CN104592152A - Amidine structure containing nitrobenzene thiazolecarboxylic acid amide dual-target inhibitor, as well as preparation method and application thereof - Google Patents

Abstract

The invention relates to the field of type 2-diabetes mellitus related medicines, and particularly relates to an amidine structure containing nitrobenzene thiazolecarboxylic acid amide non-glucoside SGLT2/SGLT1 dual-target inhibitor, as well as a preparation method and application thereof in preparation of medicines for treating type 2-diabetes mellitus.

Description

A kind of nitrophenyl thiazole carboxylic acid amides class containing amidine structure two target spot inhibitor, Preparation Method And The Use

Technical field

The present invention relates to the pharmaceutical field of the treatment of diabetes B.Specifically, the present invention relates to the two target spot inhibitor of the medicative a kind of thiazole carboxylic acid amides's class non-saccharide glycoside SGLT2/SGLT1 containing amidine structure of diabetes B, its preparation method and in purposes pharmaceutically.

Background technology

The metabolic disease of diabetes to be a kind of with hyperglycemia be feature.Hyperglycemia be then due to defect of insulin secretion or its biological action impaired, or both have concurrently and cause.Long-standing hyperglycemia during diabetes, causes various tissue, particularly eye, kidney, heart, blood vessel, neural chronic lesion, dysfunction.There is no the method for radical cure diabetes at present, but suitably can be controlled diabetes progression by multiple treatment means.Mainly comprise several aspect: the education of diabetic subject, self-monitoring of blood glucose, dietetic treatment, exercise therapy and pharmacological agent.Oral hypoglycaemic medicine by a variety of, as sulfonylurea, biguanides, thiazolidinediones, glycosidase inhibitor class, etc., but these medicines generally have various different side effect, as hepatotoxicity, hypoglycemia, abdominal distension, heart disease risk, etc.Therefore the medicine of brand-new action target spot is active demand clinically.

Sodium-glucose co-transporters body (sodium-glucose linked transporter, SGLT) be a kind of glucose transporter, there are two kinds of hypotypes and SGLT1 and SGLT2, both all have distribution in renal proximal tubules, 10% and 90% are respectively to the contribution of glucose reabsorption in kidney, in addition SGLT1 is also distributed in enteron aisle, is responsible for the absorption of glucose in enteron aisle together with GLUT.Suppress SGLT2 that the glucose in uriniferous tubules can be made heavily not absorb smoothly enter blood and discharge with urine, thus reduce blood sugar concentration, the SGLT2 inhibitor of listing at present has multiple, as dapagliflozin, canagliflozin and empagliflozin etc.

Inhibitor of these listings are all optionally SGLT2 inhibitor, very weak to SGLT1 restraining effect.At the initial stage of SGLT2 inhibitor research and development, the selectivity of SGLT2/SGLT1 was once considered to very important index, because suppress SGLT1 may cause intestines and stomach side reaction in theory.But research in recent years shows, this theoretic worry there is no need, and confirm by the clinical trial of LX4211 (Zambrowicz B, et al.Effects of LX4211, a dual sodium-dependent glucosecotransporters 1and 2inhibitor, on postprandial glucose, insulin, glucagon-likepeptide 1, and peptide tyrosine in a dose-timing study in healthy subjects, Clin Ther., 2013,35 (8), 1162-1173.e8).Because SGLT2/SGLT1 inhibitor can suppress SGLT1 further on the basis suppressing SGLT2, and this suppression can increase the discharge of glucose in urine in kidney and reduce the absorption of glucose in enteron aisle, therefore this kind of inhibitor is considered to a kind of selection of control blood sugar completely newly.

The invention discloses the two target spot inhibitor of a kind of nitrophenyl thiazole carboxylic acid amides class non-saccharide glycoside SGLT2/SGLT1 containing amidine structure, this compound can be used for the medicine preparing treatment diabetes B.

Summary of the invention

An object of the present invention is to provide and a kind of there is the two target spot inhibit activities of good SGLT2/SGLT1, there is the non-glucoside compound of one of formula I.

Another object of the present invention is to provide the method that preparation has the compound of formula I.

Another object of the present invention is to provide the application of compound in preparation treatment diabetes B medicine containing formula I.

Now in conjunction with object of the present invention, content of the present invention is specifically described.

