JP2003510352A - Compounds for the treatment of virus-borne diseases - Google Patents

Abstract

  (57) [Summary] Flavivirus, rhabdovirus, or paramyxovirus infections are inhibitors of dihydroorotate dehydrogenase enzyme, such as 6-fluoro-2- (2'-fluoro-1,1'-biphenyl-4-yl) -3- It can be treated by administering methyl-4'-quinoline carboxylic acid sodium salt (Brequinar). Interferon α2, interferon α8, interferon such as interferon β, or an inhibitor of a second enzyme selected from inosine monophosphate dehydrogenase, guanosine monophosphate synthetase, cytidine triphosphate synthetase, and S-adenosylhomocysteine hydrolase. When administered further, a synergistic effect is obtained.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to the family Flaviviridae, Rhabdovirid.
ae), or the treatment of infectious diseases caused by viruses of the Paramyxoviridae family.

BACKGROUND OF THE INVENTION Currently, an effective method for treating hepatitis C chronic absent. Until now, human interferon has been used to treat patients with chronic hepatitis C, but the efficacy of this treatment is about 20%. Recently, ribavirin and interferon have been used in combination to treat chronic hepatitis C patients. The efficacy of this combination therapy is up to about 40%. However, the high dose of ribavirin required is associated with red blood cell toxicity.

Researchers around the world are currently searching for drugs effective against hepatitis C.
Most, if not all, enzymes of the Flaviviridae hepatitis C virus and related viruses have been targeted in the search for antiviral drugs against hepatitis C. A major problem in the study of hepatitis C is the absence of in vitro cell lines and model animal systems. To date, there can be no good replicating cell line for assaying activity against hepatitis C virus. The only model animal for hepatitis C is the chimpanzee. However, establishing chronic hepatitis C infection in chimpanzees is difficult. These facts further complicate the use of chimpanzees as a model animal system.

SUMMARY OF THE INVENTION The "surrogate" virus / host cell line is used to treat specifically acute and chronic hepatitis C patients, generally flavivirus, rhabdovirus and paramyxovirus infections. We searched for antiviral compounds that could be used as antiviral drugs to Three structural and biologically similar compounds were selected to select three flaviviridae viruses (yellow fever virus, kunjin virus, dengue virus), one rhabdoviridae (Vesicular stomatitis virus: VSV) and one virus of the Paramyxoviridae (respiratory syncytial virus: RSV) were tested for antiviral activity in human and monkey cells. These are all RNA viruses that infect human cells and non-human primate cells.

6-Fluoro-2- (2′-fluoro-1,1′-biphenyl-4-yl) -3-methyl-4′-quinolinecarboxylic acid sodium salt (Brequinar) and other dihydroorotic acid dehydrogenases Inhibitors of the enzyme were found to show potent activity against the viruses tested. Ribavirin was also tested. Dihydroorotate dehydrogenase inhibitors are always more potent than interferons and, in fact, act synergistically in combination with interferons. Also, combining an inhibitor of dihydroorotate dehydrogenase with an inhibitor of a second enzyme selected from inosine monophosphate dehydrogenase, guanosine monophosphate synthetase, cytidine triphosphate synthetase and S-adenosyl homocysteine hydrolase, if Further, when administered in combination with interferon, a synergistic antiviral effect is obtained.

[0006] Accordingly, the present invention provides a method of treating a host of Flaviviridae, Rhabdoviridae, or Paramyxoviridae, which comprises administering an inhibitor of dihydroorotate dehydrogenase to the host. I will provide a.

The invention further provides: -new compounds which are structural analogues of Brequinar and methods for their preparation; -viruses of the Flaviviridae, Rhabdoviridae or Paramyxoviridae families Use of an inhibitor of dihydroorotate dehydrogenase for the manufacture of a medicament for the treatment of an infection caused by: an anti-flavivirus, an antirhabdovirus or an antiparamyxovirus which comprises an inhibitor of dihydroorotate dehydrogenase; -A dihydroorotic acid dehydrogenase inhibitor and interferon as a combined preparation for simultaneous, separate or sequential use in the treatment of infections caused by Flaviviridae, Rhabdoviridae, or Paramyxoviridae viruses. Products Containing: -Flaviwi A dihydroorotic acid dehydrogenase inhibitor and inosine monophosphate as a combined preparation for simultaneous, separate or sequential use in the treatment of infections caused by viruses of the family Rhusidae, Rhabdoviridae or Paramyxoviridae. A product containing an acid dehydrogenase, a guanosine monophosphate synthetase, a cytidine triphosphate synthetase, and an inhibitor of a second enzyme selected from S-adenosylhomocysteine hydrolase; A method of identifying an anti-flavivirus agent, an anti-rhabdovirus agent, or an anti-paramyxovirus agent, comprising testing for.

Detailed Description Inhibitors of dihydroorotate dehydrogenase Dihydroorotate dehydrogenase (DHO-DH, DHOD, EC 1.3.3.1) is de novo
It is the fourth enzyme in the pyrimidine biosynthetic pathway. Any inhibitor of this enzyme can be used in the present invention.

Methods for identifying inhibitors of dihydroorotate dehydrogenase using computer algorithms are described in Biochemical and Biophysical Research Communication.
s 223, 654-659 (1996) and Biochemical Pharmacology Vol. 49, No. 7, pp.
947-954 (1995). This technique determines the biological activity of a given compound as well as dichloroallyl lawsone and Brequinar.
Correlation with the biological activity of known DHOD inhibitors such as The COMPARE computer algorithm can be used for this correlation (J. Natl.
Cancer Inst. 81, 1088-1092, 1989).

An in vitro assay for inhibitors of mouse liver dihydroorotate dehydrogenase is described in J. Biol. Chem. 270, No. 38, pp. 22467-22472 (1995). Mouse liver dihydroorotate dehydrogenase is described in Biochem. J. (1998),
It can be prepared as described in 336, 299-303 (1998).

Inhibitors of dihydroorotate dehydrogenase are described, for example, in Douglas G.
Batt describes in Exp. Opin. Ther. Patents (1999) 9 (1), 41-54, the contents of which are incorporated herein by reference.

The finding that inhibitors of dihydroorotate dehydrogenase have activity against several classes of RNA viruses provides a means to screen for new antiviral drugs. Accordingly, the present invention provides a method for identifying an anti-flavivirus agent, an anti-rhabdovirus agent, or an anti-paramyxovirus agent, which method comprises: (a) providing a test compound; It is determined whether or not it exhibits activity as an inhibitor of dihydroorotate dehydrogenase; (c) if it is shown in step (b) that the test compound is an anti-flavivirus agent, anti-rhabdovirus agent, or Selecting as an anti-paramyxovirus drug; The dihydroorotic acid dehydrogenase inhibitory activity of a candidate test compound can be measured by any of the known techniques described above.

Four types of dihydroorotic acid dehydrogenase inhibitors are suitable for use in the present invention. These are the compounds shown below. A compound of formula (I):

[Chemical 17] [Wherein each A is independently hydrogen, halogen, perhaloalkoxy, amino C ₁ -C ₈ alkyl, NO ₂ , CN, SO ₂ CH ₃ , C ₁ -C ₈ alkyl, C ₁ -C ₈ Selected from the group consisting of alkoxy, C ₃ -C ₇ cycloalkyl, C ₃ -C ₇ cycloalkenyl, aryl, aryloxy, C ₁ -C ₆ perhaloalkyl, and Y, or adjacent 2 on ring b. The group A is
Taken together with the phenyl ring to which they are attached form a naphthalene ring system; R is cyclohexyl, phenoxy or benzoxy, or a phenyl ring optionally substituted with a group A as defined above; Alternatively, R on ring b and the adjacent group A, together with the phenyl ring to which they are attached, form a naphthalene or phenanthrene ring system; Y is from COOM, CONHR ′, SO ₃ M, and hydrogen. M is selected from the group consisting of H, Li, Na, K, and 0.5Ca; R ′ is C ₁ -C ₁₀ alkyl; n is 1 or 2; T Is = N- or = C (Z)-, where (i) Z is hydrogen, NH ₂ , OH, C ₁ -C ₈ alkyl, C ₃ -C ₇ cycloalkyl, aryl, and C ₁ -C ₆ or is selected from the group consisting of perhaloalkyl, or (ii) Z is, -VW- (wherein, V is be CH ₂ or S W is CH _2, O, S or N
H) and-(CH ₂ ) ₂ -C (= Z)-(where Z is O or H ₂ ) and the bridging moiety is an adjacent biphenyl group. Either complete one ring by attaching to the ortho position of ring b .

[0014] -A compound represented by formula (I '):

[Chemical 18] [Wherein A and Y are as defined above for formula (I); R'is hydrogen and R "is a thiophene ring or formula (i ') or (ii'):

[Chemical 19] Or R ′ and R ″ together with the carbon atom to which they are attached (represented by “C”) are of formula (iii ′) or (iv ′):

[Chemical 20] (Where R ″ is H or halogen and R ^iv is H or C ₁ -C ₆ alkoxy.
) Form a ring system].

[0015] A compound of formula (III):

[Chemical 21] [Wherein each A ¹ is independently hydrogen, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, C ₃ -C ₇ cycloalkyl , Halogen, unsubstituted aryl, X-substituted aryl, NO ₂ , CN, COOR, CONHR, and NHR; X is halogen, NO ₂ , C ₁ -C ₈ alkyl, aryl, fused aryl, and COOR
R is selected from the group consisting of hydrogen and C ₁ -C ₈ alkyl; B is selected from the group consisting of C ₁ -C ₈ alkyl, H, CF ₃ , and aryl, Aryl may be substituted with halogen, C ₁ -C ₈ alkoxy, C ₁ -C ₈ alkyl, NO ₂ , aryl or fused aryl].

[0016] A compound of formula (V):

[Chemical formula 22] [In the formula, A ² is hydrogen, C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, C ₃ -C ₇ cycloalkyl, halogen, unsubstituted aryl, halogen-substituted aryl, condensed Aryl, NO ₂ , CN, NHR ¹ , and N (R ¹ ) ₂ are selected from the group consisting of; R ¹ is selected from the group consisting of hydrogen, C ₁ -C ₈ alkyl, and OH; X ¹ is Hydrogen or halogen; B ¹ , Y ¹ and Z ¹ are each independently selected from the group consisting of hydrogen, OH, C ₁ -C ₈ alkyl, halogen, CN, NO ₂ and CF ₃ . .

[0017] -A compound of formula (VII):

[Chemical formula 23] Wherein each A ³ is independently hydrogen, C ₁ -C ₈ alkyl, C ₁ -C ₁₀ alkoxy, halogen, and N (R ²⁾ is selected from the group consisting of _2; B ² is a direct A bond, -CH = CH- or -C≡C-; X ² is selected from the group consisting of O, S and NR ² ; R ² is a group consisting of hydrogen, C ₁ -C ₄ alkyl and aryl Y ² is selected from the group consisting of COOM ¹ and SO ₃ M ¹ ; M ¹ is selected from the group consisting of H, Li, Na, K and 0.5 Ca].

The halogen atom can be fluoro, chloro, bromo or iodo. C ₁ -C ₈ alkyl group is preferably C ₁ -C ₄ alkyl group. The C ₁ -C ₄ alkyl group is typically methyl, ethyl, n-propyl, isopropyl or butyl. C ₃ -C ₇ cycloalkenyl group typically being cyclohexenyl. C ₃ -C ₇ cycloalkyl group is typically a cyclopentyl or cyclohexyl group. The C ₁ -C ₆ perhaloalkyl group can be a C ₁ -C ₄ perhaloalkyl group. The halo atom may be chloro or fluoro. A particularly preferred perhaloalkyl group is trifluoromethyl.

The aryl group is typically phenyl. C ₂ -C ₈ alkenyl groups are preferably C ₂ -
It is a C ₄ alkenyl group. C ₂ -C ₈ alkynyl group is preferably C ₂ -C ₄ alkynyl group. The fused aryl is generally naphthyl. A C ₁ -C ₁₀ alkoxy group is preferably a C ₁ -C ₆ alkoxy group, for example a C ₁ -C ₄ alkoxy group such as methoxy or ethoxy.

Perhaloalkoxy is, for example, OCF ₃ , OCCl ₃ or OCBr ₃ . Preferably it is OCF ₃ . Aryloxy is, for example, phenoxy or benzyloxy.

The compounds of formula (I) are known, and compounds prepared by or by published methods can be used to modify the compounds by routine synthetic modifications and interconversions well known in the field of organic chemistry. It can be manufactured by attaching to. A number of published method references are cited in the Douglas G. Batt article cited above. For example, compounds of formula (I) where T is = C (Z)-can be prepared as described in US-A-4680299. Compounds of formula (I) where T is = N- can be prepared as described in US-A-4639454. Compounds of formula (I) in which Z is a bridging moiety as defined above are described in US-A-4918077,
US-A-5002954, WO9506640, US-A-5371225, EP-A-721942, JP10231289, Organ. B
iol. (1997) 4 (2): 43-48 and 49-57, JP-6306079-A2, and 216 ^th ACS Mee.
Ting, Boston UAS (1998) ORGN 132. Expression (I
The compound of ') can be manufactured by the same method as the synthetic method described in US-A-4680299.

Preferred compounds of formula (I) are the following compounds: wherein each A is independently hydrogen, halogen (preferably F), amino C ₁ -C ₈ alkyl (
Preferably NH ₂ (CH ₂ )-), C ₁ -C ₈ alkyl (preferably CH ₃ ), and C ₁ -C ₆ perhaloalkyl (preferably CF ₃ ); Y is COOM, And CONHR ′, wherein M is as defined above, R ′ is C ₅ -C ₁₀ alkyl, preferably octyl; T is = C (Z)- , Wherein (i) Z is selected from the group consisting of hydrogen, C ₁ -C ₈ alkyl (preferably CH ₃ ), NH ₂ , and OH, or (ii) Z is — (CH ₂ )-,-(CH ₂ ) _3- , -SCH _2- , -CH ₂ O-, -CH ₂ S-, -CH ₂ NH-, and-(CH ₂ ) -C (= O)- It is the cross-linking moiety defined above that is more selected.

In formula (I), the substituent A on ring a is preferably OCF ₃ , halogen (most preferably F) or NH ₂ (CH ₂ ) ₂ —, preferably 6 of the quinoline ring system. It is the one that is combined with the position. Substituent A on ring b is preferably hydrogen.

[0024]   Examples of compounds of formula (I) are those of formula (Ia):

[Chemical formula 24] [Wherein each A is independently hydrogen, halogen, amino C ₁ -C ₈ alkyl, NO ₂ , CN, SO ₂ CH ₃
, C ₁ -C ₈ alkyl, C ₃ -C ₇ cycloalkyl, aryl, C ₁ -C ₆ perhaloalkyl,
And Y is selected from the group consisting of COOM, CONHR ′, SO ₃ M, and hydrogen; M is selected from the group consisting of H, Li, Na, K, and 0.5Ca ; R 'is an C ₁ -C ₁₀ alkyl; T is, = N-or = C (Z) - and is, where, (i) Z is _{hydrogen, NH 2, OH, C 1} -C ₈ alkyl, C ₃ -C ₇ cycloalkyl, aryl, and C ₁ -C ₆ perhaloalkyl, or (ii) Z is —VW— (where V is CH ₂ or S And W is CH ₂ , O, S or N
H) and-(CH ₂ ) ₂ -C (= Z)-(where Z is O or H ₂ ) and the bridging moiety is an adjacent biphenyl group. Or one of the rings completes one ring by bonding to the ortho position of ring b .].

Substituent A on ring c of formula (Ia) is preferably hydrogen or halogen in the ortho position (most preferably ortho-F).

