CN107021942A - Bark extract of pinus fenzeliana var dabeshanensis and preparation method thereof and the purposes in pharmacy - Google Patents

Bark extract of pinus fenzeliana var dabeshanensis and preparation method thereof and the purposes in pharmacy Download PDF

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CN107021942A
CN107021942A CN201610068135.0A CN201610068135A CN107021942A CN 107021942 A CN107021942 A CN 107021942A CN 201610068135 A CN201610068135 A CN 201610068135A CN 107021942 A CN107021942 A CN 107021942A
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compound
diterpene
pinus
dabeshanensis
ptp1b
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CN107021942B (en
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胡金锋
胡长玲
熊娟
杨国勋
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Fudan University
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    • C07D301/00—Preparation of oxiranes
    • C07D301/32—Separation; Purification
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C61/00—Compounds having carboxyl groups bound to carbon atoms of rings other than six-membered aromatic rings
    • C07C61/16—Unsaturated compounds
    • C07C61/39—Unsaturated compounds containing six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C62/00—Compounds having carboxyl groups bound to carbon atoms of rings other than six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C62/02—Saturated compounds containing hydroxy or O-metal groups
    • C07C62/06—Saturated compounds containing hydroxy or O-metal groups polycyclic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
    • C07D303/02—Compounds containing oxirane rings
    • C07D303/38—Compounds containing oxirane rings with hydrocarbon radicals, substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals

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Abstract

The invention belongs to the field of Chinese medicines, it is related to the bark extract of pinus fenzeliana var dabeshanensis.The present invention is from Pinaceae（Pinaceae）Pinus（Pinus）Plant pinus fenzeliana var dabeshanensis（Pinus dabeshanensis Cheng et Law）Bark in extracting and developing obtain formula have PTP 1B（PTP1B）The diterpene-kind compound of inhibitory activity, proves that such compound has significant PTP1B inhibitory activity through Bioactivity test.Available for the disease medicament for preparing prevention, delay or treating diabetes, obesity and its complication and other PTP1B are mediated.

Description

Bark extract of pinus fenzeliana var dabeshanensis and preparation method thereof and the purposes in pharmacy
Technical field
The invention belongs to the field of Chinese medicines, it is related to bark extract of pinus fenzeliana var dabeshanensis and preparation method thereof and the purposes in pharmacy, it is of the invention from pinus fenzeliana var dabeshanensis（Pinus dabeshanensis）Bark in extract isolated isopimarane type and Diterpene compound, show that such compound can significantly inhibit PTP 1B through biological activity test（PTP1B）, the lead compound available for the disease, particularly type ii diabetes, obesity and its medicine of complication or such medicine for preparing prevention or treatment PTP1B mediations.The invention further relates to the preparation method of such compound.
Background technology
Prior art discloses Protein-tyrosine-phosphatase（protein tyrosine phosphatase, PTP）It is the enzyme family for adjusting intracellular protein tyrosine residue phosphorylation level, it is closely related with cell propagation and intracellular multi-signal transductive process（Fischer et al, Science 1991, 253: 401-406）.PTP 1B（protein tyrosine phosphatase 1B, PTP1B）It is distributed widely in a variety of tissues, the KDa of total length about 50, is to be purified and determine one of PTP family members of biological property earliest（Charbonneau et al, Proc. Natl. Acad. Sci. USA. 1989, 86: 5252–5256）.The diseases such as change and human diabetes, obesity and the cancer of PTP1B expression are related.
