CN106496084B - One kind aromatic compound containing sulfydryl preparation method and its application in terms of anti HIV-1 virus - Google Patents
One kind aromatic compound containing sulfydryl preparation method and its application in terms of anti HIV-1 virus Download PDFInfo
- Publication number
- CN106496084B CN106496084B CN201510564136.XA CN201510564136A CN106496084B CN 106496084 B CN106496084 B CN 106496084B CN 201510564136 A CN201510564136 A CN 201510564136A CN 106496084 B CN106496084 B CN 106496084B
- Authority
- CN
- China
- Prior art keywords
- compound
- hiv
- independently selected
- formulas
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses the preparation method of one kind aromatic compound containing sulfydryl and its applications in terms of anti HIV-1 virus.The compound has structure shown in Formulas I, the compound of Formulas I of the present invention guards cell protein PCAF BRD as action target spot using host itself, with resistance problems brought by the variation of potential solution inhibition of HIV height, there is the preferable activity for inhibiting inhibition of HIV proliferation, and it is lower to the toxicity of people's normal lymphocytes, it can be applied to inhibit in the preparation of the drug of inhibition of HIV proliferation.
Description
Technical field
The present invention relates to biochemical field, more particularly to a kind of compound and its preparation method and application.
Background technique
AIDS, i.e. Immune Deficiency Syndrome have been human infections human immunodeficiency virus (HIV) and cause
Infectious disease.HIV is a kind of virus that can attack human immune system.It is T4 lymph group most important in human immune system
It knits as target of attack, considerable damage T4 lymphoid tissue, generates high fatefulue interior failure.This virus passes throughout one's life in region
Dye, destroys the immunologic balance of people, and human body is made to become the carrier of various diseases.
HIV itself can't cause any disease, but after immune system is destroyed by HIV, human body is due to resistivity
It is too low, the chance of duplication immunocyte is lost, and infect that other diseases lead to various compound infections and dead.
Up to the present, the drug and therapy that can effectively treat AIDS in worldwide not yet, can only use drug
Suitable control HIV is in the intracorporal proliferation of people.For the life cycle of HIV, a large amount of relevant researchs, medicine target have been done at present
Point is mainly identification fusion target spot, reverse transcriptase and the GAP-associated protein GAP target spot, integrase target spot, proteolytic enzyme of virus and cell
Target spot etc..But due to the variability of HIV, there is an urgent need to various different role approach at present, and effectively control AIDS
The drug that virus is proliferated in human body delays the growth rate of HIV with this, strives for longer life span for patient.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of compounds, so that the compound is able to suppress inhibition of HIV
Proliferation, while there are resistance problems brought by potential solution inhibition of HIV height variation.
To achieve the above object, the present invention provides a kind of compound, which belongs to sulfydryl analog derivative, has Formulas I
Shown structure:
Wherein, R1、R2、R3、R4、R5It is independently selected from-H ,-(CH2)0-4-CH3,-(CH2)0-4-CH2Cl,-(CH2)0-4-
CH2Br,-CF3,-(CH2)0-4-OH,-(CH2)0-4-O-(CH2)0-4-CH3,-(CH2)0-4-O-,CH(O)-(CH2)0-4-CH3,-
(CH2)0-4-CH-(CH3)2,-(CH2)0-4-Ar,-C(O)-(CH2)0-4-CH3,-C(O)-O-(CH2)0-4-CH3,-C(O)-NH2,-C
(O)-OH,-C(O)-O(CH2)0-4-CH3,-C(O)-O(CH2)0-4-Ar,-C(O)-NH-(CH2)0-4-CH3,-C(O)-N-
[(CH2)0-4-CH3]2,-OH,-SH,-O-(CH2)0-4-CH3,-O-C(O)-(CH2)0-4-CH3,-O-(CH2)0-4-CH-(CH3)2,-
O-(CH2)0-4-Ar,-NH2,-NH-(CH2)0-4-CH3,-NH-(CH2)0-4-Ar,-NH-(CH2)0-4-NH2,-N(CH3)2,-NH-C
(O)-(CH2)0-4-CH3,-NH-C(O)-(CH2)0-4- S-Ar ,-NH-Ts ,-F ,-Cl ,-Br ,-I ,-CN ,-CH=CH-
(CH2)0-4-CH3,-(CH2)0-4- CH=CH2,-CH=CH-Ar ,-CH=CH-CH=CH- (CH2)0-4-CH3,-(CH=CH)0-4-
CH=C (CH3)2,-C≡C-(CH2)0-4-CH3,-C≡C-Ar,-NO2;
X is independently selected from-O- ,-S- ,-NH- ,-N- (CH2)0-3-CH3,-N-(CH2)0-4-CH(CH3)2,-N-(CH2)0-4-C
(CH3)3,-COO-,-C(O)S-;
Y is independently selected from-OH ,-SH ,-NH2;
N is the integer of 1-4;
Ar is the aryl containing ten carbon atoms.
Preferably, R1、R2、R3、R4、R5It is independently selected from-H ,-CH3,-(CH2)0-4-CH3,-(CH=CH)0-4- CH=C-
(CH3)2,-(CH2)0-4-Ar,-(CH2)0-4- CH=CH2,-NO2;
X is independently selected from-O- ,-NH- ,-S- ,-N- (CH2)n-CH3,-COO-,-C(O)S-;
Y is independently selected from-OH ,-SH ,-NH2;
N is the integer of 1-3;
Ar is the aryl containing ten carbon atoms.
Further preferably, R1、R2、R3、R4、R5It is independently selected from-H ,-CH3,-(CH2)3-CH3,-CH=CH-CH=C-
(CH3)2,-NO2;
X is independently selected from-O- ,-S- ,-COO- ,-C (O) S-;
Y is independently selected from-OH ,-SH ,-NH2;
N is the integer of 1-2.
Further preferably, the compound has selected from following every structure.
The transcription of inhibition of HIV in vivo need lysine acetylation viral trans-activating factor (HIV-Tat) and
It human body trans- transcription co-activation factor PCAF BRD and is maintained with the transformation complex compound PBAF interaction of SWI/SNF chromatin
The activity of HIV, it means that in inhibition of HIV human body transcription, HIV-Tat (trans- activating transcription factor) is played and closed very much
The effect of key, and the activity of HIV-Tat needs and some cell protein complex compound collective effects are just able to achieve, such as above-mentioned PCAF
BRD etc..Therefore, as long as the activity combined between HIV-Tat and PCAF BRD can be inhibited, so that it may reach and inhibit HIV transcription
Purpose, the final proliferation for inhibiting HIV.
PCAF BRD is host cell proteins rather than virus protein, therefore the opposite guarantor of PCAF BRD protein structure domain gene
It keeps, variability is lower, and PCAF BRD can be good at solving the inhibition of HIV bring drug resistance that makes a variation itself asking as drug target
Topic.(Elisa experimental procedure bibliography J.Am.Chem.Soc.2005,127,2376-2377.) is tested by external Elisa
Prove that compound shown in Formulas I of the present invention has the preferable activity for inhibiting PCAF BRD and Tat to combine, wherein test knot
The preferable compound IC of fruit display portion inhibition50Value is at 10 μM or so.
The present invention uses international test according to SFDA (the non-clinical pharmacodynamic study technological guidance principle of inverase)
Method detects the HIV-resistant activity of compound shown in Formulas I of the present invention, obtains its cytotoxicity CC50And AntiHIV1 RT activity is living
Property EC50, the results show that compound shown in Formulas I has the activity of the preferable proliferation for inhibiting inhibition of HIV, and to people's normal lymphoid
The toxicity of cell is lower.
Therefore, the application the present invention also provides compound shown in Formulas I in the drug that preparation inhibits inhibition of HIV proliferation.
Wherein, the drug for inhibiting inhibition of HIV proliferation includes compound or pharmaceutically acceptable salt thereof and medicinal load shown in a effective amount of Formulas I
Body.According to pharmacy common sense, compound shown in Formulas I has the activity for the proliferation for inhibiting inhibition of HIV, then its pharmaceutical salts also accordingly has
There is the activity of this respect.In addition, the pharmaceutical carrier can be determined according to the dosage form specifically prepared, it is public to belong to those skilled in the art
Know, is not specifically limited.
In addition, described method includes following steps the present invention also provides the preparation method of compound shown in Formulas I:
Compound shown in step 1, Formula II and 1,3- dibromopropane or bis- bromo-n-hexane of 1,2- and the potassium tert-butoxide room in DMF
Temperature reaction, compound shown in production III;
Compound shown in step 2, formula III reacted in DMSO with vulcanized sodium or formula III shown in compound and ammonium hydroxide four
Reaction, compound shown in production I in hydrogen furans (THF);
Wherein, X is independently selected from-O- ,-S- ,-NH- ,-N- (CH2)0-3-CH3,-N-(CH2)0-4-CH(CH3)2,-N-
(CH2)0-4-C(CH3)3,-COO-,-C(O)S-;N is 1 or 2;
R1、R2、R3、R4、R5It is independently selected from-H ,-(CH2)0-4-CH3,-(CH2)0-4-CH2Cl,-(CH2)0-4-CH2Br,-
CF3,-(CH2)0-4-OH,-(CH2)0-4-O-(CH2)0-4-CH3,-(CH2)0-4-O-,CH(O)-(CH2)0-4-CH3,-(CH2)0-4-
CH-(CH3)2,-(CH2)0-4-Ar,-C(O)-(CH2)0-4-CH3,-C(O)-O-(CH2)0-4-CH3,-C(O)-NH2,-C(O)-OH,-
C(O)-O(CH2)0-4-CH3,-C(O)-O(CH2)0-4-Ar,-C(O)-NH-(CH2)0-4-CH3,-C(O)-N-[(CH2)0-4-CH3
]2,-OH,-SH,-O-(CH2)0-4-CH3,-O-C(O)-(CH2)0-4-CH3,-O-(CH2)0-4-CH-(CH3)2,-O-(CH2)0-4-
Ar,-NH2,-NH-(CH2)0-4-CH3,-NH-(CH2)0-4-Ar,-NH-(CH2)0-4-NH2,-N(CH3)2,-NH-C(O)-
(CH2)0-4-CH3,-NH-C(O)-(CH2)0-4- S-Ar ,-NH-Ts ,-F ,-Cl ,-Br ,-I ,-CN ,-CH=CH- (CH2)0-4-
CH3,-(CH2)0-4- CH=CH2,-CH=CH-Ar ,-CH=CH-CH=CH- (CH2)0-4-CH3,-(CH=CH)0-4- CH=C
(CH3)2,-C≡C-(CH2)0-4-CH3,-C≡C-Ar;
Y is independently selected from-OH ,-SH ,-NH2;
Ar is the aryl containing ten carbon atoms.
Preferably, R1、R2、R3、R4、R5It is independently selected from-H ,-(CH2)0-4-CH3,-(CH=CH)0-4- CH=C-
(CH3)2,-(CH2)0-4-Ar,-(CH2)0-4- CH=CH2,-NO2;
X is independently selected from-O- ,-NH- ,-S- ,-N- (CH2)n-CH3,-COO-,-C(O)S-;
Y is independently selected from-OH ,-SH ,-NH2;
N is the integer of 1-3;
Ar is the aryl containing ten carbon atoms.
Further preferably, R1、R2、R3、R4、R5It is independently selected from-H ,-CH3,-(CH2)3-CH3,-CH=CH-CH=C-
(CH3)2,-NO2;
X is independently selected from-O- ,-S- ,-COO- ,-C (O) S-;
Y is independently selected from-OH ,-SH ,-NH2;
N is the integer of 1-2.
Reaction equation is as follows:
Wherein reaction described in step 1 need to be stirred at room temperature 3-48 hours;Reaction described in step 2 need to be stirred at room temperature 1-12 hours.
Preferably, the amount of the substance of compound shown in the Formula II and 1,3- dibromopropane or 1,2- Bromofume and potassium tert-butoxide
The ratio between be 1:3:1.5, the ratio between amount of substance of compound shown in the formula III and vulcanized sodium is 1:2.
From the above technical scheme, compound shown in Formulas I of the present invention guards cell protein PCAF with host itself
BRD is action target spot, efficiently solves the problems, such as inhibition of HIV variability, has the preferable proliferation for inhibiting inhibition of HIV
Activity, and it is lower to the toxicity of people's normal lymphocytes, it can be applied to inhibit in the preparation of the drug of inhibition of HIV proliferation.
Note: control of abridging in this specification:
Ar is the aryl containing ten carbon atoms, and DMF is n,N-Dimethylformamide, and DMSO is dimethyl sulfoxide, and THF is four
Hydrogen furans, TBDPSCl are tert-butyl diphenyl chlorosilanes.
Detailed description of the invention
Fig. 1 show 2- [4- (4- methyl-1,3-pentylene) thiophenyl] ethyl mercaptan compound1H nuclear magnetic resonance
(400MHz) spectrogram, wherein R1For-H, R2For-H, R3For-CH=CH-CH=C (CH3)2, R4For-H, R5It is-S-, Y for-H, X
For-SH, n 1.
Fig. 2 show 3- [4- (4- methyl-1,3-pentylene) phenoxy group] propane -1- mercaptan compound1H nuclear magnetic resonance
(400MHz) spectrogram, wherein R1For-H, R2For-H, R3For-CH=CH-CH=C (CH3)2, R4For-H, R5It is-O-, Y for-H, X
For-SH, n 2.
Fig. 3 show 3- (p-methylphenyl is thio) -1- propanamine compounds1H nuclear magnetic resonance (400MHz) spectrogram, wherein R1
For-H, R2For-H, R3For-CH3, R4For-H, R5It is-S- for-H, X, Y is-NH2, n 2.
Fig. 4 show 3- mercapto propyl -2- nitric acid benzoic acid thioester compound1H nuclear magnetic resonance (400MHz) spectrogram,
In, R1For-H, R2For-H, R3For-H, R4For-H, R5For-NO2, X is-C (O) S-, and Y is-SH, n 2.
Fig. 5 show 3- (2- nitrophenylsulfenyl) propane -1- mercaptan compound1H nuclear magnetic resonance (400MHz) spectrogram,
In, R1For-H, R2For-H, R3For-H, R4For-H, R5For-NO2, X is-S-, and Y is-SH, n 2.
Fig. 6 show 3- hydroxyl -2- nitric acid propyl benzoate compound1H nuclear magnetic resonance (400MHz) spectrogram, wherein R1
For-H, R2For-H, R3For-H, R4For-H, R5For-NO2, X is-C (O) O-, and Y is-OH, n 2.
Fig. 7 show 3- (O-Nitrophenylfluorone is thio) -1- propanamine compounds1H nuclear magnetic resonance (400MHz) spectrogram,
In, R1For-H, R2For-H, R3For-H, R4For-H, R5For-NO2, X is-S-, and Y is-NH2, n 2.
Fig. 8 show 2- [4- (4- methyl-1,3-pentylene) thiophenyl] alcohol cpd1H nuclear magnetic resonance
(400MHz) spectrogram, wherein R1For-H, R2For-H, R3For-CH=CH-CH=C (CH3)2, R4For-H, R5It is-S-, Y for-H, X
For-OH, n 1.
Fig. 9 show 3- [4- vinyl benzene oxygroup]-propane -1- mercaptan compound1H nuclear magnetic resonance (400MHz) spectrum
Figure, wherein R1For-H, R2For-H, R3For-CH=CH2, R4For-H, R5It is-O- for-H, X, Y is-SH, n 2.
Figure 10 show (E) -3- [4- styryl phenoxy group]-propane -1- mercaptan compound1H nuclear magnetic resonance
(400MHz) spectrogram, wherein R1For-H, R2For-H, R3For-(E)-CH=CH-Ph, R4For-H, R5It is-O- for-H, X, Y is-SH,
N is 2.
Specific embodiment
The invention discloses a kind of compounds, also disclose the preparation method and application of the compound, those skilled in the art
Member can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications
Apparent to those skilled in the art, they are considered as being included in the present invention.Compound of the present invention, side
Method and application be described by preferred embodiment, related personnel obviously can not depart from the content of present invention, spirit and
To method described herein and application is modified or appropriate changes and combinations in range, carry out implementation and application the technology of the present invention.
Below with reference to embodiment, the present invention is further explained, and experiment agents useful for same inventory is shown in Table 1.
1 main agents inventory of table
Title | No. CAS | Producer |
Ethylene chlorhydrin | 107-07-3 | Sinopharm Chemical Reagent Co., Ltd. |
TBDPSCl | 58479-61-1 | West Asia reagent |
Imidazoles | 288-32-4 | Nine ancient cooking vessels chemistry (Shanghai) Science and Technology Ltd. |
Lawesson reagent | 19172-47-5 | Aladdin |
Parahydroxyben-zaldehyde | 123-08-0 | Nine ancient cooking vessels chemistry (Shanghai) Science and Technology Ltd. |
1,3- dibromopropane | 109-64-8 | Nine ancient cooking vessels chemistry (Shanghai) Science and Technology Ltd. |
Potassium iodide | 7681-11-0 | Sinopharm Chemical Reagent Co., Ltd. |
Sodium hydride | 7646-69-7 | Aladdin |
Vulcanized sodium | 1313-82-2 | Sinopharm Chemical Reagent Co., Ltd. |
To methylbenzene phenyl-sulfhydrate | 106-45-6 | Nine ancient cooking vessels chemistry (Shanghai) Science and Technology Ltd. |
Hydrazine hydrate | 10217-52-4 | Sinopharm Chemical Reagent Co., Ltd. |
4- nitrobenzoic acid | 62-23-7 | Nine ancient cooking vessels chemistry (Shanghai) Science and Technology Ltd. |
1,3- dimercaptopropane | 109-80-8 | Nine ancient cooking vessels chemistry (Shanghai) Science and Technology Ltd. |
O-chloronitrobenzene | 88-73-3 | Nine ancient cooking vessels chemistry (Shanghai) Science and Technology Ltd. |
1,3- propylene glycol | 504-63-2 | Sinopharm Chemical Reagent Co., Ltd. |
Embodiment 1
1, compound (R shown in Formulas I of the present invention is prepared1For-H, R2For-H, R3For-CH=CH-CH=C (CH3)2, R4
For-H, R5It is-S- for-H, X, Y is entitled 2- [4- (4- methyl-1, the 3- pentadiene) thiophenyl] ethyl mercaptan of-SH, n 1).
10mmol ethylene chlorhydrin, 10mmol TBDPSCl and 12mmol imidazoles react at room temperature 2-5h in 20mL DMF, then extract,
Column chromatography for separation is thrown in next step.First 10mmol 4- (4- methyl-1,3-pentylene) benzenethiol, 20mmol sodium bicarbonate are existed
After stirring half an hour in 25mL DMF, above-mentioned products therefrom is slowly added dropwise after entering and is reacted 5-10 hours, extracted after reaction
It takes, column chromatographs to obtain faint yellow solid.After above-mentioned solid is dissolved in DCM, two equivalent chloroacetic chlorides are added and several drop methanol, room temperature are anti-
It answers 2-8 hours, column chromatography for separation obtains product 2- [4- (4- methyl-1,3-pentylene) thiophenyl] ethyl alcohol.Finally by 2- [4- (4-
Methyl-1,3- pentadiene) thiophenyl] ethyl alcohol be dissolved in toluene and be added 0.6 equivalent lawesson reagent, at 80 DEG C react 5-10 it is small
When, cooling rotation dry chromatography can obtain final product 2- [4- (4- methyl-1,3- pentadiene) thiophenyl] second sulphur after reaction
Alcohol.
Reaction equation is as follows:
Prepared compound is carried out1H nuclear magnetic resonance (400MHz) detection, solvent CD3OD, testing result are shown in Fig. 1,
Prepared compound is consistent with structure shown in Formulas I as the result is shown.
2, cell toxicity test and HIV inhibit test material
T lymphocytes in human body system C8166 and HIV-1 is tested into strain HIV-1IIIB[1-2]To contain 10% fetal calf serum
RPMI-1640 complete medium is cultivated;After virus storage liquid packing, -70 DEG C of preservations are set;Cell line and virus are routinely square
Method freezes and recovers.
3, HIV-1 infectious titration
HIV-1IIIB storage liquid is made into 4 times of dilutions on 96 orifice plates, 10 gradients, 6 repeating holes of every gradient are set simultaneously
Set 6 hole of control wells.50 μ L (4 × 10 of C8166 cell is added in every hole5/ mL), every hole final volume is 200 μ L.37 DEG C, 5%CO2Training
It supports.Fresh 100 μ L of RPMI-1640 complete medium is added in third day, HIV- in every hole is observed under inverted microscope within the 7th day
Whether the cytopathic effect (Cytopathic effect, CPE) of 1IIIB induction, have plasomidum (Syncyti μM) with every hole
Formation determine, by Reed&Muench method calculate virus TCID50 (tissue culture infective dose).
4, the toxicity test of C8166 cell
Compound manufactured in the present embodiment (is contained 10% with RPMI-1640 complete medium on 96 hole microtest plates
FBS 5 times of doubling dilutions (originating final concentration of 100 μ g/mL, totally 6 dilutions)) is carried out, each dilution sets 3 holes, every hole 100
μL.The control wells of not drug containing are set simultaneously.Every hole is added 4 × 105The 100 μ L of C8166 cell of/mL.37 DEG C, 5%CO2Culture 3
It, using MTT colorimetric determination cytotoxicity.ELx800 microplate reader measures OD value, and measurement wavelength is 595nm, and reference wavelength is
630nm.CC is calculated50It is worth (50%Cytotoxic concentration, i.e. the normal T-lymphocytes system to 50%
C8166 generates compound concentration when toxicity) it is greater than 200 μM.
5, the Inhibition test of HIV-1IIIB cytopathogenic effect (CPE)
Compound manufactured in the present embodiment (is contained 10% with RPMI-1640 complete medium on 96 hole microtest plates
FBS 5 times of doubling dilutions (originating final concentration of 100 μ g/mL, totally 6 dilutions)) is carried out, each dilution sets 3 repeating holes,
Every 100 μ L of hole.The control wells of not drug containing are set simultaneously.Every hole is added 8 × 105The 50 μ L of C8166 cell of/mL, is then added
The HIV-1IIIB of 50 μ L dilutes supernatant, the hole 1300TCID50/.AZT (being purchased from Glaxo Wellcome drugmaker) is positive drug
Control.37 DEG C, 5%CO2Culture 3 days, (100 ×) count the formation of plasomidum and obtain EC under inverted microscope50(50%
Compound concentration when Effective concentration, i.e. inhibition Syncytium formation 50%) it is 86.80 μM.
In conjunction with CC50And EC50Result can be seen that compound shown in Formulas I of the present invention there is significant inhibit
The activity of inhibition of HIV proliferation, and it is lower to the toxicity of people's normal lymphocytes, meet the regulation of materia medica, has and be applied to system
Prospect in the standby drug for inhibiting inhibition of HIV proliferation.
Embodiment 2
1, compound (R shown in Formulas I of the present invention is prepared1For-H, R2For-H, R3For-CH=CH-CH=C (CH3)2, R4
For-H, R5It is-O- for-H, X, Y is entitled 3- [4- (4- methyl amyl -1, the 3- diene) phenoxy group] propane -1- of-SH, n 2)
Mercaptan.
10mmol parahydroxyben-zaldehyde, 11mmol 1, the potassium iodide of 3- dibromopropane, 12mmol potassium carbonate and catalytic amount exist
It is refluxed overnight in DMF, pillar layer separation obtains the bromo- propyl alcohol of 1- (4- Fonnylphenyl) -3-;Again by 6mmol sodium hydride and 5mmol
(2- methacrylic) triphenylphosphinebromide be dissolved in THF, ice bath is cooled to 0 DEG C, and 10mmol is added dropwise into above-mentioned solution
The THF solution of the bromo- propyl alcohol of 1- (4- Fonnylphenyl) -3- is added dropwise and is transferred to room temperature reaction 10-15 hours.Then column color
Spectrum separates to obtain 1- (3- bromine propoxyl group) -4- (4- methyl amyl -1,3- dialkylene) benzene.Finally product obtained above is worked as with two
The vulcanized sodium of amount reacts at room temperature overnight in DMSO, and extraction, dry, pillar layer separation can obtain product 3- [4- (4- methylpent
Base -1,3- diene) phenoxy group] propane -1- mercaptan.
Reaction equation is as follows:
Prepared compound is carried out1H nuclear magnetic resonance (400MHz) detection, solvent CDCl3, testing result is shown in Fig. 2,
Prepared compound is consistent with structure shown in Formulas I as the result is shown.
2, cell toxicity test and HIV inhibit test
Compound CC manufactured in the present embodiment is obtained according to method in embodiment 150Greater than 200 μM, EC50It is 62.90 μM.
In conjunction with CC50And EC50Result can be seen that compound shown in Formulas I of the present invention and have and significant inhibition of HIV is inhibited to increase
The activity grown, and it is lower to the toxicity of people's normal lymphocytes, meet the regulation of materia medica, has and be applied to preparation inhibition HIV
Prospect in the drug of virus multiplication.
Embodiment 3
1, compound (R shown in Formulas I of the present invention is prepared1For-H, R2For-H, R3For-CH3, R4For-H, R5For-H, X is-
S-, Y are-NH2, n 2) and entitled 3- (p-methylphenyl is thio) -1- propylamine.
10mmol toluene-ω-thiol, 30mmol 2- (3- bromopropyl) iso-indoles woods -1,3- diketone and 11mmol potassium tert-butoxide
It is reacted at room temperature 8 hours in 20mL DMF, pillar layer separation obtains 2- [3- (p-methylphenyl is thio) propyl] iso-indoles woods -1,3-
Diketone;By the hydrazine hydrate of 5mmol 2- [3- (p-methylphenyl is thio) propyl] iso-indoles woods -1,3- diketone and 20mmol in 20mL
It is refluxed overnight in ethyl alcohol, filters, is spin-dried for, then pillar layer separation obtains 3- (p-methylphenyl is thio) -1- propylamine.
Reaction equation is as follows:
Prepared compound is carried out1H nuclear magnetic resonance (400MHz) detection, solvent CDCl3, testing result is shown in Fig. 3,
Prepared compound is consistent with structure shown in Formulas I as the result is shown.
2, cell toxicity test and HIV inhibit test
Compound CC manufactured in the present embodiment is obtained according to method in embodiment 150It is 43.38 μM, EC50It is 8.97 μM.Knot
Close CC50And EC50Result can be seen that compound shown in Formulas I of the present invention and have and significant inhibit inhibition of HIV proliferation
Activity, and it is not high to the toxicity of people's normal lymphocytes, meet the regulation of materia medica, have and be applied to preparation and inhibit HIV disease
Prospect in the drug of poison proliferation.
Embodiment 4
1, compound (R shown in Formulas I of the present invention is prepared1For-H, R2For-H, R3For-H, R4For-H, R5For-NO2, X be-
C (O) S-, Y are the entitled 3- mercapto propyl -2- nitric acid benzoic acid thioesters of-SH, n 2).
5mmol o-nitrobenzoic acid is dissolved in 10mL methylene chloride, and 10mmol thionyl chloride is slowly added dropwise under condition of ice bath
Dichloromethane solution, be heated to reflux after dripping 3-6 hours.After rotation removes solvent, 15mL chloroform and 10mmol 1,3- are added
Dimercaptopropane heated overnight at reflux, rear pillar layer separation obtain 3- mercapto propyl -2- nitric acid benzoic acid thioesters.Reaction equation is as follows:
Prepared compound is carried out1H nuclear magnetic resonance (400MHz) detection, solvent CDCl3, testing result is shown in Fig. 4,
Prepared compound is consistent with structure shown in Formulas I as the result is shown.
2, cell toxicity test and HIV inhibit test
Compound CC manufactured in the present embodiment is obtained according to method in embodiment 150Greater than 200 μM, EC50It is 110.78 μM.
In conjunction with CC50And EC50Result can be seen that compound shown in Formulas I of the present invention and have and significant inhibition of HIV is inhibited to increase
The activity grown, and it is lower to the toxicity of people's normal lymphocytes, meet the regulation of materia medica, has and be applied to preparation inhibition HIV
Prospect in the drug of virus multiplication.
Embodiment 5
1, compound (R shown in Formulas I of the present invention is prepared1For-H, R2For-H, R3For-H, R4For-H, R5For-NO2, X be-
S-, Y are entitled 3- (2- nitrophenylsulfenyl) propane -1- mercaptan of-SH, n 2).
2mmol o-nitrochlorobenzene, 4mmol1,3- dimercaptopropane and 2mmol sodium bicarbonate heat reaction in 5mLDMF
5-8 hours, contact plate detection extracted after having reacted, is dry, being spin-dried for, then pillar layer separation obtains 3- (2- nitrophenylsulfenyl) propane-
1- mercaptan.Reaction equation is as follows:
Prepared compound is carried out1H nuclear magnetic resonance (400MHz) detection, solvent CDCl3, testing result is shown in Fig. 5,
Prepared compound is consistent with structure shown in Formulas I as the result is shown.
2, cell toxicity test and HIV inhibit test
Compound CC manufactured in the present embodiment is obtained according to method in embodiment 150Greater than 800 μM, EC50It is 63.06 μM.
In conjunction with CC50And EC50Result can be seen that 4- nitrobenzene-sulfonic acid 2- (N- (4- butyl phenyl) -4- nitrobenzophenone sulfonamide
Base) activity of the ethyl ester with significant inhibition inhibition of HIV proliferation, and it is lower to the toxicity of people's normal lymphocytes, meet drug
Regulation has the prospect being applied in the drug that preparation inhibits inhibition of HIV to be proliferated.
Embodiment 6
1, compound (R shown in Formulas I of the present invention is prepared1For-H, R2For-H, R3For-H, R4For-H, R5For-NO2, X be-
C (O) O-, Y are the entitled 3- hydroxyl -2- nitric acid propyl benzoate of-OH, n 2).
5mmol o-nitrobenzoic acid is dissolved in 10mL methylene chloride, and 10mmol thionyl chloride is slowly added dropwise under condition of ice bath
Dichloromethane solution, be heated to reflux after dripping 3-6 hours.Rotation is except after solvent, adding 15mL chloroform and 10mmol the third two
Alcohol heated overnight at reflux, rear pillar layer separation obtain 3- hydroxyl -2- nitric acid propyl benzoate.Reaction equation is as follows:
Prepared compound is carried out1H nuclear magnetic resonance (400MHz) detection, solvent CDCl3, testing result is shown in Fig. 6,
Prepared compound is consistent with structure shown in Formulas I as the result is shown.
2, cell toxicity test and HIV inhibit test
Compound CC manufactured in the present embodiment is obtained according to method in embodiment 150Greater than 200 μM, EC50For less than 200 μ
M.In conjunction with CC50And EC50Result can be seen that compound shown in Formulas I of the present invention with certain inhibition inhibition of HIV
The activity of proliferation, and it is lower to the toxicity of people's normal lymphocytes, meet the regulation of materia medica, has and be applied to preparation inhibition
Prospect in the drug of inhibition of HIV proliferation.
Embodiment 7
1, compound (R shown in Formulas I of the present invention is prepared1For-H, R2For-H, R3For-H, R4For-H, R5For-NO2, X be-
S-, Y are-NH2, n 2) and entitled 3- (O-Nitrophenylfluorone is thio) -1- propylamine.
10mmol ortho-nitrophenyl thiophenol, 30mmol 2- (3- bromopropyl) iso-indoles woods -1,3- diketone and the 11mmol tert-butyl alcohol
Potassium reacts at room temperature 8 hours in 20mL DMF, and pillar layer separation obtains 2- [3- (O-Nitrophenylfluorone is thio) propyl] iso-indoles woods-
1,3- diketone;By the hydrazine hydrate of 5mmol 2- [3- (O-Nitrophenylfluorone is thio) propyl] iso-indoles woods -1,3- diketone and 20mmol
It is refluxed overnight in 20mL ethyl alcohol, filters, is spin-dried for, then pillar layer separation obtains 3- (O-Nitrophenylfluorone is thio) -1- propylamine.
Reaction equation is as follows:
Prepared compound is carried out1H nuclear magnetic resonance (400MHz) detection, solvent D2O, testing result are shown in Fig. 7, knot
Fruit shows that prepared compound is consistent with structure shown in Formulas I.
2, cell toxicity test and HIV inhibit test
Compound CC manufactured in the present embodiment is obtained according to method in embodiment 150It is 14.52 μM, EC50It is 8.49 μM.Knot
Close CC50And EC50Result can be seen that compound shown in Formulas I of the present invention and have and significant inhibit inhibition of HIV proliferation
Activity, but to the toxic of people's normal lymphocytes, but still conform to the regulation of materia medica, have and be applied to preparation and inhibit
Prospect in the drug of inhibition of HIV proliferation.
Embodiment 8
1, compound (R shown in Formulas I of the present invention is prepared1For-H, R2For-H, R3For-CH=CH-CH=C (CH3)2, R4
For-H, R5It is-S- for-H, X, Y is entitled 2- [4- (4- methyl-1, the 3- pentadiene) thiophenyl] ethyl alcohol of-OH, n 1).
10mmol ethylene chlorhydrin, 10mmol TBDPSCl and 12mmol imidazoles react at room temperature 2-5h in 20mL DMF, then extract,
Column chromatography for separation is thrown in next step.First 10mmol 4- (4- methyl-1,3-pentylene) benzenethiol, 20mmol sodium bicarbonate are existed
After stirring half an hour in 25mL DMF, above-mentioned products therefrom is slowly added dropwise after entering and is reacted 5-10 hours, extracted after reaction
It takes, column chromatographs to obtain faint yellow solid.After above-mentioned solid is dissolved in DCM, two equivalent chloroacetic chlorides are added and several drop methanol, room temperature are anti-
2-8h is answered, column chromatography for separation can obtain product 2- [4- (4- methyl-1,3-pentylene) thiophenyl] ethyl alcohol.
Reaction equation is as follows:
Prepared compound is carried out1H nuclear magnetic resonance (400MHz) detection, solvent CDCl3, testing result is shown in Fig. 8,
Prepared compound is consistent with structure shown in Formulas I as the result is shown.
2, cell toxicity test and HIV inhibit test
Compound CC manufactured in the present embodiment is obtained according to method in embodiment 150It is 121.33 μM, EC50It is 41.91 μM.
In conjunction with CC50And EC50Result can be seen that compound shown in Formulas I of the present invention and have and significant inhibition of HIV is inhibited to increase
The activity grown, and it is lower to the toxicity of people's normal lymphocytes, meet the regulation of materia medica, has and be applied to preparation inhibition HIV
Prospect in the drug of virus multiplication.
Embodiment 9
1, compound (R shown in Formulas I of the present invention is prepared1For-H, R2For-H, R3For-CH=CH2, R4For-H, R5For-H,
X is-O-, and Y is entitled 3- [the 4- vinyl benzene oxygroup]-propane -1- mercaptan of-SH, n 2).
10mmol parahydroxyben-zaldehyde, 11mmol 1, the potassium iodide of 3- dibromopropane, 12mmol potassium carbonate and catalytic amount exist
It is refluxed overnight in DMF, pillar layer separation obtains the bromo- propyl alcohol of 1- (4- Fonnylphenyl) -3-;Again by 6mmol sodium hydride and 5mmol
Methyltriphenylphosphonium bromide be dissolved in THF, ice bath is cooled to 0 DEG C, into above-mentioned solution be added dropwise 10mmol 1- (4- formyl
Base phenyl) the bromo- propyl alcohol of -3- THF solution, be added dropwise be transferred to room temperature reaction 10-15 hours.Then pillar layer separation obtains 1-
(3- bromine propoxyl group) -4- vinyl benzene.Finally product obtained above is reacted in ethanol with the thiocarbamide of two equivalents and was flowed back
At night, five equivalent of sodium hydroxide back flow reaction in water is added in the product being spin-dried for, and after fully reacting, extraction is spin-dried for, column chromatography
Separation can obtain product 3- [4- vinyl benzene oxygroup]-propane -1- mercaptan.
Reaction equation is as follows:
Prepared compound is carried out1H nuclear magnetic resonance (400MHz) detection, solvent CDCl3, testing result is shown in Fig. 9,
Prepared compound is consistent with structure shown in Formulas I as the result is shown.
2, cell toxicity test and HIV inhibit test
Compound CC manufactured in the present embodiment is obtained according to method in embodiment 150Greater than 200 μM, EC50It is 32.90 μM.
In conjunction with CC50And EC50Result can be seen that compound shown in Formulas I of the present invention and have and significant inhibition of HIV is inhibited to increase
The activity grown, and it is lower to the toxicity of people's normal lymphocytes, meet the regulation of materia medica, has and be applied to preparation inhibition HIV
Prospect in the drug of virus multiplication.
Embodiment 10
1, compound (R shown in Formulas I of the present invention is prepared1For-H, R2For-H, R3For-(E)-CH=CH-Ph, R4For-H,
R5It is-O- for-H, X, Y is entitled (E) -3- [the 4- styryl phenoxy group]-propane -1- mercaptan of-SH, n 2).
10mmol parahydroxyben-zaldehyde, 11mmol 1, the potassium iodide of 3- dibromopropane, 12mmol potassium carbonate and catalytic amount exist
It is refluxed overnight in DMF, pillar layer separation obtains the bromo- propyl alcohol of 1- (4- Fonnylphenyl) -3-;Again by 6mmol sodium hydride and 5mmol
Benzyltriphenylphosphonium bromide phosphine be dissolved in THF, ice bath is cooled to 0 DEG C, into above-mentioned solution be added dropwise 10mmol 1- (4- formyl
Base phenyl) the bromo- propyl alcohol of -3- THF solution, be added dropwise be transferred to room temperature reaction 10-15 hours.Then pillar layer separation obtains E-
1- (3- bromine propoxyl group) -4- styryl benzene.Finally product obtained above is reacted back in ethanol with the thiocarbamide of two equivalents
Night is flowed through, five equivalent of sodium hydroxide back flow reaction in water is added in the product being spin-dried for, and after fully reacting, extraction is spin-dried for, column
Chromatographic isolation can obtain product (E) -3- [4- styryl phenoxy group]-propane -1- mercaptan.
Reaction equation is as follows:
Prepared compound is carried out1H nuclear magnetic resonance (400MHz) detection, solvent CDCl3, testing result is shown in Figure 10,
Prepared compound is consistent with structure shown in Formulas I as the result is shown.
2, cell toxicity test and HIV inhibit test
Compound CC manufactured in the present embodiment is obtained according to method in embodiment 150Greater than 200 μM, EC50It is 20.90 μM.
In conjunction with CC50And EC50Result can be seen that compound shown in Formulas I of the present invention and have and significant inhibition of HIV is inhibited to increase
The activity grown, and it is lower to the toxicity of people's normal lymphocytes, meet the regulation of materia medica, has and be applied to preparation inhibition HIV
Prospect in the drug of virus multiplication.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Bibliography
[1]Qiang Wang,Ruirui Wang,Baiqun Zhang,et al.Small organic molecules
targeting PCAF bromodomain as potent inhibitors of HIV-1replication[J]
.Med.Chem.Commun.,2013,4,737-740.
[2]Ping Hu,Xinghui Wang,Baiqun Zhang,et al.Fluorescence Polarization
for the Evaluation of Small-Molecule Inhibitors of PCAF BRD/Tat-
AcK50Association[J].ChemMedChem,2014,9,928-931.
Claims (12)
1. the compound of Formulas I:
Wherein, R1、R2、R4、R5For-H;
R3Selected from-(CH2)0-4-CH3,-(CH2)0-4-CH-(CH3)2,-CH=CH- (CH2)0-4-CH3,-(CH2)0-4- CH=
CH2,-CH=CH-CH=CH- (CH2)0-4-CH3Or-(CH=CH)0-4- CH=C (CH3)2;
X is independently selected from-O- or-S-;
Y is independently selected from-OH ,-SH or-NH2;
N is the integer of 1-4;
Condition is, when X is-S- or-O- and Y is-SH, R3It is not methyl;When X is-O-, Y is not-NH2;When X is-O- and R3
It is-CH=CH2When Y be not-OH;It is-SH ,-NH when X is-S- or-O- and Y2Or when-OH, n is not 1;When X be-S- and Y be-
NH2When, n is not 2;And when X is-O- and Y is-SH, n is not 3.
2. the compound of Formulas I:
Wherein, R1、R2、R3、R4For-H, and R5For-NO2;
X is independently selected from-O- ,-S- or-C (O) S-;
Y is independently selected from-OH ,-SH or-NH2;
N is the integer of 1-4;
Condition is that Y is not-NH when X is-O-2;It is-SH ,-NH when X is-S- or-O- and Y2Or when-OH, n is not 1;When X is-
S- and Y are-NH2When, n is not 2;And when X is-O- and Y is-SH, n is not 3.
3. compound according to claim 1, wherein
R3It is independently selected from-(CH2)0-4-CH3,-CH=CH-CH=CH- (CH2)0-4-CH3,-(CH=CH)0-4- CH=C (CH3)2
Or-(CH2)0-4- CH=CH2;
X is independently selected from-O or-S-;
Y is independently selected from-OH ,-SH or-NH2;
N is the integer of 1-3.
4. compound according to claim 3, wherein
R3It is independently selected from-CH3,-(CH2)3-CH3Or-CH=CH-CH=C (CH3)2;
X is independently selected from-O- or-S-;
Y is independently selected from-OH ,-SH or-NH2;
N is the integer of 1-2.
5. a kind of compound, the compound has the structure selected from any of following items:
6. a kind of compound, the compound has following structure:
7. application of any one of the claim 1-6 compound in the drug that preparation inhibits inhibition of HIV proliferation.
8. a kind of pharmaceutical composition, described pharmaceutical composition includes described in -6 any one according to claim 1 of pharmacy effective dose
Compound or pharmaceutically acceptable salt thereof and pharmaceutical carrier.
9. the preparation method of compound shown in Formulas I according to claim 1 or 2, described method includes following steps:
Compound shown in step 1, Formula II and 1,3- dibromopropane or glycol dibromide and potassium tert-butoxide room temperature in DMF are anti-
It answers, compound shown in production III;
Compound shown in step 2, formula III reacted in DMSO with vulcanized sodium or formula III shown in compound and ammonium hydroxide in THF
Reaction, compound shown in production I;
Wherein, R1、R2、R3、R4、R5It is defined with X such as claims 1 or 2, n is 1 or 2, and Y is SH or NH2。
10. preparation method according to claim 9, compound shown in the Formula II and 1,3- dibromopropane or 1,2- dibromo second
The ratio between amount of substance of alkane and potassium tert-butoxide is 1:3:1.5, the amount of the substance of compound shown in the formula III and vulcanized sodium it
Than for 1:2.
11. preparation method according to claim 9, reaction described in step 1 is is stirred at room temperature 3-48 hours.
12. preparation method according to claim 9, reaction described in step 2 is is stirred at room temperature 1-12 hours.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510564136.XA CN106496084B (en) | 2015-09-07 | 2015-09-07 | One kind aromatic compound containing sulfydryl preparation method and its application in terms of anti HIV-1 virus |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510564136.XA CN106496084B (en) | 2015-09-07 | 2015-09-07 | One kind aromatic compound containing sulfydryl preparation method and its application in terms of anti HIV-1 virus |
Publications (2)
Publication Number | Publication Date |
---|---|
CN106496084A CN106496084A (en) | 2017-03-15 |
CN106496084B true CN106496084B (en) | 2019-05-17 |
Family
ID=58287517
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201510564136.XA Active CN106496084B (en) | 2015-09-07 | 2015-09-07 | One kind aromatic compound containing sulfydryl preparation method and its application in terms of anti HIV-1 virus |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN106496084B (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102952069A (en) * | 2011-08-19 | 2013-03-06 | 中国科学技术大学 | Compound, and preparation method and application thereof |
CN104892466A (en) * | 2015-04-30 | 2015-09-09 | 中国科学技术大学 | Anilinosulfonyl compound synthesis method and use of anilinosulfonyl compound as HIV virus inhibitor |
-
2015
- 2015-09-07 CN CN201510564136.XA patent/CN106496084B/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102952069A (en) * | 2011-08-19 | 2013-03-06 | 中国科学技术大学 | Compound, and preparation method and application thereof |
CN104892466A (en) * | 2015-04-30 | 2015-09-09 | 中国科学技术大学 | Anilinosulfonyl compound synthesis method and use of anilinosulfonyl compound as HIV virus inhibitor |
Non-Patent Citations (3)
Title |
---|
Copper(II)-Catalyzed Single-Step Synthesis of Aryl Thiols from Aryl Halides and 1,2-Ethanedithiol;Yajun Liu等;《Adv. Synth. Catal.》;20141229;第357卷;2205-2212 |
Facile oxidative hydrolysis of acetals to esters using hypervalent iodine(III)/LiBr combination in water;Waraporn Panchan等;《Tetrahedron》;20100202;第66卷;2732–2735 |
Registry[Online];Columbus,Ohio,US;《STN International》;20150304;1-18 |
Also Published As
Publication number | Publication date |
---|---|
CN106496084A (en) | 2017-03-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Jin et al. | SAR and molecular mechanism study of novel acylhydrazone compounds targeting HIV-1 CA | |
RU2126794C1 (en) | Amino-derivatives of oxo- or hydroxy-substituted hydrazines, method of their synthesis and pharmaceutical compositions for inhibition of retrovirus protease | |
KR20190067890A (en) | Heterocyclic FXR modulator | |
EP0801649A2 (en) | Hydroxylamine derivatives useful for enhancing the molecular chaperon production and the preparation thereof | |
Qian et al. | Synthesis, molecular modeling and biological evaluation of guanidine derivatives as novel antitubulin agents | |
CN104903334A (en) | Tenofovir prodrug and pharmaceutical uses thereof | |
CN103159760A (en) | Azaindole derivatives as cftr modulators | |
CN107434779A (en) | Notch signal pathway inhibitors and its purposes in treatment of cancer | |
JP2009515887A (en) | Histone deacetylase inhibitors as therapeutic agents for nervous system diseases | |
CN111803501B (en) | Use of chiral chloroquine hydroxychloroquine for reducing cardiotoxicity | |
CN107253915A (en) | The assimilation compound of 1,5 diphenyl, penta Isosorbide-5-Nitrae diene 3 | |
KR101090850B1 (en) | Imidazole derivatives and their pharmaceutical compositions as inhibitors H sc 7 0 | |
CN102438612A (en) | Nitrile derivatives and their pharmaceutical use and compositions | |
Li et al. | Design, synthesis and biological evaluation of diamino substituted cyclobut-3-ene-1, 2-dione derivatives for the treatment of drug-resistant tuberculosis | |
CN106496084B (en) | One kind aromatic compound containing sulfydryl preparation method and its application in terms of anti HIV-1 virus | |
CN106892920A (en) | Aloperine derivative, Preparation Method And The Use | |
CN113999148B (en) | N- (4- (substituted sulfonyl amino) phenyl) sulfonamide compound and application thereof | |
Mu et al. | The potential role of the 5, 6-dihydropyridin-2 (1 H)-one unit of piperlongumine on the anticancer activity | |
US9682931B2 (en) | Aryloyl(oxy or amino)pentafluorosulfanylbenzene compound, pharmaceutically acceptable salt thereof, and prodrugs thereof | |
KR102591689B1 (en) | Composition for treating fibrosis and fibrosis-related diseases | |
CN104892466B (en) | A kind of synthesis of aniline sulphonyl class compound and its application as inhibition of HIV inhibitor | |
CN107501219B (en) | Asymmetric curcumin compound and application thereof in preparation of anti-gastric cancer drugs | |
Soni et al. | Synthesis and evaluation of novel quinuclidinone derivatives as potential anti-proliferative agents | |
CN103864720A (en) | Phenyl acrylic acid farnesyl thiosalicylic acid (FTA) derivative as well as preparation method and application | |
CN108186642B (en) | A kind of pharmaceutical composition of co-action treatment lung cancer |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |