyl (5-fluoro-2-(4-((2,2,2-tri-fluoro-acetamido)-methyl)- benzamido)-phenyl)-carbamate (38) Compound 4-((2,2,2-trifluoro acetamido) methyl) benzoic acid (32) (0.200 g; 0.809mmol) was dissolved in a mixture of DCM: Pyridine (1:1). Then 1-(3- Dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (0.225 g; 1.45 mmol) and a catalytic amount of 4-(dimethylamino) pyridine (DMAP) were added to the reaction mixture. tert-butyl (2-amino-5-fluorophenyl) carbamate (0.183 g; 0.809 mmol) was added, and the reaction was flushed with nitrogen gas. The reaction mixture was stirred for 12 h at room temperature to complete reaction. The solvent was evaporated and product was fractioned between ethyl acetate and saturated solution of sodium bicarbonate. The crude product was purified by column chromatography (EtOAc: Hexane; 50:50), afforded 0.307 g (yield 83%) of compound (38).1H NMR (400 MHz, DMSO-d6) δ : 10.16 (t, J = 4.0 Hz,1H), 9.83 (s, 1H), 8.81 (s, 1H), 7.99 (dd, J = 8.0 Hz,7.6 Hz, 2H), 7.50 (m, 4H), 7.04 (m,1H), 4.52 (d, J = 6.0 Hz, 2H), 1.50 (s, 9H). C21H21F4N3O4[M] : 455.41; MS (ESI) m / z: [M+H]+: 356.25.Reagents and conditions: a) K2CO3, MeOH: THF: H2O (1:1:0.5), RT, overnight. 35. Preparation of tert-butyl (2-(4-(amino methyl) benzamido)-5-fluorophenyl) carbamate (39) Compound tert-butyl (5-fluoro-2-(4-((2,2,2-trifluoroacetamido)-methyl)-benzamido)- phenyl)-carbamate (38) (0.300 g; 0.659 mmol) was dissolved in 25 mL mixture of methanol: tetra hydro-furan: water (1:1:0.5) and K2CO3(0.273 g; 1.978 mmol) was added. The mixture was stirred at room temperature for overnight. After completion, the reaction mixture was evaporated and ethylacetate was added. Ethylacetate layer was washed with saturated brine solution and was dried over sodium sulphate, concentrated under reduced pressure, afforded 0.218 g. (yield: 92%) of (39).1H NMR (400 MHz, DMSO-d6) δ : 9.76 (s, 1H), 8.78 (s, 1H), 7.92 (d, J = 8.0 Hz, 2H), 7.50 (m, 4H), 6.98 (m, 1H), 3.80 (s, 2H), 1.46 (s, 9H). C19H22FN3O3[M]:359.40; MS (ESI) m / z: [M+H]+: 360.25.Reagents and conditions: a) Succinic anhydride, 1-ethyl-3-dimethyl amino propyl carbodi imide (EDC), 4-di methyl amino pyridine (DMAP), DCM : Pyridine (1:1), RT, overnight.36. Preparation of 4-((4-((2-((tert-butoxycarbonyl)-amino)-4-fluorophenyl)- carbamoyl)-benzyl)-amino)-4-oxobutanoic acid (40) Compound tert-butyl (2-(4-(amino methyl) benzamido)-5-fluorophenyl) carbamate (39) (0.200 g; 0.557 mmol) was dissolved in 5 mL dichloromethane: pyridine (1:1). To this solution catalytic amount of 4-di methyl amino pyridine and 1-ethyl-3-dimethyl amino propyl carbodiimide (0.155 g; 1.002 mmol) were added. Reaction mixture was stirred for 20 min at room temperature. After 20 min, succinic anhydride (0.056g; 0.557mmol) was added and stirred for 5h at room temperature. Reaction was monitored by TLC. After completion of the reaction, pyridine mixture was evaporated. To the reaction mixture water was added and extracted with ethylacetate, and the separated organic layer was washed with saturated brine solution and the organic layer was dried over sodium sulphate, concentrated under reduced pressure. The crude mixture was purified by column chromatography. (methanol: DCM: acetic acid (10:90:1)) to afford 0.210 g. (yield: 74%) of (40).1H NMR (400 MHz, DMSO-d6) δ : 9.76 (s, 1H), 8.76 (s, 1H), 8.46 (s, 1H), 7.92 (d, J = 8Hz, 2H), 7.40 (m, 4H), 6.95 (m, 1H), 4.34 (s, 2H), 2.48 (t, J = 4.6 Hz, 2H), 2.42 (t, J = 4.8 Hz, 2H), 1.45 (s, 9H). C23H26FN3O6[M] : 459.47; MS (ESI) m / z : [M-H]+: 458.05; [M+Na]+: 482.25.Reagents and conditions: a) Gemcitabine hydrochloride, 1-ethyl-3-dimethyl aminopropylcarbodiimide (EDC), 1-Hydroxybenzotriazole, N-methylmorpholine, DMF: DMSO (3:1), RT, overnight. 37. Preparation of tert-butyl (2-(4-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)- 4-oxo-butanamido)-methyl)-benzamido)-5-fluorophenyl)carbamate (41) Gemcitabine hydrochloride (0.1 g; 0.380 mmol) and 8 mL mixture of DMF:DMSO (3:1) were added into a 50 mL RB flask. The reaction mixture was stirred and 1-(3-dimethylamino propyl)-3-ethylcarbodiimidehydrochloride, N-methylmorpholine, 1- hydroxy benzotriazole monohydrate, and 4-((4-((2-((tert-butoxy carbonyl)-amino)-4- fluoro phenyl)- carbamoyl)- benzyl)- amino)- 4-oxobutanoic acid (40) (0.184 g; 0.418 mmol) were added. The reaction mixture was protected under nitrogen balloon, and stirred at room temperature for overnight. The reaction was monitored by TLC. After completion 100 mL of 10% NaCl aq. solution and 50 mL of water were added slowly to the reaction solution to quench the reaction. The resulting yellow suspension was extracted with 3x 100 mL of ethylacetate. Combined ethylacetate layers were washed with 2x 60 mL of lithium chloride solution and 100 mL of saturated sodium bicarbonate solution and saturated brine solution. The ethylacetate layer was evaporated at vacuum condition to afford 0.120 g of (41) light yellow powder.1H NMR (400 MHz, DMSO-d6) δ : 11.05 (s, 1H), 9.81 (s, 1H), 8.68 (s, 1H), 8.52 (t, J = 6.0 Hz,1H), 8.25 (d, J = 7.6 Hz, 1H), 7.89 (d, J = 8.0 Hz, 2H), 7.54 (t, J = 6.4 Hz, 1H), 7.40 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 7.6 Hz, 1H), 7.16 (m, 2H), 6.33 (d, J = 6.8 Hz, 1H), 6.16 (t, J = 7.2 Hz, 1H), 5.31 (t, J = 5.2 Hz, 1H), 4.35 (d, J = 6.0 Hz, 2H), 3.89 (dt, J = 2.8 Hz, 3.8 Hz, 1H), 3.84 (dd, J = 3.6 Hz, 3.6 Hz, 2H), 3.64 (dt, J = 4.4, 3.2 Hz, 1H), 2.70 (t, J = 7.6 Hz, 2H), 1.45 (s, 8H), 1.24 (t, J = 9.2 Hz, 2H). C32H35F3N6O9[M]: 704.23; MS (ESI) m / z: [M+H]+: 705.25.Reagents and conditions: a) 4M HCl in Dioxane, DCM, 0ºC-RT, 2 h. 38. Preparation of N1-(4-((2-amino-4-fluoro phenyl) carbamoyl) benzyl)-N4-(1- (3,3-difluoro-4-hydroxy-5-(hydroxy methyl) tetra hydro furan-2-yl)-2-oxo-1,2- dihydropyrimidin-4yl) succinamide (42)Compound tert-butyl (2-(4-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl) amino)-4-oxo-butanamido)- methyl)-benzamido)-5fluoro- phenyl)-carbamate: (41) (0.05 g; 0.071 mmol) was dissolved in 3 mL dichloromethane on cooled ice bath and 3 mL of 4M HCl in dioxane was transferred slowly to the compound (41) solution at 0 °C and reaction was stirred for 1h at room temperature. After completion, the reaction mixture was dried and saturated solution of NaHCO3was added. Extracted with DCM and organic layer was separated, dried with sodium sulphate, evaporated under reduced pressure to afford 0.032 g. (yield: 74.6%) of (42).1H NMR (400 MHz, DMSO-d6) δ : 9.85 (s, 2H), 8.76 (t, 2H), 8.23 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 7.6 Hz, 2H), 7.65 (m, 2H), 7.43 (t, J = 8.4 Hz, 2H), 6.83 (dd, J = 2.4 Hz, 2.4 Hz, 2H), 6.63 (td, J = 6.0 Hz, 6.0 Hz, 2H), 6.53 (s, 3H), 6.43 (t, J = 8.0 Hz, 3H), 6.09 (d, J = 7.6 Hz, 1H), 5.51 (s, 2H), 4.63 (d, J = 5.6 Hz, 2H), 4.45 (td, J = 12.0 Hz, 12.0 Hz, 1H), 4.09 (td, J = 2.8 Hz, 2.8 Hz, 1H), 3.93 (dd, J = 2.4 Hz, 3.2 Hz, 2H), 3.64 (s, 2H), 2.80 (t, J = 1.6 Hz, 4H).13C NMR (101 MHz, DMSO-d6) δ: 167.64, 166.14, 157.20, 156.78, 155.46, 142.86, 141.49, 135.65, 130.41, 130.05, 127.84,127.75, 123.48, 121.01 – 120.81, 117.85, 115.27, 94.90, 84.02,81.32, 69.38, 59.10, 43.21.HRMS (APESI) m / z calculated for C27H27F3N6O7[M+H]+ :605.1971; found: 605.1874.Reagents and conditions: a) tert-butyl (2-amino-5-fluoro phenyl) carbamate, 4- (dimethylamino) pyridine, 1-(3-Dimethyl amino propyl)-3-ethyl carbodiimide hydrochloride, DCM: Pyridine (1:1), RT, 12 h. 39. Preparation of tert-butyl (4-(thiophen-2-yl)-2-(4-((2,2,2-trifluoroacetamido)- methyl) benzamido)-phenyl)-carbamate (43)Compound 4-((2,2,2-trifluoro acetamido) methyl) benzoic acid (32) (0.350 g; 1.420 mmol) was dissolved in a mixture of DCM: Pyridine (1:1). Then 1-(3- Dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (0.396 g; 2.556 mmol) and catalytic amount of 4-(dimethylamino) pyridine (DMAP) were added to the reaction mixture. tert-butyl (2-amino-4-(thiophene-2-yl) phenyl) carbamate (0.411 g; 1.420 mmol) was added, and the reaction was flushed with nitrogen gas. The reaction mixture was stirred for 12 h at room temperature for completion of the reaction. The solvent was evaporated and product was fractioned between ethylacetate and saturated solution of sodium bicarbonate. The crude product from the organic layer was purified by column chromatography using ethylacetate: hexane (40:60) solvent system to afford 0.378 g (yield: 51%) of compound (43).1H NMR (400 MHz, DMSO-d6) δ : 10.10 (s, 1H), 9.91 (s, 1H), 8.74 (s, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.54 (m, 2H), 7.47 (m, 3H), 7.14 (dd, J = 2.8 Hz, 2.8 Hz, 1H), 4.50 (s, 2H), 1.46 (s, 9H). C25H24F3N3O4S [M]: 519.53; MS (ESI) m / z: [M+H]+: 520.050.Reagents and conditions: a) K2CO3, MeOH: THF: H2O (1:1:0.5), RT, overnight. 40. Preparation of tert-butyl (2-(4-(amino methyl) benzamido)-4-(thiophen-2-yl) phenyl)-carbamate (44) Compound tert-butyl (4-(thiophen-2-yl)-2-(4-((2,2,2-trifluoroacetamido)-methyl) benzamido)-phenyl)-carbamate (43) (0.300 g; 0.577 mmol) was dissolved in 25 mL mixture of methanol: tetrahydrofuran: water (1:1:0.5) and K2CO3(0.240 g; 1.73 mmol) was added. The mixture was stirred at room temperature for overnight. After completion, the reaction mixture was evaporated and ethylacetate was added. Ethylacetate layer was washed with saturated brine solution and was dried over sodium sulphate, concentrated under reduced pressure, afforded 0.231 g. (yield:94.45%) of (44).1H NMR (400 MHz, DMSO-d6) δ : 9.96 (s, 1H), 8.82 (s, 1H), 7.99 (d, J = 8.4, 2H), 7.90 (d, J = 2.01H), 7.65 (d, J = 8.4, 1H), 7.58 (m, 4H), 7.51 (dd, J = 1.2 Hz, 1.4 Hz, 1H), 7.19 (dd, J = 3.6 Hz, 3.6 Hz, 1H), 3.89 (s, 2H), 1.52 (s, 9H). C23H25N3O3S [M] : 423.53; MS (ESI) m / z : [M+H]+: 424.050.Reagents and conditions: a) Succinic anhydride, 1-ethyl-3-dimethyl aminopropylcarbodiimide (EDC), 4-di methyl amino (DMAP), DCM : Pyridine (1:1), RT, overnight. 41. Preparation of 4-((4-((2-((tert-butoxycarbonyl)-amino)-5-(thiophen-2-yl)- phenyl)-carbamoyl)-benzyl)-amino)-4-oxobutanoic acid (45) Compound tert-butyl (2-(4-(amino methyl) benzamido)-4-(thiophen-2-yl) phenyl) carbamate (44) (0.200 g; 0.472 mmol) was dissolved in 5 mL dichloromethane: pyridine (1:1). To this solution catalytic amount of 4-di methyl amino pyridine and 1- ethyl-3-dimethyl amino propyl carbo-di-imide (0.132 g; 0.849 mmol) was added. Reaction mixture was stirred for 20 min at room temperature. After 20 min, succinic anhydride (0.047 g; 0.472 mmol) was added and stirred for overnight at room temperature. Reaction was monitored by TLC. After completion, the reaction mixture was evaporated. To the reaction mixture, water was added and extracted with ethyl acetate, the separated organic layer was washed with saturated brine solution, and the organic layer was dried over sodium sulfate, and concentrated under reduced pressure. The crude mixture was purified by column chromatography using methanol: DCM: acetic acid (10:90:1) to get 0.238 g (yield: 96%) of (45).1H NMR (400 MHz, MeOD4) δ : 9.84 (s, 1H), 8.70 (s, 1H), 7.87 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 2.0 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.46 (m, 4H), 7.40 (dd, J = 1.2 Hz, 1.2 Hz, 1H), 7.06 (dd, J = 3.6 Hz, 3.6 Hz, 1H), 3.77 (s, 2H), 2.43 (t, J = 1.6 Hz, 2H), 1.40 (s, 9H), 1.17 (d, J = 2.2 Hz, 2H). C27H29N3O6S [M] : 523.41; MS (ESI) m / z : [M+H]+: 524.23.Reagents and conditions: a) Gemcitabine hydrochloride, 1-ethyl-3-dimethyl aminopropyl carbodiimide (EDC), 1-Hydroxybenzotriazole, N-methylmorpholine, DMF: DMSO (3:1), RT, overnight. 42. Preparation of tert-butyl (2-(4-((4-((1-(3,3-di fluoro-4-hydroxy-5-(hydroxy methyl) tetra hydrofuran-2-yl)-2-oxo-1,2-dihydro pyrimidin-4-yl) amino)-4- oxobutanamido) methyl) benzamido)-4-(thiophen-3-yl) phenyl)carbamate (46) Gemcitabine hydrochloride (0.1 g; 0.380 mmol) and 8 mL mixture of DMF: DMSO (3:1) were added into a 50 mL flask. The reaction mixture was stirred and 1-(3- dimethylamino propyl)-3-ethylcarbodiimidehydrochloride, N-methylmorpholine, 1- hydroxybenzotriazole monohydrate, and 4-((4-((2-((tert-butoxycarbonyl)amino)-5- (thiophen-2-yl)phenyl)carbamoyl)benzyl)amino)-4-oxobutanoic acid (45) (0.184 g; 0.418 mmol) were added. The reaction mixture was protected under nitrogen balloon, and stirred at room temperature for overnight. Reaction was monitored by TLC. After completion, a 100 mL of 10% w / v aq. NaCl solution and a 50 mL of water were added slowly to quench the reaction. The resulting yellow suspension was extracted with 3x 100 mL of ethyl acetate. The combined ethyl acetate layer was washed with 2x 60 mL of lithium chloride solution and 100 mL of saturated sodium bicarbonate solution, and saturated brine solution. The ethyl acetate layer was evaporated under vacuum conditions to afford 0.120 g of (46) as a brown-yellow powder.1H NMR (400 MHz, DMSO-d6) δ : 11.06 (s, 1H), 9.89 (s, 1H), 8.74 (s, 1H), 8.52 (t, J = 6.0 Hz, 1H), 8.23 (d, J = 7.6 Hz, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 2.0 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.53 (m, 2H), 7.46 (dd, J =1.6 Hz, 1.2 Hz, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.26 (d, J =7.6 Hz, 1H), 7.14 (dd, J = 3.6, 4.0 Hz, 1H), 6.31 (d, J = 6.4 Hz, 1H), 6.18 (t, J =7.2 Hz, 1H), 5.28 (t, J = 5.6 Hz, 1H), 4.36 (d, J = 8.0 Hz, 2H), 3.90 (dt, J = 1.6Hz, 2.8 Hz, 1H), 3.83 (dt, J = 2.4 Hz, 3.6 Hz, 1H), 3.67 – 3.60 (m, 1H), 2.72 (t, J = 6.8 Hz 2H), 1.46 (s, 9H), 1.34 (s, 1H), 1.24 (t, J = 4.4 Hz, 2H).Reagents and conditions: a) 4M HCL in Dioxane, DCM, RT, 2 h. 43. Preparation of N1-(4-((2-amino-4-fluoro phenyl) carbamoyl) benzyl)-N4-(1- (3,3-di fluoro-4-hydroxy-5-(hydroxyethyl) tetra hydro furan-2-yl)-2-oxo-1,2- dihydro pyrimidin-4-yl) succinamide (47). Compound tert-butyl (2-(4-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-4-oxo-butanamido)- methyl)-benzamido)-5fluoro- phenyl)-carbamate: (46) (0.05 g; 0.071mmol) was dissolved in 3 mL dichloromethane cooled in ice bath and 3 mL 4M HCl in dioxane was transferred slowly to the compound (46) solution at 0°C and reaction was stirred for 2 h at room temperature. After completion, the reaction mixture was dried and saturated solution of NaHCO3 and extracted with DCM. The organic layer was separated, dried with sodium sulfate, and evaporated under reduced pressure to afford 0.032 g. (yield: 74.6%) of (47).1H NMR (400 MHz, DMSO-d6) δ : 9.85 (s, 1H), 8.63 (t, J = 6.8 Hz, 1H), 8.10 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 7.6 Hz, 1H), 7.63 (s, 1H), 7.52 (m, 4H), 7.45 (dd, J = 2.4 Hz, 2.4 Hz, 1H), 7.39 (dd, J = 0.8 Hz, 1.2 Hz, 1H), 7.19 (dd, J = 3.6 Hz, 3.6 Hz,1H), 6.97 (d, J = 8.4 Hz, 1H), 6.27 (t, J = 8.4 Hz, 1H), 5.93 (d, J = 7.6 Hz, 1H), 4.48 (d, J = 5.6 Hz, 2H), 4.27 (dt, J = 9.2 Hz, 8.8 Hz, 1H), 3.93 (dt, J = 2.8 Hz, 2.8 Hz, 1H), 3.89 (s, 1H), 3.77 (dd, J = 3.2 Hz, 3.6 Hz, 2H), 3.50 (s, 2H), 2.64 (t, J = 2.0 Hz, 4H), 2.05(s, 1H).13C NMR (101 MHz, DMSO-d6) δ : 167.57, 166.12, 155.18, 152.80, 142.99, 141.27, 134.62 – 134.42, 131.95, 131.14, 130.19, 129.21 – 129.0, 127.84, 127.12, 126.14, 125.42, 125.06, 124.23, 123.56,121.71, 121.09, 95.07, 80.88, 69.16, 59.42, 42.81,28.33. HRMS (APESI) m / z calculated for C31H30F2N6O7S [M+H]+ :669.1943; found: 669.2038.Reagents and conditions: a) tert-butyl 4-oxopiperidine-1-carboxylate, EtOH, Reflux, 3 h. 44. Preparation of tert-butyl 1,3,4,5-tetrahydro-2H-pyrido[4,3-b] indole-2- carboxylate (49) A mixture of phenyl hydrazine (48) (1.00 g; 9.25 mmol) and tert-butyl 4- oxopiperidine-1-carboxylate (1.84 g; 9.25 mmol) in ethanol were stirred at 80 ºC for 3 h. The reaction was monitored by TLC. After completion, to the reaction a saturated solution of NH4Cl was added and extracted with ethylacetate 3 times. The ethylacetate layers were combined and dried over sodium sulphate. Ethylacetate solution was evaporated under reduced pressure. The crude product was purified by column chromatography using methanol: DCM (10:90) solvent system to afforded 1.81 g. (yield: 72%) of (49).1H NMR (400 MHz, DMSO-d6) δ : 8.89 (s, 1H), 7.15 (t, J = 8.0 Hz, 2H), 7.05 (d, J = 7.6 Hz, 1H), 6.70 (t, J = 7.2 Hz, 1H), 3.34 (s, 2H), 2.51 (t, J = 6.0 Hz, 2H), 2.39 (t, J = 6.0 Hz, 2H), 1.43 (s, 9H). C16H20N2O2[M]: 272.35; MS (ESI) m / z : [M-H]+: 271.050.Reagents and conditions: a) methyl 4-(bromo methyl) benzoate, t-BuOK, DMF, 80οC, 3 h. 45. Preparation of tert-butyl 5-(4-(Methoxy carbonyl) benzyl)-1,3,4,5-tetrahydro- 2H-pyrido[4,3-b] indole-2-carboxylate (50) Compound tert-butyl 1,3,4,5-tetra hydro-2H-pyrido [4,3-b] indole-2-carboxylate (0.500 g; 1.84 mmol) (49) was dissolved in 3 mL of anhydrous DMF and added to a suspension of potassium tert-but oxide (0.204 g; 1.84 mmol) in 2 mL anhydrous DMF under nitrogen atmosphere at room temperature. The reaction mixture was heated to 80 °C for 15 min, after which methyl 4-(bromo methyl) benzoate (0.483 g; 1.84 mmol) dissolved in 2 mL anhydrous DMF was added at 80 °C. The reaction was stirred at 80 °C for 3 h, after which the reaction was cooled to room temperature and 15 mL saturated ammonium chloride solution was added into that. The product was extracted with EtOAc, washed with saturated brine solution, and dried over sodium sulfate. Ethylacetate was filtered and concentrated under reduced pressure. Purification was done using column chromatography using ethylacetate: hexane (80:20) solvent system afforded 0.271 g. (yield: 35%) of (50).1H NMR (400 MHz, DMSO-d6) δ :7.86 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.19 (d, J = 7.2 Hz, 2H), 6.82 (d, J = 8.3 Hz, 2H), 4.67 (s, 2H), 3.47 (s, 3H), 3.18 (t, J = 6.0 Hz, 2H), 2.43 (t, J = 6.0 Hz, 2H), 2.36 (t, J =5.6 Hz, 2H), 1.39 (s, 9H). C25H28N2O4[M]: 420.50; MS (ESI) m / z : [M-H]+: 419.050.Reagents and conditions: a) Trifluoroacetic acid: DCM (1:1), 0ºC – RT, 2 h. 46. Preparation of methyl 4-((1,2,3,4-tetra hydro-5H-pyrido [4,3-b] indol-5-yl) methyl) benzoate (51)There tert-butyl 5-(4-(Methoxy carbonyl) benzyl)-1,3,4,5-tetrahydro-2H-pyrido[4,3-b] indole-2-carboxylate (50) (0.200 g; 0.475 mmol) was dissolved in 5 mL dichloromethane on ice bath. In another round bottom flask 3 mL tri fluoro acetic acid was dissolved in 5 mL of dichloromethane. The solution was slowly transferred to the solution of compound (50) at 0°C and reaction was stirred for 2 h at room temperature. The reaction completion was monitored by TLC. After completion of the reaction, a saturated solution of sodium bicarbonate was added and extracted with ethyl acetate. The separated organic layer was washed with saturated brine solution and dried over sodium sulfate, and concentrated under reduced pressure. The crude mixture was purified by column chromatography using a DCM: methanol (90:10) solvent system that afforded 0.128g. (yield: 84%) of (51).1H NMR (400 MHz, MeOD4) δ : 7.32(d, J = 7.6 Hz, 2H), 6.83 (d, J = 8.0 Hz, 1H), 6.64 (dd, J = 8.0 Hz, 8.0 Hz, 3H), 6.59 (t, J = 7.2 Hz, 1H), 6.48 (t, J = 7.2 Hz, 1H), 4.16 (s, 2H), 3.53 (s, 3H), 2.38 (s, 4H), 1.99 (s, 2H). C20H20N2O2[M]: 320.39; MS (ESI) m / z : [M+H]+: 321.050.Reagents and conditions: a) Succinic anhydride, 1-ethyl-3-dimethyl amino propyl carbo di imide (EDC), 4-di methyl amino pyridine (DMAP), DCM: pyridine (1:1), RT, overnight. 47. Preparation of 4-(5-(4-(Methoxy carbonyl) benzyl)-1,3,4,5-tetrahydro-2H- pyrido[4,3-b] indol-2-yl)-4-oxobutanoic acid (52) Compound methyl 4-((1,2,3,4-tetrahydro-5H-pyrido [4,3-b] indol-5-yl) methyl) benzoate (51) (0.100 g; 0.312 mmol) was dissolved in 5 mL dichloromethane: pyridine (1:1). To this solution, catalytic amount of 4-di methyl amino pyridine and 1-ethyl-3- dimethyl amino propyl carbo-di-imide (0.087 g; 0.561 mmol) were added. Reaction mixture was stirred for 20 min at room temperature. After 20 min succinic anhydride(0.031 g; 0.312 mmol) was added stirred for overnight at room temperature. Reaction was monitored by TLC. After completion of the reaction, the pyridine mixture was evaporated under high vacuum and to residue, water was added and extracted with ethylacetate, separated organic layer was washed with saturated brine solution and was dried over sodium sulphate, concentrated under reduced pressure. The crude product was taken forward for next step (52).C24H24N2O5[M]: 420.47; MS (ESI) m / z : [M-H]+: 419.050.Reagents and conditions: a) Gemcitabine hydrochloride, 1-ethyl-3-dimethyl aminopropyl carbodiimide (EDC), 1-Hydroxybenzotriazole, N-methyl morpholine, DMF: DMSO (3:1), RT, overnight. 48. Preparation of methyl 4-((2-(4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxy methyl) tetra-hydrofuran-2-yl)-2-oxo-1,2-di hydro pyrimidin-4-yl)amino)-4-oxo butanoyl)-1,2,3,4-tetra-hydro-5H-pyrido[4,3-b]indol-5-yl)methyl)benzoate (53) Gemcitabine hydrochloride(0.100 g; 0.380 mmol) and 8 mL mixture of DMF:DMSO (3:1) added into a 50 mL flask. The reaction mixture was stirred and 4-(5-(4- (methoxycarbonyl)benzyl)-1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indol-2-yl)-4- oxobutanoic acid (52) (0.175 g; 0.418 mmol) was added. The reaction mixture was protected by nitrogen balloon, and stirred at room temperature for overnight. Reaction was monitored by TLC. After completion, a100 mL of 10% NaCl aq. solution and 50 mL of water were added slowly to the reaction solution to quench the reaction. The resulting yellow suspension was extracted with 3x 100 mL of ethylacetate. Combined ethylacetate layers was washed with 2x 60 mL of lithium chloride solution and a 100 mL of saturated sodium bicarbonate solution and saturated brine solution. The organic layer was evaporated at vacuum condition to afford 0.120 g of (53) brown yellow powder.1H NMR (400 MHz, MeOD4) δ : 7.75 (d, J = 7.3 Hz, 2H), 7.49 (d, J = 8.0Hz, 1H), 7.21 (d, J = 7.6 Hz, 1H), 7.17 (m, 2H), 6.96 (t, J = 7.2 Hz, 1H), 6.87 (t, J = 8.0 Hz, 1H), 6.12 (t, J = 7.6 Hz, 2H), 5.80 (d, J = 5.2 Hz, 1H), 4.60 (s, 2H), 4.13 (td, J = 3.6 Hz, 3.6 Hz, 4H), 3.99 (s, 2H), 3.85 (s, 3H), 3.82 (s, 1H), 3.79 (dt, J = 3.8 Hz, 2.8 Hz, 2H), 3.70 (d, J = 3.2 Hz, 1H), 3.67 (d, J = 3.2 Hz, 1H), 2.89 (t, J = 3.8 Hz, 4H), 2.46 (s, 2H).C33H33F2N5O8[M] : 665.47; MS (ESI) m / z : [M+H]+: 665.050.Reagents and conditions: a) aq. NH2OH, NaOH, MeOH, RT, 3 h. 49. Preparation of4-((2-(4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)- 4-oxobutanoyl)-1,2,3,4-tetrahydro-5H-pyrido[4,3-b]indol-5-yl)methyl)-N- hydroxybenzamide (54) Compound methyl 4-((2-(4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl) tetra- hydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl) amino)-4-oxobutanoyl)-1,2,3,4- tetra-hydro-5H-pyrido[4,3-b] indol-5-yl) methyl) benzoate (53) (0.055 g; 0.075 mmol) was dissolved in methanol. Then aq. solution of hydroxylamine (0.046 g; 1.384 mmol) was added and stirred for 30 min at room temperature. After 30 min, 2N NaOH solution was added into the reaction mixture and stirred at room temperature for 3 h. The reaction was monitored by TLC. After completion, water was added to the reaction mixture and neutralized with 1N HCl, and a precipitate was formed. The reaction mixture was centrifuged at 5000 rpm for 5 min and solid residue was collected after discarding the supernatant and washed with pentane to afford 0.022 g (yield: 44%) of (54).1H NMR (400 MHz, MeOD4) δ : 7.75 (d, J = 7.3 Hz, 2H), 7.49 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 7.6 Hz, 1H), 7.17 (m, 2H), 6.96 (t, J = 7.2 Hz, 1H), 6.87 (t, J = 8.0 Hz, 1H), 6.12 (t, J = 7.6 Hz, 2H), 5.80 (d, J = 5.2 Hz, 1H), 4.60 (s, 2H), 4.13 (td, J = 3.6 Hz, 3.6 Hz, 4H), 3.99 (s, 2H), 3.85 (s, 1H), 3.82 (s, 1H), 3.79 (td, J = 3.8 Hz, 2.8 Hz, 2H), 3.70 (d, J = 3.2 Hz, 1H), 3.67 (d, J = 3.2 Hz, 1H), 2.89 (t, 4H),2.46 (s, 2H).13C NMR (101 MHz, MeOD4) δ : 166.41, 156.12, 145.97, 141.07,134.75, 130.23, 129.16, 128.18, 126.72, 125.15, 122.59, 120.45, 120.02, 119.13, 117.68, 108.59, 108.06, 94.93, 84.20, 80.88, 69.23, 65.46, 59.05, 50.19, 40.30, 29.16, 20.34. HRMS (APESI) m / z calculated for C32H32F2N6O8.[M+H]+ :667.2128; found: 667.2168.Reagents and condition: a) MeOH, H2SO4, Reflux, 6 h. 50. Preparation of methyl 5-methylpyrazine-2-carboxylate (56); 5-methyl pyrazine carboxylic acid (55) (5.0 g, 36.20 mmol) was dissolved in 10 ml methanol and 0.3 mL of conc. sulfuric acid was added to the reaction mixture and refluxed for 6 h at 65 ºC. The reaction completion was monitored by TLC. After reaction completion, excess methanol was evaporated, and neutralized with diluted aq. NaHCO3solution. The reaction mixture was extracted with ethyl acetate and washed with saturated brine solution. The ethyl acetate layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude product was purified by column chromatography by using 50:50 ethyl acetate in hexane to afford 5.27 g (yield 95.68%) of compound (56) as an off-white crystalline powder.1H NMR (400 MHz, DMSO-d6) δ 8.99 (d, J = 1.4 Hz, 1H), 8.58(d, J = 1.4 Hz, 1H), 3.90 (s, 3H), 2.36 (s, 3H). C7H8N2O2[M]: 152.15; MS (ESI) m / z: [M + H]+: 153.30 [M-H]+: 151.25.Reagents and conditions: a) N-Bromo- succinimide, Azobisisobutyronitrile (AIBN), CCl4, 80ºC, 6 h. 51. Preparation of methyl 5-(bromomethyl)pyrazine-2-carboxylate (57); methyl 5-methylpyrazine-2-carboxylate (56) (2 g, 13.14 mmol) and N- Bromosuccinimide (4.68 g, 26.29 mmol) was dissolved in 10 mL of carbon tetra chloride and reflux at 80 ºC. After 20 min, AIBN (0.129 g, 0.78 mmol) was added to the reaction mixture and stirred for 6 h at 80 ºC. The residue was basified with saturated sodium bicarbonate solution and extracted with ethyl acetate. The organic layer was dried and concentrated. The crude was purified by silica gel column chromatography using 20% ethyl acetate in hexane to afford 1.746 g (yield 57.49%) methyl 5-(bromomethyl)pyrazine-2-carboxylate (57) as a fluffy crystallin powder .1H NMR (400 MHz, DMSO-d6) δ 9.18 (d, J = 1.4 Hz, 1H), 8.95 (d, J = 1.4 Hz, 1H), 4.85 (s, 2H), 3.93 (s, 3H). C7H7BrN2O2[M]: 231.04; MS (ESI) m / z: [M + H]+: 232.30.Reagents and conditions: a) Hexamethylene tetramine (HMTA), Chloroform, 18 h. b) 2N HCl, Methanol, 70 ºC, 3 h. 52. Preparation of methyl 5-(aminomethyl)pyrazine-2-carboxylate (58); To a solution of methyl 5-(bromomethyl)pyrazine-2-carboxylate (57) (1.00 g, 4.33 mmol) in chloroform (10 mL), hexamethylene tetramine (0.610 g, 4.33 mmol) was added and the reaction mixture was stirred at room temperature for 18 h. The excess chloroform was evaporated under reduced pressure and solid residue was suspended in methanol (10 mL) followed by 3 mL 2 N hydrochloric acid was added and the reaction mixture was heated at 75 ºC for 3 h. The reaction mixture was concentrated, the residue was triturated with diethyl ether and dried to afford the 0.517 g compound methyl 5-(aminomethyl)pyrazine-2-carboxylate hydrochloride (58) (yield 71.46%) as a brownish solid.1H NMR (400 MHz, DMSO-d6) δ 9.20 (d, J = 1.4 Hz, 1H), 9.00 (d, J= 1.4 Hz, 1H), 4.85 (s, 2H), 3.94 (s, 3H), 3.65 (s, 2H). C7H9N3O2[M]: 167.17; MS (ESI) m / z: [M + H]+: 168.25.Reagents and conditions: a) Trifluoroacetic anhydride, RT, 2 h. 53. Preparation of 5-((2,2,2-trifluoroacetamido)methyl)pyrazine-2-carboxylate acid (59): The solution of methyl 5-(aminomethyl)pyrazine-2-carboxylate (58) (0.5 g; 2.99 mmol) in 10 mL of trifluoroacetic anhydride was stirred at room temperature under nitrogen atmosphere for 2h. Reaction completion was monitored by TLC. After completion, the reaction mixture was neutralized by using diluted aq. NaHCO3, and extracted with ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The obtained crude product was washed with diethyl ether to afford 0.709 g (yield: 90 %) of (59) and followed to the next reaction without purification. C9H8F3N3O3[M]: 263.18; MS (ESI) m / z: [M + H]+: 264.30.Reagents and conditions: a) aq. LiOH, Methanol, RT, 2 h. 54. Preparation of methyl 5-((2,2,2-trifluoroacetamido)methyl)pyrazine-2- carboxylic acid (60); To a solution of 5-((2,2,2-trifluoroacetamido)methyl)pyrazine-2- carboxylate (59) (0.5 g, 1.90 mmol) in methanol (10 mL) was stirred at room temperature followed by aq. LiOH solution was added to the reaction mixture stirred at room temperature for 18 h. The excess methanol was evaporated under reduced pressure, and followed by 3 mL 2N hydrochloric acid was added. The reaction mixture was extracted with DCM, and evaporated under reduced pressure. The residue wastriturated with diethyl ether and dried to afford the compound 5-((2,2,2- trifluoroacetamido)methyl)pyrazine-2-carboxylic acid (60) (0.460 g, crude) as a brownish solid. C8H6F3N3O3[M]: 249.15; MS (ESI) m / z: [M + H]+: 250.30.Reagents and conditions: a) tert-Butyl (2-aminophenyl) carbamate, 4-(dimethyl amino) pyridine, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride, DCM: Pyridine (1:1), RT, 12 h. 55. Preparation of tert-butyl (2-(5-((2,2,2-trifluoroacetamido)methyl)pyrazine-2- carboxamido)phenyl)carbamate (61): Compound 5-((2,2,2-trifluoroacetamido)methyl)pyrazine-2-carboxylic acid (60) (0.2 g; 0.802 mmol) was dissolved in a mixture of DCM: Pyridine (1:1). 1-(3-Dimethyl amino propyl)-3-ethyl carbo di imide hydro chloride (0.277 g; 1.44 mmol) and catalytic amount of 4- (dimethyl amino) pyridine (DMAP) were added to the reaction mixture. tert-Butyl (2-aminophenyl) carbamate (0.251 g; 1.20 mmol) was added and the reaction was purged with nitrogen gas. The reaction mixture was stirred for 12 h at room temperature till the completion of the reaction. The excess solvent was evaporated and the product was fractioned between ethyl acetate and a saturated brine solution. The crude product was purified by column chromatography (MeOH: DCM; 10:90), afforded 0.281 g. (yield 79%) of compound (61).1H NMR (400 MHz, DMSO- d6) δ : 10.12 (s, 1H), 9.86 (d, J = 1.4 Hz, 1H), 8.68 (d, J = 1.4 Hz, 1H),), 7.95 (t, J = 7.2 Hz, 2H), 7.46 (d, J = 6.4 Hz, 4H), 7.17 (m, 2H), 4.49 (s, 2H), 1.45 (s, 9H). C19H20F3N5O4[M]: 439.39; MS (ESI) m / z : [M+H]+: 440.16.Reagents and conditions: a) K2CO3, MeOH : THF : H2O, RT, overnight . 56. Preparation of tert-butyl (2-(5-(aminomethyl)pyrazine-2- carboxamido)phenyl)carbamate (62): Compound tert-butyl (2-(5-((2,2,2- trifluoroacetamido)methyl)pyrazine-2-carboxamido)phenyl)carbamate (61) (0.2 g; 0.455 mmol) was dissolved in 5 mL mixture of methanol: tetrahydrofuran and aq. K2CO3(0.188 g, 1.365 mmol) was added. The mixture was stirred at room temperature for overnight. After completion of the reaction, the mixture was evaporated and ethyl acetate was added. The ethyl acetate layer was washed with saturated brine solution and was dried over sodium sulfate, concentrated under reduced pressure that afforded 0.143 g (yield: 90.93%) of (62).1H NMR (400 MHz, DMSO-d6) δ : 9.86 (s, 1H), 8.73 (d, J = 1.4 Hz, 1H), 7.96 (d, J = 6.8 Hz, 2H), 7.56 (m, 4H), 7.28 (m, J = 8.8 Hz, 2H), 3.86 (s, 2H), 1.50 (s, 9H). C17H21N5O3[M]: 341.42; MS (ESI) m / z: [M+H]+: 342.16.Reagents and conditions: a) Succinic anhydride, 1-ethyl-3-dimethyl amino propyl carbodiimide (EDC), 4-dimethyl aminopyridine (DMAP), DCM : Pyridine (1:1), RT, overnight. 57. Preparation of 4-(((5-((2-((tert-butoxycarbonyl)-amino)-4-phenyl)- carbamoyl)-pyrazin-2-yl)methyl)amino)-4-oxobutanoic acid (63): Compound tert- butyl (2-(5-(aminomethyl)pyrazine-2-carboxamido)phenyl)carbamate (62) (0.13 g; 0.378 mmol) was dissolved in 5 mL dichloromethane: pyridine (1:1). To this solution, catalytic amount of 4-dimethyl aminopyridine and 1-ethyl-3-dimethyl amino propyl carbo-di-imide (0.105 g, 0.680 mmol) were added. The reaction mixture was stirred for 20 min at room temperature. After 20 min, succinic anhydride (0.046 g; 0.454 mmol) was added and stirred for 5 h at room temperature. The reaction was monitored by TLC. After completion the reaction, pyridine mixture was evaporated. To the residue, water was added and extracted with ethylacetate, separated organic layer, and the organic layer was washed with saturated brine solution and was dried over sodiumsulfate, concentrated under reduced pressure to afford 0.112 g. (yield: 67%) of (63). C21H25N5O6[M] : 443.46; MS (ESI) m / z : [M+H]+: 442.16.Reagents and conditions: a) Gemcitabine hydrochloride, 1-ethyl-3-dimethyl amino propyl carbo diimide (EDC), 1-Hydroxybenzotriazole, N-Methylmorpholine, DMF: DMSO (3:1), RT, overnight. 58. Preparation of tert-butyl (2-(5-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)- 4-oxobutanamido)-methyl)pyrazine-2-carboxamido)-5-phenyl)carbamate (64): Gemcitabine hydrochloride (0.067 g; 0.225 mmol) and 5 mL mixture of DMF:DMSO (3:1) added into a 25 mL RB flask. The reaction mixture was stirred and 1-(3- dimethylamino propyl)-3-ethylcarbodiimidehydrochloride (0.063 g; 0.409 mmol), N- methylmorpholine (0.053 g; 0.45 mmol), 1-hydroxybenzotriazole monohydrate (0.030 g; 0.225 mmol), and 4-((4-((2-((tert-butoxycarbonyl) amino) phenyl) carbamoyl) benzyl) amino)-4-oxobutanoic acid (63) (0.100 g; 0.225 mmol) were added. The reaction mixture was protected with a nitrogen balloon and stirred at room temperature for overnight. The reaction was monitored by TLC. After completion of the reaction, 50 mL of 10% NaCl solution and 25 mL of water were added slowly to the reaction solution to quench the reaction. The resulting yellow suspension was extracted with 3x 100 mL of ethyl acetate. The combined ethyl acetate layer was washed with 2x 60 mL of lithium chloride solution, 100 mL of saturated sodium bicarbonate solution, and saturated brine solution. The ethyl acetate layer was evaporated at vacuum conditions and purified by column chromatography using methanol: DCM (1:9) as mobile phase to afford 0.083 g (% yeild 53.45) of (64) as yellowish-brown powder.1H NMR (400 MHz, DMSO-d6) δ : 11.05 (s, 1H), 9.81 (s, 1H), 8.68 (s, 1H), 8.50 (t, J = 5.6 Hz, 1H), 8.25 (d, J = 7.6 Hz, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.54 (d, J = 6.0, Hz, 1H), 7.43 (d, J= 8.0 Hz, 2H), 7.27 (d, J = 8.0 Hz, 1H), 7.19 (m, 2H), 6.33 (d, J = 6.4 Hz, 1H), 6.20 (t, J = 7.2 Hz, 1H), 5.28 (t, J = 5.2 Hz, 1H), 4.35 (d, J = 5.2 Hz, 2H), 3.86 (dd, J = 3.2 Hz, 3.2 Hz, 2H), 3.83 (m, 1H), 3.65 (m, 1H), 2.70 (t, J = 6.2, Hz, 2H), 1.45 (s, 9H), 1.24 (t, J = 9.2, Hz, 2H). C30H34F2N8O9[M] : 688.65; MS (ESI) m / z : M+H]+: 689.20Reagents and conditions: a) 4M HCl in Dioxane, DCM, RT, 2 h. 59. Preparation of N1-((5-((2-amino-4-phenyl)carbamoyl)pyrazin-2-yl)methyl)- N4-(1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)succinamide (65) Compound tert-butyl (2-(5-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-4-oxobutanamido)- methyl)pyrazine-2-carboxamido)-5-phenyl)carbamate (64) (0.050 g; 0.073 mmol) was dissolved in 3 mL dichloromethane and cooled in ice bath and 3 mL of 4 M HCl in dioxane was slowly transferred to compound (64) solution at 0 °C and reaction was stirred for 2 h at room temperature. After completion of the reaction, the mixture was dried and saturated solution of NaHCO3was added into it. Extracted with 1:1 mixture of n-butanol and ethyl acetate, dried with sodium sulphate, evaporated under reduced pressure to afford 0.033 g. (yield: 77.23 %) of (65).1H NMR (400 MHz, DMSO-d6) δ : 9.96 (s, 1H), 8.76 (s, 1H), 8.21 (d, J = 7.2 Hz, 2H), 8.10 (d, J = 7.6 Hz, 2H), 7.68 (d, J = 8.4 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.28 (t, J = 6.8 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 6.93 (t, J = 7.2 Hz, 1H), 6.58 (s, 1H), 6.42 (t, J = 8.0 Hz, 1H), 6.16 (d, J = 7.6 Hz, 1H), 4.65 (s, 2H), 4.44 (s, 2H), 4.13 (dt, J = 2.8 Hz, 2.8 Hz, 2H), 4.05 (s, 1H), 3.93 (dd, J = 3.2 Hz, 3.6 Hz, 2H), 2.80 (t, J = 2.4 Hz, 4H), 2.21 (s, 1H).13C NMR (101 MHz, DMSO-d6) δ : 172.67, 172.07, 163.17, 161.07, 154.05, 154.04, 153.10, 146.14, 144.74, 143.65, 142.47, 140.85, 128.67, 126.61, 124.11, 121.68, 115.58,97.83, 92.85, 92.46, 92.07. C25H26F2N8O7[M] : 588.53; MS (ESI) m / z : M+H]+: 689.20.Reagents and conditions: a) tert-butyl (2-amino-5-fluorophenyl)carbamate, 4- (dimethyl amino) pyridine, 1-(3-Dimethylaminopropyl)-3- ethylcarbodiimidehydrochloride, DCM: Pyridine (1:1), RT, 12 h. 60. Preparation of tert-butyl (5-fluoro-2-(5-((2,2,2- trifluoroacetamido)methyl)pyrazine-2-carboxamido)phenyl)carbamate (66): Compound 5-((2,2,2-trifluoroacetamido)methyl)pyrazine-2-carboxylic acid (60) (0.2 g; 0.802 mmol) was dissolved in a mixture of DCM: Pyridine (1:1). 1-(3-Dimethyl amino propyl)-3-ethyl carbo di imide hydro chloride (0.224 g; 1.44 mmol) and catalytic amount of 4- (dimethyl amino) pyridine (DMAP) were added to the reaction mixture. tert-butyl (2-amino-5-fluorophenyl)carbamate (0.182 g; 0.802 mmol) was added and reaction was purged with nitrogen gas. The reaction mixture was stirred for 12 h at room temperature till the completion of the reaction. The solvent was evaporated and product was fractioned between ethylacetate and saturated solution of sodium bicarbonate. The crude product was purified by column chromatography (MeOH: DCM; 10:90), afforded 0.274 g. (yield 74.61%) of compound (66).1H NMR (400 MHz, DMSO-d6) δ : 10.12 (s, 1H), 9.86 (s, 1H), 8.68 (s, 1H), 7.95 (t, J = 7.2 Hz, 2H), 7.46 (d, J = 6.4 Hz, 4H), 7.17 (m, 2H), 4.49 (s, 2H), 1.45 (s, 9H). C19H19F4N5O4[M] : 457.38; MS (ESI) m / z : [M+H]+: 458.16.Reagents and conditions: a) K2CO3, MeOH : THF : H2O, RT, overnight. 61. Preparation of tert-butyl (2-(5-(aminomethyl)pyrazine-2- carboxamido)phenyl)-carbamate (67): Compound tert-butyl (5-fluoro-2-(5-((2,2,2-trifluoroacetamido)methyl)-pyrazine-2- carboxamido)phenyl)-carbamate (66) (0.250 g; 0.546 mmol) was dissolved in 10 mL mixture of methanol: tetrahydrofuran followed by aq. solution of K2CO3(0.226 g; 1.638 mmol) was added and allow to stirred at room temperature for overnight. After completion of the reaction, the mixture was evaporated and ethylacetate was added. Ethylacetate layer was washed with saturated brine solution and was dried over sodium sulphate, concentrated under reduced pressure that afforded 0.186 g (yield: 94.17%) of (67).1H NMR (400 MHz, DMSO-d6) δ : 9.86 (s, 1H), 8.73 (s, H), 7.96 (d, J = 6.8 Hz, 2H), 7.56 (m, 4H), 7.28 (m, J = 8.8 Hz, 2H), 3.86 (s, 2H), 1.50 (s, 9H). C17H20FN5O3[M] : 361.38; MS (ESI) m / z : [M+H]+: 362.17.Reagents and conditions: a) Succinic anhydride, 1-ethyl-3-dimethyl amino propyl carbodiimide (EDC), 4-dimethyl aminopyridine (DMAP), DCM : Pyridine (1:1), RT, overnight. 62. Preparation of 4-(((5-((2-((tert-butoxycarbonyl)amino)-4- fluorophenyl)carbamoyl)-pyrazin-2-yl)methyl)amino)-4-oxobutanoic acid (68): Compound tert-butyl (2-(5-(aminomethyl)pyrazine-2-carboxamido)-5- fluorophenyl)carbamate (67) (0.150 g; 0.415 mmol) was dissolved in 5 mL dichloromethane: pyridine (1:1). To this solution, catalytic amount of 4-dimethyl aminopyridine and 1-ethyl-3-dimethyl amino propyl carbo-di-imide (0.143.19 g, 0.747 mmol) were added. Reaction mixture was stirred for 20 min at room temperature. After 20 min, succinic anhydride (0.050 g; 0.498 mmol) was added and stirred for overnight at room temperature. Reaction was monitored by TLC. After completion the reaction, pyridine mixture was evaporated. To the residue, water was added andextracted with ethylacetate, separated organic layer, and the organic layer was washed with saturated brine solution and was dried over sodium sulphate, and concentrated under reduced pressure. The crude residue was triturated with di-ethyl ether to afford 0.167 g (yield: 87 %) of (68). C21H24FN5O6[M] : 461.45; MS (ESI) m / z : [M+H]+: 462.25.Reagents and conditions: a) Gemcitabine hydrochloride, 1-ethyl-3-dimethyl amino propyl carbo diimide (EDC), 1-Hydroxybenzotriazole, N-Methylmorpholine, DMF: DMSO (3:1), RT, overnight. 63. Preparation of tert-butyl (2-(5-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)- 4-oxobutanamido)-methyl)pyrazine-2-carboxamido)-5-fluorophenyl)carbamate (69): Gemcitabine hydrochloride (0.097 g; 0.325 mmol) and 8 mL mixture of DMF:DMSO (3:1) added into a 50 mL RB flask. The reaction mixture was stirred and 1-(3-dimethylamino propyl)-3-ethylcarbodiimidehydrochloride (0.093 g; 0.487 mmol), N-methylmorpholine (0.076 g; 0.650 mmol), 1-hydroxybenzotriazole monohydrate (0.060 g; 0.443 mmol), and 4-(((5-((2-((tert-butoxycarbonyl)amino)-4- fluorophenyl)carbamoyl)-pyrazin-2-yl)methyl)amino)-4-oxobutanoic acid (68) (0.150 g; 0.325 mmol) were added. The reaction mixture was protected with a nitrogen balloon and stirred at room temperature for overnight. The reaction was monitored by TLC. After completion of the reaction, 50 mL of 10% NaCl solution and 50 mL of water were added slowly to the reaction solution to quench the reaction. The resulting yellow suspension was extracted with 3x 100 mL of ethyl acetate. The combined ethyl acetate layer was washed with 2x 20 mL of lithium chloride solution, 50 mL of saturated sodium bicarbonate solution, and saturated brine solution. The ethyl acetate layer was evaporated under vacuum conditions and purified by columnchromatography using methanol: DCM (1:9) as a mobile phase to afford 0.134 g of (69) pale brown foam.1H NMR (400 MHz, DMSO-d6) δ : 11.05 (s, 1H), 9.81 (s, 1H), 8.68 (s, 1H), 8.50 (t, J = 5.6 Hz, 1H), 8.25 (d, J = 7.6 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.54 (td, J = 6.0,1.6 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.0 Hz, 1H), 7.19 (m, 2H), 6.33 (d, J = 6.4 Hz, 1H), 6.20 (t, J = 7.2 Hz, 1H), 5.28 (t, J = 5.2 Hz, 1H), 4.35 (d, J = 5.2 Hz, 2H), 3.86 (dd, J = 3.2 Hz, 3.2 Hz, 2H), 3.83 (dt, J = 4.8, 4.8 Hz, 1H), 3.65 (dt, J = 3.6, 3.6 Hz, 1H), 2.70 (t, J = 6.2, Hz, 2H), 1.45 (s, 9H), 1.24 (t, J = 9.2, Hz, 2H). C30H33F3N8O9 [M] : 706.63; MS (ESI) m / z : M+H]+: 707.20.Reagents and conditions: a) 4M HCl in 1,4-dioxane, DCM, RT, 2 h. 64. Preparation of N1-((5-((2-amino-4-fluorophenyl)carbamoyl)pyrazin-2- yl)methyl)-N4-(1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2- yl)-2-oxo-1,2-dihydropyrimidin-4-yl)succinamide (70) Compound tert-butyl (2-(5-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-4-oxobutanamido)- methyl)pyrazine-2-carboxamido)-5-fluorophenyl)carbamate (69) (0.120 g; 0.170 mmol) was dissolved in 3 mL dichloromethane and cooled in ice bath and 3 mL of 4M HCl in dioxane was slowly transferred to compound (69) solution at 0 °C and reaction was stirred for 2 h at room temperature. After completion of the reaction, the mixture was dried and a saturated solution of NaHCO3was added to it. Extracted with 1:1 mixture of n-butanol and ethyl acetate, an organic layer was separated, dried with sodium sulphate, and evaporated under reduced pressure to afford 0.049 g. (yield: %) of (70).1H NMR (400 MHz, DMSO-d6) δ : 9.96 (s, 1H), 8.76 (s, 1H), 8.21 (d, J = 7.2 Hz, 2H), 8.10 (d, J = 7.6 Hz, 2H), 7.68 (d, J = 8.4 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.28 (t, J = 6.8 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 6.93 (t, J = 7.2 Hz, 1H), 6.58 (s,1H), 6.42 (t, J = 8.0 Hz, 1H), 6.16 (d, J = 7.6 Hz, 1H), 4.65 (s, 2H), 4.44 (s, 2H), 4.13 (dt, J = 2.8 Hz, 2.8 Hz, 2H), 4.05 (s, 1H), 3.93 (dd, J = 3.2 Hz, 3.6 Hz, 2H), 2.80 (t, J = 2.4 Hz, 4H), 2.21 (s, 1H).13C NMR (101 MHz, DMSO-d6) δ : 172.68, 172.22, 161.07, 159.81, 157.34, 154.05, 144.74, 141.85, 141.75, 141.02, 132.37, 128.55, 127.65, 126.61, 126.51, 124.05, 122.61, 122.52, 121.49, 107.41, 107.19, 103.42, 103.18, 97.83, 92.46, 82.74, 69.89, 61.37, 43.90, 31.82, 31.34. m / z calculated for C25H25F3N8O7[M+H]+ :606.51; found: 607.20.Reagents and Conditions: a) H2SO4, MeOH, reflux, 6 h. 65. Preparation of methyl 2-methylquinoline-6-carboxylate (72): The synthesis of methyl 2-methylquinoline-6-carboxylate was performed using the process of esterification in reflux with methanol (25 ml) as a solvent and sulfuric acid as a catalyst with 2-methylquinoline-6-carboxylic acid (71) (5 g, 26.72 mmol). After the reaction was completed while monitoring with TLC, and neutralized the reaction mixture with 10% aq. solution of NaHCO3. The reaction mixture was extracted with ethyl acetate and dried over sodium sulfate. Further evaporated the ethyl acetate layer to afford the 5.23 g (% yield 97%) of compound (72) as white crystaliine powder.1H NMR (400 MHz, DMSO-d6) δ 8.67 (d, J = 1.9 Hz, 1H), 8.54 (d, J = 8.5 Hz, 1H), 8.20 (dd, J = 8.8, 2.0 Hz, 1H), 8.05 (d, J = 8.9 Hz, 1H), 7.58 (d, J = 8.5 Hz, 1H), 3.93 (s, 3H), 2.73 (s, 3H). C12H11NO2[M] : 201.22; MS (ESI) m / z : M+H]+: 202.15.Reagents and conditions: a) N-bromosuccinamide, AIBN, CCl4, Reflux, 8 h. 66. Preparation of methyl 2-(bromomethyl) quinoline-6-carboxylate (73)The synthesis of methyl 2-(bromomethyl) quinoline-6-carboxylate was performed utilizing the process of allylic halogenation in reflux temperature of 60 °C with methyl 2-methylquinoline-6-carboxylate (72) (2 g, 9.94 mmol), and N-bromosuccinimide (2.653 g, 14.91 mmol) was suspended in 8 mL of carbon tetrachloride. The reaction mixture was heated to reflux. Catalyst azobisisobutyronitrile (0.816 g, 4.97 mmol) was added into the reaction mixture, under inert condition and stirred at 60 ºC for 6 h. Further, after reaction completion, remove excess of CCl4under reduced pressure and the residue as dissolved in ethyl acetate. The ethyl acetate layer was washed with brine solution 3 times and dried over anhydrous sodium sulfate. The ethyl acetate layer was evaporated under reduced pressure and purified by column chromatography using, up to 3:7 mixture of ethyl acetate and hexane as gradient solvent system to afford 1.83 g of (73) (% yield 66 %) as fluffy white powder.1H NMR: (400 MHz, DMSO-d6) δ 8.71 (d, J = 2.0 Hz, 1H), 8.62 (d, J = 7.6 Hz, 1H), 8.24 (dd, J = 8.9, 2.0 Hz, 1H), 8.09 (dt, J = 8.9, 0.8 Hz, 1H), 7.78 (d, J = 8.5 Hz, 1H), 4.88 (s, 2H), 3.94 (s, 3H); C12H10BrNO2[M] : 280.12; MS (ESI) m / z : M+2]+: 282.15.Reagents and Conditions: a) Hexamethylenetetramine, CHCl3, Rt, overnight. (b) MeOH, 2N aq. HCl, 70 ºC, 3 h. 67. Preparation of methyl 2-(aminomethyl)quinoline-6-carboxylate (74) The 2-(aminomethyl)quinoline-6-carboxylate (74) was synthesized by delepine reaction, of methyl 2-(bromomethyl)quinoline-6-carboxylate (73) (1.0 g, 3.57 mmol) in chloroform as solvent and hexamethylenetetramine (0.5 g, 3.57 mmol) at room temperature overnight. Further, the solvent was evaporated, and suspended the residue in methanol. Aq. 2 M hydrochloric acid was added while stirring at room temperature for 3 h, and following the completion of the reaction, excess methanol was evaporated under reduced pressure. The residue was triturated with ice cold diethyl ether to affordthe pure 0.642 g of compound (74) (% yield 83%) as brown powder.1H NMR (400 MHz, DMSO-d6) δ 8.49 (dd, J = 7.9, 2.2 Hz, 1H), 8.43 (t, J = 1.8 Hz, 1H), 8.29 (dd, J = 8.4, 1.5 Hz, 1H), 8.08 (d, J = 8.1 Hz, 1H), 7.42 – 7.36 (m, 1H), 4.57 (t, J = 6.3 Hz, 2H), 3.97 (td, J = 6.2, 0.7 Hz, 2H), 3.88 (s, 3H); C12H12N2O2[M] :216.24; MS (ESI) m / z : M+H]+: 217.15.Reagents and Conditions: a) Trifluoracetic anhydride, DCM, 0 ºC – Rt, overnight. 68. Preparation of methyl 2-((2,2,2-trifluoroacetamido)methyl)quinoline-6- carboxylate (75) The solution of methyl 2-(aminomethyl)quinoline-6-carboxylate (74) (0.50 g; 2.31 mmol) in 10 mL of trifluoroacetic anhydride was stirred at room temperature under nitrogen atmosphere for 2h. Reaction completion was monitored by TLC. After completion of the reaction, to the reaction mixture, water was added and extracted with ethyl acetate. The ethyl acetate layer was evaporated under reduced pressure. The obtained crude product was purified with column chromatography using 4:6 ethyl acetate: hexane to afford 0.604 g of compound (75) (yield : 84 %) as white amorphous powder;1H NMR (400 MHz, DMSO-d6) δ 8.47 – 8.40 (m, 2H), 8.29 (dd, J = 8.3, 1.4 Hz, 1H), 8.08 (d, J = 8.1 Hz, 1H), 8.01 – 7.93 (m, 1H), 7.55 – 7.48 (m, 1H), 4.49 (dd, J = 4.4, 0.7 Hz, 2H), 3.88 (s, 3H); C14H11F3N2O3[M] :312.25; MS (ESI) m / z : M+H]+: 213.15.Reagents and conditions: a) aq. LiOH, Methanol, RT, 18 h.69. Preparation of 2-((2,2,2-trifluoroacetamido)methyl)quinoline-6-carboxylic acid (76); To a solution of methyl 2-((2,2,2-trifluoroacetamido)methyl)quinoline-6-carboxylate (75) (0.5 g, 1.60 mmol) in Methanol (10 mL) was stirred at room temperature followed by aq LiOH solution was added to the reaction mixture stirred at room temperature for 18 h. The excess methanol was evaporated under reduced pressure, and followed by 3 mL of 2N hydrochloric acid was added. The reaction mixture was extracted with DCM, and evaporated under reduced pressure. The residue was triturated with diethyl ether and dried to afford the 0.437 g, crude of 2-((2,2,2- trifluoroacetamido)methyl)quinoline-6-carboxylic acid (76) (% yield 91 %) as a brownish solid. C13H9F3N2O3[M]: 298.22; MS (ESI) m / z: [M - H]+: 297.16.Reagents and conditions: a) tert-Butyl (2-aminophenyl) carbamate (2), 4-(dimethyl amino) pyridine, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride, DCM: Pyridine (1:1), room temperature, 12 h. 70. Preparation of tert-butyl (2-(2-((2,2,2-trifluoroacetamido)methyl)quinoline-6- carboxamido)phenyl)carbamate (77): Compound 2-((2,2,2-trifluoroacetamido)methyl)quinoline-6-carboxylic acid (76) (0.2 g; 0.670 mmol) was dissolved in a mixture of DCM: Pyridine (1:1). 1-(3-Dimethyl amino propyl)-3-ethyl carbo di imide hydro chloride (0.231 g; 1.206 mmol) and catalytic amount of 4- (dimethyl amino) pyridine (DMAP) were added to the reaction mixture. tert-butyl (2-aminophenyl) carbamate (0.139 g; 0.670 mmol) was added and the reaction was purged with nitrogen gas. The reaction mixture was stirred for 12 h at room temperature till the completion of the reaction. The solvent was evaporated and the product was fractioned between ethyl acetate and a saturated solution of sodium bicarbonate. The crude product was purified by column chromatography (MeOH: DCM; 10:90), afforded 0.263 g. (yield 80%) of compound (77) as off white solid;1HNMR (400 MHz, DMSO) δ 10.18 (t, J = 5.9 Hz, 1H), 10.08 (s, 1H), 8.73 (s, 1H), 8.64 (d, J = 2.1 Hz, 1H), 8.54 (d, J = 8.0 Hz, 1H), 8.27 (dd, J = 8.8, 2.1 Hz, 1H), 8.10 (d, J = 8.8 Hz, 1H), 7.62 – 7.54 (m, 3H), 7.21 (m, 2H), 4.75 (d, J = 5.9 Hz, 2H), 1.44 (s, 9H); C24H23F3N4O4[M]: 488.47; MS (ESI) m / z: [M + H]+: 489.26.Reagent and conditions: a) K2CO3, MeOH : THF : H2O (1:1:0.5). 71. Preparation of tert-butyl (2-(2-(aminomethyl)quinoline-6- carboxamido)phenyl)carbamate (78): Compound tert-butyl (5-fluoro-2-(5-((2,2,2- trifluoroacetamido)methyl)-pyrazine-2-carboxamido)phenyl)-carbamate (77) (0.250 g; 0.511 mmol) was dissolved in 10 mL mixture of methanol: tetrahydrofuran: water (1:1:0.5), followed by aq. solution of K2CO3(211.86 g, 0.511 mmol)was added and stirred at room temperature for overnight. After completion of the reaction, the mixture was evaporated and ethylacetate was added. Ethylacetate layer was washed with saturated brine solution and dried over sodium sulphate, evaporate under reduced pressure to afford 0.142 g (yield: 71%) of (78) as brown solid.1H NMR (400 MHz, DMSO-d6) δ 10.23 (s, 1H), 8.65 (dd, J = 7.8, 2.1 Hz, 1H), 8.35 (t, J = 1.9 Hz, 1H), 8.12 – 8.00 (m, 2H), 7.76 – 7.63 (m, 3H), 7.41 (dt, J = 8.0, 0.9 Hz, 1H), 7.33 – 7.22 (m, 2H), 3.97 (td, J = 6.2, 0.8 Hz, 2H), 1.44 (s, 9H); C22H2N4O3 [M]: 392.47; MS (ESI) m / z: [M + H]+: 393.26.Reagents and conditions: a) Succinic anhydride, 1-ethyl-3-dimethyl amino propyl carbodiimide (EDC), 4-dimethyl aminopyridine (DMAP), DCM : Pyridine (1:1), RT, overnight.72. Preparation of 4-(((6-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)quinolin-2-yl)methyl)amino)-4- oxobutanoic acid (79) : Compound tert-butyl (2-(2-(aminomethyl)quinoline-6-carboxamido)phenyl)carbamate (78) (0.100 g; 0.254 mmol) was dissolved in 5 mL dichloromethane: pyridine (1:1). To this solution, catalytic amount of 4-dimethyl aminopyridine and 1-ethyl-3-dimethyl amino propyl carbo-di-imide (0.088 g, 0.457 mmol) were added. Reaction mixture was stirred for 20 min at room temperature. After 20 min, succinic anhydride (0.031 g; 0.305 mmol) was added and stirred for overnight at room temperature. Reaction was monitored by TLC. After completion the reaction, pyridine mixture was evaporated. To the residue, water was added and extracted with ethylacetate, separated organic layer, and the organic layer was washed with water and was dried over sodium sulphate, concentrated under reduced pressure. The crude mixture was triturated with di-ethyl ether to afforded 0.104 g (yield: 83 %) of compound (79) as off white solid. C26H28N4O6 [M]: 492.47; MS (ESI) m / z: [M - H]+: 491.19.Reagents and conditions: a) Gemcitabine hydrochloride, 1-ethyl-3-dimethyl amino propyl carbo diimide (EDC), 1-Hydroxybenzotriazole, N-Methylmorpholine, DMF: DMSO (3:1), RT, overnight. 73. Preparation of tert-butyl (2-(2-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-4 oxo-butanamido)-methyl)quinoline-6-carboxamido)phenyl)carbamate (80): Gemcitabine hydrochloride (0.061 g; 0.203 mmol) and 8 mL mixture of DMF:DMSO (3:1) added into a 50 mL RB flask. The reaction mixture was stirred and 1-(3- dimethylamino propyl)-3-ethylcarbodiimidehydrochloride (0.051 g; 0.264 mmol), N-methylmorpholine (0.041 g; 0.406 mmol), 1-hydroxybenzotriazole monohydrate (0.027 g; 0.203 mmol), and 4-(((6-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)quinolin-2-yl)methyl)amino)-4-oxobutanoic acid (79) (0.100 g; 0.203 mmol) were added. The reaction mixture was protected with a nitrogen balloon and stirred at room temperature for overnight. The reaction was monitored by TLC. After completion of the reaction, 50 mL of 10% NaCl solution and 50 mL of water were added slowly to the reaction solution to quench the reaction. The resulting yellow suspension was extracted with 3x 50 mL of ethyl acetate. The combined ethyl acetate layer was washed with 2x 20 mL of lithium chloride solution and 50 mL of saturated sodium bicarbonate solution and saturated brine solution. The ethyl acetate layer was evaporated at vacuum conditions and purified by column chromatography using methanol: DCM (1:9) as mobile phase to afford 0.103 g (yield: 69 %) of (80) pale yellow amorphous powder.1H NMR (400 MHz, DMSO-d6) δ 10.24 (d, J = 5.1 Hz, 2H), 8.54 (dd, J = 8.1, 2.2 Hz, 1H), 8.40 (t, J = 5.4 Hz, 1H), 8.35 (t, J = 1.8 Hz, 1H), 8.12 – 8.00 (m, 2H), 7.84 (dd, J = 6.6, 1.8 Hz, 1H), 7.76 – 7.63 (m, 3H), 7.55 (dd, J = 8.1, 1.1 Hz, 1H), 7.33 – 7.22 (m, 2H), 6.99 (d, J = 6.4 Hz, 1H), 6.55 – 6.51 (m, 1H), 5.47 – 5.44 (m, 1H), 4.52 – 4.39 (m, 3H), 4.29 (t, J = 5.4 Hz, 1H), 4.13 (dp, J = 5.3, 2.6 Hz, 1H), 3.81 – 3.76 (m, 1H), 3.60 – 3.50 (m, 1H), 2.56 – 2.47 (m, 2H), 2.36 – 3.32 (m, 2H), 1.44 (s, 9H). C35H37F2N7O9[M]: 737.72.47; MS (ESI) m / z: [M + H]+: 738.20.Reagents and conditions: a) 4M HCl in Dioxane, DCM, RT, 2 h. 74. Preparation of N1-((6-((2-aminophenyl)carbamoyl)quinolin-2-yl)methyl)-N4- (1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2- dihydro-pyrimidin-4-yl)-succinamide (81) Compound tert-butyl (2-(2-((4-((1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)- tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-4-oxobutanamido)-methyl)quinoline-6-carboxamido)phenyl)carbamate (80) (0.090 g; 0.122 mmol) was dissolved in 3 mL dichloromethane and cooled in ice bath and 3 mL of 4 M HCl in dioxane was slowly added to compound (80) solution at 0 °C and reaction was stirred for 2 h at room temperature. After completion of the reaction, the mixture was dried and a saturated solution of NaHCO3was added to it. Extracted with 1:1 mixture of ethyl acetate and n-butanol, and organic layer was separated, dried with sodium sulphate, and evaporated under reduced pressure to afford 0.052 g. (yield: 66.85 %) of (81) as brown amorphous solid.1H NMR (400 MHz, DMSO-d6) δ 10.24 (s, 1H), 9.99 (s, 1H), 8.54 (dd, J = 8.1, 2.2 Hz, 1H), 8.44 – 8.32 (m, 2H), 8.12 – 8.00 (m, 2H), 7.84 (dd, J = 6.4, 1.8 Hz, 1H), 7.59 – 7.54 (m, 2H), 7.32 – 7.23 (m, 1H), 7.02 – 6.92 (m, 2H), 6.88 (dd, J = 7.9, 1.4 Hz, 1H), 6.56 – 6.51 (m, 1H), 5.47 – 5.44 (m, 1H), 4.87 (s, 2H), 4.52 – 4.39 (m, 3H), 4.29 (t, J = 5.4 Hz, 1H), 4.15 – 4.11 (m, 1H), 3.81 – 3.76 (m, 1H), 3.60 – 3.50 (m, 1H), 2.56 – 2.47 (m, 2H), 2.36 – 2.31 (m, J = 8.3, 1.0 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 172.67, 172.08, 166.18, 161.07, 154.56, 154.06, 154.05, 154.04, 150.09, 144.82, 144.79, 144.76, 140.82, 134.84, 130.16, 129.29, 128.48, 128.07, 127.29, 126.99, 126.61, 124.11, 124.05, 122.15, 121.81, 121.67, 121.49, 115.53, 97.83, 92.85, 92.46, 92.07, 82.76, 82.74, 70.23, 69.89, 69.56, 61.47, 61.42, 61.37, 45.68, 31.85, 31.38. C35H37F2N7O9[M]: 637.60; MS (ESI) m / z: [M + H]+: 638.20.Reagents and conditions: a) 2-amino phenol, 4-(dimethyl amino) pyridine, 1-(3- Dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride, DCM: Pyridine (1:1), RT, 12 h. 75. Preparation of N-(2-hydroxyphenyl)-2-((2,2,2- trifluoroacetamido)methyl)quinoline-6-carboxamide (82): Compound 2-((2,2,2-trifluoroacetamido)methyl)quinoline-6-carboxylic acid (76) (0.2 g; 0.670 mmol) was dissolved in a mixture of DCM: Pyridine (1:1). 1-(3-Dimethylamino propyl)-3-ethyl carbo di imide hydro chloride (0.231 g; 1.206 mmol) and catalytic amount of 4- (dimethyl amino) pyridine (DMAP) were added to the reaction mixture. 2-amino phenol (0.073 g; 0.670 mmol) was added and the reaction was purged with nitrogen gas. The reaction mixture was stirred for 12 h at room temperature till the completion of the reaction. The solvent was evaporated and the product was fractioned between ethyl acetate and a saturated solution of sodium bicarbonate. The crude product was purified by column chromatography (MeOH: DCM; 10:90), afforded 0.231 g. (yield 88 %) of compound (82) as off white solid.1H NMR (400 MHz, DMSO) δ 10.21 – 10.16 (m, 1H), 9.77 (d, J = 3.3 Hz, 2H), 8.67 (d, J = 2.1 Hz, 1H), 8.55 (d, J = 8.8 Hz, 1H), 8.29 (dd, J = 8.8, 2.1 Hz, 1H), 8.08 (d, J = 8.8 Hz, 1H), 7.71 (dd, J = 7.9, 1.7 Hz, 1H), 7.58 (d, J = 8.5 Hz, 1H), 7.10 – 7.04 (m, 1H), 6.96 (dd, J = 8.0, 1.5 Hz, 1H), 6.87 (td, J = 7.6, 1.5 Hz, 1H), 4.74 (d, J = 6.0 Hz, 2H). C19H14F3N3O3[M]: 389.33; MS (ESI) m / z: [M + H]+: 390.15.Reagents and conditions: a) K2CO3, MeOH : THF : H2O (1:1:0.5), RT, overnight. 76. Preparation of 2-(aminomethyl)-N-(2-hydroxyphenyl)quinoline-6- carboxamide (83): Compound N-(2-hydroxyphenyl)-2-((2,2,2- trifluoroacetamido)methyl)quinoline-6-carboxamide (82) (0.200 g; 0.513 mmol) was dissolved in 10 mL mixture of methanol: tetrahydrofuran: water (1:1:0.5), followed by aq. solution of K2CO3(212.68 g, 1.539 mmol) and stirred at room temperature for overnight. After completion of the reaction, the mixture was evaporated and ethylacetate was added. Ethylacetate layer was washed with saturated brine solution and was dried over sodium sulphate, concentrated under reduced pressure that afforded 0.138 g (yield: 91.58%) of (83) as brown solid.1H NMR (400 MHz, DMSO- d6) δ 9.48 (s, 1H), 8.79 (s, 1H), 8.65 (dd, J = 7.8, 2.1 Hz, 1H), 8.35 (t, J = 1.8 Hz, 1H), 8.12 – 8.00 (m, 2H), 7.75 – 7.66 (m, 1H), 7.43 – 7.40 (m, 1H), 7.11 – 7.00 (m, 2H), 6.93 – 6.83 (m, 1H), 3.99 – 3.95 (m, J = 6.2, 0.8 Hz, 2H). C17H15N3O2[M]: 293.33; MS (ESI) m / z: [M + H]+: 294.25.Reagents and conditions: a) Succinic anhydride, 1-ethyl-3-dimethyl amino propyl carbodiimide (EDC), 4-dimethyl aminopyridine (DMAP), DCM: Pyridine (1:1), RT, overnight. 77. Preparation of 4-(((6-((2-hydroxyphenyl)carbamoyl)quinolin-2- yl)methyl)amino)-4-oxobutanoic acid (84) : Compound 2-(aminomethyl)-N-(2- hydroxyphenyl)quinoline-6-carboxamide (83) (0.100 g; 0.341 mmol) was dissolved in 5 mL dichloromethane: pyridine (1:1). To this solution, catalytic amount of 4-dimethyl aminopyridine and 1-ethyl-3-dimethyl amino propyl carbo-di-imide (0.118 g, 0.614 mmol) were added. Reaction mixture was stirred for 20 min at room temperature. After 20 min, succinic anhydride (0.051 g; 0.511 mmol) was added and stirred for 5h at room temperature. Reaction was monitored by TLC. After completion of the reaction, pyridine mixture was evaporated. To the residue, water was added and extracted with ethylacetate, separated organic layer, and the organic layer was washed with saturated brine solution and was dried over sodium sulphate, concentrated under reduced pressure. The residue was triturated with di-ethyl ether to afforded 0.110 g (yield: 82 %) of (84) as brown solid. C21H19N3O5[M]: 393.40; MS (ESI) m / z: [M - H]+: 292.20.Reagents and conditions: a) Gemcitabine hydrochloride, 1-ethyl-3-dimethyl amino propyl carbo diimide (EDC), 1-Hydroxybenzotriazole, N-Methylmorpholine, DMF: DMSO (3:1), RT, overnight. 78. Preparation of N1-(1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-N4-((6- ((2-hydroxyphenyl)carbamoyl)quinolin-2-yl)-methyl)succinimide (85) : Gemcitabine hydrochloride (0.076 g; 0.254 mmol) and 8 mL mixture of DMF:DMSO (3:1) added into a 50 mL RB flask. The reaction mixture was stirred and 1-(3- dimethylamino propyl)-3-ethylcarbodiimidehydrochloride (0.088 g; 0.457 mmol), N- methylmorpholine (0.059 g; 0.508 mmol), 1-hydroxybenzotriazole monohydrate (0.034 g; 0.443 mmol), and 4-(((6-((2-hydroxyphenyl)carbamoyl)quinolin-2- yl)methyl)amino)-4-oxobutanoic acid (84) (0.100 g; 0.254 mmol) were added. The reaction mixture was protected with a nitrogen balloon and stirred at room temperature for overnight. The reaction was monitored by TLC. After completion of the reaction, 50 mL of 10% NaCl solution and 50 mL of water were added slowly to the reaction solution to quench the reaction. The resulting yellow suspension was extracted with 3x 50 mL of ethyl acetate. The combined ethyl acetate layer was washed with 2x 50 mL of lithium chloride solution, 50 mL of saturated sodium bicarbonate solution, and saturated brine solution. The ethyl acetate layer was evaporated under vacuum conditions and purified by column chromatography using methanol: DCM (1:9) as a mobile phase to afford 0.091 g (% yeild 56%) of (85) as brown solid.1H NMR (400 MHz, DMSO-d6) δ 10.24 (s, 1H), 9.48 (s, 1H), 8.54 (dd, J = 8.1, 2.2 Hz, 1H), 8.40 (t, J = 5.4 Hz, 1H), 8.35 (t, J = 1.8 Hz, 1H), 8.12 – 8.00 (m, 2H), 7.84 (dd, J = 6.4, 1.8 Hz, 1H), 7.75 – 7.66 (m, 1H), 7.55 (dd, J = 8.1, 1.1 Hz, 1H), 7.11 – 7.03 (m, 2H), 7.03 – 6.96 (m, 1H), 6.93 – 6.83 (m, 1H), 6.55 – 6.51 (m, 1H), 5.47 – 5.44 (m, 1H), 4.52 – 4.39 (m, 3H), 4.29 (t, J = 5.4 Hz, 1H), 4.15 – 4.11 (m, 1H), 3.81 – 3.76 (m, 1H), 3.60 – 3.50 (m, 1H), 2.56 – 2.47 (m, 2H), 2.34 (td, J = 8.3, 1.0 Hz, 2H).13C NMR (101 MHz, DMSO-d6) δ 172.67, 172.08, 166.10, 161.07, 154.56, 154.06, 154.05, 154.04, 150.10, 148.68, 144.82, 144.79, 144.76, 134.84, 130.55, 128.54, 128.03, 127.77, 127.29, 126.95, 126.61, 125.37, 124.05, 121.81, 121.49, 121.10, 119.77, 115.03, 97.83, 92.85, 92.46, 92.07, 82.76, 82.74, 70.23, 69.89, 69.56, 61.47, 61.42, 61.37, 45.68, 31.85, 31.38. C30H28F2N6O8[M]: 638.58 ; MS (ESI) m / z: [M + H]+: 639.30.Reagents and conditions: a) Trifluoroacetic anhydride, DCM, 0 ºC- Rt, overnight. 79. Preparation of N-(4-cyanobenzyl)-2,2,2-trifluoroacetamide (87) To the solution of 4-(aminomethyl)benzonitrile (86) (1.00 g; 7.57 mmol) in 10 mL DCM, 5 mL tri-fluoro-acetic anhydride was added and stirred at room temperature under nitrogen atmosphere for overnight. Reaction completion was monitored by TLC. After completion, the reaction mixture was evaporated under reduced pressure in rotavap. The obtained solid was washed with di-ethyl-ether to afforded 1.640 g (yield: 95 %) of (87) as an off white solid. C10H7F3N2O [M]: 228.17; MS (ESI) m / z: [M + H]+: 229.10.Reagents and conditions: a) Hydrogen balloon, 10% (w / w) Pd on activated charcoal (Pd / C), methanol, RT, overnight. 80. Preparation of N-(4-(aminomethyl)benzyl)-2,2,2-trifluoroacetamide (88) N-(4-cyanobenzyl)-2,2,2-trifluoroacetamide (87) (1.00 g; 4.38 mmol) was dissolved in 10 mL of methanol and stirred overnight at room temperature in presence of 10% Pd / C (100 mg) under hydrogen balloon. The reaction completion was monitored by thin layer chromatography (TLC). After completion of the reaction, Pd / C was removed by filtration through a pad of celite, and resulting filtrate was evaporated under reduced pressure to give 0.897 g (yield: 88%) of (88) as off white solid.1H NMR (400 MHz, DMSO-d6) δ 7.61 – 7.57 (m, 1H), 7.22 (dd, J = 1.7, 0.8 Hz, 4H), 4.56 – 4.54 (m, 2H), 4.08 – 4.00 (m, 2H). C10H11F3N2O [M]: 232.21; MS (ESI) m / z: [M + 2]+: 234.15.Reagents and conditions: a) N-(4-(aminomethyl)benzyl)-2,2,2-trifluoroacetamide (39), Et3N, Isopropyl alcohol, reflux overnight. 81. Preparation of methyl 5-((4-((2,2,2- trifluoroacetamido)methyl)benzyl)amino)pyrazine-2-carboxylate (90) Methyl 5-chloropyrazine-2-carboxylate (89) (0.500 g; 1.57 mmol) was dissolved in 10 mL isopropyl alcohol. To this solution, triethylamine (0.300 mL) was added. Reaction was stirred for 5 min and to the stirring reaction mixture N-(4-(aminomethyl)benzyl)- 2,2,2-trifluoroacetamide (88) (0.546 g; 2.355 mmol) was added and reaction was kept under nitrogen atmosphere. The reaction was refluxed for overnight. The reaction was monitored by thin layer chromatography (TLC). After the completion of the reaction, the mixture was dissolved in dichloromethane and washed with brine solution. Organic layer was separated, dried with sodium sulphate, concentrated under reduced pressure. The crude mixture was purified by column chromatography (ethylacetate: hexane (40:60)) to get pure 0.739 g (yield: 69%) of (90) as off white solid.1H NMR (400 MHz, DMSO) δ 8.50 (d, J = 1.4 Hz, 2H), 8.00 (d, J = 1.3 Hz, 1H), 7.73 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H), 4.60 (d, J = 6.1 Hz, 2H), 3.72 (s, 3H). C16H15F3N4O3[M]: 368.32; MS (ESI) m / z: [M + H]+: 369.30.Reagents and conditions: a) aq. LiOH, Methanol, RT, overnight. 82. Preparation 5-((4-((2,2,2- trifluoroacetamido)methyl)benzyl)amino)pyrazine-2-carboxylic acid (91); To asolution of methyl 5-((4-((2,2,2-trifluoroacetamido)methyl)benzyl)amino)pyrazine-2- carboxylate (90) (0.5 g, 1.20 mmol) in Methanol (10 mL) was stirred at room temperature followed by aq LiOH solution was added to the reaction mixture stirred at room temperature for overnight. The excess methanol was evaporated under reduced pressure, and followed by 3 mL 2N hydrochloric acid was added. The reaction mixture was extracted with DCM, and evaporated under reduced pressure. the residue was triturated with diethyl ether and dried to afford the 0.307 g, of compound (91) crude as a pale yellowish solid. C15H13F3N4O3 [M]: 354.29; MS (ESI) m / z: [M + H]+: 353.20.Reagents and conditions: a) tert-Butyl (2-aminophenyl) carbamate (2), 4-(dimethyl amino) pyridine, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride, DCM: Pyridine (1:1), RT, 12 h. 83. Preparation of tert-butyl (2-(5-((4-((2,2,2-trifluoroacetamido)-methyl)benzyl) amino)-pyrazine-2-carboxamido)phenyl)carbamate (92): Compound 5-((4-((2,2,2-trifluoroacetamido)methyl)benzyl)amino)pyrazine-2- carboxylic acid (91) (0.2 g; 0.705 mmol) was dissolved in a mixture of DCM: Pyridine (1:1). 1-(3-Dimethyl amino propyl)-3-ethyl carbo di imide hydro chloride (0.243 g; 1.269 mmol) and catalytic amount of 4- (dimethyl amino) pyridine (DMAP) were added to the reaction mixture. tert-Butyl (2-aminophenyl) carbamate (2) (0.176 g; 0.846 mmol) was added and the reaction was purged with nitrogen gas. The reaction mixture was stirred for 12 h at room temperature till the completion of the reaction. The solvent was evaporated and the product was fractioned between ethyl acetate and a brine solution. The crude product was purified by column chromatography (Ethyl acetate: Hexane; 70:30), afforded 0.280 g. (yield 73%) of compound (92) as pale yellow solid.1H NMR (400 MHz, DMSO-d6) δ 8.76 (s, 1H), 8.10 (s, 1H), 7.77 – 7.63 (m, 3H), 7.61 – 7.57 (m, 1H), 7.33 – 7.17 (m, 6H), 6.92 (t, J = 5.8 Hz, 1H), 4.68 – 4.66 (m, 2H), 4.56 – 4.54 (m, 2H), 1.44 (s, 9H). C26H27F3N6O4[M]: 544.54; MS (ESI) m / z: [M + H]+: 545.20.Reagents and conditions: a) K2CO3, MeOH: THF : H2O (1:1:0.5), RT, overnight. 84. Preparation of tert-butyl (2-(5-((4-(aminomethyl)benzyl)amino)pyrazine-2- carboxamido)phenyl)carbamate) (93): Compound tert-butyl (2-(5-((4-((2,2,2-trifluoroacetamido)-methyl)benzyl) amino)- pyrazine-2-carboxamido)phenyl)carbamate (92) (0.250 g; 0.459 mmol) was dissolved in 10 mL mixture of methanol: tetrahydrofuran: water (1:1:0.5) followed by aq. solution of NaOH (0.196 g; 1.377 mmol) was added and stirred at room temperature for overnight. After completion of the reaction, the mixture was evaporated and ethyl acetate was added. Ethyl acetate layer was washed with saturated brine solution and was dried over sodium sulphate, concentrated under reduced pressure that afforded 0.159 g (yield: 77 %) of (93) as pale yellow solid. C24H28N6O3 [M]: 448.53; MS (ESI) m / z: [M + 2]+: 450.10.Reagents and conditions: a) Succinic anhydride, 1-ethyl-3-dimethyl amino propyl carbodiimide (EDC), 4-dimethyl aminopyridine (DMAP), DCM : Pyridine (1:1), RT, overnight. 85. Preparation of 4-((4-(((5-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)pyrazin-2- yl)amino)methyl)benzyl)amino)-4-oxobutanoic acid (94) : Compound tert-butyl (2-(5-((4-(aminomethyl)benzyl)amino)pyrazine-2- carboxamido)phenyl)carbamate (93) (0.150 g; 0.334 mmol) was dissolved in 5 mLdichloromethane: pyridine (1:1). To this solution, catalytic amount of 4-dimethyl aminopyridine and 1-ethyl-3-dimethyl amino propyl carbo-di-imide (0.115 g, 0.601 mmol) were added. Reaction mixture was stirred for 20 min at room temperature. After 20 min, succinic anhydride (0.050 g; 0.501 mmol) was added and stirred for overnight at room temperature. Reaction was monitored by TLC. After completion the reaction, pyridine mixture was evaporated. To the residue, water was added and extracted with 1:1 mixture of ethylacetate and n-butanol, separated organic layer, and the organic layer was washed with saturated brine solution and was dried over sodium sulphate, concentrated under reduced pressure. The crude mixture triturated with di- ethyl ether to afford 0.134 g (yield: 73 %) of (94). C28H32N6O6[M]: 548.60; MS (ESI) m / z: [M - H]+: 547.16.Reagents and conditions: a) Gemcitabine hydrochloride, 1-ethyl-3-dimethyl amino propyl carbo diimide (EDC), 1-Hydroxybenzotriazole, N-Methylmorpholine, DMF: DMSO (3:1), RT, overnight. 86. Preparation of tert-butyl (2-(5-((4-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-4- oxobutanamido)methyl)benzyl)amino)pyrazine-2-carboxamido)phenyl)carbamate (95): Gemcitabine hydrochloride (0.071 g; 0.237 mmol) and 8 mL mixture of DMF:DMSO (3:1) added into a 50 mL RB flask. The reaction mixture was stirred and 1-(3-dimethylamino propyl)-3-ethylcarbodiimidehydrochloride (0.082 g; 0.426 mmol), N-methylmorpholine (0.056 g; 0.474 mmol), 1-hydroxybenzotriazole monohydrate (0.032 g; 0.237 mmol), and 4-((4-(((5-((2-((tert-butoxycarbonyl)-amino)-phenyl)- carbamoyl)pyrazin-2-yl)amino)-methyl)-benzyl)-amino)-4-oxobutanoic acid (94) (0.2 g; 0.237 mmol) were added. The reaction mixture was protected with a nitrogenballoon and stirred at room temperature for overnight. The reaction was monitored by TLC. After completion of the reaction, 50 mL of 10% NaCl solution and 50 mL of water were added slowly to the reaction solution to quench the reaction. The resulting yellow suspension was extracted with 3x 50 mL of 1:1 mixture of ethyl acetate and n- butanol. The combined orgatnic layer was washed with 2x 20 mL of lithium chloride solution and 50 mL of saturated sodium bicarbonate solution, and saturated brine solution. The organic layer was evaporated at vacuum conditions and purified by column chromatography using methanol: DCM (2:8) as mobile phase to afford 0.089 g (% yield 47 %) of (95) light yellow solid.1H NMR (400 MHz, DMSO-d6) δ 10.24 (s, 1H), 10.03 (s, 1H), 8.76 (s, 1H), 8.10 (s, 1H), 7.90 (t, J = 5.5 Hz, 1H), 7.84 (dd, J = 6.6, 1.8 Hz, 1H), 7.77 – 7.63 (m, 3H), 7.33 – 7.27 (m, 1H), 7.24 (dd, J = 18.7, 1.6 Hz, 5H), 6.99 (d, J = 6.4 Hz, 1H), 6.92 (t, J = 5.8 Hz, 1H), 6.55 – 6.51 (m, 1H), 5.47 – 5.44 (m, 1H), 4.67 (d, J = 5.8 Hz, 2H), 4.52 – 4.37 (m, 3H), 4.29 (t, J = 5.4 Hz, 1H), 4.15 – 4.11(m, 1H), 3.81 – 3.76 (, 1H), 3.60 – 3.50 (m, 1H), 2.56 – 2.47 (m, 2H), 2.40 – 2.35 (m, 2H), 1.44 (s, 9H). C37H41F2N9O9 [M]: 739.79; MS (ESI) m / z: [M + Na]+: 816.26.Reagents and conditions: a) 4M HCl in Dioxane, DCM, RT, 2 h. 87. Preparation of N1-(4-(((5-((2-aminophenyl)carbamoyl)pyrazin-2- yl)amino)methyl)benzyl)-N4-(1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4- yl)succinamide (96) Compound tert-butyl (2-(5-((4-((4-((1-(3,3-difluoro-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-4-oxo-butanamido)methyl)benzyl)amino)pyrazine-2-carboxamido)phenyl)carbamate (95) (0.080 g; 0.101 mmol) was dissolved in 3 mL dichloromethane and cooled in ice bath and 3 mL 4M HCl in dioxane was slowly transferred to compound (95) solution at 0 °C and reaction was stirred for 3 h at room temperature. After completion of the reaction, the mixture was dried and a saturated solution of NaHCO3was added to it. Extracted with 1:1 mixture of ethylacetate and n-butanol, followed by organic layer was separated, dried with sodium sulphate, and evaporated under reduced pressure to afford 0.041 g. (yield: 59 %) of (96) as light yellow solid.1H NMR (400 MHz, DMSO-d6) δ 10.24 (s, 1H), 9.82 (s, 1H), 8.76 (s, 1H), 8.10 (s, 1H), 7.94 – 7.81 (m, 2H), 7.55 (dd, J = 7.9, 1.3 Hz, 1H), 7.32 – 7.17 (m, 5H), 7.02 – 6.85 (m, 4H), 6.55 – 6.51(m, 1H), 5.47 – 5.44 (m, 1H), 4.67 (d, J = 5.7 Hz, 2H), 4.52 – 4.37 (m, 3H), 4.29 (t, J = 5.4 Hz, 1H), 4.15 – 4.11(m, 1H), 3.81 – 3.76 (, 1H), 3.61 – 3.50 (m, 1H), 2.57 – 2.47 (m, 2H), 2.40 – 2.35 (m, 2H), 1.44 (s, 9H).13C NMR (101 MHz, DMSO-d6) δ 172.67, 172.22, 163.01, 161.07, 155.60, 154.06, 154.05, 154.04, 144.82, 144.79, 144.77, 142.12, 140.86, 137.46, 136.79, 129.62, 128.70, 128.52, 128.49, 128.15, 126.61, 124.11, 124.05, 121.69, 121.63, 121.49, 115.58, 97.83, 92.85, 92.46, 92.07, 82.76, 82.74, 70.23, 69.89, 69.56, 61.47, 61.42, 61.37, 46.13, 43.87, 31.82, 31.34. C32H33F2N9O7[M]: 693.67; MS (ESI) m / z: [M + Na]+: 716.25. 1.2 Biological assay 1.2.1 HDAC inhibition activity using fluorophore coupled biochemical assay 1.2.1.1 Pan- HDAC inhibition Pan- HDAC inhibition: Table 1 and Figure 2 below shows the bifunctional compounds envisioned herein. However, the invention is not limited to these. Table 1: pan-HDAC enzyme inhibition (%) data of the synthesized bifunctional molecules with 10 µM concentration of compounds against HeLa nuclear extract (pan-HDACs). Data represents mean ± SD (n = 2).The HDAC inhibitory activity of Gemcitabine conjugate was evaluated by using HDAC colorimetric assay kit (BML-AK500-0001) as per the kit protocol. Initially, the 5 µL of HeLa nuclear extract, 10 µL of assay buffer, and 10 µL of 10 µM final concentration of the sample solution to be assayed were added to respective wells of the microtiter plate. Subsequently, 25 µL of color de Lys® substrate solution was used to initiate the reaction, and the microtiter plate was incubated at 37 ºC for 30 min. after incubation 50 µL of the developer was added and allowed 15 min of further incubation for the termination of the enzymatic reaction. The absorbance was measured at 405 nm. The assay was carried out in duplicate, and the results were analyzed using Graph Pad Prism 8.0.1. Using the same assay protocol IC50values against various was determined and the results were analyzed using Graph Pad Prism 8.0.1. (Figure 3) 1.2.1.2 Assessment of IC50against various HDAC isoforms Table 2. Inhibitory potency (IC50, µM) of compound 7, compound 15, 5 compound 47, compound65, compound 70, compound 81, compound 85 and compound 96 against human recombinant class I and class II HDACs1.2.1.3 Assessment of Anti-proliferative and cytotoxicity assay using different cancer as well as normal cell lines. 10 Table 3. Tabular representation of the IC50values ((µM)) of the conjugate in different cell linesReferring to Figure 4, To evaluate the cytotoxic activity of synthesized Gemcitabine conjugate, MTT assay was performed against six different cancer cell lines (4T1, MCF7, MDA-MB-231, PC3, DU145 and MOC2 cell line) and three normal human 5 cell lines (HEK-293, MCF10a, and HCEC). 4T1, MCF– 7, MDA-MB-231, DU145, PC3, and HEK – 293 cells were cultured in DMEM (high glucose media: AL007S, Dulbecco's modified eagle medium); MCF10a cultured in RPMI 1640 and HCEC cells were cultured in DMEM / F-12 (AT140A, Dulbecco's Modified Eagle Medium / Nutrient Mixture F12 Ham, DMEM / F-12, 1:1 mixture). Similarly the MOC2 cells 10 were cultured in IMDM media supplemented with 5 mg / mL insulin, 40 µg of hydrocartison and 5 µg of EGF with 10% fetal bovine serum (FBS)and 1% antibiotic (Pen strep: A001) under 5% CO2 at 37 ºC. MTT [3- (4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide], a yellow dye was considered for the assay. All the reagents were procured from Himedia Laboratories Pvt. Ltd., Mumbai, India. In brief, 15 the 1× 104 cells were seeded in 96 well plates with 100 µL per well in their respective complete media and incubated overnight. Subsequently, the adherent cells media were replaced with 150 µL respective fresh media containing a series of concentrations ranging from 0.078 µM to 80 μM of Gemcitabine conjugate and 0.009 µM to10 μM of Gemcitabine. Thus, the HEK-293, MCF10a, and HCEC cells (Figure 6) were treated 20 with a concentration ranging from 7.81 µM to 2000 µM for 72 h of Gemcitabine conjugate and Gemcitabine in their respective complete medium in duplicate. The cells were incubated for 72 h. After the treatment period, previous media was discarded, then 50 µL of 5 mg / mL MTT(3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) in Dulbecco's phosphate buffered saline (PBS) was 25 added and incubated for 4 h to allow the formation of formazan crystals. Subsequently, 150 µL of DMSO was added to the MTT solution added in culture to dissolve the formed formazan crystals, and the absorbance was measured using a multi-well plate reader Spectramax (Molecular Devices, USA) at two different wavelengths of 570 nmand 650 nm. The percent cell viability was computed by the equation given below. The half-maximal dose (IC50) was determined using Graph pad Prism 8.0.1 software. Referring to Figure 5, following the same above-mentioned MTT procedure, IC50 of the lead compound, compound 15 was determined against gemcitabine-resistant PC3 cells1.2.2 Cell cycle analysis Cell cycle analysis was carried out by using flow cytometry and was analyzed using FlowJo software. For this assay, PC3 cells were seeded in a flat-bottomed 12 well plate with a cell density of 0.5 million cells / well and incubated overnight. The next day, the media was aspirated, and the cells were treated with the compounds 15 (0.177 µM), 47 (0.505 µM), and Gemcitabine (0.186 µM) dissolved in the DMEM complete media and further were incubated for about 72 h. Post-treatment, the cells were washed with ice-cold PBS, trypsinized, and collected the cells in the form of a cell pellet. This cell pellet obtained was further washed twice with ice-cold PBS, and the cells were subsequently fixed using dropwise addition of ice-cold 70% ethanol vertexing gently to avoid clumping. Finally, the single-cell suspension was achieved and was confirmed under a microscope, and the sample was kept at -20 °C overnight. The following day, the cells were centrifuged, and the obtained cell pellet was resuspended in 500 µL of staining solution containing 20% w / v RNase, 2% w / v PI in about 0.1% v / v of triton X 100 solution in PBS. The samples were incubated in the dark for about 30 min at room temperature before the analysis. Flow cytometry (BDAriaTM III) and BD biosciences were obtained and analyzed in the cell cycle histograms. The data was obtained in the form of a dot plot with PI width on the X- axis and PI area on the Y-axis. The % cell population was analyzed from the histogram plotted with PI area on the X-axis and counts on the Y-axis. The data were analyzed using FlowJo software to measure the percentage of cells in each cell cycle phase. 1.2.3 Apoptosis analysis In Figure 7, Apoptosis assay was performed over PC3 cells using TACs / Annexin V kit purchased from the Biolegend US. In a flat-bottomed 12-well plate, 0.5 million / well PC3 cells were seeded and incubated overnight. The following day, the media wasaspirated, and the cells were treated with the compound 15, compound 47, and Gemcitabine in duplicate as per their IC50 values for 72 h. Following the treatment period, the cells were washed twice with ice-cold PBS, followed by trypsinization. The trypsinized cells were collected and centrifuged to obtain the cell pellet. Further, the cell pellet was washed twice with ice-cold PBS, and the obtained cell pellet was resuspended in 100 µL Annexin V incubation reagent including 10X binding buffer (10 µL), FITC (1 µL), PI (10 µL) making up to 100 µL using double distilled water. This is further kept for incubation for about 30 mins at room temperature in the dark. To this, 400 µL of 1X binding buffer was added per 100 µL of cell suspension and was analyzed by flow cytometry (BDAriaTM III), BD biosciences. The data was analyzed into four quadrants in the form of a dot plot. Each quadrant represents as follows: Q1 – necrotic cells, Q2 – late apoptosis cells, Q3 – live cells, and Q4 – early apoptotic cells. The data together in Q2 and Q4 is considered as the total apoptotic population. 1.2.4 Nuclear staining The Nuclear staining was performed to evaluate the status of nuclear disintegration of cancerous cells after treatment of compound 15, compound 47, and Gemcitabine using as standard by staining with DAPI (4′,6-diamidino-2- phenylindole) and acridine orange (Figure 8). For nuclear staining, 5×104PC3 prostate cancer cells were plated in flat bottom 12 well plate and allowed to adhere 24 h, and then they were treated with the compound 15 (0.177 µM), 47 (0.505 µM), and Gemcitabine (0.186 µM) concentrations and incubated for 72 h. After 72 h of the treatment, the control and compound 15, compound 47, and Gemcitabine treated group were fixed with 4% paraformaldehyde solution and rinsed thereafter; both control and compounds treated cells were stained with DAPI (10 µg / mL) for 5 min and acridine orange (0.1 mg / mL) for 20 min. The excess stain was rinsed with PBS, and plates were then examined under a fluorescence microscope (Leica microsystems, Germany) on 20x Magnification to detect any nuclear structural changes induced by compound 15, compound 47, and Gemcitabine. Acridine orange was detected in the green channel and red channel, while DAPI was detected in the blue channel. 1.2.5 In-vitro immunoblot analysis for various biomarkers. Assessment the cell death pathway induced by the compound when exposed to 4T1 cells incubated Gemcitabine and compound 15. The protein extracts from isolated 4T1cells pellet and cell supernatant were prepared in RIPA buffer with 1% protease inhibitor and 200nM of PMSF. For the cell supernatant, the cell supernatant was lyophilized into powder and resuspended in an RIPA cocktail. The protein from the cell pellet was collected after resuspending the pellet in the RIPA cocktail and supernatant was collected followed by centrifugation. An equal amount of proteins was loaded on SDS-PAGE gel by quantifying it through BCA protein quantification kit. The resolved proteins were transferred over PVDF membranes by wet transfer. The membrane was blocked with 5% BSA followed by incubation with primary antibody overnight at 2-8 °C. On next day the membrane was washed with (0.1% Tween 20 in PBS) and incubated with HRP-tagged secondary antibody for 2 h at room temperature. After washing the membrane with PBST(0.1% Tween 20 in PBS) immunoblots were detected with the help of chemo doc system and the intensity was quantified using Image J software. The representative blots were shown in Figure 9 & 10. 1.2.6 In vitro hemolysis study A hemolysis assay was performed to assess the toxicity of compound towards blood components. Briefly, various concentrations of compound 15 (12 µM / mL, 10 µM / mL, 8 µM / mL, 6 µM / mL, 4 µM / mL, 2 µM / mL) prepared. Triton X-100 (1%) and PBS 7.4 were maintained as the positive and negative controls, respectively. Each sample was added to RBC suspension and incubated at 37ºC for 1 hour. The samples were then centrifuged at 7000rpm, 4ºC for 20 minutes. The supernatant containing hemoglobin released due to hemolysis of the RBCs, was analyzed by measuring the Absorbance at 576nm using a Spectramax Multiplate reader (by Molecular Devices, USA). The results obtained were shown in Figure 11. 1.2.7 Plasma stability study The plasma stability of Gemcitabine conjugate was investigated in rat plasma compared with free Gemcitabine (Figure 12). Fresh rat plasma was obtained from Wistar rats (280 – 300 g) blood, collected by the retro-orbital plexus while placing rats under anesthetic conditions, followed by centrifugation of the blood at 3500 rpm at 4 ºC for 5 min. The Gemcitabine conjugate and Gemcitabine (equivalent to 1 µg / mL of Gemcitabine final conc.) were spiked into rat plasma and then incubated in a shaking incubator at 37 °C for 24 h. The 100 µL of plasma samples were drawn atpredetermined time points 0, 15, 30, 60, 120, 240, 360, 480, and 1440 min of incubation. Immediately plasma sample was precipitated using 200 µL of the ice-cold methanolic solution, vortexed for 5 min, and centrifuged (10000 rpm, 10 min). The supernatant was separated and dried. The residues were reconstituted with the mobile phase water: ACN (95:5) ratio and analyzed the samples under HPLC for determination of Gemcitabine and gemcitabine conjugate recovered from plasma sample at a particular time point. 1.2.8 In-vivo Pharmacokinetic Profiling Referring to Figure 13, the in-vivo pharmacokinetic study was carried out in male Wistar rats (250 – 350 g) and randomized into 6 groups of control, 7.87 µmol / kg and 23.51 µmol / kg for compound 15, compound 47, and Gemcitabine (n=3, male Wistar rats). The dosing solution of Gemcitabine conjugate (15 mg / kg or 23.51 µmol) and an equimole dose of Gemcitabine (23.51µmol / kg) in saline was administered intraperitoneally (IP). For the blood sample, the retro-orbital plexus, while placing rats under anesthetic conditions, was used. Blood samples (100 µL) were collected at predetermined time points (0.25 h, 0.5 h , 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h) into a tube containing 50 µL EDTA (0.4M, pH 7.4) and stored at -80 ºC until sample extraction. A fluid replacement was carried out by administering 1.5 mL of saline USP. Samples were prepared for analysis by protein precipitation and evaporation. Gemcitabine, Gemcitabine conjugate’s dFdU level were quantified by HPLC. The linear trapezoidal rule was used for the calculation of the area under the concentration and time curve (AUC values). Non-compartmental analysis was used to obtain the pk parameters by using pk solver. The clearance was estimated as CL=Dose / AUCinf; MRT=AUMCinf / AUCinf. Data obtained represents the mean ± standard deviation of the mean for n = 3. Table 4. Pharmacokinetic parameters of compound 15, compound 47 and Gemcitabine in plasma following an intraperitoneal administration in male Wistar rats1.2.9 Assessment of in vivo therapeutic efficacy The in vivo therapeutic efficacy of compound 15 was evaluated in PC3 tumor-bearing male BALB / c-nude mice (NCRNU-M), 4T1-Luc tumor-bearing female BALB / c mice, and MOC2 tumor-bearing female C57BL / 6 mice model. All the animals were adequately cared for by providing them with standard food and water. 1.2.9.1 Assessment of in vivo therapeutic efficacy in PC3 cells xenografted animal model Referring to Figure 14, to establish the subcutaneous xenograft tumor models, 100 µL of PC3 cell suspension (5 x 106cells) was injected into the dorsal flank area of each mouse. The mice were kept under observation for the sign of subcutaneous tumor development. After attaining a tumor volume of ~50 mm3. The study was conducted by dividing the mice into the following six groups, normal saline, free Gemcitabine, HDACi (23.51 µmol / kg), physical mixture (Gem 23.51 µmol+ HDACi 23.51 µmol / kg) / kg, compound 15 (11.75 µmol / kg), and compound 15 (23.51µmol / kg) was administered intraperitoneally and the control group received only normal saline on day 0, 3, 6, 9, and day 12. Tumor size [(length ×width2) / 2] was measured using a Vernier caliper every other day after the treatment started, and body weight was also recorded. Mice were sacrificed by cervical dislocation, and the surgically collected tumor masses were weighed. Mice were administered with fluorescent probe 1,10- Dioctadecyl-3,3,30,30-tetramethylindocarbocyanine perchlorate (DIL)-labeled PEG- PE Ms (2.5 mg / kg, intravenously) on days 5, 10, and 12 to check the tumorprogression (Figure 15). Dil-labeled PEG-PE Ms was injected into the mice 2 h before capturing the image using the IVIS-Lumina in vivo imaging system (PerkinElmer, Inc., USA) (Ex / Em.740 / 780 nm). 1.2.9.1.1 Immunohistochemistry analysis a. TUNEL assay TUNEL assay (Terminal deoxynucleotidyl transferase-mediated nick end labeling) was used to assess the level of apoptosis in tumor tissues as per the previously reported procedure (Figure 16) (Bhatt et al., 2020). The tumors were collected from mice and sectioned by cryostat (Leica Biosystems, Germany) at a thickness of 5 µm. Next, the tumor sections were treated with TUNEL reagent (TACS®TdT-Fluor In Situ Apoptosis Detection Kit, R&D Systems, USA) as per the manufacturer's instructions. Blue (Ex / Em. 359 / 457 nm) and green filters (Ex / Em 488nm / 520 nm) were used to observe the DAPI and the TUNEL-positive cells under a fluorescence microscope (Leica, Germany). The images were processed and analyzed using Image J software. b. Ki67 assay The Ki67 analysis was performed by the previously reported procedures. Briefly, using blocking buffer solution, the tumor tissue sections were blocked for 1 h and stored overnight at 4 ºC with Ki67 primary antibody followed by PBS washing thrice and the addition of secondary antibody (Alexa Fluor Plus 488) for 2 h at 25 ºC avoiding exposure to light. Finally, the sections were washed with PBS and imaged under the fluorescence microscope (Figure 19E). c. ROS detection assay PC3 tumor-xenografted mice were injected intratumorally with the dichloro-dihydro- fluorescein diacetate (DCFH-DA) (50 μL, 25 μM) at 5, 10 and 12 days post-treatment. After 30 min, mice were anesthetized and observed under the in vivo imaging system (IVIS® Lumina III, PerkinElmer, USA). Tumors were separated, sectioned by cryostat (Leica Biosystems, Germany) at a thickness of 5 µm, and examined under a fluorescence microscope (Figure 17). d. H & E stainingVarious organ sections of 5 µm thickness were processed with xylene and different concentration of alcohol and washed with water. Briefly, tumor tissues were dissected into multiple parts. One portion was fixed in 10% formaldehyde by dipping the tissue for 24 h. Next, the tissues were washed and processed through different concentrations of alcohols, 100–30% for 1 h each, followed by xylene (100%) incubation for 4–5 min. The nuclei were stained with hematoxylin (0.5%) followed by eosin (0.5%) for staining the cytoplasm. The slides were washed thoroughly with water, followed by ethanol solutions (30–100%), and fixed with mounting media (Fluoromount G) before visualization under an optical microscope with 10X magnification and a bright field exposure (Leica, Germany) (Figure 18). 1.2.9.1.2 Blood indicators in vivo To further detect the biosafety of the preparations, mice were divided into two groups (n = 4 / group). Four mice in each group were chosen randomly for intraperitoneal injection of 100 µL of compound 15 (corresponding to the Gemcitabine) and normal saline, respectively. Seventy-two hours after injection, the 200 µL blood of one mice group was drawn to test biochemical indicators, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN) and serum creatinine (SCr). Also, serum samples from another mice group were obtained to detect important routine blood indicators, including white blood cell (WBC) count, red blood cell (RBC) count, and hemoglobin (HGB) quantification (Figure 20). 1.2.9.1.3 Immunoblot analysis The expression level of various apoptotic and proliferative proteins biomarkers and acetylation level of histones, induced by compound 15 were estimated via western blot analysis with the PC3 tumor tissue (Figure 21 & 22). For western blotting 30 mg of tumor tissue from the xenografted tumor was homogenized with 100 μL 1X RIPA lysis buffer (Millipore, Billerica, MA, USA), supplemented with 0.5 mM phenylmethylsulfonyl fluoride (PMSF), protease inhibitor and separated the protein extract. The extracted protein was quantified using DC protein estimation kit (Bio-rad Laboratories, USA). The 20 μL (35 ng) of tumor proteins was heated at 95°C for 7 min with 5μL of loading buffer (4X). Then the denatured proteins are subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were transferred to thepolyvinylidene fluoride membranes (Bio-Rad, Laboratones, Inc.) and membranes were blocked in 5% BSA (Sisco Research Laboratories Pvt.Ltd.) in tris-buffered saline with 1% Tween 20 (TBST), and incubated with Rabbit mAb H3K9 acetylated histone H3(#9649S), H4K12 acetylated histone (#13944S), Caspase-3 (#14220), Caspase-7 (#12827), Bcl-2 (#3498), EGFR (#2132), CD44 (#3578), Cytochrome-C (#4280), p53 (#2527) and Mouse mAb beta-Actin as an internal control primary antibodies (Cell Signalling Technology, Inc.), as well as used Horseradish peroxidase (HRP)- conjugated anti-rabbit secondary antibody (Cell Signalling Technology, Inc.), visualized with a chemiluminescence kit (Bio-Rad, Laboratones, Inc.), and exposed using a Fusion plus 6 Imaging System (Vilber Lourmat, France). 1.2.9.2 In vivo antitumor therapeutic efficacy studies of compound 15 in a 4T1- Luc tumor bearing mouse model in female Balb / C mice and MOC2 tumor bearing mouse model in female C57BL / 6 mice The female BALB / c and C57BL / 6 mice 5 weeks old were purchased from the ICMR- National Animal Resource Facility for Biomedical Research, Hyderabad. After the 8- day quarantine period, the animals were shifted to the mice room in the animal facility with the room temperature and conditions maintained at 23±2 °C and at 60±10% relative humidity in a 12 h light / 12 h dark cycle. Then 1×1064T1-Luc cells were injected on the mammary pad of the female BALB / c mice and similarly 2×106MOC2 cells were injected on right flanks of the C57BL / 6 mice on. Once the tumor volume reached ~370 mm3, treatment was started with free Gemcitabine (23.51 µmol / kg), HDACi (23.51 µmol / kg), physical mixture (Gem 23.51 µmol+ HDACi 23.51 µmol) / kg), and compound 15 (23.51µmol / kg) was administered intraperitoneally and the control group received only normal saline on day 0, 3, 6, 9, and day 12. Tumor size [(length ×width2) / 2] was measured using a Vernier caliper every other day after the treatment started, and body weight was also recorded. Mice were sacrificed by cervical dislocation, and the surgically collected tumor masses were weighed. The BALB / c mice were imaged using intraperitoneally injected luciferin D (50 µL, 150 mg / kg dissolved in sterile PBS) and C57BL / 6 mice with fluorescent probe 1,10-Dioctadecyl- 3,3,30,30-tetramethylindocarbocyanine perchlorate (DIL)-labeled PEG-PE Ms (2.5 mg / Kg, intravenously) on days 5, 10, and 12 to check the tumor progression (Figure 23 & 27). The luciferin D and Dil-labeled PEG-PE Ms was injected into the mice 1hours before capturing the image using the IVIS-Lumina in vivo imaging system (PerkinElmer, Inc., USA). Referring to Figure 24, using same animal model, once the tumor volume reached ~380mm3, treatment started with compound 15 and 2 doses were given and tumor volumes were recorded every other day. The results obtained were shown in Figure 24. Referring to Figure 26, recued animals that were already given treatment with compound 15 and control animals, on 66thday they were injected with 4T1 Luc cells and observed for tumor growth. 1.2.9.3 FACS analysis of blood, spleen and tumour tissue a. Blood sample The blood was collected from animals by retro-orbital route directly into a 5% EDTA solution to prevent coagulation. A 100 µL of whole blood was blocked with 1 µL of CD16 / 32 Fc block for 15 mins at room temperature in the dark. Then the samples were incubated with a given antibody panel for myeloid derived suppressive cells (G- MDSC- CD45+CD11b+Ly6clowLy6G+and M-MDSC-CD45+CD11b+Ly6c+Ly6G-) and macrophages (M1-CD45+CD11b+F4 / 80+CD206-MHCII+and M2- CD45+CD11b+F4 / 80+CD206+MHCII-) for 45 mins. The RBC lysis was done for 10 min at room temperature (RT) with BD-RBC lysing solution, followed by multiple washing with FACS buffer and samples were analysed using BD-ARIA III (Figure 29). b. Spleen tissue samples After sacrificing the animals, the spleen was removed and kept in ice-cold media. The mouse spleen was then placed into a petri dish with 5 mL FACS buffer. Then carefully minced into small pieces (~0.2 cm2) with a razor or scalpel blade. The spleen tissue was then crushed into a small lump with a 2 mL syringe plunger and the content was passed through a 70 µM cell strainer. The samples were centrifuged at 1800 rpm for 5 minutes at 4°C to discard the supernatant. The RBCs were lysed by incubating the cells with RBC lysis buffer for 15 mins. Then the cell pellet was washed multiple times and blocked with CD16 / CD32 Fc block antibodies. Then the samples were incubated with a given antibody panel myeloid derived suppressive cells (G-MDSC-CD45+CD11b+Ly6clowLy6G+and M-MDSC-CD45+CD11b+Ly6c+Ly6G-) and macrophages (M1-CD45+CD11b+F4 / 80+CD206-MHCII+and M2- CD45+CD11b+F4 / 80+CD206+MHCII-) for 45 mins, followed by cell fixation with a 2% formaldehyde solution. The stained samples were kept in FACS buffer at 40C in dark prior to the FACS analysis (Figure 30). c. Tumour tissue samples After sacrificing the animals, the tumor was removed and kept on ice. The tissues was then mined into small pieces using the scalpel blade and digested with enzymes cocktail containing (Collagenase IV – 2mg / 1mL / sample, Dispase II - 2.5mg / mL(2U / mL) / sample and DNase I- 100 µg / mL / sample) at 370C for 1h. Then using a syringe plunger minced the tissues into small portions and FACS buffer was added. The cell suspension was then passed through the 70 µM syringe filter into a 15 mL falcon tube. Then the RBCs were lysed with RBC Lysis buffer followed by washing 2 / 3 times. Single cell suspension was then incubated with 1 µl of CD16 / CD31 mouse BD FC block for 15 mins in the dark. The fixable viability dye- 510 was used to label live cells. Then the samples were washed multiple times followed by antibody cocktail for myeloid derived suppressive cells (G-MDSC- CD45+CD11b+Ly6clowLy6G+and M-MDSC-CD45+CD11b+Ly6c+Ly6G-) and macrophages (M1-CD45+CD11b+F4 / 80+CD206-MHCII+and M2- CD45+CD11b+F4 / 80+CD206+MHCII-) and incubated for 45 mins in the dark. After completion of the incubation period wash the cell pellet 3 times with FACS buffer (1800RPM, 5 min each). The cells were fixed with 2% formaldehyde in 1 x PBS for 15 mins followed by 2X times washing with FACS buffer and samples were analyzed (Figure 31). 1.2.9.4 In vivo cardiotoxicity assessment of compound 15 in mouse model Animal experimental protocols were approved by the Institutional Animal Ethical Committee (IAEC) of National Institute of Pharmaceutical Education and Research (NIPER), Guwahati-India and were carried out as per regulations of IAEC NIPER- Guwahati guidelines on the care and welfare of laboratory animals. All animals were treated humanely and concerning the alleviation of suffering. C57BL / 6J mice weighing 20–25g were purchased from the Rodent Research Institute, Jind Haryana, India. Animals were maintained at 22 ± 2°C temperature, 50 ± 15% relative humidityand 12 h of dark and light cycle and had free access to water and diet. The compound 15 and saline were administered to mice in two groups compound 15 and Saline, respectively. compound 15 was administered in mouse six times at a dose of 15 mg / kg body weight for every 48 hours. Electrocardiography and echocardiography parameters were measured after 7- and 15-days of compound 15 and saline administration in mice (Figure 33). a. In vitro cardiac cytotoxicity assay Cell viability of H9c2 cell line was determined by using 3-(4,5- dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide (MTT) which evaluated the percentage of viable cells (Figure 32). Cells were seeded into 96-well plate with cell number of 10,000 cells / well and the following concentrations of compound 15 were tested 0, 0.1, 1, 10, 100, 200 and 400 μM. MTT assay was carried out for 24 hours after treatment. Mixing solution was prepared using 2 mg of MTT (Sigma-Aldrich) powder in 1 mL PBS. The culture medium was changed with 150 μl PBS plus 50 μl MTT reagent (2 mg / mL in PBS). Cells were incubated at 37°C with CO2 at 5% and a humidified atmosphere for 4 hours. Then, the MTT solution was removed and 50 μl of DMSO was added to each well and mixed carefully through pipetting. The plate was maintained for 30 min at 37°C and then the OD (optical density) of the wells was determined at 570 nm through a spectrophotometric microplate reader (SpectraMax iD3 Molecular devices, CA, USA). b. The performance and measurements of electrocardiography Electrocardiography (ECG) recordings (Figure 33) were conducted on mice from saline and compound 15 groups after being anesthetized with isoflurane on day 7 and day 15 of the study period. The core body temperature of the animal was maintained at 37°C by a controlled heating pad (Homeothermic blanket control unit, Harvard Apparatus ®). For the electrocardiogram (ECG), signal capture was accomplished with platinum electrodes inserted subcutaneously at three sites, namely positive (near the heart), negative (away from the heart) and ground (near right hind limb) and connected to an ECG amplifier and recorded for about 10 min (Power lab, AD Instruments, Bella Vista, NSW, Australia). Motion-altered ECG signals and artifacts were removed before analysis. Recordings were analysed for intervals with Lab Chart 8 software (AD Instruments, Bella Vista, NSW, Australia).c. Echocardiography of neonatal rat hearts To investigate the cardiac structure and function on day 7 and day 15 of the study duration (Figure 34), Fujifilm Visual Sonics Vevo LAZR-X 3100 system (Fujifilm Visual Sonics, Inc., Toronto, Canada) was used. After anaesthesia the mice were gently restrained on a three-axis micro-positioning stage to perform ultrasound precisely. Mice were placed supine on a heated pad to maintain a stable temperature (37°C). Echocardiographic examination was done with a high conductive ultrasound gel and using a linear transducer in 13-MHz (MX400) to obtain high-resolution two- dimensional and M-mode measurements. The pre-set mode used was “Mouse cardiology (small animal)” with the parasternal long-axis view (PSALX). The echocardiographic measurements were performed using a digital image analysis package (Vevo Lab version 3.2.2) (Figure 34).

[0001] Example 2: Results and discussion 2.1 In vivo antitumor activity Compound 15 was subjected to evaluation for the in vivo therapeutic efficacy at doses 23.5 µmol / kg in the PC3 cells xenograft tumor in male BALB / c nude mice, 4T1-Luc cell in female Balb / c, and MOC2 in the female C57BL / 6 mice. The results demonstrated that compound 15 exhibited potential anticancer activity by complete eradication of tumor via gradual reduction from ~400 mm3. After the treatment period, all the treated mice were healthy, and they were kept aside for survival studies. 2.2 Immunohistochemical studies 2.2.1 Ki67 activity The nuclear protein Ki67 was reduced in compound 15 treated tumors (Figure 19(E) & Figure 25). The intense green fluorescence signal in control tumors indicated the presence of the proliferative marker, Ki67, which was drastically reduced in the compound 15 treated tumor. The reduction in the rate of cell proliferation could be due to the induction of apoptosis to a greater extent in compound 15 treatment groups, as evident further in the ROS production to a greater extent than in Gemcitabine- treated groups (Figure 17(A)). 2.2.2 TUNEL assayTUNEL assay was performed to detect apoptotic cells in the tumor sections (Figure 16). During apoptosis, the DNA is cleaved into multiple strands. Each strand consists of a free 3'-OH group. TUNEL reagent acts as an apoptosis marker wherein it transfers the fluorescence-labeled dUTP, with the help of TdT, to the terminal 3'OH groups producing green fluorescence. As apoptosis leads to higher generation of the free 3'OH ends, higher apoptosis shows higher fluorescence intensity as compared to normal cells. The apoptosis increased significantly in compound 15 (higher dose) than in free Gemcitabine, HDACi, physical mixture-treated mice, as indicated by the highest green fluorescence signals in the cryo-sectioned tumor tissues. 2.2.3 ROS detection assay The generation of reactive oxygen species following treatment was indicated by the strong green fluorescence of 2′-7′ dichlorofluorescein. compound 15 (HD) treated mice displayed more fluorescence signals than the free Gemcitabine. The compound 15 generated ROS to a much greater extent than free Gemcitabine, HDACi, physical mixture, resulting in apoptosis and tumor cell death leading to superior therapeutic efficacy. (Figure 17(B)) 2.2.4 H & E staining The H&E pictures following specified treatment with compound 15 demonstrated pycnosis and karyolysis of the tumor tissue (Figure 18). More significant necrosis was observed compared to the other groups. There was no histological damage to the other organs such as the liver, lungs, spleen, heart, and kidney, which confirms the low toxicity of the compound 15. Studies using H&E staining revealed that the developed conjugate modality was safe, with no evidence of inflammation or immune response activation in isolated organs. 2.2.5 Blood indicators in vivo For further evaluation of the safety of compound 15 in vivo (Figure 20), mice were chosen, and blood was taken after intraperitoneal injection. The samples were analyzed by an automatic animal blood cell analyzer to test for blood indicators, including AST, ALT, ALP, BUN, and SCr (Figure 20), and to quantify the amounts of WBC, RBC, and HGB (Figure 20). Compared with the control group (injected withPBS), no significant differences were found among the groups injected with compound 15 (23.51 µm / kg). 2.3 Compound 15 attenuates tumour microenvironment by inhibiting accumulation of myeloid derived suppressive cells (MDSC) and increased percentage of M1-macrophages The tumour-microenvironment (TME) within the solid tumour is an essential component of growing tumour as it fulfils the nutritional demands of a growing tumour, helps in escaping from immunosurveillance, maintaining constant inflammation, promoting cell survival, growth and invasion contributing to cancer progression in oral cavity. TME composed of multiple different cell types, such as cancer-associated fibroblasts, neutrophils, macrophages, regulatory T cells, myeloid- derived suppressor cells, natural killer cells, platelets and mast cells. These cells communicate each other’s and cause secretion of cytokines, chemokines, growth factors and proteins in the extracellular matrix. Out of all the components of TME, the tumor associated macrophages and myeloid-derived suppressor cells are therapeutically important. The balance between the M1 and M2 macrophages decide the fate of tumor progression. M1 macrophages are pro-inflammatory in nature and normally activated by bacterial LPS or Th-1 driven IFN-gamma. In carcinogenic condition, M1 macrophages possess anti-tumorogenic effects and are practically beneficial against cancer. The generation of reactive oxygen or nitrogen species and production of pro-inflammatory cytokines like interleukins, TNFs and chemokines are the prime working mode of M1 macrophages mediated cancer cell death. Meanwhile, on the other side M2 macrophages are anti-inflammatory in nature they promote the immunosuppressive function of FoxP3+ regulatory T cells leading to inhibition of anti-tumour immunity mediated by Th1 cells and cytotoxic T cells and, finally, help to the escape from immune surveillance. M2 macrophages activates NF-kB and favour the production of EGF and TGF-B1 promoting tumour progression, invasiveness and metastasis. Another important player of TME is myeloid-derived suppressor cells (MDSCs), mainly creates immune suppressive microenvironment characterized by the expression of CD11b and Gr-1. MDSCs are known to inhibit immune reaction, mediate immune escape, secretes IL-10 and TGF-b1 which hampered T cell proliferation and induce angiogenesis and EMT. The high level of MDSC arecorrelated with aggressive tumour differentiation, nodal metastasis and poor survival in OSCC patients. MDSCs are derived from granulocyte macrophage-colony stimulating factor (GM-CSF), macrophage-colony stimulating factor (M-CSF), prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF), stem cell factor (SCF), interleukin-6 (IL-6), IL-10 and IL-1β. MDSCs are known to inhibits T cell proliferation through depletion of arginase by arginase enzyme (ARG), production of ROS and increased in level of inducible nitric oxide synthase (iNOS)production. iNOS and NO are responsible for increased in MDSCs accumulation and developing resistance in solid tumours. Treatment with compound 15 has significant impact on the percentage of MDSC in systemic circulation, spleen as well as in tumour tissue (Figure 27-29). A significant reduction of both G-MDSC and M-MDSC in blood, spleen and tumour tissue by compound 15 was observed while treatment with Gemcitabine had modulation of MDSC population only in blood and tumour tissue with no effect observed in spleen as shown in Figure 31. These observations suggest that modulation of anti-tumour immune response was found to be more potent with compound 15 than that of Gemcitabine. compound 15 also had more impact on M1 / M2 phenotype in tumour bearing mice. The treated mice had significantly high percentage of M1 macrophages in tumour microenvironment. 2.4 In vivo cardiotoxicity study of compound 15 The evaluation of cytotoxic effect of compound 15 at range of concentrations (0.1, 1, 10, 100, 200, and 400μM) on H9C2 cells, cell viability was performed using the MTT assay. Data showed that there is no IC50value of compound 15 on H9c2 cells in concentrations ranges from 0.1 to 400 μM. However, a reduction of cell viability was observed in H9C2 cells from 200 μM (Figure 30(A)), which is way much higher than the IC50 value for cancer cells. The overall change in heart after administration of compound 15 administration, body weight and tail length ratio in both group at the date of animal sacrifice were measured. Our study showed that there was no alteration of heart weight parameter between two groups. Similarly, we did not find any alteration of heart weight and tail length ratio between both groups [Figure 30 (B)]. The effect of compound 15 on the electrophysiology of mouse hearts, electrocardiography (ECG) of the mouse was performed at day 7 and day 15 of study duration, Alteration in the electrocardiographic parameters, i.e., RR interval, PRinterval, QT interval, P duration, R amplitude, T amplitude and QRS interval were recorded (Figure 31). As shown in Figure 31(A), the mice from compound 15 administered had not significant change in the parameters like RR interval, QT interval, P duration, and T Peak, PR interval, QRS interval, Q amplitude, R amplitude, T amplitude, P amplitude, ST height and heart rate compared to saline mice at day 7 of study duration. However, in Figure 31(C), except R amplitude which was significantly increased after 15 days administration of compound 15 in mice, no other significant changes were observed in any of the parameters like RR interval, QT interval, P duration, and T Peak, PR interval, QRS interval, Q amplitude, T amplitude, P amplitude, ST height and heart rate compared to saline treated mice at day 15 of the study duration. The data suggest that compound 15 is not affecting the electrical functioning in heart significantly in either of 7- or 15-days of study duration. Systolic, and diastolic functions in heart were assessed from M-mode ultrasound echocardiography. Representative traces of M-mode are shown in Figure 3. No significant alterations of ejection fraction (EF%), fractional shortening (FS%), cardiac output, left ventricular anterior wall thickness at end-systole (LVAWs), anterior wall thickness at end-diastole (LVAWd), left ventricular posterior wall thickness at end- systole (LVPWs), and left ventricular posterior wall thickness at end-diastole (LVPWd) were evident after administration of compound 15 in mice at day 7 of study duration (Figure 32). Similarly, no significant reduction in left ventricular internal diameter at end-systole (LVIDs), left ventricular internal diameter at end-diastole (LVIDd), left ventricular volume at end-systole (volume s) and left ventricular volume at end-diastole (volume d) were observed. Similarly, no significant changes of different echocardiography parameters except left ventricular posterior wall thickness at end-systole (LVPWs) were observed in mice after administration of compound 15 when compared to the saline mice at day 15 of study duration (Figure 32). Although, we observed a slight increase in the left ventricular mass at day 7, but completely normalised at day 15 of study period. The data suggest that the treatment of compound 15 as described in the present study did not show any cardiotoxicity.

[0052] While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment withoutdeparting from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

Claims

CLAIMS:

1. A bifunctional conjugate molecule, comprising: (a) a broad spectrum chemotherapeutic agent; (b) a linker; and (c) a histone deacetylase inhibitor (HDACi).

2. The bifunctional conjugate molecule as claimed in claim 1, wherein a broad-spectrum chemotherapeutic agent is Gemcitabine.

3. The bifunctional conjugate molecule as claimed in claim 1 having the general Formula I:or a pharmaceutically acceptable salt, hydrate, or solvate thereof. wherein; Linker is selected from -CO-CH2-CH2-CO-NH-CH2- or -CO-CH2-CH2-CO-. HDACi is selected from formula 1.1 to 1.5wherein; m is 0-5, n is 0-5, Z is selected from CH2, O, S, or NH; X1, X2and X3are independently selected from CH or N. R1, R2and R3are independently selected from hydrogen, halogen, cyano, nitro, straight or branched chain C1-C6alkyl, C1-C4alkoxy, —OH, amino, —C(O)NH2, —NHC(O)—, Aryl, heteroaryl, heterocycle, wherein alkyl, alkoxy, —OH, amino, aryl, heteroaryl and heterocycle are optionally substituted with 1-3 substituents selected from hydrogen, halogen, cyano, nitro, C1-C6alkyl, C1-C4alkoxy, —OH and amino.

4. The bifunctional conjugate molecule as claimed in claim 3 or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein aryl is selected from 5-12 membered aromatic ring preferably, phenyl and naphthyl.

5. The bifunctional conjugate molecule as claimed in claim 3 or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein heteroaryl is selected from 5-12membered heteroaromatic ring preferably, thiophenyl, furanyl, pyrrolyl, pyridyl, pyrazinyl, pyrimidinyl, imidazolyl and pyrazolyl.

6. The bifunctional conjugate molecule as claimed in claim 3 or a pharmaceutically acceptable salt, hydrate, or solvate thereof, is selected from the compounds:

7. The bifunctional conjugate molecules as claimed in claims 6, wherein the compounds are in different forms such as amorphous or crystalline solids.

8. A pharmaceutical composition comprising an effective amount of compound as claimed in claim 1-6 or a pharmaceutically acceptable salt, hydrate, or solvate thereof, and a pharmaceutical acceptable carrier, diluent, or excipient.

9. Use of compound as claimed in claim 1-6 and pharmaceutical composition as claimed in claim 8 for the treatment of cancer.

10. Use of compound as claimed in claim 1-6 and pharmaceutical composition as claimed in claim 8 as HD AC inhibitors.

11. Cancer as claimed in claim 9 is selected from the group consisting of carcinoma, lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia, squamous cell cancer, lung cancer, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, and other lymphoproliferative disorders.

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