CN103787966B - A kind of compound and the application in preparing medicine for parasitic disease thereof - Google Patents

A kind of compound and the application in preparing medicine for parasitic disease thereof Download PDF

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CN103787966B
CN103787966B CN201210436007.9A CN201210436007A CN103787966B CN 103787966 B CN103787966 B CN 103787966B CN 201210436007 A CN201210436007 A CN 201210436007A CN 103787966 B CN103787966 B CN 103787966B
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compound
formula
reaction
obtains
ndh
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CN103787966A (en
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饶燏
杨茂君
杨毅庆
冯越
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Tsinghua University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/20—Oxygen atoms
    • C07D215/22—Oxygen atoms attached in position 2 or 4
    • C07D215/233—Oxygen atoms attached in position 2 or 4 only one oxygen atom which is attached in position 4
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a kind of compound and the application in preparing medicine for parasitic disease thereof. The invention discloses the compound shown in formula (VII); In formula (VII): L is O or CH2; In R1 and R2, one is halogen, and another is H. Compound provided by the invention belongs to carbostyril compound, inventor studies and finds that it can suppress the activity of plasmodial NDH-2 albumen, therefore this compound should slow down clinical symptoms at the asexual reproduction phases of plasmodium life cycle and can act on again the syngenesis stage and block pathophoresis, and the present invention has substantial worth for the treatment of plasmodiosis.

Description

A kind of compound and the application in preparing medicine for parasitic disease thereof
Technical field
The present invention relates to a kind of compound and the application in preparing medicine for parasitic disease thereof.
Background technology
Malaria is that one is caused by plasmodium (Plasmodiumfalciparum), by malaria mosquito-borne whole world acute infectious disease, is that the mankind endanger maximum parasitic disease.Pathogenic plasmodium mainly has Plasmodium vivax, malariae, Plasmodium ovale and Plasmodium falciparum, causes tertian malaria, quartan malaria, ovale malaria and subtertian malaria respectively, former three is also called benign malaria, subtertian malaria infects the widest, and symptom is heavier, and human body health hazard is maximum. In world wide, annual malaria patients have 300,000,000 more than, cause people up to a million dead.
There is no a kind of medicine in existing antimalarial and can be had killing action each stage of plasmodium life cycle, different antimalarials acts on the different stages.
Plasmodium falciparum II type dihydrocoenzyme quinone reduction-oxidation enzyme (PfNDH2) is a kind of malaria target spot. PfNDH2 is molecular weight is the monomeric enzyme of 52kDa, is arranged in plasmodial mitochondrion, is the important electron donor of electron transport chain. Block PfNDH2 and may result in plasmodium mitochondrial dysfunction, and then kill plasmodium. It is extremely low and not yet find the existence of this enzyme in human body that its inhibitor is produced the probability of tolerance by this enzyme, this is that the side effects of pharmaceutical drugs avoided or reduce and act on this target spot provide advantage, so, the blocker of PfNDH promises to be new antimalarial medicine.
Summary of the invention
It is an object of the invention to provide a kind of compound and the application in preparing medicine for parasitic disease thereof.
Compound provided by the invention is the compound shown in formula (VII);
Formula (VII);
In formula (VII):
L is O or CH2;
In R1 and R2, one is halogen, and another is H.
Described halogen can be F, Cl, Br or I.
Compound shown in compound shown in compound shown in the described compound concretely compound shown in formula I, formula II, the compound shown in formula III, formula IV, formula (V) or the compound shown in formula VI.
The preparation method that the present invention also protects the compound shown in formula I, comprises the steps:
(1) parabromoacetophenone and to trifluoro-methoxy-phenol occur Ullamanncoupling reaction, obtain formula 1. shown in compound;
(2) dimethyl carbonate and formula 1. shown compound generation Carbonyl addition eliminates reaction, obtains formula 2. shown compound;
(3) formula 2. shown compound and m-chloroaniline generation Carbonyl addition eliminates reaction, obtains formula 3. shown compound;
(4) there is fragrance electrophilic substitution reaction in formula 3. shown compound, obtains compound shown in formula I;
Formula is 1.;Formula is 2.;
Formula is 3.;Formula I.
The preparation method that the present invention also protects the compound shown in formula II, comprises the steps:
(1) parabromoacetophenone and to trifluoro-methoxy-phenol occur Ullamanncoupling reaction, obtain formula 1. shown in compound;
(2) dimethyl carbonate and formula 1. shown compound generation Carbonyl addition eliminates reaction, obtains formula 2. shown compound;
(3) formula 2. shown compound and m-bromoaniline generation Carbonyl addition eliminates reaction, obtains formula 4. shown compound;
(4) there is fragrance electrophilic substitution reaction in formula 4. shown compound, obtains compound shown in formula II;
Formula is 1.;Formula is 2.;
Formula is 4.;Formula II.
The preparation method that the present invention also protects the compound shown in formula III, comprises the steps:
(1) melilotal and N-bromo-succinimide generation free radical substitution reaction, obtains formula 5. shown compound;
(2) formula 5. shown in compound and to trifluoromethoxy phenylboric acid occur Suzukicross-coupling reaction, obtain formula 6. shown in compound;
(3) dimethyl carbonate and formula 6. shown compound generation Carbonyl addition eliminates reaction, obtains formula 7. shown compound;
(4) formula 7. shown compound and m-chloroaniline generation Carbonyl addition eliminates reaction, obtains formula 8. shown compound;
(5) there is fragrance electrophilic substitution reaction in formula 8. shown compound, obtains compound shown in formula III;
Formula is 5.;Formula is 6.;
7.;Formula is 8.;
Formula III.
The preparation method that the present invention also protects the compound shown in formula IV, comprises the steps:
(1) melilotal and N-bromo-succinimide generation free radical substitution reaction, obtains formula 5. shown compound;
(2) formula 5. shown in compound and to trifluoromethoxy phenylboric acid occur Suzukicross-coupling reaction, obtain formula 6. shown in compound;
(3) dimethyl carbonate and formula 6. shown compound generation Carbonyl addition eliminates reaction, obtains formula 7. shown compound;
(4) formula 7. shown compound and m-bromoaniline generation Carbonyl addition eliminates reaction, obtains formula 9. shown compound;
(5) there is fragrance electrophilic substitution reaction in formula 9. shown compound, obtains compound shown in formula IV;
Formula is 5.;Formula is 6.;
Formula is 7.;Formula is 9.;
Formula IV.
The preparation method that the present invention also protects the compound shown in formula (V), comprises the steps:
(1) melilotal and N-bromo-succinimide generation free radical substitution reaction, obtains formula 5. shown compound;
(2) formula 5. shown in compound and to trifluoromethoxy phenylboric acid occur Suzukicross-coupling reaction, obtain formula 6. shown in compound;
(3) dimethyl carbonate and formula 6. shown compound generation Carbonyl addition eliminates reaction, obtains formula 7. shown compound;
(4) formula 7. shown compound and an Iodoaniline generation Carbonyl addition eliminates reaction, obtains formula 10. shown compound;
(5) there is fragrance electrophilic substitution reaction in formula 10. shown compound, obtains compound shown in formula (V);
Formula is 5.;Formula is 6.;
Formula is 7.;Formula is 10.;
Formula (V).
The preparation method that the present invention also protects the compound shown in formula VI, comprises the steps:
(1) melilotal and N-bromo-succinimide generation free radical substitution reaction, obtains formula 5. shown compound;
(2) formula 5. shown in compound and to trifluoromethoxy phenylboric acid occur Suzukicross-coupling reaction, obtain formula 6. shown in compound; (3) 2,6-methyl p-dichlorobenzene and formula 6. shown compound generation Carbonyl addition eliminates reaction, obtains formulaShown compound.
(4) formulaShown compound and liquefied ammonia generation Carbonyl addition eliminate reaction, obtain formulaShown compound;
(5) formulaShown compound generation aromatic nucleophilic substitution reaction, obtains compound shown in formula VI;
Formula is 5.;Formula is 6.;
Formula Formula
Formula VI.
The present invention also protects at least one application in preparing product in (1) to (3) as follows: (1) described compound; (2) described compound pharmaceutically acceptable salt; (3) described compound pharmaceutically acceptable precursor; The function of described product is following (a) and/or (b): (a) suppresses NDH-2 protein active; B () parasiticide is sick. Described NDH-2 albumen (two type nadh dehydrogenases) is specifically as shown in the sequence 1 of sequence table. Described NDH-2 protein active is presented as following reaction: NADH+UQ → NAD++UQH2. Described parasite concretely plasmodium.
The present invention also protects a kind of product, and its active component is at least one in following (1) to (3): (1) described compound;(2) described compound pharmaceutically acceptable salt; (3) described compound pharmaceutically acceptable precursor; The function of described product is following (a) and/or (b): (a) suppresses NDH-2 protein active; B () parasiticide is sick. Described NDH-2 albumen (two type nadh dehydrogenases) is specifically as shown in the sequence 1 of sequence table. Described NDH-2 protein active is presented as following reaction: NADH+UQ → NAD++UQH2. Described parasite concretely plasmodium.
Compound provided by the invention belongs to carbostyril compound, inventor studies and finds that it can suppress the activity of plasmodial NDH-2 albumen, this compound should slow down clinical symptoms at the asexual reproduction phases of plasmodium life cycle and can act on again the syngenesis stage and block pathophoresis, and the present invention has substantial worth for the treatment of plasmodiosis.
Accompanying drawing explanation
Fig. 1 is intermediate A1Preparation flow figure.
Fig. 2 is intermediate B1Preparation flow figure.
Fig. 3 is intermediate C1Preparation flow figure.
Fig. 4 is the preparation flow figure of compound shown in formula I.
Fig. 5 is intermediate C2Preparation flow figure.
Fig. 6 is the preparation flow figure of compound shown in formula II.
Fig. 7 is intermediate A2Preparation flow figure.
Fig. 8 is intermediate B2Preparation flow figure.
Fig. 9 is intermediate C3Preparation flow figure.
Figure 10 is the preparation flow figure of compound shown in formula III.
Figure 11 is intermediate C4Preparation flow figure.
Figure 12 is the preparation flow figure of compound shown in formula IV.
Figure 13 is intermediate C5Preparation flow figure.
Figure 14 is the preparation flow figure of compound shown in formula (V).
Figure 15 is the preparation flow figure of compound shown in formula VI.
Figure 16 is result during Km and the Kcat value measuring coenzymeQ1.
Figure 17 is result during Km and the Kcat value surveying NADH.
Figure 18 is the compound inhibitory action to NDH-2 protein active shown in formula II.
Figure 19 is the different compound inhibitory action to NDH-2 protein active.
Figure 20 is the matched curve (Ki value is 348nM, a is 0.7104) of compound shown in formula I.
Figure 21 is the matched curve (Ki value is 21nM, a is 0.4723) of compound shown in formula II.
Figure 22 is the matched curve (Ki value is 561nM, a is 0.3897) of compound shown in formula IV.
Figure 23 is the matched curve (Ki value is 27.9nM, a is 0.2143) of compound shown in formula VI.
Figure 24 is the matched curve (Ki value is 3.17 μMs, and a is 0.5646) of compound shown in formula III.
Detailed description of the invention
Below example is easy to be more fully understood that the present invention, but does not limit the present invention. Experimental technique in following embodiment, if no special instructions, is conventional method. Test material used in following embodiment, if no special instructions, is and is commercially available from routine biochemistry reagent shop. Quantitative test in following example, is respectively provided with three times and repeats experiment, results averaged. E. coli strain bl21: purchased from TIANGEN Biotech (Beijing) Co., Ltd., CB105.
Embodiment 1, compound preparation
One, intermediate A1Preparation
Preparation flow figure is shown in Fig. 1.
Round-bottomed flask add to 3g parabromoacetophenone, 570mg Hydro-Giene (Water Science)., 738mg2-pyridine carboxylic acid, 6.4g potassium phosphate, 3.9ml is to trifluoro-methoxy-phenol and 15ml dimethyl sulfoxide, 110 DEG C of heated and stirred 20 hours when argon shield, it is subsequently cooled to room temperature, add 100ml water to be diluted, then with dichloromethane extraction 3 times (adding 25ml dichloromethane) every time, merge the organic facies extracted three times, dry with anhydrous sodium sulfate after washing twice with water, then decompression is spin-dried for solvent, residue petroleum ether: ethyl acetate=25: 1(volume ratio) cross silicagel column, obtain 3.7g intermediate A1(intermediate A1For light yellow liquid), productivity is 84%.
Two, intermediate B1Preparation
Preparation flow figure is shown in Fig. 2.
The 30ml oxolane dried adds 0.5ml dimethyl carbonate and 0.2g sodium hydride, heats to 60 DEG C (make solution is micro-to boil), be slowly added dropwise 5ml intermediate A1Tetrahydrofuran solution (containing 1.0g intermediate A1), 95 DEG C are refluxed 10 hours, it is subsequently cooled to room temperature and decompression is spin-dried for solvent, residue 20ml dichloromethane dissolves, dry with anhydrous sodium sulfate after washing twice with water, then decompression is spin-dried for solvent, residue petroleum ether: ethyl acetate=40: 1(volume ratio) cross silicagel column, obtain 993mg intermediate B1(intermediate B1For light yellow liquid), productivity is 83%.
Three, the preparation of compound shown in formula I and sign
Preparation flow figure is shown in Fig. 3 and Fig. 4.
1, by 1.0g intermediate B1It is dissolved in 3ml dehydrated alcohol, add 1.2ml m-chloroaniline and 0.8ml acetic acid, 100 DEG C are refluxed about 16 hours, it is subsequently cooled to room temperature, adds 50ml water to be diluted, then use 20ml dichloromethane extraction, wash once with saturated sodium bicarbonate aqueous solution again after organic facies is washed three times with 2M aqueous hydrochloric acid solution, reduce pressure after drying with anhydrous sodium sulfate and be spin-dried for solvent, residue petroleum ether: ethyl acetate=20: 1(volume ratio) cross silicagel column, obtain 1.0g intermediate C1(intermediate C1For light yellow liquid), productivity is 77%.
