CN1831006A - Sugar substituted MAAGn synthesis, 99Tcm or 186Rc tag and tag in application of radiation medicine for treating tumor - Google Patents

Abstract

The invention relates to producing the 99TCm or 186Re tag of the 1-suger replacing MAAGn with the good effect of the knub raster display; it can't be determined the frame using the spectroscopy because of the instability of the tag (2). So the invention also deals with the composing of the 1-suger replacing MAAGn and provides the molecule of 1-suger replacing MAAGn. The molecule of 1-suger replacing MAGn of the 186Re tag is served as the therapeutical agent of curing the knub. The 99mTc-MAG3-AceA-Gla is better then the 99mTc-ECDG regarding the absorbing in the knub; Thereinto, R1 is H, OH, OAC, R2 is OH, H, OAC, (R'=H,Ac), (R'=H, Ac), R3 is OH, OAC, NHAC, R4 is OH, OAC, m is 0, 1, 2; n is 0, 1, 2.....20; M is 99Tcm, 186Re.

Description

1-sugar for MAAGn synthetic, 99Tc mOr 186The tumour radiotherapy medicinal application of Re mark and marker thereof

Technical field:

The present invention relates to have good tumor imaging, the 1-sugar of oncotherapy effect is for MAAGn's ⁹⁹Tc ^mWith ¹⁸⁶The preparation of Re marker and 1-sugar thereof are synthetic for the MAAGn molecule, because of containing radioactivity ⁹⁹Tc ^mWith ¹⁸⁶Re tag structure instability, its structure are difficult to determine by Wave Spectrum, so the present invention relates to molecule and their radioactivity ⁹⁹Tc ^mWith ¹⁸⁶The Re marker.Therefore the invention still further relates to 1-sugar synthesizing for MAAGn molecule (1). ¹⁸⁶The azathioprine peptide molecule of Re mark is a tumor therapeutic agent, ¹⁹⁹Tc ^mThe azathioprine peptide molecule of mark is a tumor developer, its tumor imaging ability is better than at present prospect very much, just clinical study ¹⁹⁹Tc ^mMark-ECDG tumor developer.

Background technology:

The early diagnosis of tumour is the key that improves patient's curative ratio, yet present diagnosing tumor medicine defective is a lot, and therefore the research and development of good tumor developer are imperative.Sugar is the basic substance and the architecture basics of life, is the basic of all vital movements, is the ideal information carrier, the division and the differentiation of control cell, and the growth of regulating cell participates in cell recognition and molecular recognition with old and feeble.Glycan molecule also has very strong target except the biological activity with self, be a kind of ideal pharmaceutical carrier.Bio-active group links to each other with sugar, by the synergy of the two, improves the target of medicine on the one hand, increase bioavailability of medicament, improve curative effect, reduce poisonous side effect of medicine, bio-active group has also activated sugared biological activity on the other hand, has strengthened sugared pharmaceutical use.So sugar can be modified the material of biologically active, target administration optionally not only solves the thorny problem of pharmacological agent poor selectivity, can also slowly discharge the drug effect group, plays the effect of control or slowly-releasing.During the nearly last ten years, molecular peptide is not only the bifunctional linking reagent of four-coordination part, also is the focus of present tumor developer research.Sugared intermediate being incorporated into the important measures that become drug research in the compound of biologically active, because they are special carriers of medicine and gene, is one of important modification mode of transforming on the biologically active polypeptides skeleton.Therefore we design and have synthesized 1-sugar for the MAAGn molecule, and further obtain radioactivity ⁹⁹Tc ^mWith ¹⁸⁶The Re marker, the tumor developer of having found to have good tumor imaging ability.

Summary of the invention:

The present invention also provides and contains The compounds of this invention as part, and this part can be determined structure by Wave Spectrum, and the integral part that closely links to each other with structural formula (2) [structural formula (2) is unstable because of containing radionuclide, is difficult to measure by spectrogram].

R wherein ¹Be H, OH, OAC, R ²For OH, H, OAC,

R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, R ⁵Be H, Tr, m is 0,1,2, and n is 0,1,2 ... 20.

R wherein ¹Be H, OH, OAC, R ²For OH, H, OAC, R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, m is 0,1,2, and n is 0,1,2 ... 20, M is ⁹⁹Tc ^m, ¹⁸⁶Re.

Compound of the present invention (1) is following compound, wherein R ²During for H, R ¹Be H, OH, OAC, R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, R ⁵Be H, Tr, m is 0,1,2, and n is 0,1,2 ... 20.

Compound of the present invention (1) is following compound, wherein R ²During for OH, R ¹Be H, OH, OAC, R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, R ⁵Be H, Tr, m is 0,1,2, and n is 0,1,2 ... 20.

Compound of the present invention (1) is following compound, wherein R ²During for OAC, R ¹Be H, OH, OAC, R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, R ⁵Be H, Tr, m is 0,1,2, and n is 0,1,2 ... 20.

Compound of the present invention (1) is following compound, wherein R ²For

The time, R ¹Be H, OH, OAC, R ³Be OH, OAC, NHAC, R ⁴Be OH, OAAC, R ⁵Be H, Tr, m is 0,1,2, and n is 0,1,2 ... 20.

Compound of the present invention (1) is following compound, wherein R ²For

The time, R ¹Be H, OH, OAC, R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, R ⁵Be H, Tr, m is 0,1,2, and n is 0,1,2 ... 20.

Compound of the present invention (1) is following compound, wherein R ²For

The time, R ¹Be H, OH, OAC, R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, R ⁵Be H, Tr, m is 0,1,2, and n is 0,1,2 ... 20.

Compound of the present invention (1) is following compound, wherein R ²For The time, R ¹Be H, OH, OAC, R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, R ⁵Be H, Tr, m is 0,1,2, and n is 0,1,2 ... 20.

Compound of the present invention (2) is following compound, wherein M= ⁹⁹Tc ^mThe time, R ¹Be H, OH, OAC, R ²For OH, H, OAC,

R ³Be OH, OAC, NHAC, R ⁴For OH, OAC, m are 0,1,2, n is 0,1,2 ... 20,

Compound of the present invention (2) is following compound, wherein M= ¹⁸⁶During Re, R ¹Be H, OH, OAC, R ²For OH, H, OAC,

R ³Be OH, OAC, NHAC, R ⁴For OH, OAC, m are 0,1,2, n is 0,1,2 ... 20,

The present invention also provides the application of preparation formula (1), (2) compound or its pharmacy acceptable salt, complex compound and corresponding pharmacological action.The preparation method may further comprise the steps:

Reaction (a):

Above-mentioned reaction (a), by earlier with compound (a1)-SH implements protection, hydroxyl activated into active ester (a2) again, forms peptide chain (a3) with the amino acid reaction again under alkaline condition, at last with glycine dipeptidase reaction formation little peptide part (a4)

The reaction of preparation compound (a2) in organic solvent (as: methylene dichloride, chloroform), generates TrSCH2COOH with compound (a1) and equimolar triphenyl muriate reaction earlier usually, and this reaction was preferably carried out one hour in room temperature.

Generate TrSCH2COOH in the organic solvent (as: methylene dichloride, chloroform, tetrahydrofuran (THF)) and condensing agent (as: DCC) of routine, with equimolar NHS react compound (a2).

This reaction is arrived room temperature reaction 18 hours preferably at anhydrous tetrahydro furan in zero degree.

Gained compound (a2) is (as: sodium hydroxide, salt of wormwood, yellow soda ash) under alkaline condition, with amino acid react compound (a3).

This reaction preferably in aqueous sodium hydroxide solution, between the control reaction temperature 50-60 degree, refluxed two hours.

Through activated carboxylic, last and glycine dipeptidase reaction forms little peptide part (a4) with compound (a3) in repetitive operation.

Reaction (b):

Form compound (b2) by the protection of saccharide compound (b1) being carried out hydroxyl, transfer the sugar that 1 halogen replaces then to

In organic solvent, transfer corresponding nitrine sugar (b4) to again, be reduced to aminosugar (b5) at last with the trinitride reaction.

The protection of hydroxyl comprises: become ester protection (as: acetic anhydride, Acetyl Chloride 98Min., carbonic ether, sulphonate), become ether protection (as: benzyl oxide, three methyl-phenoxides, dihydropyrane), acetal ketal protection (as: acetone).

(as: DMF THF) transfers corresponding nitrine sugar (b4) to the trinitride reaction to the compound that makes (b3) in organic solvent.

Reaction was preferably at room temperature reacted 12 hours with 1 mole compound (b3) and 1.5 moles trinitride.

Gained compound (b4) is reduced to aminosugar (b5).

Reduction comprises: Pd/C, Ph ₃P/H ₂

Reaction preferably in organic solvent THF, is made reductive agent with Pd/C, room temperature reaction 3 hours.

Reaction (c):

Form compound (c2) by the protection of saccharide compound (c1) being carried out hydroxyl; transfer the sugar (c3) that 1 halogen replaces then to; in organic solvent, transfer corresponding nitrine sugar (c4) to again, last catalytic hydrogenation transition bit aminosugar (c5) with the trinitride reaction.

The protection of hydroxyl comprises: become ester protection (as: acetic anhydride, Acetyl Chloride 98Min., carbonic ether, sulphonate), become ether protection (as: benzyl oxide, three methyl-phenoxides, dihydropyrane), acetal ketal protection (as: acetone).

(as: DMF THF) transfers corresponding nitrine sugar (b4) to the trinitride reaction to the compound that makes (c3) in organic solvent.

Reaction was preferably at room temperature reacted 12 hours with 1 mole compound (c3) and 1.5 moles trinitride.

Gained compound (b4) is reduced to aminosugar (b5).

Reduction comprises: Pd/C, Ph ₃P/H ₂

Reaction preferably in organic solvent THF, is made reductive agent with Pd/C, room temperature reaction 3 hours.

Reaction (d):

Form compound (d2) by the protection of saccharide compound (d1) being carried out hydroxyl; transfer the sugar (d3) that 6 halogens replace then to; in organic solvent, transfer corresponding nitrine sugar (d4) to again, last catalytic hydrogenation transition bit aminosugar (d5) with the trinitride reaction.

The protection of hydroxyl comprises: become ester protection (as: acetic anhydride, Acetyl Chloride 98Min., carbonic ether, sulphonate), become ether protection (as: benzyl oxide, three methyl-phenoxides, dihydropyrane), acetal ketal protection (as: acetone).

(as: DMF THF) transfers corresponding nitrine sugar (d4) to the trinitride reaction to the compound that makes (d3) in organic solvent.

