CN106008506B - Substituted purin analog derivative and preparation method and application - Google Patents
Substituted purin analog derivative and preparation method and application Download PDFInfo
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- CN106008506B CN106008506B CN201610486112.1A CN201610486112A CN106008506B CN 106008506 B CN106008506 B CN 106008506B CN 201610486112 A CN201610486112 A CN 201610486112A CN 106008506 B CN106008506 B CN 106008506B
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- GHQHHMQHUDIVKZ-UHFFFAOYSA-N CC[n](c1n2)c(S(CCN(C(c3ccccc33)O)C3=O)=O)nc1cnc2Cl Chemical compound CC[n](c1n2)c(S(CCN(C(c3ccccc33)O)C3=O)=O)nc1cnc2Cl GHQHHMQHUDIVKZ-UHFFFAOYSA-N 0.000 description 1
- NZJKTJIOTMASLZ-UHFFFAOYSA-N CC[n]1c2nc(Cl)ncc2nc1-c1ccc[n]1C Chemical compound CC[n]1c2nc(Cl)ncc2nc1-c1ccc[n]1C NZJKTJIOTMASLZ-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D473/00—Heterocyclic compounds containing purine ring systems
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Abstract
The invention discloses a kind of substituted purin analog derivative with structure shown in formula I and its preparation method and application.Present invention additionally comprises N oxides, stereoisomer form, stereoisomer mixture or the pharmaceutically acceptable salt of substituted purin analog derivative.Application of the composition present invention additionally comprises the preparation method of substituted purin analog derivative and containing one or more such compounds in anti-hepatitis B medicine is prepared.
Description
Technical field
The invention belongs to pharmaceutical technology field, and in particular to a kind of substituted purin analog derivative, the invention further relates to this kind of
The preparation method of derivative and its purposes in anti-hepatitis B medicine is prepared.
Background technology
Disease of viral infection is the major class disease for seriously endangering human life and health.Hepatitis B (HBV) is B-mode
The main pathogens of hepatitis.Although current hepatitis B vaccine is successfully researched and developed and is widely popularized, hepatitis B is extremely strong because of its
Infectiousness and pathogenic, social harm is still very big.In addition, the implementation of interferon and nucleoside medicine is resistance of hepatitis B
One important breakthrough for the treatment of, but the toxicity problem of the appearance and Long-term taking medicine due to drug resistance significantly limit the therapy
Application, have new construction, new mechanism anti-hepatitis B medicine research and development it is very urgent.
Heterocyclic compound has extensive antiviral activity, and they are usually female as the basic structure for forming pharmacophore
Core, to be adapted to the space requirement of medicine special role target spot, or produce as active substituent or the part of ring system
Raw corresponding bioactivity.Why medicine is because heterocycle is more fatty or aromatic compound is less susceptible in vivo dependent on heterocycle
Metabolic breakdown, and there is more preferable biocompatibility.Benzimidazole ring is a kind of important heteroaromatic, and its derivative has extensive
Bioactivity and clinical practice.The present invention utilizes bioelectronics etc. based on the good Anti-HBV effect of benzimidazole analogues
Isostere purine ring substituted benzimidazole ring, design have synthesized a series of substituted purin analog derivatives, had no in the prior art such
Compound and its application.
The content of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of substituted purin analog derivative and preparation method thereof, the present invention
The Anti-HBV effect the selection result of substituted purin analog derivative and its application in anti-hepatitis B medicine is prepared also are provided.
Technical scheme is as follows：
1. substituted purin analog derivative
Involved in the present invention is the substituted purin analog derivative with structure shown in below formula I, and its N- oxidations
Thing, stereoisomer form, stereoisomer mixture or pharmaceutically acceptable salt：
Wherein,
R is amino, chlorine atom, fluorine atom, bromine atoms, iodine atom, hydroxyl, methyl or ethyl；
R1For benzyl, substituted benzyl, various substituted hexa-member heterocycles, various substituted five-ring heterocycles, various substituted six
Member and five-ring heterocycles, various substituted hexa-atomic and hexa-member heterocycles, various substituted five yuan and five-ring heterocycles, various substituted benzos
Five-ring heterocycles or various substituted benzo hexa-member heterocycles；
Linker is the alkane chain of 0-3 carbon atom or containing heteroatomic alkane chain；
R2For various substituted hexa-member heterocycles, various substituted five-ring heterocycles, various substituted hexa-atomic and five-ring heterocycles, each
The hexa-atomic and hexa-member heterocycle of kind substitution, various substituted five yuan and five-ring heterocycles, various substituted benzo five-membered heterocycles or various
Substituted benzo hexa-member heterocycle；
According to currently preferred, in the formula I,
R is chlorine atom；
R1For methyl, ethyl, benzyl or to methyl-benzyl；
Linker is the alkane chain or 2 carbon atoms that length is the alkane chain of 0 atom, 1 carbon atom and 1 sulphur atom
With the alkane chain of 1 sulphur atom；
R2For N- methylpyrroles formaldehyde, indolecarboxaldehyde, 5- bromo indoles formaldehyde, N- bromomethyls phthalimide or N-
(2- bromoethyls) phthalimide；
Heretofore described " pharmaceutically acceptable salt " refer in reliable medicine range of value, the salt of compound
Class is suitable to be in contact without unsuitable toxicity, stimulation and allergic reaction etc. with people or compared with the tissue of lower animal, has suitable
Rational income and risk ratio, typically water or oil is solvable or dispersible, and is effectively used for its expected purposes.
Including pharmaceutically acceptable acid-addition salts and pharmaceutically acceptable base addition salts, be herein can do expected purposes and with
The chemical property of compound of formula I is compatible.The list of suitable salt is referring to S.M.Birge etc., J.Pharm.Sci., 1977, and 66,
1-19 pages.
Heretofore described " prodrug " refers to pharmaceutically acceptable derivates, so as to the biology obtained by these derivatives
Transformation product is the active medicine as defined in type I compound.
According to the present invention, it is further preferred that substituted purin analog derivative is one of following particular compound：
2. the preparation method of substituted purin analog derivative：
The preparation method of substituted purin analog derivative, by initiation material 2, the chloro- 5- nitro-pyrimidines of 4- bis- under low temperature through selecting
Property nucleophilic displacement of fluorine, obtains intermediate LN-1, intermediate LN-1 nitro obtains key intermediate LN-2 through Reduction with Stannous Chloride；It is middle
Amino in body LN-2 and the heteroaromatic generation schiff bases with aldehyde radical, then cyclization obtains part end under anhydrous ferric trichloride catalysis
Product LN-0 series；Intermediate LN-2 and thiocarbonyldiimidazole reaction generation intermediate LN-3, then obtain whole production through nucleophilic displacement of fluorine
Thing LN-S series, is then aoxidized to obtain sulfoxide product by metachloroperbenzoic acid.
Synthetic route is as follows：
Reagent and condition:(i) methylamine water solution or ethylamine solution or benzylamine or to methylbenzylamine, dichloromethane, two
Wopropyl ethyl amine, -78 DEG C；(ii) stannous chloride, absolute ethyl alcohol, backflow；(iii) the nitrogenous heteroaromatic of aldehyde radical substitution, glacial acetic acid,
Absolute methanol, anhydrous ferric trichloride, room temperature；(iv) thiocarbonyldiimidazole, tetrahydrofuran, backflow；(v) potassium carbonate or bicarbonate
Sodium, room temperature；(iv) metachloroperbenzoic acid, dichloromethane, room temperature.
Wherein, wherein, R1、R2It is defined as above described in formula I.
Room temperature of the present invention is 20-30 DEG C.
The present invention is more detailed, the preparation method of substituted purin analog derivative, and step is as follows：
(1) 2.0g 2 is weighed, the chloro- 5- nitro-pyrimidines of 4- bis- add 20mL dichloromethane room temperatures in 100mL eggplant type flasks
Stirring and dissolving, be put into low-temp reaction instrument, when being cooled to -30 DEG C, add 3.07mL diisopropylethylamine, to -78 DEG C when, slowly
The methylamine water solutions of 0.885g 40% with 5mL dchloromethanes are added dropwise, drop finishes, and continues to stir 4h, TLC monitorings, has reacted
Finish, stop reaction, recover to room temperature, three times, saturated common salt is washed once, anhydrous sodium sulfate drying for washing, and filtering, filtrate is evaporated
Mix sample, silica gel column chromatography separating purification, mobile phase selection petroleum ether：Ethyl acetate, volume ratio 8：1, obtain intermediate LNa-1；
(2) 1.4g intermediates LNa-1 is weighed in 250mL flasks, the dissolving of 100mL ethanol, 6.17g is added portionwise under stirring
Stannous chloride, finish, under nitrogen protection, be warming up to 80 DEG C of back flow reaction 9h, TLC monitorings, reaction finishes, stops reaction, treat
Reaction solution recovers to room temperature, and it is 3 to survey reaction solution pH, under ice-water bath, slowly adjusts pH to 8 with 1M sodium hydroxide solutions, uses diatomite
Filter, most of ethanol in filtrate is steamed, add ethyl acetate extraction three times, merge organic phase, saturated common salt washing one
Secondary, anhydrous sodium sulfate drying is overnight, and filtering, filtrate, which is evaporated, mixes sample, silica gel column chromatography separating purification, mobile phase selection petroleum ether：
Ethyl acetate, volume ratio 1：1 to 1：2, obtain intermediate LNa-2；
(3) 0.3g intermediates LNa-2 is weighed in 50mL eggplant type flasks, the dissolving of 15mL absolute methanols, is added respectively under stirring
Enter 0.2684g N- methyl -2- pyrrole aldehydes, 0.1mL acetic acid, 6h, TLC monitorings is stirred at room temperature, reaction finishes, and decompression steams molten
Agent methanol and acetic acid, the dissolving of 15mL absolute ethyl alcohols is added, stir lower addition 6.2g 5%FeCl3-SiO2, it is warming up to 80 DEG C and returns
1.5h is flowed, stops reaction, is cooled to room temperature, filters out insoluble matter, the ethanol in filtrate is steamed, ethyl acetate is added and redissolves,
Three times, saturated common salt is washed once, organic phase anhydrous sodium sulfate drying for washing, and filtering, filtrate, which is evaporated, mixes sample, silica gel column chromatography point
From purifying, mobile phase selection petroleum ether：Ethyl acetate, volume ratio 10：1 to 8：1, you can isolate product LN-0-1；
0.2g intermediates LNa-2 is weighed in 50mL eggplant type flasks, the dissolving of 15mL absolute methanols, is separately added under stirring
0.2197g indolals, 0.1mL acetic acid, 12h, TLC monitorings being stirred at room temperature, reaction finishes, and decompression steams solvent methanol and acetic acid,
15mL absolute ethyl alcohols or dimethylformamide dissolving are added, stirs the lower 0.2045g FeCl for adding solvent dilution3Solution, rise
Temperature stops reaction, is cooled to room temperature, directly filters out insoluble matter, filter cake is washed with water, and petroleum ether is washed, vacuum to 80 DEG C of backflow 10h
Drying box is dried, you can obtains product LN-0-4；
(4) 0.3g intermediates LNa-2 is weighed in 50mL flasks, and 15mL steams tetrahydrofuran dissolving again, stirs lower add
0.5057g thiocarbonyldiimidazoles, 0.2871g triethylamines being added, be warming up to 70 DEG C of back flow reaction 12h, TLC monitorings, reaction finishes,
Stop reaction, question response recovers to room temperature, steams tetrahydrofuran, adds ethyl acetate and redissolves, under ice-water bath, with 2M hydroxides
Sodium solution washes organic phase three times, and aqueous phase merges, and ice-water bath is slowly stirred down, and watery hydrochloric acid is added dropwise, and separates out solid, filtering, and filter cake is used
Intermediate LNa-3 is drying to obtain after washing；
(5) 0.1g intermediates LNa-3 is weighed in 50mL eggplant type flasks, is added 10mL dimethylformamides, is stirred at room temperature
Dissolving, 0.103g potassium carbonate is first added, after 0.5h is stirred at room temperature, it is sub- to add 0.139g raw materials N- (2- bromoethyls) phthalyl
Amine, it is warming up to 40 DEG C and continues to react, TLC is monitored, and after 12h, reaction finishes, and is stopped reaction, is steamed solvent dimethylformamide, second
Acetoacetic ester is redissolved, and adds washing three times, and saturated common salt is washed once, anhydrous sodium sulfate drying, and filtering, filtrate, which is evaporated, produces product
Crude product, it is then 95% ethyl alcohol recrystallization with volume fraction, obtains sterling LN-S-1；
0.1g intermediates LNa-3 is weighed in 50mL eggplant type flasks, 15mL dimethylformamides is added, is stirred at room temperature molten
Solution, 0.0460g sodium acid carbonates are added, after being warming up to 40 DEG C of stirring 1.5h, then 0.2393g N- bromomethyl neighbours benzene two is slowly added dropwise
The dimethyl formamide solution of carboximide, 2h drops finish, and TLC monitorings, reaction finishes, and stops reaction, steams solvent, ethyl acetate
Redissolving, add washing three times, saturated common salt is washed once, anhydrous sodium sulfate drying, and filtering, filtrate, which is evaporated, produces product crude product,
Then it is 95% ethyl alcohol recrystallization with volume fraction, obtains sterling LN-S-5；
(6) weigh 0.1g products LN-S-6 in 50mL eggplant type flasks, add 10mL steam again dichloromethane be stirred at room temperature it is molten
Solution, the dichloromethane solution of 0.1385g metachloroperbenzoic acids is slowly added dropwise, drop finishes, TLC monitorings, reacts and finishes after 18h, stops
Only react, the excessive hypo solution newly prepared is added into reaction solution, except remaining m-chloro peroxide benzene first in dereaction
Acid, organic phase is separated, three times, saturated common salt is washed once, anhydrous sodium sulfate drying for washing, and filtering, filtrate, which is evaporated, produces product
Crude product, then recrystallized with absolute ethyl alcohol, produce product LN-S-9.
3. Anti-HBV effect and the application of substituted purin analog derivative
External Anti-HBV effect has been carried out to the 20 substituted purin analog derivatives newly synthesized by HepG2.2.15 cell lines
Research, with Lamivudine (3TC) for positive control medicine, suppress HBsAg in vitro using ELISA method measure target compound
The activity of (surface antigen) and HBeAg (cAg) secretion；The suppression HBV DNA of part of compounds are determined by PCR methods
Replication activity；Using the cytotoxicity of CCK-8 methods detection sample compound, so as to be carried out to the Anti-HBV effect of sample compound
Screening.Their Anti-HBV effect and toxicity data is listed in table 1,2.
The substituted purin analog derivative of the present invention can be used as non-nucleoside HBV inhibitor applications.Specifically, as HBV
Inhibitor is used to prepare anti-hepatitis B medicine.
A kind of Anti-HBV drugs composition, including the substituted purin analog derivative of the present invention and one or more pharmaceutically may be used
Receive carrier or excipient.
The invention provides brand-new substituted purin analog derivative of structure and preparation method thereof, Anti-HBV effect the selection result
And its application in anti-hepatitis B medicine is prepared.It was proved that substituted purin analog derivative of the invention can conduct
HBV inhibitor is applied and has potential application value.Specifically, it is used to prepare medicine for anti hepatitis B as HBV inhibitor
Thing.
Embodiment
Contribute to understand the present invention by following examples, but present disclosure can not be limited.
Involved synthetic route is as follows in embodiment：
The intermediate LN-2 of embodiment 1. preparation method (by taking LNa-2 as an example, R1=methyl)
The chloro- 5- nitro-pyrimidines (2.0g, 10.4mmol) of 2,4- bis- are weighed in 100mL eggplant type flasks, add dichloromethane
Dissolving is stirred at room temperature in (20mL), is put into low-temp reaction instrument, when being cooled to -30 DEG C, addition diisopropylethylamine (3.07mL,
When 19.0mmol), to -78 DEG C, 40% methylamine water (0.885g, 11.4mmol) diluted with dichloromethane (5mL) is slowly added dropwise
Solution, drop is complete, continues to stir 4h, TLC monitorings, reaction finishes, and stops reaction, recovers to room temperature, washes three times (25mL), saturation
Salt is washed once (25mL), anhydrous sodium sulfate drying, and filtering, filtrate, which is evaporated, mixes sample, silica gel column chromatography separating purification, mobile phase
Select petroleum ether：Ethyl acetate, volume ratio 8：1, obtain intermediate LNa-1；
Intermediate LNa-1 (1.4g, 7.42mmol) is weighed in 250mL flasks, ethanol (100mL) dissolving, stirs lower point
Criticize and add stannous chloride (6.17g, 29.7mmol), finish, under nitrogen protection, be warming up to 80 DEG C of back flow reaction 9h, TLC prisons
Survey, reaction finishes, and stops reaction, and question response liquid recovers to room temperature, and it is 3 to survey reaction solution pH, molten with 1M sodium hydroxides under ice-water bath
Liquid slowly adjusts pH to 8, and with suction filtered through kieselguhr, most of ethanol in filtrate is steamed, and adds ethyl acetate (3 × 20mL) extraction
Three times, organic phase is merged, saturated aqueous common salt (60mL) is washed once, and anhydrous sodium sulfate drying is overnight, and filtering, filtrate, which is evaporated, mixes sample,
Silica gel column chromatography separating purification, mobile phase selection petroleum ether：Ethyl acetate, volume ratio 1：1 to 1：2, obtain intermediate LNa-2；
The chloro- N of 2-4- methylpyrimidine -4,5- diamines (LNa-2)
Light brown powder, yield 56.1%, fusing point：138-140℃；1H NMR(400MHz,CDCl3)δppm:7.61(s,
1H,purine-CH),5.09(s,1H,purine-NH-CH3), 3.07 (d, 3H, J=4.0Hz, purine-NH-CH3 ),1.57
(s,2H,purine-NH2),impurity peaks:3.79,1.25(Ethanol)；ESI-MS:m/z 159.2,161.4[M+
H]+.C5H7ClN4(158.04).
The chloro- N of 2-4- ethyl-pyrimidine -4,5- diamines (LNb-2)
Dark brown powder, yield 53.1%, fusing point：164-166℃；1H NMR(400MHz,CDCl3)δppm:7.54(s,
1H,purine-CH),5.33(s,1H,purine-NH-CH2),3.52-3.48(m,2H,NH-CH2 -CH3),3.17(s,2H,
purine-NH2),1.25(t,3H,NH-CH2-CH3 )；13C NMR(100MHz,CDCl3)δppm:156.98,152.35,
140.90,124.00(purine-C),36.03(NH-CH2 -CH3),14.58(NH-CH2-CH3 )；ESI-MS:m/z 173.3,
175.2[M+H]+.C6H9ClN4(172.05).
N4- benzyl -2- chlorine pyrimidine -4,5- diamines (LNc-2)
Light brown powder, yield 65.43%, fusing point：183-186℃；1H NMR(400MHz,CDCl3)δppm:7.64(s,
1H,purine-CH),7.37-7.32(m,5H,Ph-H),5.27(s,1H,purine-NH-CH2), 4.68 (d, 2H, J=
4.0Hz,purine-NH-CH2 -Ph),2.95(s,2H,purine-NH2),impurity peaks:1.25(Ethanol)；13C
NMR(100MHz,CDCl3)δppm:157.08,153.00,142.56,128.86(purine-C),137.87,128.23,
127.86,123.54(Ph-C),45.27(NH-CH2 -Ph)；ESI-MS:m/z 234.7,236.7[M+H]+,257.2,259.2
[M+Na]+.C11H11ClN4(234.07).
The chloro- N of 2-4- (4- methyl-benzyls) pyrimidine -4,5- diamines (LNd-2)
Buff powder, yield 51.0%, fusing point：147-150℃；ESI-MS:m/z 249.4,251.4[M+H]+
.C12H13ClN4(248.08).
The intermediate LN-3 of embodiment 2. preparation method (by taking LNa-3 as an example, R1=methyl)
Intermediate LNa-2 (0.3g, 1.89mmol) is weighed in 50mL flasks, tetrahydrofuran (15mL) dissolving is steamed again, stirs
Lower addition thiocarbonyldiimidazole (0.5057g, 2.84mmol) is mixed, triethylamine (0.2871g, 2.84mmol) is added, is warming up to 70
DEG C back flow reaction 12h, TLC monitoring, largely reaction finishes display raw material, stops reaction, question response recovers to room temperature, steamed
Most of tetrahydrofuran, add ethyl acetate and redissolve, under ice-water bath, wash organic phase three times with 2M sodium hydroxide solutions, aqueous phase
Merge, ice-water bath is slowly stirred down, and watery hydrochloric acid is added dropwise, and separates out solid, filtering, and filter cake is drying to obtain intermediate LNa- after being washed with water
3。
The chloro- 9- methyl -9H- purine -8- mercaptan (LNa-3) of 2-
Buff powder, yield 52.3%, fusing point：292-294℃；1H NMR(400MHz,DMSO-d6)δppm:13.57
(s,1H,-SH),8.39(s,1H,purine-CH),3.56(s,3H,purine-N-CH3)；13C NMR(100MHz,DMSO-
d6)δppm:173.05,153.79,152.34,136.70,124.06(purine-C),29.32(purine-N-CH3)；ESI-
MS:m/z201.2,203.2[M+H]+.C6H5ClN4S(199.99).
The chloro- 9- ethyls -9H- purine -8- mercaptan (LNb-3) of 2-
Buff powder, yield 55.3%, fusing point：263-266℃；1H NMR(400MHz,DMSO-d6)δppm:8.20
(s, 1H, purine-CH), 4.19 (q, 2H, J=8.0Hz, purine-N-CH 2-CH3),1.91(s,1H,-SH),1.25(t,
3H, J=8.0Hz, purine-N-CH2-CH 3)；13C NMR(100MHz,DMSO-d6)δppm:173.05,153.79,
152.34,136.70,124.06(purine-C),29.32(purine-N-CH3)；ESI-MS:m/z 215.3,217.4[M+
H]+.C7H7ClN4S(214.01).
The chloro- 9H- purine -8- mercaptan (LNc-3) of 9- benzyls -2-
Buff powder, yield 56.0%, fusing point：231-234℃.
The chloro- 9- of 2- (4- methyl-benzyls) -9H- purine -8- mercaptan (LNd-3)
Buff powder, yield 54.7%, fusing point：206-208℃；1H NMR(400MHz,DMSO-d6)δppm:8.05
(s, 1H, purine-CH), 7.20 (d, 2H, J=8.0Hz, Ph-H), 7.08 (d, 2H, J=8.0Hz, Ph-H), 5.31 (s, 2H,
purine-N-CH 2-Ph),2.24(s,3H,Ph-CH3),impurity peaks:1.71(Tetrahydrofuran)；13C
NMR(100MHz,DMSO-d6)δppm:177.31,174.79,147.86,137.28(purine-C),136.39,135.37,
129.13,127.99(Ph-C),45.24(purine-N-CH 2-Ph),24.86(Ph-CH3)；ESI-MS:m/z 291.3,
293.3[M+H]+.C13H11ClN4S(290.04).
Target compound LN-0-1~the LN-0-3 of embodiment 3. preparation method (by taking LN-0-1 as an example, R1=methyl, R2=
N- methyl -2- pyrroles)
Intermediate LNa-2 (0.3g, 1.89mmol) is weighed in 50mL eggplant type flasks, absolute methanol (15mL) dissolving, is stirred
Mix down and be separately added into N- methyl -2- pyrrole aldehydes (0.2684g, 2.46mol), acetic acid (0.1mL), 6h, TLC prisons is stirred at room temperature
Survey, reaction is finished, and decompression steams solvent methanol and acetic acid, adds absolute ethyl alcohol (15mL) dissolving, and 5% is added under stirring
FeCl3-SiO2(6.2g), 80 DEG C of 1~2h of backflow are warming up to, stops reaction, is cooled to room temperature, filters out insoluble matter, by filtrate
Ethanol steam, add ethyl acetate redissolve, washing (3 × 5mL) three times, saturated aqueous common salt (5mL) is washed once, and organic phase is anhydrous
Sodium sulphate is dried, and filtering, filtrate, which is evaporated, mixes sample, silica gel column chromatography separating purification, mobile phase selection petroleum ether：Ethyl acetate, body
Product ratio 10：1 to 8：1, you can isolate product LN-0-1.
With different intermediate LN-2 and N- methyl -2- pyrrole aldehydes, compound L N-0-1 is made respectively in aforementioned manners
~LN-0-3 target product, it is as a result as follows：
White powder, yield 33.7%, fusing point：152-154℃；1H NMR(400MHz,DMSO-d6)δppm:8.96(s,
1H,purine-CH),7.21-7.20,6.95-6.93,6.29-6.27(m,3×1H,pyrrole-CH),3.97(s,3H,
purine-N-CH3),3.86(s,3H,pyrrole-N-CH3)；ESI-MS:m/z 248.3,250.3[M+H]+.C11H10ClN5
(247.06).
Operation is with embodiment 3, except that intermediate LNa-2 is replaced with into LNb-2.
White powder, yield 53.6%, fusing point：140-142℃；1H NMR(400MHz,DMSO-d6)δppm:9.00(s,
1H, purine-CH), 7.22-7.21,6.84-6.83,6.30-6.29 (m, 3 × 1H, pyrrole-CH), 4.41 (q, 2H, J=
8.0Hz,purine-N-CH 2-CH3),3.94(s,3H,pyrrole-N-CH3), 1.41 (t, 3H, J=8.0Hz, purine-N-
CH2-CH 3)；ESI-MS:m/z 262.2,264.2[M+H]+.C12H12ClN5(261.08).
Operation is with embodiment 3, except that intermediate LNa-2 is replaced with into LNc-2.
Pale yellow powder, yield 28.5%, fusing point：130-132℃；1H NMR(400MHz,DMSO-d6)δppm:9.08
(s,1H,purine-CH),7.35-7.29,7.08-7.06(m,5H,Ph-H),7.17,6.46,6.16(s,3×1H,
pyrrole-CH),5.60(s,2H,N-CH 2-Ph),3.93(s,3H,pyrrole-N-CH3), impurity peak:1.23,
0.85(n-hexane)；ESI-MS:m/z 324.4,326.5[M+H]+.C17H14ClN5(323.09).
Target compound LN-0-4~the LN-0-8 of embodiment 4. preparation method (by taking LN-0-3 as an example, R1=methyl, R2=
1-H-3- indyls)
Intermediate LNa-2 (0.2g, 1.26mmol) is weighed in 50mL eggplant type flasks, absolute methanol (15mL) dissolving, is stirred
Mix down and be separately added into indolal (0.2197g, 1.51mmol), acetic acid (0.1mL), 12h is stirred at room temperature, TLC monitorings, shows raw material
React and finished, decompression steams solvent methanol and acetic acid, adds absolute ethyl alcohol or dimethylformamide (15mL) dissolving, stirring
The lower FeCl for adding solvent dilution3(0.2045g, 1.26mmol) solution, 80 DEG C of backflow 10h are warming up to, stops reaction, is cooled to
Room temperature, insoluble matter is directly filtered out, filter cake is washed with water, and petroleum ether is washed, and vacuum drying chamber is dried, you can obtains product LN-0-4.
With different intermediate LN-2 and indolecarboxaldehyde or 5- bromo indole formaldehyde, compound is made respectively in aforementioned manners
LN-0-4~LN-0-8 target product, it is as a result as follows：
Pale yellow powder, yield 35.3%, fusing point：>320℃；1H NMR(400MHz,DMSO-d6)δppm:12.11(s,
1H,indole-NH),8.95(s,1H,purine-CH),8.52,7.55(m,5H,indole-CH),4.01(s,3H,N-
CH3)；ESI-MS:m/z 284.3,286.2[M+H]+.C14H10ClN5(283.06).
Operation is with embodiment 4, except that intermediate LNa-2 is replaced with into LNb-2.
White powder, yield 41.0%, fusing point：315-317℃；1H NMR(400MHz,DMSO-d6)δppm:12.34
(s,1H,indole-NH),8.97(s,1H,purine-CH),8.51,8.25,7.58,7.28-7.23(m,5H,indole-
CH),4.55(q,2H,N-CH 2-CH3),1.42(t,3H,N-CH2-CH 3)；ESI-MS:m/z 298.5,300.4[M+H]+
.C15H12ClN5(297.08).
Operation is with embodiment 4, except that intermediate LNa-2 is replaced with into LNc-2.
White powder, yield 39.1%, fusing point：>320℃；1H NMR(400MHz,DMSO-d6)δppm:11.91(s,
1H,indole-NH),9.05(s,1H,purine-CH),8.55-8.53,7.85,7.49,7.15-7.13(m,5H,indole-
CH),7.34-7.32,7.27-7.25(m,5H,Ph-H),5.80(s,2H,N-CH 2-Ph)；ESI-MS:m/z 360.4,362.4
[M+H]+.C20H14ClN5(359.09).
Operation is with embodiment 4, except that substituent indolecarboxaldehyde is replaced with into 5- bromo indole formaldehyde.
Brown powder, yield 14.7%, fusing point：>300℃；1H NMR(400MHz,DMSO-d6)δppm:12.30(s,
1H,indole-NH),9.02(s,1H,purine-CH),9.02,8.72,8.42,7.54-7.42(m,4H,indole-CH),
4.01(s,3H,N-CH3)；ESI-MS:m/z 362.3,364.3,366.3[M+H]+.C14H9BrClN5(360.97).
Operation, except that intermediate LNa-2 is replaced with into LNb-2, substituent indolecarboxaldehyde is replaced with embodiment 4
For 5- bromo indole formaldehyde.
Pale yellow powder, yield 18.0%, fusing point：>300℃；1H NMR(400MHz,DMSO-d6)δppm:12.26(s,
1H,indole-NH),9.04(s,1H,purine-CH),8.71,8.33,7.53-7.42(m,4H,indole-CH),4.56
(m,2H,N-CH 2-CH3),1.42(t,3H,N-CH2-CH 3),impurity peaks:4.34,3.43,1.06(Ethanol)；
ESI-MS:m/z376.3,378.4,380.4[M+H]+.C15H11BrClN5(374.99).
Operation, except that intermediate LNa-2 is replaced with into LNc-2, substituent indolecarboxaldehyde is replaced with embodiment 4
For 5- bromo indole formaldehyde.
White powder, yield 24.7%, fusing point：>300℃；1H NMR(400MHz,DMSO-d6)δppm:12.10(s,
1H,indole-NH),9.13(s,1H,purine-CH),8.73,7.93,7.78,7.48-7.46,7.40-7.38(m,4H,
indole-CH),7.35-7.27,7.14-7.13(m,5H,Ph-H),5.81(m,2H,N-CH 2-Ph),impurity peaks:
4.49,3.43,1.06(Ethanol),1.44(Cyclohexane)；ESI-MS:m/z 438.4,440.4,442.4[M+H]+
.C20H13BrClN5(437.00).
The logical method of the target compound LN-S-1~LN-S-4 of embodiment 5. preparation (by taking LN-S-1 as an example, R1=methyl, R2=
N- ethyl phthalimides base)
Intermediate LNa-3 (0.1g, 0.498mmol) is weighed in 50mL eggplant type flasks, adds dimethylformamide
Dissolving is stirred at room temperature in (10mL), first adds potassium carbonate (0.103g, 0.748mmol), after 0.5h is stirred at room temperature, adds raw material N-
(2- bromoethyls) phthalimide (0.139g, 0.548mmol), is warming up to 40 DEG C and continues to react, TLC monitorings, after 12h,
Reaction finishes, and stops reaction, steams most of solvent, and ethyl acetate (6mL) redissolves, and adds washing three times (3 × 5mL), saturation
Saline solution (5mL) is washed once, anhydrous sodium sulfate drying, and filtering, filtrate, which is evaporated, produces product crude product, is then with volume fraction
95% ethyl alcohol recrystallization, obtain sterling LN-S-1.
With different intermediate LN-3 and N- (2- bromoethyls) phthalimides, chemical combination is made respectively in aforementioned manners
Thing LN-S-1~LN-S-4 target product, it is as a result as follows：
White crystal, yield 39.7%, fusing point：213-214℃；1H NMR(400MHz,DMSO-d6)δppm:8.57(s,
1H, purine-CH), 7.77-7.73 (m, 4H, Ph-H), 4.07 (t, 2H, J=8.0Hz, S-CH2-CH 2-N),3.71(t,2H,J
=8.0Hz, S-CH 2-CH2-N),3.55(s,3H,N-CH3)；13C NMR(100MHz,DMSO-d6)δppm:168.13 (N-CO=
C),157.97,155.29,151.80,131.81,123.40(purine-C),134.78,133.37,123.40
(isoindoline-1,3-dione-C),37.74(S-CH2-CH 2-N),30.57(N-CH3),29.31(S-CH 2-CH2-N)；
ESI-MS:m/z 374.3,376.4[M+H]+,396.1[M+Na]+.C16H12ClN5O2S(373.04).
Operation is with embodiment 5, except that intermediate LNa-3 is replaced with into LNb-3.
White crystal, yield 41.6%, fusing point：227-229℃；1H NMR(400MHz,DMSO-d6)δppm:8.59(s,
1H,purine-CH),7.78-7.75(m,4H,isoindoline-1,3-dione-CH),4.09-4.05(m,4H,S-CH2-CH 2-N,N-CH 2-CH3), 3.74 (t, 2H, J=8.0Hz, S-CH 2-CH2- N), 1.29 (t, 3H, J=8.0Hz, N-CH2-CH 3)
；13CNMR(100MHz,CDCl3)δppm:168.05 (N-CO=C), 156.11,154.93,152.68,145.80,133.63
(purine-C),134.13,131.88 123.29(isoindoline-1,3-dione-C),38.19(S-CH2-CH 2-N),
37.17(N-CH 2-CH3),30.18(S-CH 2-CH2-N),14.31(N-CH2-CH 3)；ESI-MS:m/z 387.9,389.9[M+
H]+,410.2[M+Na]+.C17H14ClN5O2S(387.06).
Operation is with embodiment 5, except that intermediate LNa-2 is replaced with into LNc-2.
White crystal, yield 36.0%, fusing point：186-188℃；1H NMR(400MHz,DMSO-d6)δppm:8.66(s,
1H,purine-CH),7.80-7.77(m,4H,isoindoline-1,3-dione-CH),7.35-7.28,7.18(m,5H,
Ph-H),5.29(s,2H,N-CH2- Ph), 4.09 (t, 2H, J=4.0Hz, S-CH2-CH 2- N), 3.70 (t, 2H, J=4.0Hz,
S-CH 2-CH2-N)；13C NMR(100MHz,DMSO-d6)δppm:168.12 (N-CO=C), 157.77,155.27,152.06,
146.46,127.56(purine-C),135.21,133.38,129.27(Ph-C),134.80,131.87,123.45
(isoindoline-1,3-dione-C),46.13(N-CH 2-Ph),37.49(S-CH2-CH 2-N),30.49(S-CH 2-CH2-
N)；ESI-MS:m/z 450.4[M+H]+.C22H16ClN5O2S(449.07).
Operation is with embodiment 5, except that intermediate LNa-2 is replaced with into LNd-2.
White flock crystal, yield 37.4%, fusing point：204-205℃；1H NMR(400MHz,DMSO-d6)δppm:
8.65 (s, 1H, purine-CH), 7.80-7.78 (m, 4H, isoindoline-1,3-dione-CH), 7.13 (d, 2H, J=
8.0Hz, Ph-H), 7.07 (d, 2H, J=8.0Hz, Ph-H), 5.23 (s, 2H, N-CH2- Ph), 4.09 (t, 2H, J=4.0Hz,
S-CH2-CH 2- N), 3.70 (t, 2H, J=4.0Hz, S-CH 2-CH2-N),2.25(s,3H,Ph-CH3)；13C NMR(100MHz,
CDCl3)δppm:167.99 (N-CO=C), 156.61,155.24,152.94,145.99,131.29 (purine-C),
138.28,133.18,129.54,127.98(Ph-C),134.11,131.86,123.29(isoindoline-1,3-dione-
C),46.14(N-CH 2-Ph-CH3),37.12(S-CH2-CH 2-N),30.39(S-CH 2-CH2-N),21.15(Ph-CH 3)；ESI-
MS:m/z 464.4,466.4[M+H]+,486.5[M+Na]+.C23H18ClN5O2S(463.09).
The logical method of the target compound LN-S-5~LN-S-8 of embodiment 6. preparation (by taking LN-S-5 as an example, R1=methyl, R2=
N-Methyl-o-phthalimide base)
Intermediate LNa-3 (0.1g, 0.4984mmol) is weighed in 50mL eggplant type flasks, adds dimethylformamide
Dissolving is stirred at room temperature in (15mL), after addition sodium acid carbonate (0.0460g, 0.5482mmol) is warming up to 40 DEG C of stirring 1.5h, then delays
The slow dimethyl formamide solution that N- bromomethyls phthalimide (0.2393g, 0.9968mmol) is added dropwise, drips and finishes (2h),
TLC is monitored, and reaction finishes, and stops reaction, steams most of solvent, and ethyl acetate (6mL) redissolves, add washing three times (3 ×
5mL), saturated aqueous common salt (5mL) is washed once, anhydrous sodium sulfate drying, and filtering, filtrate, which is evaporated, produces product crude product, then uses body
Fraction is 95% ethyl alcohol recrystallization, obtains sterling LN-S-5.
Compound L N- is made with different intermediate LN-3 and N- bromomethyl phthalimides respectively in aforementioned manners
S-5~LN-S-8 target product, it is as a result as follows：
Filbert acicular crystal, yield 37.8%, fusing point：210-212℃；1H NMR(400MHz,CDCl3)δppm:8.80
(s,1H,purine-CH),7.90-7.88(m,2H,Ph-H),7.78-7.75(m,2H,Ph-H),5.69(s,2H,S-CH2-
N),3.69(s,3H,N-CH3)；13C NMR(100MHz,CDCl3)δppm:166.54 (N-CO=C), 155.12,153.26,
146.95,123.87(purine-C),134.63,133.27,123.87(isoindoline-1,3-dione-C),38.85
(S-CH2-N),29.00(N-CH3)；ESI-MS:m/z 360.4,362.4[M+H]+,382.3[M+Na]+.C15H10ClN5O2S
(359.02).
Operation is with embodiment 6, except that intermediate LNa-3 is replaced with into LNb-3.
Faint yellow acicular crystal, yield 35.4%, fusing point：157-158℃；1H NMR(400MHz,CDCl3)δppm:8.80
(s,1H,purine-CH),7.89-7.77(m,4H,isoindoline-1,3-dione-CH),5.70(s,2H,S-CH2-N),
4.21 (q, 2H, J=8.0Hz, N-CH 2-CH3), 1.29 (t, 3H, J=8.0Hz, N-CH2-CH 3)；13C NMR(100MHz,
DMSO-d6)δppm:166.88 (N-CO=C), 155.13,154.92,152.38,147.45,131.87 (purine-C),
135.40,133.58,123.99(isoindoline-1,3-dione-C),39.52(S-CH2-N),38.66(N-CH 2-CH3),
14.58(N-CH2-CH3 )；ESI-MS:m/z 374.3,376.4[M+H]+,396.1[M+Na]+.C16H12ClN5O2S
(373.04).
Operation is with embodiment 6, except that intermediate LNa-3 is replaced with into LNc-3.
Faint yellow acicular crystal, yield 41.2%, fusing point：182-184℃；1H NMR(400MHz,CDCl3)δppm:8.67
(s,1H,purine-CH),7.90-7.87,7.79-7.75(m,4H,isoindoline-1,3-dione-CH),7.59-7.57
(m,2H,Ph-H),7.33-7.28(m,3H,Ph-H),6.17(s,2H,N-CH2-Ph),5.48(s,2H,S-CH2-N)；13C
NMR(100MHz,DMSO-d6)δppm:166.81 (N-CO=C), 155.75,155.31,152.69,147.79,129.20
(purine-C),133.50,131.86,123.98(isoindoline-1,3-dione-C),135.35,127.65(Ph-C),
46.34(N-CH 2-Ph),39.52(S-CH2-N)；ESI-MS:m/z 436.4,438.4[M+H]+,458.4,460.4[M+Na
]+.C21H14ClN5O2S(435.06).
Operation is with embodiment 6, except that intermediate LNa-3 is replaced with into LNd-3.
Faint yellow acicular crystal, yield 39.4%, fusing point：189-191℃；1H NMR(400MHz,CDCl3)δppm:8.83
(s,1H,purine-CH),7.87-7.84,7.78-7.75(m,4H,isoindoline-1,3-dione-CH),7.18(d,
2H, J=8.0Hz, Ph-H), 7.07 (d, 2H, J=8.0Hz, Ph-H), 5.62 (s, 2H, N-CH2-Ph),5.26(s,2H,S-
CH2-N),2.25(s,3H,Ph-CH3)；13C NMR(100MHz,DMSO-d6)δppm:166.80 (N-CO=C), 155.73,
155.25,152.67,147.78,132.43(purine-C),135.35,131.86,123.97(isoindoline-1,3-
dione-C),137.76,133.48,129.73,127.68(Ph-C),46.14(N-CH 2-Ph-CH3),39.36(S-CH2-N),
21.10(Ph-CH 3)；ESI-MS:m/z450.3,452.2[M+H]+,472.3[M+Na]+.C22H16ClN5O2S(449.07).
The logical method of embodiment 7. target compound LN-S-9, LN-S-10 and LN-S-11 preparation (by taking LN-S-9 as an example, R1=
Ethyl, R2=N-Methyl-o-phthalimide base)
Product LN-S-6 (0.1g, 0.2675mmol) is weighed in 50mL eggplant type flasks, dichloromethane is steamed in addition again
Dissolving is stirred at room temperature in (10mL), and metachloroperbenzoic acid (0.1385g, 0.8025mmol) dichloromethane solution is slowly added dropwise
(10mL), drop finish, TLC monitorings, react and finish after 18h, stop reaction, the excessive thiosulfuric acid newly prepared is added into reaction solution
Sodium solution, except remaining metachloroperbenzoic acid in dereaction, organic phase is separated, washed three times (3 × 5mL), saturated aqueous common salt
(5mL) is washed once, anhydrous sodium sulfate drying, and filtering, filtrate, which is evaporated, produces product crude product, is then recrystallized with absolute ethyl alcohol, i.e.,
Obtain product LN-S-9.
With different end-product LN-S-6, LN-S-2 or LN-S-8, be made respectively in aforementioned manners compound L N-S-9,
LN-S-10 and LN-S-11 target product, it is as a result as follows：
White flock crystal, yield 39.5%, fusing point：198-199℃；1H NMR(400MHz,CDCl3)δppm:8.74
(s,1H,purine-CH),7.76-7.69(m,4H,isoindoline-1,3-dione-CH),4.75-4.56(m,2H,N-CH 2-CH3),4.36-4.22(m,2H,SO-CH2-CH 2-N),4.16-4.10,3.89-3.83(m,2H,SO-CH 2-CH2-N),
1.59 (t, 3H, J=8.0Hz, N-CH2-CH 3)；13C NMR(100MHz,DMSO-d6)δppm:(167.10 N-CO=C),
156.32,154.39,151.88,131.59(purine-C),135.70,132.59,123.97(isoindoline-1,3-
dione-C),57.59(N-CH 2-CH3),15.52(N-CH2-CH3 )；ESI-MS:m/z 391.5[M+H]+.C16H12ClN5O3S
(389.03).
Operation is with embodiment 7, except that LN-S-6 is replaced with into LN-S-2.
White flock crystal, yield 32.7%, fusing point：221-222℃；1H NMR(400MHz,CDCl3)δppm:8.93
(s,1H,purine-CH),7.88-7.86,7.81-7.79(m,4H,isoindoline-1,3-dione-CH),5.59-5.44
(m,2H,SO-CH 2-N),4.68-4.66(m,2H,N-CH 2-CH3), 1.58 (t, 3H, J=8.0Hz, N-CH2-CH 3)；13C NMR
(100MHz,DMSO-d6)δppm:167.92 (N-CO=C), 156.83,154.34,151.84,131.85 (purine-C),
134.87,132.14,123.44(isoindoline-1,3-dione-C),50.81(SO-CH 2-CH2-N),31.08(SO-
CH2-CH 2-N,N-CH 2-CH3),15.50(N-CH2-CH 3)；ESI-MS:m/z 404.4[M+H]+,426.2[M+Na]+
.C17H14ClN5O3S(403.05).
Operation is with embodiment 7, except that LN-S-6 is replaced with into LN-S-8.
White crystal, yield 34.6%, fusing point：171-173℃；1H NMR(400MHz,CDCl3)δppm:8.96(s,1H,
), purine-CH 7.84-7.78 (m, 4H, isoindoline-1,3-dione-CH), 7.40 (d, 2H, J=12.0Hz, Ph-
), H 7.12 (d, 2H, J=8.0Hz, Ph-H), 5.78 (q, 2H, J=16.0Hz, J=12.0Hz, N-CH2-Ph),5.31(q,
2H, J=12.0Hz, J=12.0Hz, S-CH2-N),2.29(s,3H,Ph-CH3),impurity peaks:1.55(H2O),
1.25(Ethanol-CH3)；13CNMR(100MHz,CDCl3)δppm:166.52 (N-CO=C), 151.17,134.81,
129.66(purine-C),134.81,128.72(Ph-C),131.49,124.07(isoindoline-1,3-dione-C),
57.13(N-CH 2-Ph-CH3),46.82(S-CH2-N),21.36(Ph-CH 3)；ESI-MS:m/z 466.3[M+H]+,488.3[M
+Na]+,931.3[2M+H]+,953.4[M+Na]+.C22H16ClN5O3S(465.07).
Experimental example 1：The external Anti-HBV effect test experiments of target compound
Test philosophy
HBV transgenosis liver cancer cell lines -- HepG2.2.15 cell lines can secrete virion in culture supernatant, including disease
Malicious DNA, HBsAg and HBeAg.After target compound to be measured is added, cell secretion HBsAg and HBeAg contents can be varied from.
Therefore, virion content in culture supernatant is secreted into by detecting cell, contrasts non-dosing control group content, sample can be obtained
The anti-virus ability of product medicine, while toxicity size of the example pharmaceuticals to cell can also be detected.It is sun with Lamivudine (3TC)
Property comparison medicine, reached with ELISA method detection example pharmaceuticals suppress viral HBsAg's and HBeAg secretions 50% when concentration numbers
It is worth for IC50；Suppress concentration values IC during the 50% of HBV DNA replication dna amounts with PCR methods detection example pharmaceuticals50；With CCK-8 methods
Detection example pharmaceuticals cause concentration values during 50% cytotoxicity death to be CC50；Pass through formula:SI=CC50/IC50, calculate
Go out " selection coefficient " (the selectivity index, SI) of testing compound.Due to the different solubility of compound, therefore often
Individual compound primary dcreening operation concentration is also different.(Activity Results are shown in Tables 1 and 2)
Wherein, the OD values obtained by activity test are converted into inhibiting rate first, then further according to concentration gradient by its turn
It is melted into IC50And CC50Value, calculated by improveing bandit's formula method, formula is as follows：
lgIC50=Xm-I (P- (3-Pm-Pn)/4)；
Xm:Lg maximum doses；I:Lg (maximum dose/adjacent dosage)；
P:Positive reaction rate sum；Pm:Maximum positive reaction rate；Pn:Minimum positive reaction rate
Test Virus strain
HBV transgenosis liver cancer cell lines HepG2.2.15 cell lines.HepG2.2.15 can stablize expression hepatitis B
Grain, the cell line are awarded seminar by the Ma Chun Red Sect of Lamaism of Shandong University and provided.
Method of testing
The collection of culture supernatant
HepG2.2.15 cells are inoculated in 96 orifice plates, the example pharmaceuticals that next day adds various concentrations are (multiple per concentration 3
Hole), nutrient solution and the example pharmaceuticals with concentration were changed at the 4th day, it is to be measured to collect culture supernatant in the 9th day.
Cytotoxicity experiment (CCK-8 methods)
Cell into 96 orifice plates adds CCK-8, and after 4 hours plus the reaction of CCK-8 lysates is stayed overnight, and next day surveys on ELIASA
OD450.According to OD values calculate example pharmaceuticals on the toxic action of HepG2.2.15 cells and influence cell growth situation,
Finally calculating causes concentration (CC needed for half cell death amount50)。
HBeAg and HBsAg content in ELISA detection culture supernatant
50 μ L culture supernatant is separately added into the stripe board being coated with, and the HBeAg and HBsAg that add equivalent are used
Enzyme mark conjugate (blank control wells are not added with), fully mix, stick gummed paper, put and discarded after being incubated 60 minutes at 37 DEG C in stripe board
Liquid, and washed with cleaning solution, it is repeated 5 times, then pats dry.It is last that the mixed μ L of luminous substrate 50 are added per hole, fully mix
Even room temperature lucifuge adds terminate liquid (50 μ L) terminating reaction after being incubated 10 minutes, and OD is determined with ELIASA450/630, and according to OD values
Calculate half inhibiting rate (IC of the sample to HBV antigens50)。
HBV DNA content in PCR methods detection culture supernatant
After HepG2 2.2.15 cells are cultivated 4 hours in 96 porocyte culture plates, the training of various concentrations drug containing is matched somebody with somebody in addition
Nutrient solution, continue to cultivate 8 days (changing liquid once within every 4 days), collect supernatant, enter performing PCR with fluorescence probe method and detect.
The anti-hepatitis virus primary dcreening operation activity of the novel purine Non-nucleoside-type inhibitors of table 1.
Note：Reactive compound is represented with runic；aND：Undetermined.
The anti-HBsAg and HBeAg secretion activities of the part of compounds of table 2.
Note：bIC50：Suppress the concentration of compound when 50%HBsAg or HBeAg secretes；cCC50：Suppress 50% cell growth
When compound concentration；dSI：Select index, CC50/IC50Ratio；eNA：It is inactive.
Conclusion：
As can be seen from Table 1, substituted purin analog derivative of the invention is a series of novel non-nucleoside HBV suppressions of structures
Preparation, show different suppression antigen (HBsAg and HBeAg) secretion activity.Wherein, compound L N-S-4, LN-S-6,
LN-S-9 and LN-S-10 is to the inhibiting rate of two kinds of antigen more than 50%.Under maximum primary dcreening operation concentration, except compound L N-0-5, LN-
0-7, LN-0-8 and LN-S-8 DNA replication dna inhibiting rate reach 40%, and majority of compounds hardly has the suppression of HBV DNA replication dnas
System activity.Based on primary dcreening operation result, the suppression of compound L N-S-4, LN-S-6, LN-S-9 and LN-S-10 to antigen has further been screened
System is active (table 2).Wherein, preferably, the activity to HBsAg is IC to compound L N-S-10 activity50=6.63 μM, SI=1.17 is right
HBeAg activity is IC50=4.43 μM, SI=1.72.Therefore such substituted purin analog derivative has further research with opening
The value of hair, it can be used as the lead compound of Anti-HBV activity.
Claims (6)
1. substituted purin analog derivative and its pharmaceutically acceptable salt with structure shown in below formula I：
Wherein,
R is chlorine atom；
R1For methyl, ethyl, benzyl or to methyl-benzyl；
Linker is alkane chain or 2 carbon atoms and 1 that length is the alkane chain of 0 atom, 1 carbon atom and 1 sulphur atom
The alkane chain of individual sulphur atom；
R2For N- methylpyrrole -2- bases, indol-3-yl, 5- bromo indole -3- bases, phthalimide-based.
2. substituted purin analog derivative, it is characterised in that for one of following compound：
3. the preparation method of substituted purin analog derivative as claimed in claim 1, it is characterised in that by initiation material 2,4- bis-
Chloro- 5- nitro-pyrimidines obtain intermediate LN-1 through selective nucleophilic displacement of fluorine under low temperature, intermediate LN-1 nitro through stannous chloride also
Original obtains key intermediate LN-2；Amino in intermediate LN-2 and the heteroaromatic generation schiff bases with aldehyde radical, then anhydrous three
Cyclization obtains part end-product LN-0 series under Ferric Chloride；Intermediate LN-2 and thiocarbonyldiimidazole reaction generation intermediate
LN-3, then end-product LN-S series is obtained through nucleophilic displacement of fluorine, then aoxidized to obtain sulfoxide product LN- by metachloroperbenzoic acid
S’；
Synthetic route is as follows：
Reagent and condition:(i) methylamine water solution or ethylamine solution or benzylamine or to methylbenzylamine, dichloromethane, diisopropyl
Base ethamine, -78 DEG C；(ii) stannous chloride, absolute ethyl alcohol, backflow；(iii) the nitrogenous heteroaromatic of aldehyde radical substitution, glacial acetic acid are anhydrous
Methanol, room temperature；Anhydrous ferric trichloride, absolute ethyl alcohol or dimethylformamide, 80 DEG C；(iv) thiocarbonyldiimidazole, tetrahydrofuran,
Backflow；(v) potassium carbonate or sodium acid carbonate, room temperature；(iv) metachloroperbenzoic acid, dichloromethane, room temperature；
Wherein, wherein, R1、R2It is defined as above described in formula I.
4. the preparation method of substituted purin analog derivative as claimed in claim 2, it is characterised in that step is as follows：
(1) 2.0g 2 is weighed, the chloro- 5- nitro-pyrimidines of 4- bis- add 20mL dichloromethane and be stirred at room temperature in 100mL eggplant type flasks
Dissolving, be put into low-temp reaction instrument, when being cooled to -30 DEG C, add 3.07mL diisopropylethylamine, to -78 DEG C when, be slowly added dropwise
With the methylamine water solutions of 0.885g 40% of 5mL dchloromethanes, drop finishes, and continues to stir 4h, TLC monitorings, reaction is finished, stopped
Only to react, recover to room temperature, three times, saturated common salt is washed once, anhydrous sodium sulfate drying for washing, and filtering, filtrate, which is evaporated, mixes sample,
Silica gel column chromatography separating purification, mobile phase selection petroleum ether：Ethyl acetate, volume ratio 8：1, obtain intermediate LNa-1；
(2) 1.4g intermediates LNa-1 is weighed in 250mL flasks, the dissolving of 100mL ethanol, 6.17g chlorinations is added portionwise under stirring
Stannous, finish, under nitrogen protection, be warming up to 80 DEG C of back flow reaction 9h, TLC monitorings, reaction finishes, and stops reaction, question response
Liquid recovers to room temperature, and it is 3 to survey reaction solution pH, and under ice-water bath, pH to 8 is slowly adjusted with 1M sodium hydroxide solutions, with suction filtered through kieselguhr,
Most of ethanol in filtrate is steamed, adds ethyl acetate extraction three times, merges organic phase, saturated common salt is washed once, nothing
Aqueous sodium persulfate is dried overnight, and filtering, filtrate, which is evaporated, mixes sample, silica gel column chromatography separating purification, mobile phase selection petroleum ether：Acetic acid second
Ester, volume ratio 1：1 to 1：2, obtain intermediate LNa-2；
(3) 0.3g intermediates LNa-2 is weighed in 50mL eggplant type flasks, the dissolving of 15mL absolute methanols, is separately added under stirring
0.2684g N- methyl -2- pyrrole aldehydes, 0.1mL acetic acid, 6h, TLC monitorings is stirred at room temperature, reaction finishes, and decompression steams solvent
Methanol and acetic acid, the dissolving of 15mL absolute ethyl alcohols is added, stir lower addition 6.2g 5%FeCl3-SiO2, it is warming up to 80 DEG C of backflows
1.5h, stop reaction, be cooled to room temperature, filter out insoluble matter, the ethanol in filtrate is steamed, add ethyl acetate and redissolve, water
Wash three times, saturated common salt is washed once, organic phase anhydrous sodium sulfate drying, and filtering, filtrate, which is evaporated, mixes sample, silica gel column chromatography separation
Purifying, mobile phase selection petroleum ether：Ethyl acetate, volume ratio 10：1 to 8：1, you can isolate product LN-0-1；
0.2g intermediates LNa-2 is weighed in 50mL eggplant type flasks, the dissolving of 15mL absolute methanols, is separately added under stirring
0.2197g indolals, 0.1mL acetic acid, 12h, TLC monitorings being stirred at room temperature, reaction finishes, and decompression steams solvent methanol and acetic acid,
15mL absolute ethyl alcohols or dimethylformamide dissolving are added, stirs the lower 0.2045g FeCl for adding solvent dilution3Solution, rise
Temperature stops reaction, is cooled to room temperature, directly filters out insoluble matter, filter cake is washed with water, and petroleum ether is washed, vacuum to 80 DEG C of backflow 10h
Drying box is dried, you can obtains product LN-0-4；
(4) 0.3g intermediates LNa-2 is weighed in 50mL flasks, and 15mL steams tetrahydrofuran dissolving again, stirs lower addition 0.5057g
Thiocarbonyldiimidazole, 0.2871g triethylamines are added, be warming up to 70 DEG C of back flow reaction 12h, TLC monitorings, reaction is finished, stopped anti-
Should, question response recovers to room temperature, steams tetrahydrofuran, adds ethyl acetate and redissolves, under ice-water bath, with 2M sodium hydroxide solutions
Wash organic phase three times, aqueous phase merges, and ice-water bath is slowly stirred down, and watery hydrochloric acid is added dropwise, and separates out solid, filtering, after filter cake is washed with water
It is drying to obtain intermediate LNa-3；
(5) 0.1g intermediates LNa-3 is weighed in 50mL eggplant type flasks, adds 10mL dimethylformamides, dissolving is stirred at room temperature,
0.103g potassium carbonate is first added, after 0.5h is stirred at room temperature, 0.139g raw materials N- (2- bromoethyls) phthalimide is added, rises
Warm to 40 DEG C are continued to react, TLC monitorings, and after 12h, reaction finishes, and stops reaction, steams solvent dimethylformamide, acetic acid second
Ester is redissolved, and adds washing three times, and saturated common salt is washed once, anhydrous sodium sulfate drying, filtering, and filtrate is evaporated that to produce product thick
Product, it is then 95% ethyl alcohol recrystallization with volume fraction, obtains sterling LN-S-1；
0.1g intermediates LNa-3 is weighed in 50mL eggplant type flasks, 15mL dimethylformamides is added, dissolving is stirred at room temperature, adds
Enter 0.0460g sodium acid carbonates, after being warming up to 40 DEG C of stirring 1.5h, then 0.2393g N- bromomethyls phthalyl Asia is slowly added dropwise
The dimethyl formamide solution of amine, 2h drops finish, and TLC monitorings, reaction finishes, and stops reaction, steam solvent, and ethyl acetate redissolves,
Add washing three times, saturated common salt is washed once, anhydrous sodium sulfate drying, and filtering, filtrate, which is evaporated, produces product crude product, Ran Houyong
Volume fraction is 95% ethyl alcohol recrystallization, obtains sterling LN-S-5；
(6) 0.1g products LN-S-6 is weighed in 50mL eggplant type flasks, and addition 10mL steams dichloromethane and dissolving is stirred at room temperature again, delays
The slow dichloromethane solution that 0.1385g metachloroperbenzoic acids are added dropwise, drop finish, TLC monitorings, react and finish after 18h, stop anti-
Should, the excessive hypo solution newly prepared is added into reaction solution, except remaining metachloroperbenzoic acid in dereaction, is divided
From organic phase, three times, saturated common salt is washed once, anhydrous sodium sulfate drying for washing, and filtering, filtrate, which is evaporated, produces product crude product,
Then recrystallized with absolute ethyl alcohol, produce product LN-S-9.
5. application of the compound described in claim 1 or 2 in the medicine of Anti-HBV activity is prepared.
6. a kind of Anti-HBV drugs composition, pharmaceutically acceptable comprising the compound of claim 1 or 2 and one or more
Carrier or excipient.
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