Protein tyrosine phosphatase inhibitor, preparation method and uses thereof

Abstract

Disclosed in the present invention is a protein tyrosine phosphatase inhibitor. The preparation method therefor is: extracting the crude product from the Isaria Fumosorosea Wize solid or liquid fermentation broth using ethyl acetate, ethanol, methanol, or a mixed solvent of chloroform and methanol; separating the obtained extract using column chromatography on silica gel; and obtaining the target product. The inhibitor can be used to prepare pharmaceutical compositions for treating and preventing diabetes, obesity and cancers.

Claims

1. A method for treating diabetes, obesity and cancers in a subject in need thereof, the method comprising: administering a composition comprising a protein tyrosine phosphatase inhibitor and a pharmaceutically acceptable carrier to the subject, wherein the composition is administered in an effective amount, wherein the protein tyrosine phosphatase inhibitor is the following (a) or (b): (a) a compound having the following structural formula: ##STR00003## or a pharmaceutically acceptable salt of compound (a), compound (a) in the form of an ester or ether, compound (a) incorporated with a functional group of aldehyde, acid or alcohol by oxidation of double bonds, compound (a) incorporated with a sulfonic acid group in the ring, or compound (a) incorporated with a chain having from 4 to 10 carbon atoms by a Witting reaction.

2. The method according to claim 1, wherein the composition is parenterally administered in an amount ranging from 0.1 to 10,000 μg/kg body weight of the subject.

3. The method according to claim 1, wherein the composition is parenterally administered in an amount ranging from 1 to 1,000 μg/kg body weight of the subject.

4. The method according to claim 1, wherein the composition is parenterally administered in an amount ranging from 1 to 80 μg/kg body weight of the subject.

5. The method according to claim 1, wherein the composition is orally administered in an amount ranging from 0.2 to 10 mg/kg body weight of the subject.

6. The method according to claim 1, wherein the composition is orally administered in an amount ranging from 0.6 to 6 mg/kg body weight of the subject.

7. The method according to claim 1, wherein the composition is orally administered in an amount ranging from 1 to 5 mg/kg body weight of the subject.

8. The method according to claim 1, wherein the composition is in the form of powder, tablet, dragee, capsule, solution, syrup or dropping pill for oral administration.

9. The method according to claim 1, wherein the composition is in the form of a powder injection or a solution injection for injection administration.

10. The method according to claim 1, wherein the pharmaceutically acceptable salt of compound (a) maintains the biological effectiveness of said compound (a) and is an acid addition salt or base addition salt commonly used in pharmacy and derived from non-toxic organic acids, inorganic acids, organic bases or inorganic bases.

11. The method according to claim 10, wherein the inorganic acids for preparing said acid addition salt are hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid or nitric acid; the organic acids used for preparing said acid addition salt are p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, malic acid, succinic acid, lactic acid, or fumaric acid; and the basic agents for preparing said base addition salt are hydroxides of ammonium, potassium, sodium or quaternary ammonium.

12. The method according to claim 1, wherein the chain incorporated by the Witting reaction carries OR.sup.1, NR.sup.2R.sup.3 or halogen, wherein R.sup.1, R.sup.2 or R.sup.3 is H or alkyl having from 1 to 5 carbon atoms, and the halogen is F, Cl, Br or I.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The invention is illustrated by the following examples. All starting materials and equipment employed in the invention are conventional and commercially available and can be directly purchased from the market.

Example 1: Preparation of the Product

(2) The Isaria Fumosorosea Wize (=Paecilomyces fumosoroseus (Wize) A.H.S.Br.&G.Sm) used in the invention was collected from Baoding city, Hebei Province; and the strain was kept at the College of Life Science. Hebei University. For specific source, please refer to the Disclosure Form of the Origin of Genetic Resources.

