AU2007229369A1

AU2007229369A1 - Competitive binding assay for identifying inhibitors of HCV polymerase

Info

Publication number: AU2007229369A1
Application number: AU2007229369A
Authority: AU
Inventors: Pierre L. Beaulieu; George Kukolj; Ginette Mckercher
Original assignee: Boehringer Ingelheim Canada Ltd
Current assignee: Boehringer Ingelheim Canada Ltd
Priority date: 2001-08-07
Filing date: 2007-10-18
Publication date: 2007-11-08
Anticipated expiration: 2022-08-05
Also published as: AU2007229369B2; AU2007229369C1

Description

O

0 0 P/00/011 Rcgulalion 3.2

AUSTRALIA

Patents Act 1990 COMPLETE

SPECIFICATION

STANDARD

PATENT

(ORIGINAL)

Name of Applicant(s): Actual Inventor(s): Address for Service: Invention Title: Boehringer Ingelheim (Canada) Ltd., of 2100 Cunard Street, Laval, Quebec H7S 2G5, Canada George KUKOLJ; Pierre L. BEAULIEU; Ginette MCKERCHER DAVIES COLLISON CAVE, Patent Trademark Attorneys, of 1 Nicholson Street, Melbourne, 3000, Victoria, Australia Ph: 03 9254 2777 Fax: 03 9254 2770 Attorney Code: DM "Competitive binding assay for identifying inhibitors of HCV polymerase" The following statement is a full description of this invention, including the best method of performing it known to us:- O COMPETITIVE BINDING ASSAY FOR IDENTIFYING INHIBITORS OF HCV c.

POLYMERASE

0 0O This application is a divisional application of Australian Application No. 2002322243 the specification and drawings of which as originally filed are incorporated herein in their entirety by reference.

0\ C FIELD OF THE INVENTION The present invention relates generally to a method for identifying inhibitors of the HCV r RNA dependent RNA polymerase. Particularly, this method uses a novel probe in a competitive assay to identify HCV polymerase inhibitors and determine their potency.

More particularly, this invention relates to the use of a probe which binds with specificity to the polymerase, and which is capable of being displaced by inhibitors of the enzyme.

BACKGROUND OF THE INVENTION Hepatitis C virus (HCV) is the major etiological agent of post-transfusion and communityacquired non-A non-B hepatitis worldwide. It is estimated that over 200 million people worldwide are infected by the virus. A high percentage of carriers become chronically infected and many progress to chronic liver disease, so called chronic hepatitis C. This group is in turn at high risk for serious liver disease such as liver cirrhosis, hepatocellular carcinoma and terminal liver disease leading to death. The mechanism by which HCV establishes viral persistence and causes a high rate of chronic liver disease has not been thoroughly elucidated. It is not known how HCV interacts with and evades the host immune system.

HCV is an enveloped positive strand RNA virus in the Flaviviridae family. The single strand HCV RNA genome is of positive polarity and comprises one open reading frame (ORF) of approximately 9600 nucleotides in length, which encodes a linear polyprotein of approx. 3010 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce structural and non-structural (NS) proteins. The structural proteins El, E2 and E2-p7) comprise polypeptides that constitute the virus particle (Hijikata, M. et al, 1991, Proc. Natl. Acad. Sci. USA. 88, 5547-5551; Grakoui, A. et al., 1993(a), J. Virol. 67, 1385-1395). The non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) encode for enzymes or accessory factors that catalyze and regulate the replication of the HCV RNA genome. Processing of the structural proteins is catalyzed by host cell proteases (Hijikata et al., 1991, supra). The generation of the mature nonstructural proteins is catalyzed by two virally encoded proteases. The first is the NS2/3 zinc- 0 2 0 dependent metalloprotease which auto-catalyses the release of the NS3 protein from 00 the polyprotein. The released NS3 contains a N-terminal serine protease domain (Grakoui A, et al., 1993(b), Proc Natl Acad Sci USA, 90,10583-7; Hijikata, M. et al., 1993, J. Virol. 67, 4665-4675.) and catalyzes the remaining cleavages from the polyprotein. The.released NS4A protein has at least two roles. First, forming a stable complex with NS3 protein and assisting in the membrane localization of the C NS3/NS4A complex (Kim et al., Arch Virol. 1999, 144: 329-343) and second, acting as a cofactor for NS3 protease activity. This membrane-associated complex, in turn Scatalyzes the cleavage of the remaining sites on the polyprotein, thus effecting the release of NS4B, NS5A and NS5B (Bartenschlager, R. et al., 1993, J. Virol., 67, 3835-3844; Grakoui etal., 1993(a) supra; Hijikata etal., 1993 supra; Love, R.A. et al., 1996, Cell, 87, 331-342; reviewed in Kwong AD. etal., 1998, Antiviral Res., 1-18). The C-terminal segment of the NS3 protein also harbors nucleoside triphosphatase and RNA helicase activity (Kim, D.W. etal., 1995, Biochem. Biophys.

Res. Comm., 215,160-166). The function of the protein NS4B is unknown. NS5A, a highly phosphorylated protein, seems to be responsible for the Interferon resistance of various HCV genotypes (Gale Jr. et al. 1997 Virology 230, 217; Reed et al., 1997, J. Virol. 71, 7187). NS5B is an RNA-dependent RNA polymerase (RdRp) that is involved In the replication of HCV.

To better understand the mechanism of HCV RNA replication and to develop appropriate in vitro systems, biochemical analyses of the NS5B protein have been performed. Full-length NS5B has been produced and purified as a non-fusion protein from insect cells infected with recombinant baculovirus Behrens et al., 1996, EMBO 15:12-22; R. de Francesco etal., 1996, Methods Enzymol., 275:58- 67) or as a tagged protein from both insect cells Lohmann et al., 1997, J. Virol., 71:8416-8428; V. Lohmann et al., 1998, Virology 249:108-118) and E. coli Yuan et al., 1997, BBRC 232:231-235). In vitro, the RdRp activity of recombinant is dependent on an RNA template and requires RNA or DNA as a primer E. Behrens etal,, 1996, EMBO J. 15:12-22; V. Lohmann etal., 1997, J. Virol., 71:8416-8428). On RNA templates of heteropolymeric sequences, the 3'-OH of the template is used as a primer and elongation proceeds via a "snap-back" mechanism, leading to a double-stranded molecule in which template and product RNA are covalently linked Behrens et al., 1996, EMBO 15:12-22; V. Lohman et al., 1998, Virology, 249:108-118; G. Luo etal., 2000, J. Virol. 74:851-863). Recently, 3 o several groups also demonstrated that the HCV NS5B protein is able to Initiate RNA 00 synthesis de novo Oh et al., 1999, J. Virol. 73:7694-7702; X. Sun et 2000, BBRC 268:798-803; W. Zhong et al., 2000, J. Virol. 74:2017-2022).

The NS5B RdRp-has been crystallized to reveal a structure reminiscent of other nucleic acid polymerases Bressanelli et al., 1999, PNAS USA 96:13034-13039; C H. Ago et al., 1999, Structure 7:1417-1426; C.A. Lesburg et al., 1999, Nature Struct.

Biol., 6:937-943). A comprehensive understanding of the differences between HCV S and cellular polymerases will facilitate the design of specific inhibitors of HCV replication. Detailed kinetic information will also help in understanding the molecular basis of HCV NS5B-catalyzed nucleotide incorporation and subsequently the mechanistic characterization of the inhibitors.

Previous studies Behrens et al., 1996, EMBO J. 15:12-22; R. de Francesco et al., 1996, Methods Enzymol. 275:58-67; V. Lohmann et al., 1997, J. Virol. 71:8416- 8428; V. Lohmann et al., 1998, Virology 249:108-118) provided little information with regard to the proportion of the polymerase RNA complexes that are competent for catalysis. Some recent studies investigated the template and primer requirements for HCV NS5B-directed RNA replication. Templates with 3'-termini free of secondary structures and short primers 2 or 3 nucleotides (nt) long were preferred for efficient initiation of RNA synthesis Zhong et al., 2000, J. Virol. 74:9134-9143). In de novo initiation of RNA synthesis, however, NS5B needs a template with a stable secondary structure and a single-stranded sequence that contains at least one 3'cytidylate.

Viral polymerases represent attractive targets for therapeutic Inhibition of viral replication. The discovery of new antiviral agents often involves screening of large numbers of samples for inhibition of the target activity using either in vitro or in vivo assays. In general, polymerases are assayed by monitoring the incorporation of either 3 a- 32 P or a-33P-labeled mononucleotides into oligonucleotide products, or by the extension of 5'-end-labeled primers. Products incorporated into the extended primers are captured or separated using common filter assays, acid precipitation, or denaturing gel electrophoresis.

The HCV NS5B polymerase is a prime target in the search for Inhibitors of HCV 4

C.)

cj replication. Different preparations of the HCV polymerase exhibit varying efficiencies 00 of product formation with a variety of RNA substrates. Moreover, the activity of purified recombinant NS5B polymerase varies significantly with specific RNA substrates. In addition, the in vitro RNA polymerase activity of NS5B is extremely sensitive to ionic strength, and salt concentrations exceeding 100 mM inhibit the t' reaction. Hence the ability to determine the potency of inhibitors at various salt concentrations is restricted by this limitation of standard enzymatic reactions. Also, HCV polymerase enzymatic assays disclosed in the prior art provide IC 50 values as representative measurements of inhibitor potencies. For inhibitors that are competitive with either RNA or NTP, the IC50 value is proportional to the concentration of substrates in the assay and will vary depending on the concentration of these components.

In an effort to overcome the limitations of HCV polymerase assays that use suboptimal and poorly characterized RNA substrates, the Applicants have developed an assay for identifying specific inhibitors of the HCV polymerase that is independent of

RNA.

It is therefore an advantage of the present invention to provide an assay that permits a direct measurement of inhibitor potencies (reflected by Kd values as an unequivocal determination of inhibitor potency) under defined conditions, irrespective of the substrate concentration.

The direct binding assay of this invention is amenable to adjustments in salt concentration or pH levels beyond the restricted range required for RNA polymerization. This type of assay is amenable to a high sensitivity and a high throughput format.

It is a further advantage of the present invention to provide a probe that binds to the polymerase with a high affinity, and which is displaced by inhibitors of the enzyme.

It is a further advantage to provide an assay that is applicable to HCV polymerases of different genotypes.

0 O SUMMARY OF THE INVENTION oO In a first aspect of the invention, there is provided a method for identifying compounds binding to HCV polymerase comprising the steps of: a) contacting said HCV polymerase or an analog thereof with a probe being capable of binding to an HCV polymerase or an analog thereof, said probe c being displaceable by an inhibitor thereof, so as to form a complex C comprising said probe bound to said polymerase; b) measuring a signal emitted from said probe in said complex to establish a base line level; c) incubating the product of step a) with a test compound; and d) measuring the signal from said complex; and e) comparing the signal from step d) with the signal from step b); whereby a modulation In said signal is an Indication that said test compound binds to said polymerase.

In a preferred aspect the first embodiment, the probe is selected from: an isomer, enantlomer, diastereoisomer, or tautomer of a probe represented by formula I: A M

R

82 K B

(I)

wherein: A is O, S, N, NR', or CR', wherein R' is selected from the group consisting of: H, (Cl.

6 )alkyl optionally substituted with: -halogen, OR 1 1

SR

1 1 or N(R 2 2 wherein R 1 1 and each R 1 2 is independently H, (Cl.

6 )alkyl, (C 3 7 )cycloalkyl, (C,.)alkyl-(C 3 .7)cycloalkyl, (Cl.6)alkyl-aryl or (C 1 6 )alkyl-Het, said aryl or Het optionally substituted with R'O; or both R 12 are covalently bonded together and to the nitrogen to which they are both attached to form a 5, 6 or 7-membered saturated heterocycle; represents either a single or a double bond;

R

2 is selected from: H, halogen, R 21

OR

21

SR

21

COOR

2

SO

2 N(R2) 2

N(R

2 2 CON(R2) 2 NR2C(O)R 22 or NR 22

C(O)NR

2 wherein R 21 and each R 22 is independently H, (C 1 6)alkyl, haloalkyl, (C 26 )alkenyl, (C 3 7 )cycloalkyl, (C26)alkynyl, (Cs.

6

C.)

00 substituted with R 20 or both R 22 are bonded together to form a 5, 6 or 7-membered saturated heterocycle with the nitrogen to which they are attached; B Is NR 3 or CR wherein R 3 is selected from (C1.

0 )alkyl, haloalkyl, (C 7 )cycloalkyl, (Cr.

10 )blcycloalkyl, 6- or iC-membered aryl, Het, (Cl-O)alkyl-aryl or (C 1 -6)alkyl-Het, said alkyl, cycloalkyl, bicycloalkyl, aryl, Het, alkyl-aryl and alkyl-Het being optionally substituted with from 1 to 4 substituents selected from: halogen, or a) (C,_6)akyI optionally substituted with: OR 3 1 or SR 3 1 wherein R 31 is H, (Cl.

6 alkyl), (C 37 )cycloalkyl, (C,_6)akYI-(C 3 7 )CYCloalkyI, aryl, Het, (Cl.6)alkyl-aryl or (Cl- 6 )alkyl-Het; or -N(R 32 2 wherein each R 32 is independently H, (C,-6)alkyl, (C 3 7 )cycloalkyl,

(C

16 )alkyl.(C 3 .7)cycloalkyI, aryl, Het, (Cl-6)alkyl-aryl or (Cl.

6 )alkyl-Het; or both R 32 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; b) OR 33 wherein R 33 is H, (C 16 )alkyl, (C 37 )cycloalkyl or (Cl.

6 )alkyl-(C 3 .7)cycloalkyl, aryl, Het, (Cl 16 )alkyl-aryl or (Cl 6 alkyl-Het; c) SR 34 wherein R' is H, 6 )alkyl, (C 37 )cycloalkyl, or

(C

1 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 16 )alkyl-aryl or (Cl.

6 )alkyl-Het; and d) N(R 35 2 wherein each R 35 is independently H, 6 )alkyl,

(C

3 7 )cycloalkyl, (Cj_6)akyI-(C3 7 )cycloakyI, aryl, Het, (Cl.

6 )alkyl-aryl or

(C

1 .6)alkyI-Het; or both R 35 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; with the proviso that, when A is not N, then one of A or B is either CR' or CR 3 ;1 K is N or CR 4 wherein R 4 Is H, halogen, 6 )alkyl, haloalkyl, (C3.

7 )cycloalkyI or 6 )alkyl-(C3.

7 )cycloalkyl; or R 4 iS OR 4 or SR 41 COR 4 1 or NR 41 00R 4 wherein each R 4 1 is independently H, (Cl.

6 )alkyl), (C 37 )cycloalkyl or (Cj_6)akyI.(C 3 7 )CcdoalkyI or R 4 is NR 42

R

43 wherein R 1 2 and R43 are each independently H, (C,.B)alkyl, (C 3 7 o 7 )cycloaikyl, (Cl.

6 )alkyl-(C 3 7 )CyCloaikyI, or both R 42 adR r oaetybne 00 together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; sNo R 5 hri 5 hsteamdfitonsR 4 dfndaoe M is N or CR5, wherein R 7 has the same definition as R 4 defined above;

R

5 is C(Y 1 wherein Y' is 0 or S; Z is N(R 6 2)R 6 or OR 6 wherein R 6 a is H or (C 14 6)alkyl or NR 6

'R

62 wherein R 61 and R 62 are each independently H, (Cl.r 6 )alkyl, (C 3 7 )cycloalkyl, (Cl 1 o)alkyl-(C 3 7 )cycloalkyl, or both R 61 and R 62 are covalently bonded together and to the nitrogen to which they are both attached to form a 5, 6 or 7-membered saturated heterocycle; or R62 is COOR63 wherein R 63 is (C 1 .6)alkyl,

(C

3 .r 6 )cycloalkyl, said alkyl or cydloalkyl being optionally substituted with 6- or 1 0-membered aryl or Het; or R 6 2 is C0R 64 wherein R 64 is C 1 6 )alkyl,

(C

36 )CYCloalkyl -6-or 1 0-membered aryl or Het; and R 6 is selected from the group consisting of: H, (C 1 -6)alkyl, (C 3 6 )cycloalkyl,

(C

2 -6)aikenyI, 6- or lO-membered aryl, Het, (Cl-6)alkyl-aryl, (Cl.)alkyl-Het, wherein said alkyl, cycloalkyl, alkenyl, aryl, Het, alkyl-aryl, or alkyl-Het, are all optionally substituted with R 60 or R 6 is

R

7

R

8

R

Y 2 wherein R 7 and R 8 are each independently H, (C 16 )alkyl, haloalkyl, (C 37 )cycloalkyl, 6or 10-membered aryl, Het, (C 1 .6)alkyl-aryl, (C 16 )alkyl-Het, wherein said alkyl, cycloalkyl, aryl, Het, (C 1 .6)alkyl-aryl, (C 1 .6)alkyl-Het are optionally substituted with R 70 or R 7 and Ra are covalently bonded together to form second (C3.

7 )cycloalkyl or a 4, 5- or 8 o 6-membered heterocycle having from 1 to 4 heteroatom selected from 0, N, and S; 00or when Z is N(Rr'a)R either of R 7 or R 8 is covalently bonded to R 6 to form a nitrogen-containing 5-or 6-membered heterocycle; C 5 y 2 is0or S;

R

9 is H, (Cl46 alkyl), (C 37 )cycloalkyl or (Cl.

6 )alkyl-(C 3 7 )cycioalkyl, aryl, Het, (Cl-6)alkylaryl or (Cl 14 )alkyl-Het, all of which optionally substituted with R90; or

R

9 is covalently bonded to either of R 7 or RB to form a 5- or 6-membered heterocycle; o is-a 6- or 1 0-membered aryl, Het, (0146) alkyl-CONH-aryl or (C 16 alkyl-CONH-Het, all of which being optionally substituted with:

COOH

H AN "N '_COOH 0 1 ~~CGoH ,or130; or a salt or a derivative thereof; wherein Het is defined as 5- or 6-membered heterocycle having 1 to 4 heteroatoms selected from 0, N, and S, or a 9- or lO-membered heterobicycle having 1 to 4 heteroatoms selected from 0, N and S; and 113 0 R 20

R

60 R1 70 R9a and R1 00 is each defined as: 1 to 4 substituents selected from: halogen, OPO 3 H, N0 2 cyano, azido, C (=NH)NH 2 C(=NH)NH(Cl.

6 )alkyl or C(=NH)NHCO(Cj.6 )alkyl; or 1 to 4 substituents selected from: a) (0146) alkyl or haloalkyl, (C 3 7 )cycloalkyl, 03.7 splrocycloalkyl optionally containing 1 or 2 heteroatom, (C 24 6)alkenyl, (C 24 8)alkynyt, (C 14 6) alkyl-(C3- 7 )cycloalkyl, all of which optionally substituted with R 15 0 b) OR'04 wherein R1 04 is H, (C 16 alkyI), (C 37 )cycloalkyl, or (C 14 6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 1 .6alkyl)aryl or (Cl.

6 alkyl)Het, said alky, cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (Cl-6alkyl)Het being optionally substituted with 30R10 0) OCOR 1 05 wherein R' 05 is (Cl.

6 )alkyl, (0 37 )cycloalkyl, (C 1 .B)alkyl-(C 3 7 )cycloalkyl, Het, (Cl.ralkyl)aryl or (C 1 6 alkyl)Het, said alkyl, cycloalkyl, aryl, 9 Het, (Cl.

6 alkyl)aryl or (C 16 alkyl)Het bein g optionally substituted with R 150 00 d) SR 1 08

SO

3 H, SO 2

N(R'

08 2 or SO 2

N(R'

08

)C(O)R

08 wherein each RlOB is independently H, (Cl-6)aikyI, (C 37 )cycioalkyl or (Cl.

6 )aikyl-(C 37 )cycioalkyl, aryl, Het, (C 16 aikyl)aryl or (Cl.

6 alkyl)Het or both R1 08 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7- M membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C 1 6alkyi)aryl or (Cl.

6 alkyl)Het or heterocycle being optionally substituted with e) NR'R 1 wherein R 11 1 is H, (C 1 6 )alkyl, (C 37 )cycloaikyl or (Ci-e)alkyl-(C3- 7 )cycloalkyi, aryl, Het, (Cl-6alkyi)aryl or (Cl-6aikyl)Het, and R' 2 is H, ON, (Cl.

6 )alkyl, (C 37 )cycloalkyl or (C 16 )aikyl-(C 37 )cycioaikyl, aryl, Het, (Cl-6alkyl)aryl,

(C

16 alkyl)Het COOR"s or SO 2

R"

5 wherein R' 15 is (Cl-6)alkyl, (C 3 7 )cycloalkyi, or (Cl.

6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (C 14 6aikyl)aryl or (Ci.

6 aikyl)Het, or both R' 1 1 and R' 12 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said aikyl, cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl.

6 alkyi)Het, or heterocycle being optionally substituted with R 1 50 f) NR 116 00R 1 17 wherein R 11 and R' 17 is each H, (C 16 )alkyl, (C 37 )cycloalkyi,

(C

1 6 )alkyl-(C 3 7 )cycloalky, aryl, Het, (C 16 aikyl)aryl or (C 1 -6alkyI)Het, said (C 1 6 )alkyi, (C 3 7 )CYCloaikyl, (C 1 6 )alkyl-(C 37 cycloalkyl, aryl, Het, (Cl 6 alkyl)aryl or

(C

16 alkyl)Het being optionally substituted with R' 50 g) NR 118 00NR 19 R 2 wherein R 118

R

119 and R 12 is each H, (Cl-6)alkyl, (C- 7 )cycloalkyl, (Cl.

6 )alkyl-(C3aq)cycloalkyl, aryl, Het, (C 1 .6alkyl)aryl or (Ci.

6 aikyl)Het, or R 118 is covalently bonded to R 11 9 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R'19 and R 120 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (C 16 )alkyI-(C 37 )cycloakyI, aryl, Het, (C 16 alkyl)aryl or (Cl- 6 alkyl)Het or heterocycle being optionally substituted with R'1 50 h) NR 1 21 COCOR'22 wherein R 1 21 and R 1 22 is each is H, (C 16 )alkyl, (C 3 7 )cycloalkyi, (Cl 16 )alkyl-(C 3 7 )cycloalkyl, a 6- or lO-membered aryl, Het, (C 1 6 alkyl)aryl or (Cl.

6 aikyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het,

(C

1 6 alkyl)aryl or (Cl.

6 alkyl)Het being optionaily substituted with R' 50 or R 1 22 Is OR 123 or N(R 1 24 2 wherein R 1 23 and each R 1 24 is independently H, o(Cl. 6 alkyl), (C 3 7 )cycloalkyl, or (C 16 )alkyi-(C 3 .4)cyCloalkyl, aryl, Het,(C1 00 6 alkyl)aryl or (C, 6 alkyl)Hret, or R' is OH or O(C 10 6aikyl) or both R 1 2 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C 1 .6aikyl)aryl or (Cj_6alkyl)Het and heterocycle being optionally substituted with RI 50 i) C0 1 12 wherein R' 2 is H, (Cl.

6 )alkyl, (C 37 )cycloalkyl or (C 1 6 )alkyl-_(0 3 7 )cycloalkyl, aryl, Het, (C 1 .6alkyl)aryl or (C 16 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C 1 .6alkyI)aryl or (C 1 6 alkyl)Het being optionally substituted with R'; jCOOR 1 2 1 wherein R'1 2 1 is H, (Cl.

6 )alkyl, (C 37 )cycloalkyl, or(Cl-6)alky-(C3.

7 )cycloalkyl, aryl, H-et, (Ci.

6 alkyl)aryi or (Cl.6alkyl)Het, said (C 16 )alkyl, (C 3 7 )cycloalkyl, or(Cj_ 6 )akyl-(C3.

7 )CYCloalkyl, aryl, Het, (C 1 6 alkyl)aryl and (Ci.

6 alkyl)Het being optionally substituted with R 150

O;

k) CONR 12 0R'30 wherein R'1 29 and R 1 30 are independently-H, (C 16 )alkyl, (03.

7 )cycloalkyl, (Cj.

6 )alkyl.(C 3 .7)cycloalkyl, aryl, H-et, (C 1 -6alkyl)aryl or (CI_ 6 alkyl)Het, or both R 1 and R 13 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycie, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryi, (Cl.

6 alkyl)Het and heterocycle being optionally substituted with R' 50 1) aryl, Het, (C 1 6 alkyl)aryl or (Cl.

6 alkyl)Het, all of which being optionally substituted with R 150 and wherein R 150 is defined as: 1 to 3 substituents selected from: halogen, OPO 3 H, NO 2 cyano, azido, C(=NH)NH 2 C(=NH)NH(Cl- 6 )alkyl or C(=NH)NHCO(C 14 )alkyl; or 1 to 3 substituents selected from: a) (CI-6) alkyl or haloalkyl, (C 37 )cycloalkyl, C3.7 spirocycloalkyl optionally containing 1 or 2 heteroatom, (C 2 .6)aikenyl, (C 2 8 )alkynyl, (01.6) alkyl-(C 3 7 )cycloalkyl,-all of which optionally substituted with R' 60 1b) ORIM4 wherein 1 10 is H, (C1.

6 alkyl), (C 37 )cycloalkyl, or (Cl.

6 )alkyl-

(C

3 7 )cycloalkyl, aryl, Het, (Cl 16 alkyl)aryl or (Cl.

6 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C 16 aikyl)aryl or (Cl.

6 alkyl)Het being optionally substituted with R 160 c) OCOR' 05 wherein R' 05 is (Cl.

6 )alkyl, (0 3 7 )cycloalkyl, (C 1 6 )alkyl-(C 3 7 )cycloalkyl, Het, (C 1 .6alkyl)aryl or (Cl 16 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C 1 .6alkyl)aryl or (C 14 6alky!)Het being optionally substituted with R 1 60 00 d) SR 108

SO

3 H, SO 2

N(R

08 2 or SO 2

N(R'

0 8

)C(O)R'

08 wherein each R' is independently H, (Cl.

6 )aikyi, (C 37 )cycloalkyl or (Cl.

6 )aikyi-(C 3 7 )cycloaikyl, aryl, Het, (C 1 6 aikyi)aryl or (C 1 -6aikyi)Het or both R 108 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C 16 alkyi)aryi or (Cl-6aikyi)Het or heterocycle being optionally substituted with R 1 60 r.K1e)

NR

111

R'

12 wherein R' is H, (C 16 )alkyi, (C 37 )cycloalkyl or (C 1 6 )alkyl-(C 37 )CYCloalkyi, aryl, Het, (C 1 .6alkyl)aryI or (C 1 ~ealkyl)Het, and R' 1 2 is H, CN, (C 1 -6)alkyl, (C3.

7 )cycloalkyI or (C 1 6 )alkyl-(C 37 )cycIoalkyl, aryl, Het, (C 16 aikyl)aryl,

(C

1 .raikyl)Het, C00R 15 or SQ 2

R

115 wherein Is (C 16 )aikyl, (C 37 )cycloalkyl, or (C 16 )alkyI-(C3.

7 )cycloakyl, aryl, Het, (Ci.6alkyl)aryl or (Cl 16 alkyl)Het, or both R 1 11 and R' 12 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said.

aikyl, cycloalkyl, aryl, Het, (Ci- 6 aikyi)aryl or (C 16 aikyI)Het, or heterocycle being optionally substituted with R'1 60 f) NR" 6 00R 17 wherein R' 6 and R"1 7 is each H, (Cl-6)alkyl, (03.

7 )cycloaikyl, (Cl-6)alkyl-(C3.7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (C 1 6 alkyi)Het, said (Cl- 6 )alkyl, (C 37 )cycloaikyl, (Cj.

6 )ak-yI-(C. 7 )cycloakYl, aryl, Het, (C 16 alkyi)aryl or (Cl.

6 alkyl)Het being optionally substituted with R160; g) NR118CONRllBR 2 wherein R118, R 119 and R 12 is each H, (Ci.

6 )alkyl, (C 37 )cycioalkyl,

(C

16 r)aIky-(C 3 7 )CYCIoakyI, aryl, Het, (Ci.

6 alkyl)aryl or (C 1 -6alkyl)Het, or R11 8 is covalently bonded to R 1 19 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R 119 and R 1 20 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (Cl.

6 )aikyl.(C 3 7 )cycoalkyl, aryl, Het, (Ci.

6 aikyl)aryl or (Cl-6aikyl)Het or heterocycle being optionally substituted with R' 60 12 oh) NR 1 21

COCOR

1 22 wherein R 1 21 and R 1 22 is each is H, (C 1 .6)alkyl, C0 00 ~(C3. 7 )cycloalkyl, (Cl-6)alkyl-(C 3 7 )cycloalkyl, a 6- or lO-membered aryl, Het, (C 16 alkyl)aryl or (Cl.

6 alkyl)Het said alkyl, cycloalkyl, alkylcycloalkyl, aryl, Net, (C 16 alkyl)aryl or (C 16 alkyl)Het being optionally substituted with R 1 60 or R 1 22 Is OR 123 or N(R'1 24 2 wherein R 12 3 and each R 1 24 iS independently H, (Cl.

6 alkyl), (C 37 )cycloalkyl, or (C 16 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 1 .6alkyl)aryl or (C 1 -6alkyI)Het, or R 1 24 is OH or

O(C

1 6 alkyl) or both R 12 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkylcydoalkyl, aryl, Het, (C 16 aikyl)aryl or (C 1 -6alkyl)Het and heterocycle being optionally substituted with R 1 60 i) C0R 127 wherein R 1 27 is H, (Cl.

6 )alkyl, (C 37 )cycloalkyl or (Cl.

6 )alkyl-

(C

3 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (C 1 6 alkyl)Het, said alkyl, cycloalkyl, aryl, Net, (Cl-6aikyl)aryl or (C 1 .6alkyl)Het being optionally substituted with R' 60 j) tetrazole, C00R 128 wherein R 1 28 is H, (C 16 )alkyl, (C3.

7 )cycloalkyl, or(Cl 1 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (Cl.

6 alkyl)Het, said (0 1 6 ,)alkyl, (C 7 )cycloalkyl, or(Cl.

6 )alkyl-(C 37 )cyCloalkyl, aryl, Net,

(C

16 alkyl)aryl and (C 1 -6alkyl)Het being optionally substituted with R 16 1; and k) CONR'9R 1 3 1 wherein R 1 29 and R'1 30 are independently H, (C 1 6 )alkyl, (C 3 7 )cycloalkyl, (C .)alkyl-(C3.

7 )cycloalky, aryl, Het, (C- 6 alkyi)aryl or (Cl.

6 alkyl)Het, or both R129 and R 1 30 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (Cl-6alkyl)aryl, (C 16 aikyl)Het and heterocycle being optionally substituted with R' 60 wherein R 1 60 is defined as 1 or 2 substituents selected from: tetrazole, halogen, ON, C 16 alkyl, haloalkyl, C00R 6

SO

3

H,

SR

1 61 ,S0 2 R1', OR' 6

N(R'

62 2 SOANR 6 2 or CON(R 6 2 2 wherein R1 61 and each R 1 62 is independently H, (Cl.

6 )alkyl, (C3.

7 )cycloalkyl or (Cl.

6 )alkyl-(C3- 7 )cycloalkyl; or both' R' 62 are covalently bonded together and to the nitrogen to which they 13 0 are attached to form a 5, 6 or 7-membered saturated 0 heterocycle, 00 wherein said probe comprises a detectable label attached to any suitable position, whereby said probe binds to an HCV polymerase or an analog thereof and is capable of being displaced by an inhibitor thereof.

NO

SAccording to an alternative of this first embodiment, the probe used for the assay does not comprise a detectable label, and the signal measured is the change in intrinsic fluorescence of the HCV polymerase in the presence and absence of said C1 10 probe.

According to a second aspect of the invention, there is provided the use of a probe according to formula I in the development of an assay for identifying inhibitors of HCV polymerase.

According to a third aspect of the Invention, there is provided a kit for testing compounds potentially binding to HCV polymerase, said kit comprising the probe of formula and instructions on how to use said probe for identifying test compounds binding to said polymerase.

BRIEF DESCRIPTION OF THE FIGURES Having thus generally described the invention, reference will now be made to the accompanying drawings, showing byway of illustration a preferred embodiment thereof, and in which: Figure 1 illustrates the titration of probe with the NS5BA21-His polymerase.

Standard conditions for the Fluorescence anisotropy analysis are described in Example 3. The determined Kd value of probe for this polymerase is 12.6 nM.

Figure 2 illustrates Z' evaluation for the Fluorescence Polarization assay. A series of positive and negative controls were tested in the 96-well plate polarization assay, using the standard conditions, to determine the standard deviation (SD) of both controls. The Z' value was then obtained from the following calculation S 14 SZ' 1 (3 SD of pos. ctrls 3 SD of neq. ctrls) O (mean pos. ctrl- mean neg. ctrl) 00 Figure 3 illustrates Kd determination for Compounds A and B, using the Fluorescence Polarization assay. Standard conditions of the 96-well plate SPolarization assay (see Example 4) were used to determine the Kd values of the Scompounds. Kd values obtained for compound A and B are 31 and 41 nM, respectively, with Qb/Q values of 0.67 and 0.72.

C 10 Figure 4 illustrates Kd determination for Compounds C and D, using the Fluorescence Polarization assay. Standard conditions of the 96-well plate Polarization assay (see Example 4) have been used to determine the Kd values of some of our compounds. Kd values obtained for compound C and D are 231 nM and 1.08 uM, respectively, with Qb/Q values of 0.74 and 0.66.

Figures 5 to 8 illustrate the titration of probe with the NS5BA21-His in the presence of increasing (from 30 mM to 200 mM) concentration of NaCl.

Standard conditions of the Fluorescence anisotropy analysis are described in Example 3. Kd values obtained for this polymerase are 15.3 nM (30 mM NaCI), 39 nM (100 mM NaCI), 78 nM (150 mM NaCI) and finally 122 nM (200 mM NaCI).

Figure 9 illustrates the titration of probe with the NS5BA21-His in Phosphate buffer pH 6.5. Standard conditions of the Fluorescence anisotropy analysis are described in Example 3. The Kd of probe for this polymerase under these conditions is 33 nM.

Figure 10 illustrates the titration of probe with the His-NS5BA21 polymerase.

Standard conditions of the Fluorescence anisotropy analysis are described in Example 3. The Kd of probe for this N-terminally tagged polymerase is 18.1 nM.

Figure 11 illustrates the titration of probe (ii) with the GBV-BA23-His polymerase.

Standard conditions of the Fluorescence anisotropy analysis are described in Example 3. The Kd of probe (ii) for this distantly related polymerase is 1.79 uM (estimated value with an incomplete curve).

o Figure 12 Illustrates the titration of probe (ii) with the His-NS5BA21(H77c,

HCV

00 genotype la) polymerase. Standard conditions of the Fluorescence anisotropy analysis are described in Example 3. The Kd of probe (ii) for this HCV genotype la polymerase is 18.2 nM.

O c DETAILED DESCRIPTION OF THE INVENTION Definitions The following definitions apply unless otherwise noted:

O

N 10 The term "affinity tag" means a moiety whose strong affinity for a ligand can be used to extract from a solution the entity to which the tag is attached. Examples of such tags include biotin or a derivative thereof, a histidlne polypeptide, a polyarginine, an amylose sugar moiety or a defined epitope recognizable by a specific antibody. Such affinity tags are attached to the probe by well-known methods. The corresponding affinity ligands are also well known in the art.

An "analog" of the HCV NS5B polypeptide or a fragment thereof means a polypeptide modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself.

Such analogs of the natural amino acid sequence may involve insertion, addition, deletion or substitution of one or more amino acids, and may or may not alter the functional activity of the original HCV NS5B polypeptide. As mentioned above, the HCV NS5B polypeptide or protein used in the assay/method of the invention includes any fragment, derivative, variant or mutant which is derived from a HCV NS5B polypeptide and which retains at least one property or other characteristic of the HCV NS5B polypeptide.

The term "detectable label" refers to any group that is linked to a probe of the present invention such that when the probe is associated with the polymerase target, such label allows recognition either directly or indirectly of the probe such that it can be detected, measured and quantified. Examples of such "detectable labels" are intended to include, but are not limited to: photoreactive groups, fluorescent labels, chemiluminescent labels, colorimetric labels, enzymatic markers, radioactive isotopes. Such labels are attached to the probe by well known methods.

0 16 0 As used herein, the term "linker" refers to a chain of between 1 and 20 atoms O selected from the group consisting of C, N, 0, and S that covalently connects the Saforesaid label to a probe of the present invention. Examples of such a chain include, but are not limited to, the following: 0

H

Y N N HH C^ H or 0 These linkers can also comprise a pair of affinity-tag/affinity-ligand, which together, Sbind the compound to a detectable label.

The term "photoreactive group" means a group that is transformed, upon activation by light, from an inert group to a reactive species, such as a free radical. Examples of such groups Include, but are not limited to, benzophenones, azides, and the like.

As used herein, the term "probe" refer to a compound of formula that is capable of binding to an HCV polymerase in a covalent or non-covalent manner. When the probe is bound in a non-covalent manner, it can be displaced by a test compounds.

When bound in a covalent manner, the probe can be used for cross-linking experiments wherein the HCV polymerase-probe adduct formation can be quantified and inhibited by test compounds.

As used herein, the terms alkyl", alkyl" or alkyl", either alone or In combination with another radical, are intended to mean acyclic straight or branched chain alkyl radicals containing up to three, four and six carbon atoms respectively.

Examples of such radicals include methyl, ethyl, propyl, butyl, hexyl, 1-methylethyl, 1methylpropyl, 2-methylpropyl, 1,1-dimethylethyl.

As used herein, the term alkenyl", either alone or in combination with another radical, is intended to mean an unsaturated, acyclic straight chain radical containing two to six carbon atoms.

As used herein, the term (C26) alkynyl" either alone or in combination with another group, is intended to mean an unsaturated, acyclic straight chain sp hybridized radical containing 2 to six carbon atoms.

17 o As used herein, the term cycloalkyl", either alone or in combination with O another radical, means a cycloalkyl radical containing from three to seven carbon Satoms and includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

As used herein, the term "(Cs.

7 )cycloalkenyl", either alone or in combination with another radical, means an unsaturated cyclic radical containing five to seven carbon Catoms.

O As used herein, the term "aryl", or or 10-membered aryl" either alone or in C 10 combination with another radical means aromatic radical containing six or ten carbon atoms, for example phenyl or naphthyl.

As used herein, the term "COOH" refers to a carboxylic acid group. It is well known to one skilled in the art that carboxylic acid groups may be substituted by functional group equivalents. Examples of such functional group equivalents that are contemplated by this invention include, but are not limited to, esters, amides, or boronic acids.

As used herein, the term "functional group equivalent" is intended to mean an element or a substituted derivative thereof, that is replaceable by another element that has similar electronic, hybridization or bonding properties.

As used herein, the term "halo" means a halogen atom and includes fluorine, chlorine, bromine and iodine.

As used herein, the term "haloalkyl" is intended to mean an alkyl that is described above in which each hydrogen atom may be successively replaced by a halogen atom, for example CH 2 Br or CF 3 As used herein the term heteroatom means O, S or N.

As used herein, the term "heterocycle", either alone or in combination with another radical, means a monovalent radical derived by removal of a hydrogen from a five-, six-, or seven-membered saturated or unsaturated (including aromatic) heterocycle containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur.

0 18 o Furthermore, "heterobicyclic" as used herein, means a heterocycle as defined above O fused to one or more other cycle, be it a heterocycle or any other cycle. Examples of 00 such heterocycles include, but are not limited to, pyrrolldine, tetrahydrofuran, thiazolidine, pyrrole, thiophene, coumarin, hydantoln, diazepine, 1 H-imidazole, isoxazole, thiazole, tetrazole, piperidine, 1,4-dioxane, 4-morpholine, pyridine, pyridine-N-oxide, pyrimidine, thlazolo[4,5-b]-pyridine, quinoline, or indole, or the Sfollowing heterocycles:

N

As used herein, the term or 10-membered heteroblcycle" or "heterobicycle" either alone or in combination with another radical, means a heterocycle as defined above fused to one or more other cycle, be it a heterocycle or any other cycle. Examples of such heterobicycles include, but are not limited to, thiazolo[4,5-b]-pyrldlne, quinoline, or indole, or the following: I S o N H N 0 ,or As used herein, the term "Het" defines a 5- or 6-membered heterocycle having 1 to 4 heteroatoms selected from O, N, and S, or a 9- or 10-membered heterobicycle having 1 to 4 heteroatoms selected from O, N and S.

As used herein, the term "OH" refers to a hydroxyl group. It is well known to one skilled in the art that hydroxyl groups may be substituted by functional group equivalents. Examples of such functional group equivalents that are contemplated by this invention include, but are not limited to, ethers, sulfhydryls, and primary, secondary or tertiary amines.

19 SAs used herein, the term "SH" refers to a sulfhydryl group. It is intended within the O scope of the present invention that, whenever a "SH" or "SR" group Is present, it can also be substituted by any other appropriate oxidation state such as SOR,

SO

2 R, or SO 3

R.

SIt is intended that the term "substituted" when applied in conjunction with a radical having more than one moiety such as C 16 alkyl-aryl, or Cl.alkyl-Het, such substitution applies to both moieties i.e. both the alkyl and aryl or Het moieties can Sbe substituted with the defined substituents.

Description of preferred embodiments Preferably, according to the first aspect, the invention provide a method for identifying inhibitors of HCV polymerase comprising the steps of: a) contacting said HCV polymerase with a probe of formula I so as to form a complex comprising said probe bound to said polymerase; b) measuring a signal from said complex to establish a base line level; c) incubating the product of step a) with a test compound; d) measuring the signal from said complex; and e) comparing the signal from step d) with the signal from step b); whereby a decrease in said signal is an indication that said test compound is an inhibitor of said polymerase.

As will be understood by a person skilled in the art, the association of a specific probe of the invention with the NS5B polymerase can be measured directly or indirectly in a variety of ways. The probe and NS5B polymerase need not be labeled and affinity tagged respectively. The association of a specific probe with the HCV polymerase can be monitored and quantified directly by a change in the intrinsic spectral properties of a tagged or un-tagged NS5B protein and/or by a change in the intrinsic spectral properties of a specific probe. A direct measurement of inhibitor-NS5B association can also be achieved by immobilizing one of these two components on a matrix and measuring association through plasma-resonance detection technology. An assay that quantifies probe-NS5B complex association may also incorporate a photo-reactive label (such as a phenyl-azide or benzophenone) on the probe (for example probes and (vi) below) and measure the amount of label irreversibly bound to the NS5B3 (adduct) following photo-activaton of the probe.

00 Preferably, according to a first aspect of the present invention, there is provided a probe of formula:

R

5

R

N2 N 2 R RR lb or IC wherein R' is selected from the group consisting of: H or (C 14 6)alkyl; R 2 is CON(R 22 2 wherein each R22 is Independently H, (Cl-6)alkyl, (C3.

7 )cycloalkyl, (0 5 7 )cycloalkenyl, 6 or 1lO-membered aryl or Het, or both R22 are bonded together to form a 5, 6 or 7-membered saturated heterocycle with the nitrogen to which they are attached; or R 2 is selected from: H, halogen, (C 1 .6)alkyl, haloalkyl, (0 26 )alkenyl, 7 )cycloalkenyl, 6 or lo-membered aryl or Het; wherein each of said alkyl, haloalkyl,

(C

26 )alkenyl, (C 57 )cycloalkenyl, aryl or Het is optionally substituted with R 20 wherein R 20 Is defined as: -1 to 4 substituents selected from: halogen, NO 2 cyano, azido, C(=NH)NH 2 C(=NH)NH(C-)alkyl or C(=NH)NHCO(0 1 6 )alkyl; or -1 to 4 substituents selected from: a) (01.6) alkyl or haloalkyl, (0 37 )cycloalkyl, (C 26 )alkenyl, (C 2 -B)alkynyl, (01.6) alkyl-(C3.

7 )CYCloalkyl, all of which optionally substituted with R1 5 0; b) OR 1 04 wherein R1 04 is H, (Cl.

6 alkyl), (C 3 7 )cycloalkyl, or (C, 4 6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl-6alky)aryl or 6 alkyl)Het, said alkyl, cycloalky, aryl, Het, 6 alkyl)aryl or (C 1 6 alkyl)Het being optionally substituted with 25R10 c) OCOR' 05 wherein R' 05 is (C, 6 )alkyl, (0 3 -7)cycloalkyl, (Cl-6)alkyl-(C 3 7 )CYCloalkyl, Het, (C1.

6 alkyl)aryl or (0 1 6 alkyI)Het, said alkyl, cycloalky, aryl, Het, (Cl-6alkyl)aryl or (Cl.6alkyl)Het being optionally substituted with R' 50 d) SR 10 8

SO

3 H, SO 2

N(ROB)

2 or S0 2

N(R

1 1 0" wherein each R 1 0 8 is independently H, 6 ,)alkyl, (C3.

7 )cycloalkyl or (Cl, 6 )alkyl-(C 3 q)CYCloalkyl, aryl, 21 0 Het, (C 16 aikyl)aryi or (Cl.

6 aikyi)Het or both R' are covalently bonded 00 together and to the nitrogen to which they are attached to form a 5, 6 or 7membered saturated heterocycie, said alkyl, cycloalkyl, aryl, Het, (Ci.

6 alkyI)aryi or (Cl-6alkyi)Het or heterocycle being optionally substituted with e) NR" 1 R" 2 wherein R 11 1 is H, (C 1 -6)aikyI, (0 37 )CYCloalkyi or (C 16 )alkyi-(C3- 7 )CYCloaikyl, aryl, Het, (C 16 alkyl)aryl or (C 16 alkyl)Het, and R 11 2 is H, ON, (Ci.

6 )alkyi, (C 37 )cycloalkyi or (Cl.

6 )alkyi.(C 3 7 )CYCloalkyi, aryl, Het, (C,-6alkyI)aryi,

(C

16 alky)Het, COOR 1 1 5 or S0 2 191 15 wherein R" 5 6 is (0 1 6 )alkyl, (03.

7 )cycloalkyl, or (C 1 6 )aikyl-(C 37 )cycloalkyI, aryl, Het, (C 16 alkyI)aryl or (Cl.

6 alkyI)Het, or both R' 1 1 and R 1 12 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said akl, cycloalkyl, aryl, Het, (Ci 16 alkyI)aryl or (Cl-6alkyl)Het, or heterocycle being optionally substituted with R1 50 f) NR" 6 COR11 7 wherein R' 1 6 and R' 17 is each H, (C 1 .e)alkyl, (C 3 q7)CYCloalkyl,

(C

1 6 )alkyl.(C 3 7 )cycloalkyI, aryl, Het, (Ci- 6 aikyl)ary or (Cl-6alkyI)Het, said (Ci.

6 )alkyi, (C 3 -7)cycloalkyl,

(CI.

6 )aikyl.(C 37 )CycloalkW, aryl, Het, (G 14 6alkyi)arYi or (Cl.,alkyl)Het being optionally substituted with R 150 g) NR11800NRlBR 2 wherein R 118

R'

19 and R 120 is each H, (C 1

.C

6 )alkyl (P3.

7 )CYCloalkyi, (Cl.

6 )alkyi-(0 3 7 )cycioakyl, aryl, Het, (C 1 -6alkyl)ary or (C1- 6 aikyl)Het, or R 118 is covalently bonded to R' 1 9 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R1 19 and R 1 20 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl,

(C

16 )aikyi.(C3aq)cycloalkyI, aryl, Het, (C 16 alkyi)aryl or (Cl.

6 alkyi)Het or heterocycle being optionally substituted with R' 50 h) NIR121 COCORl22 wherein R 12 and R122 is each is H, (C 16 )aikyl, (C3- 7 )cycioalkyl, (0 1 6 )aikyi.(0 37 )cycloalky, a 6- or lO-membered aryl, Net, (01.

6 alkyl)aryi or (0 1 0 aikyi)Het, said alkyl, cycloalkyl, alkyl-cycloalky, aryl, Het,

(C

16 alkyI)aryl or (Cl.6alkyi)Het being optionally substituted with R 150 or R 12 2 is OR 123 or N(R 1 24 2 wherein R 1 23 and each R 1 24 is independently

H,

(Cl.

6 aikyl), (C 3 7 )cycioalkyl, or (Cj-6)alkyl.(C. 7 )Cycloaikyi, aryl, Net, (Ci.

6 alkyl)aryi or 6 aikyl)Het, or R 1 24 is OH or O(C 1 6 aikyi) or both R 1 24 are covalently bonded together to form a 5, 6 or 7-mhembered saturated 22 0 heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C 16 alkyI)aryl or C) (Cl.

6 alkyl)Het and heterocycle being optionally substituted with 13' 50 00 0) COR 1 27wherein R 1 2 7 is H, (C 16 )alky, (C 37 )cycloalkyl or (C 14 6)alkyl-(C3.

7 )CYCloalkyl, aryl, Het, (C 16 alkyl)aryl or (C 1 6 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C 1 .6alkyl)aryl or (Cl.

6 alkyl)Het being optionally substituted with R 150 jCOOR 1 2 1 wherein R 128 is H, (C 16 )alkyl, (C 37 )cycloalkyl, or(Cl.6)alkyl-(C3- 7 )cycloalkyl, aryl, Het, (C 1 .6alkyl)aryl or (C 1 6 alkyI)Het, said (Cl 14 )alkyl, (C 3 7 )cycloalkyl, or(Cl.

6 )alkyl-(C.

7 )CYCloalkyl, aryl, Het, (C 14 6alkyl)aryl and (C 1 6 alkyi)Het being optionally substituted with R' 50 k) CONR 29 R 130 wherein R 12 and R' 30 are independently H, (C 16 )alkyl, (C 3 7 )cycloalky, (C 1 6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (C 1 r 6 alkyl)Het, or both R 1 2 and are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alky-cycloalkyl, aryl, Het, (C1.

6 alkyl)aryl, (0 1 6 alkyl)Het and heterocycle being optionally substituted with R 1 6 0 1) aryl, Het, (Cl -6aikyl)aryl or (Cl-6alky)Het, all of which being optionally substituted with R 150 wherein R 150 is preferably: 1 to 3 substituents selected from: halogen, NO 2 cyano or azido; or 1 to 3 substituents selected from: a) (01.6) alkyl or haloalkyl, (C 3 7 )cycloalkyl, (C 2 .6)alkenyl, (0 28 )alkynyl,

(C

14 6) alkyl-(C 3 7 )cycloalky, all of which optionally substituted with Rl 1 60 b) OR 104 wherein R 1 04 is H, (Cl-6alkyl) or (C 3 7 )cycioalkyi, said alkyl or cycloalkyl optionally substituted with R'rao; d) SR' 08

SO

3 H, S0 2

N(R'

08 2 or SO 2

N(R'

08

)C(O)R'

0 8 wherein each 11 108 is independently H, (C 16 )alkyl, (C3.

7 )cycloalkyl or (Cl 16 )alky-(C 3 7 )cycloalky, aryl, Het, or both R 108 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het and heterocycle being optionally substituted with R'1 60 e) NR 111

R

1 12 wherein R'11 Is H, (Cl.

6 )alkyl, or (C 37 )cycloalkyl, and R' 12 is H, (C 1 6 )alkyl or (C 37 )cycloalkyl, COOR"' or S0 2

R

1 15 wherein R"' is (C 1 .6)alkyl or (C 3 7 )cycloalkyl, or both R 11 1 and R" 2 are covalently bonded together and to the nitrogen to which they are attached to 23 0 form a 5, 6 or 7-membered saturated heterocycle, said alkyl, 00 cycloalkyl and heterocycle being optionally substituted with f) NR 116 C0R 1 17 wherein R 11 6 and R1 17 is each H, (Cl.

6 )alkyl or (03.

7 )cycloalkyl said (Ci.

6 )alkyl and (C 37 )cycloalkyl being optionally substituted with R 160 g) NR11BCONRll 9

R

2 wherein R1 18

R

11 and R 12 is each H, (Cl.

6 )alkyl or (C 37 )cycloalkyl, or is covalently bonded to R 11 9 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R 11 and R'12' are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alky, cycloalkyl, and heterocycle being optionally substituted with R 1 60 h) NR 121 COCORl22 wherein R 1 21 is H, (C 16 )alkyI or (C 37 )cycloalkyl, said alkyl and cycloalkyl being optionally substituted with R'1 60 or R 1 22 is OR'23 or N(R'1 24 2 wherein RA1 23 and each RA1 24 is independently H, (Ci,,alkyl) or (C 3 7 )cycloalkyl, or both RA 12 4 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl and heterocycle being optionally substituted with R 1 60 1) COR 27 wherein R127 is H, (C 16 )alkyl or (C 3 7 )cycloalky, said alkyl and cycloalkyl being optionally substituted with R' 60 j) COOR 1 28 wherein RA 12 8 is H, (Cl, 6 )alkyl or (C 37 )cycloalkyl, said (C,-6)alkyl and (C 3 7 )CYCloalkyl being optionally substituted with RA1 60 and k) CONR 1 29 R'1 30 wherein R'1 29 and R'30 are independently H, (Ci.

6 )alkyt or (C 3 7 )cycloalkyl, or both R 1 29 and R 1 30 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl and heterocycle being optionally substituted with wherein R' 60 is defined as 1 or 2 substituents selected from: halogen, CN, C 1 -6alkyl, haloalkyl, COOR 6

OR'

61

N(R'

52 2 SOANR 1 62 2 or CON(R1 62 2 wherein R' 61 and each R16 Ils 24 U independently H, (C 1 .6)alkyl, (C 3 7 )CYCloalkyi or (C 16 )alkyl-(C 3 C0 7 )cycloalkyl; or both R 1 62 are covalently bonded together and to 00 the nitrogen to which they are attached to form a 5, 6 or 7membered saturated heterocycle; R 3 is selected from (0 3 7 )cycloalkyl, (Cr 10 )bicycloalkyl, 6- or 1 0-membered aryl, or CI Net;

R

5 is wherein CK1 Z is OR 6 wherein R6 is C 14 6alkyl substituted with: 1 to 4 substituents selected from: OPO 3 H, NO 2 cyano, azido, C(=NH)NH 2 C(=NH)NH(0 1 6 )alkyl or C(=NH)NHCO(Cl.6)alkyl; or 1 to 4 substituents selected from: a) (CI.6) alkyl or haloalkyl, (C 3 7 )cycloalkyl, C3-7 spirocycloalkyl optionally containing 1 or 2 heteroatom, (C2-)alkenyl, (C 2 8 )alkynyl, (01.6) alkyl-(0 3 ,)cycloalkyl, all of which optionally substituted with R'1 50 b) OR' 04 wherein R 104 is (CI-.

6 alkyl) substituted with R 150 (C3.

7 )CYCloalkyl, or (Cl.

6 )alkyl-(0 3 7 )cycloalkyl, aryl, Het, (Cl 16 alkyl)aryl or (Cl 16 alkyl)Het, said cycloalkyl, aryl, Het, (0 1 6 alkyl)aryl or (Cl.

4 alkyl)Het being optionally substituted with R 150 c) OC0R 1 0 5 wherein R 1 0 5 is (C 1 6 )alkyl, (C 37 )CYCloalkyl, (Cl-6)alkyl-(C 3 7 )cycloalkyl, Het, (C 16 alkyl)aryl or (Cl-6alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (C 18 6alkyl)Het being optionally substituted with R' 50 d) SR 108

SO

3 H, SO 2

N(R

08 2 or SO 2

N(R'

08

)C(O)R

1 0 8 wherein each R1 08 is independently H, (Cl.

6 )alkyl, (C 3 7 )cycloalkyl or (C 1 -6)alkyl-(C 3 7 )cycloalkyl, aryl, Net, (Cl.

6 alkyl)aryl or (Cl.

6 alkyI)Het or both Rica are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Net, (Cl.

6 alkyl)aryl or (Cl.

6 alkyl)Het or heterocycle being optionally substituted with

R'

50 e) NR 111

R

112 wherein R 1 1 1 is (0 1 6 )alkyl substituted with R' 5 0 (C3.

7 )cycloalkyl or (Cj- 6 )alkYl-(C 3 q7)cycloalkyl, aryl, Het, (C 1 -6alkyl)aryl or (0 1 6 alkyl)Het, and R' 12 is ON, (C 16 )alkyl substituted with R150, (C3- 7 )cycloalkyl or (Cl-6)alkyl-(0 3 7 )cycloalkyi, aryl, Het, (Cl.

8 aikyl)aryi, (Cl- 6 alkyl)Het,

COOR'

15 or SO 2 R' 1 C0 00 wherein R' 15 is (C 1 6 )alkyl, (C3- 7 )cycloalkyl, or (Cl.

6 )aky-(C 37 )cyciOalky, aryl, Het, (C 1 6 aikyI)aryl or (Cl-ralkyi)Het, or both R 11 and R1 12 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said cycloalkyl, aryl, Het, (C 1 6 alkyi)aryl or (Cl 16 aikyi)Het, or heterocycle being optionally substituted with R'1 50 f) N R' 16 C0R 1 17 wherein R'adR7i ahH (Cl.

6 )alkyl, (G3- 7 )cycioalkyI,

(C

1 .6)aikyi-(C3.

7 )CYCioakyI, aryl, Het, (Cl- 6 alkyl)aryl or (C 14 6alkyi)Het, said (Cl.

6 )aikyi, (C 3 7 )CYCloalkyi,

(C

1 .6)alkyi-(C 3 7 )cycioaiky, aryl, Net, (C 16 alkyi)aryl or (Cl.

6 aikyI)Het being optionally substituted with R' 50 g) NR"lACONR"OR1 2 0, wherein R 118

R

1 9 and R 1 20 is each H, (0 16 )alkyl, (0a- 7 )cycloalkyl,

(C

1 -6)aiky-(C 37 )CYCioalkyI, aryl, Het, (C 16 aikyI)aryi or (Cl.

6 alkyI)Het, or 1 1 8 1 is covalently bonded to R 119 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R' and R 12 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (Cl 16 )alky-(C 37 )cycioalkyi, aryl, Het, (C 14 6aikyi)aryi or (Cl.

6 alkyi)Het or heterocycle being optionally substituted with R' 50 h) NR 1 21 COCOR'22wherein

R

12 and R 1 22is each is H, (Cl-6)alkyl, (03.

7 )cycioaikyi, (Cl.

6 )aikyi(C 3 7 )cycioalkyi, a 6- or 1 O-membered aryl, Het, 6 alkyi)aryl or (Cl.

6 aikyI)Het, said alkyl, cycloalkyl, akl-cycloalkyl, aryl, Het,

(C

16 alkyi)aryl or (C 1 6 aikyl)Het being optionally substituted with R1 50 or R'22is OR 123 or N(R 1 24 2 wherein R 123 and each R 124 is independently

H,

(Cl.6aikyi), (C 3 7 )cycioaikyi, or (Cl-6)alkyl-(C3.7 )cycloalkyl, aryl, Net, (C 1 6 aikyi)aryl or (C 1 6 aiky)Het, or R'1 24 is OH or O(C 1 6 aikyl) or both R'1 24 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aikyl-cycloalkyl, aryl, Het, (C 16 aikyl)aqy or (Cl- 6 aikyi)Het and heterocycle being optionally substituted with R 150 i) C0R 127 wherein R' 27 is H, (Cl.

6 )aikyi, (C3.

7 )cycloalkyl or (C 1 .6)alkyl-(C 3 7 )cycloaikyl, aryl, Het, (Cl-6alkyl)aryl or (Cl-6aikyi)Het, said alkyl, cycloalkyl, aryl, Het, (C 1 .ralkyl)aryl or (C 1 6 aikyi)Het being optionally substituted with R1 50 j) COOR 12 8 wherein R 128 is (C 1 6 )alkyi substituted with R' 5 0 (C3- 7 )cycloalkyl, or(Cl-6)aky-(C3 q)cycloaikyi, aryl, Net, (C 14 6aikyi)aryi or (C 16 aikyl)Het, said 7 )cycloalkyl, or(C 16 )aky-(C 37 )cycoak-yI, aryl, Net, (C 1 .6aikyI)aqy and (Cl.

26 6 alkyl)Het being optionally substituted with R' 50 C) k) CONR 1 29R 1 3 wherein R 12 and R 13' are independently H, (C 1 6 )alkyl, A-~ 7 )cycloalkyl, (Cj.6)alkyl.(C, 7 )cycloakyI, aryl, Het, (C 16 alkyl)ary or (Ci.

6 alkyl)Het, or both R 12 and R 13' are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyi, alkyl-cycloalkyl, aryl, Het, (Cl-6alkyl)aryl,

(C

16 alkyl)Het and heterocycle being optionally substituted with R 15 0 1) aryl, Het, (Cl.

6 alkyl)aryl or (Cl.

6 alkyl)Het, all of which being optionally substituted with R 1 50 1 to 3 substituents selected from: halogen, N0 2 cyano, azido or 1 to 3 substituents selected from: a) (Cl-6) alkyl or haloalkyl, (C 3 7 )cycloalkyl, C 37 spirocycloalkyl optionally containing 1 or 2 heteroatom, (C2-)alkenyl, PC 16 alkyl-(C 37 )cycloalkyl, all of which optionally substituted with R 160 b) OR' 04 wherein R 1 04 is H, (Cl.

6 alkyl), (C 3 7 )cycloalky, or (Cl- 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl-6alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C 1 -6alkyl)aryl or (C 1 6 alkyl)Het being optionally substituted with R160 d) SOAH SO 2

N(R'

08 2 or S0 2 N(R1 0 8 )C(O)R1 0 8 wherein each R' 0 11 is independently H, (Cl.

6 )alkyl, (C 37 )cycloalkyl or (C 1 6 )alkyl-(Csq7)cycloalkyl, aryl, Het, (C 1 .6alkyl)aryl or (Cl.ralkyi)Het or both R 108 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Clr 6 alkyl)aryl or (Cl.

6 alkyl)Het or heterocycle being optionally substituted with 25R16 e) NR 111

R

112 whierein R 11 is H, (C 16 )alkyl, (C 37 )cycloalkyl or (Cl 1 6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 1 .6alkyl)aryl or (Cl.

6 alkyl)Het, and R' 12 Is H, (Cl.

6 )alkyl,

(C

3 7 )cycloalkyl or (Cl.

6 )alkyl-(C3aq)cyCloalkyl, aqy, Het, (Cl-6alwy)aryl, (Ci.

6 alkyl)Het COOR"- or SO 2

R"

6 wherein RilE is (C 1 .6)alkyl, (C3.

7 )cycloalkyl, or (C,-6)alkyl-(C 3 -7)CYCloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (Cl-ralkyl)Het, or both R11 and dR2ar covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (Cl.

6 alkyI)Het, or heterocycle being optionally substituted with Rl' 0 27

C.)

00 (Cl 16 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl-6alkyl)aryI or (Cl.

6 alkyl)Het, said (Ci.

6 )alkyl, (C3- 7 )cycloalkyl, (C 16 )aIkyl-(C3.

7 )cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (Cl.

6 al)Het being optionally substituted with R 160 g) NR" 3 00ONR' 19 R 2 wherein R"1 8

R'

19 and R' 2 is each H, (Cl.

6 )alkyl, (P3.

7 )CYCloalkyl, (CI-6)alkyl-(Cs. 7 )cycloalkyl, aryl, Het, (C.

6 alkyl)aryl or (C 1 6 alkyl)Het, or R 119 and R 1 20 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (Cl.

6 )alkyl-(C 3 7 )CYCloalkyl, aryl, Net, (Cl- 6 alkyl)aryl or (Cl-6alkyI)Het or heterocycle being optionally substituted with h) NR 1 2'COCOR'22 wherein R'1 2 1 is H, (Cl.

6 )alkyl optionally substituted with or R 122 is OR 1 23 or N(R 1 24 2 wherein R 1 23 and each R 12 4 is independently H, (Cl.

6 alkyl), (C 3 7 )cycloalkyl, or (Cl.

6 )alkyl-(C 3 -7)cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (C 1 6 alkyl)Het, or R 1 24 is OH or O(C 16 alkyl) or both R 12 4 are covalendly bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (Cl-6alkyl)ary or (Cl.6alkyl)Het and heterocycle being optionally substituted with R'1 60 J) tetrazole, C00R 12 1 wherein R' is H, (C 16 )alkyl, (C 37 )cycloalkyl, or(0 1 8 )alkyl-(C 3 7 cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (Cl.

6 alkyl)Het, said (Cj 6 )alkyl, (0 3 -7)cycloalkyl, or(C 16 r)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl and (C 1 6 alkyl)Het being optionally substituted with R 1 60 and k) C0NR 129 R 130 wherein R 1 29 and R 1 30 are independently H, (C 1 6 )alkyl, (03.

7 )cycloalkyl, (Cl- 6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (Cl- 6 alkyl)aryl or (C 1 6 alkyl)Het, or both R 129 and R 1 30 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (Cl 14 alkyI)aryl, (Cl.

6 alkyl)Het and heterocycle being optionally substituted with 13 1 wherein R 1 60 is defined as 1 or 2 substituents selected from: tetrazole, halogen, CN, C 1 -6alkyl, haloalkyl, C00R 6 S0 3

SO

2 O13 1 6 1 N(R 1 62 2 S0 2 N(R' 62 2 or CON(R 162 2 wherein R 1 6 1 and each R 1 62 is independently H, (C 16 )alkyl, (C 3 7 )cycloalkyl or (Cl.

6 )alkyl-(C 3 7 )cycloalkyl; or both R 1 6 2 are covalently bonded together and to the 28 O nitrogen to which they are attached to form a 5, 6 or 7-membered o satu rated heterocycle; 00 or Z is N(R a)R wherein R6' is H or (C 16 alkyl); and

R

6 is (C 16 )alkyl optionally substituted with: 1 to 4 substituents selected from: OPO 3 H, NO 2 cyano, azido, C(=NH)NH 2 C(=NH)NH(Cl.6)alkYI or C(=NH)NHCO(Cl. 6 )alkyl; or -1 to 4 substltuents selected from: i a) (C1.6) alkyl substituted with R 15*a, haloalkyl, (C 3 7 )cycloalkyl, C 3 7 spirocycloalkyl optionally containing 1 or 2 heteroatom, (0 26 )alkenyl, (02.

s)alkynyl, (01.6) alkyl(C3.

7 )cycloalkyl, all of which optionally substituted with

R

1 5 0 wherein R' 5 0a Is the same as R 1 50 but is not halogen, OR' O, 0 0 0 fl1b N(R 5 0b 2 wherein R1 5 0b is H or C 16 alkyl;.

b) OR' 04 wherein R' 04 IS (Cl-6alkyl) substituted with R 1 5 0

(C

3 7 )cycloalkyl, or' (Cl-6)alkYl-(C3.7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl.

6 alkyl)Het, said cycloalkyl, aryl, Het, (0 16 alkyl)aryl or (C 1 6 alkyl)Het being optionally substituted with R' 50 c) OCOR' 0 5 wherein R 105 is (C 1 .6)alkyl, (C 37 )cycloalkyl, (C 1 6 )alkYl-(C 3 7 )cycloalkyl, Het, (C 16 alkyl)aryl or (C 1 6 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (C 1 -6alkyl)Het being optionally substituted with R 15 0 d) SO 3

HSOANR

1 0") 2 or SO 2 N(ROB)C(O)ROB wherein each R' 0 8 is.

Independently H, (Cl-6)alkyl, (0 37 )cycloalkyl or (Cl.

6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C0 16 alkyl)aryl or (CI-6alkyl)Het or both R 108 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Cl- 6 alkyil)aryl or (C0 1 6 alkyl)Het or heterocycle being optionally substituted with e) IN 1 11 11 2 wherein R 1 1 is (C.

6 )alkyl substituted with R 1 50 (0 37 )cycloalkyl or (Cl.

6 )alkyl-(C 37 )cycloalk~l, aryl, Het, (C 1 6 alkyl)aryl or (0 1 6 alkyl)Het, and

R'

12 is H, ON, (Cl- 6 )alkyl, (C 37 )cycloalkyl or (CI..e)alkyl-(C3. 7 )cycloalkyl, aryl, Het, (C 14 6alkyl)aryl, (C 1 -6alkyl)Het or is H and R' 12 is S0 2

R

115 whereIn R 11 is (Cl.

6 )alkyl, (C 3 7 )cycloalkyl, or (Cl.

6 )alkyl-(C 3 q7)cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (Cj.

6 alkyl)Het, or both 29 and R' 12 are covalently bonded together and to the nitrogen to which 0 cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl.

6 alkyl)Het, or heterocycle being optionally substituted with R' 50 f) NIR 1 6 00R 1 17 wherein R' 16 and R' 1 7 is each H, (Cl 16 )aikyl, (C 37 )CYCloalkyl, (CI-6)alkYi-(C 3 7 )cycloalkyl, aryl, Het, (Cl.

6 alkyi)aryl or (Cl-6alkyl)Het, said (Ci.

6)alkyl, (C 37 )cycloalkyl, (Cl.

6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (C 14 6alkyl)aryi or

(C

16 alkyl)Het being optionally substituted with R' 50 g) NR' 18 C0NR 19 R 2 wherein R11 8

R

1 19 and R 12 is each H, (Cl.

6 alkyl, (C-1 C~110 7 )CYCloalkyl, (Cl.

6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Ci.

6 alkyl) Net, or R1 18 is covalently bonded to R' 1 9 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R 119 and R'1 20 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (C 16 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl.

6 alkyl)Het or heterocycle being optionally substituted with R 150 h) NR 1 21 COCOR'22 wherein R 1 2 and R'22 Is each is H, (C 1 6 )alkyl, (03.

7 )cycloalkyl, (Cl.

6 )alkyl-(C 3 7 )cycloalkyl, a 6- or lO-membered aryl, Het, (C 1 6 alkyi)aryl or (C 1 -6alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalky, aryl, Het,

(C

1 5 alkyI)aryl or (C 1 6 alkyl)Het being optionally substituted with R1 50 or R'22 is OR 123 or N(R 1 24 2 wherein R 1 23 and each R'1 24 is independently H,

(C

16 alkyI), (C 3 7 )cycloalkyl, or (C 1 6 )alkyl-(C 37 )CYCloaikyl, aryl, Het, (C 1 c 6 alkyl)aryl or (Cl-6alkyl)Het, or R'1 24 is OH or O(C 1 -6alkyl) or both R 1 24 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C 16 alkyl)aryl or

(C

1 -6alkyl)Het and heterocycle being optionally substituted with R1 50 0) COR'27 wherein R 127 is H, (C 1 6 )alkyl, (C 3 7 )cycloalkyl or (Cl.

6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl-6alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (Cl.

6 alkyl)Het being optionally substituted with R 150 j) COOR 1 28 wherein R 12 8 is (C 1 .6)alkyl substituted with R 15 0

(C

3 7 )cycloalkyi, or(Cl-6)alkyl-(C3.7)cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl.

6 alkyl)Net, said (C3.

7 )CYCloalkyl, or(Cl.

6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl and (Cl.

6 alkyl)Het being optionally substituted with R'1 50 k) C0NR 9 R 130 wherein R' 2 2 9 and R 1 30 are independently H, (Cl-6)alkyl,

C.)

00 6 alkyl)Het, or both R 129 and R' are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (Cl-6alkyl)aryl,

(C

16 alkyl)Het and heterocycle being optionally substituted with R 1 50 M 1I) aryl, Het, (Cl-6alkyI)aryl or (0 1 6 alkyl)Het, all of which being optionally rlsubstituted with R'1 5 1 and wherein R 150 is selected from: 1 to 3 substituents selected from: halogen, NO 2 cyano, azido or 1 to 3 substituents selected from: a) (0146) alkyl or haloalkyl, (0 37 )cycloalkyl, 03.7 spirocycloalkyl optionally containing 1 or 2 heteroatorn,

(C

26 )alkenyl, (01I) alkyl-(C 3 7 )cycloalkyl, all of which optionally substituted with R 160 b) OR 1 04 wherein R 104 is H, (C 1 6 alkyl), (C 37 )cycloalkyl, or (C 1 6 )alkyl-

(C

37 )cycloalkyl, aryl, Het, (Cl- 6 alkyl)aryl or (C 16 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (C 16 alkyl)Het being optionally substituted with R 1 60 d) SO 3 H, SO 2

N(R'

08 2 or S0 2 N(R08)C(O)ROB wherein each R 10 is independently H, (C 1 .6)alkyl, (C 37 )cycloalkyl or (Cl.

6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl.

6 alkyl)Het or both WOO 8 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (Ci-ralkyl)Het or heterocycle being optionally substituted with R 160 e) NR'Rl 12 wherein RA' 1 is H, (Cl.

6 alkyl, (C 3 7 )cycloalkyl or (Ci.

6 )alkyl-(0 3 7 )cycloalkql, aryl, Het, (Cl.

6 alkyl)aryl or (C 16 alky)Het, and R1 2 is H, (Cl-6)alkyl,

(C

37 )cycloalkyl or (0 16 )alkyl.(C3aq)cycloalkyl, aryl, Het, (Cl- 6 alkyl)aryl, (Cl 16 alkyl)Het, COOR"s or S0 2 111" 5 wherein R' is (0 14 6)alkyl, (0 37 )cycloalkyl, or (0 14 6)alkyl.(0 3 .v)cycloalkyl, aryl, Het, (0 1 6 alkyI)aryl or (Cl.

6 alkyl)Het, or both and R' 12 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Ci.

6 alkyl)aryl or (C 14 6alkyl)Het, or heterocycle being optionally substituted with Rlr 0 31 0f) N R" 6 C0OR' 7 wherein R'1 6 and R'1 7 is each H, (Cl.

6 )aikyl, (C 3 00 7 )cycloalkyl, (C 1 6 )alkyl-(C 3 7 )cycioalkyl, aryl, Het, (C 16 alkyI)aryl or (Cl.

6 alkyl)Het, said (C1.6)alkyl, (C 37 )cycloal kyl, (C 16 )alkyl-(C 37 )CYCIoalkyl, aryl, Het, (Cl, 6 alkyI)aryl or (C 16 alkyl)Het being optionally substituted with R1 6 1; g) NR" 6 8CONR"1 9

R

2 wherein R" 8 R11 9 and R12D is each H, (Cl.

c~K1 6 )alkyl, (C 37 )cycloalkyl, (Cl.

6 )alkyl-(C 37 )cycIoalkyI, aryl, Het, (C 1 6 alkyl)aryl or (C.

6 a1ky)Het, or R'" 9 and RA'2' are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (CJ.

37 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or 6 alkyl)Het or heterocycle being optionally substituted with A'16 0 h) NR 1 21 COCOR'22 wherein A'1 2 is H, (C,,)akyi optionally substituted with A'1 60 or R122 is OR' 23 or N(R'1 24 2 wherein RA' 23 and each RA 1 24 is independently H, (Cl.

6 alkyl), (C 3 7 )cycloalkyl, or (Cl-6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C1.

6 alkyl)aryl or (Cl-6alkyl)Het, or R 1 24 is OH or

O(C

16 alkyl) or both R124 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkylcycloalkyl, aryl, Het, (C1.

6 alkyl)aryi or (Cl.

6 alkyl)Het and heterocycle being optionally substituted with RA' 60 j) tetrazole, COOR 1 28 wherein R 12 1 is H, (CI-6)alkyi, (C3.

7 )cycloalkyl, or(C 6 )alkyl-(C 3 7 )CYCloalkyl, aryl, Het, 6 alkyI)aryl or (Cl.

6 alkyl)Het, said (Cl- 6 )alkyl, (C 37 )CYcloalkyl, or(C,.

6 )alWy-(C 37 )cycloalkyl, aryl, Het, 6 aikyl)aryl and (C.

6 alkyl)Het being optionally substituted with A' 60 and k) CONR 1 2 9 R 1 30 wherein RA 12 9 and R 1 30 are independently H, (C 1 6 )alkyl, (C 3 7 )CYCloalkyi, (Cl-6)alkyl-(C 37 )cycloalkyl, aryl, Het, 6 alkyl)aryi or (Cl.ralkyl)Het, or both R 12 9 and R 1 30 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl, (Cl, 4 alkyl)Het and heterocycle being optionally substituted with RA' 60 wherein R' 60 Is defined as 1 or 2 substltuents selected from: 32 tetrazole, halogen, ON,C 16 alkyl, haloalkyl, C00R 6

SO

3

H,

0 ~S0 2 R 6 OR111 1 N(R 6 2 SOANR 6 2 or CON(R 6 2 wherein and each R' 2 i independently H, (C 1 6 )alkyl, (C3.

7 )CYCloalkyl or (Cj.

6 )alkyl-(C 37 )CYCloalkyl; or both R 1 62 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle.

or R 6 is R RA I whripreferably, R 7ad eare each independently H, (0 14 6)alkyl, haloalkyl, (C3.

7 )cycloalkyl, 6- or 1lO-membered aryl, Het, (Cl.

6 )alkyl-aryl, (C 14 6)alkyl-Het, wherein said alkyl, cycloalkyl, aryl, Het, (Cl.6)alkyl-aryl, (Cl-6)alkyl-Het are optionally substituted with R 70 or

RA

7 and Re are covalently bonded together to form second (C 3 7 )cycloalkyl or a 4, 5- or 6-membered heterocycle having from 1 to 3 heteroatom selected from 0, N, and S; or when Z is N(WR 6)R, either of R 7 or Re is covalently bonded to R 6 to form a nitrogen-containing 5-or 6-membered heterocycle; wherein, preferably, R 70 is selected from: -1 to 4 substituents selected from: halogen, N0 2 cyano, azido; or -1 to 4 substituents selected from: a) (01.6) alkyl or haloalkyl, (C 37 )cycloalkyl, 03.7 spirocycloalkyl optionally containing 1 or 2 heteroatom, (0 2 6 )alkenyl, (0 2 -B)alkynyl, (C1.6) alkyl-(0 3 7 )cycloalkyl, all of which optionally substituted with b) OR 1 04wherein RA 104 is H, (Cl.

6 alkyl), (C3.

7 )cycloalkyl, or (Cj.

6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (0 1 6 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (Cl.

6 alkyl)Het being optionally substituted with d) SO 2

N(R

108 2 or SO 2

N(R'

08 )C(O)Rl' 0 wherein each R' 0 8 is independently H, (Ci.

6 )alkyl, (C3.

7 )CYCloalkyl or (Cj.

6 )alkyl-(C3.

7 )cycloalkyl, aryl, Het, Galkyl)aryl or (Cl-oalkyl)Het or both R 108 are covalently bonded together and to 33 o the nitrogen to which they are attached to form a 5, 6 or 7-membered C0 saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C 1 6 aikyl)aryl or (C.

6 alkyl)Het or heterocycle being optionally substituted with R' 50 e) NR R' 12 wherein is H, (C 1 6 alkyi, (C 3 7 )cycloalkyl or (Cl 14 )alkyi-(C 3 7 )cycloalkyl, aryl, Het, (C 1 .alkyI)aryl or (Cl.

6 aikyI)Het, and 11 1 12 is H, ON, (Cl.

6 )alkyl, (C 37 )cycloalkyl or (C 16 )alkyl-(0 37 )cycioalkyi, aryl, Het, (C 16 alkyI)aryl, (Cl.

6 alkyl)Het, COOR"1 5 or S0 2 R 115 wherein R 1 1 5 is (Cl.

6 )alky, (C3.

7 )cycloalky, or (Cj.

6 )aIkyi-(0 3 7 )CYCloaIkyI, aryl, Het, (C 16 alkyI)aryl or (Ci.

ralky)Het, or both R" 1 1 and R' 1 2 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Net, (C 16 aikyl)aryl or (C 1 6 aikyl)Het, or heterocycle being optionally substituted with R 1 50 f) NR'CO0R 17 wherein R'1 adR 7is eahH, (C 1 6 )alkyl, (C3.

7 )cycloalkyi,

(C

1 6 )alkYl-(C 3 7 )cycloalkyi, aryl, Het, (0 16 alkyl)aryl or (Cl.

6 alkyl)Het, said (Cl.

6 )alkyl, (C 3 7 )cycloalkyl, (C 1 6 )alkyl-(C 3 -7)cycioalkyl, aryl, Het, (Cl 16 alkyl)aryl or (Cl.

6 alkyl)Het being optionally substituted with R' 50 g)NR'11CONRll 9 R 2 wherein R' 15

R

11 9 and R 12 Is each H, (Cl.

6 )alkyl, (03.

7 )cycloalkyl, (Cl.

6 )alkyl-(C 3 -7)cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Ci.

6 alkyl)Het, or R' 18 is coval'ently bonded to R'" 9 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R 119 and R 120 are covalently- bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (0 1 6 )alkyl-(C 3 .7)cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (Cl.

6 alkyl)Het or heterocycle being optionally substituted with R 15 0 h) NR 121 COCOR'22wherein R'1 21 is H, (C 16 )alkyl, (C 3 7 )cycloalkyl, (Cl.

6 )alkyl-

(C

37 )cycloalkyl, a 6- or lO-membered aryl, Het, (C 16 alkyl)aryl or (Ci.

6 alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Net, (C 1 .6alkyl)aryI or

(C

1 6 alkyl)Het being optionally substituted with R' 60 and R 122 iS OR 1 23or N(R 12 4 2 wherein R'23and each R 124 is independently H,

(C

14 6alkyl), (C 3 7 )cycioalkyl, or (Cj.

6 )alkyl-(C 3 7 )cycloalkyI, aryl, Net, (Ci.

6 alkyl)aryl or (0 1 6 alkyi)Het, or R'1 24 is OH or O(C 1 6 alkyl) or both R 1 24 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alky, cycloalkyl, alkyl-cycloalkyl, aryl, Net, (C 1 .6alkyI)aryl or

(C

1 -6alkyl)Het and heterocycle being optionally substituted with Rl0; 34 0i) C0R 127 wherein R 1 is H, (0 16 )alkyl, (0 37 )cycioalkyl or (0 1 6 )alkyl-(C3.

00 7 )CYCloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (Cl-ralkyl)Het, said alkyl, cycloalkyl, aryl, Het, (Cl-6alkyl)aryl or (Cl.

6 alkyl)Het being optionally substituted with R 150 j) C00R 1 2 11 wherein R 1 28 is H, (Ci.6)alkyl, (C 3 7 )cycloalkyl, or(C 1 6 )alkyl-(C3.

IND 5 7 )CYCloalkyl, aryl, Het, (C 1 -6alkyl)aryl or (C 1 -6alkyl)Het, said (0 1 6 )alkyl, (03.

7 )CYCloalkyl, or(Cl- 6 )alkyl-(C 3 -7)cycloalkyl, aryl, Het, (0 1 6 alkyl)aryl and (Cl.

ralkyl)Het being optionally substituted with R 150 k) CONR 1 29 R'30 wherein R 129 and R 130 are independently H, (C 1 6 )alkyl, (03.

7)CYCloalkyl,

(C

14 6)aky-(C 3 .7)CyCtoalkyI, aryl, Het, (0 1 6 alkyl)ary or (Cl.

6 alkyl)Het, or both R 1 29 and R'1 30 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alky-cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl, (0 1 6 alkyl)Het and heterocycle being optionally substituted with R' 50 1) aryl, Het, (0 1 6 alkyl)aryl or (C1-6alkyl)Het, all of which being optionally substituted with R 150 wherein, preferably,

R

150 Is selected from: 1 to 3 substituents selected from: halogen, N0 2 cyano, azido; or 1 to 3 substituents selected from: a) (Ci-6) alkyl or haloalkyl, (C 37 )cycloalkyl, 03.7 spirocycloalkyl optionally containing 1 or 2 heteroatom,

(C

2 -6)alkenyl, (0 2 8 )alkynyl, all of which optionally substituted with R 1 60 b) OR 1 4 wherein R 1 04 is H, (Cl.

6 alkyl) or (0 37 )cycloalkyl, said alkyl and cycloalkyl being optionally substituted with R 1 60 ;1 d) SO 2

N(R'

08 2 wherein Rl 08 is H, (0 1 6 )alkyl or (0 37 )CYCloalkyl, said alkyl or cycloalkyl being optionally substituted with e) N R 111 R' 1 2 wherein R 11 is H, (0 1 6 )alkyl or (C3.

7 )CYCloalkyl, and R' is H, (Ci..e)alkyl, (C 3 7 )cycloalkyl or (C 1 6 )alkyl-(C 37 )cycloalkyl, aryl, Het,

(C

16 alkyl)aryl, (0 1 6 alkyl)Het, COOR 1 5 or SO 2 Rl' wherein R' 15 is (Cl- 6 )alkyl, (0 37 )cycloalkyl, or (0 1 6 )alkyl.(C 3 7 )cyCloalkyl, aryl, Het, (Cl- 6 alkyl)aryl or (Cl.