2,500mg intermediate C is taken1In sealing in pipe, add 2mlDowthermA(AlfaAesar, article No. H30347) as solvent, 240 DEG C of reaction 30min, it is subsequently cooled to room temperature, add 30ml normal hexane and stand, precipitate out solid and range estimation precipitate is not further added by rear elimination supernatant, wash gained solid 3 times (adding 10ml ethyl acetate) by ethyl acetate every time, in gained solid, then add 8ml acetone make its recrystallization, obtaining 370mg product (light yellow solid), productivity is 80%.
3, product step 2 obtained carries out hydrogen spectrum and Mass Spectrometric Identification, it is shown that this product is compound shown in formula I.
Formula I.
Sign data are as follows:
H-NMR(400MHz,d6-DMSO, ppm): 6.39 (s, 1H), 7.22-7.24 (m, 4H), 7.35 (d, J=8.48Hz, 1H), 7.45 (d, J=8.60Hz, 2H), 7.83 (d, J=8.48Hz, 1H), 7.89 (d, J=8.12Hz, 2H), 8.08 (d, J=8.20Hz, 1H), 11.77 (b, 1H).
LC-MS:calcdforC22H13ClF3NO3[M+H]+: 432.06, found432.10.
Four, the preparation of compound shown in formula II and sign
Preparation flow figure is shown in Fig. 5 and Fig. 6.
1, the m-chloroaniline in the 1 of step 3, the 1 of other Complete Synchronization rapid three is replaced with m-bromoaniline. Obtain 1.1g intermediate C2(intermediate C2For light yellow liquid), productivity is 76%.
2, intermediate C is used2Replace intermediate C1, the 2 of other Complete Synchronization rapid three. Obtaining 351mg product (light yellow solid), productivity is 75%.
3, product step 2 obtained carries out hydrogen spectrum and Mass Spectrometric Identification, it is shown that this product is compound shown in formula II.
Formula II.
Sign data are as follows:
H-NMR(400MHz,d6-DMSO, ppm): 6.36 (s, 1H), 7.22-7.25 (m, 4H), 7.44-7.50 (m, 3H), 7.88 (d, J=7.36Hz, 2H), 7.96 (s, 1H), 8.01 (d, J=8.60Hz, 1H), (11.71 b, 1H).
LC-MS:calcdforC22H13BrF3NO3[M+H]+: 476.01, found476.13.
Five, intermediate A2Preparation
Preparation flow figure is shown in Fig. 7.
4ml melilotal is dissolved in the 30ml acetonitrile dried by dry round-bottomed flask, 5.9gN-bromo-succinimide (NBS) and 500mg azodiisobutyronitrile (AIBN) is added under argon shield, continue 95 DEG C of backflow 8h under argon shield, it is subsequently cooled to room temperature and decompression is spin-dried for solvent, residue 40ml dichloromethane dissolves, use 2M aqueous hydrochloric acid solution successively, saturated sodium bicarbonate aqueous solution, saturated common salt water washing organic facies, then dry with anhydrous sodium sulfate and reduce pressure and be spin-dried for solvent, residue petroleum ether: ethyl acetate=30: 1(volume ratio) cross silicagel column, obtain 6g to bromomethyl 1-Phenylethanone. (colourless liquid), productivity is 95%.
Under argon shield; weigh 1.5g to bromomethyl 1-Phenylethanone., 2g to trifluoromethoxy phenylboric acid, 32mg palladium, 74mg triphenylphosphine and 3g potassium phosphate in 100ml round-bottomed flask; add 20ml toluene and under argon shield; 80 DEG C are reacted 8 hours, and decompression is spin-dried for solvent, and residue 20ml dichloromethane dissolves; dry with anhydrous sodium sulfate after washing twice with water; decompression is spin-dried for solvent, residue petroleum ether: ethyl acetate=30: 1(volume ratio) cross silicagel column, obtain 1.7g intermediate A2(intermediate A2For light yellow liquid), productivity is 81%.
Six, intermediate B2Preparation
Preparation flow figure is shown in Fig. 8.
Use intermediate A2Replace the intermediate A in step 21, other Complete Synchronization rapid two. Obtain 970mg intermediate B2(intermediate B2For light yellow liquid), productivity is 81%.
Seven, the preparation of compound shown in formula III and sign
Preparation flow figure is shown in Fig. 9 and Figure 10.
1, intermediate B is used2Replace the intermediate B in the 1 of step 31, the 1 of other Complete Synchronization rapid three. Obtain 943mg intermediate C3(intermediate C3For light yellow liquid), productivity is 72%.
2, intermediate C is used3Replace intermediate C1, the 2 of other Complete Synchronization rapid three. Obtaining 311mg product (white solid), productivity is 67%.
3, product step 2 obtained carries out hydrogen spectrum and Mass Spectrometric Identification, it is shown that this product is compound shown in formula III.
Formula III.
