阅读说明：本技术 一种一氧化氮供体型Netarsudil衍生物及其制备方法和用途 (Nitric oxide donor type Netarsudil derivative and preparation method and application thereof ) 是由 黄张建 张奕华 毛宇婕 吴建兵 朱明超 于 2020-05-12 设计创作，主要内容包括：本发明涉及药物化学和药物治疗学领域,具体涉及一种一氧化氮(NO)供体型Netarsudil衍生物及其制备方法和用途。这些衍生物保留了较强的ROCK抑制活性,在人小梁网细胞中可释放一定量的NO,二者协同降低新西兰白兔眼内压,且没有眼结膜的刺激性。本发明涉及此类NO供体型Netarsudil衍生物,或其药学上可接受的盐,他们的制备方法,含有这些化合物的药用组合物以及它们的医药用途,特别是在制备预防和/或治疗青光眼、高眼压等眼科疾病方面的应用。(The invention relates to the fields of medicinal chemistry and pharmacotherapeutics, in particular to a Nitric Oxide (NO) donor type Netarsudil derivative and a preparation method and application thereof. These derivatives retain strong ROCK inhibitory activity and release certain amount of NO in human trabecular meshwork cells, and the two synergistically reduce intraocular pressure of New Zealand white rabbits without irritation of conjunctiva. The invention relates to NO donor type Netarsudil derivatives or pharmaceutically acceptable salts thereof, a preparation method thereof, a medicinal composition containing the compounds and medicinal application thereof, in particular to application in preparing medicaments for preventing and/or treating glaucoma, ocular hypertension and other ophthalmic diseases.)

1. A compound of formula a or an optical isomer, enantiomer, diastereomer, racemate or racemic mixture thereof, or a pharmaceutically acceptable salt thereof:

wherein: when m is 2, X is O; when m is 1, X is S; linker represents alkylene acyl or alkylene, wherein the alkylene is C1-C10 linear chain, branched chain, alkylene containing a cyclic structure or containing ester bonds, alkenylene, alkynylene, heterocyclic radical, and the cyclic structure is saturated or unsaturated carbocyclyl or heterocyclic radical.

2. The compound of claim 1, wherein the compound of formula a is selected from the group consisting of:

wherein:

R₁selected from methylene, C2-C9 straight chain, branched chain or alkylene containing a cyclic structure, alkenylene, alkynylene and heterocyclic group, wherein the cyclic structure is saturated or unsaturated carbocyclyl or heterocyclic group.

3. The compound of claim 2, wherein said compound is selected from the group consisting of

R₂、R₃Each independently represents an alkyl group including C2-C5 straight or branched chain alkyl groups, or R₂、R₃Together form a cyclic alkyl group.

4. A compound of claim 2, wherein R is₁Represents ethylene, propylene, butylene, pentylene, or,

-R₁-is of

5. The compound of claim 1, wherein the compound is selected from the group consisting of:

6. a preparation method of the compound shown in the formula I and the formula II comprises the following steps:

reacting the compound III with a compound IV to obtain a compound V, and deprotecting the amino group of the compound V to obtain a target compound I, II; the synthetic route is as follows:

wherein: when m is 2, X is O; when m is 1, X is S; r₁Is selected from the group consisting of methylene,C2-C9 straight chain, branched chain or alkylene, alkenylene, alkynylene containing cyclic structure, heterocyclic radical, wherein the cyclic structure is saturated or unsaturated carbon ring radical, heterocyclic radical.

7. The production method according to claim 6,

in the reaction for preparing the compound V from the compound III, the solvent is selected from one or more of anhydrous acetonitrile, anhydrous dichloromethane, chloroform, ethyl acetate, redistilled acetone, anhydrous tetrahydrofuran, anhydrous N, N-dimethylformamide, dimethyl sulfoxide or dioxane; the base is selected from potassium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, pyridine, 4-methylaminopyridine, triethylamine, N, N-diisopropylmethylamine; the reaction temperature is-20 ℃ to heating reflux; and/or the presence of a gas in the gas,

in the reaction for preparing the compound I, II from the compound V, anhydrous dichloromethane is selected as a solvent; selecting trifluoroacetic acid as an acid; the reaction temperature is 0 ℃ to room temperature.

8. The production method according to claim 7,

in the reaction for preparing the compound V from the compound III, anhydrous dichloromethane is selected as a solvent, and the reaction temperature is room temperature;

in the reaction for preparing the compound I, II from the compound V, anhydrous dichloromethane is selected as a solvent, and the reaction temperature is 0 ℃ to room temperature.

9. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1 or an optical isomer, enantiomer, diastereomer, racemate or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

10. Use of a compound according to any one of claims 1 to 5, a pharmaceutical composition according to claim 9 for the manufacture of a medicament for the prevention and/or treatment of a disease associated with elevated intraocular pressure.

11. Use according to claim 10, characterized in that diseases associated with increased ocular pressure include ocular hypertension, glaucoma, diabetic complications of the eye.

Technical Field

The invention relates to the field of medicinal chemistry and pharmacotherapeutics, and relates to a Nitric Oxide (NO) donor type Netarsudil derivative, a preparation method and application thereof, in particular to a combined medication strategy for co-dilating trabecular meshwork and regulating cytoskeleton reconstruction, synthesis and evaluation of anti-glaucoma activity of the NO donor and the Netarsudil. The compound can be used for intraocular pressure increase caused by increased aqueous humor outflow resistance. The invention also relates to pharmaceutical compositions containing such compounds and to their use.

Background

Glaucoma (Glaucoma) is a group of optic nerve diseases caused by a variety of factors, and is characterized primarily by persistent elevated intraocular pressure (IOP), optic nerve damage, and visual field loss. It eventually leads to death of Retinal Ganglion Cells (RGCs) and deformation of the connective tissue supporting the optic disc, resulting in complete blindness. Glaucoma is the second most irreversible blinding disease worldwide, and 6000 or more thousands of patients are counted at present. This number is expected to grow to 8000 thousands in 2020 and to reach 1.12 billion in 2040. Clinically, lowering IOP remains the only means to prevent it from further developing into vision impairment or blindness.

At present, many studies have reported the cellular effects of ROCK inhibitors (HA-1077, H-1152P, AR-13324, Y-27632, Y-39983, AMA0076, etc.) and the IOP-lowering effect. The results of the studies indicate that ROCK inhibitors can effectively lower IOP in animal models of normal or ocular hypertension without causing serious side effects.

In 12 months 2017, ROCK inhibitor Netarsudil (AR-13324) eye drops were approved by the U.S. FDA for marketing. It is a dual ROCK kinase and norepinephrine transporter (NET) inhibitor that directly lowers IOP by relaxing the Trabecular Meshwork (TM) and contracting the ciliary muscles, reducing resistance to aqueous outflow, and it also lowers IOP by lowering scleral venous pressure and reducing aqueous humor production.

Studies prove that Netarsudil can inhibit human ROCK-1 (K)_i1nM) and ROCK-2 (K)_i1nM) is more potent than other ROCK inhibitors, such as Y27632 and fasuil. The results of the experiments carried out in rabbits and monkeys at normal intraocular pressure showed that 0.04% of Netarsudil significantly reduced intraocular pressure, and at day 3 of treatment, maximum intraocular pressure reduction values of 8.1. + -. 0.7 and 7.5. + -. 1.1mmHg were observed in rabbits and monkeys, respectively (both P. sup. + -. 0.7 and P. sup. + -. 7.5 mmHg)<0.01). It was found that a single, unilateral topical administration of different concentrations to rabbits and monkeys can result in a significant dose-dependent intraocular pressure lowering effect. In addition, the combination of Netarsudil and Latanoprost produces a stronger IOP lowering effect and the incidence of adverse events is comparable to that of Netarsudil alone. On 12.3.2019, FDA approved a new drug for Rocklatan (Netarsudil/Latanoprost) for clinical use in the treatment of glaucoma and ocular hypertension.

The classical pathway through TM and SC is the major route of outflow of aqueous humor in mammals. In both normal eyes and glaucoma, resistance to aqueous outflow is produced by the inner wall of the SC and the proximal microtubule region of the TM. Cytoskeletal structural features, adhesion interactions, SC cell permeability and TM cell secretion all play important roles in regulating aqueous outflow. Netarsudil has the ability to disrupt actin stress fibers and focal adhesions in TM cells, and therefore Netarsudil is able to relax TM, increasing SC permeability and thereby lowering IOP. In addition, Netarsudil blocks TGF- β 2-induced fiber marker expression and has anti-fibrotic activity, suggesting that Netarsudil is able to alter fibrotic disease processes associated with elevated IOP in TM. In addition, steroid glaucoma can be treated with ROCK inhibitors, the mechanism behind this type of glaucoma is through a non-canonical Wnt pathway that binds to ROR 2/RhoA/ROCK. Yuan et al determined that dexamethasone could activate this pathway, promote cross-linking of the actin network, and cause structural changes in the TM cytoskeleton. Given the critical role of Netarsudil on ROCK inhibition, it may be a viable treatment option for steroid glaucoma.

Studies have demonstrated that inhibition of NET can block the reuptake of norepinephrine in the synapse, thereby increasing the intensity and duration of endogenous norepinephrine signaling. This mechanism may involve the production of aqueous humor, and inhibition of NET reduces blood flow in the blood vessels due to noradrenaline-induced vasoconstriction, thereby reducing blood flow into the ciliary body and thus reducing aqueous humor production. Thus, Netarsudil, as a NET inhibitor, can lower IOP by inhibiting aqueous humor production.

Disclosure of Invention

The purpose is as follows: the invention provides an NO donor type Netarsudil derivative and a preparation method and application thereof. Pharmacological experiments prove that the compounds have good intraocular pressure reducing effect, so that the compounds can be used for preventing and treating glaucoma.

In recent years, NO/NO donors have been increasingly studied for the treatment of glaucoma. A large body of literature has demonstrated that NO plays a significant role in the control of IOP. The regulation of NO and its signaling pathways is closely related to aqueous humor dynamics. NO production was reduced in patients with ocular hypertension compared to healthy subjects, and exogenous administration of NO was shown to lower the ocular pressure in patients. eNOS knockout mice or animals with impaired GCs activity have higher IOP compared to their wild type, further confirming the importance of NO signaling in regulating IOP. Preliminary mechanistic studies have demonstrated that NO can reduce aqueous humor outflow resistance through a variety of pathways, the most prominent of which is relaxation TM/SC. Some studies have also demonstrated that NO reduces aqueous humor production to some extent.

Clinical research shows that compared with single-drug treatment, 2-3 eye drops with different action mechanisms can be used together to greatly reduce side effects and effectively reduce IOP.

In view of the above, the invention provides a method for coupling the nitric acid ester or nitrosothiol compound as the NO donor compound with the active metabolite of ROCK/NET for dual inhibition of Netarsudil, and the obtained compound has the effects of lowering intraocular pressure in various aspects and has better glaucoma treatment effect than a single-action medicament.

The technical scheme is as follows: in order to solve the technical problems, the technical scheme adopted by the invention is as follows:

in a first aspect, the present invention discloses a compound represented by formula a, or an optical isomer, enantiomer, diastereomer, racemate or racemic mixture thereof, or a pharmaceutically acceptable salt thereof:

wherein: when m is 2, X is O; when m is 1, X is S; linker represents alkylene acyl or alkylene, wherein the alkylene is C1-C10 linear chain, branched chain, alkylene containing a cyclic structure or containing ester bonds, alkenylene, alkynylene, heterocyclic radical, and the cyclic structure is saturated or unsaturated carbocyclyl or heterocyclic radical.

In some embodiments, the compound of formula a is selected from:

wherein:

R₁selected from methylene, C2-C9 straight chain, branched chain or alkylene containing a cyclic structure, alkenylene, alkynylene and heterocyclic group, wherein the cyclic structure is saturated or unsaturated carbocyclyl or heterocyclic group.

In some embodiments, R₁Represents ethylene, propylene, butylene, pentylene, or,

-R₁-is of

Further, the compound is selected from:

R₂、R₃each independently represents an alkyl group including C2-C5 straight or branched chain alkyl groups, or R₂、R₃Together form a cyclic alkyl group.

In particular, the compound is selected from:

4- [ 3-amino-1- (isoquinolin-6-amino) -1-oxopropan-2-yl]Benzyl-4- (nitrooxy) butanoic acid ester (I)₁) And its hydrochloride salt (I)₁·2HCl)；

4- [ 3-amino-1- (isoquinolin-6-amino) -1-oxopropan-2-yl]Benzyl-5- (nitrooxy) pentanoate (I)₂)；

4- [ 3-amino-1- (isoquinolin-6-amino) -1-oxopropan-2-yl]Benzyl-6- (nitrooxy) hexanoate ester (I)₃)；

4- [ 3-amino-1- (isoquinolin-6-amino) -1-oxopropan-2-yl]-benzyl-succinic acid isosorbide mononitrate (I)₄)

4- [ 3-amino-1- (isoquinolin-6-amino) -1-oxopropan-2-yl]Benzyl-4- [ (nitrooxy) methyl group]Benzoic acid esters (I)₅)

4- [ 3-amino-1- (isoquinolin-6-amino) -1-oxopropan-2-yl]Benzyl [ 2-methyl-2- (nitrosothiopropyl) propyl ] propyl]Succinic acid ester (II)₁)

4- [ 3-amino-1- (isoquinolin-6-amino) -1-oxopropan-2-yl]Benzyl [ 2-methyl-2- (nitrosothio) butyl]Succinic acid ester (II)₂)

4- [ 3-amino-1- (isoquinolin-6-amino) -1-oxopropan-2-yl]Benzyl [ 2-ethyl-2- (nitrosothio) butyl]Succinic acid ester (II)₃)

{1-[(λ²-azasubunit) (oxo) -lambda⁵-sulfanyl radical]Cyclohexyl } methyl {4- [ 3-amino-1- (isoquinolin-6-ylamino) -1-oxopropan-2-yl]Benzyl succinate (II)₄)

In a second aspect, a method for preparing a compound shown in formula I or formula II is provided, which comprises:

reacting the compound III with a compound IV to obtain a compound V, and deprotecting the amino group of the compound V to obtain a target compound I, II; the synthetic route is as follows:

wherein: when m is 2, X is O; when m is 1, X is S; r₁Selected from methylene, C2-C9 straight chain, branched chain or alkylene containing a cyclic structure, alkenylene, alkynylene and heterocyclic group, wherein the cyclic structure is saturated or unsaturated carbocyclyl or heterocyclic group.

In some embodiments, the method of making is characterized in that,

in the reaction for preparing the compound V from the compound III, the solvent is selected from one or more of anhydrous acetonitrile, anhydrous dichloromethane, chloroform, ethyl acetate, redistilled acetone, anhydrous tetrahydrofuran, anhydrous N, N-dimethylformamide, dimethyl sulfoxide or dioxane; the base is selected from potassium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, pyridine, 4-methylaminopyridine, triethylamine, N, N-diisopropylmethylamine; the reaction temperature is-20 ℃ to heating reflux; and/or the presence of a gas in the gas,

in the reaction for preparing the compound I, II from the compound V, anhydrous dichloromethane is selected as a solvent; selecting trifluoroacetic acid as an acid; the reaction temperature is 0 ℃ to room temperature.

Further, in some embodiments, the solvent is selected from anhydrous dichloromethane in the reaction for preparing compound V from compound III, and the reaction temperature is room temperature.

In the reaction for preparing the compound I, II from the compound V, anhydrous dichloromethane is selected as a solvent, and the reaction temperature is 0 ℃ to room temperature.

These compounds can be purified according to conventional separation techniques.

In a third aspect, there is provided a pharmaceutical composition comprising a therapeutically effective amount of said compound or an optical isomer, enantiomer, diastereomer, racemate or racemic mixture thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

In a fourth aspect, there is provided the use of a compound of the invention in the manufacture of a medicament for the prevention and/or treatment of a disease associated with elevated intraocular pressure. More specifically, the diseases associated with increased intraocular pressure include, but are not limited to, ocular hypertension, glaucoma, diabetic complications and the like.

The NO donor type Netarsudil derivative is administrated through eye drops, and releases Netarsudil and proper amount of NO in eyes, wherein the Netarsudil derivative inhibits ROCK, and the NO derivative activates an sGC-cGMP pathway and synergistically plays roles in relaxing trabecular meshwork, reducing intraocular pressure, increasing blood flow and protecting retinal ganglion cells.

Has the advantages that: the invention designs and synthesizes the NO donor type Netarsudil derivative, which is proved to generate the effects of lowering IOP and protecting retinal ganglion cells by inhibiting ROCK and releasing NO, and has NO irritation to eye conjunctiva of a New Zealand white rabbit. The medicinal composition containing the target compound and the medical application thereof have good application prospect, particularly in preventing and treating glaucoma-related diseases.

Drawings

FIG. 1 is I₁Effect of 2HCl and Netarsudil hydrochloride on the conjunctiva of the eyes of rabbits after eye-drop administration.

Detailed Description

To further illustrate the present invention, a series of examples are given below, which are purely illustrative and are intended to be a detailed description of the invention only and should not be understood as limiting the invention.

The pharmacological experimental method and the results of the compounds I and II of the invention for resisting glaucoma are as follows:

NO-donating Netarsudil Compounds Release of NO in Human Trabecular Meshwork Cells (HTMC)

NO release of compounds in HTMC cells was investigated. Discovery of Compound I Using DAF-FM DA fluorescent Probe₁And II₃NO was released in a time-dependent manner (table 1). Further, the time point of 24 hours was selected, and NO in the cells was detected by flow cytometry, whereby it was found that Compound I₁、I₂、I₄、II₃More NO was released (table 2). Wherein, ISMN is isosorbide mononitrate.

TABLE 1 Compound I₁And II₃Release of NO in HTMC cells

TABLE 2 DAF-FM DA fluorescent Probe for detecting the amount of NO released by Compounds in TM cells (flow-type)

NO donor type Netarsudil compound I₁Has effect of inhibiting ROCK-I kinase or ROCK-II kinasePreparation method of

The inhibitory effect of the compounds on ROCK-I kinase or ROCK-II kinase was investigated. Discovery of Compound I₁Has inhibitory effect on ROCK-I and ROCK-II kinases, IC₅₀24.40. + -. 3.22nM and 15.70. + -. 0.44nM, respectively (Table 3).

TABLE 3 Compounds I₁And IC for inhibiting ROCK-I and ROCK-II enzyme activities by Netarsudil₅₀

NO donor type Netarsudil derivative I₁Effect of hydrochloride and Netarsudil hydrochloride on intraocular pressure of Normal rabbits

The experimental method comprises the following steps: adopting adult New Zealand white rabbit (weight is 2-3 kg), adding normal saline into left eye, and adding compound I into right eye₁2HCl (0.3%) or Netarsudil hydrochloride (0.3%). Intraocular pressure (IOP) changes were measured using a non-contact tonometer at 0h, 1h, 2h, 4h, 8h, 24h, respectively. Found through experiments, I₁The IOP can be quickly reduced within 0-4h, and the reduction amplitude is equivalent to that of Netarsudil. (Table 4).

It is noted that, the Netarsudil hydrochloride (0.3%) causes conjunctival congestion of white rabbit eyes 2h after eye drop administration, and white flocculent secretion is increased (see fig. 1, arrow part), and the side effect lasts for 24 h; and I₁The 2 HCl-administered group did not suffer from such side effects. Prompt I₁2HCl is safer than Netarsuidil.

TABLE 4 Compounds I₁Effect of 2HCl and Netarsudil HCl on intraocular pressure in rabbits

The pharmacological data show that the NO donor type Netarsudil derivative I related to the invention₁Can exertHas obvious anti-glaucoma effect and higher safety, and is worthy of further research.