Reaction was preferably at room temperature reacted 12 hours with 1 mole compound (d3) and 1.5 moles trinitride.

Gained compound (b4) is reduced to aminosugar (b5).

Reduction comprises: Pd/C, Ph ₃P/H ₂

Reaction preferably in organic solvent THF, is made reductive agent with Pd/C, room temperature reaction 3 hours.

Reaction (e):

Reaction (f):

Reaction (g):

Reaction (h):

Above-mentioned reaction (e) (f), (g), is in the presence of the peptide condensing agent (h), and aminosugar and little peptide part form sugar for MAAGn.

The peptide condensing agent comprises: BOP and DIPEA, DCC and DMAP

Reaction is preferred with condensing agent BOP and DIPEA, reacts 5 hours.

Embodiment:

Further specify the present invention by the following examples, but do not limit the present invention.

Embodiment 1 Tr-MAAG ₂-Glu (Ace) ₄

Following sugared structure abbreviates as:

(1) preparation of little peptide part:

The activation of carboxyl

At the 20mL there-necked flask, load onto prolong and the thermometer of being furnished with drying tube, stir adding TrCH down ₂The anhydrous THF solution (4mL) of COOH (3mmol) and NHS (3mmol), cryosel are bathed and to be cooled to-5～0 ℃, after the 10min, add anhydrous THF (3mL) solution of DCC (3mL) gradually, keep then-5-0 ℃ reaction 2 hours, have a large amount of white precipitates to generate.Continue at room temperature to stir stopped reaction 16 hours.Filter precipitation, use cold CH ₂Cl ₂Thorough washing revolves and steams solvent, gets thick product.Recrystallization in ethyl acetate gets colourless rib shape crystal 1.10g, productive rate 91%.M.p.177.5-178.3 ℃, IR (KBr, cm ^-1): 3070 (w), 1790,1745 (active ester characteristic peaks): 1630,1580,1495,1445.

Compound Tr-MAAG's is synthetic

In the 500mL flask of prolong and whipping appts is housed, add active ester (0.012mol) and 250mL acetonitrile, slow elevated temperature after the stirring and dissolving.Be controlled between 50-60 ℃, slowly add the 0.2MNaOH aqueous solution of amino acid (0.012mol), 2h then refluxes.TLC follows the tracks of reaction, and raw material disappears.Stopped reaction, solution become neutral.Use rare H behind the ice bath ₂SO ₄Regulate pH value and be about 1, obtain white precipitate.

Compound Tr-MAAG ₂

By synthetic logical method, active ester 5g (0.012mol) and excessive Gly react.Use re-crystallizing in ethyl acetate, get white crystal 5.1g, productive rate 91.0%.IR(KBr，cm ^-1)：3350(s)，3100-2500(w)，1740，1640

¹H NMR(500MHz，DMSO)，δ(ppm)：12.15(1H，br，COOH)，8.27(1H，NH)，7.37-7.26(15H，m，Ar)，3.67(2H，d，J＝4.7Hz，CH ₂)，2.82(2H，s，CH ₂)

The activation once more of carboxyl

Be equipped with thermometer, in the 25mL there-necked flask of prolong (adding drying tube) and agitator, adding previous step product 0.002mol, NHS (0.002mol) and anhydrous THF (7.0mL) stir 10min under the room temperature.Ice bath is cooled to 0-5 ℃, adds anhydrous THF (4mL) solution of DCC (0.002mol), white precipitate occurs, and holding temperature continues reaction 2h for 0～5 ℃ then, and stirring is spent the night, termination reaction.Filtration is spin-dried for, and after the ether development, washs with massive laundering.Oven dry then.IR shows 1820cm ^-1, 1780cm ^-1And 1740cm ^-1The feature ester absorption peak that active ester is arranged.

Synthesizing of tetradentate ligands

Tr-MAAG ₂: triphenyl thioacetyl-triglycine

In the 100mL flask that is equipped with prolong and agitator, add active ester (0.028mol) and the 60mL acetonitrile of Tr-MAG, be warming up to then about 50 ℃, slowly add the 0.2MNaOH aqueous solution of equimolar glycine dipeptidase.Reflux 2h, TLC follows the tracks of reaction.Raw material disappears, and solution is alkalescence, adds the entry dilution, regulates about pH value to 1 with dilute sulphuric acid again, has a large amount of white precipitates to produce, and collects solid.Productive rate 70%.Tr-MAG ₃：C ₂₇H ₂₇N ₃O ₅S FW：505

(2) osamine is synthetic

2,3,4,6-four-O-ethanoyl-1-glucosamine

In the reaction flask of 100mL, add the 20mL acetic anhydride, dropwise add 103 μ L perchloric acid (70%) under 4 ℃, return to room temperature, add 5g (27.75mmol) 1a in batches, 30～40 ℃ are reacted 30min down and obtain 2a, the 2a mixed solution is cooled to 20 ℃, add 1.55g red phosphorus, slowly add 2.9mL liquid bromine with dropping funnel, keep temperature of reaction to be no more than 20 ℃, approximately adding 3.6mL distilled water in the 30min then, keep temperature of reaction to be lower than 20 ℃, add the back and return to room temperature reaction 2h naturally, TLC detection reaction process, after reaction finishes, with 30mL methylene dichloride dilute reaction solution, with sand filter funnel insolubles is filtered out, with a small amount of dichloromethane rinse reactor and funnel, merging filtrate and washing lotion, wash fast with 20mL * 2 frozen water, then organic layer being changed over to 25mL is stirring to be neutralized in the sodium bicarbonate frozen water liquid and is no longer producing till the bubble, with separating funnel water layer is divided to fall, the organic layer anhydrous sodium sulfate drying filters, revolve to steam and obtain yellow oil, change oily matter over to mortar and grind up to the white solid powder with the mixed solution of sherwood oil and ether [V/V=1: 1] and occur, will wash drying behind the powder filter with a little ether, get 3a (9.30g), C with ether-sherwood oil recrystallization ₁₄H ₁₉BrO ₉FW:411 yield 81.5%, products therefrom instability directly carry out next step reaction.