[0026]   Among them, preferred compounds of formula (I) are those of formula (II):

[Chemical 25] [Wherein R ¹ is H, halogen or OCF ₃ ; M is as defined above; R ² is H or C ₁ -C ₆ alkyl; R ³ is H or OR ⁶ (where R ⁶ is H or C ₁ -C ₆ alkyl); R ⁴ is H or C ₁ -C ₆ alkyl, or R ⁴ and R ³ are phenyl to which they are attached. Taken together with ring b to form a naphthalene ring; R ⁵ is cyclohexyl, phenoxy, or benzoxy, or an unsubstituted or halogen-substituted phenyl ring, or R ⁴ and R ⁵ are bound to each other Forms a naphthalene ring together with the phenyl ring b ].

Specific substitution patterns for compounds of formula (II) are as follows:

[0028]   Particularly preferred are those of formula (IIb):

[Chemical formula 26] Is a sodium salt of Brequinar represented by. Breqinal
uinar) can be prepared as described in US-A-4680299 (Example 28).

Some of the compounds of formula (II) are new. Therefore, the present invention further provides formula (IIa)
:

[Chemical 27] Wherein M is selected from the group consisting of H, Li, Na, K, and 0.5Ca; R ²² is H or C ₁ -C ₆ alkyl; R ³³ is H or OR ⁶ . , R ⁶ is H or C ₁ -C ₆ alkyl; R ⁴⁴ is H or C ₁ -C ₆ alkyl; R ⁵⁵ is phenyl, cyclohexyl, phenoxy or benzoxy] To provide a compound.

Preferred examples of compounds of formula (IIa) are: 2- (4-biphenylyl) -6-trifluoromethoxy-quinoline-4-carboxylic acid (compound I2K5); 2- (4-biphenylyl) ) -3-Methyl-6-trifluoromethoxy-quinoline-4-carboxylic acid (Compound I2K55); 2- (4-cyclohexylphenyl) -6-trifluoromethoxy-quinoline-4-carboxylic acid (Compound I2K46); 2 -(4-Benzyloxy-2-methoxy-3-methyl-phenyl) -6-trifluoromethoxy-quinoline-4-carboxylic acid (Compound I2K51); and 2- (4-phenoxyphenyl) -6-trifluoromethoxy -Quinoline-4-carboxylic acid (compound I2K52).

The compounds of formula (II), including the novel compounds of formula (IIa), are described in US-A-4680299 above.
Can be produced by the synthetic route described in. This involves the Pfitzinger reaction, whereby an appropriately substituted isatin is condensed with an appropriately substituted ketone (J. Org. Chem. 18, 1209, 1953).

Accordingly, the present invention further provides a process for the preparation of a compound of formula (IIa) as defined above, which process comprises (a) formula (IX):

[Chemical 28] A trifluoromethoxy-substituted isatin compound represented by the formula (X):

[Chemical 29] [Wherein R ²² , R ³³ , R ⁴⁴ and R ⁵⁵ are as defined above for formula (IIa)]
Condensation with a ketone of formula (II) in the presence of a base; (b) optionally obtaining the resulting compound of formula (IIa) wherein M is H, wherein M is Li, Na, K or 0.5Ca; Converting to a pharmaceutically acceptable salt;

The base used in step (a) may be an inorganic base or an organic base. Preferred bases include potassium hydroxide and sodium hydroxide. This reaction is generally carried out in a solvent, preferably ethanol.

Preferred compounds of formula (I ′) have the following substitution pattern:

Preferred compounds of formula (III) are the following compounds: each A ¹ is independently selected from the group consisting of hydrogen, halogen, C ₁ -C ₈ alkyl, and C ₁ -C ₈ alkoxy. B is selected from the group consisting of hydrogen, C ₁ -C ₈ alkyl, phenyl, and benzyl, which phenyl and benzyl are halogen (preferably Cl or Br), C ₁ -C ₈ alkoxy (preferably C ₁ -C ₄ alkoxy, such as OCH ₃ ), or C ₁ -C ₈ alkyl (
It may be optionally substituted with C ₁ -C ₄ alkyl, such as CH ₃ .

When B is a substituted phenyl or benzyl as defined above, the substituent is preferably in the 4-position of the phenyl ring. The group A ^{1 of the} ring a is preferably H. The group A ^{1 of} ring b is preferably H or is para-C ₁ -C ₈ alkoxy or para-halogen (preferably Cl or Br).

[0037]   Particularly preferred are those of formula (IV):

[Chemical 30] 1- (p-bromophenyl) -2-methyl-1H-naphtho [2,3-d] imidazole-4,9 represented by
-It's Zeon (BNID).

The compound of formula (III) is well known in the field of organic chemistry as a compound prepared by the method described in J. Med. Chem. 1996, 39, 1447-1451 or by the method described in this document. It can be prepared by subjecting it to routine synthetic modifications and interconversions. BNID can be prepared as described in J. Med. Chem. 1964, 7, 362-364.

Preferred compounds of formula (V) are the following compounds: A ² is the group consisting of hydrogen, C ₁ -C ₈ alkyl (preferably C ₁ -C ₄ alkyl, eg CH ₃ ), and halogen. B is hydrogen or OH; X ¹ is hydrogen; Y ¹ and Z ¹ are each independently hydrogen, halogen (preferably Cl), and
It is selected from the group consisting of C ₁ -C ₈ alkyl (preferably C ₁ -C ₄ alkyl, eg CH ₃ ).

[0040]   Particularly preferred are those of formula (VI):

[Chemical 31] Is dichloroallyl lawson.

The compound of the formula (V) is prepared by the method described in US-A-3655699 or WO9106863, or the compound produced by the method described in these documents, in a routine manner well known in the field of organic chemistry. It can be produced by subjecting it to synthetic modification and interconversion. Dichloroallyl lawson can be prepared as described in US-A-3655699.

Preferred compounds of formula (VII) are: each A ³ is hydrogen or C ₁ -C ₁₀ alkoxy (preferably C ₁ -C ₆ alkoxy, eg methoxy); B ² is , A direct bond; X ² is NR ² , where R ² is hydrogen or C ₁ -C ₄ alkyl; Y ² is COOH.

Preferably, in formula (VII) each A ³ is in the meta position relative to the linking group B ² and each Y ²
Is in the ortho position relative to the linking group X ² .

[0044]   Particularly preferred are those of formula (VIII):

[Chemical 32] 2,2 '-[3,3'-dimethoxy [1,1'-biphenyl] -4,4'-diyl] diimino] bis-benzoic acid (redoxal) represented by Redoxal is Zhur. A
Nal. Khim. 15, pp. 671-675, 1960 and Chemical Abstracts 1961, vol. 5, 1
It is described in 8447b. Other compounds of formula (VII) can be prepared by routine synthetic modifications and interconversions of the compounds prepared in this reference.

Interferon The interferon used in the present invention is interferon α such as interferon α2 or α8, or interferon β. The term "interferon" includes fragments that have interferon activity and mutant forms of interferon that retain interferon activity. For example, the sequence of interferon α or β may be modified to increase activity or stability as reported in US-A-5582824, US-A-5593667 or US-A-5594107.

The interferon may be purified from natural sources or may be a recombinant interferon. The species of interferon is generally the same as the species of the host that administers the interferon. The invention has particular application in the treatment of human flavivirus infections. Therefore, human interferons such as human interferon α2 or α8 or human interferon β are preferably used.

The specific activity of interferon α8, especially human interferon α8, is typically
Greater than 0.3x10 ⁹ , generally 0.3x10 ^{9 to} 3x10 ⁹ , and preferably 0.5x10 ^{9 to} 3x
10 ⁹ IU / mg protein. The specific activity of human interferon β is typically 4x
10 ⁸ ~8x10 ^8, preferably 4.8x10 ⁸ ~6.4x10 ⁸ IU / mg protein. The specific activities of interferon alpha and interferon beta are determined by reference standards MRC 69/19 and Gb-23-902-531, respectively. Specific activity is 0.2 μg /
Armstrong, Applied Microbiology 21 , 723,1 characterized by the inclusion of ml of actinomycin D and a direct reading of the virus-induced cytopathic effect.
Determined by the amendment of 971.

