AN IMPROVED PROCESS TO PREPARE MALONIC ACID DERIVATIVES USING
MELDRUM’S ACID
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] None.
FIELD OF THE INVENTION
[0002] This invention relates generally to cancer diagnostics and therapy and in
particular to methods of preparation of antitumor ligands for use in personalized medicine (PM) in treatment of cancer patients.
DESCRIPTION OF THE RELATED ART
[0003] Personalized medicine (PM) is an emerging trend in medical practices that
proposes the customization of drugs based on the genetic profile of the patience. Since the genetic constitution of each patient varies from the other, the gene mutations occurring in each individual may also differ. Pharmacogenomics deals with the study of how an individual‟s genetic inheritance affects the body‟s response to drugs. This causes some patients to respond lesser or greater to a common drug used for certain diseases.
[0004] In this customized therapeutics, the diagnostic testing is used for selecting
appropriate therapies and drugs tailored to suit individual patients. Based on the patient factors such as genetics, age, gender, concurrent disease, concurrent drug therapy, environmental agents and drug factors such as pharmacokinetics, pharmacodynamics, adverse effects and drug interactions, physicians can determine the course and prescribe drug dosages required to minimize diseases with less adverse effects. This technique follows the „best fit drug‟ for an individual. The risk in generating the best fit drug can be simplified using computational techniques of modeling and simulation.
[0005] Single nucleotide polymorphism (SNPs) is accounted for almost 90% of all
genetic variations. Of all SNPs, rs1800053, rs9332969 and rs9332971 are the three most

deleterious SNPs responsible for the susceptibility of prostate cancer. All these SNPs are found to be thermodynamically stable as the respective mutations produce mutated protein molecules which are thermodynamically more stable (Exothermic) than the wild forms as shown in Table. 1 Aand 1B. Hence patients with the above SNPs as genetic signature are more prone to diseases caused by the specific gene mutation.
Table 1A: Thermodynamic Stability of Deleterious SNPs
I Allele I Residue I Variant I Mutated Energy (kJ/Mol)
| Change | Change | Position 1GS4 I 1Z95 I 2Q7K
Initial Energy -11860.25 -106405.51 -23691.88
rs1800053 GCT A [Ala] =˃ [646 -15568.83 -184040.44 -34760.34
=˃ GAT D [Asp]
rs9332969 CGT R [Arg] =˃ 841 -15490.54 -183678.56 -34775.46
=˃ CAT H [His]
rs9332971 CGC R [Arg] =˃ 856 -15295.85 -183469.52 -34741.92
| =˃ CAC | H [His] | | | |
Table 1B: Thermodynamic Stability of Deleterious SNPs
I Allele I Residue I Variant I Mutated Energy (kJ/Mol)
| Change | Change | Position 2YHD I 2YLP I 3L3X
Initial Energy -9837.372 10437.021 -12334.126
rs1800053 GCT A [Ala] =˃ [646 -9847.793 -10450.229 -12345.103
=˃ GAT D [Asp]
rs9332969 CGT R [Arg] =˃ 841 -9582.993 -10191.678 12031.017
=˃ CAT H [His]
rs9332971 CGC R [Arg] =˃ 856 -9374.969 -10146401 -11943.587
| =˃ CAC | H [His] | | | |
[0006] U.S patent 8859625 to Suarez et al., discloses synthetic methods of preparation
and purification of 1,5-bis(aryl)penta-1,4-dien-3-one derivatives for in-vivo and in-vitro biological applications such as anti-tumoral agents and anti-parasite agents, respectively.
[0007] PCT Patent application 2012175046 to Wang et al., describes preparation of
water-soluble platinum complexes for use as drugs in the prevention and treatment of tumors. European Patent application 2098507 to Hasuoka et al., relates to cyclic amine compound having a superior androgen receptor regulating action.