6 alkyl)Het, or both R 111 and R 1 1 2 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Cl.6alkyl)aryl or (C 1 6 alkyi)Het, or heterocycle being optionally substituted with R' 60 o;

C.)

00 7 )cycloalkyl, said (C 16 )alkyl or (C 3 7 )cycloalkyl being optionally substituted with Rle 60 g) NR118CONR" 9 R 2 wherein R 118

R

1 1 and R 12 is each H, (C 1 IND 5 6 )aikyl or (C 3 7 )CYCioalkyi; or 111 1 9 and R 12 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 (Ni or 7-membered saturated heterocycle; said alkyl, cycloalkyl or heterocycle being optionally substituted with R' 80 h) NR 1 21 COC0R 12 2 wherein R 12 1 is H, (C 16 )alkyl or (C 37 )cycloalkyl, said alkyl or cycloalkyl being optionally substituted with R 1 or R'22 is OR 123 or N(R 124 2 wherein R 1 23 and each R'1 24 is independently H, (CI-6alkyl) or (C 3 7 )cycloalkyl, or R 124 is OH or O(Cl.

6 alkyl) or both R 12 4 are covalently bonded together to form a 5, 6 or 7membered saturated heterocycle, said alkyl, cycloalkyl and heterocycie being optionally substituted with R 1 60 J) tetrazole, COOR 1 2 8 wherein R 1 28 is H, (CI.

6 )alkyl or (C 3 7 )cycloalkyl, said (0 1 6 )alkyl and (C 3 7 )cycloalkyl being optionally substituted with R'1 60 and k) CONR'29R 30 wherein R 129 and R 1 30 are independently H, (C 1 6 )alkyl or (C 37 )cycloalkyl, or both R 1 2 1 and R 1 30 are covalendly bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalky and heterocycle being optionally substituted with R1 60 wherein R 1 60 is defined as 1 or 2 substituents selected from: tetrazole, halogen, CN, C 1 .ealkyl, haloalkyl, COOR 6

OR',

N(R'1 62 2 or CON(R 162 2 wherein R161 and each R 1 6 2 is independently H or (C 1 6 )alkyl; Re is H; or Re is covalently bonded to either of R 7 or Re to form a 5- or 6-membered heterocycle; and 0 is a 6- or 1 0-membered aryl, Het, all of which being optionally substituted with: 36 00 H 0 r

COCH

0 ~COOH orR'0 wherein R 100 is: IND 1 to 4 substituents selected from: halogen, N0 2 cyano or azido; or 1 to 4 substituents selected from: a) (C 1 6 alkyl or haloalkyl, (C3- 7 )cycloalkyl, (C 24 6)alkenyl, (C 2 8 )alkynYl, (01.6) C)alkyl-(C3. 7 )cycloalkyl, all of which optionally substituted with R 1 (Nib) OR' wherein R'04 is H, (Cl.

6 alkyl), (C 37 )cycloalkyl, or (Cl.6)alkyl-(C3.

7 )cycloalkyl, aryl, Het, (C 16 aiky)aryl or (C 1 6 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C1.6aikyl)aryi or (Cl.

6 alkyl)Het being optionally substituted with 10R15 e) NR 111 R' 12 wherein R 1 11 is H, (Ci.

6 )alkyi, (C 3 7 )cycloalkyi or (C 1 6 )aikyi-(C 3 7 )cycloalkyl, aryl, Het, (C 16 aikyi)aryl or (C 1 6 alkyi)Het, and R' 12 is H, CN, (Cl.

6 )aikyl, (C 3 7 )cycioalkyi or (C 16 )alkyi-(C 3 7)cycioalkyi, aryl, Het, (C 16 alkyi)aryl, (Cl.

6 aikyl)Het COOR 15 or SO 2 R' wherein R"1 5 Is (Cl.

6 aikyl, (C3.

7 )cycioalkyl, or (Cl.

6 )aikyl-(C 37 )cycioalkYi, aryl, Het, (C 1 6 alkyi)aryi or (Cl.

6 aikyl)Het, or both and R 112 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycie, said aikyl, cycloalkyl, aryl, Het, (C 1 6 aikyi)aryi or (Cl.

6 aikyl)Het, or heterocycie being optionally substituted with R1 50 f) NR 11 r3C0R 117 wherein R" 5 6 and R 1 17 is each H, (C 16 )aikyl, (C 37 )cycloaikyi,

(C

16 )aikyl.(C 3 7 )cycioalkyi, aryl, Het, (C 1 .6aikyi)aryl or (Cl.

6 alkyi)Het, said (Cl.

6 )aikyi, (C 3 7 )cycioalkyl, (Ci 6 )aikyi- (C 37 )cycloalkyi, aryl, Het, (C 1 6 alkyi)aryi or

(C

1 .6aikyl)Het being optionally substituted with R'w; g) NR11SCONRllBR 2 wherein R 118

R'

19 and R' 2 is each H, (Ci.

6 )aikyl, (03.

7 )cycloalkyl,

(C

16 )aIkyI-(C 3 7 )cycioalkyl, aryl, Het, (C 16 aikyl)aryl or (Ci.

6 aikyi)Het, or 13 11 8 is covalently bonded to R' 19 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R 119 and R 1 20 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl,

(CI-

6 )alkYl-(C 37 )cycioaikyi, aryl, Het, (C 16 aikyi)aryl or (Ci.

6 alkyi)Het or heterocycie being optionally substituted with R1 50 h) NR 121 00OCOR 1 22 wherein R'1 2 1 and R 1 2' is each is H, (C 16 )aikyl, (03.

37

C.)

00 6 alkyI)aryl or (C 16 calkyl)Net, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (Cl-6alkyl)aryi or (C 16 alkyI)Het being optionally substituted with R 150 or R 1 22 is OR'1' or N(R 1 24 2 wherein R 1 23 and each R 1 2 4 is independlently H,

(C

1 -6alkyl), (C 3 7 )cycloalkyl, or (Cl.

6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (C 16 alkyl)Het, or R'1 24 is OH or O(C 1 .6alkyI) or both R 1 24 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (Cl.

6 alkyI)aryl or (C 16 alkyI)Het and heterocycle being optionally substituted with R' 5 j) COOR 12 8 wherein R 1 28 is H, (Cl.

6 )alkyl, (C 3 7 )cycioalkyl, or(C 1 6 )alkyl-(C3.

7 )cycloalkyl, aryl, Het, (Cl.6aikyl)aryl or (C 16 alkyl)Het, said (C 16 )alkyl, (C 3 7 )CYCloaikyl, or(Ci.

6 )alkyl-(C 3 7 )CYCioaikyl, aryl, Net, (C 16 alkyl)aryl and (Cl- 6 alkyl)Het being optionally substituted with R' 50 k) CONR 129 R 13 1 wherein R' 29 and R 1 30 are independently H, (Cl-6)alkyl, (C 3 7 )cycloalkyl, (C 16 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 1 .6alkyl)aryi or (Cl.

6 alkyl)Het, or both R 1 29 and R 1 30 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (Cl.

6 alkyI)aryl,

(C

1 6 alkyl)Het and heterocycle being optionally substituted with R'1 50 1) aryl, Het, (C 16 alkyl)aryl or (Cl-6alkyl)Het, all of which being optionaldly substituted with R1 50 wherein R 15 is selected from: -1 to 3 substituents selected from: halogen, NO 2 cyano or azido; or -1 to 3 substituents selected from: a) (C 16 alkyl or haloalkyl, (C 37 )cycloalkyl, C 37 spirocycloalkyl optionally containing 1 or 2 heteroatom, (C 2 .6)alkenyl, (C 28 )alkynyl,

(C

1 6 alkyl-(C 37 )cycloalkyl, all of which optionally substituted with R 1 6 0 b) OR' 04 wherein R 1 04 is H, (Cl.

6 alkyI), (C 37 )cycloalkyl, or (Cl.

6 )alkyl- (C3.

7 )cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (CI-6alkyl)Het, said alkyl, cycloalky, aryl, Het, (Cl.

6 alkyl)aryl or (C 1 6 alkyl)Het being optionally substituted with R1 60 d) S0 3 H, S0 2 N(R1 0 8) 2 or SO 2 N(R1' 0

)C(O)R

108 wherein each R 1 08 is independently H, (Cl.

6 )alkyl, (C 3 7 )cycloalkyl or (C 16 )alkyl-(C 3 7 )CYCloalkyl, aryl, Het, (C 16 alkyI)aryl or (C 1 6 alkyl)Het or both R10 are 38

C.)

00 attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (C 16 alkyl)Het or heterocycle being optionally substituted with R 160 e) NR 111

R'

1 wherein R 111 is H, (C 16 )alkyl, (C 3 7 )cycloalkyl or (C 1 6 )alkyl-(C 37 )CYCloalkyl, aryl, Het, (Cl.ralkyl)aryl or (Cl-6alkyl)Het, and

R

1 12 is H, ON, (C 1 6 alkyl, (C 37 )cycloalkyl or (Cl.

6 )alkyl-(C 3 .7)cycloalkyl, aryl, Het, (C 16 alkyl)aryl, (C 1 6 alkyI)Het or S0 2

R

15 wherein R' 1 5 is (C 1 6 )allyl, (C 37 )cycloalkyl, or (Cl.

6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (Cl- 6 alkyl)aryl or (Cl.6alkyl)Het, or both R' 11 and R' are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Cl- 6 alkyl)aryl or (C 15 6alkyl)Het, or heterocycle being optionally substituted with R1 60 f) NR' 1 6 00R 17 wherein R" 6 and R'1 7 is each H, (0 1 6 )alkyl, (03.

7 )cycloalkyl, (Cl.

6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (Ci.

.,alkyi)Het, said 6 ,)alkyl, (C 37 )cycloalkyl, (C 1 6 )alky-(C 37 )cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (Ci.

6 alkyl)Het being optionally substituted with R 1 60 g) NRI1SCONR'l 9

R

2 wherein Rl" 3 R"1 9 and R 12 is each H, (Ci.

6 )alkyl, (C 3 7 )cycloalkyl, (Cl.

6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl.

6 alkyI)aryl or (Cl.

6 alkyl)Het, or Rl"' is cova lently bonded to R'1 9 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R11 9 and R 1 20 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (Cl.

6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl.

6 alkyI)aryl or (C1.

6 alkyl)Het or heterocycle being optionally substituted with R' 60 h) NR 1 21 00C0R 1 22 wherein R 1 2 1 is H, (Cl.

6 )alkyl optionally substituted with R1 60 or R'22is OR 1 23 or N(R'1 24 2 wherein R 1 23 and each R 1 2 1 is independently H, (C 1 .6alkyl), (C 37 )cycloalkyl, or (Ci 6 )alkyl-(C 3 39 o 7 )cycloalkyl, aryl, Het, (Cl.ralkyl)aryl or (C 16 alkyl)Het, or R'1 4 is OH or 0 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkylcycloalkyl, aryl, Het, (Cl.ralkyl)aryl or (C 16 alkyI)Het and heterocycle being optionally substituted with R 160 jtetrazole, 0CR1 28 wherein R128 is H, (C,)alkyl, (C 37 )cycloalkyl, CK1or(C 1 6 )alkyl-(C.

7 )cycloalkYl, aryl, Het, (C1.

6 alkyl)aryl or (Cl.

6 alkyl)Het, said (Cl.

6 )alkyl, (C 37 )cycloalkyl, or(C 1 6 )alkyl-(C 3 7 )CYCloalkyl, aryl, Het,

(C

16 alkyl)ary and (Cl.6alkyl)Het being optionally substituted with R' 60 and k) CONR 129 R 1 30 wherein R' 2 2 9 and R 1 30 are independently H, (Ci.

)alkyl, (C 3 .4)cycloalkyl,

(C

1 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (C 1 6 alkyl)Het, or both R 12' and R 1 3 1 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (Cl.6alkyl)aryl, (Cl.

6 alkyl)Het and heterocycle being optionally substituted with R1 60 wherein R1 60 is defined as 1 or 2 substituents selected from: tetrazole, halogen, ON, C 1 -6alkyl, haloalkyl, CR 6

SO

3

H,

SR 6 S0 2

R'

6

OR'

6 N(R 6 2 SOANR 6 2 or CON(R 1 62 2 wherein R'1 6 1 and each R 1 62 is independently H, (C1.

6 )alkyl, (C 3 7 )cycloalkyl or (Cl.

6 )alkyl-(C 3 7 )cycloalkyl; or both R 1 62 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or a salt thereof; wherein said probe comprises a detectable label attached to any suitable position, whereby said probe binds to an HCV polymerase or an analog thereof and is capable of being displaced by an inhibitor thereof.

More preferably, the probe of the invention is a compound of formula: C.0

R

3 00 N N R HN H

NN

R H 1 (llb), R (Ild)

O

7

A

8 R' 3 8R RR(lie) orR (f) wherein R' is (C 56 )cycloalkyl; R 2 is phenyl, or Het both being optionally substituted with R R 3 R 7 Re 113 9

R

100 and R 1 50 are as defined above;

R

1 is OPO 3 H-1, NO 2 cyano, azido, C(=NH)NH 2

C(=NH)NH(C

1 .6)alkyl or C(=NH)NHCO(C,.)alkyl; or a) (C.

6 alkyl substituted with R 15 oa, haloalkyl, (C3.

7 )cycloalkyl, C 37 spirocycloalkyl optionally containing 1 or 2 heteroatom, (0 2 6 )alkenyl, (C 2 8 )alkynyl, (C1.6) alkyl-(C 3 7 )cycloalkyl, all of which optionally substituted with

R'

5 wherein R1 5 09 is the same as R 1 50 but is not halogen, OR1l0b, COOR N (R1 5b 2 wherein R 15ob is H or 0 16 alkyl; b) OR'04 wherein R' 04 is (Cl-6alkyl) substituted with R' 50

(C

3 7 )CYCloalkyl, or (C1.

6 )aky-(C 37 )cycloaky, aryl, Het, (C1.

6 alkyl)aryl or (C.

6 alkyl)Het, said cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (Cl.ralkyl)Het being optionally substituted with R 15 0 c) OCOR' 05 wherein R' 05 is (C1.

6 )alkyl, (C,3 7 )cycloalkyl, (Cj.

6 )alkyI-(C 3 7 )cycloalkyl, Het, (C 16 alkyl)aryl or (Cl.ralkyl)Het, said alkyl, cycloalky, aryl, Het, (Cl.

6 alkyl)aryl or (Cl.Balkyl)Het being optionally substituted with R' 5 0 d) S0 3 1-1, SO 2

N(R'

08 2 or SON(R' 08 )C(O)Rl 08 wherein each R 1 is independently H, (Cl.

6 )alkyl, (C 3 7 )cycloalkyl or (Cj- 6 )aky-(C 3 7 )cycloalkyI, aryl, 41

C.)

00 together and to the nitrogen to which they are attached to form a 5, 6 or 7membered saturated heterocycle, said alkyl, cycloalky, aryl, Het, (Ci.

6 alkyl)aryl or (0 1 6 alkyl)Het or heterocycle being optionally substituted with R.0 e) NR 11 wherein p111 is (C 1 6 )alkyl substituted with R 150

(C

37 )cycloalkyl or (0 1 6 )alkyl..(C 3 7 )cycloalkyl, aryl, Het, (Cl 16 alkyI)aryl or (Cl.

6 alkyl)Het, and

R'

1 2 is H, ON, (C 1 6 )alkyl, (0 3 7 )cycloalkyl or (Cl.

6 )alkyl-(0 3 7 )cycloalkyl, aryl, Het, (Cl 16 alkyl)aryl, (C 16 alkyI)Het or

R

1 11 is H and R 112 is SO 2

R

115 wherein R 115 is (C 1 6 )alkyl, (C 37 )cycloalkyl, or (CI-6)alkyl-(C 3 .7)cycloalkyl, aryl, Het, (C 1 .ralkyl)aryl or (C 1 8 alkyl)Het, or both and R 112 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said cycloalkyl, aryl, Het, (Cl.ralkyI)aryl or (Cl.

6 alkyl)Het, or heterocycle being optionally substituted with R150; f) NR' 16 0COW 17 wherein R 116 and R"' 7 is each H, (C 1 6 alkyl, (C 37 )cycloalkyl,

(CI.

6 )alkyl.(C 3 .7)cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (Cl.

6 alkyl)Het, said (Cl- 6 )alkyl, (C 3 7 )cycloalkyl, (Cl.

6 )alkyl.(C 3 7 )cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (Cl.

6 alkyl)Het being optionally substituted with g) NR11CONRllOR1 2 0, wherein R 113

R

11 and R 120 is each H, (C 1 6 ')alkyl, (03.

7 )cycloalkyl, (C,.r)alkyl-(C 37 )CYCloaikyl, aryl, Het, (C 16 ralkyl)aryl or (Ci..

6 alkyi)Het, or R11 8 is covalently bonded to R11 9 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R 119 and R 12 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocyc le; said alkyl, cycloalkyl, (Cl- 6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (C 16 alkyI)aryl or (Cj- 6 alkyl)Het orheterocycle being optionally substituted with R 150 NR 121

COCOR

1 22 wherein R 1 21 and R 1 22 is each is H, (C 16 )alkyl, (03.

7 )cycloalkyl, (Cl.

6 )alkyl.(C 37 )cycloalkyl, a 6- or 1 0-ffiembered aryl, Het, (Cl- 6 alkyl)aryl or (Cl.Balkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (CI-ralkyl)Het being optionally substituted with R 150 or R 122 is OR'23 or N(R 1 24 2 wherein R 123 and each R 124 is independently

H,

(C

1 6 alkyl), (C 3 7 )cycloalkyl, or (Cl.

6 )alkyI-(C3.7cycloalky, aryl, Het, (Cir 6 alkyl)aryl or (Cl.

6 alkyl)Het, or R 1 24 is OH or O(0 1 .ralkyl) or both R 1 24 are 42 0 covalently bonded together to form a 5, 6 or 7-membered saturated C0 00 heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (Ci.

6 alkyl)Het and heterocycle being optionally substituted with R 150 1) C0R 127 wherein R 1 27 is H, (C 1 6 alkyl, (C 3 7 )cycloalkyl or (Cj.6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl or (C 16 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (Cl-6alkyl)aryl or (C 14 6alkyl)Het being optionally substituted with R 1

O;

)C00R 128 wherein R 1 28 is (Cl-B)alkyl substituted with R1 5 0

(C

3 .7)CYCloalkyl, or(C 16 )alkyl-(C 37 )cycloalkyl. aryl, Het, (Ci 6 alkyl)aryl or (C 1 6 alkyl) Het, said

(C

3 7 )cycloalkyl, or(C 1 6 )alkyl-(C 3 7 )cycoakyl, aryl, Het, (C1.

6 alkyl) aryl and (C 1 ealkyl)Het being optionally substituted with R' 5 0 k) C0NR 12 9 R 130 wherein R 1 and R 1 are independently H, (C 1 6 )alkyl, (03.

7 )CYCloalkyl, (C 16 )alkyl-(C 37 )Cycloalkyl, aryl, H-et, (C.

6 alkyl)aryl or (Ci.

6 alkyl)Het, or both R 129 and R 130 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (Cl.

6 alkyl)aryl, (Cl.

6 alkyl)Het and heterocycle being optionally substituted with R 1 1) aryl, Het, (0 1 6 alkyl)aryl or (Cl-6alkyl)Het, all of which being optionally substituted with R' 50 wherein R 1 50 is as defined herein; or a salt thereof; wherein said compound Is either: a) marked with a radioactive isotope at any suitable position; b) linked to a detectable moiety by a suitable linker at any suitable position, except R' and R 3 or c) linked to an affinity tag at any suitable position, except R' and R.

Even more preferably, the probe of the invention is a compound of formula: R 1 50 (IlIa), 43 0 OH 0 3 0 H SR

H

R

1 R 1

II

5 S(lic).

wherein R' is (Cs.

6 )cycloalkyl;

R

2 is phenyl, or Het both being optionally substituted with R 2 0 O 5 R 3 and R 5 is are as defined above;

O

C or a salt thereof; wherein said compound is optionally: a) marked with a radioactive isotope at any suitable position; b) linked to a detectable moiety by a suitable linker at any suitable position, except R 1 and R 3 or c) linked to an affinity tag at any suitable position, except R 1 and R 3 Specifically, according to a first aspect of the invention, the probe of the present invention is selected from the group consisting of: 0 No

,CQOH

(IV);

and o

OOH

0 N NH1 OH

SO

(vi) According to an alternative aspect of this first embodiment, there Is provided a method for identifying compounds that inhibit HCV polymerase comprising the steps of: a) contacting said HCV polymerase or an analog thereof with a probe of formula I, as defined herein, so as to form a complex having said probe bound to said polymerase; b) measuring the signal from said complex to establish a base line level; c) incubating the product'of step a) with a test compound; and d) measuring the signal from said complex; and e) comparing the signal from step d) with the signal from step b); whereby a modulation in said signal is an indication that said test compound inhibits said polymerase.

Preferably, the method for identifying compounds capable of inhibiting HCV polymerase, comprises: f) repeating steps to as defined above in a high throughput screen.