Interferons such as interferon α2 or α8 or interferon β can preferably be obtained by the method of WO 96/30531. Interferon thus contains (i) a coding sequence which encodes interferon and is operably linked to a promoter capable of directing the expression of the coding sequence in mammalian cells in the presence of heavy metal ions; (ii) a metallothionein gene. And a first selectable marker sequence operably linked to a promoter capable of directing the expression of the metallothionein gene in the presence of heavy metal ions in the cell; and (iii) containing the neo gene and the neo gene in the cell A second selectable marker sequence operably linked to a promoter capable of directing the expression of the expressed mammalian cell without the condition of allowing the expression of the coding sequence. Culture; and obtained by a method comprising recovering the interferon thus produced You can

The transfected mammalian cells may be human or animal cell lines. They may be fibroblastoid cells such as BHK, COS, Vero, CHO, HeLa, or human lymphoblastoid cells or human tumor cell lines. However, preferably
CHO cells, especially wild type CHO cells.

The transfected cells preferably have all or part of such a vector integrated into their genome. Such cells are preferred because they stably express the coding sequence contained in the vector. It is preferred to incorporate more than one copy of the total vector, multiple copies of the vector, eg 20-100.
Particularly preferred are cells that have incorporated more than one copy. This is because these cells express a stable high level of the coding sequence contained in the vector.

However, even if a cell integrates a region smaller than the complete region of the vector into the genome, if the cell is functionally different from the cell that integrates the complete vector into the genome (incorporation of the vector Such cells can also be used, provided the regions are identical (in the sense that they allow the expression of the coding sequences of the cell and can be selected by the use of heavy metals). Therefore, even in cells that exhibit partial integration of the vector,
It may be used if the incorporated elements include a coding sequence operably linked to the associated promoter and a metallothionein marker sequence operably linked to the associated promoter.

Any promoter capable of increasing expression in mammalian cells in the presence of heavy metal ions such as Cd ²⁺ , Cu ²⁺ and Zn ²⁺ is operably linked to the interferon coding sequence. Good. A suitable promoter is the metallothionein gene promoter. The mouse metallothionein gene I (mMT1) promoter is preferred.

Suitable promoter / enhancer combinations for the coding sequences include the mmT1 promoter (MV1 promoter (MVV1 enhancer flanked by the
SV-mMT1), and the Rous sarcoma virus (RSV) enhancer upstream of the mouse mammary tumor virus (MMTV) promoter. MSV-mMT1 is preferred.

As far as the first selectable marker sequence is concerned, any promoter capable of increasing the expression in mammalian cells in the presence of heavy metal ions such as Cd ²⁺ , Cu ²⁺ and Zn ²⁺ may be used as the human metallothionein gene. May be operably linked to the metallothionein gene of. Preferably, the marker sequence gene is a human metallothionein gene, such as human metallothionein gene IIA, which has its own promoter.

The second selectable marker is the neo gene. One or more forms of this gene occur naturally: any particular neo gene may be used in the vectors of the invention. One of the preferred neo genes is the E. coli neo gene.

The promoter for the neo gene can direct the expression of this gene in mammalian cells. Suitable promoters are cytomegalovirus (CMV) early promoter, SV40 promoter, mouse mammary tumor virus promoter, human elongation factor 1α-P promoter (EF-1α-P), SRα promoter, and metallothionein gene promoters such as mMT1. The promoter is also capable of expressing the neo gene in bacteria such as Escherichia coli with which vectors can be constructed.

Therefore, the interferon coding sequence (i) and the marker sequences (ii) and (ii
i) is operably linked to a promoter capable of directing the expression of the relevant sequences. The term “operably linked” means that the promoter and coding / marker sequences are juxtaposed in a relationship such that expression of the coding / marker sequence is under the control of the promoter. Therefore, between the promoter and the coding / marker sequence
There may be elements such as 5'non-coding sequences. Such sequences may be included in the construct if they increase or do not impair the correct control of the coding / marker sequence by the promoter.

The vector may be a DNA or RNA vector, preferably a DNA vector. Typically the vector is a plasmid. Each of the sequences (i)-(iii) is typically associated with other elements that control their expression. For each sequence, the following elements are generally present, usually located 5'to 3 ': a promoter that directs expression of the sequence and optionally promoters of the promoter, translation initiation codons, code / markers. Sequences, polyadenylation signals and transcription terminators.

In addition, the vector typically comprises one or more origins of replication, for example a bacterial origin of replication such as the pBR322 origin, which allows replication in bacterial cells. Alternatively or additionally, the vector may include one or more eukaryotic origins of replication that allow replication in, for example, yeast cells and / or mammalian cells.

The vector may also include a 1 on the 3 ′ or 5 ′ side of the coding sequence or one or more marker sequences.
The above introns or other non-coding sequences may be included. Such non-coding sequences may be from any organism and may be synthetic in nature. Therefore, these may be any sequences. Such sequences may be included provided they do not enhance or impair the correct expression of the coding or marker sequences.

Transfected cells are typically cultured in the presence of heavy metal ions selected from Cd ²⁺ , Cu ²⁺ and Zn ²⁺ , in particular in a non-cytotoxic amount. This can result in higher expression of the desired interferon. The concentration of heavy metal ions in the medium is typically 100-200 μM. Thus, the cells may be cultured in the presence of 100-200 μM of a heavy metal ion selected from Cd ^{2 +} , Cu ²⁺ and Zn ²⁺ , for example 130-170 μM of a heavy metal ion. A useful concentration is about 150 μM, especially when the heavy metal ion is Zn ²⁺ .

The interferon produced can be recovered by any suitable method, which may vary depending, for example, on the cell type utilized and the culture conditions used. It is desirable to purify the produced interferon after recovery. Thus, substantially pure interferon can be obtained.

Human β-interferon provided by WO96 / 30531 has a high degree of sialylation. It is a natural human β- produced by primary diploid human fibroblasts.
It is fully glycosylated, like interferon. But that is
It has a higher degree of bioavailability than either native β-interferon or recombinant β-interferon produced in E. coli (betaseron).

Β-interferon can be characterized by high bioavailability. Subcutaneous injection of 1.5 x 10 ⁶ IU of interferon into the back of approximately 2 kg of rabbit: (a) One hour later, ≧ 128 IU / ml of interferon can be detected in the serum of the rabbit, and / or (b ) After 5 hours,> 64 IU / ml interferon can be detected in rabbit serum.

Maximum levels of interferon are typically observed after 1 hour. Therefore, according to (a), 128-256 IU / ml, for example 140-1 in rabbit serum after 1 hour.
90 IU / ml interferon can be detected. After 5 hours, according to (b), ≧ 70 IU / ml, eg ≧ 80 IU / ml interferon can be detected in rabbit serum. Typically, according to (b), an amount of interferon in the range of 64-128 IU / ml, for example 80-110 IU / ml, can be detected.

[0066]   Additionally or alternatively, the human interferon beta is characterized by its specific activity.
Can be attributed. This is 4.8x10, as above⁸~ 6.4x10⁸ IU / mg equivalent (
Bovine serum albumin protein). Its specific activity is 5x10⁸~ 6x10 ⁸ , For example, 5.3x10⁸~ 5.5x10⁸Like 5.2x10⁸~ 5.8x10⁸ IU / mg equivalent (bovine serum
Albumin protein).

The human interferon beta can also be characterized by one or more of the following properties: 1. The interferon beta is typically 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It has an apparent molecular weight of 26,300 as measured by.

2. When infused into rabbits as an undiluted intravenous bolus, the half-life of the interferon is typically 12-15 minutes, eg about 13.5 minutes. A bolus is injected into the rabbit ear vein and a blood sample is collected from the rabbit ear artery. Rabbit serum, Armstr
Assay interferon for antiviral activity according to the modified method of Ong (1971).

3. The antiviral activity of the interferon in the human hepatoblastoma cell line (HepG2) is at least equal to that of native interferon β from primary diploid human fibroblasts, typically about 1.5 times that. Is. The interferon is also about 2.2-fold more effective than beta-serone in protecting Hep2 cells from viral challenge. Antiviral activity is again measured according to the modified method of Armstrong (1971). H
Actinomycin D was omitted in the antiviral assay in epG2 cells.