Diabetes（diabetes mellitus）And obesity（obesity）It is that the serious endocrine disturbance for threatening human body health is metabolized class disease, the often reduction along with peripheral tissues' cell to insulin sensitivity（That is insulin resistance）, cause the glycolipid metabolism dysequilibrium in muscle, liver and adipose tissue.According to the pathogenesis of diabetes, diabetes are typically divided into type i diabetes at present（Insulin-dependent, IDDM）With type ii diabetes（Non-insulin-depending type, NIDDM）.Wherein type ii diabetes account for more than 90%, and insulin resistance is the key factor in type ii diabetes generation, evolution.Research shows that PTP1B can make the insulin receptor and IRS dephosphorylation of activation, and important negative regulation effect is played during the signal transduction of insulin and leptin（Kenndy et al, Science 1999, 283: 1544–1548）.PTP1B overexpression will cause the change of insulin and Leptin signaling, so as to cause the generation of diabetes and obesity.Therefore, PTP1B can be considered the important drug target of the treatment relevant disease such as diabetes and obesity（Johnson et al., Nat. Rev. Drug. Discov. 2002, 1: 696–709）.PTP1B specific inhibitor is found, sensitiveness of the peripheral tissues to insulin is improved by suppressing PTP1B activity, there is important application prospect to diabetes and bariatrician.
Separately studies have found that PTP1B is including having overexpression and level to improve phenomenon in some cancer cells including chronic granulocytic leukemia, breast cancer, oophoroma and prostate cancer（Liu et al., J. Biol. Chem. 1996, 271, 31290–31295; Kenneth et al., Mol. Cell. Biol. 1998, 18, 2965–2975; Weiner te al., J. Natl. Cancer Inst. 1996, 86, 372–378）, illustrate that PTP1B plays a part of regulating and controlling kinase activity in these cancer cells.Therefore, PTP1B inhibitor can be used for treating or preventing cancer or be used to slow down its progress in cancer development.Also there are some researches show PTP1B inhibitor can be used for treating or preventing nerve degenerative diseases.
PTP1B turns into a focus of biology and innovation drug research in recent years as new drugable target.With the extensive utilization of High Throughput Screening Assay, it has now been found that compared with the PTP1B inhibitor of multiple types.But so far, not yet there is any PTP1B inhibitor to be able to successfully list as medicine, the first cause for limiting its patent medicine is that PTP1B has identical catalytic center with other enzymes in PTP families, and it is of crucial importance for PTP1B selectivity to improve it while PTP1B activity is suppressed.In addition, carry phosphoric acid, carboxylic acid, sulfonic acid isopolarity group the high inhibitor of the expression activitiy having now been found that more, with higher negative electrical charge, it is unfavorable for passing through cell membrane, causes poor bioavailability.Therefore efficient, high selectivity is found, while the small molecule PTP1B inhibitor for having good pharmacokinetic property concurrently is significant, the treatment for diseases such as diabetes and obesity has broad application prospects.
The characteristics of natural products has structural complexity and structure diversity, is the important sources of new drug discovery.The unique chemical constitution of natural products and its derivative, the advantages of making it have high-drug-effect and advantage to specific target spot high selectivity and potential unique mechanism of action（Newman, et al., Nat. Prod. Rep.2000, 17: 215–34; Newman et al., J. Nat. Prod. 2012, 75: 311–335）.Therefore new, the efficient PTP1B inhibitor of exploitation is found from active skull cap components has important researching value.
Statistics shows that the druggability of endangered plants secondary metabolite is higher, is the important sources for finding the novel drugs with novel structure and unique mechanism of action, just attracts great attention in the world（Ibrahim et al., Proc. Natl. Acad. Sci. U S A. 2013: 110, 16832–16837; Zhu et al., Proc. Natl. Acad. Sci. U S A. 2011, 108: 12943–12948）.Pinus fenzeliana var dabeshanensis（Pinus dabeshanensis）It is subordinate to Pinaceae（Pinaceae）Pinus（Pinus）Plant, is up to more than 30 rice after a kind of aiphyllium, adult, diameter, up to 50 cm, is one of distinctive rare tree species for facing extinction in imminent danger of China, and national rare tree species first second class protection seeds are listed in October, 1992（Fu et al., China Plant Red Data Book, Science Press: Beijing; New York, 1992）；Its natural distributed is in the ridge cliff abrupt slope and cheuch both sides of height above sea level 800-1350 meters of Dabie Mountains Region.Distribution is narrow, and solid maternal plant is rare, and pollination rate is extremely low, and ten nine, cone seed is empty, seeds abortion rate is high, and germination percentage is extremely low, and often because cone seed endangers by squirrel, therefore sylvan life treelet is few, natural renovation is very difficult, and its chemical composition not yet has any report.