Alternatively, there is provided a probe of formula I: M R 6 B. K// A is O, S, NR 3 or CR 3 B is NR 1 or CR'; with the proviso that, when A is CR 3 B is NR', and when A is O or S, B is CR'; 0 46 represents either a single or a double bond; 00

R

1 is selected from the group consisting of: (C 4 7 )cycloalkyl optionally substituted with (C1.6 alkyl); norbornane, 6- or 7-membered heterocycle having 1 to 4 heteroatoms selected from O, N, and S, all of which optionally substituted with 1 to 4 substituent selected from the group consisting of: Shalo, OH and C1-6 alkyl optionally substituted with hy'droxy;

R

2 is selected from the group consisting of: phenyl, pyridine-N-oxide, 5- or 6-membered -aromatic heterocycle having 1 to 4 heteroatoms selected from O, N, S and S, and 9- or 10-membered aromatic heterobicycle having 1 to 4 heteroatoms selected from O, N and S; said phenyl, pyridine-N-oxide, aromatic heterocycle and aromatic heterobicycle being optionally substituted with from 1 to 4 substituents selected from the group consisting of: halogen, C1-6 haloalkyl, 6 )alkyl, C.6 alkoxy, OH, amino optionally mono- or di-substituted with Ci.8 alkyl;

R

3 is selected from the group consisting of: H, (Cl.e)alkyl, (01-6 alkyl)-(C 6 1 oaryl), (C1.6 or 6-membered heterocycle having 1 to 4 heteroatoms selected from O, N, and S, and 5- or 6-membered heterocycle having 1 to 4 heteroatoms selected from O, N and S, wherein said aryl and said heterocycle are optionally substituted with from 1 to 4 substituents selected from the group consisting of: COOH, COO(C.6 alkyl), halogen, and (C1.6 alkyl); M is N, CR 4 a, or COR 4 b, wherein R 4 a is selected from the group consisting of: H, halogen, and (C0.6 alkyl); and R 4 b is selected from the group consisting of: H and (C.

6 alkyl); K and L is each independently N or CR 6 wherein R 6 is H, halo, C1.6 alkyl, OH, or C1.

6 alkoxy;

R

5 is wherein Y is O or S; and Z is NHR s a or OR 5 a; wherein: is selected from the group consisting of: H, (C1.

6 )alkyl, (C2.

6 )alkenyl,

(C

3 6 )cycloalkyl optionally substituted with C 1 6 alkyl or C 2 6 alkenyl, (C 6 .,o)aryl optionally substituted with C.s 6 alkyl or C 2 6 alkenyl, N{(C0 1 alkyl} 2 NHCOO(Ci.

6 )alkyl(Cs.o0)aryl, NHCO(C6.

1 0 )aryl, or 6-atom heterocycle, having 1 to 4 heteroatoms selected from O, N and S, and or 10-atom heterobicycle having 1 to 4 heteroatoms selected from O, N and S; 47 0 wherein said alkyl, alkenyl, cycloalkyl, aryl, heterocycle or C0 00 heterobicycle are all optionally substituted with from 1 to 4 substituents selected from: OH, COOH, (0 1 6 )alkyl, (C 2 4)alkenyl,

(C

1 .6)alkyl-hydroxy, COO(C 16 )alkyl, C3-7 cycloalkyl, benzyloxy, halogen, (C 2 .4alkenyl-(0 1 .B)alkyl-COOH, coumarln, (Cl-6)alkyl-amino,

NH(C

1 6 alkyl), C(halogen) 3 -C(O)N H(Cl-4)alkyl, and

-C(O)NH(C

6 10 )aryl, 5- or 6-membered heterocycle having 1 to 4 heteroatoms selected from 0, N and S, 9- or lO-membered heterobicycle having 1 to 4 heteroatoms selected from 0, N and S, and 6- or lO-membered aryl; wherein said alkyl, alkenyl, cycloalkyl, aqyl, heterocycle and heterobicycle are all optionally substituted with from 1 to 4 substituents selected from: halogen, OPO 3 H, sulfonamido,

SO

3 H, SO 2

CH

3

-CONH

2

-COCH

3 (Cl.

3 )alky, (02.

4 alkenyl)COOH, tetrazolyl, COOH, -CONH 2 triazolyl, OH, NO 2

NH

2 -0(01.6, alkyl)COOH, hydantoin, benzoyleneu rea,

(C

1 .4alkoxy, cyano, azido, -O-(C 16 r)alkyl COOH, -O-(Cl- 6 )alkyl COO-(Cl 1 .)alkyl, NHCO-(Cl-ralkyl), -NHCOCOOH,

-NHCOCONHOH,-NHCOCONH

2

-NHCOCONHCH

3 -NHCO(Cl 16 )alky-COOH, -NHCOCONH(Cl.

6 )alkyl-COOH,

-NHCO(C

37 )cycloalkyl-COOH, -NH CON H (C 610 )aryl-COOH,

NHCONH(C

610 )aryl-COO(Cl- 6 )alkyl, NHCONH(C 1 6 )alkyl- COOH,- NHCONH(Cl.

6 alkyl-COO(0 14 6)alkyl, NHCONH(0 1 e)al kyl-(C 2 alkenyl -COO H, NH(C 16 )alkyl-(Cr 10 )aryl-O(Cj_ 6 )alkyl COOH, NH(0 16 )alkyl-(0 6 1 o)aryl-COOH,

-NHCH

2 000H, -NHCONH 2 -NHCO(Cl.

6 )hydroxyalkyl COOH,

-OCO(C

16 )hydroxyalky COOH, (C3.

6 )cycloalW COOH, 0 OH ~jJ 0 s ,-NHCN, -NHCHO, -NHSO 2

CH

3 -NHS0 2

CF

3 and -O(Cl-6alkyl)-tetrazol; or R58ls 48 o

BR

00

N,

0 wherein RWand Reare each independently H, (01.6 alkyl), (03.7 cycloalkyl), (016 alkyl)phenyl, (01.6 alkyl)-(0 3 7 cycloalkyl), (03.7 cycloalkyl)-( C1.6alkyl), (03.7 cycloalkyl)-(0 2 4 alkenyl), (0.6 alkq)-OH, phenyl, CH- 2 blphenyl, 5- or 6-membered heterocycle having fromi to 4 heteroatoms selected from 0, N, and S, 9- or lO-membered heterobicycle having i to 4 heteroatoms selected from 0, N, and S, or 6-membered heterocycle having fromi to 4 heteroatoms selected from 0, N, and S, or (01.6 alkyl)-9- or lO-membered heterobicycle having 1 to 4 heteroatoms selected from 0, N, and S, or R 7 and R 8 are covalently bonded together to form cycloal kyl), 5- or 6-membered heterocycle having fromi to 4 heteroatoms selected from 0, N, and S; or one of R 7 or R 8 is covalently bonded to R 9 to form a pyrrolidine; wherein said alkyl, cycloalkyl, heterocycle, heterobicycle, phenyl are optionally substituted with from 1 to 4 substituents selected from the group consisting of: OH, COOHI P-6alkyl), (C2-4alkenyl), CONH- 2

NH-

2 NH(Cj-r, alkyl), N(0 1 6 alkyl) 2 NHCOCOOH, NHCOCON(Cl.

6 alkyl) 2

NHCOCONH(C

1 6 alkyl), SH, S(Cl-.

6 alkyl), NHC(=NH)NH 2 halogen, and COO(0 1 6 alkyl); is H or (01-6 alkyl); and 0 is selected from the group consisting of: (C 13 alkyl)CONHaryl, 6- or 1lO-membered aryl, biphenyl, 5- or 6-atom heterocycle having 1 to 4 heteroatoms selected from 0, N and S, 9- or 1lO-membered heterobicycle having 1 to 4 heteroatoms selected from 0, N and S; wherein said aryl, biphenyl, heterocycle and heterobicycle are all optionally substituted with from 1 to 4 substltuents selected from: OH, COOH, COO(Cl.

6 )alkyl, (0 1 6 )alkyl, (Cl.

6 )alkylCOOH, (C~r alkyl)(0 24 alkynyl),

(C

1 .6)alkyl-hydroxy, phenyl, benzyloxy, halogen, (C 2 4alkenyl, (C 2 4)alkenyl-

(C

1 6 )alkyl-COOH, 5- or 6-membered second heterocycle having 1 to 4 heteroatoms selected from 0, N and S, NH-5- or 6- membered second heterocycle having 1 to 4 heteroatoms selected from 0, N, and S, wherein said second heterocycle and phenyl being optionally substituted with from 1 to 4 substituents selected from: (01.6 alkyl),

CF

3 ,01OH, (0 1 .ralkyl) COOH, O(0,.

6 alkyl)OOOH, (0 1 6 alkyl) CO0(Cj- 49 raklCChny,.)c- lyl,(l6ly)(C-aklCO C) NCa lky l p yl 00(1.6 alkyl), NH .alk yl),( 1 al ogen, COOH, N0(0 6 ly),NOC. ly) H 2 HC. ly) aoeNC.

alkyl) 2 and C2-6 alkenyl-COOH halogen, OPO 3 H, benzyl, sulfonamido, SH, SOCH 3

SO

3 H, SO 2

CH

3

S(C

1 6 alkyl)COOH,

-CONH

2

-GOGH

3 (Cl.

3 )alkyl, (C 2 4 alkenyl)COOH wherein said alkenyl is optionally substituted with from 1 to 2 (01.6 alkyl) substituents,

(C

24 alkenyi)COO(C.salkyl), tetrazolyl, COOH, triazolyl, OH, NO 2

NH

2 0(01.6 alkyl)COOH, hydantoin, benzoyleneurea, (Cl.4alkoxy, (C-4)alkoxy(C- 6 alkyl)COOH, cyano, azido, -O-(Cl.

6 )alkyl COOH, -O-(Cl.

6 )alkyl

COO-(C

16 )alkyl, -NHCOCOOH, -NHCOCONHOH ,-NHCOCONH 2 -NHCOCONHCH 3

-NHCO(C

1 6 )alkyl-COOH, -NHCOCON H(Cl.

6 )alkyl-COOH,

-NHCO(C

37 )cycloalkyl-COOH,

-NHCONH(C

6 10 )aryl-COOH,

NHCONH(C

6 10 )aryl-COO(Cl-6 )alkyl, NHCONH(Ci-6 )alkyl-COOH NHCONH(C 1 .r)alkyl-

COO(C

16 )alkyl, NHCONH(0 1 6 )alkyl-(C 2 s)alkenyl-COOH, NH(Cl-r)alkyl-(C6 1 0 )aryl-O(C 1 6 )alkyl COOH, NH(Cl-r 6 )alkyl-(Cr 6 .lo)aryl-COOH, -NHCH 2 C00H,

-NHCONH

2

-NHCO(C

1 6 )hydroxyalkyl COOH, -OCO(CI- 6 )hydroxyalkyl COOH, (C 3 -r)cycloalkyl COOH, 0 0 0 NHCN, -NHCHO, -NHSO 2

CH

3

-NHSO

2

CF

3 coumnarin, (Cl 16 )alkyl-amino, NH(Cl.6alkyl) 2 C(halogen) 3

-NH(C

2 4acyl, -NH(C 6 -io)aroyl, -CONH(Ci.

6 alkyl), -CO(C 1 6 )alky-COOH, -CON H(C 1 6 )alkyl-COOH, -CO-NH-alanyl, .CONH(C 2 4alkylN(C.

6 alkyl) 2 -CON H(C 24 alkyl-Het, -CONH(C 2 4 alkyl-(COOH)-Het,

-CONH(C

1 2 alkyl)

(OH)(C

1 2 alkyl)OH, -CONH(C 1 6 alkyl-COQH, -CONH(C 6 10 aryl), -CONH-Het,

-CONH(C

6 10 aryl-COOH, -CONH(C 6 10 aryl-COO(C-6,) alkyl, -CONH(Cl-.

6 alkyl-COO(Cl-6) alkyl, -CONH(C 6 10 aryl-(Cl.

6 )alkyl-COOH, and -CONH(C6_ 10 aryl-(C 2 .6)alkenyl-COOH; or a salt thereof; said probe comprises a detectable label, whereby said probe binds to an HCV polymnerase or an analog thereof and is capable of being displaced by an Inhibitor thereof.

o Labels incorporated into the probe may be paired with appropriate labels attached to 00 the tagged NS5B polymerase such that the close proximity of the two pairs of labels upon probe-polymerase association results in a measurable signal; examples of such detection techniques include, but are not limited to, fluorescence resonance energy transfer (FRET), and time resolved fluorescence (TRF).

SPreferably, the detectable label is selected from the group consisting of: a Sfluorescent label (such as fluorescein, Oregon green, dansyl, rhodamine, Texas-red, Sphycoerythrin or Eu 3 a radioactive atom (such as 3 H, 14C, a chemiluminescent CN 10 label (such as luciferase), colorimetric produced by an enzymatic marker (such as Pgalactosidase or horseradish peroxidase).

Alternatively, a fluorescent reporter and quencher may be used as pair of labels to monitor association of the probe with the HCV NS5B polymerase. Commonly known reporter/quencher pair may be selected from, for example: EDANS/DABCYL, tryptophan/2,4-dinitrophenyl, tryptophan/DANSYL, 7-methoxycoumarin/2,4dinitrophenyl, 2-aminobenzoyl/2,4-dinitrophenyl and 2-aminobenzoyl/3-nitrotyrosine.

As will be readily understood by a person skilled in the art, a radioactive label can be incorporated within the probe of formula I at any suitable position. For example, a 3

H,

or 14C isotope can replace any hydrogen or carbon present in the molecule. Similarly, a 5 I isotope can be substituted on any aromatic ring.

In principle, these tracer methodologies can easily be adapted for the purpose of high-volume screening. Scintillation proximity assay (SPA) methods for radioactive detection have been developed which do not require a separation step and are easily adapted for robotics and microtiter plate format.

Preferably, the detectable label is a fluorescent label or a chemiluminescent label.

More preferably, the label is a fluorescent label. Most preferably, the detectable label is a fluorescein.

Non-radioactive detection methods have become increasingly widespread in screening assay because of the costs associated with radiolabeled reagents and their disposal. Fluorescence spectroscopy is one of the most prevalent non- 51 O radioactive detection methods. One type of assay in which fluorescence may be 00 used is fluorescence polarization. Polarization is independent of total fluorescence intensity; therefore, this technique may not be as prone to interference as fluorescence amplitude measurements. As disclosed herein, the new type of assay 0 5 developed uses a fluorescein-labeled inhibitor, though other fluorescent labels or non-fluorescent techniques can also be applied.

Preferably, the polymerase used in the assay may comprise an affinity tag by which 0 the polymerase can be attached to a solid support, and the probe may be labeled so as to provide a detectable signal. An affinity tag Incorporated into the probe maybe a biotin that is used to indirectly measure the association of this biotinylated probe to the NS5B polymerase through the secondary use of an avidin-coupled detection technique.

Preferably, the HCV polymerase used in the present assay is selected from the group consisting of: NS5B; NS5BA21; NS5BA57 or analogs thereof from a variety of genotypes including HCV-1a or lb strains having optionally a histidine tag at either the N- or C-terminal. Particularly, as will be understood by a person skilled in the art, this binding assay does not require the polymerase activity of the NS5B to be optimal or functional for such a binding assay to perform according to the invention.

EXAMPLES

Example 1A) probe (iii): (S)-3-(5-Carboxymethoxy-1H-indol-3-yl)-2-({1-[1-cyclohexyl-2-(4-{[2-(5dimethylamino-naphthalene-1-sulfonylamino)-ethylcarbamoyl]-methoxy-phenyl)- 1 Hacid

O

52 Sa) Ib) d) SO Me 00 H BcHN O CHO H N rIHNa) h) TFA Pd(OH) 2 (0 g) oxone DMF 1 K)OJ COOMe c ooMe OO COOMe

SCOOH

a) 4-Chloro-3-nitrobenzoic acid (40.40 g, 0.20 mole) was suspended in DCM (100 mL) containing 3 drops of DMF. Oxalyl chloride (1.5 equivalents, 0.3 mole, 27 mL) was added in small portions and the mixture stirred overnight at room temperature.

After refluxing for an additional hour to complete the reaction, volatiles were removed under reduced pressure and the residue was co-evaporated twice with hexane to give the title compound as a light yellow solid.

b) (S)-5-Hydroxytryptophan methyl ester hydrochloride (1.55 g, 5 mmol) was dissolved in 80% aqueous MeCN (25 mL) and the solution cooled in ice. Sodium bicarbonate (0.850 g, 10 mmol) was added followed by di-tert-butyldicarbonate (1.10 g, 5.1 mmol). The mixture was stirred for 2 h at room temperature, poured into water (200 mL) and extracted with EtOAc (3 The combined extracts were washed with water and brine, dried (MgSO 4 and concentrated to give a beige solid (1.65 g).

The crude product from above (1.50 g, 4.83 mmol) was dissolved In acetone (20 mL) and anhydrous potassium carbonate (1.5 g, 11 mmol) and methyl bromoacetate (0.76 g, 5 mmol) were added. The mixture was reflux for 4 h after which point additional methyl bromoacetate was added to complete the reaction (15 mg portions 2 TFA 6 OCOOMe _R9-y

(COOH

a) 4-Chloro-3-nitrobenzoic acid (40.40 g, 0.20 mole) was suspended in DCM (100 mL) containing 3 drops of DMF. Oxalyl chloride (1.5 equivalents, 0.3 mole, 27 m) was added in small portions and the mixture stirred overnight at room temperature.

b) (S)-5-Hydroxytryptophan methyl ester hydrochloride (1.55 g, 5 mmol) was dissolved in 80% aqueous MeCN (25 mL) and the solution cooled in ice. Sodium bicarbonate (0.850 g, 10 mmol) was added followed by di-tert-butyldicarbonate (1.10 g, 5.1 mmol). The mixture was stirred for 2 h at room temperature, poured into water (200 mL) and extracted with EtOAc (3 The combined extracts were washed with water and brine, dried (MgSC4) and concentrated to give a beige solid (1.65 g).

The crude product from above (1.50 g, 4.83 mmol) was dissolved in acetone (20 mL) and anhydrous potassium carbonate (1.5 g, 11 mmol) and methyl bromoacetate (0.76 g, 5 mmol) were added. The mixture was reflux for 4 h after which point additional methyl bromoacetate was added to complete the reaction (15 mg portions 53 o until complete by HPLC). The reaction mixture was then cooled and filtered to 00 remove solid. Evaporation of the filtrate gave the desired carbamate as an oil The crude carbamate from above (2.0 g) was deprotected by stirring with 4N HCI dioxane for 1 h at room temperature. Removal of volatiles in yacuo gave the desired 1 5 tryptophan ester derivative as a tan-colored solid (1.51 g).

c) The tryptophan derivative from step b) (0.343 g, 1 mmol) was dissolved in aqueous MeCN (10 mL) and sodium bicarbonate (3 equivalents, 0.260 g) was added. The solution was cooled in ice and 4-chloro-3-nitrobenzoyl chloride from O step a) (0.220 g, 1 mmol) was added. The mixture was stirred for one hour at room temperature, concentrated under reduced pressure and the residue purified by flash chromatography (1:2 hexane EtOAc as eluent) to give compound c) as a yellow foam (0.391 g).

d) The 4-chlorobenzamide derivative from above (0.214 g, 0.45 mmol) was dissolved in DMSO (1 mL) and DIEA (0.2 mL) was added followed by cyclohexylamine (3 equivalents, 0.16 mL). The mixture was stirred at 60-65 oC for 4 h and subsequently diluted with water. The orange precipitate that formed was collected, washed with water and dried (0.200 g).

e) The crude material from above (0.200 g, 0.36 mmol) was hydrogenated (1 atm 12) over 20% Pd(OH) 2 on charcoal (60 mg) in MeOH (15 mL). After 2 h, the suspension was filtered to remove the catalyst and concentrated in vacuo to give the title compound as a foam (0.16 g).

f) 4-Formylphenoxyacetic acid (0.306 g, 1.70 mmol) was dissolved in DCM (5 mL).

DIEA (0.524 g, 4 mmol) and TBTU (0.550 g, 1.70 mmol) were added followed by tert-butyl N-(2-aminoethyl)carbamate (0.250 g, 1.56 mmol). The mixture was stirred 2 h at room temperature, dissolved in EtOAc and washed sequentially with aqueous K 2

CO

3

KHSO

4 water and brine. The extract was dried (MgSO 4 and concentrated under reduced pressure to give a yellow solid (0.350 g).

g) The diamine derivative from step e) (0.026 g, 0.05 mmol) and aldehyde from step f) (0.020 g, 0.06 mmol) were dissolved in DMF (0.3 mL) and water (0.03 mL) was added followed by oxone@ (0.024 g, 0.04 mmol). The mixture was stirred 1 h at room temperature and then diluted with water. The resulting precipitate was collected by filtration, washed with water and dried to give a beige solid (0.020 g).

h) The crude carbamate from above was stirred with TFA for 30 min at room temperature. Volatiles were removed under reduced pressure and the residue was purified by preparative C18 reversed-phase HPLC to give the bis TFA salt.