[0008] Greene et al., Journal of Neurochemistry, 1993 reports on the effect of malonic
acid on neurodegenerative disorders by inhibition of succinate dehydrogenase, an enzyme, central to both the tricarboxylic acid cycle and the electron transport chain which in turn blocks the NMDA antagonist MK-801.
[0009] An efficient process for preparing target compounds which utilizes non-
hazardous chemicals and facilitates easy synthesis and purification of substituted malonic acids is desired. Furthermore, a process which minimizes the cost of preparing the target compound is useful.
[0010] The present disclosure relates to design and development of potential ligands
suitable for treating patients with a specified genetic signature and also relates to processes of synthesis of potential ligands with improved yield efficiencies.

SUMMARY OF THE INVENTION
[0011] An improved process for the preparation of substituted malonic acids using
Meldrum‟s acid is provided. In various embodiments, sodium hydrate is not used in the process for the preparation of substituted malonic acids using Meldrum‟s acid.
[0012] In some embodiments, the process of preparing a malonic acid derivative
includes step a) reacting 4-nitrobenzyl bromide with Meldrum‟s acid to obtain an intermediate; and step b) hydrolyzing the intermediate in an acid medium to obtain a malonic acid derivative of formula I:

wherein R1 and R2 are independently selected from -H or -methyl and R3 is a -NO2.
[0013] In other embodiments, the process includes step c) esterifying the malonic acid
derivative. In some embodiments, step c) is performed in an ethanolic medium and the malonic acid derivative comprises -ethyl as R1, R2 or both. In some embodiments, the malonic acid derivative comprises -n-propyl or -iso-propyl as R1, R2or both.
[0014] In some embodiments, step a) is performed in the presence of potassium
carbonate in acetonitrile. In various embodiments, step a) is performed at room temperature. In certain embodiments, the acid medium comprises methonolic hydrogen chloride or aqueous hydrogen chloride. In different embodiments, the process further comprises substituting R3 with a -NH2 group to obtain the substituted malonic acids.

[0015] The present synthetic process of preparing malonic acid derivatives 1a-c
provides for a simple and efficient process. The use of sodium hydride which is flammable, highly reactive, powerful oxidising agent, and reacts violently with moisture causing explosion is excluded. Handling of such chemicals, which requires utmost care with difficulty is circumvented. The substituted malonic acids or derivatives thereof are used in anti-breast/-prostate cancer compositions and therapies.

BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The invention has other advantages and features which will be more readily
apparent from the following detailed description of the invention and the appended claims, when taken in conjunction with the accompanying drawings, in which:
[0017] FIG. 1 illustrates the structure of the designed molecules.
[0018] FIG. 2 illustrates the structure of Meldrum‟ s acid
[0019] FIG. 3 A illustrates the synthesis of molecules 1a-e by proposed pathway 1.
[0020] FIG. 3B illustrates the synthesis of molecules 1a-e by proposed pathway 2.
[0021] FIG. 4A illustrates the synthesis of an intermediate (I) from 4-nitrobenzyl
bromide and Meldrum‟s acid.
[0022] FIG. 4B illustrates the synthesis of compounds 1b and 1c by the improved
process.
[0023] FIG. 4C illustrates the synthesis of compound 1a by the improved process.
[0024] FIG. 5 A illustrates the retrosynthesis of molecules 2a-c by proposed pathway 1.
[0025] FIG. 5B illustrates the retrosynthesis of molecules 2a-c by proposed pathway 2.
[0026] FIG. 5C illustrates the retrosynthesis of molecules 2a-c by proposed pathway 3.
[0027] FIG. 6 illustrates the detailed synthesis of molecules 1a-e.
[0028] FIG. 7A illustrates the synthesis of half acids.
[0029] FIG. 7B illustrates the detailed synthesis of molecules 2a-c.