5.0g (12.16mmol) 3a is dissolved among the 10mLDMF, adds 1.00g (15.38mmol) sodiumazide, react 12h under the room temperature, TLC detection reaction process, after reaction finishes, filter, filtrate is to going in a large amount of middle frozen water, a large amount of white precipitates appear, filter thick product 4a (3.43g), C ₁₄H ₁₉N ₃O ₉FW:373 yield 75.6% gets rib shape crystal with re-crystallizing in ethyl acetate, m.p.129～130 ℃.

500mg (1.34mmol) 4a is dissolved among the 43mLTHF, add 130mgPd/C, hydrogenation 3h under the room temperature, TLC detection reaction process, reaction finishes the back and with husky filter funnel Pd/C is filtered out, solvent is steamed except that obtaining white solid, get 326mg white needle-like crystals 5a yield 70.0%C with the dehydrated alcohol recrystallization ₁₄H ₂₁NO ₉FW:347, the products therefrom instability is directly carried out next step reaction.

IR(KBr，cm ^-1)：3411.3(NH ₂)1754.9，1734.0，993.1；

(3) the glycopeptide molecule is synthetic

Logical method: in the 25ml reaction flask, add the 10ml DMF of drying treatment, add little peptide part (10mmol), stir the 5min dissolving, add BOP reagent (15mmol), reaction 5min, add (10mmol) osamine and several DIPEA again, reaction 5h joins mixed solution in the 25ml frozen water mixed solution then, stir 5min, there are a large amount of white precipitates to generate, filter drying, column chromatography (methylene dichloride: methyl alcohol=10: 1), obtain target product.

β-N-trityl thioacetyl propionyl two glycyl-2,3,4, the tetra-acetylated glucose (Tr-MAAG of 6- ₂-Glu (Ace) ₄)

β-N-triphenylmethylthioacetyl alanyl diglycyl-2，3，4，6-tetra-O-acetyl glucose

By the logical method of reaction, and behind the column chromatography (methylene dichloride: methyl alcohol=10: 1), yield 53.2%C ₄₂H ₄₈N ₄O ₁₃S FW:848 m.p.:116～118 ℃

MS：m/z C ₄₂H ₄₈N ₄O ₁₃SNa[M+Na] ⁺871 found 871.0[α] _D ²⁰+12.5(c 1.1，CH ₃OH)

IR (cm ^-1): 3315.6 (NH), 3059.0 (Ar-H), 2936.2 (CH ₂), 1756.2 (ester groups), 1655.1 (acid amides I bands), 1523.2 (acid amides II bands), 907.2

¹H NMR(500MHz，DMSO-d ₆)d：8.62-8.60(d，1H，NH)，8.15-8.14(m，2H，NH)，8.06(m，2H，NH)，7.35-7.25(m，15H，Ar)，5.413-5.375(t，J＝9.5H，1H’，H-1’)，5.368-5.330((t，J＝9.5Hz，1H，H-3’)，4.920-4.880(t，J＝9.4，1H，H-4’)，4.874-4.836(t，J＝9.4，1H，H-2’)，4.18-4.10(m，3H，CH(NH)，H-6’，H-5’)，3.98-3.96((d，J＝12.4，H-6”)，3.70-3.69(br，，4H，CH ₂)，2.86(m，2H，H)，3.76-3.70(br，4H，CH ₂)，2.86(m，2H，SCH ₂)，2.00-1.93(m，12H，CO(CH ₃))，1.16-1.15(dd，J＝7.0，3H，CH ₃) ¹³C NMR(125MHz，DMSO-d ₆)d：170.5(1C＝O)，170.0-168.1(6C＝O)，168.0(1C＝O)，144.6(3C，Ar)，129.6(6C，Ar)，128.5(6C，Ar)，127.30(3C，Ar)，77.2(C ³)，72.6(C ⁵)，71.07(C2)，68.3(C ⁴)，66.5(CPh ₃)，62.2(C ⁶)，58.5(CH(NH))，42.2-42.1(2CH ₂)，36.2(SCH ₂)，30.72(CH(CH ₃) ₂)，21.0-20.8(CO(CH ₃))，19.6(CH(CH ₃) ₂)，18.5(CH(CH ₃) ₂)。

β-N-trityl thioacetyl propionyl two glycyl-2,3,4, the tetra-acetylated glucose (Tr-MAAG of 6- ₂-Glu)

IR (cm ^-1) 3360.9 (OH), 3059.0 (Ar-H), 2936.2 (CH ₂), 1655.1 (acid amides I bands) 1523.2 (acid amides II band)

¹H NMR(500MHZ，DMSO-d ₆)d：8.62-8.60(d，1H，NH)，8.15-8.14(m，2H，NH)，7.35-7.25(m，15H，Ar)，5.413-5.375(t，J＝9.5HZ，1H，H-1’)，5.368-5.330(t，J＝9.5HZ，1H，H-3’)，4.920-4.880(t，J＝9.4Hz，1H，H-4’)，4.874-4.836(t，J＝9.4HZ，1H，H-2’)，4.18-4.10(m，3H，CH(NH)，H-6’，H-5’)，3.98-3.96(d，J＝12.4，1H，H-6’)，3.80-3.75(m，4H，OH)，3.70-3.69(br，4H，CH ₂)，2.86(m，2H，SCH ₂)，1.16-1.15(dd，J＝7.0Hz，3H，CH ₃)

Embodiment 2 Tr-MMG ₂-Gla (Ace) ₄

(1) preparation of little peptide part:

Prepare Tr-MAAG as stated above ₂

(2) osamine is synthetic:

2,3,4,6-four-O-ethanoyl-1-galn

In the reaction flask of 100mL, add the 20mL acetic anhydride, dropwise add 103 μ L perchloric acid (70%) under 4 ℃, return to room temperature, add 5g (27.75mmol) 1b in batches, 30～40 ℃ are reacted 30min down and obtain 2b, the 2b mixed solution is cooled to 20 ℃, add 1.55g red phosphorus, slowly add 2.9mL liquid bromine with dropping funnel, keep temperature of reaction to be no more than 20 ℃, approximately adding 3.6mL distilled water in the 30min then, keep temperature of reaction to be lower than 20 ℃, add the back and return to room temperature reaction 2h naturally, TLC detection reaction process, after reaction finishes,, insolubles is filtered out with husky filter funnel with 30mL methylene dichloride dilute reaction solution, with a small amount of dichloromethane rinse reactor and funnel, merging filtrate and washing lotion are washed fast with 20mL * 2 frozen water, then organic layer are changed over to 25mL and are stirring to be neutralized in the sodium bicarbonate frozen water liquid and no longer produce till the bubble, with separating funnel water layer is divided to fall, the organic layer anhydrous sodium sulfate drying filters, and revolves to steam to obtain yellow oil, changing oily matter over to mortar grinds up to the white solid powder with the mixed solution of sherwood oil and ether [V/V=1: 1] and occurs, with the powder filter after drying, get 3b (9.10g), C ₁₄H ₁₉BrO ₉FW:411 yield 79.8%, products therefrom instability directly carry out next step reaction.

5.0g (12.16mmol) 3b is dissolved among the 10mLDMF, adds 1.00g (15.38mmol) sodiumazide, react 12h under the room temperature, TLC detection reaction process, after reaction finishes, filter, filtrate is to going in a large amount of middle frozen water, a large amount of white precipitates appear, filter thick product 4b (3.62g), C ₁₄H ₁₉N ₃O ₉FW:373 yield 79.8% gets rib shape crystal with re-crystallizing in ethyl acetate, m.p.99～100 ℃.

IR(KBr，cm ^-1)：2124.9(N ₃)，1750.8 1，1382.4，1224.4，1089.0

500mg (1.34mmol) 4b is dissolved among the 43mLTHF, adds 130mgPd/C, hydrogenation 3h under the room temperature, TLC detection reaction process, reaction finishes the back and with husky filter funnel Pd/C is filtered out, and solvent is steamed to remove obtain oily matter 5b C ₁₄H ₂₁NO ₉FW:347, the products therefrom instability is directly carried out next step reaction.

(3) glycopeptide is micromolecular synthetic

β-N-trityl sulfo-propionyl two glycyl-2,3,4, the tetra-acetylated semi-lactosi (Tr-MAAG of 6- ₂-Gla (Ace) ₄)

β-N-triphenylmethylthioacetyl alanyl diglycyl-2，3，4，6-tetra-O-acetyl glalacose

By the logical method of reaction, and behind the column chromatography (methylene dichloride: methyl alcohol=10: 1), yield 56.1%C ₄₂H ₄₈N ₄O ₁₃S FW:848., m.p.:117.9～119.5 ℃

MS m/z：C ₄₂H ₄₈N ₄O ₁₃SNa[M+Na] ⁺871 found 871.1[α] _D ²⁰+20.8(c 1.2，CH ₃OH)

IR (cm ^-1): 3325.9 (NH), 3059.0 (Ar-H), 2933.4 (CH ₂), 1751.6 (ester groups), 1653.3 (acid amides I bands), 1521.1 (acid amides II bands), 908.8

¹H NMR(500MHz，DMSO-d ₆)d：8.70-8.68(d，1H，NH)，8.14(m，2H，NH)，8.07-8.06(m，1H，NH)，7.35-7.26(m，15H，Ar)，5.36-5.34(t，J＝9.4，1H，H-1’)，5.29(m，2H，H-3’，H-4’)，5.06-5.04(t，J＝9.4，1H，H-2’)，4.34-4.31(t，J＝6.0Hz，1H，H-5’)，4.19-4.18(m，1H，CHNH)，4.06-4.04(dd，6.1Hz，11.3Hz，1H，H-6’)，3.98-3.95(dd，J＝6.8Hz，11.3Hz，1H，H-6”)，3.70-3.67(br，4H，CH ₂)，2.87(m，2H，SCH ₂)，2.10-1.91(m，12H，4CO(CH ₃))，1.16-1.15(dd，J＝7.0，3H，CH ₃)。

¹³C NMR(125MHz，DMSO-d ₆)d：172.7(1C＝O)，172.6-167.7(6C＝O)，167.6(1C＝O)，144.6(3C，Ar)，129.6(6C，Ar)，128.6(6C，Ar)，127.3(3C，Ar)，77.7(C ¹)，71.8(C ⁵)，71.3(C ⁴)，68.7(C ²)，68.0(C ³)，66.4(CPh ₃)，61.9(C ⁶)，48.9(CH(NH))，42.3(2CH ₂)，36.3(SCH ₂)，21.0-20.8(4CO(CH ₃)，18.4(CH ₃)。

β-N-trityl sulfo-propionyl two glycyl-semi-lactosi (Tr-MAAG ₂-Gla)

IR (cm ^-1) 3377.9 (OH), 3058.1 (Ar-H), 2928.7 (CH ₂), 1658.1 (acid amides I bands) 1533.9 (acid amides II band)

¹H NMR(500MHz，DMSO-d ₆)：8.48-8.46(m，1H，NH)，8.21-8.19(m，1H，NH)，8.16-8.15(d，1H，NH)，7.96(S，1H，NH)，7.36-7.25(m，15H，Ar)，4.86(m，1H，H-1’)，4.69-4.66(m，2H，H-3’，H-4’)，4.50(m，1H，H-6’)，4.20-4.17(m，.1H，H-5’)，3.79-3.69(m，4H，OH)，3.48-3.47(m，1H，CH(CH ₃))，3.43-3.37(m，4H，CH ₂)，2.89-2.83(m，2H，SCH ₃)，2.53-2.50(m，1H，CH(CH ₃))，1.24-1.14(m，3H，CH(CH ₃))

Embodiment 3 Tr-MAAG ₂-Mal (Ace) ₇

(1) preparation of little peptide part:

Prepare Tr-MAAG as stated above ₂

(2) osamine is synthetic:

2-N-ethanoyl-3,4,6-three-O-ethanoyl-1-glucosamine

In the reaction flask of 50mL, add the 10mL Acetyl Chloride 98Min., stir fast in 2～3min, to add down and react 16h under 5.0g (22.60mmol) the 1c room temperature, TLC detection reaction process, reaction finishes back adding 40mL chloroform in reaction system and dilutes, then in the beaker of going into to be equipped with 40g ice and 10mL water, vigorous stirring, the separating funnel separatory, organic layer is transferred to rapidly in the 40mL saturated sodium bicarbonate solution and neutralizes, the separating funnel separatory, the organic layer anhydrous sodium sulfate drying, above aftertreatment will be finished in 15min, and drying is finished after-filtration, rotation is evaporated to the about 7.5mL solvent of residue under 50 ℃, add rapidly anhydrous diethyl ether, begin to occur crystallization after about 30, place 12h to crystallization fully 3c (5.3g) C ₁₄H ₂₀ClNO ₈FW:365.7, yield 64.1%, the products therefrom instability is directly carried out next reaction.

4.6g (12.58mmol) 3c is dissolved among the 12.3mLDMF, adds the 1.31g sodiumazide, react 12h under the room temperature, TLC detection reaction process after reaction finishes, is filtered, under reduced pressure steam and remove DMF, residual looking into is dissolved in the 30mL chloroform, with 2.5mL * 3 frozen water washing, washing lotion 15mL * 2 chloroform back extractions, the combined chloroform layer is used anhydrous sodium sulfate drying, filters, rotary evaporation gets solid, carries out recrystallization with ethyl acetate-ether mixed solvent and gets 3.23g white needle-like crystals 3c C ₁₄H ₂₀N ₄O ₈FW:372 yield 69.0%, m.p.172～173 ℃.

500mg (1.34mmol) 4c is dissolved among the 43mLTHF, adds 130mgPd/C, hydrogenation 3h under the room temperature, TLC detection reaction process, reaction finishes the back and with husky filter funnel Pd/C is filtered out, and solvent is steamed to remove obtain oily matter 5c C ₁₄H ₂₂N ₂O ₈FW:346, the products therefrom instability is directly carried out next step reaction.

(3) glycopeptide is micromolecular synthetic

β-N-trityl thioacetyl propionyl two glycyl-seven ethanoyl maltose (Tr-MAAG ₂-Mal (Ace) ₇)

β-N-triphenylmethylthioacetyl alanyl diglycyl-sept-O-acetyl maltose

By the logical method of reaction, and behind the column chromatography (methylene dichloride: methyl alcohol=10: 1), yield 55.6%C ₅₄H ₆₄N ₄O ₂₁S FW:1136, mp:123～125 ℃

MS m/z：C ₅₄H ₆₄N ₄O ₂₁SNa[M+Na] ⁺1159found 1159.1[α] _D ²⁰+53.7(c 1.3，CH ₃OH)

IR (cm ^-1): 3376.2 (NH), 3060.0. (Ar-H), 2933.4 (CH ₂), 175156 (ester groups), 1653.2 (acid amides I bands), 1521.1 (acid amides II bands), 903.8.

¹H NMR(500MHz，DMSO-d ₆)d：8.56-8.54(d，1H，NH)，8.15-8.14(m，2H，2NH)，8.02(m，1H，2NH)，7.35-7.25(m，15H，Ar)，5.38-5.23(m，3H，J＝9.2Hz，H-4’，H-3’)，5.00(m，1H，H-3’)，4.90-4.88(dd，J＝8.0，10.1，Hz，1H，H-2”)，4.72(t，J＝9.5Hz，H-2’)，4.18-4.15(m，3H，CHNH，H-6”，H-5”)，4.04-3.68(m，5H，H-6”-2，H-6-5’，H-6-1’，H-6-2’，H-6-4’)，3.69-3.68(br，4H，2CH ₂)，2.87(m，2H，SCH ₂)，2.06-1.90(m，21H，7CO(CH ₃))，1.16-1.14(dd，J＝7.0Hz，3H，CH ₃)。

¹³C NMR(125MHz，DMSO-d ₆)d：172.7(1C＝O)，170.6-167.6(9C＝O)，167.6(1C＝O)，144.6(3C，Ar)，129.6(6C，Ar)，128.5(6C，Ar)，127.3(3C，Ar)，95.8(C-1”)，76.9(C-1’)，75.5(C-4’)，74.0(C-5’)，73.2(C-3’)，71.6(C-2’)，69.9(C-3”)，69.4(C-5”)，68.4(C-2”)，68.2(C-4”)，66.4(CPh ₃)，63.5(C-6”)61.8(C-6’)，48.9(CH(NH))，42.3(2CH ₂)，3639(SCH ₂)，21.1-20.7(7CO(CH ₃)，18.3(CH ₃)。

Utilize above-mentioned synthetic method can synthesize remaining target compound

As all target compounds in the structure (1):

R wherein ¹Be H, OH, OAC, R ²For OH, H, OAC,

R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, R ⁵Be H, Tr, m is 0,1,2, and n is 0,1,2 ... 20.

The check of deprotection, mark and radiochemicsl purity

Deprotection: in the 10mL flask, add sample 5.11 * 10 ^-5Mol and TFA (3mL) are behind the stirring 5min.Add HSiEt again ₃(1mL), solution is clarified immediately.Continue to stir 5min, revolve the steaming solvent, add the 4mL0.1MNaOH aqueous solution, use CH ₂Cl ₂Tritane is removed in extraction, the organic phase of skimming, the protection of water inflated with nitrogen.

The mark of part: adopt the direct labelling method preparation.The 10mg calglucon adds in the little penicillin bottle, with 100 μ L ligand solution (concentration: 10g.L ^-1), adding 50 μ L acetic acid-sodium acetate buffer solution then, the pH value of regulator solution is 4～5, adds 25 μ L tartrate-SnCl ₂Solution (tartrate: SnCl ₂=0.8mg/mL: 0.4mg/mL), the adding proper amount of fresh ⁹⁹Tc ^mO ₄ ^-React 30min under the leacheate, room temperature.Reaction can be finished.

The chromatography evaluation of marker and the check of radiochemicsl purity: as support, make developping agent with No. 1 filter paper of Xinhua, identify to record the radiochemicsl purity of marker all greater than 95% with physiological saline.

Biological activity

1) lotus sarcoma S ₁₈₀Experiment distributes in γ video picture and the body in the mouse body

Lotus sarcoma S ₁₈₀γ video picture in the mouse body: Zhi Bei Different Complex solution is as tail vein injection liquid as stated above.Every lotus sarcoma S ₁₈₀Mouse tail vein injection 100 μ L marking fluids (0.56-0.74MBq), the Kunming mouse body weight is 20-24g, different time utilizes GE MPR γ camera to carry out total body opacification after injection respectively.

Distribute in the body: lotus sarcoma S ₁₈₀The disconnected neck of different time is put to death behind the marking fluid of mouse tail vein injection Different Complex solution, gets blood and internal organs and carries out radiometry, and the weight of blood is calculated the uptake ratio (ID/%g of each tissue respectively in 7% of body weight ^-1) and the ratio of T and NT.

2) biological activity

Glycopeptide ^99mThe Tc marking target ^99mTc-Gal-MAAG ₂Distribution at mouse organ, blood and tumour S180 thereof sees Table 1, and clinical studies show is reasonable now ^9mThe Tc marker ^99mThe data of Tc-ECDG are listed in table 2, data presentation in the table ^99mTc-MAG ₃-AceA-Gla is better than in tumour ^99mThe absorption of Tc-ECDG, with the pertinent literature comparative result also be like this (DavidJ.Yang, Chang-Guhn Kim, Naomi R.Schechter, Imaging with 99mTc ECDC Targeted at theMultifunctional Glucose Transport System:Feasiility Study with Rodens, Radiology2003; 226:465-473); The ratio that absorption in tumour and other tissue absorb, ^99mTc-Gal-MAAG ₂With ^99mTc-ECDG is close, and the both can make the clear video picture of tumour, therefore ^99mTc-MAG _nThe class marker of-AceA-Gla has the possibility that becomes the novel tumor developer.

Table 1 ^99mTc-MAAG ₂-Gal lotus behind administration 2h has the distribution (%ID/g) of the mouse of S180 knurl

Tabl 1 Biodistribution of ^99mTc-MAAG ₂-Gal in mice bearing S180 tumor.(％ID/g)

Organ	ID	ID/g	T/NT
				The bladder wall	0.019±0.004	1.37±0.293	1.1±0.189
Stomach-intestinal kidney liver lung muscle heart spleen brain neoplastic hematologic disorder	0.249±0.133 0.296±0.074 4.79±1.01 12.3±1.65 0.392±0.106 1.99±0.462 0.107±0.047 0.104±0.023 0.048±0.006 l.840.336 1.22±0.497	0.461±0.278 1.03±0.209 14.4±2.55 5.94±0.919 1.81±0.395 0.262±0.061 0.875±0.237 0.489±0.077 0.117±0.016 1.38±0.253 1.46±0.164	3.8±1.36 1.44±0.167 0.103±0.009 0.25±0.042 0.828±0.144 5.75±1.21 1.73±0.289 3.02±0.379 12.6±1.36 1.07±0.098 1±0

Table 2, ^99mTc-ECDG has the distribution of the mouse of S180 knurl (%ID/g) lotus behind administration 2h

Tabl 2 Biodistribution of ^9mTc-ECDG in mice bearing S180 tumor.(％ID/g

Organ	Time after the injection
							15 minutes	30 minutes	1 hour	2 hours	4 hours	8 hours
	The heart	1.15±0.34	0.57±0.13	0.33±0.99	0.35±0.10	0.22±0.05							0.19±0.05
Liver spleen lung brain kidney intermuscular bone small intestine stomach neoplastic hematologic disorder							1.47±0.50 0.82±0.31 1.72±0.42 0.10±0.33 11.32±2.54 0.74±0.29 1.50±0.23 1.68±0.72 1.23±0.34 3.17±0.73 0.93±0.20	1.09±0.26 0.50±0.13 0.92±0.22 0.06±0.01 8.86±2.08 0.40±0.13 1.00±0.10 0.69±0.09 0.61±0.20 2.09±0.28 0.79±0.22	1.11±0.19 0.40±0.15 0.61±0.16 0.05±0.01 7.44±1.43 0.33±0.12 0.66±0.30 0.44±0.13 0.53±0.09 1.45±0.25 0.66±0.17	0.89±0.11 0.30±0.05 0.46±0.21 0.04±0.01 7.20±1.35 0.30±0.08 0.65±0.32 0.47±0.13 0.76±0.14 1.11±0.26 0.72±0.13	0.99±0.21 0.27±0.08 0.36±0.05 0.03±0.01 6.33±0.72 0.12±0.02 0.59±0.15 0.32±0.06 0.64±0.10 0.72±0.16 0.73±0.09	1.23±0.36 0.30±0.11 0.35±0.10 0.03±0.01 6.34±1.28 0.13±0.05 0.60±0.27 0.28±0.11 0.59±0.30 0.40±0.12 0.58±0.18

Claims (5)

1. the compound that has formula (1) structure is a mark ⁹⁹Tc ^mWith ¹⁸⁶The crucial ligand moiety of Re [being comprehensively to determine structure and the integral part that closely links to each other with structural formula (2) (structural formula (2) is difficult to measure by spectrogram because of the radioactivity instability)].

R wherein ¹Be H, OH, OAC, R ²For OH, H, OAC,

R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, R ⁵Be H, Tr, m is 0,1,2, and n is 0,1,2 ... 20.

2. the compound of claim (1)

R ²During for H, R ¹Be H, OH, OAC, R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, m is 0,1,2, and n is 0,1,2 ... 20.

R ²During for OH, R ¹Be H, OH, OAC, R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, m is 0,1,2, and n is 0,1,2 ... 20.

R ²During for OAC, R ¹Be H, OH, OAC, R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, m is 0,1,2, and n is 0,1,2 ... 20.

R ²For

The time, R ¹Be H, OH, OAC, R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, m is 0,1,2, and n is 0,1,2 ... 20.

R ²For

The time, R ¹Be H, OH, OAC, R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, m is 0,1,2, and n is 0,1,2 ... 20.

R ²For The time, R ¹Be H, OH, OAC, R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, m is 0,1,2, and n is 0,1,2 ... 20.

R ²For The time, R ¹Be H, OH, OAC, R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, m is 0,1,2, and n is 0,1,2 ... 20.

3. claim has the compound of structure (2), be by the compound of structure (1) through with ⁹⁹Tc ^mOr ¹⁸⁶Re mark and the instability that obtains, be difficult to determine by wave spectrum etc. the marker of its structure, they have good tumor imaging ability.

R wherein ¹Be H, OH, OAC, R ²For OH, H, OAC,

R ³Be OH, OAC, NHAC, R ⁴Be OH, OAC, m is 0,1,2, and n is 0,1,2 ... 20, M is ⁹⁹Tc ^m, ¹⁸⁶Re.

4. the compound of claim (2)

Compound of the present invention (2) is following compound, wherein M= ⁹⁹Tc ^mThe time, R wherein ¹Be H, OH, OAC,

R ²For OH, H, OAC,

R ³Be OH, OAC, NHAC, R ⁴For OH, OAC, m are 0,1,2, n is 0,1,2 ... 20,

Compound of the present invention (2) is following compound, wherein M= ¹⁸⁶During Re, R wherein ¹Be H, OH, OAC, R ²For OH, H, OAC,

R ³Be OH, OAC, NHAC, R ⁴For OH, OAC, m are 0,1,2, n is 0,1,2 ... 20.

5. the compound of claim (2) is applied to tumour radiotherapy medicine, M= ⁹⁹Tc ^mMarker is as tumor developer; M= ¹⁸⁶The Re marker is as tumor therapeutic agent.