54 0i) The amine salt (0.019 g, 0.02 mmol) was dissolved in DM50S (0.3 mL) and D IEA 00 (0.06 mL) was added followed by dansyl chloride (0.065 g, 0.02 mmol). The mixture was stirred for 1 h at room temperature. 5N NaOH (0.12 mL) and water (0.05 mL) were added and the saponification was allowed to proceed for 1 h at room temperature. Following acidification with TFA, the probe (iii) was directly isolated from the reaction mixture by preparative 018 reversed-phase HPLC: MS mlz *930 Example 1 B) probe (ii):5-(3-(2-[2-(4-(5-[(S)-l1-Carboy-2-(5-ca.bQxyethoxy-l1H-lndol-3-yI)ethylcarbamoyl]- 1 -cyclohexyl- 1 H-benzimidazol-2-y)-pheoxy)-ethaloylaio]-e thyl)thioureido)-2(6-hydroxy-3-oxo-3H-xalthel-9-yI)-belzoI C acid a COO

C

qO0 The amine salt from step h) of Exampie 1lA (0.06 mmol) was dissolved in IDM50S (0.6 mL) and DIEA (0.3 mL) was added followed by fluorescein isothiocyanate Isomer 1 (0.026 g, 0.066 mmol). The mixture was stirred for 1 h at room temperature. NaOH (0.3 mL) and water (0.15 mL) were added and stirring resumed for an additional 30 min. Foliowing acidification with TEA, probe (Ii) was Isoiated directly by preparative 01 8 reversed-phase H PLC: MS mlz 1086 Example 1C) probe (S)-2-ff 1-(4-Azido-phenyl)-mthanoyl-aJ'llfo)ethylcarbamoyl)-methoxy]-phel)- 1 -cyclohexyl- 1H-indol-3-yI)-propionic acid 00 HO o CHO 0 0 BocHN NH N

H

NH

2 b)

COOH

COOH COOMe

SHN

0 o (example 1A) O COOMe N- )CHO c C(v) a) 4-Azidobenzoic acid (0.160 g, 1 mmol) was dissolved in DCM (3 mL). DIEA mL, 2.5 mmol) and TBTU (0.337 g, 1.05 mmol) were added followed by tert-butyl N- (2-aminoethyl)carbamate (0.165 g, 1.03 mmol). The mixture was stirred 2.5 h at room temperature, dissolved in EtOAc and washed sequentially with 5% aqueous

K

2

CO

3

KHSO

4 water and brine. The extract was dried (MgSO 4 and concentrated under reduced pressure to give a yellow solid (0.257 The crude carbamate (0.257 g, 0.84 mmol) was deprotected by stirring in 4N HCI dioxane (15 mL) for 2 h at room temperature. Volatiles were removed under reduced pressure to give a pinkish solid.

b) 4-Formylphenoxyacetic acid (0.200 g, 1.1 mmol) was dissolved in DCM (3 mL) and DIEA (0.5 mL) was added followed by TBTU (0.350 g, 1,1 mmol) and the amine salt from above (0.240 g, 1 mmol). The mixture was stirred 4 h at room temperature, dissolved in EtOAc and washed sequentially with 5% aqueous K 2

CO

3

KHSO

4 water and brine. The extract was dried (MgSO 4 and concentrated under reduced pressure to give an off-white solid (0.162 g).

c) The benzaldehyde derivative from above (0.044 g, 0.12 mmol) and the diamine derivative from step e) of Example 1A (0.052 g, 0.1 mmol) were dissolved in DMF (0.6 mL) and water (0.1 mL). Oxone (0.050 g, 0.8 mmol) was added and the mixture stirred for 1 h at room temperature. 5N NaOH (0.2 mL) and water (0.1 mL) were added and saponification allowed to proceed for 1 h. Probe was isolated 56 o directly by preparative C18 reversed-phase HPLC (12.5 mg): MS mn/z 842 00 Example 1 D) probe (S)-3-(5-Carboxymethoxy-l1H-indol-3-yl)-2-([1 -cyclohexyl-2-(4-([2-((1 .phenyl-methanoyl)pheyl]-methaoyl)-amio)-ethylCarbamoylrmethoxy)phenyl)- 1 H-benzoimldazol-5-y]-methaloyl)-amilo)-propiofliC acid 0\ OH 0 0J -IOH (ES0 m/(95 MH) Example IlE) probe (1y) (S)-3-(5-Carboxymethoxy-l1H-indol-3-yI)-2-([l1-(1 -cyclohexyl-2-(4-[2-(5dlmethylamino-naphthalefle- 1-sulfonylamino)-ethylcarbamoyl-phenl)-

H-

acid

/N

0 a) Following the procedures described for step f)in Example 1A, 4carboxybe nzaldehyde was coupled to tert-butyi N-(2-aminoethyl)carbamate.

b) Following benzimidazoie ring formation with the diamine derivative of Example 1lA step e) and the aldlehyde from above using oxone® as described in Example 1lA step the Boc protecting group was removed and the resulting amine condensed with dansyl chloride as described in Example 1lA step i).

C^ 57

C-)

Q c) Probe (iv) was obtained following saponification of the ester groups under the 00 usual conditions and isolation by preparative C18 reversed-phase HPLC: MS (ES+) m/z 900 O 5 Example 1F) C\ (probe 1-Carboxy-2-(5-carboxymethoxy- H-indol-3-yl)ethylcarbamoyl]- 1-cyclohexyl- 1H-benzimidazol-2-yl}-phenyl)-methanoyl]-amino}ethyl)-thioureido]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid 0 o o COO

H

HO o COOH o COOH o (i) The procedure described for Example 1 E) was used except that fluorescein isothiocyanate isomer 1 was used instead of dansyl chloride. Probe was obtained after purification by preparative C18 reversed-phase HPLC: MS m/z 1056 Example 2 Production and purification of HCV NS5B polymerase A21-His The recombinant HCV NS5B polymerase can be produced In soluble form by expression of a variant that lacks the C-terminal 21 amino acids normally found on the mature NS5B (Yamashita etal. 1998, J. Biol. Chem. 273:15479-15486; Ferrari et al., 1999, J. Virol. 73:1649-1654). We have expressed this so called NS5BA21 with a C-terminal hexa-histidine (termed NS5BA21-His; SEQ ID. NO. 1) and with an Nterminal hexa-histidine tag (termed His-NS5BA21; SEQ ID NO. 2) (either proteins being referred to as "his-tag NS5B"). Expression of these genes from pET vectors in E. coli strain JM109 (DE3) is induced with 0.4 mM IPTG for 3 hours at 22 OC. Cells are harvested and lysed in a microfluidizer in lysis buffer (Tris-HCI pH 7.5, 10 glycerol, 1 mM EDTA, 2 mM 2-mercaptoethanol, 500 mM NaCI, 1 mM PMSF, 1 g/ml antipain, 1 Ilg/ml pepstatin A and 1 upg/ml leupeptin). The lysate is clarified by a 30 000 g centrifugation and then supplemented with imidazole to final concentration of 10 mM. The lysate is then loaded onto a metal-chelating resin (Ni- NTA; Qiagen) previously equilibrated with buffer A (Tris-HCI pH 7.5, 10 glycerol, 0 58 o 500 mM NaCI, 10 mM imidazole), washed extensively and then the protein is eluted Oo with gradient of buffer A containing 500 mM imidazole. Peak fractions containing the his-tag NS5BA21 are pooled and diluted with buffer C (20 mM Tris-HCI pH 7.5, 10 glycerol, 5 mM DTT) to reduce the NaCI concentration to 300 mM and then applied to a DEAE-Sepharose column to remove any nucleic acid. The flow-through from the SDEAE-Sepharose column is diluted with buffer C to reduce the NaCI to 200 mM and Sthen applied to a heparin-Sepharose column. The his-tag NS5B is eluted from the Sheparin-Sepharose in buffer C with a 200 mM to 1 M NaCI gradient. Peak fractions Scontaining the his-tag NS5B are pooled and diluted with buffer C to achieve a final NaCI of 200 mM and loaded onto a Resource S column. Concentrated His-tag is eluted from the resource S, loaded and size fractionated on a Superdex 200 column in buffer C containing 300 mM NaCI. Peak fractions contain highly pure histag NS5B and are stored at -80 "C until use.

Example 3 Fluorescence anisotrovp analysis Titration of the probe with the enzyme was performed as follows: The fluorescein labeled probe was diluted to the desired concentration in 20 mM Tris-HCI pH 7.5, 1mM EDTA, 5 mM MgCI 2 1 mM DTT and 10% DMSO. The NS5BA21 -His protein was serially diluted in 25 mM Tris-HCI pH 7.5, 300 mM NaCI, mM DTT, 1 mM EDTA, 30% glycerol and 0.1% IGEPAL. Total volume of the reaction was 500 R.L and final assay buffer was 20 mM Tris-HCI pH 7.5, 1 mM EDTA, 5 mM MgCI 2 1 mM DTT, 30 mM NaCI, 3% glycerol, 0.01% IGEPAL and 5% DMSO.

Anisotropy measurements were performed on a SLM Aminco 8100 Spectrofluorometer equipped with a 450-W xenon arc lamp and a T-optics configuration. Excitation wavelength was at 493 nm and emission was monitored at 530 nm. In each anisotropy measurement, the parallel and perpendicular intensities of the background buffer solution was subtracted from the measured values of the sample and the anisotropy was calculated. Data were processed on SAS program (SAS Institute Inc., NC, USA) for a non linear regression to obtain the direct binding equilibrium constant and other parameters, and the plot of the regressed fit over the experimental data. An example of a titration curve obtained with probe is shown on Figure 1. Kd values for probes and (ii) with the polymerase were respectively of nM and 6 nM.

S59 0 Example 4 00 96-well plate Polarization assay To obtain Kd values of different compounds competing with these probes (test compounds), this assay was transformed to a more amenable format and a binding I 5 assay was made suitable for a 96-well microplate reader. The probe was diluted in Sorder to obtain the desired final concentration (from 4 to 25 nM, depending on its Kd

C

towards the enzyme and on the conditions of the assay). The tested compounds l were serially diluted by a factor of 2 or 3-fold In 20 mM Trs-HCL pH 7.5, 1 mM

O

O EDTA, 5 mM MgCI 2 1 mM DTT and 15% DMSO. The NS5BA21-His concentration in the assay was calculated to obtain 70% of binding of the probe; these conditions allowed for the displacement of the probe by test compounds. The assay reactions finally contained 50 jL of the serial dilutions of the tested compounds that were transferred In 96-well black plates (Packard); a complete row was however free of compound to obtain a positive control value and verify real percent of bound probe in the experiment. 50 .LL of the probe were then added to each well, except in one column for blank subtraction. Lastly, 150 LL of enzyme were added to all wells, except in one row, which was used to determine the 0% and 100% bound values. In this row of 8 wells, enzyme buffer was added to the first 4 wells (to determine the anisotropy value of the free probe or rf) and a 10-fold excess of the concentration of the enzyme used in the assay was added to the other 4 wells (to determine the anisotropy value when 100% of the probe is bound i.e. the rb value). These values were required to calculate the Kd values. The final buffer conditions of the assay were identical to the ones used for Kd determination of the probes, i.e. 20 mM Tris- HCI pH 7.5, 1 mM EDTA, 5 mM MgCI 2 1 mM DTT, 30 mM NaCI, 3% glycerol, 0.01% IGEPAL and 5% DMSO. The reactions were incubated for 90 minutes at room temperature in the dark. Readings of polarization were then performed on a POLARstar Galaxy, equipped with a high-energy xenon flash lamp, using an excitation filter of 485 nm and an emission filter of 520 nm. Polarization values can be converted easily to anisotropy values with the following calculation (Owicki etal., 2000, J. Biomol. Screen. 5:297-306): a=2xP/(3-P)where a: anisotropy value P: Polarization value 0 Anisotropy values can then be used to obtain two types of results 00 fitted to SAS nonlinear regression analysis to obtain apparent Kd values, using for the calculations as positive control the anisotropy value at 70% binding, and as negative control the anisotropy value of the free probe (rf); fitted to the Anisotropy equation: S(-Kd ((Kd+I-E 2 4*K* d

E

a0 aQ= (aQr b +KP r (KP a Q) where a: anisotropy Kd: dissociation constant for the inhibitor I: Concentration of compound (or inhibitor) tested Eo: NS5B concentration (Eo has to be [probe]) Q Qb/Q total fluorescence for probe 100% bound/ total fluorescence for free probe rb: anisotropy value when the probe is 100% bound rf: anisotropy value when the probe is free Kp: dissociation constant for the probe This high throughput assay was evaluated and validated by the determination of the statistical parameter Z' Zhang et al., 1999, J. of Blomol. Screening, 4:67-73).

Results of this experiment are illustrated on Figure 2. The anisotropy values for a series of positive and negative controls were very similar, resulting in very low standard deviations; 0.2186±0.0036 A units for the positive controls and 0.0738±0.0037 A units for the negative controls. The Z' value obtained for the assay was of 0.85, implying that we have excellent conditions to detect compounds that would compete with the probe.

Example Inhibitor testing We have identified potent compounds that can effectively displace the probe in this binding assay. Figures 3 and 4 show examples of some of them, with Kd values ranging from 31 nM to 1 iM. The anisotropy equation was defined in the Grafit Software (Erithacus Software Ltd., UK) and plotted such that inhibitor concentration was the X-variable and anisotropy was the Y variable; parameters calculated by the 00 (61 O software were the inhibitor Kd and Qb/Qf ratio. Supplied constants were the Kp, Eo, rb and rf.

SExample 6 Modified conditions for the Polarization assay N The usefulness of this polarization assay is evident when binding of compounds has to be studied under different conditions. For example, binding constants of the O probes have been determined at different concentrations of salts and pH. Figures N to 8 show the binding curves of probe in final NaCI concentrations ranging from mM to 200 mM. All other reagents in the assay were as described in the standard protocol (Example As shown on these Figures, Kd values gradually increase with salt concentration from Kd=15 nM (at 30 mM NaCI) to Kd=122 nM (at 200 mM NaCI).

Studies at pH 6.5 were also performed to determine the Kd of the probe at lower pH. For these assays, 20 mM Phosphate buffer pH 6.5 was used in place of Tris; all other reagents of the assay were as described in the 96-well Polarization assay (Example An example of these types of experiments is shown in Figure 9. The Kd value obtained at pH 6.5 with probe was of 33 nM. Having established these Kd values under different experimental conditions, it is then trivial to determine what concentrations of probe and enzyme should be used to obtain 70% of binding of the probe with the equilibrium equation. Once these values are obtained, compounds of interest can easily be studied under the new conditions to determine their Kd values.

Example 7 Fluorescence Polarization assay with a modified enzyme The Fluorescence polarization assay was also used with other constructs of our HCV polymerase enzyme. In addition to the C-terminally tagged NS5BA21-His polymerase, the NS5B enzyme with the His-tag at the N-terminal position was also used in the fluorescence polarization assay. Determination of the Kd for the probe (i) with this enzyme was performed, using the same conditions described in the standard 96-well format assay. Figure 10 shows that the Kd obtained with probe (i) was similar, i.e. 18 nM. A comparison was made between the IC 5 0 and the Kd for three compounds, using these two different constructs of the enzymes (NS5BA21- His and His-NS5BA21).

0 62 o

IC

50 's are determined using the Scintillation Proximity Assay (SPA) according to the 00 following assay: The substrates are: a 12 nucleotide RNA oligo-uridylate (or oligo-uridinemonophosphate) (oligo-U) primer modified with biotin at the free 5'C position; (ii) a complementary poly-adenylate (or adenosine monophospahte) (polyA) template of Sheterogeneous length (1000-10000 nucleotides); and (iii) UTP-[5,6 3 Polymerase C activity is measured as the incorporation of UMP-[5,6 3 H] into the chain elongated from the oligo-U primer. The 3 H-labelled reaction product is captured by SPA-beads Scoated with streptavidin and quantified on the TopCount (Packard). Inhibitors are tested at various concentrations In a reaction containing: 1 to 5 nM of the his-tagged 1 gIg/ml of blotlnylated ollgo U primer, 10 [ig/ml of polyA template, 20 mM Tris-HCI pH 7.5, 5 mM MgCI 2 25 mM KCI, 1 mM EDTA, 1 mM DTT, 0.33 ndodecyl maltoside, 5% DMSO, 0.0083 .Ci/pl [0.25 rIM] UTP-[5,6- 3 0.75 RM UTP, 1.67 U/ pI RNAsinTM. The reaction was incubated at room temperature for 1.5 hours.

STOP solution (20 Il; 0.5 M EDTA, 150 ng/ ld tRNA) was added, followed by 30 I.i streptavidin coated PVT beads (8mg/ml in 20 mM Tris-HCI, pH 7.5, 25 mM KCI, 0.025% NaN 3 The plate was then shaken for 30 minutes. A solution of CsCI was added (70 p.I, 5 to bring the CsCI concentration to 1.95 M. The mixture was then allowed to stand for 1 hour. The beads were then counted on a Hewlett Packard TopCount TM instrument. Based on the results at ten different concentrations of test compound, standard concentration-% inhibition curves were plotted and analysed to determine ICso's for the compounds.

Results of this experiment are illustrated in Table I. The Kd values were similar with both enzymes for the three compounds tested, whereas the ICso values obtained with the two enzymes show significant differences and reflect the differences in substrate affinity.

Example 8 Specificity of the Fluorescence Polarization assay The utility of the Fluorescence polarization assay was examined with another distantly related viral polymerase and with a closely related genotype (la) HCV polymerase.

The GBV-B polymerase enzyme (termed GBV-BA23-His; SEQ ID NO. 3) (Simons, 63 o J.N. etal., 1995, Proc. Natl. Acad. Sci. USA 92, 3401-3405; Bukh, J. etal., 1999, 00 Virology 262, 470-478) was produced and purified as described in Example 2 with the following modifications: Expression of the gene from pET vectors in E. coli strain JM109 (DE3) was induced with 0.5 mM IPTG for 3 hours at 22 Cells were harvested and lysed in a Smicrofluidizer in buffer A (Tris-HCI pH 7.5, 10 glycerol, 1 mM EDTA, 2 mM 2- Smercaptoethanol, 500 mM NaCI, 1 mM PMSF, 1 ug/ml antipain, 1 ug/ml pepstatin A, S1 ug/ml leupeptin and 0.5% dodecyl-p-D-maltoside). The lysate was clarified by a S000 g centrifugation and then supplemented with imidazole to a final concentration of 10 mM. The lysate was then loaded onto a metal-chelating resin (NI-NTA; Qiagen) previously equilibrated with buffer A containing 10 mM imidazole, washed extensively and then the protein was eluted with a gradient of buffer A containing 500 mM imidazole. Peak fractions containing the his-tag GBV-BA23 were pooled and diluted with buffer C (20 mM Tris-HCI pH 7.5, 10 glycerol, 5 mM DTT, 0.01% dodecyl-p-D-maltoside) to reduce the NaCI concentration to 300 mM and then applied to a DEAE-Speharose column to remove any nucleic acid. The flow-through from the DEAE-Speharose column was diluted with buffer C to reduce the NaCI to 200 mM and then applied to a heparin-Sepharose column. The his-tag GBV-B was eluted from the heparin-Sepharose in buffer C with a 200 mM to 1 M NaCI gradient.

Peak fractions containing the pure his-tag GBV-B were then pooled and stored at OC until use.

The HCV genotype la NS5B polymerase [termed His-NS5BA21(H77c,1a); SEQ ID NO. 4] (Yanagi, M. etal., 1997, Proc. Natl. Acad. Sci. USA 94, 8738-8743) was produced and purified as described in Example 2 with the following modifications: Expression of the gene from pET vectors in E. coli strain JM109 (DE3) was induced with 0.4 mM IPTG for 3 hours at 22 OC. Cells were harvested and lysed in a microfluidizer in buffer A (Tris-HCI pH 8.0, 10 glycerol, 1 mM EDTA, 2 mM 2mercaptoethanol, 500 mM NaCI, 1 mM PMSF, 1 ug/ml antipain, 1 ug/ml pepstatin A, 1 ug/ml leupeptin, 1% dodecyl-p-D-maltoside, 1% Triton X-100 and 0.1% CHAPS).

The lysate was clarified by a 30 000 g centrifugation and then supplemented with imidazole to a final concentration of 10 mM. The lysate was then loaded onto a metal-chelating resin (Ni-NTA; Qiagen) previously equilibrated with buffer A containing 10 mM imidazole, 0.1% NP-40, without CHAPS, and with lower 64 o concentrations of the other detergents dodecyl-p-D-maltoside, 0.05% Triton X- 00 100); after extensive washing, the protein was eluted with a gradient of buffer A containing 500 mM imidazole. Peak fractions containing the his-tag NS5BA21 (H77c,1a) were pooled and diluted with buffer C (20 mM Tris-HCI pH \O 5 10 glycerol, 5 mM DTT, 0.2% dodecyl-p-D-maltoside) to reduce the NaCI concentration to 300 mM and then applied to a DEAE-Sepharose column to remove any nucleic acid. The flow-through from the DEAE-Sepharose column was diluted with buffer C to reduce the NaCI to 200 mM and then applied to a heparin- 0 Sepharose column. The his-tag NS5BA21(H77c,1a) was eluted from the heparin- Sepharose in buffer C with a 200 mM to 1 M NaCI gradient. Peak fractions containing the polymerase were then pooled and diluted with buffer C to achieve a final NaCI of 200 mM and loaded onto a Resource S column. Peak fractions containing the his-tag NS5B(H77c,1a) were pooled, loaded and size fractionated on a Superose 12 column In buffer C containing 600 mM NaCI. Peak fractions contain highly pure his-tag NS5B were pooled and stored at -80 oC until use.

