CN106806366B - New application of isoindole-1, 3-diketone compound - Google Patents

Abstract

The invention provides an application of a novel skeleton compound used as a calcium-dependent protein kinase in preparing medicaments for preventing and treating parasitic infection diseases; the isoindole-1, 3-diketone compound disclosed by the invention has the effect of inhibiting the activity of calcium-dependent protein kinase, can be used for preparing a medicament for preventing or/and treating protozoan infectious diseases, and has a potential application prospect in acute and chronic parasitic diseases; through comparison tests, the isoindole-1, 3-dione compound provided by the invention has obviously better inhibitory activity on CDPK1 than pyrazolopyrimidine compound NM-PP 1.

Description

New application of isoindole-1, 3-diketone compound

Technical Field

The invention relates to a medical technology, in particular to a new application of isoindole-1, 3-diketone compounds in medicines.

Background

Toxoplasma gondii (Toxoplasma gondii) is an intracellularly propagating parasite, one of the most widespread host species. Toxoplasma gondii infections are currently one of the most common foodborne infections worldwide requiring hospitalization, with a total number of infections in excess of 20 million people worldwide. Toxoplasma infection poses a fatal threat to immunocompromised people, such as organ transplantation, cancer patients or AIDS patients. In addition, toxoplasmosis causes problems such as abnormal development of human embryos and abortion. At present, medicaments for treating toxoplasmosis are mainly broad-spectrum antibacterial compounds such as pyrimethamine, sulfadiazine and the like, but the medicaments are easy to generate medicament resistance and side effects, so that long-term curative effect is reduced or the patients are difficult to tolerate, and novel effective medicaments are urgently needed to be developed.

Type I Calcium-dependent protein kinase I (CDPK 1) is a potential drug target recently discovered by researchers for parasitic infections such as Toxoplasma (Toxoplasma), cryptosporidium (cryptosporidium), and malaria (plasmodium). The CDPK1 protein is orthologous in Toxoplasma gondii (T.gondii), Cryptosporidium parvum (C.parvum) and Plasmodium falciparum (P.falciparum), has important regulation and control functions on cell invasion and transfer of parasites, and can achieve the aim of effectively treating toxoplasmosis, Cryptosporidiosis and malaria by inhibiting CDPK 1.

The CDPK1 inhibitors discovered at present mainly have two types of skeleton structures, namely pyrazolopyrimidine (pyrazolopyrimidine) and 5-Aminopyrazole 4-carboxamide (5-Aminopyrazole-4-carboxamid), but in order to obtain inhibitor drugs with high selectivity, the CDPK1 inhibitors with novel compound skeletons still need to be developed clinically urgently.

At present, the isoindole-1, 3-diketone compound is not reported to be used as a medicament for resisting toxoplasmosis and cryptosporidiosis.

Disclosure of Invention

The invention aims to provide a new application of isoindole-1, 3-diketone compounds as I-type calcium dependent protein kinase inhibitors.

Another purpose of the invention is to provide application of isoindole-1, 3-dione in preparation of medicines for treating Protozoan (Protozoan) parasite infectious diseases.

Preferably, the protozoal parasitic infectious disease is toxoplasmosis.

The chemical structural formula of the isoindole-1, 3-diketone compound is as follows:

wherein:

R ₁，R ₆represents H, Br, phenyl, 4-chlorophenyl, preferably R ₁Represents a phenyl group.

R ₂，R ₅Represents H, a benzene bridge, a 1-formyl bridge, a 1-phenylformate-2-methylethenyl bridge, a methyl group, preferably R ₂，R ₅Represents a formyl bridge.

R ₃，R ₄Represents H, phenyl, 4-methoxyphenyl, benzo, N-2-methylbenz-1, 3-ketopyrrolo, preferably R ₃，R ₄Represents 4-methoxyphenyl.

R ₇Represents 1-substituted naphthalene, 2-substituted N- (4-bromophenyl) -4-methylthiobutanamide, 2-bromophenyl, benzyl, 4-benzoic acid methyl ester, 3-Cl-4-tolyl, 2-methyl-5-azocarboxylic acid phenyl, 2-substituted 3-carbonitrile-4, 5, 6, 7-tetrahydrothiophene, preferably R ₇Is naphthalene substituted at the 1-position.

The invention has the following beneficial effects: the invention provides an application of a novel skeleton compound used as a calcium-dependent protein kinase in preparing medicaments for preventing and treating parasitic infection diseases; the isoindole-1, 3-diketone compound disclosed by the invention has the effect of inhibiting the activity of calcium-dependent protein kinase, can be used for preparing a medicament for preventing or/and treating protozoan infectious diseases, and has a potential application prospect in acute and chronic parasitic diseases.

In addition to the objects, features and advantages described above, other objects, features and advantages of the present invention are also provided. The present invention will be described in further detail below.

Detailed Description

The following is a detailed description of embodiments of the invention, but the invention can be implemented in many different ways, as defined and covered by the claims.

Example 1: preparation of isoindole-1, 3-dione compounds I.

The synthetic route of 8, 9-di (4-methoxyphenyl) -4- (1-naphthyl) -1, 7-diphenyl-4-azatricyclo [ 5.2.1.0-2, 6- ] decaalkane-8-alkene-3, 5, 10-trione is as follows:

wherein, the step 1 reference [1], the step 2 reference [2] and the step 3 reference [3,4 ].

1.Cesari,C.,et al.,Microwave-Assisted Synthesis of FunctionalizedShvo-Type Complexes.Organometallics,2014.33(11):p.2814–2819.

2.e-EROS Encyclopedia of Reagents for Organic Synthesis,I.John Wileyand Sons,Editor.2001.

3.Lai,Y.-H.,et al.,Structural studies on phenanthro[9,10-e]pyrene andits 9,10-dihydro-derivative:dependence of chemical shift on degree ofplanarity of and buttressing effect in the polycyclic aromaticcompounds.Journal of the Chemical Society,Perkin Transactions 2,1992(8):p.1315-1319.

4.Carroll,W.R.,et al.,A Molecular Balance for Measuring Aliphatic CH-πInteractions.Organic Letters,2011.13(16):p.4320-4323.

Example 2: the inhibitory activity of isoindole-1, 3-dione compounds I, II and III prepared by the invention on CDPK1 was determined.

The compound II is N- (4-bromophenyl) -2- (16, 18-dioxo-17-aza-pentacyclo [ 6.6.5.0-2, 7-0-9, 14-0-15, 19-nonadecane-2, 4,6,9,11, 13-hexene-17 position substituted) -4- (methylthio) butyramide, and the structural formula is as follows:

the compound III is phenyl 1, 14-dimethyl-4, 10-di (2-tolyl) -3,5,9, 11-tetraoxo-4, 10-diazepicyclo [ 5.5.2.0-2, 6-0-8, 12-tetradecane-13-ene-13-carboxylate, and the structural formula is as follows:

both compound II and compound III were purchased from Specs.

1 laboratory apparatus

Spectrofluorometer (Shimadzu RF-5301PC, Shimadzu, Japan), electric heating thermostat water bath (kovia), low speed centrifuge (flying pigeon brand), 1mL, 200uL, 100uL, 10uL pipette gun (Biohit), 1.5mL EP tube (Solebao), 200uL PCR tube (Solebao), pH regulator (Sartorius)

2 Experimental reagent

DMSO (Haikouln), Kinase-Glo fluorescence Kinase kit (Promega), ATPNa2 (Solay), TgCDPK1 protein (the Shanghai Sheng chemical industry company expresses and purifies), substrate polypeptide (the Genscript company synthesizes), HEPES (Solay), NaOH (Xinhua), MgCl2.6H2O (Cramar), BSA (Solay), EGTA (Solay), CaCl2 (Shanghai Ficus)

3 preparation of reagents

3.1 kinase buffer

Medicine and food additive	Dosage of
		20mM HEPES	2.3831g
0.1％BSA	0.5g
		10mM MgCl 2·6H 2O	1.0165g
1mM EGTA	0.19017g
		2mM CaCl 2	0.111g
dd H2O	500mL

2.3831g HEPES was first dissolved in 300mL dd H ₂In O, the pH is adjusted to 7.5 with 1M NaOH, and then other drugs are added and dissolved, and the volume is adjusted to 500mL after complete dissolution.

3.2 preparation of Kinase-Glo reagent

The buffer and substrate in the Kinase-Glo fluorogenic Kinase kit were centrifuged at 2000rpm for 2min at room temperature, all the buffer was transferred to the substrate, recombined with the substrate to a lyophilized luciferase/substrate mixture, and stored at-20 ℃.

3.310 mmol/mLATP Na2

121mg of ATP Na2 was weighed out and dissolved in 20mL of 25mmol/mL Tris-HCl (pH8.0) to obtain a mother solution with a concentration of 10mmol/mL, and the solution was dispensed at-20 ℃.

3.46 mol/mL substrate polypeptide formulation

9.2mg of the substrate polypeptide was dissolved in 1mL of ddH2O to give a 6mol/mL stock solution, which was stored at-20 ℃.

3.520 μmol/mL Tg-calcium dependent kinase formulation

1.172mg of Tg-calcium dependent kinase was dissolved in 1mL of kinase buffer to give a mother liquor having a concentration of 20 μm/mL and was dispensed at-20 ℃.

4 materials of experiment

A, dissolving the 18 compounds in DMSO with different amounts, and preparing the final concentration to be 6 mu mol/mL;

b, adding 10 mul of mother liquor substrate polypeptide diluted by water and having the concentration of 9.3mg/mL into 290 mul of kinase buffer solution to obtain 200 mul mol/mL of front solution;

c, adding 5 mu l of ATP Na2 mother solution with the concentration of 10mmol/mL into 995 mu l of kinase buffer solution to obtain 50 mu mol/mL front solution;

d, taking 5 mu l of Tg-calcium dependent kinase with the concentration of 1.172mg/mL, and adding the Tg-calcium dependent kinase into 2mL of kinase buffer solution to obtain a 50nmol/mL front solution;

e adding the prepared medicine precursor liquid into a 1.5mL EP tube according to the volume

Name (R)	Dosage of	Final concentration
			Tg-calcium dependent kinases	10μl	20nm/mL
Polypeptide substrates	5μl	40μmol/mL
			Medicine	5μl	400nmol/mL
ATP Na 2	5μl	10μmol/mL

A, centrifuging and uniformly mixing the added system, and incubating for 40min at 30 ℃;

adding 25 mu l of Kinase-Glo solution into the mixture, uniformly mixing, and reacting at room temperature for 10 min;

and C, adding 650 mu l of kinase buffer solution, mixing uniformly, and measuring the fluorescence optical density value (excitation wavelength of 488nm, emission wavelength of 525 nm).

5 results of the experiment

Table 1: inhibitory activity of compounds against CDPK 1.

Name of Compound	IC50(μM)
		Compound I	0.048
Compound II	0.145
		Compound III	0.177
Pyrazolopyrimidine compound NA-PP1	0.989

The pyrazolopyrimidine compound NA-PP1 in Table 1 is referred to as reference [5], which is a comparative example; as can be seen from the test results in Table 1, the isoindole-1, 3-dione compounds I, II and III of examples 1 and 2 of the present invention are superior in inhibitory activity against CDPK1 to those of the comparative examples.

5.Ojo,K.K.,et al.,Toxoplasma gondii calcium-dependent protein kinase1 is a target for selective kinase inhibitors.Nat Struct Mol Biol,2010.17(5):p.602-607.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (1)

1. An application of isoindole-1, 3-diketone compounds in preparing medicaments for treating toxoplasma diseases, wherein the isoindole-1, 3-diketone compounds are selected from the following three compounds:

the compound I is 8, 9-di (4-methoxyphenyl) -4- (1-naphthyl) -1, 7-diphenyl-4-azatricyclo [ 5.2.1.0-2, 6- ] decaalk-8-ene-3, 5, 10-trione, and the structural formula is as follows:

the compound II is N- (4-bromophenyl) -2- (16, 18-dioxo-17-aza-pentacyclo [ 6.6.5.0-2, 7-0-9, 14-0-15, 19-nonadecane-2, 4,6,9,11, 13-hexene-17 position substituted) -4- (methylthio) butyramide, and the structural formula is as follows:

the compound III is phenyl 1, 14-dimethyl-4, 10-di (2-tolyl) -3,5,9, 11-tetraoxo-4, 10-diazepicyclo [ 5.5.2.0-2, 6-0-8, 12-tetradecane-13-ene-13-carboxylate, and the structural formula is as follows: