CN103897042A - Glycosylated derivative of cyclic peptide compounds and preparation method and application thereof - Google Patents

Abstract

The invention relates to a glycosylated derivative of cyclic peptide compounds. The chemical structural formula of the glycosylated derivative of cyclic octapeptide TunicyclinD is shown in the specification, wherein n is equal to 0, 1 or 2; Glycosyl is monosaccharide or disaccharide, the monosaccharide is glucose, galactose, xylose, rhamnose, mannose, ribose, glucosamine or n-acetylglucosamine; the disaccharide is maltose or lactose. The invention also provides a preparation method and application of the glycosylated derivative of the cyclic peptide compounds. The compounds provided by the invention have relatively strong in-vitro antifungal activity, thereby being capable of being used for preparing antifungal drugs; the preparation method provided by the invention is used for successfully introducing different glycosyls on carboxyl of TunicyclinD, and is simple in process and high in yield. The formula is shown in the specification.

Description

Glycosylated derivative of a kind of cyclic peptide compound and preparation method thereof and purposes

Technical field

The present invention relates to medical compounds technical field, specifically, is glycosylated derivative about a kind of cyclic peptide compound and preparation method thereof and purposes.

Background technology

In recent years, due to the increase of cancer radiation, chemotherapy and organ transplantation and interventional therapy patient number, being widely used of broad-spectrum antifungal medicine, cortin and immunosuppressor clinically, and acquired immune deficiency syndrome (AIDS) is popular, deep fungal infections case is sharply increased, and nearly 300,000 examples of systemic fungal infection patient that the whole world is diagnosed out every year, in AIDS patient, having 60% the direct cause of death is systemic fungal infection (Markus, R. drugs, 2004,64,1163-1180.).The mycosis serious threat mankind's life and health.In the medicine for the treatment of deep fungal infection, nitrogen azole compounds is most widely used, and modal fluconazole and itraconazole are exactly azole antifungals clinically at present.But fluconazole day by day serious resistance problem has seriously limited its application., as polyenoid class antifungal drug, propylamine, morpholine class and 5-FU class etc., there is many deficiencies (Owens, the J. such as toxicity, side effect large (as amphotericin B), narrow antimicrobial spectrum, easy recurrence (as 5-flurocytosine) more in other antifungal drug nat. Rev.Drug. Discov.2005,1,884-885.).Development is efficient, wide spectrum, the anti-deep fungal infection medicine of low toxicity have become a urgent world subject, day by day causes attention both domestic and external.

In the 70's of last century, scientist extracts cyclic peptide compound---echinocandin (Echinocandins) (Denning, the W. D. that a class can suppress multiple fungal growth breeding from aspergillus fumigatus lancet, 2003,362,1142-1151.).Further research is found, the mechanism of action of this compounds is by β-1 in Antifungi cytolemma, 3-D-glucan synthase (Wiederhold, N. P. expert. Opin. Investig. Drugs. 2003, 12, 1313-1333.), make main component β-1 of fungal cell wall, the biosynthesis block of 3-D-dextran, and the cytoskeleton that finally causes fungi cannot form and death, such antifungal compound and amphotericin B and azole antifungal medicine medicine are without cross resistance, because mammalian cell is not containing cell walls, do not produce β-1, 3-D-glucan synthase, so this type of drug selectivity is high, act on powerful, toxicity is lower, add easy to use, better tolerance, have broad application prospects, focus and the new direction of current searching novel antifungal drugs at present the research of this compounds are become.

Ring octapeptide molecule Tunicyclin D is from Root of Tuniclike Psammosilene by my institute's Zhang Weidong teach problem group psammosilene tunicoidesroot in extract and separate the coupling reagent with anti-mycotic activity (Tian, the J. that obtain j. Nat. Prod.2010, 73, 1987-1992.).In Vitro Anti fungi is tested and shows, Tunicyclin D has very strong extracorporeal antifungal activity, and it is to fungal bacterial strain candida tropicalis, candida parapsilosis, cryptococcus neoformans (BLS108), Candida albicans (SC5314),with candida albicans (Y0109)mIC80 value be respectively 0.25,1.0,1.0,4.0 and 16.0

mg/mL?。

In recent years, to glycosylation modified one of pharmaceutical chemistry study hotspot that become of medicine, by introduce glycan molecule in drug molecule, can obviously improve the lipid of medicine, increase the water-soluble of medicine, thereby the sterie configuration that even can change medicine strengthens the activity of medicine to a certain extent, the toxicity that reduces medicine has certain effect.

Therefore, Tunicyclin D carries out glycosylation, reduces that it is fat-soluble, it is water-soluble to improve, and investigates the impact on its anti-mycotic activity simultaneously.Yet there are no similarly report.

