COMPOUND FOR TREATING RESPIRATORY SYNCYTIAL VIRUS INFECTION AND PREPARATION METHOD AND USE THEREOF

Abstract

The present invention declares a kind of compound to treat respiratory syncytial virus infection and its preparation method and application. The compound stated is cyclopamine's chemical analogs, has the property of inhibiting respiratory syncytial virus replication, and does not have the property of inhibiting Hedgehog signaling pathways. The preparation method stated is to get the cyclopamine's chemical analogs through drug chemical synthesis, then to screen the analogs with two parallel in vitro experiment. The compound described can be used to treat respiratory virus infection, paramyxovirus, respiratory syncytial virus infection, capillary bronchitis/pneumonia/tympanitis caused by respiratory syncytial virus. Furthermore, the compound described never cause the side effects of fetal malformation, and it overcame the teratogenicity of cyclopamine, and filled the gap of anti-human respiratory syncytial virus drug, especially of the field of pediatric drug.

Claims

1-13. (canceled)

14. A kind of compound to treat respiratory syncytial virus infection, whose character is: the compound described posses with the following structure: ##STR00005##

15. The compound described in claim 14, whose character is that the preparation protocol is as following: 1) Preparation of intermediates Firstly, Cyclopamine (498.06 mg, 1.21 mmol, 1.00 eq) and Et3N (489.76 mg, 4.84 mmol, 4.00 eq) were dissolved in dry DCM (4 ml); Secondly, TFAA (762.41 mg, 3.63 mmol, 3.00 eq) in DCM (1 ml) was added at 0 C.; Thirdly, the reaction mixture was warmed slowly to 15 C. and stirred for another 16 h; Fourthly, LCMS showed started material was almost consumed completely. (Desired product RT=0.996 min, 25%; di-CF.sub.3CO byproduct RT=1.125 min, 47%); Fifthly, the reaction mixture was concentrated and the residue was diluted with MeOH (15 ml); Sixthly, the resulted suspension was heated to 70 C. for 1 h; Seventhly, then the reaction mixture was cooled to 15 C.; Lastly, the undissolved solid was collected by filtration. Intermediate (346.00 mg, 681.60 mol, 56.33% yield) was obtained as white solid; 2) Preparation of compounds, Firstly, 130.00 mg, 256.09 umol, 1.00 eq) and TEA (182.00 mg, 1.80 mmol, 7.02 eq) were dissolved in DCM (2 ml), cooled by ice-bath; Secondly, isocyanatoethane (isocyanato ethane) (125.00 mg, 1.76 mmol, 6.87 eq) in DCM (0.5 ml) was added; Thirdly, the reaction mixture was stirred at 45 C. for 48 h. TLC showed started material was consumed completely; Fourthly, the reaction was concentrated under vacuum; Fifthly, the residue was washed with MeOH (2.5 ml); and Sixthly, desired product was collected by filtration, and 30.00 mg, 51.84 mol, 20.24% yield) was obtained as white solid.

16. The usage of the compound described in claim 14 is that described compounds can be used in the treatment of respiratory tract infection caused by disease, and never cause side effects on patients, which act as a character of the compound usage.

17. As stated in claim 16, the character of application is that the compound described is used for treatment of respiratory tract infection caused virus.

18. As stated in claim 16, the character of application is that the compound described is used for treatment of paramyxovirus infection.

19. As stated in claim 16, the character of application is that the compound described is used for treatment of respiratory syncytial virus infection.

20. As stated in claim 16, the character of application is that the compound described is used for treatment of capillary bronchitis caused by respiratory syncytial virus.

21. As stated in claim 16, the character of application is that the compound described is used for treatment of pneumonia caused by respiratory syncytial virus.

22. As stated in claim 16, the character of application is that the compound described is used for treatment of tympanitis caused by respiratory syncytial virus.

Description

DESCRIPTION OF FIGURES

[0068] A further explanation of the invention is illustrated by the attached figures and example.

[0069] FIG. 1 shows the preparation method flow diagram described in the invention.

[0070] FIG. 2 shows the flow diagram of intermediate preparation described in the invention.

[0071] FIG. 3 shows the flow diagram of compound preparation described in the invention.

[0072] FIG. 4 shows the effect of compounds described in the invention on RSV replication.

[0073] As shown in the figure, compounds were tested at 2 concentrations (2 M, 8 M) in the RSV replication assay. The control (compound-free) is scored at 100% replication. Replication in the presence of CPM is scored at 0% replication. Replication inhibition by analogs S2-6 and C2-2 to C2-4 are scored % inhibition of control.

[0074] FIG. 5 shows the effect of compounds described in the invention on Smo associated induced gene expression.

[0075] Figure shows that compounds were tested at 10 uM in the hedgehog (Hh) assay. The control (compound-free, presence of SAG) is scored at 100% gene expression. The gene expression in the presence of CPM is significantly reduced by 56%. Gene expression inhibition by analogs S1-2 and S1-4 however are not significant, with 36% and 5% respectively. These data show the proof of principle that the anti-RSV activity of steroidal alkaloid molecules derived from CPM can be dissociated from the inhibitory activity of the Smo-dependent Hh pathway.

[0076] FIG. 6 shows the Inhibition of coefficient (ICrh) of CPM and its analogs.

EXAMPLE 1

[0077] The preparation of CPM analog S1-2, referring to FIGS. 1, 2, and 3, and the procedure is as following: [0078] 1) Cyclopamine (498.06 mg, 1.21 mmol, 1.00 eq) and Et3N (489.76 mg, 4.84 mmol, 4.00 eq) were dissolved in dry DCM (4 ml). Then TFAA (762.41 mg, 3.63 mmol, 3.00 eq) in DCM (1 ml) was added at 0 C. The reaction mixture was warmed slowly to 15 C. and stirred for another 16 h. LCMS showed started material was almost consumed completely. (Desired product RT=0.996 min, 25%; di-CF3CO byproduct RT=1.125 min, 47%).The reaction mixture was concentrated and the residue was diluted with MeOH (15 ml). The resulted suspension was heated to 70 C. for 1 h. Then the reaction mixture was cooled to 15 C. The undissolved solid was collected by filtration. Intermediate (346.00 mg, 681.60 mol, 56.33% yield) was obtained as white solid, used for next step without further purification. [0079] 2) Preparation of Compound S1-2.Firstly, 130.00 mg, 256.09 umol, 1.00 eq) and TEA (182.00 mg, 1.80 mmol, 7.02 eq) were dissolved in DCM (2 ml), cooled by ice-bath. Then isocyanatoethane (isocyanato ethane) (125.00 mg, 1.76 mmol, 6.87 eq) in DCM (0.5 ml) was added. Then the reaction mixture was stirred at 45 C. for 48 h. TLC showed started material was consumed completely. The reaction was concentrated under vacuum. The residue was washed with MeOH (2.5 ml). Desired product was collected by filtration. Series 1-2 (30.00 mg, 51.84 mol, 20.24% yield) was obtained as white solid. The filtrate was concentrated. The crude product was used for next step. [0080] 3) Detection of effect of compound on RSV replication. Human respiratory syncytial virus Long strain (ATCC reference VR-26) and HEp-2 cells (ATCC reference CCL-23) were used for infection assays. Cells were maintained in DMEM, supplemented with penicillin/streptomycin and 10% FBS, and incubated at 37 C., 5% CO.sub.2. Virus was passaged in HEp-2 cells in the same conditions, but with 2% FBS. Virus stocks were prepared by infecting confluent HEp-2 cells at a low multiplicity of infection (MOI) of 0.1 for two to three days. Cells and virus-containing supernatant were then subjected to a single freeze/thaw cycle at 80 C. to release cell-bound virus, and the sample was clarified by centrifugation at 2000g for 10 min at 4 C. The supernatant was homogenized, aliquoted and stored at 20 C. The anti-hRSV potency of compounds was evaluated by focus reduction assay following the focus forming assay method, using 50 to 100 focus forming units per well. Compounds were tested either during virus adsorption for 1 h at 4 C. to investigate viral entry, post-adsorption for 72 h at 37 C. to investigate viral propagation and replication, or during all stages of infection. The concentration of compound resulting in 50% inhibition (IC.sub.50) of virus replication was determined by focus counting (viral entry) or focus size measurement (viral propagation) and was determined using non-linear regression analysis using GraphPad Prism (GraphPad Software, La Jolla Calif. USA). To assess the cytotoxicity of compounds, a CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, Wis.) was carried following the manufacturer's instruction in the conditions of the focus reduction assay.

[0081] The detailed protocol is as following: [0082] Day 1: Seed 210.sup.5 cells/well in 24-well plate with 0.5 ml 10% FBS DMEM. Incubate at 37 C., 5% CO.sub.2 for 1624 h. [0083] Day 2: Compound treatment and virus infection. [0084] 1. dilution: the diluent is 25% DMSO, 2% FBS, DMEM. The Compound is diluted to 800 M and 200 M; [0085] 2. Virus dilution: the diluent is DMEM with 2% fetal bovine serum (FBS), hRSV virus stock is P9 (2.910.sup.7pfu/ml) (stored at 80 C.), both thaw it and use it on ice, MOI=0.1. [0086] 3.compound treatment and virus infection:Pour off the medium in the 24-well plate (Or rinse once with PBS), add the diluented virus 0.5 ml, and 5 l diluented drug to make the virus infection moi=0.1, and the drug concentration is 8 M, 2M; [0087] 4. The plates were incubated at 37 C., 5% CO.sub.2 for 3 days (observe the CPE) [0088] Day 4: Seed new Hep2 cells for titration Seed HEp-2 cells in new 24-well plates with 1.510.sup.5cells/well. Incubate at 37 C., 5% CO.sub.2 for 1624 h [0089] Day 5: Collect virus and virus titration [0090] 1. Put the 24-wells plates in 80 C. freezer for 1 h, and then thaw them on ice, collect the virus in wells into tubes (on ice). Centrifugate these tubes with 8000 rpm, 5 min, and then pipette out 50 l into a new tubes to test virus titer and then the rest virus samples were pipetted into 2 ml prechilled tube (on ice) then fast frozen in liquid nitrogen and storaged in 80 C. freezer. [0091] 2. Virus dilution: Prepare six 1.5 ml EP tube, mark, on ice. Add 270 l 2% medium in each tube. From stock, 30 l virus in 270 l 2% medium serial dilution to get 10.sup.-1 to 10.sup.-6 virus dilutions [0092] 3. Remove cell culture medium, and rinse once with PBS (on ice); [0093] 4. Infect monolayer HEp-2 cells with 200 l virus dilutions correspondingly. [0094] 5. Incubate for about 1.5 h in the incubator at 37 C., 5% CO.sub.2. Shake every 15 min [0095] 6. Water bath the overlay medium (DMEM with 2% FBS and 0.8% CMC) at 37 C. for 1 h, then pipette out virus solution after the 1.5 h of virus incubation time, and wash twice with PBS. Add DMEM with 2% FBS and 0.8% CMC per well. Incubate for 3-4 days at 37 C., 5% CO.sub.2. [0096] Day 8: [0097] 1. Pipette out CMC and wash once with PBS (on ice); [0098] 2. Fix the cells with 4% PFA for at least 20 min, then wash with PBS, and air dry; [0099] 3. Incubate the cells with 0.25% tritonX-100 in PBS for 20 minutes. Wash the plate with PBS for 3 times, and every wash lasts for 4 min; [0100] 4. Incubate the cells for about 1 hour at RT with 200 l of primary anti-hRSV F antibody (mouse-Fizgerald, Acton, Mass.) diluted 1:1000 in PBS-5% skim milk; [0101] 5. Wash 34 min with PBS-0.02% Tween 20; [0102] 6. Apply a 1:6000 dilution (200 l) of secondary HRP-conjugated antibody (goat anti-mouse-Bethyl, Montgomery, Tex.) in PBS-5% skim milk and incubate at room temperature for 1 h. [0103] 7. Wash the monolayers with PBS-0.02% Tween 20 for 3 times, and every wash lasts for 4 min [0104] 8. Apply 200 l of True Blue substrate and leave at room temperature in the dark until blue spots appear (more than 10 min); [0105] 9. Rinse with running water (PBS will fade the colour); [0106] 10. Dry in oven, scan plate and count plaques; [0107] From FIG. 4, compound S1-2 has the maxium inhibition effect on RSV replication is 99% (compared with blank control.) [0108] 4) Detection of inhibition effect of compounds on Hh pathway. NIH3T3 cells are seeded cells in 6 well plates (310.sup.5 cell/well), or 12 well plate (1.510.sup.5 cell/well) one day in advance, with 5% FBS DMEM, without antibiotics per well so that they will be 9095% confluent at the time of transfection. Plasmids are diluted with Lipofectamine 2000 (total volume is 1001000 l) and added to each well containing cells and medium, followed by incubation at 37 C. in a CO.sub.2 incubator for 48 h. After 48 h transfection, SAG is added to reach a final concentration of 0.5 M and CPM analogs at various concentrations. After 48 h of incubation with compounds, cells are washed, lysed and lysate transferred to a 96-well white plate. LAR II reagent is added (Promega, Dual-luciferase reporter assay system, E1910) and samples read in a luminescence reader. [0109] The detailed protocol is as following: [0110] Day 1: Seed NIH3T3 cells in 6 well plate (310.sup.5cell/well), or 12 well plate (1.510.sup.5cell/well) one day advance, with 5% FBS DMEM, without antibiotics per well so that they will be 9095% confluent at the time of transfection. [0111] Day 2: For each transfection sample, prepare DNA-Lipofectamine 2000 complexes as follows: (the reagent volume and DNA quantity, see the following table); [0112] 1. Dilute DNA in 250 l of Opti-MEM reduced serum medium with serum. And the DMA is Gli-binding site-luciferase expressing plasmid and Renilla luciferase expressing plasmid, with ratio of 50:1. Mix gently. [0113] 2. Mix Lipofectamine 2000 gently before use, then dilute the appropriate amount in 250 l of Opti-MEM. Mix gently. [0114] 3. After the 5 min incubation (not longer than 30 min), combine the diluted DNA with the diluted Lipofectamine 2000 (total volume is 100-1000 l). Mix gently and incubate for 20 min at room temperature to allow the DNA-Lipofectamine 2000 complexes to form. [0115] 4. Add the DNA-Lipofectamine 2000 complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth. [0116] 5. After 46 h, pipette out the medium from the well, and growth medium should be replaced. Then incubate the cells at 37 C. in a 5% CO.sub.2 incubator for 48 h. [0117] Day 4: After 48h transfection, prepare drug diluent, which is DMEM, with 0.5% FBS, 0.25% DMSO. Then drug preserving fluid was prepared. SAG will be diluted to 1 M. And Cyclopamine (CPM) and its analogs will be diluted to 20 M. The same well will be added with 250 l 1 M SAG and 250 l 20 M CPM or its analogs. The final concentration of SAG is 0.5M, and the final concentration of CPM or its analogs is 10 M, [0118] Day 5: [0119] 1.Remove growth media from cultured cells. Rinse cultured cells in 1PBS. Remove all rinse solution. [0120] 2. According to the operating manual of the Dual-luciferase reporter kit (Promega, E 1910), dispense the recommended volume 100 l of 1PLB into each culture vessels. Gently rock/shake the culture vessel for 15 min at room temperature. Transfer lysate to a 96-well white plate. [0121] 3.Add cell lysate 20 l to a well and then add 50 l LAR II in kit. Read the plate and measure the firefly luciferase activity level. [0122] 4. Add 50 l stop&Glo reagent, mix them, no bubbles. Read the plate and measure the Renilla luciferase activity level.

[0123] Record all the data and calculate the ratio. All the luminicence data will be deducted with blank data. The firefly luciferase activity data in the group with SAG and with DMSO control will be seen as 1, and the data in other group will be normalized. The ratio above will be named as ratio 1. Then, the Renilla luciferase activity data in the group with SAG and with DMSO control will be seen as 1, and the data in other group will be normalized. The ratio above will be named as ratio 2. Finally a new ratio can be calculated, which is the ratio of ratio 1 to ratio 2, and it means that normalized expression level (percentage) of Renilla luciferase.

[0124] CPM analog S1-2 shows unsignificant inhibition effect on gene expression, with inhibition percentage 36%. The data above indicated that anti-RSV effect can be disassociated with anti-Hh signal pathway the Steroid alkaloids derived from CPM, see the FIG. 5. [0125] 5) Calculate the coefficient of inhibition on RSV replication and Hh activity (ICrh). The ICrh of S1-2 is 1.44596, which is larger than 1 and means that it owns benifical inhibition property, see the FIG. 6. [0126] 6) Secreening compound S1-2. compound S1-2, with benifical inhibition property, can be developed to treat RSV infection see the FIG. 6. Example 2 [0127] The preparation of CPM analog S1-4, and the procedure is as following: see the FIGS. 1, 2, and 3. [0128] 1) Preparation of intermediate. The procedure is as example 1. [0129] 2) Preparation of compound S1-4.The procedure is as example 1. [0130] 3) Detection of inhibition effect on RSV replication of S1-4.The detection protocol is as example 1. see the diagram 4. The maxium inhibition percentage of S1-4 on RSV replication is 88%, see the FIG. 4. [0131] 4) Detection of inhibition effect on Hh signal pathway of S1-4. The detection protocol is as example 1. see the diagram 5. The inhibition effect of CPM analog S1-4 on gene expression is not significant, with inhibition percentage 5%. The data above indicated that, the anti-RSV effect can be disassociated with anti-Hh signal pathway the Steroid alkaloids derived from CPM, see the FIG. 5. [0132] 5) Calculate the coefficient of inhibition on RSV replication and Hh activity (ICrh). The ICrh of S1-4 is 1.6172823, which is larger than 1 and means that it owns benifical inhibition property, see the FIG. 6. [0133] 6) Secreening compound S1-4. compound S1-4, with benifical inhibition property. Furthermore,within the compounds tested, S1-4 owns the largest ICrh data, suggesting that it can be developed to treat RSV infection, see the FIG. 6. [0134] Finally, it should be illustrated that, the examples above is only to explain clearly the case in the invention, and is not to limit the mode of execution. For the normal technician in the field, it can be changed and modified based on the illustration above. Here, it is not necessary and no way to state all the examples with different mode of execution. However, all the obvious change and modification derived from the invention should be protected by the invention.