JP2003504334A - 2-Amino-benzoxazinones for treating herpes simplex virus - Google Patents

Abstract

  (57) [Summary] Antiviral drugs and methods for treating herpes simplex virus.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention is in the field of antiviral agents and more particularly relates to compounds, compositions and methods for treating herpes simplex virus.

[0002] there exists a great need for new treatment methods for the treatment of a background virus disease of the invention. Although various therapies have been developed and greatly advanced for the treatment of bacterial infections, there are few types of treatment for the treatment of viruses. Zidovudine is the major approved treatment for the human immunodeficiency virus. Ganciclovir, acyclovir, and foscarnet are currently used for the treatment of herpes virus infections. However, these therapies can have substantial side effects based on either having a deleterious effect on the DNA replication of the host cell or acting only on a limited number of viral infections. In addition, the virus has been shown to become resistant to therapies, gradually diminishing efficacy.

Viruses are classified into broad categories based on whether they incorporate RNA or DNA. Important virus families classified into this DNA type include adenovirus, poxvirus, papovavirus and herpes virus.

Herpes simplex virus includes herpes simplex virus type-1 (HSV-1), herpes simplex virus type-2 (HSV-2), cytomegalovirus (CMV), and varicella zoster virus, among others.
(VZV), Epstein-Barr virus, human herpes virus-6 (HHV6), human herpes virus-7 (HHV7), human herpes virus-8 (HHV8), pseudorabies and nasal hepatitis, DNA viruses including others Is a family of.

Herpes virus is known to express gene products by directing the synthesis of many proteins encoded by herpes virus DNA contained in host cells. One of the important viral encoded proteins is made as a precursor consisting of a protease located at the amino terminus and an assembly protein located at the carboxy terminus. This precursor is proteolytically processed in an autocatalytic manner with a specific amino acid sequence known to be the "release" site that results in a separate protease and assembly protein. This assembly protein is further cleaved by a protease at another specific amino acid sequence known to be the "mature" cleavage site. Most recently, European Patent No. 514,830 published November 25, 1992.
In the publication, virus-specific serine proteases involved in the replication of herpes virus are described. In addition, Lui and Roizman (J. Virol. 65, 514
9 (1991)) describe the sequence and action of proteases and related assembly proteins encoded by the U _L 26 of HSV-1. Welsh (AR We
lch) et al. (Proc. Natl. Acad. Sci. USA, 88, 10792 (1991) and WO 93/01291 published Jan. 21, 1993) described related proteases (Assemblin. )), And the assembly protein encoded by CMV U _L 80. An approach currently being investigated for potential use in the treatment of herpes virus infections is the development of inhibitors of herpes virus proteases.

The 4H-3,1-benzoxazinones are described in the literature as having, inter alia, serine protease activity. For example, Teshima et al. (J. Biol. Chem. 257, 5085-509.
1 (1982)) are various 2-alkyl-4H-3,1-benzoxazines as enzyme inhibitors.
-4-ones are described. Moorman and Abeles
(J. Amer. Chem, Soc., 104, 6785-6786 (1982)) describe 4H-3,1-benzoxazine-2,4-diones with some enzyme inhibitory activity. Stain (R. S
tein) et al. (Biochemistry, 26, 4126-4130, (1987)) describe a 2-alkyl-4H-benzoxazin-4-one further substituted at the 5th, 6th and 7th positions so as to inhibit the elastase enzyme. It describes the type. International Publication No.92 / 18488 (October 29, 1992)
(Published on the day), 2-alkyl-4H-3,1-benzoxazin-4-ones substituted at the 5 and 7 positions are described as selective inhibitors of elastase. European Patent Publication (EP Pub.) No. 206,323 (published on December 30, 1985) discloses that 2-alkoxy-substituted at position 5, 6, 7 and 8 as an enzyme inhibitor. , 2-aryloxy-
, And 2-aralkoxy-4H-3,1-benzoxazin-4-one. U.S. Pat. No. 4,745,116 by Kranz et al. Describes enzyme inhibitors such as 2-alkoxy, 2-aryloxy- and 2-alkoxy, 2-aryloxy and
2-Aralkoxy-4-H-3,1-benzoxazin-4-ones are described. US Pat. No. 5,428,021 to Hiebert et al. Describes 6- (amino acid) amino-2-alkoxybenzoxazinones as elastase inhibitors. WO 96/07648 published March 14, 1996 describes 2-phenylamino-benzoxazinones for treating Alzheimer's disease,
In particular 6-chloro-2- (2-iodophenylamino) -benzo [d] [1,3] oxazin-4-one is described.

2-Amino-4H-3,1-benzoxazinones have been described. Kranz
antz) et al. (J. Med. Chem., 33, 464-479 (1990)) describes that the 2-position is substituted with alkyl, alkylamino, alkoxy and alkylthio substituents, and further 5-, 6- and
7-substituted 4H-3,1-benzoxazin-4-ones are described as elastase inhibitors. Uejima et al. (J. Pharm. Exp. Ther., 265,
516-522 (1993)) is a highly selective elastase inhibitor with significant plasma stability, 2-alkylamino-5-methyl-7-acylamino-4H-3,1-benzoxazines.
-4-ones are described. US Patent No. 4,6 to Krantz et al.
No. 57,893 describes 2-alkylamino- and 2-alkylurido-4H-3,1-benzoxazin-4-ones as enzyme inhibitors, which further have substituents at 5-, 7- and 8-positions. ing.

FLM Alvarez (An. Quim., 79, 115-17 (1983)) describes the preparation of 2-sulfonylamino-4H-3,1-benzoxazinones. JG Trcero et al. (An. Quim., 83, 247-50, (1987)) describe the preparation of 2-arylsulfonylamino-4H-3,1-benzoxazinones.

[0009] I. Butula et al. (Croat. Chem. Acta, 54, 105-8 (1981)) describe the synthesis of 2-alkylamino-4H-3,1-benzoxazinones. Urich (
H. Urich) et al. (J. Org. Chem., 32, 4052-53 (1967)) describes 2-alkylamino-4H-3.
It describes the synthesis of 1,1-benzoxazinones. E. Papa
dopoulos) (J. Heterocyclic. Chem., 21, 1411-14 (1984)) used 2-haloalkylamino-4H-3,1-benzoxazin-4-one as a starting material for the synthesis of phenylureas. It describes what to do. European Patent Application (EP Appln) No. 466
, 944 (published on January 22, 1992) describes 2-alkylamino-7-acylamino-5-alkyl-4H-benzoxazin-4-ones, which are selective enzyme inhibitors of elastase. Has been done.

[0010] M. Badawy et al. (J. Heterocyclic. Chem., 21, 1403-4 (1984)) used N-phenyl-2-amino-4H- as a starting material for synthesizing quinazolines. 3,1
-The use of benzoxazin-4-one is described. Khan (R. Kha
n) et al. (J. Chem. Research (S), 342-43 (1992)) describes 2- [5-aryl-1,3,4-oxadiazol-2-yl] amino-4H-3,1. -Describes the synthesis of benzoxazin-4-ones.

WO 96/37485 describes antiviral drugs and compounds, compositions, and methods for treating herpes-related disorders. This report does not describe compounds with properties for HSV.

[0012] Certain compounds falling general scope of formula II Overview WO 96/37485 the invention, at lower concentrations than the compounds of the embodiments specifically disclosed in WO 96/37485
It has been found to be very effective against HSV. The compounds of the present invention have specificity for HSV. It is believed that this specificity derives from the structural morphology of compounds with R ²⁸ and R ²⁹ substituents.

The present invention is directed to compounds of formula II, or pharmaceutically acceptable salts thereof.

[Chemical 15] In this formula, R ²⁸ is selected from amino optionally substituted with two radicals selected from alkyl, aralkyl, heterocyclylalkyl, heterocyclyl, and aryl, wherein R ²⁹ is

[Chemical 16]

[Chemical 17]

[Chemical 18] Wherein R ³⁰ is alkyl, alkoxy, alkylamino, carboxyalkyl,
Alkoxyalkyl, alkylaminoalkyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aralkoxy, aryloxy, cycloalkyloxy, arylamino, aralkenyl, heterocyclylalkoxy, alkylaminoalkoxy, alkylaminoalkylamino, heterocyclylalkylamino, N-aryl -N-alkylamino, and N-aralkylamino, wherein R ³¹ is alkyl, R ³² is selected from alkyl and aryl, and R ³³ is from hydride, halo and alkyl. To be selected.

The present invention is further directed to pharmaceutical compositions comprising a therapeutically effective amount of a compound of formula II and a pharmaceutically acceptable carrier or diluent.

The present invention is further directed to a method of treating a herpes simplex virus in a patient, ie, a therapeutic or prophylactic treatment method, the method comprising treating the patient with an effective amount of a compound of formula II. Including.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a class of substituted benzoxazinones, as defined by Formula II, useful for the therapeutic and prophylactic treatment of viral infections of herpes simplex virus, Or, it relates to a pharmaceutically acceptable salt thereof.

[Chemical 19] Wherein R ²⁸ is selected from amino optionally substituted with two radicals selected from alkyl, aralkyl, heterocyclylalkyl, heterocyclyl, and aryl, or where the nitrogen can form a member of the heterocycle, R ²⁹ is

[Chemical 20]

[Chemical 21]

[Chemical formula 22] Wherein R ³⁰ is alkyl, alkoxy, alkylamino, carboxyalkyl,
Alkoxyalkyl, alkylaminoalkyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aralkoxy, aryloxy, cycloalkyloxy, arylamino, aralkenyl, heterocyclylalkoxy, alkylaminoalkoxy, alkylaminoalkylamino, heterocyclylalkylamino, N-aryl -N-alkylamino, and N-aralkylamino, wherein R ³¹ is alkyl, R ³² is selected from alkyl and aryl, and R ³³ is from hydride, halo and alkyl. To be selected.

An even more preferred class of compounds, wherein R ²⁸ is selected from amino optionally substituted with two radicals selected from lower alkyl, lower aralkyl, lower heterocyclylalkyl, heterocyclyl, and aryl, wherein R ²⁹ is

[Chemical formula 23]

[Chemical formula 24]

[Chemical 25] Wherein R ³⁰ is lower alkyl, lower alkoxy, lower alkylamino, lower carboxyalkyl, lower alkoxyalkyl, lower alkylaminoalkyl, lower cycloalkyl, heterocyclyl, lower heterocyclylalkyl, lower heterocyclylalkoxy, lower Aralkenyl, lower aralkyl, lower aralkoxy, phenyloxy, phenylamino, lower cycloalkyloxy, lower N-phenyl-N-
Alkylamino, lower alkylaminoalkoxy, lower alkylaminoalkylamino, lower heterocyclylalkylamino, and lower N-aralkylamino, wherein R ³¹ is lower alkyl and R ³² is lower alkyl and aryl. And those compounds of formula II, wherein R ³³ is selected from hydride and lower alkyl, or a pharmaceutically acceptable salt thereof.

Most preferred R ²⁸ is methyl (phenylmethyl) amino, methyl [(4-methoxyphenyl) -methyl] amino, 1- (1,2,3,6-tetrahydro-pyridyl), isopropyl (methyl ) Amino, (4-furoyl) piperazinyl, (2-cyano) ethyl, (4-thenoyl) piperazinyl, (4-benzenesulfonyl) -piperazinyl, diisopropylamino, [methyl (4-dimethylamino) phenylmethyl] amino, methyl (2-pyridylmethyl) -amino, methyl [2- (3-indolyl) ethyl] amino, 4-morpholyl, allyl
Selected from (methyl) amino, 1-decahydroquinolyl, and 4- (1-acetylpiperazinyl), R ²⁹ is [(1,1-dimethylethoxy) carbonyl] amino, benzoylamino, (phenylmethoxyacetyl) Amino, and (2,4,6-trifluorobenzoyl) amino.

[0019]   The term "hydride" refers to a single hydrogen atom (H). This hydrad deer
Is, for example, bonded to one oxygen atom to form one hydroxy radical,
, And two hydride radicals are bound to one carbon atom to give one methylene (-CH ₂ -) Can form radicals. Alone, or "haloalkyl
], “Alkylsulfonyl”, “alkoxyalkyl”, “hydroxyalkyl”
And other terms such as “aralkyl”, when used
The term "alkyl" refers to 1 to about 20 carbon atoms, preferably 1 to about 12 carbon atoms.
Included are straight or branched radicals having 3 carbon atoms. More preferred
Rukyl radicals are "lower" radicals having one to about ten carbon atoms. 1
Most preferred are lower radicals having 1 to about 6 carbon atoms. Such a boom
Examples of groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobuty
, Sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, etc.
Be done.

The term “halo” means halogens such as fluorine, chlorine, bromine, or iodine.

The terms “alkoxy” and “alkoxyalkyl” include straight or branched oxygen containing radicals each having an alkyl moiety of 1 to about 10 carbon atoms. More preferred alkoxy radicals are "lower radicals" having 1 to 6 carbon atoms. Examples of such radicals include methoxy, ethoxy, propoxy, butoxy and tert-butoxy.

The term “alkoxyalkyl” also includes 2 attached to an alkyl radical.
Included are alkyl radicals having one or more alkoxy radicals, ie, forming monoalkoxyalkyl and dialkoxyalkyl radicals. More preferred alkoxyalkyl radicals have 1 to 6 carbon atoms and 1
A "lower alkoxyalkyl" radical having one or two alkoxy radicals. Examples of such radicals include methoxymethyl, methoxyethyl, ethoxyethyl, methoxybutyl and methoxypropyl. "Alkoxy"
Alternatively, the "alkoxyalkyl" radical may be further substituted with one or more halogen atoms such as fluorine, chlorine or bromine to form a "haloalkoxy" or haloalkoxyalkyl radical. More preferred haloalkoxy radicals are "lower haloalkyl" having 1 to 6 carbon atoms and one or more halogen radicals. Examples of such radicals include fluoromethoxy, chloromethoxy, trifluoromethoxy, trifluoroethoxy, fluoroethoxy and fluoropropoxy.

The term “aryl”, used alone or in combination, means a carbocyclic aromatic system containing one, two or three rings, in which case such rings are in the protruding form. And may be fused with each other.

The term “aryl” includes aromatic radicals such as phenyl, naphthyl, tetrahydronaphthyl, indane and biphenyl. The aryl moiety also includes alkyl, aralkyl, alkoxyalkyl, alkylaminoalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl,
Alkoxy, aralkoxy, heterocyclylalkoxy, alkylaminoalkoxy, carboxyamino, carboxyaminoalkyl, carboxyaminoaralkyl, amino, halo, nitro, alkylamino, alkylcarbonylamino, arylcarbonylamino, alkoxycarbonylamino, aralkoxycarbonylamino, amino One or more substituents independently selected from carbonylamino, alkylaminocarbonylamino, alkylsulfonylamino, arylsulfonylamino, acyl, cyano, carboxy, aminocarbonyl, alkylaminocarbonyl, alkoxycarbonyl and aralkoxycarbonyl The positions that can be replaced with may be replaced.

The term “heterocyclyl” or “heterocyclic” includes ring-shaped radicals containing a heteroatom that can be selected from saturated, partially saturated and unsaturated nitrogen, sulfur and oxygen. Examples of saturated heterocyclic radicals are saturated 5- to 7-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms (eg, pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl, tropanyl, homotropanyl, etc.) Saturated of 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms
5-membered to 7-membered heteromonocyclic group (eg, morpholinyl), saturated 5-membered to 7-membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms (Eg, thiazolidinyl and the like). Examples of partially saturated heterocyclic radicals include dihydrothiophene, dihydropyran, oxazolinyl, dihydrofuran and dihydrothiazole. Unsaturated heterocyclic radicals are also referred to as "heteroaryl" radicals, for example, unsaturated 5- to 7-membered heteromonocyclic radicals containing 1 to 4 nitrogen atoms, such as pyrrolyl. , Pyrrolinyl,
Imidazolyl, pyrazolyl, pyridyl, pyrimidyl, azepinyl, pyrazinyl,
Pyridazinyl, triazolyl (eg 4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl etc.), tetrazolyl (eg 1H-tetrazolyl, 2H-tetrazolyl etc. ) Etc., an unsaturated fused heterocyclic group containing from 1 to 5 nitrogen atoms, such as indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl ( (Eg, tetrazolo [1,5-b] pyridazinyl), etc., and unsaturated 3-membered to 6-membered heteromonocyclic groups containing one oxygen atom, such as pyranyl, furyl, etc., containing one sulfur atom. A saturated 5-membered to 7-membered heteromonocyclic group, such as thienyl, is an unsaturated 5-membered to 7-membered heteromonocyclic ring containing 1 or 2 carbon atoms and 1 to 3 nitrogen atoms. 1 to 2 oxygen atoms and 1 ring group such as oxazolyl, isoxazolyl, oxadiazolyl (eg 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, etc.) Unsaturated fused heterocyclic groups containing from 3 to 3 nitrogen atoms (eg, benzoxazolyl, benzoxadiazolyl, etc.), unsaturated groups containing from 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms 5- to 7-membered heterocyclic groups, such as thiazolyl, thiadiazolyl (eg, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.) 1 to 2 Unsaturated fused heterocyclic groups containing one sulfur atom and one to three nitrogen atoms (eg, benzothiazolyl,
Benzothiadiazolyl and the like), and the like. This term also
Also included are radicals in which a heterocyclic radical is fused with an aryl radical. Examples of such fused bicyclic radicals include benzofuryl, benzothienyl, and the like. The above "heterocyclyl" radicals also include alkyl, aralkyl,
Alkoxyalkyl, alkylaminoalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, alkoxy, aralkoxy, alkylaminoalkoxy, aminocarboxy, alkylaminocarboxy,
Aralkylaminocarboxy, amino, halo, nitro, alkylamino, alkylcarbonylamino, arylcarbonylamino, alkoxycarbonylamino, aralkoxycarbonylamino, aminocarbonylamino, alkylaminocarbonylamino, alkylsulfonylamino, arylsulfonylamino, acyl, Substitutable positions may be substituted with one or more substituents independently selected from cyano, carboxy, aminocarbonyl, alkoxycarbonyl and aralkoxycarbonyl. More preferred heteroaryl radicals include 5-membered to 6-membered heteroaryl radicals.

The term “cycloalkyl” embraces radicals having 3 to 10 carbon atoms. More preferred cycloalkyl radicals are "lower cycloalkyl" radicals having 3 to 7 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

The term “sulfonyl” used alone or in combination with other terms such as alkylsulfonyl means a divalent radical —SO ₂ — respectively. "Alkylsulfonyl" embraces alkyl radicals attached to a sulfonyl radical, where alkyl is as defined above. More preferred alkylsulfonyl radicals are "lower alkylsulfonyl" radicals having one to six carbon atoms. Examples of such lower alkylsulfonyl radicals include methylsulfonyl, ethylsulfonyl and propylsulfonyl. “Alkylsulfonyl” radicals may also be radicals such as fluorine, chlorine or bromine.
By substituting one or more halogen atoms, "haloalkylsulfonyl" radicals can be formed. Further preferred haloalkylsulfonyl radicals are "lower haloalkylsulfonyl" radicals having one or more halogen atoms attached to a lower alkylsulfonyl radical as described above. Examples of such lower haloalkylsulfonyl radicals include fluoromethylsulfonyl, trifluoromethylsulfonyl, and chloromethylsulfonyl. The term "arylsulfonyl" includes an aryl radical as defined above linked to a sulfonyl radical. Phenylsulfonyl is mentioned as an example of such a radical. The term "sulfamyl", "aminosulfonyl" and "sulfonamidyl" denotes a NH ₂ O ₂ S.

The term “carboxy” or “carboxyl” is used alone
It is also used in combination with other terms such as “carboxycarbonyl” to represent —CO ₂ H.

The term “carbonyl” is used alone or in combination with other terms such as “alkoxycarbonyl” to represent — (C═O) —. "
The term "alkoxycarbonyl" means a radical containing an alkoxy radical as defined above attached to a carbonyl radical through an oxygen atom.
Preferably "lower alkoxycarbonyl" includes alkoxy radicals having 1 to 6 carbon atoms. Examples of such "lower alkoxycarbonyl" ester radicals include substituted and unsubstituted methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, and hexyloxycarbonyl.

The term “aralkyl” includes aryl-substituted alkyl radicals.
Preferred aralkyl radicals are "lower aralkyl" radicals which contain an aryl radical attached to an alkyl radical having 1 to 6 carbon atoms. Examples of such radicals include benzyl, diphenylmethyl, triphenylmethyl, phenylethyl and diphenylethyl. The aryl included in this aralkyl may be optionally substituted as described above.

The term “aralkenyl” includes aryl-substituted alkenyl radicals. Preferred aralkenyl radicals are "lower phenylalkenyl" radicals including a phenyl radical attached to an alkenyl radical having 1 to 6 carbon atoms. Examples of such radicals include phenylethenyl and phenylpropenyl. The aryl included in this aralkyl may be optionally substituted as described above. The terms benzyl and phenylmethyl are interchangeable.

The term “alkylcarbonyl” includes radicals having an alkyl radical as defined above attached to a carbonyl radical. More preferred alkylcarbonyl radicals are "lower alkylcarbonyl" radicals having one to six carbon atoms. Examples of such radicals include methylcarbonyl and ethylcarbonyl.

The term “carboxyalkyl” embraces radicals having a carboxy radical as defined above attached to an alkyl radical. Alkanoyl radicals are, for example, formyl, acetyl, propionyl, butyryl, isobutyryl,
Substituted or unsubstituted valeryl, isovaleryl, pivaloyl, hexanoyl, trifluoroacetyl and the like, and among them, formyl, acetyl, propionyl or trifluoroacetyl is preferable.

The term “heterocyclylalkyl” embraces heterocyclic substituted alkyl radicals. More preferred heterocyclic alkyl radicals are “lower heterocyclic alkyl” radicals that include a 5- to 6-membered heterocyclic radical attached to a lower alkyl radical having 1 to 6 carbon atoms. Examples of such radicals include pyrrolidinylmethyl, piperidinylmethyl, morpholinylmethyl, piperazinylmethyl, oxazolylmethyl, oxazolylethyl, oxazolinylmethyl, oxazolinylethyl, indine. Drill ethyl, indolyl methyl,
Mention may be made of pyridylmethyl, quinolylmethyl, thienylmethyl, furylethyl and quinolylethyl. The heterocycle contained in the heterocyclic alkyl may be optionally substituted as described above.

The term “aryloxy” embraces aryl radicals as defined above attached to an oxygen atom. The aryl included in the aryloxy may be optionally substituted as described above. An example of such a radical is phenoxy.

The term “aralkoxy” embraces oxygen-containing aralkyl radicals attached to another radical through an oxygen atom. The "aralkoxy" radical may be further substituted at the aryl ring portion of the radical.

The term “alkylamino” means an amino group substituted with one or two alkyl radicals. Further preferred alkylamino radicals are "lower alkylamino" containing alkyl radicals of 1 to 6 carbon atoms attached to the nitrogen atom of the amine. Suitable "lower alkylamino" may be monoalkylamino or dialkylamino, such as N-methylamino, N-ethylamino, N, N-dimethylamino, N, N-diethylamino and the like. May be.

The term “alkylaminoalkyl” means an alkylamino group, as defined above, attached to an alkyl radical. More preferred alkylaminoalkyl radicals are "lower alkylaminoalkyl" having 1 to 6 carbon atoms attached to a lower aminoalkyl radical as described above. Suitable "lower alkylaminoalkyl" means N-methylaminomethyl, N-ethylaminomethyl, N, N-dimethylaminomethyl, N, N-dimethylaminoethyl, N, N-dimethylaminopropyl, etc. It may be a monoalkylaminoalkyl radical or a dialkylaminoalkyl radical.