Sign data are as follows:
H-NMR(400MHz,d6-Acetone, ppm): 4.12 (s, 2H), 6.33 (s, 1H), 7.26 (d, J=7.96Hz, 2H), 7.30 (d, J=8.68Hz, 1H), 7.41 (d, J=8.64Hz, 2H), 7.45 (d, J=8.08Hz, 2H), 7.75-7.77 (m, 3H), 8.17 (d, J=8.60Hz, 1H), 10.64 (b, 1H).
LC-MS:calcdforC23H15ClF3NO2[M+H]+: 430.08, found430.17.
Eight, the preparation of compound shown in formula IV and sign
Preparation flow figure is shown in Figure 11 and Figure 12.
1, intermediate B is used2Replace the intermediate B in the 1 of step 31, the m-chloroaniline in the 1 of step 3, the 1 of other Complete Synchronization rapid three is replaced with m-bromoaniline. Obtain 1.0g intermediate C4(intermediate C4For light yellow liquid), productivity is 70%.
2, intermediate C is used4Replace intermediate C1, the 2 of other Complete Synchronization rapid three. Obtaining 393mg product (light yellow solid), productivity is 84%.
3, product step 2 obtained carries out hydrogen spectrum and Mass Spectrometric Identification, it is shown that this product is compound shown in formula IV.
Formula IV.
Sign data are as follows:
H-NMR(400MHz,d6-Acetone, ppm): 4.16 (s, 2H), 7.00 (s, 1H), 7.28 (d, J=8.46Hz, 2H), 7.44 (d, J=8.68Hz, 2H), 7.51 (d, J=8.42Hz, 2H), 7.63 (d, J=8.68Hz, 1H), 7.90 (d, J=8.42Hz, 2H), 8.20 (d, J=8.64Hz, 1H), 8.30 (s, 1H).
LC-MS:calcdforC23H15BrF3NO2[M+H]+: 474.03, found474.13.
Nine, the preparation of compound shown in formula (V) and sign
Preparation flow figure is shown in Figure 13 and Figure 14.
1, intermediate B is used2Replace the intermediate B in the 1 of step 31, between using, Iodoaniline replaces the m-chloroaniline in the 1 of step 3, the 1 of other Complete Synchronization rapid three. Obtain 1.1g intermediate C5(intermediate C5For light yellow liquid), productivity is 69%.
2, intermediate C is used5Replace intermediate C1, the 2 of other Complete Synchronization rapid three. Obtaining 367mg product (yellow solid), productivity is 76%.
3, product step 2 obtained carries out hydrogen spectrum and Mass Spectrometric Identification, it is shown that this product is compound shown in formula (V).
Formula (V).
Sign data are as follows:
H-NMR(400MHz,d6-DMSO, ppm): 4.08 (s, 2H), 6.33 (s, 1H), 7.30 (d, J=8.16Hz, 2H), 7.40 (d, J=8.40Hz, 2H), 7.46 (d, J=7.88Hz, 2H), 7.63 (d, J=8.56Hz, 1H), 7.76 (d, J=7.72Hz, 2H), 7.81 (d, J=8.36Hz, 1H), 8.15 (s, 1H), 11.57 (b, 1H).
LC-MS:calcdforC23H15IF3NO2[M+H]+: 522.02, found522.10.
Ten, the preparation of compound shown in formula VI and sign
Preparation flow figure is shown in Figure 15.
1,2.2g2,6-methyl p-dichlorobenzene is dissolved in 20ml dry tetrahydrofuran (THF), adds 400mgNaH, then drip dissolved with 1.62g intermediate A2THF solution 5ml, 2h is stirred at room temperature, then heat to 80 DEG C of backflow 16h, being cooled to room temperature, decompression is spin-dried for solvent, and residue 30ml dichloromethane dissolves, successively by water, 1M aqueous hydrochloric acid solution, saturated common salt water washing organic facies, then dry with anhydrous sodium sulfate and reduce pressure and be spin-dried for solvent, residue petroleum ether: ethyl acetate=25: 1(volume ratio) cross silicagel column, obtain 1.2g intermediate B3(intermediate B3For light yellow liquid), productivity is 47%.
2, in round-bottomed flask, 1g intermediate B is added3, it is subsequently adding the 10ml alcoholic solution containing 2M liquefied ammonia, 100 DEG C are refluxed 12 hours, it is subsequently cooled to room temperature, add 20ml dichloromethane, wash organic facies with water, then reduce pressure after drying with anhydrous sodium sulfate and be spin-dried for solvent, residue joins in 20ml dimethylformamide (DMF), add 1.4g potassium carbonate, 105 DEG C of backflow 24h, add 100ml water to be diluted, then 50ml dichloromethane extraction is used, take organic facies and wash three times with water, reduce pressure after drying with anhydrous sodium sulfate and be spin-dried for solvent, residue acetone recrystallization, obtain 478mg product (white solid), productivity is 52%.
