CN107827940B

CN107827940B - Uncaria amide A and pharmaceutical composition and application thereof

Info

Publication number: CN107827940B
Application number: CN201711159741.4A
Authority: CN
Inventors: 耿长安; 陈纪军; 张建钢; 黄晓燕; 马云保; 李天泽; 杨通华; 张雪梅
Original assignee: Kunming Institute of Botany of CAS
Current assignee: Kunming Institute of Botany of CAS
Priority date: 2017-11-20
Filing date: 2017-11-20
Publication date: 2020-10-09
Anticipated expiration: 2037-11-20
Also published as: CN107827940A

Abstract

The invention provides a novel indole alkaloid trisaccharide shown in a structural formula (I), uncaria amide A (1), a pharmaceutical composition consisting of a therapeutically effective amount of compound 1 and a pharmaceutically acceptable carrier or excipient, a preparation method of the compound 1 and the pharmaceutical composition thereof, a melatonin receptor agonist and application of the compound as the melatonin receptor agonist in preparing medicaments for treating or improving central nervous system diseases related to melatonin receptors.

Description

Uncaria amide A and pharmaceutical composition and application thereof

The technical field is as follows:

the invention belongs to the technical field of medicines. In particular to a novel indole alkaloid trisaccharide glycoside, uncaria amide A (1), a pharmaceutical composition taking the compound 1 as an active ingredient, application of the pharmaceutical composition as a melatonin receptor agonist, and application of the pharmaceutical composition in preparing a medicament for treating or improving central nervous system diseases related to melatonin receptors.

Background art:

melatonin (MT) is an endogenous hormone secreted by the pineal body of mammals, has obvious circadian and seasonal rhythmicity, and has important regulation effects on circadian rhythms, reproduction, aging and the like of organisms. Melatonin acts by activating specific melatonin receptors in vivo, has physiological activities of improving sleep, resisting aging, enhancing immunity, and the like, and also has regulating effects on depression, anxiety and pain. Human beingThe melatonin receptor includes MT₁And MT₂The two subtypes belong to G protein coupled receptors, have 7 transmembrane segments in total, have high affinity to melatonin, and are main action sites of the melatonin in a human body. MT (multiple terminal)₁And MT₂Receptors are widely distributed in the central nervous system and peripheral tissues of a human body, and are mainly distributed in the upper nucleus of the visual cross, pituitary, olfactory bulb, midbrain, hypothalamus and the like in the central nervous system; in peripheral tissues, they are mainly distributed in the cardiovascular system, spleen, thymus, lymph nodes, gonads, gastrointestinal tract, kidneys, etc. After the melatonin receptor is activated, the melatonin receptor mediates intracellular Ca through a series of signal transduction cascades²⁺Messenger levels such as NO, cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cGMP), etc., activate the MEK/ERK signaling pathway, and further exert biological effects such as sleep arousal, immunoregulation, cardiovascular system regulation, etc. Current research indicates that MT is activated₁The receptor can inhibit the neuron discharge of nucleus at the visual cross and the release of prolactin, and is related to the sleep promoting and vasoconstriction activity of melatonin; activating MT₂Receptors can cause proliferation of splenocytes and relaxation of coronary arteries, which are associated with the circadian rhythm of melatonin and vasodilatory function. The melatonin receptor stimulant is a research hotspot of the current novel antidepressant and sedative hypnotic, and can avoid side effects such as drug dependence caused by acting on GABA receptors, opioid receptors and the like. Successful cloning of MT since the eighties of the twentieth century₁And MT₂Since the receptor, several MT receptor agonists have been synthesized, some of which have been marketed as antidepressants and sleep-improving agents, such as agomelatine, remeltaon, tasimelteon, and the like.

With the intensive research on natural products, more and more natural small molecules are attracting the interest of medicinal chemists. Especially, traditional Chinese medicines are important sources for finding natural active molecules. Uncaria rhynchophylla (Uncaria rhynchophylla) belonging to Uncaria genus of Rubiaceae family is an important plant as a basic source of traditional Chinese medicine Uncaria rhynchophylla. The traditional Chinese medicine composition is slightly cold in nature and sweet in taste, enters liver and pericardium channels, has the effects of calming wind, arresting convulsion, clearing heat, calming liver and the like, is clinically used for treating liver wind stirring, convulsive epilepsy, convulsion, febrile convulsion, common cold with convulsion, infantile cry, gestational eclampsia, headache, dizziness and the like, and is a common traditional Chinese medicine for clinically lowering blood pressure and treating nervous system diseases. Modern pharmacological research shows that the uncaria has the activities of reducing blood pressure, calming, resisting convulsion, calcium antagonism, removing free radicals, protecting nerves and the like. The uncaria plant mainly contains indole alkaloid, flavone, triterpene, organic acid and other components, and particularly the indole alkaloid is a research hotspot of the traditional Chinese medicine uncaria. The antihypertensive activity of the traditional Chinese medicine uncaria is still the focus of the current research, and the research shows that the rhynchophylline and the isocorynine are main active ingredients of the uncaria playing the role of reducing blood pressure.

To date, the prior art has no report of uncaria amide A (1), no report of a pharmaceutical composition with uncaria amide A (1) as an active ingredient, no report of the application of uncaria amide A (1) and the pharmaceutical composition thereof as melatonin receptor agonists and the application of treating or improving the central nervous system diseases related to melatonin receptors.

The invention content is as follows:

the invention aims to provide a new indole alkaloid trisaccharide shown in formula (I), namely uncaria amide A (1), which has medicinal value, and the uncaria amide A (1) with effective dose is used as a melatonin receptor agonist, and the uncaria amide A (1) and a pharmaceutical composition thereof are applied to treating or improving the central nervous system diseases related to melatonin receptors.

In order to achieve the above purpose of the present invention, the present invention provides the following technical solutions:

a compound uncaria amide A (1) shown in a structural formula (I),

the application of the compound 1 of the formula (I) in preparing melatonin receptor stimulant.

The application of the compound 1 of the formula (I) in preparing medicines for treating or improving central nervous system diseases.

The use as described, wherein the disease is a central nervous system disease associated with melatonin receptors.

The invention also provides a pharmaceutical composition containing a therapeutically effective amount of compound 1 of formula (I) and a pharmaceutically acceptable carrier.

The application of the pharmaceutical composition in preparing melatonin receptor stimulant.

The application of the pharmaceutical composition in preparing medicines for treating or preventing central nervous system diseases.

The invention also provides a method for preparing the compound 1 shown in the formula (I), which comprises the steps of taking dry stems with hooks of uncaria, crushing, carrying out reflux extraction twice by using 70% ethanol for 3 hours each time, merging ethanol extract, and recycling ethanol under reduced pressure to obtain extract. Dissolving the extract with 5 times of water, performing D101 macroporous resin column chromatography, and performing ethanol-water gradient elution. Mixing the 50% ethanol eluate, concentrating, dissolving with methanol, adsorbing on silica gel, standing at room temperature to volatilize solvent, grinding, sieving, performing silica gel column chromatography, and eluting with chloroform-methanol-water (7: 3: 0.3) to obtain 4 fractions A-D. And continuously performing medium-pressure preparation on the fraction C through an MCI CHP-20P gel column, and performing gradient elution with methanol-water (1: 9-9: 1) to obtain 5 fractions, namely C-1-C-5. Purifying fraction C-2 by HPLC using Rp-C₁₈And (3) carrying out acetonitrile-water gradient elution (1: 9-3: 7) on the column to obtain a target compound 1.

The method for preparing the pharmaceutical composition containing the compound 1 is to take the compound 1 as a raw material and add a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carrier or excipient is one or more of solid, semi-solid and liquid diluents, fillers and pharmaceutical adjuvants.

When the compound 1 of the present invention is used as a melatonin receptor agonist or drug, it can be used as it is or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99%, preferably 0.5-90% of compound 1, and the balance pharmaceutically acceptable carriers and/or excipients which are nontoxic and inert to human and animals. The pharmaceutical composition of the present invention is used in the form of a dose per unit body weight. The medicine of the present invention may be administrated through injection and oral taking.

Description of the drawings:

FIG. 1 is a structural formula of Compound 1 of the present invention;

fig. 2 shows the activation rate of melatonin receptor of compound 1 of the present invention, which is determined as the maximum activation rate of Melatonin (MT) set as 100%, and the test concentration of the compound is 1.0mM, and the activation rate is Mean ± SD (n ═ 3).

The specific implementation mode is as follows:

in order to better understand the essence of the present invention, the preparation method, structural identification and pharmacological action of the compound uncaria amide A (1) are further described below by using the test examples and examples of the present invention in combination with the drawings, and the preparation method and pharmaceutical composition of the present invention are not limited by the test examples and examples.

Example 1:

preparation of compound 1:

pulverizing dried stem and branch of ramulus Uncariae cum uncis, extracting with 70% ethanol under reflux twice (each for 3 hr), mixing ethanol extractive solutions, and recovering ethanol under reduced pressure to obtain extract. Dissolving the extract with 5 times of water, performing D101 macroporous resin column chromatography, and performing ethanol-water gradient elution. Mixing the 50% ethanol eluate, concentrating, dissolving with methanol, adsorbing on silica gel, standing at room temperature to volatilize solvent, grinding, sieving, performing silica gel column chromatography, and eluting with chloroform-methanol-water (7: 3: 0.3) to obtain 4 fractions A-D. And continuously performing medium-pressure preparation on the fraction C through an MCI CHP-20P gel column, and performing gradient elution with methanol-water (1: 9-9: 1) to obtain 5 fractions, namely C-1-C-5. Purifying fraction C-2 by HPLC using Rp-C₁₈And (3) carrying out acetonitrile-water gradient elution (1: 9-3: 7) on the column to obtain a target compound 1.

Structural characterization of compound 1:

the optical rotation was determined by a Jasco model 1020 polarimeter (Horiba, Tokyo, Japan); infrared Spectrum (IR) was measured by a Bio-Rad FTS-135 type Infrared spectrometer (Hercules, California, USA) using KBr pellet method; the ultraviolet spectrum was measured by a UV-2401PC type ultraviolet spectrometer (Shimadzu, Kyoto, Japan); ECD spectra were determined by an Applied Photophysics circular dichroism spectrometer (Agilent, Santa Clara, United States), and nuclear magnetic resonance spectra (1D and 2D NMR) were determined by a model AVANCE III-600 superconducting nuclear magnetic resonance spectrometer (Bruker, Bremerhaven, Germany) using deuterated water as solvent; high Resolution Mass Spectrometry (HRMS) was performed using LCMS-IT-TOF type mass spectrometer (Shimadzu, Kyoto, Japan); thin-layer chromatography silica gel and column chromatography silica gel (200-300 mesh) were purchased from Qingdao Meigao and Qingdao ocean chemical group, Inc. CHP20P MCI gel was purchased from Mitsubishi Chemical Corporation (Tokyo, Japan).

Compound 1

The molecular formula is as follows: c₃₈H₅₀N₂O₁₉

Molecular weight: 838.30

The characteristics are as follows: white powder

HRESIMS(+)m/z:839.3081([M+H]⁺,0mDa)。

HRESIMS(–)m/z:883.2983([M+HCOO]^-,–0.7mDa)

IR(KBr)v_max:3420,2924,1613,1519,1446,1384,1284,1073,592cm^-1。

UV/Vis (methanol) lambda_max(log):206(4.76),279(4.06)nm。

¹H-NMR and¹³the C-NMR data are shown in Table 1.

[α]_D ²³-45.5(c 0.13, methanol).

TABLE 1 preparation of Compound 1¹H-NMR (600MHz) and¹³C-NMR (150MHz) data (D)₂O)

Example 2:

compound 1 for melatonin receptor MT₁And MT₂Agonistic activity of the receptor.

1 materials and methods

1.1 materials:

melatonin receptor MT₁And MT₂The cell strains used for the screening of the agonistic activity respectively correspond to human kidney epithelial cells HEK293-MT₁And HEK293-MT₂(ii) a Cell culture medium (Dulbecco's Modified EagleMedium, DMEM) containing 10% fetal bovine serum; a wash-free calcium flux kit.

1.2 Instrument: CO 2₂A constant temperature incubator Thermo Forma 3310 (usa); inverted biomicroscope model XD-101 (nanjing); flexstation 3Benchtop Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, California, USA).

1.3 Experimental procedures

Coating 96-well black-wall cell culture plate with matrix BD Matrigel, incubating at 37 deg.C for 1h, sucking supernatant, and culturing with 4 × 10⁴Density per well, corresponding HEK293 cells were seeded in 96-well black-wall-primed cell culture plates in CO₂Culturing for 16-24 h in a constant-temperature incubator with the concentration of 5% and the temperature of 37 ℃; the original medium was discarded, and 100. mu.l/well of freshly prepared dye solution was added, followed by incubation at 37 ℃ in the dark for 60 min. Preparing a sample to be tested: preparing samples to be detected with different concentrations. Adding a sample to be detected with a specific volume into cells, adding the sample with the volume of 50 mu l/hole, and measuring the excitation effect of the sample on a melatonin receptor by using a Flexstation 3 multifunctional microplate reader. The results of the experiment were analyzed using Graphpad prism 5 software.

2. As a result:

compound 1 at 1.0mM concentration, on MT₁And MT₂The receptor agonism rates were 79.6 and 46.3%, respectively (see FIG. 2 for Compound 1 vs. MT₁And MT₂The receptor agonism was determined to be 100% of the maximum Melatonin (MT) agonism, and the test concentration of the compound was 1.0mM, and the agonism was Mean ± SD (n ═ 3)).

3. And (4) conclusion:

the experimental results show that Compound 1 is on MT₁And MT₂The receptors all showed better agonism, at 1.0mM concentration, to MT₁And MT₂The agonism of the receptor was 79.6 and 46.3%, respectively. The above results indicate that compound 1 can be a novel melatonin receptor agonist, and can treat or ameliorate central nervous system diseases associated with melatonin receptorsAnd (6) treating the disease.

Example 3:

formulation examples:

1. compound 1 was prepared as in example 1, dissolved in a small amount of DMSO, and then injected with water for injection, fine filtered, filled and sterilized to prepare an injection.

2. Compound 1 was prepared by the method of example 1, dissolved in a small amount of DMSO, dissolved in sterile water for injection, stirred to dissolve, filtered through a sterile suction funnel, fine filtered aseptically, sub-packaged in ampoules, freeze dried at low temperature, aseptically melt sealed to obtain powder for injection.

3. Compound 1 was prepared according to the method of example 1, and excipients were added in a weight ratio of 9:1 to make a powder.

4. Compound 1 was prepared according to the method of example 1, and excipients were added in a weight ratio of 5:1 to excipients, granulated and tabletted.

5. Compound 1 was prepared according to the method of example 1 and prepared into oral liquid according to the conventional oral liquid preparation method.

6. Compound 1 was prepared according to the method of example 1, and the excipient was added in a weight ratio of 5:1 to the excipient to make capsules.

7. Compound 1 was prepared according to the method of example 1, and the excipients were added in a weight ratio of 3:1 to make capsules.

8. Compound 1 was prepared according to the method of example 1, and excipients were added in a weight ratio of 5:1 to make granules.

Claims

1. A compound uncaria amide A shown in a structural formula (I),

2. use of a compound of formula (I) as defined in claim 1 for the preparation of a melatonin receptor agonist.

3. Use of a compound of formula (I) according to claim 1 for the preparation of a medicament for the treatment or amelioration of a central nervous system disorder.

4. The use as claimed in claim 3, wherein the disease is a central nervous system disease associated with melatonin receptors.

5. A process for the preparation of a compound of formula (I) according to claim 1, taking dried stem branches of Uncaria rhynchophylla, crushing, extracting twice with 70% ethanol under reflux for 3 hours each time, combining the ethanol extract, recovering ethanol under reduced pressure to obtain an extract, dissolving the extract with 5 times of water, performing D101 macroporous resin column chromatography, performing ethanol-water gradient elution, combining 50% ethanol elution parts, concentrating, dissolving methanol, adsorbing on silica gel, standing at room temperature to volatilize the solvent, grinding, sieving, performing silica gel column chromatography, eluting with chloroform-methanol-water at a ratio of 7: 3: 0.3 to obtain 4 fractions A-D, continuously performing medium-pressure preparation on the fraction C through an MCI CHP-20P gel column, performing methanol-water gradient elution at a ratio of 1: 9-9: 1 to obtain 5 fractions, purifying the fractions C-1-C-5 and the fraction C-2 by HPLC, and utilizing Rp-C.₁₈And (3) carrying out acetonitrile-water gradient elution on the column at a ratio of 1: 9-3: 7 to obtain the compound shown in the formula (I).

6. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) according to claim 1 and a pharmaceutically acceptable carrier.

7. Use of the pharmaceutical composition of claim 6 for the preparation of a melatonin receptor agonist.

8. Use of the pharmaceutical composition of claim 6 for the manufacture of a medicament for the treatment or prevention of a central nervous system disorder.

9. The use as claimed in claim 8, wherein the disease is a central nervous system disease associated with melatonin receptors.

10. Preparation of claim 6The method of the pharmaceutical composition comprises the steps of taking dry stems and branches with hooks of uncaria, crushing, carrying out reflux extraction twice with 70% ethanol for 3 hours each time, merging ethanol extract, recycling ethanol under reduced pressure to obtain extract, dissolving the extract with 5 times of water, carrying out D101 macroporous resin column chromatography, carrying out ethanol-water gradient elution, merging elution parts with 50% ethanol, concentrating, dissolving methanol, adsorbing on silica gel, standing at room temperature to volatilize solvent, carrying out silica gel column chromatography after grinding and sieving, carrying out chloroform-methanol-water 7: 3: 0.3 elution to obtain 4 fractions A-D, carrying out medium pressure preparation on the fraction C through an MCI CHP-20P gel column, carrying out methanol-water gradient elution with the ratio of 1: 9-9: 1 to obtain 5 fractions, carrying out C-1-C-5, carrying out HPLC purification on the fraction C-2, and carrying out Rp-C gel column chromatography₁₈Performing gradient elution on the column by acetonitrile-water at a ratio of 1: 9-3: 7 to obtain a compound shown in the formula (I), and adding a medicinal carrier in a certain proportion into the compound shown in the formula (I) serving as a raw material.