(3) A. Fermentation and Cultivation: Isaria Fumosorosea Wize strain was inoculated with an inoculation needle into a 500 ml Erlenmeyer flask containing 100 ml of modified potato dextrose culture medium (formulated by dissolving 200.0 g of peeled potato, 20.0 g of dextrose, 3.0 g of KH.sub.2PO.sub.4. 1.5 g of MgSO.sub.4, 0.1 g of citric acid, and 10.0 mg of vitamin B.sub.1 in 1 l of sterile water, pH 6.5 and sterilizing at 121° C. for 20 minutes). The mixture was cultured in a shaker at 150 rpm and at 26° C. for 7 days, to obtain a seed broth, 10 ml of the seed broth was inoculated into a 500 ml Erlenmeyer flask containing rice culture medium (formulated by dissolving 80 g of rice in 100 ml of sterile water, soaking for 12 hours, and then at natural pH sterilizing at 121° C. for 30 minutes). There were 50 Erlenmeyer flasks in total. The mixture was further cultured under light at 26° C. for 30 days.
B. Extraction and Separation: the fermented product was extracted by soaking with equal volume of ethyl acetate at room temperature for 48 hours, and evaporated to dryness by vacuum distillation with a rotatory evaporator at 45° C. for three times, to obtain 200 g of extractum sample. Said extractum sample was thoroughly mixed with 240 g of silica gel (with a mesh size of 100 to 200) under stirring, dried, ground uniformly to obtain powders, rendering said extractum sample sufficiently absorbed onto the silica gel particles. The mixture was then subjected to atmospheric column chromatography on silica gel (with a mesh size ranging from 300 to 400), and eluted gradiently with petroleum ether/ethyl acetate system (100:0, 95:5, 90:10, 80:20, 60:40 and 50:50 (v/v)). The eluents were collected in 500 ml triangular flasks, subjected to vacuum distillation at 45° C., concentrated and washed with a small amount of organic solvent. The resulting samples were placed in 5 ml flaskets and the flaskets were numbered and labeled, 37 g of the fraction obtained from the elution section with 80:20 of petroleum ether/ethyl acetate was further subjected to atmospheric column chromatography on silica gel under gradient elution with petroleum ether/ethyl acetate system (100:0, 50:1, 20:1, 15:1, 10:1 and 5:1 (v/v)). The eluents were collected in 200 ml triangular flasks and subjected to vacuum distillation at 45° C., to obtain 10 g of Fumosorinone at the 5:1 elution section, namely, to obtain the protein tyrosine phosphatase inhibitor (a).

Example 2: Preparation of the Product

(4) This example differed from example 1 in the following procedures: the extractum sample, after being sufficiently adsorbed on the silica gel particles, was subjected to atmospheric column chromatography on silica gel (with a mesh size ranging from 300 to 400), and eluted gradiently with petroleum ether/ethyl acetate system in ratios of 100:0, 95:5, 90:10, 80:20, 60:40 and 50:50 (v/v). The eluents were collected, subjected to vacuum distillation at 45° C., concentrated and washed with an organic solvent. The eluents from each elution section were combined and subjected to the above-mentioned atmospheric column chromatography on silica gel again, and eluted gradiently again with petroleum ether/ethyl acetate system in ratios of 100:0, 50:1, 20:1, 15:1, 10:1 and 5:1, (v/v). The eluents were collected and subjected to vacuum distillation at 45° C. The eluents from each elution section were combined again and further subjected to the gradient elution process with petroleum ether/ethyl acetate in ratios of 100:0, 50:1, 20:1, 15:1, 10:1 and 5:1 (v/v), to obtain Fumosorinone at the 5:1 elution section, namely, to obtain the protein tyrosine phosphatase inhibitor (a).

(5) The yield of product was advantageously further increased by eluting the product in such manner.

Example 3: Preparation of the Product

(6) This example differed from example 1 in the following procedures: the extractum sample, after being sufficiently adsorbed on the silica gel particles, was subjected to atmospheric column chromatography on silica gel (with a mesh size ranging from 300 to 400), and eluted gradiently with petroleum ether/ethyl acetate system in ratios of 100:0, 95:5, 90:10, 80:20, 60:40 and 50:50, (v/v). The eluents were collected, subjected to vacuum distillation at 45° C., concentrated and washed with an organic solvent. The fraction obtained from the above elution section with 80:20 of petroleum ether/ethyl acetate was further subjected to the above-mentioned atmospheric column chromatography on silica gel, and eluted gradiently with chloroform/methanol system in ratios of 100:0, 95:5 and 90:10 (v/v), to obtain Fumosorinone at the 90:10 elution section, namely, to obtain the protein tyrosine phosphatase inhibitor (a).

Example 4: Determination of the Structure of the Product

(7) The structure of Fumosorinone was determined according to the spectral data of mass spectrum. NMR spectrum and the like.

(8) Data of UV spectrum: UV (MeOH) λ.sub.max nm: 367 (6.87), 265 (7.20), 209 (7.19).

(9) Data of Infrared spectrum: IRν.sup.KBr.sub.maxcm.sup.−1: 3267, 2960, 1643, 1516, 1446, 1268, 1218, 837, 758.

(10) Data of Mass Spectrum: HR-ESI-MS m/z: 500.24100 ([M+Na].sup.+, 500.24074 calcd. for C.sub.29H.sub.35NNaO.sub.5).

(11) For NMR data, see Table 2.

(12) TABLE-US-00002 TABLE 2 .sup.1H-(600 MHz) and .sup.13C-NMR (150 MHz) Data of Fumosorinone in CD.sub.3OD.sup.1 δ (H) δ (C) HMBC(H .fwdarw. C) C(2) 158.6 C(3) 111.1 C(4) 166.5 C(5) 114.9 H—C(6) 7.87 (s) 137.9 1′, 2, 4, 5 C(7) 199.8 C(8) 137.1 H—C(9) 6.94 (d, J = 9.8) 141.8 7, 10, 24 H—C(10) 6.67(m) 128.2 11 H—C(11) 6.67(m) 142.9 10 H—C(12) 6.41 (dd, J = 15.2, 127.6 13 9.3) H—C(13) 6.45 (d, J = 15.3) 143.4 12, 15 C(14) 134.1 H—C(15) 5.39 (d, J = 9.8) 144.2 13, 16, 17 H—C(16) 2.68 (m) 31.9 15 CH.sub.2(17) 1.16 (m), 1.34 (m) 46.1 18 H—C(18) 1.34 (m) 33.7 17, 19, 21 CH.sub.2(19) 1.16 (m), 1.34 (m) 31.3 18, 20 Me(20) 0.88 (m) 11.7 18, 19 Me(21) 0.88 (m) 19.6 17, 18, 19 Me(22) 0.98 (d, J = 6.6) 21.8 15, 16, 17 Me(23) 1.84 (s) 12.7 13, 14, 15 Me(24) 2.06 (s) 12.4 7, 8, 9 C(1′) 125.0 H—C(2′) 7.32 (d, J = 8.6) 131.7 5, 2′, 4′ H—C(3′) 6.84 (d, J = 8.6) 116.4 1′, 4′ C(4′) 159.1 H—C(5′) 6.84 (d, J = 8.6) 116.4 1′, 4′ H—C(6′) 7.32 (d, J = 8.6) 131.7 5, 4′, 5′

(13) It can be determined from the above spectral data that Fumosorinone, i.e., the protein tyrosine phosphatase inhibitor (a) of the invention, has the following structure:

(14) ##STR00002##

Example 5: Use of the Product

(15) According to the experiments described above, the use of the protein tyrosine phosphatase inhibitor (a) of the invention lies in the manufacture of a medicament, in particular a pharmaceutical composition for the treatment and prevention of diabetes, obesity and cancers. When it is used as a medicament, this compound can either be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition may contain 0.1% to 99%, preferably 0.5% to 90% of the compound of the invention, and the balance of pharmaceutically acceptable carriers or excipients which are non-toxic and inert to human and animals. The pharmaceutically acceptable carriers or excipients may be one or more selected from the group consisting of fillers of solid, semi-solid and liquid diluents, or auxiliaries for drug products. The pharmaceutical composition is administered in the form of dosage per unit body weight. The medicament of the invention may be administered orally or by injection (intravenous or intramuscular injection). Oral administration may employ solid or liquid formulations thereof, such as powders, tablets, dragees, capsules, solutions, syrups and dropping pills. Injection administration may employ solid or liquid formulations thereof, such as powder injections and solution injections.

(16) In the case of parenteral administration for human body, in order to achieve optimal effects, it is advantageous to employ an amount ranging from 0.1 to 10,000 μg/kg, preferably from 1 to 1,000 μg/kg, and in particular from 1 to 80 μg/kg body weight. In the case of oral administration, the amount is ranging from 0.2 to 10 mg/kg, preferably 0.6 to 6 mg/kg, and in particular from 1 to 5 mg/kg body weight. Nevertheless, depending on the body weight, administration route, individual response to the active compound, formulation type, and the time and intervals of administration, an amount deviated from said ranges may be necessary.

Example 6: Preparation of the Medicament

(17) The compound Fumosorinone prepared according to example 1, was mixed with excipients at a weight ratio of 1:1 of the compound in crystal form to the excipients, and then granulated and compressed into tablets; or

(18) The compound Fumosorinone prepared according to example 1, was formulated into capsules by conventional methods for preparing capsules; or

(19) The compound Fumosorinone prepared according to example 1, was mixed with excipients at a weight ratio of 1:2 of the compound in crystal form to the excipients, and then granulated and compressed into tablets; or

(20) The compound Fumosorinone prepared according to example 1, was mixed with excipients at a weight ratio of 1:3 of the compound in crystal form to the excipients, and then granulated and compressed into tablets.

Example 7: Preparation of the Medicament

(21) TABLE-US-00003 Tablet: Fumosorinone 100 mg Starch 100 mg 17% of Corn syrup appropriate amount Magnesium stearate appropriate amount

Example 8: Preparation of the Medicament

(22) TABLE-US-00004 Capsule: Fumosorinone 100 mg Starch 100 mg Magnesium stearate appropriate amount

(23) Preparation method: Fumosorinone was mixed with auxiliaries, and then sieved and thoroughly mixed in a proper container. The resulting mixture was placed into hard gelatin capsules.

Example 9: Preparation of the Medicament

(24) TABLE-US-00005 Ampoule: Fumosorinone 50 mg

(25) Preparation method: Fumosorinone was dissolved in 2 ml of propylene glycol. After filtration, the resulting solution was placed into ampoules under sterile condition.

(26) In addition, the protein tyrosine phosphatase inhibitor (a) of the invention may further be an isomer of the above-mentioned compound (a) or pharmaceutically acceptable derivative thereof. The pharmaceutically acceptable derivative is a commonly used pharmaceutical derivative which maintains the biological effectiveness of said compound (a). The selectable range and preparation method for such pharmaceutically acceptable derivative are common knowledge in the field of pharmacy. The illustrations as described above are only proposed as implementable technical solutions of the invention and would not serve as a single limitation to the technical solutions per se.