HK1104044B - Hcv ns-3 serine protease inhibitors - Google Patents

Description

Hcv NS-3 serine protease inhibitors

Technical Field

The present invention relates to novel inhibitors of the flavivirus HCV NS3 serine protease and methods for their use in the treatment or prevention of HCV.

Background

The HCV NS3 serine protease is a multifunctional protein comprising a serine protease domain and an RNA helicase domain. The protease cofactor NS4A, a relatively small protein, is a protein essential for enhancing serine protease activity. The NS3 serine protease is essential in the viral life cycle. Matrix binding site analysis revealed from the X-ray crystal structure revealed that the binding site for the NS3 protease was significantly shallow and solvent exposed, making small molecule inhibitor design difficult.

Two HCV protease inhibitors are believed to have entered clinical trials, Boehringer Ingelheim's BILN-2061 disclosed in WO0059929 and Vertex' VX-950 disclosed in WO 0387092. A number of similar peptidomimetic HCV protease inhibitors have also been proposed in the academic and patent literature. Most of the above prior art peptidomimetics are usually present as L-proline derivatives at position P2 of the inhibitor and interact with the S2 subsite of the HCV protease. In the case of BILN-2061, the L-proline is 4-substituted with quinolineether, however there is one carbocyclic ring fused to the L-proline ring in VX-950. Most peptidomimetics also include other L-amino acid derivative peptides linked at position P3, and many of the above-mentioned proposed inhibitors also include L-amino acid derivatives that are additionally extended to P4, P5 and P6.

It has become apparent that continuous administration of BILN-2061 or VX-950 selects for HCV mutants which are resistant to the corresponding drug, so-called drug escape mutants. These drug escape mutants have characteristic mutations in the HCV protease genome, particularly D168V, D168Y and/or a 165S. Thus, the therapeutic paradigm for HCV has to resemble HIV therapy, where drug escape mutations are also likely to occur. Accordingly, there is a continuing need for other drugs with different resistance properties in order to provide a treatment regimen for non-effective patients, and combination therapy with multiple drugs is likely to be a normal form in the future, even for the first treatment.

The practice of using HIV drugs, and HIV protease inhibitors in particular, is further emphasized that sub-optimal pharmacokinetics and complex dosage regimes will soon result in compliance being unintentionally compromised. This in turn means that in HIV conditions, the 24 hour trough concentration (minimum plasma concentration) of the corresponding drug tends to decrease in IC for most of the day₉₀Or ED₉₀Below the limit. It is generally accepted that at least IC₅₀And more realistic, IC₉₀Or ED₉₀The 24 hour trough concentration is essential to delay the production of drug escape mutants and to obtain the necessary pharmacokinetics and drug metabolism, which makes the trough concentration a powerful challenge for drug design. The strong peptidomimetic nature of prior art HCV protease inhibitors, as well as the multiple peptide bonds of their own structure, form a pharmacokinetic hurdle to effective dosage regimes.

Brief description of the invention

According to a first aspect of the present invention there is provided a compound of formula I, or a pharmaceutically acceptable salt or prodrug thereof.

Wherein:

a is C (═ O) OR¹、C(＝O)NHSO₂R²、C(＝O)NHR³Or CR⁴R⁴', wherein:

R¹is hydrogen, C₁-C₆Alkyl radical, C₀-C₃Alkyl carbocyclyl, C₀-C₃An alkyl heterocyclic group;

R²is C₁-C₆Alkyl radical, C₀-C₃Alkyl carbocyclyl, C₀-C₃An alkyl heterocyclic group;

R³is C₁-C₆Alkyl radical, C₀-C₃Alkyl carbocyclyl, C₀-C₃Alkylheterocyclyl, -OC₁-C₆Alkyl, -OC₀-C₃Alkyl carbocyclyl, -OC₀-C₃An alkyl heterocyclic group;

R⁴is halogen, amino or OH; or R⁴And R⁴Together are ═ O;

R⁴' is C₁-C₆Alkyl radical, C₀-C₃Alkyl carbocyclyl, C₀-C₃An alkyl heterocyclic group;

wherein R is²、R³And R⁴' each optionally substituted with 1 to 3 substituents independently selected fromSubstituted by the following groups: halogen, oxo, nitrile, azido, nitro, C₁-C₆Alkyl radical, C₀-C₃Alkyl carbocyclyl, C₀-C₃Alkyl heterocyclic group, NH₂CO-、Y-NRaRb、Y-O-Rb、Y-C(＝O)Rb、Y-(C＝O)NRaRb、Y-NRaC(＝O)Rb、Y-NHSO_pRb、Y-S(＝O)_pRb、Y-S(＝O)_pNRaRb, Y-C (═ O) ORb, and Y-NRaC (═ O) ORb;

y is independently a bond or C₁-C₃An alkylene group;

ra is independently H or C₁-C₃An alkyl group;

rb is independently H, C₁-C₆Alkyl radical, C₀-C₃Alkyl carbocyclyl or C₀-C₃An alkyl heterocyclic group;

p is independently 1 or 2;

m is CR⁷R⁷' or NRu;

R⁷is C₁-C₆Alkyl radical, C₀-C₃Alkyl radical C₃-C₇Cycloalkyl, or C₂-C₆Alkenyl, each of which is optionally substituted by 1 to 3 halogen atoms or by amino, -SH or C₀-C₃Alkyl cycloalkyl substituted; or R⁷Is J;

R⁷' is H or with R⁷Together form optionally substituted R^7′aSubstituted C₃-C₆A cycloalkyl ring,

wherein;

R^7′ais C₁-C₆Alkyl radical, C₃-C₅Cycloalkyl radical, C₂-C₆Alkenyl, each of which may be optionally substituted with halogen; or R^7′aCan be J;

q is 0 to 3 and k is 0 to 3; wherein q + k is more than or equal to 1;

w is-CH₂-、-O-、-OC(＝O)NH-、-OC(＝O)-、-S-、-NH-、-NRa、-NHSO₂-, -NHC (═ O) NH — or-NHC (═ O) -, -NHC (═ S) NH — or a bond;

R⁸is a ring system comprising 1 or 2 saturated, partially unsaturated or unsaturated rings each having 4 to 7 ring atoms and each having 0 to 4 heteroatoms independently selected from S, O and N, optionally via C₁-C₃Alkyl and W are interrupted; or R⁸Is C₁-C₆An alkyl group; any of the above R⁸All of which may be optionally substituted by R⁹Mono-, di-or tri-substituted,

wherein:

R⁹independently selected from: halogen, oxo, nitrile, azido, nitro, C₁-C₆Alkyl radical, C₀-C₃Alkyl carbocyclyl, C₀-C₃Alkyl heterocyclic group, NH₂C(＝O)-、Y-NRaRb、Y-O-Rb、Y-C(＝O)Rb、Y-(C＝O)NRaRb、Y-NRaC(＝O)Rb、Y-NHSO_pRb、Y-S(＝O)_pRb、Y-S(＝O)_pNRaRb, Y-C (═ O) ORb, and Y-NRaC (═ O) ORb; wherein said carbocyclyl or heterocyclyl moiety is optionally substituted with R¹⁰Substituted; wherein

R¹⁰Is C₁-C₆Alkyl radical, C₃-C₇Cycloalkyl radical, C₁-C₆Alkoxy, amino, sulfonyl, (C)₁-C₃Alkyl) sulfonyl, NO₂OH, SH, halogen, haloalkyl, carboxyl, amido;

e is-C (O) -, -C (S) -, -S (O)₂-、-S(＝O)-、-C(＝N-Rf)-；

Rf is H, -CN, -C (═ O) NRaRb, -C (═ O) C₁-C₃An alkyl group;

x is-NRx-wherein Rx is H, C₁-C₅Alkyl or J; or in case E is-C (═ O)X may also be-O-or-NRjNRj-;

one of Rj is H and the other is H, C₁-C₅Alkyl or J;

R¹¹is H, C₁-C₆Alkyl radical, C₀-C₃Alkyl carbocyclyl, C₀-C₃(ii) an alkylheterocyclyl group, each of which may be substituted with a group selected from: halogen, oxo, nitrile, azido, nitro, C₁-C₆Alkyl radical, C₀-C₃Alkyl carbocyclyl, C₀-C₃Alkyl heterocyclic group, NH₂CO-、Y-NRaRb、Y-O-Rb、Y-C(＝O)Rb、Y-(C＝O)NRaRb、Y-NRaC(＝O)Rb、Y-NHSO_pRb、Y-S(＝O)_pRb、Y-S(＝O)_pNRaRb, Y-C (═ O) ORb, and Y-NRaC (═ O) ORb; or R¹¹Is J;

j, if present, is a single 3-to 10-membered saturated or partially unsaturated alkylene chain derived from R⁷/R⁷' cycloalkyl or from R⁷The carbon atom to which it is attached extends to Rj, Rx, Ry or R¹¹One thereby forming a macrocycle, said chain being optionally substituted with one to three groups independently selected from-O-, -S-or-NR¹²-is interrupted by a heteroatom, and wherein 0 to 3 carbon atoms in the chain are optionally interrupted by R¹⁴Substitution;

wherein;

R¹²is H, C₁-C₆Alkyl radical, C₃-C₆Cycloalkyl or-C (═ O) R¹³；

R¹³Is C₁-C₆Alkyl radical, C₀-C₃Alkyl carbocyclyl, C₀-C₃An alkyl heterocyclic group;

R¹⁴independently selected from: H. c₁-C₆Alkyl radical, C₁-C₆Haloalkyl, C₁-C₆Alkoxy, hydroxy, halogen, amino, oxo, thio and C₁-C₆ThioalkaneA group;

ru is independently H or C₁-C₃An alkyl group;

m is 0 or 1; n is 0 or 1;

u is ═ O or absent;

R¹⁵is H, C₁-C₆Alkyl radical, C₀-C₃Alkyl carbocyclyl, C₀-C₃An alkylheterocyclyl group, each of which may be substituted with: halogen, oxo, nitrile, azido, nitro, C₁ -C₆Alkyl radical, C₀-C₃Alkyl carbocyclyl, C₀-C₃Alkyl heterocyclic group, NH₂CO-、Y-NRaRb、Y-O-Rb、Y-C(＝O)Rb、Y-(C＝O)NRaRb、Y-NRaC(＝O)Rb、Y-NHS(＝O)_pRb、Y-S(＝O)_pRb、Y-S(＝O)_pNRaRb、 Y-C(＝O)ORb、Y-NRaC(＝O)ORb；

G is-O-, -NRy-, -NRjNRj-; one of Rj is H and the other is H, C₁-C₅Alkyl or J;

ry is H, C₁-C₃An alkyl group; or Ry is J;

R¹⁶is H, C₁-C₆Alkyl radical, C₀-C₃Alkyl carbocyclyl, C₀-C₃An alkylheterocyclyl group, each of which may be substituted with: halogen, oxo, nitrile, azido, nitro, C₁ -C₆Alkyl radical, C₀-C₃Alkyl carbocyclyl, C₀-C₃Alkyl heterocyclic group, NH₂CO-、Y-NRaRb、Y-O-Rb、Y-C(＝O)Rb、Y-(C＝O)NRaRb、Y-NRaC(＝O)Rb、Y-NHSO_pRb、Y-S(＝O)_pRb、Y-S(＝O)_pNRaRb、Y-C(＝O)ORb、Y-NRaC(＝O)ORb；

Provided that when m ═ n ═ 0 and G is O, then R¹⁶Not tert-butyl or phenyl.

Is not desirableBeing bound in any way by a model of temporary constraints, either theoretical or specific, the ideographic concepts P1, P2, P3 and P4 applied herein are provided for convenience only and basically have, for example, Schechter&Berger, (1976) Biochem Biophys ResComm 27157-162, which refers to those portions of inhibitors identified as filling the S1, S2, S3 and S4 subsites, respectively, with S1 proximal and S4 distal to the cleavage site. Regardless of the manner of attachment, the components defined by formula I are intended to be included within the scope of the present invention. For example, it is contemplated that the end-capping group R is especially when m and/or n is 0¹⁶G can interact with S3 and S4 subsites.

Various embodiments of the present invention may be symbolically represented as R¹⁶-G-P4-P3-link-P2-P1, wherein P3 and/or P4 may be absent, and wherein P1, P3 and P4 each represent a structural unit constituting a natural or non-natural amino acid derivative, P2 is a heterocyclic residue and G-R¹⁶Is a capping group. The linker is a carbonyl or other functional group as defined by E. Thus, the above-mentioned P1 and P2 building blocks and P3 and P4 building blocks are typically linked together by amide bonds, whereas the P2 and P3 building blocks are linked by the above-mentioned links. Thus, in the compounds of the invention, the amide bonds are generally inverted relative to each other on each side of the linker.

Another aspect of the invention includes a pharmaceutical composition comprising a compound of the invention as defined above together with a pharmaceutically acceptable carrier or diluent therefor.

The compounds and compositions of the present invention may be used in methods of medical treatment or prevention of HCV infection in humans. Accordingly, another aspect of the invention is the use of a compound of the invention as defined above in therapy, such as in the manufacture of a medicament for the prevention or treatment of flavivirus infection in humans or animals. Exemplary flaviviruses include BVDV, dengue and in particular HCV.

Compounds of the invention are represented by the structural units P2 and P3With a non-peptide linkage between them, which results in the P3 and P4 residues being positioned in an inverted orientation relative to the original substrate. Such non-peptide linkages are also generally longer than the corresponding pre-existing peptide bonds, meaning that the P3 and/or P4 groups (including R)¹⁶Capped to the extent that it interacts with S3 or S4) will migrate outward relative to the original peptide matrix. It is expected that such reversal and transfer will facilitate the formation of P3 and/or P4 and/or R¹⁶Non-natural D-type stereochemistry of pocket filling groups (e.g., side chains). Indeed, the above compounds are generally highly active compounds and are within the scope of the present invention. However, it has now surprisingly been found that even though the compounds according to the invention with L-amino acid side chains at positions P3 and/or P4 show good activity, the corresponding side chain units have to approach the S3 or S4 pocket from different angles with respect to the original peptidic matrix. Accordingly, R¹¹And/or R¹⁵At L-stereochemistry and/or R corresponding to pseudo-L-stereochemistry¹⁶The configurations all represent preferred aspects of the invention.

The different approach angles to the S3 and/or S4 pockets also imply the ability of the compounds of the present invention to avoid the resistance exhibited by prior art HCV protease inhibitors, which to date have a conventional peptide backbone of natural or non-natural L-amino acid residues. RNA-associated HCV RNA polymerase NS5A has very poor read-ability, as well as the well-known HIV reverse transcriptase which rapidly generates drug escape mutants under antiviral therapy selection pressure. This also means that HCV polymerase is very error prone and may develop characteristic resistance when HCV antiviral agents are administered for long periods of time. Even before the input, it is clear that BILN 2061 with a substantially peptide backbone (even if macrocyclization is performed) and the Vertex' NS3 protease inhibitor VX-950 with a linear peptide backbone at P3 and P4 rapidly develop characteristic resistance mutations at positions 155, 156 or 168 of the NS3 protease (Lin et al, J Biol Chem 2004279 (17): 17808-17).

A preferred group of compounds of the invention include those wherein P1 represents a hydrazine derivative, i.e. M is NRu wherein Ru is typically H orC is₁-C₃Alkyl wherein M is CR⁷R⁷The compounds of' form a further preferred aspect of the invention.

In formula I M is CR⁷R⁷Preferred embodiments of' include formula IA:

preferably in formula I the values of q and k include 2: 1, 2: 2, 2: 3, 3: 2, 3: 3, more preferably 1: 2 and 1: 0; and most preferably 1: 1, in which case preferred compounds have the following partial structure:

wherein e is 1 or 2.

It is currently preferred that E is-C (═ O) -or-C ═ N-Rf, for example where Rf is-CN or-C (═ O) NH₂。

The compounds of the invention may contain two functional groups, P3 and P4, i.e. m and n are each 1. Preferred embodiments within formula I at this time include the following formulae Ida-Idd:

further embodiments include structures corresponding to Ida, Idb, Idc, and Idd, where M is NRu.

Another structure of the compounds of the present invention comprises the P3 functional group, but does not include the P4 functional group, i.e., m is 1 and n is 0. Preferred embodiments within formula I include the following formulas Iea-Iee:

additional embodiments include structures corresponding to Iea, Ieb, Iec, Ied, and Iee, where M is NRu.

Further structures of the compounds of the invention include those in which m and n are both 0 and thus the radical R¹⁶G is adjacent to the structure of P2, but as mentioned above, the end capping group R¹⁶G may advantageously interact with S3 and/or S4.

Preferred embodiments within formula I include the following formula Ifa-Ife:

in Ifb and elsewhere, R¹⁶Generally H, C₁-C₃Alkyl radical, C₅-C₆Alkyl radical, C₀-C₃Alkyl heterocyclic group, C₁-C₃Alkyl carbocyclyl or C₃-C₇Cycloalkyl groups, each of which is optionally substituted, as described above. For example, R¹⁶May be phenyl substituted as described above.

Additional embodiments include structures corresponding to Ifa, Ifb, Ifc, Ifd, and Ife, wherein M is NRu.

The compounds of the invention may include linear molecules, as described above. In addition, in which R⁷And R⁷' together as a spirocycloalkyl (such as spirocyclopropyl), the compounds of the invention may be configured as macrocycles, wherein the linking group J is Rj, Rx, Ry or R in formula I¹¹One group extending between them. In addition, the macrocyclic ring J can be substituted with R⁷Adjacent carbon extending to Rj, Rx, Ry or R¹¹One of them.

Within formula I, where m is 0 and n is 1, preferred embodiments of the above macrocyclic structures include the following formulae Iga-Igd:

also preferred is a group wherein the J chain is linked to R⁷The corresponding structure of adjacent carbon atoms.

Within formula I, where m is 0 and n is 1, additional preferred embodiments of the above macrocyclic structures include the following formulas Ige-Igf:

also preferred is a group wherein the J chain is linked to R⁷The corresponding structure of adjacent carbon atoms.

Within formula I, which includes the P3 and P4 functional groups, i.e., where m and n are each 1, preferred macrocyclic structures include those of the following formulae Iha-Ihd:

also preferred is a group wherein the J chain is linked to R⁷The corresponding structure of adjacent carbon atoms.

Within compounds of formula I in which both the P3 and P4 functional groups are absent, i.e. in which m and n are each 0, preferred macrocyclic structures include those of the formulae Ihe-Ihh, especially Ihe and Ihf:

also preferred is a group wherein the J chain is linked to R⁷The corresponding structures of adjacent carbon atoms, in particular of formulae Ihe and Ihf.

Generally, in optional macrocyclic structures, such as those illustrated above, the linker J is a saturated or partially unsaturated alkylene chain having from 3 to 10 chain atoms, preferably from 5 to 8 chain atoms, such as 6 or 7 chain atoms, i.e., an alkylene chain with from 1 to 3 unsaturated bonds between adjacent carbon atoms, typically one unsaturation. The length of the chain will naturally depend on whether J is selected from Rd, Rj, Rx, Ry, R¹¹Or from R⁷Adjacent carbon extensions. Suitable chains are described in detail in WO 00/59929. Typically J is of a size that provides a macrocycle of 13 to 16 ring atoms (including those atoms contained within the ring in the P1, P2, and P3 groups if present). Suitably J is of a size to provide a macrocycle of 14 or 15 ring atoms.

Desirably, the J chain contains one or two heteroatoms selected from: o, S, NH, NC₁ -C₆Alkyl or N-C (═ O) C₁-C₆An alkyl group. More preferably, the J chain optionally comprises one of the following heteroatoms: NH or N-C (═ O) C₁-C₆Alkyl, most preferably n (ac). Most preferably, the chain comprising nitrogen atoms is a saturated chain. In another embodiment, J comprises one heteroatom selected from O or S. The chain may be substituted with R¹⁴Substitution, such as H or methyl.

Typically the J-link is saturated. In addition, J contains 1-3 double bonds, preferably 1 double bond, and is generally bonded to cycloalkyl R⁷The functional groups being separated by one carbon atom, if presentIf so. The double bond may be a cis-or trans-double bond.

Thus, representative examples of J include pentene, hexene, heptene, each of which is substituted with: c₁-C₆Alkyl radical, C₁-C₆Haloalkyl, C₁-C₆Alkoxy, hydroxy, halogen, amino, oxo, thio or C₁-C₆A thioalkyl group; penten-3-yl, hexen-4-yl, hepten-5-yl, wherein 3, 4 or 5 refers to the double bond between the carbon atoms in positions 3 and 4, 4 and 5, and the like.

Suitable R⁷And R⁷' the group includes wherein R⁷' is H, and R⁷Those which are n-ethyl, n-propyl, cyclopropylmethyl, cyclopropyl, cyclobutylmethyl, cyclobutyl, 2-difluoroethyl or mercaptomethyl. Preferred embodiments include those wherein R⁷Those which are n-propyl or 2, 2-difluoroethyl.

R⁷And R⁷Another preferred structure of' includes wherein R⁷' is H and R⁷Is C₃-C₇Cycloalkyl or C₁-C₃Alkyl radical C₃-C₇Those structures of cycloalkyl groups.

For R⁷And R⁷', further preferred structures include those wherein R is⁷' is H and R⁷Those structures that are J.

In addition, R⁷And R⁷Together,' are defined as a spiro-cycloalkyl functional group, such as a spiro-cyclobutyl ring, and more preferably a spiro-cyclopropyl ring. In the context of the present invention, "spiro" simply means that the cycloalkyl ring shares one carbon atom with the peptide backbone of the compound. The ring is a substituted or unsubstituted ring. Preferred substituents include R^7′aMono-or di-substituted, wherein R^7′aIs C₁-C₆Alkyl radical, C₃-C₅Cycloalkyl or C₂-C₆Alkenyl, each optionally substituted by halogenAnd (4) substituting. Alternatively, the substituent may be a J-linker as described above. For spiro-cyclopropyl rings, the generally preferred stereochemistry is defined as follows.

Particularly preferred substituents include R^7′aIs ethyl, vinyl, cyclopropyl (i.e. to R)⁷/R⁷The spiro-cyclopropyl substituent of the 'spiro' cycloalkyl ring), 1-or 2-bromoethyl, 1-or 2-fluoroethyl, 2-bromovinyl or 2-fluoroethyl.

In one embodiment of the invention, A is-CR as detailed in PCT/EP03/10595⁴R⁴', the contents of which are incorporated herein by reference.

Thus, R is appropriate⁴' the group includes C₁-C₆Alkyl radicals, such as methyl, ethyl, propyl, vinyl and-CHCHCH₃. Further preferred R⁴' groups include aryl or heteroaryl, such as optionally substituted phenyl, pyridyl, thiazolyl or benzimidazolyl or C₁-C₃Alkylaryl or C₁-C₃Alkylheteroaryl, wherein the alkyl moiety is methyl, ethyl, propyl, vinyl and-CH ═ CHCH₃. Preferred aryl moieties include optionally substituted phenyl, benzothiazole and benzimidazole.

Preferred R⁴The radicals comprising-NH₂Fluorine or chlorine. Further preferred R⁴The group includes-OH and in particular ═ O.

Another embodiment of a is C (═ O) NH³Wherein R is³Is optionally substituted C₀-C₃Alkylaryl group, C₀-C₃Alkylheteroaryl, OC₀-C₃Alkylaryl or OC₀-C₃An alkyl heteroaryl group. Suitable substituents appear in the definitions section below.

For a, a currently preferred structure is C (═ O) OR¹In particular wherein R¹Is C₁-C₆Alkyl radicals, such as the methyl, ethyl or tert-butyl radical, andmost preferably hydrogen.

A particularly preferred structure for a is C (═ O) NHSO₂R²In particular wherein R²Is optionally substituted C₁-C₆Alkyl (preferably methyl), or optionally substituted C₃-C₇Cycloalkyl (preferably cyclopropyl) or optionally substituted C₀-C₆Alkylaryl (preferably optionally substituted phenyl). Suitable substituents appear in the definitions section below.

substituent-W-R on cyclic P2 radical⁸Any proline substituent may be used, which is widely described in the following documents: WO00/59929, WO00/09543, WO 00/09558, WO 99/07734, WO 99/07733, WO 02/60926, WO 03/35060, WO 03/53349, WO 03/064416, WO 03/66103, WO03/064455, WO 03/064456, WO 03/62265, WO 03/062228, WO03/87092, WO 03/99274, WO 03/99316, WO 03/99274, WO 04/03670, WO 04/032827, WO 04/037855, WO 04/43339, WO 04/92161, WO04/72243, WO 04/93798, WO 04/93915, WO 04/94452, WO 04/101505, WO 04/101602, WO 04/103996, WO 04113365 and the like.

Preferred W functional groups include, W is-OC (═ O) NH-, -OC (═ O) -, -NH-, -NR⁸′-、-NHS(O)₂-or-NHC (═ O) -, in particular-OC (═ O) NH-or-NH-. Preferred R for the above W functional group⁸The radicals including optionally substituted C₀-C₃Alkyl carbocyclyl or C₀-C₃Alkyl-heterocyclyl including those groups described in WO 0009543, WO 0009558 and WO 00/174768. For example, an ester substituent on a cyclic P2 group, -W-R⁸Including those substituents disclosed in WO 01/74768, such as C₁-C₆Alkanoyloxy group, C₀-C₃Alkylaryloxy, especially (optionally substituted) benzoyloxy or C₀-C₃Alkyl heterocyclic acyloxy, in particular the following groups:

this publication also describes the possibility of further-W-R⁸E.g. C₁-C₆Alkyl (e.g. ethyl, isopropyl), C₀-C₃An alkylcyclo group (such as cyclohexyl), 2-difluoroethyl, -C (═ O) NRc where Rc is C₁-C₆Alkyl radical, C₀-C₃Alkyl cyclopropyl, C₀ -C₃Alkylaryl or C₀-C₃An alkyl heterocyclic group.

Presently preferred W functional groups include-S-and in particular-O-. In said embodiment, R⁸Suitable meanings of (A) include C₀-C₃Alkylaryl or C₀-C₃Alkyl heteroaryl, each of which is optionally substituted by R⁹Mono-, di-, or tri-substituted, wherein:

R⁹is C₁-C₆Alkyl radical, C₁-C₆Alkoxy group, NO₂OH, halogen, trifluoromethyl, amino or amido (e.g. optionally substituted by C)₁-C₆Alkyl mono-or di-substituted amido or amino), C₀-C₃Alkylaryl group, C₀-C₃Alkylheteroaryl or carboxy, wherein the aryl or heteroaryl moiety is optionally substituted with R¹⁰Substitution, wherein:

R¹⁰is C₁-C₆Alkyl radical, C₃-C₇Cycloalkyl radical, C₁-C₆Alkoxy, amino, amido, sulphonyl C₁-C₃Alkyl radical, NO₂OH, halogen, trifluoromethyl, carboxyl or heteroaryl.

In general, R⁸Is C₀-C₃Alkylaryl or C₀-C₃C in alkyl heteroaryl₀-C₃The alkyl moiety being methyl and being particularly absent, i.e. C₀. Said aryl group orThe heteroaryl moiety is as broadly described in the definitions section below.

Preferred R⁹Comprising C₁-C₆Alkyl radical, C₁-C₆Alkoxy, amino (e.g. di-C)₁ -C₃Alkylamino, amido (e.g., -NHC (O) C)₁-C₆Alkyl or C (═ O) NHC₁ -C₆Alkyl), aryl or heteroaryl, wherein aryl or heteroaryl is optionally substituted by R¹⁰Substitution;

wherein:

R¹⁰is C₁-C₆Alkyl radical, C₃-C₇Cycloalkyl radical, C₁-C₆Alkoxy, amino (e.g. mono-or di-C)₁-C₃Alkylamino, amido (e.g., -NHC (O) C)₁-C₃Alkyl or C (═ O) NHC₁-C₃Alkyl), halogen, trifluoromethyl or heteroaryl.

Preferred R¹⁰Comprising C₁-C₆Alkyl radical, C₁-C₆Alkoxy, amino, amido (e.g. -NHC (O) C₁-C₆Alkyl or C (═ O) NHC₁-C₆Alkyl), halogen, or heteroaryl.

Particularly preferred R¹⁰Including methyl, ethyl, isopropyl, t-butyl, methoxy, chloro, amino, amido (e.g., -NHC (O) C)₁-C₆Alkyl radicals, e.g. -NC (═ O) CHC (CH)₃)₃Or C (═ O) NHC₁-C₃Alkyl) or C₁-C₃An alkyl thiazole.

R⁸Preferred embodiments include 1-naphthylmethyl, 2-naphthylmethyl, benzyl, 1-naphthyl, 2-naphthyl or quinolyl, each unsubstituted or substituted by R as defined above⁹Mono-or di-substituted, especially 1-naphthylmethyl, or unsubstituted, with R as defined above⁹Mono-or di-substituted quinolinyl.

Currently preferred R⁸Comprises the following steps:

wherein R is^9aIs C₁-C₆Alkyl radical, C₁-C₆Alkoxy, thio C₁-C₃Alkyl, optionally substituted by C₁-C₆Alkyl-substituted amino, C₀-C₃Alkylaryl or C₀-C₃Alkyl heteroaryl, C₀-C₃Alkylheterocyclyl, wherein said aryl, heteroaryl or heterocyclyl is optionally substituted with R¹⁰And wherein:

R¹⁰is C₁-C₆Alkyl radical, C₃-C₇Cycloalkyl radical, C₁-C₆Alkoxy, amino, amido, heteroaryl and heterocyclyl; and

R^9bis C₁-C₆Alkyl radical, C₁-C₆Alkoxy, amino, amido, NO₂OH, halogen, trifluoromethyl, carboxyl.

Suitable R^9aIncluding aryl or heteroaryl, each optionally substituted with R as defined above¹⁰Substituted, especially wherein R^9aSelected from:

wherein R is¹⁰Is H, C₁-C₆Alkyl or C₀-C₃alkyl-C₃-C₆Cycloalkyl, optionally substituted by C₁-C₆Alkyl mono-or di-substituted amino, amido (e.g. -NHC (O) C)₁ -C₆Alkyl or C (═ O) NHC₁-C₆Alkyl), heteroarylOr a heterocyclic group.

R^9aConveniently phenyl, and whereby R⁸Comprises the following steps:

wherein R is^10aIs H, C₁-C₆Alkyl radical, C₁-C₆Alkoxy or halogen; and R^9bIs C₁ -C₆Alkyl radical, C₁-C₆Alkoxy, amino (e.g. di- (C)₁-C₃Alkyl) amines), amido (e.g., -NHC (O) C₁-C₃Alkyl or C (═ O) NHC₁-C₃Alkyl), NO₂OH, halogen, trifluoromethyl, carboxyl.

Further preferred R⁸Comprises the following steps:

wherein R is^10aIs H, C₁-C₆Alkyl or C₀-C₃alkyl-C₃-C₆Cycloalkyl, amines (e.g. by C)₁-C₆Alkyl mono-or di-substituted amines), amido (e.g., -NHC (O) C₁ -C₆Alkyl or C (═ O) NHC₁-C₆Alkyl), heteroaryl or heterocyclyl; and R^9bIs C₁-C₆Alkyl radical, C₁-C₆Alkoxy, amino (e.g. di- (C)₁-C₃Alkyl) amino), amido (e.g., -NHC (O) C₁-C₃Alkyl or C (═ O) NHC₁-C₃Alkyl), NO₂OH, halogen, trifluoromethyl or carboxyl.

In the embodiments described immediately above, R^9bConveniently is C₁-C₆-alkoxy, preferably methoxy.

For example, when W is an ether, R is additionally suitable⁸Is of the formula:

wherein W 'is N or CH, r is 0 or 1, Ra' is H, C₁-C₆Alkyl radical, C₀-C₃Alkyl cycloalkyl radical, C₁-C₆Alkoxy, hydroxy or amine, and Rb' is H, halogen, C₁ -C₆Alkyl radical, C₀-C₃Alkyl cycloalkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Thioalkyl, cycloalkyl C₀-C₃Alkoxy radical, C₁-C₃Alkoxy radical C₁-C₃Alkyl radical, C₀-C₃Alkylaryl or C₀-C₃An alkyl heterocyclic group. A particularly preferred ether substituent is 7-methoxy-2-phenyl-quinolin-4-yloxy.

When W is a bond, then R⁸Preferred are substituted or unsubstituted heterocyclic ring systems, as described in WO2004/072243 or WO 2004/113665.

When W is a bond, R⁸Representative examples include the following aromatic compounds which may optionally be substituted: 1H-pyrrole, 1H-imidazole, 1H-pyrazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, pyridine, pyridazine, pyrimidine, pyrazine, phthalazine, quinoxaline, quinazoline, quinoline, cinnoline, 1H-pyrrolo [2, 3-b ]]Pyridine, 1H-indole, 1H-benzimidazole, 1H-indazole, 7H-purine, benzothiazole, benzoxazole, 1H-imidazo [4, 5-c ]]Pyridine, 1H-imidazo [4, 5-b ]]Pyridine, 1, 3-dihydro-benzimidazol-2-one, 1, 3-dihydro-benzimidazol-2-thione, 2, 3-dihydro-1H-indole, 1, 3-dihydro-indol-2-one, 1H-indol-2, 3-dione, 1, 3-dihydro-benzimidazol-2-one, 1H-pyrrolo [2, 3-c]Pyridine, benzofuran, benzo [ b ]]Thiophene, benzo [ d ]]Isoxazoles, benzols [ d ]]Isothiazole, 1H-quinolin-2-one, 1H-quinolin-4-one, 1H-quinazolin-4-one, 9H-carbazole, 1H-quinazolin-2-one.

When W is a bond, R⁸Further representative examples include the following non-aromatic compounds which may optionally be substituted: aziridine, azetidine, pyrrolidine, 4, 5-dihydro-1H-pyrazole, pyrazolidine, imidazolidin-2-one, imidazolidin-2-thione, pyrrolidin-2-one, pyrrolidine-2, 5-dione, piperidine-2, 6-dione, piperidin-2-one, piperazine-2, 6-dione, piperazin-2-one, piperazine, morpholine, thiomorpholine-1, 1-dioxide, pyrazolidin-3-one, imidazolidine-2, 4-dione, piperidine, tetrahydrofuran, tetrahydropyran, [1, 4 ] pyrrolidine]Dioxane and 1, 2, 3, 6-tetrahydropyridine.

When W is a bond, R is preferably⁸The meaning of (a) includes tetrazole and derivatives thereof. The tetrazole moiety is attached to a cyclic P2 scaffold and is optionally substituted as shown below:

wherein Q^＊Selected from: absence of-CH₂-、-O-、-NH-、-N(R^1＊)、-S-、-S(＝O)₂-and- (C ═ O) -; q^＊Selected from: absence of-CH₂-and-NH-; y is^＊Selected from: H. c₁-C₆Alkyl radical, C₀-C₃Aryl radical, C₀-C₃A heterocyclic group; r^1＊Selected from: H. c₁-C₆Alkyl, carbocyclic radical, C₀-C₃Aryl radical, C₀-C₃A heterocyclic group.

Representative examples of substituted tetrazoles are described in table 1 and subsequent structures in WO2004/072243 or in WO 2004/113665.

Further, when W is a bond,preferably R⁸The meaning of (a) includes triazoles and derivatives thereof. The triazole moiety is attached to a cyclic P2 scaffold and is optionally substituted as shown below:

wherein X^＊And Y^＊Independently selected from: H. halogen, C₁-C₆Alkyl radical, C₀-C₃Carbocyclyl, -CH₂-amino, -CH₂Arylamino, -CH₂-diarylamino, - (C ═ O) -amino, - (C ═ O) -arylamino, - (C ═ O) -diarylamino, C₀-C₃Aryl radical, C₀ -C₃Heterocyclyl or, in addition, X^＊And Y^＊Together with the carbon atoms to which they are attached form a cyclic moiety selected from aryl and heteroaryl.

Representative examples of substituted triazoles are set forth in table 2 in WO2004/072243 and the structures that follow and in table in WO 2004/113365.

Further, when W is a bond, R is preferably⁸The meaning of (a) includes pyridazinones and derivatives thereof. The pyridazinone moiety is attached to a cyclic P2 scaffold and is optionally substituted as shown below:

wherein X^＊、Y^＊And Z^＊Independently selected from: H. n is a radical of₃Halogen, C₁-C₆Alkyl, carbocyclyl, amino, C₀-C₃Aryl, -S-aryl, -O-aryl, -NH-aryl, diarylamino, diheteroarylamino, C₀-C₃Heterocyclyl, -S-heteroaryl, -O-heteroaryl, -NH-heteroaryl, or alternatively, X and Y or Y and Z and their derivativesThe attached carbon atoms are joined to form an aryl or heteroaryl ring moiety.

Representative examples of substituted pyridazinones are described in table 3 and subsequent structures in WO2004/072243 and tables in WO 2004/113365.

The P3 group is preferred, i.e. when m is 1, analogous to a natural or unnatural amino acid, especially an aliphatic amino acid, such as L-valyl, L-leucyl, L-isoleucyl or L-tert-leucyl. Further preferred P3 groups, as shown in WO 02/01898, comprise C₀ -C₃Alkylcycloalkylalanines, in particular cyclohexylalanine, optionally coated with CO₂Rg is H, C₁-C₆Alkyl radical, C₀-C₃Alkylaryl group, C₀-C₃Alkyl heterocyclic group, C₀-C₃An alkylcycloalkyl or an amine; or N-acetylpiperidine or tetrahydrofuran. Thus, R is preferred¹¹The radicals including C₁-C₆Alkyl radical, C₀-C₃Alkyl carbocyclyl (e.g. C)₀-C₃Alkyl radical C₃-C₇Cycloalkyl), C₀-C₃Alkylaryl or C₀-C₃Alkylheteroaryl, each of which is optionally substituted by hydroxy, halogen, amino, C₁-C₆Alkoxy radical, C₁-C₆Thioalkyl, C (═ O) OR¹⁴Carboxyl group, (C)₁-C₆Alkoxy) carbonyl, aryl, heteroaryl OR heterocyclyl, wherein the substituents are in particular hydroxy OR C (═ O) OR¹⁴。

Particularly preferred R¹¹Including t-butyl, isobutyl, cyclohexyl, phenethyl, 2-dimethyl-propyl, cyclohexylmethyl, benzyl, 2-pyridylmethyl, 4-hydroxy-phenylmethyl, or carboxypropyl. Most preferred R¹¹Meaning tert-butyl, isobutyl or cyclohexyl.

Embodiments of the invention include compounds wherein P4 is absent (i.e., n is 0) and wherein the P3 functional group does not bear a carbonyl group (i.e., U is absent). Representative substructures include those of formula Ii:

wherein

Rx and Ry are as defined above, preferably H;

R¹¹' is C₁-C₆Alkyl, preferably C₃-C₅Branched alkyl groups such as the side chains of L-valyl, L-leucyl, L-isoleucyl, L-tert-leucyl; or C₀-C₂Alkyl radical C₃ -C₇Cycloalkyl groups such as cyclohexyl or cyclohexylmethyl;

R^16ais-Rba, -S (═ O)_pRba、-C(＝O)Rba；

Rba is C₁-C₆Alkyl radical, C₀-C₃Alkyl heterocyclic group, C₀-C₃An alkyl carbocyclyl group.

Alternatively, compounds with partial structure Ii may be in the appropriate R⁷The meanings of Rx, Ry or R¹¹One of which forms a macrocycle.

Representative examples of P3 groups that do not bear a carboxyl functionality (i.e., the variable U is absent) include the following formulas Iia-Iid:

wherein Ar is a carbocyclic or heterocyclic group, especially an aryl or heteroaryl group, each of which is optionally substituted with R⁹And (4) substituting. Although the partial structure of formula Iia-Iid has been elucidated within the context of the compounds of formula I, it is clear that the above structure of Ii can also be applied to compounds at other values of q and k. Similarly, although the formulas Iic and Iid are partial junctionsStructure represents R corresponding to leucine¹¹Groups, but it is clear that these structures can also be used for other R' s¹¹Among the groups, in particular those analogous to the side chains of natural or unnatural L-amino acids, for example tert-butylalanine/tert-leucine.

In the compounds of the invention in which n is 1, R¹⁵Preferably optionally substituted C₁-C₆Alkyl or C₀-C₃Alkyl carbocyclyl (e.g. C)₀-C₃Alkyl radical C₃-C₇Cycloalkyl) which may each be optionally substituted. The P4 group is preferably a generic analogue of a natural or unnatural amino acid, in particular an aliphatic amino acid, such as L-valyl, L-leucyl, L-isoleucyl, L-tert-leucyl or L-cyclohexylalanine, and R is thus preferably R¹⁵The group includes cyclohexyl, cyclohexylmethyl, t-butyl, isopropyl or isobutyl.

Preferred G meanings include-NRy- (especially where Ry is methyl or preferably H) or hydrazine.

It is further preferred that G means O, so as to define an ester with the carbonyl group of P4 (if present) or P3 (if present), or an ether in variants where the group U is absent. For R¹⁶Conventional pharmaceutically acceptable ether or ester end capping groups include C₁-C₆Alkyl (especially methyl or tert-butyl), C₀-C₃Alkylheterocyclyl (especially pyridyl, benzimidazolyl, piperidyl, morpholinyl, piperazinyl) or C₀-C₃An alkyl carbocyclyl (in particular phenyl, benzyl, 2, 3-indanyl), each of which is optionally substituted by hydroxy, halogen, amino or C₁-C₆Alkoxy groups.

It is clear that for compounds of formula I, when m ═ n ═ 0, then R¹⁶G-is not a BOC or CBz protecting group, but this limitation does not apply to other m and n substitution values. Thus, Boc or CBz protected-4-substituted proline synthesis intermediates as described in e.g. WO0059929 are not within the scope of the present invention.

Preferably, the compounds of the present invention may comprise a hydrazine functional group, for example wherein X is-NHNH-and m is 1; while n is 0 or 1. In addition, particularly where m is 0, G may be-NRjNRj-such as-NHNH-. The compounds will generally not contain hydrazine on both G and X. Hydrazines of compounds of formula I wherein m and n are 0 include compounds having the following partial structures Ija-Ijb:

in formulae Ija and Ijb, R¹⁶' can be considered to be alkyl (or C)₁-C₃Alkyl heterocyclic radical or C₁-C₃Alkyl carbocyclyl) wherein the first alkyl carbon is substituted with an oxo group to produce a ketone functional group and R¹⁶' is the remaining alkyl, alkylheterocyclyl or alkylcarbocyclyl moiety. Formula Ijb shows a variant wherein R¹⁶Is a methylene group whose carbon is substituted by an oxy group and-ORb, wherein Rb is as defined above and is generally C₁-C₆Alkyl (e.g. tert-butyl), C₀-C₃Alkyl heterocyclic radical (e.g. pyridyl) or C₀-C₃An alkyl carbocyclyl (such as benzyl or phenyl), each of which is optionally substituted, as defined above. The compounds having partial structures Ija and Ijb may be linear molecules as described above (both Rj are H), or preferably one of the Rj groups shown may be joined to the appropriate R through J⁷The group macrocyclates.

Further hydrazines of formula I wherein m is 1 include those having the following partial structures Ijc and Ijd:

wherein R is¹⁶、G、R¹¹、R¹⁵Rj and Ru are as aboveI is defined as follows. The compounds having partial structures Ijc and Ijd may be linear molecules as described above (both Rj are H), or preferably one of the Rj groups shown or R¹¹The group may be formed by J with the appropriate R⁷The group macrocyclates.

Although formulae Ija-Ijd are illustrated with proline analogues as P2, it is clear that this aspect of the invention is equally applicable to other q and k structures.

When G is amino, and m and n are both 0, and R¹⁶When an N-linked unsaturated heterocycle as defined below (e.g. pyridyl or pyrimidinyl) or an N-linked saturated heterocycle as defined below (such as piperazinyl, piperidinyl and especially morpholinyl), another hydrazine-like structure results. Examples of the above embodiments include those with formula Ije:

the compound with partial structure Ije may be a linear molecule as shown above, or preferably Rx may be coupled to the appropriate R via J⁷The group undergoes macrocyclization. Although these partial structures are illustrated using P2 five-membered rings, it is clear that such structures can be extended to other values of q and k. Similarly, these structures may be applied to others as R¹⁶The N-linked heterocycle of (1).

Returning now to formula I, in general, R is preferred for the compounds of the present invention¹⁶The group includes 2-indanol, 2, 3-indanyl, 2-hydroxy-1-phenyl-ethyl, 2-thienylmethyl, cyclohexylmethyl, 2, 3-methylenedioxybenzyl, cyclohexyl, phenyl, benzyl, 2-pyridylmethyl, cyclobutyl, isobutyl, n-propyl, methyl or 4-methoxyphenylethyl.

Currently preferred R¹⁶The gene includes 2-indanol, indane, 2-hydroxy-1-phenyl-ethyl, 2-thienylmethyl, 2, 3-methylenedioxybenzyl orA cyclohexylmethyl group.

Unnatural amino acids include L-amino acids, where the side chain is not one of the 20 naturally occurring amino acids. Examples of the unnatural amino acids include L- β -methanesulfonyl methylalanine, L-cyclohexylalanine, L-tert-leucine (L-tert-leucine), L-norleucine, L-norvaline, L-ornithine, L-sarcosine, L-citrulline, L-homophenylalanine, L-homoserine, L- β - (1-naphthyl) alanine, L- β - (2-naphthyl) alanine and the like. Unnatural amino acids also include D-amino acids corresponding to the 20 natural amino acids and D-amino acids with other side chains, such as those listed above.

"C" as used herein₁-C₆Alkyl "(also abbreviated as C)₁-C₆alk, or for compound expressions such as C₁-C₆Alkoxy, etc.) are meant to include straight or branched aliphatic carbon chains such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl, heptyl, and simple isomers thereof. In addition, C₁-C₆Any C atom in the alkyl group may be optionally substituted with one, two or three halogen atoms as allowed by the valence bond and/or the alkyl chain is interrupted by a heteroatom S, O, NH. If the heteroatom is at the end of the chain, it may suitably be substituted by one or two hydrogen atoms. C1-C4 alkyl and C₁-C₅Alkyl having C adjusted as required by carbon number₁-C₆The corresponding meaning of alkyl.

"C" as used herein₁-C₃Alkyl "includes methyl, ethyl, propyl, isopropyl, cyclopropyl, each of which may be optionally substituted or interrupted by heteroatoms as described in the preceding paragraph.

"C" as used herein₁-C₃Alkylene "describes a divalent C₁-C₃Alkanediyl moieties include propylene, ethylene and in particular methylene. For J, the generally longer alkylene chain may include 1 to 3 unsaturations and/or be substituted as described aboveThe heteroatom is interrupted.

"amino" includes NH₂、NHC₁-C₆Alkyl or N (C)₁-C₆-alkyl groups)₂In particular C₁-C₃Alkyl variants.

"amido" includes C (═ O) NH₂And alkylamido radicals, such as C (═ O) NHC₁-C₆Alkyl, C (═ O) N (C)₁-C₆Alkyl radical)₂Especially C (═ O) NHC₁-C₃Alkyl, C (═ O) N (C)₁-C₃Alkyl radical)₂or-NH (C ═ O) C₁-C₆Alkyl, for example-NHC (═ O) CHC (CH)₃)₃including-NH (C ═ O) C₁-C₃An alkyl group.

"halogen" as used herein is meant to include F, Cl, Br, I, especially chlorine and preferably fluorine.

"C" as used herein₀-C₃Alkylaryl "is meant to include aryl moieties such as phenyl, naphthyl or fused to C₃-C₇Phenyl of cycloalkyl (e.g. 2, 3-indanyl), wherein the aryl is directly bonded (i.e. C)₀) Or by C as above₁-C₃An intermediate methyl, ethyl or propyl linkage as defined by alkylene. Unless otherwise specified, the aryl group and/or its fused cycloalkyl moiety is optionally substituted with 1 to 3 substituents selected from the group consisting of: halogen, hydroxy, nitro, cyano, carboxy, C₁-C₆Alkyl radical, C_1-C₆Alkoxy radical, C₁-C₆Alkoxy radical C₁-C₆Alkyl radical, C₁-C₆Alkanoyl, amino, azido, oxo, mercapto, nitro C₀-C₃Alkyl carbocyclyl, C₀-C₃An alkyl heterocyclic group. "aryl" has the corresponding meaning, i.e. where C₀-C₃Alkyl linkages are absent.

"C" as used herein₀-C₃Alkyl radical C₃-C₇Cycloalkyl radicals"is intended to include C₃-C₇Cycloalkyl radicals, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl, in which the cycloalkyl radicals are bonded directly (i.e. C)₀Alkyl) or by C as above₁-C₃The middle methyl, ethyl, propyl (proyl) or isopropyl linkage as defined by alkylene. The cycloalkyl group may include an unsaturated bond. Unless otherwise specified, the cycloalkyl moiety is optionally substituted with 1 to 3 substituents selected from the group consisting of: halogen, hydroxy, nitro, cyano, carboxy, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Alkoxy radical C₁-C₆Alkyl radical, C₁-C₆Alkanoyl, amino, azido, oxo, mercapto, nitro C₀-C₃Alkyl carbocyclyl, C₀-C₃An alkyl heterocyclic group.

"C" as used herein₀-C₃Alkylcarbocyclyl "is meant to include C₀-C₃Alkylaryl and C₀ -C₃Alkyl radical C₃-C₇A cycloalkyl group. Unless otherwise specified, the aryl or cycloalkyl group is optionally substituted with 1 to 3 substituents selected from: halogen, hydroxy, nitro, cyano, carboxy, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Alkoxy radical C₁-C₆Alkyl radical, C₁-C₆Alkanoyl, amino, azido, oxo, mercapto, nitro, C₀-C₃Alkyl carbocyclyl and/or C₀ -C₃An alkyl heterocyclic group. "carbocyclyl" has the meaning corresponding thereto, i.e. wherein C₀-C₃Alkyl linkages are absent.

"C" as used herein₀-C₃Alkylheterocyclyl "is intended to include monocyclic, saturated or unsaturated, heteroatom-containing rings, such as piperidinyl, morpholinyl, piperazinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazinyl (thiazinoyl), isothiazolyl (isothiazoloyl), thiazolyl, oxadiazolyl, 1, 2, 3-triazolyl, 1,2, 4-triazolyl, tetrazolyl, furyl, thienyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazolyl, or any of the foregoing fused to a phenyl ring, such as quinolinyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazinyl, benzisothiazinyl, benzothiazolyl, benzoxadiazolyl, benzo-1, 2, 3-triazolyl, benzo-1, 2, 4-triazolyl, benztetrazolyl, benzofuranyl, benzothienyl, benzopyridyl, benzopyrimidinyl, pyridazinyl, benzopyrazolyl, and the like, the rings may be directly bonded (i.e., C. sub.l. C.sub.₀) Or by C as above₁-C₃An intermediate methyl, ethyl, propyl or isopropyl linkage as defined by alkylene. Any of the above unsaturated rings having aromatic character may be referred to herein as heteroaryl. Unless otherwise specified, the heterocycle and/or its fused phenyl moiety is optionally substituted with 1 to 3 substituents selected from the group consisting of: halogen, hydroxy, nitro, cyano, carboxy, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Alkoxy radical C₁-C₆Alkyl radical, C₁-C₆Alkanoyl, amino, azido, oxo, mercapto, nitro, C₀-C₃Alkyl carbocyclyl, C₀-C₃An alkyl heterocyclic group. "Heterocyclyl" and "heteroaryl" have the corresponding meanings, i.e. wherein C₀-C₃Alkyl linkages are absent.

Thus, general heterocyclyl and carbocyclyl moieties within the above definitions are monocyclic having 5 or, especially, 6 ring atoms, or bicyclic structures comprising a 6-membered ring fused to a 4-, 5-or 6-membered ring.

Typically the above groups include C₃-C₈Cycloalkyl, phenyl, benzyl, tetrahydronaphthyl, indenyl, 2, 3-indanyl, heterocyclyl, such as azepanyl, azocanyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, thiopyranyl, furanyl, tetrahydrofuranyl, thienyl, dihydrogenated benzyl, azacycloheptyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydroindolyl, pyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, thiopyranyl, furanyl, tetrahydrofuranyl, thienyl, dihydro,Pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, tetrazolyl, pyrazolyl, indolyl, benzofuranyl, benzothienyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, quinazolinyl, tetrahydroquinazolinyl, and quinoxalinyl, each of which may be optionally substituted, as defined herein.

Thus, the saturated heterocyclic moiety includes groups such as: pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyranyl, thiopyranyl, piperazinyl, indolinyl, azetidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrofuranyl, hexahydropyrimidyl, hexahydropyridazinyl, 1, 4, 5, 6-tetrahydropyrimidinyl amine, dihydro-oxazolyl, 1, 2-thiazinyl-1, 1-dioxide, 1, 2, 6-thiadiazinany1, 1-dioxide, isothiazolidinyl-1, 1-dioxide, and imidazolidinyl-2, 4-dione, however, the unsaturated heterocycle includes groups having aromatic character such as furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, oxazolyl, isothiazolyl, oxazolyl, triazolyl, Tetrazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, indolyl, isoindolyl. In each case, the heterocyclic ring may be fused to a phenyl ring, thereby forming a bicyclic ring system.

Synthesis of

The compounds of the invention can be synthesized in solution or in solid phase or a combination of both by different chemical strategies. The appropriately protected individual building blocks can be prepared first and subsequently coupled together, i.e., P2+ P1 → P2-P1. Alternatively, precursors of the building blocks may be coupled together and then altered in a later step in the sequential synthesis of the inhibitor. Additional building blocks, building block precursors or presynthesized larger fragments of the desired structure may then be formedCoupled to a chain-lengthening chain, e.g. R¹⁶-G-P3+E-P2-p1→R¹⁶-G-P3-P2-P1 or R¹⁶-G-P4-P3+E-P2-P1→R¹⁶-G-P4-P3-E-P2-P1。

The coupling between two amino acids, between an amino acid and a peptide or between two peptide fragments can be carried out using standard coupling methods, such as the azide method, the mixed carbon-carboxylic anhydride (isobutyl chloroformate) method, the carbodiimide (dicyclohexylcarbodiimide, diisopropylcarbodiimide or water-soluble carbodiimide) method, the active ester (p-nitrophenyl ester, N-hydroxysuccinimide ester) method, the Woodward reagent K-method, the carbonyldiimidazole method, the phosphorous reagent or the redox method. Some of these methods, particularly the carbodiimide method, can be improved by adding 1-hydroxybenzotriazole or 4-DMAP. These coupling reactions can be carried out in solution (liquid phase) or in solid phase.

More specifically, the coupling step comprises dehydrative coupling of a free carboxyl group of one reactant with a free amino group of another reactant in the presence of a coupling agent to form an amide linkage. Descriptions of the above coupling agents are disclosed in textbooks of general Peptide Chemistry, such as m.bodanszky, "Peptide Chemistry", second revised edition, Springer-Verlag, Berlin, Germany, (1993), hereinafter abbreviated Bodanszky, the contents of which are incorporated herein by reference. Examples of suitable coupling agents are N, N ' -dicyclohexylcarbodiimide, 1-hydroxybenzotriazole in the presence of N, N ' -dicyclohexylcarbodiimide or N-ethyl-N ' - [ (3-dimethylamino) propyl ] carbodiimide. A practical and effective coupling agent is commercially available (benzotriazol-1-yloxy) tris- (dimethylamino) phosphonium hexafluorophosphate, used either alone or in the presence of 1-hydroxybenzotriazole or 4-DMAP. Another useful and effective coupling agent is the commercially available 2- (1H-benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium tetrafluoroborate. Another useful and effective coupling agent is commercially available O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate.

The coupling reaction is carried out in an inert solvent, such as dichloromethane, acetonitrile or dimethylformamide. An excess of a tertiary amine (e.g., diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, or 4-DMAP) is added to maintain the pH of the reaction mixture at about 8. The reaction temperature is usually 0 ℃ to 50 ℃, and the reaction time is usually 15min to 24 h.

During the coupling reaction, the functional groups of the amino acid component must generally be protected to avoid the formation of undesirable bonds. Protecting groups that may be used are listed in Greene, "protective groups in Organic Chemistry", John Wiley & Sons, New York (1981) and "The Peptides: analysis, Synthesis, Biology ", Vol.3, academic Press, New York (1981), hereinafter simply Greene, the disclosure of which is incorporated herein by reference.

The alpha-carboxy group of the C-terminal residue is typically protected as an ester, which can be cleaved to form the carboxylic acid. Protecting groups that may be used include 1) alkyl esters such as methyl, trimethylsilyl and tert-butyl esters, 2) aralkyl esters such as benzyl and substituted benzyl esters, or 3) esters such as trichloroethyl and acetophenone esters, which may be decomposed by mild base or mild reduction methods.

The alpha-amino group of each amino acid to be coupled is generally protected. Any protecting group known in the art may be used. Examples of the above groups include: 1) acyl groups such as formyl, trifluoroacetyl, phthaloyl and p-toluenesulfonyl; 2) aromatic carbamate groups such as benzyloxycarbonyl (Cbz or Z) and substituted benzyloxycarbonyl and 9-fluorenylmethyloxycarbonyl (Fmoc); 3) aliphatic urethane groups such as a tert-butoxycarbonyl group (Boc), an ethoxycarbonyl group, a diisopropylmethoxycarbonyl group, and an allyloxycarbonyl group; 4) cyclic alkyl carbamate groups such as cyclopentyloxycarbonyl and adamantyloxycarbonyl; 5) alkyl groups such as trityl and benzyl; 6) trialkylsilyl groups such as trimethylsilyl; and 7) thiol-containing groups such as phenylthiocarbonyl and dithiosuccinyl. Preferably, the alpha-amino protecting group is Boc or Fmoc. Many amino acid derivatives are commercially available with appropriate protection for peptide synthesis.

The alpha-amino protecting group is cleaved prior to the next coupling step. When using a Boc group, the method of choice is trifluoroacetic acid alone or in dichloromethane, or HCl in dioxane or ethyl acetate. The resulting ammonium salt is then neutralized with an alkaline solution (such as an aqueous buffer solution, or a tertiary amine in dichloromethane or acetonitrile or dimethylformamide) either before coupling or in situ. When the Fmoc group is used, the reactant of choice is piperidine or substituted piperidine in dimethylformamide, but any secondary amine may be used. The deprotection is carried out between 0 ℃ and room temperature, and is usually 20-22 ℃.

During the preparation of the peptides using any of the above groups, any natural or unnatural amino acid having a side chain functionality is typically protected. It will be appreciated by those skilled in the art that the selection and use of appropriate protecting groups for these side chain functionalities will depend on the presence of amino acids and other protecting groups on the peptide. In selecting the above protecting groups, it is desirable that the groups are not removed during deprotection and coupling of the α -amino group.

For example, when Boc is used as the α -amino protecting group, the following side chain protecting groups are suitable: the p-toluenesulfonyl moiety can be used to protect amino acid side chains such as lysine and arginine; acetaminomethyl, benzyl (Bn) or tert-butylsulfonyl moieties can be used to protect the sulfide-containing side chain of cysteine; benzyl (Bn) ethers may be used to protect the hydroxyl-containing side chain of serine, threonine or hydroxyproline; and benzyl esters can be used to protect the carboxyl-containing side chains of aspartic acid and glutamic acid.

When Fmoc is chosen for alpha-amine protection, a protecting group based on typically a tert-butyl group is acceptable. For example, Boc may be used to protect lysine and arginine, t-butyl ether may be used to protect serine, threonine and hydroxyproline and t-butyl ester may be used to protect aspartic acid and glutamic acid. The Trityl (Trityl) moiety can be used to protect the sulfide-containing side chain of cysteine.

Once the sequential synthesis of the inhibitor is complete, any protecting groups are removed in any manner determined by the choice of protecting group. These methods are well known to those skilled in the art.

In the compounds of formula I, the P2 unit is encompassed by W and R⁸A partially substituted nitrogen-containing cyclic residue.

Synthesis of heterocyclic P2 building Block

R⁸The groups may be coupled to the P2 scaffold in any suitable step of the synthesis of compounds according to the invention. One method is to firstly carry out R⁸The group is coupled to a P2 scaffold, followed by the addition of other desired building blocks, i.e. P1 and optionally P3 and P4. Another approach is to couple P1 and P3 and P4, if present, using an unsubstituted P2 scaffold, then add R⁸A group.

Wherein W is O and R⁸Is alkyl, C₀-C₃Alkyl carbocyclyl, C₀-C₃The compounds of the invention of alkylheterocyclyl groups may be prepared according to the methods described by e.m. smith et al (j.med. chem. (1988), 31, 875-885), as shown in scheme 1, which illustrates the process for the section where q and k are 1.

Scheme 1

Commercially available Boc-4- (R) -hydroxyproline or any suitable hydroxy-substituted proline analogue (such as hydroxypiperidinic acid) is treated with a base such as sodium hydride or potassium tert-butoxide in a solvent such as dimethylformamide and the resulting alkoxide is reacted with an alkylating agent, R⁸-X, reaction wherein X is a suitable leaving group (such as halide, mesylate, triflate)Acid salts or tosylate salts, etc.) to yield the desired substituted proline derivative.

In addition, when W is O or S and R⁸In the case of carbocyclic rings (e.g., phenyl or heterocyclic (e.g., heteroaryl)), the P2 building block may also be prepared by a Mitsunobu reaction (Mitsunobu, 1981, Synthesis, 1 month, 1-28; Rano et al, Tetrahedron Lett., 1995, 36, 22, 3779-.

Scheme 2

In the presence of triphenylphosphine and an activator, such as diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD), etc., by reaction with the desired alcohol or thiol (R)⁸WH) to an appropriately hydroxy-substituted proline analog (such as hydroxypiperidinoic acid, herein commercially available Boc-4-hydroxyproline methyl ester) to yield ester compound (2 b). The ester is hydrolyzed to the acid by standard methods to form the P2 structural unit (2 c).

Alternatively, the alcohol (2a) is treated with phosgene, thereby yielding the corresponding chloroformate, which is reacted with an amine, R, in the presence of a base such as sodium bicarbonate or triethylamine⁸NH₂Reacted to form the carbamate, i.e., W is-OC (═ O) NH-, and alcohol (2a) is reacted with an acylating agent, R⁸-CO-X (such as an acid anhydride or acid halide (e.g. acid chloride)) to form an ester, i.e. W is-OC (═ O) -.

Plural alcohols R⁸-OH and alkylating agent R⁸X is described in WO00/09543 and WO 00/59929. Wherein R is⁸An example of the synthesis for substituted quinoline derivatives is shown in scheme 3.

Scheme 3

The appropriate substituted aniline (3a), either commercially available or available in the literature, is subjected to Friedel-Craft acylation with an acylating agent such as acetyl chloride or the like in the presence of boron trichloride and aluminum trichloride in a solvent such as dichloromethane to yield (3 b). Under basic conditions (such as in pyridine), in carboxylic ester group activators (e.g. POCl)₃) Coupling of (3b) to heterocyclic carboxylic acid (3c) in the presence of a base, followed by ring closure and dehydration in t-butanol under basic conditions such as potassium t-butoxide, yields quinoline derivative (3 e). In the Mitsunobu reaction, the quinoline derivative (3e) may be coupled to an alcohol as described above, or wherein the hydroxy group may be replaced by a suitable leaving group (such as a halogen, e.g. chlorine, bromine or iodine) by treating quinoline (3e) with a suitable halogenating agent (e.g. phosphoryl chloride or the like).

A variety of carboxylic acids having the general structure (3c) can be used in scheme 3. These acids are commercially available or available in the literature. An example of the preparation of 2- (substituted) -amino-carboxy-aminothiazole derivatives is shown below according to Berdikhina et al chem. heterocyclic. Compd. (Engl. Transl.) (1991), 427-433.

Scheme 4

Thioureas (4c) having different alkyl substituents R' can be formed by: the appropriate amine (4a) is reacted with tert-butyl isothiocyanate in the presence of a base such as diisopropylethylamine in a solvent such as dichloromethane, followed by removal of the tert-butyl group under acidic conditions. The thiourea derivative (4c) is then condensed with 3-bromopyruvic acid, thereby forming acid (4 d).

Wherein R is⁸The P2 building block with the substituent attached via an amine, amide, urea or sulfonamide can be prepared from an analog of aminoproline derived from a suitable commercially available aminoproline and the like derivative, or by converting the hydroxyl group of the corresponding hydroxyl derivative to the azido group, for example, by converting the hydroxyl group to a suitable leaving group such as methanesulfonate or a halogen such as chloride and then replacing the leaving group with an azide or by reducing the azide using an azide transfer agent such as Diphenylphosphorylazide (DPPA) by catalytic hydrogenation or any other suitable reduction method to form the amine⁸An alkylating agent of X, in which R is⁸And X is as described in scheme 1, thereby forming the P2 structural element useful in the preparation of compounds of formula I, wherein W is-NH-. Reacting an aminoproline analog with a compound of formula R⁸-COOH acid is reacted under standard amide coupling conditions to form a compound wherein R⁸By compounds in which the substituents are linked via an amide bond, the aminoproline analogues being coupled to the appropriate sulphonic acid derivatives R⁸-S(O)₂-X, wherein X is a leaving group (e.g. chloro), in the presence of a base, thereby forming a sulfonamide. Wherein the ring-shaped scaffold and R⁸Compounds in which the link between the substituents consists of a urea group can be obtained, for example, by the following method: the aminoproline analog is treated with phosgene to provide the corresponding chlorocarbamate, which is subsequently reacted with the desired amine. In addition, the aminoproline analogs may be reacted with carbamoyl chlorides or have the desired R⁸The isocyanates of the substituents react to form the urea linkages. Obviously, the corresponding reactions apply to P2 groups with other ring sizes and substitution characteristics.

Wherein W is-CH₂4-substituted heterocyclyl derivatives of the P2 building block of (E) -such as 4-substituted prolines can be as shown in scheme 5 illustrating the process for the part where q and k are 1, according to J.Ezquerra et al, tetrahedreron, 1993, 38, 8665-.

Scheme 5

Treatment of an appropriately acid-protected pyrrolidone or piperidone (such as the commercially available Boc-pyroglutamic acid (5a)) with a strong base such as lithium diisopropylamide in a solvent such as tetrahydrofuran, followed by addition of an alkylating agent R⁸-CH₂-X, wherein X is a suitable leaving group (such as halogen, e.g. chloro or bromo), followed by reduction of the amide and deprotection of the resulting ester to give the desired compound (5 d).

Wherein the heterocyclic ring R⁸Compounds of the invention, in which the group is directly attached to a cyclic P2 scaffold, i.e. W is a bond in formula I, may be prepared by, for example, a displacement reaction in which a suitable leaving group on the P2 scaffold is replaced by a desired R8 group, such as a heterocyclic group.

In addition, the R is⁸Groups can be introduced by way of a Mitsunobu reaction, in which the hydroxyl group in the P2 scaffold is reacted with a heterocycle R⁸The nitrogen atoms in the group react.

Compounds in which the tetrazole derivative is attached via a heterocyclic carbon atom may suitably be prepared by constructing the tetrazole moiety directly onto the P2 precursor. This can be achieved, for example, by converting the hydroxyl group of the P2 precursor to a cyano group, which is then reacted with an azide reagent, such as sodium azide. Triazole derivatives can also be built directly on the P2 precursor, for example by converting the hydroxyl group of the P2 precursor to the azido group, followed by a 3+2 cycloaddition reaction of the provided azide with the appropriate alkyne derivative.

Structurally different tetrazoles for the above substitution reaction and Mitsunobu reaction can be prepared by reacting commercially available nitrile compounds with sodium azide. Triazole derivatives can be prepared by reacting an alkyne compound with trimethylsilylazide. The alkyne compounds used are either commercially available or they can be prepared, for example, according to the Sonogashira reaction, i.e.in PdCl₂(PPh)₃And CuI, as described in A.Elanggovan, Y. -H.Wang, T. -I.Ho, org.Lett., 2003, 5, 1841-propan 1844. The heterocyclic substituent may also be altered when attached to the P2 building block, either before or after the P2 building block is coupled to other building blocks.

Preparation of a compound in which W is a bond and R⁸These and other methods for compounds that are optionally substituted heterocycles are broadly described in WO 2004/072243.

W-R with proline derivatives in schemes 1, 2 and 5⁸Compounds of additional ring sizes of substituents and/or their position elsewhere may also be used in the preparation of compounds according to the invention. For example, alkylation of commercially available 3-hydroxyproline forms a compound of formula (I) wherein k is 0 and q is 2. Accordingly, alkylation of 5-hydroxyproline, for example as prepared as described by Hallberg et al, J.Med.Chem. (1999), 4524-4537, provides compounds of formula (I) wherein k is 2 and q is 0.

Various methods for preparing hydroxylated 2-piperidinecarboxylic acids are described in the literature, for example Celestini et al, org.Lett., (2002), 1367-: asymmetry, (1996), 2585-. For example, the corresponding pyridine carboxylic acid may be reduced to form a hydroxylated 2-piperidine carboxylic acid. For the preparation of hydroxylated proline analogues, enzymatic methods may also be used. For example, 3-hydroxy substituents can be introduced onto commercially available 4-, 5-and 6-membered heterocyclic acids by using proline 3-hydroxylase, as described by Ozaki et al, Tet.letters, 40, (1999), 5227-.

Synthesis and introduction of P1 building Block

The amino acids used to prepare the P1 fragment are either commercially available or are available in the literature, see for example WO00/09543 and WO00/59929 by Boehringer-Ingelheim or US 2004/0048802 by BMS.

Scheme 6 shows an example of the preparation of sulfonamide derivatives that serve as P1 fragments and are subsequently coupled to Boc protected P2 building blocks.

Scheme 6

By treating the amino acid with a coupling reagent (e.g., N' -Carbonyldiimidazole (CDI), etc.) in a solvent such as THF, followed by a strong base (e.g., 1, 8-diazabicyclo [5.4.0 ]]Undec-7-ene (DBU)) with the desired sulfonamide (6b), the sulfonamide group can be introduced onto the appropriately protected amino acid (6 a). Alternatively, the amino acid is treated with the desired sulfonamide (6b) in the presence of a base such as diisopropylethylamine followed by treatment with a base such as PyBOPThe coupling reagent of (a) is treated to effect the introduction of the sulfonamide group. The amino protecting group is removed by standard methods and subsequently coupled to the reagent O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) prepared as above using standard methods of amide bond formation in a solvent such as dimethylformamide in the presence of a base such as diisopropylamineThe P2 building block was prepared, thus giving Boc protected P2-P1 compound (6 e). Alternatively, the sulfonamide group may be introduced at a later step of the synthesis, for example as the last step. In this case, an amino acid having a reverse protected form, i.e., an amino acid having an unprotected amino function and a protected acid function, is coupled to the acid function of the P2 building block using, for example, standard peptide coupling conditions as described above. Removal of the acid protecting group by using appropriate conditions appropriate for the protecting group applied, followed by coupling of the sulfonamide as described above, affords compound 6 e.

The P1 building block for the preparation of compounds according to formula I, wherein A is an ester or amide, can be prepared by reacting the amino acid (6a) with the appropriate amine or alcohol, respectively, under standard conditions for amide or ester formation. Wherein A is CR⁴R⁴' Compounds according to formula I may be prepared by coupling an appropriate P1 building block to a P2 building block as described in Oscarsson et al Bioorg Med Chem 200311 (13)2955-2963 and in PCT/EP03/10595, 09/23 of 2003, the contents of which are incorporated herein by reference.

Compounds comprising the residue of the azapeptide P1, i.e. of formula I wherein Q is NRu, can be prepared by using the appropriate P1 aza-aminoacyl moiety in the coupling to the P2 fragment. The preparation of aza-aminoacyl moieties is described by m.d. bailey et al in j.med.chem., 47, (2004), 3788-.

Scheme 6A

The appropriate N-linked side chain Ru can be introduced onto commercially available tert-butylhydrazine, for example by reductive amination with the appropriate aldehyde or ketone as described in scheme 19 below, thereby producing an N-alkylated hydrazinoformate (6 Aa). In the presence of a base such as triethylamine or diisopropylethylamineIn the presence of a solvent such as THF, 6Aa is condensed with the desired chloroformate to produce 6 Ab. Then, the utilization depends on the specific R¹' suitable conditions, e.g. R¹' when it is benzyl, catalytic hydrogenation conditions are employed, optionally with R¹' partial removal, thereby giving the corresponding acid. The resulting acid as described above is then reacted with the desired sulfonamide derivative as described in scheme 6 to give a sulfonamide-capped building block. In addition, the hydrazinoformate 6Aa is reacted with an isocyanate R³-N ═ C ═ O reaction, forming the structural units used to prepare the compounds according to general formula I, where M is NRu and a is CONHR³。

The P2 and P3 moieties may be joined together before or after introduction of the P1 building block.

Synthesis of end-capped P3 and P3-P4 building blocks

Structural unit R¹⁶-G-P3 and R¹⁶the-G-P4-P3 can be prepared by the general method shown in scheme 7.

Scheme 7

The appropriate N-protected amino acid (7a) may be reacted with an amino-capping group (R) in the presence of a base such as DIEA or DMAP, in a solvent such as dichloromethane, chloroform or dimethylformamide or mixtures thereof, by applying standard peptide coupling conditions (e.g.using coupling reagents such as HATU, DCC or HOBt) and ester formation conditions, etc¹⁶-NHRy) to form an amide, i.e. G is NHRy (7 b). Further, the amino acid (7a) is reacted with the general formula R in the presence of a base such as cesium carbonate or silver oxide (I)¹⁶-X compound reaction, wherein R¹⁶As defined above and X is a leaving group such as halogen, forming an ester, i.e. G is O (7 b). On the other hand, amino acid (7a) can be coupled to the appropriate O-Protected secondary amino acid (7d), to form (7 e). Substitution of the ester group with a suitable end-capping group (7b) forms a moiety (7f) useful in the preparation of compounds according to the invention, where m and n are both 1.

When G is N-Ry, the capped P3 or P2 building blocks may also be prepared on a solid support as illustrated in scheme 8.

Scheme 8

An appropriately N-protected (e.g. Boc-protected) amino acid (8a) may be immobilised on a solid support by reacting the amino acid with the desired solid support in a solvent such as dichloromethane and dimethylformamide in the presence of a coupling reagent such as N, N' -diisopropylcarbodiimide and a base such as DMAP, the latter being exemplified herein by the Agronaut resin PS-TFP. The above-described immobilised amino acids can then be isolated from the support by using a suitable end-capping group (8a), thereby giving a fragment which can be used to prepare compounds according to the invention, where m or n is 1. Optionally, the amino protecting group may be removed and subsequently coupled to the appropriate amino acid using standard methods, thereby forming a fragment useful in the preparation of compounds according to the invention, wherein m and n are 1.

Coupling of end capping groups or end capping structural units to P2-P1 structures

R linked to the P2-P1 structure via a urea function¹⁶-G、R¹⁶-G-P3 or R¹⁶the-G-P4-P3 building block can be introduced as shown in scheme 9, which illustrates the process with a variant in which the P2 scaffold is a 5-membered ring.

Rx 'and R11' have the same definitions as Rx and R11, respectively, but are not macrocyclic rings.

A 'is a protected carboxylic acid, substituted amide or sulfonamide or CR4R 4'.

Scheme 9

The chlorourethane groups can be formed on the P2-P1 structural cyclic amine (9a) by removal of the amine protecting group using standard methods such as acidic treatment with, for example, TFA in dichloromethane or the like when Boc groups are used, followed by reaction of the free amine with phosgene in toluene in the presence of a base such as sodium bicarbonate or triethylamine in a solvent such as tetrahydrofuran. Subsequently, the electrophilic center formed as described above is reacted with R in a solvent such as dichloromethane in the presence of a base such as sodium bicarbonate¹⁶-NH₂、R¹⁶-NH-NH₂、R¹⁶-G-P3 or R¹⁶The amino group of the structural unit (9c) of G-P4-P3 reacts to form (9 d). Wherein E is C ═ S, S (═ O) or S (═ O)₂The compounds of the general formula (I) can be prepared according to the above-described processes, but reagents such as thiocarbonyldiimidazole, thionyl chloride or sulfuryl chloride are used instead of phosgene, respectively.

Compounds containing a hydrazine group attached to the P2 unit, i.e. where X is-NRjNRj-in formula I, or when P3 and P4 units are absent and G is nrjnrjnjj, can be prepared as shown below. Scheme 10 demonstrates the introduction of hydrazine derivatives into the 5-membered P2 building block.

Scheme 10

Tert-butyl carbazate (10a) (optionally substituted by alkyl on one or both nitrogens) is reacted with P-nitrophenyl chloroformate in the presence of a base such as sodium bicarbonate followed by addition with P2 building block (10b) to form urea derivative 10 c. Alternatively, the phosgene process described in scheme 9 can also be used to effect ligation of fragments 10a and 10 b. The boc group is optionally removed by standard procedures, such as acidic treatment with TFA in a suitable solvent, such as dichloromethane, to form the hydrazine-containing derivative (10 d). In addition, any suitable hydrazine derivative other than t-butyl carbazate derivatives, such as morpholin-1-ylamine or piperidin-1-ylamine, and the like, may be attached to the 9 Ab.

Thus, by coupling the P3 or P4-P3 building blocks to the primary amine compound 9Ad, the resulting compound can be further expanded, for example, as shown in scheme 11.

R11' has the same definition as R11, but is not a macrocyclic moiety.

A 'is a protected carboxylic acid, substituted amide or sulfonamide or CR4R 4'.

Scheme 11

Alpha-amino compound (11a) (Yang et al J. org. chem. (2001), 66, 7303-7312) was treated with sodium nitrite, potassium bromide and sulfuric acid to form the corresponding alpha-bromo compound (11b), which was reacted with the above-mentioned derivative (10d) to form the hydrazine-containing derivative (11 c).

The link between the P2 and P3 building blocks can also be made up of carbamate groups, and the general route to the synthesis of this compound is shown in scheme 12, which illustrates the process with a variant in which P2 is a proline derivative.

Scheme 12

The desired, optionally protected, amino-capping group (12a) is coupled to the hydroxy acid (10b) using standard peptide coupling procedures, followed by reaction and optional deprotection with the electrophilic P2 building block (12d) as described above, to form structure (12 e).

Compounds in which no carboxyl group is present in the P3 unit can be prepared as illustrated in scheme 13, which illustrates a procedure applied to compounds of formula I.

R11' has the same definition as R11, but is not a macrocyclic moiety.

A 'is a protected carboxylic acid, substituted amide or sulfonamide or CR4R 4'.

Scheme 13

The chlorocarbamoyl derivative (13a) can undergo substitution reaction with an azide derivative (13b) prepared by a known method in the literature in the presence of a base such as sodium hydrogencarbonate to give (13 c). X is shown as the general formula (I). The azide functionality is reduced, for example by using a polymer bound triphenylphosphine in a solvent such as methanol or by any other suitable reduction method, to form intermediate (13d), which can then be reacted with an acid under peptide coupling conditions or an amine in a reductive amination reaction to form an amide and a secondary amine, respectively.

Scheme 14 represents another route to compounds in which no carboxyl group is present in the P3 structural unit.

R11' has the same definition as R11, but is not a macrocyclic moiety.

A 'is a protected carboxylic acid, substituted amide or sulfonamide or CR4R 4'.

Scheme 14

Instead of using the azide derivative (13b) in scheme 13, the corresponding optionally protected hydroxy derivative (14b) is used to perform a substitution reaction with the chlorocarbamate (14a), thereby introducing a primary alcohol. The alcohol (14c) is then oxidized, after optional deprotection, with a suitable oxidizing agent, such as Dess-Martin periodinane (periodinate), to form the corresponding aldehyde. The aldehyde is reductively aminated with the desired amine in a solvent such as THF by using a reagent such as, for example, polystyrene bound to cyanoborohydride, to form the amine derivative (14 e).

In addition, under appropriate conditions, the alcohol (14c) may react with a suitable acylating or alkylating agent to form an ester and ether compound, respectively, i.e., a compound of formula (I) wherein G is O.

Subsequently, by reacting the formed alcohol with a suitable acylating or alkylating agent using appropriate conditions, ester and ether compounds, respectively, i.e. compounds of formula (I) wherein G is O, are formed.

Additionally, the P2 and P3 building blocks may be linked via a guanidine group, and a general route to the synthesis of the compounds is shown in scheme 15.

R11' has the same definition as R11, but is not a macrocyclic moiety.

A 'is a protected carboxylic acid, substituted amide or sulfonamide or CR4R 4'.

Scheme 15

In solvents such as dimethylformamideThiocarbonyldiimidazole, etc. to the P2-structural unit (15a), followed by condensation with sodium cyanamide in a solvent such as ethanol, to give the thiolate intermediate (15 b). Intermediate (15b) is reacted with the desired structural unit, represented herein as capped P3 structural unit (12c), to form cyanoguanidine derivative (15 d). In addition, other structural units R¹⁶-G or R¹⁶-G-P4-P3 may also be coupled to intermediate (15 b). Hydrolysis of the cyano group in (15d) by treatment with dilute hydrochloric acid gives the guanylurea derivative (15 e).

When R is⁷、R⁷'and A' contain functional groups, which groups are suitably protected by methods recognized by those skilled in the art, see, e.g., Bodanzky or Greene, cited above.

Formation of macrocyclic Compounds

Wherein the alkylene chain is taken from R⁷/R⁷` Cycloalkyl extension to Rx or R¹¹The compounds according to the invention, which thus form a macrocycle, can be prepared as described below. By using the strategy described above, followed by a ring closure reaction (macrocyclization reaction), the appropriate P1, P2 and P3 building blocks or precursors thereof can be coupled together. Substituents W-R of P2 structural unit before or after macrocycle formation⁸May be incorporated via the Mitsunobu reaction as described above, and the desired building blocks may be coupled together by using appropriately substituted P2-building blocks. For the slave R⁷/R⁷' cycloalkyl extends to R¹¹The macrocyclic structure of (a), P3 amino acid containing an appropriate side chain may be prepared as described in WO 00/59929.

A general route for the synthesis of macrocyclic compounds is shown in scheme 16, which illustrates a process applied to a compound having a 5-membered P2 scaffold and a spiro-cyclopropyl group in the P1 moiety, wherein the macrocycle protrudes from the P3 side chain.

Scheme 16

Proline derivative (16a) is coupled with the appropriate acid-protected amino acid (16b) using, for example, phosgene process conditions as described above to form (16 c). Macrocycle formation is then carried out via olefin metathesis using a Ru-based catalyst such as that reported in Miller, s.j., Blackwell, h.e.; grubbs, r.h.j.am.chem.soc.118, (1996), 9606-; kingsbury, j.s., Harrity, j.p.a.bonitatebus, p.j., Hoveyda, a.h., j.am.chem.soc.121, (1999), 791-. It should also be recognized that catalysts containing other transition metals (such as Mo) may also be used for this reaction. The double bond is optionally reduced and/or the ethyl ester is hydrolyzed by standard hydrogenation methods and/or standard hydrolysis methods, respectively, well known in the art. Alternatively, the methyl ester may be selectively hydrolyzed and then coupled to R by standard peptide coupling conditions¹⁶-G-P4 building block coupling. The macrocyclization step described in scheme 16 can also be applied to the corresponding carbocyclic analogs as described above. When the linker contains a nitrogen atom, ring closure may be effected by reductive amination as described in WO 00/59929.

Macrocyclic compounds in which the cyclopropyl moiety is absent from the P1 moiety, i.e. the macrocycle is directly linked to R by a peptidic backbone⁷Compounds with adjacent carbon atoms extended can be prepared using the methods described herein. An example in which a proline derivative is used as a cyclic P2 scaffold is shown in scheme 17.

Scheme 17

The amide derivative (17c) is obtained by coupling the appropriate allylglycine derivative (17a) to the acid functionality of P2 building block (17b) using standard peptide coupling conditions. The Boc protecting group is removed by acidic treatment, followed by treatment with phosgene in the presence of sodium bicarbonate to form a chlorocarbamate, which is subsequently reacted with the alkene-substituted amino acid (17d) to form the urea compound (17 e). By using, for example, a Hoveyda-Grubbs catalyst, a ring closure metathesis reaction is effected, whereby the macrocyclic compound (17f) is obtained.

Although scheme 17 shows the use of where R⁸Synthesis sequence of P2 building blocks with substituents attached to P2 scaffold, however, it is clear that unsubstituted P2 scaffold can also be used, and R⁸Groups may be introduced in any suitable step of the synthesis using any of the methods described herein.

Building blocks for the preparation of compounds in which the macrocycle extends from the nitrogen atom in the linkage between the P2 and P3 moieties (i.e. X is NRx in formula I) or for the preparation of compounds in which the P3 and P4 moieties are absent (i.e. m and n are 0 and G is NRj in formula I) may generally be prepared as outlined in scheme 18B.

Scheme 18

Carbamate 18a, which is commercially available or readily prepared, for example, by reacting the desired alkylamine with di-tert-butyl dicarbonate, can be reacted with the appropriate omega unsaturated alcohol under Mitsunobu conditions to form the alkylated carbamate (18 b). 18b is subjected to acidic conditions, for example treatment with trifluoroacetic acid in a solvent such as dichloromethane, to give the free amine (18c), which can be attached to the P2 fragment using any of the previously described strategies.

Macrocyclic structures containing hydrazine groups, i.e. where X is NRjNRj or m and N are 0 and G is NRjNRj in formula I, can be prepared by attaching an appropriate N-alkylated carbazate derivative to the P2 fragment. Alkylated carbazate derivatives may be prepared, for example, as described in scheme 19.

Scheme 19

The aldehyde (19b) is formed by oxidation of the appropriate alcohol (19a) by a suitable oxidation process, for example using N-methylmorpholine oxide and tetrapropylammonium ferulate in a solvent such as dichloromethane. Reductive alkylation of t-butyl carbazate with the resulting aldehyde provides the desired N-alkylated structural unit (19 c). In addition, any desired hydrazine derivative other than t-butyl carbazate may be used in the reaction with the aldehyde 19b, such as morpholin-1-ylamine or piperidin-1-ylamine, and the like.

Scheme 20 illustrates a synthetic sequence of building blocks suitable for preparing compounds in which the "external" nitrogen atom of the hydrazine group is alkylated with an omega-unsaturated alkyl chain suitable for subsequent macrocyclization, or any other suitable alkyl.

Scheme 20

An appropriately protected hydrazine derivative, such as tert-butyl (1, 3-dioxo-1, 3-dihydro-isoindol-2-yl) -carbamate (20a), which can be readily prepared by one skilled in the art, is reacted with the desired alcohol R-OH under Mitsunobu conditions to form the N-alkylated hydrazine compound (20 b). Removal of the phthalimido group may be achieved by treatment with hydrazine or a derivative thereof, such as hydrazine hydrate or hydrazine acetate, to form the carbazate ester (20 c). The resulting primary amine can then be attached to the desired P2 fragment using any of the methods previously described to give the urea derivative (20d), or it can be further alkylated using, for example, the reductive amination method described in scheme 19, followed by coupling to the previously described P2 fragment to give 20 e.

Scheme 21 illustrates the coupling of a hydrazine containing P3 building block to a cyclopentane scaffold, followed by macrocyclization.

Scheme 21

The carbazate derivative (21b) was coupled to the P2-P1 building block (21a) using standard peptide coupling conditions to form intermediate (21 c). (21c) is ring-closed by olefin metathesis as depicted in scheme 18, to give the macrocyclic compound (21 d).

The term "N-protecting group" or "N-protected" as used herein refers to those groups intended to protect the N-terminus of an amino acid or peptide or to protect the amino group from undesirable reactions during the synthetic process. Commonly used N-protecting Groups are disclosed in Greene, "Protective Groups in Organic Synthesis" (John Wiley & Sons, New York, 1981), which is incorporated herein by reference. The N-protecting group includes acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthaloyl, o-nitrophenoxyacetyl, α -chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl and 4-nitrobenzoyl and the like; sulfonyl groups such as benzenesulfonyl and p-toluenesulfonyl, and the like; carbamate-form groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3, 4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4, 5-dimethoxybenzyloxycarbonyl, 3, 4, 5-trimethoxybenzyloxycarbonyl, 1- (p-biphenyl) -1-methylethoxycarbonyl, alpha-dimethyl-3, 5-dimethoxybenzyloxycarbonyl, diphenylmethoxycarbonyl, tert-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2, 2, 2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; alkyl groups such as benzyl, trityl, benzyloxymethyl, and the like; and silyl groups such as trimethylsilyl and the like. Preferred N-protecting groups include formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl, t-Butoxycarbonyl (BOC) and benzyloxycarbonyl (Cbz).

As used herein, "hydroxyl-protecting group" refers to a substituent that protects a hydroxyl group from undesirable reactions during the synthetic process, such as those O-protecting Groups disclosed in Greene, "Protective Groups Inorganic Synthesis" (John Wiley & Sons, New York (1981)). Hydroxy protecting groups include substituted methyl ethers, for example, methoxymethyl, benzyloxymethyl, 2-methoxyethoxymethyl, 2- (trimethylsilyl) ethoxymethyl, tert-butyl and other lower alkyl ethers, lower alkyl such as isopropyl, ethyl and, in particular, methyl, benzyl and trityl; tetrahydropyranyl ether; substituted ethers, such as 2, 2, 2-trichloroethyl ether; silyl ethers such as trimethylsilyl ether, t-butyldimethylsilyl ether and t-butyldiphenylsilyl ether; and esters prepared by reacting a hydroxyl group with a carboxylic acid, such as acetates, propionates, and benzoates, among others.

In treating conditions caused by flaviviruses, such as HCV, the compounds of formula I are typically administered in amounts to achieve plasma concentrations of about 100-5000 nM, such as 300-2000 nM. This corresponds to a dosage rate of 0.01-10 mg/kg/day, preferably 0.1-2 mg/kg/day, of administration, depending on the bioavailability of the formulation. For normal adults, the dosage rate is typically about 0.05 to 5g, preferably 0.1 to 2g, such as 500 to 750mg, per day in one to four dosage units per day. As with all medicaments, the dose rate will vary with the weight and metabolic condition of the patient and the severity of the infection, and will need to be adjusted according to the accompanying medication.

Based on good prescription practices for antiviral therapy, the compounds of formula I are generally co-administered with other HCV therapeutic agents to avoid the generation of drug escape mutants. Examples of such additional HCV antiviral therapeutic agents include ribavirin, interferons (including pegylated interferons). In addition, a number of nucleoside analogs and protease inhibitors are in clinical or preclinical research settings and are suitable for co-administration with the compounds of the present invention.

Accordingly, another aspect of the present invention provides a composition comprising a compound of formula I and at least one other HCV antiviral agent in a universal dosage unit, such as any of the dosage forms described below, but in particular a tablet or capsule for oral administration or a liquid suspension or a solution for oral or injectable use. Another aspect of the invention provides a method of treating or preventing flavivirus (such as HCV) infection comprising sequentially or simultaneously administering a compound of formula I and at least one other HCV antiviral agent. A related aspect of the invention provides a patient pack comprising: a first pharmaceutical composition, preferably in unit dosage form, of a compound of formula I, and a second pharmaceutical composition of a second HCV antiviral agent, typically in separate containers in unit dosage form and usually in a patient pack. The patient pack is also suitably provided with instructions printed on the package or on a container therein or on a package insert for simultaneous or sequential administration of the respective pharmaceutical compositions.

Many HCV patients are co-infected or prone to superinfection with other infectious diseases. Accordingly, another aspect of the invention provides combination therapies comprising co-formulating the compound of the invention with at least one other anti-infective agent in the same dosage unit or co-packaged. The compounds of the present invention are administered simultaneously or sequentially with at least one other anti-infective agent, generally at dosages equivalent to the dosages of the agents involved in the monotherapy. However, certain anti-infective agents may induce a synergistic effect, which results in the administration of a lower dose of one or both active ingredients than the corresponding monotherapy. For example, in drugs that tend to be rapidly metabolized by Cyp3a4, co-dosing with the HIV protease inhibitor ritonavir may be administered in lower doses.

Common co-infections or superinfections with HCV include hepatitis b virus or HIV. Accordingly, the compounds of the present invention are advantageously co-administered (either in the same dosage unit, or in co-packaged or separately prescribed dosage units) with at least one HIV antiviral agent and/or at least one HBV antiviral agent.

Representative HIV antivirals include NRTI such as alovudine (FLT), zidovudine (AZT, ZDV), stavudine (D4T, Zerit), zalcitabine (ddC), didanosine (ddI, Videx), abacavir (ABC, Zigen), lamivudine (3TC, Epivir), emtricitabine (FTC, Emtriva), racevir (racemic FTC), Adefovir (ADV), entacavir (BMS 200475), alovudine (FLT), tenofovir disoproxil (TNF, Viread), amdoxavir (DAPD), D-D4FC (DPC-817),. dOTC (ShiSPD 754), elvucitabine (AchilliaceAchillion ACH-126443), BCH 36 (RV) 756, Racivir, GS, INK 7340, thioethers (3-20-D-3 ', 3' -D, and their prodrugs such as FLT-3-D-3-D, D-20 ', 3' -D-539, and its prodrug, pharmasset DPC-817).

Representative NNRTIs include delavirdine (Rescriptor), efavirenz (DMP-266, Sutiva), nevirapine (BIRG-587, Viramune), (+) Cikara Nalimide A and B (advanced Life sciences), caspovirin (AG1549f S-1153; Pfizer), GW-695634 (GW-8248; GSK), MIV-150(Medivir), MV026048 (R-1495; Medivir AB/Roche), NV-0522(IdenixPharm.), R-278474(Johnson & nsson), RS-1588(IdenixPharm.), TMC-120/125(Johnson & Johnson), TMC-125 (R-165335; Johnson & Johnson), Johnson-389, (Bio yshi 215215215).

Representative HIV protease inhibitors include PA-457(Panacos), KPC-2(Kucera Pharm.), 5HGTV-43(Enzo Biochem), amprenavir (VX-478, Agenerase), atazanavir (Reyataz), indinavir sulfate (MK-639, Crixivan), Lexiva (calcium fosamprenavir, GW-433908 or 908, VX-175), ritonavir (Norvir), lopinavir + ritonavir (ABT-378, Kaletra), tipranavir, nelfinavir mesylate (Viracept), saquinavir (Invirase, Fortovase), AG1776(JE-2147, KNI-764; Nippon MiningHoldings), AG-Pf9 (Pfizer), DPC-681/684(BMS, GS, Sciencesin 338), Gilen I-272 (KNI-194272), Nappan-P-100P (Biophyr P-100P), Naringavir-378, Lexiva (Naxiva, Lexan-433908, VX-175, Lexan-7, and Nazeri (Napylen-P-100, Naringson, Napylen, Nalcine, Naphi-Na, P-1946(Procyon Biopharma), R-944(Hoffmann-LaRoche), RO-0334649(Hoffmann-LaRoche), TMC-114(Johnson & Johnson), VX-385(GW 640385; GSK/Vertex), VX-478 (Vertex/GSK).

Other HIV antivirals include entry inhibitors including fusion inhibitors, CD4 receptor inhibitors, CCR5 co-receptor inhibitors, and CXCR⁴A co-receptor inhibitor, or a pharmaceutically acceptable salt or prodrug thereof. Examples of entry inhibitors are AMD-070(AMD 11070; AnorMed), BlockAide/CR (ADVENTRXRX Pharm.), BMS 806 (BMS-378806; BMS), Enfurvirtide (T-20, R698, Fuzeon), KRH1636(Kureha Pharmaceuticals), ONO-4128(GW-873140, AK-602, E-913; ONO Pharmaceuticals), Pro-140 (Progenics Pharm), PRO542(Progenics Pharm.), SCH-D (SCH-417690; Schering-Pluough), T-1249 (R724; Roche/Trimeris), TAK-220(Takeda Chem.Ind.), TNX-355(Tanox) and UK-427,857 (Uizer). An example of an integrase inhibitor is L-870810 (Merck)&Co.)、c-2507(Merck&Co.) and S (RSC) -1838 (shionogi/GSK).

Examples of HBV antiviral agents include adefovir dipivoxil (hepsera), and in particular lamivudine and 2 ', 3' -dideoxy-3 '-Fluoroguanosine (FLG) and prodrugs thereof, such as MIV-210, the 5' -O-valinyl-L-lactyl prodrug of FLG. These latter HBV antivirals are particularly suitable because they also have anti-HIV activity.

Although the active agent may be administered alone, it is preferably present as an integral part of a pharmaceutical formulation. Such formulations will comprise an active agent as defined above together with one or more acceptable carriers or excipients and optionally other therapeutic ingredients. The carrier must be acceptable in the sense that it is compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The formulations include those suitable for rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, but preferably the formulations are oral administration formulations. The formulations may conveniently be presented in unit dosage form, for example as tablets and sustained release capsules, and may be prepared by any of the methods well known in the art of pharmacy.

The above method comprises the step of bringing into association the active agent as defined above with a carrier. Generally, the formulation is prepared by the following method: the active agent is homogenized and intimately associated with liquid carriers or finely divided solid carriers or both, and the product is then shaped if necessary. The present invention provides a process for preparing a pharmaceutical composition comprising combining or associating a compound of formula I, or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable carrier or excipient. If the production of a pharmaceutical formulation involves homogeneous mixing of pharmaceutical excipients and the active ingredient in salt form, it is generally preferred to use excipients which are not basic in nature, i.e. acidic or neutral excipients. The formulations of the present invention for oral administration may be presented as discrete units, such as capsules, sachets or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution or suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion, as well as a pill, and the like.

For compositions for oral administration (e.g., tablets and capsules), the term "suitable carrier" includes excipients such as common excipients, for example binding agents, e.g., syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, such as corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metal stearates, stearic acid, glyceryl stearate, silicone oil, talc, oils and silica colloids. Flavoring agents such as peppermint, methyl salicylate, or cherry flavoring, and the like, may also be used. Colorants may desirably be added to make the dosage form easily identifiable. Tablets may also be coated by methods well known in the art.

Tablets may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Molded tablets may be made by molding in suitable equipment a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide sustained or controlled release of the active agent.

Other formulations suitable for oral administration include lozenges comprising the active agent in a flavoured base (usually sucrose and acacia or tragacanth); pastilles comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes containing the active agent in a suitable liquid carrier.

The compounds of formula I may form salts, which form a further aspect of the invention. Suitable pharmaceutically acceptable salts of the compounds of formula I include salts of organic acids, particularly carboxylates, including but not limited to acetates, trifluoroacetates, lactates, gluconates, citrates, tartrates, maleates, malates, pantothenate, isethionates, adipates, alginates, aspartates, benzoates, butyrates, digluconates, cyclopentonates, glucoheptonates, glycerophosphates, oxalates, heptanoates, hexanoates, fumarates, nicotinates, palmitates, pectinates, 3-phenylpropionates, picrates, pivalates, propionates, tartrates, lactobonates, pilates, camphorates, undecanoates and succinates, organic sulfonates such as methanesulfonates, ethanesulfonates, 2-isethionates, camphorsulfonates, 2-naphthalenesulfonates, camphorsulfonates, and salts of succinic acid, Benzene sulfonate, p-chlorobenzene sulfonate, and p-toluene sulfonate; and inorganic acid salts such as hydrochloride, hydrobromide, sulfate, bisulfate, hemisulfate, thiocyanate, persulfate, phosphoric acid, and sulfonate. In addition, the present invention provides salts of the compounds of formula I, which may or may not be pharmaceutically acceptable salts, but which may be useful as synthetic intermediates, with the salt moiety being replaced or substituted as desired.

The present invention includes prodrugs of the compounds of formula I. Prodrugs of compounds of formula I are those which release the compounds of formula I in vivo upon administration to a patient, typically upon hydrolysis in the digestive tract, liver or plasma. Prodrugs are typically pharmaceutically acceptable ethers and especially esters (including phosphates), amides or carbamates, or esters, of hydroxy functional groups, or of carboxy functional groups. Preferred pharmaceutically acceptable esters include alkyl esters (including acetate, butyrate, t-butyrate, octadecyl ester and pivalate), phosphate esters and sulfonates (i.e., those derived from RSO)₂Esters of OH, wherein R is lower alkyl or aryl). Pharmaceutically acceptable esters include lower alkyl ethers as well as the ethers disclosed in WO 00/47561, in particular methoxyaminoacyl and ethoxyaminoacyl.

The compounds of the present invention have multiple stereocenters, and the present invention provides racemates and enantiomers at each of these stereocenters.

In general, the side chains corresponding to P3 and P4 (i.e., R)¹⁵And/or R¹¹) The stereochemistry of the groups of (a) will correspond to the L-amino acid configuration, although the invention also provides D-isomers at one or both of these centers. It should be noted that although the nature of the E moiety is that P3 and P4 are generally transferred with one atom and indeed flip the peptide residue relative to conventional polypeptides, the L configuration remains active, assuming that P3 and P4 subsequently tilt the amine acid side chain to the other side as compared to conventional peptide substrates.

The stereochemistry of the backbone components of the cyclic P2 group (i.e., the carbonyl group bridging the P1 amide bond and the carbonyl group or E extending from P3) will generally correspond to L-proline. The stereochemistry of the P2 ring atom to which W is bonded is generally as follows:

in which R is⁷And R⁷' together as a spirocycloalkyl group in the compounds of the invention, the aforementioned spiro-cycloalkyl group will typically comprise R on the spiro-cyclopropyl ring, which is cis to A^7′aSubstituent(s):

orAnd

or in reverse to A:

orAnd

desirably, the spiro carbon atom of the aforementioned spiro-cyclopropyl ring has the R configuration:

desirably, in the absolute configuration below, R on the spiro-cyclopropyl ring adjacent to a^7′aThe substituents are in the cis direction:

particular preference is given to the variable R^7′aIncluding ethyl groups, whereby the asymmetric carbon atoms in the 1 and 2 positions have the R, R configuration. Further preferred is R^7′aIncluding vinyl groups, whereby the asymmetric carbon atoms in the 1 and 2 positions have the R, S configuration.

In the case where the compound of the invention is a macrocycle comprising a J group, J is preferably a diastereomer represented by part of the structure (i) or (ii):

or

J and amide Forward (i) J and A Forward (ii)

In particular wherein J and A are in the forward direction.

Detailed description of the embodiments

Various embodiments of the present invention will now be described by way of illustration only with reference to the following non-limiting examples.

Example 1

7-methoxy-2-phenyl-quinolin-4-ol (1)

To a stirred round bottom flask charged with toluene (100mL) were added ethyl benzoylacetate (18.7g, 97mmol) and m-anisidine (12g, 97 mmol). 4M HCl in dioxane (0.5mL) was then added and the reaction mixture was refluxed for 6 hours (140 ℃). The resulting mixture was co-evaporated with toluene. To the resulting crude mixture was added diphenyl ether (50mL) and the resulting mixture was heated to 280 ℃ for 2 h. When the theoretical amount of ethanol (6mL) was collected in the Dean Stark trap, the heating was stopped and the mixture was cooled to room temperature. Dissolving the crude mixture in CH₂Cl₂(100mL) and stirred for 30 minutes. The precipitate formed was filtered off and dried, thus giving 1(4.12g, 16.4mmol, 17%): light yellow powder.

¹H(300 MHz，DMSO-D₆)：δ3.8(s，3H)，6.24(s，1H)，6.88-6.96(dd，1H，J＝9.07Hz，J＝2.47Hz)，7.19(d，1H，J＝2.19Hz)，7.56(t，3H，J＝2.19Hz)，7.8(dd，2H，J＝7.14Hz，J＝2.19Hz)，8.0(d，1H，J＝9.06Hz)； ¹³C(75.5MHz，DMSO-D₆)：δ 55.3，99.6，106.9，113.1，119.1，126.4， 127.5，128.8，130.2，134.1，142.2，149.4，161.8，176.4。

Example 2

Boc-L-tert-leucine-OH (2)

Triethylamine (890. mu.L, 6.40mmol) was added dropwise to a stirred 1: 1(8mL) solution of L-tert-leucine (300mg, 2.29mmol) and di-tert-butyl dicarbonate (599mg, 2.74mmol) in dioxane/water, and the resulting solution was stirred overnight. The resulting mixture was extracted with petroleum ether (2 ×), the aqueous phase was cooled to 0 ℃ and the precipitate was removed by slow addition of 4M NaHSO₄·H₂O carefully acidify it to pH 3. The acidified aqueous phase was extracted with EtOAc (3 ×), the combined organic phases were washed with brine (2 ×) and then dried, filtered and concentrated to give the title compound as a colourless powder (522mg, 99%). No further purification was required.

¹H-NMR(300MHz，CD₃OD)δ0.99(s，9H)，1.44(s，9H)，3.96(s，1H)；¹³C-NMR(75.5 MHz，CD₃OD)δ27.1，28.7，34.9，68.0，80.5，157.8，174.7。

Example 3

((S) -cyclohexyl-methylcarbamoyl-methyl) -carbamic acid tert-butyl ester (3)

Boc-Chg-OH (387mg, 1.50mmol) was coupled to methylamine hydrochloride (111mg, 1.65mmol) using the same HATU coupling conditions as in the synthesis of compound 7.

The resulting crude product was extracted with EtOAc, washed with brine and concentrated. Purification was performed by flash column chromatography (EtOAc) to give the title compound as a colourless solid (307mg, 76%).

¹H-NMR(300MHz，CDCl₃)δ0.91-1.13(m，2H)，1.14-1.31(m， 3H)，1.44(s，9H)，1.61-1.80(m，6H)，2.80(d，J＝4.7Hz，3H)，3.91(dd，J＝7.1，9.1Hz，1H)，5.23(b，1H)，6.52(bs，1H)；¹³C-NMR(75.5MHz，CDCl₃)δ25.9，26.0，26.1，28.3，28.5，29.6，40.5，59.5，79.7，155.9，172.4。

Example 4

{ (S) -1- [ ((S) -cyclohexyl-methylcarbamoyl-methyl) -carbamoyl ] -2, 2-dimethyl-propyl } -carbamic acid tert-butyl ester (4)

To a solution of compound 3(98mg, 0.362mmol) in dichloromethane (3mL) was added triethylsilane (115mL, 0.742mmol) and TFA (3 mL). The resulting mixture was stirred at room temperature for 2h and then evaporated and co-evaporated with toluene. The deprotected amine was dissolved in DMF (5mL) and coupled to compound 2(84mg, 0.363mmol) using the same HATU coupling conditions as in synthesis 7. The resulting crude product was extracted with EtOAc, washed with brine, dried, filtered and concentrated. It was purified by flash column chromatography (toluene/EtOAc 1: 1) to give the title compound as a colorless solid (128mg, 92%).

¹H-NMR(300MHz，CDCl₃)δ0.99(s，9H)，1.02-1.30(m，5H)，1.44(s，9H)，1.58-1.77(m，4H)，1.78-1.89(m，2H)，2.79(d，J＝4.7Hz，3H)，4.11(d，J＝9.3Hz，1H)，4.33(app.t，J＝8.5Hz，1H)，5.65(b，1H)，7.25(b，1H)，7.39(b，1H)；¹³C-NMR(75.5MHz，CDCl₃)δ25.9，25.9，26.0，26.2，26.8，28.4，29.0，29.7，34.5，39.7，58.4，62.4，79.4，156.0，171.4，171.8。

Example 5

Hepta-6-enal (5)

To a solution of hept-6-en-1-ol (1mL, 7.44mmol) and N-methylmorpholine N-oxide (1.308g, 11.17mmol) in DCM (17mL) was added the milled molecular sieve (3.5g,). The mixture was stirred at room temperature for 10 minutes under nitrogen before tetrapropylammonium ferulate (TPAP) (131mg, 0.37mmol) was added. After stirring for an additional 2.5 hours, the resulting solution was filtered through celite. The solvent was then carefully evaporated and the remaining liquid was purified by flash column chromatography (DCM) to give volatile aldehyde 5 as an oil (620mg, 74%).

Example 6

N' -hept-6-en- (E) -ylidene-hydrazinecarboxylic acid tert-butyl ester (6)

To a solution of 5(68mg, 0.610mmol) and tert-butyl carbazate (81mg, 0.613mmol) in methanol (5mL) was added ground molecular sieve (115mg,). The resulting mixture was stirred for 3h, after which it was filtered through celite and evaporated. The resulting residue was dissolved in anhydrous THF (3mL) and acetic acid (3 mL). Reacting NaBH₃CN (95mg, 1.51mmol) was added thereto and the resulting solution was stirred overnight. With saturated NaHCO₃The reaction mixture was diluted with solution (6mL) and EtOAc (6 mL). The organic phase was washed with brine, saturated NaHCO₃Washed with brine and MgSO₄Dried and evaporated. The cyanoborane adduct was hydrolyzed by treatment with methanol (3mL) and 2M NaOH (1.9 mL). The resulting mixture was stirred for 2h and the methanol was evaporated. H is to be₂O (5mL) and DCM (5mL) were added and the resulting aqueous phase was extracted three times with DCM. The combined organic phases were dried and evaporated. Purification was performed by flash column chromatography (toluene/ethyl acetate 9: 1 with 1% triethylamine and toluene/ethyl acetate 6: 1 with 1% triethylamine) to give the title compound as an oil (85mg, 61%).

Example 7

((S) -1-Cyclopentylcarbamoyl-2, 2-dimethyl-propyl) -carbamic acid tert-butyl ester (7)

To a cold solution of 2(133mg, 0.575mmol), cyclopentylamine (64. mu.L, 0.648mmol) and DIEA (301. mu.L, 1.73mmol) in DMF (3mL) was added the coupling reagent HATU (240mg, 0.631 mmol). The resulting mixture was stirred for half an hour and for an additional 2 hours at room temperature. The solvent was removed by heating the reaction flask in a water bath under reduced pressure and the resulting residue was dissolved in ethyl acetate, after which the resulting organic phase was washed three times with brine, dried, filtered and evaporated. Purification by flash column chromatography (toluene/ethyl acetate 4: 1) gave the title compound as colorless crystals (140mg, 82%).

¹H-NMR(300MHz，CDCl₃): δ 0.95(s, 9H), 1.28-1.48(m, overlap peak, 2H), 1.40(s, 9H), 1.49-1.71(m, 4H), 1.86-2.01(m, 2H), 3.76(b, 1H), 4.09-4.23(m, 1H), 5.32(b, 1H), 5.91(b, 1H);¹³C-NMR(75.5MHz，CDCl₃)：δ23.6，23.7，26.5，28.3，32.6，33.1，34.5，51.0，62.2，79.4，155.9，170.3。

example 8

(S) -tert-Butoxycarbonylamino-cyclohexyl-acetic acid methyl ester (8)

To a solution of Boc-Chg-OH (53mg, 0.206mmol) in acetone (3mL) was added iodomethane (195. mu.L, 3.1mmol) and silver (I) oxide (53mg, 0.229 mmol). The mixture was allowed to stir overnight in a reaction flask covered with aluminum foil. After this time, the solution was filtered through celite and evaporated. Purification by flash column chromatography (toluene/ethyl acetate 15: 1) gave methyl ester 8(56mg, 100%) as a colourless oil.

¹H-NMR(300MHz，CDCl₃)：δ1.00-1.34(m，5H)，1.44(s，9H)，1.54-1.82(m，6H)，3.73(s，3H)，4.20(dd，J＝2.8，5.0Hz，1H)，5.05(bs，1H)；¹³C-NMR(75.5MHz，CDCl₃)：δ26.0，28.2，28.3，29.5，41.1，52.0，58.3，79.7，155.6，172.9。

Example 9

(S) - ((S) -2-benzyloxycarbonylamino-3-methyl-butyrylamino) -cyclohexyl-acetic acid methyl ester (9)

Compound 8(93mg, 0.343mmol) was deprotected and coupled with Z-Val-OH (95mg, 0.378mmol) according to preparation of 39. Purification by flash chromatography (toluene/ethyl acetate 4: 1) gave the title compound as a colourless solid (131mg, 94%).

¹H-NMR(300MHz，CDCl₃)：δ0.92-1.30(m，11H)，1.54-1.88(m，6H)，2.02-2.18(m，1H)，3.72(s，3H)，4.05-4.18(m，1H)，4.52(dd，J＝3.0，5.5Hz，1H)，5.12(s，2H)，5.49(bs，1H)，6.52(bs，1H)，7.34(s，5H)； ¹³C-NMR(75.5MHz，CDCl₃)：δ17.8，19.0，25.8，28.2，29.3，31.2，40.5，51.9，56.8，60.0，66.8，127.7，127.9，128.1，128.3，136.2，156.3，171.3，172.2。

Example 10

N-Boc-4R- (2-phenyl-7-methoxyquinoline-4-oxo) proline (10)

To a stirred solution of N-Boc-trans-4-hydroxy-L-proline (3.9g, 16.9mmol) in DMSO (90mL) was added potassium tert-butoxide (4.5g, 40.1 mmol). After 1 hour, 4-chloro-2-phenyl-7-methoxyquinoline (4.5g, 16.7mmol) was added thereto and stirred at room temperature for 12 hours. The resulting mixture was diluted with water (180mL) and washed with waterEthyl acetate (1X 30mL) was washed and neutralized with 1N HCl. The resulting solid was filtered, washed with water and dried to give (4.65g, 10mmol) of the product. Purity > 95% by HPLC. M + H⁺464.2。

Example 11

2- (1-ethoxycarbonyl-2-vinyl-cyclopropylcarbamoyl) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-1-carboxylic acid tert-butyl ester (11)

To a solution of ethyl 1-amino-2-vinylcyclopropanecarboxylate (41mg, 0.26mmol), 10(11mg, 0.22mmol), HATU (204mg, 0.54mmol) in DMF (4mL) was added diisopropylethylamine (187. mu.L, 1.08 mmol). After stirring at room temperature for 1 hour, dichloromethane (4mL) was added thereto. The resulting solution is treated with NaHCO₃The aqueous solution (saturated) and two portions of water were washed. The resulting organic layer was dried and concentrated. The product obtained was of sufficient purity (> 95% by HPLC) to be used in the next step. M + H⁺602.2。

Example 12

1- { [4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid ethyl ester (12)

Compound 11 was held in TFA-DCM 1: 2(3mL) for 60 min at room temperature. Toluene (3mL) was added. The above samples were co-evaporated to dryness. By HPLPurity > 95% by C. M + H⁺502.4。

Example 13

1- { [1- [1- (2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid ethyl ester (13)

To a solution of compound 12(0.13mmol) in THF (2mL) was added a large excess of NaHCO₃(s) and phosgene in toluene (1.6M, 600. mu.L). After stirring for 10 minutes, the resulting slurry was filtered and concentrated to dryness. The resulting solid was redissolved in dichloromethane and a large excess of NaHCO was added₃(s) and 2-amino-N- (2-hydroxy-indan-1-yl) -3, 3-dimethyl-butyramide (0.65mmol) were added thereto. The resulting slurry was stirred at room temperature for 24 to 40 hours. The resulting slurry was filtered, concentrated and subjected to column chromatography on silica (gradient elution 100% DCM to MeOH/DCM 2: 98) to give the title compound (89.6mg, 0.11 mmol). Purity > 95% by HPLC. M + H⁺790.3。

Example 14

1- [1- [1- (2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propyl ] -4- (6-methoxy-3-phenyl-naphthalen-1-yloxy) -pyrrolidin-2-yl ] -2-vinyl-cyclopropanecarboxylic acid (14)

To a solution of compound 13(76.7mg, 0.097mmol) in THF-MeOH 2: 3(2mL) was added 5 equivalents of 1M LiOH. The resulting solution was held at 60 ℃ for 60 minutes. After cooling to room temperature, 15-30 equivalents of HOAc were added followed by toluene (2mL) and then concentrated to dryness. The resulting residue was taken up in DCM and washed with water. The resulting organic layer was dried and concentrated to give the title compound (72mg, 0.094 mmol). Purity > 95% by HPLC. M + H⁺762.2。

Example 15

N- (2-hydroxy-indan-1-yl) -2- [4- (6-methoxy-3-phenyl-naphthalen-1-yloxy) -2- (1-benzylsulfonylaminocarbonyl-2-vinyl-cyclopropyl) -pyrrolidin-1-yl ] -3, 3-dimethyl-butyramide (15)

To a solution of compound 14(25mg, 0.033mmol) in chloroform (1mL) was added benzenesulfonamide (10.5mg, 0.066mmol), followed by diisopropylethylamine (34. mu.L, 0.197 mmol). The resulting solution was stirred at room temperature for 10 minutes, then at-20 ℃ for 30 minutes. Then PyBOP (76mg, 0.13mmol) was added as a solid. The resulting solution was held at-20 ℃ for 48 hours. The resulting solution was then poured into NaHCO₃Aqueous solution (saturated) and washed with water. The resulting organic layer was dried, concentrated and subjected to HPLC purification to give the title compound as a white solid.

Example 16

Resin bound 2-tert-butyloxycarbonylamino-3, 3-dimethylbutyric acid (16)

To Argonaut resin PS-TFP (1.38mmol/g, 10g) and 2-tert-butoxycarbonylamino-3, 3-dimethyl-butyric acid (4.5g, 20.7mmol) was added dichloromethane (40mL) and DMF (10 mL). DMAP (1g, 8.28mmol) was added to the mixture, followed by DIC (9.5mL, 60.7 mmol). After stirring at room temperature for 3 hours, the resin was filtered and washed sequentially with DMF, THF, DCM and diethyl ether, then dried in vacuo.

Example 17

[1- (2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propyl ] -carbamic acid tert-butyl ester (17)

To a portion of compound 16(200mg) in DCM was added aminoindanol (0.14 mmol). The mixture was stirred for 2 hours. The liquid was filtered and the resin was washed with 2 × DCM. The combined liquids were combined and concentrated to dryness to give the title compound (20.5mg, 0.055 mmol). Purity > 95% by HPLC. M + H⁺363.15。

¹³C NMRδ_C(100MHz；CDCl₃；Me₄Si)27.0，28.5，34.2，39.8，50.8，57.9，68.2，73.7，124.8，125.6，127.4，128.5，140.4，171.6。¹H NMRδ_H(400MHz；CDCl₃；Me₄Si)1.07(9H，s，CCH₃)，1.44(9H，s，OCCH₃)，2.93(1H，dd，J_gem16.4Hz，J_3，2 2.3Hz，CH₂)，3.15(1H，dd，J_gem16.4Hz，J_3，2 5.2Hz，CH₂)。

Example 18

2-amino-N- (2-hydroxy-indan-1-yl) -3, 3-dimethylbutanamide (18)

Compound 17 was held in DCM-TFA 2: 1(2mL) for 60 min at room temperature. The solution was co-evaporated to dryness with toluene.

Example 19

(2-tert-Butoxycarbonylamino-3, 3-dimethyl-butyrylamino) -cyclohexyl-acetic acid methyl ester (19)

To a solution of 2-tert-butoxycarbonylamino-3, 3-dimethylbutyric acid (500mg, 2.16mmol), amino-cyclohexyl-acetic acid methyl ester (444mg, 2.59mmol) and HATU (2g, 5.40mmol) in DMF (20mL) was added diisopropylethylamine (1.88mL, 10.8 mmol). The solution was stirred at room temperature for 1 hour and diluted with dichloromethane (40 mL). NaHCO for the above solution₃The aqueous solution (saturated) and water (× 2) were washed, dried and concentrated. The purity of the obtained product is more than 95 percent. M + H⁺385.4。

Example 20

{1- [ (cyclohexyl-methylcarbamoyl-methyl) -carbamoyl ] -2, 2-dimethyl-propyl } -carbamic acid tert-butyl ester (20)

To compound 19 in EtOH-THF 1: 2 was added a large excess of methylamine (30% in water) and it was left at room temperature for 2 weeks. The above solution was concentrated to dryness and the resulting residue was passed through a short silica gel column eluting with 2% methanol in dichloromethane to give pure product (> 95%). M + H⁺384.5。

Example 21

2-amino-N- (cyclohexyl-methylcarbamoyl-methyl) -3, 3-dimethyl-butyramide (71)

Compound 20 was kept in dichloromethane-trifluoroacetic acid 2: 1 for 1 hour at room temperature and concentrated to dryness. The resulting residue was dried in vacuo for 16 h. Reversed phase C18HPLC indicated > 95% purity. M + H⁺283.1。

Example 22

(1R, 2S) -1- { [ (2S, 4R) -1- ((1S, 2R) -2-hydroxy-indan-1-ylcarbamoyl) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (22)

Compound 12 was treated as described for preparation 13, but using (1S, 2R) -cis-1-amino-2-indanol instead of 2-amino-N- (2-hydroxyindan-1-yl) -3, 3-dimethylbutanamide, followed by ester hydrolysis as described for preparation 14 to give the title compound. Purity > 95% by HPLC. M + H⁺649.1。

Example 23

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- (cyclohexylmethyl-carbamoyl) -2-methyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (23)

N- (tert-Butoxycarbonyl) -L-valine was attached to the resin as described for preparation 16, subsequently reacted with cyclohexylamine as described for preparation 17 and the Boc group was removed as described for preparation 18. Subsequently, the compound obtained above was reacted with a chlorocarbamate obtained from compound 12 as described for the preparation of compound 13, thereby giving the title compound. Purity > 95% by HPLC. M + H⁺712.3。

Example 24

(1R, 2S) -1- { [ (2S, 4R) -1- ((1R) -2-hydroxy-1-phenyl-ethylcarbamoyl) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (24)

Compound 12 was treated as described for preparation 13 but with (R) -2-phenylglycoll instead of 2-amino-N- (2-hydroxyindan-1-yl) -3, 3-dimethylbutanamide followed by ester hydrolysis as described for preparation 14 to give the title compound. Purity > 95% by HPLC. M + H⁺637.1。

Example 25

(1R, 2S) -1- { [ (2S, 4R) -1- { [ (1S) -cyclohexyl- (cyclohexylmethyl-carbamoyl) -methyl ] -carbamoyl } -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (25)

N- (tert-butoxycarbonyl) -L-cyclohexylglycine was attached to the resin as described for preparation of compound 16, followed by reaction with cyclohexanemethylamine as described for preparation of compound 17 and removal of the Boc group as described for preparation of compound 18. Subsequently, the compound obtained above was reacted with a chlorocarbamate obtained from compound 12 as described for the preparation of compound 13, thereby giving the title compound. Purity > 95% by HPLC. M + H⁺752.4。

Example 26

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -2-cyclohexyl-1- (cyclohexylmethyl-carbamoyl) -ethylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (26)

N- (tert-butoxycarbonyl) -L-cyclohexylalanine was attached to the resin as described for preparation of compound 16, followed by reaction with cyclohexylmethylamine as described for preparation of compound 17 and removal of the Boc group as described for preparation of compound 18. Subsequently, the compound obtained above was reacted with a chlorocarbamate obtained from compound 12 as described for the preparation of compound 13, thereby giving the title compound. Purity > 95% by HPLC. M + H⁺766.4。

Example 27

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- (cyclohexylmethyl-carbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (27)

N- (tert-butoxycarbonyl) -L-tert-butylglycine was attached to the resin as described for preparation of compound 16, followed by reaction with cyclohexanemethylamine as described for preparation of compound 17 and removal of the Boc group as described for preparation of compound 18. Subsequently, the compound obtained above was reacted with a chlorocarbamate obtained from compound 12 as described for the preparation of compound 13, thereby giving the title compound. Purity > 95% by HPLC. M + H⁺726.3。

Example 28

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- (cyclohexylmethyl-carbamoyl) -2-phenyl-ethylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (28)

N- (tert-butoxycarbonyl) -L-phenylalanine was attached to the resin as described for preparation of compound 16, followed by reaction with cyclohexane methylamine as described for preparation of compound 17 and removal of the Boc group as described for preparation of compound 18. Subsequently, the compound obtained above was reacted with a chlorocarbamate obtained from compound 12 as described for the preparation of compound 13, thereby giving the title compound. Purity > 95% by HPLC. M + H⁺760.4。

Example 29

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- ((1S, 2R) -2-hydroxy-indan-1-ylcarbamoyl) -3-phenyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (29)

N- (tert-butyloxycarbonyl) -L-phenethylglycine was attached to the resin as described for preparation 16, followed by reaction with (1S, 2R) -cis-1-amino-2-indanol as described for preparation 17 and removal of the Boc group as described for preparation 18. Subsequently, the compound obtained above was reacted with a chlorocarbamate obtained from compound 12 as described for the preparation of compound 13, thereby giving the title compound. Purity > 95% by HPLC. M + H⁺810.4。

Example 30

(1R, 2S) -1- { [ (2S, 4R) -1- ((1S) -1-benzylcarbamoyl-2-methyl-propylcarbamoyl) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl } -amino } -2-vinyl-cyclopropanecarboxylic acid (30)

N- (tert-Butoxycarbonyl) -L-valine was attached to the resin as described for preparation 16, subsequently reacted with benzylamine as described for preparation 17 and Boc group was removed as described for preparation 18. Subsequently, the compound obtained above was reacted with a chlorocarbamate obtained from compound 12 as described for the preparation of compound 13, thereby giving the title compound. Purity > 95% by HPLC. M + H⁺706.2。

Example 31

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- ((1R) -2-hydroxy-1-phenyl-ethylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (31)

N- (tert-Butoxycarbonyl) -L-tert-butylglycine was attached to the resin as described for preparation of compound 16, followed by reaction with (R) -2-phenylglycinol as described for preparation of compound 17 and removal of the Boc group as described for preparation of compound 18. Subsequently, e.g. preparing the compounds13 with the chlorocarbamate obtained from compound 12, thereby giving the title compound. Purity > 95% by HPLC. M + H⁺750.3。

Example 32

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- ((1R) -indan-1-ylcarbamoyl) -2-methyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (32)

(2S) -tert-Butoxycarbonylamino-3-methylbutyric acid was attached to the resin as described for preparation of Compound 16, followed by reaction with (1R) -1-aminoindan as described for preparation of Compound 17 and removal of the Boc group as described for preparation of Compound 18. The compound obtained above was then reacted with the chlorocarbamate obtained from compound 12 as described for preparation 13 to give the title compound (12.5mg, 28% yield) after HPLC purification with a purity of > 90% by HPLC. M + H⁺732.2。

Example 33

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- ((1S) -indan-1-ylcarbamoyl) -2-methyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (33)

(2S) -tert-Butoxycarbonylamino-3-methylbutyric acid was attached to the resin as described for preparation of Compound 16, followed by reaction with (1S) -1-aminoindan as described for preparation of Compound 17 and removal of the Boc group as described for preparation of Compound 18. The compound obtained above was then reacted with the chlorocarbamate obtained from compound 12 as described for preparation 13 to give the title compound (22mg, 49% yield) after HPLC purification with a purity of > 90% by HPLC. M + H⁺732.2。

Example 34

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- (2-hydroxyethylcarbamoyl) -2-methyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (34)

(2S) -tert-Butoxycarbonylamino-3-methylbutyric acid was attached to the resin as described for preparation of Compound 16, subsequently reacted with 2-aminoethanol as described for preparation of Compound 17, and Boc group was removed as described for preparation of Compound 18. The compound obtained above was then reacted with the chlorocarbamate obtained from compound 12 as described for preparation 13 to give the title compound (3mg, 8% yield) after HPLC purification with a purity of > 90% by HPLC. M + H⁺660.2。

Example 35

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- ((1S, 2R) -2-hydroxy-indan-1-ylcarbamoyl) -2-methyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (35)

(2S) -tert-Butoxycarbonylamino-3-methylbutyric acid was attached to the resin as described for preparation of Compound 16, followed by reaction with (1S, 2R) -1-amino-2-indanol as described for preparation of Compound 17 and removal of the Boc group as described for preparation of Compound 18. The compound obtained above was then reacted with the chlorocarbamate obtained from compound 12 as described for preparation 13 to give the title compound (10mg, 22% yield) after HPLC purification with a purity of > 90% by HPLC. M + H⁺748.2。

Example 36

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- ((1R, 2S) -2-hydroxy-indan-1-ylcarbamoyl) -2-methyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (36)

(2S) -tert-Butoxycarbonylamino-3-methylbutyric acid was attached to the resin as described for preparation of Compound 16, followed by reaction with (1R, 2S) -1-amino-2-indanol as described for preparation of Compound 17 and removal of the Boc group as described for preparation of Compound 18. The compound obtained above was then reacted with the chlorocarbamate obtained from compound 12 as described for preparation 13 to give the title compound (11mg, 24% yield) after HPLC purification with a purity of > 75% by HPLC. M + H⁺748。

Example 37

(1R, 2S) -1- { [ (2S, 4R) -1- { [ cyclohexyl- (S) - ((1S, 2R) -2-hydroxy-indan-1-ylcarbamoyl) -methyl ] -carbamoyl } -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (37)

(2S) -tert-Butoxycarbonylamino-cyclohexylacetic acid was attached to the resin as described for preparation 16, followed by reaction with (1S, 2R) -1-amino-2-indanol as described for preparation 17 and removal of the Boc group as described for preparation 18. The compound obtained above was then reacted with the chlorocarbamate obtained from compound 12 as described for preparation 13 to give the title compound (7.5mg, 16% yield) after HPLC purification with a purity of > 95% by HPLC. M + H⁺788.3。

Example 38

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- ((1S, 2R) -2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (38)

(2S) -tert-Butoxycarbonylamino-3, 3-dimethylbutyric acid was attached to the resin as described in preparation 16, followed by reaction with (1S, 2R) -1-amino-2-indanol as described in preparation 17 and removal of the Boc group as described in preparation 18. Then theThe compound obtained above was reacted with the chlorocarbamate obtained from compound 12 as described for preparation 13 to give the title compound (12mg, 26% yield) after HPLC purification with > 95% purity by HPLC. M + H⁺762.3。

Example 39

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- ((1S, 2R) -2-hydroxy-indan-1-ylcarbamoyl) -3, 3-dimethyl-butylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (39)

(2S) -tert-Butoxycarbonylamino-4, 4-dimethylpentanoic acid was attached to the resin as described for preparation 16, followed by reaction with (1S, 2R) -1-amino-2-indanol as described for preparation 17 and removal of the Boc group as described for preparation 18. The compound obtained above was then reacted with the chlorocarbamate obtained from compound 12 as described for preparation 13 to give the title compound (14.2mg, 30% yield) after HPLC purification with a purity of > 95% by HPLC. M + H⁺776.3。

Example 40

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- ((1S, 2R) -2-hydroxy-indan-1-ylcarbamoyl) -2-phenyl-ethylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (40)

(2S) -tert-Butoxycarbonylamino-3-phenylpropionic acid was attached to the resin as described for preparation of Compound 16, followed by reaction with (1S, 2R) -1-amino-2-indanol as described for preparation of Compound 17 and removal of the Boc group as described for preparation of Compound 18. The compound obtained above was then reacted with the chlorocarbamate obtained from compound 12 as described for preparation 13 to give the title compound (2.4mg, 5% yield) after HPLC purification with a purity of > 95% by HPLC. M + H⁺796.2。

EXAMPLE 41

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -2-cyclohexyl-1- ((1S, 2R) -2-hydroxy-indan-1-ylcarbamoyl) -ethylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (41)

(2S) -tert-Butoxycarbonylamino-3-cyclohexylpropionic acid was attached to the resin as described for preparation 16, followed by reaction with (1S, 2R) -1-amino-2-indanol as described for preparation 17 and removal of the Boc group as described for preparation 18. The compound obtained above was then reacted with the chlorocarbamate obtained from compound 12 as described for preparation 13 to give the title compound (12.3mg, 25% yield) after HPLC purification with a purity of > 95% by HPLC. M + H⁺802.3。

Example 42

(1R, 2S) -1- { [ (2S, 4R) -1- { (1S) -1- [ (S) - (cyclohexyl-methylcarbamoyl-methyl) -carbamoyl ] -2, 2-dimethyl-propylcarbamoyl } -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (42)

Treatment of compound 12 as described for preparation 13 but using compound 21 instead of 2-amino-N- (2-hydroxy-indan-1-yl) -3, 3-dimethylbutanamide followed by ester hydrolysis as described for preparation 14 gave the title compound (8.6mg, 18% yield) after purification by HPLC. Purity > 95% by HPLC. M + H⁺ 783.3。

Example 43

1- (2-amino-4-methoxyphenyl) ethanone (ethanone) (43)

M-methoxyaniline (10.0g, 82mmol) was dissolved in CH₂Cl₂(50mL) and the solution was cooled to-50 ℃. The BCl is added over a period of 20 minutes₃(in CH)₂Cl₂1M in 82mL, 82mmol) was added slowly, after which the mixture was stirred at-50 ℃ for 30 minutes, then AcCl (6.0mL, 84mmol) and AlCl were added sequentially to it₃(11g, 82 mmol). The mixture was stirred at-50 ℃ for 1 hour and then allowed to stand at room temperature. After stirring at room temperature overnight, the above solution was heated at 40 ℃ for 4h, after which the resulting mixture was poured onto ice. The resulting aqueous mixture was basified with 10% NaOH (w/v) and extracted with EtOAc (4X 200 mL). The combined organic phases were washed with brineDried (MgSO)₄) And evaporated to give a black solid which was purified by flash column chromatography (ether/CH)₂Cl₂20: 80) was purified. The resulting solid was recrystallized from ether/hexane to give compound 93(5.6g, 42%) as bright tan leaves.

Example 44

N- (tert-butyl) -N' -isopropylthiourea (44)

To tert-butyl isothiocyanate (5.0mL, 39mmol) in CH₂Cl₂(200mL) to the solution were added isopropylamine (4.0mL, 47mmol) and Diisopropylethylamine (DIEA) (6.8mL, 39mmol), and the resulting mixture was stirred at room temperature for 2 h. The resulting reaction mixture was diluted with EtOAc and diluted with 10% citric acid (2X), saturated NaHCO₃(2x)、H₂O (2x) and brine (1 x). The resulting organic layer was dried (MgSO)₄) And evaporated to give compound 94(3.3g, 52%) as a white solid, which was used without further purification.

Example 45

N-isopropylthiourea (45)

Compound 44(3.3g, 20mmol) was dissolved in concentrated HCl (45mL) and the solution was refluxed for 40 minutes. The mixture was cooled to room temperature, then cooled in an ice bath and quenched with solid NaHCO₃And saturated NaHCO₃It was basified to pH 9.5, after which the product was extracted into EtOAc (3 ×). Combined organic phases with H₂O (2x) and brine (1x) were washed, dried (MgSO)₄) And evaporated to give crude compound 95(2.1g, 90%) which was used without further purification.

Example 46

2- (isopropylamino) -1, 3-thiazole-4-carboxylic acid hydrobromide salt (46)

A suspension of compound 45(2.1g, 18mmol) and 3-bromopyruvic acid (3.0g, 18mmol) in dioxane (180mL) was heated to 80 ℃. Upon reaching 80 ℃ the mixture became clear and soon the product began to precipitate as a white solid. After heating for 2h, the reaction mixture was cooled to room temperature and the precipitate was filtered off and collected. This gave pure compound 46(4.4g, 94%).

Example 47

N- (2-acetyl-5-methoxyphenyl) -2- (isopropylamino) -1, 3-thiazole-4-carboxamide (47)

A mixture of compound 46(4.4g, 16.5mmol) and aniline derivative 93(2.75g, 16.5mmol) in pyridine (140mL) was cooled to-30 deg.C (the clear solution partially became a suspension by cooling) and POCl was added over a period of 5 minutes₃(3.3mL, 35mmol) was added slowly. The mixture was stirred at-30 ℃ for 1 hour and then allowed to reach room temperature. After stirring at room temperature for 1.5h, the resulting reaction mixture was poured onto ice and washed with solid NaHCO₃And saturated NaHCO₃Adjusting the pH value to about 9-10. Extracting the obtained crude product with CH₂Cl₂(3X) and the combined organic phases are dried (MgSO)₄) And evaporation. The resulting crude dark beige solid was purified by flash column chromatography (hexane/EtOAc 55: 45) to afford the compound as a pale yellow solid47(5.6g，76％)。

Example 48

2- [2- (isopropylamino) -1, 3-thiazol-4-yl ] -7-methoxyquinolin-4-ol (48)

A solution of t.BuOK (2.42g, 21mmol) in dry t.BuOH (40mL) was heated to reflux. Compound 47(1.8g, 5.4mmol) was added thereto in portions over a period of 5 minutes, and the resulting dark red solution was stirred under reflux for a further 20 minutes. The mixture was cooled to room temperature and HCl (4M in dioxane, 8.0mL, 32mmol) was added, after which the resulting reaction mixture was concentrated in vacuo. To ensure that all HCl and dioxane were removed, the crude product was redissolved in CH₂Cl₂Twice and evaporated thoroughly to give the slightly impure compound 98 hydrochloride salt as a brown solid (1.62 g). Dissolving the above product in CH₂Cl₂Neutralized and saturated NaHCO₃Washing is carried out, after which the aqueous phase is washed with CH₂Cl₂And extracting for several times. The combined organic phases were dried (MgSO)₄) And evaporated to give the title compound as a light brown solid (1.38g, 81%) (purity > 95% according to HPLC test).¹H-NMR(MeOH-d₄，400MHz)：δ1.30(d，J＝6.0Hz，6H)，3.93(s，3H)，3.95-4.07(m，1H)，6.73(s，1H)，6.99(dd，J＝2.4，9.2Hz，1H)，7.26(d，J＝2.4Hz，1H)，7.37(s，1H)，8.10(d，J＝9.2Hz，1H)。

Example 49

(1S) -1- { [ (2S, 4R) -2- (1-methoxycarbonyl-butylcarbamoyl) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) ] -pyrrolidine } -carboxylic acid tert-butyl ester (49)

Compound 10 was reacted with Nva-OMe hydrochloride according to the method described in example 11 to give the title compound. Purity > 95% by HPLC. M + H⁺578.24。

Example 50

(1S) -1- { [ (2S, 4R) -2- [4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -pentanoic acid methyl ester (50)

Compound 49 was held in TFA-DCM 1: 2(3mL) for 60 min at room temperature. Toluene (3mL) was added. The samples were co-evaporated to dryness. Purity > 95% by HPLC. M + H⁺478.21。

Example 51

(1S) -2- { [ (2S, 4R) -1- [ (1S) -1- (cyclohexylmethyl-carbamoyl) -2-methyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -pentanoic acid methyl ester (51)

To compound 50(0.1 m) cooled to 0 deg.Cmol) in THF (4mL) was added a large excess of NaHCO₃(s) and phosgene in toluene (0.2mmol, 21. mu.L). After stirring for 10 minutes, the resulting slurry was filtered and concentrated to dryness. The resulting solid was redissolved in dichloromethane and a large excess of NaHCO was added₃(s) and 2-amino-N-cyclohexylmethyl-3-methyl-butyramide (described in example 23) (0.15mmol) were added thereto. The resulting slurry was stirred at room temperature for 30 hours. The slurry was filtered, concentrated and subjected to column chromatography on silica (gradient elution from 100% DCM to MeOH/DCM 2: 98) to give the title compound (30mg, 0.042 mmol). Purity > 95% by HPLC. M + H⁺716.40。

Example 52

(1S) -2- { [ (2S, 4R) -1- [ (1S) -1- (cyclohexylmethyl-carbamoyl) -2-methyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -pentanoic acid (52)

To a solution of compound 51(26mg, 0.036mmol) in THF-MeOH 2: 3(2mL) was added 1.5 equivalents of 1M LiOH. The solution was kept at 60 ℃ for 60 minutes. After cooling to room temperature, HOAc was added thereto, followed by toluene (2mL), which was then concentrated to dryness to give the title compound (25mg, 0.035 mmol). Purity > 95% by HPLC. M + H⁺702.34。

Example 53

(1R, 2S) -1- { [ (2S, 4R) -1- [2- (2-methoxy-phenoxy) -ethylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid ethyl ester (53)

To a solution of compound 12(0.06mmol) in THF (2mL) was added a large excess of NaHCO₃(s) and phosgene in toluene (0.078 mmol). After stirring for 10 minutes, the resulting slurry was filtered and concentrated to dryness. The resulting solid was redissolved in dichloromethane and a large excess of NaHCO was added₃(s) and 2- (2-methoxy-phenoxy) -ethylamine (15mg, 0.09mmol) were added thereto. The resulting slurry was stirred at room temperature for 30 hours. The slurry was filtered, concentrated to dryness, redissolved in methanol and subjected to HPLC purification to give the title compound (10.6mg, 0.015 mmol). Purity > 95% by HPLC. M + H⁺ 695.17。

Example 54

(1R, 2S) -1- { [ (2S, 4R) -1- [2- (2-methoxy-phenoxy) -ethylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (54)

To a solution of compound 53(10.6mg, 0.0153mmol) in THF-MeOH 2: 3(2mL) was added 10 equivalents of 1M LiOH. The solution was kept at 50 ℃ for 60 minutes. After cooling to room temperature, 25 equivalents of HOAc were added followed by toluene (2mL) and then concentrated to dryness. The resulting residue was taken up in ethyl acetate, filtered and concentrated to dryness to give the title compound (9.4mg, 0.014 mmol). Purity > 95% by HPLC. M + H⁺667.14。

Example 55

(1R, 2S) -1- { [ (2S, 4R) -1- ((1S, 2R) -5-hydroxy-4, 5, 6, 7-tetrahydro-benzo [ b ] thiophen-4-yl-carbamoyl)) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (55)

Prepared according to the procedure described in example 53, but using 2-amino-4, 5, 6, 7-tetrahydro-benzo [ b ]]Thiophen-5-ol instead of 2- (2-methoxy-phenoxy) -ethylamine followed by hydrolysis of the ethyl ester as described in example 54 gave the title compound (7.5mg, 0.011 mmol). Purity > 95% by HPLC. M + H⁺669。

Example 56

(1R, 2S) -1- { [ (2S, 4R) -1- [ (3R-3-hydroxy-pyrrolidine-1-carbonyl) ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (56)

Prepared according to the procedure described in example 53, but using (R) -3-pyrrolidinol instead of 2- (2-methoxy-phenoxy) -ethylamine, followed by hydrolysis of the ethyl ester as in example 54 to give the title compound (4mg, 0.007 mmol). Purity > 95% by HPLC. M + H⁺587.1。

Example 57

(1R, 2S) -1- { [ (2S, 4R) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -1- [ (thiophen-2-yl-methyl) -carbamoyl ] -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (57)

Prepared according to the procedure described in example 53, but using thiophene-2-methylamine instead of 2- (2-methoxy-phenoxy) -ethylamine and then performing hydrolysis of the ethyl ester as described in example 54 to give the title compound (8mg, 0.013 mmol). Purity > 95% by HPLC. M + H⁺613.08。

Example 58

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1, 1-dioxo-tetrahydro-1-. lamda. ] -1⁶-thien-3-yl-carbamoyl]-4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl]-amino } -2-vinyl-cyclopropanecarboxylic acid (58)

Prepared according to the procedure described in example 53, but using 3-aminotetrahydro-1H-. lambda.⁶-thiophene-1, 1-dione instead of 2- (2-methoxy-phenoxy) -ethylamine followed by hydrolysis of the ethyl ester as described in example 54 to give the title compound (13mg, 0.02 mmol). Purity > 95% by HPLC. M + H⁺635.05。

Example 59

2-amino-3, 3-dimethyl-N-thiophen-2-yl-methyl-butyramide (59)

The title compound was prepared as follows: the Boc group was subsequently removed as described in example 18, but using thiophene-2-methylamine instead of aminoindanol.

Example 60

2-amino-N- (6-hydroxy-4, 5, 6, 7-tetrahydro-benzo [ b ] thiophen-5-yl) -3, 3-dimethylbutanamide (60)

The title compound was prepared as follows: as described in example 17, but using 2-amino-4, 5, 6, 7-tetrahydro-benzo (b) thiophen-5-ol instead of aminoindanol, the Boc group was subsequently removed as described in example 18.

Example 61

2-amino-N- (2-diethylamino-ethyl) -3, 3-dimethyl-butyramide (61)

The title compound was prepared as follows: as described in example 17, but using N, N-diethylethylenediamine instead of aminoindanol, the Boc group was subsequently removed as described in example 18.

Example 62

2-amino-N- [2- (2-methoxy-phenoxy) -ethyl ] -3, 3-dimethyl-butyramide (62)

The title compound was prepared as follows: as described in example 17, but using 2-methoxyphenoxyethylamine instead of aminoindanol, the Boc group was subsequently removed as described in example 18.

Example 63

2-amino-1- (3-hydroxy-pyrrolidin-1-yl) -3, 3-dimethyl-butan-1-one (63)

The title compound was prepared as follows: the Boc group was subsequently removed as described in example 18, but using (R) -3-pyrrolidone instead of aminoindanol.

Example 64

2-amino-N- (1, 1-dioxo-tetrahydro-1-lambda)⁶-thiophen-3-yl) -3, 3-dimethyl-butyramide (64)

The title compound was prepared as follows: as described in example 17, but using 2-methoxyphenoxyethylamine instead of aminoindanol, the Boc group was subsequently removed as described in example 18.

Example 65

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- (2, 2-dimethyl-1- [ (thiophen-2-yl-methyl) -carbamoyl ] -propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid ethyl ester (65)

To a solution of compound 12(0.06mmol) in THF (2mL) was added a large excess of NaHCO₃(s) and phosgene in toluene (0.078 mmol). After stirring for 10 minutes, the resulting slurry was filtered and concentrated to dryness. The resulting solid was redissolved in dichloromethane and a large excess of NaHCO was added₃(s) and compound 59(0.09mmol) were added thereto.

The resulting slurry was stirred at room temperature for 30 hours. The resulting slurry was filtered, concentrated to dryness, redissolved in methanol and subjected to HPLC purification to give the title compound (15.5mg, 0.02 mmol). Purity > 95% by HPLC. M + H⁺754.2。

Example 66

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- (2, 2-dimethyl-1- [ (thiophen-2-ylmethyl) -carbamoyl ] -propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (66)

To a solution of compound 65(14mg, 0.017mmol) in THF-MeOH 2: 3(2mL) was added 10 equivalents of 1M LiOH. The solution was kept at 50 ℃ for 60 minutes. After cooling to room temperature, 20 equivalents of HOAc were added, followed by toluene (2mL), which was then concentrated to dryness. The resulting residue was taken up in ethyl acetate, filtered and concentrated to dryness to give the title compound (13mg, 0.017 mmol). Purity > 95% by HPLC. M + H⁺748.13。

Example 67

(1R, 2S) -1- { [ (2S, 4R) - (1S) -1- [ (1S, 2R) -1- [1- (5-hydroxy-4, 5, 6, 7-tetrahydro-benzo [ b ] thiophen-4-yl-carbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (67)

Following the procedure described for example 65 but using compound 60 instead of compound 59, the hydrolysis of the ethyl ester was followed as described in example 66 to give the title compound (4mg, 0.005 mmol). Purity > 95% by HPLC. M + H⁺782.16。

Example 68

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- (2-diethylamino-ethylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (68)

Following the procedure described for example 65 but using compound 61 instead of compound 59, the hydrolysis of the ethyl ester was followed as described in example 66 to give the title compound (6mg, 0.008 mmol). Purity > 95% by HPLC. M + H⁺729.24。

Example 69

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- [2- (2-methoxy-phenoxy) -ethylcarbamoyl ] -2, 2-dimethyl-propylcarbamoyl } -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (69)

Following the procedure described for example 65 but using compound 62 instead of compound 59, hydrolysis of the ethyl ester was then carried out as described for example 66 to give the title compound (3mg, 0.004 mmol). Purity > 95% by HPLC. M + H⁺780.19。

Example 70

(1R, 2S) -1- { [ (2S, 4R) - (1S) -1- [ (3R) -1- (3-hydroxy-pyrrolidine-1-carbonyl) -2, 2-dimethyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (70)

Following the procedure described in example 65, but using compound 63 instead of compound 59, the hydrolysis of the ethyl ester was followed as described in example 66 to give the title compound (12.4mg, 0.02 mmol). Purity > 95% by HPLC. M + H⁺700.16。

Example 71

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- (1, 1-dioxo-tetrahydro-1-. lamda. -)⁶-thien-3-yl-carbamoyl) -2, 2-dimethyl-propylcarbamoyl]-4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl]-amino } -2-vinyl-cyclopropanecarboxylic acid (71)

Following the procedure described for example 65 but using compound 64 instead of compound 59, hydrolysis of the ethyl ester was then performed as described for example 66 to give the title compound (13mg, 0.014 mmol). Purity > 95% by HPLC. M + H⁺748.13。

Example 72

(4R-1- (tert-butyl)Butoxycarbonyl) -4- [ (7-methoxy-2-phenylquinolin-4-yl) oxy]-L-prolyl-N¹- (benzenesulfonyl) -L-norvalinamide (72)

To a solution of compound 10(60mg, 0.13mmol) in DMF were added HATU (124mg, 0.325mmol) and diisopropylethylamine (114. mu.L, 0.65mmol) and stirred at room temperature for 30 min. A solution of compound 75(0.157mmol) in DMF was added. The slurry was stirred at room temperature for 16 hours and then concentrated to dryness. The resulting residue was taken up in DCM and washed with NaHCO₃(saturated) and water. The organic layer was dried, concentrated and subjected to silica column chromatography (gradient elution from 100% DCM to 2% MeOH/DCM) to give the title compound (61mg, 0.087 mmol). Purity > 90% by HPLC. M + H⁺703.23。

Example 73

(4R) -4- [ (7-methoxy-2-phenylquinolin-4-yl) oxy]-L-prolyl-N¹- (benzenesulfonyl) -L-norvalinamide (73)

Compound 72 was held in DCM-TFA 2: 1(2mL) for 2.5h at room temperature. The solution was co-evaporated to dryness with toluene. The yield was 100%. M + H603.12.

Example 74

Carbamic acid, [ (1S) -1- [ [ (phenylsulfonyl) amino ] carbonyl ] butyl ] -, phenylmethyl ester (74)

To a stirred solution of Z-Nva-OH (150mg, 0.59mmol) in THF (6mL) was added CDI (400mg, 2.4 mmol). The slurry was stirred at room temperature for 30 minutes, to which DBU (200. mu.L, 1.3mmol) and a solution of benzenesulfonamide (250mg, 1.59mmol) in THF (2mL) were then added. The mixture was stirred at 60 ℃ for 48 hours and then concentrated to dryness. The resulting residue was dissolved in methanol and subjected to HPLC purification to give the title compound (118.5mg, 0.304 mmol). Purity > 95% by HPLC. M-H⁺389.0，+Na 412.96。

Example 75

(2S) -2-amino-N- (phenylsulfonyl) pentanamide (75)

Compound 74 was dissolved in methanol (5mL), followed by the addition of Pd/C and hydrogenation for 2 hours. The slurry was filtered through celite, washed with methanol and concentrated to dryness to give the title compound. The yield was 100%. M + H⁺257.3。

Example 76

4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-1, 2-dicarboxylic acid 1- ({1- [ (cyclohexyl-methylcarbamoyl-methyl) -carbamoyl ] -2, 2-dimethyl-propyl } -amide) -2- [ (1-benzylsulfonylaminocarbonyl-2-vinyl-cyclopropyl) -amide ] (76)

To a solution of compound 42(8.7mg, 0.011mmol) in chloroform (1ml) was added α -toluenesulfonamide (7mg, 0.04mmol) followed by diisopropylethylamine (21 μ L, 0.12 mmol). The resulting solution was stirred at room temperature for 10 minutes, then at-20 ℃ for 30 minutes. Then PyBOP (46.5mg, 0.08mmol) was added as a solid. The resulting solution was held at-20 ℃ for 48 hours. The resulting solution was then poured into NaHCO₃Aqueous solution (saturated) and washed with water. The resulting organic layer was dried, concentrated and purified by HPLC to give the title compound as a white solid (> 95% purity by HPLC) (2.8mg, 0.0049 mmol). M + H⁺936.26。

Example 77

N- (2-hydroxy-indan-1-yl) -2- [4- (6-methoxy-3-phenyl-naphthalen-1-yloxy) -2- (1-methanesulfonylaminocarbonyl-2-vinyl-cyclopropyl) -pyrrolidin-1-yl ] -3, 3-dimethyl-butyramide (77)

The title compound was prepared as described in example 76, but using compound 14 as the carboxylic acid starting material and methanesulfonamide instead of α -toluenesulfonamide. Yield 13%, purity > 95% by HPLC. M + H⁺839.16。

Example 78

4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-1, 2-dicarboxylic acid 1- { [1- (cyclohexylmethyl-carbamoyl) -2-methyl-propyl ] -amide }2- [ (1-phenylmethanesulfonylaminocarbonyl-2-vinyl-cyclopropyl) -amide ] (76)

The title compound was prepared as described for example 76, using compound 23 as the carboxylic acid starting material. The yield was 2%. Purity > 95% by HPLC. M + H⁺865.28。

Example 79

4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-1, 2-dicarboxylic acid 1- { [1- (cyclohexylmethyl-carbamoyl) -2-methyl-propyl ] -amide }2- [ (1-benzenesulfonylaminocarbonyl-butyl) -amide ] (79)

The title compound was prepared as described in example 76, using compound 52 as the carboxylic acid starting material. The yield was 8%. Purity > 95% by HPLC. M + H⁺855.28。

Example 80

4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-1, 2-dicarboxylic acid 2- [ (1-benzenesulfonylaminocarbonyl-butyl) -amide ]1- { [1- (cyclohexylmethyl-carbamoyl) -2-methyl-propyl ] -amide } (80)

The title compound was prepared as described in example 76, but using compound 52 as the carboxylic acid starting material and benzenesulfonamide instead of α -toluenesulfonamide. Product produced by birthThe rate was 21.5%. Purity > 95% by HPLC. M + H⁺841.28。

Example 81

4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-1, 2-dicarboxylic acid 2- [ (1-benzenesulfonylaminocarbonyl-2-vinyl-cyclopropyl) -amide ]1- ({1- [ (cyclohexyl-methylcarbamoyl-methyl) -carbamoyl ] -2, 2-dimethyl-propyl } -amide) (81)

The title compound was prepared as described in example 76 using benzenesulfonamide instead of α -toluenesulfonamide. The yield was 26%. Purity > 95% by HPLC. M + H⁺922.23。

Example 82

4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-1, 2-dicarboxylic acid 2- [ (1-benzenesulfonylaminocarbonyl-butyl) -amide ]1- { [1- (2-hydroxy-indan-1-ylcarbamoyl) -2-methyl-propyl ] -amide } (82)

To a solution of compound 73(24.1mg, 0.04mmol) in DCM (2ml) was added a large excess of NaHCO₃(s) and phosgene in toluene (50. mu.L, 0.096 mmol). After stirring for 10 minutes, the resulting slurry was filtered and concentrated to dryness. The resulting solid was redissolved in DCM and a large excess of NaHCO was added₃(s) and 2-amino-N- (2-hydroxy-indan-1-yl) -3-methyl-butanamide (described in example 35) (0.1mmol) plusAnd put into it. The resulting slurry was stirred at room temperature for 40 hours. The resulting slurry was filtered, concentrated and subjected to HPLC purification to give the title compound (1.6mg, 0.0018 mmol). Purity > 95% by HPLC. M + H⁺877.21。

Example 83

4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-1, 2-dicarboxylic acid 2- [ (1-benzenesulfonylaminocarbonyl-butyl) -amide ]1- ({1- [ (cyclohexyl-methylcarbamoyl-methyl) -carbamoyl ] -2, 2-dimethyl-propyl } -amide) (83)

The title compound was prepared as described in example 82, but using compound 21 instead of 2-amino-N- (2-hydroxy-indan-1-yl) -3-methyl-butyramide. The yield was 2%. Purity > 95% by HPLC. M + H⁺912.25。

Example 84

(1R, 2S) -1- { [ (4R, 2S) -1- (1- (1S) -hydroxymethyl-2, 2-dimethyl-propylcarbamoyl) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid ethyl ester (84)

Compound 12 was treated as described for preparation of compound 13, but using (S) -tert-leucinol instead of 2-amino-N- (2-hydroxy-indan-1-yl) 3, 3-dimethyl-butyramide to give the title product. M + H⁺645.2。

Example 85

(1R, 2S) -1- { [ (4R, 2S) -1- (1- (1S) -formyl-2, 2-dimethyl-propylcarbamoyl) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid ethyl ester (85)

To a stirred solution of compound 84(64mg) in dichloromethane was added Dess-Martin periodinane (80mg) at ambient temperature. After 4 hours, the slurry was filtered through basic alumina and concentrated to dryness. M + H⁺643.2。

Example 86

(1R, 2S) -1- { [ (4R, 2S) -1- {1- [ ((1S, 2R) -2-hydroxy-indan-1-ylamino) -methyl ] -2, 2-dimethyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid ethyl ester (86)

To a solution of compound 85 in THF (2mL) and HOAc (0.5mL) was added polystyrene bound cyanoborohydride (2.36mmol/g, 100mg) and (1S, 2R) -1-aminoindan-2-ol (18mg), and it was stirred for 4 hours. The resulting mixture was filtered, concentrated and purified on preparative HPLC. Purity > 90% by HPLC. M + H⁺776.5。

Example 87

(1R, 2S) -1- { [ (4R, 2S) -1- {1- [ ((1S, 2R) -2-hydroxy-indan-1-ylamino) -methyl ] -2, 2-dimethyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (87)

To a solution of compound 86 in THF (2mL) and methanol (1mL) was added 1N LiOH (0.2mL), and the resulting solution was left at 60 ℃ for 1.5 hours. The resulting slurry was neutralized to pH7 with 1N HCl, concentrated and purified on preparative HPLC to give pure product, > 95% by HPLC. M + H⁺748.4。

Example 88

(1R, 2S) -1- { [ (4R, 2S) -1- (1- { [ (1S) - (cyclohexyl-methylcarbamoyl-methyl) -amino ] -methyl } -2, 2-dimethyl-propylcarbamoyl } -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (88)

Compound 85 was treated as described for preparation of compound 86, but 2-amino-2-cyclohexyl-N-methyl-acetamide (17mg) was used instead of (1S, 2R) -1-aminoindan-2-ol, followed by ethyl ester hydrolysis as described in example 87 to form the title compound. Purity > 95% by HPLC. M + H⁺769.5。

Example 89

Acetic acid (1S, 2R) -1- ((2S) -2-amino-3, 3-dimethyl-butyrylamino) -indan-2-yl ester (89)

Compound 17(4g) was placed in pyridine-acetic anhydride 2: 1 for 30 minutes. DCM was added to the solution and the resulting solution was treated with citric acid (aq) and NaHCO₃(aqueous solution) washing was performed. The resulting organic layer was concentrated to dryness to give the acetylated product with a purity of > 90% by HPLC. The resulting compound was then placed in 30% TFA in DCM for 1.5 hours, and then concentrated to dryness. Coevaporation from toluene twice gave the title product in an HPLC purity of > 90%,

example 90

(2S, 4R) -2- ((1S, 2R) 1-ethoxycarbonyl-2-vinyl-cyclopropylcarbamoyl) -4-hydroxy-pyrrolidine-1-carboxylic acid tert-butyl ester (90)

A solution of HATU (6g), diisopropylethylamine (6.8mL), (1R, 2S) -1-amino-2-vinyl-cyclopropanecarboxylic acid ethyl ester (1.5g), and BOC-L-hydroxyproline (1.6g) in dichloromethane was stirred for 1 hour. The resulting mixture was diluted with DCM-NaHCO₃(aqueous solution), dried and concentrated. HPLC purity was approximately 90%. M + H⁺369.1。

Example 91

(1S, 2R) -1- [ (2S, 4R) - (4-hydroxy-pyrrolidine-2-carbonyl) -amino ] -2-vinyl-cyclopropanecarboxylic acid ethyl ester (91)

Compound 90 was placed in 30% trifluoroacetic acid in dichloromethane and 1% methanol for 2 hours before it was concentrated to dryness. The residue obtained is redissolved in dichloromethane and 1N NaOH is added thereto during stirring to give a pH of 10 to 11. The organic layer was separated and concentrated to give 1.6g of the titled product. HPLC purity was approximately 90%. M + H⁺ 269.1。

Example 92

(1R, 2S) -1- ({ (2S, 4R) -1- [ (1S) -1- ((1S, 2R) -2-acetoxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4-hydroxy-pyrrolidine-2-carbonyl } -amino) -2-vinyl-cyclopropanecarboxylic acid ethyl ester (92)

To a stirred solution of compound 89(1.81g) in acetonitrile was added solid NaHCO at 0 deg.C₃(800mg) and p-nitrophenylchloroformate (1.2 g). The slurry was adjusted to ambient temperature and stirred for an additional 30 minutes. To this slurry was added compound 91(1.6g) in acetonitrile (5mL) and diisopropylethylamine (1 mL). After 10 minutes, the mixture was concentrated, redissolved in ethyl acetate and washed with K₂CO₃Washed (aq) then with 0.5N HCl. Drying and concentration gave a product with a purity of > 80% by HPLC. M + H⁺ 599.6。

Example 93

(1R, 2S) -1- ({ (2S, 4R) -1- [ (1S) -1- ((1S, 2R) -2-acetoxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4-phenylcarbamoyloxy-pyrrolidine-2-carbonyl } -azo) -2-vinyl-cyclopropanecarboxylic acid ethyl ester (93)

To a stirred solution of compound 92(20mg) in DCM was added solid K₂CO₃(200mg) and a 20% phosgene in toluene (1 mL). After 6 hours, the slurry was filtered and concentrated to dryness. To the residue was added aniline (30mg), DCM (3mL) and solid NaHCO₃(50mg) of the mixture and stirred for 10 hours. The resulting mixture was filtered, concentrated and purified on preparative HPLC to give the title product. The purity is more than 95 percent. M + H⁺718.6。

Example 94

(1R, 2S) -1- ({ (2S, 4R) -1- [1- ((1S, 2R) -2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4-phenylcarbamoyloxy-pyrrolidine-2-carbonyl } -amino) -2-vinyl-cyclopropanecarboxylic acid (94)

To compound 93 in THF-MeOH 2: 1(3mL) was added 1N LiOH (0.2 mL). The above solution was heated to 60 ℃ for 2 hours. After cooling to ambient temperature, acetic acid (0.5mL) was added and the solution was concentrated toAnd (5) drying. The remaining residue was purified on preparative HPLC to give the title product in > 95% purity. M + H⁺ 648.5。

Example 95

(5S, 3R) -3, 4-dihydro-1H-isoquinoline-2-carboxylic acid 5- ((1R, 2S) -1-carboxy-2-vinyl-cyclopropylcarbamoyl) -1- [1- ((1S, 2R) -2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -pyrrolidin-3-yl ester (95)

Compound 92 was treated as described for the preparation of compound 93 but using 1, 2, 3, 4-tetrahydroisoquinoline instead of aniline followed by hydrolysis of the ethyl ester as described in example 94 to give the title compound. The purity is more than 90 percent. M + H⁺688.6。

Example 96

(5S, 3R) -3, 4-dihydro-2H-quinoline-1-carboxylic acid 5- ((1R, 2S) -1-carboxy-2-vinyl-cyclopropylcarbamoyl) -1- [1- ((1S, 2R) -2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -pyrrolidin-3-yl ester (96)

Compound 92 was treated as described for the preparation of compound 93 but using 1, 2, 3, 4-tetrahydro-quinoline instead of aniline followed by hydrolysis of the ethyl ester as described in example 94 to give the title compound. The purity is more than 90 percent. M + H⁺688.6。

Example 97

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- ((1S, 2R) -2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4- (pyridin-3-ylmethylcarbamoyloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (97)

Compound 92 was treated as described for the preparation of compound 93 but using 2-pyridin-3-yl-ethylamine instead of aniline followed by hydrolysis of the ethyl ester as described in example 94 to give the title compound. The purity is more than 90 percent. M + H⁺663.5。

Example 98

(1R, 2S) -1- { [ (2S, 4R) -1- [ (1S) -1- ((1S, 2R) -2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4- (methyl-phenethyl-carbamoyloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (98)

Compound 92 was treated as described for the preparation of compound 93 but with N-methylphenethylamine instead of aniline followed by hydrolysis of the ethyl ester as described in example 94 to give the title compound. The purity is more than 90 percent. M + H⁺690.6。

Example 99

(1R, 2S) -1- ({ (2S, 4R) -4-Benzylcarbamoyloxy-1- [ (1S) -1- ((1S, 2R) -2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -pyrrolidine-2-carbonyl } -amino) -2-vinyl-cyclopropanecarboxylic acid (99)

Compound 92 was treated as described for the preparation of compound 93 but with benzylamine instead of aniline followed by hydrolysis of the ethyl ester as described in example 94 to give the title compound. The purity is more than 90 percent. M + H⁺662.4。

Example 100

(1R, 2S) -1- ({ (2S, 4R) -1- [ (1S) -1- ((1S, 2R) -2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4-phenethylcarbamoyloxy-pyrrolidine-2-carbonyl } -amino) -2-vinyl-cyclopropanecarboxylic acid (100)

Compound 92 was treated as described for the preparation of compound 93 but with phenethylamine instead of aniline followed by hydrolysis of the ethyl ester as described in example 94 to give the title compound. The purity is more than 90 percent. M + H⁺676.5。

Example 101

(1R, 2S) -1- ({ (4R) -1- { [2- (tert-Butoxycarbonyl) hydrazino ] carbonyl } -4- [ (7-methoxy-2-phenylquinolin-4-yl) oxy ] -L-prolyl } amino) -2-vinylcyclopropanecarboxylic acid ethyl ester (101)

To a solution of tert-butyl carbazate (0.3mmol) and p-nitrophenyl chloroformate (0.3mmol) in acetonitrile (6ml) was added solid sodium bicarbonate (0.48 mmol). The solution was stirred at room temperature for 5 hours and then cooled to 0 ℃. Compound 62(0.3mmol) dissolved in acetonitrile (10mL) was mixed together with diisopropylethylamine (0.75mmol) at 0 deg.C and then added to the above solution. The resulting mixture was stirred at room temperature overnight, then concentrated to dryness. The residue obtained is dissolved in DCM and then treated with citric acid at pH 4 and subsequently with NaHCO₃It was washed with water, dried over anhydrous sodium sulfate, filtered and concentrated to dryness. The crude product was dissolved in DCM and purified by column chromatography eluting with 0.1-0.2% MeOH in DCM to give the title compound (101 mg). Purity > 95% by HPLC. M + H⁺660.1。

Example 102

(1R, 2S) -1- ({ (4R) -1- { [2- (tert-Butoxycarbonyl) hydrazino ] carbonyl } -4- [ (7-methoxy-2-phenylquinolin-4-yl) oxy ] -L-prolyl } amino) -2-vinylcyclopropanecarboxylic acid (159)

The method A comprises the following steps: to a solution of compound 101(0.0115mmol) in THF-MeOH 2: 3(2ml) was added 1M LiOH (10 equivalents). The resulting solution was held at 50 ℃ for 60 minutes. After cooling to room temperature, HOAc (20 equiv.) was added thereto, followed by toluene (20mL), which was then concentratedAnd drying. The resulting residue was taken up in methanol and then purified by preparative LCMS to give the title compound (0.7 mg). Purity > 95% by HPLC. M + H⁺732.2。

The method B comprises the following steps: to a solution of tert-butyl carbazate (0.07mmol) and p-nitrophenyl chloroformate (0.07mmol) in acetonitrile (3ml) was added solid sodium bicarbonate (0.112 mmol). The solution was stirred at room temperature for 2.5 hours and then cooled to 0 ℃. Compound 103 (described below) (0.07mmol) dissolved in acetonitrile (10mL) was mixed together with diisopropylethylamine (0.175mmol) at 0 ℃ and then added to the above solution. The resulting mixture was stirred at room temperature overnight, then concentrated to dryness. The resulting crude product was dissolved in methanol and then purified by preparative LCMS to give the title compound (4.8 mg). Purity > 95% by HPLC. M + H⁺632.2。

Example 103

(1R, 2S) -1- { [ (2S, 4R) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (103)

To a solution of compound 12(0.067mmol) in THF-MeOH 2: 3(2mL) was added 10 equivalents of 1M LiOH. The solution was kept at 50 ℃ for 2.5 hours. After cooling to room temperature, 20 equivalents of HOAc were added, followed by toluene (2mL), which was then concentrated to dryness. The resulting residue was taken up in DCM and filtered to form the salt giving the title compound (0.07 mmol). Purity > 95% by HPLC. M + H⁺474。

Example 104

(1R, 2S) -1- ({ (4R) -1- (hydrazinocarbonyl) -4- [ (7-methoxy-2-phenylquinolin-4-yl) oxy ] -L-prolyl } amino) -2-vinylcyclopropanecarboxylic acid (104)

Compound (102) was placed in TFA-DCM 1: 2(3mL) for 60 min at room temperature. Toluene (1mL) was added. The sample was co-evaporated to dryness to give the title compound as a trifluoroacetate salt (10.5 mg). Purity > 95% by HPLC. M + H⁺532。

Example 105

(1R, 2S) -1- ({ (4R) -1- (hydrazinocarbonyl) -4- [ (7-methoxy-2-phenylquinolin-4-yl) oxy ] -L-prolyl } amino) -2-vinylcyclopropanecarboxylic acid ethyl ester (105)

Compound 101(50mg) was placed in TFA-DCM 1: 2(3mL) for 60 min at room temperature. Toluene (1mL) was added. The above samples were co-evaporated to dryness, then taken up in DCM and taken up with K₂CO₃Washing was conducted, drying was conducted over anhydrous sodium sulfate, and concentration to dryness, whereby the title compound (41.8mg) was given. Purity > 95% by HPLC. M + H⁺560。

Example 106

(1R, 2S) -1- ({ (4R) -1- [ (2-Benzylhydrazino) carbonyl ] -4- [ (7-methoxy-2-phenylquinolin-4-yl) oxy ] -L-prolyl } amino) -2-vinylcyclopropanecarboxylic acid ethyl ester (106)

To a solution of compound 105(0.037mmol) in MeOH: THF (4: 1) was added benzaldehyde (0.0448 mmol). The solution was stirred at room temperature for 18 hours. Borane-pyridine complex (0.37mmol) was added thereto, followed by HCl (37%, 400. mu.l). This solution was stirred for 1.5 hours, then filtered and concentrated to dryness. . The resulting crude product was dissolved in methanol and then purified by preparative LCMS to give the title compound (0.01 mmol). Purity > 95% by HPLC. M + H⁺650。

Example 107

(1R, 2S) -1- ({ (4R) -1- [ (2-Benzylhydrazino) carbonyl ] -4- [ (7-methoxy-2-phenylquinolin-4-yl) oxy ] -L-prolyl } amino) -2-vinylcyclopropanecarboxylic acid (107)

To a solution of compound 106(0.0101mmol) in THF-MeOH 2: 3(3mL) was added 10 equivalents of 1M LiOH. The solution was kept at 50 ℃ for 18 hours. After cooling to room temperature, the resulting sample was neutralized with HCl and concentrated to dryness. The resulting crude product was dissolved in DCM (2ml) and a solution of TFA: TES 1: 1(1ml) was added thereto. The resulting mixture was stirred at room temperature for 3 hours, then concentrated to dryness. The resulting crude product was dissolved in methanol and then purified by preparative LCMS to give the title compound (0.6 mg). Purity > 95% by HPLC. M + H⁺622。

Example 108

(1R, 2S) -1- { [ (2S, 4R) -1- ((1S) -1-azidomethyl-3-methyl-butylcarbamoyl) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid ethyl ester (108)

i) (2S) -methanesulfonic acid 2-tert-butoxycarbonylamino-4-methyl-pentyl ester

To a solution of ((1S) -1-hydroxymethyl-3-methyl-butyl) -carbamic acid tert-butyl ester (25g, 115mmol) in dichloromethane (500ml) cooled by an ice-water bath was added diisopropylethylamine (35.7g, 276mmol) and methanesulfonyl chloride (15.81g, 138mmol) in that order. The resulting solution was stirred overnight, during which time the mixture was allowed to warm gradually to ambient temperature. The resulting mixture was sequentially washed with water, 10% citric acid (aq), water and saturated NaHCO₃(aqueous solution) washing, then with Na₂SO₄Dried and concentrated to give a brown solid (32.6g, 96%) which was used in the next reaction without further purification.

ii) ((1S) -1-Azidomethyl-3-methyl-butyl) carbamic acid tert-butyl ester

In DMF at 80 ℃ fromThe mesylate from step i (32.6g, 110mmol) was treated with sodium azide (21.45g, 330mmol) for 24 h. The solvent was evaporated, the resulting residue was taken up in DCM, filtered and washed with saturated NaHCO₃(aqueous solution) washing. The obtained solution was treated with Na₂SO₄Drying and concentration were performed to give a brown oil, which was purified by flash chromatography using gradient ethyl acetate and hexane to give the title compound as a white solid (19.55g, 73%).

iii) (1S) -1-azidomethyl-3-methyl-butylamine

((1S) -1-Azidomethyl-3-methyl-butyl) -carbamic acid tert-butyl ester (9.64g, 39.78mmol) was treated with TFA (30ml) in DCM (150ml) for 3h, the mixture was evaporated under reduced pressure, the resulting residue was dissolved in ethyl acetate and taken up with 1MK₂CO₃Washing with aqueous solution and adding Na₂SO₄Dried and concentrated to give a yellow liquid (4.55g, 80%).

Compound 12 was treated with phosgene as described in example 13 to give the corresponding chlorocarbamate compound. The resulting chlorocarbamate (568mg, 1.13mmol) was dissolved in DCM-THF (1: 1, 10ml) solution and (1S) -1-azidomethyl-3-methyl-butylamine (401mg, 2.82mmol) and a large excess of NaHCO₃(s) adding thereto. The resulting mixture was stirred for 18 hours, filtered and washed with dilute citric acid (aq, pH 5). The organic layer was washed with Na₂SO₄Drying and evaporation were carried out to give the desired product as a pale yellow oil (837mg, 99%) with sufficient purity for the next step.

M+H⁺670.1。

Practice ofExample 109

(1R, 2S) -1- { [ (2S, 4R) -1- ((1S) -1-aminomethyl-3-methyl-butylcarbamoyl) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid ethyl ester (109)

A solution of compound 108(717mg, 1.07mmol) in THF (25ml) was reacted with PS-triphenylphosphine resin (diphenylphosphine polystyrene) (3.24g, 1.65mmol PPh)₃/g) and methanol (2.5ml) were shaken together for 78 hours. The mixture was filtered and the polymer was washed repeatedly with DCM and methanol. The combined filtrates were evaporated to give the title compound as a pale beige solid foam (685mg, 99%) which was more than 95% pure as determined by reverse phase HPLC. M + H⁺644.1。

General Process 1A for preparing Compounds 110 to 116

To a solution of the acid chloride (0.075mmol) in DCM (0.5ml) was added NaHCO₃(s) (60mg, 0.7mmol) and amine 109(25mg, 0.037mmol) in THF (1ml) the resulting mixture was stirred at room temperature overnight, filtered and shaken in the presence of PS-tris (tris) resin (tris- (2-aminoethyl) aminomethyl polystyrene) (3.91mmol/g, 50mg, 0.2mmol) for 5h the resulting mixture was filtered and evaporated, the resulting solid residue was dissolved in MeOH-THF (2: 1, 1.5ml) and treated with 1M LiOH (aq) (170. mu.l) at 50 ℃ for 2-16 h the reaction was monitored by HPLC-MS, the resulting mixture was acidified with acetic acid and evaporated to dryness, the resulting residue was dissolved in methanol and purified by reverse phase HPLC.

General procedure 1B for the preparation of Compounds 110-116

To the acid (0.039mmol) was added sequentially a solution of HATU (14.7mg, 0.039mmol) in DMF (0.5ml),A solution of amine 109(20mg, 0.031mmol) in DMF (0.5ml) and diisopropylethylamine (30. mu.l, 0.155 mmol). The resulting mixture was stirred for 16h, then the solvent was evaporated, the resulting residue was dissolved in DCM and water and saturated NaHCO₃The aqueous solution is washed. The solvent was evaporated and the resulting residue was dissolved in methanol-THF (2: 1, 1.5 ml). To this solution 1M LiOH (aq) (155. mu.l) was added and the resulting mixture was stirred at 60 ℃ for 3-5 hours. Glacial acetic acid (50 μ l) was added to it and the resulting mixture was concentrated, dissolved in methanol and purified by reverse phase HPLC.

Example 110

(1R, 2S) -1- { [ (2S, 4R) -1- { (1S) -1- [ (3-fluoro-benzoylamino) -methyl ] -3-methylbutylcarbamoyl } -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (110)

Preparation was carried out according to general method 1A, using 3-fluorobenzoyl chloride (12mg) as the acid chloride to give the title compound as a solid (13.6mg, 50%). M + H⁺738.1。

Example 111

(1R, 2S) -1- { [ (2S, 4R) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -1- ((1S) -3-methyl-1- { [ (pyridine-3-carbonyl) -amino ] -methyl } -butylcarbamoyl) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (111)

Preparation was carried out according to general method 1A, using nicotinoyl chloride (10.5mg) as the acid chloride, to give the title compound as a solid (10mg, 37%). M + H⁺721.1。

Example 112

(1R, 2S) -1- { [ (2S, 4R) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -1- ((1S) -3-methyl-1- { [ (pyrazine-2-carbonyl) -amino ] -methyl } -butylcarbamoyl) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (112)

Preparation was carried out according to general method 1B, using pyrazine-2-carboxylic acid (5mg) as the acid to give the title compound as a solid (5.7mg, 25%). M + H⁺722.1。

Example 113

(1R, 2S) -1- { [ (2S, 4R) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -1- ((1S) -3-methyl-1- { [ (thiophene-3-carbonyl) -amino ] -methyl } -butylcarbamoyl) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (113)

Preparation was carried out according to general method 1A, using thiophene-3-carbonyl chloride (11mg) to give the title compound as a solid (4.3mg, 16%). M + H⁺726.1。

Example 114

(1R, 2S) -1- { [ (2S, 4R) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -1- ((1S) -3-methyl-1- { [ (tetrahydro-furan-2-carbonyl) -amino ] -methyl } -butylcarbamoyl) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (114)

Preparation according to general method 1B, using tetrahydrofuran-2-carboxylic acid (4.5mg) as the acid gave the title compound as a solid (7.9mg, 36%). M + H⁺714.1。

Example 115

(1R, 2S) -1- { [ (2S, 4R) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -1- ((1S) -3-methyl-1- { [ (5-phenyl-oxazole-4-carbonyl) -amino ] -methyl } -butylcarbamoyl) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (115)

Preparation was carried out according to general method 1B, using 5-phenyl-oxazole-4-carboxylic acid (7.5mg) as the acid to give the title compound as a solid (7.5mg, 31%). M + H⁺787.1。

Example 116

(1R, 2S) -1- { [ (2S, 4R) -1- ((1S) -1- { [ (benzofuran-2-carbonyl) -amino ] -methyl } -3-methyl-butylcarbamoyl) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (116)

Preparation according to general method 1B, using benzofuran-2-carboxylic acid (6.5mg) as the acid gave the title compound as a solid (5.4mg, 23%). M + H⁺760.1。

General Process 2 for preparing Compounds 117 to 119

To a solution of sulfonyl chloride (0.075mmol) in DCM (0.5ml) was added NaHCO₃(s) (60mg) and a solution of amine 109(25mg, 0.037mmol) in THF (1 ml). The resulting mixture was stirred at room temperature for 18 hours, filtered and shaken with PS-tris (hydroxymethyl) aminomethane (trisamine) (tris- (2-aminoethyl) aminomethyl polystyrene, 3.91mmol/g,. about.50 mg) for 5 hours.

The mixture was filtered and the polymer was washed sequentially with DCM, THF and methanol. The solid residue obtained by evaporation of the combined filtrates was dissolved in methanol-THF (2: 1, 1.5ml) and treated with 1M LiOH (aq) (170. mu.l) at 50 ℃ with reaction times varying depending on the actual structure from 18 hours to one week. The reaction was monitored by HPLC-MS. The resulting mixture was acidified with acetic acid and evaporated to dryness. The resulting residue was dissolved in methanol and purified by reverse phase HPLC.

Example 117

(1R, 2S) -1- ({ (2S, 4R) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -1- [ (1S) -3-methyl-1- (phenylmethanesulfonylamino-methyl) -butylcarbamoyl ] -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (117)

Preparation was carried out according to general method 2, using α -toluenesulfonyl chloride (14mg) as the sulfonyl chloride to give the title compound as a white solid (4.9mg, 17%). M + H⁺770.1。

Example 118

(1R, 2S) -1- [ ((2S, 4R) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -1- { (1S) -3-methyl-1- [ (5-methyl-isoxazole-4-sulfonylamino) -methyl ] -butylcarbamoyl } -pyrrolidine-2-carbonyl ] -nitrogen } -2-vinyl-cyclopropanecarboxylic acid (118)

Preparation according to general method 2 using 5-methyl-isoxazole-4-sulfonyl chloride (14mg) as the sulfonyl chloride gave the title compound as a white solid (1.6mg, 6%). M + H⁺761.0。

Example 119

(1R, 2S) -1- [ ((2S, 4R) -1- { (1S) -1- [ (5-isoxazol-3-yl-thiophene-2-sulfonylamino) -methyl ] -3-methyl-butylcarbamoyl } -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (119)

Preparation was carried out according to general method 2, using 5-isoxazol-3-yl-thiophene-2-sulfonyl chloride (19mg) as the sulfonyl chloride,the title compound was given as a white solid (3.0mg, 10%). M + H⁺828.98。

Example 120

1- { [1- (N' -tert-Butoxycarbonyl-N-hept-6-enyl-hydrazinocarbonyl) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid ethyl ester (120)

Compound 12(200mg, 0.4mmol) was dissolved in tetrahydrofuran (10 ml). A teaspoon of sodium bicarbonate was added, followed by phosgene (1.8. mu.l, 1.9M in toluene). The resulting mixture was stirred for 30 minutes and filtered. The solvent was evaporated and the resulting crude chloride was redissolved in dichloromethane (10 ml). Sodium bicarbonate (1 teaspoon) and tert-butyl N' -hept-6-enyl-hydrazinocarboxylate (182mg, 0.8mmol) were added thereto. The reaction mixture was stirred at room temperature for 40 h. Then filtered and purified by silica chromatography (1% methanol in ether → 2% methanol in ether) to give the pure title product (240mg, 79%).

Example 121

14-tert-Butoxycarbonylamino-18- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2, 15-dioxo-3, 14, 16-triaza-tricyclo [14.3.0.0^＊4，6^＊]Nineteen-carbon-7-ene-4-carboxylic acid ethyl ester (121)

Compound 120(200mg, 0.26mmol) was dissolvedIn degassed dichloromethane (30 ml). Hoveyda-Grubbs catalyst II (16mg, 0.026mmol) was then added thereto and the mixture was refluxed overnight under an argon atmosphere. The solvent was then evaporated and the resulting crude product was purified by silica chromatography (1% methanol in ether) to give 39mg (20%) of the title product. MS (M + H)⁺)728.2。

Example 122

14-tert-Butoxycarbonylamino-18- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2, 15-dioxo-3, 14, 16-triaza-tricyclo [14.3.0.0^＊4，6^＊]Nineteen-carbon-7-ene-4-carboxylic acid (122)

Compound 121(39mg, 0.054mmol) was dissolved in tetrahydrofuran (3.5ml), water (1.75ml) and methanol (1.75 ml). Lithium hydroxide (430. mu.l, 1M in water) was then added thereto and the reaction was stirred at room temperature for 24 hours. The volume was reduced to half and water (10ml) was added. Acidification (pH 5) followed by extraction with chloroform gave 34mg (90%) of pure acid 179. MS (M + H)⁺)700.2。

Example 123

1- { [1- (N' -tert-Butoxycarbonyl-N-hex-5-enyl-hydrazinocarbonyl) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid ethyl ester (123)

The title compound was prepared from compound 12(800mg, 1.6mmol) and tert-butyl N' -hex-5-enyl-hydrazinecarboxylate (620mg, 2.9mmol) according to the method described in example 120 to give 1g (85%) of product. MS (M + H)⁺)742.37。

Example 124

13-tert-Butoxycarbonylamino-17- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2, 14-dioxo-3, 13, 15-triaza-tricyclo [13.3.0.0^＊4，6^＊]Octadecan-7-ene-4-carboxylic acid ethyl ester (124)

Compound 123(400mg, 0.54mmol) was treated according to the method described in example 121 to give the crude product. Purification by silica gel chromatography (1% methanol in ether) gave the title product (67mg, 17%). MS (M + H)⁺)714.29。

Example 125

13-tert-Butoxycarbonylamino-17- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2, 14-dioxo-3, 13, 15-triaza-tricyclo [13.3.0.0^＊4，6^＊]Octadecan-7-ene-4-carboxylic acid (125)

The title compound was prepared from compound 124(67mg, 0.09mmol) by the same procedure as described for compound 122 to give 46mg (71%) of the pure acid. To prepare this compound, 1, 2-dichloroethane is substituted in the extraction stepChloroform. MS (M + H)⁺)686.33。

Example 126

13-Tertiary amino-17- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2, 14-dioxo-3, 13, 15-triaza-tricyclo [13.3.0.0^＊4，6^＊]Octadecan-7-ene-4-carboxylic acid (126)

Compound 125(10mg) was dissolved in dichloromethane (4 ml). Trifluoromethanesulfonic acid (4ml) was added thereto and the resulting mixture was left at 50 ℃ for 6 hours. The solvent was removed and the resulting residue was washed with acetonitrile to give 3mg of pure title product (35%). MS (M + H)⁺)586.25。

Example 127

1- { [1- (1-methoxycarbonyl-oct-7-enylcarbamoyl) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid ethyl ester (127)

The title compound was prepared from compound 12(380mg, 0.758mmol) and methyl 2-aminonon-8-enyl-carboxylate (250mg, 1.89mmol) using the conditions described in example 120 to give the pure product (405mg, 75%).

Example 128

19- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2, 16-dioxo-3, 15, 17-triaza-tricyclo [15.3.0.0^＊4，6^＊]Eicosa-7-ene-4, 14-dicarboxylic acid 4-ethyl ester 14-methyl ester (128)

Compound 127(170mg, 0.2385mmol) was dissolved in dichloromethane (40ml) and degassed by bubbling nitrogen gas for 20 minutes. Hoveyda-Grubbs catalyst II (10mg, 0.016mmol, 6.7 mol%) was then added thereto and the mixture was refluxed under a nitrogen atmosphere overnight. The solvent was then evaporated, the catalyst and salts removed by flash chromatography (5% methanol in chloroform) and the crude product (120mg, 73% yield, 85-90% purity) used in the next step. MS (M + H)⁺)685

Example 129

19- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2, 16-dioxo-3, 15, 17-triaza-tricyclo [15.3.0.0^＊4，6^＊]Eicosa-7-ene-3, 14-dicarboxylic acid 3-ethyl ester (129)

Compound 128(120mg, 0.175mmol) was dissolved in dioxane (9ml) and water (6 ml). Lithium hydroxide (12mg, 0.526mmol) was added to it and the reaction was stirred at room temperature for 3.5 h. The mixture was acidified with acetic acid to pH 5 and co-evaporated with toluene. The crude product obtained was used in the next step. MS (M + H)⁺)671。

Example 130

14- [ (cyclohexyl-methylcarbamoyl-methyl) -19- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2, 16-dioxo-3, 15, 17-triaza-tricyclo [15.3.0.0^＊4，6^＊]Eicosa-7-ene-4-carboxylic acid 3-ethyl ester (130)

Compound 129 (crude, 100mg), indanolamine (33mg, 0.209mmol) and Hunig's base (DIEA) (0.2ml) were dissolved in DMF (14 ml). After stirring at 0 ℃ for 10 minutes, HATU was added thereto. The reaction was monitored by LC-MS. The conversion after 5 hours was 100%. DMF and DIEA were removed in vacuo. The resulting residue was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried and concentrated under vacuum. 120mg of crude product are obtained, which is purified by preparative HPLC to give 21mg (25%) of the title product. MS (M + H)⁺)802。

Example 131

14- [ (cyclohexyl-methylcarbamoyl-methyl) -19- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2, 16-dioxo-3, 15, 17-triaza-tricyclo [15.3.0.0^＊4，6^＊]Eicosa-7-ene-4-carboxylic acid (131)

To a solution of ester 130(19mg, 0.024mmol) in a mixture of THF (0.2ml) and methanol (0.3ml) was added a solution of LiOH (6mg, 0.237mmol) in 0.15ml of water. The resulting mixture was stirred at 60 ℃ for 3.5 hours. After cooling to room temperature, acetic acid was added thereto (30 equivalents). The mixture was co-evaporated with toluene. The resulting residue was partitioned between chloroform and an aqueous phase, the aqueous phase was extracted with chloroform and ethyl acetate, and the organic phases were combined, dried over sodium sulfate and evaporated to give 15mg of pure product.

MS(M+H⁺)774。

Example 132

[ [ 14-cyclopropanesulfonylaminocarbonyl-17- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2, 14-dioxo-3, 13, 15-triaza-tricyclo [13.3.0.0 ]^＊4，6^＊]Octadec-7-en-13-yl]-carbamic acid tert-butyl ester (132)

To acid 125(19mg, 0.028mmol) in 0.5ml DMF was added 5.5mg (0.044mmol) DMAP and 10.7mg (0.056mmol) EDC. After stirring for 6.5h, 20mg of cyclopropylsulfonamide and 0.04ml of DBU were added. The mixture was stirred overnight, acidified with 5% citric acid (in water) and extracted with ethyl acetate. Drying, evaporation, purification by 5% to 10% methanol in chloroform (or preparative LC-MS) gave 8mg of the title compound (37%).

MS(M+H⁺)783。

Example 133

4- [2- (2-isopropylamino-thiazol-4-yl) -7-methoxy-quinolin-4-yloxy ] -pyrrolidine-1, 2-dicarboxylic acid 1-tert-butyl ester (133)

To a stirred solution of N-Boc-trans-4-hydroxy-L-proline (221mg, 0.96mmol) in DMSO was added potassium tert-butoxide (320mg, 2,9 mmol). After 1 hour, 2- [2- (isopropylamino) -1, 3-thiazol-4-yl ] -7-methoxyquinolin-4-ol (319mg, 0,96mmol) was added to it and the mixture was stirred at 70 ℃ for 72 hours. The resulting mixture was diluted with water and extracted with ethyl acetate. The product was used without further purification. Yield 429mg, 85%.

Example 134

2- (1-ethoxycarbonyl-2-vinyl-cyclopropylcarbamoyl) -4- [2- (2-isopropylamino-thiazol-4-yl) -7-methoxy-quinolin-4-yloxy ] -pyrrolidine-1-carboxylic acid tert-butyl ester (134)

Compound 133(300mg, 0.56mmol) was reacted with ethyl 1-amino-2-vinyl-cyclopropanecarboxylate (130mg, 0.84mmol) as described in example 11 to give the title compound (302mg, 80%).

Example 135

1- ({4- [2- (2-isopropylamino-thiazol-4-yl) -7-methoxy-quinolin-4-yloxy ] -pyrrolidine-2-carbonyl } -amino) -2-vinyl-cyclopropanecarboxylic acid ethyl ester (135)

Compound 134(302mg, 0.45mmol) was treated as described in example 12, giving the title compound (195mg, 76%).

Example 136

1- ({1- [1- (2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4- [2- (2-isopropylamino-thiazol-4-yl) -7-methoxy-quinolin-4-yloxy ] -pyrrolidine-2-carbonyl } -amino-2-vinyl-cyclopropanecarboxylic acid ethyl ester (136)

Compound 135(80mg, 0.14mmol) was treated as described in example 13, giving the title product (87mg, 72%).

Example 137

1- ({1- [1- (2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4- [2- (2-isopropylamino-thiazol-4-yl) -7-methoxy-quinolin-4-yloxy ] -pyrrolidine-2-carbonyl } -amino-2-vinyl-cyclopropanecarboxylic acid (137)

Ethyl ester compound 136(80mg, 0.09mmol) was hydrolyzed as described in example 14 to give the title product after preparation of LC-MS (7.5mg, 10%).

Example 138

1- { [ 1-ethylcarbamoyl-4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid ethyl ester (138)

Compound 12(330mg, 0.66mmol), phosgene (1.6ml, 1.9M in toluene, 3mmol) and hex-5-enylamine hydrochloride (500mg, 3.68mmol) were reacted as described in example 120 to give the pure title product (328mg, 80%). MS (M + H)⁺)627。

Example 139

17- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2, 14-dioxo-3, 13, 15-triaza-tricyclo [13.3.0.0^＊4，6^＊]Octadecan-7-ene-4-carboxylic acid ethyl ester (139)

Compound 138(200mg, mol) was dissolved in degassed anhydrous dichloromethane (200ml) and bubbled with nitrogen. Hoveyda-Grubbs (second generation) catalyst (5mg, 2 mol%) was then added thereto and the reaction mixture was refluxed for 20h under a nitrogen atmosphere. The resulting mixture was cooled to room temperature and concentrated by rotary evaporation. The resulting oil was purified by column chromatography on YMC silica (ethyl acetate-toluene 1: 1 to 9: 1) to give 55mg of the title compound as a beige solid. The yield was 29%. MS (M + H)⁺)599。

Example 140

17- (7-methoxy-2-phenyl-quinolin-4-yloxy) -2, 14-dioxo-3, 13, 15-triaza-tricyclo [13.3.0.0^＊4，6^＊]Octadecan-7-ene-4-carboxylic acid (140)

In a closed vial, compound 139(55mg, mol) was dissolved in 2ml methanol, mixed with 3 equivalents of aqueous NaOH and heated at 60 ℃ for 2 h. Then, the resulting reaction mixture was extracted into ethyl acetate. The aqueous solution was collected and acidified to pH 2 with 1N HCl solution. The resulting solution was concentrated by rotary evaporation, dissolved in methanol and purified by preparative HPLC (acetonitrile-water) to give 34mg of the title product. The yield was 65%. MS (M + H)⁺)571。

Example 141

1- { [1- {1- [ (cyclohexyl-methoxycarbonyl-methyl) -carbamoyl ] -2, 2-dimethyl-propylcarbamoyl ] -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (141)

Compound 103 was dissolved in dichloromethane (3ml), and solid sodium bicarbonate (100mg) and 20% phosgene toluene solution (0.1ml) were added thereto. After 30 minutes at room temperature, the mixture was concentrated to dryness. To this was added (S) - (2S-2-amino-3, 3-dimethyl-butyrylamino) -cyclohexyl-acetic acid methyl ester (12mg in 2ml dichloromethane). After stirring at room temperature for 3 days, the reaction mixture was filtered, concentrated to dryness and purified on preparative HPLC-MS, thus giving the title product (4.4 mg). M + H⁺784.7。

Example 142

1- { [1- (1-aminomethyl-2, 2-dimethyl-propylcarbamoyl) -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid ethyl ester (142)

The title compound was prepared from compound 12(1.22g, 2.43mmol) by the method described for the preparation of compound 108, but using 2-tert-butoxycarbonylamino-3, 3-dimethyl-butyl methanesulfonate instead of 2-tert-butoxycarbonylamino-4-methyl-pentyl methanesulfonate, in example 165, step 1. Reduction of the azide as described in example 109 gave the title compound (1.49g, 95%). Purity > 95% according to HPLC. M + H⁺644.2。

Example 143

1- { [1- (2, 2-dimethyl-1- { [ thiophene-3-carbonyl) -amino ] -methyl } -propylcarbamoyl } -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (143)

Compound 142(100mg, 0,155mmol) was reacted according to general method 1A for the preparation of compounds 110-116 using thiophene-3-carbonyl chloride (28.5mg, 0.194mmol) as the acid chloride to give the title compound as a white solid (45mg, 40%). Purity > 95% according to HPLC. M + H⁺726。

Example 144

1- { [1- {1- [ (5-isoxazol-3-yl-thiophene-2-sulfonylamino) -methyl ] -2, 2-dimethyl-propylcarbamoyl } -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (144)

Compound 142(25mg, 0.039mmol) was reacted according to general method 1A for the preparation of compounds 110-116 using 5-isoxazol-3-yl-thiophene-2-sulfonyl chloride (14.5mg, 0.058mmol) as the acid chloride to give the title compound as a white solid (1.8mg, 6%). Purity > 94% according to HPLC. M + H⁺829。

Example 145

1- { [1- (3-fluoro-benzoylamino) -methyl ] -propylcarbamoyl } -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (145)

Compound 142(25mg, 0.039mmol) was reacted according to general method 1A for the preparation of compounds 110-116 using 3-fluorobenzoyl chloride (12.3mg, 0.078mmol) as the acid chloride to give the title compound as a white solid (4.1mg, 14%). Purity > 94% according to HPLC. M + H⁺738。

Example 146

1- { [1- (1- { [ (-furan-3-carbonyl) -amino ] -methyl ] -2, 2-dimethyl-propylcarbamoyl } -4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (146)

Compound 142(25mg, 0.039mmol) was reacted according to general method 1B for the preparation of compounds 110-116 using 3-furanoic acid (5.5mg, 0.049mmol) as the acid chloride to give the title compound as a white solid (4.1mg, 14%). Purity > 99% according to HPLC. M + H⁺710。

Example 147

4- (7-methoxy-2-phenyl-quinolin-4-yloxy) -pyrrolidine-1, 2-dicarboxylic acid 2- [ (1-cyclopropanesulfonylaminocarbonyl-2-vinyl-cyclopropyl) -amide ]1- [ (2, 2-dimethyl-1- { [ (thiophene-3-carbonyl) amino ] -methyl } -propyl) -amide (147)

To a solution of compound 143(42.2mg, 0.058mmol) in chloroform (3mL) was added cyclopropylsulfonamide (14mg, 0.116mmol) followed by diisopropylethylamine (60.5. mu.l, 0.17 mmol). The resulting solution was stirred at room temperature for 10 minutes, then at-20 ℃ for 30 minutes. Then PyBOP (121mg, 0.116mmol) was added as a solid. The solution was kept at-20 ℃ for 10 days. The resulting solution was then poured into NaHCO₃In aqueous solution (saturated) and washed with water. The resulting organic layer was dried, concentrated and purified by HPLC to give the title compound as a white solid (2.3mg, 0.0028mmol), which was determined by HPLCThe purity is more than 95 percent. M + H⁺830。

Example 148

Fmoc-4-amino-2- (1-ethoxycarbonyl-2-vinyl-cyclopropylcarbamoyl) -pyrrolidine-1-carboxylic acid tert-butyl ester (148)

(2S, 4R) Fmoc-4-amino-1-Boc-pyrrolidine-2-carboxylic acid (5.3g, 11.8mmol) was dissolved in DCM (100ml) and HATU (4.94g, 12.99mmol), DIEA (4.63ml, 26.57mmol) and ethylvinylcyclopropylglycine (2.26g, 11.81mmol) were added sequentially thereto. The mixture was stirred at room temperature for 16h, then diluted with DCM (50ml) and citric acid (10% aq), water, NaHCO₃(saturated aqueous solution) and water. The organic phase obtained is treated with Na₂SO₄Drying and concentration were performed to give a beige solid foam (8.11g), which was subjected to silica gel column chromatography to give the title compound (7.14g, 12.11 mmol).

Example 149

1- [ (Fmoc-4-amino-pyrrolidine-2-carbonyl) -amino ] -2-vinyl-cyclopropanecarboxylic acid ethyl ester (149)

Compound 148(3.65g, 6.04mmol) was treated with a solution of TFA/DCM (10ml TFA, 50ml DCM) for 2.5h and then concentrated to give the title compound (2.99g, 6.12 mmol).

Example 150

1- ({ Fmoc-4-amino-1- [1- (2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -pyrrolidine-2-carbonyl } -amino) -2-vinyl-cyclopropanecarboxylic acid ethyl ester (150)

The aminoproline derivative 149(2.96g, 6.04mmol) was stirred with phosgene (1.93M in toluene, 4ml, 7.55mmol) for 10 minutes. The solvent and excess phosgene were evaporated. The resulting residue was dissolved in DCM (30ml) and a solution of t-Bug-aminoindanol (1.9g, 7.24mmol) in DCM (30ml) was added followed by NaHCO₃(2g) In that respect The mixture was stirred for 48h, then diluted with DCM, water, 10% citric acid and NaHCO₃(saturated aqueous solution) and Na₂SO₄Dried and evaporated to dryness. The resulting residue was subjected to column chromatography (EtOAc-hexane 0-30%) to provide the title compound (1g, 1.3 mmol).

Example 151

1- ({ 4-amino-1- [1- (2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -pyrrolidine-2-carbonyl } -amino) -2-vinyl-cyclopropanecarboxylic acid ethyl ester (151)

Compound 150(595mg, 0.765mmol) was dissolved in dinDMF (20ml) and treated with Si-piperazine (0.08mmol/g, 4.78g, 3.82mmol) for 48 h.

The silica was filtered and washed once with DMF and then with several portions of DCM. The solvent was evaporated and the resulting residue was subjected to column chromatography to give the title compound (170mg, 0.3 mmol).

Example 152

1- ({1- [1- (2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4- [ (pyridine-3-carbonyl) -amino ] -pyrrolidine-2-carbonyl } -amino) -2-vinyl-cyclopropanecarboxylic acid (152)

To a stirred solution of compound 151(35mg, 0.064mmol) in DCM (1ml) were added DIEA (0.12mmol, 19. mu.l) and nicotinoyl chloride hydrochloride (0.12mmol, 17 mg). The solution was stirred at room temperature for 18h, PS-tris (hydroxymethyl) aminomethane (trisamine) was added thereto, and then stirred at room temperature for 4 h. After filtration, the resulting solution was treated with citric acid (10% aqueous solution) and NaHCO₃(saturated aqueous solution) and the organic phase obtained was washed with Na₂SO₄Dried and concentrated. The resulting residue was dissolved in THF: MeOH (2: 1, 1.5 ml). LiOH (1N aqueous solution, 3.2mmol, 320. mu.l) was added thereto. The resulting solution was stirred at 60 ℃ for 24 hours. Acetic acid was added thereto, which was then concentrated. The resulting residue was used for methanol neutralization and subjected to HPLC purification to give the title compound (19.5mg, 0.03 mmol). Purity > 98% by HPLC. M + H⁺633.1。

Example 153

1- ({1- [1- (2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4-phenylacetamido-pyrrolidine-2-carbonyl } -amino) -2-vinyl-cyclopropanecarboxylic acid (153)

Prepared as described in example 152, but using phenylacetyl chloride instead of nicotinoyl chloride hydrochloride, to give the title compound (12.7mg, 0.019 mmol). Purity > 90% by HPLC. M + H⁺646.1。

Example 154

1- ({1- [1- (2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4- [ (5-methyl-3-phenyl-isoxazole-4-carbonyl) -amino ] -pyrrolidine-2-carbonyl } -amino) -2-vinyl-cyclopropanecarboxylic acid (154)

Prepared as described in example 152, but using benzene 5-methyl-3-phenylisoxazole-4-carbonyl chloride instead of nicotinoyl chloride hydrochloride, to give the title compound (3.6mg, 00055 mmol). Purity > 98% by HPLC. M + H⁺713.1。

Example 155

1- { [1- [1- (2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4- (3-phenyl-ureido) -pyrrolidine-2-carbonyl ] -amino } -2-vinyl-cyclopropanecarboxylic acid (155)

To a stirred solution of compound 151(30mg, 0.054mmol) in acetonitrile/dichloromethane (2: 1, 3ml) were added triethylamine (0.0648mmol, 9. mu.l) and phenyl isocyanate (0.0648mmol, 7. mu.l). The resulting solution was stirred at room temperature for 3h, and then methanol (1ml) was added thereto, followed by concentration thereof. The resulting residue was dissolved in methanol and purified by HPLC to give the ester compound as a white solid (32.7mg, 0.047mmol) with a purity of > 95% by HPLC. M + H⁺675.31. 1N aqueous LiOH (0.47mmol, 475. mu.l) was added to the above ester in THF: MeOH (2: 1). The reaction was stirred at 50 ℃ for 15 minutes and then at 8 ℃ for 12h, then acetic acid (0.98mmol, 53. mu.l) was added to it and concentrated. The resulting residue was dissolved in methanol and purified by HPLC to give the title compound as a white solid (3.8mg, 0.006mmol) with a purity of > 98% by HPLC. M + H⁺675.31。

Example 156

1- ({ 4-benzenesulfonylamino-1- [1- (2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -pyrrolidine-2-carbonyl } -amino) -2-vinyl-cyclopropanecarboxylic acid (156)

To a stirred solution of compound 151(30mg, 0.054mmol) in DCM (2ml) was added DIEA (0.0648mmol, 11.5. mu.l) and benzenesulfonyl chloride (0.0648mmol, 11.5. mu.l) in that order. The solution was stirred at room temperature for 3h, then concentrated. The resulting residue was dissolved in methanol and purified by HPLC to give the ester compound as a white solid (17.9mg, 0.0257mmol) with a purity of > 95% by HPLC. M + H⁺696.24. 1N aqueous LiOH solution (0.25mmol, 257. mu.l) was added to the above ester dissolved in THF: MeOH (2: 1). The reaction was stirred at 50 ℃ for 1.5h, then acetic acid (0.98mmol, 53. mu.l) was added. The above solution was concentrated. The resulting residue was dissolved in DCM and washed with water; the aqueous phase was acidified to pH 5 and then extracted with dichloromethane and ethyl acetate. The combined organic phases are washed with Na₂SO₄Dried and concentrated to give the title compound as a white solid (7.1mg, 0.01mmol) with a purity of > 98% by HPLC. M + H⁺668.19。

Example 157

1- { [1- [1- (2-hydroxy-indan-1-ylcarbamoyl) -2, 2-dimethyl-propylcarbamoyl ] -4- (3-phenyl-thioureido) -pyrrolidine-2-carbonyl } -amino) -2-vinyl-cyclopropanecarboxylic acid (157)

To a stirred solution of compound 151(30mg, 0.054mmol) in acetonitrile (3ml) were added TEA (0.0648mmol, 9. mu.l) and phenyl isothiocyanate (0.0648mmol, 7.8. mu.l) sequentially. The solution was stirred at room temperature for 16h, then concentrated. The resulting residue was dissolved in methanol and purified by HPLC to give the ester compound as a white solid (22.7mg, 0.0328mmol) with a purity of > 95% by HPLC, M + H⁺691.2. 1N aqueous LiOH (0.33mmol, 328. mu.l) was added to the above ester in THF: MeOH (2: 1). The reaction was stirred at 50 ℃ for 2.5h, then acetic acid (0.98mmol, 53. mu.l) was added. The solution was concentrated. The resulting residue was dissolved in dichloromethane and washed with water, and the resulting aqueous phase was extracted with EtOAc. The combined organic phases are washed with Na₂SO₄Dried and concentrated to give the title compound as a white solid (7.2mg, 0.01mmol) as determined by HPLCThe purity is more than 98 percent. M + H⁺663.26。

Measurement of

The activity of the compounds of the present invention against the flavivirus (e.g., HCV) NS3 protease is conveniently determined using conventional in vitro (enzyme) assays or cell culture assays.

One useful assay is the bartenshellager replicon assay disclosed in EP 1043399. Another replicon assay is described in WO 03064416.

Suitable enzyme assays involving inhibition of full-length hepatitis C NS3 are essentially described in Poliakov, 2002Prot Expression&Purification 25363371. Briefly, the depsipeptide substrate Ac-DED (Edans) EEAbu Ψ [ COO [ ]]ASK(Dabcyl)-NH₂Hydrolysis of (Anaspec, SanJos de, USA) was measured by spectrofluorimetry in the presence of the peptide cofactor KKGSVVVIVGRIVLSGK, as described by Landro, 1997 Biochem 369340-. The enzyme (1nM) is incubated with 25. mu.M cofactor and the inhibitor in a buffer (such as 50mM HEPES, pH7.5, 10mM DTT, 40% glycerol, 0.1% n-octyl-beta-D-glucoside) for 10 minutes at about 30 ℃ where the reaction is initiated by the addition of a matrix, typically 0.5. mu.M matrix. Inhibitors are typically dissolved in DMSO, sonicated for 30s and spun. The above solutions are generally stored at-20 ℃ between measurements.

Another enzyme activity assay is described in WO 0399316, which utilizes the HCV NS3/4A protease complex FRET peptide assay. The purpose of this in vitro assay was to determine the inhibitory effect of the compounds of the invention on HCV NS3 protease complexes derived from BMS, H77C or J416S strains, as described below. This assay provides an indication of how effective the compounds of the present invention are in inhibiting HCV proteolytic activity.

Sera were taken from patients infected with HCV. Design of HCV genome (BMS strain) a full-length cDNA template was constructed from a DNA fragment obtained by reverse transcription-PCR (RT-PCR) of serum RNA, and using primers selected according to homology between other genotype la strains. Genotype Ia was determined for HCV isolates according to the classification of Simmonds et al (see P Simmonds, KA Rose, S Graham, SW Chan, F McOmish, BC Dow, EA Follett, PL Yap and H Marsden, J.Clin. Microbiol., 31(6), 1493-1503 (1993)). The amino acid sequence of the non-structural region NS2-5B was shown to be > 97% identical to HCV genotype Ia (H77C) and 87% identical to genotype Ib (J4L 6S). Infectious clones H77C (Ia genotype) and J4L6S (Ib genotype) can be obtained from r.purcell (NIH) and are disclosed in their sequence in Genbank (AAB67036, see Yanagi, m., Purcell, r.h., Emerson, s.u., and bukh.proc.natl.acad.sci.u.s.a.94(16) 8738-.

BMS, H77C, and J4L6S strains are conventional strains that produce recombinant NS3/4A protease complex. DNA encoding the recombinant HCV NS3/4A protease complex (amino acids 1027-1711) for these strains was manipulated as described in P.Gallinari et al (see Gallinari P, Paolini C, Brennan D, Nardi C, Steinkuhler C, De France sco R.biochemistry.38 (17): 562032, (1999)). Briefly, a lytic tail of tri-lysine was added to the 3' -end of the 30 NS4A coding region. The cysteine at position P1 of the NS4A-NS4B cleavage site (amino acid 1711) was converted to glycine to avoid proteolytic cleavage of the lysine tag. In addition, a cysteine to serine mutation at amino acid 1454 can be introduced by PCR to prevent autolytic cleavage of the NS3 helicase domain. Variant DNA fragments can be cloned in pET21b bacterial expression vector (Novagen) and NS3/4A complex can be expressed in Escherichia coli strain BL21(DE3) (Invitrogen) as described in P.Galliari et al (see Galliari P, Brennan D, Nardi C, Brunettim, Tomei L, Steinkuhler C, De France sco R., J Virol.72 (8): 6758-69(1998)) and modifications thereof. Briefly, NS3/4A expression was induced by treatment with 0.5mM isopropyl-. beta. -D-thiogalactopyranoside (IPTG) for 22 hours at 20 ℃. General fermentation (10l) yieldApproximately 80g of wet cell slurry was produced. The cells were resuspended in 25mM N-2- (hydroxyethyl) piperazine-N' -2- (ethanesulfonic acid) (HEPES), pH7.5, 20% glycerol, 500mM sodium chloride (NaCl), 0.5% Triton-X100, 1. mu.g/mL lysozyme, 5mM magnesium chloride (MgCl)₂)1 μ g/mL DnaseI, 5mM β -mercaptoethanol (BME), protease inhibitor-ethylenediaminetetraacetic acid (EDTA) free (Roche) in lysis buffer (10mL/g), homogenized and incubated in VC for 20 min. The homogenate was sonicated and clarified by ultracentrifugation at 235000g for 1 hour at 4 ℃.

Imidazole was added to the supernatant to a final concentration of 15mM and its pH was adjusted to 8. The crude protein extract was loaded onto a nickel nitrilotriacetate (Ni-NTA) column pre-equilibrated with buffer B (25n-tM 20 HEPES, pH 820% glycerol, 500mM NaCl, 0.5% Triton-XIOO, 15mM imidazole, 5mM BMME). The sample was loaded at a flow rate of 1 mL/min. The column was washed with 15 column volumes of buffer C (same as buffer B except that it contained 0.2% Triton-X100). The protein was eluted with 5 column volumes of buffer D (same as buffer C except 200mM imidazole).

Fractions containing the NS3/4A protease complex were pooled and loaded onto desalting column Superdex-S200 pre-equilibrated with buffer D (25MM HEPES, pH7.5, 20% glycerol, 300mM NaCl, 0.2% Triton-X1OO, lOmM BME). The sample was loaded at a flow rate of 1 mL/min. Fractions containing the NS3/4A protease complex 30 were collected and concentrated to approximately 0.5 mg/mL. The purity of the NS3/4A protease complex obtained from BMS, H77C and J4L6S strains was generally judged to be greater than 90% by SDS-PAGE and mass spectrometry analysis.

The enzyme is typically stored at-80 ℃, thawed on ice and diluted before use in assay buffer. The substrate used in the NS3/4A protease assay is suitably RET S1 (resonance energy transfer depsipeptide substrate; AnaPec, Inc. cat # 22991) (FRET peptide), described in anal. biochem.240 (2): 6067 (1996). The sequence of this peptide is presumably based on the natural cleavage site of NS4A/NS4B, except that an ester bond rather than an amide bond is present at the cleavage site. The peptide matrix was incubated with one of three recombinant NS3/4A complexes in the absence or presence of the compounds of the invention and the formation of fluorescent reaction products was followed in real time using Cytofluor Series 4000. The reagents used are listed below: HEPES and glycerol (ultrapure) are available from GIBCO-BRL. Dimethyl sulfoxide (DMSO) was obtained from Sigma. Beta-mercaptoethanol was obtained from Bio Rad.

Determination of buffer: 50mM HEPES, pH 7.5; 0.15M NaCl; 0.1% Triton; 15% of glycerol; 10mM BME. Matrix: mu.M final concentration (from 2mM stock solution 20 in DMSO, stored at-20 ℃ C.). HCV NS3/4 type A la (lb) at 2-3 nM final concentration (5. mu.M stock solution in 25mM HEPES, pH7.5, 20% glycerol, 300m.M NaCl, 0.2% Triton-X100, 10mM BME solution). For compounds whose potency is near the assay limit, the assay can be made more sensitive by adding 50 μ g/mL BSA to the assay buffer and/or reducing the terminal protease concentration to 300 pM.

The assay is desirably performed in a 96-well polystyrene blackboard (plate) from Falcon. Each well contained 25. mu.l of NS3/4A protease complex in assay buffer, 50. mu.l of the compound of the invention in 10% DMSO/assay buffer, and 25. mu.l of matrix in assay buffer. A control (no compound) was also prepared on the same assay plate. The enzyme complex is mixed with the compound or control solution, typically for 1 minute before the enzymatic reaction is initiated by the addition of the substrate. The assay plate is typically read instantaneously using a spectrophotometer such as the Cytofluor Series 4000 (persictive biosystems). The instrument is desirably set up to read a 340nm emission and 490nm excitation at 25 ℃. The reaction generally lasts about 15 minutes.

Percent inhibition can be calculated using the following equation.

100-[(dF_inh/dF_con)×100]

Where dF is the change in fluorescence over the linear range of the curve. Nonlinear curve fitting is applied to the inhibition-concentration data and 50% effective concentration (IC) is determined by using software such as Excel XI-fitting software using the following equation₅₀) And (3) calculating:

y＝A+((B-A)/(1+((C/x)^D)))。

the enzyme assay desirably employs the principle of Fluorescence Resonance Energy Transfer (FRET) to produce a spectral response to the result of NS4A/4B cleavage catalyzed by the HCV NS3 serine protease. The activity is typically measured in a continuous fluorescence assay using an excitation wavelength of 355nm and an emission wavelength of 500 nm. The rate of initiation was determined from 10 minute consecutive readings of the enhanced fluorescence intensity resulting from the NS3 protease catalyzed cleavage event.

Another enzyme assay may be performed as follows:

material

Recombinant HCV NS3 full-length enzyme can be prepared as shown in Poliakov et al Protein Expression & purification 25(2002) 363-371.

The NS4A cofactor desirably has the amino acid sequence KKGSVVIVGRIVLSGK (commercially available), typically prepared as a 10mM stock in DMSO.

FRET-matrix (Ac-Asp-Glu-Asp (EDANS) -Glu-Glu-Abu-phi- [ COO) Ala-Ser-Lys (DABCYL) -NH₂MW1548.60, available from naspec RET S1, ca. usa) and is typically prepared as a 1.61mM stock in DMSO. Aliquots (50. mu.l/tube) should be covered with aluminum foil to prevent direct light and stored at-20 ℃.

Reference compound-1, N-1725 with the sequence AcAsp-D-Gla-Leu-Ile-Cha-Cys, MW 830.95, commercially available from BACHEM, Switzerland, is typically prepared as a 2mM stock DMSO solution and stored as an aliquot at-20 ℃.

1M HEPES buffer, commercially available from Invitrogen Corporation, was stored at 20 ℃.

Glycerol was purchased from Sigma, 99% pure.

CHAPS, 3- [ (3-cholamidopropyl) dimethylammonium ] -1-propanesulfonate: available from Research Organics, Cleveland, OH44125, USA. MW614.90

DTT, DL-dithiothreitol (Cleland Reagent: DL-DTT) 99% pure, MW.154.2. And (3) storage: +4 ℃.

DMSO was purchased from SDS, 13124 Peypin, France. 99.5% purity.

TRIS, ultrapure (TRIS) is commercially available from ICN biomedicals inc.

N-dodecyl- β -D-maltoside, 98% minimum, available from Sigma, storage: at 20 ℃.

Device

Microtiter plate (Cliniplate, ThermoLab System scat No.9502890)

Eppendorf pipette

Biohit pipette, multiple dose

Ascent fluorometer, color filter pair ex 355nm, em 500nm

Method of producing a composite material

The test method comprises the following steps:

a10 mM stock solution of a compound of the present invention was prepared in DMSO. The stock solutions were stored at room temperature during the test, but were left at-20 ℃ during long-term storage.

Assay buffer a:

50mM HEPES buffer, pH7.5, 40% glycerol, 0.1% CHAPS

And (3) storage: at room temperature

10mM DTT (stored in aliquots at-20 ℃ C. and fresh added in each assay) assay buffer B:

25mM TRIS pH7.5, 0.15M NaCl, 10% glycerol, 0.05% n-dodecyl-beta-D-maltoside

5mM DTT (stored in aliquots at-20 ℃ C. and fresh added in each assay) assay sequence:

preparation of reaction buffer (for one plate, 100 reactions) (buffer A)

1. Prepare 9500 μ l assay buffer (HEPES, pH7.5, 40% glycerol and 0.1% CHAPS) in deionized water. DTT was added to give a final concentration of 10mM (freshly prepared for each experiment).

2. Rapid melting of NS3 protease

3. 13.6. mu.l NS3 protease and 13.6. mu.l NS4A peptide were added and mixed appropriately. The mixture was left at room temperature for 15 minutes.

4. The enzyme stock solution was returned to liquid nitrogen or-80 ℃ as soon as possible.

Preparation of reaction buffer (for one plate, 100 reactions) (buffer B)

5. Prepare 9500 μ l assay buffer (TRIS, pH7.5, 0.15m nacl, 0.5mM EDTA, 10% glycerol and 0.05% n-dodecyl β -D-maltoside) in deionized water. Addition of DTT gave a final concentration of 5mM (freshly prepared for each experiment).

6. Rapid melting of NS3 protease

7. Add 27.2. mu.l NS3 protease and 13.6. mu.l NS4A peptide and mix appropriately. The mixture was left at room temperature for 15 minutes.

8. The enzyme stock solution was returned to liquid nitrogen or-80 ℃ as soon as possible.

Suppression ofPreparation of agent/reference Compound

Serial inhibitor dilutions were prepared and the inhibitor in DMSO diluted to 100 x final concentrations of 10, 1, 0.1, 0.01 and 0.001 μ M. The final DMSO concentration was 1% in the total reaction volume of 100. mu.l.

Serial dilutions of the reference compound were prepared and N-1725 was diluted to 100 x final concentrations of 120, 60, 30, 15, 7.5 and 3.75nM in DMSO.

Eight enzyme control wells were required for each experiment.

Blank wells contained 95 μ L buffer (no NS3 PR present), 1 μ L DMSO, and 5 μ L matrix.

Preparation of FRET matrix

The stock matrix (1.61mM) was diluted with assay buffer to 40. mu.M working solution. Preventing exposure to light.

Order of measurement

A96-well format plate was used, and the total assay volume per well was 100. mu.l.

1. Add 95. mu.l of assay buffer to each well

2. Add 1. mu.l inhibitor/reference Compound

3. Preincubation for 30 minutes at room temperature

4. The reaction was started by adding 5. mu.40. mu.M matrix solution (final concentration of 2. mu.M)

5. Readings were continued at ex-355 nm and em-500 nm for 20 minutes, and the increased fluorescence was monitored every minute.

6. Continuous curves (in the linear range, 8-10 time points) were plotted and the slope determined as the onset rate versus each individual inhibitor concentration.

7. % inhibition was calculated from enzyme control data.

Result processing

Results are expressed as% inhibition at a certain concentration (filter) or as Ki values in nM or μ M.

Calculation of% inhibition

The initial rate was determined from 10 minute consecutive readings of increased fluorescence intensity due to the NS3 protease catalyzed cleavage event. The change in the slope of the inhibitor compared to the enzyme control data gives the% inhibition at a certain concentration.

Calculation of Ki

All inhibitors were treated, assuming they all obeyed the competitive inhibition rules.

IC according to inhibition values at a series of inhibitor concentrations₅₀The value is calculated. The calculated values are used in the following equations:

Ki＝IC₅₀/(1+S/Km)

the plots were performed with the aid of two calculation procedures: grafit and Graphpad

Various compounds exemplified above according to the invention all show an IC of 1nM to 6.9 micromolar₅₀Value and sub-micromolar to micromolar ED₅₀The value is obtained.

Drug escape resistance forms and rates

Replicons cultured on microtiter plates can be used to determine the rate of resistance development and to select for drug escape mutants. The compounds tested were tested at about their ED₅₀Concentrations were added, 8 replicates per concentration. After an appropriate replicon incubation period, protease activity in the supernatant or lysed cells is assayed.

The following operations were performed in subsequent subcultures. Viruses produced at concentrations where the test compound showed > 50% protease activity on untreated infected cells (SIC), were passaged to fresh replicon cultures. Aliquots, say 15 μ l of supernatant, taken from each of the eight replicates were transferred to replicon cells without test compound (control) and to cells with the same concentration of test compound, and in addition, two concentrations were each five-fold higher. (see the following Table)

When the viral components of the replicon were allowed to multiply at the highest non-toxic concentration (5-40 μ M), e.g., as determined by HCV protease activity, 2-4 parallel wells were collected and expanded to yield material for sequence analysis and cross-resistance.

The key is as follows:

allowing viral growth

Inhibition of virus production

125×SIC

125×SIC 25×SIC→

25×SIC 5×SIC

25×SIC 5×SIC→ No compound

25×SIC 5×SIC→ No compound

5×SIC SIC

S/C→ No compound

S/C→ No compound

Pass1 Pass2 Pass3 Pass4 Pass5

Another method of assessing activity against drug escape mutants involves the preparation of mutant enzymes that produce specific mutations for use in standard Ki determinations as shown above. For example, WO04/039970 describes the structure of HCV proteases that can be directed to produce 155, 156 and/or 168 drug escape mutants that result from the selection pressure of BILN-2061 and VX-950. Thus, the above-described structures can be designed to replace the replicon vectors of natural-type proteases, thereby allowing for convenient evaluation of whether a given compound is active on a given drug escape mutant in a cellular assay.

P450 metabolism

The metabolism of the compounds of the invention by the major isoform of the human cytochrome system P450 is desirably determined in insect cells infected with baculovirus transfected with human cytochrome P450 cdna (supersomes) genttest corp.

Test compounds at concentrations of 0.5, 5 and 50 μ M were cultured in duplicate in the presence of supersomes overexpressing multiple cytochrome P450 isoforms including CYP1A2+ P450 reductase, CYP2A6+ P450 reductase, CYP2C9-Arg144+ P450 reductase, CYP2C19+ P450 reductase, CYP2D6-Val 374+ P450 reductase and CYP3A4+ P450 reductase. The culture includes a fixed concentration of cytochrome P450 (e.g., 50pmol) and is performed for 1 hour or more. The involvement of a given isoform in the metabolism of the test compound was determined by the disappearance of the parent compound as determined by UV HPLC chromatographic analysis.

Claims (21)

1. A compound of formula I, or a pharmaceutically acceptable salt or prodrug thereof

Wherein

A is C (═ OO) R¹Or C (═ O) NHSO₂R²Wherein:

R¹is H;

R²is methyl, cyclicPropyl or phenyl;

m is CR⁷R^7′；

R⁷Is n-propyl or 2, 2-difluoroethyl;

R^7′is H;

or R⁷And R^7′Together form a spiro-cyclopropyl or spiro-cyclobutyl ring, optionally substituted by R^7′aMono-or di-substituted; wherein:

R^7′ais C₁-C₆Alkyl radical, C₃-C₅Cycloalkyl or C₂-C₆Alkenyl, each optionally substituted with halogen; or R^7′aIs J;

q is 1 and k is 1;

w is-O-;

R⁸comprises the following steps:

wherein R is^9aIs optionally substituted by R¹⁰Substituted phenyl orWherein:

R¹⁰is C₁-C₆Alkyl radical, C₀-C₃Alkyl radical, C₃-C₇Cycloalkyl radical, C₁-C₆Alkoxy, optionally substituted by C₁-C₆Alkyl mono-or di-substituted amino, acylamino, C₁-C₃An alkylamide; and

R^9bis C₁-C₆Alkyl radical, C₁-C₆Alkoxy, amino, di (C)₁-C₃Alkyl) amino, (C)₁-C₃Alkyl) amides, NO₂OH, halogen, trifluoromethyl, carboxyl;

e is-C (═ O) -;

x is-NRx-wherein Rx is H, C₁-C₅Alkyl or J; or in the case where E is-C (═ O),x may also be-O-or-NRjNRj-;

one of Rj is H and the other is H, C₁-C₅Alkyl or J;

R¹¹is tert-butyl, isobutyl or cyclohexyl;

j, if present, is a 4-7 membered saturated or monounsaturated all-carbon alkylene chain, having a size providing a macrocycle with 14 or 15 ring atoms;

ru is independently H or C₁-C₃An alkyl group;

m is 0 or 1; n is 0 or 1;

u is ═ O or absent;

R¹⁵cyclohexyl, cyclohexylmethyl, tert-butyl, isopropyl or isobutyl;

g is-O-, -NRy-, -NRjNRj-; one of Rj is H and the other is H, C₁-C₅Alkyl or J;

ry is H, C₁-C₃An alkyl group; or Ry is J;

R¹⁶is H, C₃-C₆Cycloalkyl radical, C₁-C₆Alkyl or is a 5 or 6 membered heterocyclic ring;

provided that when m ═ n ═ 0 and G is O, then R¹⁶Not tert-butyl or phenyl.

2. A compound according to claim 1, wherein m is 0 and n is 0.

3. A compound according to claim 2, wherein G is-NRy-or-NRjNRj-.

4. A compound according to claim 3, wherein Ry or one Rj group is J, thereby defining a macrocyclic compound.

5. A compound according to claim 1, wherein R¹⁶Morpholine, piperidine or piperazine.

6. A compound according to claim 1, wherein m is 1.

7. The compound according to claim 6, wherein X is-NRx-.

8. The compound according to claim 6, wherein U is O.

9. The compound according to claim 6, wherein one of Rx or R¹¹Is J, thereby defining a macrocyclic compound.

10. The compound according to claim 6, wherein n is 1.

11. A compound according to claim 6, wherein G is NRy or-NRjNRj-,

wherein Ry or one Rj is H or methyl and the other Rj is H.

12. A compound according to claim 1, wherein R^9aComprises the following steps:

wherein R is¹⁰Is H, C₁-C₆Alkyl or C₀-C₃Alkylcycloalkyl, optionally substituted by C₁-C₆Alkyl mono-or di-substituted amino, acylamino, (C)₁-C₃Alkyl) amides.

13. A compound according to claim 1, wherein R^9aIs phenyl, optionally substituted by C₁-C₆An alkyl group; c₁-C₆An alkoxy group; or halogen substitution.

14. According to the claimsThe compound of claim 1, wherein R⁸Comprises the following steps:

wherein R is^10aIs H, C₁-C₆Alkyl or C₀-C₃Alkyl carbocyclyl, optionally substituted by C₁-C₆Alkyl mono-or di-substituted amino, amido, heteroaryl or heterocyclyl; and R^9bIs C₁-C₆Alkyl radical, C₁-C₆Alkoxy, amino, di (C)₁-C₃Alkyl) amino, amido, NO₂OH, halogen, trifluoromethyl or carboxyl.

15. A compound according to claim 1, wherein R^9bIs C₁-C₆-alkoxy groups.

16. A compound according to claim 15, wherein R^9bIs methoxy.

17. A compound according to claim 2, wherein R⁷And R^7′Together form a spiro-cyclopropyl or spiro-cyclobutyl ring, optionally substituted by R^7′aMono-or di-substituted; wherein:

R^7′ais C₁-C₆Alkyl radical, C₃-C₅Cycloalkyl or C₂-C₆Alkenyl, each optionally substituted with halogen; or R^7′aIs J.

18. The compound according to claim 17, wherein said ring is substituted with R^7′aA substituted spiro-cyclopropyl ring, wherein:

R^7′ais ethyl, vinyl, cyclopropyl, 1-or 2-bromoethyl, 1-or 2-fluoroethyl or 2-bromovinyl.

19. A pharmaceutical composition for preventing or treating a flavivirus infection comprising a compound as defined in claim 1 and a pharmaceutically acceptable carrier therefor.

20. The pharmaceutical composition according to claim 19, additionally comprising other HCV antiviral agents selected from the group consisting of nucleoside analogue polymerase inhibitors, protease inhibitors, ribavirin and interferons.

21. Use of a compound as defined in claim 1 in the manufacture of a medicament for the prevention or treatment of a flavivirus infection, said flavivirus comprising HCV.