WO2013015661A2

WO2013015661A2 - Novel prodrugs of 5-(2,4-dihydroxy-5-isopropylphenyl)-n-ethyl-4-(5-methyl1-1,2,4-oxadiazol-3-yl)isoxazole-3-carboxamide

Info

Publication number: WO2013015661A2
Application number: PCT/KR2012/006043
Authority: WO
Inventors: Jae-Hoon Kang; Hong-Sub Lee; Jin-Sun Kwon; Joon-Tae Park; Jin-Ah Jeong; Sung-Wook Kwon
Original assignee: Ildong Pharm Co.,Ltd.
Priority date: 2011-07-28
Filing date: 2012-07-27
Publication date: 2013-01-31
Also published as: WO2013015661A3

Abstract

The present invention relates to the novel compound of Formula I. The compound of Formula I are used as a prodrug of the parent compound, Formula II, and have valuable pharmacological properties. The compound of Formula I have improved solubility by 1,000~20,000 times or more, and increased bioavailability compared to those of the compound of Formula II. Furthermore, they can be absorbed into the body with increased concentration by improved value of AUC_0-120minand C_max, more by 3~5 times and 10~20 times respectively compared to the compound of Formula II. Thus, those improved pharmacokinetic properties can be used very effectively to treat various diseases by inhibition of HSP90 activity, especially for ovarian and gastric cancer.

Description

NOVEL PRODRUGS OF 5-(2,4-DIHYDROXY-5-ISOPROPYLPHENYL)-N-ETHYL-4-(5-METHYL1-1,2,4-OXADIAZOL-3-YL)ISOXAZOLE-3-CARBOXAMIDE

This present invention relates to the compound of Formula I.

wherein,

both R₁ and R₂ are PO(OH)₂ or

, or wherein R₁ or R₂ is hydrogen and the other R₁ or R₂ is

, PO(OH)₂ or

, wherein M is Na⁺, K⁺, Mg²⁺, or Ca²⁺, and n is 1 or 2, wherein n is 1 and M is Mg²⁺ or Ca²⁺ , or wherein n is 2 and M is Na⁺or K⁺.

It has been found that the compound of Formula Ⅰ may be as a prodrug of the compound of Formula Ⅱ, 5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazole-3-carboxamide, which is HSP90 inhibitory activity, is stated in the following patent, WO 2011/102660.

A prodrug is in most cases a pharmacologically inactive derivative of a parent drug molecule that requires spontaneous or enzymatic transformation within the body in order to release the active drug, and that has improved delivery properties over the parent drug molecule. It has been shown that a molecule with optimal structural configuration and physicochemical properties for eliciting the desired therapeutic response at its target site does not necessarily possess the best molecular from and properties for its delivery to its point of ultimate action. Usually, only a minor fraction of doses administered reaches the target area and since most agents interact with non-target sites as well, an inefficient delivery may result in undesirable side effects. This fact of differences in transport and in situ effect characteristics for many drug molecules is the basic reason why bioreversible chemical derivatization of drugs, i.e., prodrug formation is a means by which a substantial improvement in the overall efficacy of drugs can often be achieved. Prodrugs are designed to overcome pharmaceutically and/or pharmacokinetically based problems associated with the parent drug molecule.

In recent years several types of bioreversible derivatives have been exploited for utilization in designing prodrugs. Using esters as a prodrug type for drugs containing carboxyl or hydroxyl functional group is most popular. Further well-known are prodrug derivatives of peptides, 4-imidazolidinones and the like, described in Drugs of the Future, 1991, 16(5), 443-458 or N-oxides, described for example in US 5.691.336.

On the other hand, molecular chaperones are a general term for proteins that form a complex temporally with client proteins to promote the formation of the conformation of the client proteins. These proteins, the activity of which is to help folding and association of protein and to prevent aggregation are broadly defined as molecular chaperones.

Exposure of cells to a number of environmental stresses, including heat shock, alcohol, heavy metals and oxidative stress, results in the cellular accumulation of a number of chaperones, commonly known as heat shock proteins (HSPs). Molecular chaperones of the "heat shock proteins" family (HSPs), classified according to their molecular mass (HSP70, HSP90, HSP27, etc.,), protect the cell against the initial stress, enhances recovery and leads to maintenance of a stress tolerant state. It has also become clear, however, that certain HSPs may also play a major molecular chaperone role under normal, stress-free conditions by regulating the correct folding, degradation, localization and function of a growing list of important cellular proteins.

Several diseases in humans can be acquired as a result of protein misfolding. In some conditions (e.g., Alzheimer's disease, prion diseases and Huntington's disease), misfolded proteins can cause protein aggregation resulting in neurodegenerative disorders ([Tytell M. and Hooper P.L., Emerging Ther. Targets (2001), 5, 3788-3796]). HSPs, and in particular HSP90, are also involved in the regulation of various major functions of the tumor cell, via their association with various client proteins involved in cell proliferation or apoptosis. In these pathologies, approaches aimed at breaking up or at disturbing the function of chaperones could be available for treatment of disease. Especially, HSP90 chaperons has recently been demonstrated as a particularly promising target in anticancer therapy ([Moloney A. and Workman P., Expert Opin. Biol. Ther. (2002), 2(1), 3-24]; [Choisis et al, Drug Discovery Today (2004), 9, 881-888]).

HSP90 (Heat Shock Protein 90) family proteins included HSP90α HSP90β GRP94 and HSP75/TRAP1. These proteins represent approximately 1-2% of the total cellular protein mass. It is usually in the form of a dimer in the cell and is associated with multiplicity of proteins, so-called co-chaperones. HSP90 plays a key role in the response to cellular stress by interaction with many proteins whose native folding has been modified by external stress, such as, for example, heat shock, in order to restore the original folding or to prevent aggregation of the proteins ([Smith D.F. et al., Pharmacological Rev. (1998), 50, 493-513]). There are also indications that HSP90 is of importance as buffer against the effects of mutations, presumably through correction of incorrect protein folding caused by the mutation ([Rutherford and Lindquist, 1998]). HSP90 also has a regulatory importance. Under physiological conditions, HSP90, together with its homologue in the endoplasmatic reticulum, GRP94, plays a role in the cell balance for ensuring the stability of the conformation and maturing of various client key proteins, such as, EGFR R/HER2, Src, Akt, Raf, MEK, Bcr-Abl, Flt-3, mutated p53, Akt, survivin, Cdk4, Plk, Wee1, VEGF-R, FAK, HIF-1, hTert and c-Met, etc. These client proteins are involved in the six mechanisms of tumour progression. i) An ability to proliferate in the absence of growth factor(EGFR-R/HER2, Src, Akt, Raf, MEK, Bcr-Abl, Flt-3, etc.,); ii) An ability to evade apoptosis (mutated form of p53, Akt, survivin, etc.,); iii) An insensitivity to proliferation stop signal(Cdk4, Plk, Wee1, etc.,); iv) An ability to activate angiogenesis (VEGF-R, FAK, HIF-1, Akt, etc.,); v) An ability to proliferate with no replicative limit (hTert, etc.,); vi) An ability to evade new tissue and to metastasize(c-Met);(Hanahan D. and Weinberg R.A., Cell (2002), 100, 57-70). Therefore, the client protein-induced tumor formation can be blocked by inhibition of HSP90 activity.

The first known HSP90 inhibitors are compounds of the ansamycin family, in particular geldanamycin and herbimycin A. X-ray studied have shown that geldanamycin binds to the ATP site of the N-terminal domain of HSP90, where it inhibits the ATPase activity of the chaperone (Prodromou C. et al, Cell (1997), 90, 65-75). Currently, the NIH and Kosan BioScience are carrying out the clinical development of 17AAG, which is a geldanamycin-derived HSP90 inhibitor.

Radicicol is also a Hsp90 inhibitor of natural origin ([Roe S.M. et al, J. Med Chem. (1999),42, 260-66]). However, although the latter is by far the best in vitro HSP90 inhibitor, its metabolic instability with respect to sulphur-containing nucleophiles makes it difficult to use in vivo. One Hsp90 inhibitor of natural origin, novobiocin, binds to a different ATP site located in the C-terminal domain of the protein ([Itoh H. et al, Biochem J. (1999), 343, 697-703]). Purines, such as the compound PU3 ([Chiosis et al, Chem. Biol. (2001),8, 289-299]), have also been described as small molecule Hsp90 inhibitors.

In addition, the analogues, such as 8-heteroaryl-6-phenylimidazo [1,2-a]pyrazines (WO 2004/072080), pyrazole derivatives (WO 2004/050087), isoxazole derivatives (WO 2004/07051) and benzophonone derivatives (WO 2005/00778) have also been described as HSP90 inhibitor, that are useful for the treatment of tumors.

The known HSP90 inhibitors involve binding to HSP90 at the ATP binding site located in the N-terminal domain of the protein, leading to inhibition of the intrinsic ATPase activity of HSP90. Inhibition of HSP90 ATPase activity prevents recruitment of co-chaperons, which these client proteins are targeted for degradation via the ubiquitin proteasome pathway. An attractive rationale for developing drugs against this target for use in the clinic is that by simultaneously depleting tumor and associated with the client proteins, one may obtain a strong antitumor effect and achieve a therapeutic advantage against cancer versus normal cells.

A cited compound in the present invention, the compound of Formula Ⅱ, 5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazole-3-carboxamide, having a HSP90 inhibitory activity, is stated in the following patent, WO 2011/102660.

The compound of Formula II has high activities in vitro, but it has low solubility in water. As a result, many problems like low bioavailability and pharmacokinetic parameter are caused for developing medicine.

However, it has been shown that the compound of Formula I of the present invention fulfill all requirements of good prodrug and have significantly improved the feature in solubility and pharmacokinetic property.

Accordingly, this present invention is designed to provide a novel prodrug of 5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazole-3-carboxamide, the compound of Formula Ⅱ, having to improve pharmacokinetic property

To solve the problem described above, the present invention provides the novel compound of Formula I.

wherein,

both R₁ and R₂ are PO(OH)₂ or

, or wherein R₁ or R₂ is hydrogen and the other R₁ or R₂ is

, PO(OH)₂ or

Particularly preferred examples of the compound of Formula I according to the present invention include the following.

4-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-phenylene bis(dihydrogen phosphate); (I-1)

(S)-2-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl 2,6-diaminohexanoate dihydrochloride; (I-2)

Sodium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-phenylene diphosphate; (I-3)

2-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl dihydrogen phosphate; (I-4)

Sodium 2-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl phosphate; (I-5)

4-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl dihydrogen phosphate; (I-6)

Sodium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl phosphate; (I-7)

Potassium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-phenylene diphosphate; (I-8)

Potassium 2-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl phosphate; (I-9)

Potassium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl phosphate; (I-10)

Calcium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-phenylene diphosphate; (I-11)

Calcium 2-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl phosphate; (I-12)

Calcium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl phosphate; (I-13)

Magnesium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-phenylene diphosphate; (I-14)

Magnesium 2-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl phosphate; (I-15)

Magnesium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl phosphate; (I-16)

In another aspect, the present invention provides a process for preparing a compound of Formula I.

A process for preparing a compound of Formula I is shown in the following scheme 1.

The compound of Formula I of the present invention, as shown in scheme 1, can be prepared by a series of steps from the compound of Formula Ⅱ, parent drug.

Each steps of the above preparation process is described in more detail as follows.

1) Preparing the compound of Formula Ⅱ

The compound of Formula Ⅱ used as a start substance can be prepared using a known preparation process(WO 2011/102660).

2) Preparing the compound of Formula I

First of all, the compound of Formula Ia can be prepared R₁ or R₂ is

by deprotection with HCl after synthesizing with intermediate compound, boc(tert-butoxy carbonyl) protected lysine by esterification the compound of Formula II with Boc-Lys(Boc)-OH in methylene chloride solvent.

wherein R₁ and R₂ are differently selected from each other hydrogen or

.

The reaction scheme as below shows an example according to the process for preparation as above.

Secondly, the compound of Formula Ib, wherein R₁ and/or R₂ are PO(OH)₂can be prepared by hydrolysis reaction with trifluoroacetic acid or HCl after synthesizing bis(tetramethylphosphorodiamidate)-intermediate compound by substitution reaction of the compound of Formula II with tetramethylphsphorodiamidic chloride in H₂O or dioxane solvent.

wherein R₁ and R₂ are both PO(OH)₂, or wherein R₁ and R₂are differently selected from each other hydrogen or PO(OH)₂.

Thirdly, the compound of Formula Ic as below wherein R₁ and/or R₂ are/is

, can be prepared by base reaction of the compound of Formula Ib as above with inorganic base, NaOH, KOH, Ca(OH)₂, or Mg(OH)₂. The compound of Formula II, the compound of salt as above, can be included in the compound of Formula I according to the present invention

wherein R₁ and R₂ are both

, or wherein R₁ and R₂ are differently selected from each other hydrogen or

, wherein n and M are defined in Formula I.

The reaction formula as below shows an example according to according to the process for preparation as above.

As mentioned earlier, the compound of Formula I may be used as a prodrug of the parent compound of Formula II, which process valuable pharmacological properties.

The compound was examined in accordance with the test given hereinafter. The evidence, that the compound of Formula I can be used as prodrugs of their parent compound of Formula II is shown in accordance with the description given hereinafter.

The compound of Formula I have improved solubility by 1,000~20,000 times or more, and increased bioavailability compared to the compound of Formula II. Furthermore, the compound of Formula I can be absorbed into the body with increased concentration by improved value of AUC_0-120minand C_max, more by 3~5 times and 10~20 times respectively compared to the compound of Formula II and can be improved pharmacokinetic properties.

The compound represented by the parent drug of Formula Ⅱ could be useful in the treatment of diseases which are responsive to inhibition of HSP90 activity such as immunosupression, Rheumatoid arthritis, Asthma, MS, Type I Diabetes, Lupus, Psoriasis, inflammatory Bowel Diseases, viral Diseases; diabetic retinopathy, hemangiomas, endometriosis; normal cells protection against chemotherapy-induced toxicity; protection from hypoxia-ischemic injury due to elevation of HSP70 in the heart and brain, scrapie/CJD, Huntingdon's and Alzhiemer's. Especially, it could be useful in the treatment of cancer.

Therefore, in this invention, the compound of Formula I, prodrug which involved variable functional group induced resorcinol derivatives, show effective anti-tumor activity in the many cancer cell line.

A pharmaceutical composition of the present invention for use in the treatment of cancer can be administered by any convenient passage; for example, oral, paranteral, oral cavity, hypoglossal, the nasal cavity, rectal, and hypodermic administration, and can be prepared by containing pharmaceutically acceptable excipient and the compound of Formula I.

The doses of pharmaceutical composition of the present invention may vary depending on the patient's weight, age, gender, physical condition, diet, the time and mode of administration, excretion rates, and the severity of illness. The doses of detailed drug composition may be administered in an effective amount ranging from 0.1 to 1000㎎ on adult.

The present invention provides the novel compound of Formula I. The compound of Formula I are used as a prodrug of the parent compound, Formula II, which process valuable pharmacological properties.

The compound of Formula I have improved solubility by 1,000~20,000 times or more, and increased bioavailability compared to those of the compound of Formula II. Furthermore, they can be absorbed into the body with increased concentration by improved value of AUC_0-120minand C_max, more by 3~5 times and 10~20 times respectively compared to the compound of Formula II. Thus, those improved pharmacokinetic properties can be used very effectively to treat various diseases by inhibition of HSP90 activity, especially for ovarian and gastric cancer.

The present invention will be described more particularly by the Examples but the present invention is not limited at all by these examples.

Example 1 4-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-phenylene bis(dihydrogen phosphate) (I-1)

Step 1: 4-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-phenylenebis(tetramethylphosphorodiamidate)

5-(2,4-Dihydroxy-5-isopropylphenyl)-N-ethyl-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazole-3-carboxamide(250㎎, 0.67mmol) was dissolved in methylene chloride(7㎖). Tetramethylphosphorodiamidic chloride(0.35㎖, 2.35mmol), 4-(dimethylamino)pyridine(41㎎, 0.34mmol) and 1,8-diazabicylco{5.4.0]undec-7-ene(0.25㎖, 1.68mmol) were added sequentially. The reaction mixture was stirred at RT for overnight. Saturated ammonium chloride was added to the mixture. The residue was extracted between methylene chloride and water. The organic phase was washed water, brine, dried with magnesium sulfate, and evaporated in vacuo. The residue was purified by silica gel column chromatography to afford the intermediate compound 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-phenylene bis(tetramethylphosphorodiamidate)(428㎎, 0.67mmol) in a yield 99%.

¹H-NMR (400 MHz, CDCl₃) δ 7.54 (s, 1H), 7.44 (s, 1H), 6.97 (br t, 1H), 3.49 (m, 2H), 3.27 (sept, 1H), 2.80 (s, 6H), 2.77 (s, 6H), 2.66 (s, 6H), 2.63 (s, 6H), 2.61 (s, 3H), 1.26 (t, 3H), 1.19 (d, 6H)

step 2: 4-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-penylene bis(dihydrogen phosphate)

The intermediate compound(Step 1)(428㎎, 0.67mmol) was dissolved in trifluoroacetic acid(1.8㎖)/H₂O(0.2㎖). The reaction mixture was stirred at RT for overnight. Solvents were removed in vacuo, and methanol was added to the mixture. The mixture was filtered through a pad of Celite, and the filtrate was concentrated. The residue was diluted by water and washed by ethyl acetate. The aqueous phase was evaporated in vacuo. The residue was purified by Dowex 50WX4 cation exchange resin to afford the title compound (250㎎, 0.47mmol) in a yield 70%.

¹H-NMR (400 MHz, D₂O) δ 7.49 (s, 1H), 7.22 (s, 1H), 3.35 (q, 2H), 3.24 (sept, 1H), 2.50 (s, 3H), 1.14 (t, 3H), 1.11 (d, 6H)

Example 2 (S)-2-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl2,6-diaminohexanoate dihydrochloride (I-2)

Step 1:(S)-2-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl2,6- bis((tertbutoxycarbonyl)amino)hexanoate (I-9)

5-(2,4-Dihydroxy-5-isopropylphenyl)-N-ethyl-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazole-3-carboxamide(138㎎, 0.37mmol) was dissolved in DMF(3.0㎖). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide(248㎎, 1.29mmol), 4-(dimethylamino)pyridine(22㎎, 0.19mmol) and Boc-Lys(Boc)-OH(384㎎, 1.11mmol) were added sequentially. The reaction mixture was stirred at RT for 12h. The residue was evaporated in vacuo and purified by silica gel column chromatography to afford the intermediate compound (S)-2-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl 2,6-bis((tert-butoxycarbonyl)amino)hexanoate(200㎎, 0.19mmol) in a yield 53%.

¹H-NMR (400 MHz, CDCl₃) δ 7.65 (s, 1H), 7.43 (s, 1H), 5.21 (br, 2H), 4.55 (br, 2H), 4.40 (br, 2H), 3.48 (m, 2H), 3.18-3.06 (m, 3H), 2.67 (s, 3H), 1.98 (br, 2H), 1.84 (br, 1H), 1.48 (m, 18H), 1.28-1.23 (m, 9H)

Step 2: (S)-2-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl2,6-diaminohexanoate dihydrochloride (I-10)

To a solution of the intermediate compound(Step 1)(50㎎, 0.05mmol) in CH₃CN(2㎖) was added 4.0M HCl in dioxane(0.4㎖, 1.46mmol). The reaction mixture was stirred at RT for 12h. The reaction mixture was evaporated in vacuo to afford the title compound(28㎎, 0.05mmol) in a yield 99%.

¹H-NMR (400 MHz, CDCl₃) δ 7.85 (s, 1H), 7.58 (s, 1H), 4.55 (m, 1H), 4.31 (m, 1H), 3.44 (m, 2H), 3.41 (m, 3H), 3.13 (m, 1H), 3.04-2.95 (m, 5H), 2.61 (s, 3H), 1.82 (m, 2H), 1.73 (m, 1H), 1.28-1.21 (m, 9H)

Example 3 Sodium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-phenylene diphosphate (I-3)

4-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl dihydrogen phosphate(4.90g, 9.20mmol) was dissolved in H₂O(350㎖). 1N NaOH(36.59㎖, 36.59mmol) was added. The reaction mixture was stirred at RT for 30 min, and evaporated in vacuo to afford the title compound(5.63g, 9.08mmol) in a yield of 99%.

¹H-NMR (400 MHz, D₂O) δ 7.40 (s, 1H), 7.33 (s, 1H), 3.46 (q, 2H), 3.38 (m, 1H), 2.63 (s, 3H), 1.26 (t, 3H), 1.16 (d, 6H)

Example 4 2-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl dihydrogen phosphate (I-4)

Step 1: 5-(4-(Ethoxymethoxy)-2-hydroxy-5-isopropylphenyl)-N-ethyl-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazole-3-carboxamide

To a solution of 5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazole-3-carboxamide(5.0g, 13.4mmol) in acetone(300㎖) cooled to 0℃ was added triethylamine(2.25㎖, 16.1mmol) and ethoxymethyl chloride(1.31㎖, 14.1mmol) sequentially, and the reaction mixture was stirred at RT for overnight. Solvent was evaporated in vacuo, and the residue was extracted between ethyl acetate and brine. The organic phase was dried with anhydrous magnesium sulfate, and evaporated in vacuo. The residue was purified by silica gel column chromatography to afford the intermediate compound 5-(4-(ethoxymethoxy)-2-hydroxy-5-isopropylphenyl)-N-ethyl-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazole-3-carboxamide(1.40g, 3.26mmol) in a yield of 24.3%.

¹H-NMR (400 MHz, CDCl₃) δ 7.44 (s, 1H), 6.99(brt, 1H), 6.67 (s, 1H), 5.87 (s, 1H), 4.90 (s, 2H), 3.54-3.45 (m, 4H), 3.10 (sept, 1H), 2.60 (s, 3H), 1.38-1.21 (m, 12H).

Step 2: 5-(Ethoxymethoxy)-2-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-4-isopropylphenyltetramethyl phosphorodiamidate

The intermediate compound(Step 1)(1.40g, 3.26mmol) was dissolved in methylene chloride(40㎖). 4-(dimethylamino)pyridine(199㎎, 1.63mmol), 1,8-diazabicyclo[5.4.0]-undec-7-ene(0.56㎖, 3.91mmol) and tetramethylphosphorodiamidic chloride(0.56㎖, 3.91mmol) were added sequentially. The reaction mixture was stirred at RT for overnight. And the residue was extracted by methylene chloride after the reaction was completed by saturated ammonium chloride. The organic phase was washed by brine, was dried with magnesium sulfate, and was evaporated in vacuo. After concentrated with decompression, the residue was purified by silica gel column chromatography to afford the intermediate compound 5-(ethoxymethoxy)-2-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-4-isopropylphenyltetramethyl phosphorodiamidate(1.82g, 3.23mmol) in a yield of 99%.

¹H-NMR (400 MHz, CDCl₃) δ 7.52 (s, 1H), 7.23 (s, 1H), 6.98 (brt, 1H), 4.93 (s, 2H), 3.52-3.43 (m, 4H), 3.26 (sept, 1H), 2.77 (s, 6H), 2.74 (s, 6H), 2.60 (s, 3H), 1.27-1.22 (m, 9H), 1.18(t, 3H)

Step 3: 2-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl dihydrogen phosphate

The intermediate compound (Step 2)(1.82g, 3.23mmol) was dissolved in 1,4-dioxane(100㎖) was added to 12N HCl(100㎖). The reaction mixture was stirred at RT for 3h, and evaporated in vacuo. After the solvent and HCl were removed, the residue was purified by reversed phase chromatography to afford the title compound(1.10g, 2.42mmol)in a yield of 75%.

¹H-NMR (400 MHz, D₂O) δ 7.33 (s, 1H), 6.79 (s, 1H), 3.30 (q, 2H), 3.07 (sept, 1H), 2.47 (s, 3H), 1.10 (t, 3H), 1.04(d, 6H)

Example 5 Sodium 2-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl phosphate (I-5)

2-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl dihydrogen phosphate(60mg, 0.13mmol) was dissolved in H₂O(4.8㎖). 1N NaOH(0.26㎖, 0.26mmol) was added. The reaction mixture was stirred at RT for 30 min, and evaporated in vacuo. The solvent was concentrated by decompression and was removed to afford the title compound(66㎎, 0.13mmol) in a yield of 99%.

¹H-NMR (400 MHz, D₂O) δ 7.22 (s, 1H), 6.96 (s, 1H), 3.33 (q, 2H), 3.06 (sept, 1H), 2.49 (s, 3H), 1.12 (t, 3H), 1.02 (d, 6H)

Example 6 4-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl dihydrogen phosphate (I-6)

Step 1: 5-(2-(Ethoxymethoxy)-4-hydroxy-5-isopropylphenyl)-N-ethyl-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazole-3-carboxamide

To a solution of 5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazole-3-carboxamide(5.0g, 13.4mmol) in acetone(300㎖) cooled to 0℃ was added Cs₂CO₃(5.25g, 16.1mmol), and ethoxymethyl chloride(1.31㎖, 14.1mmol) sequentially. The reaction mixture was stirred at RT for overnight. Solvent was evaporated in vacuo, and the residue was extracted between ethyl acetate and brine. The organic phase was dried with magnesium sulfate, and evaporated in vacuo. The residue was purified by silica gel column chromatography to afford the intermediate compound 5-(2-(ethoxymethoxy)-4-hydroxy-5-isopropylphenyl)-N-ethyl-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazole-3-carboxamide(3.02g, 7.01mmol) in a yield of 52.3%.

¹H-NMR (400 MHz, CDCl₃) δ 7.42 (s, 1H), 6.64 (s, 1H), 5.85 (s, 1H), 4.89 (s, 2H), 3.52~3.44 (m, 4H), 3.15~3.09 (m, 1H), 2.60 (s, 3H), 1.64 (s, 3H), 1.35~1.05 (m, 16H), 0.90~0.82 (m, 2H).

Step 2: 5-(Ethoxymethoxy)-4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-2-isopropylphenyltetramethyl phosphorodiamidate

The intermediate compound(Step 1)(3.02g, 7.01mmol) was dissolved in methylene chloride(80㎖). 4-(dimethylamino)pyridine(428㎎, 3.50mmol), 1,8-diazabicyclo[5.4.0]-unde-7-sen(1.21㎖, 8.41mmol) and tetramethylphosphorodiamidic chloride(1.21㎖, 8.41mmol) were added sequentially. The reaction mixture was stirred at RT for overnight. And the residue was extracted between methylene chloride, was saturated ammonium chloride and was washed by brine. The organic phase was dried with magnesium sulfate, and evaporated in vacuo. After concentrated by decompression, the residue was purified by silica gel column chromatography to afford the intermediate compound 5-(ethoxymethoxy)-4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-2-isopropylphenyltetramethyl phosphorodiamidate(3.95g, 6.99mmol) in a yield of 99%.

¹H-NMR (400 MHz, CDCl₃) δ 7.50 (s, 1H), 7.24 (s, 1H), 6.96 (brt, 1H), 4.95 (s, 2H), 3.52-3.44 (m, 4H), 3.26 (sept, 1H), 2.78 (s, 6H), 2.74 (s, 6H), 2.60 (s, 3H), 1.27-1.22 (m, 9H), 1.17(t, 3H)

Step 3: 4-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl dihydrogen phosphate

The intermediate compound(Step 2)(3.95g, 6.99mmol) was dissolved in 1,4-dioxane(200㎖), 12N HCl(200㎖) was added. The reaction mixture was stirred at RT for 3h, and evaporated in vacuo. After concentrated by decompression, the residue was purified by reversed phase chromatography to afford the intermediate compound(2.53g, 5.59mmol) in a yield of 80%.

¹H-NMR (400 MHz, D₂O) δ 7.30 (s, 1H), 6.76 (s, 1H), 3.26 (q, 2H), 3.08 (sept, 1H), 2.46 (s, 3H), 1.08 (t, 3H), 1.00(d, 6H)

Example 7 Sodium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl phosphate (I-7)

4-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl dihydrogen phosphate(177mg, 0.39mmol) was dissolved in H₂O(3.91㎖), and 1N NaOH(0.78㎖, 0.78mmol) was added. The reaction mixture was stirred at RT for 30 min, and evaporated in vacuo. After concentrated by decompression, the residue was dissolved in H₂O and was lyophilized to afford the title compound(196㎎, 0.39mmol) in a yield of 99%.

¹H-NMR (400 MHz, D₂O) δ 7.27 (s, 1H), 6.87 (s, 1H), 3.27 (q, 2H), 3.14 (sept, 1H), 2.47 (s, 3H), 1.07 (t, 3H), 1.01(d, 6H)

Example 8 Potassium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-phenylene diphosphate (I-8)

4-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl dihydrogen phosphate(1.00g, 1.87mmol) was dissolved in H₂O(70㎖), and 1N KOH(36.59㎖, 36.59mmol) was added. The reaction mixture was stirred at RT for 30 min, and evaporated in vacuo. It was concentrated to afford the title compound(1.11g, 1.63mmol) in a yield of 87%.

Example 9 Potassium 2-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl phosphate (I-5)

2-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl dihydrogen phosphate(60mg, 0.13mmol) was dissolved in H₂O(4.8㎖), and 1N KOH(0.26㎖, 0.26mmol) was added. After concentrated by decompression, the reaction mixture was stirred at RT for 30 min, and evaporated in vacuo to afford the title compound was freeze dried in vacuo to afford the title compound(64㎎, 0.12mmol) in a yield of 94%.

Example 10 Potassium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl phosphate (I-10)

4-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl dihydrogen phosphate(150㎎, 0.33mmol) was dissolved in H₂O(3.31㎖), and 1N KOH(0.66㎖, 0.66mmol) was added. The reaction mixture was stirred at RT for 30 min, and evaporated in vacuo. After concentrated by decompression, the solvent was removed. The residue was dissolved in H₂O and was freeze dried to afford the title compound (162㎎, 0.30mmol) in a yield of 93%.

Example 11 Calcium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-phenylene diphosphate (I-11)

4-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl dihydrogen phosphate(1.00g, 1.87mmol) was dissolved in H₂O(70㎖) and 1N Ca(OH)₂(3.73㎖, 3.73mmol) was added. The reaction mixture was stirred at RT for 30 min, and evaporated in vacuo. The reaction mixture was concentrated by decompression to afford the title compound(1.01g, 1.66mmol) in a yield of 89%.

Example 12 Calcium 2-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl phosphate (I-12)

2-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl dihydrogen phosphate(60mg, 0.13mmol) was dissolved in H₂O(4.8㎖), and 1N Ca(OH)₂(0.13㎖, 0.13mmol) was added. The reaction mixture was stirred at RT for 30 min, and evaporated in vacuo. After concentrated by decompression, the solvent was removed to afford the title compound(52㎎, 0.11mmol) in a yield of 82%.

Example 13 Calcium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl phosphate (I-13)

4-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl dihydrogen phosphate(100㎎, 0.22mmol) was dissolved in H₂O(2.2㎖). 1N Ca(OH)₂(0.22㎖, 0.22mmol) was added. The reaction mixture was stirred at RT for 30 min, and evaporated in vacuo. After concentrated by decompression, the solvent was removed. The residue was dissolved in H₂O and was free dried to afford the title compound(96㎎, 0.19mmol) in a yield of 89%.

Example 14 Magnesium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-phenylene diphosphate (I-14)

4-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl dihydrogen phosphate(100㎎, 0.187mmol) was dissolved in H₂O(7㎖). 1N Mg(OH)₂(0.37㎖, 0.37mmol) was added. The reaction mixture was stirred at RT for 30 min, and evaporated in vacuo. It was concentrated by decompression to afford the title compound(79.8㎎, 0.14mmol) in a yield of 74%.

Example 15 Magnesium 2-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl phosphate (I-15)

2-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl dihydrogen phosphate(40㎎, 0.087mmol) was dissolved in H₂O(3.2㎖). 1N Mg(OH)₂(0.087㎖, 0.087mmol) was added. The reaction mixture was stirred at RT for 30 min, and evaporated in vacuo. After concentrated by decompression, the solvent was removed to afford the title compound(28.5㎎, 0.06mmol) in a yield of 82%.

Example 16 Magnesium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl phosphate (I-16)

4-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl dihydrogen phosphate(62㎎, 0.14mmol) was dissolved in H₂O(1.36㎖), and 1N Mg(OH)₂(0.14㎖, 0.14mmol) was added. The reaction mixture was stirred at RT for 30 min, and evaporated in vacuo. After concentrated by decompression, the solvent was removed. The residue was dissolved in H₂O and was freeze dried to afford the title compound (42㎎, 0.11mmol) in a yield of 81%.

<EXPERIMENT 1> Solubility test

The solubility of the compound of Formula Ⅰ was examined to identify whether or not its solubility was superior to the compound of Formula Ⅱ(parent compound). The results are given as shown in Table 1.

The compound of Formula Ⅰ and the compound of Formula Ⅱ(parent compound) were dissolved respectively in deionized water. The solution was vigorously stirred for 30 seconds at 20±5℃ every 5 minutes and the solubility tests were performed within 30 minutes.

Table 1

As shown in table 1, more than 10,000㎖ of water was required to dissolve the compound of Formula Ⅱ(parent compound). According to The Korean Pharmacopoeia, the compound of Formula Ⅱ can be a practically insoluble compound. The present invention relates to the compound of Formula Ⅰ which shows significant improvement in solubility because the solubility of the compound of Formula I was 1,000 to 20,000 times higher than that of the compound of Formula Ⅱ(parent compound).

Therefore, modified physicochemical characteristics of the compound of Formula Ⅰ may have positive effects on both developing formulations for a drug candidate and increasing bioavailability, due to its enhanced solubility.

<EXPERIMENT 2> Mouse Pharmacokinetics

Pharmacokinetic studies were performed to determine whether the compound of Formula Ⅰcould be converted to the compound of Formula Ⅱ(parent compound) in vivo and efficacious plasma concentration of the compound of Formula Ⅱ(parent compound) could be obtained by administrating the compound of Formula Ⅰ. Oral single dose(10㎖/㎏) of the compound of Formula Ⅰ was administered to male BALB/C mice. Blood was collected in tubes coated with lithium heparin at 10, 20, 30, 60, and 120 minutes. After centrifugation, plasma was harvested and kept frozen at -20℃.

Liquid-liquid extraction with ethyl acetate was used for plasma sample preparation. LC-MS/MS analysis was conducted to analyze the compound of Formula Ⅰ and Formula Ⅱ(parent compound). Pharmacokinetic parameters were determined by noncompartmental analysis(WinNonLin®), and PK results are given as shown in Table 2.

Table 2

As shown in table 2, AUC_0-120min(Area under the plasma concentration-time curve) and C_maxof the compound of Formula Ⅱ(parent compound) were 33,529 ng·min·㎖ ^-1 and 420 ng/㎖, respectively. AUC_0-120minof the compound of Formula Ⅰ was 3 to 5 times higher than that of the compound of Formula Ⅱ as compared to its parent compound on a molecular weight and doses. Furthermore, the compound of Formula Ⅰ was absorbed more rapidly than its parent compound because C_maxof the compound of Formula Ⅰ was 10 to 20 times higher than that of the compound of Formula Ⅱ(parent compound) as compared to its parent drug on a molecular weight and doses.

Therefore, the present invention relates to the compound of Formula Ⅰ which displays much more improved PK profile than the compound of Formula Ⅱ( parent compound).

<EXPERIMENT 3> In vivo anticancer activity of compound

To determine the in vivo antitumor efficacy, the compound of Formula I was performed as follows.

A2780 Human ovarian cancer cells were established as subcutaneous xenografts by injection of 8×10⁶ cells into the flanks of adult female Balb/c nude mice. Mice with established tumors (50-200 mm³) were selected for study (n=3~6/treatment group). The compound of Formula I (I-1, I-2, I-3, I-7) were formulated in deionized water and administered orally (po) at a dose of 200㎎/㎏. The vehicle alone was administered to control groups. Animals were dosed 5 days per week (Monday through Friday) for 2 consecutive weeks.

Animals were weighed and tumor size was determined twice weekly by digital caliper measurements, and tumor volumes were calculated(volume = [length×width²]/2(mm³), where length and width refer to the larger and smaller dimensions collected at each measurement). The mean tumor volumes of each group were calculated. The change in mean treated tumor volume was divided by the change in mean control tumor volume, multiplied by 100 and subtracted from 100% to give the tumor growth inhibition for each group. (Table 3)

Tumor growth inhibition (TGI) was calculated as follows:

TGI(%) = (1-DT/DC) x 100

wherein DT and DC represent the mean tumor volume changes in treatment and control groups, respectively.

Table 3

As shown in table 3, the compound of Formula Ⅰ significantly inhibited tumor growth in vivo.

According to experiment 1, 2, and 3, the novel compound of Formula Ⅰexhibits more improved solubility by approximately 1,000 to 20,000 times compared to the compound of Formula Ⅱ(parent compound). Moreover, the compound of Formula Ⅰ has more favorable PK profile. As compared to its parent drug on a molecular weight and doses, AUC_0-120minand C_maxof the compound of Formula Ⅰ were 3 to 5 times and 10 to 20 times higher than those of the compound of Formula Ⅱ, respectively. Therefore, the improved PK profile of the compound of Formula Ⅰ can be useful in developing a new anticancer drug candidate.

Claims

A compound of Formula I.

<Formula Ⅰ>

wherein

both R₁ and R₂ are PO(OH)₂ or
, or wherein R₁ or R₂ is hydrogen and the other R₁ or R₂ is
, PO(OH)₂ or
, wherein M is Na⁺, K⁺, Mg²⁺, or Ca²⁺, and n is 1 or 2, wherein n is 1 and M is Mg²⁺ or Ca²⁺ , or wherein n is 2 and M is Na⁺or K⁺.
The compound of Formula I according to claim 1, wherein R₁ and R₂ are differently selected from each other hydrogen or
.
The compound of Formula I according to claim 1, wherein R₁ and R₂ are both PO(OH)₂, or wherein R₁ and R₂are differently selected from each other hydrogen or PO(OH)₂.
The compound of Formula I according to claim 1, wherein R₁ and R₂ are both
, or wherein R₁ and R₂ are differently selected from each other hydrogen or
, wherein n and M are defined in claim 1.
The compound of Formula I according to claim 1, which is selected from the group consisting of the following compounds:

4-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-phenylene bis(dihydrogen phosphate),

(S)-2-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl 2,6-diaminohexanoate dihydrochloride,

Sodium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-phenylene diphosphate,

2-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl dihydrogen phosphate,

Sodium 2-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl phosphate,

4-(3-(Ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl dihydrogen phosphate,

Sodium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl phosphate,

Potassium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-phenylene diphosphate,

Potassium 2-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl phosphate,

Potassium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl phosphate,

Calcium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-phenylene diphosphate,

Calcium 2-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl phosphate,

Calcium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl phosphate,

Magnesium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-6-isopropyl-1,3-phenylene diphosphate,

Magnesium 2-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-4-isopropylphenyl phosphate, or

Magnesium 4-(3-(ethylcarbamoyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)isoxazol-5-yl)-5-hydroxy-2-isopropylphenyl phosphate.
A pharmaceutical composition for use in the treatment of tumor, comprising pharmaceutically acceptable excipient and a compound of Formula I in any one of claims 1-5.
The use of a compound of Formula I in any one of claims 1-5 for the treatment of tumor.
The use of a compound of Formula I in any one of claims 1-5 as prodrug for a parent compound of Formula II.

<Formula Ⅱ>
A process for preparing a compound of Formula I as defined in claim 2, which process comprises deprotection of the obtained intermediate compound with HCl after synthesizing it with boc(tert-butoxy carbonyl) protected lysine by esterification the compound of Formula II as below with Boc-Lys(Boc)-OH in methylene chloride solvent.

<Formula Ⅱ>
A process for preparing a compound of Formula I as defined in claim 3, which process comprises hydrolyzing the obtained bis(tetramethylphosphorodiamidate) intermediate compound with trifluoroacetic acid or HCl after synthesizing it by substitution reaction of the compound of Formula II as below with tetramethylphosphorodiamidic chloride in H₂O or dioxane solvent

<Formula Ⅱ>
A process for preparing a compound of Formula I as defined in claim 4, which process comprises base reaction of compound of Formula Ib as below with an inorganic base selected from NaOH, KOH, Ca(OH)₂ or Mg(OH)₂

<Formula Ib>

,wherein R₁ and R₂ are both PO(OH)₂ ,or wherein R₁ and R₂ are differently selected from each other hydrogen or PO(OH)₂ .