The term “dialkylaminoalkyl” also includes radicals where the bridging alkyl moiety is optionally substituted with alkylsulfonyl, alkoxy, aralkoxy, heterocyclyl, and aryl.

The term “alkylaminoalkoxy” means an alkylamino group as defined above linked to an alkoxy radical. Suitable "alkylaminoalkoxy" means mono-, such as N-methylaminomethoxy, N-ethylaminomethoxy, N, N-dimethylaminomethoxy, N, N-dimethylaminoethoxy, N, N-dimethylaminopropoxy, It may be an alkylaminoalkoxy radical or a dialkylaminoalkoxy radical.

The term “alkylaminocarbonyl” embraces alkylamino radicals attached to a carbonyl radical as described above. More preferred alkylaminocarbonyl radicals are "lower alkylaminocarbonyl" having a lower alkylamino radical as described above attached to a carbonyl radical.
Is. Examples of such radicals include N-methylaminocarbonyl and N, N-
Dimethylaminocarbonyl may be mentioned.

The term “arylamino” means an amino group substituted with one or two aryl radicals such as N-phenylamino. The "arylamino" radical may be further substituted at the aryl ring position of the radical.

“N-Arylaminoalkyl” and “N-aryl-N-alkyl-aminoalkyl”
The term means an amino group, each substituted with one aryl radical or one aryl and one alkyl radical, having an amino group attached to the alkyl radical. More preferred arylaminoalkyl radicals are
A "lower arylaminoalkyl" having an arylamino radical attached to one to six carbon atoms. Examples of such radicals include N-phenylaminomethyl and N-phenyl-N-methylaminomethyl.

The term “aminocarbonyl” means an amide group of formula —C (═O) NH ₂ . The term "aminocarbonylalkyl" means an aminocarbonyl group attached to an alkyl radical. More preferred are "lower aminocarbonylalkyl" having lower aminocarbonyl radicals as described above attached to an alkyl of 1 to 6 carbon atoms. The term "alkylaminocarbonylalkyl" means an aminocarbonyl group substituted with one or two alkyl radicals and attached to an alkyl radical. More preferably, it is a "lower alkylaminocarbonylalkyl" having a loweralkylaminocarbonyl radical as described above attached to an alkyl radical of 1 to 6 carbon atoms.

The term “aryloxy” forms an aryl radical attached to a divalent oxygen atom, ie a monoaryloxy radical and a diaryloxy radical. More preferred aryloxy radicals are "lower aryloxy". Examples include phenoxy.

“Amino acid residue” means any of the naturally occurring α-aminocarboxylic acids, β-aminocarboxylic acids and γ-aminocarboxylic acids, which includes the D and L optical isomers and their Included are racemic mixtures, synthetic amino acids, and derivatives of these natural and synthetic amino acids. Naturally occurring amino acids that can be incorporated into the present invention include, but are not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, Ornithine, phenylalanine, proline, serine, threonine, thyroxine, tryptophan, tyrosine, valine, β-alanine, and γ-aminobutyric acid are included. Derivatives of amino acids that can be incorporated into the present invention include, but are not limited to, protected and modified carboxylic acids, such as acid esters and acid amides, protected amines, and substituted phenyl rings. That is, but is not limited to, amino acids having alkyl, alkoxy and halogen substituted tyrosine and phenylalanine.

The present invention includes a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula II together with at least one pharmaceutically acceptable carrier, adjuvant or diluent.

The present invention also includes a method for the therapeutic and prophylactic treatment of viral infections in patients, in particular herpes viral infections, wherein a therapeutically effective amount of a compound of formula II is used to treat such herpes. A step of treating a patient with an infectious disease is performed.

The invention also includes a method of inhibiting a viral protease, the method comprising administering a therapeutically effective amount of a compound of formula II.

The family of compounds of formula II also includes their stereoisomers and tautomers. The compounds of the present invention may have one or more asymmetric carbon atoms,
As such, it can exist in the form of optical isomers, as well as in the form of racemic or non-racemic mixtures of those isomers. Therefore, some of the compounds of this invention may exist as racemic mixtures and are also included in this invention. The optical isomers can be obtained by decomposition of the racemic mixture by conventional methods, for example by treatment with optically active acids or bases to form diastereoisomeric salts. Examples of suitable acids are tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, ditoluoyl tartaric acid and camphorsulfonic acid, in which case a mixture of diastereoisomers by crystallization to liberate the optically active base from their salt. Can be separated.

Another method for performing the separation of optical isomers involves using a chiral chromatography column that is optically selected to maximize the separation of the enantiomers. Yet another possible method is to use an optically pure acid in the active form or an optically pure acid in the active form, i.e. an optically pure isocyanate, to give a compound of formula II It involves the synthesis of covalently linked diastereoisomeric molecules by reacting the amine functionality of the precursor to Alternatively, diastereomeric derivatives can be synthesized by reacting the carboxy functionality of the precursor to the compound of formula II with an optically pure amine base. The synthesized diastereoisomers are chromatographed, distilled,
Separation by simple methods such as crystallization or sublimation, followed by hydrolysis yields the enantiomerically pure compound. The optically active compounds of formula II can likewise be obtained using optically active starting materials. These isomers may take the form of free acids, free bases, esters, or salts. Additional methods known to those of skill in the art for separating optical isomers are described, for example, by Jaques et al., “Enantiomers, Racemates, and Degraders (Enantiomers, Ra.
cemates, and Resolutions) ", John Wiley and Sons, New York (1981).

The family of compounds of formula II also includes pharmaceutically acceptable salts thereof.
The term "pharmaceutically acceptable salt" includes salts commonly used to form alkali metal salts and to form additional salts of free acids and free bases. It will be pharmaceutically acceptable that the nature of this salt is not critical.
Additional pharmaceutically acceptable acid addition salts of the compounds of formula II may be formed with inorganic acids or organic acids. Examples of such inorganic acids include hydrochloric acid, bromic acid, iodic acid, nitric acid, carboxylic acids, sulfuric acid and phosphoric acid. Suitable organic acids include aliphatic, cycloaliphatic, aromatic, araliphatic (a
raliphatic), heterocyclic, carboxylic and sulfonic acid classes of organic acids, examples of which are formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid. , Citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, mesylic acid, salicylic acid, p-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid ( (Pamoic acid), methanesulfonic acid, ethylsulfonic acid, benzenesulfonic acid, pantothenic acid, toluenesulfonic acid, 2-hydroxyethanesulfonic acid, sulfanilic acid, stearic acid, cyclohexylaminosulfonic acid, algenic acid, β-hydroxy Butyric acid, galacturic acid and galacturon There is. Suitable pharmaceutically acceptable base addition salts of compounds of formula II include metal salts formed from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc, or N, N′-dibenzylethylenediamine, Included are organic salts formed from chlorine, chloroprocaine, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts can be synthesized from the corresponding compound of formula II by simple methods, for example by reacting the compound of formula II with a suitable acid or base.

This invention includes one or more non-toxic pharmaceutically acceptable carriers and / or
Or a class of pharmaceutical compositions comprising a diluent and / or an adjuvant (collectively referred to herein as “carrier” materials), and optionally one or more compounds of formula II, together with other active ingredients. To be done. The compounds of the present invention may be administered by any suitable route, preferably in the form of a pharmaceutical composition suitable for such a route, and in a dose effective for the intended treatment. The compounds and compositions can be administered, for example, orally, intravenously, intraperitoneally, subcutaneously, intramuscularly or topically.

Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, suspensions or solutions, for example. The pharmaceutical composition is preferably formed into dosage unit forms containing a specified amount of the active ingredient. Examples of such dosage units are tablets or capsules. The active ingredient can also be administered by injection, for example as a composition in which saline, dextrose or water can be used as a suitable carrier.

The amount and dosing regimen of a therapeutically effective compound administered in treating a disease state using the compounds and / or compositions of the present invention will include the age, weight, sex and medical condition of the patient, It will depend on a variety of factors such as the severity of the illness, the route and frequency of administration, and the particular compound used, and will therefore vary considerably. The pharmaceutical composition is in the range of about 0.1 mg to 2000 mg, preferably in the range of about 0.5 mg to 500 mg,
Most preferably the active ingredient may be included in the range of between about 1 mg and 100 mg. The daily dose is preferably about 0.01 mg / kg to 100 mg / kg body weight, preferably about 0.1 mg / kg to about 50 mg / kg, and most preferably about 1 mg / kg to 20 mg / kg. Will. This daily dose may be administered once to four times a day.

For therapeutic purposes, the compounds of this invention are ordinarily combined with one or more adjuvants appropriate to the indicated route of administration. When administered orally, the compounds are lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium salts of phosphoric acid and sulfuric acid and calcium. It may be administered with salt, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol, in which case tablets or capsules may be used for ease of administration. Such capsules or tablets which may contain a controlled release formulation may be used in a dispersion of the active compound in hydroxypropylmethylcellulose. Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions can be prepared from sterile powders or granules having one or more of the carriers or diluents mentioned for use in the formulation for oral administration. This compound is water, polyethylene glycol, propylene glycol,
Ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol,
It may be dissolved in sodium chloride and / or various buffers. Other adjuvants and modes of administration are well and widely known in the pharmaceutical arts.

For injection into the eye or other external tissues, for example mouth and skin, the combination may comprise a total amount of, for example, 0.075% w / w to 30% w / w, preferably 0.2% w / w to 20%. It is preferably applied as a topical ointment or cream containing the active ingredient at% w / w, and most preferably 0.4% w / w to 15% w / w. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base. The active ingredient can also be incorporated into a cream using an oil-in-water cream base. The aqueous phase of the cream base, if desired, is, for example, at least 30% w / w polyhydric alcohol, i.e. polypropylene glycol, butane-1,3-diol, mannitol,
Sorbitol, glycerol, polyethylene glycol and mixtures thereof and the like may be included. It may be desirable for topical formulations to include compounds that enhance absorption and penetration of the active ingredient through the skin or other affected areas. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogs. The compounds of this invention can also be administered by a transdermal device. Preferably topical administration will be achieved using either a patch consisting of a reservoir and a porous membrane type, or a patch consisting of a solid matrix type. In either case, the active agent is continuously delivered through the membrane from a reservoir or microcapsule to the permeable adhesive bond of the active agent in contact with the skin or mucous membranes of the recipient. When the active drug is absorbed through the skin,
A controlled, predetermined amount of the active agent is administered to the recipient. In the case of microcapsules, the encapsulant of the capsule will also function as a membrane.

The oily phase of the emulsions of the invention may be composed of known ingredients in a known manner. This phase may only contain emulsifiers, but it may contain a mixture of at least one emulsifier with fats or oils or both fats or oils. Preferably hydrophilic emulsifiers are included along with lipophilic emulsifiers which act as stabilizers. It is also preferable that both oil and fat are contained. At the same time, the emulsifier (s) with or without the stabilizer (s) make up the so-called emulsified wax, and the wax with oil and fat together make up the so-called emulsified ointment base, ie cream formulation. Make a base that forms an oily dispersed phase of the agent. Suitable emulsifiers and emulsion stabilizers for use in the formulations of the present invention include, among others, Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate,
And sodium lauryl sulfate.

Suitable oils or fats for this combination may be used in pharmaceutical emulsification formulations very slowly due to the solubility of the active compounds contained in most oils, thus making it a desirable cosmetic product. Selected based on the ability to achieve the above properties. Therefore, the cream is preferably a non-greasy, non-greasy and washable product with suitable consistency to prevent leakage from tubes and other containers. Diisoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acid, isopropyl myristate,
Linear or branched, monobasic alkyl esters or dibasic alkyls, such as decyl oleate, isopropyl palmitate, butyl stearate, 2-tylhexyl palmitate, or a mixture of branched esters. Esters can be used. These may be used alone or in combination depending on the properties required. Alternatively, high melting point liquids such as white soft paraffin and / or liquid paraffin or other mineral oils can be used.

Formulations suitable for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially the aqueous solvent for the active ingredient. . Antiviral active ingredient 0.5% to 20%, advantageously 0.5% to 10%
It is also preferred to be present in such formulations at a concentration of about 1.5% w / w, in particular.

Examples The following examples include a detailed description of how to prepare compounds of formula II. These detailed descriptions are included within the scope and are set forth to illustrate the General Synthetic Procedures above which form part of the invention. These detailed descriptions are presented for purposes of illustration only and are not intended to limit the scope of the invention. All parts are by weight and temperatures are in degrees Celsius unless otherwise noted.

[0062]   The following abbreviations are used:

[Table 1]

[0063]   The following is a list of compounds of the invention and comparative compounds.

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Table 8]

Example 1 Compound 1 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2- [methyl (phenylmethyl) amino] -4H-3,1-benzoxazine-4- on

[0065] A. 3-[[(1,1-dimethylethoxy) carbonyl] amino] -2-methyl-6-[[[methyl (phenyl
Phenylmethyl) -amino] carbonyl] amino] benzoic acid, 2- (trimethylsilyl) eth
Preparation of chill ester   6-amino-3-[[(1,1-dimethylethoxy) carbonyl] amino] -2-methylbenzoic acid
, 2- (trimethylsilyl) ethyl ester (3 ml of CH₂Cl₂0.11 mmol), CH₂Cl ₂ P-nitrophenyl chloroformate (0.1 mmol, 20.2 mg) in (0.2 ml)
The solution was added. After stirring at room temperature for 3 hours, the obtained solution was washed with 1N-HCl and water.
, Followed by MgSO_FourDried on. To this solution of activated carbamate, methyl (
Phenylmethyl) amine (0.016 ml, 0.12 mmol) was added. Stir at room temperature for 15 hours
And then tetrafluorophthalic anhydride (15 mg, 0.07 mmol) in CH₂Cl₂(0.7 ml)
The solution was added and stirred for 3 hours, followed by L. Flynn et al. (J. Amer. Chem. Soc.
, 119, 4874-4881 (1997)), a polyamine resin (200 mg, 0.6
Mmol) is added. After 1 hour, the mixture was filtered, concentrated and CH₂C
l₂Amberlyst A-21 (50 mg) was added, and the mixture was stirred for 1 hour.
. The resin was filtered off and the solution was concentrated to give white crystals.
(38.7 mg).

B. Preparation of 3-[[(1,1-dimethylethoxy) carbonyl] amino] -2-methyl-6-[[[methyl (phenylmethyl) -amino] carbonyl] amino] benzoic acid Product of Step A (38.7 mg ) In 3 ml of THF, TBAF in THF (1.0 M, 0.
(12 ml, 0.12 mmol) was added and stirred at room temperature for 1 hour. Ambelist A-15 H type (
200 mg), Ambellist A-15 calcium form (200 mg), shaken overnight, filtered and then concentrated to give white crystals (26.0 mg).

C. 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2- [methyl (phenylmethyl) -amino] -4H-3,1-benzoxazin-4-one Product of Step B ( P-EDC (300 mg, 0.3 mmol) was added to a solution of 26.0 mg) in 4 ml DMF and stirred for 2 hours. White crystals (2
1.5 mg) was obtained.

[0068]   Similar methods were used to synthesize the following compounds.

Compound 3 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2- [methyl [(4-methoxyphenyl) methyl] amino] -4H-3,1-benzoxazine- 4-on

Compound 8 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2- [1- (1,2,3,6-tetrahydropyridyl)]-4H-3,1 -Benzoxazin-4-one HPLC (A) retention time = 18.4 min, MS (MH +) = 358.

Compound 10 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2- [isopropyl (
Methyl) amino] -4H-3,1-benzoxazin-4-one HPLC (A) retention time = 22.9 minutes.

Compound 14 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2-[(4-furoyl) piperazinyl] -4H-3,1-benzoxazin-4-one HPLC ( A) Retention time = 20.0 minutes.

Compound 13 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2-[(2-cyanoethyl
)-(Cyclopropyl) amino] -4H-3,1-benzoxazin-4-one HPLC (A) retention time = 21.0 minutes.

Compound 17 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2-[(4-thenoyl) piperazinyl] -4H-3,1-benzoxazin-4-one HPLC ( B) Retention time = 7.02 minutes.

Compound 15 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2-[(4-benzenesulfonyl) -piperazinyl] -4H-3,1-benzoxazin-4-one HPLC (B) retention time = 7.88 minutes.

Example 2 Compound 2 6- (benzoylamino) -5-methyl-2- [methyl (phenylmethyl) amino] -4H-3,1-
Benzoxazin-4-one

A. Preparation of 3-amino-2-methyl-6-[[[methyl (phenylmethyl) -amino] carbonyl] amino] -benzoic acid, 2- (trimethylsilyl) ethyl ester The product of Example 1-Step A (40.2 mg ) In 1,4-dioxane (2 ml),
A solution of 4N-HCl in 4,4-dioxane (2 ml) was added. After stirring at room temperature for 2 hours, the reaction mixture was concentrated. CH ₂ Cl ₂ (4 ml) and DMF (0.5 ml) were added and dissolved, and Ambelist A-21 (100 mg) was added. After shaking for 1 hour, the resin was filtered off and concentrated to give 35.7 mg of product.

B. Preparation of 3- (benzoylamino) -2-methyl-6-[[[methyl (phenylmethyl) amino] -carbonyl] amino] -benzoic acid, 2- (trimethylsilyl) ethyl ester Product of Step A (35.7 mg) Was added to a solution of CH ₂ Cl ₂ in ₂ ml of pyridine (0.011 ml, 0
.14 mmol), benzoyl chloride (0.014 ml, 0.12 mmol) was added. 14 at room temperature
After stirring for an hour, polyamine resin (200 mg, 0.6 mmol) was added and stirred for 2 hours. Filtration and concentration gave 32.3 mg of product.

C. Preparation of 3- (benzoylamino) -2-methyl-6-[[[methyl (phenylmethyl) -amino] -carbonyl] amino] -benzoic acid The product of Step B (32.3 mg) was placed in 3 ml THF. To the solution, TBAF in THF (1.0M, 0.
(12 ml, 0.12 mmol) was added and stirred at room temperature for 1 hour. Ambelist A-15 H type (
200 mg), the calcium form of Amberlyst A-15 (200 mg), shaken overnight, filtered and subsequently concentrated to give white crystals (25.4 mg).

D. 6- (benzoylamino) -5-methyl-2- [methyl (phenylmethyl) -amino] -4H-3
, 1-Benzoxazin-4-one P-EDC (300 mg, 0.3 mmol) was added to a solution of the product of Step C (25 mg) in 4 ml of DMF.
) Was added and stirred for 2 hours. White crystals (15.
8 mg) was obtained.

Compound 9 6-[(phenylmethoxyacetyl) amino] -5-methyl-2- (diisopropylamino) -4
H-3,1-benzoxazin-4-one HPLC (A) retention time = 24.1 minutes.

Compound 12 6-[(2,4,6-trifluorobenzoyl) amino] -5-methyl-2- (diisopropylamino)
-4H-3,1-benzoxazin-4-one HPLC (A) retention time = 23.3 minutes.

Example 3 Compound 4 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2-[[[(4-dimethylamino) phenylmethyl] -methylamino] -4H- 3,1-benzoxazin-4-one

A. 3-[[(1,1-Dimethylethoxy) carbonyl] amino-2-methyl-6-[[[N-[[4- (dimethylamino) phenyl] methyl] methylamino] carbonyl] amino] benzoic acid, 2
Preparation of-(Trimethylsilyl) ethyl Ester In a manner similar to Example 1A, the methyl (phenylmethyl) amine is replaced with N-[(4-dimethylamino) phenyl] methyl] methylamine to proceed.

B. 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2-[[[4- (dimethylamino) phenyl] methyl] methylamino] -4H-3,1-benzoxazine-4- Preparation of ON To a solution of the product of Step A (521 mg, 0.935 mmol) in 10 ml of THF was added a solution of tetrabutylammonium fluoride in THF (1.0 M, 1.12 ml, 1.12 mmol). Stir at room temperature for 45 minutes, add acetyl chloride (0.12 ml, 1.7 mmol) and add 1.5.
Stir for hours. Concentration, purification on silica gel and washing with saturated aqueous NaHCO ₃ solution gave a white amorphous solid (241 mg).

Compound 18 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2- [methyl (2-pyridylmethyl) -amino] -4H-3,1-benzoxazine-4- On HPLC (B) retention time = 6.71 mins, MS (MH +) = 397.

Compound 5 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2- [methyl [2- (3-indolyl) ethyl] amino] -4H-3,1-benzoxazine -4-on

Compound 6 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2- (4-morpholyl) -4
H-3,1-benzoxazin-4-one

Compound 7 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2- [allyl (methyl)]
Amino] -4H-3,1-benzoxazin-4-one

Compound 11 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2- (1-decahydroquinolyl) -4H-3,1-benzoxazin-4-one HPLC (B ) Retention time = 8.63 minutes, MS (MH ⁺ ) = 414.

Compound 16 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2- [4- (1-acetylpiperazinyl)]-4H-3,1-benzoxazine-4 -On HPLC (B) retention time = 4.98 min, MS (MH ⁺ ) = 403.

BIOLOGICAL EVALUATION The compounds of the invention exhibited antiviral activity as demonstrated by inhibiting herpesvirus protease and HCMV infections in vitro. The antiviral activity of the compounds of the present invention exemplified in the examples was measured by the following method.

Enzymatic Assay for HSV-1 Protease (Assembly) Inhibition FP Assay Assemin protease activity was detected using fluorescence polarizability (FP). The fluorescent substrate is biotin-gamma-aminobutyrate-based on the HSV-1 free cleavage site. Met. Incubation of this substrate with assemblyin results in cleavage between alanine and serine. The difference in the molecular size of the fluorescent substrate molecule becomes larger when a reaction reagent, avidin, is used, and is cleaved well so that it can be measured by FP.
The protease-inhibiting inhibitor was dissolved in DMF and then diluted 5-fold with assay buffer. Blocking solution (10 mM Tris-HCl, pH 8.0, 150 mM NaCl
6.5 μL was added to the wells of a 96-well plate (U-bottom 96-well black plate, Dynatech or Costar) previously blocked with 0.05% Tween 20, 1 mg / mL BSA). The enzyme was added to the assay buffer (1 M sodium citrate, 50 mM sodium phosphate, pH 7.4, 100 mM NaCl, 20% glycerol, 2 mM).
TECP) and diluted to 21.3 ug / mL, and 48.5 uL was added to each well. 3
After incubating for 0 minutes at room temperature, 10 uL of 62.5 ug / mL substrate was added. After incubating at room temperature for about 90 minutes, phosphate buffer (50 mM sodium phosphate, 100 mM
50 mg of avidin at 2 mg / mL in NaCl, pH 7.4) was added to each well. The plate was read by a fluorescence polarizable plate reader. Inhibitory capacity was detected by comparison with that incubated without inhibitor.

HPLC assay peptide substrate, Was used to carry out the assay method. This substrate is the HSV-1-amide UL26 open reading frame (242-255), which is derived from the free site of HSV-1 protease. The HSV-1 protease cleaves between alanine and serine. Product Was quantified by HPLC with fluorescence detection of tryptophan residues. The enzyme was assayed in buffer (1 M sodium citrate, 50 mM sodium phosphate, pH 7.3, 100 mM Na).
Cl, 20% glycerol, 2 mM TCEP) diluted to 4.3 ug / mL and 48.5 uL was added to the tube. The inhibitory protease inhibitors were dissolved in DMF and then diluted 10-fold with assay buffer. 6.5 uL of inhibitor solution was added to each tube. After incubation for 30 minutes at room temperature, 10 uL of substrate in phosphate buffer (50 mM sodium phosphate, 100 mM NaCl, pH 7.4) was added to all tubes. The final concentration of substrate contained in the reaction mixture was 10 uM. After incubating for 15 minutes at room temperature, the assay was quenched with 50 uL of 50% TCA. The potency of the inhibitor is that it is incubated without inhibitor, i.e.
Twenty percent of the substrate was detected compared to that which was cleaved.

Assay Components Recombinant HSV-1 Protease HSV-1 protease is a baculovirus expressing a DNA construct encoding residues 1-288 of the HSV-1 UL26 open reading frame and 32 heterologous amino acids. Purified from. This construct also encoded 6 additional histidine residues at the amino terminus of the protease. These additional histidine residues provided affinity ligands by which the protein could be purified using Ni-NTA agarose gel (Qiagen). The purified protease was stored in a stock solution (20 mM HEPES buffer, pH 8.5 containing 20% (v / v) glycerol). By diluting this stock with assay buffer, the concentration of the enzyme assay could be adjusted.

Substrate FP Assay Specific substrates were synthesized based on the cleavage specificity of HSV-1 protease at the “cleavage site” of the assembly protein (DiIanni, CL, et al., J. Biol. Chem. 26.
8, 2048, (1993)). The free site of this assembly protein is a sequence, have. The substrate used is It was prepared by standard peptide synthesis methods such as those described in Bodansky and Bodansky, “The Practice of Peptide Synthesis” (1984), and 2.5% in DMF. Stored as a mg / mL stock solution. Immediately before use, it was diluted to 62.5 ug / mL with phosphate buffer (50 mM sodium phosphate, 100 mM NaCl, pH 7.4).

HPLC Assay This substrate was HSV-1-amide UL 26 open reading frame (242-255) (Product No. M-2160) obtained from Bachem.

Assay buffer Assay buffer (1 M sodium citrate, 50 mM sodium phosphate, pH 7.
4, 100 mM NaCl, 20% glycerol, 2 mM TCEP) was used to dilute the enzyme and inhibitor stock solutions.

Antiviral and Cytotoxicity Assays These complementary assays tested the ability of the compounds to inhibit the development of new viruses and the toxicity of the compounds to host cells. Since virulence can indirectly reduce viral replication, it was important to run both assays simultaneously so that the results could be compared directly.

Abbreviations: DMEM-Dulbecco's Modified Eagle Medium, commercially available. FBS-fetal bovine serum, commercially available, is an unknown factor essential for growth of cells in culture. HSV-herpes simplex virus.

Antiviral Assay Antiviral assay was estimated by plaque reduction assay performed by the following method. Overnight culture of 1 x 10 ⁵ Vero cells (African Green Monkey kidney cells) in 48-well plates and this medium contains 2x the desired final concentration of test compound or no compound as a control Replaced with 200 μL of 2% FBS DMEM. The culture contained 2% FBS D together with a unit amount of HSV-1 which forms approximately 50 plaques.
Add 50 μL of MEM, followed by 250 μl of 2% FBS DMEM with 1% methylcellulose
Was added. These infected cultures were incubated for 3 days at 37 ° C. in 5% CO ₂ until plaques were visible. The cells were fixed and simultaneously stained with 0.025% crystal violet in 5% formalin solution and plaques were then counted. 50 plaque formation compared to control without compound
The concentration of test compound showing% inhibition was interpolated from the observed data and defined as the IC ₅₀ . The results are contained in Table 2.

Cytotoxicity Assay 4 × 10 ⁴ Vero cells in 96 wells were cultured overnight in 100 μl of 10% FBS DMEM. These cultured cells were treated with 10% FBS DMEM containing 50 μM test compound.
Alternatively, 100 μl of 10% FBS DMEM without compound was added as a control. 37 cells
The cells were incubated at 3 ° C in 5% CO ₂ for 3 days and cultured. 20 μl of alamarBlue® was added to the cells to measure the proliferation of the cells,
The control was incubated for 8 hours until the color changed. The cells were then analyzed by optical spectroscopy (570 nm and 60 nm) using a BIO-RAD model 3550 microplate reader.
Absorption at 0 nm). The results were expressed as the ratio with and without compound concentration of 50 μM.

Chymotrypsin Assay The chymotrypsin assay is based on the method of Delmar et al. (Anal. Biochem. 99).
, 316-320 (1979)). Bovine pancreatic α-chymotrypsin (type II, Si
gma) in 0.001 N HCl to 1 mg / ml and use assay buffer (0.1 M) before use.
Further diluted 1/1000 with 0.1 M Tris, pH 7.8) containing CaCl ₂ . 0.75 μl of test compound in DMF (or DMF alone), 50 μl of assay buffer and 50 μl of enzyme were added to 96-well plate, mixed and pre-incubated for 30 minutes at ambient temperature. The reaction is 0.2 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma, 2 mM in DMSO, which is diluted 1/10 with assay buffer before use. ), Started by adding 50 μl. The increase in absorbance at 405 nm was monitored for 3 minutes using a BIO-RAD model 3550 microplate reader.

Human Leukocyte Elastase Assay Human leukocyte elastase (HLE) (CALBIOCHEM) was dissolved in saline to 1 mg / ml and further diluted with assay buffer (0.2 M Tris, pH 8.0) before use. Diluted to / 10. 0.75 μl of test compound in DMF (or DMF alone), 50 μl of assay buffer and 50 μl of enzyme were added to 96 well plates, mixed and pre-incubated for 30 minutes at ambient temperature. The reaction was 2.5 mM methoxysuccinyl-
Start by adding 50 μl of Ala-Ala-Pro-Val-p-nitroanilide (Sigma, 25 mM in DMSO, diluted 1/10 in assay buffer before use) did. The increase in absorbance at 405 nm was monitored for 3 minutes using a BIO-RAD model 3550 microplate reader.

[0105]

[0106]

[Table 9]

It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions and methods of the present invention without departing from the spirit or scope of the invention. Accordingly, it is intended that the present invention cover the modifications and variations of this invention made within the scope of the appended claims and their equivalents.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, C H, CN, CR, CU, CZ, DE, DK, DM, EE , ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, K P, KR, KZ, LC, LK, LR, LS, LT, LU , LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, S E, SG, SI, SK, SL, TJ, TM, TR, TT , TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (71) Applicant Asahi Kasei Corporation             632-1 Mifuku, Ohito-cho, Takata-gun, Shizuoka Prefecture (72) Inventor Kawanishi Masashi             632-1 Mifuku, Ohito-cho, Takata-gun, Shizuoka Prefecture (72) Inventor Takahashi Wataru             632-1 Mifuku, Ohito-cho, Takata-gun, Shizuoka Prefecture F-term (reference) 4C056 AA02 AB01 AC02 AD03 AE03                       DA01 DB07 DC01                 4C063 AA01 BB02 BB09 CC54 CC78                       CC92 DD06 DD11 DD12 DD14                       DD54 EE01                 4C086 AA01 AA02 AA03 BC72 GA02                       GA03 GA07 GA08 GA12 MA04                       MA52 MA55 NA14 ZB33

Claims (9)

[Claims] 1. A chemical formula II, that is, In this formula, R ²⁸ is selected from amino optionally substituted with two radicals selected from alkyl, aralkyl, heterocyclylalkyl, heterocyclyl, and aryl, wherein R ²⁹ is [Chemical 3] [Chemical 4] Wherein R ³⁰ is alkyl, alkoxy, alkylamino, carboxyalkyl,
Alkoxyalkyl, alkylaminoalkyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aralkoxy, aryloxy, cycloalkyloxy, arylamino, aralkenyl, heterocyclylalkoxy, alkylaminoalkoxy, alkylaminoalkylamino, heterocyclylalkylamino, N-aryl -N-alkylamino, and N-aralkylamino, wherein R ³¹ is alkyl, R ³² is selected from alkyl and aryl, and R ³³ is from hydride, halo and alkyl. A compound of formula II, or a pharmaceutically acceptable salt thereof, as selected. 2. R ²⁸ is selected from (one) amino optionally substituted with two radicals selected from lower alkyl, lower aralkyl, lower heterocyclylalkyl, heterocyclyl, and aryl, wherein R ²⁸ is ²⁹ is [Chemical 5] [Chemical 6] [Chemical 7] Wherein R ³⁰ is lower alkyl, lower alkoxy, lower alkylamino, lower carboxyalkyl, lower alkoxyalkyl, lower alkylaminoalkyl, lower cycloalkyl, heterocyclyl, lower heterocyclylalkyl, lower heterocyclylalkoxy, lower Aralkenyl, lower aralkyl, lower aralkoxy, phenyloxy, phenylamino, lower cycloalkyloxy, lower N-phenyl-N-
Alkylamino, lower alkylaminoalkoxy, lower alkylaminoalkylamino, lower heterocyclylalkylamino, and lower N-aralkylamino, wherein R ³¹ is selected from lower alkyl, R ³² is selected from lower alkyl and aryl, And a compound of claim 1, wherein R ³³ is selected from hydride and lower alkyl, or a pharmaceutically acceptable salt thereof. 3. 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-
2- [methyl (phenylmethyl) amino] -4H-3,1-benzoxazin-4-one, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2- [methyl [( 4-Methoxyphenyl) methyl] amino] -4H-3,1-benzoxazin-4-one, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2- [1- (1 , 2,3,6-Tetrahydropyridyl)]-4H-3,1-benzoxazin-4-one, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2- [isopropyl ( Methyl) amino] -4H-3,1-benzoxazin-4-one, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2-[(4-furoyl)
Piperazinyl] -4H-3,1-benzoxazin-4-one, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2-[[(2-cyano) ethyl]-(cyclo Propyl) amino] -4H-3,1-benzoxazin-4-one, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2-[(4-thenoyl)
Piperazinyl] -4H-3,1-benzoxazin-4-one, 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2-[(4-benzenesulfonyl) -piperazinyl] -4H -3,1-benzoxazin-4-one, 6- (benzoylamino) -5-methyl-2- [methyl (phenylmethyl) amino] -4H-3,1-
Benzoxazin-4-one, 6-[(phenylmethoxyacetyl) amino] -5-methyl-2- (diisopropylamino)
-4H-3,1-benzoxazin-4-one, 6-[(2,4,6-trifluorobenzoyl) amino] -5-methyl-2- (diisopropylamino) -4H-3,1-benzoxazine-4 -One, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2-[[methyl (4-dimethylamino) phenylmethyl] -amino] -4H-3,1-benzoxazine- 4-one, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2- [methyl (2-pyridylmethyl) -amino] -4H-3,1-benzoxazin-4-one , 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2- [methyl [2- (3-
(Indolyl) ethyl] amino] -4H-3,1-benzoxazin-4-one, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2- (4-morpholyl)
-4H-3,1-Benzoxazin-4-one, 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2- [allyl (methyl
) Amino] -4H-3,1-benzoxazin-4-one, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2- (1-decahydroquinolyl) -4H-3 , 1-Benzoxazin-4-one, and 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2- [4- (1-acetylpiperazinyl)]-4H-3, The compound of claim 2 selected from the group consisting of 1-benzoxazin-4-one and pharmaceutically acceptable salts thereof. 4. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 1 and a pharmaceutically acceptable carrier or diluent. 5. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 2 and a pharmaceutically acceptable carrier or diluent. 6. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 3 and a pharmaceutically acceptable carrier or diluent. 7. A method for treating herpes simplex virus, or a therapeutic or prophylactic method for treating a patient, which method is represented by formula II: In this formula, R ²⁸ is selected from (one) amino optionally substituted with two radicals selected from alkyl, aralkyl, heterocyclylalkyl, heterocyclyl, and aryl, wherein R ²⁹ is [Chemical 10] [Chemical 11] Wherein R ³⁰ is alkyl, alkoxy, alkylamino, carboxyalkyl,
Alkoxyalkyl, alkylaminoalkyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aralkoxy, aryloxy, cycloalkyloxy, arylamino, aralkenyl, heterocyclylalkoxy, alkylaminoalkoxy, alkylaminoalkylamino, heterocyclylalkylamino, N-aryl -N-alkylamino, and N-aralkylamino, wherein R ³¹ is alkyl, R ³² is selected from alkyl and aryl, and R ³³ is hydride, halo and alkyl. Treating the patient with an effective amount of a compound of formula II, or a pharmaceutically acceptable salt thereof, as selected from: 8. wherein R ²⁸ is selected from (one) amino optionally substituted with two radicals selected from lower alkyl, lower aralkyl, lower heterocyclylalkyl, heterocyclyl, and aryl, wherein R ²⁸ is ²⁹ is [Chemical formula 12] [Chemical 13] [Chemical 14] Wherein R ³⁰ is lower alkyl, lower alkoxy, lower alkylamino, lower carboxyalkyl, lower alkoxyalkyl, lower alkylaminoalkyl, lower cycloalkyl, heterocyclyl, lower heterocyclylalkyl, lower heterocyclylalkoxy, lower Aralkenyl, lower aralkyl, lower aralkoxy, phenyloxy, phenylamino, lower cycloalkyloxy, lower N-phenyl-N-
Alkylamino, lower alkylaminoalkoxy, lower alkylaminoalkylamino, lower heterocyclylalkylamino and lower N-aralkylamino, wherein R ³¹ is selected from lower alkyl, R ³² is selected from lower alkyl and aryl, And the method of claim 7 wherein R ³³ is selected from hydride and lower alkyl,
Or a pharmaceutically acceptable salt thereof. 9. The compound is 6-[[(1,1-dimethylethoxy) carbonyl] amino]
-5-methyl-2- [methyl (phenylmethyl) amino] -4H-3,1-benzoxazin-4-one, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2 -[Methyl [(4-methoxyphenyl) methyl] amino] -4H-3,1-benzoxazin-4-one, 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2- [1- (1,2,3,6-Tetrahydropyridyl)]-4H-3,1-benzoxazin-4-one, 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl- 2- [isopropyl (methyl) amino] -4H-3,1-benzoxazin-4-one, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2-[(4-furoyl) )
Piperazinyl] -4H-3,1-benzoxazin-4-one, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2-[[(2-cyano) ethyl]-(cyclo Propyl) amino] -4H-3,1-benzoxazin-4-one, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2-[(4-thenoyl)
Piperazinyl] -4H-3,1-benzoxazin-4-one, 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2-[(4-benzenesulfonyl) -piperazinyl] -4H -3,1-benzoxazin-4-one, 6- (benzoylamino) -5-methyl-2- [methyl (phenylmethyl) amino] -4H-3,1-
Benzoxazin-4-one, 6-[(phenylmethoxyacetyl) amino] -5-methyl-2- (diisopropylamino)
-4H-3,1-benzoxazin-4-one, 6-[(2,4,6-trifluorobenzoyl) amino] -5-methyl-2- (diisopropylamino) -4H-3,1-benzoxazine-4 -One, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2-[[methyl (4-dimethylamino) phenylmethyl] -amino] -4H-3,1-benzoxazine- 4-one, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2- [methyl (2-pyridylmethyl) -amino] -4H-3,1-benzoxazin-4-one , 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2- [methyl [2- (3-
(Indolyl) ethyl] amino] -4H-3,1-benzoxazin-4-one, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2- (4-morpholyl)
-4H-3,1-Benzoxazin-4-one, 6-[[(1,1-Dimethylethoxy) carbonyl] amino] -5-methyl-2- [allyl (methyl
) Amino] -4H-3,1-benzoxazin-4-one, 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2- (1-decahydroquinolyl) -4H-3 , 1-Benzoxazin-4-one, and 6-[[(1,1-dimethylethoxy) carbonyl] amino] -5-methyl-2- [4- (1-acetylpiperazinyl)]-4H-3, 9. The method of claim 8 selected from the group of compounds consisting of 1-benzoxazin-4-one and pharmaceutically acceptable salts thereof.