DETAILED DESCRIPTION
[0030] While the invention has been disclosed with reference to certain embodiments, it
will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope.
[0031] Throughout the specification and claims, the following terms take the meanings
explicitly associated herein unless the context clearly dictates otherwise. The meaning of "a", "an", and "the" include plural references. The meaning of "in" includes "in" and "on." Referring to the drawings, like numbers indicate like parts throughout the views. Additionally, a reference to the singular includes a reference to the plural unless otherwise stated or inconsistent with the disclosure herein.
[0032] The word “exemplary” is used herein to mean “serving as an example, instance,
or illustration.” Any implementation described herein as “exemplary” is not necessarily to be construed as advantageous over other implementations. The ingredients are indicated to be in weight % throughout this specification.
[0033] In various embodiments, the invention discloses novel processes for preparing a
ligand useful in treating an individual with prostate cancer having at least one genetic variation of rs1800053, rs9332969 or rs9332971 as already disclosed in 5417/CHE/2015. In some embodiments, the synthesized ligands are one or more of EVO15, EVO16, EVO17, EVO20, EVO21, EVO23, EVO24 and EVO25, as illustrated in FIG. 1 (henceforth named as 1a, 1b, 1c, 1d, 1e, 2a, 2b and 2c, respectively for reference).
[0034] In one embodiment a process for the preparation of a ligand of formula (I) is
disclosed. In the formula compound, R1 and R2 are independently selected from H or a lower alkyl group selected from methyl or ethyl, R3 is a substituent selected from –NH2 or –NO2.


[0035] In one embodiment of the disclosure, when R3 is NO2 the compound of formula
(I) is selected from the group of diethyl (4-nitrobenzyl)propanedioate [1a], dimethyl (4-nitrobenzyl)propanedioate [1b], (4-nitrobenzyl)propanedioic acid [1c], 3-ethoxy-2-(4-nitrobenzyl)-3-oxopropanoic acid [1d] and 3-methoxy-2-(4-nitrobenzyl)-3-oxopropanoic acid [1e].



[0036] In another embodiment of the disclosure, when R3 is NH2 the compound of
formula (I) is selected from the group of diethyl (4-aminobenzyl)propanedioate [2a], dimethyl (4-aminobenzyl)propanedioate [2b] and (4-aminobenzyl)propanedioic acid [2c].


[0037] In one other embodiment, a process of preparing a ligand of formula (I) is
disclosed. The process comprises reacting para-substituted benzyl halide of formula (II) with a dicarboxylic acid derivative of formula (III), wherein R1 and R2 are independently selected from H or a lower alkyl group selected from methyl or ethyl group; R3 is selected from -NH2 or -NO2 group; and X is a halide selected from the group consisting of bromide, fluoride, iodide, or chloride.
[0038] In one embodiment, when para-substituted benzyl halide of formula (II) is para-
nitrobenzyl bromide and dicarboxylic acid derivative of formula (III) is malonic acid the reaction yields ligands of formula (I) selected from the group of diethyl (4-nitrobenzyl) propanedioate [1a], dimethyl (4-nitrobenzyl)propanedioate [1b], (4-nitrobenzyl)propanedioic acid [1c], 3-ethoxy-2-(4-nitrobenzyl)-3-oxopropanoic acid [1d] and 3-methoxy-2-(4-nitrobenzyl)-3-oxopropanoic acid [1e].
[0039] In one other embodiment, when para-substituted benzyl halide of formula (II) is
para-aminobenzyl bromide and dicarboxylic acid derivative of formula (III) is malonic acid the reaction yields ligands of formula (I) selected from the group of diethyl (4-aminobenzyl)propanedioate [2a], dimethyl (4-aminobenzyl)propanedioate [2b] and (4-aminobenzyl)propanedioic acid [2c].
[0040] In one embodiment, Meldrum‟s acid which is represented by the formula (IV) is
used as one of the starting material. In certain embodiments, Meldrum‟s acid in the starting material undergoes nucleophilic attack at position 4 and 6, electrophilic attack (via the anion) at position 5, or both, as illustrated in FIG. 2.


[0041] In one embodiment, the process of preparing a malonic acid derivative includes
step a) reacting 4-nitrobenzyl bromide with Meldrum‟s acid to obtain an intermediate; and step b) hydrolyzing the intermediate in an acid medium to obtain a malonic acid derivative of formula I, wherein R1 and R2 are independently selected from –H or –methyl and R3 is a – NO2.
[0042] In other embodiments, the process includes step c) esterifying the malonic acid
derivative. In some embodiments, step c) is performed in an ethanolic medium and the malonic acid derivative comprises –ethyl as R1, R2 or both. In some embodiments, the malonic acid derivative comprises –n-propyl or –iso-propyl as R1, R2 or both.
[0043] In some embodiments, step a) is performed in the presence of potassium
carbonate in acetonitrile. In various embodiments, step a) is performed at room temperature. In certain embodiments, the acid medium comprises methonolic hydrogen chloride or aqueous hydrogen chloride. In different embodiments, the process further comprises substituting R3 with a –NH2 group to obtain the substituted malonic acids.
[0044] The present synthetic process of preparing malonic acid derivative compounds
1a-c excludes the use of sodium hydride, to make the route simpler. The use of sodium hydride which is flammable, highly reactive, powerful oxidizing agent, reacts violently with moisture causing explosion is excluded. Handling of such hazardous chemicals, which requires utmost care with difficulty is circumvented. The raw materials which are used in the preparation are cheaper and hence the improved process also provides for a low cost solution. The improved process allows for efficient synthesis and purification which is easily scalable for bulk manufacturing. The substituted malonic acids or derivatives thereof are used in anti-breast/-prostate cancer compositions and therapies.

EXAMPLES
[0045] Example 1
[0046] Retro Synthesis
[0047] Two pathways involving nucleophilic substitution reaction of two synthones
were used for the synthesis of molecules1a-e. FIG. 3A illustrates the synthesis of 1a-e by the reaction of p-nitrobenzyl bromide with malonic acid/acid derivatives (pathway 1). FIG. 3B illustrates the synthesis of 1a-c molecules by the reaction of nitrobenzene with 2-(bromomethyl) malonic acid/acid derivatives (pathway 2). However the product yields of pathway 1 was found to be greater than pathway 2 and hence pathway 1 was followed for the synthesis of 1a-e. Alternatively, an improved process is described for synthesis of molecules 1a-1c.
[0048] Improved process for synthesis
[0049] The intermediate (I) was obtained by the reaction of 4-nitrobenzyl bromide with
Meldrum‟s acid in the presence of potassium carbonate in acetonitrile medium at room
temperature, as illustrated in FIG. 4A.
[0050] The intermediate (I) thus obtained was subjected to hydrolysis in acid medium,
as illustrated in FIG. 4B. Dimethyl malonate derivative 1b was obtained when methonolic
hydrogen chloride was used, while diacid 1c was obtained when aqueous hydrogen chloride
was used. Simple esterification of 1c in ethanolic medium with trace of sulphuric acid yields
1a, as illustrated in FIG. 4C.
[0051] For the synthesis of 2a-c molecules three retrosynthesis pathways were
experimented. FIG. 5A illustrates the synthesis of 2a-e through a reduction reaction involving corresponding compounds 1a-c. FIG. 5B illustrates the second pathway involving nucleophilic substitution reaction of p-aminobenzyl bromide with malonic acid/acid derivatives. However the starting material p-aminobenzylbromide was found to be unstable under normal conditions due to polymer formation. FIG. 5C illustrates the third pathway involving nucleophilic substitution reaction of aniline with 2-(bromomethyl)malonic acid /acid derivatives. However the reaction involving aniline was found to be difficult to carry out. Hence due to the above mentioned difficulties oriented with pathway 2 and pathway 3,

pathway 1 was found to be most preferred for the chemical synthesis of molecules 2a-c. Thus after evaluating the possibilities of synthesis based on availability and cost of raw materials pathway 1 was carried out for synthesis of both the compounds 1a-e and 2a-c.
[0052] Synthesis of Molecules 1a-e
[0053] FIG. 6 illustrates the detailed synthesis of molecules 1a-e. To a suspension of
sodium hydride (60%, 0.38g, 137.5mmol) in dry DMF (8ml), malonic acid/acid derivative (125mmol) was slowly added portion wise with constant stirring at ice-cooling temperature under inert atmosphere. Formation of white foam indicated the reaction of malonic acid/acid derivative with sodium hydride. After complete addition of NaH, a solution of p-nitrobenzyl bromide (1g, 62.5mmol) in dry DMF (2ml) was added slowly to the reaction mixture. The color of the reaction mixture changed from white to yellowish brown. The resultant mixture was allowed to stir for 5-20 hours at 50-80oC.
[0054] The mixture was cooled to room temperature and monitored by TLC and was
quenched with ammonium chloride and extracted using ethyl acetate (4×50 ml). The yellow color organic layer was separated, washed with 100ml of brine (saturated sodium chloride solution) and distilled water (2×100ml) and dried over sodium sulphate. The solvent was removed using rotary evaporator under reduced pressure to get a thick yellow solution. The thick oil was kept at -50 C to get crude product as yellow color solid. The crude product was purified by column chromatography using a mixture of hexane and ethyl acetate as mobile phase. The synthesized compounds were characterized by means of 1H and 13C-NMR, mass analysis, IR studies and melting point.
[0055] Synthesis of Half Acid
[0056] For the synthesis of molecules 1d-e, the precursor half acids were prepared as
illustrated in FIG. 7A. In a 50ml RB flask equipped with a magnetic stirrer, malonic ester (1.2mol) was dissolved in acetonitrile (1.5ml). The reaction mixture was cooled to 0°C with an ice bath and 15ml of water was added followed by drop wise addition of 5M aqueous NaOH (1.2mol). The reaction mixture was stirred for one hour at 0 to 4°C.

[0057] The mixture was monitored by TLC using Hexane: Ethyl acetate in a ratio of 3:2,
and was acidified with 12M aqueous HCl solution in the ice bath. After saturation of the mixture, it was extracted using dichloromethane (3×50ml) in a 500ml separatory funnel. The extract was washed with 200ml of a saturated aqueous NaCl (Brine) solution, distilled water (2×50ml) and dried over anhydrous sodium sulfate. After the drying agent was separated by filtration, the organic solvent was removed from the mixture by using rotary evaporator under reduced pressure. The final product was obtained as light yellow color oil. The synthesized compounds were characterized by means of1H and 13C-NMR, mass analysis, IR studies and melting point
[0058] Synthesis of Molecules 2a-c
[0059] FIG. 7B illustrates the synthesis of molecules 2a-c. Ina 100ml conical flask, a
solution of 1a-c (1g, 3.74mmol) in ethanol (30ml) was prepared. To this solution, stannous chloride (SnCl2.2H2O) (8.43g, 10mmol) was added and kept under ultrasonic irradiation at 30°C until the reaction was complete as indicated by TLC analysis. After the completion of the reaction, the solution was poured into ice water. Saturated sodium bicarbonate solution was added to the above mixture till the pH of the solution become 8. Then the mixture was extracted with ethyl acetate (4×200ml) and the organic layer was separated. The separated layer was washed with brine (250ml), water (2×250ml) and dried over magnesium sulphate. The solvent was removed in reduced pressure using rotaevaporator. Purification was done by column chromatography using hexane and ethylacetate as solvent system. The synthesized compounds were characterized by means of1H and13C-NMR, mass analysis, IR studies and melting point.
[0060] Example 2
[0061] Synthesis of intermediate I:
[0062] The 5-benzyl substrate was prepared in single step by reacting Meldrum‟s acid
(0.1g, 4.6x10-4mol) with one equivalent of 4-nitrobenzyl bromide (0.07g, 4.6x10-4mol), using
K2CO3 (0.08g, 5.5x10-4mol) in acetonitrile (5 ml).The reaction was performed at temperature
ranging between 25°C to 35°C for ~18 hours.

[0063] Workup: After the completion of the reaction, the product was monitored by
TLC, and the solvent was distilled to dryness. The crude product was dissolved in ethyl acetate (50 ml) and washed with 10 ml of distilled water. The layers were separated; organic layer was dried over sodium sulphate and distilled under reduced pressure. The orange brown solid thus obtained was purified by recrystallization process.
[0064] Recrystallisation: To the crude product, ethanol was added drop wise till 50% of
the solid was dissolved. The resulting mixture was gently warmed to get a clear solution. The clear solution was kept undisturbed for three hours at room temperature and overnight at 0-5°C in a refrigerator. The solid obtained was centrifuged and supernatant liquid was separated. The product was characterized by GC-MS.
[0065] I. Synthesis of dimethyl ester1b:
[0066] To the intermediate I (0.1g, 3.6x10-4mol) in a single neck round bottom flask,
methanolic HCl (10 ml) was added and refluxed for a period of 3-4 hours. The acid was removed under low pressure; the yellow solid thus obtained was recrystallised using ethanol.
[0067] II. Synthesis of diacid 1c:
[0068] To the intermediate I (0.1g, 3.6x10-4mol) in a single neck round bottom flask,
1:1 HCl (10ml) was added and refluxed for a period of 3-4 hours. The acid was removed under low pressure; the yellow solid thus obtained was recrystallised using ethanol.
[0069] III. Synthesis of diethyl ester 1a:
[0070] To a solution of 1c (0.1g, 4.2x10-4mol) in ethanol (10 ml) in a single neck round
bottom flask, concentrated sulphuric acid was added and stirred at room temperature for overnight. After the completion of the reaction, the solvent was removed under low pressure. The resultant crude product was recrystallised using ethanol.
[0071] The end products dimethyl ester 1b, diethyl ester 1a and diacid 1c were
characterized by NMR, IR and MASS analyses. The results of these analyses exactly match with those molecule obtained by the other synthetic route which was filed for patenting earlier.

[0072] Cost comparison: The new process involves in cost effective synthesis of diester
and diacid when compared to the methodology adopted in the previous work (Indian Patent application No: 5417/CHE/2015), as illustrated in Table. 3. The usage of malonic acid was replaced with Meldrum‟s acid. Similarly NaH, which is a strong base, hazardous and difficult to handle was replaced by potassium carbonate or sodium carbonate.


WE CLAIM:
1. A process of preparing a malonic acid derivative, comprising the steps:
a) reacting 4-nitrobenzyl bromide with Meldrum‟s acid to obtain an intermediate; and
b) hydrolyzing the intermediate in an acid medium to obtain a malonic acid derivative of formula I:

wherein R1 and R2 are independently selected from -H or -methyl and R3 is a -NO2.
2. The process of claim 1, wherein step a) is performed in the presence of potassium
carbonate in acetonitrile.
3. The process of claim 2, wherein step a) is performed at room temperature.
4. The process of claim 1, wherein sodium hydride is not used in the process.
5. The process of claim 1, wherein said acid medium comprise methonolic hydrogen chloride.

6. The process of claim 1, wherein said acid medium comprise aqueous hydrogen chloride.
7. The process of claim 6, further comprising: c) esterifying the malonic acid derivative, and wherein R1, R2or both are –ethyl.
8. The process of claim 6, further comprising: c) esterifying the malonic acid derivative, and wherein R1, R2or both are n-propyl or iso-propyl.
9. The process of claim 1, further comprising: substituting R3 with a –NH2 group.