CN105884645B - A kind of rhein compound and application thereof - Google Patents

A kind of rhein compound and application thereof Download PDF

Info

Publication number
CN105884645B
CN105884645B CN201610297815.XA CN201610297815A CN105884645B CN 105884645 B CN105884645 B CN 105884645B CN 201610297815 A CN201610297815 A CN 201610297815A CN 105884645 B CN105884645 B CN 105884645B
Authority
CN
China
Prior art keywords
rhein
dmso
bone
nmr
osteoclast
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN201610297815.XA
Other languages
Chinese (zh)
Other versions
CN105884645A (en
Inventor
江敏
徐醒
漆学宇
杨春皓
邓廉夫
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SHANGHAI INSTITUTE OF TRAUMATOLOGY AND ORTHOPEDICS
Shanghai Institute of Materia Medica of CAS
Original Assignee
SHANGHAI INSTITUTE OF TRAUMATOLOGY AND ORTHOPEDICS
Shanghai Institute of Materia Medica of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SHANGHAI INSTITUTE OF TRAUMATOLOGY AND ORTHOPEDICS, Shanghai Institute of Materia Medica of CAS filed Critical SHANGHAI INSTITUTE OF TRAUMATOLOGY AND ORTHOPEDICS
Priority to CN201610297815.XA priority Critical patent/CN105884645B/en
Publication of CN105884645A publication Critical patent/CN105884645A/en
Application granted granted Critical
Publication of CN105884645B publication Critical patent/CN105884645B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/70—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/84—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/10—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with radicals containing only carbon and hydrogen atoms attached to ring carbon atoms
    • C07D211/16—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with radicals containing only carbon and hydrogen atoms attached to ring carbon atoms with acylated ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D211/20—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms
    • C07D211/22—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members 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
    • C07D211/56—Nitrogen atoms
    • C07D211/58—Nitrogen atoms attached in position 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen 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
    • C07D213/72—Nitrogen atoms
    • C07D213/75—Amino or imino radicals, acylated by carboxylic or carbonic acids, or by sulfur or nitrogen analogues thereof, e.g. carbamates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/125—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/13—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
    • C07D295/182—Radicals derived from carboxylic acids
    • C07D295/192—Radicals derived from carboxylic acids from aromatic carboxylic acids

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention relates to a kind of rhein compounds and application thereof.In particular it relates to a kind of rhein compound with following general structural formula.The rhein compound of the present invention can be used for the drug for preparing prevention and/or the treatment extremely caused disease of osteoclast activity.

Description

Rhein compounds and application thereof
Technical Field
The invention relates to a rhein compound and application thereof.
Background
Rhein (Rhein): the chemical name is 1, 8-dihydroxy-3-carboxyl anthraquinone, which is derived from rhizomes and other parts of various plants such as rhubarb, tuber fleeceflower root and the like, and rhein and derivatives thereof have attracted much attention in recent years due to the antibacterial, antitumor and anti-inflammatory effects and the pharmacological effects on digestive systems, kidneys, cardiovascular systems, bones and the like (such as dawn red and the like, new progress in research on the pharmacological effects of rhein and derivatives thereof, modern medicines and clinics, 2010, 25(6), 417-.
Rhein has a core structure of polyhydroxy anthraquinone, and various natural products containing the core structure are known at present, such as emodin, chrysophanol, physcion, aloe-emodin and other series compounds. Compounds with this characteristic structure tend to have multiple biological activities, such as aloe-emodin inhibition of inflammatory responses by blocking mRNA expression of Inducible Nitric Oxide Synthase (iNOS) and cyclooxygenase-2 (COX-2) (Park MY et al, biosci.biotechnol.biochem.,2009,73(4), 828-; emodin can inhibit osteoclast generation and promote osteoblast formation at a certain concentration, and through the dual action, emodin shows good anti-osteoporosis performance in vivo experiments (Kim JY et al, J. bone. Miner. Res.,2014,29(7), 1541-1553).
Osteoclasts are derived from mononuclear precursor cells in bone marrow hematopoietic stem cells, and are differentiated under the regulation of cytokines such as macrophage colony stimulating factor and nuclear factor kappa B receptor activator ligand to form activated osteoclasts, so that the osteoclasts in bone tissues of patients with osteoporosis are formed far more than normal persons, and the bone resorption is over-active, which is one of important pathological mechanisms of osteoporosis. Although there are many osteoporosis therapeutic drugs for inhibiting osteoclast formation in clinic, most drugs still have limited effect on osteoporosis, and some drugs have large toxic and side effects and many adverse reactions. For example, bisphosphonates have low bioavailability, are easy to generate gastrointestinal reaction, and are easy to cause esophageal cancer, mandibular necrosis and the like after long-term use; estrogens and selective estrogen receptor modulators are mainly targeted at women in the early menopause stage, and have the risk of increasing deep vein thrombosis and the like; calcitonin analogues may cause allergic reactions. In view of the inhibiting effect of polyhydroxy anthraquinone compounds such as rhein, emodin and the like on osteoclast formation, the design of novel rhein derivatives has important significance and can lay a foundation for novel anti-osteoporosis medicines.
The application reserves the parent structure of the rhein polyhydroxy anthraquinone, introduces acetyl at the position of phenolic hydroxyl and introduces amide active group at the position of carboxylic acid to form a novel rhein compound with unique physiological activity.
Disclosure of Invention
The invention discloses a rhein compound.
The invention also aims to provide the application of the rhein compound.
According to the first object of the invention, the invention provides rhein compounds with the following structural general formula:
wherein,
R1selected from hydrogen or acetyl;
R2，R3are the same or different and are each independently hydrogen, C1-C6 straight or branched chain hydrocarbyl, C3-C8 saturated heterocyclyl C1-C4 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted 5-7 membered heteroaryl; preferably hydrogen, piperidyl C1-C2 alkyl, morpholinyl C1-C2 alkyl, pyrrolyl C1-C2 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl; or R2、R3Together with the nitrogen atom to which they are attached form a C3-C8 saturated heterocyclic group substituted with a hydroxyl-substituted or unsubstituted C1-C6 straight or branched chain hydrocarbon group, a C3-C8 saturated heterocyclic group substituted with a C5-C6 saturated heterocyclic group, a substituted or unsubstituted aryl-substituted C3-C8 saturated heterocyclic group, a C1-C6 alkyl-substituted amino-substituted C3-C8 saturated heterocyclic group; the definition of "substituted or unsubstituted" refers to substitution by one or more substituents selected from the group consisting of: hydroxy, halogen, trifluoromethyl, C1-C6 alkyl.
Preferably, R2And R3One of which is hydrogen and the other is hydrogenSelected from 2-chloro-4-trifluoromethylphenyl, 1-pyrrylethyl, 4-morpholinoethyl, phenyl, 4-methylphenyl, 4-fluorophenyl, 3-trifluoromethylpyridin-2-yl; or R2、R3Together with the nitrogen atom to which they are attached form a 4-methyl-1-piperazinyl group, a 4-ethyl-1-piperazinyl group, a 4-phenyl-1-piperazinyl group, a 4- (morpholin-1-yl) -1-piperidinyl group, a 4-hydroxymethyl-1-piperidinyl group, a 4- (2-isopropanolyl) -1-piperidinyl group.
In the present invention, the term "aryl" refers to an aromatic ring group containing no hetero atom, preferably an aryl group having 6 to 14 carbon atoms, more preferably an aryl group having 6 to 10 carbon atoms, such as phenyl, naphthyl, biphenyl; examples of such substituted aryl groups include, but are not limited to, 4-methylphenyl, 4-fluorophenyl, 2-chloro-4-trifluoromethylphenyl.
In the present invention, the term "C1-C6 alkyl" means a straight or branched chain alkyl group having 1 to 6 carbon atoms in the main chain, including, but not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, and the like; methyl, ethyl, propyl, isobutyl are preferred. The term "C1-C4 alkyl" has a similar meaning.
In the present invention, the term "C3-C8 saturated heterocycle" means a 3-8 membered saturated cycloalkane having at least one heteroatom selected from N, O and S, and includes, without limitation, pyrrolidinyl, tetrahydroimidazolyl, piperidinyl, morpholinyl, piperazinyl, etc.; pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl are preferred.
In the present invention, "C5-C6 saturated heterocycle" means a 5-6 membered saturated cycloalkane having at least one heteroatom selected from N, O and S.
In the present invention, the term "5-7 membered heteroaryl" refers to a 5-7 membered aromatic ring group having at least one heteroatom selected from N, O and S in the ring, including without limitation furyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, pyrazolyl and pyridyl; preferably a pyridyl group.
The rhein compound is further preferably the following specific compound:
the rhein compound is prepared by the following steps: stirring compound a in the presence of dichloromethane, oxalyl chloride and N, N-dimethylformamide at room temperature for 1 hour, spin-drying the solvent, dissolving with dichloromethane, and adding NHR2R3Reacting with triethylamine, and stirring for 4 hours at room temperature to obtain the rhein compound (namely the compound shown in the formula I), wherein the reaction formula is as follows:
R1、R2、R3the definition of (A) is as above.
The preparation can also be carried out by referring to patent publication No. CN 101508645.
When R is1When the radical is hydrogen, the compound a is rhein, and when R is1When the compound is acetyl, the compound a can be prepared by the reaction of rhein and acetic anhydride at 130 ℃ for 2h, the catalyst is zinc trifluoromethanesulfonate, and the reaction formula is as follows:
the invention also discloses application of the rhein compound in preparing a medicament for preventing and/or treating diseases caused by abnormal osteoclast activity.
The diseases caused by the abnormal activity of the osteoclast comprise osteoporosis, rheumatoid arthritis, periodontitis, tooth loss, Paget's bone diseases, rickets, giant cell tumor of bones, myeloma bone diseases, bone destruction caused by cancer bone metastasis and the like.
Drawings
FIG. 1 shows the effect of rhein compounds of the present invention on osteoclastogenesis (TRAP staining).
Fig. 2 shows the effect of rhein b6 on osteoclastic bone resorption function. P <0.01, significant differences were seen in pairwise comparisons.
FIG. 3 shows the effect of rhein b6 on the expression of mRNA related to the bone resorption function of osteoclasts. # p <0.001, with significant differences in pairwise comparisons.
Detailed Description
The invention is further described in the following examples, which are not intended to limit the scope of the invention.
Preparation examples
Example 1
N- (2-chloro-4- (trifluoromethyl) -phenyl) -4, 5-dihydro-9, 10-dioxo-9, 10-dihydroanthracene-2-carboxamide (b1)
Rhein (284mg,1mmol) was dissolved in dichloromethane (5mL), oxalyl chloride (254mg,2mmol) was added, one drop of N, N-dimethylformamide was added, stirring was performed at room temperature for 1 hour, the solvent was spin-dried, then dissolved in dichloromethane, 2-chloro-4-trifluoromethylaniline (200mg,1.1mmol) and triethylamine (1mL) were added, stirred at room temperature for 4 hours, the reaction was quenched with a saturated sodium chloride solution and extracted three times with dichloromethane, the organic phases were combined, washed with saturated brine, filtered, spin-dried, and separated by column chromatography to obtain 380mg of a product (yield 82%).
1H NMR(500MHz,DMSO-d6)δ11.98(s,1H),11.91(s,1H),10.77(s,1H),8.26(d,1H),8.03(s,1H),7.92(s,1H),7.90(d,J＝8.4Hz,1H),7.86(t,J＝7.9Hz,1H),7.82(d,J＝8.3Hz,1H),7.78(d,J＝7.4Hz,1H),7.44(d,J＝8.3Hz,1H).
13C NMR(126MHz,DMSO-d6)δ191.40,181.06,163.53,161.39,161.03,140.59,138.48,137.60,133.82,133.30,129.92,128.80,127.84(d,J＝32.6Hz),126.80,126.77,124.60,123.29(d,J＝272.5Hz),123.00,119.47,118.28,117.88,116.21.
Example 2
1, 8-dihydroxy-3- (4-methylpiperazine-1-carbonyl) anthracene-9, 10-dione hydrochloride (b2)
The synthesis was as in example 1, replacing "2-chloro-4-trifluoromethylaniline" with "N-methylpiperazine".
1H NMR(500MHz,DMSO-d6)δ12.01(s,1H),11.90(s,1H),11.30(s,1H),7.83(t,J＝7.9Hz,1H),7.73(d,J＝7.5Hz,1H),7.71(d,J＝1.3Hz,1H),7.46(d,J＝1.4Hz,1H),7.42(d,J＝8.3Hz,1H),3.80–3.46(m,4H),3.25–3.00(m,4H),2.76(s,3H).
13C NMR(126MHz,DMSO-d6)δ191.26,181.03,166.60,161.35,161.16,142.99,137.55,133.91,133.19,124.64,121.94,119.42,117.22,116.71,115.98,51.73,51.69,42.07.
Example 3
3- (4-ethylpiperazine-1-carbonyl) -1, 8-dihydroxyanthracene-9, 10-dione (b3)
The synthesis was as in example 1, replacing "2-chloro-4-trifluoromethylaniline" with "N-ethylpiperazine".
1H NMR(500MHz,DMSO-d6)δ12.01(s,1H),11.90(s,1H),11.17(s,1H),7.83(t,J＝7.9Hz,1H),7.77–7.71(m,2H),7.47(d,J＝1.4Hz,1H),7.44–7.38(m,1H),3.83–3.44(m,6H),3.18–3.04(m,4H),1.26(t,J＝7.3Hz,3H).
13C NMR(126MHz,DMSO-d6)δ191.30,181.03,166.53,161.35,161.15,142.98,137.55,133.91,133.20,124.64,121.95,119.42,117.26,116.72,116.00,50.72,49.62,43.68,8.74.
Example 4
1, 8-dihydroxy-3- (4-methylpiperidine-1-carbonyl) anthracene-9, 10-dione (b4)
The synthesis was as in example 1, replacing "2-chloro-4-trifluoromethylaniline" with "4-methylpiperidine".
1H NMR(500MHz,DMSO-d6)δ11.97(s,1H),11.91(s,1H),7.83(t,J＝7.8Hz,1H),7.73(d,J＝7.3Hz,1H),7.59(s,1H),7.41(d,J＝8.3Hz,1H),7.35(s,1H),4.44(d,J＝11.3Hz,1H),3.48(d,J＝12.2Hz,1H),3.09(s,1H),2.80(s,1H),1.82–1.48(m,3H),1.29–0.99(m,2H),0.94(d,J＝6.0Hz,3H).
13C NMR(126MHz,DMSO-d6)δ191.87,181.54,166.67,161.83,161.74,145.20,137.98,134.32,133.74,125.05,121.94,119.88,117.18,116.85,116.49,47.59,42.11,34.44,33.69,30.83,21.98.
Example 5
4, 5-dihydroxy-N- (2-morpholinylethyl) -9, 10-dioxo-9, 10-dihydroxyanthracene-2-carboxamide hydrochloride (b5)
The synthesis was as in example 1, replacing "2-chloro-4-trifluoromethylaniline" with "2-morpholinoethylamine".
1H NMR(600MHz,DMSO-d6)δ11.86(s,3H),8.85(t,J＝5.4Hz,1H),8.10(d,J＝1.4Hz,1H),7.81(t,J＝7.9Hz,1H),7.73(d,J＝7.7Hz,2H),7.39(d,J＝8.3Hz,1H),3.65–3.51(m,4H),3.41(q,J＝6.5Hz,2H),2.51(d,J＝6.7Hz,2H),2.44(s,4H).
13C NMR(126MHz,DMSO-d6)δ192.67,182.30,165.10,162.55,162.32,142.89,138.72,134.78,134.50,125.70,123.55,120.61,118.74,118.69,117.28,67.34,58.25,54.41,37.93.
Example 6
1, 8-dihydroxy-3- (4-morpholinylpiperidine-1-carbonyl) anthracene-9, 10-dione (b6)
The synthesis was as in example 1, replacing "2-chloro-4-trifluoromethylaniline" with "4-morpholinopiperidine".
1H NMR(400MHz,Deuterium Oxide)δ11.92(s,2H),7.82(dd,J＝8.3,7.6Hz,1H),7.71(dd,J＝7.5,1.1Hz,1H),7.59(d,J＝1.5Hz,1H),7.40(dd,J＝8.4,1.1Hz,1H),7.37(d,J＝1.5Hz,1H),4.44(d,J＝13.6Hz,1H),3.66–3.43(m,5H),3.09(t,J＝12.6Hz,1H),2.85(t,J＝12.0Hz,1H),2.45(d,J＝13.7Hz,5H),1.88(d,J＝12.8Hz,1H),1.73(d,J＝13.2Hz,1H),1.52–1.32(m,2H).
13C NMR(126MHz,DMSO-d6)δ192.57,182.24,167.32,162.53,162.43,145.70,138.68,135.02,134.43,125.75,122.72,120.58,117.97,117.58,117.19,67.65,62.01,50.53,47.23,41.78,29.31,28.58.
Example 7
4, 5-hydroxy-9, 10-dioxo-N- (2-pyrrolidine-ethyl) -9, 10-dihydroxyanthracene-2-carboxamide (b7)
The synthesis was as in example 1, replacing "2-chloro-4-trifluoromethylaniline" with "2-pyrrolidinylethylamine".
MS＝381.2
Example 8
4, 5-hydroxy-9, 10-dioxo-N-phenyl-9, 10-dihydroxyanthracene-2-carboxamide (b8)
The synthesis was as in example 1, replacing "2-chloro-4-trifluoromethylaniline" with "aniline".
1H NMR(500MHz,DMSO-d6)δ11.92(s,2H),10.61(s,1H),8.22(s,1H),7.90(s,1H),7.84(t,J＝7.9Hz,1H),7.80(d,J＝7.9Hz,2H),7.76(d,J＝7.3Hz,1H),7.42(d,J＝8.4Hz,1H),7.38(t,J＝7.7Hz,2H),7.15(d,J＝7.3Hz,1H).
13C NMR(126MHz,DMSO-d6)δ192.65,182.28,164.38,162.58,162.26,143.23,139.77,138.77,134.80,134.51,129.86,125.75,125.41,124.05,121.77,120.66,119.16,118.98,117.35.
Example 9
4, 5-dihydroxy-9, 10-dioxo-N-p-tolyl-9, 10-dihydroxyanthracene-2-carboxamide (b9)
The synthesis was as in example 1, replacing "2-chloro-4-trifluoromethylaniline" with "p-toluidine".
1H NMR(600MHz,DMSO-d6)δ11.95(s,1H),11.89(s,1H),10.52(s,1H),8.20(s,1H),7.89(s,1H),7.83(t,J＝7.7Hz,1H),7.75(d,J＝7.1Hz,1H),7.67(d,J＝7.7Hz,2H),7.41(d,J＝8.2Hz,1H),7.17(d,J＝7.7Hz,2H),2.29(s,3H).
13C NMR(151MHz,DMSO-d6)δ191.92,181.52,163.34,161.87,161.56,142.54,138.06,136.55,134.02,133.76,133.71,129.53,125.02,123.29,121.02,119.95,118.47,118.14,116.57,21.01.
Example 10
4, 5-dihydroxy-9, 10-dioxo-N- (4-trifluoromethylphenyl) -9, 10-dihydroxyanthracene-2-carboxamide (b10)
The synthesis was as in example 1, replacing "2-chloro-4-trifluoromethylaniline" with "p-trifluoromethylaniline".
1H NMR(600MHz,DMSO-d6)δ11.95(s,2H),10.95(s,1H),8.24(s,1H),8.05(d,J＝8.4Hz,2H),7.94(s,1H),7.86(t,J＝7.9Hz,1H),7.78(t,J＝9.0Hz,3H),7.44(d,J＝8.4Hz,1H).
13C NMR(151MHz,DMSO-d6)δ191.92,181.60,164.25,161.88,161.53,142.74,141.95,138.10,134.20,133.82,126.49,126.47,125.10,124.80(d,J＝271.4Hz),124.63(d,J＝32.0Hz),123.54,120.93,119.97,118.57,118.45,116.70.
Example 11
4, 5-dihydroxy-9, 10-dioxo-N- (5- (trifluoromethyl) pyridin-2-yl) -9, 10-dihydroxyanthracene-2-carboxamide (b11)
The synthesis was as in example 1, replacing "2-chloro-4-trifluoromethylaniline" with "2-amino-5-trifluoromethylpyridine".
1H NMR(500MHz,DMSO-d6)δ11.95(s,1H),11.90(s,1H),11.73(s,1H),8.87–8.76(m,1H),8.40(d,J＝8.8Hz,1H),8.28(dd,J＝8.8,2.2Hz,1H),8.23(d,J＝1.7Hz,1H),7.96(d,J＝1.7Hz,1H),7.90–7.81(m,1H),7.77(d,J＝8.4Hz,1H),7.43(d,J＝8.3Hz,1H).
13C NMR(126MHz,DMSO-d6)δ192.63,182.20,165.93,162.58,162.13,156.13,146.59(d,J＝3.8Hz),142.04,138.80,137.17(d,J＝3.3Hz),134.69,134.52,125.76,124.98(d,J＝271.7Hz),124.72,122.26(d,J＝32.5Hz),120.65,119.64,119.34,117.43,115.55.
Example 12
4, 5-dihydroxy-9, 10-dioxo-N- (4- (trifluoromethyl) pyridin-2-yl) -9, 10-dihydroxyanthracene-2-carboxamide (b12)
The synthesis was as in example 1, replacing "2-chloro-4-trifluoromethylaniline" with "2-amino-4-trifluoromethylpyridine".
1H NMR(300MHz,DMSO-d6)δ11.92(s,1H),11.86(s,1H),11.74(s,1H),8.72–8.64(m,1H),8.49(s,1H),8.25–8.17(m,1H),7.97–7.89(m,1H),7.87–7.79(m,1H),7.78–7.70(m,1H),7.61–7.53(m,1H),7.46–7.33(m,1H).
Example 13
1, 8-dihydroxy-3- (4- (hydroxymethyl) piperidine-1-carbonyl) anthracene-9, 10-dione (b13)
The synthesis was as in example 1, replacing "2-chloro-4-trifluoromethylaniline" with "4-hydroxymethylpiperidine".
1H NMR(500MHz,DMSO-d6)δ11.95(s,1H),11.89(s,1H),7.81(t,J＝7.9Hz,1H),7.71(d,J＝7.2Hz,1H),7.61–7.53(m,1H),7.39(d,J＝8.3Hz,1H),7.36–7.30(m,1H),4.54–4.43(m,2H),3.51(d,J＝12.8Hz,1H),3.32–3.21(m,2H),3.08(t,J＝12.3Hz,1H),2.79(t,J＝11.8Hz,1H),1.76(d,J＝12.7Hz,1H),1.70–1.59(m,2H),1.23–1.02(m,2H).
13C NMR(126MHz,DMSO-d6)δ192.56,182.22,167.38,162.53,162.45,145.90,138.67,135.01,134.41,125.75,122.65,120.57,117.88,117.54,117.17,66.56,48.13,42.61,39.53,30.17,29.31.
Example 14
1, 8-dihydroxy-3- (4-piperidinylpiperidine-1-carbonyl) anthracene-9, 10-dione (b14)
Synthesis procedure as in example 1, 2-chloro-4-trifluoromethylaniline was replaced with "4-piperidylpiperidine".
MS＝435.2
Example 15
1, 8-dihydroxy-3- (4- (2-hydroxypropyl-2-yl) piperidine-1-carbonyl) anthracene-9, 10-dione (b15)
Synthesis procedure as in example 1, the "2-chloro-4-trifluoromethylaniline" was replaced with "4- (2-hydroxypropan-2-yl) piperidine".
1H NMR(500MHz,DMSO-d6)δ11.96(s,1H),11.90(s,1H),7.86–7.79(m,1H),7.72(dd,J＝7.5,1.0Hz,1H),7.57(d,J＝1.5Hz,1H),7.40(dd,J＝8.4,1.0Hz,1H),7.33(d,J＝1.5Hz,1H),4.55(d,J＝12.2Hz,1H),4.16(s,1H),3.54(d,J＝12.1Hz,1H),3.02(t,J＝12.3Hz,1H),2.68(t,J＝12.2Hz,1H),1.80(d,J＝12.3Hz,1H),1.65(d,J＝12.0Hz,1H),1.43(dt,J＝12.1,3.0Hz,1H),1.18(ddd,J＝30.0,12.2,3.0Hz,2H),1.03(s,6H).
13C NMR(126MHz,DMSO-d6)δ192.57,182.27,167.29,162.53,162.43,145.86,138.68,135.03,134.45,125.76,122.68,120.58,117.92,117.58,117.22,71.25,48.66,48.00,43.17,28.18,28.04,27.38.
Example 16
1, 8-dihydroxy-3- (4- (N, N-dimethyl) piperidine-1-carbonyl) anthracene-9, 10-dione (b16)
Synthesis procedure as in example 1, the "2-chloro-4-trifluoromethylaniline" was replaced with "4- (N, N-dimethyl) piperidine".
1H NMR(400MHz,Methanol-d4)δ11.66(s,2H),7.84(t,J＝7.9Hz,1H),7.74(d,J＝7.4Hz,1H),7.65(s,1H),7.42(t,J＝4.0Hz,2H),4.60(d,J＝12.5Hz,1H),3.62(d,J＝13.0Hz,1H),3.45–3.27(m,1H),3.14(t,J＝13.2Hz,1H),2.82(t,J＝12.7Hz,1H),2.67(s,6H),2.11(d,J＝11.8Hz,1H),1.94(d,J＝11.4Hz,1H),1.75–1.58(m,2H).
Example 17
3- ((2-chloro-4- (trifluoromethyl) phenyl) carbamoyl) -9, 10-dioxo-9, 10-dihydroanthracene-1, 8-diacetate (c1)
Dissolving rhein (284mg,1mmol) in acetic anhydride (612mg,6mmol), adding zinc trifluoromethanesulfonate (2mg,0.005mmol), reacting at 130 ℃ for 2h, cooling to room temperature after the reaction is finished, filtering, and recrystallizing the crude product in acetic acid to obtain 4, 5-acetoxy-9, 10-dioxo-9, 10-dihydroanthracene-2-carboxylic acid. 4, 5-acetoxy-9, 10-dioxo-9, 10-dihydroanthracene-2-carboxylic acid (368mg,1mmol) was dissolved in dichloromethane (5mL), oxalyl chloride (254mg,2mmol) was added, a drop of N, N-dimethylformamide was added, stirring was performed at room temperature for 1 hour, the solvent was spun dry, then dissolved in dichloromethane, 2-chloro-4-trifluoromethylaniline (200mg,1.1mmol) and triethylamine (1mL) were added, stirring was performed at room temperature for 4 hours, the reaction was quenched with a saturated sodium chloride solution and extracted three times with dichloromethane, the organic phases were combined, washed with a saturated brine, filtered, spun dry, and isolated by column chromatography to give 420mg of the product (77% yield).
1H NMR(600MHz,DMSO-d6)δ10.91(s,1H),8.72(d,J＝1.8Hz,1H),8.19(dd,J＝7.7,1.1Hz,1H),8.16(d,J＝1.8Hz,1H),8.06–8.03(m,1H),7.98(t,J＝7.9Hz,1H),7.90(d,J＝8.3Hz,1H),7.84–7.81(m,1H),7.68(dd,J＝8.0,1.1Hz,1H),2.44(s,3H),2.42(s,3H).
13C NMR(151MHz,DMSO-d6)δ181.41,180.77,169.56,169.51,163.56,150.15,150.06,139.38,138.98,136.02,135.01,134.67,131.17,130.52,129.68,129.42,128.43(d,J＝32.9Hz),127.94,127.33(d,J＝4.1Hz),125.76,125.51,125.16(d,J＝3.9Hz),124.36,123.80(d,J＝272.4Hz),21.32,21.30
Example 18
3- (4-methylpiperidine) -1-carbonyl) -910-dioxo-9, 10-dihydroanthracene-1, 8-diacetate (c2)
Synthesis procedure as in example 17, substituting "2-chloro-4-trifluoromethylaniline" for "4-methylpiperidine".
1H NMR(500MHz,DMSO-d6)δ8.13(d,J＝7.7Hz,1H),8.01(d,J＝1.5Hz,1H),7.94(t,J＝7.9Hz,1H),7.65(d,J＝1.5Hz,1H),7.64(d,J＝8.0Hz,1H),4.44(d,J＝12.6Hz,1H),3.46(d,J＝12.1Hz,1H),3.08(t,J＝11.7Hz,1H),2.80(t,J＝12.2Hz,1H),2.39(s,6H),1.72(d,J＝12.4Hz,1H),1.67–1.59(m,1H),1.55(d,J＝11.8Hz,1H),1.21–1.03(m,2H),0.92(d,J＝6.5Hz,3H).
13C NMR(126MHz,DMSO-d6)δ180.93,180.18,168.99,168.93,165.73,149.62,149.48,142.55,135.38,134.48,134.08,130.55,128.08,125.46,125.20,124.90,122.42,47.20,41.76,33.87,33.19,30.31,21.47,20.81,20.79.
Example 19
9, 10-dioxo-3- ((4-trifluoromethyl) pyridin-2-yl) carbamoyl) -9, 10-dihydroanthracene-1, 8-diacetate (c3)
Synthesis procedure as in example 17, substituting "2-chloro-4-trifluoromethylaniline" for "2-amino-4-trifluoromethylpyridine".
1H NMR(500MHz,DMSO-d6)δ11.83(s,1H),8.71(d,J＝5.1Hz,1H),8.69(d,J＝1.8Hz,1H),8.52(s,1H),8.20(d,J＝1.8Hz,1H),8.17(dd,J＝7.8,1.1Hz,1H),7.96(t,J＝7.9Hz,1H),7.65(dd,J＝8.0,1.1Hz,1H),7.59(d,J＝5.0Hz,1H),2.42(s,3H),2.40(s,3H).
13C NMR(126MHz,DMSO-d6)δ180.82,180.24,169.01,168.91,164.18,152.84,150.04,149.53,149.44,139.13,138.34(q,J＝33.3Hz),135.49,134.17,134.12,130.62,129.46,127.34,125.26,124.97,124.46,122.86(q,J＝273.1Hz),115.61(d,J＝3.0Hz),110.02(d,J＝3.8Hz),20.80,20.75.
Example 20
9, 10-dioxo-3- (4- (trifluoromethyl) phenyl) carbamoyl) -9, 10-dihydroanthracene-1, 8-diacetate (c4)
Synthesis procedure as in example 17, substituting "2-chloro-4-trifluoromethylaniline" for "4-trifluoromethylaniline".
1H NMR(300MHz,DMSO-d6)δ10.99(s,1H),8.66(s,1H),8.15(d,J＝8.1Hz,2H),8.01(d,J＝8.3Hz,2H),7.94(t,J＝7.8Hz,1H),7.74(d,J＝8.6Hz,2H),7.63(d,J＝7.9Hz,1H),2.42(s,3H),2.39(s,3H).
13C NMR(126MHz,DMSO-d6)δ181.42,180.73,169.52,169.45,163.63,150.07,150.05,142.67,140.21,135.98,134.87,134.63,131.15,129.63,127.70,126.48,126.46,125.72,125.48,124.78(q,J＝271.4Hz),124.69(d,J＝31.8Hz),124.37,120.96,21.29,21.27.
Example 21
9, 10-dioxo-3-p-toluylcarbamoyl-9, 10-dihydroanthracene-1, 8-diacetate (c5)
The synthesis was carried out as in example 17, replacing "2-chloro-4-trifluoromethylaniline" with "4-methylaniline".
1H NMR(500MHz,DMSO-d6)δ10.65(s,1H),8.66(d,J＝1.8Hz,1H),8.17(dd,J＝7.8,1.2Hz,1H),8.13(d,J＝1.8Hz,1H),7.96(t,J＝7.9Hz,1H),7.68(d,J＝8.4Hz,2H),7.66(dd,J＝8.1,1.2Hz,1H),7.19(d,J＝8.3Hz,2H),2.43(s,3H),2.40(s,3H),2.29(s,3H).
13C NMR(126MHz,DMSO-d6)δ182.39,181.68,170.43,170.37,163.81,150.95,150.94,141.73,137.39,136.88,135.72,135.57,134.74,132.02,130.49,130.35,128.33,126.64,126.38,125.11,122.00,22.21,22.19,21.91.
Example 22
9, 10-dioxo-3- (4- (trifluoromethyl) phenyl) carbamoyl) -9, 10-dihydroanthracene-1, 8-diacetate (c6)
Synthesis procedure as in example 17, substituting "2-chloro-4-trifluoromethylaniline" for "4-trifluoromethylaniline".
1H NMR(300MHz,DMSO-d6)δ10.99(s,1H),8.66(s,1H),8.15(d,J＝8.1Hz,2H),8.01(d,J＝8.3Hz,2H),7.94(t,J＝7.8Hz,1H),7.74(d,J＝8.6Hz,2H),7.63(d,J＝7.9Hz,1H),2.42(s,3H),2.39(s,3H).
13C NMR(126MHz,DMSO-d6)δ181.42,180.73,169.52,169.45,163.63,150.07,150.05,142.67,140.21,135.98,134.87,134.63,131.15,129.63,127.70,126.48,126.46,125.72,125.48,124.78(q,J＝271.4Hz),124.69(d,J＝31.8Hz),124.37,120.96,21.29,21.27.
Example 23
9, 10-dioxo-3- (4-methyl-3-chlorobenzoyl) -9, 10-dihydroanthracene-1, 8-diacetate (c5)
The synthesis was carried out as in example 17, replacing "2-chloro-4-trifluoromethylaniline" with "4-methyl-3-chloroaniline".
Example 24
9, 10-dioxo-3- ((5-trifluoromethyl) pyridin-2-yl) carbamoyl) -9, 10-dihydroanthracene-1, 8 diacetate (c8)
Synthesis procedure as in example 17, the "2-chloro-4-trifluoromethylaniline" was replaced with "2-amino-5-trifluoromethylpyridine".
1H NMR(600MHz,DMSO-d6)δ11.83(s,1H),8.83(s,1H),8.68(d,J＝1.8Hz,1H),8.41(d,J＝8.7Hz,1H),8.29(dd,J＝8.9,2.5Hz,1H),8.22(d,J＝1.9Hz,1H),8.17(dd,J＝7.8,1.2Hz,1H),7.97(t,J＝7.9Hz,1H),7.66(dd,J＝8.1,1.2Hz,1H),2.43(s,3H),2.41(s,3H).
13C NMR(151MHz,DMSO-d6)δ181.32,180.74,169.54,169.43,164.64,155.43,150.04,149.95,145.93(d,J＝4.1Hz),139.61,136.49(d,J＝3.7Hz),136.01,134.64,134.61,131.14,130.06,127.86,125.75,125.48,125.04,124.28(d,J＝271.3Hz),121.58(d,J＝32.7Hz),114.83,21.31,21.26.
Test examples
Anti-osteoporosis effect
The invention provides an effect of rhein compounds in inhibiting osteoclast formation.
1. Main experimental materials and instruments
Materials:fetal bovine serum,. alpha. -MEM medium, 0.25% pancreatin and penicillin/streptomycin were purchased from Gibco, tartrate-resistant acid phosphatase (TRAP) enzyme activity detection kit and total protein assay kit were purchased from Picoma, 17 β -estradiol (17 β -estradiol, E2), DMSO, toluidine blue, MTT, TRAP staining kit was purchased from sigma, cytokines such as M-CSF and mRANKL were purchased from peprotech, cell lysate was purchased from promega, PBS was purchased from WISENT, dexamethasone, β -glycerophosphate and ascorbic acid were purchased from CAYMAN, RNAiso Reagent, reverse transcription and PCR kit were purchased from TaKaRa, and 4 week old C57BL/6 mice were purchased from Slick.
Cell lines:bone marrow osteoclast precursor cells are obtained from femur and tibia medullary cavities of C57BL/6 mice, mesenchymal stem cells mMSC are obtained from Setaria Biotech, Inc., osteoblast cell strains MC3T3-E1 are obtained from cell banks of Shanghai academy of sciences, and complete culture medium, namely α -MEM + 10% fetal bovine serum + 1% penicillin/streptomycin, is adopted for normal subculture of the cells.
The instrument comprises the following steps:CO, Thermo scientific Co2An incubator; LEICA SP 1600 saw microtome (germany); olympus inverted microscope; shanghai New Ganoderma biotechnological research institute ultrasonic cleaning machine, Tecan corporation enzyme labeling instrument.
2. The experimental method comprises the following steps:
1) preparation of test cell lines:
c57BL/6 mouse is killed by breaking neck, soaked in 75% alcohol for disinfection, the long bones (femur and tibia) of limbs are stripped under aseptic condition, the marrow cavity is washed repeatedly by using complete culture medium after the attached soft tissue is removed, the cells in the marrow cavity are completely fallen off, the cell suspension is filtered by using cell sieve and cultured in 10cm cell culture dish. The next day, the supernatant cells were centrifuged and the cells were resuspended in a new 10cm cell culture dish using complete medium supplemented with 30ng/mL M-CSF. Two days later adherent osteoclast precursor cells were collected with a cell scraper, resuspended and quantitatively seeded in cell culture plates at 5% CO2And cultured overnight under saturated humidity conditions, and the next day is replaced with fresh complete culture medium containing M-CSF and mRANKL and the specified drug or drug solvent DMSO for testing.
2) The MTT method is used for detecting the influence of the rhein compounds on the survival rate of bone marrow osteoclast precursor cells:
osteoclast precursor cells were seeded at a concentration of 5000 cells/well in 96-well plates and cultured overnight. The next day, the experimental group diluted the compound with complete medium containing M-CSF (30ng/mL) to a final drug concentration of 5mmol/L and added cells at 100. mu.L per well, the control group added an equal amount of medium and DMSO at the corresponding ratio, and the blank group added no cells but an equal amount of medium and DMSO at the corresponding ratio. After 48h incubation, 10. mu.L MTT (5g/L) was added to each well and placed in the incubator for further 2 h. After 2h, the cell culture medium was removed, 100. mu.L of DMSO was added to each well, and the mixture was shaken for 10 minutes to dissolve the purple crystals sufficiently. Using a TECAN microplate reader, the absorbance value was measured at 490nm, and the cell viability was calculated as the survival rate (PR%) (a)Experimental group－ABlank control group)/(AControl group－ABlank control group)×100％。
3) The MTT method is used for detecting the influence of the rhein compounds on the survival rate of osteoblasts:
MC3T3-E1 osteoblasts were seeded at 5000/well in 96-well plates and cultured overnight. The next day, the experimental groups will compoundThe cells were diluted with complete medium to a final drug concentration of 5. mu. mol/L and added to each well at 100. mu.L, and the control group was supplemented with the same amount of medium and the corresponding proportion of DMSO, while the blank group was supplemented with no cells, but with the same amount of medium and the corresponding proportion of DMSO. After 48h incubation, 10. mu.L MTT (5g/L) was added to each well and placed in the incubator for further 2 h. After 2h, the cell culture medium was removed, 100. mu.L of LDMSO was added to each well, and the mixture was shaken for 10 minutes to dissolve the purple crystals in the cells sufficiently. Using a TECAN microplate reader, the absorbance value was measured at 490nm, and the cell viability was calculated as the survival rate (PR%) (a)Experimental group－ABlank control group)/(AControl group－ABlank control group)×100％。
4) Testing of the effect of rhein on osteoclast TRAP activity:
bone marrow osteoclast precursor cells were seeded at a concentration of 5000 cells/well in 96-well plates and cultured overnight. The next day, the experimental group diluted the compound with complete medium containing M-CSF and mRANKL to a final drug concentration of 5mmol/L and added cells at 100. mu.L per well, and the control group added an equal amount of medium and DMSO in the corresponding ratio. After 2 days of cell culture, the culture solution was removed, after the cells were washed with PBS, the operation was carried out according to the TRAP enzyme activity detection kit instructions, and the reaction was terminated after 30min of reaction. The absorbance value is measured at the wavelength of 405nm, and the enzyme activity is converted.
5) Test of the effect of rhein on osteoclastogenesis (TRAP staining method):
bone marrow osteoclast precursor cells were seeded into 96-well plates at a concentration of 5000 cells/well, 100 μ L per well, and cultured overnight. The next day, the compounds were diluted with complete medium containing M-CSF and mRANKL and added to cells to a final concentration of 5. mu. mol/L, 100. mu.L per well, and control wells were supplemented with equal amounts of medium and DMSO in the corresponding proportions. After 2 days of cell culture, the fresh culture medium containing the drug was replaced once. After 4 days osteoclasts fused and formed bubbles, and the cells were washed with PBS, fixed with 4% paraformaldehyde for 10min and washed with PBS. Preparing TRAP staining solution according to a TRAP staining kit, adding the staining solution into the fixed cells, reacting at 37 ℃ for about 1h, observing by an inverted microscope, photographing, and counting osteoclast-like cells with the number of nuclei being more than or equal to 3.
6) Analysis of influence of rhein compounds on osteoclast differentiation process related genes (real-time fluorescent quantitative PCR (RT-PCR)):
bone marrow osteoclast precursor cells were seeded into 6-well plates and cultured overnight. The next day, the rhein drug was diluted with complete medium containing M-CSF and mRANKL and added to the cells to a final concentration of 5 μ M, and the same amount of medium and DMSO in the corresponding ratio were added to the control wells. Total RNA was extracted 3 days after drug culture, and expression levels of TRAP, NFATC1, c-fos, mmp-9, and CTSK were determined by RT-PCR. The primers were synthesized by TaKaRa. The extraction of total RNA is carried out by adopting a Trizol one-step method, an ultraviolet spectrophotometer is used for detecting the purity and the concentration, a reverse transcription system, a PCR amplification system and reaction conditions are set according to the instruction of a kit, and the relative expression quantity of a target gene is obtained through internal reference correction.
7) Effect of rhein on osteoclastic bone resorption function (bone resorption lacunae assay):
taking fresh cow femur, stripping adhered soft tissue, cutting cortex into bone pieces with thickness of 100 μm with saw type slicer, trimming to proper size, ultrasonically cleaning, and soaking in 75% alcohol. Soaking in 100% ethanol for 10min before use, air drying, irradiating with ultraviolet lamp, placing in 96-well plate, and adding complete culture medium for culturing for 30 min. After 30min the medium was removed and bone marrow osteoclast precursor cells were added. After the cells are attached to the wall, the culture solution is changed into a complete culture medium containing M-CSF and RAN KL and containing rhein compounds with specified concentration, and an equal amount of culture medium and DMSO in a corresponding proportion are added into a control hole. After culturing for 7 days, the culture solution is discarded (wherein the solution is changed every two days), 2.5% glutaraldehyde is used for fixing the bone fragments for 7min, ultrasonic cleaning is carried out in 0.25mol/L ammonium hydroxide for 5 min, after ultrasonic cleaning is carried out for 3 times, 1% toluidine blue dye solution is used for dyeing for 3 min, and the bone fragments are cleaned by distilled water, observed under a mirror and photographed. The area of bone resorption lacunae on the bone plate was analyzed with Image Pro Plus 6.0 software (USA Media Cybernetics Co.).
3. As a result:
(1) the rhein compounds of the invention are used for researching the toxic effect of the rhein compounds on bone marrow osteoclast precursor cells.
Rhein compounds at 5 μ M were selected for co-incubation with bone marrow osteoclast precursor cells and viable cells were detected by MTT, with the results shown in table 1. The test result shows that: the rhein compounds b4, b6, b13, b15 and c2 have no obvious toxicity to bone marrow osteoclast precursor cells.
TABLE 1 Effect of rhein compounds on the survival of bone marrow osteoclast precursor cells
(2) Research on the inhibitory effect of rhein compounds on the TRAP activity of osteoclast differentiation
According to the toxicity test result, the rhein compounds without obvious toxicity to bone marrow osteoclast precursor cells are selected for testing, and the influence of the rhein compounds on TRAP activity in the differentiation process is detected by inducing osteoclastic differentiation of the bone marrow osteoclast precursor cells through M-CSF and mRANKL. As shown in table 2, the results show that: the rhein compounds b6, b13 and b15 have very good inhibition effect on TRAP activity in the process of bone marrow osteoclast differentiation, wherein the inhibition rate of b6 on TRAP activity reaches 98.22%.
TABLE 2 inhibitory Effect of rhein compounds on TRAP Activity of bone marrow osteoclast precursor cell differentiation
(3) Effect of the Rhein Compounds of the present invention on osteoclastogenesis
Selecting rhein compounds b6 with optimal TRAP activity inhibition effect, intervening osteoclast differentiation of bone marrow osteoclast precursor cells, staining osteoclasts through TRAP after differentiation is finished, and counting the osteoclast-like cells by ≧ 3 cell nuclei, as shown in figure 1 and table 3, the test results show that: the rhein compound b6 of the invention can effectively inhibit the formation of osteoclast at the concentration of 5 mu M.
TABLE 3 Effect of rhein compounds on the amount of osteoclast formation
(4) The rhein compound of the invention is used for researching the toxic effect of osteoblast
To investigate whether rhein b6 had an effect on osteoblast survival, 5 μ M rhein was co-incubated with MC3T3-E1 osteoblasts and the survival cells were tested by MTT, the results are shown in table 4. The test result shows that: the rhein compound b6 has no obvious toxicity to osteoblast under the concentration of 5 mu M.
TABLE 4 Effect of rhein b6 on osteoblast survival
(5) The rhein compound of the invention is used for researching the toxic effect on mesenchymal stem cells.
To investigate whether rhein b6 affects the survival of mesenchymal stem cells, 5 μ M rhein was selected to co-incubate with mesenchymal stem cells MSC and the survival was tested by MTT, with the results shown in table 5. The test result shows that: the rhein compound b6 has no obvious toxicity to MSC at 5 mu M concentration.
TABLE 5 Effect of rhein compounds b6 on survival of mesenchymal stem cells
(6) The effect of the rhein compounds on the bone resorption function of osteoclast
Selecting rhein compounds b6 with optimal TRAP activity inhibition effect, and interfering bone absorption effect of bone marrow osteoclast precursor cells on bovine bone fragments during osteoclastic differentiation. After the differentiation was completed, the bone fragments were stained with toluidine blue, and the area of bone resorption was counted. As shown in fig. 2, the test results show that: the rhein compound b6 of the invention can effectively inhibit osteoclast bone resorption at a concentration of 5 mu M.
(7) The effect of the rhein compound on the expression of osteoclast specific mRNA is shown.
Selecting rhein compounds b6 with optimal TRAP activity inhibition effect, intervening bone marrow osteoclast precursor cell osteoclast differentiation, and studying the influence of rhein compounds b6 on osteoclast differentiation and key gene mRNA during osteoclast function. As shown in fig. 3, the test results show that: the rhein compound b6 of the invention can effectively inhibit the expression of osteoclast differentiation and bone resorption key genes such as NFATc1, c-fos, TRAP, MMP-9 and Cathepsin K under the concentration of 5 mu M.

Claims (4)

1. A rhein compound with the following structural general formula:
wherein,
R1selected from hydrogen;
R2、R3together with the nitrogen atom to which they are attached form a piperidinyl group substituted with a hydroxy-substituted C1-C6 straight or branched chain hydrocarbyl group or a morpholinyl-substituted piperidinyl group.
2. The rhein compound according to claim 1,
wherein,
R1selected from hydrogen;
R2、R3together with the nitrogen atom to which they are attached form a 4- (morpholin-1-yl) -1-piperidinyl group or a 4-hydroxymethyl-1-piperidinyl group or a 4- (2-isopropanolyl) -1-piperidinyl group.
3. Use of the rhein compound according to any one of claims 1 to 2 for the preparation of a medicament for the prevention and/or treatment of a disease caused by abnormal osteoclast activity.
4. Use according to claim 3, characterized in that: the diseases caused by the abnormal activity of the osteoclast comprise osteoporosis, rheumatoid arthritis, periodontitis, tooth loss, Paget's bone diseases, rickets, giant cell tumor of bone, myeloma bone diseases and bone destruction caused by cancer bone metastasis.
CN201610297815.XA 2016-05-06 2016-05-06 A kind of rhein compound and application thereof Expired - Fee Related CN105884645B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610297815.XA CN105884645B (en) 2016-05-06 2016-05-06 A kind of rhein compound and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610297815.XA CN105884645B (en) 2016-05-06 2016-05-06 A kind of rhein compound and application thereof

Publications (2)

Publication Number Publication Date
CN105884645A CN105884645A (en) 2016-08-24
CN105884645B true CN105884645B (en) 2018-06-26

Family

ID=56703294

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610297815.XA Expired - Fee Related CN105884645B (en) 2016-05-06 2016-05-06 A kind of rhein compound and application thereof

Country Status (1)

Country Link
CN (1) CN105884645B (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107298648B (en) * 2017-06-06 2019-01-25 上海市伤骨科研究所 A kind of Rhein thioamide analog compound, preparation method and the usage
CN112920141B (en) * 2019-12-05 2023-03-17 中检科医药科技(北京)集团有限公司 Rhein derivatives and antiviral application thereof
US10941120B1 (en) * 2020-06-23 2021-03-09 Shaanxi University Of Science And Technology 6-aminouracil cassic acid ester with antibacterial activity and a method of preparing the same

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU666704B2 (en) * 1992-02-28 1996-02-22 Lilly Industries Limited Anthraquinone compounds and pharmaceutical compositions containing them
CN102060809B (en) * 2009-05-01 2015-05-20 常州高新技术产业开发区三维工业技术研究所有限公司 Rhein derivatives and preparation and application thereof

Also Published As

Publication number Publication date
CN105884645A (en) 2016-08-24

Similar Documents

Publication Publication Date Title
CA2986930C (en) Chemical modulators of signaling pathways and therapeutic use
CN105884645B (en) A kind of rhein compound and application thereof
JP2012529432A (en) Aminopyrrolidinone derivatives and uses thereof
Thakur et al. Novel 3, 5-bis (arylidiene)-4-piperidone based monocarbonyl analogs of curcumin: anticancer activity evaluation and mode of action study
AU2020213346B2 (en) Phenylallyl cyclohexenone derivatives and their preparation method and application
CN113234116B (en) Tripterine derivative, preparation method and medical application thereof
CN108884124A (en) Caborane compounds and its application method
JP2016185947A (en) IRE-1α INHIBITORS
EA036984B1 (en) 4-substitued coumarin derivatives, preparation methods and uses thereof
CN108276373B (en) Flavonoid compound and application thereof in anti-cancer drugs
CN104761572B (en) Flueggea suffruticosa-type alkaloid dimer-type compound or pharmaceutically acceptable salt thereof, preparation method and application thereof
CN113149942A (en) Rockmilanol phenolic hydroxyl derivative, preparation method and application thereof
CN107298648B (en) A kind of Rhein thioamide analog compound, preparation method and the usage
CN111108083B (en) Use of aminomethylene cyclohexane 1, 3-dione compounds
CN113387867B (en) Carbamate anthranilic tryptamine derivative and preparation and application thereof
CN112430223B (en) Substituted benzoyl piperazine compounds and application thereof
Thakor et al. Synthesis and cell line study of pyrazole substituted coumarin derivatives
CN110872263B (en) Compound, preparation method and application
CN114409522A (en) Chalcone derivative and preparation method and application thereof
Radwan et al. Synthesis and Characterization of bis-3, 5-disubstituted thiadiazine-2-thione derivatives as anticancer agents
CN112174872A (en) Indole benzoquinone derivative as fluorescent and photoacoustic imaging agent and application thereof
CN107019687B (en) Application of N- (4-chlorphenyl) -3-hydroxy-2-naphthamide compound
Tripathi et al. Isolation, Cytotoxicity, and In‐silico Screening of Coumarins from Psoralea corylifolia Linn.
CN108186642B (en) A kind of pharmaceutical composition of co-action treatment lung cancer
CN117624015B (en) Substituted isatin-melatonin derivative and preparation method and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20180626

Termination date: 20200506

CF01 Termination of patent right due to non-payment of annual fee