Methotrexate derivatives and uses thereof

Abstract

A pharmaceutical compound is provided. The pharmaceutical compound includes a first component as methrotrexate and a second component as cholesterol, wherein the first component is covalently conjugated to the second component having a structure represented by the following chemical formula (I): ##STR00001##
wherein α carboxyl group of methrotrexate is conjugated to cholesterol using spermidine as a linker.

Claims

1. A pharmaceutical compound comprising a first component as methotrexate and a second component as cholesterol, wherein the first component is covalently conjugated to the second component having a structure represented by the following chemical formula (I): ##STR00004## wherein the double lines indicate the bond that connects the first part of formula (I) to the second part, wherein α carboxyl group of methotrexate is conjugated to cholesterol using spermidine as a linker, and a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition comprising a compound as defined in claim 1 and a pharmaceutically acceptable carrier, excipient and/or adjuvant.

3. Method of treating an indication treatable with methotrexate by administering the compound as defined in claim 1.

4. Method of treating an indication treatable with methotrexate by administering the composition as defined in claim 2.

5. Method of treating a cancer comprising cancer cells or an autoimmune disease by administering the compound as defined in claim 1.

6. The method of treatment according to claim 5, wherein the cancer is breast cancer, lung cancer, multiple myeloma, malignant melanoma, malignant glioma, lymphoma, leukemia, or other hematologic malignancy.

7. The method of treatment according to claim 5, wherein the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus (SLE), psoriasis, pemphigus, pemphigoid, sarcoidosis, or vitiligo.

8. Method of treating a cancer comprising cancer cells or an autoimmune disease by administering the compound as defined in claim 2.

9. The method of treatment according to claim 8, wherein the cancer is breast cancer, lung cancer, multiple myeloma, malignant melanoma, malignant glioma, lymphoma, leukemia, or other hematologic malignancy.

10. The method of treatment according to claim 9, wherein the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus (SLE), psoriasis, pemphigus, pemphigoid, sarcoidosis, or vitiligo.

11. The method of treatment according to claim 3, wherein the administering is to a mammal.

12. The method of treatment according to claim 4, wherein the administering is to a mammal.

13. The method of treatment according to claim 5, wherein the administering is to a mammal.

14. The method of treatment according to claim 6, wherein the administering is to a mammal.

15. The method of treatment according to claim 7, wherein the administering is to a mammal.

16. The method of treatment according to claim 8, wherein the administering is to a mammal.

17. The method of treatment according to claim 9, wherein the administering is to a mammal.

18. The method of treatment according to claim 10, wherein the administering is to a mammal.

19. The method of treatment according to claim 11, wherein the mammal is a human.

20. The method of treatment according to claim 13, wherein the mammal is a human.

Description

BRIEF DESCRIPTIONS OF THE DRAWINGS

(1) FIG. 1 shows the chemical structure and 1H NMR of a disclosed compound, (4S)-4-{[3-({4-[({[(1R,3aS,3bS,7S,9aR,9bS,11aR)-9a,11a-dimethyl-1-[(2R)-6-methylheptan-2-yl]-1H,2H,3H,3aH,3bH,4H,6H,7H,8H,9H,9aH,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-7-yl]oxy}carbonyl)amino]butyl}amino)propyl]carbamoyl}-4-[(4-{[(2,4-diaminopteridin-6-yl)methyl](methyl)amino}phenyl)formamido]butanoic acid, according to one embodiment of the disclosure.

(2) FIG. 2 shows inhibitory effect on dihydrofolate reductase enzyme of a compound according to one embodiment of the disclosure (compound A).

(3) FIG. 3 shows cytotoxicity of MTX (3A.) and a compound according to one embodiment of the disclosure (3B.).

(4) FIG. 4 shows an image of embodied compound in O/W droplets, visualized under light microscopy.

DETAILED DESCRIPTION OF THE INVENTION

(5) Various aspects of the present invention are described in detail in the following, each attached as an individual Example. The Examples are provided for the purpose of illustration and are not intended to limit the scope of the present invention.

(6) This invention relates to molecule and methods of treating diseases and disorders utilizing a Compound: (4S)-4-{[3-({4-[({[(1R,3aS,3bS,7S,9aR,9bS,11aR)-9a,11a-dimethyl-1-[(2R)-6-methylheptan-2-yl]-1H,2H,3H,3aH,3bH,4H,6H,7H,8H,9H,9aH,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-7-yl]oxy}carbonyl)amino]butyl}amino)propyl]carbamoyl}-4-[(4-{[(2,4-diaminopteridin-6-yl)methyl](methyl)amino}phenyl)formamido]butanoic acid and pharmaceutically acceptable salts, hydrates, solvates, clathrates, prodrugs and polymorphs thereof.

(7) The present invention encompasses the in vitro and in vivo use of the said compound and the incorporation of the said compound into pharmaceutical compositions for the treatments and preventions of a variety of diseases and disorders. Such treatments include, particularly, the inhibition of tumor cell proliferation, the treatment or prevention of cancer, including, but not limited to, solid tumors, blood-born tumors, leukemias, and in particular, acute lymphoblastic leukaemia (ALL).

(8) The present invention encompasses the in vitro and in vivo use of the said compound and the incorporation of the said compound into pharmaceutical compositions for the treatments and preventions of a variety of diseases and disorders. Such treatments include, particularly, the inhibition of an inflammation in autoimmune disease, including, but not limited to, rheumatoid arthritis, systemic lupus erythematosus (SLE), psoriasis, pemphigus, pemphigoid, sarcoidosis, vitiligo.

(9) The pharmaceutical composition comprising the disclosed compound is adjunctively administered with at least one additional therapeutic agent. Examples of additional therapeutic agents include, but are not limited to, anti-cancer drugs, anti-inflammatories, antihistamines, and decongestants.

(10) According to one aspect of the disclosure, a pharmaceutical compound comprising a first component as methotrexate and a second component as cholesterol is disclosed, wherein the first component is covalently conjugated to the second component having a structure represented by the following chemical formula (I):

(11) ##STR00003##
wherein the α carboxyl group of methrotrexate is conjugated to cholesterol using spermidine as a linker.

(12) In some embodiments, a pharmaceutical compound comprising a compound as defined above and a pharmaceutically acceptable carrier, excipient and/or adjuvant is provided.

(13) In one exemplification, the synthesis of the disclosed compound which is a methrotrexate-spermidine-cholesterol conjugate, (4S)-4-{[3-({4-[({[(1R,3aS,3bS,7S,9aR,9bS,11aR)-9a,11a-dimethyl-1-[(2R)-6-methylheptan-2-yl]-1H,2H,3H,3aH,3bH,4H,6H,7H,8H,9H,9aH,9bH,10H, 11H,11 aH-cyclopenta[a]phenanthren-7-yl]oxy}carbonyl)amino]butyl}amino)propyl]carbamoyl}-4-[(4-{[(2,4-diaminopteridin-6-yl)methyl](methyl)amino}phenyl)formamido]butanoic acid, utilizes an activation of the hydroxyl group and follows Scheme A. Cholesterol (1.93 g, 5 mmol) and 1,1′-Carbonyldiidazole (CDI) (as disclosed by Greg T. Hermanson., Bioconjugate. 2013, p 229) which were suspended in dimethyl sulfoxide (DMSO)(10 ml). was mixed. The mixture obtained was incubated to form active substances for 1 hour at room temperature. After 1 hour of incubation, spermidine (0.73 g, 5 mmol) was added to the mixtures. Subsequently, all mixtures were incubated at room temperature overnight. Reaction Scheme B, Methotrexate (2.27 g, 5 mmol) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (0.96 g, 5 mmol) were suspended in DMSO. Then, the mixtures from the Scheme A were added to the solution and incubated for 24 hours at room temperature. The new product was purified by flash chromatography on silica column. The purified product was further dried in vacuo, resulting a yellowish material, the final yield was 3.25 g (65.92%) ES(−)-MS: M-994.4.

(14) According to another aspect of the disclosure, the use of the compound as defined above in medicine, such as the use of the embodied compound in a medicament, is provided herein. In some embodiments, the medicament including the compound as defined herein is suitable for treating mammals, such as a human. Moreover, another aspect of the disclosure includes the use of the compound as defined above for the manufacture of a medicament for the treatment of a cancer comprising cancer cells, wherein the examples of the cancer include leukemia, lymphoma, breast, lung, cervical, brain, and other hematologic cancer. In addition, another aspect of the disclosure includes the use of the compound as defined above for the manufacture of a medicament for the treatment of connective tissue and autoimmune disease, wherein the examples of connective tissue and autoimmune diseases include systemic lupus erythematosus (SLE), psoriasis, pemphigus, pemphigoid, sarcoidosis, vitiligo.

(15) Further examples of the tests of the composition comprising the disclosed compound will now be described.

Example 1: Dihydrofolate Reductase Inhibitor Assay of Embodied Compound and MTX

(16) Embodied compound (compound A) and MTX at final concentrations 5, 50, 50 nM were tested for DHFR inhibiting activity with Dihydrofolate reductase (DHFR) assay kit (Sigma-Aldrich, Mo., USA). According to the supplier protocol, the activity of DHFR reductase was measured at 340 nM, every 15 seconds for 2.5 minutes, with the Biochrom Anthos 2010 microplate reader (Biochrom Ltd., Cambourne, Cambridge, UK). The enzymatic activity was calculated by the following formula:

(17) $\mathrm{Units}/\mathrm{mg}P=\frac{\left(\mathrm{\Delta O}D/\mathrm{minsample}-\mathrm{\Delta O}D/\mathrm{minblank}\right)\times d}{12.3\times V\times \mathrm{mg}P/\mathrm{ml}}$$\underset{¯}{\Delta }\mathrm{OD}/\mathrm{minblank}=\underset{¯}{\Delta }OD/\min .\mathrm{for}\mathrm{the}\mathrm{blank}$$\underset{¯}{\Delta }OD/\mathrm{minsample}=\underset{¯}{\Delta }OD/\min .\mathrm{for}\mathrm{the}\mathrm{reaction}$$12.3=\mathrm{Extinction}\mathrm{coefficient}\left(\underset{¯}{\Delta },{\mathrm{mM}}^{-1}{\mathrm{cm}}^{-1}\right)\mathrm{for}\mathrm{the}$$\mathrm{DHFR}\mathrm{reaction}\mathrm{at}340\mathrm{nm}$$V=\mathrm{Enzyme}\mathrm{volume}\mathrm{in}\mathrm{ml}\mathrm{used}\mathrm{in}\mathrm{the}\mathrm{assay}d=\mathrm{The}\mathrm{dilution}\mathrm{factor}\mathrm{of}\mathrm{enzyme}\mathrm{sample}$$\mathrm{mg}P/\mathrm{ml}=\mathrm{Enzyme}\mathrm{concentration}\mathrm{of}\mathrm{the}\mathrm{original}\mathrm{sample}$$\mathrm{before}\mathrm{dilution}$$\mathrm{Units}/\mathrm{mgP}=\mathrm{Specific}\mathrm{activity}\mathrm{in}\mu \mathrm{mole}/\min /\mathrm{mg}\mathrm{protein}$

(18) The changing of OD 340 is shown in FIG. 2 and the enzymatic activities are shown in Table 1.

(19) TABLE-US-00001 TABLE 1 Dihydrofolate reductase activity Dihydrofolate reductase (DHFR) activity Unit/mgP % No inhibitor 14.998 100 MTX 5 mM 0.981 6.54 MTX 50 mM 0.14 0.93 MTX 500 mM 0.14 0.93 Embodied compound 1.261 8.41 5 mM Embodied compound 0.561 3.74 50 mM Embodied compound 0.561 3.74 500 mM

Example 2: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide (MTT) Assay for Cytotoxicity Effect of Embodied Compound on Tumor Cells

(20) The human breast cancer cell line, MCF-7, was obtained from American Type Cell Collection (ATCC) (Rockville, Mo., U.S.A.). The cells were cultured to 70-80% confluence in Dulbecco's modified Eagle medium (DMEM) (Gibco, Grand Island, N.Y., U.S.A.) with 10% fetal bovine serum (FBS) (Gibco) at 37° C. and 5% CO2. MCF-7 cells were plated overnight in a 96-well plate (4,000 cells per well). Cells were treated with either embodied compound or MTX at a final concentration 0, 0.01, 0.1, 1 and 10 mM for 24 hours. Next, 10 μl/well of WST-1/ECS solution from the Quick Cell Proliferation Colorimetric Assay Kit (BioVision, Inc., Milptas, Calif., U.S.A.) was added, and the solution was incubated for 2 hours at 37° C. and 5% CO2. Absorbance was measured at 480 nm with the Biochrom Anthos 2010 microplate reader (Biochrom Ltd., Cambourne, Cambridge, UK). The half maximal inhibitory concentrations (IC50) were calculated from log-linear regression from the absorbance optical density (OD). The IC50 of MTX is 3.27 mM, whereas the IC50 of embodied compound is 0.06 mM. The 24 h cell viability is shown in FIG. 3A for MTX and FIG. 3B for embodied compound.

Example 3: Lipid Solubility and Oil in Water (O/W) Emulsion Analysis

(21) Embodied compound or Methrotrexate in DMSO (3 μg/μl) were dissolved in olive oil, canola oil and sunflower oil(Sigma-Aldrich (St. Louis, Mo., USA)) at 40° C. The suspended were vortexed and centrifuged at 3000 RPM, 10 mins periodically. Oil solvent volume was stepwise increased till completely soluble was observed and the soluble concentration were determined (Table 2). The solutions were left at 24° C. overnight and were centrifuged at 5000 RPM for 20 mins. Ten microliter of embodied compound in oils were added to 100 μl deionized (DI) water. The suspensions were vigorously vortex and left at room temperature. The distribution of embodied compound in O/W droplets were visualized under light microscope (as shown in FIG. 4). FIG. 4 shows an image of embodied compound in O/W droplets, visualized under light microscopy.

(22) TABLE-US-00002 TABLE 2 Solubility in Oil (40° C.) Disclosed compound Methrotrexate (mg/100 ml) (mg/100 ml) Olive Oil 48.1 less than 0.1 Canola Oil 81.3 less than 0.1 Sunflower Oil 103.6 less than 0.1

(23) The foregoing description of the present invention has been presented for the purposes of illustration and description. Furthermore, the description is not intended to limit the invention to the form disclosed herein. Consequently, variations and modifications commensurate with the above teachings, and the skill or knowledge of the relevant art, are within the scope of the present invention. The embodiment described hereinabove is further intended to explain the best mode known for practicing the invention and to enable others skilled in the art to utilize the invention in such, or other, embodiments and with various modifications required by the particular applications or uses of the present invention. It is intended that the appended claims be construed to include alternative embodiments to the extent permitted by the prior art.