The oligosaccharides that bind to interferon β of the present invention can also characterize the interferon β. The interferon β has an oligosaccharide that can be characterized by one or more of the following properties: 1. Neutral (no acidic substituents): 5-15%, preferably about 10% or less Acidic: 95-85%, preferably > 90% 2. All desialylated oligosaccharide pools are heterogeneous with at least 6 different structural components in the pool 3. Matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry Method and high resolution gel permeation chromatography data are summarized below: detection mass composition calculated mass gu equivalent 1786.2 5Hex, 4HexNAc, 12AB, Na 1782 11.1 1929.9 5Hex, 1dHex, 4HexNAc, 12AB, Na 1928 12.2 2295.5 6Hex, 1dHex , 5HexNAc, 12AB, Na 2293 14.5 2660.1 7Hex, 1dHex, 6HexNAc, 12AB, Na 2658 17.6 3019.1 8Hex, ldHex, 7HexNAc, 12AB, Na 3023 20.7

The carbohydrate portion of human interferon-β of WO96 / 30531 consists of N-linked oligosaccharides of the bi-, tri- and tetra-antennary complex type. These oligosaccharides contain repetitive lactosamines. About 20-50%, for example 20-30%, 30-40% or 35
~ 50% of oligosaccharides are biantennary oligosaccharides. About 30-65%, for example 40-60%
Or 50-60% of the oligosaccharides are tri-antennary oligosaccharides. About 2-15%, for example 2-8%, 4-10% or 5-15% oligosaccharides are tetra-antennary oligosaccharides. Percentages are calculated by weight of total analyzable oligosaccharide content.

Compounds that are inhibitors of the second enzyme inhibitor inosine monophosphate dehydrogenase, guanosine monophosphate synthetase, cytidine triphosphate synthetase and S-adenosyl homocysteine hydrolase are described in WO 00/50064 (the content of which is (Incorporated herein by reference), and having antiviral activity against the Flaviviridae and Rhabdoviridae family of viruses. Examples of these compounds include 5-membered carbocyclic nucleosides and mycophenolic acid compounds as described below.

Some of these antiviral compounds were tested as inhibitors of dihydroorotate dehydrogenase and were found to be inactive. This means that (a) intracellular replication of flaviviruses, rhabdoviruses and paramyxoviruses involves more than inhibition of dihydroorotate dehydrogenase, and (b) dihydroorotate dehydrogenase is required for viral replication. It is suggested that it is only one of multiple nucleotide synthases.

This finding indicates that infections caused by flaviviridae, rhabdoviridae and paramyxoviridae family viruses are associated with dihydroorotate dehydrogenase inhibitors, inosine monophosphate dehydrogenase, guanosine monophosphate synthetase, and cytidine trihydrate. It offers the opportunity for combination therapy to be treated with an inhibitor of a second enzyme selected from phosphate synthetase and S-adenosyl homocysteine hydrolase. If desired, two inhibitors may be used in combination with interferon. Strategies using combination therapy aim to solve the problem of multidrug resistance that typically occurs when a single agonist is used to treat a disease or disorder.

[0075]   The 5-membered carbocyclic nucleoside has the following formula (XI):

[Chemical 33] Wherein B is selected from the group consisting of purines, pyrimidines and 5- or 6-membered aglycones, R ₂ and R ₃ are independently H, halogen, OH, O-acyl, O-aryl and O-silyl. Selected from the group consisting of, and R ₁ is as defined for R ₂ and R ₃ or is O-phosphate], or a pharmaceutically acceptable metabolite, metabolite derivative or salt thereof. be able to.

[0076]   Preferred compounds of formula (XI) are those in which B has the following groups (i) to (viii):

[Chemical 34] [Wherein R ₂₅ is Cl or NH ₂ and R ₂₆ is H, CH ₃ , CF ₃ , F, Cl, Br or I].

The 5-membered carbocyclic nucleosides of formula (XI) are known compounds and can be synthesized by the disclosed methods or by methods analogous to those disclosed. For example, (-
) -Neplanocin A synthesis is described by Arita et al., In J. Am. Chem. Soc. (1983), 105 (12), 404.
9-4055.

The synthesis of other suitable cyclopentenyl carbocyclic nucleosides is described in US-A-4975434, the contents of which are incorporated herein by reference. A table of specific nucleosides is listed in the patent.

Substitution patterns for preferred compounds of formula (XI) are shown in the table below. A particularly preferred compound is cyclopentenyl cytidine (CPE-C).

Substitution pattern R ₁ R ₂ in formula (XI) R ₃ Nucleoside Formula Compound OH OH OH R ₂₅ is NH ₂ (ii) 3-Deazaneplanocin A OH OH OH R ₂₅ is NH ₂ (i) Neplanocin A OH OH OH R ₂₆ is H (iii ) Cyclopentenyl cytidine (CPE-C)

[0081]   The mycophenolic acid compound has the following structure (XII):

[Chemical 35] [Wherein R ₄ is OR ₆ or —N (R ₇ ) R ₈ where R ₆ , R ₇ and R ₈ are independently selected from the group consisting of hydrogen and C ₁ -C ₆ alkyl. , And R ₅ is selected from the group consisting of hydrogen, phenyl and C ₁ -C ₆ alkyl which is unsubstituted or substituted by a 5 or 6 membered saturated or unsaturated heterocycle]] .

Preferred compounds of formula (XII) are those compounds in which R ₄ is hydroxy or NH ₂ . When one or more of R ₅ , R ₆ , R ₇ and R ₈ is a C ₁ -C ₆ alkyl group, it is preferably a C ₁ -C ₄ alkyl group such as methyl or ethyl. A 5- or 6-membered saturated or unsaturated heterocycle generally has 1, 2 or 3 N atoms and optionally 1
It contains O and / or S atoms. Suitable such rings include pyridino, piperidino, pyrrolo, pyrrolidno and morpholino rings. Therefore, an N-morpholino ring may be present.

Suitable mycophenolic acid compounds for use in combination with a dihydroorotic acid dehydrogenase inhibitor in the present invention are mycophenolic acid, mycophenolate mofetil, a morpholinoethyl ester of mycophenolic acid.
ate mofetil), and the individual mycophenolic acid derivatives described in US-A-5380879. The contents of US-A-5380879 are incorporated herein by reference.

Therapeutic Uses Dihydroorotic acid dehydrogenase inhibitors are used to treat flavivirus, rhabdovirus and paramyxovirus infections, especially in humans. The infection may be acute or chronic. Flavivirus is a hepatitis virus such as yellow fever virus, Kunjin virus, West Nile virus, dengue virus, hepatitis C virus, or encephalitis virus such as St. Louis encephalitis virus, Japanese encephalitis virus, Mary Valley encephalitis virus and tick-borne encephalitis. It may be. The rhabdovirus may be vesicular stomatitis or rabies virus. The paramyxovirus may be respiratory syncytial virus (RSV).

A therapeutically effective amount of a dihydroorotic acid dehydrogenase inhibitor is administered to the subject to be treated. In this way, the subject's symptoms can be improved. The infection is completely eliminated from the subject.

The inhibitors may be administered orally in various dosage forms, such as tablets, capsules, dragee- or film-coated tablets, solutions or suspensions, or parenterally, eg intramuscularly, intravenously or It may be administered subcutaneously. Therefore, it may be administered by injection or infusion.

The method and regimen of administration of the inhibitor will depend on various factors such as the particular inhibitor involved, the age, weight and condition of the patient, and the nature of the viral infection. However, typically, the dose applied to each route of administration for humans, eg adults, will be in the range 0.001-30 mg / kg body weight, most usually 0.01-5 mg / kg body weight. Such a dose is administered, for example, daily. The dose may be given orally or by bolus infusion, infusion over several hours and / or repeated administration.

Interferon can also be administered to a subject being treated. The dihydroorotate dehydrogenase inhibitor and interferon may be given simultaneously. Alternatively, they may be given to each other up to 5 days apart, for example up to 2 days apart or up to 1 day apart or up to 4 hours apart. The relative timing of administration of the inhibitor and interferon may be determined by monitoring their respective serum levels. The interferon may be given before the dihydroorotic acid dehydrogenase inhibitor or vice versa.