3, product step 2 obtained carries out hydrogen spectrum and Mass Spectrometric Identification, it is shown that this product is compound shown in formula VI.
Formula VI.
Sign data are as follows:
H-NMR(400MHz,d6-Acetone, ppm): 4.13 (s, 2H), 6.29 (s, 1H), 7.25-7.29 (m, 3H), 7.41 (d, J=8.48Hz, 2H), 7.46 (d, J=8.04Hz, 2H), 7.52 (t, J=8.56Hz, 1H), 7.67 (d, J=8.56Hz, 1H), 7.76 (d, J=8.08Hz, 2H), 10.57 (b, 1H).
LC-MS:calcdforC23H15ClF3NO2[M+H]+: 430.08, found430.21.
11, compound shown in formula I is summarized as below formula to compound shown in formula VI:
Formula (VII);
L is O or CH2;
In R1 and R2, one is halogen, and another is H.
Described halogen is F, Cl, Br or I.
Embodiment 2, compound the inhibitory action to NDH-2 protein active
One, experimental principle
NDH-2(bis-type nadh dehydrogenase) chemical reaction of catalysis is: NADH+UQ → NAD++UQH2; Wherein NADH has light to absorb at 340nm, and that therefore can pass through 340nm place absorbance value reduces the minimizing speed evaluating NADH.
Two, the preparation of experiment material
(1) preparation of NDH-2 albumen
At the NDH-2 albumen of expression in escherichia coli Plasmodium falciparum 3D7 strain, (NDH-2 albumen is made up of 533 amino acid residues, as shown in the sequence 1 of sequence table, it is signal peptide from N-terminal the 1st to 24 amino acids residue, for by protein localization to plasmodium mitochondrial inner membrane; NDH-2 gene is made up of 1602 nucleotide, as shown in the sequence 2 of sequence table), specifically comprise the following steps that
1, the double chain DNA molecule shown in sequence 2 of composition sequence table.
2, with the double chain DNA molecule of step 1 for template, the primer pair formed with NDH-2 forward primer and NDH-2 downstream primer carries out pcr amplification, obtains pcr amplification product.
NDH-2 forward primer: 5 '-ACCGAGGATCCAATGTAGCAAAAAATAATTTA-3’
NDH-2 downstream primer: 5 '-cgtagctcgagTCATTTGATGAAAGGACGCCCATA-3’。
3, with the pcr amplification product of restricted enzyme BamHI and XhoI double digestion step 2, digestion products is reclaimed.
4, with restricted enzyme BamHI and XhoI double digestion PQE80L carrier (purchased from QIAGEN company), carrier framework (about 4700bp) is reclaimed.
5, the carrier framework of the digestion products of step 3 and step 4 is connected, obtain recombiant plasmid. According to sequencing result, recombiant plasmid is carried out structure and describes as follows: between BamHI and the XhoI restriction enzyme site of PQE80L carrier, insert the double chain DNA molecule shown in the 73rd to 1602 nucleotide of sequence 2 of sequence table. In recombiant plasmid, external source insertion sequence merges with the His label coding sequence on carrier framework, expressed fusion protein.
6, recombinant bacterium step 5 obtained converts e. coli strain bl21, obtains recombinant bacterium.
7, picking step 6 obtains recombinant bacterium is also seeded to 50mlLB culture medium, 37 DEG C, 220rpm shaken cultivation 7-8 hour, then takes 10mL bacterium solution and is seeded to 1LLB culture medium, and 37 DEG C are cultured to OD600nm=1.0, in cultivating system, add IPTG(isopropyl-beta D-thio galactopyranoside, a kind of derivant so that it is the initial concentration in cultivating system is 0.5mM), 23 DEG C, 80rpm shaken cultivation is after 12 hours, centrifugal collect thalline.
8, thalline step 7 collected is in the 50mMTris buffer (pH8.0) containing 1mMEDTA and middle ultrasonication, and 11000rpm is centrifuged 15min, collects supernatant.
9, centrifugal 1 hour of supernatant 35000rpm step 8 obtained, collects precipitation.
10, the precipitation that step 9 is collected is resuspended in buffer A (pH7.6,50mMTris buffer containing 200mMNaCl, 0.1mMEDTA and 10% glycerol) and crushes, being subsequently adding TritonX-100 makes its concentration be 0.3g/100ml, 2 hours are hatched at 4 DEG C, then centrifugal 1 hour of 35000rpm, collects supernatant.
11, the supernatant that step 10 obtains is splined on nickel metal chelation resin (Ni-NTA, Qiagen), pillar is cleaned to remove foreign protein, eluent after then cleaning pillar by the 12ml buffer A containing 0.02g/100mlTritonX-100 and 200mM histidine and collected post by the 15ml buffer A containing 0.1g/100mlTritonX-100 and 8mM histidine.