The compound that the present invention has formula I has following structural formula:

Formula I of the present invention is synthesized by following route:

Compound II per uses HCl gas processing in presence of methyl alcohol, obtains compound III; Compound III is reacted with amine IV in the presence of a base, obtains the compound V containing amidine structure; Compound V and compound VI are reacted, and obtain compound VI I; Compound VI I DCC exist under with compound VI II condensation, obtain Compound I.

Formula I of the present invention has the double inhibition effect of SGLT2/SGLT1, can be used as the medicine of effective constituent for the preparation of diabetes B.The activity of formula I of the present invention is verified by receptor binding assays.

Formula I of the present invention is effective in quite wide dosage range.The dosage that such as every day takes, within the scope of 1mg-1000mg/ people, is divided into once or administration for several times.The actual dosage taking formula I can be decided according to relevant situation by doctor.

Embodiment

Below in conjunction with embodiment, the present invention is further illustrated.It should be noted that, following embodiment be only for illustration of, and not for limiting the present invention.The various changes that those skilled in the art's training centre according to the present invention is made all should within the protection domain required by the application's claim.

The synthesis of embodiment 1 Compound I

A. the synthesis of compound III

7.55g (100mmol) Compound II per is dissolved in 100mL anhydrous methanol, slowly passes into dry HCl gas under ice-water bath cooling, and substantially saturated to solution, then overnight at room temperature stirs.Collected by suction solid, ambient temperature in vacuum is dry, obtains white solid, is compound III.

B. the synthesis of compound V

12.96g (90mmol) compound III and 8.91g (90mmol) IV are dissolved in the DMF of 100mL drying, stirred at ambient temperature, add 12.14g (120mmol) triethylamine, then be warming up to 80 DEG C of reactions, until TLC checks that reaction completes (usual 3 hours).After having reacted, be poured into after reaction mixture is slightly cold in 300mL frozen water, stir, use the CH of 100mL × 3 ₂cl ₂extraction, merges extraction phase, anhydrous sodium sulfate drying.Suction filtration removing siccative, filtrate is evaporate to dryness on a rotary evaporator, and the resistates obtained uses column chromatography purification, obtains compound V, white solid, ESI-MS, m/z=175 ([M+H] ⁺).

C. the synthesis of compound VI I

10.44g (60mmol) compound V and 6.13g (60mmol) compound VI is dissolved in the THF of 50mL drying, stirred at ambient temperature, until TLC detection reaction completes (usual one week).After having reacted, reaction mixture is poured in 200mL frozen water, stirs, and uses the CH of 50mL × 3 ₂cl ₂extraction, merges extraction phase, anhydrous sodium sulfate drying.Suction filtration removing siccative, filtrate is evaporate to dryness on a rotary evaporator, and the resistates obtained uses column chromatography purification, obtains compound VI I, white solid, ESI-MS, m/z=241 ([M+H] ⁺).

D. the synthesis of Compound I

7.20g (30mmol) compound VI I and 7.50g (30mmol) VIII is dissolved in the THF of 100mL drying, stirred at ambient temperature, then add 6.81g (33mmol) N, N'-dicyclohexyl carbodiimide (DCC) and 1.00g DMAP (DMAP), then room temperature for overnight.After having reacted, add 100mL methyl tertiary butyl ether in reaction mixture, then stir 1 hour, suction filtration, filtrate pours in 500mL frozen water, stirs, and uses the CH of 100mL × 3 ₂cl ₂extraction, merges extraction phase, anhydrous sodium sulfate drying.Suction filtration removing siccative, filtrate is evaporate to dryness on a rotary evaporator, and the resistates obtained uses column chromatography purification, obtains Compound I, white solid, ESI-MS, m/z=473 ([M+H] ⁺).

Embodiment 2-3

With reference to embodiment 1 method, synthesize compound listed in Table.

Embodiment 4 Compound ira vitro is to the suppression of people SGLT2 and people SGLT1

Prepared by people SGLT2 expression vector

To full length cDNA clone (purchased from GenScript company) (having put Hind III and Not I site at the cDNA two ends) subclone of people SGLT2 be expressed between the Hind III and Not I site of pEAK15 expression vector (Theracos company of the U.S.).The method of the clone's digestion with restriction enzyme containing target gene is determined.

Prepared by people SGLT2 stably transfected cell line

Utilize restriction enzyme Nsi I to digest the plasmid containing people SGLT2, make it linearizing, with agarose gel electrophoresis, purifying is carried out to linear DNA.With transfection reagent Lipofectamine2000 (Invitrogen company), the DNA after purifying is proceeded to HEK293 cell (Theracos company of the U.S.).By the cell after transfection containing 10% foetal calf serum DMEM substratum in 37 DEG C, 5%CO ₂cultivate under condition after 24 hours, in identical growth medium, add purine mycin (Invitrogen company) continue cultivation 2 weeks, the cell after screening with puromycin-resistant is inoculated in (one, every hole cell) in 96 new orifice plates, cultivate in the substratum containing purinase element, until cell grows to converging state.There is the methyl-α-D-[U-of cell clone by reflection SGLT2 activity of puromycin-resistant ¹⁴c] pyranoside picked-up test screening (experimental technique has detailed description hereinafter) further.Select the cell clone with highest signal to noise ratio for follow-up methyl-α-D-[U- ¹⁴c] pyranoside picked-up test.

Prepared by people SGLT1 express cell

PDream2.1 expression vector containing total length people SGLT1cDNA is purchased from GenScript company.Plasmid increases in bacillus coli DH 5 alpha, and this strain culturing is in Luria-Bertani (LB) substratum containing penbritin.With QIAGEN Plasmid Midi test kit (QIAGEN company) extracting plasmid.With Lipofectamine 2000 transfection reagent, according to the method for operational manual, people SGLT1 expression vector plasmid is proceeded to COS-7 cell (purchased from American Type Tissue Collection).Transfectional cell in containing the DMEM of 10%DMSO in-80 DEG C of preservations.

Methyl-α-D-[U- ¹⁴c] pyranoside picked-up test

Before test, the DMEM of cell containing 10%FBS expressing SGLT1 and SGLT2 is respectively inoculated in 96 holes ScintiPlate liquid sudden strain of a muscle plate (PerkinElmer company), and (every hole adds 100 μ L nutrient solutions, containing 1 × 105 cell), and in 37 DEG C, cultivate 48 hours under 5%CO2 condition.Cell 150 μ L contain sodium damping fluid (137mM NaCl, 5.4mM KCl, 2.8mM CaCl ₂, 1.2mM MgCl ₂10mM Tutofusin tris/N-2-hydroxyethyl piperazine-N '-ethane sulfonic acid [Tris/Hepes], pH7.2) or without sodium damping fluid (137mM N-methyl-glucamine, 5.4mM KCl, 2.8mM CaCl2,1.2mM MgCl2,10mMTris/Hepes, pH7.2) wash twice.Testing compound is dissolved in containing 25% human plasma and 40 μ Ci/mL methyl-α-D-[U- ¹⁴c] pyranoside (Amersham Biosciences/GE Healthcare) containing sodium or without in sodium damping fluid, be prepared into the testing compound solution of a series of suitable concn.The 96 every holes of orifice plate add 50 μ L testing compound solutions, shaking culture 2 hours (SGLT1 analysis) or 1.5 hours (SGLT2 analysis).Cell 150 μ L cleaning buffer solution (137mM N-METHYL-ALPHA-L-GLUCOSAMINEs, 10mM Tris/Hepes, pH7.2) wash twice, with TopCount liquid scintillation counter (Perkin Elmer company), quantitative analysis is carried out to the picked-up of methyl-α-D-[U-14C] pyranoside.Sodium dependency pyranoside intake is the difference (mean value of three wells) deducting the intake obtained without the process of sodium damping fluid by the intake obtained containing the process of sodium damping fluid.

Shown in the following list of result.

The IC that part of compounds of the present invention suppresses people SGLT2 and people SGLT1 ₅₀value

Compound	IC 50(hSGLT2,nM)	IC 50(hSGLT1,nM)
			Dapagliflozin	1.3	1219
Compound I-1	7.7	9.1
			Compound I-2	9.0	11.3
Compound I-3	9.6	10.8

Above-mentioned IC ₅₀measurement result show, compare single target spot inhibitor, compound of the present invention is strong SGLT2 target spot inhibitor and SGLT1 target spot inhibitor simultaneously, can be used for prepare treatment diabetes B medicine.

Claims (3)

1. there is the compound of formula I structure,

2. synthesize the method for the compound of formula I described in claim 1:

Compound II per uses HCl gas processing in presence of methyl alcohol, obtains compound III; Compound III is reacted with amine IV in the presence of a base, obtains the compound V containing amidine structure; Compound V and compound VI are reacted, and obtain compound VI I; Compound VI I DCC exist under with compound VI II condensation, obtain Compound I.

3. the application of compound described in claim 1 in preparation treatment diabetes B medicine.