Present inventor is based on protectively gathering a little pinus fenzeliana var dabeshanensis plant sample, actively promotes the objective serviced using this rare resources in imminent danger for the mankind, intends providing bark extract of pinus fenzeliana var dabeshanensis and preparation method thereof and the purposes in pharmacy.
The content of the invention
Purposes it is an object of the present invention to provide bark extract of pinus fenzeliana var dabeshanensis and preparation method thereof and in pharmacy, it is of the invention from pinus fenzeliana var dabeshanensis（Pinus dabeshanensis）Bark in extract isolated isopimarane type and Diterpene compound, show that such compound can significantly inhibit PTP 1B through biological activity test（PTP1B）, the lead compound available for the disease, particularly type ii diabetes, obesity and its medicine of complication or such medicine for preparing prevention or treatment PTP1B mediations.
The described diterpene-kind compound of the present invention has following chemical structural formula：
Wherein compound1With2For isopimarane type diterpene, compound3–5For Diterpene,
In the present invention, described diterpene-kind compound, wherein C-18 are oxidized to carboxyl, while isopimarane type diterpene1With2Side chain have three membered oxygen rings；Described Diterpene compound3–5C cyclophanes in structure.
It is a further object of the present invention to provide the preparation method of described compound.
Compound of the present invention extraction separation method conventional as involved by pinus fenzeliana var dabeshanensis bark via this area is prepared, and it includes step：The pinus fenzeliana var dabeshanensis bark of dried and crushed is extracted with methanol soaking at room temperature, and solvent is recovered under reduced pressure in extract solution, and medicinal extract is obtained after merging.Extracted successively with petroleum ether and ethyl acetate after medicinal extract is water-dispersible, obtain oil ether moiety, ethyl acetate portion and water-soluble portion, ethyl acetate portion prepares HPLC through silica gel, Sephadex LH-20 and half and separated, and obtains compound1–5。
Compound of the present invention can be obtained by being isolated and purified from plant；Also acquisition can be chemically synthesized through well known to those skilled in the art.
The present invention has carried out PTP 1B to obtained diterpene-kind compound（PTP1B）Inhibitory activity is tested, as a result show that such compound is respectively provided with significant inhibitory activity, further, described compound can be used as Protein-tyrosine-phosphatase PTP1B inhibitor, for prevention is made, delay or treats the disease mediated by PTP1B, particularly type ii diabetes and obesity medicine or the lead compound as such medicine.
Compound of the present invention can be used alone or share, and can also be combined with pharmaceutically acceptable carrier or excipient, and oral or non-oral dosage forms is conventionally made.
The invention has the advantages that：The target compound1For new isopimarane type diterpene compound, and compound2For first from nature it is isolated；Find that the diterpene-kind compound has significant PTP1B enzyme inhibition activities first, there is application prospect to diabetes occurred frequently in Modern human populations, obesity etc..
With reference to embodiment, the present invention is further elaborated, but these embodiments have absolutely not any limitation to the present invention.Any variation that those skilled in the art are made in implementing under the enlightenment of this specification to the present invention will all be fallen within the scope of the appended claims.
Embodiment
In following preparation examples,
, specific rotation is tested to be completed by JASCO P-1020 polarimeters；Ultraviolet and ir data passes through Shimadzu respectively UV-2550 ultraviolet spectrometers and the type infrared spectrometers of Nicolet AVATAR 360 are obtained；NMR Bruker Avance II 400 type instrument are determined；LR-MS is by Agilent 1100 Series LC/MSD G1946D types instrument are determined, and HR-MS is determined by the type instrument of AB Sciex TripleTOF 5600；Used silica gel and lamellae produce for Yantai Kombi promise company, and Sephadex LH-20 gels are Switzerland GE Healthcare Bio-Sciences companies produce；Half prepares HPLC for Waters e2695, is equipped with 2998 PDA and 2424 evaporative light-scattering dual detectors and SunFire ODS (5μM, 250 × 10 mm) semi-preparative column, Japanese Shimadzu LC-2010A HT SHIMADZU normal-phase chirality column (5 μm, 250 × 4.6 mm, ChiRaLPAK, AD-H)；All reagents are the production of Shanghai Chemical Reagent Co., Ltd., Sinopharm Group.
Embodiment 1：The preparation of diterpene-kind compound
The g of pinus fenzeliana var dabeshanensis bark 180 is taken, is extracted 5 times with 90% methanol room temperature cold soaking after crushing, merges extract solution, is concentrated under reduced pressure, obtains the g of medicinal extract 50.Extracted successively with petroleum ether with ethyl acetate after medicinal extract is water-dispersible, obtain the g of ethyl acetate portion 40.0.Ethyl acetate portion is through 100-200 mesh silica gel column chromatographies, with petroleum ether：Ethyl acetate 30:1-0:1 and ethyl acetate：Methanol 15:1-0:1 gradient elution, obtains 8 components（Fr.A-Fr.H）, component Fr.B（1.9 g）Again through 200-300 mesh silica gel column chromatographies（Eluant, eluent is petroleum ether：Ethyl acetate 10:1-2:1）Obtain 3 subfractions（Fr.B1-3）, wherein subfraction Fr.B1 (30.6 mg) is further separated using semi-preparative HPLC, with 73% acetonitrile isocratic elution（Flow velocity：3 mL/min）, obtain compound3（8.0 mg,t R= 21.3 min）, component Fr.C（1.9 g）Through 200-300 mesh silica gel column chromatographies（Eluant, eluent is petroleum ether：Ethyl acetate 7:1-3:1）Obtain 2 subfractions（Fr.C1-2）, wherein subfraction Fr.C1 is by gel Sephadex LH-20 column chromatographies（Eluant, eluent is methanol）Obtain compound4（28.0 mg）, subfraction Fr.C2 is further separated by semi-preparative HPLC, with 90% methanol isocratic elution（Flow velocity：3 mL/min）, respectively obtain compound1With2Mixture, the mixture passes through chiral column again（N-hexane：Isopropanol 94:6, flow velocity：1 mL/min）Further isolated compound1（2.2 mg,t R= 9.5 min）With2（2.5 mg,t R= 12.5 min）, component Fr.F（0.5 g）Eluted through Sephadex LH-20 with pure methanol, obtain compound5（120.0 mg）.
Compound1–5Nuclear-magnetism and physicochemical data it is as follows：
Compound 1 (15(R*),16-Epoxy-13-epi-pimara-7-en-18-oic acid):Colorless oil, [α]D 20 +5.0° (c 0.1, CHCl3); UV (CHCl3) λmax (log ε ) 217 (0.97) nm; IR (KBr) νmax 2922, 2857, 1698, 1452, 1442, 1386, 1366, 1024, 786 cm−1; 1H NMR (400 MHz, CDCl3): δ 0.75 (3H, s, Me-17), 0.90 (3H, s, Me-20), 1.13 (1H, m, H-1a), 1.28 (3H, s, H-19), 1.36 (1H, m, H-11a), 1.39 (1H, m, H-12a), 1.45 (1H, m, H-12b), 1.56 (2H, m, H-2), 1.58 (1H, m, H-11b), 1.68 (1H, m, H-3a), 1.71 (1H, m, H-6), 1.76 (1H, m, H-5), 1.77 (1H, m, H-3b), 1.85 (1H, d, J = 13.6 Hz, H-14a), 1.86 (1H, m, H-1b), 1.94 (1H, d, J = 13.6 Hz, H-14b), 1.96 (1H, m, H-9), 1.99 (1H, m, H-6b), 2.64 (2H, br d, J = 3.6 Hz, H-16), 2.72 (1H, dd, J = 3.6, 3.2 Hz, H-15), 5.34 (1H, br d, J = 5.2 Hz, H-7). 13C NMR (100 MHz, CDCl3): δ 38.8 (C-1), 17.9 (C-2), 36.9 (C-3), 46.3 (C-4), 52.0 (C-5), 25.2 (C-6), 121.3 (C-7), 134.7 (C-8), 44.9 (C-9), 35.0 (C-10), 19.4 (C-11), 33.3 (C-12), 33.9 (C-13), 42.0 (C-14), 60.6 (C-15), 43.4 (C-16), 18.1 (C-17), 184.7 (C-18), 17.1 (C-19), 15.3 (C-20); (+) ESIMS m/z 319 [M+H]+, 341 [M+Na]+; HRESIMS m/z 341.2095 [M + Na]+ (calcd for C20H30O3Na, calcd 341.2093, the ppm of Δ=- 2.7)
Compound 2 (15(S*),16-Epoxy-13-epi-pimara-7-en-18-oic acid):Colorless oil, [α]D 20 +8.0° (c 0.1, CHCl3); UV (CHCl3) λmax (log ε ) 217 (0.83) nm; IR (KBr) νmax 2922, 2857, 1698, 1452, 1442, 1386, 1366, 1024, 786 cm−1; 1H NMR (400 MHz, CDCl3): δ 0.78 (3H, s, Me-17), 0.90 (3H, s, Me-20), 1.13 (1H, m, H-1a), 1.28 (3H, s, H-19), 1.36 (1H, m, H-11a), 1.37 (1H, m, H-12a), 1.45 (1H, m, H-12b), 1.56 (2H, m, H2-2), 1.58 (1H, m, H-11b), 1.68 (1H, m, H-3a), 1.71 (1H, m, H-6), 1.76 (1H, m, H-5), 1.77 (1H, m, H-3b), 1.85 (1H, d, J = 13.6 Hz, H-14a), 1.86 (1H, m, H-1b), 1.94 (1H, d, J = 13.6 Hz, H-14b), 1.96 (1H, m, H-9), 1.99 (1H, m, H-6b), 2.64 (1H, dd, J = 4.8, 4.0 Hz, H-16), 2.68 (1H, dd, J = 4.8, 3.2 Hz, H-16), 2.72 (1H, dd, J = 4.0, 3.2 Hz, H-15), 5.34 (1H, br d, J = 5.2 Hz, H-7). 13C NMR (100 MHz, CDCl3): δ 38.8 (C-1), 17.9 (C-2), 36.9 (C-3), 46.3 (C-4), 52.0 (C-5), 25.2 (C-6), 121.3 (C-7), 134.7 (C-8), 44.9 (C-9), 35.0 (C-10), 19.4 (C-11), 32.0 (C-12), 33.9 (C-13), 42.9 (C-14), 60.7 (C-15), 43.5 (C-16), 18.5 (C-17), 184.7 (C-18), 17.1 (C-19), 15.3 (C-20); (+) ESIMS m/z 319 [M+H]+, 341 [M+Na]+;HRESIMS m/z 341.2095 [M+Na]+ (calcd for C20H30O3Na, calcd 341.2093, the ppm of Δ=- 2.5)
Compound 3 (Abieta-8,11,13,15-tetraen-18-oic acid):White amorphous powder, [α]D 20 +55.0 (c 0.1, MeOH). 1H NMR (400 MHz, CDCl3): δ 1.25 (3H, s, Me-20), 1.29 (3H, s, Me-19), 1.50 (1H, m, overlapped, H-6a), 1.50 (1H, dd, overlapped, H-1a), 1.72 (1H, m, H-3a), 1.75 (2H, m, H2-2), 1.80 (1H, m, H-3b), 1.84 (1H, m, H-6b), 2.14 (3H, br s, H-17), 2.25 (1H, dd, J = 12.4, 2.1 Hz, H-5), 2.33 (1H, br d, J = 13.5 Hz, H-1b), 2.94 (2H, m, H2-7), 5.04 (1H, br s, H-16a), 5.34 (1H, br s, H-16b), 7.16 (1H, d, J = 2.0 Hz, H-14), 7.20 (1H, d, J = 8.0 Hz, H-11), 7.26 (1H, dd, J = 8.0, 2.0 Hz, H-12); (+) ESIMS m/z 299 [M+H]+, 321 [M+Na]+.
Compound 4 (12-Hydroxydehydroabietic acid):Faint yellow oily, [α]D 20 +35.0° (c 0.1, CDCl3). 1H NMR (400 MHz, CDCl3): δ 1.22 (3H, s, Me-20), 1.25 (3H, d, J = 7.2 Hz, Me-16), 1.27 (3H, d, J = 7.2 Hz, Me-17), 1.29 (3H, s, H-19), 1.52 (1H, m, overlapped, H-6a), 1.52 (1H, dd, overlapped, H-1a), 1.70 (1H, m, H-3a), 1.74 (2H, m, H2-2), 1.79 (1H, m, H-3b), 1.82 (1H, m, H-6b), 2.22 (1H, dd, J = 12.5, 2.2 Hz, H-5), 2.25 (1H, br d, J = 13.5 Hz, H-1b), 2.83 (2H, m, H2-7), 3.12 (1H, sept., J = 7.2 Hz, H-15), 6.64 (1H, s, H-11), 6.85 (1H, s, H-14), 13C NMR (100 MHz, CDCl3): δ 37.9 (C-1), 18.5 (C-2), 36.7 (C-3), 47.4 (C-4), 44.5 (C-5), 21.9 (C-6), 29.2 (C-7), 127.0 (C-8), 147.8 (C-9), 36.8 (C-10), 110.8 (C-11), 150.7 (C-12), 131.8 (C-13), 126.7 (C-14), 26.8 (C-15), 22.7 (C-16), 22.5 (C-17), 184.5 (C-18), 16.2 (C-19), 25.0 (C-20); (+) ESIMS m/z 317 [M+H]+, 339 [M+Na]+.
Compound 5 (15-Hydroxy-7-oxo-8,11,13-abietatrien-18-oic acid):Pale yellow powder, [α]D 20 +16.5 (c 0.1, MeOH). 1H NMR (400 MHz, CDCl3): δ 1.27 (3H, s, Me-20), 1.29 (3H, s, Me-19), 1.58 (6H, s,Me-16, 17), 1.68 (1H, m,H-1a), 1.80 (5H, m, H-1b, H2-2, H2-3), 2.42 (1H, dd, J = 17.0, 3.2 Hz, H-6a), 2.68 (1H, dd, J = 13.5, 3.2 Hz, H-5), 2.86 (1H, dd, J = 17.0, 13.5 Hz, H-6b), 7.38 (1H, d, J = 8.4 Hz, H-11), 7.76 (1H, d, J = 8.4, 2.1 Hz, H-12), 8.06 (1H, d, J = 2.1 Hz, H-12), 13C NMR (100 MHz, CDCl3): δ 37.7 (C -1), 18.1 (C-2), 37.0 (C-3), 46.3 (C-4), 43.5 (C-5), 36.4 (C-6), 198.8 (C-7), 130.4 (C-8), 153.8 (C-9), 37.3 (C-10), 123.6 (C-11), 130.7 (C-12), 147.3 (C-13), 123.3 (C-14), 72.4 (C-15), 31.7 (C-16), 31.5 (C-17), 182.5 (C-18), 16.1 (C-19), 23.6 (C-20); (+) ESIMS m/z 331 [M+H]+, 353 [M+Na]+. 。
Embodiment 2：PTP 1B inhibitory activity is determined
Experimental method：Using the method for molecular biology, the hGST-PTP1B-BL21 of genetic recombination is builtE. coliMankind's PTP1B engineering bacterias, it is purified after hGST-PTP1B recombinant proteins can hydrolyze substratepara-Nitrophenyl Phosphate (pNPP) phosphatide key, obtained dephosphorization acid product pNP has very strong light absorbs at the nm of wavelength 405, therefore can be by directly detecting 405 The change of light absorbs is to observe the suppression situation of Enzyme activities and compound to enzymatic activity at nm；Primary dcreening operation selects the compound concentration to be 20µThe percent inhibition of PTP1B enzymatic activitys is investigated during g/ml, result of the test shows compound4With5Inhibiting rate is higher than 95%, compound1-3Inhibiting rate is higher than 60%.Further determine IC50Value：Sample is dissolved in DMSO and is made into suitable concn before use, and 3 times of dilutions, 7 dilution factors, three wells takes 2 μ L sample solution to be added to the live body system of standard（30 nM GST-hPTP1B, 2 mM pNPP, 50 mM 3- N-morpholinyls (MOPS), pH 6.5,2 mM DTT, 1mM EDTA, 2% DMSO）.Reaction temperature is 30oC, dynamically measures the light absorbs at 405 nm on VERSAmax, and the time is 3 min, the slope of its kinetic curve first order reaction as enzyme activity index.Compound concentration is mapped with relative activity, through formulav/v 0=100/ (1+b*[I]/IC50) Fitting obtains IC50Value.Experiment in triplicate, as a result takes the average value of three times, positive control oleanolic acid（oleanolic acid）IC50It is worth for 1.45µg/ml；
Table 1.Test compound suppresses PTP1B activity datas
Compound IC50 (µg/ml)
1 13.04 ± 1.21
2 11.17 ± 3.09
3 16.10 ± 1.44
4 1.61 ± 0.26
5 3.42 ± 0.28
Compound1–5Suppress PTP1B IC50Value is as shown in table 1, and test result shows, 5 compounds show significant inhibitory activity, especially compound4With5Action intensity it is substantially suitable with positive control oleanolic acid, show that compound of the present invention can be used for preparing treatment diabetes, the medicine or the lead compound as such medicine of obesity and its complication.

Claims (5)

1. the diterpene-kind compound of following formula chemical constitution, wherein, compound1With2For isopimarane type diterpene, compound3–5For Diterpene,
。
2. diterpene-kind compound as claimed in claim 1, it is characterised in that described C-18 positions are oxidized to carboxyl, while isopimarane type diterpene1With2Side chain have three membered oxygen rings；Described Diterpene compound3–5C cyclophanes in structure.
3. diterpene-kind compound as claimed in claim 1 or 2, it is characterised in that described isopimarane type and Diterpene class compound is prepared by following methods：Using pinus fenzeliana var dabeshanensisPinus dabeshanensisDry bark, is extracted after crushing with methanol, extract solution concentration, and add water suspension, is extracted respectively with petroleum ether and ethyl acetate；Ethyl acetate extract is through silica gel, Sephadex LH-20 gels and partly prepares HPLC chromatogram separation, and compound is made1–5。
4. purposes of the diterpene-kind compound as protein-tyrosine phosphatase 1B inhibitor in medicine is prepared described in claim 1.
5. purposes as claimed in claim 4, it is characterised in that described medicine is prevention, delay or treat diabetes, obesity and other medicines with PTP1B mediation relevant diseases.
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