Patent Description:
In order to keep the eyeball in a certain shape to maintain the optical function and metabolism of the eye, the ciliary body will secrete aqueous humor to perform this work. When the drainage route of aqueous humor is blocked and aqueous humor accumulates, an increase in intraocular pressure will be the result, which is the biggest risk factor for glaucoma.

At present, it is known that aqueous humor drainage mainly occurs in the following three ways: (<NUM>) Schlemm's canal pathway (major, conventional pathway); (<NUM>) Uvea-sclera pathway (small amount), about <NUM>%-<NUM>%; and (<NUM>) Absorption on the surface of the iris (minor amount). The mechanisms of reducing intraocular pressure of commonly used glaucoma drugs are: (a) reducing the production of aqueous humor, such as β-receptor blockers, carbonic anhydrase inhibitors, and α-receptor agonists; and (b) increasing the drainage of aqueous humor (Uveoscleral pathway), such as prostaglandin analogs, alpha receptor agonists, etc..

The Rho/ROCK pathway plays an important role in the regulation of the cytoskeleton. Rho/ROCK inhibitors can regulate the functions of the actin cytoskeleton, extracellular matrix and Schlemm's tube endothelial cells in the trabecular meshwork tissue, thereby reducing the intraocular pressure.

Currently, some pharmaceutical companies have begun to explore the impact of ROCK inhibitors on reducing intraocular pressure in humans, and have successively developed new drugs. After evaluating the clinical safety and effectiveness for humans, current commercial ROCK inhibitors for reducing intraocular pressure include Ripasudil (K-<NUM>) and Netarsudil (AR-<NUM>). In the phase III clinical trial of Ripasudil, it was found that mild conjunctival hyperemia was the most common adverse reaction, with an incidence of up to <NUM>%, and conjunctivitis and punctate keratitis also occurred. Netarsudil (AR-<NUM>) is a ROCK/norepinephrine transporter (NET) inhibitor compound. In addition to acting as a ROCK inhibitor, it also has the effect of inhibiting norepinephrine and has the ability to continuously reduce intraocular pressure, and has good local tolerance, but it still has conjunctival hyperemia. In addition, Netarsudil (AR-<NUM>) in Phase III clinical trials ROCKET1 and ROCKET2 (total <NUM>,<NUM> patients) is not as effective as the conventional glaucoma treatment drug Timolol in patients with intraocular pressure > <NUM> mmHg, but in patients < <NUM> mmHg, the efficacy is comparable to Timolol.

Currently known ROCK inhibitors still have various side effects when applied to reducing intraocular pressure, and the long-term use of single-mechanism drugs may lead to the problem of reduced drug efficacy, making it impossible for patients to use a single drug for a long time. Therefore, it is still needed to develop new intraocular pressure reducing drugs with new targets or multiple targets to effectively reduce intraocular pressure and reduce side effects.

Document <CIT> describes compounds, compositions, and methods for treating diseases and conditions, wherein an inhibitor of a kinase, such as rho kinase (ROCK), and an inhibitor of one or more of the monoamine transporters, such as NET or SERT, act in concert to improve the condition.

Document <CIT> relates to certain amides and heterocyclic compounds and to the uses of these compounds to treat several diseases including autoimmune disorders, cardiovascular disorders, inflammation, central nervous system disorders, arterial thrombotic disorders, fibrotic disorders, glaucoma, and neoplastic disorders.

The full prescribing information on RHOPRESSA® "RHOPRESSA (netarsudil ophthalmic solution) Label, NDA, <NUM>, Reference ID: <NUM>" describes that the chemical compound (S)-<NUM>-(<NUM>-amino-<NUM>-(isoquinolin-<NUM>-yl-amino)-<NUM>-oxopropan-<NUM>-yl) benzyl <NUM>,<NUM>-dimethylbenzoate dimesylate is a Rho kinase inhibitor.

The inventon is defined by the features of the independent claims.

The present disclosure provides a compound represented by Formula (I) -(IV), a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form thereof:
<CHM>
<CHM>
<CHM>
<CHM>
The compound represented by Formulae (I) to (IV) are a β-amino acid derivative. In Formula (I) : X is a single bond; Y is NH; Z is C=O, C=S; W is CH; A is a single bond, O, OH, OCH<NUM>, or N<NUM>; R<NUM> is H or F; R<NUM> is H, F, OH, CF<NUM>, CH<NUM>OH, CHO or
<CHM>
and; n is <NUM> or <NUM>.

The present disclosure also provides a kinase inhibitor comprising the compound represented by Formulae (I)-(IV), or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form thereof mentioned above.

The present disclosure further provides a pharmaceutical composition comprising the compound represented by Formulae (I)-(IV), or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form thereof mentioned above.

Moreover, the present disclosure provides a compound represented by Formulae (I)-(IV), or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form thereof mentioned above for use as a kinase inhibitor.

The present disclosure also provides a use of the compound represented by Formulae (I)-(IV), or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form thereof mentioned above in the manufacture of a medicament, wherein the medicament is used for an in vivo related application that benefits from the inhibition of a kinase, and the kinase is at least one selected from a group consisting of: myosin light chain kinase <NUM>; mitogen-activated protein kinase <NUM>; and a Rho-associated protein kinase.

In addition, the present disclosure also provides a use of the compound represented by Formulae (I)-(IV), or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form thereof mentioned above in the manufacture of a medicament for reducing intraocular pressure.

The present disclosure provides a novel β-amino acid derivative and provides a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β-amino acid derivative at the same time.

The β-amino acid derivative mentioned above may comprise a compound represented by Formulae (I)-(IV), but it is not limited thereto:
<CHM>
<CHM>
<CHM>
<CHM>.

The compound represented by Formulae (I) to (IV) are a β-amino acid derivative. In Formula (I) X is a single bond; Y is NH; Z is C=O, C=S; W is CH; A is a single bond, O, OH, OCH<NUM>, or N<NUM>; R<NUM> is H or F; R<NUM> is H, F, OH, CF<NUM>, CH<NUM>OH, CHO or
<CHM>
and; n is <NUM> or <NUM>. In the present disclosure, the compound represented by Formulae (I)-(IV) mentioned above may be present in the form of the individual optical isomers, a mixture of the individual enantiomers or a racemate, and may comprise, but is not limited to, a compound shown in the following Table <NUM>.

In one embodiment, the compound represented by formula (I) of the present disclosure may be the Compound <NUM> mentioned above, which is a racemic compound. In another embodiment, the compound represented by formula (I) of the present disclosure may be the Compound <NUM> mentioned above, which is an S-form compound, and more specifically, is an S-enantiomer.

In one embodiment, the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative may have an effect of inhibiting a kinase, but it is not limited thereto. Example of the aforementioned kinase may comprise, but is not limited to, myosin light chain kinase <NUM> (MYLK-<NUM>), mitogen-activated protein kinase <NUM> (MAPK19, YSK-<NUM>), a Rho-associated protein kinase (ROCK) or any combination thereof. The Rho-associated protein kinase (ROCK) may comprise, but is not limited to, Rho-associated protein kinase-<NUM> (ROCK-<NUM>).

In one embodiment, the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative may have an effect of inhibiting myosin light chain kinase <NUM>.

Furthermore, the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative may have a synergistic target inhibiting effect. Therefore, in another embodiment, the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative may have an effect of inhibiting mitogen-activated protein kinase <NUM>.

Moreover, in another embodiment, the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative may have an effect of simultaneously inhibiting myosin light chain kinase <NUM> and a Rho-associated protein kinase.

Also, in another embodiment, in another embodiment, the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative may have an effect of simultaneously inhibiting mitogen-activated protein kinase <NUM> and a Rho-associated protein kinase.

In addition, the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative may have an effect of simultaneously inhibiting myosin light chain kinase <NUM>, mitogen-activated protein kinase <NUM> and a Rho-associated protein kinase.

In another embodiment, the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative may have an effect of reducing intraocular pressure. In a specific embodiment, the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative may achieve an effect of reducing intraocular pressure through myosin light chain kinase <NUM>, mitogen-activated protein kinase <NUM>, a Rho-associated protein kinase or any combination thereof.

Since the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative may have an effect of reducing intraocular pressure, it may be applied to a treatment and/or prevention of ocular hypertension or a disease with ocular hypertension. The above-mentioned ocular hypertension refers to a symptom of an intraocular pressure greater than a normal range. For example, human normal intraocular pressure is about <NUM>-<NUM> mmHg, and ocular hypertension is a symptom of intraocular pressure greater than about <NUM> mmHg, such as greater than about <NUM> mmHg, greater than about <NUM> mmHg, greater than about <NUM> mmHg, etc., but it is not limited thereto.

In one embodiment, the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative may be applied to a treatment and/or prevention of ocular hypertension with an intraocular pressure greater than about <NUM> mmHg, such as greater than about <NUM> mmHg.

Based on the foregoing, the present disclosure also provides a kinase inhibitor, which may comprise any of the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative, but it not limited thereto.

In the kinase inhibitor of the present disclosure, the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative may have an effect of inhibiting a kinase, and the kinase described herein may comprise, but is not limited to, myosin light chain kinase <NUM>, mitogen-activated protein kinase <NUM>, a Rho-associated protein kinase or any combination thereof. The Rho-associated protein kinase may comprise, but is not limited to, Rho-associated protein kinase-<NUM>.

In the kinase inhibitor of the present disclosure, in one embodiment, the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative may have an effect of inhibiting myosin light chain kinase-<NUM> and/or mitogen-activated protein kinase-<NUM>. In the kinase inhibitor of the present disclosure, in another embodiment, the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative, in addition to an effect of inhibiting myosin light chain kinase-<NUM> and/or mitogen-activated protein kinase-<NUM>, may also have an effect of inhibiting a Rho-associated protein kinase.

In one embodiment, the kinase inhibitor of the present disclosure may comprise the foregoing Compound <NUM> (which is a racemic compound) or Compound <NUM> (which is an S-form compound). In this specific embodiment, the kinase inhibitor of the present disclosure may have an effect of simultaneously inhibiting myosin light chain kinase <NUM>, mitogen-activated protein kinase <NUM> and a Rho-associated protein kinase.

In addition, based on the foregoing, the present disclosure can also provide a use of the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative as a kinase inhibitor.

In the use of the present disclosure, regarding the relevant description for the kinase inhibitory effect of the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative is the same as that described above, and thus is not repeated herein.

In this use of the present disclosure, in a specific embodiment, the novel β amino acid derivative of the present disclosure mentioned above may be the foregoing Compound <NUM> (which is a racemic compound) or Compound <NUM> (which is an S-form compound). In this specific embodiment, the kinase inhibitor mentioned above may have an effect of simultaneously inhibiting myosin light chain kinase <NUM>, mitogen-activated protein kinase <NUM> and a Rho-associated protein kinase.

In addition, the present disclosure also provides a pharmaceutical composition, which may comprise, but is not limited to, any of the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative, but it not limited thereto.

In the pharmaceutical composition of the present disclosure, regarding all for the kinase inhibitory effect of the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative are the same as those described above, and thus are not repeated herein.

In one embodiment, the pharmaceutical composition of the present disclosure may comprise the foregoing Compound <NUM> which is a racemic compound. In another embodiment, the pharmaceutical composition of the present disclosure may comprise the foregoing Compound <NUM> which is an S-form compound.

Moreover, in one embodiment, the above-mentioned pharmaceutical composition of the present disclosure may also comprise a pharmaceutically acceptable carrier or salt, but it is not limited thereto.

The pharmaceutically acceptable carrier mentioned above may comprise, but is not limited to, a solvent, a dispersion medium, a coating, an antibacterial and antifungal agent, or an isotonic and absorption delaying agent, etc. which is suitable for pharmaceutical administration. The pharmaceutical composition can be formulated into dosage forms for different administration routes utilizing conventional methods.

Moreover, the pharmaceutically acceptable salt mentioned above may comprise, but is not limited to, salts including inorganic cation, such as alkali metal salts such as sodium salt, potassium salt or amine salt, such as alkaline-earth metal salt such as magnesium salt or calcium salt, such as the salt containing bivalent or quadrivalent cation such as zinc salt, aluminum salt or zirconium salt. In addition, the pharmaceutically acceptable salt may also be organic salt, such as dicyclohexylamine salt, methyl-D-glucamine, and amino acid salt such as arginine, lysine, histidine, or glutamine.

Furthermore, the pharmaceutical composition of the present disclosure can be administered to a subject in need thereof, but is not limited thereto. The administration route of the pharmaceutical composition of the present disclosure may include parenteral manner, oral manner, via inhalation spray, or by implanted reservoir, but is not limited thereto. The parenteral methods may comprise, but is not limited to, smearing affected region, subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intraleaional injection, external ophthalmic use, and intraocular injection, as well as infusion techniques, etc..

Topical use form for smearing may include ointment, emulsion, liquid, gel, etc., but it is not limited thereto. In addition, external use form for eye may include, but is not limited to, eye drops, eye ointment, eye gel, etc..

The subject in need to be administrated the pharmaceutical composition mentioned above may comprise, but is not limited to, a vertebrate. The vertebrate mentioned above may comprise a fish, an amphibian, a reptile, a bird or a mammal, but it is not limited thereto. Examples of the mammal may comprise, but are not limited to a human, an orangutan, a monkey, a horse, a donkey, a dog, a cat, a rabbit, a guinea pig, a rat and a mouse. In one embodiment, the said subject may be a human.

In one embodiment, the above-mentioned pharmaceutical composition of the present disclosure can be used to any in vivo-related application that benefits from the inhibition of a kinase, such as benefiting from treatment and/or prevention of any disease or symptom that benefits from the inhibition of a kinase, and example of the kinase described herein may include, but are not limited to, myosin light chain kinase-<NUM>, mitogen-activated protein kinase-<NUM>, a Rho-associated protein kinase, or any combination thereof.

In addition, the aforementioned in vivo-related application may include, but are not limited to, an ophthalmology-related application and/or a lung-related application, etc. Example of the ophthalmology-related application may include, protection of optic nerve, and/or prevention and/or treatment of high intraocular pressure, glaucoma, ocular stroke, macular degeneration, macular edema, diabetic retinopathy, Fuchs endothelial corneal dystrophy (FECD), corneal fibrosis or any combination thereof, etc., but it is not limited to thereto. Among them, glaucoma may include exfoliation glaucoma (XFG), open angle glaucoma, angle-closure glaucoma, secondary glaucoma, congenital glaucoma, etc., but not it is not limited to thereof. In addition, example of the lung-related application mentioned above may include, but is not limited to, prevention and/or treatment of pulmonary hypertension, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), pulmonary emphysema, lung cancer, or any combination thereof, etc..

Moreover, the pharmaceutical composition of the present disclosure can be formulated into a pharmaceutical preparation, but is not limited thereto. In one embodiment, the pharmaceutical composition of the present disclosure can be formulated into an ophthalmic preparation, but it is not limited thereto. Example of the aforementioned ophthalmic preparation mentioned above may include, but is not limited to, an eye drop, an ophthalmic ointment, an ophthalmic gel, an intraocular injection formulation, etc. In a specific embodiment, the pharmaceutical composition of the present disclosure can be formulated into an eye drop.

In addition, the present disclosure can also provide a use of any of the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative in the manufacture of a medicament, wherein the medicament is used for an in vivo related application that benefits from the inhibition of a kinase. Example of the above-mentioned kinase may include, but is not limited to, myosin light chain kinase-<NUM>, mitogen-activated protein kinase-<NUM>, a Rho-associated protein kinase, or any combination thereof.

In one embodiment, in the above-mentioned use of the present disclosure, what is used is Compound <NUM>, which is a racemic compound. In one embodiment, in the above-mentioned use of the present disclosure, what is used is the Compound <NUM>, which is an S-form compound.

Furthermore, in the above-mentioned use of the present disclosure, the foregoing in vivo-related application may include, but are not limited to, an ophthalmology-related application and/or a lung-related application, etc. Example of the ophthalmology-related application may include, protection of optic nerve, and/or prevention and/or treatment of high intraocular pressure, glaucoma, ocular stroke, macular degeneration, macular edema, diabetic retinopathy, Fuchs endothelial corneal dystrophy (FECD), corneal fibrosis or any combination thereof, etc., but it is not limited to thereto. Among them, glaucoma may include exfoliation glaucoma, open angle glaucoma, angle-closure glaucoma, secondary glaucoma, congenital glaucoma, etc., but not it is not limited to thereof. In addition, example of the lung-related application mentioned above may include, but is not limited to, prevention and/or treatment of pulmonary hypertension, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), pulmonary emphysema, lung cancer, or any combination thereof, etc..

In one embodiment, in the above-mentioned use of the present disclosure, the foregoing in vivo-related application may be the prevention and/or treatment of glaucoma.

In the above-mentioned use of the present disclosure, in another embodiment, a pharmaceutically acceptable carrier or salt may be together with any of the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative to prepare the medicament mentioned above.

With regard to the pharmaceutically acceptable carrier or salt described herein, please refer to the relevant description of the pharmaceutically acceptable carrier or salt in the pharmaceutical composition of the present disclosure above, and thus is not repeated herein.

Moreover, the present disclosure also provides a use of any of the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative in the manufacture of a medicament for reducing intraocular pressure.

Furthermore, in one embodiment, in the above-mentioned use of the present disclosure, the foregoing medicament for reducing intraocular pressure may be used for prevention and/or treatment of ocular hypertension or a disease with ocular hypertension. Regarding the relevant descriptions of the ocular hypertension or a disease with ocular hypertension, please refer to the above descriptions, and thus are not repeated herein.

In one embodiment, in the above-mentioned use of the present disclosure, the foregoing medicament for reducing intraocular pressure may be a medicament for treating glaucoma. The glaucoma mentioned above may include, but is not limited to, exfoliation glaucoma, open angle glaucoma, angle-closure glaucoma, secondary glaucoma, congenital glaucoma, etc..

In addition, in one embodiment, in the above-mentioned use of the present disclosure, the foregoing medicament for reducing intraocular pressure may be an ophthalmic preparation. The ophthalmic preparations may include, but are not limited to, an eye drop, an ophthalmic ointment, an ophthalmic gel, an intraocular injection formulation, etc. In a specific embodiment, the foregoing medicament for reducing intraocular pressure may be may be an eye drop.

In the above-mentioned use of the present disclosure, in another embodiment, a pharmaceutically acceptable carrier or salt may be together with any of the novel β amino acid derivative of the present disclosure mentioned above or a pharmaceutically acceptable salt or ester, hydrate, solvate or crystalline form of the novel β amino acid derivative to prepare the medicament for reducing intraocular pressure mentioned above.

The synthesis scheme of Compound <NUM> and Compound <NUM> is shown in the following Scheme <NUM>. <CHM>
<CHM>.

To a mixture of <NUM>-bromo-<NUM>-nitrobenzene (<NUM>, <NUM> mmol), <NUM>-Boc-<NUM>-pyrazoleboronic acid pinacol ester (<NUM>, <NUM> mmol), PdCl<NUM>(dppf) (<NUM>, <NUM>µmol) and Cs<NUM>CO<NUM> (<NUM>, <NUM> mmol) in a sealed tube, a mixed solvent (dioxane/H<NUM>O =<NUM>/<NUM>, <NUM>) was injected under argon, and then the mixture mentioned above was stirred at <NUM> for <NUM> hours. After cooling to room temperature, the solvent was removed by rotary evaporation, and the residue was added with water and extracted with EtOAc (<NUM> × <NUM>). The combined organic layers were washed with brine, dried over anhydrous Na<NUM>SO<NUM> and filtered. The filtrate was concentrated and the residue was purified by flash chromatography (EtOAc/Hex = <NUM>%) on silica gel to give tert-butyl <NUM>-(<NUM>-(<NUM>-(dimethylamino)ethoxy)-<NUM>-nitrophenyl)-<NUM>-pyrazole (1a) which was a white solid (<NUM>, <NUM> %).

To a solution of tert-butyl <NUM>-(<NUM>-(<NUM>-(dimethylamino)ethoxy)-<NUM>-nitrophenyl)-<NUM>-pyrazole (1a) (<NUM>) in MeOH (<NUM>), <NUM>% Pd/C was added, and the reaction mixture mentioned above was stirred at room temperature under H<NUM> balloon atmosphere for <NUM> hour. The mixture was filtered, and the filtrate was evaporated by rotary evaporation to give tert-butyl-(<NUM>-amino-<NUM>-(<NUM>-(dimethylamino)ethoxy)phenyl)-<NUM>-pyrazole (1b) as brown solid (<NUM>, <NUM> %). <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

<NUM>-azido-<NUM>-((tert-butoxycarbonyl)amino)propanoic acid (<NUM> eq), HATU (<NUM> eq) and DIPEA (<NUM> eq) were dissolved in DMF (<NUM>), and tert-butyl-(<NUM>-amino-<NUM>-(<NUM>-(dimethylamino)ethoxy)phenyl)-<NUM>-pyrazole (1b) (<NUM> eq) was added thereto to form a mixture and stirred at room temperature for <NUM> hour. The reaction was workup by water and extracted with EtOAc. The organic layer was collected and dried over Na<NUM>SO<NUM>, and the extract was condensed under reduced pressure. The residue was purified with silica gel (EtOAc /Hex = <NUM>%), and the desired compound tert-butyl <NUM>-(<NUM>-(<NUM>-azido-<NUM>-((tert-butoxycarbonyl)amino)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (1c) was given. <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J =<NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

<NUM> HCl in <NUM>,<NUM>-dioxane (<NUM> eq) and tert-butyl <NUM>-(<NUM>-(<NUM>-azido-<NUM>-((tert-butoxycarbonyl) amino)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (1c) (<NUM> eq) were stirred at room temperature for <NUM> hour. The white solid was collected by filtration, washed with <NUM>,<NUM>-dioxane and DCM. The white solid was dried by vacuum to give N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-azidopropanamide dihydrochloride (<NUM>). <NUM>H-NMR (<NUM>, D2O) δ: <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM> (m, <NUM>).

