Compound for enhancing the coupling degree of complex TRPV4-KCa2.3 and anti-hypertension applications thereof

Abstract

The applicant provides a compound for enhancing the space coupling degree of an endothelial cell ion channel complex TRPV4-KCa2.3 and anti-hypertension applications thereof. By finding the structural domains of the interacting sites of the endothelial cell ion channel complex TRPV4-KCa2.3, a compound with specificity which can act at the two interacting sites is prepared in the present invention. It is found that the compound can enhance the space coupling degree of the TRPV4-KCa2.3 complex and has great significance for the research and development of anti-hypertension drugs.

Claims

1. A preparation method of a compound for enhancing the space coupling degree of an endothelial cell ion channel complex TRPV4-KCa2.3, wherein structural domains of interacting sites of the endothelial cell ion channel complex TRPV4-KCa2.3 are structural domain AR2 of protein TRPV4 and structural domain 17C of protein KCa2.3 and the compound is represented by structure formula (1): ##STR00009## the method comprising: providing propane diamine with t-butyloxycarboryl to protect a single amino first: ##STR00010## an exposed amino reacting with diphenylacetyl chloride to generate an amide: ##STR00011## removing a t-butyloxycarboryl protection from the amide under an acidic condition: ##STR00012## a compound obtained after removing the t-butyloxycarboryl protection performing an amino-ester exchange with 4-quinazolone-2-carboxylic acid ethyl ester to obtain a target compound: ##STR00013##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram of the three-dimensional structures and functional zones of the protein TRPV4 and the protein KCa2.3;

(2) FIG. 2 is a diagram of results of finding the interacting sites of the protein TRPV4 and the protein KCa2.3 by using fluorescence resonance energy transfer and stochastic optical reconstruction microscopy in Embodiment 2 of the present invention;

(3) FIG. 3 is a diagram of the results of preparing a compound with a dual-target co-immunoprecipitation effect in Embodiment 1;

(4) FIG. 4 is a diagram of the variation of the coupling degrees of the complex TRPV4-KCa2.3 detected using the fluorescence resonance energy transfer technology after the compound prepared in Embodiment 1 acts on mice with hypertension in Embodiment 3;

(5) FIG. 5 is a diagram of the variation of the blood pressure detected after the compound prepared in Embodiment 1 acts on three types of mice with hypertension.

DETAILED DESCRIPTION

(6) The present invention is described in further detail with reference to the attached drawings and embodiments.

(7) Experimental materials of the following embodiments are as follows:

(8) Cell system: HEK293 cells, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

(9) Experimental animals: C57BL/6 J male mice, 8 weeks aged, SPF grade, purchased from Changzhou Cavens Experimental Animal Co., Ltd.

(10) Plasmids and primers: Plasmid templates containing whole-genome CFP-TRPV4 or whole-genome YFP-KCa2.3 donated by the K S Lo College of the Chinese University of Hong Kong; primers all purchased from Sangon Biotech (Shanghai) Co., Ltd.

(11) Oligopeptide:

(12) TABLE-US-00001 AR2: YGRKKRRQRRRTGKTCLPKALLNLSNGRNDTIPVLLDIAERTGNMREFINS PFRDIYY; 17C: YGRKKRRQRRRRKLELTKAEKHVHNFMM allpurchasedfromSangonBiotech(Shanghai)Co., Ltd.

(13) Reagents: primary antibody goat anti-TRPV4 (sc-47527), purchased from Santa Cruz; primary antibody rabbit anti-KCa2.3 (APC-025), purchased from Alomone; secondary antibody Alexa Fluor 647 and Alexa Fluor 488, purchased from Invitrogen.

(14) Point mutation kit QuickChange purchased from Stratagene; DNA product purification kit and plasmid extraction kit, both purchased from Tiangen Biotech (Beijing) Co., Ltd.; DH5 competent cells, purchased from Tiangen Biotech (Beijing) Co., Ltd.

(15) The synthesis procedures of the biotinylated JNc-440 are as follows: (1) The compound JNc-440 is treated with phosphorus oxychloride to generate a carbonyl chloride:

(16) ##STR00006## (2) The chloride and 5-amino-1-pentanol perform nucleophilic substitution:

(17) ##STR00007## (3) Biotin and the product obtained in the previous step perform an esterification reaction to obtain biotinylated JNc-440:

(18) ##STR00008##

(19) 8% High-salt feed (TP6032-S8), purchased from Trophic Animal Feed High-tech Co., Ltd; L-NAME (Nitro-L-arginine) (N5501-5G), purchased from Sigma; Angiotensin 2 (ANG-2) (A107852), purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd;

(20) All other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

(21) Experimental instruments: blood pressure gauge for rats and mice (BP98A), purchased from SINSI; OSMOTIC PUMPS (implantable capsule osmotic pump), purchased from ALZET; gel imaging system, purchased from GENE; laser scanning confocal microscope, purchased from Leica.

Embodiment 1: Preparation Method of the Compound

(22) (1) A compound 1-diamine propylene (30 g, 405 mmol, 1 eq) is dissolved in 150 mL of dichloromethane, ice bathed and stirred; 2-tertiary butyl sodium bicarbonate (16.1 g, 73 mmol, 0.18 eq) is dissolved and diluted in 50 mL of dichloromethane; the mixture is poured into a flask slowly and stirred for 3 h at room temperature; after the materials react completely, detection is carried out by using thin layer chromatography (TLC), diluted with 50 mL of dichloromethane, washed with water several times, washed with saturated NaCl solution, and then dried with hydrous Na.sub.2SO.sub.4; the obtained product is concentrated to obtain a compound 2 (19 g, 27%);

(23) (2) The compound 2 (10 g, 57 mmol, 1 eq) and triethylamine (TEA) (8.7 g, 86 mmol, 1.5 eq) are dissolved in 100 mL of dichloromethane, ice bathed and stirred; diphenylacetyl chloride (13.1 g, 57 mmol, 1 eq) is dissolved in 30 mL of dichloromethane, the mixed materials are slowly poured into a flask, and stirred for 2.5 h at room temperature; after the materials react completely, TLC detection is carried out, followed by concentration of the crude product; the concentrated product is separated with column chromatography (dichloromethane/methanol 20:1) to obtain a compound 3 (13.2 g, 63%);

(24) (3) The compound 3 (8 g, 22 mmol, 1 eq) is dissolved in a mixed solvent (40 mL:dichloromethane:trifluoroacetic acid=4:1); the mixture is oil bathed, heated until the temperature reaches 35 C., maintained at the temperature for 1 h; after the materials react completely, TLC detection is carried out; ammonia water is added to adjust the pH value of the system to 8-9 until solid precipitates are generated; the precipitates are filtered and dried to obtain a compound 4 (5.3 g, 90%);

(25) (4) The compound 4 (2 g, 7 mmol, 2 eq) and 4-quinazolinone-2-ethyl formate (0.8 g, 3.5 mmol, 1 eq) are dissolved in 6 mL of ethanol; the mixed materials react for 45 min at a temperature of 100 C. in a microwave reactor; after the materials react completely, TLC detection is carried out; the solution is naturally cooled to obtain white solid precipitates, and the precipitates are filtered and dried to obtain a compound 5. The compound 5 is the compound for hypertension treatment in the present invention.

Embodiment 2: Find the Structural Domains of the Interacting Sites of the Protein TRPV4 and the Protein KCa2.3

(26) Experimental method: Possible binding sites (as shown in FIG. 1) are selected according to the three-dimensional structures and functional characteristics of the protein TRPV4 and the protein KCa2.3. The selected structural domains are mainly used to adjust the protein-protein relation, and are platforms where the proteins interact with each other. The selected binding sites are mutated such that the binding sites are lost. The used primers can be seen in Table 1.

(27) TABLE-US-00002 TABLE1 Genename Primersequence TRPV4AR1 5-gccaccccccatcctcaaaacggggaaga-3 5-tcttccccgttttgaggatggggggtggc-3 TRPV4AR2 5-cgggagccgtcccgaggccagaca-3 5-tgtctggcctcgggacggctcccg-3 TRPV4AR3 5-cagagacatctactactttggggagctgccct-3 5-agggcagctccccaaagtagtagatgtctctg-3 TRPV4AR4 5-gggaggctacttctacagggggaacacggtg-3 5-caccgtgttccccctgtagaagtagcctccc-3 TRPV4AR5 5-ggcgacaggactcggatggcctttcgcc-3 5-ggcgaaaggccatccgagtcctgtcgcc-3 TRPV4AR6 5-acctggagacagttctcaacaatgatgaggacaccc-3 5-gggtgtcctcatcattgttgagaactgtctccaggt-3 TRPV4CaMBD 5-ctaccagtactatggcttcgagctgaacaagaactcaa-3 5-ttgagttcttgttcagctcgaagccatagtactggtag-3 KCa.sub.2.317c 5-tggtgagctgagtgtcaaccacagctaccacaa-3 5-catgtgcacaacttcatgatgctaaagaagattgaccatgcc-3 KCa.sub.2.31 5-ggcatggtcaatcttctttagcatcatgaagttgtgcacatg-3 5-gctccgtgattaagtcatacatgtcaatcttctttagcagcttt-3 Kca.sub.2.3loop 5-gtctataaacatacaaagctgcatgccaaagtcaggaaacac-3 5-gtgtttcctgactttggcatgcagctttgtatgtttatagac-3 KCa.sub.2.32 5-aaagctgctaaagaagattgacatgtatgacttaatcacggagc-3 5-gctccgtgattaagtcatacatgtcaatcttctttagcagcttt-3 KCa.sub.2.3CaMBD 5-ctccgtgattaagtcatacatcatcatgaagttgtgcacatg-3 5-catgtgcacaacttcatgatgatgtatgacttaatcacggag-3

(28) The used PCR reaction system is as follows: 0.5 l template (whole-genome CFP-TRPV4 or whole-genome YFP-KCa2.3), 25 l Prim Star HS, upstream and downstream primers, each 0.5 l, and H.sub.2O added to 50 l. PCR reaction process: 2 min predegeneration at a temperature of 95 C., 30 s degeneration at a temperature of 95 C., 30 s annealing at a temperature of 55 C., 5 min extension at a temperature of 68 C., 30 circulations, 10 min full extension at a temperature of 68 C. After the PCR ends, the obtained product is purified with a PCR product purification kit; the purified product is digested with the enzyme DpnI. The digestion system includes 0.4 l enzyme DpnI, 5 l PCR purified product, 2 l 10 buffer, and 12.6 l ddH2O. The digestion is carried out for 1 h at a temperature of 37 C., and deactivation is carried out for 20 min at a temperature of 80 C. The digestion product is converted into competent DH5a cells. Screened monoclone cells undergo expanding culture and then plasmids are extracted using a plasmid extraction kit. Samples are sent to test the gene sequence.

(29) The mutated plasmids are transferred into the HEK293 cells. After transfection, the cells are laid onto a confocal vessel. After 24 h, the coupling of the two proteins before and after the mutation is detected using a laser scanning confocal microscope in FRET AB mode.

(30) Plasmids TRPV4-AR2H and plasmids KCa2.317C are constructed by the above mentioned gene mutation method, and then transferred into the HEK293 cells. After 24 h, the cells are immobilized with the PBS solution which contains 3% paraformaldehyde and 0.1% glutaraldehyde, and then washed with 0.1% sodium borohydride which is diluted with PBS. Cells undergo confining and osmosis treatment in a confining liquid (3% BSA and 0.2% Triton X-100 added into the PBS), incubated with the primary antibody at a temperature of 4 DEGC for a whole night, washed three times, and then incubated with a secondary antibody at room temperature for 45 min. Goat anti-TRPV4 and rabbit anti-KCa2.3 are used as the primary antibody, while Alexa Fluor 647 and Alexa Fluor 488 are used as the secondary antibody. At the same time, the two proteins are co-located by using stochastic optical reconstruction microscopy (STORM) to further verify the accuracy of the FRET results. Samples are prepared using the above method. Approximately 4 L imaging buffer is added into the center of a clean confocal vessel. The buffer contains 5% (w/v) glucose, 100 mM aminothiopropionic acid, 0.8 mg/mL notatin and 40 g/mL catalase, and is dissolved in Tris-HCL with a pH value of 7.5 or 8. The STORM imaging buffer represents spectral-resolved unimolecule image.

(31) Experimental results: FIG. 2a shows, compared with other mutation sites, the FRET phenomenon is obviously reduced in the structural domain AR2 where the TRPV4 is mutated or in the structural domain 17C where KCa2.3 is mutated. FIG. 2b shows further verification of the ultra-high-definition imaging experiment. According to the verification result, the structural domain AR2 of the TRPV4 and the structural domain 17C of the KCa2.3 are binding sites where two proteins are bound.

(32) In FIG. 2a, the colors, blue-green-yellow-red, represent that the FRET efficiency increases in turn.

(33) In FIG. 2b, red represents the protein TRPV4, green the protein KCa2.3, and yellow is the superimposing of both green and red.

Embodiment 3: The Compound Prepared in Embodiment 1 Takes the Structural Domain AR2 of the TRPV4 and the Structural Domain 17C of KCa2.3 as Target Points

(34) Experimental Method:

(35) The endothelial cells of C57BL/6 J mouse mesentery are primarily isolated, and then incubated in a constant-temperature cell incubator. Biotinylated JNc-440 (10 M/L) is added or oligopeptide and biotinylated JNc-440 (10 M/L) are added into the incubator to jointly incubated with the cells for 96 h. RIPA Lysis is carried out to obtain cellular protein. The protein supernatant is added with 10 L streptavidin magnetic beads, and the mixture is incubated at a temperature of 4 C. for a whole night. Suspension generated after the incubation is absorbed using a magnetic stand to obtain an avidin magnetic bead-biotinylated JNc-440-protein complex. The complex is suspended with 50 L of 1 Loading buffer, and boiled for 5 min in a boiling bath. Samples are separated through SDS-PAGE. Proteins are transferred onto a PVDF membrane. The PVDF membrane with the proteins are confined in 5% BSA for 4 h at room temperature, then incubated with a primary antibody at a temperature of 4 C. for a whole night, and next incubated with a secondary antibody for 2 h at room temperature. ECL color developing agent is added. A protein imaging system is used to detect and analyze the obtained product.

(36) Experimental Results:

(37) Experimental results can be seen in FIG. 3. The biotinylated JNc-440 can be bound with the proteins TrpV4 and KCa2.3; after the oligopeptide AR2 is added, the biotinylated JNc-440 is obviously reduced in the capability of binding with the protein TRPV4. On the other hand, compared with the reference control, after the oligopeptide 17C is added, the biotinylated JNc-440 is obviously reduced in the capability of binding with the protein KCa2.3. Results show that the biotinylated JNc-44 can be bound with TRPV4 and KCa2.3, and the binding zones are AR2 and 17C.

Embodiment 4: The Compound Prepared in Embodiment 1 can Enhance the Space Coupling Degree of the Endothelial Cell Ion Channel Complex TRPV4-KCa2.3

(38) Experimental Method:

(39) C57BL/6J Male mice, 8 weeks aged, are selected, and freely fed for one week. Then, the mice are classified into four groups, respectively 8% high-salt group, L-NAME group, ANG-2 group and reference group. The basic blood pressures of the four groups of mice are measured first to determine the base line of the blood pressure of the mice. The conditions of the reference group are not changed, and the blood pressure is measured every day. The high-salt group is fed with 8% high-salt feed, and then the blood pressure is measured every day. The L-name group is fed with 5% L-NAME solution, and then the blood pressure is measured every day. The ANG-2 group is injected with 4% chloral hydrate to perform anesthesia; then, the mice are respectively operated by implanting a pump on the back, wherein each pump is injected with 200 ul of 2.88 mg/ml ANG-2 solution; then, the mice are sutured and allowed to recover; after approximately 3 days, the measurement of the blood pressure begins, and then continues for about 4 weeks. The blood pressure of the high-salt group is stabilized at 120 mmHg; the blood pressures of the L-NAME and the ANG-2 group are stabilized at approximately 130 mmHg. When the blood pressure is maintained for approximately one week without rising, this represents that the modeling has succeeded.

(40) The mesentery endothelial cells of the mice in the high blood pressure model are primarily isolated and incubated in a constant-temperature cell incubator. JNc-440 (10 M) is added to be jointly incubated with cells for 96 h. The cells are dyed using the immunostaining method as described in Embodiment 2. Then, the coupling of the two proteins before and after the mutation is detected using the laser scanning confocal microscope in the FRET AB mode.

(41) Experimental results: as shown in FIG. 4, the space coupling degree of the proteins TRPV4 and KCa2.3 is increased obviously after the compound acts on the endothelial cells of the three different high blood pressure types of mice models. This represents that the compound can enhance the space coupling degree of the ion channel complex TRPV4-KCa2.3.

Embodiment 5: Effects of the Compound Prepared in Embodiment 1 on Three High Blood Pressure Types of Mice Models

(42) Experimental method: three high blood pressure types of mice models are constructed according to the method in Embodiment 4. After modeling succeeds, JNc-440 (1 mg/kg) is injected via the caudal veins, and then the blood pressure of the mice is measured and observed.

(43) Experimental results: As shown in FIG. 5, the blood pressure of the mice drops obviously after the compound acts on the three high blood pressure types of mice models, which represents that the compound has an obvious anti-hypertension effect.