Summary of the invention

The object of the invention is, for deficiency of the prior art, provides a kind of glycosylated derivative of cyclic peptide compound.

One object more of the present invention is that a kind of preparation method of glycosylated derivative of cyclic peptide compound is provided.

Another object of the present invention is that a kind of purposes of glycosylated derivative of cyclic peptide compound is provided.

For achieving the above object, the technical scheme that the present invention takes is:

The glycosylated derivative of one class ring octapeptide Tunicyclin D, the glycosylated derivative general structure of described Tunicyclin D is as follows:

Wherein, n equals 1,2 or 3;

Glycosyl is monose or disaccharides;

Described monose is glucose, semi-lactosi, wood sugar, rhamnosyl, seminose, ribose, glucosamine or acetylglucosamine;

Described disaccharides is maltose or lactose.

For realizing above-mentioned second object, the technical scheme that the present invention takes is:

A preparation method for glycopeptide class antifungal compound, the method comprises the following steps:

A) key intermediate glycosyl amino acid: taking chloroethanol, chloroethoxy ethanol as raw material, make corresponding side chain nitrine ethanol and nitrine ethoxy ethanol by azido reaction, then received on corresponding acetyl sugar and obtain key intermediate, in key

Azido-in mesosome is reduced into after amino, with the aspartic acid condensation of protection, then through a step deprotection, obtains key intermediate glycosyl amino acid;

B) prepare target glycopeptide class antifungal compound: first with the method for solid phase synthesis; glycosyl amino acid and other amino acid are coupled together to a synthetic straight chain octapeptide; then it is scaled off from solid-phase resin; in liquid phase, it is carried out to cyclization; then through two step deprotections, can obtain target compound.

For realizing above-mentioned the 3rd object, the technical scheme that the present invention takes is:

The application of a kind of glycopeptide class antifungal compound in preparation treatment fungi infestation medicine.

Described fungi is Candida albicans, cryptococcus neoformans, Candida glabrata, Candida parapsilosis, trichophyton, gypsum shape sporidiole bacteria or Candida albicans.

The invention has the advantages that:

Compound glycopeptide class antifungal compound of the present invention is to have introduced different glycosyls on the primary amino in Tunicyclin D structure, has solved the shortcomings such as Tunicyclin D poorly water-soluble; Meanwhile, on this Tunicyclin D molecule, introduce glycosyl and can strengthen the interaction between it and target enzyme, thereby improved anti-mycotic activity.Pharmacological evaluation proves, compound of the present invention shows stronger inhibition activity to part fungi, compared with the antifungal drug of existing clinical application, has the advantages such as efficient, low toxicity, has a broad antifungal spectrum, therefore can be used for preparing antifungal drug.

Embodiment

Below embodiment provided by the invention is elaborated.

The high resolution mass spectrum of embodiment 1 the compounds of this invention 1a-1g and nucleus magnetic hydrogen spectrum data

The high resolution mass spectrum of the compounds of this invention 1a-f and nucleus magnetic hydrogen spectrum data are in table 1 and table 2.

The high resolution mass spectrum table of table 1 the compounds of this invention 1a-f

Table 2 the compounds of this invention 1a-fnucleus magnetic hydrogen spectrum data sheet

Embodiment 2the compounds of this invention 1a-gpreparation

2.1 the compounds of this invention 1a-fpreparation can slightly be divided into two steps:

2 .1.1 prepare key intermediate glycosyl Acibenzolar 5a-g, reaction scheme is as follows:

2.1.2 prepare target compound 1a-g, reaction scheme is as follows:

2.2 the compounds of this invention 1a- gconcrete preparation method is as follows:

2.2.1 the preparation of nitrine side chain

Take sodiumazide (45 g, 0.69 mol) in 250 mL three-necked bottles, add ethylene chlorhydrin (32 mL, 0.58 mol), under mechanical stirring, add catalyzer Tetrabutyl amonium bromide (TBAB, 4 g), reflux 12 h, reaction solution becomes brown suspension from a khaki color suspension.Mention coolingly, add anhydrous diethyl ether 60 mL, suction filtration, filter cake washs with 30 mL anhydrous diethyl ethers, merging filtrate.Decompression is revolved in filtrate must obtain brown liquid by ether, and underpressure distillation, obtains product chloroethanol, is a colourless liquid, and sealing is preserved, for subsequent use.

Intermediate 3a- gpreparation

We are with compound 3cfor row, specifically introduce its synthetic method, the synthetic method of other compounds therewith method is basic identical, by acetylizad glucose 2c(10g, 25.62 mmol) and 4 molecular sieve 10g be placed in 250 mL eggplant-shape bottles, vacuumize, argon shield, under condition of ice bath, add anhydrous methylene chloride 100 mL, stir after 10 min, dropwise add 2-nitrine ethanol (3.32 g, 38.42 mmol), continue to stir after 30min, dropwise add boron trifluoride diethyl etherate (BF3.Et2O, 8.0 mL, 64.05 mmol), continue to stir after 1 h, remove ice bath, stirring at room temperature is reacted after 8 h, TLC (sherwood oil: ethyl acetate=1:1) checks, react complete, so suction filtration, filter cake washed with dichloromethane, then filtrate is poured in saturated NaHCO3, till being stirred to and not producing bubble, separate organic layer, organic layer is respectively washed once with saturated NaHCO3 and saturated NaCl again, anhydrous Na 2SO4 is dry, after concentrating under reduced pressure, cross purification by silica gel column chromatography, developping agent is sherwood oil and ethyl acetate, after purifying, obtain intermediate 3c, be white powder solid, weigh 8.6 g, yield 80 %.

Glycosyl amino acid 4a- gsynthetic

We are with compound 4cfor row, specifically introduce its synthetic method, the synthetic method of other compounds therewith method is identical.Take dried nitrine acetyl glucosamine 3c(1.0 g, 2.40 mmol) are in 100 mL eggplant-shape bottles, after adding anhydrous methylene chloride 10 mL to make it to dissolve completely, add again 10% Pd-C(150 mg, w/w=15%) and methyl alcohol 40mL, vacuumize, be filled with hydrogen, after stirring at room temperature 1 h, TLC (methylene dichloride: methyl alcohol=20:1) checks, reacts complete, so suction filtration, after filtrate concentrating drained, obtain an oily matter, not purified, directly carry out next step reaction.Take 1-hydroxy benzo triazole (HOBT, 476 mg, 3.52 mmol) in 25mL eggplant-shape bottle, after adding dry DMF 0.5 mL to make it to dissolve completely, add again Fmoc-L-Asp-1-OtBu(1.156g, 2.8mmol) with anhydrous methylene chloride 5mL, after dissolving completely under vibration, drip DIC (0.6 mL, 3.52 mmol), after 10 min, adularescent solid is separated out, vibrate after 40 min and be filtered in the 50mL eggplant-shape bottle that the thick product of step is housed, add methylene dichloride 20 mL, sealing, vacuumize, stirred overnight at room temperature under argon shield.Next day, TLC (methylene dichloride: methyl alcohol=20:1) inspection, reacts complete, so reaction solution washes twice with saturated sodium carbonate solution, saturated sodium chloride solution is washed one time, anhydrous sodium sulfate drying.After concentrated, through silica gel column chromatography, obtain intermediate glycosyl amino acid 4c, be a white foam shape thing, weigh 920 mg, two step total recovery 49 %.

Key intermediate glycosyl amino acid 5a- gpreparation

We are with compound 5cfor row, specifically introduce its synthetic method, the synthetic method of other compounds therewith method is identical.Take glycosyl amino acid 4c(500 mg; 0.64 mmol) in 50 mL eggplant-shape bottles; add the methylene dichloride that now prepares and mixing solutions (v/v=5:1) 30 mL of trifluoroacetic acid; under argon shield after stirring at room temperature 4 h; TLC (methylene dichloride: methyl alcohol=20:1) checks; react complete, so concentration of reaction solution revolves trifluoroacetic acid with toluene band.After concentrated, through silica gel column chromatography, obtain the intermediate glycosyl amino acid of deprotection 5c, be a white foam shape thing, weigh 370 mg, yield 79%.

Straight chain glycopeptide 6a- gsynthetic

This walks us and adopts the method for solid phase synthesis to complete the synthetic of linear peptides.Below taking linear peptides Tunicyclin D(T-1) as example, introduce in detail its synthetic method, straight chain glycopeptide 6a- gsynthetic herewith method, just change amino acid Fmoc-L-Asp (Trt)-OH into corresponding glycosyl amino acid 5a- g.First be to connect first amino acid to resin: take 2-chlorine trityl chloride resin (200 mg; 0.26 mmol) and Fmoc-L-Gly-OH (89 mg; 0.30 mmol) in solid phase oscillator tube; add methylene dichloride 5 mL; under oscillating condition, drip diisopropylethylamine (DIPEA) 0.5 mL; after room temperature oscillatory reaction 1 h; suction filtration; bonding has amino acid whose resin to wash three times with methyl alcohol and methylene dichloride vibration successively; drain, then carry out the Fmoc protection in deaminizating acid.Secondly de-Fmoc protection: upwards walk DMF and piperidine solution (v/v=4:1) that dried solid phase synthesis oscillator tube adds 5 mL to prepare, after room temperature oscillatory reaction 2 h, suction filtration, methylene dichloride vibration washing three times for resin, drains for subsequent use.Then connect second amino acid: take successively Fmoc-L-His (Trt)-OH (248 mg; 0.4 mmol), HOBT (81 mg; 0.6 mmol), HBTU (228 mg; 0.6 mmol) in 25 mL eggplant-shape bottles; add 5 mL DMF to make it to dissolve completely; lower DIPEA (0.2 mL that drips of vibration; 1.2 mmol); after room temperature vibration shakes up, added in the dried resin of supreme step, after room temperature oscillatory reaction 4 h; suction filtration; resin washs after three times with DMF and methylene dichloride vibration successively, drains, de-Fmoc protection.We access Fmoc-L-Trp-OH successively with aforesaid method, Fmoc-L-Pro-OH, Fmoc-L-Pro-OH, Fmoc-L-Ile-OH, Fmoc-L-Asp (Trt)-OH, Fmoc-L-Val-OH, after de-Fmoc protection, to the acetic acid/trifluoroethanol/dichloromethane solution (v/v/v=1:2:16) that adds 5 mL now to join in solid phase synthesis oscillator tube, room temperature 1 h that vibrates, then by eggplant-shape bottle clean solution filter to, methylene dichloride vibration washing three times for resin, merging filtrate is also concentrated, between diakinesis, revolve acetic acid with toluene band, the oily matter finally obtaining first dissolves with appropriate methylene dichloride, after adding again sherwood oil to solidify, be spin-dried for to obtain a white powder thing, it is straight chain octapeptide t-1.

The cyclization of linear peptides ( t-2with 7a- g)

This step completes in liquid phase, below taking linear peptides Tunicyclin D(T-1) as example, introduce in detail its cyclization method, other straight chain glycopeptides 6a- gherewith method of cyclization.Take successively HOBT (147 mg, 1.09 mmol), PyBOP (567 mg, 1.09 mmol) in 1 L eggplant-shape bottle, add methylene dichloride 306 mL, under agitation condition, drip DIPEA (360 μ L, 2.18 mmol), after 0 DEG C of stirring reaction 10 min, by constant pressure funnel, linear peptides solution 306 mL (linear peptides 306 mg, 0.218 mmol that slowly dropping has prepared; Anhydrous methylene chloride 306 mL), after dripping off, move to stirred overnight at room temperature, next day, concentration of reaction solution, crosses gel filtration chromatography and obtains the not cyclic peptide Tunicyclin D of deprotection, i.e. and T-2, is a yellow foam.

Deprotection

This step is carried out in two steps, first sloughs the unsettled trityl of acid (Trt) protecting group, and then sloughs the ethanoyl protecting group on sugar.The method of sloughing of Trt we with T-3, target cyclic peptide Tunicyclin D is example, specifically introduces working method, other compounds 8a- gsynthetic herewith method; to be placed in 50 mL eggplant-shape bottles through the T-2 of gel filtration chromatography purifying; add methylene dichloride/trifluoroacetic acid solution (v/v=5:1) 24 mL of existing preparation; under argon shield, after stirring at room temperature 5 h, TLC (methylene dichloride: methyl alcohol=10:1) checks; react complete; so revolve trifluoroacetic acid three times with toluene band, be spin-dried for the rear gel filtration chromatography of first crossing and carry out preliminary purification, carry out purifying after HPLC.By crude product 8cbe placed in 50 mL eggplant-shape bottles, adding pH is sodium methylate/methanol solution of 8, stirred overnight at room temperature, next day, ESI-MS detected, react complete, then with acetic acid adjust pH be about 7, concentrate under reduced pressure at low temperature, revolve gained oily matter after acetic acid with toluene band, first cross gel filtration chromatography and carry out preliminary purification, after preparative HPLC purifying, moving phase is acetonitrile and water (0.1% trifluoroacetic acid, v/v=20:80-80:20), after lyophilize, obtain white powder 1c.

Embodiment 3the pharmacological evaluation of the compounds of this invention

3.1 experimental techniques: the Herbs By Broth Microdilution that adopts the U.S. (Clinical and Laboratory Standards Institute, the CLSI) CLSI-M27A3 of Association for Standardization of clinical labororatory and M38-A2 file to recommend.

3.1.1 experimental strain

This experiment has selected the common human body cause illness's standard fungal bacterial strain of following 6 kind of 7 strain as screening object:

Two strain ATCC type strains:

Candida albicans (Candida albicans) SC5314

Cryptococcus neoformans (Cryptococcus neoformans) 32609

Five strain clinical strains:

Candida glabrata (Candida krusei) 537

Candida parapsilosis (Candida parapsilosis) 22019

Trichophyton (Trichophyton rubrum) Cmccftla

Gypsum shape sporidiole bacteria (Microsporum gypseum) Cmccfmza

Candida albicans (Candida albicans) Y0109

3.1.2 test method

Bacteria suspension preparation: above-mentioned candidiasis is cultivated 24 hours through 35 DEG C of SDA substratum, filamentous fungus was through PDA substratum activation 7 days, and twice activation, with blood cell counting plate counting, adjusts bacteria concentration to 1 × 10 with RPMI 1640 liquid nutrient mediums ³-2 × 10 ⁴individual/mL.

Liquid preparation: get testing compound of the present invention and be dissolved in methyl-sulphoxide, be made into the medicine storage liquid of 6.4 mmol/L ,-70 DEG C of preservations.Before experiment, be diluted to 640 μ mol/L with RPMI 1640 substratum.

Inoculation: 96 No. 1, orifice plate holes add RPMI 1,640 200 μ l and make blank, 3-12 hole respectively adds RPMI 1,640 100 μ l, No. 2 hole adds RPMI 1,640 180 μ l and 640 μ mol/L liquid 20 μ l, 10 grades of doubling dilutions of drug level in 2-11 hole, each hole drug level is respectively 64,32,16,8,4,2,1,0.5,0.25,0.125 μ mol/L, No. 12 hole does not add liquid, makes positive control.Medicine contrast is fluconazole (FCZ.).The bacteria suspension preparing is seeded to 2nd ~ No. 12 holes of 96 orifice plate with multichannel pipettor by 100 μ l bacterium liquid, and final inoculum density is 0.5 × 10 ³-2.5 × 10 ³cFU/ml; Last row of 96 orifice plates is Quality Control bacterial strain Candida parapsilosis ATCC22019.

Cultivate and detect: 96 orifice plates of inoculating are left standstill and cultivated after 1-7 days in 35 DEG C, observations.If positive control hole optical density value (OD value) is 100%, taking optical density value than positive control hole lower than 80% lowest concentration of drug as minimal inhibitory concentration value (MIC80).

3.2 experimental result

In Vitro Bacteriostasis experimental result is in table 3.

Table 3 compound 1 of the present invention a- gin Vitro Anti fungi experimental result

As seen from the above table, the compounds of this invention has and suppresses active the selected fungi of major part, although glycosyl derivatives molecular weight increases, but the target compound of synthesized has kept the vitro inhibition activity to part bacterial strain substantially, some compound is if 1c, 1e and 1f isoreactivity are than control compound Tunicyclin D better effects if, and therefore the compounds of this invention can be used for preparing new antifungal drug.

The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the inventive method; can also make some improvement and supplement, these improvement and the supplementary protection scope of the present invention that also should be considered as.

Claims (4)

1. the glycosylated derivative of a class ring octapeptide Tunicyclin D, is characterized in that, the glycosylated derivative general structure of described Tunicyclin D is as follows:

Wherein, n equals 1,2 or 3;

Glycosyl is monose or disaccharides;

Described monose is glucose, semi-lactosi, wood sugar, rhamnosyl, seminose, ribose, glucosamine or acetylglucosamine;

Described disaccharides is maltose or lactose.

2. a preparation method for glycopeptide class antifungal compound as claimed in claim 1, is characterized in that, the method comprises the following steps:

A) key intermediate glycosyl amino acid: taking chloroethanol, chloroethoxy ethanol as raw material, make corresponding side chain nitrine ethanol and nitrine ethoxy ethanol by azido reaction, then received on corresponding acetyl sugar and obtained key intermediate, azido-in key intermediate is reduced into after amino, aspartic acid condensation with protection, through a step deprotection, obtain key intermediate glycosyl amino acid again;

B) prepare target glycopeptide class antifungal compound: first with the method for solid phase synthesis; glycosyl amino acid and other amino acid are coupled together to a synthetic straight chain octapeptide; then it is scaled off from solid-phase resin; in liquid phase, it is carried out to cyclization; then through two step deprotections, can obtain target compound.

3. the application of glycopeptide class antifungal compound as claimed in claim 1 in preparation treatment fungi infestation medicine.

4. application as claimed in claim 1, is characterized in that, described fungi is Candida albicans, cryptococcus neoformans, Candida glabrata, Candida parapsilosis, trichophyton, gypsum shape sporidiole bacteria or Candida albicans.