Tert-butyl <NUM>-(<NUM>-(<NUM>-azido-<NUM>-((tert-butoxycarbonyl)amino)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (1c) (<NUM> eq), phenyl acetylene (<NUM> eq), copper(II) sulfate (<NUM> eq) and (+)-Na-L-ascorbate (<NUM> eq) were stirred in THF and <NUM>-<NUM> drops H<NUM>O. The mixture was stirred at room temperature overnight, and then the solvent was removed. The residue was purified with silica gel (EtOAc /Hex = <NUM>%), and the desired compound tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-phenyl-<NUM>-<NUM>,<NUM>,<NUM>-triazol-<NUM>-yl) propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate was given. <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

The preparation method for Compound <NUM> was similar to that for Compound <NUM>. The distinction therebetween was that the compound tert-butyl <NUM>-(<NUM>-(<NUM>-azido-<NUM>-((tert-butoxycarbonyl)amino)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate was replace with the compound tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-phenyl-<NUM>-<NUM>,<NUM>,<NUM>-triazol-<NUM>-yl)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate to obtain the product N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-(<NUM>-phenyl-<NUM>-<NUM>,<NUM>,<NUM>-triazol-<NUM>-yl) propenamide dihydrochloride (<NUM>). <NUM>H-NMR (<NUM>, CD<NUM>OD) δ: <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>).

The synthesis scheme of Compound <NUM> to Compound <NUM> is shown in the following Scheme <NUM>. <CHM>
<CHM>.

The preparation method for Compound 3a was similar to that for Compound 1c. The distinction therebetween was that the compound <NUM>-azido-<NUM>-((tert-butoxycarbonyl)amino)propanoic acid was replace with the compound <NUM>-((tert-butoxycarbonyl)amino)-<NUM>-hydroxypropanoic acid to obtain the product tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-hydroxypropanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (3a). The synthesis method for the final product N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-hydroxypropanamide dihydrochloride (<NUM>) synthesis was similar to that for Compound <NUM>. <NUM>H-NMR (<NUM>, D<NUM>O) δ: <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>).

To a zero degree solution of compound 3a (1eq), phenol (<NUM> eq), PPh<NUM> (<NUM> eq) in <NUM> THF, DIAD (<NUM> eq) was added dropwise. The mixture mentioned above was stirred at room temperature for <NUM> hours. The solvent was removed by reduced pressure, and the crude product was purified by column chromatography (EtOAc/Hex = <NUM>%) to get tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-phenoxypropanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (4a) which was an oil. <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (brs, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>). The synthesis method for the final product N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-phenoxypropanamide dihydrochloride (<NUM>) was similar to that for Compound <NUM>. <NUM>H-NMR (<NUM>, CD<NUM>OD) δ: <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>).

The synthesis method for the compound N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-(<NUM>-(trifluoromethyl) phenoxy)propenamide dihydrochloride (<NUM>) was similar to that for Compound <NUM>. <NUM>H-NMR (<NUM>, CD<NUM>OD) δ: <NUM> (br, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>). <NUM>-NMR (<NUM>, CD<NUM>OD) δ: <NUM> (br, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>).

The synthesis scheme of Compound <NUM> is shown in the following Scheme <NUM>:
<CHM>
<CHM>.

To a zero degree solution of compound 3a (<NUM> eq) in <NUM> THF, NaH (<NUM> eq) was add. The mixture mentioned above was stirred at zero degree for <NUM> hour, and then BnBr (<NUM> eq) was added dropwise thereto. The reaction mixture mentioned above was stirred at room temperature for <NUM> hours. The reaction was workup by water and extracted with EtOAc. The organic layer was collected and dried over Na<NUM>SO<NUM>, and then the extract was removed under reduced pressure. The residue was purified with silica gel (EtOAc /Hex = <NUM>% to <NUM>%) to give the desired compound tert-butyl <NUM>-(<NUM>-(<NUM>-(benzyloxy)-<NUM>-((tert-butoxycarbonyl)amino)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (6a). <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (brs, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

The synthesis method for the final product N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-(benzyloxy) propanamidedihydrochloride (<NUM>) was similar to that for Compound <NUM>. <NUM>H-NMR (<NUM>, CD<NUM>OD) δ: <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>).

The synthesis scheme of Compound <NUM> and Compound <NUM> is shown in the following Scheme <NUM>.

The preparation methods for the intermediate compounds 7a and 8a were similar to that for Compound 1c.

The intermediate compound tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-phenylpropanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate 7a was white powder. <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

The intermediate compound tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-fluorophenyl)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate 8a was white powder. <NUM>H-NMR (CDCl<NUM>, <NUM>) <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM> (brs, NH, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

The preparation methods for the final Compounds <NUM> and <NUM> were similar to that for Compound <NUM>.

The final compound N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-phenylpropanamide dihydrochloride (<NUM>) was white powder. <NUM>H-NMR (<NUM>, CD<NUM>OD) δ: <NUM> (br, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM> (m, <NUM>).

The final compound N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-(<NUM>-fluorophenyl) propanamide dihydrochloride (<NUM>) was white powder. <NUM>H-NMR (<NUM>, D<NUM>O) δ: <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>).

The synthetic scheme of Compound 9f is shown in the following Scheme <NUM>.

To a solution of iodophenol (9a) (<NUM>, <NUM> mmol) in DCM (<NUM>), imidazole (<NUM>, <NUM> mmol) was added, followed by dropwise addition of TIPSCl (<NUM>, <NUM> mmol). The mixture mentioned above was stirred for <NUM> hours and poured into ice/water, and extracted with DCM. The organic layers were washed with brine, dried over Na<NUM>SO<NUM> and concentrated. The residue was purified by silica gel column chromatography (eluting solvent: ethyl acetate/petroleum ether = <NUM>/<NUM>) to give Compound 9b (<NUM>, <NUM> %), which was colorless oil. <NUM>H-NMR (CDCl<NUM>, <NUM>): δ: <NUM>-<NUM> (d, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>).

To a solution of Compound 9b (<NUM>, <NUM> mmol) in dioxane (<NUM>), ethyl <NUM>-cyanoacetate (<NUM>, <NUM> mmol), picolinic acid (<NUM>, <NUM> mmol), Cs<NUM>CO<NUM> (<NUM>, <NUM> mmol) and CuI (<NUM>, <NUM> mmol) were added. The mixture was mentioned above stirred for <NUM> hours at <NUM> and filtered. The organic layer was concentrated and purified by silica gel column chromatography (eluting solvent: ethyl acetate/petroleum ether = <NUM>/<NUM>) to give Compound 9c (<NUM>, <NUM> %), which was white solid.

To a solution of compound 9c (<NUM>, <NUM> mmol) in MeOH/THF (<NUM>:<NUM>, <NUM>), CoCl<NUM><NUM><NUM>O (<NUM>, <NUM> mmol) and NaBH<NUM> (<NUM>, <NUM> mmol) were added at -<NUM>. The mixture mentioned above was stirred for <NUM> minutes and filtered to obtain a filtrate. To the filtrate, (Boc)<NUM>O (<NUM>, <NUM> mmol) was added, and stirred for <NUM> hours at room temperature. Next, the mixture mentioned above was poured into ice/water and extracted with EtOAc. The organic layer was washed with brine, dried over Na<NUM>SO<NUM> and concentrated. The residue was purified by silica gel column chromatography (eluting solvent: ethyl acetate/ petroleum ether = <NUM>/<NUM>) to give compound 9e (<NUM>, <NUM> %), which was yellow oil. <NUM>H-NMR (CDCl<NUM>, <NUM>): δ: <NUM>-<NUM> (t, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (t, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (d, <NUM>), <NUM>-<NUM> (d, <NUM>).

To a solution of Compound 9e (<NUM>, <NUM> mmol) in EtOH/H<NUM>O (<NUM>:<NUM>, <NUM>), NaOH (<NUM>, <NUM> mmol) was added and stirred for <NUM> hour at room temperature. The mixture mentioned above was neutralized with <NUM> N HCl and extracted with EtOAc. The organic layer was washed with brine, dried over Na<NUM>SO<NUM> and concentrated. The residue was purified by silica gel column chromatography (eluting solvent: ethyl acetate/petroleum ether = <NUM>/<NUM>) to give compound 9f (<NUM>, <NUM> %), which was a white solid. <NUM>H-NMR (DMSO-d6, <NUM>): δ: <NUM>-<NUM> (d, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (t, <NUM>), <NUM>-<NUM> (t, <NUM>), <NUM>-<NUM> (d, <NUM>), <NUM>-<NUM> (d, <NUM>).

The synthesis scheme of Compound <NUM> is shown in the following Scheme <NUM>.

The preparation method for the intermediate Compounds <NUM> was similar to that for Compound 1c.

The intermediate compound tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-((triisopropylsilyl)oxy)phenyl)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate <NUM> was white powder. <NUM>H-NMR (CDCl<NUM>, <NUM>) <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (brs, NH, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (brs, NH, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>).

To a stirred solution of tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-((triisopropylsilyl)oxy)phenyl)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (<NUM>) (<NUM>, <NUM> mmol) in THF (<NUM>), <NUM> TBAF in THF solution (<NUM>, <NUM> mmol) was added. The resulting mixture was stirred at room temperature for <NUM> hour. The reaction was workup by water and extracted with EtOAc. The organic layer was collected and dried over Na<NUM>SO<NUM>, and the extract was condensed under reduced pressure. The residue was purified with preparative reverse HPLC (<NUM> % ACN, <NUM> % H<NUM>O), and then lyophilized to get tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-hydroxyphenyl)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (<NUM>) (<NUM>, <NUM> %), which was white powder. <NUM>H-NMR (CDCl<NUM>, <NUM>) <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (brs, NH, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (brs, OH, <NUM>), <NUM> (s, <NUM>).

The preparation method for the final Compound <NUM> was similar to that for Compound <NUM>.

The final compound N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-(<NUM>-hydroxyphenyl) propanamide dihydrochloride (<NUM>) was white powder. <NUM>H-NMR (<NUM>, D<NUM>O) δ: <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>).

The synthesis scheme of compound <NUM> is shown in the following Scheme <NUM>. <CHM>
<CHM>.

To a stirred solution of tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-hydroxyphenyl)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (<NUM>) (<NUM>, <NUM> mmol), <NUM>,<NUM>-dimethylbenzoic acid (<NUM>, <NUM> mmol) and NEt<NUM> (<NUM> ul, <NUM> mmol) in CH<NUM>Cl<NUM> (<NUM>), EDCI (<NUM>, <NUM> mmol) and DMAP (<NUM>, <NUM> mmol) were added. The resulting mixture was stirred for <NUM> hours. The reaction was workup by saturated citric acid and extracted with EtOAc. The organic layer was collected and dried over Na<NUM>SO<NUM>, and the solvent was removed under reduced pressure. The residue was purified with preparative reverse HPLC (<NUM> % ACN), and then lyophilized to get tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-((<NUM>,<NUM>-dimethylbenzoyl)oxy)phenyl)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (10a) (<NUM>, <NUM> %), which was white powder. <NUM>H-NMR (CDCl<NUM>, <NUM>) <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (brs, NH, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

In the <NUM> vial, tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-((<NUM>,<NUM>-dimethylbenzoyl)oxy)phenyl)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (10a) (<NUM>, <NUM> mmol) and <NUM> HCl in dioxane (<NUM>) were charged. The resulting mixture was stirred at room temperature for <NUM> hour. The solvent was removed under reduced pressure to give the desired product <NUM>-(<NUM>-((<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)amino)-<NUM>-amino-<NUM>-oxopropan-<NUM>-yl)phenyl <NUM>,<NUM>-dimethylbenzoate dihydrochloride (<NUM>) which was as white powder (<NUM>, <NUM> %). <NUM>H-NMR (<NUM>, DMSO) δ: <NUM> (s, <NUM>), <NUM> (brs, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

To a zero degree solution of <NUM>-(<NUM>-(hydroxymethyl)phenyl)acetic acid (<NUM>, <NUM> mmol) in CH<NUM>Cl<NUM> (<NUM>) and MeOH (<NUM>), TMS diazomethane (<NUM> of a <NUM> solution in hexanes, <NUM> mmol) was added dropwise. After <NUM> minutes, the reaction was quenched by the addition of HOAc (<NUM>). The reaction mentioned above was diluted with EtOAc (<NUM>) and washed with saturated NaHCO<NUM> (<NUM> x <NUM>) and brine (<NUM>). The organic layer was dried (Na<NUM>SO<NUM>), filtered and concentrated. The residue was purified by flash chromatography (<NUM> to <NUM> percent EtOAc/hexanes) to afforded methyl <NUM>-(<NUM>-(hydroxymethyl)phenyl)acetate (11b). <NUM>H-NMR (<NUM>, CDCl<NUM>): δ: <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

To a zero degree solution of methyl <NUM>-(<NUM>-(hydroxymethyl)phenyl)acetate (11b) (<NUM>, <NUM> mmol) in CH<NUM>Cl<NUM> (<NUM>), <NUM>,<NUM>-lutidine (<NUM>, <NUM> mmol) and TIPS-OTf (<NUM>, <NUM> mmol) were added. The ice bath was removed and the solution was allowed to warm to room temperature and stirred. After <NUM> hours, the reaction was quenched by the addition of NH<NUM>Cl(aq) (<NUM>). The reaction was diluted with CH<NUM>Cl<NUM> (<NUM>) and washed with H<NUM>O (<NUM> x <NUM>) and brine (<NUM>). The organic layer was dried (Na<NUM>SO<NUM>), filtered and concentrated. The residue was purified by flash chromatography (<NUM> to <NUM> percent EtOAc/hexanes) to afforded methyl <NUM>-(<NUM>-(((triisopropylsilyl)oxy)methyl)phenyl)acetate (11c). <NUM>H-NMR (<NUM>, CDCl<NUM>): δ <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>).

To a -<NUM> solution of methyl <NUM>-(<NUM>-(((triisopropylsilyl)oxy)methyl)phenyl)acetate (11c) (<NUM>, <NUM> mmol) in THF (<NUM>), LiHMDS (<NUM>, <NUM> mmol) was added dropwise. After <NUM> minutes, the bromo-methyl phthalimide (<NUM>, <NUM> mmol) in THF (<NUM>) was added dropwise at the same temperature. The -<NUM> bath was removed and the solution was allowed to warm to room temperature and stirred. After <NUM> hours, the reaction was quenched by the addition of NH<NUM>Cl(aq) (<NUM>) and extracted with EtOAc (<NUM>). The organic layer was dried (Na<NUM>SO<NUM>), filtered and concentrated. The residue was purified by flash chromatography (<NUM> to <NUM> percent EtOAc/hexanes) to afforded methyl <NUM>-(<NUM>,<NUM>-dioxoisoindolin-<NUM>-yl)-<NUM>-(<NUM>-(((triisopropylsilyl)oxy)methyl)phenyl) propanoate (11d). <NUM>H-NMR (<NUM>, CDCl<NUM>): δ <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>).

To a stirred solution of methyl <NUM>-(<NUM>,<NUM>-dioxoisoindolin-<NUM>-yl)-<NUM>-(<NUM>-(((triisopropylsilyl) oxy)methyl)phenyl)propanoate (11d) (<NUM>, <NUM> mmol) in MeOH (<NUM>) and EtOH (<NUM>), hydrazine hydrate (<NUM>, <NUM> mmol) was added, and the solution was refluxed for <NUM> hours. The solids were filtered and the solvents were evaporated. The residue was purified by flash chromatography (<NUM> to <NUM> percent EtOAc/hexanes) to afforded methyl <NUM>-amino-<NUM>-(<NUM>-(((triisopropylsilyl)oxy)methyl)phenyl)propanoate (11e).

To a stirred solution of methyl <NUM>-amino-<NUM>-(<NUM>-(((triisopropylsilyl)oxy)methyl) phenyl)propanoate (11e) (<NUM>, <NUM> mmol) in CH<NUM>Cl<NUM> (<NUM>), TEA (<NUM>, <NUM> mmol) and (Boc)<NUM>O (<NUM>) were added. The solution was stirred at room temperature for <NUM> hours, and then poured into CH<NUM>Cl<NUM>/NaHCO<NUM>(aq). The aqueous layers were further extracted with CH<NUM>Cl<NUM>, dried (Na<NUM>SO<NUM>), filtered and evaporated. The residue was purified by flash chromatography (<NUM> to <NUM> percent EtOAc/hexanes) to afforded methyl <NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-(((triisopropylsilyl)oxy)methyl)phenyl)propanoate (11f). <NUM>H-NMR (<NUM>, CDCl<NUM>): δ <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>).

To a solution of methyl <NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-(((triisopropylsilyl) oxy)methyl)phenyl)propanoate (11f) (<NUM>, <NUM> mmol) in THF/MeOH (<NUM>:<NUM>, <NUM>), NaOH (<NUM>, <NUM> mmol) was added, and stirred for <NUM> hours at room temperature. The mixture was neutralized with <NUM> N HCl and extracted with EtOAc. The organic layer was washed with brine, dried over Na<NUM>SO<NUM> and concentrated. The residue was purified by silica gel column chromatography (eluting solvent: EtOAc/Hexanes = <NUM>/<NUM>) to give <NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-(((triisopropylsilyl)oxy)methyl)phenyl)propanoic acid (<NUM>) (<NUM>, <NUM> %), which was a white solid. <NUM>H-NMR (<NUM>, CDCl<NUM>): δ <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (brs, <NUM>), <NUM> (brs, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>).

The synthesis method for the final Compound <NUM> was similar to that for Compound <NUM>.

The compound tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-(((triisopropylsilyl)oxy)methyl)phenyl)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (<NUM>) was white powder. <NUM>H-NMR (<NUM>, CDCl<NUM>): δ <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (brs, <NUM>), <NUM> (s, <NUM>), <NUM> (brs, <NUM>), <NUM> (brs, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>).

The compound tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-(hydroxymethyl)phenyl)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (11i) was white powder. <NUM>H-NMR (<NUM>, CDCl<NUM>): δ <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (brs, <NUM>), <NUM> (brs, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>).

The final compound N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-(<NUM>-(hydroxymethyl) phenyl)propenamide dihydrochloride (<NUM>) was white powder. <NUM>H-NMR (<NUM>, D<NUM>O) δ: <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>).

The compound tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-(((<NUM>,<NUM>-dimethylbenzoyl)oxy)methyl)phenyl)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (12a) was white powder. <NUM>H-NMR (CDCl<NUM>, <NUM>) <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (brs, <NUM>), <NUM> (brs, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

The compound <NUM>-(<NUM>-((<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)amino)-<NUM>-amino-<NUM>-oxopropan-<NUM>-yl) benzyl <NUM>,<NUM>-dimethylbenzoate dihydrochloride (<NUM>) was white powder. <NUM>H-NMR (<NUM>, DMSO) δ: <NUM> (s, NH, <NUM>), <NUM> (brs, NH, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

To a stirred solution of tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-(hydroxymethyl)phenyl)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (11i) (<NUM>, <NUM> mmol) in CH<NUM>Cl<NUM> (<NUM>), Dess-Martin reagent (<NUM>, <NUM> mmol) was added. The resultant solution was stirred at room temperature for <NUM> hour. The solvent was evaporated under reduced pressure, and the crude product was purified by column chromatography (EtOAc/Hexanes = <NUM>% to <NUM>%) to get tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-formylphenyl)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (13a) which was white powder. <NUM>H-NMR (CDCl<NUM>, <NUM>) <NUM> (s, CHO, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (brs, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (brs, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

In the <NUM> vial, tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-formylphenyl)propanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (13a) (<NUM>, <NUM> mmol) and <NUM> HCl in dioxane (<NUM>) was charged. The resulting mixture was stirred at room temperature for <NUM> hour. The solvent was removed under reduced pressure to give the desired product N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-(<NUM>-formylphenyl)propanamide dihydrochloride (<NUM>) (<NUM>, <NUM> %). <NUM>H-NMR (<NUM>, D<NUM>O) δ: <NUM> (s, CHO, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>).

A mixture of tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-phenylpropanamido)phenyl)-<NUM>H-pyrazole-<NUM>-carboxylate (7a) (<NUM>, <NUM> mmol, <NUM> equiv) and Lawesson reagent (<NUM>, <NUM> mmol, <NUM> equiv) in toluene (<NUM>) was heated to <NUM> for <NUM> hours under N<NUM> atmosphere. The reaction mixture mentioned above was cooled and concentrated to afford the crude compound tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl) amino)-<NUM>-phenylpropanethioamido)phenyl)-<NUM>H-pyrazole-<NUM>-carboxylate (14a) (<NUM>, yield: <NUM>%) which could be used to the next step without further purification. LCMS (ES, m/z): [M +H]+=<NUM>.

Tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-phenylpropanethioamido)phenyl)-<NUM>H-pyrazole-<NUM>-carboxylate (14a) (<NUM>, <NUM> mmol, <NUM> eq) was dissolved in <NUM> MeOH to be added to HCl/MeOH (<NUM>, <NUM>), and stirred at room temperature for <NUM> hours. The reaction mixture mentioned above was concentrated. The residue was purified by flash (DCM/MeOH from <NUM>% to <NUM>%) to obtain the crude product (<NUM>), and that was crystallized with DCM and MeOH and adjusted pH to <NUM> with <NUM> N HCl to afford N-(<NUM>-(<NUM>H-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-phenylpropanethioamide dihydrochloride (Compound <NUM>) (<NUM>, yield: <NUM>% for two steps), which was a yellow solid. LCMS(ES, m/z): [M+H]+=<NUM>. <NUM>H-NMR (<NUM>, DMSO-d<NUM>): <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>).

The synthesis scheme of Compound <NUM> is shown in the following Scheme <NUM>. <CHM>
<CHM>.

To the solution of tert-butyl-(<NUM>-amino-<NUM>-(<NUM>-(dimethylamino)ethoxy)phenyl)-<NUM>-pyrazole (<NUM>, <NUM> mmol, <NUM> eq) in CH<NUM>Cl<NUM> (<NUM>), a saturated solution of NaHCO<NUM> (<NUM>, <NUM>) was added. Next, the thiophosgene (<NUM>, <NUM> mmol, <NUM> eq) was added slowly and stirred for <NUM> hours. The organic layer was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to obtain a crude product 15a. In the residue 15a, tert-butyl (<NUM>-amino-<NUM>-phenylethyl)carbamate (<NUM>, <NUM> mmol, <NUM> eq) and K<NUM>CO<NUM> (<NUM>, <NUM> mmol, <NUM> eq) in CH<NUM>Cl<NUM> (<NUM>) were added. The reaction was workup by water and extracted with CH<NUM>Cl<NUM>. The organic layer was collected and dried over Na<NUM>SO<NUM>, the extract solution was condensed under reduced pressure. The residue was purified with silica gel (EtOAc/Hex = <NUM>%) to obtain the compound tert-butyl <NUM>-(<NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-phenylethyl)thioureido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (15b) (<NUM>, <NUM> %) which was a pale yellow solid. <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

In the <NUM> vial, the compound tert-butyl <NUM>-(<NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-phenylethyl)thioureido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (15b) (<NUM>, <NUM> mmol) and <NUM> HCl in dioxane (<NUM>) were charged. The resulting mixture was stirred at room temperature for <NUM> hour. The solvent was removed under reduced pressure to obtain the compound <NUM>-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-(<NUM>-amino-<NUM>-phenylethyl)thiourea dihydrochloride (<NUM>) which presented as pale yellow powder (<NUM>, <NUM> %). <NUM>H-NMR (<NUM>, CD<NUM>OD) δ: <NUM> (brs, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>).

For illustrative purpose the synthesis of compound <NUM> not falling under the scope of claim <NUM> is explained. (<NUM>) Synthesis example <NUM>.

The synthesis Scheme of Compound <NUM> is shown in the following Scheme <NUM>.

A mixture of tert-butyl <NUM>-(<NUM>-aminophenyl)-<NUM>H-pyrazole-<NUM>-carboxylate (1b) (<NUM>, <NUM> mmol, <NUM> equiv) and (isothiocyanatomethyl)benzene (<NUM>, <NUM> mmol, <NUM> equiv) in t-BuOH (<NUM>) was stirred at <NUM> overnight under N<NUM>. The reaction mixture mentioned above was cooled and concentrated. The residue was purified by silica gel column and eluted with ethyl acetate/petroleum ether (<NUM>:<NUM>) to afford tert-butyl <NUM>-(<NUM>-(<NUM>-benzylthioureido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (16a) (<NUM>, yield: <NUM>%), which was a yellow solid. LCMS(ES, m/z): [M+H]+ = <NUM>.

A mixture of tert-butyl <NUM>-(<NUM>-(<NUM>-benzylthioureido)phenyl)-<NUM>H-pyrazole-<NUM>-carboxylate (16a) (<NUM>, <NUM> mmol, <NUM> equiv) and CH<NUM>I (<NUM>, <NUM> mmol, <NUM> equiv) in acetone (<NUM>) was stirred at <NUM> for <NUM> hours under N<NUM>. The reaction mixture mentioned above was filtered. The filter cake was collected to afford tert-butyl <NUM>-(<NUM>-(((benzylamino)(methylthio)methylene)amino)phenyl)-<NUM>H-pyrazole -<NUM>-carboxylate (16b) (<NUM>, yield: <NUM>%) as a white solid. LCMS (ES, m/z): [M+H]+=<NUM>.

A mixture of tert-butyl <NUM>-(<NUM>-(((benzylamino)(methylthio)methylene)amino)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (16b) (<NUM>, <NUM> mmol, <NUM> equiv), cyanamide (<NUM>, <NUM> mmol, <NUM> equiv) and <NUM>,<NUM>-diazabicyclo[<NUM>. <NUM>]octane (<NUM>, <NUM> mmol, <NUM> equiv) in t-BuOH (<NUM>) was stirred at <NUM> overnight under N<NUM> and then stirred at <NUM> for <NUM> hours. The reaction mixture mentioned above was cooled and concentrated. The residue was treated with dichloromethane (<NUM>) and filtered. The filtered cake was purified by Prep-HPLC (H<NUM>O:CH<NUM>CN = <NUM>:<NUM>) to afford <NUM>-(<NUM>-(<NUM>H-pyrazol-<NUM>-yl)phenyl)-<NUM>-benzyl-<NUM>-cyanoguanidine hydrochloride (<NUM>) (<NUM>, yield: <NUM>%), which was a white solid. LCMS (ES, m/z): [M+H]+= <NUM>∘ <NUM>H-NMR (<NUM>, DMSO-d<NUM>): <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>).

To a stirred solution of <NUM>-bromobenzoic acid (<NUM>, <NUM> mmol) and tert-butyl (<NUM>-((<NUM>-fluorobenzyl)amino)ethyl)carbamate (<NUM>, <NUM> mmol) in CH<NUM>Cl<NUM> (<NUM>), DIPEA (<NUM>µL, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added. The reaction mixture mentioned above was stirred at room temperature for <NUM> hours. The solvent was removed by reduced pressure, and the residue was purified by column chromatography to afford tert-butyl (<NUM>-(<NUM>-bromo-N-(<NUM>-fluorobenzyl)benzamido)ethyl)carbamate (17a) (<NUM>, <NUM> %) as oil. <NUM>H-NMR (CDCl<NUM>, <NUM>) <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>).

A mixture of tert-butyl (<NUM>-(<NUM>-bromo-N-(<NUM>-fluorobenzyl)benzamido)ethyl)carbamate (17a) (<NUM>, <NUM> mmol), tert-butyl <NUM>-(<NUM>,<NUM>,<NUM>,<NUM>-tetramethyl-<NUM>,<NUM>,<NUM>-dioxaborolan-<NUM>-yl)-<NUM>-pyrazole-<NUM>-carboxylate (<NUM>, <NUM> mmol), PdCl<NUM>(dppf) (<NUM>, <NUM> mmol) and Cs<NUM>CO<NUM> (<NUM>, <NUM> mmol) was added in a sealed tube, a mixed solvent (dioxane/H<NUM>O = <NUM>/<NUM>, <NUM>) was then injected thereto under argon, and the mixture was stirred at <NUM> for <NUM> hours. After cooling to room temperature, the solvent was removed by rotary evaporation, and the residue was added with water and extracted with EtOAc (<NUM> × <NUM>). The combined organic layers were washed with brine, dried over anhydrous Na<NUM>SO<NUM> and filtered. The filtrate was concentrated and the residue was purified by flash chromatography on silica gel (EtOAc/Hex = <NUM>% to <NUM>%) to give tert-butyl <NUM>-(<NUM>-((<NUM>-((tert-butoxycarbonyl)amino)ethyl)(<NUM>-fluorobenzyl)carbamoyl)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (17b) which was white powder (<NUM>, <NUM> %). <NUM>H-NMR (CDCl3, <NUM>) <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

To a mixture of tert-butyl <NUM>-(<NUM>-((<NUM>-((tert-butoxycarbonyl)amino)ethyl)(<NUM>-fluorobenzyl)carbamoyl)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (17b) (<NUM>, <NUM> mmol) and <NUM>,<NUM>-dioxane (<NUM>), <NUM> HCl in <NUM>,<NUM>-dioxane (<NUM>) was added. The reaction mixture mentioned above was stirred at room temperature for <NUM> hour. The reaction mixture was concentrated. The residue was treated with DCM (<NUM>) and filtered. The filtered cake was collected to afford N-(<NUM>-aminoethyl)-N-(<NUM>-fluorobenzyl)-<NUM>-(<NUM>-pyrazol-<NUM>-yl)benzamide dihydrochloride (<NUM>) (<NUM>, <NUM> %) which was a pale yellow solid. <NUM>H-NMR (<NUM>, D<NUM>O) δ: <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

For illustrative purpose the synthesis of compound <NUM> not falling under the scope of claim <NUM> is explained (<NUM>) Synthesis example <NUM>.

To a stirred solution of tert-butyl <NUM>-(<NUM>,<NUM>,<NUM>,<NUM>-tetramethyl-<NUM>,<NUM>,<NUM>-dioxaborolan-<NUM>-yl)-<NUM>-pyrazole-<NUM>-carboxylate (<NUM>, <NUM> mmol) in THF (<NUM>) at <NUM>, <NUM> NaOH(aq) (<NUM>, <NUM> mmol) was added, and then <NUM>% hydrogen peroxide (<NUM>, <NUM> mmol) was added. The reaction mixture mentioned above was stirred at <NUM> for <NUM> minutes and then room temperature for <NUM> hour. The reaction mixture was cooled to <NUM> and diluted with DCM, and <NUM> HCl(aq) was added thereto till pH <NUM> was reached. The organic layer were collected and dried, and the solvent was removed under reduced pressure to give tert-butyl <NUM>-hydroxy-<NUM>-pyrazole-<NUM>-carboxylate (18a) (<NUM>, <NUM> %) which was a yellow solid. <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (s, OH, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

To a stirred solution of tert-butyl <NUM>-hydroxy-<NUM>-pyrazole-<NUM>-carboxylate (18a) (<NUM>, <NUM> mmol) and <NUM>-fluoro-<NUM>-nitrobenzene (<NUM>, <NUM>) in DMF (<NUM>), K<NUM>CO<NUM> (<NUM>, <NUM> mmol) was added. The reaction mixture mentioned above was stirred at <NUM> for <NUM> hours. The solvent was removed under reduced pressure, and the residue was purified by column chromatography (EtOAc/Hexane = <NUM>%) to give tert-butyl <NUM>-(<NUM>-nitrophenoxy)-<NUM>-pyrazole-<NUM>-carboxylate (18b) (<NUM>, <NUM> %) which was a yellow solid. <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (s, <NUM>).

To a solution of tert-butyl <NUM>-(<NUM>-nitrophenoxy)-<NUM>-pyrazole-<NUM>-carboxylate (18b) (<NUM>, <NUM> mmol) in MeOH (<NUM>) and EtOAc (<NUM>), <NUM>% Pd/C (<NUM>, <NUM> mmol) was added. The reaction mixture mentioned above was stirred at room temperature under H<NUM> balloon atmosphere for <NUM> hours. The mixture was filtered, and the filtrate was evaporated by rotary evaporation to give tert-butyl <NUM>-(<NUM>-aminophenoxy)-<NUM>-pyrazole-<NUM>-carboxylate (18c) (<NUM>, <NUM> %) which was a brown solid.

To a stirred solution of <NUM>-((tert-butoxycarbonyl)amino)-<NUM>-phenylpropanoic acid (<NUM>, <NUM> mmol) and tert-butyl <NUM>-(<NUM>-aminophenoxy)-<NUM>-pyrazole-<NUM>-carboxylate (<NUM>, <NUM> mmol) in CH<NUM>Cl<NUM> (<NUM>), DIPEA (<NUM>µL, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added. The reaction mixture mentioned above was stirred at room temperature for <NUM> hours. The solvent was removed by reduced pressure, then the residue was purified with preparative reverse HPLC (<NUM>% ACN, <NUM>% H<NUM>O), and then lyophilized to get tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-phenylpropanamido)phenoxy)-<NUM>-pyrazole-<NUM>-carboxylate (18d) (<NUM>, <NUM> %) which was white powder. <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (brs, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

To a mixture of tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-phenylpropanamido)phenoxy)-<NUM>-pyrazole-<NUM>-carboxylate (18d) (<NUM>, <NUM> mmol) and <NUM>,<NUM>-dioxane (<NUM>), <NUM> HCl in <NUM>,<NUM>-dioxane (<NUM>) was added. The reaction mixture mentioned above was stirred at room temperature for <NUM> hour. The reaction mixture was concentrated. The residue was treated with DCM (<NUM>) and filtered. The filtered cake was collected to afford N-(<NUM>-((<NUM>-pyrazol-<NUM>-yl)oxy)phenyl)-<NUM>-amino-<NUM>-phenylpropanamide dihydrochloride (<NUM>) (<NUM>, <NUM> %) which was white solid. <NUM>H-NMR (<NUM>, CD<NUM>OD) δ : <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>),<NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>).

To a mixture of <NUM>-bromo-<NUM>-fluoroaniline (<NUM>, <NUM> mmol), <NUM>-Boc-<NUM>-pyrazoleboronic acid pinacol ester (<NUM>, <NUM> mmol), PdCl<NUM>(dppf) (<NUM>, <NUM> mmol) and Cs<NUM>CO<NUM> (<NUM>, <NUM> mmol) in a sealed tube, a mixed solvent (dioxane/H<NUM>O = <NUM>/<NUM>, <NUM>) was injected under argon, and then the mixture was stirred at <NUM> for <NUM> hours. After cooling to room temperature, the solvent was removed by rotary evaporation, and the residue was added with water and extracted with EtOAc (<NUM> × <NUM>). The combined organic layers were washed with brine, dried over anhydrous Na<NUM>SO<NUM> and filtered. The filtrate was concentrated, and the residue was purified by flash chromatography on silica gel (EtOAc/Hex = <NUM>% to <NUM>%) to give tert-butyl <NUM>-(<NUM>-amino-<NUM>-fluorophenyl)-<NUM>-pyrazole-<NUM>-carboxylate (19a) which was oil (<NUM>, <NUM> %). <NUM>H-NMR (<NUM>, CDCl<NUM>) δ : <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM> (s, <NUM>).

To a stirred solution of <NUM>-((tert-butoxycarbonyl)amino)-<NUM>-phenylpropanoic acid (<NUM>, <NUM> mmol) and tert-butyl <NUM>-(<NUM>-amino-<NUM>-fluorophenyl)-<NUM>-pyrazole-<NUM>-carboxylate (19a) (<NUM>, <NUM> mmol) in DMF (<NUM>), DIPEA (<NUM>µL, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added. The reaction mixture mentioned above was stirred at room temperature for <NUM> hours. The solvent was removed by reduced pressure, then the residue was purified with preparative reverse HPLC (<NUM>% ACN, <NUM>% H<NUM>O), and then lyophilized to get tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-phenylpropanamido)-<NUM>-fluorophenyl)-<NUM>-pyrazole-<NUM>-carboxylate (19b) (<NUM>, <NUM> %) which was white powder. <NUM>H-NMR (<NUM>, CDCl<NUM>) δ: <NUM> (t, J=<NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (brs, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

To tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-phenylpropanamido) -<NUM>-fluorophenyl)-<NUM>-pyrazole-<NUM>-carboxylate (19b) (<NUM>, <NUM> mmol) dissolved in <NUM>,<NUM>-dioxane (<NUM>), <NUM> HCl in <NUM>,<NUM>-dioxane (<NUM>) was added. The reaction mixture mentioned above was sonicated at room temperature for <NUM> minutes. The white powder was collected by filtration, and that was washed with DCM and dried to afford <NUM>-amino-N-(<NUM>-fluoro-<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-phenylpropanamide dihydrochloride (<NUM>) (<NUM>, <NUM> %). <NUM>H-NMR (<NUM>, D<NUM>O) δ: <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM> (t, J=<NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>), <NUM> (dd, J=<NUM>, <NUM>, <NUM>). LCMS (ES, m/z): [M-2HCl+H]+=<NUM>, [M-2HCl+Na]+=<NUM>.

To a solution of tert-butyl <NUM>-(<NUM>-(<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-phenylpropanamido)phenyl)-<NUM>-pyrazole-<NUM>-carboxylate (<NUM>, <NUM> mmol, <NUM> eq) in MeOH (<NUM>), HCl/MeOH (<NUM>) was added. The mixture mentioned above was stirred at room temperature overnight. The mixture was concentrated and the residue was dissolved in H<NUM>O (<NUM>), adjusted pH to <NUM> with NaHCO<NUM> aqueous solution. The mixture was filtered, and the filtrated cake was dried in vacuo to obtain N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-phenylpropanamide (<NUM>, <NUM>%) which was a white solid. The solid was purified by chiral resolution to obtain (S)-N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-phenylpropanamide (20a) and (R)-N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-phenylpropanamide (21a).

To a solution of (S)-N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-phenylpropanamide in H<NUM>O (<NUM>), concentrated HCl (<NUM>) was added, and the mixture was concentrated to obtain (S)-N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-phenylpropanamide dihydrochloride (<NUM>) (<NUM>). <NUM>H-NMR (<NUM>, DMSO-d<NUM>): <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>)∘ LCMS: [M+H]+=<NUM>.

To a solution of (R)-N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-phenylpropanamide in H<NUM>O (<NUM>) concentrated HCl (<NUM>) was added, and the mixture was concentrated to obtain (R)-N-(<NUM>-(<NUM>-pyrazol-<NUM>-yl)phenyl)-<NUM>-amino-<NUM>-phenylpropanamide dihydrochloride (<NUM>) (<NUM>). <NUM>H-NMR (<NUM>, DMSO-d6): <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>). LCMS: [M+H]+=<NUM>.

Inhibitory effect of each compound synthesized above on Rho-associated protein kinase (ROCK) was tested.

The results are shown in Table <NUM> - compounds <NUM> and <NUM> not falling under the scope of claim <NUM> are included for illustrative purpose.

The experimental procedure referred to the LanthaScreen Eu Kinase Binding Assay Screening Protocol and Assay Conditions provided by ThermoFisher Scientific.

<NUM>-fold serial dilution was performed on the test compound stock solution (<NUM>) prepared in <NUM>% DMSO was for <NUM> times for ready to use to determine the IC<NUM> of the test compound on MYLK-<NUM>.

Kinase/Antibody Mixture was pre-diluted with Kinase Buffer to 2X working concentration.

In addition, a control group for the binding analysis of LanthaScreen Eu kinase was also set up in the analysis mentioned above: Inhinitoin <NUM>% control (<NUM>% displacement control): the reaction of which contained no known inhibitor, and was used as the maximum emission ratio; Inhinitoin <NUM>% control (<NUM>% displacement control): which contained a known inhibitor with highest concentration, and was used as the minimum emission ratio; wherein the known inhibitor was Sunitinib.

The reaction components of the binding analysis of LanthaScreen Eu Kinase are shown in Table <NUM>. The IC<NUM> of the known inhibitor, sunitinib, was <NUM>, which was in line with the expected IC<NUM> range.

The inhibition rate of the test compound at different concentrations on MYLK-<NUM> could be obtained according to the following formula: <MAT>.

IC<NUM> of the test compound on MYLK-<NUM> was calculated by the above determined inhibition rates of the test compound at different concentrations on MYLK-<NUM>.

The results are shown in Table <NUM> - compound <NUM> not falling under the scope of claim <NUM> is included for illustrative purpose.

The experimental procedure referred to the Z'-LYTE Screening Protocol and Assay Conditions provided by ThermoFisher Scientific.

<NUM>-fold serial dilution was performed on the test compound stock solution (<NUM>) prepared in <NUM>% DMSO was for <NUM> times for ready to use to determine the IC<NUM> of the test compound on YSK-<NUM>.

Peptide/Kinase Mixture was pre-diluted with Kinase Buffer to 2X working concentration.

ATP solution was pre-diluted with Kinase Buffer (<NUM> HEPES pH <NUM>, <NUM>% BRIJ-<NUM>, <NUM> MgCl<NUM>, <NUM> EGTA) to 4X working concentration.

The development reagent solution is Novel PKC Lipid Mix, which contains <NUM>/mL Phosphatidyl Serine, <NUM>/mL DAG in <NUM> HEPES, pH <NUM> and <NUM>% CHAPS, and was pre-diluted <NUM> fold with development buffer.

In addition, for the kinase, the following control group was prepared and placed on the same culture plate as the kinase:.

The maximum Emission Ratio is established by the <NUM>% Phosphorylation Control (<NUM>% Inhibition Control), which contains no ATP and therefore exhibits no kinase activity. This control yields <NUM>% cleaved peptide in the Development Reaction.

A synthetically phosphorylated peptide of the same sequence as the peptide substrate is used as the <NUM>% Phosphorylation Control. This control yields a very low percentage of cleaved peptide in the Development Reaction.

The Phosphorylation percent achieved in a specific reaction well (Experimental group) was calculated based on the <NUM>% Phosphorylation Control and <NUM>% Phosphorylation Control.

The Z'-LYTE substrate used in the binding analysis reaction for Z'-LYTE Screening Kinase was Ser/Thr <NUM> peptide, and ATP reaction concentration was <NUM> (Km of YSK4). The known inhibitor used as a control in this analysis was Staurosporine. The IC<NUM> in the system was <NUM>, which was in line with the expected IC<NUM> range. The inhibition rate of the test compound on YSK-<NUM> at different concentrations was calculated based on the measured value of each hole according to the formula below, and IC<NUM> of the test compound on YSK-<NUM> was calculated thereby. <MAT> <MAT> <MAT> <MAT>.

The results are described as Table <NUM> - compound <NUM> not falling under the scope of claim <NUM> is included for illustrative purpose.

According to the foregoing results, it can be known that the compounds synthesized in the present disclosure have inhibitory effects on ROCK, MYLK-<NUM> and YSK4, and have synergistic target inhibitory effects, especially Compound <NUM> and Compound <NUM>.

Moreover, since it is currently known that through inhibiting ROCK expression, the effects of protection of optic nerve (see for example, <NPL>and <NPL>. ) and alleviation and/or treatment of high intraocular pressure (see for example,<NPL>), glaucoma (see, for example, <NPL>), ocular stroke (see for example, <NPL>and<NPL>. ), macular degeneration (see, for example,<NPL>. ), macular edema (see, for example, <NPL>. ), diabetic retinopathy (see, for example,<NPL>), Fuchs endothelial corneal dystrophy (FECD) (see, for example, <NPL>. ), corneal fibrosis (see, for example, S<IMG>oniecka et al. , Substance P induces fibrotic changes through activation of the RhoA/ROCK pathway in an in vitro human corneal fibrosis model. J Mol Med (Berl). <NUM>; <NUM>(<NUM>): <NUM>-<NUM>), etc. can be achieved, the compounds with the effect of inhibiting ROCK of the present disclosure mentioned above, can also be used in ophthalmology related applications, such as protection of optic nerve, and/or prevention and/or treatment of high intraocular pressure, glaucoma, ocular stroke, macular degeneration, macular edema, diabetic retinopathy, Fuchs endothelial corneal dystrophy (FECD) and/or corneal fibrosis, etc..

Furthermore, since it is also currently known that through inhibiting ROCK expression, the effects of alleviation and/or treatment of pulmonary hypertension (see, for example,<NPL>), chronic obstructive pulmonary disease (COPD) (see, for example,<NPL>), idiopathic pulmonary fibrosis (IPF) (see, for example,<NPL>. ), pulmonary emphysema (see, for example, <NPL>. ), lung cancer (see, for example, <NPL>). ), etc. can be achieved, the compounds with the effect of inhibiting ROCK of the present disclosure mentioned above, can also be used in lung-related applications such as prevention and/or treatment of pulmonary hypertension, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), pulmonary emphysema and/or lung cancer, etc..

The references to methods of treatment in the subsequent paragraphs of this description are to be interpreted as references to the compounds, kinase inhibitors, pharmaceutical composition and medicaments of the present invention for use in a method for treatment of the human or animal body by therapy or for diagnosis.

The maximal effect (Emax) dose evaluation of the intraocular pressure reduction model in normal rabbit.

The intraocular pressure of each animal was measured by Tono-Vet (iCare) before administration (<NUM> hours) and <NUM>, <NUM>, <NUM> and <NUM> hours after administration on Day <NUM> and Day <NUM>.

The results are shown in <FIG> and Table <NUM> and Table <NUM>.

<FIG> and Table <NUM> show the results of intraocular pressure measurement on Day <NUM> of administration. <FIG> and Table <NUM> show the results of intraocular pressure measurement on Day <NUM> of administration.

The results show that Compound <NUM> exhibits a dose dependent effect in reduction of intraocular pressure, wherein the group administered with <NUM>% Compound <NUM> and the group administered with <NUM>% Compound <NUM> have similar magnitude and trend in lowering intraocular pressure.

In addition, after continuous administration for <NUM> days, the group administered <NUM>% Compound <NUM> had a slight accumulation of drug effect (the reduction in intraocular pressure on Day <NUM> increased by nearly <NUM> mmHg on average compared to Day <NUM>) (see Table <NUM> and Table <NUM>). This result shows that Compound <NUM> at a concentration of <NUM>% should be close to the intraocular pressure test limit of this model, and thus the cumulative difference of medicinal effect (Emax) shown thereby it is not significant.

The data presented in this experiment is the data that has removed the unqualified data (white rabbits with an intraocular pressure difference ≥<NUM> mmHg (n=<NUM>) in the two eyes on Day <NUM> and ><NUM> mmHg in the two eyes before the administration on the Day <NUM> (n=<NUM>) have been removed) and removed the rebound high intraocular pressure (n=<NUM>). The n value of each group ≥<NUM>; in addition, the n value of the group administered with <NUM>% Compound <NUM> was <NUM>.

Based on the results mentioned above, it can be known that the concentration of <NUM>% should be the maximum effect (Emax) dose of Compound <NUM> in the normal intraocular pressure rabbit model.

Using a similar method to Compound <NUM>, the maximum effective doses of compounds <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> in a normal intraocular pressure rabbit model were confirmed.

The results show that the maximum effective doses of compounds <NUM>, <NUM>, <NUM> and <NUM> were all <NUM>%, and the maximum effective doses of compounds <NUM>, <NUM> and <NUM> were <NUM>%, <NUM>% and <NUM>%, respectively.

Evaluation of the effect of decreasing intraocular pressure in a model of rabbit with normal intraocular pressure.

Experimental animal: New Zealand white rabbit, male, weighing more than <NUM>. New Zealand white rabbits were purchased from Huijun Farm (Changhua, Taiwan). After a one-week quarantine period, they were raised in MASTER LABORATORY Co. , Ltd under the environmental conditions of <NUM>-<NUM>, <NUM>-<NUM>% relative humidity (RH), <NUM> hours day/<NUM> hours night.

After weighing <NUM> New Zealand white rabbits (<NUM>-<NUM>) and grouping them (n=<NUM>-<NUM> in each group), the white rabbits were fixed with a wrap. After the white rabbit was in a stable state, the lower eyelid of its right eye were opened, and <NUM>µL of the test sample (eye drops containing <NUM>% or <NUM>% Compound <NUM>) was instilled into the conjunctival sac of the right eye of the white rabbit, and the eyelid was closed, and the white rabbit was kept in a stable state for at least <NUM> minutes to avoid the white rabbit shaking its head causing the eye drops to flow out of the eye; the left eye was administered by vehicle (without test compound) as a control group. <NUM>% AR-<NUM> was used as a benchmark to evaluate the medicinal effect competitiveness of the tested compounds. The intraocular pressure detection time points were before administration (<NUM> hour) and <NUM>, <NUM>, <NUM>, <NUM> and <NUM> hours after administration.

After measuring the intraocular pressure, the appearance of rabbit eyes were photographed, and adverse event on the cornea, iris, or conjunctiva of the rabbit's eyes resulted from the test substance were evaluated according to the eye irritation test guidelines (OECD/OCDE <NUM>) stipulated by the Organization for Economic Cooperation and Development (OECD).

The scoring manner of OECD/OCDE <NUM> for cornea, iris or conjunctiva is shown in Table <NUM>.

Eye drops of <NUM>% Compound <NUM> was used as a test sample while the experimental method was the same as the experimental method described in the above "(<NUM>) Evaluation of the effect of reducing intraocular pressure". Administration was performed continuously for <NUM> days (once a day), an then the corneal state was observed with a slit lamp.

Compound <NUM> was administered to normal rabbits, and the content of the compound in aqueous humor was confirmed at different time points.

The results of evaluation of the effect of reducing intraocular pressure are shown in <FIG>.

According to <FIG>, it is known that the eye drops containing <NUM>% Compound <NUM> and the eye drops containing <NUM>% Compound <NUM> show the maximum effects (Emax) at <NUM>-<NUM> hours after administration, and the maximum magnitude of the reduction in intraocular pressure thereof are about <NUM> -<NUM> mmHg, and eye irritation thereof is slight.

In addition, the result also shows that the eye drops containing <NUM>% Compound <NUM> in the normal intraocular pressure rabbit model has reached the maximum effect of reducing intraocular pressure.

The results of eye irritation evaluation are shown in <FIG>, <FIG>.

Based on <FIG> and <FIG>, it is known that the eye irritation of Compound <NUM> at the maximum dose effect dose (<NUM>%) is still lower than AR-<NUM> (<NUM>%).

In addition, <FIG> shows that the cornea is still free of damage and turbidity after continuous administration for <NUM> days.

The content analysis results of Compound <NUM> presenting in the iris and ciliary body and aqueous humor after administration are shown in <FIG>. For the iris and ciliary body, the concentration of Compound <NUM> was measured in unit of ng/g; for aqueous humor, the concentration of Compound <NUM> is measured in unit of ng/mL.

According to <FIG>, it is known that after administration, the content of Compound <NUM> in the aqueous humor can reach the requirement of ROCK inhibition.

Using a similar method to Compound <NUM>, the effects of reducing intraocular pressure of compounds <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> (using the maximum effect dose) in a normal intraocular pressure rabbit model were confirmed.

Compound <NUM> was taken as a representative, the eye drops containing <NUM>% Compound <NUM> were administered to rabbit eyes (<NUM> doses a day, with an interval of <NUM> hours each time, and observation was performed before administration and <NUM> hour after each administration). In addition, Eye drops containing <NUM>% Compound <NUM> were administered to rabbit eyes (one dose a day, and observation was performed before administration and <NUM> hour and <NUM> hours after administration). According to the eye irritation test guidelines (OECD/OCDE <NUM>) stipulated by the Organization for Economic Cooperation and Development (OECD) mentioned above, the eye irritation of Compound <NUM> was evaluated.

The results of Evaluation of the effect of reducing intraocular pressure are shown in Table <NUM>.

The results of safety margin assay are shown in <FIG> and Table <NUM>.

Based on <FIG> and Table <NUM>, it is known that when AR-<NUM> is at a therapeutic index of <NUM>, the total score of OECD405 is <NUM>, and when Compound <NUM> is at a therapeutic index of <NUM>, the total score of OECD405 is <NUM>. Therefore, the safety margin of Compound <NUM> is better than AR -<NUM>.

Eye drop containing <NUM>% or <NUM>% Compound <NUM> was administered to normal macaques. <NUM>% AR-<NUM> was used as a benchmark to evaluate the medicinal effect competitiveness of the tested compounds. The intraocular pressure of animals was measured by a pneumatic tonometer (Model <NUM>™ Pneumatonometer). Animal anesthesia was required before intraocular pressure measurement. After topical administration (once a day), the intraocular pressure of the animals was measured at the set time points.

Based on <FIG>, it can be known that Compound <NUM> can achieve an effect of reducing intraocular pressure which is equivalent to or better than that of AR-<NUM> in normal intraocular pressure macaque without obvious side effects, such as pink eye.

Evaluation of the effect of reducing intraocular pressure in model of hypertonic saline induced high intraocular pressure rabbit (acute high intraocular pressure model).

Experimental animal: New Zealand white rabbit, male, weighing <NUM>-<NUM>. New Zealand white rabbits were purchased from a qualified laboratory animal rabbit farm in Taiwan before the experiment and were raised in the Animal Center of National Chiao Tung University.

The results are shown in <FIG> and Table <NUM>.

The results show that in the rabbit model of hyperosmolar saline-induced high intraocular pressure, the eye drop containing <NUM>% Compound <NUM> can reduce the intraocular pressure by about <NUM>±<NUM> mmHg while the eye drop containing <NUM>% AR-<NUM> can reduce the intraocular pressure by about <NUM> ±<NUM> mmHg. It shows that the effect of Compound <NUM> in reducing intraocular pressure is significantly better than that of AR-<NUM> (t-test , p<<NUM>). Moreover, compared with the physiological saline, the vehicle has no statistically significant difference (t-test). Compared with the physiological saline or the vehicle, AR-<NUM> also has no statistically significant difference (t-test).

Evaluation of the effect of reducing intraocular pressure in model of magnetic bead induced high intraocular pressure rabbit (high intraocular pressure model with intraocular pressure> <NUM> mmHg).

Exfoliation glaucoma (XFG) is considered to be more serious than primary open angle glaucoma. The maximum intraocular pressure (<NUM> vs <NUM> mmHg), minimum intraocular pressure (<NUM> vs <NUM> mmHg), and mean intraocular pressure change (<NUM> vs <NUM> mmHg) in exfoliative glaucoma and primary corner open glaucoma are statistically significant difference. Furthermore, there are currently no known diet therapies, drugs or other interventions that can prevent the occurrence of exfoliation syndrome or slow down its development (<NPL>). According to the foregoing, it is known that the intraocular pressure of exfoliative glaucoma can reach> <NUM> mmHg, and there is currently no effective drug for it, and thus a high intraocular pressure model with an intraocular pressure> <NUM> mmHg is provided here to evaluate the feasibility of the compound of the present disclosure in the treatment of exfoliative glaucoma and ocular hypertension with intraocular pressure> <NUM> mmHg.

Experimental animal: New Zealand white rabbit, male, body weight <NUM>-<NUM>. New Zealand white rabbits were purchased from Huijun Farm (Changhua, Taiwan) before the experiment and were raised in the Animal Center of National Chiao Tung University.

The results are shown as Table <NUM> and <FIG>.

The results show that in the model of magnetic bead induced high intraocular pressure rabbit (the high intraocular pressure model with intraocular pressure> <NUM> mmHg), the eye drop containing <NUM>% Compound <NUM> can reduce the intraocular pressure by about <NUM>±<NUM> mmHg (by about <NUM>%), and the eye drop containing <NUM>% Compound <NUM> can reduce intraocular pressure by about <NUM>±<NUM> mmHg (by about <NUM>%). Namely, the eye drop containing <NUM>% Compound <NUM> and the eye drop containing <NUM>% Compound <NUM> have better intraocular pressure reducing effects than those containing <NUM>% AR-<NUM> (reducing intraocular pressure by about <NUM>±<NUM> mmHg (reducing by about <NUM>%)), and the eye drop containing <NUM>% Compound <NUM> can even be more than twice as effective as eye drops containing <NUM>% AR-<NUM>.

Using a similar method to that for Compound <NUM>, the effect of Compound <NUM> (using maximum effect dose of <NUM>% and <NUM>%) in reducing intraocular pressure in a model of normal intraocular pressure rabbit was confirmed.

According to Table <NUM>, it is known that in the a model of magnetic bead induced high intraocular pressure rabbit (the high intraocular pressure model with intraocular pressure> <NUM> mmHg), the eye drop containing <NUM>% Compound <NUM> and the eye drop containing <NUM>% Compound <NUM> have better intraocular pressure reducing effects than those containing <NUM>% AR-<NUM>, wherein the eye drop containing <NUM>% Compound <NUM> can even be more than <NUM> times as effective as eye drops containing <NUM>% AR-<NUM>.

Human Trabecular Meshwork (HTM) cells (Cat. <NUM>) were obtained from ScienCell Research Laboratories. HTM cells were maintained in Trabecular Meshwork Cell Medium (TMCM) (Cat. TMCM formulated from <NUM> basal medium, <NUM> FBS (Cat. <NUM>), <NUM> trabecular meshwork cell growth supplement (TMCGS, Cat. <NUM>) and <NUM> penicillin/streptomycin solution (P/S, Cat. When the cell growth reached <NUM>-<NUM>% saturation, the HTM cells were treated overnight with <NUM>µg/mL dexamethasone (Cat. <NUM>, Sigma).

The expression of MYLK4 and GAPDH in cell lysates was analyzed by Western blotting method. The expression of MYLK4 and GAPDH in cell lysates was analyzed by Western blotting method. First, the cells were collected and washed with 1x RIPA (<NUM> Tris-HCl, pH <NUM>, <NUM> NaCl, <NUM>% deoxycholic acid, <NUM>% NP-<NUM>, <NUM> EDTA, phosphatase inhibitor and protease inhibitor mixture). Next, the cell lysates were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred to PVDF membrane (iBlot™ <NUM> Transfer Stacks, polyvinylidene fluoride membrane, Invitrogen). Immunoblotting was performed on the membrane with primary antibodies (mouse anti-MYLK4 antibody (<NUM>:<NUM>,<NUM>, Cat. SAB1412951) and mouse anti-GAPDH antibody (<NUM>:<NUM>,<NUM>, Cat. G8795)), wherein GAPDH was used as a loading control. After that, the membrane was washed <NUM> times with 1X TBST. Next, the film and the secondary antibody (Cat. <NUM>-<NUM>, Cat. <NUM>-<NUM>-<NUM>) were incubated for <NUM> hour at room temperature. The membrane was visualized by an enhanced chemiluminescence (WBKLS0500, Millipore) detection system (Fisher Scientific, US).

According to <FIG>, it is known that the expression level of MYLK-<NUM> in HTM cells treated with dexamethasone (disease state cells) is higher than that of normal HTM cells.

<NUM>µL of magnetic bead solution (<NUM>/mL, magnetic bead size of <NUM>) was injected into the anterior chamber of the left and right eyes of the rabbit under anesthesia, so that the rabbit's eyes became a state of high intraocular pressure. The white rabbits not treated with the magnetic bead solution were used as the control group. After that, the rabbits were sacrificed, the eyeballs were taken out, and histopathological analysis was performed.

GLOBAL VIEW BIOTECHNOLOGY INC. was entrusted to perform immunohistochemical (IHC staining) for rabbit eyeball paraffin tissue sections, and in addition to the interpretation of the results of the trabecular meshwork, a score sheet of interpretation results and photomicrographs of different magnifications were also provided.

After the removed eyeball tissues were fixed with formalin, they were dehydrated and embedded in paraffin, and then <NUM> thick paraffin tissue sections were made. The biomarkers for IHC staining are MYLK-<NUM> and MLC-<NUM>.

According to the histopathological sections made by the specimen, the tissue lesions were observed and recorded under a microscope at <NUM> times, <NUM> times, <NUM> times, <NUM> times, and <NUM> times. According to the severity of the lesion, the range of distribution and the percentage of the tissue it occupies, the lesion is scored based on the <NUM>-level grading method recommended by INHAND (International Harmonization of Nomenclature and Diagnostic Criteria): Grade <NUM> means that there is no obvious pathological change and the lesions account for less than <NUM>% of the total tissue; Grade <NUM> means that the disease is minimal (<NUM>-<NUM>%); Grade <NUM> means that the disease is mild (mild, <NUM> -<NUM>%); Grade <NUM> Represents moderate disease (moderate, <NUM>-<NUM>%); Level <NUM> represents moderately severe disease (<NUM>-<NUM>%); Level <NUM> represents severe disease (severe/high, ><NUM>%).

The immunohistochemical staining results are shown in <FIG>, and the scoring results are shown in Table <NUM>.

According to <FIG> and Table <NUM>, it can be known that whether it is diseased cells or diseased animal tissues (rabbit eyes induced by magnetic beads), the expression level of MYLK-<NUM> is higher than that of normal cells and tissues. Therefore, it is presumed that the compound of the present disclosure with MYLK-<NUM> inhibitory effect can effectively reduce intraocular pressure by inhibiting MYLK-<NUM> in addition to by ROCK inhibitory effect.

Claim 1:
A compound represented by one of Formulae (I)-(IV), or a pharmaceutically acceptable salt or ester, hydrate, individual optical isomers, a mixture of the individual enantiomers or a racemate thereof:
<CHM>
<CHM>
<CHM>
<CHM>
wherein the compound represented by Formula (I) is a β-amino acid derivative, and
in Formula (I):
X is a single bond;
Y is NH;
Z is C=O, C=S;
W is CH;
A is a single bond, -O-, -OH, -OCH<NUM>-, or -N<NUM>;
R<NUM> is H or F;
R<NUM> is H, F, OH, CF<NUM>, CH<NUM>OH, CHO or
<CHM>
and
n is <NUM> or <NUM>.