Interferon may be administered in a variety of ways, such as orally, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, and subcutaneously. The specific mode of administration and dosing regimen will be determined by the attending physician, including the age, weight and condition of the patient, the nature of the viral infection, the co-administered dihydroorotate dehydrogenase inhibitor, and, if necessary, the need for synergistic effects. Select in consideration of the factors of.

In addition to the dihydroorotate dehydrogenase inhibitor, an inhibitor of a second enzyme selected from inosine monophosphate dehydrogenase, guanosine monophosphate synthetase, cytidine triphosphate synthetase and S-adenosyl homocysteine hydrolase, and In addition to or instead of interferon, it can be administered to the subject being treated. Suitable dosages and formulations for the inhibitor of the second enzyme are given above.
It is described in WO 00/50064.

In general, the patient will be provided with a separate formulation of a dihydroorotate dehydrogenase inhibitor on the one hand and an inhibitor of said second enzyme and / or interferon on the other hand. However, a dihydroorotate dehydrogenase inhibitor and the second
If the enzyme inhibitor and / or interferon are stable in the presence of each other and do not otherwise interfere with each other, a single formulation containing the respective components may be administered. .

Pharmaceutical compositions containing interferon as the active principle can usually be formulated with suitable pharmaceutically acceptable carriers or diluents depending on the particular mode of administration utilized. For example, parenteral formulations are typically injectable solutions that use pharmaceutically and physiologically acceptable fluids, such as saline, balanced salt solutions, as a vehicle, and physiologically acceptable doses of DMSO and the like. It may contain an organic diluent.
On the other hand, oral formulations may be solids, eg tablets or capsules, or solutions or suspensions.

The dihydroorotate dehydrogenase inhibitor and interferon, or the dihydroorotate dehydrogenase inhibitor and the inhibitor of the second enzyme are typically administered in a synergistic amount. Lower doses of the two inhibitors and interferon provide cost savings and reduced or eliminated side effects that can occur at higher doses. Interferon is usually formulated as a unit dosage form containing 10 ⁴ to 10 ⁹ , more usually 10 ⁶ to 10 ⁷ IU / dose. Typically 3x10 ^{6 to} 36x1 per day
The 0 ⁶ IU interferon, particularly administered by intravenous or subcutaneous injection. This dose may be administered daily, for example for 5-20 weeks.

The invention will be illustrated by the following examples. Example 1 A test compound was weighed and newly dissolved in dimethyl sulfoxide (DMSO). DMSO
A stock solution of each chemical dissolved in was kept at 4 ° C or -80 ° C for long-term storage. The stock solution was diluted in standard medium and serially diluted 2-fold so that the chemical concentration of each chemical decreased from 100 μM to reach the nM concentration.

Human liver (HuH7) cells, human primary fibroblasts (MRC5) and transformed monkey cells (Vero) cultured in 96-well microtiter plates were each grown to near confluency in standard medium. . The medium was removed, using 1-2 ml of fetal bovine serum, various concentrations of each compound dissolved in standard medium, and 0.1 ml of standard medium containing a fixed amount of each virus listed in Tables 1 and 2, The cells in each well were reincubated. Controls were cell controls (treated with compound but no virus) as well as virus controls (treated with virus but no compound).
Was made of.

Almost all of the virus control cells (90-100%) were virtually killed by viral challenge after incubation with the virus at 37 ° C. for several days (1-2 days for VSV (vesicular stomatitis virus), yellow 5-6 days for fever virus, and 7-8 days for dengue and Kunjin virus). Cytotoxicity is defined as days 1-2, 5-6, or 7-8 days after treatment of cells with a given concentration of each compound and virus at the same time (VSV, yellow fever or dengue and Kunjin virus, respectively). Corresponding to the number of days it takes to kill the cells), and gross morphological changes monitored by microscopy.
Evaluation was by cell lysis and / or cytopathic effect.

The results are shown in Tables 1 and 2. The antiviral activity of a compound was indicated by its ED ₅₀ value. Here, the ED ₅₀ value is based on the 100 TCID ₅₀ challenge dose of yellow fever virus, dengue virus or Kunjin virus or RSV, or the multiplicity of infection (MOI).
..) The concentration of test compound that confers 50% protection against a vesicular stomatitis virus challenge of 2.0.

[0098]

[Table 1]

[0099]

[Table 2]

Example 2 Vero and HuH7 cells were grown in 96 microwells to about 99% confluency with minimal essential medium (MEM) containing 10% fetal bovine serum. Growth medium was removed from the microwells and incubated with one of the following: (1) 6, 3, 0.6, 0.3 per ml of MEM containing 2.5 mg / ml human serum albumin.
, 0.06, or 0.03 IU of interferon (Natl. Inst. Allergy and Infecti
ous Diseases, NIH, USA distributed NIH standard, see Gb 23-902-531), 100 μl interferon α8, (2) per ml of MEM containing 2.5 mg / ml human serum albumin , 6, 3, 0.6, 0.3
, 0.06, or 0.03 IU of interferon (MRC 69/19 and Gb-23-902-531
100 μl of interferon α2, (3) 2.5 mg / ml human serum albumin, 60, 30, 6, 3 or 0.6 IU of interferon (Gb-23) per ml of MEM containing 2.5 mg / ml human serum albumin. -902-531 standard), 100 μl of interferon β.

Experiments were performed in 4 or 2 replicates. After adding interferon to Vero cells in microwells, 100 μl of virus challenge consisting of 100 TCLD ₅₀ of yellow fever virus, Kunjin virus, Dengue virus or VSV was immediately added to each microwell. The virus challenge also contained varying concentrations of test compound. Without test compound (ie viral control),
In a well containing 100 μl MEM containing 2.5 mg / ml human serum albumin, 100 μl
Virus challenge was added. The test compound is Brequinar
And

In control cells treated with test compound alone, 2.5 mg /
100 μl MEM containing ml human serum albumin was added to the cells, followed by an additional 100 μl MEM / human serum albumin (2.5 mg / ml) containing the test compound. Cells should be subjected to virus-induced cytopathic effects on days 1-2, 5-6, or 7-8 (corresponding to the number of days VSV, yellow fever or dengue and Kunjin virus require to kill the cells, respectively). Tested to assess the antiviral activity of interferon or test compounds. The results are shown in Table 3.

[0103]

[Table 3]

Example 3: Preparation of a novel compound of formula IIa A mixture of trifluoromethoxy-substituted isatin of formula (IX) as defined above (0.3 mmol) and potassium hydroxide (1.8 mmol in 1 ml of water) is added to 125 Heated in ethanol (2 mL) for 2 hours at ° C. A ketone of formula (X) as defined above (wherein R ²² , R ³³ , R ⁴⁴ and R ⁵⁵ are all hydrogen) (0.6 mmol in 1 mL ethanol) is added and the mixture is further added to 12
Reflux for hours.

The mixture was cooled and concentrated under reduced pressure. The residue was taken up in water (25 mL) and extracted with diethyl ether (2x20 mL). The aqueous layer was acidified with glacial acetic acid to cause precipitation. The resulting precipitate of 2- (4-biphenylyl) -6-trifluoromethoxy-
The quinoline-4-carboxylic acid (Compound I2K5) was filtered, washed well and dried in high vacuum for 2 days.

The characterization data of the compound are as follows: NMR: (Acetone-d6,400 MHz) δ: 7.42-7.54 (m, 3H), 7.76-7.79 (m, 3H), 7.88 (d,
J = 8.44Hz, 2H), 8.30 (d, J = 9.16Hz, 1H), 8.44-8.47 (m, 2H), 8.76 (s, 1H), 8.94 (s, 1
_{H), C 23 H 14 F} 3 NO 3 Calculated 409.1; Found 410.1 (M ⁺ H) +.

