# Patent

## Title
Antiviral indoles

## Abstract

Compounds of the formula (I):




wherein A, B, D, M, Ar, W, X, Y, Z and R1 are as defined herein, are useful in the prevention and treatment of hepatitis C infections. The compounds, their preparation, pharmaceutical compositions containing them and their use in medicine are disclosed.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to British Provisional application GB 0625345.4, filed Dec. 20, 2006, which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to macrocyclic indole compounds, to pharmaceutical compositions containing them, to their use in the prevention and treatment of hepatitis C infections and to methods of preparation of such compounds and compositions.
BACKGROUND OF THE INVENTION
Hepatitis C (HCV) is a cause of viral infections. There is as yet no adequate treatment for HCV infection but it is believed that inhibition of its RNA polymerase in mammals, particularly humans, would be of benefit.
Published International patent application WO 93/00334 (Fidia-Georgetown Institute for the Neurosciences) discloses the following indole derivatives:




where A, Z, R1, R2, R3, R4 and n are defined therein, as useful in compositions and methods for treating psychiatric and neurological disorders. However, this document does not disclose the use of tetracyclic indole derivatives in treating or preventing viral infections.

Published International patent application WO 2005/080399 (Japan Tobacco Inc.) discloses the following fused heterotetracyclic compounds:




where A, X, Cy, G1, G2, G3, G4, G5, G6, R1, R2, R3, R4, R5, R6 and a are defined therein, and their use as HCV polymerase inhibitors.

Published International patent application WO 2006/020082 (Bristol-Myers Squibb Company) discloses the following fused tetracyclic compounds:




where A, B, R1, R2, R3 and n are defined therein, and their use in treating hepatitis C.

Published International applications WO2006/046030 and WO2006/046039 (both Istituto Di Ricerche Di Biologia Molecolare P. Angeletti SpA) disclose certain tetracyclic indole derivatives:




wherein R1, R2, A, Ar, W, X, Y, and Z are defined therein, useful for the treatment or prevention of infection by hepatitis C virus.

SUMMARY OF THE INVENTION
Thus, the present invention provides the compound of the formula (I):




wherein

Ar is a moiety containing at least one aromatic ring and possesses 5-, 6-, 9- or 10-ring atoms optionally containing 1, 2 or 3 heteroatoms independently selected from N, O and S, which ring is optionally substituted by groups Q1 and Q2;
Q1 is halogen, hydroxy, C1-6alkyl, C1-6alkoxy, aryl, heteroaryl, CONRaRb, (CH2)0-3NRaRb, O(CH2)1-3NRaRb, O(CH2)0-3CONRaRb, O(CH2)0-3aryl, O(CH2)0-3heteroaryl, O(CReRf)aryl, O(CReRf)heteroaryl or OCHRcRd;
Ra and Rb are each independently selected from hydrogen, C1-4alkyl and C(O)C1-4alkyl;
or Ra, Rb and the nitrogen atom to which they are attached form a heteroaliphatic ring of 4 to 7 ring atoms, where said ring is optionally substituted by halogen, hydroxy, C1-4alkyl or C1-4alkoxy;
Rc and Rd are each independently selected from hydrogen and C1-4alkoxy;
or Rc and Rd are linked by a heteroatom selected from N, O and S to form a heteroaliphatic ring of 4 to 7 ring atoms, where said ring is optionally substituted by halogen, hydroxy, C1-4alkyl or C1-4alkoxy;
and wherein said C1-4alkyl, C1-4alkoxy and aryl groups are optionally substituted by halogen or hydroxy;
Re is hydrogen or C1-6alkyl;
Rf is C1-6alkyl;
Q2 is halogen, hydroxy, C1-4alkyl or C1-4alkoxy, where said C1-4alkyl and C1-4alkoxy groups are optionally substituted by halogen or hydroxy;
or Q1 and Q2 may be linked by a bond or a heteroatom selected from N, O and S to form a ring of 4 to 7 atoms, where said ring is optionally substituted by halogen, hydroxy, C1-4alkyl or C1-4alkoxy;
A is C3-6alkyl or C2-6alkenyl,
or A is a non-aromatic ring of 3 to 8 ring atoms where said ring may contain a double bond and/or may contain a O, S, SO, SO2 or NH moiety,
or A is a non-aromatic bicyclic moiety of 4 to 8 ring atoms,
and A is optionally substituted by halogen, hydroxy, C1-4alkyl or C1-4alkoxy;
D is N or CRg;
Rg is hydrogen, fluorine, chlorine, C1-4alkyl, C2-4alkenyl or C1-4alkoxy, where said C1-4alkyl, C2-4alkenyl and C1-4alkoxy groups are optionally substituted by hydroxy or fluorine;
W is a bond, C═O, O, S(O)0-2 or —(CR10R11)—(CR12R13)0-1—;
Y is a bond, C═O, O, —CR14R15— or NR14;
Z is a bond, C═O, O, S(O)0-2, —(CR10R11)—(CR12R13)0-1— or NR10;
and none, one or two of W, Y and Z are a bond;
X is —CR14a— or N;
R10, R11, R12, R13, R14, R14a and R15 are each independently selected from hydrogen, hydroxy, C1-6alkyl, C2-6alkenyl, C1-6alkoxy, C(O)C1-6alkyl, Het, (CH2)0-3NR16R17, C(O)(CH2)0-3NR16R17, NHC(O)(CH2)0-3NR16R17, O(CH2)1-3NR16R17, S(O)0-2(CH2)0-3R16R17 and C(O)(CH2)0-3OR16;
or one of R10, R14 and R14a is linked to R22 or R23 to form a ring of 4 to 10 atoms, where said ring is optionally substituted by halogen, hydroxy, C1-4alkyl or C1-4alkoxy; Het is a heteroaliphatic ring of 4 to 7 ring atoms, which ring may contain 1, 2 or 3 heteroatoms selected from N, O or S or a group S(O), S(O)2, NH or NC1-4alkyl;
R16 and R17 are independently selected from hydrogen, C1-6alkyl and (CH2)0-4NR18R19;
or R16, R17 and the nitrogen atom to which they are attached form a heteroaliphatic ring of 4 to 7 ring atoms, which ring may optionally contain 1 or 2 more heteroatoms selected from O or S or a group S(O), S(O)2, NH or NC1-4alkyl, and which ring is optionally substituted by halogen, hydroxy, C1-4alkyl or C1-4alkoxy;
R18 and R19 are independently selected from hydrogen and C1-6alkyl;
or R18, R19 and the nitrogen atom to which they are attached form a heteroaliphatic ring of 4 to 7 ring atoms, which ring may optionally contain 1 or 2 more heteroatoms selected from O or S or a group S(O), S(O)2, NH or NC1-4alkyl, and which ring is optionally substituted by halogen, hydroxy, C1-4alkyl or C1-4alkoxy;
R1 is hydrogen or C1-6alkyl;
B is —CR20R21—, —C(═O)—, —SO— or —SO2—;
R20 and R21 are independently selected from hydrogen and C1-6alkyl;
or R20 and R21, together with the carbon atom to which they are joined, form a C3-6cycloalkyl group;
M is C4-8alkylene or C4-8alkenylene, optionally substituted by R22, where 1, 2 or 3 of the carbon atoms in the C4-8alkylene or C4-8alkenylene groups is optionally replaced by O, NR23, S, SO, SO2, piperidinyl, piperazinyl or pyrrolidinyl,
where R23 is hydrogen or C1-6alkyl, or R23 is linked to one of R10, R14 and R14a to form a ring of 4 to 10 atoms as hereinbefore described;
where R22 is halo, C1-4alkyl, (CH2)0-3C3-8cycloalkyl, (CH2)0-3aryl, (CH2)0-3heteroaryl, (CH2)0-3Het or oxo,
or R22 is linked to one of R10, R14 and R14a to form a ring of 4 to 10 atoms as hereinbefore described;
and pharmaceutically acceptable salts thereof.


DETAILED DESCRIPTION OF THE INVENTION
In one embodiment of the present invention, Ar is a five- or six-membered aromatic ring optionally containing 1, 2 or 3 heteroatoms independently selected from N, O and S, and which ring is optionally substituted by groups Q1 and Q2 as hereinbefore defined.
Preferably, Ar is a five- or six-membered aromatic ring optionally containing 1 or 2 heteroatoms independently selected from N, O or S, such as phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, furanyl, pyrazolyl, imidazolyl and thienyl, which ring is optionally substituted by groups Q1 and Q2 as hereinbefore defined. More preferably, Ar is phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furanyl or 3-furanyl, particularly phenyl, optionally substituted by groups Q1 and Q2 as hereinbefore defined.
Preferably, Q1 is halogen, hydroxy, C1-4alkyl or C1-4alkoxy. More preferably, Q1 is fluorine, chlorine, methyl or methoxy. Most preferably, Q1 is methoxy.
Preferably, Q2 is absent.
In a further embodiment, A is C3-6alkyl, C2-6alkenyl or C3-8cycloalkyl, where A is optionally substituted by halogen, hydroxy, C1-4alkyl or C1-4alkoxy. Preferably, A is C3-8cycloalkyl, more preferably cyclopentyl or cyclohexyl, most preferably cyclohexyl, optionally substituted by halogen, hydroxy, C1-4alkyl or C1-4alkoxy.
Preferably, A is unsubstituted or substituted by fluorine, chlorine, methyl or methoxy, particularly fluorine. More preferably, A is unsubstituted or substituted by fluorine. Examples of suitable A groups include cyclohexyl and fluorocyclohexyl, especially 2-fluorocyclohexyl.
In a further embodiment, D is CRg where Rg is as hereinbefore defined. Preferably, Rg is hydrogen or C1-4alkyl. More preferably, Rg is hydrogen.
In a further embodiment, W is a bond, C═O or —(CR10R11)—(CR12R13)0-1— where R10, R11, R12 and R13 are as hereinbefore defined. Preferably, W is —(CR10R11)—(CR12R13)0-1—. More preferably, W is —CH2— or —CH2CH2—. Most preferably, W is —CH2—.
In a further embodiment, Z is a bond, C═O, O, —(CR10R11)—(CR12R13)0-1— or NR10 where R10, R11, R12 and R13 are as hereinbefore defined. Preferably, Z is a bond, O or —(CR10R11)—(CR12R13)0-1. More preferably, Z is a bond, O, —CH2— or —CH2CH2—. Most preferably, Z is O.
In a further embodiment, Y is C═O, O, —CR14R15— or NR14 where R14 and R15 are as hereinbefore defined. Preferably, Y is O, —CR14R15— or NR14. More preferably, Y is —CH2—, NH, N(C1-6alkyl), NCH2CH2N(C1-6alkyl)2 or NC(O)(CH2)1-2N(C1-6alkyl)2. Most preferably, Y is —CH2—, NH, N(C1-4alkyl), N(CH2)2N(C1-4alkyl)2 or NC(O)CH2N(C1-4alkyl)2. Especially, Y is —CH2—, NCH3 or N(CH2)2N(CH3)2. More especially, Y is —CH2—.
In a further embodiment, X is —CR14—, where R14 is as hereinbefore defined. Preferably, X is —CH— or —C(C1-6alkyl)-. More preferably, X is —CH—.
In a further embodiment, R1 is hydrogen or methyl. Preferably, R1 is hydrogen.
In a further embodiment, B is —CH2— or —SO2—. Preferably, B is —SO2—.
In a further embodiment, M is C4-8alkylene, optionally substituted by halo, C1-4alkyl or oxo, where 1 or 2 of the carbon atoms in the C4-8alkylene group is optionally replaced by O, NR23, S, SO or SO2, where R23 is as hereinbefore defined. Preferably, M is C5-8alkylene, optionally substituted by C1-4alkyl or oxo, where 1 or 2 of the carbon atoms in the C5-8alkylene group is replaced by O, NH or N(C1-4alkyl). Examples of suitable M groups include:




In one embodiment of the present invention, there is provided the compound of formula (Ia):




or a pharmaceutically acceptable salt thereof, wherein W, X, Y, Z and M are as defined in relation to formula (I).

When any variable occurs more than one time in formula (I) or in any substituent, its definition on each occurrence is independent of its definition at every other occurrence.
As used herein, the term “alkyl” or “alkoxy” as a group or part of a group means that the group is straight or branched. Examples of suitable alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl and t-butyl. Examples of suitable alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy and t-butoxy.
The cycloalkyl groups referred to herein may represent, for example, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.
As used herein, the term “alkenyl” and “alkynyl” as a group or part of a group means that the group is straight or branched. Examples of suitable alkenyl groups include vinyl and allyl. A suitable alkynyl group is propargyl.
As used herein, the term “alkylene” means that the alkyl group links two separate groups and may be straight or branched. Examples of suitable alkylene groups include ethylene [—CH2—CH2—] and propylene [—CH2—CH2—CH2—, —CH(CH3)—CH2— or —CH2—CH(CH3)—]. The terms “alkenylene” and “alkynylene” shall be construed in an analogous manner.
When used herein, the term “halogen” means fluorine, chlorine, bromine and iodine.
When used herein, the term “aryl” as a group or part of a group means a carbocyclic aromatic ring. Examples of suitable aryl groups include phenyl and naphthyl.
When used herein, the term “heteroaryl” as a group or part of a group means a 5- to 10-membered heteroaromatic ring system containing 1 to 4 heteroatoms selected from N, O and S. Particular examples of such groups include pyrrolyl, furanyl, thienyl, pyridyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazolyl, oxadiazolyl, thiadiazolyl, triazinyl, tetrazolyl, indolyl, benzothienyl, benzimidazolyl, benzofuryl, quinolinyl and isoquinolinyl.
Where a compound or group is described as “optionally substituted” one or more substituents may be present. Furthermore, optional substituents may be attached to the compounds or groups which they substitute in a variety of ways, either directly or through a connecting group of which the following are examples: amine, amide, ester, ether, thioether, sulfonamide, sulfamide, sulfoxide, urea, thiourea and urethane. As appropriate an optional substituent may itself be substituted by another substituent, the latter being connected directly to the former or through a connecting group such as those exemplified above.
Specific compounds within the scope of this invention include:

    101: (7R)-14-cyclohexyl-21,24-dimethyl-7,8-dihydro-6H-7,11-(epiminoethanoiminopropanothio iminomethano)indolo[1,2-e][1,5]benzoxazocin-15-one 17,17-dioxide,
    102: (7R)-14-cyclohexyl-24-methyl-7,8-dihydro-6H-7,11-(epiminoethanoiminopropanothioimino methano)indolo[1,2-e][1,5]benzoxazocin-15-one 17,17-dioxide,
    201: (7R)-14-cyclohexyl-25-methyl-7,8-dihydro-6H-7,11-(epiminoethanoiminobutanothioimino methano)indolo[1,2-e][1,5]benzoxazocine-15,21-dione 17,17-dioxide,
    301: (7R)-14-[(1R,2S) or (1S,2R)-2-fluorocyclohexyl]-21,24-dimethyl-7,8-dihydro-6H-7,11-(epiminoethanoiminopropanothioiminomethano)indolo[1,2-e][1,5]benzoxazocin-15-one 17,17-dioxide,

and pharmaceutically acceptable salts thereof.



For use in medicine, the salts of the compounds of formula (I) will be non-toxic pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compounds according to the invention or of their non-toxic pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds of this invention include acid addition salts which may, for example, be formed by mixing a solution of the compound according to the invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, fumaric acid, p-toluenesulfonic acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid, carbonic acid, phosphoric acid or sulfuric acid. Salts of amine groups may also comprise quaternary ammonium salts in which the amino nitrogen atom carries a suitable organic group such as an alkyl, alkenyl, alkynyl or aralkyl moiety. Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include metal salts such as alkali metal salts, e.g. sodium or potassium salts; and alkaline earth metal salts, e.g. calcium or magnesium salts.
The salts may be formed by conventional means, such as by reacting the free base form of the product with one or more equivalents of the appropriate acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is removed in vacuo or by freeze drying or by exchanging the anions of an existing salt for another anion on a suitable ion exchange resin.
The present invention includes within its scope prodrugs of the compounds of formula (I) above. In general, such prodrugs will be functional derivatives of the compounds of formula (I) which are readily convertible in vivo into the required compound of formula (I). Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in “Design of Prodrugs”, ed. H. Bundgaard, Elsevier, 1985.
A prodrug may be a pharmacologically inactive derivative of a biologically active substance (the “parent drug” or “parent molecule”) that requires transformation within the body in order to release the active drug, and that has improved delivery properties over the parent drug molecule. The transformation in vivo may be, for example, as the result of some metabolic process, such as chemical or enzymatic hydrolysis of a carboxylic, phosphoric or sulfate ester, or reduction or oxidation of a susceptible functionality.
The present invention includes within its scope solvates of the compounds of formula (I) and salts thereof, for example, hydrates.
The present invention also includes within its scope any enantiomers, diastereomers, geometric isomers and tautomers of the compounds of formula (I). It is to be understood that all such isomers and mixtures thereof are encompassed within the scope of the invention.
The present invention further provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in therapy.
In another aspect, the invention provides the use of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treatment or prevention of infection by hepatitis C virus in a human or animal.
A further aspect of the invention provides a pharmaceutical composition comprising a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier. The composition may be in any suitable form, depending on the intended method of administration. It may for example be in the form of a tablet, capsule or liquid for oral administration, or of a solution or suspension for administration parenterally.
The pharmaceutical compositions optionally also include one or more other agents for the treatment of viral infections such as an antiviral agent, or an immunomodulatory agent such as α-, β- or γ-interferon.
In a further aspect, the invention provides a method of inhibiting hepatitis C virus polymerase and/or of treating or preventing an illness due to hepatitis C virus, the method involving administering to a human or animal (preferably mammalian) subject suffering from the condition a therapeutically or prophylactically effective amount of the pharmaceutical composition described above or of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof. “Effective amount” means an amount sufficient to cause a benefit to the subject or at least to cause a change in the subject's condition.
The dosage rate at which the compound is administered will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age of the patient, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition and the host undergoing therapy. Suitable dosage levels may be of the order of 0.02 to 5 or 10 g per day, with oral dosages two to five times higher. For instance, administration of from 1 to 50 mg of the compound per kg of body weight from one to three times per day may be in order. Appropriate values are selectable by routine testing. The compound may be administered alone or in combination with other treatments, either simultaneously or sequentially. For instance, it may be administered in combination with effective amounts of antiviral agents, immunomodulators, anti-infectives or vaccines known to those of ordinary skill in the art. It may be administered by any suitable route, including orally, intravenously, cutaneously and subcutaneously. It may be administered directly to a suitable site or in a manner in which it targets a particular site, such as a certain type of cell. Suitable targeting methods are already known.
An additional aspect of the invention provides a method of preparation of a pharmaceutical composition, involving admixing at least one compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable adjuvants, diluents or carriers and/or with one or more other therapeutically or prophylactically active agents.
The present invention also provides a process for the preparation of compounds of formula (I).
According to a general process (a), compounds of formula (I) may be prepared by internal ring closure of the compound of formula (II):




where A, Ar, B, D, R1, W, X, Y and Z are as defined in relation to formula (I), and M′ and M′′ have suitable precursor functionality to form group M as defined in relation to formula (I). For instance, when M is —CH2—CH2—CH2—NH—CH2—CH2—N(CH3)—, M′ can be —CH2—CH2—CH2Cl and M′′ can be —N(CH3)—CH2—CH2—NH2, where the reaction is carried out in the presence of a mild base, such as diisopropylethylamine, in a suitable solvent, such as acetonitrile or DMF, under microwave irradiation. Alternatively, when M is —CH2—CH2—CH2—C(═O)—NH—CH2—CH2—N(CH3)—, M′ can be —CH2—CH2—CH2—CO2H and M′′ can be —N(CH3)—CH2—CH2—NH2, where the reaction is carried out in the presence of a coupling reagent, such as HATU, and a base, such as diisopropylethylamine, in a suitable solvent, such as DMF.

Compounds of formula (II) are either known in the art or may be prepared by conventional methodology well known to one of ordinary skill in the art using, for instance, procedures described in the accompanying Descriptions and Examples, or by alternative procedures which will be readily apparent.
Compounds of formula (I) can be converted into other compounds of formula (I) using synthetic methodology well known in the art. For instance, the compound of formula (I) where M comprises an N—H group may be converted into the compound of formula (I) where M comprises an N—CH3 group by methylation using formaldehyde followed by a mild reducing agent, such as sodium borohydride.
General Synthetic Schemes
Two general strategies were employed for assembly of compounds from the macrocyclic class (Methods A and B); Method B can be regarded as an extension of Method A.




A suitably functionalised tether was assembled first (as described in published International applications WO 2006/046030 and WO 2006/046039). A precursor fragment to one section of the macrocycle was installed on the tether, with subsequent unmasking of the acid at C6 and functionalisation to introduce a precursor fragment to the remaining segment of the macrocycle. Functional group manipulation and macrocyclisation (e.g., via amide bond formation, alkylation, reductive amination, metathesis etc.) set up the macrocycle. Potentially, the bond formed in ring closure could be at almost any point around the macrocyclic linker (e.g., forming the acylsulfonamide bond could also be the ring closing step).




Functional groups on the macrocycle were manipulated post-closure, e.g., via reductive amination, alkylation, amide reduction, amide formation etc. Potentially, sidechains can branch from any point around the macrocyclic linker.
During any of the above synthetic sequences it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W. Greene & P. G. M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 3rd edition, 1999. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.
The present invention is illustrated further by the following non-limiting examples.
The compounds of the invention were tested for inhibitory activity against the HCV RNA dependent RNA polymerase (NS5B) in an enzyme inhibition assay (example i)) and in a cell based sub-genomic replication assay (example ii)). The compounds generally have IC50's below 1 μM in the enzyme assay and several examples have EC50's below 2 μM in the cell based assay.
Compound names in the examples were generated using software from ACDLabs (version 8.0).
i) In-Vitro HCV NS5B Enzyme Inhibition Assay
WO 96/37619 describes the production of recombinant HCV RdRp from insect cells infected with recombinant baculovirus encoding the enzyme. The purified enzyme was shown to possess in vitro RNA polymerase activity using RNA as template. The reference describes a polymerisation assay using poly(A) and oligo(U) as a primer or an heteropolymeric template. Incorporation of tritiated UTP or NTPs is quantified by measuring acid-insoluble radioactivity. The present inventors have employed this assay to screen the various compounds described above as inhibitors of HCV RdRp.
Incorporation of radioactive UMP was measured as follows. The standard reaction (50 μl) was carried out in a buffer containing 20 mM tris/HCl pH 7.5, 5 mM MgCl2, 1 mM DTT, 50 mM NaCl, 0.03% N-octylglucoside, 1 μCi [3H]-UTP (40 Ci/mmol, NEN), 10 μM UTP and 10 μg/ml poly(A) or 5 μM NTPs and 5 μg/ml heteropolymeric template. Oligo(U)12 (1 μg/ml, Genset) was added as a primer in the assay working on Poly(A) template. The final NS5B enzyme concentration was 5 nM. The order of assembly was: 1) compound, 2) enzyme, 3) template/primer, 4) NTP. After 1 h incubation at 22° C. the reaction was stopped by adding 50 μl of 20% TCA and applying samples to DE81 filters. The filters were washed thoroughly with 5% TCA containing 1M Na2HPO4/NaH2PO4, pH 7.0, rinsed with water and then ethanol, air dried, and the filter-bound radioactivity was measured in the scintillation counter. Carrying out this reaction in the presence of various concentrations of each compound set out above allowed determination of IC50 values by utilising the formula:

% Residual activity=100/(1+[I]/IC50)S 

where [I] is the inhibitor concentration and “s” is the slope of the inhibition curve.

ii) Cell Based HCV Replication Assay

Cell clones that stably maintain subgenomic HCV replicon were obtained by transfecting Huh-7 cells with an RNA replicon identical to I377neo/NS3-3′/wt described by Lohmann et al. (1999) (EMBL-GENBANK No. AJ242652), followed by selection with neomycin sulfate (G418). Viral replication was monitored by measuring the expression of the NS3 protein by an ELISA assay performed directly on cells grown in 96 wells microtiter plates (Cell-ELISA) using the anti-NS3 monoclonal antibody 10E5/24 (as described in published International application WO 02/59321). Cells were seeded into 96 well plates at a density of 104 cells per well in a final volume of 0.1 ml of DMEM/10% FCS. Two hours after plating, 50 μl of DMEM/10% FCS containing a 3× concentration of inhibitor were added, cells were incubated for 96 hours and then fixed for 10′ with ice-cold isopropanol. Each condition was tested in duplicate and average absorbance values were used for calculations. The cells were washed twice with PBS, blocked with 5% non-fat dry milk in PBS+0.1% Triton X100+0.02% SDS (PBSTS) and then incubated o/n at 4° C. with the 10E5/24 mab diluted in MiLk/PBSTS. After washing 5 times with PBSTS, the cells were incubated for 3 hours at room temperature with Fc specific anti-mouse IgG conjugated to alkaline phosphatase (Sigma), diluted in Milk/PBSTS. After washing again as above, the reaction was developed with p-Nitrophenyl phosphate disodium substrate (Sigma) and the absorbance at 405/620 nm read at intervals. For calculations, we used data sets where samples incubated without inhibitors had absorbance values comprised between 1 and 1.5. The inhibitor concentration that reduced by 50% the expression of NS3 (IC50) was calculated by fitting the data to the Hill equation,

Fraction inhibition=1−(Ai−b)/(A0−b)=[I]n/([I]n+IC50)

where

    
    
        Ai=absorbance value of HBI10 cells supplemented with the indicated inhibitor concentration.
        A0=absorbance value of HBI10 cells incubated without inhibitor.
        b=absorbance value of Huh-7 cells plated at the same density in the same microtiter plates and incubated without inhibitor.
        n=Hill coefficient.

iii) General Procedures

    
    


All solvents were obtained from commercial sources (Fluka, puriss.) and were used without further purification. With the exception of routine deprotection and coupling steps, reactions were carried out under an atmosphere of nitrogen in oven dried (110° C.) glassware. Organic extracts were dried over sodium sulfate, and were concentrated (after filtration of the drying agent) on rotary evaporators operating under reduced pressure. Flash chromatography was carried out on silica gel following published procedure (W. C. Still et al., J. Org. Chem. 1978, 43, 2923) or on commercial flash chromatography systems (BIOTAGE Corporation and JONES FLASHMASTER II) utilising pre-packed columns.
Reagents were usually obtained directly from commercial suppliers (and used as supplied) but a limited number of compounds from in-house corporate collections were utilised. In the latter case the reagents are readily accessible using routine synthetic steps that are either reported in the scientific literature or are known to those skilled in the art.
1H NMR spectra were recorded on BRUKER AM series spectrometers operating at (reported) frequencies between 300 and 600 MHz. Chemical shifts (δ) for signals corresponding to non-exchangeable protons (and exchangeable protons where visible) are recorded in parts per million (ppm) relative to tetramethylsilane and are measured using the residual solvent peak as reference. Signals are tabulated in the order: multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; b, broad, and combinations thereof); coupling constant(s) in hertz (Hz); number of protons. Mass spectral (MS) data were obtained on a PERKIN ELMER API 100, or WATERS MICROMASS ZQ, operating in negative (ES−) or positive (ES+) ionization mode and results are reported as the ratio of mass over charge (m/z) for the parent ion only. Preparative scale HPLC separations were carried out on a WATERS DELTA PREP 4000 separation module, equipped with a WATERS 486 absorption detector or on an automated WATERS FRACTION LYNX or GILSON preparative system. In all cases compounds were eluted with linear gradients of water and MeCN both containing 0.1% TFA using flow rates between 15 and 40 mL/min.
The following abbreviations are used in the examples, the schemes and the tables: Ac: acetyl; aq.: aqueous; Ar: aryl; atm: atmosphere; cat.: catalytic; dioxan(e): 1,4-dioxane; dppf: (1,1′-bisdiphenylphosphino)ferrocene; 1,2-DCE: 1,2-dichloroethane; DCM: dichloromethane; DIPEA: diisopropylethyl amine; DMAP: N,N-dimethylpyridin-4-amine; DME: dimethoxyethane; DMF: dimethylformamide; DMS: dimethylsulfide; DMSO: dimethylsulfoxide; DMP: Dess-Martin Periodinane; EDAC.HCl: 1-ethyl-(3-dimethylaminopropyl)carbodiimide HCl salt; eq.: equivalent(s); Et3N: triethylamine; EtOAc: ethyl acetate; Et2O: diethyl ether; EtOH: ethanol; Et3SiH: triethylsilane; FC: Flash Chromatography; h: hour(s); HOAc: acetic acid; HATU: O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophophate; Me: methyl; MeCN: acetonitrile; MeOH: methanol; min: minutes; MS: mass spectrum; NBS: N-bromo succinimide; PE: petroleum ether; Ph: phenyl; quant.: quantitative; RP-HPLC: reversed phase high-pressure liquid chromatography; RT: room temperature; sat.: saturated; sec: second(s); SFC: Super-critical fluid chromatography; s.s.: saturated solution; TBAF: tetrabutyl ammonium fluoride; TBTU: O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborate; TFA: trifluoroacetic acid; THF: tetrahydrofuran; THP: tetrahydropyranyl; TMS: trimethylsilyl.
Example 1
10-cyclohexyl-2-(dimethylamino)-1,2,3,3a,4,14b-hexahydrocyclopenta[d]indolo[2,1-a][2]benzazepine-7-carboxylic acid
Step 1: benzyl 4-(aminosulfonyl)butanoate
Cs2CO3 (0.51 eq) was added to a solution of 4-(aminosulfonyl)butanoic acid (1 M) in DMF. After 1 h, benzyl bromide (1 eq) was introduced and the reaction left to stir overnight before diluting with DCM and filtering. The filtered liquor was concentrated in vacuo, the residue taken up in DCM and washed with saturated aqueous NaHCO3, water and brine, before being dried over Na2SO4, filtered and concentrated in vacuo. Trituration of the residue with Et2O afforded the title compound as a white solid (27%). (ES+) m/z 280 (M+Na)+
Step 2: tert-butyl (2R)-2-({[(4-nitrophenyl)sulfonyl]oxy}methyl)aziridine-1-carboxylate
A solution of tert-butyl (2R)-2-({[tert-butyl(dimethyl)silyl]oxy}methyl)aziridine-1-carboxylate (prepared following literature procedures: Travins, J. M.; Etzkorn, F. A. Tetrahedron Lett. 1998, 39, 9389-9392) in THF/Et2O (1/1) (0.17 M) was cooled in an ice bath and treated dropwise over 20 min with 1 M TBAF in THF (1.05 eq). The resulting solution was stirred in the ice bath for 30 min, before being quenched by the addition of sat. aq. NaHCO3 and extracted into Et2O/PE (4/1). The organic layers were collected, washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was taken up in dry DCM (0.17 M) and TEA (1.3 eq) introduced prior to cooling to 0° C. DMAP (0.1 eq) and 4-nitrobenzenesulfonyl chloride (1.1 eq) were added and the resulting mixture left to stir at RT overnight. The reaction mixture was diluted with DCM and washed with sat. aq. NaHCO3, water and brine before drying over Na2SO4, filtering and concentrating in vacuo. The crude was purified by FC (PE/EtOAc 80:20) to afford the title compound as an off-white solid (57%). (ES+) m/z 359 (M+H)+
Step 3: methyl (7R)-7-[(tert-butoxycarbonyl)amino]-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylate
A solution of methyl 3-cyclohexyl-2-(2-hydroxyphenyl)-1H-indole-6-carboxylate (prepared as described in international patent application WO2006/046030) (0.15 M) in DMF was treated with CsF (4 eq) in one portion; the resulting mixture was stirred for 20 min at RT then treated via dropping funnel over 30 min with a solution of tert-butyl (2R)-2-({[(4-nitrophenyl)sulfonyl]oxy}methyl)aziridine-1-carboxylate (1.3 eq) in DMF (0.5 M). The resulting solution was stirred at RT overnight. The reaction mixture was then placed into an ice bath and powdered KOtBu (1.4 eq) added slowly to the reaction mixture. After 1.5 h, the reaction was quenched with sat. aq. NH4Cl and extracted into EtOAc. The combined organic layers were washed with water and brine, before being dried over Na2SO4, filtered and concentrated in vacuo. The crude material was purified by FC (PE/EtOAc 80:20) affording the product as an off-white foam (85%). (ES+) m/z 505 (M+H)+
Step 4: methyl (7R)-7-amino-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylate
Methyl (7R)-7-[(tert-butoxycarbonyl)amino]-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylate (0.14 M) in DCM was treated with TFA (10 eq) and stirred at RT for 1 h. The reaction was diluted with DCM and cautiously basified with aq. NaHCO3, before separating the phases and extracting the aqueous with DCM. The combined organics were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo to afford the product as an off-white foam (100%) that was used without further purification. (ES+) m/z 405 (M+H)+; [α]D+42.3, c=1, MeOH
Step 5: methyl (7R)-14-cyclohexyl-7-(methylamino)-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylate
A solution of methyl (7R)-7-amino-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylate (0.35 M) in THF was treated drop-wise with 2,2,2-trifluoroethyl formate (1.2 eq) and stirred overnight at RT. The volatiles were removed in vacuo and the residue dissolved (0.11 M) in THF and treated drop-wise with BH3.DMS complex (2 M in THF; 5 eq). The resulting solution was stirred at RT for 20 h. The reaction was quenched by the careful addition of HCl/MeOH (1.25 M) and the resulting solution refluxed for 2 h. The volatiles were then removed in vacuo and the residue partitioned between sat. aq. NaHCO3 and EtOAc. The combined organics were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude was purified by FC (EtOAc/PE 80:20+1% NEt3) to afford the product (79%). (ES+) m/z 419 (M+H)+; [α]D+47.4, c=0.46, CHCl3 
Step 6: methyl (7R)-7-[{2-[(tert-butoxycarbonyl)amino]ethyl}(methyl)amino]-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylate
To a solution of tert-butyl (2-oxoethyl)carbamate (1 eq; 0.38 M) in dry methanol was added a mixture of methyl (7R)-14-cyclohexyl-7-(methylamino)-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylate (0.14 M), acetic acid (2 eq) and sodium acetate (1 eq) in dry methanol, and the mixture stirred at RT for 15 min. Then Pd/C (0.3 weight eq) was added as a slurry in MeOH under N2. The atmosphere in the reaction vessel was charged with H2 and the reaction stirred vigorously under a H2 atmosphere (balloon) at 60° C. overnight. The reaction was allowed to cool to RT, flushed with N2 and filtered through a plug of CELITE. The filtrate was concentrated in vacuo and the residue purified by FC (PE/EtOAc 2.5:1 to 1.5:1 gradient) to afford the title compound (82%). (ES+) m/z 562 (M+H)+; [α]D+67.1, c=0.67, CHCl3 
Step 7: (7R)-7-[{2-[(tert-butoxycarbonyl)amino]ethyl}(methyl)amino]-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic acid
Lithium hydroxide monohydrate (4.4 eq) was added to a solution of methyl (7R)-7-[{2-[(tert-butoxycarbonyl)amino]ethyl}(methyl)amino]-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylate (0.02 M) in MeOH/THF/H2O (1/1/1). The reaction was heated at 60° C. for 4 h prior to introducing further lithium hydroxide monohydrate (5 eq) and continuing heating for 2 h. The reaction was allowed to cool to RT, and the solvent volume reduced in vacuo. The residue was partitioned between 1N HCl (aq) and EtOAc, extracting the aqueous fraction a further two times with EtOAc. The combined organics were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo to afford the product as an off white foam (98%). (ES+) m/z 548 (M+H)+
Step 8: benzyl 4-{[({7R)-7-[{2-[(tert-butoxycarbonyl)amino]ethyl}(methyl)amino]-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocin-11-yl}carbonyl)amino]sulfonyl}butanoate
Benzyl 4-(aminosulfonyl)butanoate (1.3 eq) (prepared as described in step 1), DMAP (2.5 eq) and EDAC.HCl (1.5 eq), were added to a solution of (7R)-7-[{2-[(tert-butoxycarbonyl)amino]ethyl}(methyl)amino]-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic acid (0.03 M) in DCM. The reaction was stirred under N2 at RT for 24 h, before volatiles were removed in vacuo to leave the crude product as a yellow gum which was purified by automated RP-HPLC (WATERS XTERRA column; MeCN/H2O/0.1% TFA gradient). Fractions containing the pure compound were combined and lyophilized to afford the product as a white powder (37%). (ES+) m/z 787 (M+H)+
Step 9: 4-{[({(7R)-7-[{2-[(tert-butoxycarbonyl)amino]ethyl}(methyl)amino]-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocin-11-ylcarbonyl)amino]sulfonyl}butanoic acid
Pd/C (10 wt %) was added as a slurry in MeOH under N2 to a solution of benzyl 4-{[({(7R)-7-[{2-[(tert-butoxycarbonyl)amino]ethyl}(methyl)amino]-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocin-11-yl}carbonyl)amino]sulfonyl}butanoate (0.003 M) in MeOH. The atmosphere in the reaction vessel was exchanged for H2 and the reaction stirred vigorously at RT for 1 h. The reaction vessel was flushed with N2 and the reaction mixture filtered through a plug of CELITE (washing well with MeOH). Volatiles were removed in vacuo to afford the crude product as a yellow oil. (ES+) m/z 697 (M+H)+
Step 10: 4-{[({(7R)-7-[(2-aminoethyl)(methyl)amino]-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocin-11-yl}carbonyl)amino]sulfonyl}butanoic acid
4-{[({(7R)-7-[{2-[(tert-butoxycarbonyl)amino]ethyl}(methyl)amino]-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocin-11-yl}carbonyl)amino]sulfonyl}butanoic acid was dissolved in DCM (0.01 M), and ethereal HCl added (2M, >100 eq). The reaction was stirred with heating at 40° C. for 1 h. Volatiles were removed in vacuo, and the residue diluted with Et2O and reconcentrated in vacuo (twice) to drive off excess HCl and afford the crude product as the bis hydrochloride salt. (ES+) m/z 597 (M+H)+
Step 11: (7R)-14-cyclohexyl-25-methyl-7,8-dihydro-6H-7,11-(epiminoethanoiminobutanothioiminomethano)indolo[1,2-e][1,5]benzoxazocine-15,21-dione 17,17-dioxide
DIPEA (6 eq) and HATU (1.2 eq) were introduced to a solution of 4-{[({(7R)-7-[(2-aminoethyl)(methyl)amino]-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocin-11-yl}carbonyl)amino]sulfonyl}butanoic acid (0.005 M) in DMF and the reaction stirred under N2 at 45° C. for 1 h. The volatiles were evaporated in vacuo and the residue purified by automated RP-HPLC (WATERS XTERRA column; MeCN/H2O/0.1% TFA gradient). Fractions containing the pure compound were combined and lyophilized to afford the product as a white powder (30% overall for steps 4, 5, 6). 1H NMR (600 MHz, DMSO-d6+TFA, 335 K) 1.14-1.22 (m, 1H), 1.31-1.40 (m, 2H), 1.53-1.57 (m, 1H), 1.68-1.75 (m, 2H), 1.84-1.87 (m, 1H), 1.91-2.07 (m, 5H), 2.10-2.16 (m, 1H), 2.29-2.34 (m, 1H), 2.38-2.45 (m, 1H), 2.71-2.77 (m, 1H), 2.94 (s, 3H), 3.37-3.55 (m, 6H), 3.88-3.95 (m, 1H), 4.26-4.31 (m, 1H), 4.36-4.42 (m, 1H), 4.81-4.86 (m, 1H), 7.28-7.32 (m, 2H), 7.38 (dd, J 7.7, 1.5, 1H), 7.47 (d, J 8.7, 1H), 7.54-7.57 (m, 1H), 7.92 (d, J 8.7, 1H), 8.16 (s, 1H), 8.19 (b s, 1H), 11.57 (b s, 1H); (ES+) m/z 579 (M+H)+.
Example 2
(7R)-14-cyclohexyl-24-methyl-7,8-dihydro-6H-7,11-(epiminoethanoiminopropanothioiminomethano)indolo[1,2-e][1,5]benzoxazocin-15-one 17,17-dioxide
Step 1: 3-chloropropane-1-sulfonamide
A 0.5 M solution of ammonia (15 eq) in dioxane was added slowly at RT under nitrogen to a solution of 3-chloropropane sulfonyl chloride in dioxane (0.56 M). The reaction was left to stir for 2 h before removing volatiles in vacuo. The residue was taken up in CHCl3, filtered to remove ammonium chloride and the filtered liquor concentrated in vacuo to afford the title compound as a colourless oil that solidified on standing. 1H NMR (300 MHz, DMSO-d6, 300 K) 2.09-2.17 (m, 2H), 3.06-3.11 (m, 2H), 3.72-3.77 (m, 2H), 6.87 (s, 2H).
Step 2: tert-butyl {2-[[(7R)-11-({[(3-chloropropyl)sulfonyl]amino}carbonyl)-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocin-7-yl](methyl)amino]ethyl}carbamate
3-chloropropane-1-sulfonamide (1.8 eq), DMAP (2.9 eq) and EDAC.HCl (1.8 eq) were added to a solution of (7R)-7-[{2-[(tert-butoxycarbonyl)amino]ethyl}(methyl)amino]-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic acid (prepared as described in Example 1, Step 7) (0.025 M) in DCM. The reaction was stirred under N2 at 40° C. for 2 h, before being allowed to cool. Volatiles were removed in vacuo to leave the crude product as a yellow gum which could be taken on without further purification. (ES+) m/z 687 (M+H)+; 689 (M+H)+
Step 3: (7R)-7-[(2-aminoethyl)(methyl)amino]-N-[(3-chloropropyl)sulfonyl]-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxamide
tert-butyl {2-[[(7R)-11-({[(3-chloropropyl)sulfonyl]amino}carbonyl)-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocin-7-yl](methyl)amino]ethyl}carbamate was dissolved in DCM (0.025 M), and ethereal HCl added (2M, >50 eq). The reaction was stirred with heating at 40° C. for 1 h. Volatiles were removed in vacuo, and the residue diluted with Et2O and reconcentrated in vacuo (twice) to drive off excess HCl and afford the crude product as the bis hydrochloride salt. Purification was by automated RP-HPLC (Waters xterra column; MeCN/H2O/0.1% TFA gradient). Fractions containing the pure compound were combined and lyophilized to afford the product as a white powder (37% over steps 2, 3). (ES+) m/z 587 (M+H)+; 589 (M+H)+
Step 4: (7R)-14-cyclohexyl-24-methyl-7,8-dihydro-6H-7,11-(epiminoethanoiminopropanothioiminomethano)indolo[1,2-e][1,5]benzoxazocin-15-one 17,17-dioxide
iPr2NEt (20 eq) was added to a solution of (7R)-7-[(2-aminoethyl)(methyl)amino]-N-[(3-chloropropyl)sulfonyl]-14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxamide in MeCN (0.003 M). The reaction was heated in a microwave at 150° C. for 300 sec. The volatiles were removed in vacuo and the residue was purified by automated RP-HPLC (WATERS XTERRA column; MeCN/H2O/0.1% TFA gradient). Fractions containing the pure compound were combined and lyophilized to afford the product as a white powder (8.5%). 1H NMR (400 MHz, DMSO-d6, 300 K) 1.10-1.20 (m, 1H), 1.28-1.37 (m, 2H), 1.42-1.47 (m, 1H), 1.65-1.73 (m, 2H), 1.81-2.09 (m, 5H), 2.17-2.28 (m, 1H), 2.31 (s, 3H), 2.62-2.69 (m, 1H), 2.80-2.91 (m, 1H), 3.02-3.18 (m, 3H), 3.44-3.62 (m, 5H), 3.84 (dd, J 14.8, 9.4, 1H), 4.02 (t, J 11.4, 1H), 4.34 (dd, J 11.4, 5.4, 1H), 4.54 (d, J 14.8, 1H), 7.31-7.38 (m, 3H), 7.48 (d, J 8.4, 1H), 7.54-7.59 (m, 1H), 7.92 (d, J 8.4, 1H), 8.09 (s, 1H), 8.65 (b s, 1H), 11.85 (b s, 1H); (ES+) m/z 551 (M+H)+.
Example 3
(7R)-14-cyclohexyl-21,24-dimethyl-7,8-dihydro-6H-7,11-(epiminoethanoiminopropanothioiminomethano)indolo[1,2-e][1,5]benzoxazocin-15-one 17,17-dioxide
Formaldehyde (37 wt % in water; 15 eq) was added to a solution of (7R)-14-cyclohexyl-24-methyl-7,8-dihydro-6H-7,11-(epiminoethanoiminopropanothioiminomethano) indolo[1,2-e][1,5]benzoxazocin-15-one 17,17-dioxide (prepared as described in Example 2; Step 4) in MeOH (0.004 M). The pH was adjusted to pH 5-6 with AcOH and, after 5 min, NaBH (20 eq) introduced. The reaction was stirred at RT for 15 min before quenching with 1N HCl (aq) and concentrating to dryness in vacuo. The residue was purified by automated RP-HPLC (WATERS XTERRA column; MeCN/H2O/0.1% TFA gradient). Fractions containing the pure compound were combined and lyophilized to afford the product as a white powder (65%). 1H NMR (400 MHz, DMSO-d6, 300 K) 1.10-1.20 (m, 1H), 1.29-1.38 (m, 2H), 1.42-1.46 (m, 1H), 1.67-1.74 (m, 2H), 1.83-1.94 (m, 4H), 2.12-2.29 (m, 2H), 2.33 (s, 3H), 2.63-2.69 (m, 1H), 2.80 (s, 3H), 2.84-2.95 (m, 1H), 3.08-3.20 (m, 3H), 3.44-3.68 (m, 5H), 3.84 (dd, J 14.9, 9.8, 1H), 4.02 (t, J 11.7, 1H), 4.29 (dd, J 11.7, 5.2, 1H), 4.53 (d, J 14.9, 1H), 7.31-7.39 (m, 3H), 7.49 (d, J 8.4, 1H), 7.54-7.59 (m, 1H), 7.93 (d, J8.4, 1H), 8.11 (s, 1H), 11.98 (s, 1H); (ES+) m/z 565 (M+H)+.
Example 4
(7R)-14-[(1R,2S) or (1S,2R)-2-fluorocyclohexyl]-21,24-dimethyl-7,8-dihydro-6H-7,11-(epiminoethanoiminopropanothioiminomethano)indolo[1,2-e][1,5]benzoxazocin-15-one 17,17-dioxide
Step 1: methyl 3-cyclohex-1-en-1-yl-1H-indole-6-carboxylate
A solution (0.1 M) of 3-cyclohex-1-en-1-yl-1H-indole-6-carboxylic acid (prepared as described in published International patent application WO 2004/087714) in dry DMF was cooled to 0° C. and treated with K2CO3 (1.05 eq). A solution (3 M) of MeI (1.05 eq) in DMF was then added over 0.5 h and the temperature was raised to 20° C. After 18 h the reaction was quenched with aqueous HCl (1 N) and diluted with EtOAc. The organic phase was separated and washed several times with aqueous HCl (1 N), then with brine. The dried organics were concentrated to give the title compound (99%) as a solid; (ES+) m/z 256 (M+H)+.
Step 2: (±)-methyl 3-[(trans)-2-hydroxycyclohexyl]-1H-indole-6-carboxylate
A solution (0.2 M) of the preceding material in dry THF was treated over 1 h at 0° C. with BH3.SMe2 (2 M in THF, 1.1 eq). The mixture was stirred at 20° C. for 12 h, then cooled to 0° C. and treated sequentially with aqueous NaOH (3 M, 5.7 eq) and H2O2 (30% in H2O 8.4 eq). This mixture was stirred at 20° C. for 3 h then diluted with EtOAc and neutralized with s.s. NH4Cl. The organic phase was washed with s.s. NaHCO3 and brine, then dried and concentrated. The residue was washed several times with Et2O to give the title compound (73%) as a white powder; (ES+) m/z 274 (M+H)+.
Step 3: (±)-methyl 3-[(trans)-2-fluorocyclohexyl]-1H-indole-6-carboxylate
A solution (0.08 M) of the foregoing material in dry EtOAc was treated with DAST (1.2 eq) over 15 min at −50° C. The mixture was stirred for 1 h then warmed to 20° C. After 3 h the mixture was quenched with s.s. NaHCO3 and diluted with EtOAc. The organic phase was washed with brine, dried and concentrated under reduced pressure. The residue was crystallized from hot EtOAc to give the title compound (61%). The filtrate was concentrated and the residue purified by flash chromatography (10% to 30% EtOAc: PE) to give a second crop of the title compound (17%) as a solid; (ES+) m/z 276 (M+H)+.
Step 4: (±)-methyl 2-bromo-3-[(trans)-2-fluorocyclohexyl]-1H-indole-6-carboxylate
A solution (0.16 M) of the foregoing material in CH2Cl2 was treated with NBS (1.1 eq) over 2 h. The resulting mixture was stirred for 4 h then diluted with aqueous Na2S2O3 (1 N) and stirred for 12 h. The organic phase was separated and washed with aqueous Na2S2O3 (1 N) and brine. The dried organics were concentrated to afford a residue that was purified by flash chromatography (1:9 to 2:8 EtOAc:PE) to give the title compound (56%) as a pale solid; (ES+) m/z 354 (M+H)+.
Step 5: methyl 2-bromo-3-[(1R,2S)-2-fluorocyclohexyl]-1H-indole-6-carboxylate and methyl 2-bromo-3-[(1S,2R)-2-fluorocyclohexyl]-1H-indole-6-carboxylate
The preceding material was dissolved in MeOH and the enantiomers were separated by SFC chromatography (stationary phase: CHIRALCEL OJ-H 250×10 mm; mobile phase: 25% MeOH containing 0.2% diethylamine/CO2; flow rate 10 mL/min; column pressure: 100 bar; column temperature: 35° C.; detection UV 254 nm). The enantiomeric excess of the two fractions thus obtained (compound recovery 95%) were determined by chiral phase analytical HPLC (stationary phase: CHIRALPAK AD 250×4.6 mm; mobile phase 95:5 n-hexane:isopropyl alcohol containing 0.2% TFA; flow rate 1 mL/min; detection: JV 300 nM; sample concentration: 1 mg/mL; injection volume 10 uL): Isomer A (retention time 37.82 min, e.e. 99.8%, [α]D20=−8.0 (c=0.77, CHCl3)); Isomer B (retention time 43.89 min, 99%, [α]D20=+8.0 (c=0.77, CHCl3)).
Step 6: methyl 3-[(1R,2S) or (1S,2R)-2-fluorocyclohexyl]-2-(2-hydroxyphenyl)-1H-indole-6-carboxylate
A solution (0.16 M) of (−)-methyl 2-bromo-3-[(trans)-2-fluorocyclohexyl]-1H-indole-6-carboxylate (isomer A from Step 5, above) in dioxane was treated with aqueous Na2CO3 (2 N, 4.6 eq), 2-hydroxyphenylboronic acid (1.8 eq) and Pd(PPh3)4 (0.1 eq). The mixture was stirred at 80° C. for 2 h, then diluted with EtOAc, washed with aqueous HCl (1 N) and brine. The dried organic layer was concentrated in vacuo to give a residue that was purified by flash chromatography (8:2 PE:EtOAc) to give the title compound (90%) as a solid. 1H NMR (300 MHz, DMSO-d6, 300 K) δ 1.21-1.65 (m, 3H), 1.68 (m, 4H), 2.05-2.19 (m, 1H), 2.75-2.97 (m, 1H), 3.87 (s, 3H), 5.00 (dm, JHF 49.0, 1H), 6.93 (t, J 7.5, 1H), 7.01 (d, J 7.5, 1H), 7.28 (t, J 7.5, 1H), 7.29 (d, J7.5, 1H), 7.59 (d, J8.4, 1H), 7.82 (d, J8.4, 1H), 8.02 (s, 1H), 9.74 (s, 1H), 11.34 (s, 1H).
Step 7: methyl (7R)-7-[(tert-butoxycarbonyl)amino]-14-[(1R,2S) or (1S,2R)-2-fluorocyclohexyl]-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylate
The preceding material was treated as described in Example 1, Step 3 to furnish the title compound (96%) as a pale yellow oil. (ES+) m/z 523 (M+H)+
Step 8: methyl (7R)-7-amino-14-[(1R,2S) or (1S,2R)-2-fluorocyclohexyl]-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylate
The preceding material was treated as described in Example 1, Step 4 to furnish the title compound (100%) as a yellow foam. (ES+) m/z 423 (M+H)+
Step 9: methyl (7R)-14-[(1R,2S) or (1S,2R)-2-fluorocyclohexyl]-7-(methylamino)-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylate
The preceding material was treated as described in Example 1, Step 5 to furnish the title compound (73%) as a yellow foam. (ES+) m/z 437 (M+H)+
Step 10: methyl (7R)-7-[{2-[(tert-butoxycarbonyl)amino]ethyl}(methyl)amino]-14-(1R,2S) or (1S,2R)-2-fluorocyclohexyl]-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylate
The preceding material was treated as described in Example 1, Step 6 to furnish the title compound (80%). (ES+) m/z 580 (M+H)+
Step 11: (7R)-7-[{2-[(tert-butoxycarbonyl)amino]ethyl}(methyl)amino]-14-[(1R,2S) or (1S,2R)-2-fluorocyclohexyl]-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic acid
The preceding material was treated as described in Example 1, Step 7 to furnish the title compound (80%). (ES+) m/z 566 (M+H)+
Step 12: tert-butyl {2-[{(7R)-11-({[(3-chloropropyl)sulfonyl]amino}carbonyl)-14-[(1R,2S) or (1S,2R)-2-fluorocyclohexyl]-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocin-7-yl}(methyl)amino]ethyl}carbamate
The preceding material was treated as described in Example 2, Step 2 to furnish the title compound used as crude in the next step. (ES+) m/z 705 (M+H)+; 707 (M+H)+
Step 13: (7R)-7-[(2-aminoethyl)(methyl)amino]-N-[(3-chloropropyl)sulfonyl]-14-[(1R,2S) or (1S,2R)-2-fluorocyclohexyl]-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxamide
The preceding material was treated as described in Example 2, Step 3 to furnish the title compound (39% two steps). (ES+) m/z 605 (M+H)+; 607 (M+H)+
Step 14: (7R)-14-[(1R,2S) or (1S,2R)-2-fluorocyclohexyl]-24-methyl-7,8-dihydro-6H-7,11-(epiminoethanoiminopropanothioiminomethano)indolo[1,2-e][1,5]benzoxazocin-15-one 17,17-dioxide
The preceding material was treated as described in Example 2, Step 4 to furnish the title compound (4%). (ES+) m/z 569 (M+H)+.
Step 15: (7R)-14-[(1R,2S)) or (1S,2R)-2-fluorocyclohexyl]-21,24-dimethyl-7,8-dihydro-6H-7,11-(epiminoethanoiminopropanothioiminomethano)indolo[1,2-e][1,5]benzoxazocin-15-one 17,17-dioxide
The preceding material was treated as described in Example 3, Step 1 to furnish the title compound (15%). 1H NMR (400 MHz, DMSO-d6+TFA, 330 K) δ 1.00-1.40 (m, 2H), 1.50-1.70 (m, 4H), 1.70-1.80 (m, 1H), 1.90-2.10 (m, 1H), 2.20-2.40 (m, 3H), 2.30 (s, 3H), 2.85 (s, 3H), 3.10-3.40 (m, 3H), 3.40-3.80 (m, 6H), 3.90-4.00 (m, 1H), 4.00-4.10 (m, 1H), 4.30-4.40 (m, 1H), 4.55-4.70 (m, 1H), 4.95-5.10 (m, 1H), 7.25-7.35 (m, 2H), 7.45-7.60 (m, 3H), 7.95 (d, J 8.5, 1H), 8.16 (s, 1H); (ES+) m/z 583 (M+H)+.
The following tables show these examples:







TABLE 1







16 membered macrocycles











Example



m/z


no.
Compound name
Structure
procedure
(ES+)





3 ≡ 101
(7R)-14-cyclohexyl-21,24- dimethyl-7,8-dihydro-6H-7,11- (epiminoethanoiminopropanothio- iminomethano)indolo[1,2- e][1,5]benzoxazocin-15-one 17,17-dioxide




A, B
565


 


2 ≡ 102
(7R)-14-cyclohexyl-24-methyl- 7,8-dihydro-6H-7,11- (epiminoethanoiminopropanothio- iminomethano)indolo[1,2- e][1,5]benzoxazocin-15-one 17,17-dioxide




A
551
















TABLE 2







17 membered macrocycle











Example



m/z


no.
Compound name
Structure
procedure
(ES+)





1 ≡ 201
(7R)-14-cyclohexyl-25-methyl- 7,8-dihydro-6H-7,11- (epiminoethanoiminobutanothio- iminomethano)indolo[1,2- e][1,5]benzoxazocine-15,21-dione 17,17-dioxide




A
579
















TABLE 3







2-Fluorocyclohexyl macrocycles











Example



m/z


no.
Compound name
Structure
procedure
(ES+)





4 ≡ 301
(7R)-14-[(1R,2S) or (1S,2R)-2- fluorocyclohexyl]-21,24- dimethyl-7,8-dihydro-6H-7,11- (epiminoethanoiminopropanothio- iminomethano)indolo[1,2- e][1,5]benzoxazocin-15-one 17,17-dioxide




A, B
583

## Claims

1. A compound of the formula (I):




wherein
Ar is a moiety containing at least one aromatic ring and possesses 5-, 6-, 9- or 10-ring atoms optionally containing 1, 2 or 3 heteroatoms independently selected from N, O and S, which ring is optionally substituted by groups Q1 and Q2;
Q1 is halogen, hydroxy, C1-6alkyl, C1-6alkoxy, aryl, heteroaryl, CONRaRb, (CH2)0-3NRaRb, O(CH2)1-3NRaRb, O(CH2)0-3CONRaRb, O(CH2)0-3aryl, O(CH2)0-3heteroaryl, O(CReRf)aryl, O(CReRf)heteroaryl or OCHRcRd;
Ra and Rb are each independently selected from hydrogen, C1-4alkyl and C(O)C1-4alkyl;
or Ra, Rb and the nitrogen atom to which they are attached form a heteroaliphatic ring of 4 to 7 ring atoms, where said ring is optionally substituted by halogen, hydroxy, C1-4alkyl or C1-4alkoxy;
Rc and Rd are each independently selected from hydrogen and C1-4alkoxy;
or Rc and Rd are linked by a heteroatom selected from N, O and S to form a heteroaliphatic ring of 4 to 7 ring atoms, where said ring is optionally substituted by halogen, hydroxy, C1-4alkyl or C1-4alkoxy;
and wherein said C1-4alkyl, C1-4alkoxy and aryl groups are optionally substituted by halogen or hydroxy;
Re is hydrogen or C1-6alkyl;
Rf is C1-6alkyl;
Q2 is halogen, hydroxy, C1-4alkyl or C1-4alkoxy, where said C1-4alkyl and C1-4alkoxy groups are optionally substituted by halogen or hydroxy;
or Q1 and Q2 may be linked by a bond or a heteroatom selected from N, O and S to form a ring of 4 to 7 atoms, where said ring is optionally substituted by halogen, hydroxy, C1-4alkyl or C1-4alkoxy;
A is C3-6alkyl or C2-6alkenyl,
or A is a non-aromatic ring of 3 to 8 ring atoms where said ring may contain a double bond and/or may contain a O, S, SO, SO2 or NH moiety,
or A is a non-aromatic bicyclic moiety of 4 to 8 ring atoms,
and A is optionally substituted by halogen, hydroxy, C1-4alkyl or C1-4alkoxy;
D is N or CRg;
Rg is hydrogen, fluorine, chlorine, C1-4alkyl, C2-4alkenyl or C1-4alkoxy, where said C1-4alkyl, C2-4alkenyl and C1-4alkoxy groups are optionally substituted by hydroxy or fluorine;
W is a bond, C═O, O, S(O)0-2 or —(CR10R11)—(CR12R13)0-1—;
Y is a bond, C═O, O, —CR14R15— or NR14;
Z is a bond, C═O, O, S(O)0-2, —(CR10R11)—(CR12R13)0-1— or NR10;
and none, one or two of W, Y and Z are a bond;
X is —CR14a— or N;
R10, R11, R12, R13, R14, R14a and R15 are each independently selected from hydrogen, hydroxy, C1-6alkyl, C2-6alkenyl, C1-6alkoxy, C(O)C1-6alkyl, Het, (CH2)0-3NR16R17, C(O)(CH2)0-3NR16R17, NHC(O)(CH2)0-3NR16R17, O(CH2)1-3NR16R17, S(O)0-2(CH2)0-3R16R17 and C(O)(CH2)0-3OR16;
or one of R10, R14 and R14a is linked to R22 or R23 to form a ring of 4 to 10 atoms, where said ring is optionally substituted by halogen, hydroxy, C1-4alkyl or C1-4alkoxy;

Het is a heteroaliphatic ring of 4 to 7 ring atoms, which ring may contain 1, 2 or 3 heteroatoms selected from N, O or S or a group S(O), S(O)2, NH or NC1-4alkyl;
R16 and R17 are independently selected from hydrogen, C1-6alkyl and (CH2)0-4NR18R19;
or R16, R17 and the nitrogen atom to which they are attached form a heteroaliphatic ring of 4 to 7 ring atoms, which ring may optionally contain 1 or 2 more heteroatoms selected from O or S or a group S(O), S(O)2, NH or NC1-4alkyl, and which ring is optionally substituted by halogen, hydroxy, C1-4alkyl or C1-4alkoxy;
R18 and R19 are independently selected from hydrogen and C1-6alkyl;
or R18, R19 and the nitrogen atom to which they are attached form a heteroaliphatic ring of 4 to 7 ring atoms, which ring may optionally contain 1 or 2 more heteroatoms selected from O or S or a group S(O), S(O)2, NH or NC1-4alkyl, and which ring is optionally substituted by halogen, hydroxy, C1-4alkyl or C1-4alkoxy;
R1 is hydrogen or C1-6alkyl;
B is —CR20R21—, —C(═O)—, —SO— or —SO2—;
R20 and R21 are independently selected from hydrogen and C1-6alkyl;
or R20 and R21, together with the carbon atom to which they are joined, form a C3-6cycloalkyl group;
M is C4-8alkylene or C4-8alkenylene, optionally substituted by R22, where 1, 2 or 3 of the carbon atoms in the C4-8alkylene or C4-8alkenylene groups is optionally replaced by O, NR23, S, SO, SO2, piperidinyl, piperazinyl or pyrrolidinyl,

where R23 is hydrogen or C1-6alkyl, or R23 is linked to one of R10, R14 and R14a to form a ring of 4 to 10 atoms as hereinbefore described;
where R22 is halo, C1-4alkyl, (CH2)0-3C3-8cycloalkyl, (CH2)0-3aryl, (CH2)0-3heteroaryl, (CH2)0-3Het or oxo,
or R22 is linked to one of R10, R14 and R14a to form a ring of 4 to 10 atoms as hereinbefore described;
or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1 in which Ar is phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furanyl or 3-furanyl, optionally substituted by groups Q1 and Q2 as defined in claim 1.

3. A compound according to claim 2 in which Q1 is methoxy and Q2 is absent.

4. A compound according to claim 1 in which A is cyclopentyl or cyclohexyl optionally substituted by halogen, hydroxy, C1-4alkyl or C1-4alkoxy.

5. A compound according to claim 1 in which D is CRg where Rg is hydrogen or C1-4alkyl.

6. A compound according to claim 1 in which W is —CH2— or —CH2CH2—.

7. A compound according to claim 1 in which W is Z is a bond, O, —CH2— or —CH2CH2—.

8. A compound according to claim 1 in which Y is —CH2—, NH, N(C1-4alkyl), N(CH2)2N(C1-4alkyl)2 or NC(O)CH2N(C1-4alkyl)2.

9. A compound according to claim 1 in which X is —CH— or —C(C1-6alkyl)-.

10. A compound according to claim 1 in which R1 is hydrogen or methyl.

11. A compound according to claim 1 in which B is —CH2— or —SO2—.

12. A compound according to claim 1 in which M is selected from:

13. A compound according to claim 1 of the formula (Ia):




or a pharmaceutically acceptable salt thereof, wherein W, X, Y, Z and M are as defined in relation to formula (I).

14. A compound according to claim 1 selected from:
(7R)-14-cyclohexyl-21,24-dimethyl-7,8-dihydro-6H-7,11-(epiminoethanoiminopropanothio iminomethano)indolo[1,2-e][1,5]benzoxazocin-15-one 17,17-dioxide,
(7R)-14-cyclohexyl-24-methyl-7,8-dihydro-6H-7,11-(epiminoethanoiminopropanothioimino methano)indolo[1,2-e][1,5]benzoxazocin-15-one 17,17-dioxide,
(7 R)-14-cyclohexyl-25-methyl-7,8-dihydro-6H-7,11-(epiminoethanoiminobutanothioimino methano)indolo[1,2-e][1,5]benzoxazocine-15,21-dione 17,17-dioxide,
(7R)-14-[(1R,2S) or (1S,2R)-2-fluorocyclohexyl]-21,24-dimethyl-7,8-dihydro-6H-7,11-(epiminoethanoiminopropanothioiminomethano)indolo[1,2-e][1,5]benzoxazocin-15-one 17,17-dioxide,
and pharmaceutically acceptable salts thereof.

15. A pharmaceutical composition comprising an effective amount of a compound of the formula (I), or a pharmaceutically acceptable salt thereof, according to claim 1 and a pharmaceutically acceptable carrier.