CN113350344A - Application of levorotatory chiral chloroquine phosphate in preparation of drugs for treating coronavirus - Google Patents
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- 229960002328 chloroquine phosphate Drugs 0.000 title claims abstract description 39
- 241000711573 Coronaviridae Species 0.000 title claims abstract description 30
- AEUAEICGCMSYCQ-UHFFFAOYSA-N 4-n-(7-chloroquinolin-1-ium-4-yl)-1-n,1-n-diethylpentane-1,4-diamine;dihydrogen phosphate Chemical compound OP(O)(O)=O.ClC1=CC=C2C(NC(C)CCCN(CC)CC)=CC=NC2=C1 AEUAEICGCMSYCQ-UHFFFAOYSA-N 0.000 title claims abstract description 29
- 239000003814 drug Substances 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 229940079593 drug Drugs 0.000 title description 12
- 229910019142 PO4 Inorganic materials 0.000 claims description 19
- WHTVZRBIWZFKQO-AWEZNQCLSA-N (S)-chloroquine Chemical compound ClC1=CC=C2C(N[C@@H](C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-AWEZNQCLSA-N 0.000 claims description 18
- WHTVZRBIWZFKQO-UHFFFAOYSA-N chloroquine Natural products ClC1=CC=C2C(NC(C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-UHFFFAOYSA-N 0.000 claims description 18
- 239000010452 phosphate Substances 0.000 claims description 18
- 229960003677 chloroquine Drugs 0.000 claims description 17
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 17
- 230000005764 inhibitory process Effects 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 12
- 230000000120 cytopathologic effect Effects 0.000 abstract description 10
- 238000002474 experimental method Methods 0.000 abstract description 5
- 238000000338 in vitro Methods 0.000 abstract description 3
- 241001678559 COVID-19 virus Species 0.000 abstract description 2
- 208000001528 Coronaviridae Infections Diseases 0.000 abstract 1
- 238000000034 method Methods 0.000 abstract 1
- 239000007787 solid Substances 0.000 description 39
- 238000003756 stirring Methods 0.000 description 34
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 30
- 238000001914 filtration Methods 0.000 description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- 238000001816 cooling Methods 0.000 description 18
- 235000021317 phosphate Nutrition 0.000 description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 14
- 238000001035 drying Methods 0.000 description 13
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 12
- 239000012074 organic phase Substances 0.000 description 12
- 239000000047 product Substances 0.000 description 11
- 239000013078 crystal Substances 0.000 description 10
- 238000010438 heat treatment Methods 0.000 description 10
- 238000004296 chiral HPLC Methods 0.000 description 9
- 238000001704 evaporation Methods 0.000 description 9
- 239000002904 solvent Substances 0.000 description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 8
- 239000000706 filtrate Substances 0.000 description 8
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 6
- 241000700605 Viruses Species 0.000 description 6
- 230000000144 pharmacologic effect Effects 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- DVWQNBIUTWDZMW-UHFFFAOYSA-N 1-naphthalen-1-ylnaphthalen-2-ol Chemical compound C1=CC=C2C(C3=C4C=CC=CC4=CC=C3O)=CC=CC2=C1 DVWQNBIUTWDZMW-UHFFFAOYSA-N 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 4
- 230000005587 bubbling Effects 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 239000012043 crude product Substances 0.000 description 4
- 239000002932 luster Substances 0.000 description 4
- 239000012452 mother liquor Substances 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000008346 aqueous phase Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000005191 phase separation Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 231100000491 EC50 Toxicity 0.000 description 2
- HEFNNWSXXWATRW-UHFFFAOYSA-N Ibuprofen Chemical compound CC(C)CC1=CC=C(C(C)C(O)=O)C=C1 HEFNNWSXXWATRW-UHFFFAOYSA-N 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- 231100000645 Reed–Muench method Toxicity 0.000 description 2
- 239000003907 antipyretic analgesic agent Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000004821 distillation Methods 0.000 description 2
- 239000012065 filter cake Substances 0.000 description 2
- 229960001680 ibuprofen Drugs 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 238000001953 recrystallisation Methods 0.000 description 2
- 238000007670 refining Methods 0.000 description 2
- 230000000241 respiratory effect Effects 0.000 description 2
- 208000023504 respiratory system disease Diseases 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 208000025721 COVID-19 Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- -1 chloroquine phosphates Chemical class 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- 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
- C07D215/38—Nitrogen atoms
- C07D215/42—Nitrogen atoms attached in position 4
- C07D215/46—Nitrogen atoms attached in position 4 with hydrocarbon radicals, substituted by nitrogen atoms, attached to said nitrogen atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
Abstract
The invention discloses application of levorotatory chiral chloroquine phosphate in preparation of a medicament for treating coronavirus. The invention is based on the discovery of the inventor that in vitro cell experiments, the levorotatory chiral chloroquine phosphate has obvious inhibition effect on cytopathic effect of novel coronavirus SARS-CoV-2 infected Vero E6 cells, and IC50The value is 1.45 mu g/mL, the inhibition concentration is obviously lower than that of dextro chloroquine phosphate and racemic chloroquine phosphate, and the method has important significance for preventing, controlling and treating the novel coronavirus infection.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to application of levorotatory chiral chloroquine phosphate in preparation of a medicine for treating coronavirus.
Background
Chloroquine phosphate is an old medicine which has been applied as early as the three and four decades of the last century and is mainly used for treating malaria. Recent studies show that chloroquine has a certain inhibitory effect on viruses (2019-nCoV) causing the novel coronavirus pneumonia COVID-19. However, based on the current clinical application effect, cloquine phosphate, which is pointed out by the academy of China, in the southern Stadium, is not enough to be called as a specific drug for treating the new coronary pneumonia, but has the treatment effect and little side effect, and is worthy of discussion and research.
The side chain of the chloroquine contains a chiral carbon, and the chloroquine in various dosage forms in clinical application at present is a racemate. The existing research shows that the chiral structure of some medicines has close relation with the biological activity thereof, for example, ibuprofen antipyretic analgesic commonly used in daily life mainly plays a role in S configuration, and R configuration of the ibuprofen antipyretic analgesic has no activity at all. For chloroquine, no related report of chiral chloroquine in resisting coronavirus exists at present.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the application of the levorotatory chiral chloroquine phosphate in preparing the medicaments for treating the coronavirus.
The purpose of the invention is realized by the following technical scheme: the application of the levorotatory chiral chloroquine phosphate in preparing the medicaments for treating the coronavirus is based on the discovery of the inventor of the invention that the levorotatory chiral chloroquine phosphate has obvious inhibition effect on novel coronavirus (SARS-CoV-2) in vitro cell experiments, and the inhibition concentration is obviously lower than that of dextrorotatory chloroquine phosphate and racemic chloroquine phosphate.
The levorotatory chiral chloroquine phosphate is R- (-) -chloroquine phosphate, and the structural formula is shown as follows:
the coronavirus is preferably a novel coronavirus (2019-ncov, also known as SARS-CoV-2).
The concentration of the levo chloroquine phosphate is preferably at least 1.45 mu g/mL; more preferably 1.88 to 15. mu.g/mL.
Compared with the prior art, the invention has the following advantages and effects:
the invention relates to a novel coronavirus (SARS-COV-2) resistance assay method, which comprises the steps of preparing R- (-) -chloroquine phosphate (chloroquine levorotatory phosphate) and S- (+) -chloroquine phosphate (chloroquine dextrorotary phosphate), evaluating the effects of the chloroquine levorotatory phosphate, the dextrorotatory phosphate and the racemate phosphate on the level of in vitro cells, discussing the application possibility of the chiral chloroquine in preparing a novel coronavirus (SARS-COV-2) resistance medicament, finding that the chloroquine levorotatory phosphate has obvious inhibition effect on cytopathic effect of the novel coronavirus (SARS-CoV-2) infected Vero E6 cells, and the inhibition concentration is obviously lower than that of the chloroquine dextroquine phosphate and the racemate phosphate, and has important significance for the prevention, control and treatment of the novel coronavirus (SARS-COV-2) infection.
Drawings
FIG. 1 is a graph showing the results of the detection of the pharmacological effects of chloroquine levorotatory phosphate on the novel coronavirus.
FIG. 2 is a graph showing the results of the test of the pharmacological effects of dexchloroquine phosphate on the novel coronavirus.
FIG. 3 is a graph showing the results of the test of the pharmacological effects of racemic chloroquine phosphate on the novel coronavirus.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
EXAMPLE 1 preparation of chiral chloroquine phosphate
(1) Preparation of S- (+) -chloroquine binaphthol phosphate
Taking 8.3g chloroquine racemate, adding 9.7g of (S) - (+) -binaphthol phosphate, adding 90g of isopropanol and 30g of acetone, heating and dissolving at 70 ℃, keeping the temperature for 1h, stirring and cooling to room temperature, cooling to 0-4 ℃, standing for 18h, separating out crystals, filtering, draining, storing filtrate as R-rich mother liquor, obtaining a light yellow solid filter cake, and drying at 60 ℃ to obtain 11.8g of S- (+) -chloroquine binaphthol phosphate.
(2) Recrystallization
Adding 11.8g (17.7mmol) of the solid obtained in the step (1) into 75g of isopropanol and 25g of acetone, heating to dissolve at 70 ℃, slowly stirring to room temperature, cooling to 0-4 ℃, standing for 6 hours, separating out large-particle crystals, filtering, draining to obtain a loose solid with luster, and drying at 60 ℃ to obtain 8.6g of the solid.
(3) Preparation of S- (+) -chloroquine and recovery of (S) - (+) -binaphthol phosphate
Stirring 8.6g (12.9mmol) of the solid obtained in the step (2) with 15g of methanol and 10g of sodium hydroxide solution with the concentration of 30 percent by mass for 1h, adding 80ml of dichloromethane and 80ml of water, stirring to separate out a large amount of solid with good crystal form, filtering to obtain wet white crystalline solid with the wet weight of 10.2g, wherein the solid is released (S) - (+) -binaphthol phosphate sodium salt, separating the filtrate, back-extracting the aqueous phase with 60ml of dichloromethane, washing the organic phase with 60ml of 3 water, combining the organic phases, adding anhydrous sodium sulfate, stirring and drying for 1h, filtering, distilling and recovering the solvent under normal pressure, evaporating to dryness under 10kPa under reduced pressure at 90 ℃ until no bubbling is generated to obtain 3.9g of light yellow oily matter, cooling to obtain a yellow solid, wherein the solid is S- (+) -chloroquine, and the enantioselectivity of the product is analyzed by chiral HPLC, and the ee value is 99.6%; [ alpha ] to]24 D＝+107.8°(c＝1,EtOH)。
(4) Preparation of S- (+) -chloroquine phosphate
Taking 3.2g of S- (+) -chloroquine prepared in the step (3), adding 60g of ethanol, heating and stirring at 70 ℃ for dissolving, adding 4.2g of phosphoric acid with the concentration of 50% by mass, immediately separating out a white solid, keeping the temperature and stirring for 1h, stirring and cooling to 10 ℃, filtering, pumping out, and drying in vacuum at 80 ℃ to obtain 4.7g of white solid which is S- (+) -chloroquine phosphate, wherein the product enantioselectivity is analyzed by chiral HPLC, and the ee value is 99.5%; [ alpha ] to]24 D＝+86.7°(c＝1,H2O)。
(5) Preparation of R- (-) -chloroquine
Adding 7.2g of sodium hydroxide solution with the concentration of 30 percent by mass into the R-enriched mother liquor obtained in the step (1), distilling and recovering the solvent under normal pressure, adding 80ml of dichloromethane and 80ml of water into the distillation residue, stirring, carrying out phase separation, carrying out back extraction on the water phase by using 60ml of dichloromethane, washing the organic phase by using 60ml of 3 water, combining the organic phases, adding anhydrous sodium sulfate and activated carbon, stirring and drying for 1 hour, filtering, and distilling the filtrate under normal pressure to obtain filtrateRecovering solvent, evaporating to dryness at 90 deg.C under 10kPa under reduced pressure, evaporating to obtain 3.8g pale yellow oily substance as crude product of R- (-) -chloroquine, and analyzing enantioselectivity of product by chiral HPLC, wherein ee value is 53%; [ alpha ] to]24 D＝-58.1°(c＝ 1,EtOH)。
(6) Refining of R- (-) -chloroquine and recovery of (R) - (-) -binaphthol phosphate
Adding 4.1g of (R) - (-) -binaphthol phosphate into the crude product of R- (-) -chloroquine obtained in the step (5), adding 40g of isopropanol, 10g of acetone and 2g of water, heating and dissolving at 60 ℃, keeping the temperature for 1h, stirring and cooling to room temperature, cooling to 0-4 ℃, standing for 12h, separating out crystals, filtering, draining to obtain a loose solid with luster, adding 10g of methanol and 5g of sodium hydroxide solution with the concentration of 30 percent by mass, stirring for 1h, adding 60ml of dichloromethane and 80ml of water, stirring, separating out a large amount of solid with good crystal forms, filtering to obtain a wet white crystalline solid with the weight of 4.1g, separating the phase of the filtrate, back extracting the water phase by using 70ml of dichloromethane, washing the organic phase by using 50ml of 3 water, combining organic phases, adding anhydrous sodium sulfate, stirring and drying for 1h, filtering, distilling at normal pressure to recover the solvent, evaporating at 90 ℃ under 10kPa under reduced pressure until dryness is realized, evaporating until no bubbling occurs to obtain 2.7g of light yellow oily matter, cooling to obtain a yellow solid, wherein the solid is R- (-) -chloroquine, and the enantioselectivity of a product is analyzed by chiral HPLC, and the ee value is 99%; [ alpha ] to]24 D＝-107.0°(c＝1,EtOH)。
(7) Preparation of R- (-) -chloroquine phosphate
Taking 1.6g of R- (-) -chloroquine prepared in the step (6), adding 10g of methanol and 20g of isopropanol, heating and stirring at 70 ℃ for dissolving, adding 1.2g of phosphoric acid with the concentration of 85% by mass, immediately precipitating a white solid, keeping the temperature and stirring for 1h, stirring and cooling to 10 ℃, filtering, draining, and drying in vacuum at 80 ℃ to obtain 2.2g of the white solid which is the R- (-) -chloroquine phosphate, wherein the product enantioselectivity is analyzed by manual HPLC, and the ee value is 99%; [ alpha ] to]24 D＝-86.0°(c＝1,H2O)。
EXAMPLE 2 preparation of chiral chloroquine phosphate
(1) Preparation of R- (-) -chloroquine binaphthol phosphate
Taking 4.6g chloroquine racemate (CAS No.54-05-7), adding 5.4g of (R) - (-) -binaphthol phosphate, adding 50g of isopropanol and 16g of acetone, heating and dissolving at 60 ℃, keeping the temperature for 1h, stirring and cooling to room temperature, cooling to 0-4 ℃, standing for 12h, precipitating crystals, filtering, draining, storing the filtrate as S-enriched mother liquor, wherein the filter cake is light yellow solid, and drying at 60 ℃ to obtain 6.2g of solid which is R- (-) -chloroquine binaphthol phosphate.
(2) Recrystallization
Adding 30g of isopropanol and 9g of acetone into 6.2g of the solid obtained in the step (1), heating and dissolving at 60 ℃, slowly stirring to room temperature, cooling to 0-4 ℃, standing for 6h, separating out large-particle crystals, filtering, draining to obtain a loose solid with luster, and drying at 60 ℃ to obtain 4.7g of solid.
(3) Preparation of R- (-) -chloroquine and recovery of (R) - (-) -binaphthol phosphate
Adding 10g of methanol and 7g of sodium hydroxide solution with the concentration of 20 percent by mass into 4.7g (7mmol) of the solid obtained in the step (2), stirring for 1h, then adding 50ml of dichloromethane and 50ml of water, stirring, separating out a large amount of solid with good crystal form, filtering to obtain a wet white crystalline solid with the weight of 5.31g, wherein the solid is released (R) - (-) -binaphthol phosphate sodium salt, separating the filtrate, back extracting the aqueous phase with 40ml of dichloromethane, washing the organic phase with 40ml of 3 water, combining the organic phases, adding anhydrous sodium sulfate, stirring and drying for 1h, filtering, distilling and recovering the solvent at normal pressure, evaporating to dryness at 90 ℃ under 10kPa under reduced pressure until no bubbling occurs to obtain 2.1g of light yellow oily matter, cooling to obtain a yellow solid, wherein the solid is R- (-) -chloroquine, and the enantioselectivity of the product is analyzed by chiral HPLC, and the ee value is 99%; [ alpha ] to]24 D＝-107.1°(c＝1,EtOH)。
(4) Preparation of R- (-) -chloroquine phosphate
Taking 1.6g of R- (-) -chloroquine prepared in the step (3), adding 25g of ethanol, heating and stirring at 70 ℃ for dissolving, adding 1.2g of phosphoric acid with the concentration of 85% by mass, immediately separating out a white solid, keeping the temperature and stirring for 1h, stirring and cooling to 20 ℃, filtering, draining, and drying in vacuum at 80 ℃ to obtain 2.3g of white solid which is R- (-) -chloroquine phosphate, wherein the product enantioselectivity is analyzed by chiral HPLC, and the ee value is 99%; [ alpha ] to]24D＝-86.8°(c＝1,H2O)。
(5) Preparation of S- (+) -chloroquine
Adding 6g of sodium hydroxide solution with the concentration of 20% in mass into the S-enriched mother liquor obtained in the step (1), distilling and recovering the solvent at normal pressure, adding 40ml of dichloromethane and 40ml of water into the residue of distillation, stirring, carrying out phase separation, carrying out back extraction on the water phase by using 40ml of dichloromethane, washing the organic phase by using 40ml of 3 water, combining the organic phases, adding anhydrous sodium sulfate and active carbon, stirring and drying for 1h, filtering, distilling and recovering the solvent at normal pressure of the filtrate, evaporating to dryness at 90 ℃ and 10kPa under reduced pressure until bubbling does not occur to obtain 2.15g of light yellow oily matter, wherein the product is a crude product of S- (+) -chloroquine, and the enantioselectivity of the product is analyzed by chiral HPLC, and the ee value is 49%; [ alpha ] to]24 D＝+52.5°(c＝ 1,EtOH)。
(6) S- (+) -chloroquine refining and (S) - (+) -binaphthol phosphate recovery
Adding 2.3g of (S) - (+) -binaphthol phosphate into the S- (+) -chloroquine crude product obtained in the step (5), adding 20g of isopropanol and 7g of acetone, heating and dissolving at 60 ℃, keeping the temperature for 1h, stirring and cooling to room temperature, cooling to 0-4 ℃, standing for 12h, separating out crystals, filtering, draining to obtain a loose solid with luster, adding 7g of methanol and 5g of sodium hydroxide solution with the concentration of 20% by mass, stirring for 1h, adding 30ml of chloroform and 30ml of water, stirring, separating out a large amount of solid with good crystal forms, filtering to obtain a white crystalline solid with the wet weight of 2.31g, separating the solid which is released (S) - (+) -binaphthol phosphate sodium salt, carrying out phase separation on the filtrate, carrying out back extraction on the aqueous phase by using 30ml of chloroform, washing the organic phase by using 30ml of 3 water, merging the organic phases, adding anhydrous sodium sulfate, stirring and drying for 1h, filtering, distilling under normal pressure to recover solvent, evaporating at 90 deg.C under 10kPa under reduced pressure to dryness, evaporating to obtain 1.5g pale yellow oily substance, cooling to obtain yellow solid, which is S- (+) -chloroquine, and analyzing enantioselectivity of product by chiral HPLC, wherein ee value is 98%; [ alpha ] to]24 D＝+104.9°(c＝1,EtOH)。
(7) Preparation of S- (+) -chloroquine phosphate
Taking 1g of S- (+) -chloroquine prepared in the step (6), adding 20g of ethanol, heating and stirring at 70 ℃ for dissolving, adding 0.72g of phosphoric acid with the concentration of 85 percent by mass, immediately separating out white solid, keeping the temperature and stirring for 1h, stirring and cooling to 20 ℃, filtering, draining, and vacuum drying at 80 ℃ to obtain 1.4g of white solid which is S- (+) -phosphorusChloroquine hydrochloride, and the enantioselectivity of the product is analyzed by chiral HPLC, and the ee value is 98%; [ alpha ] to]24 D＝+85.1°(c＝1,H2O)。
EXAMPLE 3 evaluation of the Effect of levorotatory, dextrorotatory and racemic chloroquine phosphates on the resistance to the novel coronavirus (SARS-COV-2) at the cellular level
The experiment for evaluating the effect of a drug against the novel coronavirus (SARS-COV-2) at the cellular level was conducted by the Guangzhou respiratory health institute. Wherein the used cells are VeroE6 cells, and are preserved by a virus laboratory in the respiratory disease national key laboratory of Guangzhou respiratory health research institute; virus SARS-CoV-2, titer TCID50＝10-6100 μ L, stored at 80 ℃ in BSL-3 laboratory (respiratory disease national center laboratory high pathogenic microorganism research laboratory). Using a Virus titre of 100TCID50。
The levorotatory chiral chloroquine phosphate was prepared according to the step (1) of example 2, and the dextrorotatory chiral chloroquine chloride was prepared according to the step (1) of example 1.
2.1 test drug antiviral Experimental procedures
(1) The tested drugs are:
table 1 drug name, experimental concentration and grouping
(2) Sterile 96-well culture plate, 100. mu.L of 2X 10 concentration per well5cells/mL VeroE6 cells, 37 ℃ and 5% CO2Culturing for 24 hours;
(3) adding 100TCID into culture plate experimental group and virus control group50Virus liquid 100 mul/well, 37 deg.C, 5% CO2Adsorbing for 2 hours by an incubator;
(4) after 2h, discarding the cell culture solution in the 96-well culture plate; diluting the tested medicine into each concentration in table 1, wherein each concentration is 3 multiple holes, and adding the liquid medicine into each hole at 100 mu L;
(5) simultaneously setting a cell control, a blank control (a solvent control), a virus control (a negative control) and a positive drug control (chloroquine or Reidesvir);
(6) at 37 ℃ with 5% CO2Incubating in an incubator for 3-4 days;
(7) cytopathic effect (CPE) was observed under an optical microscope and the degree of cytopathic effect was recorded according to the following 6-point scale: "-" no lesions present; "±" means less than 10% cytopathic effect; "+" is about 25% of the cellular pathology; "+ +" indicates about 50% of cellular pathology; "+ + + +" indicates that about 75% of the cells are diseased: "+ ++" indicates that 75% or more of the lesions are diseased. The half maximal effective concentration (IC50) was calculated using the Reed-Muench method or GraphPad prism 5.0. Judging the drug effect standard: concentrations that inhibit viral CPE by 50% are considered effective concentrations.
(8) The experimental conditions are as follows: the experimental operations are all completed in a BSL-3 level biological safety laboratory.
2.2 Experimental results and analysis
The half maximal effective concentration (IC50) was calculated by observing cytopathic effect (CPE) and recording the results of the experiment, as shown in tables 2-4 and FIGS. 1-3, using the Reed-Muench method or GraphPad prism 5.0:
TABLE 2 results of pharmacological effects of chloroquine levo-phosphate against novel coronaviruses
| Drug concentration (μ g/mL) | Inhibition ratio (%) |
| 15 | 90.00±5.00 |
| 7.5 | 86.67±2.89 |
| 3.75 | 81.67±2.89 |
| 1.88 | 70.00±8.66 |
| 0.94 | 38.33±10.41 |
TABLE 3 results of the pharmacological effects of dexchloroquine phosphate against the novel coronavirus
| Drug concentration (μ g/mL) | Inhibition ratio (%) |
| 60 | 93.33±2.89 |
| 30 | 88.33±2.89 |
| 15 | 83.33±2.89 |
| 7.5 | 81.67±2.89 |
| 3.75 | 78.33±2.89 |
| 1.88 | 46.67±10.41 |
| 0.94 | 16.67±2.89 |
TABLE 4 results of the pharmacological effects of racemic chloroquine phosphate against novel coronaviruses
The experimental result shows that the levorotatory chiral chloroquine phosphate has obvious inhibition effect on cytopathic effect of novel coronavirus (SARS-CoV-2) infected Vero-E6 cells within the concentration range of 1.88-15 mu g/mL, and the inhibition rate reaches 70% when the medicine concentration is 1.88 mu g/mL; the concentration of the dextrorotatory chiral chloroquine phosphate is 1.88 mu g/mL, the inhibition rate is only 46.67 percent, the concentration of the racemic chloroquine phosphate is 3.13 mu g/mL, and the inhibition rate is only 56.67 percent.
Therefore, the inhibition concentration of the levorotatory chiral chloroquine phosphate on the novel coronavirus (SARS-COV-2) is obviously lower than that of the dextrorotatory chloroquine phosphate and the racemic chloroquine phosphate.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (3)
1. Application of levorotatory chiral chloroquine phosphate in preparing medicine for treating coronavirus is disclosed.
2. The use of levorotatory chiral chloroquine phosphate according to claim 1 for the preparation of a medicament for the treatment of coronavirus, wherein: the coronavirus is a novel coronavirus.
3. The use of levorotatory chiral chloroquine phosphate according to claim 2 in the preparation of a medicament for the treatment of coronavirus, wherein: the concentration of the chloroquine levorotatory phosphate is 1.88-15 mu g/mL.
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