The following compounds were prepared using the appropriately substituted ketone of formula (X) by the method described above: 2- (4-biphenylyl) -3-methyl-6-trifluoromethoxy-quinoline-4 -Carboxylic acid (I2K55); NMR: (acetone-d6,400 MHz) δ: 2.44 (s, 3H), 7.49 (t, J = 7.31Hz, 1H) (t, J = 7.59
Hz, 2H), 7.62 (d, J = 8.57Hz, 1H), 7.72-7.82 (m, 6H), 7.92 (s, 1H), 8.09 (d, J = 9.16
Hz, 1H), C ₂₄ H ₁₆ F ₃ NO ₃ calculated 423.1; measured 424.1 (M + H) ⁺ ;

2- (4-Cyclohexylphenyl) -6-trifluoromethoxy-quinoline-4-carboxylic acid (I2K46); NMR: (acetone-d6,400 MHz) δ: 1.29-1.34 (m, 1H), 1.42 -1.58 (m, 4H), 1.75-1.
78 (m, 1H), 1.85-1.96 (m, 4H), 2.62-2.68 (m, 1H), 7.46 (d, J = 8.20Hz, 2H), 7.78 (d,
J = 9.16Hz, 1H), 8.28-8.31 (m, 3H), 8.69 (s, 1H), 8.91 (s, lH), C ₂₃ H ₂₀ F ₃ NO ₃ Calculated value
415.1; measured value 416.1 (M + H) ⁺ ;

2- (4-benzyloxy-2-methoxy-3-methyl-phenyl) -6-trifluoromethoxy
-Quinoline-4-carboxylic acid (I2K51); NMR: (acetone-d6,400 MHz) δ: 2.29 (s, 3H), 3.58 (s, 3H), 5.27 (s, 2H), 7.07 (
d, J = 8.64Hz, lH), 7.35 (t, J = 7.23Hz, 1H), 7.43 (t, J = 7.48Hz, 2H), 7.55 (d, J = 7.39H
z, 1H), 7.77 (d, J = 8.82Hz, 1H), 7.87 (d, J = 8.73Hz, 2H), 7.77 (d, J = 8.82Hz, 1H), 7.
87 (d, J = 8.65Hz, 1H), 8.27 (d, J = 9.15Hz, 1H), 8.79 (s, 1H, 8.96 (s, 1H), C ₂₆ H ₂₀ F ₃ N
O ₅ calculated 483.1; measured 484.1 (M + H) ⁺ ；

2- (4-phenoxyphenyl) -6-trifluoromethoxy-quinoline-4-carboxylic acid (I2
K52); NMR: (acetone-d6,400 MHz) δ: 7.12-7.21 (m, 5H), 7.43-7.47 (m, 2H), 7.78 (d,
J = 9.20Hz, 1H), 8.27 (d, J = 9.22Hz, 1H), 8.39 (d, J = 8.89Hz, 2H), 8.68 (s, 1H), 8.92
(s, 1H), C ₂₃ H ₁₄ F ₃ NO ₄ Calculated 425.1: Measured 426.1 (M + H) ⁺ .

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 31/437 A61K 31/437 31/47 31/47 31/513 31/513 38/21 45/06 45 / 06 A61P 1/02 A61P 1/02 1/16 1/16 11/00 11/00 25/00 25/00 29/00 29/00 31/14 31/14 43/00 111 43/00 111 121 121 121 C07D 215/52 C07D 215/52 G01N 33/15 Z G01N 33/15 33/50 Z 33/50 C07D 235/02 D // C07D 235/02 A61K 37/66 G (81) Designated country EP (AT, BE) , CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA , GN, GW, ML, MR, NE, SN, TD, TG), AP (GH GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL , AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD , MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Muimui, Shim Singapaw Republic of Republic 117609 Singapore, Medical Drive 30 F Term (Reference) 2G045 AA40 4C084 AA02 AA17 AA19 AA20 AA22 AA23 AA24 BA44 DA21 DA22 DA23 MA02 NA14 ZA02 ZA59 BCZA67 ZA75 ZB11 AB20 ZC75 MAB02A02 MA03 MA04 NA14 ZA02 ZA59 ZA67 ZA75 ZB11 ZB33 ZC20 ZC75 4C206 AA01 AA02 CB28 FA33 MA01 MA02 MA03 MA04 NA14 ZA02 ZA59 ZA67 ZA75 ZB11 ZB33 ZC20 ZC75

Claims (34)

[Claims] 1. Use of an inhibitor of dihydroorotate dehydrogenase for the manufacture of a medicament for the treatment of infectious diseases caused by Flaviviridae, Rhabdoviridae or Paramyxoviridae viruses. 2. The inhibitor is of formula (I): [Wherein each A is independently hydrogen, halogen, perhaloalkoxy, amino C ₁ -C ₈ alkyl, NO ₂ , CN, SO ₂ CH ₃ , C ₁ -C ₈ alkyl, C ₁ -C ₈ Selected from the group consisting of alkoxy, C ₃ -C ₇ cycloalkyl, C ₃ -C ₇ cycloalkenyl, aryl, aryloxy, C ₁ -C ₆ perhaloalkyl, and Y, or adjacent 2 on ring b. The group A is
Taken together with the phenyl ring to which they are attached form a naphthalene ring system; R is cyclohexyl, phenoxy or benzoxy, or a phenyl ring optionally substituted with a group A as defined above; Alternatively, R on ring b and the adjacent group A, together with the phenyl ring to which they are attached, form a naphthalene or phenanthrene ring system; Y is from COOM, CONHR ′, SO ₃ M, and hydrogen. M is selected from the group consisting of H, Li, Na, K, and 0.5Ca; R ′ is C ₁ -C ₁₀ alkyl; n is 1 or 2; T Is = N- or = C (Z)-, where (i) Z is hydrogen, NH ₂ , OH, C ₁ -C ₈ alkyl, C ₃ -C ₇ cycloalkyl, aryl, and C ₁ -C ₆ or is selected from the group consisting of perhaloalkyl, or (ii) Z is, -VW- (wherein, V is be CH ₂ or S W is CH _2, O, S or N
H) and-(CH ₂ ) ₂ -C (= Z)-(where Z is O or H ₂ ) and the bridging moiety is an adjacent biphenyl group. Or one of which completes one ring by bonding to the ortho position of ring b. ]. 3. The inhibitor is of formula (Ia): [Wherein each A is independently hydrogen, halogen, amino C ₁ -C ₈ alkyl, NO ₂ , CN, SO ₂ CH ₃
, C ₁ -C ₈ alkyl, C ₃ -C ₇ cycloalkyl, aryl, C ₁ -C ₆ perhaloalkyl,
And Y is selected from the group consisting of COOM, CONHR ′, SO ₃ M, and hydrogen; M is selected from the group consisting of H, Li, Na, K, and 0.5Ca ; R 'is an C ₁ -C ₁₀ alkyl; T is, = N-or = C (Z) - and is, where, (i) Z is _{hydrogen, NH 2, OH, C 1} -C ₈ alkyl, C ₃ -C ₇ cycloalkyl, aryl, and C ₁ -C ₆ perhaloalkyl, or (ii) Z is —VW— (where V is CH ₂ or S And W is CH ₂ , O, S or N
H) and-(CH ₂ ) ₂ -C (= Z)-(where Z is O or H ₂ ) and the bridging moiety is an adjacent biphenyl group. Or one of which completes one ring by being bonded to the ortho position of ring b. ]. 4. The inhibitor is of formula (II): [Wherein R ¹ is H, halogen, or OCF ₃ ; R ² is H, or C ₁ -C ₆ alkyl; R ³ is H, or OR ⁶ (wherein R ⁶ is H or C ₁ -C ₆ alkyl); R ⁴ is H, or C ₁ -C ₆ alkyl, or R ⁴ and R ³ together with the phenyl ring b to which they are attached. Form a naphthalene ring; R ⁵ is cyclohexyl, phenoxy, or benzoxy, or an unsubstituted or halogen-substituted phenyl ring, or R ⁴ and R ⁵ are taken together with the phenyl ring b to which they are attached. Forming a naphthalene ring]. The use according to claim 2 or 3, which is a compound represented by the formula: 5. The inhibitor is of formula (IIb): [In formula, M is H or Na] The use of any one of Claims 2-4 which is a compound represented by these. 6. The inhibitor has the formula (I ′): [Wherein A and Y are as defined above for formula (I); R'is hydrogen and R "is a thiophene ring or formula (i ') or (ii'): Or R'and R "together with the carbon atom to which they are attached (represented by" C ") are of formula (iii ') or (iv'): (Where R ″ is H or halogen and R ^iv is H or C ₁ -C ₆ alkoxy.
Forming a ring system), the use of claim 1. 7. The inhibitor is of formula (III): [Wherein each A ¹ is independently hydrogen, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, C ₃ -C ₇ cycloalkyl , Halogen, unsubstituted aryl, X-substituted aryl, NO ₂ , CN, COOR, CONHR, and NHR; X is halogen, NO ₂ , C ₁ -C ₈ alkyl, aryl, fused aryl, and COOR
R is selected from the group consisting of hydrogen and C ₁ -C ₈ alkyl; B is selected from the group consisting of C ₁ -C ₈ alkyl, H, CF ₃ , and aryl, Aryl is a compound represented by halogen, C ₁ -C ₈ alkoxy, C ₁ -C ₈ alkyl, NO ₂ , aryl or fused aryl]. 8. The inhibitor is of formula (IV): The use according to claim 7, which is a compound represented by: 9. The inhibitor is of formula (V): [In the formula, A ² is hydrogen, C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, C ₃ -C ₇ cycloalkyl, halogen, unsubstituted aryl, halogen-substituted aryl, condensed Aryl, NO ₂ , CN, NHR ¹ , and N (R ¹ ) ₂ are selected from the group consisting of; R ¹ is selected from the group consisting of hydrogen, C ₁ -C ₈ alkyl, and OH; X ¹ is Hydrogen or halogen; B ¹ , Y ¹ and Z ¹ are each independently selected from the group consisting of hydrogen, OH, C ₁ -C ₈ alkyl, halogen, CN, NO ₂ and CF ₃ . The use according to claim 1, which is a compound represented by: 10. The inhibitor is of formula (VI): The use according to claim 1, which is a compound represented by: 11. The inhibitor is of formula (VII): Wherein each A ³ is independently hydrogen, C ₁ -C ₈ alkyl, C ₁ -C ₁₀ alkoxy, halogen, and N (R ²⁾ is selected from the group consisting of _2; B ² is a direct A bond, -CH = CH- or -C≡C-; X ² is selected from the group consisting of O, S and NR ² ; R ² is a group consisting of hydrogen, C ₁ -C ₄ alkyl and aryl Y ² is selected from the group consisting of COOM ¹ and SO ₃ M ¹ ; M ¹ is selected from the group consisting of H, Li, Na, K and 0.5Ca] The use according to claim 1, wherein: 12. The inhibitor is of formula (VIII): The use according to claim 11, which is a compound represented by: 13. The virus is selected from the group consisting of hepatitis virus, yellow fever virus, West Nile virus, Kunjin virus, dengue virus, St. Louis encephalitis virus, Japanese encephalitis virus, Mary Valley encephalitis virus, and tick-borne encephalitis virus. Use according to any one of claims 1 to 12, which is a flavivirus of choice. 14. The method according to claim 1, wherein the virus is a rhabdovirus selected from vesicular stomatitis virus and rabies virus, or paramyxovirus RSV. 15. Use according to any one of claims 1 to 14, wherein the medicament is for administration with interferon. 16. The method of claims 1-1, wherein the medicament further comprises interferon.
Use according to any one of 5 above. 17. The use according to claim 15 or 16, wherein the interferon is human interferon. 18. The use according to claim 17, wherein the interferon is selected from the group consisting of interferon α2, interferon α8, and interferon β. 19. Interferon is 0.3 × 10 5 per 1 mg of protein.⁹~ 3 x 10 ⁹ The use according to claim 18, which is human interferon α8 having a specific activity of IU.
for. 20. The use according to claim 18, wherein the interferon is human interferon β having a specific activity of 2 × 10 ⁸ to 8 × 10 ⁸ IU / mg protein. 21. The use according to any one of claims 15 to 20, wherein the inhibitor and interferon are each used in a synergistic amount. 22. Use of the medicament with an inhibitor of a second enzyme selected from inosine monophosphate dehydrogenase, guanosine monophosphate synthetase, cytidine triphosphate synthetase, and S-adenosyl homocysteine hydrolase. 22. Use according to any one of claims 1 to 21, which is 23. The use according to claim 22, wherein the medicament further comprises an inhibitor of the second enzyme. 24. The use according to claim 22 or 23, wherein the inhibitor is mycophenolic acid, cyclopentenylcytosine (CPE-C), or 3-deazaneplanocin A. 25. The use according to any one of claims 22 to 24, wherein the inhibitor of the second enzyme and the inhibitor of dihydroorotic acid dehydrogenase are each used in a synergistic amount. 26. Formula (IIa): Wherein M is selected from the group consisting of H, Li, Na, K, and 0.5Ca; R ²² is H or C ₁ -C ₆ alkyl; R ³³ is H or OR ⁶ . , R ⁶ is H or C ₁ -C ₆ alkyl; R ⁴⁴ is H or C ₁ -C ₆ alkyl; R ⁵⁵ is phenyl, cyclohexyl, phenoxy or benzoxy] Or a metabolite or prodrug precursor thereof. 27. The following compound: 2- (4-biphenylyl) -6-trifluoromethoxy-quinoline-4-carboxylic acid (Compound I2K5); 2- (4-biphenylyl) -3-methyl-6-trifluoro Methoxy-quinoline-4-carboxylic acid (Compound I2K55); 2- (4-Cyclohexylphenyl) -6-trifluoromethoxy-quinoline-4-carboxylic acid (Compound I2K46); 2- (4-Benzyloxy-2-methoxy -3-Methyl-phenyl) -6-trifluoromethoxy-quinoline-4-carboxylic acid (Compound I2K51); and 2- (4-phenoxyphenyl) -6-trifluoromethoxy-quinoline-4-carboxylic acid (Compound I2K52 ); The compound of claim 26, selected from: 28. A method for producing a compound represented by the formula (IIa) according to claim 26, which comprises (a) the formula (IX): A trifluoromethoxy-substituted isatin compound represented by the formula (X): [Wherein R ²² , R ³³ , R ⁴⁴ and R ⁵⁵ are as defined in claim 26] in the presence of a base; (b) if M is H Converting the resulting compound of formula (IIa) to a pharmaceutically acceptable salt thereof, wherein M is Li, Na, K or 0.5Ca. 29. A method of treating a host, which comprises administering an inhibitor of dihydroorotate dehydrogenase to a host infected with a Flaviviridae, Rhabdoviridae, or Paramyxoviridae virus. 30. An anti-flavivirus drug, an anti-rhabdovirus drug, or an anti-paramyxovirus drug, which comprises an inhibitor of dihydroorotic acid dehydrogenase. 31. Dihydroorotic acid dehydrogenase as a combined preparation for simultaneous, separate or sequential use in the treatment of infections caused by Flaviviridae, Rhabdoviridae, or Paramyxoviridae viruses. Products containing inhibitors and interferons. 32. A dihydroorotic acid dehydrogenase as a combination preparation for simultaneous, separate or sequential use in the treatment of infections caused by Flaviviridae, Rhabdoviridae or Paramyxoviridae viruses. A product containing an inhibitor and an inhibitor of a second enzyme selected from inosine monophosphate dehydrogenase, guanosine monophosphate synthetase, cytidine triphosphate synthetase, and S-adenosyl homocysteine hydrolase. 33. The product of claim 32, further comprising interferon. 34. A method for identifying an anti-flavivirus drug, an anti-rhabdovirus drug, or an anti-paramyxovirus drug, comprising: (a) preparing a test compound; (b) inhibiting the dihydroorotic acid dehydrogenase from the test compound. (C) If it is shown to be active in step (b), the test compound is treated with an anti-flavivirus drug, anti-rhabdovirus drug, or anti-paramyxovirus drug. The above method comprising: