阅读说明：本技术 上调神经肽pacap及其受体pac1-r的小分子化合物spam1及制备方法和用途 (Small molecule compound SPAM1 for up-regulating neuropeptide PACAP and receptor PAC1-R thereof, and preparation method and application thereof ) 是由 余榕捷 于 2020-05-12 设计创作，主要内容包括：本发明公开了上调神经肽PACAP及其受体PAC1-R的小分子化合物SPAM1及制备方法和用途,其结构如式(Ⅰ)所示,其中R=无或H<Sub>2</Sub>0或HCl；本发明的SPAM1分子量小,能高效穿越生物学屏障,包括血脑屏障、血睾屏障等；本发明的SPAM1正反馈上调下丘脑-垂体分泌的神经递质/调质PACAP及其特异受体PAC1-R的表达,作用于性腺轴和肾上腺轴的下游腺体,因此其调控作用是全面的；本发明的SPAM1特异靶向PAC1-R1,仅作用于天然表达PAC1-R神经系统与内分泌系统的细胞和组织,副作用较小。因此,SPAM1将成为有效治疗与预防与神经肽PACAP密切相关的,神经系统、内分泌系统及免疫系统功能衰老与紊乱的新型小分子化合药物。(The invention discloses a small molecular compound SPAM1 for up-regulating neuropeptide PACAP and a receptor PAC1-R thereof, and a preparation method and application thereof, and the structure of the small molecular compound SPAM1 is shown as a formula (I), wherein R = none or H 2 0 or HCl; the SPAM1 has small molecular weight, and can efficiently cross biological barriers including blood brain barrier, blood testis barrier and the like; SPAM1 positive of the inventionThe feedback up-regulates the expression of a neurotransmitter/modified PACAP secreted by hypothalamus-pituitary and a specific receptor PAC1-R thereof, acts on downstream glands of a gonadal axis and an adrenal axis, so that the regulation and control effects are comprehensive; the SPAM1 of the invention specifically targets PAC1-R1, only acts on cells and tissues of a nerve system and an endocrine system of naturally expressed PAC1-R, and has small side effect. Therefore, SPAM1 will become a novel small molecule compound drug for effectively treating and preventing aging and disorder of nervous system, endocrine system and immune system functions closely related to the neuropeptide PACAP.)

1. A small molecular compound SPAM1 for up-regulating neuropeptide PACAP and a receptor PAC1-R thereof has a structure shown in a formula (I), wherein R ═ isNone or H₂0 or HCl;

2. the process for preparing SPAM1 as a small molecule compound in claim 1, which comprises the following steps:

(1) 2-aminobenzamide and glutaric anhydride are polymerized to obtain a basic compound 4- (4-oxo-3, 4-dihydroquinazoline-2-yl) butyric acid;

(2)4- (4-oxo-3, 4-dihydroquinazolin-2-yl) butyric acid, benzo [ d ] [1,2,3] triazol-1-ol, 4- (aminomethyl) benzoic acid and triethylamine.

3. The use of the small molecule compound SPAM1 according to claim 1 in the preparation of a medicament for the prevention or treatment of aging and disorders of the nervous system, endocrine system or immune system function closely related to the neuropeptide PACAP.

4. Use of the small molecule compound SPAM1 of claim 1 for the manufacture of a medicament for the prevention or treatment of hypothalamic-pituitary-adrenal axis dysfunction and disorder, hypothalamic-pituitary-gonadal axis dysfunction and disorder, or thymic function decline and disorder.

5. The use of the small molecule compound SPAM1 according to claim 1 in the manufacture of a medicament for the prevention or treatment of life extension, the prevention and treatment of aging with age, male and female climacteric syndrome, age-related decline in male and female sexual function, decline in male and female reproductive function, immune dysfunction and decline, or sepsis.

Technical Field

The invention relates to the field of chemistry and biochemistry, in particular to a small molecular compound SPAM1 which can effectively up-regulate the expression of neuropeptide Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) and a specific receptor PAC1-R thereof: 4- ((4- (4-oxo-3, 4-dihydroquinazolin-2-yl) butyryl) methylbenzoic acid and derivatives thereof, a high-efficiency preparation method of SPAM1 and application of SPAM1 in preventing and treating physiological and pathological diseases and functional decline of nervous system, endocrine system and immune system related to PACAP and specific receptor PAC1-R thereof.

Background

The neuropeptide PACAP, originally isolated from the hypothalamic-pituitary gland of cattle, belongs to the vasoactive intestinal peptide/secretagogue/growth hormone releasing hormone/glucagon superfamily; the PACAP has the extremely conservative active peptide in the evolution process, and the difference of only one amino acid between frog and human PACAP^【1】. PACAP mediates the important functions of regulating and controlling the nervous system, endocrine system and immune system through three B-class G protein-coupled receptors: one PACAP-specific receptor PAC1-R and two vasoactive intestinal peptide-sharing receptors VPAC1-R and VPAC 2-R. As an important neurotransmitter and neuromodulator, age-dependent downregulation of PACAP is considered to be one of the causes of aging.

PACAP and its receptor, especially its specific PAC1-R, are not only distributed in high density in hypothalamus-pituitary, but also in neuroendocrine tissues and glands such as pituitary-adrenal axis, pituitary-gonadal axis, pineal body and thymus, including adrenal gland, testis and ovary, mediating the secretion and regulation of related stress hormones, sex hormones and immune factors; including upregulation of Corticotropin Releasing Hormone (CRH) and Corticosterone (COR), involved in the regulation of melatonin and gonadotropin releasing hormone (GnRH), upregulation of Testosterone (TE) and estradiol (E2); and the thymus of animals with PACAP deletion is declined, and the immune function is reduced. In summary, existing cell and animal studies have shown that: PACAP and PAC1-R mediate stress regulation of organisms, enhance sexual function and reproductive capacity of males and females, promote immunologic function and effectively reduce sepsis mortality.

Although PACAP has potential anti-aging pharmaceutical development value, direct development of PACAP into a drug is greatly limited due to the extremely poor in vivo stability of PACAP, for example, PACAP38 has an in vivo half-life of less than 2min and a limited function of crossing biological barriers.

Small molecule SPAM1(4- ((4- (4-oxo-3, 4-dihydroquinazolin-2-yl) butyryl) methylbenzoic acid) is a PAC1-R small molecule allosteric modulator obtained by targeting an allosteric regulatory site located in the N-terminal extracellular domain of PAC1-R, virtual screening in silico, and validation at the cellular and animal levels.

The latest cytological and animal research of the inventor firstly discovers that SPAM1 can positively feedback and concentration-dependently up-regulate the expression of PACAP specific receptor PACAP 1-R in vivo PACAP and target organs thereof, and the subsequent animal experiment of a mouse aging model induced by D-galactose firstly confirms that SPAM1 can play the functions of regulating pituitary-adrenal axis, pituitary-gonadal axis and thymus by up-regulating PACAP/PAC1-R signal channels and resist the organism decline caused by the down-regulation of PACAP, and the specific functions comprise: 1) up-regulating the stress ability of the organism, 2) enhancing the sexual function and reproductive capacity, and 3) resisting the hypoimmunity.

Disclosure of Invention

The invention aims to provide a small molecule SPAM1 which can effectively up-regulate the expression of PACAP and a specific receptor PAC1-R thereof in vivo.

In order to achieve the purpose, the invention adopts the following technical scheme:

a small molecular compound SPAM1 for up-regulating neuropeptide PACAP and a receptor PAC1-R thereof has a structure shown in a formula (I), wherein R is nothing or H₂0 or HCl (no modification or hydration modification or hydrochlorination modification);

compound SPAM1-3 (formula (II), A-C) having the following structure;

the preparation method of the small molecular compound SPAM1 is a two-step synthesis (formula (III) I, II), and comprises the following steps:

(1) 2-aminobenzamide (A) and glutaric anhydride (B) are subjected to polymerization reaction to obtain a basic compound 4- (4-oxo-3, 4-dihydroquinazolin-2-yl) butyric acid (C);

(2)4- (4-oxo-3, 4-dihydroquinazolin-2-yl) butyric acid (C) and benzo [ D ] [1,2,3] triazol-1-ol (D), 4- (aminomethyl) benzoic acid (E) and triethylamine (F) by condensation;

the small molecular compound SPAM1 can be used for preparing medicines for preventing or treating aging and disorder of nervous system, endocrine system or immune system, which are closely related to the neuropeptide PACAP.

The small molecular compound SPAM1 can be used for preparing medicines for preventing or treating hypothalamus-pituitary-adrenal axis function deterioration and disorder, hypothalamus-pituitary-gonad axis function deterioration and disorder or thymus function deterioration and disorder.

The small molecular compound SPAM1 can be used for preparing medicines for prolonging life, and preventing and treating aging of body with age, male and female climacteric syndrome, male and female sexual function decline with age, male and female reproductive function decline, immune dysfunction and decline or sepsis.

Compared with the prior art, the invention has the following beneficial effects:

(1) the SPAM1 has small molecular weight, and can efficiently cross biological barriers including blood brain barrier, blood testis barrier and the like;

(2) the SPAM1 positive feedback of the invention can up-regulate the neurotransmitter/modified PACAP secreted by hypothalamus-pituitary and the expression of the specific receptor PAC1-R thereof, and acts on the downstream glands of the gonadal axis and the adrenal axis, so that the regulation and control effect is comprehensive;

(3) the SPAM1 of the invention specifically targets PAC1-R1, only acts on cells and tissues of a nerve system and an endocrine system of naturally expressed PAC1-R, and has small side effect.

Therefore, SPAM1 will become a novel small molecule compound drug for effectively treating and preventing aging and disorder of nervous system, endocrine system and immune system functions closely related to the neuropeptide PACAP.

Drawings

FIG. 1: a synthesis process (two-step method) of micromolecular SPAM 1; a: 2-aminobenzamide; b: glutaric anhydride; c: 4- (4-oxo-3, 4-dihydroquinazolin-2-yl) butanoic acid; d: benzo [ d ] [1,2,3] triazol-1-ol; e: 4- (aminomethyl) benzoic acid; f: triethylamine.

FIG. 2: detecting the nuclear magnetic resonance of the intermediate product C;¹HNMR (400MHz, DMSO): 12.16 (meter, 2H), 8.08(d, J ═ 5.8Hz, 1H), 7.79-7.76 (meter, 1H), 7.60(d, J ═ 6.6 Hz, 1H), 7.46(d, J ═ 6.0Hz, 1H), 2.64(t, J ═ 6.0Hz, 2H), 2.32(t, J ═ 5.6 Hz, 2H), 2.00-1.96 (meter, 2H).

FIG. 3: nuclear magnetic resonance detection of SPAM 1;¹HNMR (400MHz, DMSO-d6) ═ 12.79(s, 1H), 12.18(s, 1H), 8.42(t, J ═ 6.0Hz, 1H), 8.09(d, J ═ 7.9Hz, 1H), 7.90(d, J ═ 7.9Hz, 2H), 7.78(t, J, 1H), J ═ 7.6Hz, 1H), 7.61(d, J ═ 8.2Hz, 1H), 7.47(t, J ═ 7.5Hz, 1H), 7.36(d, J ═ 7.9Hz, 2H), 4.33(d, J ═ 5.9Hz, 2H), 2.64(t, J ═ 7.4, 2H), 2.27(t, J ═ 7.5, 2H), 2.95 (t, J ═ 2H, 1H), 2.09 (m).

FIG. 4: SPAM1 modulated plasma concentrations of PACAP in vivo (, P <0.01, SPAM1+ vs. SPAM 1-).

FIG. 5: westernblot assay SPAM1 concentration-dependently upregulated PAC1-R expression in neural cells Neuro2 a.

FIG. 6: the immunofluorescence detection SPAM1 effectively up-regulates the expression of PAC1-R in nerve cells SHSY-5Y.

FIG. 7: the enzyme-linked immunosorbent assay detects that the concentration of the intraperitoneal injection of SPAM1 dependently increases the serum corticosterone level (P <0.01, SPAM1vs. control group).

FIG. 8: SPAM1 was effective in upregulating estradiol and luteinizing hormone and in resisting D-galactose-induced ovarian failure (. beta., P <0.01, aging model group vs. normal; # P <0.01, SPAM1 intervention group vs. aging model group).

FIG. 9: SPAM1 was effective in upregulating testosterone and resisting D-galactose-induced testicular degeneration (. about.P <0.01, aging model vs. normal; # P <0.01, SPAM1 intervention group vs. aging model).

FIG. 10: SPAM1 was effective in upregulating thymus index and resisting dexamethasone-induced thymus decline (. about.P <0.01, aging model group vs. normal; # P <0.01, SPAM1 intervention group vs. aging model group).

FIG. 11: SPAM1 is effective in reducing death rate of sepsis induced by LPS, and improving survival rate.

Detailed Description

The present invention will be further described with reference to specific examples, which are not intended to limit the present invention in any way.