CN102816197B - Novel pyrimidopyrimidine nucleoside analogue, preparation method thereof, supramolecular structure formed by novel pyrimidopyrimidine nucleoside analogue and application of novel pyrimidopyrimidine nucleoside analogue - Google Patents

Abstract

The present invention relates to a new pyrimidopyrimidine nucleoside analogue and its preparation method, in particular to the nucleoside analogue represented by Formula I, its preparation method and its application in anti-tumor and anti-virus, etc., belonging to Medicinal Chemistry field. The application of such compounds and their salts and the formed nanostructures in drug loading, regenerative medicine and catalysis.

Description

A new pyrimidopyrimidine nucleoside analogue, preparation method and its supramolecular structure and application

technical field

The invention relates to a novel pyrimidopyrimidine nucleoside analogue and a preparation method, and the application of the nanostructure formed by the compound in drug loading, regenerative medicine, catalysis and the like.

Background technique

In the field of nanotechnology, DNA and RNA molecules have been proved to be the easiest to tailor because of their good biological function, nanoscale, biocompatibility, biodegradability, molecular recognition ability, good thermal stability and controllable length. and general-purpose materials with a wide range of uses. Scientists have successfully used DNA and RNA to build a variety of amazing nanostructures, such as regular grids, origami, supramolecular aggregates, and even three-dimensional structures. In addition, deoxyribonucleosides and ribonucleosides, the basic building blocks of DNA and RNA, are also used to construct various supramolecular structures. In addition to the classic purine and pyrimidine nucleosides, structurally modified nucleosides have also been widely used to construct supramolecular structures of different shapes. In order to find better nanomaterials, we designed and synthesized a series of pyrimidopyrimidine nucleoside compounds with self-assembly ability for the first time, hoping that these compounds can provide a new way for the development of new drug delivery systems, and hope that As a scaffold material, it has new applications in regenerative medicine and catalysis.

Contents of the invention

The object of the present invention is to provide novel nucleoside analogues modified at the base or sugar moiety. Each group of the general formula (I) defined below will be described in more detail below:

Wherein A is selected from O, S, CH ₂ ; R ₂ , R _2' , R ₃ , and R _3' are independently selected from H, F, OH, NH ₂ , SH, CN, N ₃ and R, wherein R is lower Alkyl, lower alkenyl, lower alkynyl or lower acyl, and optionally contain at least one heteroatom; R ₄ is H, OH, NH ₂ , SH, CN, N ₃ , and R, wherein R is lower alkane group, lower alkenyl group, lower alkynyl group or lower acyl group, and optionally contains at least one heteroatom; R ₅ is H, OH, OR ₆ , SH, SR ₇ , R ₈ , OP(O)(OH) ₂ , OP(O)(OR") ₂ , SP(O)(OH) ₂ , SP(O)(OR") ₂ wherein R ₆ , R ₇ , R ₈ , and R" are functional groups; and B is selected from formula (II):

Wherein X, Y, Z, U are independently selected from H, halogen, SH, SR, NH ₂ , NHR, NHC(O)OR, OH or OR, wherein R is lower alkyl, lower acyl.

Wherein said lower alkyl, lower alkenyl, lower alkynyl or lower acyl refers to:

Lower alkyl groups include: methyl, ethyl, propyl, isopropyl, butyl and other alkyl groups within C18.

Lower olefins include: olefins within C18 such as ethylene and propylene.

Lower alkynyl groups include alkynes within C18 such as acetylene and propyne.

Lower acyl groups include: acyl groups within C18 such as acetyl groups.

The heteroatom refers to: O, S, N

The functional group refers to, for example, a fluorescent group (coumarin, fluorine).

At the same time, it should be noted that the above three substituents may be R, and when these three substituents are all R, the substituents may be different.

Specifically, the pyrimidopyrimidine nucleoside analogs are:

The object of the present invention is also to provide the preparation method of above-mentioned nucleoside;

The object of the present invention is also to provide the above-mentioned nucleoside compounds numbered 1-6 obtained by the following synthetic method;

The purpose of the present invention is also to provide a supramolecular structure composed of the above-mentioned compounds, such as nanotubes, nanorods and nanoflowers composed of nanotubes;

The purpose of the present invention is also to provide applications of such nanostructures in drug carrier systems, as scaffolds in regenerative medicine and in catalysis;

Description of drawings

Figure 1: Flower-like and tubular nanostructures formed by pyrimidopyrimidine nucleoside analogs in aqueous solution, among which are the supramolecular structures formed by nucleosides 1-6 in aqueous solution, respectively.

Figure 2: SEM comparison images of drug encapsulation, which are the drug encapsulation images of nucleoside 2 and 6 respectively.

Figure 3: UV comparison diagram of drug encapsulation, in which are the drug encapsulation comparison pictures of nucleoside 2 and 6 respectively.

Detailed ways

The following examples will illustrate the present invention in more detail without limiting its scope in any sense

Example 1: 4,7-diamino-1-(D-ribofuranose)-5-methoxy-pyrimido[4,5-d]pyrimidine-2,5(1H,2H)-dione (compound 1)

4,7-Diamino-1-(2',3'-O-isopropylidene-D-ribofuranose)-5-methoxy-pyrimido[4,5-d]pyrimidine-2(1H )-ketone 3.8g (purchased, 10mmol) was dissolved in acetone, 4.5g sodium iodide and 3.3g (30mmol) tetramethylchlorosilane were added, and stirred at room temperature for 20 hours. Consider the precipitate generated by the reaction, and wash with acetonitrile (30ml*3) and acetone (30ml*3). Next, 30 ml of trifluoroacetic acid was added to the obtained yellow precipitate under ice-bath conditions, and after stirring for 20 minutes, the trifluoroacetic acid was distilled off under reduced pressure. The resulting product was repeatedly co-distilled with ethanol to remove residual acid. Finally, the obtained product is heated and dissolved in water, and left to stand for several hours to obtain a pure white solid product 4,7-diamino-1-(D-ribofuranose)-5-methoxy-pyrimido[4,5 -d] Pyrimidine-2,5(1H,2H)-dione (3 g, 91% yield).

λmax(MeOH)/nm(ε/dm ³ mol ^-1 cm ^-1 ):228(2359),283(1216).δ _H (400MHz; d ₆ -DMSO):3.41-3.45(1H,m,4' H), 3.59-3.65 (2H, t, 5'CH ₂ ), 4.19 (1H, s, 3'H), 4.54-4.56 (1H, d, J=8.0Hz, 3'OH), 4.68 (1H, s,2'OH),4.80-4.81(1H,bd,J＝2.0Hz,2'H),5.02(1H,s,5'OH),6.47(1H,s,1'H),7.57-8.25 (4H,m,NH ₂ ×2),11.72(1H,br,NH).HRMS(ESI-)m/z: Calc.for C ₁₁ H ₁₄ N ₆ O ₆ :325.0896[MH] ^- .Found325.0891 [MH] ^-

Example 2: Preparation of 5-amino-8-(2,3,5-tri-oxo-benzoyl-D-ribofuranose)pyrimido[4,5-d]pyrimidine-2,4(3H,8H) - dione

Add 600mg base (3.3mmol) into 30mL hexamethyldisilazane (HMDS), stir at 140°C for about 2-3 minutes, then add trimethylchlorosilane (TMSCl). The reaction was carried out under reflux for 18 hours. Then, the remaining HMDS is removed to obtain the silanized base. Use it immediately for the next glycosylation reaction. Add 20ml of anhydrous 1,2-dichloroethane into the reactor containing the compound, stir and dissolve at room temperature, then dissolve 630mg of 3,5-di-O-benzoyl-β-D- in 20ml of anhydrous acetonitrile Ribofuranose (purchased) was added to the above reactor. 550 μL (mmol) of tin tetrachloride was added to the above mixture as a catalyst. Until 3,5-di-O-benzoyl-β-D-ribofuranose disappeared. Addition of 40 ml saturated aqueous NaHCO ₃ was used to stop the reaction. Dichloromethane was added to extract the organic phase (40×3), the organic phases were combined and dried over anhydrous sodium sulfate, and finally rotary evaporated under reduced pressure. The product after rotary evaporation was separated by column chromatography, the silica gel column was 4×8cm, and the ribonucleoside was obtained as a white powder after vacuum drying: 5-amino-8-(2,3,5-tri-oxo-benzoyl -D-ribofuranose)pyrimido[4,5-d]pyrimidine-2,4(3H,8H)-dione 940 mg (45%). λmax(MeOH)/nm(ε/dm3mol-1cm-1):225(95658),251(27263),273(15395),278(14355);1H-NMR(DMSO-d6,600MHz):δ4.73 ~4.75(2H,t,J=6Hz,5'-H2),4.82~4.84(1H,t,J=6Hz,4'-H),6.12~6.13(1H,d,J=6Hz,3'- H),6.17-6.20(1H,t,J1＝12Hz,J2＝6Hz,2'-H),6.51(1H,s,1'-H),7.40～7.99(15H,m,H-arom), 8.66(1H,s,CH),9.07(1H,s,NHα)9.14(1H,s,NHβ),10.87(1H,s,NH).13C NMR(600MHz; DMSO-d6)δ64.41,71.12, 16.16

Example 3: Preparation of 5-amino-8-(D-ribofuranose)pyrimido[4,5-d]pyrimidine-2,4(3H,8H)-dione (Compound 2)

80mg of 5-amino-8-(2,3,5-tri-oxo-benzoyl-D-ribofuranose)pyrimido[4,5-d]pyrimidine-2,4( 3H, 8H)-diketone (0.13mmol), was dissolved in 10ml 0.5M NaOMe/MeOH, and the reactant changed from a suspension state to a jelly, after cooling to room temperature, neutralized with diluted glacial acetic acid, filtered, and Washed three times with methanol and dried in vacuo to obtain light yellow powder 5-amino-8-(D-ribofuranose)pyrimido[4,5-d]\pyrimidine-2,4(3H,8H)-dione (32mg, 80%). λmax(MeOH)/nm(ε/dm3mol-1cm-1) 251(25943), 277(5681); 1H-NMR(DMSO-d6,600MHz): δ3.60～3.80(2H,m,5'-H2 ),3.94～3.97(1H,m,4'-H),4.08～4.17(2H,m,3'-H,2'-H),5.08～5.09(1H,d,J＝6Hz,5'- OH), 5.27～5.29(1H,t,J=6Hz,3'-OH),5.55～5.56(1H,d,J=6Hz,2'-OH),6.17～6.18(1H,d,J=6Hz ,1'-H),8.88(1H,s,CH),8.92(1H,s,NHα)8.98(1H,s,NHβ),10.77(1H,s,NH).13C NMR(600MHz; DMSO-d6 ) δ60.04, 69.04, 74.98, 85.06, 87.44, 90.26, 151.88, 157.00, 158.18, 162.00, 165.44.

Example 4: 4,6-dichloropyrimidine-5 formaldehyde oxime

Dissolve 1.8g of 4,6-dichloropyrimidine-5 formaldehyde (purchased) in 20ml of glacial acetic acid, then drop into 40ml of ethanol solution of hydroxylamine hydrochloride (1.4g), and react at room temperature for 15 hours. After the solution was spin-dried, the organic phase was washed with saturated NaHCO ₃ and water, and finally the organic phase was dried with anhydrous sodium sulfate and spin-dried to obtain the white product 4,6-dichloropyrimidine-5-carbaldehyde oxime (1.8 g, 84% yield). 1H NMR (400MHz; DMSO-d6): δ = 8.19(s, 1H), 8.91(s, 1H), 12.27(s, 1H). 13C NMR (600MHz; DMSO-d6) δ = 113.63, 142.03, 149.50, 154.25. Anal. Calcd for C5H3Cl2N3O: C, 31.28; H, 1.57; N, 21.89. Found C, 31.44; H, 1.46; N, 22.17.

Example five: 4-(5'-Oacetyl-2',3'-O-isopropylidene-β-D ribofuranose)amine-5 cyano-6-chloropyrimidine

1.7 g of the compound 4,6-dichloro-5-cyanopyrimidine (purchased) was dissolved in 50 ml of dry tetrahydrofuran (THF). Add 7.2g 2,3-O-isopropylidene-D-furanosamine p-toluenesulfonate (purchased), 1.7g NaHCO ₃ , 3.4ml ipr ₂ EtN (N,N-diisopropylethylamine ,Buy). After the mixed solution was reacted at room temperature for 10 minutes, it was heated to 60° C., and the reaction was continued for 60 minutes. After cooling, the product was redissolved with 50 ml of dichloromethane (DCM). The product was washed twice with water and dilute hydrochloric acid (0.1N), dried over anhydrous sodium sulfate, and spin-dried to obtain the crude product 4d. Dissolve unpurified 4d in 50ml DCM, add 2.6ml ipr2EtN, 1.4ml acetic anhydride, 12.2mg DMAP (4-dimethylaminopyridine, the same below). After reacting for about 10 minutes, 1 ml of dilute hydrochloric acid was added to stop. The resulting solution was diluted to 150ml with DCM, washed with dilute hydrochloric acid and water, dried over anhydrous sodium sulfate, and spin-dried to obtain a crude foamy product. Column chromatography separation (eluent: DCM, Rf=0.9) gave colorless product 4-(5'-Oacetyl-2',3'-O-isopropylidene-β-D ribofuranose)amine- 5 cyano-6-chloropyrimidine (2.1 g, 58% yield). 1H NMR (400MHz; DMSO-d6) δ=1.295(s,3H),1.462(s,3H),2.021(s,3H),4.125-4.185(m,3H)4.753-4.775(m,1H)4.998- 5.019(m,1H),5.861-5.885(m,1H),8.619(s,1H),8.996-9.015(d,1H,);13C NMR(600MHz; DMSO-d6)δ=21.06,25.56,27.21, 64.48, 81.80, 83.13, 83.84, 87.91, 113.20, 159.97, 162.23, 170.57. HR-MS (ESI+): calcd for C15H17ClN4O5: 368.0877 Found: 369.0973 [M+H].

Example 6: 4-amino-1-(2',3'-O-isopropylidene-β-D ribofuranose)-5-methoxypyrimido[4,5-d]pyrimidine-2(1H )-ketone

3.7g of 4-(5'-Oacetyl-2',3'-O-isopropylidene-β-D ribofuranose)amine-5cyano-6-chloropyrimidine prepared in Example 4 was dissolved DCM was dried in 100ml, and 6.5ml ipr ₂ EtN (N,N-diisopropylethylamine, purchased) was added under ice-bath conditions. After stirring for a while, 5.2 ml of CCI (N-(chloroformyl)isocyanate, purchased) was added dropwise. After CCI was added completely, 5ml of HCl (0.1N) was added to terminate the reaction. After the precipitate was filtered, the filtrate was washed twice with water, dried over anhydrous sodium sulfate, and spin-dried to obtain a crude urea-added product. The crude product was reacted in 50ml of sodium methoxide (0.15N) to obtain the cyclized product. The resulting mixture was separated by column chromatography to obtain the white product 4-amino-1-(2',3'-O-isopropylidene-β-D ribofuranose)-5-methoxypyrimido[4,5- d] Pyrimidin-2(1H)-one (1.1 g, 30% yield). 1H NMR (400MHz; DMSO-d6)δ=1.280(s,3H),1.492(s,3H),3.474-3.33(m,1H),3.579-3.636(m,1H),3.956-3.997(m,1H ),4.104(s,3H),4.720-4.749(t,1H),4.871-4.898(t,1H),5.197-5.217(m,1H),6.852(s,1H),7.818(s,1H), 8.521(s,1H),8.716,8.729(s,1H);13C NMR(600MHz;DMSO-d6)δ=25.74,27.68,55.84,62.68,82.67,84.53,88.21,112.96,154.09,159.42,160.12,161.10 , 166.85.HR-MS(ESI-):calcdfor[C15H19N5O6]:365.1335Found:364.0982[MH].

Example 7: 4-amino-1-(β-D ribofuranose) pyrimido[4,5-d]pyrimidine-2,5(1H,6H)-dione (compound 3)

3.6g of the compound 4-amino-1-(2',3'-O-isopropylidene-β-D ribofuranose)-5-methoxypyrimido[4,5- d] Pyrimidin-2(1H)-one was dissolved in 30 ml acetone. Add 4.5 g of sodium iodide and 3.3 g of TMSi-Cl (trimethylchlorosilane, purchased), and stir at room temperature for 20 hours. The precipitate generated by the reaction was filtered, and the precipitate was washed 3 times with 30 ml of acetonitrile and 3 times with 30 ml of acetone. Then, 30 ml of trifluoroacetic acid (containing 10% water) was added to the obtained yellow precipitate (3.3 g, 94% yield) under ice-cooling conditions. After stirring for 20 minutes, trifluoroacetic acid was distilled off under reduced pressure. The resulting product was repeatedly co-distilled with ethanol to remove residual acid. Finally, the obtained product is heated and dissolved in water, and after standing at room temperature for several hours, a pure white solid product precipitate 4-amino-1-(β-D ribofuranose)pyrimido[4,5-d]pyrimidine-2 can be obtained , 5(1H, 6H)-diketone (2.66g, 91% yield) 1H NMR (400MHz; DMSO-d6) δ=3.45(br,1H); 3.608-3.699(m,2H); 4.196(s, 1H); 4.561(s,1H); 4.636(s,1H); 4.838(s,1H); 5.042(s,1H); 6.512(s,1H); 8.221-8.304(t,1H); (d,2H); 13.150(br,1H).HR-MS(ESI-):calcd for[C11H13N5O6]:311.0866Found 310.0782[M-H].

Example 8: 5-amino-8-(2-deoxy-D-ribofuranose) pyrimido[4,5-d]pyrimidine-2,4.(3H,8H)-dione (compound 5)

According to the method similar to Example 2, the product 4-aminopyrimido[4,5-d]pyrimidine-5,7(6H,8H)-dione (synthesized according to Document 1) was used to obtain 5-amino-8-(2- deoxy-D-ribofuranose)pyrimido[4,5-d]pyrimidine-2,4.(3H,8H)-dione. λmax(MeOH)/nm(ε/dm3mol-1cm-1), 251(28571), 277(6158); 1H-NMR(DMSO-d6, 600MHz): δ2.18～2.22(1H,m,2'- Hα),2.35-2.39(1H,m,2'-Hβ),3.59～3.61(1H,d,J＝12Hz,5'-Hα),3.67-3.69(1H,d,J＝12Hz,5'- Hβ), 3.89～3.91(1H,t,J＝6HZ,4'-H),4.27(1H,s,3'-H),5.20(1H,s,5'-OH),5.34(1H,s ,3'-OH),6.41～6.43(1H,t,J＝6Hz,1'-H),8.77(1H,s,CH),8.89～8.93(2H,2s,NH2),10.76(1H,s , NH).13C NMR (600MHz; DMSO-d6).13C NMR (600MHz; DMSO-d6) δ 41.60, 60.95, 69.99, 86.75, 87.46, 88.59, 151.39, 157.11, 157.78, 162.07, 165.43.

Example 9: 5-Amino-1-(2-deoxy-D-ribofuranose)pyrimido[4,5-d]pyrimidine-2,4.(1H,3H)-dione (compound six)

According to the method similar to Example 2, the product 4-aminopyrimido[4,5-d]pyrimidine-5,7(6H,8H)-dione (synthesized according to Document 1) was used to obtain 5-amino-1-(2- Deoxy-D-ribofuranose)pyrimido[4,5-d]pyrimidine-2,4.(1H,3H)-dione.1H NMR(400MHz,d6-DMSO)δ(ppm):2.31-2.38(1H ,m,2'-CH),2.51-2.63(1H,m,2'-CH),3.36-3.60(2H,m,5'-CH2),4.07-4.14(2H,m,3'-CH and 4'-CH),4.61-4.63(1H,t,J=5.2Hz,5'-OH),5.16-5.18(1H,d,J=2.4Hz,3'-OH),6.48-6.50(1H, t,J=7.6Hz,1'-CH),8.22-8.23(2H,d,J=5.2Hz,-NH2),8.25(1H,s,7-CH),11.89-11.90(1H,d,J ＝2.4Hz,-NH);13C NMR(100MHz,d6-DMSO)δ(ppm):36.39,61.81,70.89,81.45,86.06,89.56,150.26,157.68,161.95,162.66,163.56; UV(H2O):λmax (ε): 227 (10000); 289 (1559); HRMS (ESI) calcd for (M+Na+)/z: 318.0815, found: 318.0807.

Example 10: 5-Amino-1-(D-ribofuranose)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (Compound 4)

According to the method similar to Example 2, the product 4-aminopyrimido[4,5-d]pyrimidine-5,7(6H,8H)-dione (synthesized according to Document 1) was used to obtain 5-amino-1-(D- Ribofuranose) pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione. 1H NMR(600MHz,d6-DMSO)δ(ppm):3.18(1H,s,3'-CH),3.44-3.47(1H,m,4'-CH),3.61-3.72(4H,m,5' -CH2,2'-CH and 5'-OH),4.17(1H,t,J＝6Hz,3'-OH),4.52-4.54(1H,q,J＝3.6Hz,2'-OH),6.17 (1H,d,J＝3.6Hz,1'-CH),7.79-8.19(2H,m,-NH2),8.14(1H,s,7-CH);13C NMR(150MHz,d6-DMSO)δ( ppm): 22.83, 48.09, 62.90, 70.73, 71.58, 84.86, 89.87, 161.77, 163.18, 173.30; UV (H2O): λmax (ε): 227 (10412); 289 (1777); HRMS (ESI) calcd for ( M+Na+)/z:334.0764,found:334.0766.

Embodiment 11: Such nucleoside compounds can form a good nanostructure through self-assembly in aqueous solution. Weigh 1-60.2 mg of nucleosides in the above structure diagram and dissolve them in water, then heat to boiling, and stand at room temperature After 48 hours, the results of scanning electron microscopy and transmission electron microscopy are shown in Figure 1.

Embodiment 12: All these nanostructures can be used to encapsulate medicines.

Weigh compounds 2 and 6 respectively (as shown in the previous structural diagram, other additional compounds have the same double-sided base structure as these two compounds and can self-assemble in water, preliminary scanning electron microscopy to show that such cores Glycoside compounds can form a similar supramolecular structure. As shown in Figure 1) 0.2mg, heated and dissolved in water, then left to stand for 24h, added 1mg of honokiol respectively, and then left at room temperature for 2 days, scanning electron microscope results before and after drug wrapping As shown in Figure 2, the UV comparison before and after wrapping the drug is shown in Figure 3.

Moreover, through the MTT cytotoxicity test, it is confirmed that the IC50 of these nucleoside compounds is greater than 3000umol, indicating that the biological toxicity is very low and can be used in vivo. (It is not necessary to specify the experimental scheme, and the method of citing literature can be used as reference 2). At the same time, the drug loading of the drug was studied by NMR, showing that the drug loading of compounds 2 and 6 reached 40% (see Table 1).

Table 1: Drug loading studies

Packing ratio 5:1 5:3 5:5 5:7 Points before pack 0.3 1.0 2.5 4.5 Points after pack 0.2 0.6 1.5 2.65 average drug loading 33.3% 40% 40% 41%

The above experiments preliminarily show that this type of nucleoside compound has good drug encapsulation ability and low toxicity, so it has a good application prospect as a new type of drug delivery system. great application potential.

literature:

1 Y. Tominaga, S. Ohno, S. Kohra, H. Fujito, H. Mazurae, J. Heterocycl. Chem. 1991, 28, 1039-1042.

2 Geise Ribeiro et al. Polyhedron, 2008, 27, 1131-1137.

Claims (3)

1. A pyrimidopyrimidine nucleoside analogue, characterized in that it has the following structure: 2. The supramolecular structure composed of the pyrimidopyrimidine nucleoside analogs according to claim 1, the supramolecular structure is: nanotubes, nanorods and nanoflowers composed of nanotubes. 3. The application of the supramolecular structure described in claim 2 in drug carrier system.