12, after the post excessively step 11 collected, eluent is splined on molecular sieve (Superdex-20010/30, GEHealthcare), with buffer B (containing 300mMNaCl, 5mMDTT, 0.1mMEDTA and 0.02g/100mlTritonX-100 pH7.5,25mMTris-HCl buffer) carry out eluting, flow velocity is 0.5ml/ minute, collecting retention volume is eluent (peak value is positioned at 13.5 milliliters of places) after the post excessively between 12-15 milliliter, is destination protein solution (NDH-2 protein liquid).
(2) other experiment material
NADH(is water-soluble): purchased from Sigma, catalog number is N8129.
CoenzymeQ1(DMSO is molten): purchased from Sigma, catalog number is C7956.
Three, the compound inhibitory action to NDH-2 protein active
1, Km and the Kcat value of coenzymeQ1 and NADH
Reaction system (1.6ml): solvent is 50mMMOPS-NaOH buffer (pH6.5), solute and concentration thereof is: 150mMNaCl, 1mMEDTA, 0.01g/100mlTritonX-100,1nMNDH-2 albumen, NADH and coenzymeQ1.
Reaction temperature is 25 DEG C. In reaction system preparation process, it is eventually adding NDH-2 protein liquid, starts to count the response time from adding NDH-2 protein liquid, altogether record 1 minute. Adopt Lambda45 spectrophotometer (PerkinElmerLifeSciences; Include magnetic stirring apparatus) detect the absorbance value at 340nm place. NADH is 6220cm at the specific absorbance of 340nm-1M-1。
When measuring Km and the Kcat value of coenzymeQ1, NADH concentration in reaction system is 200 μMs, and coenzymeQ1 adopts variable concentrations. Result is shown in Figure 16, and what vertical coordinate represented is that the NADH of average catalysis per second how many μMs is degraded (because NADH is 6220cm at the extinction coefficient of 340nM-1M-1, therefore it is light absorption value is multiplied by 1000 and divided by 6.22 by the conversion method of light absorption value to μM NADH/s). Km (UQ1)=21.8uM, Kcat (UQ1)=418s-1。
When surveying Km and the Kcat value of NADH, coenzymeQ1 concentration in reaction system is 100 μMs, and NADH adopts variable concentrations. Result is shown in Figure 17, and what vertical coordinate represented is that the NADH of average catalysis per second how many μMs is degraded. Km(NADH)=47uM, Kcat(NADH)=482s-1。
2, the compound inhibitory action to NDH-2 protein active
Reaction system first (1.6ml): solvent is 50mMMOPS-NaOH buffer (pH6.5), solute and concentration thereof is: 150mMNaCl, 1mMEDTA, 0.01g/100mlTritonX-100,1nMNDH-2 albumen, 80 μMs of NADH, 80 μMs of coenzymeQ1 and testing compound.
Reaction system second (1.6ml): solvent is 50mMMOPS-NaOH buffer (pH6.5), solute and concentration thereof is: 150mMNaCl, 1mMEDTA, 0.01g/100mlTritonX-100,1nMNDH-2 albumen, 80 μMs of NADH, 30 μMs of coenzymeQ1 and testing compound.
Reaction system third (1.6ml): solvent is 50mMMOPS-NaOH buffer (pH6.5), solute and concentration thereof is: 150mMNaCl, 1mMEDTA, 0.01g/100mlTritonX-100,1nMNDH-2 albumen, 20 μMs of NADH, 30 μMs of coenzymeQ1 and testing compound.
Described testing compound is compound shown in formula II, the varying concentrations of testing compound.
Reaction temperature is 25 DEG C. In reaction system preparation process, it is eventually adding NDH-2 protein liquid, starts to count the response time from adding NDH-2 protein liquid, altogether record 2 minutes. Adopt Lambda45 spectrophotometer (PerkinElmerLifeSciences; Include magnetic stirring apparatus) detect the absorbance value at 340nm place. NADH is 6220cm at the specific absorbance of 340nm-1M-1。
Result is shown in Figure 18, and what vertical coordinate represented is that the NADH of average catalysis per second how many μMs is degraded. The suppression that enzyme is lived by testing compound should belong to Noncompetition inhibition.
3, the different compound inhibitory action to NDH-2 protein active of detection
Reaction system (1.6ml): solvent is 50mMMOPS-NaOH buffer (pH6.5), solute and concentration thereof is: 150mMNaCl, 1mMEDTA, 0.01g/100mlTritonX-100,1nMNDH-2 albumen, 50 μMs of NADH, 30 μMs of coenzymeQ1 and 1 μM of testing compound. Testing compound is compound shown in compound, formula IV shown in compound, formula III or compound shown in formula VI shown in compound, formula II shown in formula I. The control treatment being added without testing compound is set.
Reaction temperature is 25 DEG C. In reaction system preparation process, it is eventually adding NDH-2 protein liquid, starts to count the response time from adding NDH-2 protein liquid, altogether record 2 minutes.Adopt Lambda45 spectrophotometer (PerkinElmerLifeSciences; Include magnetic stirring apparatus) detect the absorbance value at 340nm place. NADH is 6220cm at the specific absorbance of 340nm-1M-1。
Result is shown in Figure 19, and (calculating and initiate the time phase to reaction end from reaction, the NADH of average catalysis per second how many μMs is degraded, and is protein active; Using the protein active of control treatment as 1.0, other protein active ratio processed with control treatment is its relative activity). Compound shown in compound, formula IV shown in compound shown in formula I, formula II and compound shown in formula VI are stronger to the inhibitory action of NDH-2 protein active.
4, the different compound inhibiting dynamic analyses to NDH-2 protein active
Reaction system (1.6ml): solvent is 50mMMOPS-NaOH buffer (pH6.5), solute and concentration thereof is: 150mMNaCl, 1mMEDTA, 0.01g/100mlTritonX-100,1nMNDH-2 albumen, 50 μMs of NADH, 30 μMs of coenzymeQ1 and testing compound. The varying concentrations of testing compound. Testing compound is compound shown in compound, formula IV shown in compound, formula III or compound shown in formula VI shown in compound, formula II shown in formula I. The control treatment being added without testing compound is set.
Reaction temperature is 25 DEG C. In reaction system preparation process, it is eventually adding NDH-2 protein liquid, starts to count the response time from adding NDH-2 protein liquid, adopt Lambda45 spectrophotometer (PerkinElmerLifeSciences; Include magnetic stirring apparatus) detect the absorbance value at 340nm place. NADH is 6220cm at the specific absorbance of 340nm-1M-1。
Use Noncompetition inhibition model: v=v0* (Ki+a*I)/(Ki+I) is fitted. Wherein V and V0 respectively adds the response speed (response speed V refers to the initial velocity of reaction in 20 seconds) in the system of testing compound and the response speed in control treatment system (referring to the initial velocity of reaction in 20 seconds equally). Ki is inhibition constant, and a is parameter, and I is inhibitor concentration.
Figure 20 is shown in the matched curve (Ki value is 348nM, a is 0.7104) of compound shown in formula I.
Figure 21 is shown in the matched curve (Ki value is 21nM, a is 0.4723) of compound shown in formula II.
Figure 22 is shown in the matched curve (Ki value is 561nM, a is 0.3897) of compound shown in formula IV.
Figure 23 is shown in the matched curve (Ki value is 27.9nM, a is 0.2143) of compound shown in formula VI.
Figure 24 is shown in the matched curve (Ki value is 3.17 μMs, and a is 0.5646) of compound shown in formula III.

Claims (5)

1. compound shown in formula II or formula VI;
2. the preparation method of the compound shown in formula II, comprises the steps:
(1) parabromoacetophenone and to trifluoro-methoxy-phenol occur Ullamanncoupling reaction, obtain formula 1. shown in compound;
(2) dimethyl carbonate and formula 1. shown compound generation Carbonyl addition eliminates reaction, obtains formula 2. shown compound;
(3) formula 2. shown compound and m-bromoaniline generation Carbonyl addition eliminates reaction, obtains formula 4. shown compound;
(4) there is fragrance electrophilic substitution reaction in formula 4. shown compound, obtains compound shown in formula II;
3. the preparation method of the compound shown in formula VI, comprises the steps:
(1) melilotal and N-bromo-succinimide generation free radical substitution reaction, obtains formula 5. shown compound;
(2) formula 5. shown in compound and to trifluoromethoxy phenylboric acid occur Suzukicross-coupling reaction, obtain formula 6. shown in compound;
(3) 2,6-methyl p-dichlorobenzene and formula 6. shown compound generation Carbonyl addition eliminates reaction, obtains formulaShown compound;
(4) formulaShown compound and liquefied ammonia generation Carbonyl addition eliminate reaction, obtain formulaShown compound;
(5) formulaShown compound generation aromatic nucleophilic substitution reaction, obtains compound shown in formula VI;
4. at least one application in preparing product in following (1) to (2): compound described in (1) claim 1; (2) compound pharmaceutically acceptable salt described in claim 1; The function of described product is following (a) and/or (b): (a) suppresses NDH-2 protein active; B () parasiticide is sick.
5. a product, its active component is at least one in following (1) to (2): compound described in (1) claim 1; (2) compound pharmaceutically acceptable salt described in claim 1; The function of described product is following (a) and/or (b): (a) suppresses NDH-2 protein active; B () parasiticide is sick.
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WO2008028427A1 (en) * 2006-09-04 2008-03-13 Univerzita Palackeho Derivatives of 2-phenyl-3-hydroxyquinoline-4(1h)-one and methods of their preparation and utilization
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