Protease inhibitors

Background to the invention

Two distinct retroviruses, human immunodeficiency virus (HIV) type-1 (HIV-I) or type- 2 (HIV-2), have been etiologically linked to the immunosuppressive disease, acquired immunodeficiency syndrome (AIDS). HIV seropositive individuals are initially asymptomatic but typically develop AIDS related complex (ARC) followed by AIDS. Affected individuals exhibit severe immunosuppression, which predisposes them to debilitating and ultimately fatal opportunistic infections.

The disease AIDS is the end result of an HIV-I or HIV-2 virus following its own complex life cycle. The virion life cycle begins with the virion attaching itself to the host human T-4 lymphocyte immune cell through the bonding of a glycoprotein on the surface of the virion's protective coat with the CD4 glycoprotein on the lymphocyte cell. Once attached, the virion sheds its glycoprotein coat, penetrates into the membrane of the host cell, and uncoats its RNA. The virion enzyme, reverse transcriptase, directs the process of transcribing the RNA into single-stranded DNA. The viral RNA is degraded and a second DNA strand is created. The now double-stranded DNA is integrated into the human cell's genes and those genes are used for virus reproduction.

At this point, RNA polymerase transcribes the integrated DNA into viral RNA. The viral RNA is translated into the precursor gag-pol fusion polyprotein, the polyprotein is then cleaved by the HIV protease enzyme to yield the mature viral proteins. Thus, HIV protease is responsible for regulating a cascade of cleavage events that lead to the virus particle's maturing into a virus that is capable of full infectivity.

The typical human immune system response, killing the invading virion, is taxed because the virus infects and kills the immune system's T cells. In addition, viral reverse transcriptase, the enzyme used in making a new virion particle, is not very specific, and causes transcription mistakes that result in continually changed glycoproteins on the surface of the viral protective coat. This lack of specificity decreases the immune system's effectiveness because antibodies specifically produced against one glycoprotein may be useless against another, hence reducing the number of antibodies available to fight the virus. The virus continues to reproduce while the immune response system continues to weaken. Eventually, the HIV largely holds free reign over the body's

immune system, allowing opportunistic infections to set in and without the administration of antiviral agents, immunomodulators, or both, death may result.

There are at least three critical points in the virus's life cycle which have been identified as possible targets for antiviral drugs: (1 ) the initial attachment of the virion to the T-4 lymphocyte or macrophage site, (2) the transcription of viral RNA to viral DNA (reverse transcriptase, RT), and (3) the processing of gag-pol protein by HIV protease.

The genomes of retroviruses encode a protease that is responsible for the proteolytic processing of one or more polyprotein precursors such as the pol and gag gene products. Retroviral proteases most commonly process the gag precursor into the core proteins, and also process the pol precursor into reverse transcriptase and retroviral protease. The correct processing of the precursor polyproteins by the retroviral protease is necessary for the assembly of the infectious virions. It has been shown that in vitro mutagenesis that produces protease-defective virus leads to the production of immature core forms which lack infectivity. Therefore, retroviral protease inhibition provides an attractive target for antiviral therapy.

As evidenced by the protease inhibitors presently marketed and in clinical trials, a wide variety of compounds have been studied as potential HIV protease inhibitors. The first inhibitor of so-called retroviral aspartate protease to be approved for combating the infection was saquinavir. Since then others have followed including indinavir (Merck), ritonavir (Abbott), amprenavir and its prodrug amprenavir phosphate (Vertex/GSK), lopinavir (Abbott), nelfinavir (Aguoron/Pfizer), tipranavir (Pharmacia/Boehringer) and atazanavir (Novartis/BMS).

Each of these prior art compounds has liabilities in the therapeutic context resulting in sub-optimal treatment regimes, side effects such as lipodystrophy and poor patient compliance. In conjunction with the replicative infidelity of the HIV genetic machinery and the very high viral turnover in vivo, the sub-optimal performance and pharmacokinetics of prior art HIV protease inhibitors enable the rapid generation of drug escape mutants. This in turn dramatically limits the effective treatment length of current HIV drugs as HIV quickly becomes resistant and/or patients develop physical or psychological aversions to the drugs themselves or their side effects.

The aim of the present invention is to provide a novel type of compound that is equipped, especially, with a high degree of inhibitory activity against virus replication in cells, high antiviral activity against numerous virus strains, including those which are resistant to known compounds, such as saquinavir, ritonavir and indinavir, and especially advantageous pharmacological properties, for example good pharmacokinetics, such as high bioavailability and high blood levels, and/or high selectivity.

In accordance with the invention, there is provided a compound of the formula I:

wherein

R ¹ is -R ^{1 '} , -OR ^1' , -SR ^{1 '} ,

R ¹ is Ci-C ₆ AIk, C ₀ -C ₃ alkanediylcarbocyclyl or Co- ₃ alkanediylheterocyclyl, any of which is optionally substituted with up to 3 substituents independently selected from R ¹⁰ ; R ² is Cj-C ₆ AIk, C ₀ -C ₃ alkanediylcarbocyclyl, C ₀ -C ₃ alkanediylheterocyclyl, any of which is optionally substituted with up to 3 substituents independently selected from R ¹⁰ ;

X is H, F, OH, Ci-C ₃ AIk or C ₀ -C ₃ alkanediyl-O-Ci-C ₃ alkyl;

L is F, NH ₂ , -NHd-C ₃ AIk; -N(C ₁ -C ₃ AIk) ₂ ; n is 1 or 2; A ¹ is a bicyclic ring system comprising a first 5 or 6 membered saturated ring optionally containing an oxygen hetero atom and optionally substituted with hydroxy and/or methyl, having fused thereto a second 5 or 6 membered unsaturated ring optionally containing one or two hetero atoms selected from S, O and N, and optionally substituted with mono- or di-fluoro; or A' is a group of formula (II), (H'), (HI) or (IV):

(M ¹ ) (ill) (IV)

wherein;

R ³ is H; or R ³ is Q-C ₆ AIk, Co-C ₃ alkanediylcarbocyclyl, C ₀ -C ₃ alkanediylheterocyclyl, any of which is optionally substituted with up to three substituents independently selected from R ¹¹ ; R ⁴ is Ci-C ₆ AIk, C ₀ -C ₃ alkanediylcarbocyclyl, C ₀ -C ₃ alkanediylheterocyclyl, any of which is optionally substituted with up to three substituents independently selected from R ¹⁰ ;

R ⁵ is Ci-C ₆ AIk, Co-C ₃ alkanediylcarbocyclyl, Co-C ₃ alkanediylheterocyclyl, any of which is optionally substituted with up to three substituents independently selected from R ¹⁰ ;

Z is a bond, -NH- or -O-; Rx is H, Ci-C ₃ alkyloxy, Ci-C ₃ straight or branched alkyl optionally substituted with halo, hydroxy, Ci-C ₃ alkyloxy; or Rx, together with the adjacent carbon atom, defines a fused furanyl or pyranyl ring which is optionally substituted with halo or Ci-C ₃ Alk; t is 0 or 1;

A" is a group of formula (V), (VI) (VII) or (VIII);

(V) (Vl) (VII) (VIII) wherein;

R is H; or R is Ci-C ₆ AIk, Co-C ₃ alkanediylcarbocyclyl, Co- ₃ alkanediylheterocyclyl, any which is optionally substituted with up to three substituents independently selected from R"

R ⁹ is Ci-C ₆ AIk, C ₀ -C ₃ alkanediylcarbocyclyl, C ₀ - ₃ alkanediylheterocyclyl, any of which is optionally substituted with up to three substituents independently selected from R ¹⁰ ;

W is a bond, -NR ¹³ - or -O-;

R ¹³ is H, Ci-C ₆ AIk or R ¹³ and R ⁹ together with the N atom to which they are attached define a saturated, partially saturated or aromatic N-containing ring containing 5 or 6 ring atoms, which is optionally substituted with up to three substituents selected from R ¹⁰ '

D is O or NH;

Ry is H or Ry, together with the adjacent C atom defines a fused furan or pyran ring;

Q is O, CHR ⁸ or a bond; R ¹⁵ is carbocyclyl or heterocyclyl, any of which is optionally substituted with up to three substituents independently selected from Ci-C ₃ Alk, hydroxy, oxo, halo; r and q are independently 0 or 1 ;

R ¹⁰ is halo, oxo, cyano, azido, nitro, Ci-C ₆ AIk, C ₀ -C ₃ alkanediylcarbocyclyl, C ₀ - C ₃ alkanediylheterocyclyl, Y-NRaRb, Y-O-Rb, Y-C(=O)Rb, Y-(C=O)NRaRb, Y- NRaC(=O)Rb, Y-NHSOpRb, Y-S(=O) _p Rb, Y-S(=O) _p NRaRb, Y-C(=O)ORb or Y- NRaC(=O)ORb; wherein; Y is a bond or Ci-C ₃ alkanediyl;

Rb is H or C)-C ₆ AIk, C ₀ -C ₃ alkanediylcarbocyclyl or C ₀ -C ₃ alkanediylheterocyclyl; p is 1 or 2;

R ^{1 1} is halo, oxo, cyano, azido, nitro, Ci-C ₃ AIk, Y-NRaRa', Y-O-Ra; wherein; Ra' is H or Ci-C ₃ AIk; or Ra and Ra' and the nitrogen atom to which they are attached define pyrrolidine, morpholine, piperidine or piperazine which is optionally 4-substitued with methyl or acetyl; and pharmaceutically acceptable salts thereof.

A further aspect of the invention embraces a pharmaceutical composition comprising a compound as defined above and a pharmaceutically acceptable carrier or diluent therefore. A still further aspect of the invention envisages the use of a compound as defined above in the manufacture of a medicament for the prophylaxis or treatment of HIV infection. An additional aspect of the invention provides a method of medical treatment or prophylaxis for HIV infection comprising the administration of an effective amount of a compound as defined in above to an individual infected or threatened with HIV infection.

Without in any way wishing to be bound by theory, or the ascription of tentative binding modes for specific variables, the notional concepts Pl, Pl ', P2 and P2' as used herein are provided for convenience only and have substantially their conventional meanings, as illustrated by Schechter & Berger, (1976) Biochem Biophys Res Comm 27 157-162, and denote those portions of the inhibitor believed to fill the Sl ₅ Sl ', S2 and S2' subsites respectively of the enzyme, where Sl is adjacent and S2 remote from the cleavage site on one side and Sl ' is adjacent and S2' remote from the cleavage site on the other side. Regardless of binding mode, the compounds defined by Formula I are intended to be within the scope of the invention. It is conceivable that R ¹ and R ² respectively fill the Sl and Sl ' subsites, whereas A' and A" interact with the S2 and S2', but also conceivable with the inverse arrangement.

Conveniently, the compounds of the invention display at least 75%, preferably at least 90%, such as in excess of 95%, enantiomeric purity around the carbon shared by the hydroxyl group and the R ¹ methylene function depicted in formula I. It is currently preferred that the compounds exhibit a high degree of enantiomeric purity of the steroisomeres as shown in the partial structure:

The steric center whereto group X is attached can be of either R or S stereochemistry. Preferably the compounds of the invention display at least 75%, preferably at least 90%, such as in excess of 95%, enantiomeric purity around the carbon whereto group X is attached. It is currently preferred that the compounds exhibit a high degree of enantiomeric purity of the stereoisomers as shown in the partial structure:

For example compounds of the invention include those having the stereochemistry shown in the partial structure:

As defined above X is H, OH, Ci-C ₃ AIk or C ₀ -C ₃ alkanediyl-O-Ci-C ₃ alkyl. Convenient values for X include OH and Co-C ₃ alkanediyl-0-Ci-C ₃ alkyl especially methoxy (i.e. Co) and hydroxymethyl. A currently favoured value for X is F or OH and especially H.

As recited above, L is F, NH ₂ , NHCi-C ₃ AIk, N(Ci-C ₃ Alk) ₂ ,wherein the NHCi-C ₃ AIk and N(Ci-C ₃ AIk) ₂ preferably are NHMe and NHMe ₂ respectively. A currently preferred value for L is fluoro and a more preferred value is NH ₂ .

The compounds of the invention can have 3 or 4 chain atoms between the carbonyl depicted in formula I and the β-nitrogen of the hydrazide function (i.e. n is 1 or 2). In favoured embodiments of the invention the compounds have 3 chain atoms between the carbonyl and the β-nitrogen of the hydrazide function, i.e. n is 1.

As defined above, R ¹ is R ^r , OR ^1' or SR ^{1 '} wherein R ^{1 '} is Ci-C ₆ allkyl, but is especially C ₀ - C ₃ alkanediylcarbocyclyl or Co- ₃ alkanediylheterocyclyl. Typical examples of such species are recited below. Any of these species is optionally substituted with up to 3 substituents independently selected from R ¹⁰ as defined above. Convenient optional substituents to R ¹ include one or two substituents selected from halo, oxo, cyano, Ci-C ₆ AIk, C ₀ -

C ₃ alkanediylcarbocyclyl, C ₀ -C3alkanediyiheterocyclyl, Y-NRaRb, Y-O-Rb; where Y is a bond or Ci-C ₃ AIk, Ra is H or Ci-C ₃ AIk and Rb is H or Ci-C ₃ AIk. Particularly preferred substituents include fluoro, Cj-C ₃ AIk, C ₀ -Cialkanediylcarbocyclyl, C ₀ - Cialkanediylheterocyclyl.

Conveniently, the C ₀ -C ₃ alkanediyl linker moiety of such C ₀ -C ₃ alkanediylcarbocyclyl or C ₀ - ₃ alkanediylheterocyclyl species as R ¹ or the optional substituent thereto defines methylene or even more preferably a bond, i.e. R ¹ or the substituent is simply an optionally substituted carbocyclyl or heterocyclyl, such as optionally substituted phenyl or optionally substituted pyridyl, pyrazinyl, pyrimidinyl or pyridazinyl. Preferably R ¹ is R ^{1 '} or OR ^{1 '} .

In one embodiment of the present invention the R ¹⁰ substituent of R ¹ is Y-O-Rb where Y is a bond and Rb is an optionally substituted C ₀ -C ₃ alkanediylaryl or Co- C ₃ alkanediylheteroaryl. The optional substituent is preferably Ci-C ₃ AIk, such as methyl

Preferred structures for R ¹ according to this embodiment include:

especially in the para position of a phenyl R ¹ group.

Accordingly, other suitable values for R ¹ include phenyl, pyrid-2-yl, pyrid-3-yI, pyrid-4- yl, pyrimidin-2-yl, pyrimidiny-4-yl, pyrazin-2-yl, pyrazin-3-ylyl or pyridazin-3-yl, pyridazin-4-yl or triazinyl; or mono- or di-halo substituted phenyl, such mono- or di- fluoro substituted phenyl. Currently preferred substituents to a phenyl R ¹ include H, methyl, methoxy, fluoromethyl or trifluoromethyl. Favoured R ¹ groups include H, m- fluoro, o-fluoro or p-fluoro. When R represents two non-H substituents, typical configurations include 2,5-difluoro or -dimethyl, 2,4-difluoro or -dimethyl, 2.3-difluoro or -dimethyl or 3,4 difluoro or -dimethyl and especially 2,6-difluoro or 3,5 difluoro.

As defined above, R ² is Ci-C ₆ AIk, but especially C ₀ -C ₃ alkanediylcarbocyclyl, C ₀ -

₃ alkanediylheterocyclyl, any of which species can be substituted with up to 3 substituents independently selected from R ¹⁰ . The optional substituent is preferably one or two members chosen from halo, oxo, cyano, Ci-C ₆ AIk, C ₀ -C ₃ alkanediylcarbocyclyl, C ₀ - C ₃ alkanediylheterocyclyl, Y-NRaRb, Y-O-Rb; where Y is a bond or Ci-C ₃ AIk, Ra is H or Ci-C ₃ AIk and Rb is H or Ci-C ₃ AIk. Currently favoured substituents include fluoro, Ci- C ₃ AIk, methylenecarbocyclyl or methyleneheterocyclyl, but especially a substituent such as optionally substituted carbocyclyl or heterocyclyl, for example in the para position of the R ² cyclic group.

Conveniently, the Co-C ₃ alkanediyl linker moiety of such Co-C ₃ alkanediylcarbocyclyl or Co-C ₃ alkanediylheterocyclyl species as R ² or the optional substituent thereto defines methylene or even more preferably a bond, i.e. R ² or the substituent is simply an optionally substituted carbocyclyl or heterocyclyl, such as optionally substituted phenyl or optionally substituted pyridyl, pyrazinyl, pyrimidinyl or pyridazinyl

Accordingly suitable values for R ² include phenyl, pyrid-2-yl, pyrid-3-yl, pyrid-4-yl, pyrimidin-2-yl, pyrimidiny-4-yl, pyrazin-2-yl, pyrazin-3-ylyl or pyridazin-3-yl, pyridazin-4-yl or triazinyl; or phenyl substituted, especially in the para position with an aryl carbocyclic ring such as phenyl or heterocyclic ring, such as heteroarylic group as defined below, for example pyrid-2-yl, pyrid-3-yl and especially pyrid-4-yl.

Preferred values for R ² include:

especially at the para position of a phenyl R ² group.

Turning now to the terminal amide A', one convenient embodiment comprises a bicyclic ring system comprising a first 5 or 6 membered saturated ring optionally containing an oxygen hetero atom, and optionally substituted with hydroxy or methyl, having fused thereto a second 5 or 6 membered unsaturated ring optionally containing one or two hetero atoms selected from S, O and N, and optionally mono- or di-fluoro substituted.

Conveniently in this embodiment the bond to the amide and rest of the molecule extends from carbon 1 of said saturated ring. Suitably the optional hydroxy substituent in this embodiment is at carbon 2 of said saturated ring. Alternatively an oxygen hetero atom is provided, typically at position 3 of a 5 membered saturated ring or position 4 of a 6 membered saturated ring.

The second ring in this embodiment of A' is conveniently 5-membered and comprises a sulphur hetero atom or an oxygen hetero atom. Alternatively, the said second ring is typically a fused pyridyl as described in WO9845330 or an optionally substituted phenyl, for example a fused phenyl wherein the substituent is mono- or di-fluoro.

Representative A' groups in this embodiment of the invention include:

and especially

Particularly preferred variants within this embodiment include cis-indanol-yl and cis chromanolyls as depicted in the partial structure:

An alternative embodiment of the compounds of the invention includes those wherein A' is a group of formula (II), thereby defining a compound of the formula:

A further alternative embodiment of the compounds of the invention includes those wherein A' is a group of formula (H'), thereby defining a compound of the formula:

As recited above R ³ is H; or R ³ is Ci-C ₆ AIk, C ₀ -C ₃ alkanediylcarbocyclyl, Co- ₃ alkanediylheterocyclyl, any of which is optionally substituted with up to three substituents independently selected from R ¹¹ Convenient values for R ³ include optionally substituted C ₀ -C ₃ aklylheterocycylyl and especially H or optionally substituted Ci-C ₆ AIk. Favoured R ³ values include Ci-C ₆ AIk such as /sopropyl or /-butyl optionally substituted with hydroxy or methoxy or halo, such as fluoro.

Preferred values for R ³ are /.sopropyl, /-butyl, 2-fluoro-l-methylethyl, 2-hydroxy-l- methylethyl, 2-methoxy- 1 -methylethyl, 2-fluoro- 1 , 1 -dimethylethyl, 2-hydroxy- 1,1- dimethylethyl and 2-methoxy- 1,1 -dimethylethyl.

The optional substituent to R ³ is as defined above. Representative values include oxo, cyano or especially halo or Y-O-Ra, where Y is a bond or Ci-C ₃ AIk and Ra is H or Ci- C ₃ AIk.

As recited above R in Formulae I, Ha and H'a is Ci-C ₆ AIk, Co-C ₃ alkanediylcarbocyclyl or Co-C ₃ alkanediylheterocyclyl, any of which is optionally substituted with up to three substituents independently selected from R ¹⁰ . Favoured values of R ⁴ include optionally substituted Ci-C ₆ AIk, especially methyl or ethyl or optionally substituted methyl or ethyl.

Convenient optional substituents to R ⁴ include halo, oxo, cyano, azido, nitro, Ci-C ₆ AIk, Co-C ₃ alkanediylcarbocyclyl, C ₀ -C ₃ alkanediylheterocyclyl, Y-NRaRb or Y-O-Rb wherein; Y is a bond or Ci-C ₃ AIk; Rb is H or Ci-C ₆ AIk, Co-C ₃ alkanediylcarbocyclyl or Co-C ₃ alkanediylheterocyclyl.

Preferred values for R ⁴ are fluoroethyl, difluoroethyl, trifluoroethyl and methoxyethyl.

Preferred optional substituents to R ⁴ include halo, oxo, Ci-C ₆ AIk, C ₀ - C ₃ alkanediylcarbocyclyl, Co-C ₃ alkanediylheterocyclyl or Y-O-Rb, especially halo or Y- O-Rb.

Formula II may comprise the S or R sterochemistry at the chiral centre to which R ³ is attached, or a racemate thereof, but it is currently preferred that it has the stereochemistry shown in the partial structure:

(H)

Alternatively A' may comprise the substructure:

CD where R ³ is H; or R ³ is Ci-C ₆ AIk, Co-C ₃ alkanediylcarbocyclyl, Co-

₃ alkanediylheterocyclyl, any of which is optionally substituted with up to three substituents independently selected from R ¹¹ ; R ⁵ is Ci-C ₆ AIk, C ₀ - C ₃ alkanediylcarbocyclyl, Co- ₃ alkanediylheterocyclyl, any of which is optionally substituted with up to three substituents independently selected from R ¹⁰ ; and Z is bond, NH-, -O-; Preferred values for R ³ are as defined above in respect of formula II.

Formula III may comprise the S or R stereochemistry at the chiral centre to which R ³ is attached, or a racemate thereof, but it is currently preferred that it has the stereochemistry shown in the partial structure:

(III)

Currently preferred values for Z is O. Favou urreedd values of R ⁵ include optionally substituted Ci-C ₆ AIk, especially methyl or optionally substituted methyl.

A favoured value for A' is formula IV, thus defining a compound of the formula

Representative values for formula IV include monocyclic furans where Rx is H, Ci- C ₃ alkyloxy, Ci-C ₃ straight or branched alkyl optionally substituted with halo, hydroxy, Ci-C ₃ alkyloxy. Representative values within this series include those wherein Rx is H, or wherein Rx is Ci-C ₃ AIk substituted at chain carbon 1 with halo, hydroxy or Ci-C ₂ AIk. Favoured values include those wherein Rx is hydroxymethyl, 1-hydroxyethyl, 1- hydroxypropyl, fiuoromethyl, 1-fluoroethyl or 1-fluoropropyl and those wherein Rx is methoxymethyl, ethoxymethyl, 1-methoxyethyl, 1 -ethoxyethyl, 1 -methoxypropyl or 1- ethoxypropyl. Specially preferred compounds according to formula IVa are those wherein n is 1 and/or L is OH.

Alternatively Rx defines a further furanyl or pyranyl ring fused to the depicted furan and optionally substituted with halo or Ci-C ₃ AIk. Representative examples include those wherein the heterocyclic oxygen is located as follows:

Turning now to the other terminal amide A", as defined above, this is selected from formula V, VI, VII or VIII.

Representative values for formula VI, especially when A' is of formula II, IV or a bicyclic ring system, include those of the formula:

Favoured compounds according to this embodiment include compounds according to formulae Via and VIb:

Specially preferred compounds according to formula Via and VIb, are those wherein n is 1 , R ¹ is phenyl and/or L is NH ₂ .

Suitable building blocks for the preparation of compounds according to this embodiment of the invention are described herein and in WO99/48885 and WO94/05639. The stereochemic synthesis of hexahydrofurofuranol is described in WO2003 22853 and WO05 63770 with corresponding methodology being applicable to the mirror. Preferred stereochemistries for formula VI include those shown in the partial structures:

Conveniently A" is of formula V, thus defining a compound of the formula:

As recited above, R ⁸ is H; or R ⁸ is Ci-C ₆ AIk, Co-C ₃ alkanediylcarbocyclyl, C ₀ - ₃ alkanediylheterocyclyl, any which is optionally substituted with up to three substituents independently selected from R . Conveniently R is H, optionally substituted Ci-C ₆ AIk or optionally substituted C ₀ -C ₃ alkanediylcarbocyclyl. Currently favoured values for R ⁸ include H or optionally substituted Ci-C ₆ AIk, especially isopropyl or t-butyl.

R is optionally substituted with 1 to 3 members independently selected from R . Representative optional substituents include oxo, cyano, Ci-C ₃ AIk or especially halo or Y-O-Ra, where Y is a bond or Ci-C ₃ AIk and Ra is H or Ci-C ₃ AIk.

As recited above, R ⁹ is Ci-C ₆ AIk, C ₀ -C ₃ alkanediylcarbocyclyl, C ₀ - ₃ alkanediylheterocyclyl, any of which is optionally substituted with up to three substituents independently selected from R ¹⁰ ; and W is a bond, -NH- or -O-. Conveniently, R ⁹ is optionally substituted Ci-C ₆ AIk or Co-C ₃ alkanediylcarbocyclyl, especially optionally substituted methyl, or unsubstituted methyl.

Representative optional substituents to R ⁹ include halo, oxo, cyano, azido, nitro, Q- C ₆ AIk, Co-C ₃ alkanediylcarbocyclyl, Co-C ₃ alkanediylheterocyclyl, Y-NRaRb or Y-O-Rb where Y is a bond or Ci-C ₃ AIk, Ra is H or Ci-C ₃ AIk and Rb is H or Ci-C ₆ AIk, C ₀ -

C ₃ alkanediylcarbocyclyl or C ₀ -C ₃ alkanediylheterocyclyl. Particularly preferred optional

substituents, for example when R ⁹ is methyl include halo, oxo, Ci-C ₆ AIk, Co- C ₃ alkanediylcarbocyclyl, Co-C ₃ alkanediylheterocyclyl or Y-O-Rb.

When A" is of formula V, it is currently preferred that W is -O-.

Formula V may comprise the S or R stereochemistry at the chiral centre to which R ⁸ is attached, or a racemate thereof, but it is currently preferred that it has the stereochemistry shown in the partial structure:

(V)

One embodiment when A" is according to formula V includes compounds wherein R ⁹ is an optionally substituted heterocyclyl either directly bonded to W, (i.e. C ₀ ) or bonded to W via an Ci-C ₃ alkanediyl chain for example a methylene chain (i.e. Ci).

Preferred compounds according to this embodiment include those having the structure according to formulae Va:

Specially preferred compounds according to formulae Va are those wherein n is 1, R ¹ is phenyl and/or L is NH ₂ .

Suitable building blocks for the preparation of compounds according to this embodiment of the invention are described herein and in WO98/00410 and WO96/039398.

Another embodiment when A" is according to formula V includes compounds wherein W is a bond and R ⁹ is Co-C ₃ alkanediylcarbocyclyl or Co-C ₃ alkanediylheterocyclyl, the carbocyclyl and heterocyclyl being optionally substituted.

Preferred compounds according to this embodiment include those having the structure according to formulae Vc:

Specially preferred compounds according to formula Vc and Vd are those wherein n is 1, R ¹ is phenyl and/or L is NH ₂ .

Suitable building blocks for the preparation of compounds according to this embodiment of the invention are described herein and in US5196438.

When A" is of formula VII, it is currently preferred that R ⁸ is as described above and R ⁹ is Ci-C ₆ AIk such as methyl.

Conveniently A" is of formula VIII, thus defining compounds of formula Villa:

As recited above, R ¹⁵ is carbocyclyl or heterocyclyl, any of which is optionally substituted with up to three substituents independently selected from Ci-C ₃ AIk, hydroxy, oxo, halo, Q is O, NR ⁸ or a bond and r and q are independently 0 or 1.

Representative values for R ¹⁵ are 5 to 6 membered, optionally substituted, aromatic rings containing 0 to 2 heteroatoms, the heteroatoms being independently selected from N, O and S.

Convenient optional substituents to R ¹⁵ include Ci-C ₃ AIk, such as methyl, ethyl, propyl or isopropyl.

Representative compounds in this embodiment of the invention are those wherein Q is a bond and r and q are both zero.

Preferred compounds according to this embodiment are those with the structures according to formulae VIlIb:

Specially preferred compounds according to formula VIIIb are those wherein n is 1, R ¹ is phenyl and/or L is NH ₂ .

Suitable building blocks for the preparation of compounds according to this embodiment of the invention are described herein and in US 5484926 and US 5952343.

Further favoured compounds wherein A" is according to formula VIII are those wherein Q is O.

Preferred compounds according to this embodiment include those having the structures according to formulae VIIIc and VIIId:

Specially preferred compounds according to formula VIIIc and VIIId are those wherein n is 1, R ¹ is phenyl and/or L is NH ₂ .

Suitable building blocks for the preparation of compounds according to this embodiment of the invention are described herein and in WO98/00410 and WO96/39398.

Further favoured compounds wherein A" is according to formula VIII are those wherein Q is CR ⁸ .

Preferred compounds according to this embodiment include those having the structure according to formula VIIIe:

(VIIIe)

Specially preferred compounds according to formula VIIIh and Villi are those wherein n is 1, R ¹ is phenyl and/or L is NH ₂ .

Suitable building blocks for the preparation of compounds according to this embodiment of the invention are described herein and in US6372905 and WO97/21685.

Further preferred compounds of the invention are those having general formula IX:

wherein

R ¹ is one or two substituents independently selected from H, halo, amine, OH, cyano, nitro, Ci-C ₄ alkyl, OCi-C ₄ alkyl, Ci-C ₄ haloalkyl;

R ² is halo, phenyl or pyridyl;

X is H or OH;

L is F, NH ₂ , NHC-C ₃ AIk Or N(Ci-C ₃ AIk) ₂ ;

A is chromanol, indanolyl which is optionally substituted with fluoro and/or methyl, or A is a group of formula (a):

(a) wherein;

R ³ is H or Ci-C ₆ AIk;

R ⁴ is C-C ₆ AIk;

A" has the partial structure

or

R ⁸ Is H Or C-C ₆ AIk,

W is -NR ¹³ - or -O-;

R ¹³ is H, C ₁ -C ₆ AIk; the stereoisomeric forms and pharmaceutically acceptable salts thereof.

'C ₀ -C ₃ alkanediyl-O-Ci-C ₃ alkyr as applied herein is meant to include Ci-C ₃ alkoxy groups such as methoxy, ethoxy, n-propoxy, isopropoxy directly bonded (i.e. C ₀ ) or through an intermediate methylene, ethanediyl, 1 ,3-propanediyl or 1,3-propanediyl chain.

'Ci-C ₆ AIk' as applied herein is meant to include straight and branched aliphatic carbon chain substituents containing from 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, isopentyl and hexyl and any simple isomers thereof. The AIk group may have an unsaturated bond. Additionally, any C atom in Ci- C ₆ AIk may optionally be substituted by one, two or where valence permits three halogens and/or a heteroatom S, O, NH. If the heteroatom is located at a chain terminus then it is appropriately substituted with one or 2 hydrogen atoms, such as OH or NH _2. Preferably the Ci-C ₆ AIk is small, saturated and unsubstituted or substituted with halo such as fluoro. Ci-C ₄ AIk and Ci-C ₅ AIk have the corresponding meaning to Ci-C ₆ AIk adjusted as necessary for the carbon number. Me denotes a methyl group.

'Ci-C ₃ AIk' as applied herein is meant to include methyl, ethyl, propyl, isopropyl, cyclopropyl, any of which may be optionally substituted as described in the paragraph above or in the case of C ₂ or C ₃ , bear an unsaturated bond such as CH=CH ₂ .

'C ₀ -C ₃ alkanediyP as applied herein is meant to include bivalent straight and branched aliphatic carbon chains such as methylene, ethanediyl, 1,3-propanediyl, 1,2-propanediyl.

'Amino' includes NH ₂ , NHCi-C ₃ AIk or N(Ci-C ₃ AIk) ₂ .

'Halo' or halogen as applied herein is meant to include F, Cl, Br, I, particularly chloro and preferably fluoro.

'Co-C ₃ alkanediylaryr as applied herein is meant to include a phenyl, naphthyl or phenyl fused to C ₃ -C ₇ cyclopropyl such as indanyl, which aryl is directly bonded (i.e. C ₀ ) or through an intermediate methylene, ethanediyl, 1,2-propanediyl, or 1,3-propanediyl group as defined for Co-C ₃ alkanediyl above. Unless otherwise indicated the aryl and/or its fused cycloalkyl moiety is optionally substituted with 1-3 substituents selected from halo, hydroxy, nitro, cyano, carboxy, Ci-C ₆ AIk, Ci-C ₆ alkoxy, Ci-C _ό alkoxy-Ci-C _δ Alk, C)- C ₆ alkanoyl, amino, azido, oxo, mercapto, nitro Co-C ₃ alkanediylcarbocyclyl _> C ₀ - C ₃ alkanediylheterocyclyl. "Aryl" has the corresponding meaning.

'C ₀ -C ₃ alkanediylcarbocyclyF as applied herein is meant to include C _o -C ₃ alkanediylaryl and C ₀ -C ₃ alkanediylC ₃ -C ₇ cycloalkyl. Unless otherwise indicated the aryl or cycloalkyl group is optionally substituted with 1-3 substituents selected from halo, hydroxy, nitro, cyano, carboxy, Ci-C ₆ AIk, Ci-C ₆ alkoxy, Ci-C ₆ alkoxyC)-C ₆ Alk, Ci-C ₆ alkanoyl, amino, azido, oxo, mercapto, nitro, C ₀ -C ₃ alkanediylcarbocyclyl and/or Co- C ₃ alkanediylheterocyclyl. "Carbocyclyl" has the corresponding meaning, i.e. where the C ₀ -C ₃ alkanediyl linkage is absent

'Co-C ₃ alkanediylheterocycylyl' as applied herein is meant to include a monocyclic, saturated or unsaturated, heteroatom-containing ring such as piperidinyl, morpholinyl, piperazinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazinolyl, isothiazinolyl, thiazolyl, oxadiazolyl, 1,2,3-triazolyl, 1 ,2,4-triazolyl, tetrazolyl, furanyl, thienyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, pyrazolyl, or any of such groups fused to a phenyl ring, such as quinolinyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazinolyl, benzisothiazinolyl, benzothiazolyl, benzoxadiazolyl, benzo- 1,2,3-triazolyl, benzo- 1,2,4- triazolyl, benzotetrazolyl, benzofuranyl, benzothienyl, benzopyridyl, benzopyrimidinyl, benzopyridazinyl, benzopyrazinyl, benzopyrazolyl etc, which ring is bonded directly i.e. (Co),or through an intermediate methyl, ethyl, propyl, or isopropyl group as defined for C ₀ -C ₃ alkanediyl above. Any such non-saturated rings having an aromatic character may be referred to as heteroaryl herein. Unless otherwise indicated the hetero ring and/or its fused phenyl moiety is optionally substituted with 1-3 substituents selected from halo, hydroxy, nitro, cyano, carboxy, Ci-C ₆ AIk, Ci-C ₆ alkoxy, Ci-C _ό alkoxyCi-C _ό Alk, Q- Cβalkanoyl, amino, azido, oxo, mercapto, nitro, Co-Cscarbocycly^ Co-Csheterocyclyl. "Heterocyclyl" and "Heteroaryl" has the corresponding meaning, i.e. where the C ₀ - C ₃ alkanediyl linkage is absent.

Typically the terms 'optionally substituted C ₀ -C ₃ alkanediylcarbocyclyP and 'optionally substituted Co-C ₃ alkanediylheterocyclyP refers preferably to substitution of the carbocyclic or heterocyclic ring.

Typically heterocyclyl and carbocyclyl groups are thus a monocyclic ring with 5 or especially 6 ring atoms, or a bicyclic ring structure comprising a 6 membered ring fused to a 4, 5 or 6 membered ring.

Typical such groups include C3-Cgcycloalkyl, phenyl, benzyl, tetrahydronaphthyl, indenyl, indanyl, heterocyclyl such as from azepanyl, azocanyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, thiopyranyl, furanyl, tetrahydrofuranyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, tetrazolyl, pyrazolyl, indolyl, benzofuranyl, benzothienyl, benzimidazolyl, benzthiazolyl, benzoxazolyl, benzisoxazolyl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, quinazolinyl, tetrahydroquinazolinyl and quinoxalinyl, any of which may be optionally substituted as defined herein.

The saturated heterocycle thus includes radicals such as pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyranyl, thiopyranyl, piperazinyl, indolinyl, azetidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrofuranyl, hexahydropyrimidinyl, hexahydropyridazinyl, 1,4,5,6- tetrahydropyrimidinylamine, dihydro-oxazolyl, 1 ,2-thiazinanyl- 1,1 -dioxide, 1,2,6- thiadiazinanyl- 1,1 -dioxide, isothiazolidinyl-1,1 -dioxide and imidazolidinyl-2,4-dione, whereas the unsaturated heterocycle include radicals with an aromatic character such as furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, tetrazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, indolyl, isoindolyl. In each case the heterocycle may be condensed with a phenyl ring to form a bicyclic ring system.

The compounds of the invention can form salts which form an additional aspect of the invention. Appropriate pharmaceutically acceptable salts of the compounds of Formula I include salts of organic acids, especially carboxylic acids, including but not limited to acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, isethionate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, proprionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate, organic sulphonic acids such as methanesulphonate, ethanesulphonate, 2-hydroxyethane sulphonate, camphorsulphonate, 2-napthalenesulphonate, benzenesulphonate, p-chlorobenzenesulphonate and p-toluenesulphonate; and inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoric and sulphonic acids. The compounds of Formula I may in some cases be isolated as the hydrate.

It will be appreciated that the invention extends to prodrugs, solvates, complexes and other forms releasing a compound of formula I in vivo.

While it is possible for the active agent to be administered alone, it is preferable to present it as part of a pharmaceutical formulation. Such a formulation will comprise the above defined active agent together with one or more acceptable carriers/excipients and optionally other therapeutic ingredients. The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.

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 formulation is an orally administered formulation. The formulations may conveniently be presented in unit dosage form, e.g. tablets and sustained release capsules, and may be prepared by any methods well known in the art of pharmacy.

Such methods include the step of bringing into association the above defined active agent with the carrier. In general, the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. The invention extends to methods for preparing a pharmaceutical composition comprising bringing a compound of Formula I or its pharmaceutically acceptable salt in conjunction or association with a pharmaceutically acceptable carrier or vehicle. If the manufacture of pharmaceutical formulations involves intimate mixing of pharmaceutical excipients and the active ingredient in salt form, then it is often preferred to use excipients which are non-basic in nature, i.e. either acidic or neutral.

Formulations for oral administration in the present invention may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution or a 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 and as a bolus etc.

With regard to compositions for oral administration (e.g. tablets and capsules), the term suitable carrier includes vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example 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 metallic stearates, glycerol stearate stearic acid, silicone fluid, talc waxes, oils and colloidal silica. Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring or the like can also be used. It may be desirable to add a colouring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art.

A tablet may be made by compression or moulding, 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 a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be optionally be coated or scored and may be formulated so as to provide slow 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 comprising the active agent in a suitable liquid carrier.

The appropriate dosage will depend upon the indications and the patient, and is readily determined by conventional animal drug metabolism and pharmacokinetics (DMPK) or clinical trials and in silico prediction software.

In treating HIV, the compounds of formula I are typically administered in an amount to achieve a plasma level of around 100 to 5000 nM, such as 300 to 2000 nM. This corresponds to a dosage rate, depending on the bioavailability of the formulation, of the order 0.01 to 10 mg/kg/day, preferably 0.1 to 2 mg/kg/day. A typical dosage rate for a normal adult will be around 0.05 to 5 g per day, preferably 0.1 to 2 g such as 500-750 mg,

in one to four dosage units per day. As with all pharmaceuticals, dosage rates will vary with the size and metabolic condition of the patient as well as the severity of the infection and may need to be adjusted for concomitant medications.

In general dosages of from about 3 mg to approximately 1.6 grams per person per day, divided into 1 to 3 single doses, are suitable. A typical dosage for adult patients is 50-800, more preferably 400-600 twice, or most preferably once daily. As elaborated below HIV inhibitors are typically co-administered in a unit dosage form with other HIV inhibitors or metabolism modifying agents and the dosage regime (QD, BiD TiD, fast/with food etc) for such co-administered drugs will of course necessitate concomitant adjustment of the dosage regime for formula I

As is good prescribing practice with antiviral therapy, the compounds of formula I are typically co-administered with other HIV therapies to avoid the generation of drug escape mutants. However, certain antifectives can induce a synergistic response, allowing one or both of the active ingredients to be administered at a lower dose that the corresponding monotherapy. For example in drugs prone to rapid metabolism by Cyp3A4, co-dosing with the HIV protease inhibitor ritonavir can allow lower dosage regimes to be administered. The compound of the invention and the or each further antiviral agent are typically co-administered at molar ratios reflecting their respective activities and bioavailabilities. Generally such ratio will be of the order of 25:1 to 1 :25, relative to the compound of formula I, but may be lower, for instance in the case of cytochrome antagonists such as ritonavir.

Representative HIV antivirals include NRTI such as alovudine (FLT), zudovudine (AZT, ZDV), stavudine (d4T, Zerit), zalcitabine (ddC), didanosine (ddl, Videx), abacavir, (ABC, Ziagen), lamivudine (3TC, Epivir), emtricitabine (FTC, Emtriva), racevir (racemic FTC), adefovir (ADV), entacavir (BMS 200475), alovudine (FLT), tenofovir disoproxil fumarate (TNF, Viread), amdoxavir (DAPD), D-d4FC (DPC-817), -dOTC (Shire SPD754), elvucitabine (Achillion ACH- 126443), BCH 10681 (Shire), SPD-756, racivir, MIV-606 (Medivir), D-FDOC, GS7340, INK-20 (thioether phospholipid AZT, Kucera), 2'3'-dideoxy-3'-fluoroguanosine (FLG) & its prodrugs such as MIV-210, reverset (RVT, D-D4FC, Pharmasset DPC-817) .

Representative NNRTI include delavirdine (Rescriptor), efavirenz (DMP-266, Sustiva), nevirapine (BIRG-587, Viramune), (+)calanolide A and B (Advanced Life Sciences),

capravirine (AG1549f S-1 153; Pfizer), GW-695634 (GW-8248; GSK), MIV- 150 (Medivir), MV026048 (R-1495; Medivir AB/Roche), NV-05 2 2 (Idenix Pharm.), R- 278474 (Johnson & Johnson), RS-1588 (Idenix Pharm.), TMC-120/125 (Johnson & Johnson), TMC- 125 (R- 165335; Johnson & Johnson), UC-781 (Biosyn Inc.) and YM215389 (Yamanoushi).

Representative HIV protease inhibitors include PA-457 (Panacos), KPC-2 (Kucera Pharm.), 5 HGTV-43 (Enzo Biochem), amprenavir (VX -478, Agenerase), atazanavir (Reyataz), indinavir sulfate (MK-639, Crixivan), Lexiva (fosamprenavir calcium, GW - 433908 or 908, VX- 175), ritonavir (Norvir), lopinavir, lopinavir + ritonavir (ABT-378, Kaletra), tipranavir, nelfinavir mesylate (Viracept), saquinavir (Invirase, Fortovase), AG 1776 (JE-2147, KNI-764; Nippon Mining Holdings), AG- 1859 (Pfizer), DPC- 681/684 (BMS), GS224338 (Gilead Sciences), KNI-272 (Nippon Mining Holdings), Nar- DG-35 (Narhex), P(PL)-IOO (P- 1946; Procyon Biopharma), P- 1946 (Procyon Biopharma), R-944 (Hoffmann-LaRoche), RO-0334649 (Hoffmann-LaRoche), TMC- 114/Darunavir (Tibotec/Johnson & Johnson), VX-385 (GW640385; GSK/Vertex), VX- 478 (Vertex/GSK).

Other HIV antivirals include entry inhibitors, including fusion inhibitors, inhibitors of the CD4 receptor, inhibitors of the CCR5 co-receptor and inhibitors of the CXCR4 coreceptor, or a pharmaceutically acceptable salt or prodrug thereof. Examples of entry inhibitors are AMD-070 (AMDl 1070; AnorMed), BlockAide/CR (ADVENTRX Pharm.), BMS 806 (BMS-378806; BMS), Enfurvirtide (T-20, R698, Fuzeon), KRH 1636 (Kureha Pharmaceuticals), ONO-4128 (GW-873140, AK-602, E-913; ONO Pharmaceuticals), PRO- 140 (Progenies Pharm), PRO-542 (Progenies Pharm.), SCH-D (SCH-417690; Schering-Plough), T- 1249 (R724; Roche/Trimeris), TAK-220 (Takeda Chem. Ind.), TNX-355 (Tanox) and UK-427,857 (Pfizer). Examples of integrase inhibitors are L-870810 (Merck & Co.), c-2507 (Merck & Co.) and S(RSC)-1838 (shionogi/GSK).

Many HIV patients are co-infected, or prone to superinfection, with other infectious diseases. Accordingly, a further aspect of the invention provides combination therapies comprising the compound of the invention co-formulated in the same dosage unit or co- packaged with at least one further anti-infective pharmaceutical. The compound of the invention and the at least one further antinfective are administered simultaneously or

sequentially, typically at doses corresponding to the monotherapy dose for the agent concerned.

Typical coinfections or superinfections include hepatitis B virus (HBV) or Hepatitis C virus (HCV). Accordingly the compound of the invention is advantageously coadministered (either in the same dosage unit, co-packaged or separately prescribed dosage unit) with at least one HCV antiviral and/or at least one HBV antiviral. Accordingly the compound of the invention is advantageously co-administered (either in the same dosage unit, co-packaged or separately prescribed dosage unit) with at least one HCV antiviral and/or at least one HBV antiviral.

Examples of HBV antivirals include lamivudine and 2'3'-dideoxy-3'-fluoroguanosine (FLG) & its prodrugs such as the 5'-O-lactylvalyl prodrug MIV-210. These HBV antivirals are particularly convenient as they are simultaneously active against both HBV and HIV.

Examples of HCV antiviral for co-administration with formula I include immune modifiers such as ribavirin or interferons, nucleoside HCV polymerase inhibitors or HCV protease inhibitors, many of which are currently under development.

The compounds of the invention are believed to counteract elevated LDL-cholesterol and/or triglyceride levels often appearing as a side effect of prior art HIV protease inhibitors. Accordingly the compounds of the invention are useful for replacing such prior art inhibitors in the ongoing dosage regimes of patients. Typically such patient has been or is undergoing antiretroviral therapy with one or more conventional HIV protease inhibitors and exhibits elevated plasma LDL-cholesterol and/or triglyceride levels. Such other HIV protease inhibitor(s) may be given as monotherapy or as part of an antiretroviral therapy which also includes one or more other antiretroviral drugs such as reverse transcriptase inhibitors or nonnucleoside reverse transcriptase inhibitors. Such candidates, although they may exhibit satisfactory viral suppression, may be of increased risk for hyperlipidemia and premature cardiovascular events.

The term "elevated plasma LDL-cholesterol and triglyceride levels" as used herein is based on the National Cholesterol Education Program (NCEP) clinical practice guidelines for the prevention and management of high cholesterol in adults.

In the latest guidelines issued in 2001, plasma levels of > 130 mg/dL of LDLcholesterol and >150 mg/dL of triglycerides are considered elevated or "high". The process of the present invention is particularly useful for those patients having plasma triglyceride levels of >200 mg/dL and for those patients with no risk factors or previous cardiovascular events having LDL-cholesterol levels of >160 mg/dL.

The definition of "elevated" LDL-cholesterol and triglyceride levels may, of course, change in the future as the NCEP continues to evaluate heart attack risk factors. It is intended, then, that the term "elevated LDL-cholesterol and triglyceride levels" as used will be consistent with current NCEP guidelines.

In one of its aspects, the present invention involves discontinuing the offending (the drug responsible for the elevated plasma LDL-cholesterol and/or triglyceride levels) HIV protease inhibitor from the above regimen and substituting therefore an amount of the compound of formula I which is effective to inhibit HIV and to reduce plasma LDL- cholesterol and/or triglyceride levels.

The dose of the compound of the invention to be employed depends on such factors as the body weight, age and individual condition of the patient to be treated and the mode of administration.

It is believed that the compounds according to some embodiments of the invention can in certain formulations interact favourably with cytochrome P450 monooxygenase and can improve the pharmacokinetics of drugs metabolized by this enzyme, including particularly other HIV protease inhibitors such as saquinavir, indinavir, nelfinavir, araprenavir, tipanavir and lopinavir. Thus, it may act in a similar way to ritonavir described in U.S. Patent 6,037,157 to increase blood levels of the coadministered HlV protease inhibitor. Conveniently and in contradistinction to ritonavir it is believed that the compound of the invention may be employed in combination therapy with other HIV protease inhibitors at its normal therapeutic dose level instead of the sub-therapeutic dose levels used with ritonavir. Any such potentiating effect on other HIV protease inhibitors which are metabolized by cytochrome P450 monooxygenase, may allow the use of the compounds of the invention concomitantly with such other HIV protease inhibitors thereby allowing reduced dosages of such other HIV protease inhibitors to be used while maintaining the same degree of viral suppression. Conceivably the compound of the invention can be used in combination with other HIV protease inhibitors to reduce LDL-

cholesterol and triglyceride levels in AIDS patients undergoing protease inhibitor therapy while still maintaining the desired level of viral suppression.

The appropriate dose of the HIV protease inhibitor being combined with the compounds of the invention can be determined by the following method which was used for the atazanavir/saquinavir combination, as disclosed in WO03020206. Atazanavir is a moderate inhibitor of the cytochrome P450 3A enzyme comparable to nelfinavir and indinavir, with a Ki of 2.4 μM. The latter two compounds increase the exposure of saquinavir (dosed at 1200 mg thrice-daily (TID) by 392 and 364%, respectively, at steady-state. A multiple-dose pharmacology study was completed to evaluate if a similar increase could be expected for the combination of atazanavir and saquinavir. This study showed a greater than 3 -fold increase in exposure, due to combination with atazanavir, supporting a 1200 mg once-daily saquinavir dosing, was equivalent to the currently marketed saquinavir regimen of 1200 mg TID. Using a constant dose of atazanavir the range of saquinavir doses were studied to target the saquinavir exposure (AUC (area under the curve) and CMlN (minimum concentration)) similar to those in the literature. Similarly, appropriate dosing of other HIV protease inhibitors to be used in combination with the compound of the invention can be calculated.

Compounds of the invention are typically synthesized outlined below.

The preparation of cis-aminoindanols are well described in the literature and many are commercially available. The preparation of cis aminochrominols is shown in Biorg Med Chem Lett 2005 15 531 1-5314 and Biorg Med Chem Lett 2004 14 4651-4654.

A useful intermediate for the preparation of compounds of general formula (I) wherein L is NH ₂ , is an α,α-disubstituted amino acid, which can be prepared according to a procedure adapted from the method descried by Khalil et al. in J. Med. Chem., 42, (1999), 2977-2989 as illustrated in scheme 1.

A suitably protected amino acid carrying the desired side chain, Rl can be converted to the oxazolidinone (Ib) by treatment with benzaldehyde dimethyl acetal in the presence of a Lewis acid such as BF ₃ XOEt ₂ . Alkylation of the oxazolidinone with an olefinic alkylating agent for example the alkenyl halide such as the chloride, bromide or iodide of the appropriate chain length, i.e. allyl halide for the preparation of compounds wherein n is 1 and but-3-enyl halide for the preparation of compounds wherein n is 2 provides α,α- dialkyl oxazolidinone (Ic). Basic hydrolysis of the lactone effected by treatment with a hydroxide solution for example sodium hydroxide in methanol or the like gives the α,α- dialkylated amino acid (Id).

Compounds of formula (I) can then be prepared from the unsaturated acid as illustrated in scheme 2.

Scheme 2

Coupling of the above described acid (Id) to a desired amine A'-NH ₂ , where A' is as defined above, using standard peptide coupling conditions using for example reagents like O-(l H-benzotriazol- 1 -yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) in the presence of a base like N,N-diisopropylethyIamine (DIEA) in a solvent like dimethylformamide (DMF) or any other suitable conditions well known in the art, provides the amide derivative (2a). Oxidative cleavage of the double bond effected by any suitable reagents such as sodium periodate in the presence of a catalytic amount of osmium tetroxide provides the aldehyde (2b), which subsequently can be reacted with a desired hydrazide derivative (2c) in a reductive amination reaction using any suitable reducing such as NaBH(OAc) ₃ or the like, to give hydrazide derivative (2d). Removal of the amino protecting group using standard methods well known in the art, for example, when a Boc or Cbz protecting group is used, acidic treatment e.g. TFA optionally in the presence of anisole gives the primary amino derivative (2e).

As is recognized by the skilled person, the synthetic steps and the introduction of the various moieties of the compounds of the invention may be performed in any convenient order. For example the hydrazide moiety (2c) can be introduced prior to the amine A'-

NH ₂ , as illustrated in scheme 3.

Scheme 3

Oxidation of the double bond of the acid (Id) to the corresponding aldehyde (3a) followed by a reductive amination with the hydrazide (2c) as described in scheme 2, provides the hydrazide (3b). The amide (2e) is then achieved by a peptide coupling with the desired amine A'-NH ₂ as described in scheme 2.

An unsubstituted or optionally temporarily N-protected hydrazide derivative may alternatively be used in the reductive amination step in schemes 2 and 3 and the substituent -CH ₂ -R ² of the hydrazide nitrogen is introduced afterwards as exemplified with the aldehyde 2b, in scheme 4.

Scheme 4

Reaction of aldehyde (2b) with a primary hydrazide derivative (4a) in a reductive amination reaction as described in scheme 1 provides the hydrazide (4b). The N- substituent CH ₂ -R ² can then be introduced by alkylation of the β-nitrogen of the hydrazide with a desired alkylating agent, R ² -CH ₂ -X wherein R ² is as defined above for R ² or R ² is a group that subsequently can be converted to R ² , and X is a leaving group such as a halide like chloride, bromide or iodide or a derivative of sulphonic acid such as a triflate, mesylate or tosylate, thus providing the N-alkylated compound (4c). The introduction of the N-substituent may alternatively be achieved by reaction with an aldehyde in a reductive amination using any suitable reductive agent such as NaB(OAc) ₃ or the like.

If desired, the primary amine of compound If can be alkylated once or twice by way of an alkylation or by a reductive amination or any other suitable method described in the literature in order to achieve compounds of formula (I) wherein L is NH(Ci-C ₃ )AIk or N(Ci-C ₃ )AIk ₂ .

Compounds of formula (I) wherein n is 1, L is F and X is F or OH are conveniently prepared from an intermediate cyclic mono or di fluoro derivative which can be synthesized as shown in scheme 5.

Scheme 5

Alkylation of commercially available ethyl acetoacetate by standard methods well known in the art, and a subsequent ring closure effected by treatment with bromine optionally under elevated temperature gives the lactone (5a). Amination with pyrrolidine followed by a reduction effected by treatment with a reducing agent such as NaCNBH ₃ or the like gives the olefine (5b) which subsequently can be dihydroxylated using any suitable oxidation system, for instance OsO ₄ -NMMO, providing the dihydroxy lactone (5c). Treatment of the afforded dihydroxy lactone (5c) with thionyl chloride in the presence of triethylamine followed by tetrabutylammonium fluoride in the presence Of RuCl ₃ and NaIO ₄ provides the fluorohydrine (5d) whereas treatment of the dihydroxy lactone with DAST followed by diisopropylamine-HF-complex provides the difluoro compound (5e).

The afforded fluoro derivatives (5d) and (5e) can then be further reacted as shown in scheme 6.

Scheme 6

Opening of the cyclic fluoro derivative (6a) with a desired amine A'-NH ₂ provides the alcohol (6b). Selective oxidation of the primary alcohol to the corresponding aldehyde using any convenient reagent such as Dess-Martin periodinane followed by a reductive amination with a suitable hydrazide derivative as described above, provides the hydrazide (6c)

Various amines, A'-NH ₂ , used in the above schemes are available commercially or alternatively they can be prepared according to literature procedures. For example, amines wherein A' is according to formula (IV) can be prepared as described by B. Samuelsson et al. in Bioorg. Med. Chem., 1 1, 2003, p. 1107-1 1 15. Alternatively, they can be prepared from the corresponding alcohols A'-OH by transforming the hydroxy group to an amino group. This transformation can be effected by any suitable method known by the skilled person, for instance by converting the hydroxy group to a leaving group such as a halide like a bromide, chloride or iodide or to a derivative of sulphonic acid such as a mesylate, triflate or tosylate, followed by a nucleophilic displacement reaction with azide and finally reduction of the azide to the amine using any suitable reduction method such as catalytic hydrogenation. Suitable alcohols are described for example by A. K. Gosh et al. in J. Med. Chem., 1996, 39, 3278-3290.

A further alternative to prepare amines, A'-NH ₂ , wherein A' is according to formula (IV) is illustrated in scheme 7.

X is O or S, n is 1 or 2

Scheme 7

Addition of a bromide and a propargyloxy group to the double bond of the unsaturated ring (7a) effected for instance by reaction with N-bromosuccinimide and propargyl alcohol followed by a reductive ring closure reaction promoted by tri-n-butyltin hydride in the presence of a radical initiator for example l,l '-azobis(isobutyronitrile) or the like yields bicyclic olefin (7c). The exocyclic double bond can then be cleaved oxidatively by subjecting the olefinic compound to the appropriate oxidation conditions such as treatment with osmium tertoxide in combination with sodium periodate which gives the keto derivative (7d). Reaction of the formed keto group with O-benzylhydroxyl amine followed by reduction with a reducing agent like lithium aluminium hydride gives the corresponding amine (7f) as a racemic mixture. The racemic mixture can thereafter be separated according to procedures known in the art. For example, a diastereomeric mixture which can be separated by chromatographic methods, can be prepared by coupling of a chiral auxiliary compound such as a chiral amino acid for example Boc-L- phenylalanine, using standard peptide coupling methods. Separation of the mixture and thereafter cleavage of the auxiliary amino acid then provides the pure diastereomers of the desired amine (7f).

An example of the preparation of amine derivatives A'-NH ₂ used i.a. in scheme 1 wherein A' is according to formula (II) is shown in scheme 8 below.

8a NMM _{τ 8c 8d}

Scheme 8

Coupling of a suitably N-protected, for example Boc protected, amino acid (8a), carrying the desired side chain R ³ to an amino derivative (8b), where R ³ and R ⁴ are as defined above, using standard peptide coupling conditions, like using coupling reagents such as EDAC, NMM and HOBT in an inert solvent like dimethylformamide gives the amide (8c). Removal of the N-protecting group, by acidic treatment in the case of a Boc protecting group, for example by using trifluoroacetic acid in dichloromethane, gives the amine (8d). Amino acids (8a) used in the above scheme are commercially available or they can be prepared according to literature procedures. A method to prepare amino acids carrying a branched side chain is exemplified in Scheme 9.

9d 9e

Scheme 9

Treatment of the amino acid (9a), achieved as described by Rapoport et al. in J. Org. Chem., 55, (1990) p. 5017-5025, with one or two successive additions of a base such as potassium bis-(trimethylsilyl) amide (KHMDS) and methyl iodide provides mono or dimethylated amino acid (9b) respectively. Reduction of the side chain ester using a reagent like DIBAL followed by interchanging of the PhFl group for a Boc group effected by catalytic hydrogenation in the presence of BoC ₂ O and a catalyst like Pd/C, provides the alcohol (9c). If desired, the hydroxy group of the afforded alcohol can subsequently be methylated for instance by treatment with a suitable methylating agent such as methyl iodide and a base like NaH which gives the methoxy compound (9e). Alternatively, the alcohol can be converted to the corresponding fluorocompound (9d) by treatment with a fluorinating agent such as DAST or the like, or any other suitable fluorinating method described herein or elsewhere can be used.

Amines, A'-NH ₂ , wherein A' is according to formula (III) can be prepared as exemplified in scheme 10.

10a 10b 10c

1Od

Scheme IO

Reaction of a natural or non-natural amino acid (10a) carrying the appropriate side chain R ³ defined as above, with a desired acylating agent; a chloroformate (i) for the formation of compounds wherein W is O, an acid chloride (ii) for the formation of compounds wherein W is a bond or an isocyanate (iii) for the formation of compounds wherein W is NH, provides the acid (10b). The amine A'-NH ₂ (1Od) can then be achieved by transforming the acid (10b) to the corresponding primary amide (10c) for example by treatment with an ammonia solution in the presence of isobutyl chloroformate and N- methylmorpholine in a solvent like dimethoxy ethane, followed by a rearrangement reaction brought about by treatment with [bis(trifluoroacetoxy)iodo]benzene optionally in the presence of pyridine as described e.g. by J-A. Fehreentz in J. Med. Chem., 2003, 46, 1 191-1203.

Hydrazide derivatives R ² CH ₂ NHNHC(=O)A" used in the above schemes can be prepared by reaction of an acid A"COOH or a derivative thereof, for instance an acid chloride or an acid anhydride, with a hydrazine R ² CH ₂ NHNH ₂ under standard peptide coupling conditions. Scheme 1 1 shows an example wherein A" in the acid, A"COOH is according to formula (V) as defined above.

Scheme 1 1

Reaction of a natural or non-natural amino acid (1 Ia) carrying the appropriate side chain R ⁸ defined as above, with a desired acylating agent as described in scheme 3 provides the acid (8b). The hydrazide derivative (l id) can then be achieved by coupling of a hydrazine derivative (1 Ic) which is available either commercially or in the literature, using standard peptide coupling conditions as described above. Compounds wherein A" is according to formula (VII) can conveniently be prepared according to the above described route but with the use of a suitable sulphonylating agent like alkylsulphonyl chloride, R ⁹ -S(=O) ₂ C1, in the presence of a base like sodium hydroxide, instead of any of the depicted acylating agents i, ii or iii, in the reaction with amino acid 1 Ia.

Hydrazides wherein A" is according to formula (VI) can be prepared by reaction of an appropriate electrophilic carbonyl compound such as a chloroformate or an activated carbonate with the hydrazine derivative R ² CH ₂ NHNH ₂ as illustrated in scheme 12.

Scheme 12

The alcohol (12a) can be converted to the corresponding activated carbonate (12b) or chloroformate by reaction of the hydroxy group with a suitable acylating agent like a carbonate such as dipyridyl carbonate or para-nitrophenyl chloroformate optionally in the presence of a base such as triethylamine or imidazole, or to a chloroformate by reaction with phosgene optionally in the presence of base like sodium hydrogen carbonate. The afforded electrophilic compound can then be reacted with a desired hydrazine derivative (12c) to give the corresponding hydrazide (12d). Alcohol (12a) is either commercially available or can be prepared for example as described by A. K. Ghosh et al. in J. Med. Chem., 1996, 39, 3278-3290. For example, the stereochemic synthesis of a bis-THF alcohol, i.e. an alcohol according to formula 12a wherein Ry together with the adjacent C atom defines a fused furan ring, is described in WO2003/22853 and WO05/63770 with corresponding methodology being applicable to the mirror image derivative.

The procedure described in scheme 12 can also be applied to other alcohols for instance optionally substituted carbocyclylmethanol, optionally substituted heterocyclylmethanol, optionally substituted carbocyclylalcohol or optionally substituted heterocyclylalcohol thus providing hydrazides wherein A" is according to formula (VIII) as defined above.

Synthesis of unsubstituted hydrazides (4a) are described in the literature, se for example J. Med. Chem. 1998, 41, p. 3387, and a general example thereof is shown in scheme 13.

γ JL-

13a ^13b 13c 4a

Scheme 13

Commercially available t-butyl carbazate (13a) can be coupled to an acid (13b) wherein A" is as defined above, in a peptide coupling reaction using standard procedure to give the corresponding Boc protected hydrazide (13c). Removal of the Boc group using standard conditions like acidic treatment, for example with TFA in dichloromethane provides the unprotected hydrazide (4a).

Compounds of formula (I) wherein the R ² group is a substituted aryl can be prepared by using the appropriate hydrazide derivative carrying the desired R ² group in any of the above schemes, or alternatively, the substituent can be introduced at a later stage of the synthesis, using any suitable method known from the literature. A method wherein a heteroaryl group is added to an aryl group is exemplified in scheme 14.

14a 14b

Scheme 14

The aryl group of compound (14a) can be substituted with for example an aryl or heteroaryl group such as a pyridyl group by reacting the tri-n-butyltin derivative of the desired substituent in a coupling reaction using a palladium(O) reagent such as Pd(PPh ₃ ) ₂ Cl ₂ or the like in the presence of CuO in a solvent like dimethylformamide at an elevated temperature effected for instance by heating with microwaves.

Even though scheme 14 exemplifies the aromatic substitution on an amide derivative (A'- NH-C(=0)-), the skilled person will realize that the same procedure is applicable to the corresponding acid derivatives. It should further be recognized that the strategy described in scheme 13 is not restricted to pyridyl groups but is also applicable to other, optionally substituted, alkyl, aryl or heteroaryl groups. It should also be recognized that other methods, many of which are extensively described in the literature, may be used for the substitution of the R ² -group.

Compounds in which R or R are substituted with a 2-methyl-thiazol-4-ylmethoxy residue, typically at the para position of a phenyl R ¹ or R ² moiety are typically prepared by alkylation of the corresponding phenol with 2-methylthiazol-4-ylmethylenechloride. The substituent may be introduced to R ¹ or R ² prior to coupling of the P1/P2 and Pl 7P2' building blocks, for example when preparing lactones Ib or 5a or hydrazide 1 Ic or 12 c.

A convenient synthon for a 2-methyl-thiazol-4-ylmethoxy substituent to Rl or R2 is hydroxyl-protected phenol, in the benzyl protecting group as this group is readily removed, for example with H ₂ /palladium catalysis at atmospheric pressure in neutral conditions to activate the phenolic hydroxy!, without substantial concomitant activation of other hydroxyl functions on the backbone, thereby allowing coupling with an alkylating agent. The following general conditions are typically used: 2-methylthiazol-4- ylmethylenechloride, 2.5 Eq, CsCO ₃ , dioxane, 8OC, 2-4 hours. Similar alkylating conditions are reported in J Med Chem 1992 1688 along with representative routes for synthesis of R ² methylenechloride reagents. Further guidance is provided in WO00/76961 and JOC 2000, 65(22) page 7464. The presence of other primary or secondary hydroxyl groups, such as in an indanolamine A' may however require protection.

Any functional groups present on any of the constituent compounds used in the preparation of the compounds of the invention are appropriately protected where necessary. For example functionalities on the natural or non-natural amino acids are typically protected as is appropriate in peptide synthesis. Those skilled in the art will appreciate that the selection and use of appropriate protecting groups depend upon the reaction conditions. Suitable protecting groups are described in Greene, "Protective Groups in Organic Synthesis", John Wiley & Sons, New York (1981) and "The Peptides: Analysis, Synthesis, Biology", Vol. 3, Academic Press, New York (1981), the disclosure of which are hereby incorporated by reference.

Detailed Description

Various embodiments of the compounds of the invention and key intermediates towards such compounds will now be described by way of illustration only with reference to the accompanying non-limiting chemistry and biology examples.

Chemistry. General Information. Analytical RP-LC-MS was performed on a Gilson HPLC system with a Finnigan AQA quadropole mass spectrometer using a Chromolith Performance RP- 18e 4.6 x 100 mm (Merck KGaA) column, with MeCN in 0.05% aqueous HCOOH as mobile phase at a flow rate of 4 mL/min. Preparative RP-LC-MS was performed on a Gilson HPLC system with a Finnigan AQA quadropole mass spectrometer using a Zorbax SB-C8, 5 μm 21.2 x 150 mm (Agilent technologies) column, with MeCN in 0.05% aqueous HCOOH as mobile phase at a flow rate of 15 mL/min. Optical rotations were obtained on a Perkin-Elmer 241 polarimeter, specific rotations ([α]o) are reported in deg/dm and the concentration (c) is given in g/100 mL in

the specified solvent. ¹ H and ¹³ C NMR spectra were recorded on Varian Mercury Plus instruments at 300 and 75.45 MHz or 399.78 and 100.53 MHz respectively. Chemical shifts are reported as δ values (ppm) indirectly referenced to TMS via the solvent residual signal. Flash column chromatography was performed on Merck silica gel 60 (40-63 μm) or Merck silica gel 60 RP-18 (40-63 μm). Analytical thin layer chromatography was done using aluminum sheets precoated with silica gel 60 F ₂₅₄ . UV light and an ethanolic solution of phosphomolybdic acid followed by heating visualized components. Analytische Laboratorien, Lindlar, Germany, performed elemental analyses.

Example 1. synthesis of π-(SVrλ^-r3-(5Vamino-3-((15'J2;?)-2-hydroxy-indan-l- ylcarbamoyl)-4-phenyl-butyl1-λ' ⁷ -(4-pyridin-3-yl-benzyl)-hydrazinocarbonyl1-2,2- dimethyl-propyll-carbamic acid methyl ester step a

[MiSVBenzyl-MπS^iO^-hydroxy-indan-l-ylcarbamoyD-but-S-enyli -carbarnic acid benzyl ester (Ia)

A solution of (25)-N-(benzyloxycarbonyl)-2-allylphenylalanine, prepared as described in J. Med. Chem. 42, (1999), 2977-2987, (0.97 g, 2.86 mmol) and (lS,2R)-c/s- l-amino-2-indanol (0.427 mg, 2.86 mmol) in DMF (20 ml) was cooled to 0 °C. HATU (1.03 g, 2.86 mmol) was added in one portion followed by N,τV-diisopropylethylamine (2.5 ml, 14.3 mmol). The reaction mixture was stirred at 0 °C for 2 h and then diluted with DCM. The resulting solution was washed with water, sat. NaHCO ₃ and concentrated. The residue was purified by silica gel flash chromatography eluting with 1% of EtOH in DCM to give the title compound (1.15g, 86%). ¹ H NMR (CDCl ₃ ): 7.36 (m, 4H), 7.3-7.15 (m, 8H), 7.0 (m, 2H), 6.52 (m, IH), 5.85 (m, IH), 5.44 (m, IH), 5.25-5.12 (m, 3H), 4.95 (m, 2H), 4.71 (m, IH), 3.38 (A-B q, 2H), 3.06 (A-B m, 2H), 2.72 (br, IH), 2.56 (A-B m, 2H)

( l-f^-Benzyl-l-fπ.S'^^-σ-hvdroxy-indan-l-ylcarbamovn-S-rN'- Q-^-methoxy- carbonylamino-33-dimethyl-butyryl)-N-(4-pyridin-3-yl-benzyl) -hydrazino1-propyU- carbamic acid benzyl ester (I b)

To a solution of compound Ia (0.6 g, 1.28 mmol) in THF/H ₂ O (10 ml, 4: 1) at 0 ⁰ C was added NaIO ₄ (0.544 g, 3.2 mmol) and a catalytic amount (~2.5 mol%) of OsO ₄ . After being stirred for 4h, the solution was extracted with Et ₂ O, the organic extracts were dried over Na ₂ SO ₄ and concentrated under vacuum.

The afforded aldehyde and { l-(S)-[N'-(4-pyrid-3-yl-benzyl)-hydrazinocarbonyl]-2,2- dimethyl-propyl} -carbamic acid methyl ester (0.472 g, 1.28 mmol) were dissolved in 1,2- dichloroethane (20 ml) and stirred for 20 min. NaBH(OAc) ₃ (0.81 g, 3.82 mmol) was added in three portions over 1 h and the mixture was stirred for 1 h. The reaction was quenched by addition of water, then washed with brine and evaporated in vacuum. The residue was purified by RP (C ₈ ) prep. HPLC (15 min gradient of 15-100% CH ₃ CN in water) to give 0.095g of the title compound (9% overall yield). MS (ESI ⁺ ): m/z 827 (M+ 1) ⁺ .

( l-(^-[λP-r3-(5^-amino-3-f(15'.2i?)-2-hvdroxy-indan-l-ylcarb amoyl)-4-phenyl-butyl1-N'- (4-pyridin-3-yl-benzyl)-hydrazinocarbonvn-2,2-dimethyl-propy l} -carbamic acid methyl ester (Ic)

A solution of compound Ib (95 mg, 0.12 mmol), anisole (26 μl, 0.24 mmol) and trifluoromethanesulfonic acid (36 μl, 0.42 mmol) in DCM (3.5 ml) were stirred at ambient temperature for 2h. The reaction mixture was diluted with DCM washed with

sat. NaHCO ₃ and evaporated in vacuum. The residue was purified by RP (C ₈ ) prep. HPLC (15 min gradient of 10-100% CH ₃ CN in water) to give the title compound (64 mg, 81%). MS (ESI ⁺ ): m/z 693 (M+ 1) ⁺ .

¹ H NMR (CDCl ₃ ): 8.76 (br. s, IH), 8.58 (br. s, I H), 7.94 (d, IH), 7.78 (d, IH), 7.5-7.05 (m, 15H), 6.76 (br. s, I H), 5.28 (m, 2H), 4.38 (m, IH), 4.16 (d, IH), 3.95 (d, IH), 3.61 (m, 3+1 H), 3.26 (d, IH), 3.1 1 (m, 2H), 2.92 (m, 2H), 2.61 (d, IH), 2.44 (m, IH), 1.70 (br. m, 4H), 0.82 (s, 9H).

Example 2. synthesis of ( l-(S)-rλ^-r3-(5 ^r )-amino-3-(d5' _< 2/?)-2-hvdroxy-indan-l- ylcarbamoyl)-4-phenyl-butyll-λ^-(4-pyridin-2-yl-benzyl)-hyd razinocarbonyl1-2,2- dimethyl-propyU-carbamic acid methyl ester: step a

{ 1 -(SVBenzyl- 1 -(( 1 S2R)-2-h vdroxy-indan- 1 -ylcarbamoyD-S-rN'^-ffl-methoxy- carbonylamino-SJ-dimethyl-butyrvD-N-^-bromo-benzyO-hydrazino i-propyU-carbamic acid benzyl ester (2a)

{(iS)-l-[N'-(4-Bromo-benzyl)-hydrazinocarbonyl]-2,2-dimethyl -propyl}-carbamic acid methyl ester was reacted with compound 1 a according to the procedure described in example 1, step b, which gave the title compound (33%). MS (ESI ⁺ ): m/z 828, 830 (M+ 1) ⁺ .

¹ H NMR (CDCI ₃ ): 7.6-7.0 (m, 22H), 5.42 (m, IH), 5.14 (m, 3H), 4.65 (m, IH), 3.87 (d, I H), 3.65 (d, IH), 3.59 (m, I H), 3.55 (s, 3H), 3.47 (m, IH), 3.3-2.9 (m, 5H), 2.6 (br.m, 2H), 2.00 (m, 2H), 0.64 (s, 9H).

step b

( l-f^-Benzyl-l-fd^ig^-hvdroxy-indan-l-ylcarbamovn-S-rN'^-^-me thoxy- carbonylamino-3,3-dimethyl-butyryl)-N-(4-pyridin-2-yl-benzyl )-hydrazino1-propyl>- carbamic acid benzyl ester (2b) and ( l-(5 ^r )- rN'-(2-r4-ffl-benzyl-l -((l.S.2/?)-2-hvdroxy-indan-l-vn-2,5-dioxo-imidazolidin-4- yl1-ethyl}-NM4-pyridin-2-yl-benzyl)-hvdrazinocarbonyl1-2,2-d imethyl-propyl}-carbamic acid methyl ester (2bb)

A mixture of compound 2a (58 mg, 0.07 mmol), 2-(l,l,l-tri-n-butylstannyl)pyridine

(51 mg, 0.14mmol) and CuO (11 mg, 0.14 mmol) in DMF (2 ml) was degassed with argon for 5 min. Pd(PPh ₃ ) ₂ Cl ₂ (2.4 mg, 5 mol%) was added and reaction mixture was heated at 120C for 2 h. The mixture was diluted with ethyl acetate, washed with sat.

NH ₄ Cl and evaporated. The residue was re-dissolved in CH ₃ CN and washed with hexane.

The acetonitrile phase was concentrated and the crude reaction mixture was purified by

RP (C ₈ ) prep. HPLC (15 min gradient of 10-100% CH ₃ CN in water) to give compound 5 ( 19 mg, 33 %) and compound 6 (8 mg, 15 %).

Compound 5 MS (ESI ⁺ ): m/z 827 (M+ 1) ⁺ , compound 6 MS (ESI ⁺ ): m/z 719 (M+ 1) ⁺ .

¹ H NMR (CDCl ₃ ): 8.67 (d, I H), 8.44 (br.s, IH), 7.94 (d, 2H), 7.73 (dt, IH) 7.66 (d, 2H),

7.29 (m, 4H), 7.21 (m, 5H), 7.04 (m, IH), 6.54 (br.s, IH), 6.23 (d, IH), 5.29 (d, IH), 5.19

(d, 1H)/4.51 (m, IH), 4.08 (d, IH), 3.68 (d, IH), 3.61 (s, 3H), 3.58 (d, I H), 3.09 (m, 3H), 2.87 (m, 4H), 2.00 (m, 2H), 0.72 (s, 9H).

ster.

( l-(S)-rN ^; -f3-(^-amino-3-((15'.2/?)-2-hvdroxy-indan-l-ylcarbamoyl)-4-p henyl-butyll- N"-(4-pyridin-2-yl-benzyl)-hvdrazinocarbonvn-2,2-dimethyl-pr opyU-carbamic acid methyl ester (2c)

The title compound was prepared by treatment of compound 2b according to the method described in Example 1, step c. MS (ESI ⁺ ): m/z 693 (M+ 1) ⁺ .

¹ H νMR (CDCl ₃ ): 8.63 (d, IH), 8.06 (br. d, IH), 7.72-7.66 (m, 3H), 7.52 (d, IH), 7.40 (d, 2H), 7.32-7.19 (m, 7H), 7.02 (m, 2H), 6.92 (m, 2H), 6.79 (br. s, IH), 5.33 (m, IH), 5.10

(m, I H), 4.28 (t, IH), 4.05 (d, IH), 3.94 (d, IH), 3.7-3.6 (s+m, 3H+1H), 3.27 (d, IH), 3.04 (m, 2H), 2.79 (m, 2H), 2.57 (d, IH), 2.34 (br. m, IH), 1.85 (br. m, 3H), 0.92 (s, 9H).

Example 3 Synthesis of 1 -(^-Benzyl- 1 -[I -(S^-methylcarbamoyl^^-dimethylpropyllcarbamoyl-S- fN'-(2-(5Vmethoxycarbonylamino-3,3-dimethylbutyryl)-N-(4- bromobenzyl)hydrazinolpropylamine: Step a

{ 1 -dSVBenzyl-1 -carboxy-S-rNW^SVmethoxycarbonylaminoOJ-dimethylbutyryQ-iV- (4-bromobenzyQhydrazinoipropyUcarbamic acid benzyl ester (3a) To a solution of (25)-N-(benzyloxycarbonyl)-2-allylphenylalanine (prepared as described in in J. Med. Chem. 42, (1999), 2977-2987, (373 mg, 1.00 mmol) in THF/H ₂ O (15 ml, 4: 1 ) at 0° C was added NaIO ₄ (512 mg, 2.5 mmol) and a catalytic amount (-2.5 mol%) of OsO ₄ . After being stirred for 5h, the solution was evaporated to small volume, diluted with water (10 ml), extracted with EtOAc (20 ml), and the organic extracts were combined and dried over Na ₂ SO ₄ and concentrated under vacuum to give crude aldehyde. MS (ESI ⁺ ): m/z 342 (M+ 1) ⁺ . The afforded aldehyde and { l-(5)-[N'-(4-bromobenzyl)hydrazinocarbonyl]-2,2- dimethylpropyl}carbamic acid methyl ester (447 mg, 1.20 mmol) were dissolved with acetic acid (72 mg, 1.20 mmol) in 1,2-dichloroethane (8 ml) and stirred at RT for Ih. NaBH(OAc) ₃ (318 mg, 1.5 mmol) was added in three portions over 0.5 h and the mixture stirred for 2 h. The reaction mixture was diluted with EtOAc (5 ml), CHCl ₃ (10 ml) and water (10 ml) and stirred for 15 min. The organic phase was washed with brine, dried with Na ₂ SO ₄ and evaporated in vacuum. Column chromatography on SiO ₂ with 2.5-5% MeOH in CH ₂ Cl ₂ gave the title compound (656 mg, 94%) as a white foam.. MS (ESI ⁺ ): m/z 697/699 (M+ 1) ⁺ .

¹ H NMR (CDCl ₃ ): 7.4-7.0 (m, 16H), 6.3 (br s, IH), 5.6 (br s, IH), 5.22 and 5.06 (AB q, 2H), 3.90 and 3.75 (AB q, 2H), 3.70-3.62 (m, 3+1 H), 3.47 and 3.16 (AB q, 2H), 2.95 (diffuse m, IH), 2.72 (diffuse m, IH), 2.53 (m, IH), 2.14 (m, IH), 0.79 (s, 9H).

( l -C^-Benzyl-l-ri-f^-methylcarbamoyl^^-dimethylpropyncarbamoyl -S-rN'^-^- methoxycarbonylamino-3,3-dimethylbutyryl)-N-(4-bromobenzyDhy drazino1- propyl jcarbamic acid benzyl ester (3b)

Compound 3a (210 mg, 0.30 mmol), (5)-t-butylglycine N-methylamide (90 mg, 0.36 mmol), HOBT.H ₂ O (27 mg, 0.20 mmol) and triethylamine (101 mg, 1.00 mmol) were dissolved in dichloromethane (10 ml), and EDAC (92 mg, 0.48 mmol) was added. After stirring over night, ethyl acetate (20 ml) and aq. NaHCO ₃ (5 ml) were added. After stirring a few minutes, brine (5 ml) was added and the organic phase was washed with 2 x 5 ml of saturated aq. KH ₂ PO ₄ , dried (Na ₂ SO ₄ ) and evaporated. Column chromatography on SiO ₂ with 2.5-5% MeOH in CH ₂ Cl ₂ gave title compound (123 mg, 50 %). MS (ESI ⁺ ): m/z 823/825 (M+ 1) ⁺ . ¹ H NMR (CDCl ₃ ): 8.17 (br s, IH), 8.0 (br d, IH), 7.4-7.0 (m, 14H), 6.74 (br s, IH), 6.44 (br s, IH), 5.71 (br d, IH), 5.17-5.02 (AB q, 2H), 4.28 (d, IH), 3.91 (d, I H), 3.84 (d, IH), 3.63 (s+d, 3+1H), 3.55 (d, IH), 3.35 (d, IH), 2.92-2.80 (m, 2H), 2.78 (d, 3H), 2.23 (m, IH), 2.02 (m, 2H), 1.06 (s, 9H), 0.73 (s, 9H).

1 -(SVBenzyl- 1 -F 1 -(5Vmethylcarbamoyl-2.2-dimethylpropyllcarbamoyl-3-rN'-(2-(. SV methoxycarbonylamino-3,3-dimethylbutyryl)-N-(4-bromobenzyl)h ydrazino1propylamine

(3c)

Compound 3b (80 mg, 0.097 mmol), anisole (21 mg, 0.194 mmol) and trifluoromethanesulfonic acid (81 mg, 0.54 mmol) in DCM (3 ml) were stirred at ambient temperature over night. The reaction mixture was diluted with DCM, washed with sat.

NaHCO ₃ and evaporated in vacuum. Column chromatography on SiO ₂ with 3-10 %

MeOH in CH ₂ Cl ₂ gave the title compound (59 mg, 88 %).

MS (ESI ⁺ ): m/z 689/691 (M+ 1) ⁺ .

¹ H NMR (CDCl ₃ ): 8.2 (br. d, IH), 7.7 (br. s, IH), 7.40-7.07 (m, 2+5+2H), 6.54 (br. s,

IH), 5.47 (d, IH), 4.24 (br. d, IH), 3.93 and 3.74 (AB q, 2H), 3.87 (d, I H), 3.65 (s, 3H),

3.20 (d, IH), 2.98 (diffuse m, IH), 2.75 (d, 3H), 2.68 (d, IH), 2.6 (diffuse m, I H), 2.25

(m, IH), 1.9 (br. s, NH ₂ ), 1.76 (m, I H), 0.98 (s, 9H), 0.83 (s, 9H).

Example 4

( l-(.S^-Benzyl-l-carboxy-3-rN ^> -(2-(.$^-methoxycarbonylamino-3,3-dimethylbutyryl)-N-

(4-(4-pyridyl)benzyl)hydrazinolpropyl}carbamic acid benzyl ester (4)

A mixture of compound 3a (70 mg, 0.10 mmol), 4-pyridylboric acid (31 mg, 0.25 mmol), triethylamine (66 mg, 0.66 mmol) and PEPPSI (7 mg, 0.01 mmol) in DME+H ₂ O+EtOH 7:3:2 (1 ml) was run at 140 °C for Ih in a microwave oven. After cooling, ethyl acetate (3 ml) was added and the mixture washed with 2 x 1 ml saturated aq. KH ₂ PO ₄ , dried (Na ₂ SO ₄ ) and evaporated to small volume. Column chromatography on SiO ₂ with 2-5% MeOH in CH ₂ Cl ₂ gave the title compound (20.4 mg, 29.3 %). MS (ESI ⁺ ): m/z 696 (M+ 1) ⁺ . ¹ H NMR (CDCl ₃ ): 8.6 (br.s, IH), 8.45 (d, 2H), 7.5-7.0 (m, 17H), 6.15 (br. s, IH), 5.45 (br. d, IH), 5.28 and 5.04 (AB q, 2H), 4.56 (br. d, IH), 3.89 (d, IH), 3.7-3.6 (m, 3+2 H), 3.41 (d, IH), 3.11 (d, IH), 2.88 (t, IH), 2.70 (d, I H), 2.40 (t, IH), 0.70 (s, 9H).

Example 5

{ l-(S ^r )-Benzyl-l-carboxy-3-[N'-(2-(5 ^f )-methoxycarbonylamino-3.3-dimethylbutyryπ-N- (4-(3-pyridyl)benzvDhydrazino]propyl}carbamic acid benzyl ester (5)

Compound 3a was reacted with 3-pyridylboric acid according to the procedure described in example 4, which gave the title compound (25 mg, 36 %) 14. MS (ESI ⁺ ): m/z 696 (M+ 1) ⁺ .

¹ H νMR (CDCl ₃ ): 8.54-8.49 (m, 2H), 7.78 (d, IH), 7.45-7.3 and 7.2-7.0 (m, 17H), 6.22 (br. s, I H), 5.47 (br. d, I H), 5.26 and 5.07 (AB q, 2H), 4.36 (d, IH), 3.95 (d, IH), 3.7- 3.55 (m, 3+2 H), 3.31 (d, IH), 3.13 (d, I H), 2.90 (t, IH), 2.65 (d, I H), 2.34 (t, IH), 0.75 (s, 9H).

Example 6

Synthesis of 1 -CSVBenzyl- !-(( 1&2 in-2-hvdroxyindan-l-ylcarbamoyl)-3-|W-f2-CSV methoxycarbonylamino-3,3-dimethylbutyryl)-N-(4-bromobenzyl)h ydrazino1-propyl-N- methylamine: Step a

l-(.S^-Benzyl-l-(carboxy)allyl-N-methylcarbamic acid benzyl ester (6a)

Sodium hydride, 60% in oil (440 mg, 11 mmol) was first washed with n-pentane and then added in small portions with ice cooling to a stirred solution of (2S)-N- (benzyloxycarbonyl)-2-allylphenylalanine (prepared as described in in J. Med. Chem. 42, (1999), 2977-2987, (144; 680 mg, 2.0 mmol) and methyl iodide (1.0 ml, 16 mmol) in 6 ml of dry THF. After 3 days at RT the mixture was cooled with ice-water and 0.5 ml of water was added dropwise. After 0.5h of stirring at RT, 10 drops of aqueous 2M NaOH and 1 ml of MeOH were added to get a homogenous solution. After a further 2h, part of the solvents were removed in vacuum and the residue was washed with 3 portions of diethyl ether, acidified to pH 3 with solid citric acid and then extracted with 3 portions of diethyl ether. Drying with Na ₂ SO ₄ and evaporation in vacuum gave an oil which solidified to a light yellow solid. Yield 644.5 mg (91%) of title compound. MS (ESI ⁺ ): m/z 354 (M+ 1) ⁺ .

2-Benzyl-2-(benzyloxycarbonyl-methyl-amino)-4-oxo-butyric acid (6b)

To a solution of the alkene (6a, 623 mg, 1.763 mmol) in THF/H ₂ O (25 ml, 4:1) at 0 ⁰ C was added NaIO ₄ (943 mg, 4.4 mmol) and a catalytic amount (-2.5 mol%) Of OsO ₄ . After being stirred for 5h, the solution was evaporated to a small volume, diluted with water (15 ml) and extracted with EtOAc (30 ml). The organic extracts were dried over Na ₂ SO ₄ and concentrated under vacuum to give crude aldehyde as an off-white solid. MS (ESI ⁺ ): m/z 356 (M+ 1) ⁺ .

Ster

{ l-(.S)-Benzyl-l-carboxy-3-[N'-(2-( ₁ S^-methoxycarbonylamino-3,3-dimethylbutyryπ-N- (4-(4-pyridyl)benzyl)hvdrazino1propyU-N-methylcarbamic acid benzyl ester (6c) Crude aldehyde (6b) and { 1 -(5)-[ν'-(4-bromobenzyl)hydrazinocarbonyl]-2,2- dimethylpropyl}carbamic acid methyl ester (745 mg, 2.0 mmol) were dissolved with acetic acid (120 mg, 2.0 mmol) in 1,2-dichloroethane (15 ml) and stirred at RT for Ih. Sodium triacetoxyborohydride (530 mg, 2.5 mmol) was added in three portions over 0.5 h and the mixture stirred for 2 h. The reaction mixture was diluted with EtOAc (7 ml), CHCl ₃ (15 ml) and water (15 ml) and stirred for 15 min. The organic phase was washed with brine, dried with Na ₂ SO ₄ and evaporated in vacuum. Column chromatography on SiO ₂ with 2.5-5% MeOH in CH ₂ Cl ₂ gave the title compound as a white foam (359 mg). MS (ESI ⁺ ): m/z 71 1/713 (M+l) ⁺ .

( l-(^-Benzyl-l-((15.2i?)-2-hvdroxyindan-l-ylcarbamovn-3-rN'-( 2-(5 ^> )- methoxycarbonylamino-3,3-dimethylbutyryπ-N-(4-bromobenzyπh ydrazino1propyU-iV- methylcarbamic acid benzyl ester (6d)

The acid (6d, 78 mg, 0.1 1 mmol), (lS,2R)-(-)-cis- l-amino-2-indanol (25 mg, 0.164 mmol), HOBT H ₂ O (10 mg, 0.074 mmol) and triethylamine (33 mg, 0.33 mmol) were dissolved in dichloromethane (3 ml), and EDAC (30 mg, 0.154 mmol) was added. After stirring over night, ethyl acetate (5 ml) and NaHCO ₃ aq. (2 ml) were added. After stirring a few minutes, brine (2 ml) was added and the organic phase was washed with 2 x 2 ml of saturated KH ₂ PO ₄ aq, dried (Na ₂ SO ₄ ) and evaporated to colourless glass. Column chromatography on SiO ₂ with 2.5-5% MeOH in CH ₂ Cl ₂ gave the title compound (19 mg, 20.5 %). MS (ESI ⁺ ): m/z 842/844 (M+ 1) ⁺ .

1 -ISVBenzyl- 1 -(T 1 £2i?)-2-hvdroxyindan- 1 -ylcarbamovn-3-riv"-(2-(.S)- methoxycarbonylamino-3,3-dimethylbutyryl)-N-(4-bromobenzyl)h ydrazino]propyl-A ^f - methylamine (6e)

Compound (6d, 13 mg, 0.0154 mmol), anisole (30 mg, 0.28 mmol) and trifluoromethanesulfonic acid (50 mg, 0.33 mmol) in DCM (1 ml) were stirred at ambient temperature over night. The reaction mixture was diluted with EtOAc, washed with sat. NaHCO ₃ and evaporated in vacuum. Column chromatography on SiO ₂ with 3-5 % MeOH in CH ₂ Cl ₂ gave the title compound (7.67 mg, 70 %). MS (ESI ⁺ ): m/z 708/710 (M+ 1) ⁺ . ¹ H NMR (CDCl ₃ ): 8.2 (br. d, IH), 7.7 (br. s, IH), 7.40-7.07 (m, 2+5+2H), 6.54 (br. s, IH), 5.47 (d, IH), 4.24 (br. d, IH), 3.93 and 3.74 (AB q, 2H), 3.87 (d, IH), 3.65 (s, 3H),

3.20 (d, IH), 2.98 (m, IH), 2.75 (d, 3H), 2.68 (d, I H), 2.6 (m, IH), 2.25 (m, IH), 1.9 (br. s, NH ₂ ), 1.76 (m, IH), 0.98 (s, 9H), 0.83 (s, 9H).

General procedure for the preparation of hydrazides

Benzylhydrazineχ2HCl and Et ₃ N in EtOAc (20 mL) were allowed to stir for 30 min at room temperature and then added to a solution of N-functionalised amino acid (below), EDAC, HOBT and νMM in EtOAc (40 mL) after which the reaction mixture was allowed to stir overnight at room temperature. Dilution with EtOAc, washing with saturated NaHCO ₃ (aq.), H ₂ O and brine followed by drying (Na ₂ SO ₄ ), filtration and concentration of the organic phase under vacuum afforded the crude product which was purified by column chromatography (silica, CHCl ₃ /MeOH, 100:0-95:5).

Example 7

IYlSVl -(N'-Benzyl-hydrazinocarbonvD^-methyl-propyli-carbamic acid benzyl ester (7)

The general procedure for the preparation of hydrazides described above was followed using Cbz-(L)-valine (0.540 g, 2.15 mmol), EDAC (0.450 g, 2.35 mmol), HOBT (0.320 g, 2.37 mmol), NMM (0.260 mL, 2.36 mmol), benzyl hydrazinex 2HCl (0.500 g, 2.56 mmol) and Et ₃ N (0.710 mL, 5.09 mmol) which gave the title compound (0.502 g, 66%) as a white solid.

[α] _D ²¹ -41.7° (c 0.35, MeOH/CH ₂ Cl ₂ 50:50); MS (m/z 356, M + H ⁺ ); Anal. (C ₂₀ H ₂₅ N ₃ O ₃ ) C, H, N.

¹ H NMR (DMSO-d ₆ + 2 drops Of D ₂ O) δ 7.42-7.18 (m, 10H), 5.01 (s, 2H), 3.82 (s, 2H), 3.72 (d, J= 7.61, IH), 1.83 (m, IH), 0.78 (d, J= 6.86, 3H), 0.76 (d, J= 6.86, 3H); ¹³ C NMR (DMSO-d ₆ + 2 drops OfD ₂ O) δ 170.8, 156.7, 139.2, 137.7, 129.1, 129.0, 128.8, 128.5, 128.3, 127.6, 66.1, 59.5, 55.0, 30.9, 19.7, 19.0.

Example 8

[(lSVMA^-Benzyl-hvdrazinocarbonvO^.σ-dimethyl-propyli-ca rbarnic acid benzyl ester

(9)

The general procedure for the preparation of hydrazides described above was followed using Cbz-(L)-ter/-leucine (2.00 g, 4.48 mmol), EDAC (0.969 g, 5.05 mmol), HOBT (0.669 g, 4.95 mmol), NMM (0.542 mL, 4.93 mmol), benzylhydrazineχ2HCl (0.962 g, 4.93 mmol) and Et ₃ N (1.38 mL, 9.85 mmol) which gave the title compound (1.1 1 g, 67%) as a low melting solid.

[α] _D ¹⁹ -17.5° (c 1.0, CHCl ₃ ); MS (mlz 370, M + H ⁺ ); Anal. (C ₂ iH ₂₇ N ₃ O ₃ ) C, H, N. ¹ H NMR (CD ₃ OD) δ 7.38-7.15 (m, 10H), 5.05 (d, J= 12.3 Hz, IH), 4.99 (d, J= 12.3 Hz, I H), 3.99 (s, IH), 3.90 (s, 2H), 0.92 (s, 9H); ¹³ C NMR (CD ₃ OD) δ 171.3, 158.0, 138.6, 137.9, 129.8, 129.3, 129.2, 128.9, 128.7, 128.3, 67.6, 62.5, 56.2, 35.2, 27.0.

Example 9

rπ.SVl-fA^-Benzyl-hvdrazinocarbonvD-σ-methyl-propyll-carba mic acid methyl ester

(10)

The general procedure for the preparation of hydrazides described above was followed using N-(methoxycarbony l)-(L)-val ine (J. Med. Chem., 39, 3203-3216, 1996)

(2.1 1 g, 12.0 mmol), EDAC (2.41 g, 12.6 mmol), HOBT (1.70 g, 12.6 mmol), NMM (1.38 mL, 12.6 mmol), benzylhydrazineχ2HCl (2.45 g, 12.6 mmol) and Et ₃ N (3.52 mL,

25.0 mmol). which gave the title compound (2.08 g, 65%) as a light yellow solid.

[α] _D ¹⁹ -45.5° (c 1.0, CHCl ₃ ); MS (mlz 280, M + H ⁺ , 559); Anal. (Ci ₄ H ₂₁ N ₃ O ₃ ) C, H, N.

¹ H NMR (CDCl ₃ ) δ 8.00 (s, IH) 7.40-7.25 (m, 5H), 5.50 (d, J= 9.04 Hz, IH), 4.85 (s,

I H), 3.96 (s, 2H), 3.89 (dd, J= 7.04, 9.04, IH), 3.64 (s, 3H), 2.05 (m, IH), 0.94 (d, J = 4.94 Hz, 3H), 0.92 (d, J= 4.94 Hz, 3H); ¹³ C NMR (CDCl ₃ ) δ 171.2, 157.3, 137.5, 129.2,

128.7, 127.9, 59.4, 56.1, 52.6, 31.2, 19.4, 18.2.

Example 10

[(lSVMN'-Benzyl-hydrazinocarbonyO^^-dimethyl-propyl^-carb arnic acid methyl ester

OD

The general procedure for the preparation of hydrazides described above was followed using 7V-(methoxycarbonyl)-(L)-/er/-leucine (J. Med. Chem., 41, 3387-3401, 1998) (1.56 g, 8.24 mmol), EDAC (1.74 g, 9.08 mmol), HOBT (1.22 g, 9.03 mmol), νMM (0.995 mL, 9.05 mmol), benzylhydrazineχ2HCl (1.61 g, 8.25 mmol) and Et ₃ N (2.53 mL, 18.0 mmol) which gave the title compound (1.21 g, 50%) as a light yellow solid.

[α] _D ¹⁹ -40.7° (c 0.98, CHCl ₃ ); MS (m/z 294, M + H ⁺ ); Anal. (Ci ₅ H ₂₃ N ₃ O ₃ ) C, H, N. ¹ H NMR (CDCl ₃ ) δ 8.07 (s, IH) 7.38-7.24 (m, 5H), 5.63 (d, J= 9.64 Hz, IH), 4.95 (s, I H), 4.00 (d, J= 12.4 Hz, IH), 3.95 (d, J= 12.4 Hz, IH), 3.92 (d, J= 9.64 Hz, IH), 3.68 (s, 3H), 0.98 (s, 9H); ¹³ C NMR (CDCl ₃ ) 8170.2, 157.1, 137.3, 128.9, 128.4, 127.6, 61.1, 55.8, 52.3, 34.5, 26.4;

Example 12

l-Benzyl-3-|Y IS)-I -(TV -benzyl-hvdrazinocarbonyn-2,2-dimethyl-propyl1-urea (13)

(L)-terZ-Leucine (0.500 g, 3.81 mmol) was dissolved in dioxane (23 mL) and 2M NaOH (6.3 mL, 12.6 mmol) was added. After stirring for 10 min, phenyl isocyanate (0.900 mL, 7.29 mmol) was added drop wise to yield a clear solution. The reaction mixture was stirred at room temperature for 18h and then made acidic by addition of concentrated HCl and thereafter extracted with EtOAc. The organic phase was dried and evaporated to afford (2S)-2-(3-benzyl-ureido)-3,3-dimethyl-butyric acid (0.36 g, 36% yield), which was analysed by NMR and then used without further purification. The general procedure for the preparation of hydrazides described above was then followed using the crude (25)-2-(3-benzyl-ureido)-3 ,3 -dimethyl-butyric acid (0.646 g, 2.44 mmol), EDAC (0.515 g, 2.67 mmol), HOBT (0.363 g, 2.69 mmol), NMM (0.300 mL, 2.73 mmol), benzylhydrazinex2HCl (0.528 g, 2.71 mmol) and Et ₃ N (0.753 mL, 5.38 mmol). The product was filtered through a short silica column (CHCl ₃ /MeOH, 100:0-95:5) and then used without further purification in the next step.

Example 13

{(15 ^r )-l-[N ^r -(4-Bromo-benzvπ-hydrazinocarbonyl1-2,2-dimethyl-propyll-ca rbamic acid methyl ester (14)

N-(Methoxycarbonyl)-(L)-terMeucine (J. Med. Chem., 41, 3387-3401, 1998) (1.74 g, 9.20 mmol) was dissolved in EtOAc (50 mL) and EDAC ( 1.94 g, 10.1 mmol), HOBT (1.37 g, 10.1 mmol), and NMM (1.1 1 mL, 10.1 mmol) were added. The reaction mixture was stirred at room temperature for 30 min and then 4-bromo-benzylhydrazine (prepared as described in Zh. Org. Khim., 28, 43-50, 1992) (2.31 g, 1 1.5 mmol) in EtOAc (20 mL) was added and the stirring was continued over night. The reaction mixture was washed with saturated NaHCO ₃ (aq.), H ₂ O and brine and then the organic phase was dried (Na ₂ SO ₄ ), filtered and evaporated. The crude product was purified by column chromatography (silica, CHCl ₃ /MeOH, 100:0-96:4) yielding the title compound (1.85 g, 54%) as a white solid. [α] _D ²² -26.4° (c 0.84, MeOH); Anal. (Ci ₅ H ₂₂ BrN ₃ O ₃ ) C, H, N. ¹ H NMR (CD ₃ OD) δ 7.45 (m, 2H), 7.29 (m, 2H), 3.90 (s, 2H), 3.81 (s, IH), 3.64 (s, 3H), 0 0..9900 ((ss,, 99HH));; ¹¹³³ CC NNMMRR ((CCDD ₃₃ OODD)) δδ 117700..55,, 115577..99,, 113377..22, 131.3, 130.8, 121.0, 61.7, 54.2, 51.5, 33.9, 25.8; MS (m/z 372, M + H ⁺ , 374, M + H ⁺ );

Example 14

{(15 ^r )-l-[N ^> -(4-Bromo-benzyl)-hvdrazinocarbonyll-2-methyl-propyl}-carbam ic acid benzyl ester (15)

Cbz-(L)- Valine (1.04 g, 4.14 mmol) was dissolved in EtOAc (50 mL) and EDAC (0.870 g, 4.54 mmol), HOBT (0.610 g, 4.51 mmol), and NMM (0.500 mL, 4.55 mmol) were added. The reaction mixture was stirred at room temperature for 30 min and then 4- bromo-benzylhydrazine (1.00 g, 4.97 mmol) in EtOAc (5 mL) was added and the stirring was continued for 2 h. After evaporation of the solvent, CHCl ₃ was added and the solution was washed with saturated NaHCO ₃ (aq.) and brine followed by drying (Na ₂ SO ₄ ), filtration and evaporation of the organic solvent. The crude product was

purified by column chromatography (silica, CHCl ₃ /MeOH, 100:0-95:5) yielding the title compound (1.42 g, 79%) as a white solid.

[α] _D ²¹ +6.2° (c 0.47, DMF); MS (m/z 434, M + H ⁺ , 436, M + H ⁺ ); Anal. (C ₂₀ H ₂₄ BrN ₃ O ₃ )

C, H, N.

¹ H NMR (DMSOd ₆ + 2 drops OfD ₂ O) δ 7.53-7.18 (m, 9H), 4.98 (s, 2H), 3.79 (s, 2H),

3.69 (d, J= 7.65 Hz, IH), 1.80 (m, IH), 0.75 (d, J= 6.92 Hz, 3H), 0.72 (d, J= 6.92 Hz,

3H); ¹³ C NMR (DMSO-d ₆ + 2 drops Of D ₂ O) δ 170.8, 156.6, 138.8, 137.7, 131.6, 131.4,

129.0, 128.5, 128.3, 120.6, 66.1, 59.5, 54.2, 30.8, 19.7, 19.0;

Example 15

Synthesis of 1 -(S)-benzy 1- 1 -[ 1 -(S)-rnethylcarbarnoyl-2,2-dimethylpropyl]carbamoyl-3-

[N'-(2-(S)-methoxycarbonylamino-3,3-dimethylbutyryl)- iV-(4-(4- pyridyl)benzyl)hydrazino]propylamine

Step a

( l-r^-Benzyl-l-n-^-methylcarbamoyl^^-dimethylpropyllcarbamoyl -S-rN'-Q-r^- methoxycarbonylamino-3,3-dimethylbutyryl)-N-(4-(4-pyridyl)be nzyl)hvdrazinol- propyPcarbamic acid benzyl ester (15a)

The procedure described for the synthesis of compound 3b was followed but using acid 4 (14.8 mg, 0.0213 mmol), instead of acid 3a. Column chromatography on SiO ₂ with 2.5-5% MeOH in CH ₂ Cl ₂ gave the title compound (6.3 mg, 36 %). MS (ESI ⁺ ): m/z 822 (M+ 1) ⁺ .

l-ffl-Benzyl-l-ri-r^-methylcarbamoyl-l^-dimethylpropyllcarba moyl-S-rN'-q-ry)- methoxycarbonylamino-3,3-dimethylbutyryl)- N-(4-(4-pyridyl)benzyl)hydrazino1- propylamine (15b) The Cbz group was removed from compound 15b (6.3 mg, 0.00766 mmol) according to the procedure described in example 6 step e. Column chromatography on SiO ₂ with 3- 10 % MeOH in CH ₂ Cl ₂ gave the title compound (4.6 mg, 93 %). MS (ESI ⁺ ): m/z 688 (M+ 1) ⁺ .

¹ H NMR (CDCl ₃ ): 8.2 (br. d, I H), 7.7 (br. s, I H), 7.40-7.07 (m, 2+5+2H), 6.54 (br. s, I H), 5.47 (d, IH), 4.24 (br. d, I H), 3.93 and 3.74 (AB q, 2H), 3.87 (d, IH), 3.65 (s, 3H), 3.20 (d, IH), 2.98 (m, IH), 2.75 (d, 3H), 2.68 (d, IH), 2.6 (m, IH), 2.25 (m, IH), 1.9 (br. s, NH ₂ ), 1.76 (m, IH), 0.98 (s, 9H), 0.83 (s, 9H).

Example 16 Synthesis of { 1 -(5)-benzyl- 1 -(( 15,2i?)-2-hydroxyindan- 1 -ylcarbamoyl)-3-[N '-(2-(S)- methoxycarbonylamino-3,3-dimethylbutyryl)-N-(4-bromobenzyl)h ydrazino]- propyl}carbamic acid benzyl ester: Step a

( l-^-Benzyl-l-^l^^^^-hvdroxyindan-l-ylcarbamovn-S-rN^-^- methoxycarbonylamino-33-dimethylbutyryl)-N-(4-bromobenzyl)hv drazino]- propyUcarbamic acid benzyl ester (16a)

Compound 3a (31.5 mg, 0.0452 mmol) was treated as described in example 3 step b but using (lS,2R)-(-)-cis- l-amino-2-indanol (1 1 mg, 0.074 mmol) instead of (5)-t- butylglycine N-methylamide. Column chromatography on SiO ₂ with 2.5-5% MeOH in

CH ₂ Cl ₂ gave the title compound (23.6 mg, 63 %). MS (ESI ⁺ ): m/z 828/830 (M+ 1) ⁺ .

{ l-(^-Ben2yl-l-(π5.2^V2-hvdroxyindan-l-ylcarbamovn-3-rN'-(2- (y)- methoxycarbonylamino-3.3-dimethylbutyryl)-N-(4-( ^' 4-pyridvπbenzvπhydrazinol- propyU carbarn ic acid benzyl ester (16b):

A mixture of compound 16a (22.8 mg, 0.0275 mmol), 4-pyridylboronic acid (17.5 mg, 0.142 mmol), triethylamine (15 mg, 0.15 mmol) and PEPPSI (2 mg, 0.003 mmol) in DME+H ₂ O+EtOH 7:3:2 (0.6 ml) was run at 140 ⁰ C for Ih in a microwave oven. After cooling, ethyl acetate (2 ml) was added and the mixture washed with 2 x 0.5 ml saturated aq. KH ₂ PO ₄ and 1 ml of 1% aqueous EDTA solution, dried (Na ₂ SO ₄ ) and evaporated to a small volume. Column chromatography on SiO ₂ with 2-5% MeOH in CH ₂ Cl ₂ gave the title compound (8.0 mg, 35 %). MS (ESI ⁺ ): m/z 827 (M+ 1) ⁺ .

Ster

l-(.y)-Benzyl-l-((15.2/?)-2-hvdroxyindan-l-ylcarbamoyl)-3-rN '-(2-(^- methoxycarbonylamino-3,3-dimethylbutyryl)-iV-(4-(4-pyridyl)b enzyl)hvdrazino1- propylamine (16c)

Compound 16b (8.0 mg, 0.00967 mmol) was deprotected according to the method described in Example 6 step e. Column chromatography on SiO ₂ with 3-10 % MeOH in CH ₂ Cl ₂ gave the title compound (4.2 mg, 62.7%). MS (ESI ⁺ ): m/z 693 (M+ 1) ⁺ . ¹ H NMR (CDCl ₃ ): 8.2 (br. d, IH), 7.7 (br. s, IH), 7.40-7.07 (m, 2+5+2H), 6.54 (br. s, IH), 5.47 (d, IH), 4.24 (br. d, IH), 3.93 and 3.74 (AB q, 2H), 3.87 (d, IH), 3.65 (s, 3H), 3.20 (d, IH), 2.98 (m, IH), 2.75 (d, 3H), 2.68 (d, IH), 2.6 (m, IH), 2.25 (m, IH), 1.9 (br. s, NH ₂ ), 1.76 (m, IH), 0.98 (s, 9H), 0.83 (s, 9H).

Example 23 Step a)

5,6-Dihydro-cyclopentafl>1thiophen-4-one (23a) Over a period of 10 minutes a solution of triflic anhydride (84.7g, 0.30 mol) in DCE

(300 mL) was added to a cold solution of N,N-dimethylacrylamide (29,8 g, 0.30 mol) in DCE (270OmL). The mixture was stirred for 15 minutes at O ⁰ C. A solution of thiophene (25,3 g, 0.30 mol) was added and the mixture was refluxed for seven hours. A solution of potassium carbonate (150g in 200OmL of water) was added. The mixture was extracted two times with DCM dried over sodium sulphate and evaporated under reduced pressure. The compound was purified by silica gel chromatography with ethyl acetate/hexane. Yield: 36.8 g = 53% ¹ H-NMR CDCl ₃ 7.32 (dd, IH), 7.14 (dd, I H), 3.20 (m, 2H), 3.00 (m, 2H)

Step b

5-Hydroxy-5,6-dihydro-cyclopenta[b 1 thiophen-4-one (23b)

5,6-dihydro-cyclopenta[6]thiophen-4-one (36.8 g, 0.266 mol) in MeOH (100OmL) was added at about 5°C to a solution of potassium hydroxide 85% (52.7 g, 0.798 mol) in MeOH (50OmL). Between 0 ⁰ C and 5 "C iodobenzene diacetate (94.4g, 0.293 mol) was added in portions and the mixture was allowed to come to room temperature. The mixture was stirred overnight at room temperature. The mixture was evaporated and a 20% solution of potassium carbonate (50OmL) was added. The mixture was extracted for times with DCM dried with sodium sulphate and evaporated under reduced pressure. The residue was dissolved in 1,4-dioxane (400 mL) and water (150 mL) and concentrated hydrochloric acid (15OmL) was added. The mixture was stirred for two hours at room temperature. The mixture was neutralized by the addition of potassium carbonate and extracted four times with dichloromethane. The organic phase was dried with sodium sulphate and evaporated under reduced pressure. The product was crystallized with ether ethyl acetate and the mother liquid was purified by silica gel chromatography with toluene and acetone. Yield: 33.5 g = 81,6%. ¹ H-NMR CDCl ₃ δ 7.36 (dd, IH), 7.18 (d, IH), 4.76 (m, IH), 3.64 (m, 2H), 3.10 (m, IH)

ster

5-Hvdroxy-5.6-dihydro-cvclopenta[l>1thiophen-4-one Obenzyl-oxime (23c)

To a solution of S-hydroxy-Sjό-dihydro-cyclopenta [ό]thiophen-4-one (33.4 g , 0.216 mol) in pyridine (300 mL) was added Obenzylhydroxylamine hydrochloride (38.3 g, 0.240 mol) and the mixture was stirred at room temperature over weekend. The mixture was evaporated under reduced pressure and co-evaporated two times with toluene. Ethyl acetate was added and the organic phase was washed with 5% citric acid and brine. The organic phase was dried with sodium sulphate and evaporated under reduced pressure. Yield . 55.1 g = 98% ¹ H-NMR CDCl ₃ δ 7.40-7.20 (m, 7H), 5.20(m, 3H), 3.45 (m, 2H), 3.0 (m, IH)

step d

c/.s-4-Amino -5,6-dihydro-4H-cyclopenta[6]thiophene-5-ol (racemate ) (23d)

A solution of 5-hydroxy-5,6-dihydro-cyclopenta[6]thiophen-4-one O-benzyl-oxime (55.1 g, 0.212 mol) was added drop wise at about 5°C to 1.0 M solution of borane in TηF (650 mL) and the mixture was stirred at room temperature overnight. The mixture was refluxed for two hours and cooled to about 5°C. Water (70 mL) and 20% potassium hydroxide solution (80 mL) was added dropwise. The mixture was refluxed for two hours and cooled. Brine was added and the TηF removed under reduced pressure. The mixture was extracted five times with DCM, dried with sodium sulphate and evaporated under reduced pressure. The product was purified by silica gel chromatography with DCM and 10% methanol. Yield: 17.8 g = 54%

¹ H-NMR DMSO-d ₆ δ 7.30 (d, I H), 6.92 (d, IH), 4.46 (m, IH), 4.20 (m, IH) 3.99-3.84 (dd, 2H)

Example 24

Separation of the enantiomeres from example 23

Stet

[l-fS-Hydroxy-S.ό-dihydro^H-cyclopentafblthiophen^-ylcar bamovπ^-phenyl-ethyll- carbamic acid tert-butyl ester (24a)

To a mixture of the racemic cw^-amino-S.ό-dihydro^H-cyclopenta^lthiophen-S-ol (17.5 g, 0.1 12 mol) in dry DMF (400 mL) was added Boc-L-phenylalanin (30.51 g, 0.1 15 mol) ηOBT (15.6 g, 0.1 15 mol) and EDAC (22,0 g, 0.1 15 mol). To the stirred mixture was added TEA (16 mL, 0.115 mol) and the mixture was stirred at room temperature overnight. The mixture was added to 5% citric acid and extracted three times with ethyl acetate. The organic phase was washed with brine and saturated sodium hydrogen carbonate (two times). The organic phase was dried with sodium sulphate and evaporated under reduced pressure. Yield: 43 g = 95%

2-Amino-N-(5-hvdroxy-5,6-dihvdro-4η-cvclopentafb1thiophe n-4-yl)-3-phenyl- propionamide (24b)

Compound 24a was dissolved in chloroform (400 mL) and TFA (100 mL) was added and the mixture was stirred for three hours at room temperature. The organic phase was washed two times with 15 % ammonia solution (300 mL) and with brine. The organic phase was dried over sodium sulphate and evaporated. The product was purified by silica gel chromatography with DCM with three to ten percent methanol. Yield A : 12,5 g first diastereomere = 40% Yield B : 12,5 g second diastereomere = 40%

Step c)

4-Amino-5,6-dihydro-4H-cyclopenta[b]thiophen-5-ol (24c)

The first diastereomere (12.4 g, 41 mmol) was dissolved in EtOH (400 mL) and a solution of sodium hydroxide (21.0 g, 525 mmol) water (300 mL) was added. The mixture was refluxed overnight. The ethanol was removed and the alkaline phase was extracted six times with DCM. The organic phase was washed with brine, dried with sodium sulphate and evaporated under reduced pressure. Yield: 6.2 g = 97%. ¹ H-NMR DMSOd ₆ δ 7.30 (d,lH), 6.92 (d, IH), 4.46 (m, IH), 4.20 (m, I H), 3.99-3.84 (dd, 2H).

Example 25

{( 1 S)- 1 -[N'-(3-Bromo-benzyl)-hydrazinocarbonyl1-2.2-dimethyl-propyl }-carbamic acid methyl ester (25)

N-(Methoxycarbonyl)-(L)-ter/-leucine (3.25 g, 17.1 mmol) was dissolved in EtOAc (40 mL) and HOBT (2.55 g, 18.9 mmol), EDAC (3.62 g, 18.9 mmol) and νMM (2.08 mL, 18.9 mmol) were added subsequently. 3-Bromo-benzylhydrazine (4.14 g, 20.6 mmol), dissolved in EtOAc (20 mL) was added to the reaction mixture, which thereafter was stirred at room temperature over night. The organic phase was washed with saturated NaHCO ₃ (aq., 50 mL), H ₂ O (50 mL) and brine (50 mL). The combined aqueous phases were extracted with EtOAc (3x50 mL). The combined organic phases were dried (Na ₂ SO ₄ ), filtered and concentrated under reduced pressure. The crude product was purified by column chromatography (silica, CHCl ₃ /MeOH, 100:0-95:5) to afford 2 (4.88 g, 76%). RP-LC-MS (35 min gradient of 35-80% CH ₃ CN in 0.05% aqueous formic acid) was performed on a small fraction of the residue to obtain a sample of higher purity for characterization and the product was isolated as a white solid. [α] _D ²⁰ -28.0° (c 1.2, CH ₃ OH); ¹ H NMR (CD ₃ OD) δ 7.56 (m, IH), 7.40 (m, IH), 7.32 (m, IH), 7.22 (m, IH), 3.93 (s, 2H), 3.81 (s, IH), 3.63 (s, 3H), 0.89 (s, 9H); ¹³ C NMR (CD ₃ OD) δ 171.7, 159.0, 141.8, 132.9, 131.5, 131.1, 128.8, 123.3, 62.9, 55.5, 52.7, 35.1, 26.9; MS (m/z 372, M + H ⁺ , 374, M + H ⁺ ).

General procedures for the Pd-catalyzed reactions:

Method A. Aryl bromide 19 or 26, tin reagent, Pd(PPh) ₃ Cl ₂ , CuO and DMF (2 mL) were stirred in a heavy-walled Smith process vial at 130 °C for 20 min in the microwave cavity. CH ₂ Cl ₂ (30 mL) was added to the mixture followed by washing with saturated NaHCO ₃ (aq., 3x20 mL). The organic phase was dried (Na ₂ SO ₄ ), filtered and evaporated. The residue was redissolved in CH ₃ CN (70 mL) and washed with isohexane (3x20 mL) after which the CH ₃ CN phase was evaporated and the crude product was purified using RP-LC-MS. Method B. Aryl bromide 19 or 26, boronic acid, Pd(PPh) ₃ Cl ₂ , 2 M Na ₂ CO ₃ (aq.),

EtOH and DME were stirred in a heavy-walled Smith process vial at 120 °C for 30 min in the microwave cavity. Five drops of formic acid were added to the mixture and then the solvent was evaporated. The residue was redissolved in CH ₃ CN/H ₂ O/DMF and filtered before purification by RP-LC-MS.

Method C. Aryl bromide 26, acetylene, Et ₂ NH, Pd(PPh ₃ ) ₂ Cl ₂ , CuI and DMF were stirred in a heavy-walled Smith process vial at 140 ⁰ C for 30-40 min. Work up was performed by extracting the mixture with CH ₂ Cl ₂ (2 mL) and H ₂ O (2x2 mL). The organic phase was filtered and evaporated before the product was purified by RP-LC-MS. Method D. Aryl bromide 19, acetylene, Et ₃ N, Pd(PPh ₃ ) ₂ Cl ₂ , CuI and DMF were stirred in a heavy-walled Smith process vial at 130 °C for 60 min. Filtration and evaporation of most of the solvent yielded the crude product which was purified by RP- LC-MS.

Example 26

Hydrazide building block

Step a

A mixture of 1 (1.2 g, 5.22 mmol) and Rany-Nickel (400 mg) in 70% formic acid was heated to 100 ⁰ C for 3 hrs in a high pressure, microwave oven. The cooled mixture was filtered and concentrated, diluted with H2O and made alkaline with KOH and then extracted with EtOAc. The organic layer was dried and concentrated to give a pure product 2 in a near quantitive yield.

A solution of 2 (900 mg, 3.86 mmol) and 3 (785 mg, 3.86 mmol) in 2-propanol (15 ml) was heated to 80 ⁰ C for 4 hrs to give 4 which was purified on a silica gel column with methanol-dichloromethane 0 to 5 %. Compound 4 was collected and concentrated. To a stirred solution of 4 (1.18 g, 2.82 mmol) in THF (20 ml), NaBH ₃ CN (186 mg, 2.96 mmol) was added. To this mixture a solution of camphor sulfonic acid (654 mg, 2.82 mmol) in THF (10 ml) was added drop wise. After 16 hrs the mixture was diluted with EtOAc, stirred and then filtered through a pad of Na2SO4 + Celite. The organic solution was concentrated and redissolved in THF. About 7 ml of a solution of NaOH (200 mg) in MeOH (10 ml) was added to break up the borne complex. The mixture was diluted with

EtOAc and H2O. The organic layer was separated, dried and concentrated. Pure compound was obtained after a SiO2-column eluted with 0 to 5% MeOH in DCM.

Biological Examples Extensive guidance on the assay of test compounds at the enzyme level and in cell culture, including the isolation and/or selection of mutant HIV strains and mutant RT are found in DAIDS Virology Manual for HIV Laboratories complied by Division of AIDS, NIAID USA 1997. Resistance studies, including rational for various drug escape mutants is described in the HIV Resistance Collaborative Group Data Analysis Plan for Resistance Studies, revised 31 August 1999 and subsequently.

Cellular assay

Compounds of the invention are assayed for HIV activity, for example using multiple determinations with XTT in MT-4 cells (Weislow et al, J Nat Cancer Inst 1989, vol 81 no 8, 577 et seq), preferably including determinations in the presence of 40-50% human serum to indicate the contribution of protein binding. In short the XTT assay uses human T cell line MT4 cells grown in RPMI 1640 medium supplemented with 10% fetal calf serum (or 40-50% human serum as appropriate), penicillin and streptomycin seeded into 96 well microplates (2- 10 ⁴ cells/well) infected with 10-20 TCID ₅₀ per well of HIV-I _HIB (wild type) or mutant virus, such as those bearing RT He 100, Cys 181 or Asn 103 mutations. Serially diluted test compounds are added to respective wells and the culture incubated at 37°C in a CO ₂ enriched atmosphere and the viability of cells is determined at day five or six with XTT vital dye. Results are typically presented as ED ₅₀ μM.

Expression of HIV-I protease suitable for enzyme determination is also described in Danielsson et al. Adv. Exp. Med. Biol., 1998, 436, 99-103.

Fluorometric assays for Ki determinations are also described in Antimicrob. Agents Chemother., 1997, 41, 2383-2388. This journal also describes a cellular assay for ED50 using MT4 cells and a colorimetric XTT assay.

The following table shows the Ki and EDs ₀ figures for a representative selection of compounds according to the invention. Category A indicates a Ki of < 10 nM inhibition, category B indicates 1 1-50 nM inhibition and category C indicates 50-100 nM inhibition, category D indicates an ED ₅₀ < 2 μM, category E indicates 2-10 μM and category E indicates >10 μM:

Table 3. Enz me inhibition and antiviral activity in cell culture.

Time to resistance 2 x 10 ⁴ MT4 cells per well in a microtitre plate are infected with 5-10 TCID ₅₀ of HIV-I me. The compounds being tested are added at concentrations around ED ₅₀ using 8 duplicates per concentration. After 6 days of incubation the RT activity in lOμL supernatant is measured.

The following procedure is followed at subsequent passages of the cultures once per week. Virus produced at the concentration of test compound showing > 50% of the RT activity of untreated infected cells (SIC, Starting Inhibitory Concentration) are passaged to fresh MT4 cells. 15μL supernatant from each of the eight duplicates are transferred to cells without the test compound (control) and to cells with test compound at the same concentration, and additionally two respectively fivefold higher concentrations. (See Table 2 below)

When viral growth is permitted at the highest non-toxic concentration (5 - 40 μM), 2-4 parallel wells are collected and expanded to give material for sequence analysis and cross-wise resistance.

TABLE 2

Viral growth permitted

Virus production inhibited

125 x SIC

125 x SIC 25 x SIC →

25 x SIC 5 x SIC

25 x SIC 5 x SIC → No compound

25 x SIC 5 x SIC → No compound

5 x SIC SIC

SIC -> No compound

SIC → No compound

Pass 1 Pass 2 Pass 3 Pass 4 Pass 5

P450 metabolism

The metabolism of compounds of the invention through the main isoforms of the human cytochrome system P450 are conveniently determined in baculovirus infected insect cells transfected with human cytochrome P450 cDNA (supersomes) Gentest Corp. Woburn USA.

The test compounds at concentrations 0.5, 5 and 50 μM are incubated in duplicate in the presence of supersomes overexpressing various cytochrome P450 isoforms, including CYPl A2 + P450 reductase, CYP2A6 + P450 reductase, CYP2C9-Arg 144 + P450 reductase, CYP2C19 + P450 reductase, CYP2D6-Val 374 + P450 reductase and CYP3A4 + P 450 reductase. Incubates contain a fixed concentration of cytochrome P450 (eg 50 pmoles) and are conducted over 1 hour. The involvement of a given isoform in the metabolism of the test compound is determined by UV HPLC chromatographically measuring the disappearance of parent compound.

Plasma and Human Liver Metabolism

Stability in plasma or whole blood is estimated by following disappearance of compound by MS or HPLC according to conventional techniques. Susceptibility to first pass metabolism is measured in human liver microsomes, such as HLM 1037 from Invitro

Technologies Inc according to the manufacturers' specifications. Again, disappearance of compound incubated in the HLM preparation is typically monitored with MS or HPLC.

Permeability This example measures transport of inhibitors through the cells of the human gastroenteric canal. The assay uses the well known Caco-2 cells with a passage number between 40 and 60.

Apical to basolateral transport Generally every compound will be tested in 2-4 wells. The basolateral and the apical wells will contain 1.5 mL and 0.4 mL transport buffer (TB), respectively, and the standard concentration of the tested substances is 10 μM. Furthermore all test solutions and buffers will contain 1% DMSO. Prior to the experiment the transport plates are pre-

coated with culture medium containing 10% serum for 30 minutes to avoid nonspecific binding to plastic material. After 21 to 28 days in culture on filter supports the cells are ready for permeability experiments.

Transport plate no I comprises 3 rows of 4 wells each. Row 1 is denoted Wash, row 2 "30 minutes" and row 3 "60 minutes". Transport plate no 2 comprises 3 rows of 4 wells, one denoted row 4 "90 minutes", row 5 "120 minutes and the remaining row unassigned. The culture medium from the apical wells is removed and the inserts are transferred to a wash row (No. 1) in a transport plate (plate no.1) out of 2 plates without inserts, which have already been prepared with 1.5 mL transport buffer (HBSS, 25 mM HEPES, pH 7.4) in rows 1 to 5. In A— »B screening the TB in basolateral well also contains 1% Bovine Serum Albumin.

0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to the inserts and the cell monolayers equilibrated in the transport buffer system for 30 minutes at 37 ⁰ C in a polymix shaker. After being equilibrated to the buffer system the Transepithelial electrical resistance value (TEER) is measured in each well by an EVOM chop stick instrument. The TEER values are usually between 400 to 1000 ω per well (depends on passage number used).

The transport buffer (TB, pH 6.5) is removed from the apical side and the insert is transferred to the 30 minutes row (No. 2) and fresh 425 μL TB (pH 6.5), including the test substance is added to the apical (donor) well. The plates are incubated in a polymix shaker at 37°C with a low shaking velocity of approximately 150 to 300 rpm. After 30 minutes incubation in row 2 the inserts will be moved to new pre-warmed basolateral (receiver) wells every 30 minutes; row 3 (60 minutes), 4 (90 minutes) and 5 (120 minutes) . 25 μL samples will be taken from the apical solution after ~2 minutes and at the end of the experiment. These samples represent donor samples from the start and the end of the experiment.

300 μL will be taken from the basolateral (receiver) wells at each scheduled time point and the post value of TEER is measured at the end the experiment. To all collected samples acetonitrile will be added to a final concentration of 50% in the samples. The collected samples will be stored at -20 ⁰ C until analysis by HPLC or LC-MS.

Basolateral to apical transport

Generally every compound will be tested in 2-4 wells. The basolateral and the apical wells will contain 1.55 mL and 0.4 mL TB, respectively, and the standard concentration of the tested substances is 10 μM. Furthermore all test solutions and buffers will contain

1% DMSO. Prior to the experiment the transport plates are precoated with culture medium containing 10% serum for 30 minutes to avoid nonspecific binding to plastic material.

After 21 to 28 days in culture on filter supports the cells are ready for permeability experiments. The culture medium from the apical wells are removed and the inserts are transferred to a wash row (No.l) in a new plate without inserts (Transport plate). The transport plate comprises 3 rows of 4 wells. Row 1 is denoted "wash" and row 3 is the "experimental row". The transport plate has previously been prepared with 1.5 mL TB (pH 7.4) in wash row No. 1 and with 1.55 mL TB (pH 7.4), including the test substance, in experimental row No. 3 (donor side).

0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to the inserts in row No. 1 and the cell monolayers are equilibrated in the transport buffer system for 30 minutes, 37 °C in a polymix shaker. After being equilibrated to the buffer system the TEER value is measured in each well by an EVOM chop stick instrument. The transport buffer (TB, pH 6.5) is removed from the apical side and the insert is transferred to row 3 and 400 μL fresh TB, pH 6.5 is added to the inserts. After 30 minutes 250 μL is withdrawn from the apical (receiver) well and replaced by fresh transport buffer. Thereafter 250 μL samples will be withdrawn and replaced by fresh transport buffer every 30 minutes until the end of the experiment at 120 minutes, and finally a post value of TEER is measured at the end of the experiment. A 25 μL samples will be taken from the basolateral (donor) compartment after ~2 minutes and at the end of the experiment. These samples represent donor samples from the start and the end of the experiment. To all collected samples acetonitrile will be added to a final concentration of 50% in the samples. The collected samples will be stored at -20°C until analysis by HPLC or LC- MS.

Calculation

Determination of the cumulative fraction absorbed, FA _cum , versus time. FA _cum is calculated from:

_F ^rλ _A cum ₌ γ δJ£ _r κ.

Where CRJ is the receiver concentration at the end of the interval i and CγJJ is the donor concentration at the beginning of interval i. A linear relationship should be obtained. The determination of permeability coefficients (P _a pn, cm/s) are calculated from:

(k - V _R )

³ PP (A - 60) where k is the transport rate (min"l) defined as the slope obtained by linear regression of cumulative fraction absorbed (FA _cum ) as a function of time (min), VR is the volume in the receiver chamber (mL), and A is the area of the filter (cm^).

Re erence com ounds