Hybrid molecules between pyrimidine derivatives and idasanutlin

Abstract

This invention relates to hybrid molecules between pyrimidine derivatives and idasanutlin, designed to induce the degradation of dihydrofolate reductase-thymidylate synthase through the ubiquitin-proteasome system. The hybrid compounds according to the present invention are different from P218 and idasanutlin, as they can stimulate the degradation of dihydrofolate reductase-thymidylate synthase in the malaria parasite, Plasmodium falciparum, through the ubiquitin-proteasome system. This capability holds significant promise for various developments of new forms of drug intended for treatment and prevention of malaria. It is also served as a fundamental concept for the development of new antimalarial drugs using PROTAC technology to target and degrade other target proteins.

Description

[0001] HYBRID MOLECULES BETWEEN PYRIMIDINE DERIVATIVES AND IDASANUTLIN

[0002] FIELD OF INVENTION

[0003] This invention relates to organic chemistry, biotechnology and cellular and molecular biology, and a field relating to hybrid molecules between pyrimidine derivatives and idasanutlin. BACKGROUND OF THE INVENTION

[0004] Malaria is caused by parasites in the Plasmodium group, which are transmitted to humans through the bites of Anopheles mosquitoes, acting as disease vectors. In Thailand, there is a goal to eliminate malaria infections entirely by 2024. Unfortunately, the prevalence of malaria remains high due to failures in controlling mosquito vectors and the emergence of drug-resistant parasites (Nat. Commun., vol. 13, no. 1, p. 2678, Dec. 2022, doi: 10.1038 / s41467-022-30133-w., Nat. Commun, vol. 13, no. 1, p. 2677, Dec. 2022, doi: 10.1038 / s41467-022-30267-x.).

[0005] The current treatment for malaria is Artemisinin-based combination therapy. However, reports indicate drug resistance in Plasmodium parasites, particularly Plasmodium falciparum in Southeast Asian countries, including Thailand, leading to a decline in the efficacy of Artemisininbased combination therapy (Chin. J. Nat. Med., vol. 17, no. 5, pp. 331-336, May 2019, doi: 10.1016 / S1875-5364(19)30038-X.). Antimalarial drugs in the antifolate group, such as Pyrimethamine, Chloroquine, 3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoic acid or P218, are developed to inhibit dihydrofolate reductasethymidylate synthase (DHFR-TS) activity in Plasmodium falciparum. DHFR-TS has been identified in reports as a crucial target for antimalarial drug development due to its involvement in the growth and survival processes of Plasmodium parasites (Proc. Natl. Acad. Sci., vol. 109, no. 42, pp. 16823-16828, Oct. 2012, doi: 10.1073 / pnas.1204556109., ACS Med. Chem. Lett., vol. 9, no. 12, pp. 1235-1240, Dec. 2018, doi: 10.1021 / acsmedchemlett.8b00389.). However, at present, the problems in drug resistance of Plasmodium falciparum to the DHFR-TS drugs have been found. As a result, the efficacy of antifolate drugs decreases. Therefore, exploring and designing antimalarial drugs with novel mechanisms is imperative to address the issue of drug-resistant parasites both now and in the future.

[0006] Currently, the design and synthesis of drugs using PROTAC technology (PRoteolysis TArgeting Chimeras) have gained popularity and are employed in molecular design to develop treatments for various diseases. PROTAC compounds, in the treatment of diverse ailments, can accurately and precisely degrade target proteins by leveraging the mechanism already existing in living things, i.e. the ubiquitin-proteasome system. PROTACs are formed by linking two compounds with a linker. The first compound is specific to the protein of interest, while the other binds to E3 ligase. PROTAC compounds stimulate the close proximity of these two proteins, leading to the addition of ubiquitin to the target protein (substrate ubiquitination). Substrate ubiquitination serves as a signal for transportation of these proteins to the proteasome, a vital cellular component responsible for protein degradation. Therefore, PROTAC is an important tool for stimulating the degradation of target proteins through this mechanism (Figure 1). PROTAC is a rational design compound depending on the target proteins that needs to be degraded (Eur. J. Med. Chem., vol. 235, p. 114290, May 2022, doi: 10.1016 / j.ejmech.2022.114290., Mol. Microbiol., vol. 117, no. 3, pp. 670-681, Mar. 2022, doi: 10.1111 / mmi.l4849., Nat. Rev. Drug Discov., Jan. 2022, doi: 10.1038 / s41573-021-00371-6., Nat. Biotechnol., pp. s41587-021-01173- 2, Dec. 2021, doi: 10.1038 / s41587-021-01173-2.). A significant advantage of PROTAC compounds is their utility as a tool to address issues related to drug resistance. This is because the mechanism of action of synthesized PROTAC compounds does not necessitate tight binding between compounds and target proteins. Therefore, even in cases where target proteins undergo mutations, if compounds can bind to those target proteins, they can stimulate the ubiquitination to target proteins and ultimately degrade those proteins through the proteasome.

[0007] Upon reviewing patent documents related to antimalarial compounds, the US Patent Nos. US9000003B2 and US11028072B2 disclose pyrimidine derivatives demonstrating the ability to inhibit the activity of dihydrofolate reductase in the malaria parasite, Plasmodium falciparum. In addition, the Thai patent application No. 1801006040 discloses 2,4-diamino-6-alkylpyrimidine derivatives hybridized with artemisinin derivatives demonstrating the ability to inhibit Plasmodium falciparum. However, it is noted that the derivatives in these documents inhibit the activity of dihydrofolate reductase but do not exhibit the property of degrading disease-causing proteins. Also, there is no disclosure or indication of the mechanism of action of PROTAC compounds in the development of antimalarial drugs.

[0008] Additionally, in the doctoral dissertation of Duquesne University (Quadery, T. (2021). DESIGN AND SYNTHESIS OF FUSED PYRIMIDINES AS SINGLE AGENTS WITH COMBINATION CHEMOTHERAPY POTENTIAL, AS TUBULIN POLYMERIZATION AND ONE-CARBON METABOLISM INHIBITORS FOR CANCER (Doctoral dissertation, Duquesne University), Retrieved from https: / / dsc.duq.edu / etd / 2115.) discloses the structure of various E3 ligase degraders, including idasanutlin. However, the document specifies the use of PROTAC mechanism in developing compounds for cancer treatment, without any disclosure or indication of the mechanism of action of PROTAC in the context of antimalarial drug development.

[0009] Hence, this invention designs hybrid molecules between pyrimidine derivative and idasanutlin by taking advantage of PROTAC technology and the ubiquitin-proteasome system within Plasmodium parasite in order to degrade dihydrofolate reductase-thymidylate synthase of malaria parasite, Plasmodium falciparum. The synthesized hybrid compound represents a connection between P218 derivatives and idasanutlin with various linking bonds. P218 can bind to dihydrofolate reductase-thymidylate synthase, inhibiting the activity of this enzyme. On the other hand, idasanutlin can bind to a specific type of E3 ligase. When these two proteins come into close proximity, E3 ligase activates the ubiquitin-proteasome system, initiating the degradation of dihydrofolate reductase-thymidylate synthase. It is noteworthy that the connection between P218 and idasanutlin proves more efficacious than P218 alone. The synthesized hybrid compound not only inhibits the activity of dihydrofolate reductase-thymidylate synthase, but also degrades these proteins. This marks the first report dealing the utilization of PROTAC technology in the design and development of antimalarial drugs. In addition, the hybrid molecules between pyrimidine derivatives and idasanutlin according to this invention, could be an alternative approach to address the challenges associated with antimalarial drug resistance. The mechanism of action of synthetic PROTAC compounds does not require tight binding between compounds and target proteins. Therefore, even in the presence of mutations in target proteins, if compounds can still bind to them, they can stimulate ubiquitination to target proteins and degradation of these proteins through the proteasome.

[0010] SUMMARY OF THE INVENTION

[0011] This invention relates to hybrid molecules between pyrimidine derivatives and idasanutlin, designed to induce the degradation of dihydrofolate reductase-thymidylate synthase through the ubiquitin-proteasome system.

[0012] The hybrid molecules between pyrimidine derivatives and idasanutlin according to this invention, is of a Formula (A), wherein:

[0013] Ri is selected from a linear alkyl group having 1 to 10 carbon atoms, a branched alkyl group having 3 to 10 carbon atoms, or a cycloalkyl group having 3 to 10 carbon atoms;

[0014] R2 is selected from a linear hydrocarbon having 1 to 10 carbon atoms, a branched hydrocarbon having 3 to 10 carbon atoms, or a cyclic hydrocarbon having 3 to 10 carbon atoms;

[0015] R3 is selected from hydrogen (H), an alkyl group, or halogen;

[0016] R4 is selected from hydrogen (H), ortho-propanoic acid, or ortho-propanoate (-CH2CH 2 COOR'); wherein R' of said propanoate is selected from a linear alkyl group having 1 to 10 carbon atoms or a branched alkyl group having 3 to 10 carbon atoms;

[0017] Rs is selected from hydrogen (H), an alkyl group, or halogen;

[0018] L is selected from -O-R"-, -NH-R"-, -CH2CH 2CONH-R"-, or -R"-; wherein R" is selected from -(CH2CH2O)nCH2CH2-, -(CH2)m-, group containing carbocyclic, group containing heterocyclic, group containing alcohol acetylene, or group containing amino acetylene; n is an integer in the range from 1 to 6 and m is an integer in the range from 2 to 10;

[0019] Z is selected from -O-, -NH-, a linear hydrocarbon having 1 to 10 carbon atoms or a branched hydrocarbon having 3 to 10 carbon atoms.

[0020] The objective of this invention is to design hybrid molecules between pyrimidine derivatives and idasanutlin, leveraging PROTAC technology and the ubiquitin-proteasome system, and to utilize the synthesized compounds for the development of active compounds aimed at inhibiting malaria parasite, Plasmodium falciparum. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 shows the mechanism of action of PROTAC compound.

[0022] Figure 2 shows red blood cells infected with Plasmodium falciparum. (1) shows the shape of Plasmodium in the ring stage, occurring approximately 12 to 15 hours post-infection. This is considered premature stage, and the expression of dihydrofolate reductase-thymidylate synthase is low. After the parasites progress for about 24 hours (2), they enter the trophozoite stage, characterized by a high expression of dihydrofolate reductase-thymidylate synthase. Subsequently, parasites undergo parasitic division or schizont and generate offspring that disperse from older red blood cells and search for new red blood cells to initiate the cycle anew. The total duration of these processes spans approximately 48 hours.

[0023] Figure 3 shows the protein levels of human dihydrofolate reductase (hDHFR) and human thymidylate synthase (hTS) in representative human cells, named KB cells, after being stimulated with various compounds for about 24 hours. Methotrexate (MTX) is used as a positive control in the experiment because it has been proven to stimulate the expression of both proteins. The levels of target proteins from each group are normalized to the levels of total protein to yield accurate expression values.

[0024] Figure 4 shows the simulated structure of mouse double minute 2 homolog protein of Plasmodium falciparum and the binding between the simulated protein and pyrrolidine, nutlin-3a, and idasanutlin by computer method. (A) is a comparison of simulated structure and template structure available in the Protein Data Bank. (B) is a prediction of the binding site of simulated protein. (C) is the binding region between various compounds and proteins, including associated amino acids.

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] This invention relates to hybrid molecules between pyrimidine derivatives and idasanutlin, designed to induce the degradation of dihydrofolate reductase-thymidylate synthase through the ubiquitin-proteasome system.

[0027] Any aspects described herein shall encompass modification to other aspects of this invention, unless stated otherwise.

[0028] Definition

[0029] Technical terms or scientific terms used herein are defined to the understanding of persons skilled in the art, unless stated otherwise. Any apparatus, equipment, methods, or chemicals named here refer to apparatus, equipment, methods, or chemicals being commonly operated or used by persons skilled in the art, unless stated otherwise. Any mention of equipment, apparatus, methods, or chemicals specifically used in this invention will be clearly specified.

[0030] When the term “comprising” is used in the claims or the specification, singular or plural nouns should be interpreted as “one” or “one or more,” “at least one,” and “one or more than one.” All compositions and / or methods disclosed and claimed in this application are intended to encompass variations involving any actions, operations, modifications, or adjustments to any parameters, provided that they do not significantly deviate from experiments conducted, examples provided, or data presented in this invention and they achieve similar objectives, utilities and results as described in the present invention, as understood by persons skilled in the art although without specifically stated in claims. Therefore, substitutable or similar modifications to the present invention, including any minor modifications or adjustments that are apparent to persons skilled in the art should be construed as remains in spirit, scope, and concept of invention as appeared in appended claims.

[0031] Throughout this application, term “about” refers to any value appeared or presented herein that may vary or deviate due to factors such as equipment deviation, methodological error, or differences between individual operators implementing the apparatus or methods.

[0032] Hereafter, the following embodiments of the present invention are presented without intending to limit the scope of the invention in any manner.

[0033] The hybrid molecules between pyrimidine derivatives and idasanutlin, designed to induce the degradation of dihydrofolate reductase-thymidylate synthase through the ubiquitin-proteasome system, according to this invention is of a Formula (A), wherein:

[0034] Ri is selected from a linear alkyl group having 1 to 10 carbon atoms, a branched alkyl group having 3 to 10 carbon atoms, or a cycloalkyl group having 3 to 10 carbon atoms;

[0035] R2 is selected from a linear hydrocarbon having 1 to 10 carbon atoms, a branched hydrocarbon having 3 to 10 carbon atoms, or a cyclic hydrocarbon having 3 to 10 carbon atoms;

[0036] R3 is selected from hydrogen (H), an alkyl group, or halogen;

[0037] R4 is selected from hydrogen (H), ortho-propanoic acid, or ortho-propanoate (-CH2CH 2 COOR'); wherein R' of said propanoate is selected from a linear alkyl group having 1 to 10 carbon atoms or a branched alkyl group having 3 to 10 carbon atoms;

[0038] Rs is selected from hydrogen (H), an alkyl group, or halogen;

[0039] L is selected from -O-R"-, -NH-R"-, -CH2CH 2CONH-R"-, or -R"-; wherein R" is selected from -(CH2CH2O)nCH2CH2-, -(CH2)m-, group containing carbocyclic, group containing heterocyclic, group containing alcohol acetylene, or group containing amino acetylene; n is an integer in the range from 1 to 6 and m is an integer in the range from 2 to 10;

[0040] Z is selected from -O-, -NH-, a linear hydrocarbon having 1 to 10 carbon atoms or a branched hydrocarbon having 3 to 10 carbon atoms.

[0041] In an embodiment of the invention, group containing carbocyclic is selected from a cyclic hydrocarbon, not limited to a saturated hydrocarbon, but also including an unsaturated hydrocarbon, or an aromatic hydrocarbon having C3 to C12 atoms for 1 ring, or having C7 to C12 atoms for 2 rings.

[0042] Examples are shown as the structures below.

[0043] In another embodiment of the invention, group containing heterocyclic is selected from a hydrocarbon having at least one heteroatom (nitrogen, oxygen, phosphorus, or sulfur), not limited to a saturated hydrocarbon, but also including an unsaturated hydrocarbon having C3 to C12 atoms for 1 ring (C2 to Cl 1, and 1 to 6 atoms may contain heteroatom (N, O, P, S)), or C5 to C15 atoms for 2 rings (C4 to C14, and 1 to 10 atoms may contain heteroatom (N, O, P, S)). Examples are shown as the structures below.

[0044] In another embodiment of the invention, examples of group containing alcohol acetylene are shown as the structures below.

[0045] In another embodiment of the invention, examples of group containing amino acetylene are shown as the structures below.

[0046] In another embodiment of the invention, Ri is a linear alkyl group having 1 to 4 carbon atoms.

[0047] In another embodiment of the invention, R2 is a linear alkyl group having 1 to 4 carbon atoms. In another embodiment of the invention, R3 is hydrogen (H).

[0048] In another embodiment of the invention, R4 is hydrogen (H).

[0049] In another embodiment of the invention, R5 is hydrogen (H).

[0050] In another embodiment of the invention, L is ortho-CFhCFhCONFHCFhCFhO^CFhCFh- , where n is an integer in the range from 1 to 6.

[0051] In another embodiment of the invention, Z is -NH-.

[0052] In another embodiment of the invention, this invention also relates to the method for preparing the hybrid compound between pyrimidine derivatives and idasanutlin according to a

[0053] Formula (A), where R4 and R5 are hydrogen (H), L is ortho-CH2CH2CONH-R"-, comprising steps of: a) amidation between 3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)proproxy)phenyl)propanoic acid and azido-polyethylene glycoln-amine in the presence of an amidation reagent and a first base in a first organic solvent to obtain a product of N-(2-(2-azidoethoxy )nethyl)-3 -(2-(3 -((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanamide of a Formula (III); b) staudinger reduction between the obtained N-(2-(2-azidoethoxy)nethyl)-3-(2-(3-((2,4- diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanamide of a Formula (III) obtained from step a) and triphenylphosphine (PPI13) in a second organic solvent to obtain a product of N-(2-(2-aminoethoxy)nethyl)-3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy) phenyl)propanamide of a Formula (IV);

[0054] (III) (IV) c) amidation between the obtained N-(2-(2-ammoethoxy)nethyl)-3-(2-(3-((2,4-diamino-6- ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanamide of a Formula (IV) and 4- ((2R,3 S ,4R,5S)-3 -(3 -chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cy ano-5- neopentylpyrrolidine-2-carboxamido)-3-methoxybenzoic acid (or idasanutlin) in the presence of an amidation reagent and a first base in a first organic solvent to obtain a product of (2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4- cyano-N-(4-((n-(2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanamido)-n-ethoxy)carbamoyl)-2-methoxyphenyl)-5- neopentylpyrrolidine-2-carboxamide of a Formula (I),

[0055] (IV) (I) wherein n is an integer in the range from 1 to 6.

[0056] In another embodiment of the invention, step a) comprises the steps of: a-1) mixing 3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5-yl)oxy)proproxy)phenyl) propanoic acid solution with an amidation reagent in a first organic solvent, wherein a concentration of 3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5-yl)oxy)proproxy) phenyl)propanoic acid solution is in the range from 0.01 mM to 1 M; a concentration of the amidation reagent is in the range from 0.15 mM to 3 M; a-2) adding a first base to the mixture obtained from step a-1) and stirring the obtained mixture for 5 to 60 minutes; a-3) adding azido -polyethylene glycoln-amine to the mixture obtained from step a-2), wherein n is an integer in the range from 1 to 6, and stirring the obtained mixture at a temperature ranging from 25 °C to 40 °C for 8 to 24 hours.

[0057] In another embodiment of the invention, step a), a molar ratio of 3-(2-(3-((2,4-diamino-6- ethylpyrimidin-5-yl)oxy)proproxy)phenyl)propanoic acid : amidation reagent : first base : azidopolyethylene glycoln-amine is in the range from 1.0 : 1.5 : 4.0 : 1.2 to 1.0 : 3.0 : 7.0 : 2.5.

[0058] In another embodiment of the invention, step a) further comprises the step of extracting the mixture obtained from step a-3) with ethyl acetate and water, and eliminating water residue using sodium sulfate (Na2SO4), and evaporating the first organic solvent under low pressure, followed by purification through column chromatography.

[0059] In another embodiment of the invention, step b) comprises the steps of: b-1) mixing the mixture of compound obtained from steps a) with triphenylphosphine in a second organic solvent at a temperature ranging from 25 °C to 40 °C for 15 to 60 minutes; b-2) adding ammonium hydroxide solution (NH4OH) to the mixture obtained from step b- 1), and allow it to react for 8 to 24 hours.

[0060] In another embodiment of the invention, step b), a molar ratio of N-(2-(2- azidoethoxy)nethyl)-3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanamide : triphenylphosphine is in the range from 1.0 : 2.0 to 1.0 : 7:0.

[0061] In another embodiment of the invention, step b) further comprises the step of eliminating the second organic solvent contained in the mixture obtained from step b-2) under low pressure, followed by purification through column chromatography.

[0062] In another embodiment of the invention, step c) comprises the steps of: c-1) mixing idasanutlin with an amidation reagent in a first organic solvent, wherein a concentration of idasanutlin is in the range from 0.01 mM to 1 M; a concentration of the amidation reagent is in the range from 0.15 to 3 M; c-2) adding a first base to the mixture obtained from step c-1) and stirring the obtained mixture for 5 to 60 minutes; c-3) adding N-(2-(2-aminoethoxy)nethyl)-3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanamide and stirring at a temperature ranging from 25 °C to 40 °C for 8 to 24 hours. In another embodiment of the invention, step c), a molar ratio of idasanutlin : amidation reagent : first base : N-(2-(2-aminoethoxy)ethyl)-3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanamide is in the range from 1.0 : 1.5 : 4.0 : 1.2 to 1.0 : 3.0 : 7.0 : 2.5.

[0063] In another embodiment of the invention, step c) further comprises the step of extracting the mixture obtained from step c-3) with ethyl acetate and water, and eliminating water residue using sodium sulfate (Na2SO4), and evaporating the first organic solvent under low pressure, followed by purification through column chromatography.

[0064] In another embodiment of the invention, this invention also relates to the method for preparing the hybrid molecules between pyrimidine derivatives and idasanutlin according to the structure (A), R4 is ortho-propanoic acid, R5 is hydrogen (H), and L is para-O-R”-, comprising steps of: a) alkylation between ethyl 3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)-

[0065] 5-hydroxyphenyl)propanoate and dibromo alkane in the presence of a second base in a first organic solvent to obtain a product of ethyl3-(bromoalkoxy)m-2-(3-((2,4-diamino-

[0066] 6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoate of a Formula (V); b) nucleophilic substitution between the obtained ethyl3-(brornoalkoxy)m-2-(3-((2,4- diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoate and azide in a first organic solvent to obtain a product of ethyl3-(azidoalkoxy)m-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy) phenyl)propanoate of a Formula (VI); c) staudinger reduction between the obtained ethyl3-(azidoalkoxide)-2-(3-((2,4-diamino- 6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoate and triphenylphosphine (PPhs) in a second organic solvent to obtain a product of ethyl3-(arninealkoxy)m-2-(3-((2,4-diamino- 6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoate of a Formula (VII); d) amidation (step d-1) and hydrolyzation (step d- 2), wherein step d-1) is amidation between the obtained ethyl3-(aminealkoxy)m-2-(3-((2,4- diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoate and idasanutlin in the presence of an amidation reagent and a first base in a first organic solvent to obtain an intermediate of ethyl 3-(5-(m-(4-((3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2- fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamido)-3- methoxybenzamido)alkoxy)m-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoate;

[0067] Step d-2) is hydrolyzation between the intermediate of ethyl3-(5-(m-(4-((3S,4R,5S)-3-(3- chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2- carboxamido)-3-methoxybenzamido)alkoxy)m-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoate obtained from step d-1) and a second base to obtain a product of 3-(5-(m-(4-((3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)- 4-cyano-5-neopentylpyrrolidine-2-carboxamido)-3-methoxybenzamido)alkoxy)m-2-(3-((2,4- diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoic acid of a Formula (II),

[0068] (VII) (II) wherein X is selected from oxygen or carbon; wherein, when X is carbon, m is an integer in the range from 1 to 3, and when X is oxygen, m is an integer in the range from 1 to 6.

[0069] In another embodiment of the invention, step a) is performed by mixing ethyl3-(2-(3-((2,4- diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)-5-hydroxyphenyl)propanoate, dibromo alkane and a base, in the first organic solvent, and stirring the mixture for 5 to 10 minutes, and then increasing a temperature of the mixture to 35 °C to 60 °C for 1 to 4 hours, wherein a concentration of ethyl3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)-5-hydroxyphenyl)propanoate is in the range from 0.01 mM to 1 M; a concentration of dibromo alkane is in the range from 0.02 mM to 5 M; a concentration of the first base is in the range from 0.015 mM to 6 M.

[0070] In another embodiment of the invention, step a), a molar ratio of ethyl3-(2-(3-((2,4- diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)-5-hydroxyphenyl)propanoate : dibromo alkane : first base is in the range from 1.0 : 2.0 : 1.5 to 1.0 : 5.0 : 6.0.

[0071] In another embodiment of the invention, step a) further comprises the step of extracting the mixture obtained from step a with ethyl acetate and water; and eliminating water residue using sodium sulfate (Na2SO4), and evaporating the first organic solvent under low pressure, followed by purification through column chromatography.

[0072] In another embodiment of the invention, step b) is performed by mixing ethyl3- (bromoalkoxy)m-2-(3-((2,4-diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoate with azide in the first organic solvent, and stirring the mixture at the temperature ranging from 20 °C to 40 °C for 8 to 24 hours, wherein a concentration of ethyl3-(bromoalkoxy)m-2-(3-((2,4-diamino-6-ethylpyrimidin- 5-yl)oxy)propoxy)phenyl)propanoate is in the range from 0.01 mM to 1 M; a concentration of azide is in the range from 0.015 mM to 5 M.

[0073] In another embodiment of the invention, step b) further comprises the step of extracting the mixture obtained from step b) with ethyl acetate and water; and eliminating water residue using sodium sulfate (Na2SO4), and evaporating the first organic solvent under low pressure, followed by purification through column chromatography.

[0074] In another embodiment of the invention, step c) comprises the steps of: c- 1) mixing ethyl3-(azidoalkoxy)m-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy) phenyl)propanoate with triphenylphosphine (PPhs) in a second organic solvent at a temperature ranging from 25 °C to 40 °C for 15 to 60 minutes; c-2) adding ammonium hydroxide (NH4OH) to the mixture obtained from steps c-1), and allow it to react for 8 to 24 hours.

[0075] In another embodiment of the invention, step c), a molar ratio of ethyl3 -(azidoalkoxy )m-2- (3-((2,4-diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoate : triphenylphosphine is in the range from 1.0 : 2.0 to 1.0 : 7.0.

[0076] In another embodiment of the invention, step c) further comprises the step of eliminating the second organic solvent of the mixture obtained from step c-2) under low pressure, followed by purification through column chromatography.

[0077] In another embodiment of the invention, step d-1) comprises the steps of: d-1-1) mixing idasanutlin with an amidation reagent in a first organic solvent, wherein a concentration of idasanutlin is in the range from 0.01 mM to 1 M; a concentration of the amidation reagent is in the range from 0.015 mM to 3 M; d -1-2) adding base to the mixture obtained from steps d-1-1) and stirring the obtained mixture for 5 to 60 minutes; d- 1-3) adding ethyl3-(aminealkoxy)m-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy) phenyl)propanoate to the mixture obtained from steps d-1 -2) and stirring at a temperature ranging from 25 °C to 40 °C for 8 to 24 hours.

[0078] In another embodiment of the invention, step d-1), a molar ratio of idasanutlin : amidation reagent : first base : ethyl3-(aminealkoxy)m-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoate is in the range from 1.0 : 1.5 : 4.0 : 1.2 to 1.0 : 3.0 : 7.0 : 2.5.

[0079] In another embodiment of the invention, step d-1) further comprises the step of extracting the mixture obtained from step d-1-3) with ethyl acetate and water; and eliminating water residue using sodium sulfate (Na2SO4), evaporating the first organic solvent under low pressure, followed by purification through column chromatography.

[0080] In another embodiment of the invention, step d-2) is performed by mixing ethyl3-(5-(m- (4-((3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5- neopentylpyrrolidine-2-carboxamido)-3-methoxybenzamido)alkoxy)m-2-(3-((2,4-diamino-6- ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoate intermediate obtained from step d-1) with the second base, and stirring the obtained mixture at a temperature ranging from 25 °C to 40 °C for 0.5 to 4 hours, wherein a concentration of the second base is in the range from 0.5 to 3.0 M in the mixed solvent of water and ethanol, wherein a volume ratio of water : ethanol is in the range from 1.0 :1.0 to 1.0:1.5.

[0081] In another embodiment of the invention, step d-2) further comprises the step of eliminating ethanol under low pressure, and adjusting pH of the mixture to 2 to 3 using acid solution to obtain the white product, and filtering to collect the product, and washing with water.

[0082] In another embodiment of the invention, the first organic solvent is selected from N,N- dimethyl formamide (DMF), tetrahydrofuran anhydrous, dimethyl sulfoxide (DMSO), acetonitrile, or a combination thereof.

[0083] In another embodiment of the invention, the first organic solvent is N,N-dimethyl formamide (DMF).

[0084] In another embodiment of the invention, the second organic solvent is selected from pyridine, pyrimidine, or a combination thereof.

[0085] In another embodiment of the invention, wherein a concentration of pyridine is in the range from 10% w / v to 30% w / v.

[0086] In another embodiment of the invention, the amidation reagent is selected from carbodiimides, phosphonium salts, uronium and guanidinium salts, benzotriazole, or a combination thereof.

[0087] In another embodiment of the invention, carbodiimide is selected from l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC1.HC1), N,N'-dicyclohexane carbodiimide (DCC), diisopropylcarbodiimide (DIC), or a combination thereof.

[0088] In another embodiment of the invention, carbodiimide is l-(3-Dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride.

[0089] In another embodiment of the invention, phosphonium salt is benzotriazol- 1- yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP).

[0090] In another embodiment of the invention, uronium and guanidinium salt is hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU).

[0091] In another embodiment of the invention, benzotriazole is selected from hydroxybenzotriazole (HOBt), l-hydroxy-7-azabenzotriazole (HOAt), 2-(lH-benzotriazol-l-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), or a combination thereof. In another embodiment of the invention, benzotriazole is hydroxybenzotriazole (HOBt).

[0092] In another embodiment of the invention, the first base is selected from N,N- diisopropylethylamine (DIPEA), triethylamine (TEA), or a combination thereof

[0093] In another embodiment of the invention, the first base is N,N-diisopropylethylamine.

[0094] In another embodiment of the invention, the second base is metal hydroxide.

[0095] In another embodiment of the invention, metal hydroxide is selected from lithium hydroxide (LiOH) or sodium hydroxide (NaOH).

[0096] In another embodiment of the invention, azide is metal azide.

[0097] In another embodiment of the invention, metal azide is sodium azide (NaNs).

[0098] In another embodiment of the invention, dibromo is selected from dibromo alkane, dibromo ethylene glycol, or a combination thereof.

[0099] In another embodiment of the invention, the hybrid molecules between pyrimidine derivatives and idasanutlin of a Formula (I) is selected from

[0100] BION186 (3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-N-(4-

[0101] ((2-(2-(3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl) propanamido)ethoxy)ethyl)carbamoyl)-2-methoxyphenyl)-5-neopentylpyrrolidine-2- carboxamide or BION187 (3 S ,4R,5S )-3 -(3 -chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cy ano-N-

[0102] (4-((2-(2-(2-(3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanamido)ethoxy)ethoxy)ethyl)carbamoyl)-2-methoxyphenyl)-5- neopentylpyrrolidine-2-carboxamide or BION106 (3 S ,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-N- (4-((15-(2-(3-((2,4-diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)-13-oxo-3,6,9-trioxa-12- azapentadecyl)carbamoyl)-2-methoxyphenyl)-5-neopentylpyrrolidine-2-carboxamide or BION258 (3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-N-

[0103] (4-((18-(2-(3-((2,4-diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)-16-oxo-3,6,9,12- tetraoxa- 15-azaoctadecyl)carbamoyl)-2-methoxyphenyl)-5-neopentylpyrrolidine-2-carboxamide

[0104] In another embodiment of the invention, the hybrid molecules between pyrimidine derivatives and idasanutlin of a Formula (II) is selected from

[0105] BION188 3-(5-((5-(4-((2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-

[0106] 4-cyano-5-neopentylpyrrolidine-2-carboxamido)-3-methoxybenzamido)pentyl)oxy)-2-(3-((2,4- diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoic acid or BION257 3-(5-(2-(2-(4-((3R,4S,5R)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2- fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamido)-3- methoxybenzamido)ethoxy)ethoxy)-2-(3-((2,4-diamino-6-ethylpyrimidin-5-yl)oxy)propoxy) phenyl)propanoic acid

[0107] or BION189 3-(5-(2-(2-(2-(4-((3R,4S,5R)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2- fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamido)-3- methoxybenzamido)ethoxy)ethoxy)ethoxy)-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoic acid or BION190 3-(5-((l-(4-((3R,4S,5R)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-

[0108] 4-cyano-5-neopentylpyrrolidine-2-carboxamido)-3-methoxyphenyl)-l-oxo-5,8,l l-trioxa-2- azatridecan-13-yl)oxy)-2-(3-((2,4-diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoic acid or BION259 3-(5-((l-(4-((3R,4S,5R)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)- 4-cy ano-5 -neopentylpyrrolidine-2-carboxamido)-3 -methoxyphenyl)- 1 -oxo-5 , 8,11, 14-tetraoxa-2- azahexadecan-16-yl)oxy)-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoic acid

[0109]

[0110] In any embodiments of the invention, the hybrid molecules according to the invention, or pharmaceutically acceptable salt thereof can be used as an active ingredient in pharmaceuticals.

[0111] In another embodiment of the invention, this invention relates to a pharmaceutical composition comprising the hybrid molecules or pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or diluent.

[0112] The following will describe examples of the hybrid molecules between pyrimidine derivatives and idasanutlin, including preparation method according to this invention. It should be understood that examples of the invention described hereinafter have been presented by way of explanatory and exemplary embodiments of the present invention only. Thus, the breadth and scope of the present invention should be defined only in accordance with the claims and their equivalents issuing from this disclosure.

[0113] EXAMPLES

[0114] Examples of the preparation method of the hybrid molecules between pyrimidine derivatives and idasanutlin having a Formula (I) are shown in diagram 1 below.

[0115] Diagram 1

[0116]

[0117] The preparation method of the hybrid molecules between pyrimidine derivatives and idasanutlin having a Formula (I), comprises the steps of:

[0118] Step 1 Preparation of molecules between a Formula B.

[0119] 3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoic acid (or P218) was mixed with 1 -(3 -dimethylaminopropyl)-3 -ethylcarbodiimide hydrochloride (EDCI.HCI) and hydroxybenzotriazole (HOBt) in N,N-dimethyl formamide (DMF) solvent and reacted at a temperature ranging from 25 °C to 40 °C. Then, N,N-diisopropylethylamine (DIPEA) was added and stirred for about 5 to 60 minutes, followed by adding azido-polyethylene glycoln- amine to the reaction mixture, wherein n = 1 to 6, but not limited to this. The mixture was further stirred at the temperature in the range from 25 °C to 40 °C for 8 to 24 hours. The molar ratio of P218 : 1 -(3 -dimethylaminopropyl)-3 -ethylcarbodiimide hydrochloride : hydroxybenzotriazole : N,N-diisopropylethylamine : azido-polyethylene glycoln-amine was in the range from 1.0 : 1.5 : 1.5 : 4.0 : 1.2 to 1.0 : 3.0 : 3.0 : 7.0 : 2.5, respectively. P218 was dissolved in the organic solvent with the concentration in the range from 0.1 to 1 M. The organic solvent was selected from, but not limited to N,N-dimethyl formamide (DMF), tetrahydrofuran anhydrous, dimethyl sulfoxide (DMSO), acetonitrile, or a combination thereof.

[0120] Step 2 Preparation of molecules between a Formula C

[0121] Molecules between a Formula (C) can be prepared as shown in the above diagram. Molecules between a Formula (B) was reacted with triphenylphosphine (PPhs) in organic solvent at the temperature ranging from 25 °C to 40 °C for 15 to 60 minutes, followed by adding ammonium hydroxide (NH4OH) to the reaction mixture. The reaction was further proceeded for 8 to 24 hours. The molar ratio of molecules between a Formula (B) : triphenylphosphine was in the range from 1.0 : 2.0 to 1.0 : 7:0. The organic solvent was not limited to pyridine with a concentration of 10% w / v to 30% w / v.

[0122] Step 3 Preparation of molecules between a Formula D.

[0123] Wherein n = 1 to 6.

[0124] Molecules between a Formula (D) can be prepared as shown in the above diagram, starting from reacting 4-((2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4- cyano-5-neopentylpyrrolidine-2-carboxamido)-3-methoxybenzoic acid (or idasanutlin), l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC1.HC1) and hydroxybenzotriazole (HOBt) in organic solvent at the temperature ranging from 25 °C to 40 °C. Then N,N- diisopropylethylamine (DIPEA) was added and stirred for about 5 to 60 minutes, followed by adding molecules between a Formula C. The mixture was further stirred at temperatures ranging from 25 °C to 40 °C for 8 to 24 hours. The molar ratio of idasanutlin : l-(3-dimethylaminopropyl)- 3 -ethylcarbodiimide hydrochloride : hydroxybenzotriazole : N,N-diisopropylethylamine : molecules between a Formula (C) was in the range from 1.0 : 1.5 : 1.5 : 4.0 : 1.2. to 1.0 : 3.0 : 3.0 : 7.0 : 2.5, respectively. The organic solvent was selected from, but not limited to N,N-dimethyl formamide (DMF), tetrahydrofuran anhydrous, dimethyl sulfoxide (DMSO), acetonitrile, or a combination thereof.

[0125] Examples of the preparation method of the hybrid molecules between pyrimidine derivatives and idasanutlin having a Formula (II) are shown in diagram 2 below. Diagram 2

[0126] The preparation method of the hybrid molecules between pyrimidine derivatives and idasanutlin having a Formula (II), comprises the steps of: Step 1 Preparation of molecules between a Formula (E) v v

[0127] Molecules between a Formula (E) can be prepared as shown in the above diagram. Ethyl 3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)-5-hydroxyphenyl)propanoate (or P218 derivative) was reacted with dibromo alkanenor dibromo ethylene glycoln, and base in organic solvent. The mixture was stirred for about 5 to 10 minutes and the temperature was raised to about 35 °C to 60 °C for 1 to 4 hours. The molar ratio of P218 derivatives : dibromo alkanenor dibromo ethylene glycoln : base was in the range from 1.0 : 2.0 : 1.5 to 1.0 : 5.0 : 6.0. The concentration of the organic solvent was in the range from 0.1 to 1 M. The organic solvent was selected from, but not limited to, N,N-dimethyl formamide (DMF), tetrahydrofuran anhydrous, dimethyl sulfoxide (DMSO), acetonitrile or a combination thereof.

[0128] Step 2 Preparation of molecules between a Formula F

[0129] Molecules between a Formula (F) was prepared by mixing molecules between a Formula (E) and azide, which was selected from metal azide, but not limited to sodium azide (NaNs), in organic solvent. The mixture was stirred at the temperature ranging from 20 °C to 40 °C for 8 to 24 hours. The concentration of organic solvent was in the range from 0.1 to 1 M. The organic solvent was selected from, but not limited to N,N-dimethyl formamide (DMF), tetrahydro furan anhydrous, dimethyl sulfoxide (DMSO), acetonitrile, or a combination thereof.

[0130] Step 3 Preparation of molecules between a Formula G

[0131] Molecules between a Formula (G) can be prepared as shown in the above diagram. Molecules between a Formula (F) was reacted with triphenylphosphine in organic solvent at temperature in the range from 25 °C to 40 °C for 15 to 60 minutes, followed by adding ammonium hydroxide (NH4OH) to the reaction mixture. The reaction was further proceeded for 8 to 24 hours. The molar ratio of molecules between a Formula (F) : triphenylphosphine was in the range 1.0 : 2.0 to 1.0 : 7.0. The organic solvent was not limited to pyridine with a concentration of 10% w / v to 30% w / v.

[0132] Step 4 Preparation of molecules between a Formula H

[0133] Molecules between a Formula (H) can be prepared by 2 sub-steps, i.e. step 4.1 and 4.2.

[0134] Step 4.1: An intermediate of ethyl3-(5-(5-(4-((3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4- ( 4-chl or o-2-fluoro phenyl )-4-cy ano-5-neopentylpyrro lidin e-2-carb ox amido) -3- methoxybenzamido)oxy)-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoate can be prepared by reacting 4-((2R,3S,4R,5S)-3-(3-chloro-2- fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamido)-3- methoxybenzoic acid (or idasanutlin), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC1.HC1) and hydroxybenzotriazole (HOBt) in organic solvent with stirring at a temperature ranging from 25 °C to 40 °C for about 5 to 60 minutes, followed by adding molecules between a Formula (G) and N,N-diisopropylethylamine (DIPEA) to the reaction mixture. The mixture was further stirred at the temperature ranging from 25 °C to 40 °C for 8 to 24 hours. The concentration of organic solvent was in the range from 0.1 to 1 M. The molar ratio of idasanutlin : l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride : hydroxybenzotriazole : N,N- diisopropylethylamine : molecules between a Formula (G) was in the range from 1.0 : 1.5 : 1.5 : 4.0 : 1.2 to 1.0 : 3.0 : 3.0 : 7.0 : 2.5. The organic solvent was selected from, but not limited to N,N- dimethyl formamide (DMF), tetrahydrofuran anhydrous, dimethyl sulfoxide (DMSO), a c e t o n i t r i l e , o r a c o m b i n a t i o n t h e r e o f .

[0135] Step 4.2: Ethyl3-(5-(5-(4-((3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2- fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamido)-3- methoxybenzamido)alkyl)oxy)-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoate intermediate obtained from step 4.1 was mixed with base solution having the concentration in the range from 0.5 to 3.0 M in the solvent of water : ethanol in the ratio of 1.0 : 1.0 to 1.0 : 1.5. The base was selected from metal hydroxide, but not limited to sodium hydroxide (NaOH). The mixture was stirred at the temperature of 25 °C to 40 °C for about 0.5 to 4 hours.

[0136] The followings will illustrate examples of the hybrid compound synthesized based on two aforementioned diagrams. P218 and 2- (2- azidoethoxy) ethan- 1- amine were used as the reactants for Diagram 1. P218 derivative and l-bromo-2-(2-bromoethoxy)ethane were used as the reactants for Diagram 2 to obtain (3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4- cyano-N-(4-((2-(2-(3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanamido)ethoxy)ethyl)carbamoyl)-2-methoxyphenyl)-5- neopentylpyrrolidine-2-carboxamide (BION286) and 3-(5-(2-(2-(4-((3R,4S,5R)-3-(3-chloro-2- fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamido)-3- methoxybenzamido)ethoxy)ethoxy)-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoic acid (BION257), respectively.

[0137] Example 1 (step 1 of Diagram 1): Preparation of N-( 2- (2- azidoethoxy) ethyl) -3- (2- (3- ((2,4- diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanamide of a Formula (Illa).

[0138] P218 (200 mg, 0.5 mmol) was mixed with l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (EDC1.HC1) (241.5 mg, 1.3 mmol) and hydroxybenzotriazole (HOBt) (189.2 mg, 1.4 mmol) in N,N-dimethyl formamide (DMF) (5 ml). N,N- diisopropylethylamine (DIPEA) (0.5 ml, 3.0 mmol) was added and stirred for about 5 minutes, followed by adding 2-(2-azidoethoxy)ethan-l -amine (130.2 mg, 1.0 mmol). The mixture was further stirred at room temperature for 18 hours. Then the resulting mixture was subjected to multiple extractions with ethyl acetate and water. Water residue was eliminated using sodium sulfate (Na2SO4). The organic solvent was evaporated under low pressure, followed by purification through column chromatography to obtain the yellow liquid product of N-(2-(2- azidoethoxy)ethyl)-3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanamide (199.8 mg, 85%):!H NMR (500 MHz, MeOD) 6 7.17 (td, J = 7.8, 1.7 Hz, 1H), 7.13 (dd, J = 7.4, 1.6 Hz, 1H), 6.95 (d, J = 8.2 Hz, 1H), 6.85 (t, J = 7.4 Hz, 1H), 4.22 (t, J = 5.9 Hz, 2H), 3.96 (t, J = 6.1 Hz, 2H), 3.60 - 3.57 (m, 2H), 3.48 (t, J = 5.5 Hz, 2H), 3.33 (t, J = 5.6 Hz, 4H), 2.92 (dd, J = 8.7, 6.8 Hz, 2H), 2.52 - 2.41 (m, 4H), 2.27 (p, J = 6.0 Hz, 2H), 1.10 (t, J = 7.6 Hz, 3H). ^C H } NMR (125 MHz, MeOD) 8 175.6, 161.2, 160.6, 160.0, 157.9, 130.9, 130.3, 128.7, 121.6, 112.3, 70.9, 70.9, 70.5, 65.3, 51.7, 40.3, 37.2, 31.0, 27.7, 24.8, 13.3.

[0139] Example 2 (step 2 of Diagram 1): Preparation of N-(2-(2-aminoethoxy)ethyl)-3-(2-(3-((2,4- diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanamide of a Formula (IVa).

[0140] The obtained N-(2-(2-azidoethoxy)ethyl)-3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanamide of a Formulae (Illa) (199.8 mg, 0.4 mmol) was mixed with triphenylphosphine (554.5 mg, 2.0 mmol) in pyrimidine solvent (5 ml) at room temperature for 30 minutes, followed by adding ammonium hydroxide (NH4OH) to the reaction mixture. The reaction was further proceeded for 18 hours. Then, solvent was evaporated under low pressure, followed by purification through column chromatography to obtain the yellow liquid product of N-(2-(2- aminoethoxy)ethyl)-3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanamide (168.8 mg, 94%): NMR (400 MHz, MeOD) 67.22 - 7.11 (m, 2H), 6.97 (d, J = 8.1 Hz, 1H), 6.86 (t, J = 7.4 Hz, 1H), 4.23 (t, J = 5.9 Hz, 2H), 3.96 (t, J = 6.1 Hz, 2H), 3.49 (dt, J = 15.4, 5.2 Hz, 4H), 3.34 (t, J = 5.5 Hz, 2H), 2.93 (dd, J = 8.7, 6.8 Hz, 2H), 2.87 (t, J = 5.1 Hz, 2H), 2.52 - 2.40 (m, 4H), 2.27 (p, J = 6.0 Hz, 2H), 1.10 (t, J = 7.6 Hz, 3H). ^CpH} NMR (100 MHz, MeOD) 6 175.7, 161.7, 160.5, 160.3, 158.0, 130.9, 130.3, 128.7, 121.7, 112.3, 71.2, 70.8, 70.7, 65.3, 41.6, 40.2, 37.2, 31.0, 27.6, 25.0, 13.4.

[0141] Example 3 (step 3 of Diagram 1): Preparation of (3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4- chloro-2-fluorophenyl)-4-cyano-N-(4-((2-(2-(3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanamido)ethoxy)ethyl)carbamoyl)-2-methoxyphenyl)-5- neopentylpyrrolidine-2-carboxamide of a Formula (la). This compound was specified the name as BION186.

[0142] Idasanutlin (125.7 mg, 0.2 mmol) was mixed with l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (EDC1.HC1) (97.7 mg, 0.5 mmol) and hydroxybenzotriazole (HOBt) (75.7 mg, 0.6 mmol) in 5 ml of N,N-dimethyl formamide (DMF) solvent. Then mixture was stirred for about 15 minutes, followed by adding N-(2-(2-aminoethoxy)ethyl)-3-(2-(3-((2,4- diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanamide (134.0 mg, 0.3 mmol) and N,N-diisopropylethylamine (DIPEA) (0.2 ml, 1.2 mmol), respectively. The reaction mixture was further stirred at room temperature for 18 hours. Then, the resulting mixture was subjected to multiple extractions with ethyl acetate and water, and water residue was removed using sodium sulfate (Na2SO4). The organic solvent was evaporated under low pressure, followed by purification through column chromatography to obtain the white foam-like product of a formula I (185.5 mg, 89%): ’ H NMR (500 MHz, MeOD) 8 8.32 (d, J = 8.4 Hz, 1H), 7.73 (ddd, J = 8.0, 6.2, 1.5 Hz, 1H), 7.50 (d, J = 1.9 Hz, 1H), 7.44 - 7.37 (m, 2H), 7.36 - 7.31 (m, 2H), 7.28 - 7.22 (m, 2H), 7.15 (ddd, J = 8.2, 7.4, 1.7 Hz, 1H), 7.06 (dd, J = 7.5, 1.7 Hz, 1H), 6.90 (dd, J = 8.3, 1.1 Hz, 1H), 6.82 (td, J = 7.4, 1.1 Hz, 1H), 4.77 - 4.72 (m, 1H), 4.62 (d, J = 8.4 Hz, 1H), 4.16 (t, J = 5.9 Hz, 2H), 4.07 (dd, J = 9.9, 1.3 Hz, 1H), 3.90 (d, J = 2.7 Hz, 5H), 3.61 (ddd, J = 6.6, 5.4, 1.5 Hz, 2H), 3.57 - 3.53 (m, 2H), 3.50 (t, J = 5.3 Hz, 2H), 3.35 - 3.32 (m, 2H), 2.89 - 2.81 (m, 2H), 2.48 - 2.38 (m, 4H), 2.21 (p, J = 6.1 Hz, 2H), 1.76 - 1.66 (m, 1H), 1.36 (dd, J = 14.3, 1.3 Hz, 1H), 1.09 (t, J = 7.6 Hz, 3H), 1.03 (s, 9H).13C{ 1H} NMR (125 MHz, MeOD) 8 175.7, 173.2, 169.6, 161.4 (d, JCF = 250.0 Hz),

[0143] 161.3, 160.5, 160.0, 1579, 157.8 (d, JCF = 248.4 Hz), 149.7, 137.4 (d, JCF = 11.3 Hz), 132.6 (d, JCF = 4.7 Hz), 131.3, 131.1 (d, JCF = 1.8 Hz), 130.8, 130.3, 130.2, 129.4 (d, JCF = 2.5 Hz), 128.7, 126.9 (d, JCF = 13.6 Hz), 126.5 (d, JCF = 3.2 Hz), 126.0 (d, JCF = 4.6 Hz), 121.9 (d, JCF = 19.0 Hz), 121.6, 121.1, 120.2 (d, JCF = 9.5 Hz), 119.2, 118.8 (d, JCF = 27.5 Hz), 118.5, 112.3, 110.5, 70.9, 70.5, 70.4, 66.2 (d, JCF = 7.3 Hz), 65.9, 65.3, 64.4 (d, JCF = 7.8 Hz), 56.4, 51.4 (d, JCF = 4.1 Hz), 45.9,

[0144] 40.9. 40.3, 37.2, 31.3, 31.0, 30.3, 27.7, 24.9, 13.3.19F NMR (471 MHz, MeOD) 8 -109.41, -121.42. Table 1 shows theXH,13C,19F NMR spectrum and molecular weight (ESI-HRMS) of the hybrid compounds, which were synthesized according to Diagram 1.

[0145] Example 4 (step 1 of Diagram 2): Preparation of ethyl3-(5-(2-(2-bromoethoxy)ethoxy)-2-(3- ((2,4-diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoate of a Formula (Vb).

[0146] Ethyl3)-2)-3))-2,4-diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)-5- hydroxyphenyl)propanoate (or P218 derivative) (416.1 mg, 1.0 mmol) was mixed with 1-bromo- 2-(2 -bromoethoxy )ethane (0.4 ml, 3.0 mmol) and lithium hydroxide (LiOH) (71.9 mg , 1.0 mmol) in 5 ml of N,N-dimethyl formamide (DMF). The mixture was stirred at room temperature for 10 minutes, and then increased the temperature to 50 °C and reacted for 1 hour. Then, the resulting mixture was subjected to multiple extractions with ethyl acetate and water, and water residue was removed using sodium sulfate (Na2SO4). The organic solvent was evaporated under low pressure, followed by purification through column chromatography to obtain the colorless liquid products of a Formula Vb (350.7 mg , 63%):1H NMR (500 MHz, MeOD) 8 6.88 (d, J = 8.7 Hz, 1H), 6.80 - 6.74 (m, 2H), 4.17 (t, J = 5.8 Hz, 2H), 4.10 - 4.03 (m, 4H), 3.94 (t, J = 6.1 Hz, 2H), 3.85 (t, J = 6.0 Hz, 2H), 3.83 - 3.80 (m, 2H), 3.52 (t, J = 6.0 Hz, 2H), 2.88 (t, J = 7.7 Hz, 2H), 2.58 (t, J = 7.7 Hz, 2H), 2.46 (q, J = 7.6 Hz, 2H), 2.23 (p, J = 6.0 Hz, 2H), 1.18 (t, J = 7.1 Hz, 3H), 1.12 (t, J = 7.6 Hz, 3H). ^CpH} NMR (125 MHz, MeOD) 8 174.9, 161.6, 160.5, 160.2, 154.1, 152.4, 131.2, 130.3, 118.2, 114.0, 113.3, 72.5, 70.9, 70.8, 69.2, 65.9, 61.5, 35.3, 31.3, 31.1, 27.3, 25.0, 14.6, 13.4. Example 5 (step 2 of Diagram 2): Preparation of ethyl3-(5-(2-(2-azidoethoxy)ethoxy)-2-(3-((2,4- diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoate of a Formula (Vib).

[0147] Ethyl3-(5-(2-(2-bromoethoxy)ethoxy)-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoate (350.7 mg, 0.6 mmol) was mixed with sodium azide (205.2 mg, 3.2 mmol) in 0.6 ml of N,N-dimethyl formamide (DMF). The mixture was stirred at room temperature for 18 hours. Then, the resulting mixture was subjected to multiple extractions with ethyl acetate and water, and the water residue was removed using sodium sulfate (Na2SO4). The organic solvent was evaporated under low pressure, followed by purification through column chromatography to obtain the colorless liquid products of a Formula (Vib) (329.5 mg, >100%): ’ H NMR (500 MHz, MeOD) 8 6.87 (d, J = 8.6 Hz, 1H), 6.80 - 6.73 (m, 2H), 4.16 (t, J = 5.8 Hz, 2H), 4.11 - 4.03 (m, 4H), 3.93 (t, J = 6.1 Hz, 2H), 3.83 - 3.78 (m, 2H), 3.74 - 3.69 (m, 2H), 3.39 (t, J = 5.0 Hz, 2H), 2.88 (t, J = 7.7 Hz, 2H), 2.57 (dd, J = 8.2, 7.2 Hz, 2H), 2.46 (q, J = 7.6 Hz, 2H), 2.22 (p, J = 6.0 Hz, 2H), 1.18 (t, J = 7.1 Hz, 3H), 1.11 (t, J = 7.6 Hz, 3H). ^CpH } NMR (125 MHz, MeOD) 8 174.9, 161.7, 160.4, 160.2, 154.1, 152.3, 131.2, 130.3, 118.2, 113.9, 113.3, 71.3, 70.9, 70.9, 69.2, 65.9, 61.5, 51.8, 35.3, 31.1, 27.2, 25.0, 14.5, 13.4.

[0148] Example 6 (step 3 of Diagram 2): Preparation of ethyl3-(5-(2-(2-aminoethoxy)ethoxy)-2-(3- ((2,4-diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoate of a Formula (VUb).

[0149] Ethyl3-(5-(2-(2-azidoethoxy)ethoxy)-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoate (329.5 mg, 0.6 mmol) was mixed with triphenylphosphine (834.9 mg, 3.2 mmol) in pyridine solvent (10% w / v) at room temperature for 60 minutes, followed by adding ammonium hydroxide (NH4OH) to the reaction mixture. The reaction was further proceeded for 18 hours. The organic solvent was eliminated under low pressure, followed by purification through column chromatography to obtain the light yellow liquid products of a Formula (Vllb) (53.6 mg, 61%): %): ’ H NMR (400 MHz, MeOD) 8 6.89 (d, J = 8.4 Hz, 1H), 6.77 (d, J = 8.6 Hz, 2H), 4.17 (t, J = 5.8 Hz, 2H), 4.11 - 4.03 (m, 4H), 3.94 (t, J = 6.1 Hz, 2H), 3.83 - 3.69 (m, 2H), 3.59 (t, J = 5.3 Hz, 2H), 2.95 - 2.81 (m, 4H), 2.58 (t, J = 7.6 Hz, 2H), 2.46 (q, J = 7.6 Hz, 2H), 2.23 (p, J = 6.0 Hz, 2H), 1.18 (t, J = 7.1 Hz, 3H), 1.11 (t, J = 7.6 Hz, 3H).13C{ NMR (100 MHz, MeOD) 6 174.9, 161.8, 160.4, 160.3, 154.1, 152.4, 131.2, 130.3, 118.2, 113.9, 113.3, 73.0, 70.9, 70.8, 69.1, 65.9, 61.5, 42.0, 35.3, 31.1, 27.3, 25.0, 14.5, 13.4.

[0150] Example 7 (step 4 of Diagram 2): Preparation of 3-(5-(2-(2-(4-((3R,4S,5R)-3-(3-chloro-2- fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamido)-3- methoxybenzamido)ethoxy)ethoxy)-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoic acid of a Formula (lib). This compound was specified the name

[0151] Step 4-1: Idasanutlin (67.4 mg, 0.1 mmol) was mixed with l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (EDC1.HC1) (48.0 mg, 0.25 mmol) and hydroxybenzotriazole (HOBt) (37.8 mg, 0.3 mmol) in 0.1 ml of N,N-dimethyl formamide (DMF). Then, N,N- diisopropylethylamine (DIPEA) (0.1 ml, 0.6 mmol) was added to the mixture and stirred for about 5 minutes, followed by adding ethyl3-(5-(2-(2-aminoethoxy)ethoxy)-2-(3-((2,4-diamino-6- ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoate (62.9 mg, 0.15 mmol). The mixture was further stirred at room temperature for 18 hours. Then, the resulting mixture was subjected to multiple extractions with ethyl acetate and water, and water residue was removed using sodium sulfate (Na2SO4). The organic solvent was evaporated under low pressure to obtain an intermediate of ethyl3-(5-(2-(2-(4-((3R,4S,5R)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4- cyano-5-neopentylpyrrolidine-2-carboxamido)-3-methoxybenzamido)ethoxy)ethoxy)-2-(3-((2,4- diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoate. Step 4-2: The obtained ethyl3-(5-(2-(2-(4-((3R,4S,5R)-3-(3-chloro-2-fluorophenyl)-4-(4- chloro-2-fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamido)-3- methoxybenzamido)ethoxy)ethoxy)-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoate intermediate was mixed with 1 ml of sodium hydroxide (NaOH) (2 M in mixed solvent of water : ethanol in the ratio of 1.0 : 1.0). Then, the mixture was stirred at room temperature for 3 hours. Ethanol was eliminated under low pressure. pH was adjusted to the range of about 2 to 3 with hydrochloric acid. The resulting mixture was filtered and washed with water to obtain the white solid product of a Formula II (89.8 mg, 79%): ’ H NMR (500 MHz, MeOD) 8 8.33 (d, J = 8.4 Hz, 1H), 7.72 (ddd, J = 8.0, 6.3, 1.6 Hz, 1H), 7.52 (d, J = 1.9 Hz, 1H), 7.41 - 7.36 (m, 2H), 7.35 - 7.31 (m, 2H), 7.26 - 7.20 (m, 2H), 6.80 (d, J = 8.9 Hz, 1H), 6.78 (d, J = 3.0 Hz, 1H), 6.71 (dd, J = 8.8, 3.0 Hz, 1H), 4.75 (dd, J = 8.4, 1.2 Hz, 1H), 4.62 (d, J = 8.3 Hz, 1H), 4.11 (t, J = 5.7 Hz, 2H), 4.07 - 4.05 (m, 3H), 3.97 (t, J = 6.1 Hz, 2H), 3.94 (s, 3H), 3.82 - 3.79 (m, 2H), 3.72 (t, J = 5.5 Hz, 2H), 3.59 (t, J = 5.4 Hz, 2H), 2.87 - 2.81 (m, 2H), 2.52 (q, J = 7.6 Hz, 2H), 2.48 - 2.43 (m, 2H), 2.22 (p, J = 5.9 Hz, 2H), 1.69 (dd, J = 14.3, 9.9 Hz, 1H), 1.35 (dd, J = 14.4, 1.3 Hz, 1H), 1.12 (t, J = 7.6 Hz, 3H), 1.03 (s,9H). ^CpH} NMR (125 MHz,

[0152] MeOD) 8 179.3, 173.2, 169.5, 162.5, 161.4 (d, JCF = 251.0 Hz), 157.8 (d, JCF = 247.5 Hz), 156.6, 154.2, 152.9, 152.3, 149.7, 137.3 (d, JCF = 11.3 Hz), 132.5 (d, JCF = 4.4 Hz), 132.1, 131.3, 131.1 (d, JCF = 8.2 Hz), 129.8, 129.4 (d, JCF = 1.8 Hz), 127.0 (d, JCF = 13.4 Hz), 126.8 (d, JCF = 3.2 Hz), 126.0 (d, JCF = 4.5 Hz), 121.9 (d, JCF = 18.9 Hz), 121.2, 120.2 (d, JCF = 9.6 Hz), 119.2, 118.8 (d, JCF = 27.3 Hz), 118.5, 118.0, 113.8, 113.3, 110.5, 71.8, 70.8, 70.7, 69.3, 66.3 (d, JCF = 7.3 Hz), 65.9, 65.6, 64.4 (d, JCF = 7.6 Hz), 56.4, 51.4 (d, JCF = 3.6 Hz), 45.8, 41.0, 36.9, 31.3, 30.8, 30.3, 27.8, 224, 12.9.19F NMR (470 MHz, MeOD) 8 -109.38, -121.34.

[0153] Table 2 shows theXH,13C,19F NMR spectrum and molecular weight (ESI-HRMS) of the hybrid compounds, which were synthesized according to Diagram 2. Efficacy test in reducing amounts of dihydrofolate reductase-thymidylate synthase (DHFR- TS) in Plasmodium falciparum.

[0154] Efficacy test of nine synthesized hybrid compounds of P218 and idasanutlin, including P218 alone and idasanutlin alone, in the reduction of dihydrofolate reductase-thymidylate synthase (DHFR-TS) in Plasmodium falciparum was conducted using Western blot analysis.

[0155] Plasmodium falciparum in the ring stage, approximately 12 to 15 hours after entering red blood cells (Figure 2), was used to evaluate the effect of the synthesized hybrid compounds. Plasmodium falciparum was treated with the synthesized hybrid compounds at a concentration of 1 pM for 24 hours. During this timeframe, the parasites would be matured or in the trophozoite stage, and the expression of target protein, dihydrofolate reductase-thymidylate synthase, increased. The results, shown in Table 3 as the remaining amount of dihydrofolate reductase- thymidylate synthase, indicated that the certain hybrid compounds particularly, BION187, BION106, BION258, BION189, BION190 and BION259 could reduce the amount of dihydrofolate reductase-thymidylate synthase. This was in contrast with P218 alone or idasanutlin alone, which did not exhibit a clear reduction of protein level in Plasmodium falciparum. The results demonstrated that the hybrid compounds, formed by combining P218 and idasanutlin, possessed a superior ability to stimulate the degradation of target protein compared to P218 alone or idasanutlin alone.

[0156] Table 3 shows percentage of the remaining dihydrofolate reductase-thymidylate synthase (DHFR- TS) in Plasmodium falciparum after treating with the synthesized hybrid compounds of P218 and idasanutlin compared to P218 alone or idasanutlin alone.

[0157] 1Plasmodium falciparum, ring stage, approximately 12 to 15 hours after entering red blood cells (Figure 2), was treated with the synthesized hybrid compounds according to Table 1 at a concentration of 1 pM or dimethyl sulfoxide -control for 24 hours.

[0158] The values are presented as mean ± standard deviation, derived from at least three independent biological samples.

[0159] Efficacy test in inhibiting dihydrofolate reductase (DHFR) enzyme of Plasmodium falciparum and human.

[0160] The results of inhibition of dihydrofolate reductase enzyme using the competitive enzyme inhibition assay are shown in Table 4. It was found that all nine synthesized hybrid compounds demonstrated high efficacy in inhibiting the activity of dihydrofolate reductase enzyme of Plasmodium falciparum at levels below 10 nM. In contrast, none of them at the highest concentration (>100 pM) could inhibit the activity of human dihydrofolate reductase enzyme. However, P218 alone could inhibit the activity of human dihydrofolate reductase, indicating lower specificity toward the target enzyme compared to the synthesized hybrid compounds.

[0161] The results demonstrated that all nine hybrid compounds exhibited greater specificity towards the dihydrofolate reductase enzyme in malaria parasite, Plasmodium falciparum, as opposed to human cells. Moreover, all synthesized hybrid compounds also improved the specificity of P218 towards the dihydrofolate reductase enzyme in Plasmodium falciparum. Table 4 shows the inhibition constant of dihydrofolate reductase (Ki) of nine synthesized hybrid compounds of P218 and idasanutlin, P218 alone, or idasanutlin alone in wild type strain of Plasmodium falciparum (TM4 / 8.2) and human cells.

[0162] The values are presented as mean ± standard deviation, derived from at least three independent biological samples.

[0163] Efficacy test of compounds on toxicity in Plasmodium falciparum and mammalian cells.

[0164] The results of toxicity or killing efficiency in Plasmodium falciparum of nine synthesized hybrid compounds, P218 alone, and idasanutlin alone, compared with mammalian cells using the [3H] -hypoxanthine incorporation assay are shown in Table 5. Cells derived from epidermoid carcinoma of human oral cavity, named KB cells, were employed as representative mammalian cells because they expressed dihydrofolate reductase, the main target of the studied compounds.

[0165] From the comparison, it was found that all synthesized hybrid compounds had a lower half maximal inhibitory concentration (IC50) in wild type strain of Plasmodium falciparum (TM4 / 8.2) compared to human cells, indicating that the synthesized hybrid compounds effectively inhibited the growth of Plasmodium falciparum. They also demonstrated selectivity in killing Plasmodium falciparum over representative mammalian cells, particularly the hybrid compounds BION106, 258, 188, 257, 189,190, and 259. The observed selective killing underscored the importance of this properties in antimalarial drug development.

[0166] Table 5 shows the IC50 of the synthesized hybrid compounds, P218 alone and idasanutlin alone against Plasmodium falciparum and mammalian cells.

[0167] The values are presented as mean ± standard deviation, derived from at least three independent biological samples.

[0168] $The selectivity value is a proportion derived from mean value of toxicity in mammalian cells and the mean value of toxicity in wild type strain of Plasmodium Falciparum (IC50 ratio). Activity of the synthesized hybrid compound through the ubiquitin-proteasome system in Plasmodium falciparum.

[0169] The synthesized hybrid compound BION 106 (Ic), which demonstrated a high ability to degrade dihydrofolate reductase-thymidylate synthase in Plasmodium falciparum, (% Degradation = 43. 81 ± 11.63, as shown in Table 3), and the ability to killed Plasmodium falciparum at a low concentration (IC50 = 0.20 pM, as shown in Table 5) was selected as a representative for studying the mechanism of action of the synthesized hybrid compound through the ubiquitin-proteasome system in Plasmodium falciparum.

[0170] To study the mechanism of action of the representative hybrid compound, Plasmodium falciparum, in growing trophozoite stage (aged 30 to 40 hours after entering red blood cells, as shown in Figure 2), was cultured for the study. As the expression of dihydrofolate reductasethymidylate synthase was higher in the growing trophozoite stage than in other stages, it was deemed suitable for studying the mechanism of action of compounds in a shorter period of time. In this experiment, the cultured parasites were treated with different concentrations of BION106 for 4 hours, and then protein extraction was performed to measure the remaining protein through Western blot analysis. The results are shown in Table 6. It was found that BION106 demonstrated the ability to degrade dihydrofolate reductase-thymidylate synthase with dose-dependent degradation. BION106 can significantly reduce the amount of target protein at a concentration of 10 pM within 4 hours (P-value < 0.0001).

[0171] The ability of BION 106 in reducing the levels of dihydrofolate reductase-thymidylate synthase in Plasmodium falciparum, is shown in Table 7. The cultured parasites were treated with BION106 at a concentration of 10 pM for varying durations, as outlined in Table 7 (30 to 240 minutes). The results indicated that BION106 showed the ability to rapidly reduce the amount of target protein. Additionally, the results revealed that BION106 could reduce the amount of dihydrofolate reductase-thymidylate synthase in Plasmodium falciparum. After the cultured parasites were treated with BION106 for only 30 minutes, it reduced a haft amount of dihydrofolate reductase-thymidylate synthase (% Degradation = 46. 76 ± 6.77).

[0172] To prove whether the observed reduction in dihydrofolate reductase-thymidylate synthase, as presented in the above results, is resulted from BION106 activating the ubiquitin-proteasome system in Plasmodium falciparum in accordance with its mechanism of action of PROTAC hybrid compound as shown in Figure 1, two chemical proteasome inhibitors, bortezomib and epoxomicin, were selected for investigation. Both chemicals were selected based on their established ability to selectively inhibit the activity of proteasome. Moreover, these chemicals are commonly employed to study the mechanism of compound through ubiquitin-proteasome.

[0173] Growing trophozoite stage Plasmodium falciparum was treated with proteasome inhibitor, 10 pM of bortezomib or 0.5 pM of epoxomicin, as defined in Table 8. This pre-treatment spanned a duration of 2 hours, aiming for the complete inhibition of proteasome activity. Subsequently, the parasites were exposed to BION106 at a concentration of 10 pM for 30 minutes. Then, protein was extracted and used for quantifying the remaining amount of dihydrofolate reductasethymidylate synthase through Western blot analysis.

[0174] The results revealed that inhibition of proteasome activity using bortezomib or epoxomicin significantly prevented the reduction of dihydrofolate reductase-thymidylate synthase stimulated by BION106 (P-value < 0.0001). This indicated that the synthesized hybrid compounds provided activity in reducing the levels of target proteins related to ubiquitin-proteasome system in Plasmodium falciparum.

[0175] From the above biological experiments results, it can be concluded that the synthesized hybrid compounds of P218 and idasanutlin provided additional effects distinct from P218 alone or idasanutlin alone. It had an effective degradation of dihydrofolate reductase-thymidylate synthase enzyme in Plasmodium falciparum, through the ubiquitin-proteasome system. In addition, these hybrid compounds exhibited a more selective inhibition of the activity of dihydrofolate reductase- thymidylate synthase in Plasmodium falciparum, compared to human. Additionally, the toxicity was also high in Plasmodium falciparum.

[0176] Table 6 shows the dose-dependent degradation of BION106 in reducing the amount of dihydrofolate reductase-thymidylate synthase in Plasmodium falciparum, over 4 hours.

[0177] 1Plasmodium falciparum, growing trophozoite, approximately age 30-40 hours after entering red blood cells were treated with BION106 at various concentrations (pM) for 4 hours.

[0178] The values are presented as mean ± standard deviation, derived from at least three independent biological samples.

[0179] 2The statistical significance value was analyzed using One-way ANOVA. Dunnett’s multiple comparisons test was performed with GraphPad Prism version 8.

[0180] Table 7 shows the effect of BION106 on the time-course degradation of dihydrofolate reductasethymidylate synthase in Plasmodium falciparum.

[0181] 1Plasmodium falciparum, growing trophozoite, approximately age 30-40 hours after entering red blood cells were treated with BION106 at concentration of 10 pM or dimethyl sulfoxide-control for the defined period of time (minutes).

[0182] The values are presented as mean ± standard deviation, derived from at least three independent biological samples.

[0183] 2The statistical significance value was analyzed using One-way ANOVA. Tukey’s multiple comparisons test was performed with GraphPad Prism version 8. Table 8 shows percentage of the remaining dihydrofolate reductase-thymidylate synthase (DHFR- TS) in Plasmodium falciparum, after treating with various compounds to prove the action of synthesized hybrid BION106 through the ubiquitin-proteasome system.

[0184] 1Plasmodium falciparum, growing trophozoite, approximately age 30-40 hours after entering red blood cells were treated with proteasome inhibitors, bortezomib (10 pM) or epoxomicin (0.5 pM), for 2 hours. Then parasites were treated with BION 106 at a concentration of 10 pM or dimethyl sulfoxide-control for 30 minutes.

[0185] 1values are presented as mean ± standard deviation, derived from at least three independent biological samples.

[0186] 2The statistical significance value was analyzed using One-way ANOVA. Tukey’s multiple comparisons test was performed with GraphPad Prism version 8.

[0187] Specificity of the hybrid compound to target proteins (target specificity) of human cells.

[0188] The synthesized hybrid compounds underwent further investigation to determine whether they specifically influenced the degradation of target proteins only in Plasmodium falciparum, without affecting the target proteins, dihydrofolate reductase and thymidylate synthase, in human cells. To assess this specificity, the inventor designed an assay to examine the targeted degradation of proteins by hybrid PROTAC compounds in human cells. The representative human cells, named KB cells, the cultured cells developed from epidermoid carcinoma of the human oral cavity, were selected as a model due to their expression of the proteins of interest, namely human dihydrofolate reductase (hDHFR) and human thymidylate synthase (hTS). In order to ensure accuracy and reliability in the experiment, methotrexate (MTX), which was an anti-cancer and antifolate drug, was used. Similar to P218, MTX also inhibited the activity of dihydrofolate reductase enzyme (DHFR inhibitor). It exhibited specific binding and inhibition of the activity of dihydrofolate reductase enzyme in human cells. Notably, MTX stimulated the up-regulation of the targeted proteins, including thymidylate synthase protein in human cells. Given its well-established impact on increasing the expression of both target proteins in human cells, methotrexate (MTX) was used in this experiment as a positive control in this experiment [EE Abali, NE Skacel, H. Celikkaya, and Y. Hsieh, “Chapter 9 Regulation of Human Dihydrofolate Reductase Activity and Expression,” in Vitamins & Hormones, Elsevier, 2008, pp. 267-292. doi: 10.1016 / S0083- 6729(08)00409-3.].

[0189] As shown in Figure 3 and Table 9, the results showed that all concentrations of BION106 (0.1, 1, 10 pM) could stimulated the expression of human dihydrofolate reductase (hDHFR) in human KB cells compared to dimethyl sulfoxide-control group. However, BION 106 did not significantly changed the levels of human thymidylate synthase (hTS). Importantly, the inventor proved that P218 could significantly increase the expression of both proteins (P-value < 0.0001), following the same trend as positive control (methotrexate (MTX)). This provided new evidence that P218, one of the folate pathway inhibitors, can increase levels of human dihydrofolate reductase (hDHFR) and human thymidylate synthase (hTS) as well.

[0190] Idasanutlin alone did not induce any significant changes in the levels of both proteins in representative human cells. Therefore, the observed increase in level of human dihydrofolate reductase (hDHFR) by BION 106, synthesized through the combination of P218 with idasanutlin, could be attributed primarily to the influence of P218. The results in human KB cells indicated important information about BION106. While BION106 can stimulate the degradation of dihydrofolate reductase-thymidylate synthase in Plasmodium falciparum through the ubiquitin- proteasome system (Table 6 to 8), it was found that this phenomenon could not be observed in representative human cells. This information indicated that BION106 PROTAC had target specificity to P218 between malaria parasite and human cells. This is an important feature of PROTAC, which targets only parasite proteins rather than host cells. Table 9 shows relative quantity of human dihydrofolate reductase (hDHFR) and human thymidylate synthase (hTS) in representative human KB cells after treating with each compound for 24 hours. This analysis aims to demonstrate the specificity of BION106, P218 and idasanutlin in influencing the expression of target proteins in human cells. Methotrexate (MTX) was used as positive control for the expression of human dihydrofolate reductase (hDHFR) and human thymidylate synthase (hTS).

[0191] Approximately 1 to 2 million cultured human KB cells per group were used to test each compounc at a defined concentration for 24 hours. Cells from each group were subjected to protein extraction to test the levels of human dihydrofolate reductase (hDHFR) and human thymidylate synthase (hTS) through Western blot analysis. The level of target proteins of each group was analyzed through relative quantification compared with dimethyl sulfoxide-control using the Image Studio (IS) program from Li-COR, version 5.2.5. The values are presented as mean ± standard deviation, derived on at least three independent biological samples.2The statistical significance value was analyzed using One-way ANOVA. Holm-Sidak’s & Dunnett’s multiple comparisons test was performed with GraphPad Prism version 8.

[0192] Simulation of the structure of mouse double minute 2 homolog (P / MDM2) protein in Plasmodium falciparum and the binding interaction between the protein and idasanutlin.

[0193] Figure 1 illustrated the mechanism of action of PROTAC compounds with a Formula (1) formed by the connection of P218 and idasanutlin. Upon entering the Plasmodium parasite, the hybrid compound binds to dihydrofolate reductase-thymidylate synthase and proteins in E3 ligase group, respectively (2). This binding brings two proteins into close proximity. As a result, a mechanism known as target ubiquitination is activated (3), signaling that these proteins are to be transported to proteasome-a cellular component having an important role in protein degradation (4). Therefore, dihydrofolate reductase-thymidylate synthase undergo degradation resulting in a decrease in the level of this protein. In addition, hybrid PROTAC can stimulate the degradation of target proteins according to this mechanism for several cycles.

[0194] As shown in Figure 1, the mechanism of action of PROTAC relies on the proximity of the ternary complex formation of target proteins that need to be degraded and proteins that act as E3 ligase, stimulating the process of target ubiquitination. This is a signal for the target proteins to be degraded by proteasome. It is known that P218 mainly targets dihydrofolate reductase-thymidylate synthase of Plasmodium falciparum. On the contrary, there is no evidence indicating that idasanutlin targets any type of E3 ligase in the studied malaria parasite. Therefore, this invention has provided further studies to elucidate this aspect.

[0195] Initially, there were evidences indicating that Plasmodium falciparum has 54 types of E3 ligase enzymes, less than the 600 proteins in E3 ligase group found in mammalian cells [MN Aminake, H.-D. Arndt, and G. Pradel, “The proteasome of malaria parasites: A multi-stage drug target for chemotherapeutic intervention?,” Int. J. Parasitol. Drugs Drug Resist., vol. 2, pp. 1-10, Dec. 2012, doi: 10.1016 / j.ijpddr.2011.12.001]. Idasanutlin was reported as an inhibitor for the activity of human mouse double minute 2 homolog protein (hMDM2), which has E3 ligase enzyme properties [Q. Ding et al., “Discovery of RG7388, a Potent and Selective p53-MDM2 Inhibitor in Clinical Development,” J. Med. Chem., vol. 56, no. 14, pp. 5979-5983, Jul. 2013, doi: 10.1021 / jm400487c]. This invention utilized a database to predict proteins in Plasmodium falciparum with a structure similar to human mouse double minute 2 homolog (hMDM2) and employed this protein for studying its ability to bind to idasanutlin. The structure of this protein was simulated based on the amino acid sequence from the PlasmoDB database (https: / / plasmodb.org / plasmo / app) using the Homology modeling method and referenced to the template structure of human mouse double minute 2 homolog (hMDM2) protein. The results indicate that the SWIB / MDM2 domain-containing protein (reference no. PF3D7_0518200) from the 3D7 strain of Plasmodium falciparum is a protein with an amino acid sequence that is the most similar to human mouse double minute 2 homolog (hMDM2). This protein is named Plasmodium falciparum mouse double minute 2 homolog (P MDM2) in this invention. Then a computational method called protein docking was used for simulating the binding between Plasmodium falciparum mouse double minute homolog protein (PfMDM2) and idasanutlin. The binding sites were determined in the same region with template structure to study the differences of overall structure and amino acids that important for affinity.

[0196] The results are shown in Figure 4, indicating that the simulated protein structure of Plasmodium falciparum closely resemble the template structure in Protein Data Bank database number 4JSC and 4HG7. These entries correspond to human mouse double minute 2 homolog bind to nutlin-3a and pyrrolidine, which is an initial structure of idasanutlin (Figure 4A). According to the prediction of the binding site of simulated protein, it was found that the most probable binding groove was located in the same region as human Sitel, spanning 124 angstroms (Figure 4B). Site2 and Site3, spanning 61 and 58 angstroms, respectively, may provide less potential for protein and compound binding. However, the amino acids involved in these regions are different from humans, resulting in a narrower surface area and grooves utilized for reactions. The direction in which the compound enters the binding site may vary from the template structure.

[0197] Pyrrolidine, nutlin-3a, and idasanutlin can interact with Site 1 region of the simulated protein with different affinities (Figure 4C). The important amino acids involved in binding these compounds are leucine at position 23, alanine at position 26, tryptophan at position 27, isoleucine at position 30, arginine at position 42, isoleucine at position 61, leucine at position 62 and glutamate at position 63. It can be seen that these amino acids have large side chains or functional groups. Consequently, the surface area and groove of Sitel would be narrow. For binding with nutlin-3a, it can stimulate the entry and binding of arginine at position 19, an amino acid with a positive charge. This differs from pyrrolidine and idasanutlin which introduce non-polar amino acids into that area, i.e. valine at position 20, lysine at position 24, and lysine at position 28.

[0198] When comparing the binding energy values between idasanutlin and protein, it was found that idasanutlin exhibits the best binding to the protein with an energy value of -9.64 kCal / mole compared to pyrrolidine is used as a model, having an energy value of -7.90 kCal / mole. Meanwhile, nutlin-3a shows a higher protein binding efficiency than pyrrolidine but is surpassed by idasanutlin, which has an energy value of -8.28 kcal / mole. The results show that the Plasmodium falciparum mouse double minute 2 homolog can interact with idasanutlin in the same binding site as in human.

[0199] Therefore, this simulation suggested that idasanutlin has the potential to bind to E3 ligase enzyme of Plasmodium, even though it had not been studied before the disclosure of this invention. BEST MODE OF THE INVENTION

[0200] The best mode or preferred embodiment of the invention is as described in the detailed description.

Claims

CLAIMS1. Hybrid molecules between pyrimidine derivatives and idasanutlin having a Formula (A),wherein:Ri is selected from a linear alkyl group having 1 to 10 carbon atoms, a branched alkyl group having 3 to 10 carbon atoms, or a cycloalkyl group having 3 to 10 carbon atoms;R2 is selected from a linear hydrocarbon having 1 to 10 carbon atoms, a branched hydrocarbon having 3 to 10 carbon atoms, or a cyclic hydrocarbon having 3 to 10 carbon atoms;R3 is selected from hydrogen (H), an alkyl group, or halogen;R4 is selected from hydrogen (H), ortho-propanoic acid, or ortho-propanoate (-CH2CH 2 COOR'); wherein R' of said propanoate is selected from a linear alkyl group having 1 to 10 carbon atoms or a branched alkyl group having 3 to 10 carbon atoms;Rs is selected from hydrogen (H), an alkyl group, or halogen;L is selected from -O-R"-, -NH-R"-, -CH2CH 2CONH-R"-, or -R"-; wherein R" is selected from -(CH2CH2O)nCH2CH2-, -(CH2)m-, group containing carbocyclic, group containing heterocyclic, group containing alcohol acetylene, or group containing amino acetylene; n is an integer in the range from 1 to 6 and m is an integer in the range from 2 to 10;Z is selected from -O-, -NH-, a linear hydrocarbon having 1 to 10 carbon atoms or a branched hydrocarbon having 3 to 10 carbon atoms.

2. The hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 1, wherein Ri is a linear alkyl group having 1 to 4 carbon atoms.

3. The hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 1, wherein R2 is a linear alkyl group having 1 to 4 carbon atoms.

4. The hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 1, wherein R3 is hydrogen (H).

5. The hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 1, wherein R4 is hydrogen (H).

6. The hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 1, wherein R5 is hydrogen (H).

7. The hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 1, wherein L is ortho-CH2CH2CONH-(CH2CH2O)nCH2CH2-, where n is an integer in the range from 1 to 6.

8. The hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 1, wherein Z is -NH-.

9. The method for preparing the hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 1, wherein the hybrid molecules between pyrimidine derivatives and idasanutlin has a Formula (A), where R4 and R5 are hydrogen (H). L is ortho-CH2CH2CONH-R"-,comprising steps of: a) amidation between 3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)proproxy)phenyl)propanoic acid and azido-polyethylene glycoln-amine in the presence of an amidation reagent and a first base in a first organic solvent toobtain a product of N-(2-(2-azidoethoxy)nethyl)-3-(2-(3-((2,4-diamino-6- ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanamide of a Formula (III);b) staudinger reduction between the obtained N-(2-(2-azidoethoxy)nethyl)-3-(2-(3- ((2,4-diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanamide of a Formula (III) obtained from step a) and triphenylphosphine (PPI13) in a second organic solvent to obtain a product of N-(2-(2-aminoethoxy)nethyl)-3-(2-(3-((2,4- diamino-6-ethylpyrimidin-5-yl)oxy)propoxy) phenyl)propanamide of a Formula (IV);c) amidation between the obtained N-(2-(2-aminoethoxy)nethyl)-3-(2-(3-((2,4- diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanamide of a Formula (IV) and idasanutlin in the presence of an amidation reagent and a first base in a first organic solvent to obtain a product of (2R,3S,4R,5S)-3-(3-chloro-2- fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-N-(4-((n-(2-(3-((2,4-diamino- 6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanamido)-n-ethoxy)carbamoyl)-2-methoxyphenyl)-5-neopentylpyrrolidine-2-carboxamide of a Formula (I),wherein n is an integer in the range from 1 to 6.

10. The method for preparing the hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 9, wherein step a) comprises the steps of: a- 1 ) mixing 3 -(2-(3 -((2,4-diamino-6-ethylpyrimidin-5-yl)oxy)proproxy)phenyl) propanoic acid solution with an amidation reagent in a first organic solvent, wherein a concentration of 3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)proproxy) phenyl)propanoic acid solution is in the range from 0.01 mM to 1 M; a concentration of the amidation reagent is in the range from 0.15 mM to 3 M; a-2) adding a first base to the mixture obtained from step a-1) and stirring the obtained mixture for 5 to 60 minutes; a-3) adding azido -polyethylene glycoln-amine to the mixture obtained from step a-2), wherein n is an integer in the range from 1 to 6, and stirring the obtained mixture at a temperature ranging from 25 °C to 40 °C for 8 to 24 hours.

11. The method for preparing the hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 9, wherein the amidation reagent is selected from carbodiimides, phosphonium salts, uronium and guanidinium salts, benzotriazole, or a combination thereof.

12. The method for preparing the hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 9, wherein the first base is selected from N,N- diisopropylethylamine (DIPEA), triethylamine (TEA), or a combination thereof13. The method for preparing the hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 9, wherein the first organic solvent is selected from N,N- dimethyl formamide (DMF), tetrahydrofuran anhydrous, dimethyl sulfoxide (DMSO), acetonitrile, or a combination thereof.

14. The method for preparing the hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 9, wherein the second organic solvent is selected from pyridine, pyrimidine, or a combination thereof.

15. The method for preparing the hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 1, wherein the hybrid molecules between pyrimidinederivatives and idasanutlin has a Formula (A), where R4 is ortho-propanoic acid, R5 is hydrogen (H), and L is para-O-R”-,comprising steps of: b) alkylation between ethyl 3-(2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)-5-hydroxyphenyl)propanoate and dibromo alkane in the presence of a second base in a first organic solvent to obtain a product of ethyl3- (bromoalkoxy)m-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoate of a Formula (V);b) nucleophilic substitution between the obtained ethyl3-(brornoalkoxy)m-2-(3-((2,4- diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoate and azide in a first organic solvent to obtain a product of ethyl3-(azidoalkoxy)m-2-(3-((2,4-diamino-6- ethylpyrimidin-5-yl)oxy)propoxy) phenyl)propanoate of a Formula (VI);c) staudinger reduction between the obtained ethyl3-(azidoalkoxide)-2-(3-((2,4- diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoate and triphenylphosphine(PPhs) in a second organic solvent to obtain a product of ethyl3 -(aminealkoxy )m-2-(3- ((2,4-diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoate of a Formula (VII);d) amidation (step d-1) and hydrolyzation (step d- 2), wherein step d-1) is amidation between the obtained ethyl3-(aminealkoxy)m-2-(3-((2,4- diamino-6-ethylpyrimidin-5-yl)oxy)propoxy)phenyl)propanoate and idasanutlin in the presence of an amidation reagent and a first base in a first organic solvent to obtain an intermediate of ethyl 3-(5-(m-(4-((3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2- fluorophenyl)-4-cy ano-5-neopentylpyrrolidine-2-carboxamido)-3 - methoxybenzamido)alkoxy)m-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoate;Step d-2) is hydrolyzation between the intermediate of ethyl3-(5-(m-(4-((3S,4R,5S)-3- (3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5-neopentylpyrrolidine- 2-carboxamido)-3-methoxybenzamido)alkoxy)m-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoate obtained from step d- 1 and a second base to obtain a product of 3 -(5-(m-(4-((3 S ,4R,5S )-3 -(3 -chloro-2-fluorophenyl)-4-(4-chloro-2- fluorophenyl)-4-cy ano-5-neopentylpyrrolidine-2-carboxamido)-3 - methoxybenzamido)alkoxy)m-2-(3-((2,4-diamino-6-ethylpyrimidin-5- yl)oxy)propoxy)phenyl)propanoic acid of a Formula (II);(VII) (II) wherein X is selected from oxygen or carbon; wherein, when X is carbon, m is an integer in the range from 1 to 3, and when X is oxygen, m is an integer in the range from 1 to 6.

16. The method for preparing the hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 15, wherein the amidation reagent is selected from carbodiimides, phosphonium salts, uronium and guanidinium salts, benzotriazole, or a combination thereof.

17. The method for preparing the hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 15, wherein the first base is selected from N,N- diisopropylethylamine (DIPEA), triethylamine (TEA), or a combination thereof18. The method for preparing the hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 15, wherein the second base is metal hydroxide.

19. The method for preparing the hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 15, wherein the first organic solvent is selected from N,N- dimethyl formamide (DMF), tetrahydrofuran anhydrous, dimethyl sulfoxide (DMSO), acetonitrile, or a combination thereof.

20. The method for preparing the hybrid molecules between pyrimidine derivatives and idasanutlin according to claim 15, wherein the second organic solvent is selected from pyridine, pyrimidine, or a combination thereof.

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