The GBV-B and the HCV la polymerases were used to titrate probe ii, using the protocol described in Example 3. Figures 11 and 12 show the titration curves observed with the GBV-B polymerase and the NS5B(H77c,1a) polymerase, respectively. The Kd value of probe ii for the GBV-B enzyme was 1.8 uM (estimated value with an incomplete curve and an rb value of 0.21), illustrating the weak binding of the probe to this distantly related polymerase. In contrast, the Kd for the HCV la polymerase was 18 nM, revealing that the 1 a genotype enzyme binds probe ii with the same affinity as the HCV lb genotype polymerase.

Kd values for a series of compounds were determined with these two HCV (genotypes la and 1b) polymerases, using the assay format described in Example 4.

Results of this experiment are illustrated In Table 2. These results show that the Kd values for this series of inhibitors are in the same range with the two genotypically related HCV enzymes.

TABLE 1 Comparison of compound Kd and ICso values with two different HCV polymerases

\O

Cc, Cpd Kd value (nM)

IC

5 0 value (nM) NS5BA21-His His-NS5BA21 NS5BA21-HIs 867 348 735 His-NS5BA21 66 68 34 TABLE 2 Comparison of compound Kd values with NS5B polymerases from two HCV genotypes Cpd Kd values (nM) His-NS5BA21(1 b) His-NS5BA21(H77c,1a) 1.8 7.2 7.1 2.7

DISCUSSION

The HCV NS5B polymerase is a prime target in the search for inhibitors of HCV replication. The HCV NS5B enzymatic activity has been studied in vitro with a variety of RNA substrates (Behrens et al., 1996; and many references thereafter). Different preparations of the HCV polymerase exhibit varying efficiencies of product formation with a variety of RNA substrates. Estimations are that only a small fraction 1%) of the common preparations of purified recombinant HCV NS5B polymerase interact (N 66 0 with RNA substrate to reconstitute processive RNA product synthesis (Carroll SS, et 00 al., 2000. Biochemistry, 39:8243-8249). Moreover, the activity of purified recombinant NS5B polymerase varies significantly with specific RNA substrates; a characteristic that presumably reflects the capability of the NS5B of forming productive replication-competent complexes with these substrates (Zhong W, et al., S2000, J Virol, 74, 9134-9143).

In an effort to overcome the limitations of HCV polymerase assays that use sub- Soptimal and poorly characterized RNA substrates, the Applicants have developed an assay for specific inhibitors of the HCV polymerase that is independent of the presence of RNA. The assay is based upon the use of a characterized Inhibitor specific for the HCV polymerase. In the examples presented above, the inhibitor was labeled with a fluorescein moiety and the Interaction of this probe with the was measured and quantified by fluorescence polarization. However, the interaction can also be measured by the use of a radiolabel, or other common labels placed on the inhibitor and applying common techniques for assessing the association of the labeled probe with an appropriately tagged target HCV polymerase. Binding equilibrium with the fluorescein labeled probe is clearly evident in Example 3, as the fraction of bound probe increased with the amount of HCV polymerase. An HCV polymerase assay with components at equilibrium is an advantage over previous assays with RNA substrates, as the active HCV polymerase that stably associates with RNA substrates in processive complexes does not readily dissociate (Carroll SS, et 2000 Biochemistry, 39:8243-8249; Zhong W, et al., 2000 J Virol, 74, 9134- 9143; Tomei L, et al. 2000 J Gen. Virol. 81, 759-767.). Though these labeled probes readily dissociate from the HCV polymerase, they do so with low nM dissociation constants and provide the required sensitivity (in the low nM range) to detect potent and specific inhibitors. The assay format is adaptable to screening in 96-well (or higher density) plate format as demonstrated in Example 4. A particular advantage of this high throughput screening format is the extremely stable signal and minimal well-to-well variation that the assay provides, particularly in a convenient nonradioactive format. Specific inhibitors of the HCV polymerase were identified and potencies easily determined with this assay (Figures 3 and 4).

The direct binding assay described herein overcomes other limitations of the enzymatic HCV polymerase assay. The in vitro RNA polymerase activity of NS5B is 0 67 0 extremely sensitive to ionic strength, and KCI or NaCI concentrations exceeding 100 00 mM inhibit the reaction (Lohmann V, et al., 1998 Virology 249, 108-118; Luo G, et al., 2000, J Virol., 74, 851-63.) Hence the ability to determine the potency of inhibitors at various salt concentrations is restricted by this limitation of standard enzymatic reactions. The direct binding assay of this Invention is amenable to c adjustments in salt concentration or pH levels as demonstrated in Example 6. The N potencies and interaction of specific inhibitors with the NS5B target can easily be determined under conditions not suitable for enzymatic RNA polymerization studies 0(such as the absence of divalent cation).

Established HCV polymerase enzymatic assays provide ICso values as representative measurements of inhibitor potencies. For inhibitors that are competitive with either RNA or NTP, the ICso value is proportional to the concentration of substrates in the assay and will vary depending on the concentration of these components. The assay described herein permits a direct measurement of inhibitor potencies (reflected by Kd values), under defined conditions, irrespective of the substrate concentration. In enzymatic reactions that use either the N-terminal tag His-NS5BA21 or the C-terminal tag NS5BA21-His, significantly disparate IC5o values are obtained for identical compounds assayed under identical conditions. The His-NS5BA21 and NS5BA21-His polymerases have different affinities for the primer/template RNA substrate thereby resulting in the disparate IC50 for the identical compounds (Example 7, Table A major advantage that is exemplified by the direct binding assay described in this invention is that these differences are reconciled by the relatively similar Kd values that the individual inhibitors display with the two different HCV polymerases.

The direct binding assay described herein has also been shown to be specific for HCV polymerase enzymes. Example 8, in which a Kd at least 100-fold higher for the probe ii was obtained with the GBV-B polymerase, illustrates the weak binding of the probe to this polymerase and the specificity of binding to the HCV polymerases.

Moreover, Example 8 also demonstrates that the polymerases from two distinct and clinically relevant HCV genotypes bind the probe with similar affinities.

-68- O The direct inhibitor-binding assay of this invention alleviates many restrictions of O_ conventional HCV polymerase enzymatic assays described to date. The Applicants have exemplified how the use of a characterized inhibitor as a competitive probe provides a number of improvements and advancements in the search for specific inhibitors of the S 5 NS5B polymerase. This assay may accelerate the identification and characterization of candidate therapeutics for the treatment of HCV related diseases.

(Ni O The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Claims

1. A method for identifying compounds binding to HCV polymerase comprising the steps of: O a) contacting said HCV polymerase or an analog thereof with a probe being capable of binding to an HCV polymerase or an analog thereof, said probe being displaceable by an inhibitor thereof, so as to form a complex comprising said probe bound to said polymerase; Sb) measuring a signal emitted from said probe in said complex to establish a base line level; c) Incubating the product of step a) with a test compound; and d) measuring the signal from said complex; and e) comparing the signal from step d) with the signal from step b); whereby a modulation in said signal is an indication that said test compound binds to said polymerase.

2. The method according to claim 1, wherein said probe is selected from: an isomer, enantiomer, diastereoisomer, or tautomer of a probe represented by formula A M' Rs R 2 'B KL (1) wherein A is O, S, N, NR 1 or CR 1 wherein R' is selected from the group consisting of: H, (C 1 8 )alkyl optionally substituted with: -halogen, OR 1 SR 1 or N(R12) 2 wherein R" and each R 12 is independently H, (C1. 6 )alkyl, (C 3 7 )cycloalkyl, (C1.6)alkyl-(C3. 7 )cycloalkyl, (C 16 )alkyl-aryl or (C 1 e)alkyl-Het, said aryl or Het optionally substituted with R 10 or both R 12 are covalently bonded together and to the nitrogen to which they are both attached to form a 5, 6 or 7-membered saturated heterocycle; represents either a single or a double bond; R 2 is selected from: H, halogen, R 21 OR 21 SR 21 COOR 21 SO 2 N(R) 2 N(R2) 2 CON(R 22 2 NR2C(O)R 22 or NR2C(O)NR 2 2 wherein R 2 and each R 2 is Q ~~~independently H, (Cl. 6 )alkyl, haloalkyl, (C 26 )alkenyl, (C 3 7 )CYCloalkyl, (C 2 alyy,(. 00 7 )cycloalkenyl, 6 or lo-membered aryl or Het, said R21 and R22 being optionally substituted with R 20 or both R22 are bonded together to form a 5, 6 or 7-membered saturated heterocycle IND 5 with the nitrogen to which they are attached; MN is NR 3 or CR, wherein R 3 i selected from (Cl. 6 alkyl, haloalkyl, (C3- 7 )cycloalkYt, (C 610 )bicycloalkyl, 6- or lO-membered aryl, Het, (C 1 .6)alkyl-aryl or (C 16 alkyl-Het, said alkyl, cycloalkyl, bicycloalkyl, aryl, Het, alkyl-aryl and alkyl-Het being optionally substituted with from 1 to 4 substituents selected from: halogen, or a) (C 16 )alkyl optionally substituted with: OR 31 or SR 31 wherein R 31 is H, (C 1 6 alkyI), (C 3 7 )cycloalkyl, (Ci 16 )alkyl-(C3.7)cycloaky, aryl, Het, (C 16 )alky-aryl or (Cl- 6 )alkyl-Het; or N(R 3 2 wherein each R32 is independently H, (C 1 0 )alkyl, (C 3 7 )cycloalkyl, (Cl-6)alkyl-(C 3 q7)cycloalkyl, aryl, H-et, (0 1 6 )alkyl-aryl or (Cl. 6 )alkyl-Het; or both R 32 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or

7-membered saturated heterocycle; b) OR 33 wherein R 3 3 is H, (C 16 )alkyt, (C 3 7 )cycloalkyl or (C 1 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl. 6 )alkyl-aryl or .)alkyl-Het; c) SR34 wherein R34 is H, (Cl. 6 )alkyl, (C 3 7 )cycloalkyl, or (C 18 6)alkYl-(C 3 7 )cycloalky, aryl, Het, (Cl. 6 alkyl-aryl or (C 1 6 )alkyl-Het; and d) N(R 35 2 wherein each R 35 1IS independently H, (C 1 .6)alkyl, (C3- 7 )cycloalkyl, (C 1 .8)alkyI-(C 37 )cycloalkyl, aryl, Het, (Cl. 6 )alkyl-aryl or (C 1 .6)alkyl-Het; or both R 35 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; with the proviso that when A is not N, then one of A or B is either CR 1 or CR 3 K is N or CR wherein R 4 Is H, halogen, (C 14 6)alkyl, haloalkyl, (C 37 )cycloalkyl or (C 1 B)alkyl-(C 37 )cycloalkyl; or R 4 is OR 41 or SR 41 COR 41 or NR 41 00R 4 1 wherein each R 41 is independently H, (C 1 .e)alkyl), (C 3 7 )cycloalkyl or (Cl. 8 )alkyl-(C 3 7 )cycloalkyl; 71 Q) or R 4 is NR 42 R' wherein and R' are each independently H, (Cl-6)alkyl, (C 3 00 7 )cycloalkyl, (C 16 )alkyl-(C 37 )cycloalkyI, or both R 1 2 and R' are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated hete rocycle; L sNo R 6 hri 5 hsteamdeninasR 4 dfndaoe M is N or CR 7 wherein R 7 has the same definition as R 4 defined above; R 5 is C(Y')Z wherein Y' is 0 or S; Z is N(R6B)R6 or OR 6 wherein R 6 a is H or (C 1 .6)alkyl or NR 61 R 62 wherein R 6 1 and R62 are each independently H, (C 16 )alkyl, (C 37 )cycloalkyl, (Cl. 6 )alkyl-(C 37 )cycloalkyl. or both R 61 and R 62 are covalently bonded together and to the nitrogen to which they are both attached to form a 5, 6 or 7-membered saturated heterocycle; or R 62 IS COO R63wherein R 6 3 is (Cl. 6 )alkyl, (C 3 6 )cycloalkyl, said alkyl or cycloalkyl being optionally substituted with 6- or lO-membered aryl or Het; or R 62 is CO R 6 whereln R64is C 1 6 ,)alkyl, (C 3 6 )cycloalkyl -6-or 1 O-membered aryl or Het; and R 6 is selected from the group consisting of: H, (Cl.e)alkyl, (C 36 )cycloalkyl, (C 26 )alkenyl, 6- or 1 0-membered aryl, Het, (C 1 6 )alkyl-aryl, (Cl. 6 )alkyl-Het, wherein said alkyl, cycloalkyl, alkenyl, aryl, Het, alkyl-aryl, or alkyl-Het, are all optionally substituted with R 60 or Rr'is 7 R 8 Y 2 wherein R 7 and R 8 are each independently H, (C 14 6)alkyl, haloalkyl, (C 3 7 )cycloalkyl, 6- or 1i0-membered aryl, Het, (C 16 )alkyl-aryl, (C 14 6)alkyl-Het, wherein said alky, cycloalkyl, aryl, Het, (Cl. 6 )alkyl-aryl, (Cl. 6 )alkyl-Het are optionally substituted with R 7 0 or R 7 and Ra are covalently bonded together to form second (C 37 )cycloalkyl or a 4, 5- or 72 C0 6-membered heterocycle having from 1 to 4 heteroatomn selected from 0, N, and S; 00 or when Z is N(R60)R 6, either of R 7 or R 8 is covalently bonded to R 6a to form a nitrogen-containing 5-or 6-membered heterocycle; IND 5 y 2 is or S; c-i R 9 is H, (C 15 alkyl), (C 37 )cycloalkyl or (Cl. 6 )alkyl-(0 3 7 )cycloalkyI, aryl, Het, (Cle)alkyl- aryl or (C 16 )alkyl-Het, all of which optionally substituted with R 90 or ciR 9 is covalently bonded to either of R 7 or R' to form a 5- or 6-membered heterocycle; Q Is a 6- or 1 0-membered aryl, Het, (C1.6) alkyl-CONH-aryl or (01.6) alkyl-CONH-Het, all of which being optionally substituted with: OHO 0 0 H GKOoH or a salt or a derivative thereof; wherein Hellis defined as 5- or 6-membered heterocycle having 1 to 4 heteroatoms selected from 0, N, and S, or a 9- or lO-membered heterobicycle having 1 to 4 heteroatoms selected from 0, N and S; and R1 0 R 20 R 60 R 70 R 9 0 and WOO 0 is each defined as: 1 to 4 substituents selected from: halogen, OPO 3 H, N0 2 cyano, azido, C(=NH)NH 2 C(=NH)NH(C 1 6 )alkyl or C(=NH)NHCO(C 1 .a)alkyl; or 1 to 4 substituents selected from: a) (C 1 alkyl or haloalkyl, (C 37 )CYCloalkyl, 03.7 spirocycloalkyl optionally containing 1 or 2 heteroatom, (C _)alkenyl, (C 2 8 )alkynyl, (C1.6) alkyl-(C 3 7 )cycloalkyl, all of which optionally substituted with R 150 b) OR 1 04wherein R 1 1 4 is H, (Cl.6alkyl), (C 3 7 )CYCloalkyl, or (Cl. 8 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (C 14 6alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (C 16 alkyl)Het being optionally substituted with 30R10 c) OCOR 1 05 wherein R 1 05is (0 16 )alkyl, (C 37 )cycloalkyl, (0 1 6 )alkyl-(C3. 7 )cycloalkyl, Het, (Cl- 5 alkyl)aryl or (Cl- 6 alkyl)Het, said alkyl, cycloalkyl, aryl, 73 O Het, (C 16 alkyl)aryl or (Cl. 6 alkyl)I-et being optionally substituted with R' 50 00 d) SR 10 8 SO 3 H, SO 2 N(Rl 1 2 or S0 2 N(R' 0 8 )C(O)R 1 08 wherein each R 1 0 8 is independentlyH, (Cl. 6 )alkyl, (0 3 7 )CYCloalkyl or (C i: 6 )alkyl-(C3.7)cycloalkyl, aryl, Het, (Cl-ralkyl)aryl or (Cl-6alkyl)Het or both R 10 8 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7- membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (C 1 .6alkyl)Het or heterocycle being optionally substituted with e) NR" 1 R' 1 2 wherein R'1 1 is H, (C 16 )alkyl, (C 3 7 )cycIOalkyl or (Cl. 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl. 6 alkyI)aryl or (Cl. 6 alkyl)Het, and R1 2 is H, CN, (C 1 6 )alkyl, (C 37 )cycloalkyl or (C 1 -6)alkyi-(C 3 7 )cycloalkyl, aryl, Het, (0 1 6 alkyl)aryl, (Cl,. 6 alkyl)Het COOR" 5 or SO 2 R" 5 wherein R11 5 is (Cl. 6 )alkyl, (03. 7 )cycloalkyl, or (Cj-6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C1. 6 alkyl)aryl or (Ci. r 6 alkyl)Het, or both R 11 and R' 12 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (C 1 6 alkyl)Het, or heterocycle being optionally substituted with R 1 50 f) NR 116 00R" 7 wherein R1 16 and R 1 17 is each H, (C 1 .6)alkyl, (0 3 7 )cycloalkyl, (Cl- 6 )alkyl-(C 3 .7)cycloalkyl, aryl, Het, (G 1 6 alkyl)aryl or (Cl. 6 alkyl)I-et, said (Ci. 6 )alkyl, (C 3 7 )cycloalkyl, (Cl-6)alkyl-(C 37 )cycloalkyl, aryl, Het, (C 16 allkyl)aryl or (Cl-6aikyl)Het being optionally substituted with R 150 g) NR' 18 00NR' 19 R 0 wherein R"1 8 R" 9 and R' 2 is each H, (C 16 )alkyl, (03. 7 )cycloalkyl, (C 1 6 )alkyl-(C 3 7 )cycloalkyl, ary, Het, 6 alkyl)aryl or (Cl- 6 alkyl)Het, or 111" is covalently bonded to R' 19 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R11 9 and R 1 20 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (Cl-6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 1 .&alkyl)aryI or (Cl. 6 alkyl)Het or heterocycle being optionally substituted with R1 50 h) NR 121 COCOR 1 22 wherein RA' 2 and R"2 is each is H, (Cl. 6 )alkyl, (03. 7 )cycloalkyl, (Cj. 6 )alkyl-(C 3 7 )cycloalkyl, a 6- or 1O-membered aryl, Het, (Cl. 6 alkyl)aryl or (C 1 .6alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, 6 alkyl)aryl or (C 1 -6alkyl)Het being optionally substituted with F' 5 orI A' 22 is OR'1 23 or N(R 1 24 2 wherein R'1 23 and each R'1 24 is independently H, 74 C0 (Cl. 6 alkyI), (C 37 )cycloalkyl, or (C 16 )alkyl-(C 37 )cycloalkyl, aryl, Het, (Cl. 00 6 alkyl)aryl or (Cl. 6 alky)Het, or R 124 is OH or O(Cl. 6 alkyl) or both R 124 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C 1 .calkyl)aryl or (0 1 6 alkyl)Het and heterocycle being optionally substituted with R1 50 i) COR 1 z? wherein R 1 27 is H, (0 16 )alkyl, (C 37 )cycloalkyl or (C 1 6 )alkyl-(C3- 7 )cycloalkyl, aryl, Het, (C 1 6 alkyl)ary or (C 1 -6alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (Cl 16 alkyl)aryl or (Cl 15 alkyI)Het being optionally substituted with R'1 50 (ij) COOR 1 28 wherein 1 2 is H, (C 1 -6)alkyl, (C 3 7 )cycloalkyl, or(Cl. 6 )alky-(C3- 7 )cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (C 1 .calkyl)Het, said (0 1 6 )alkyl, (03. 7 )cycloalkyl, or(Cl. 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 1 .ralkyl)aryl and (Cl. 6 alkyl)Het being optionally substituted with R' 50 k) CONR 12 9 R 1 30 wherein R 12 and R 130 are independently H, (C 1 6 alkyl, (C3. 7 )cycloalkyl, (Cl. 6 )alky-(C 3 7 )cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (C 1 6 alkyi)Het, or both R 129 and R 1 30 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl, (C 16 alkyl)Het and heterocycle being optionally substituted with R 150 1) aryl, Het, (C 1 6 alkyl)aryl or (Cl. 6 alkyl)Het, all of which being optionally substituted with R 15 and wherein R150 is defined as: 1 to 3 substituents selected from: halogen, OPO 3 H, N0 2 cyano, azido, C(=NH)NH 2 C(=NH)NH(C 16 )alkyl or C(=NH)NHCO(C 1 0 )alkyl; or 1 to 3 substituents selected from: a) (C1.6) alkyl or haloalkyl, (C 3 7 )cycloalkyl, 03.7 spirocycloalkyl optionally containing 1 or 2 heteroatom, (C 2 .e)alkenyl, (C 2 -8)alkynyl, (CI-6) alkYl-(C 37 )cycloalkyl, all of which optionally substituted with R 1 60 b) OR 104 wherein R 1 4 is H, (C 16 alkyl), (C 3 7 )cycloalkyl, or (CI. 6 )alkyl- (C 3 7 )CYCloalkyl, aryl, Het, (C 16 alkyl)aryl or (0 1 6 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (Cl-6alkyl)aryl or (C 1 -6alkyl)Het being optionally substituted with R'1 60 c) OCOR 1 06 wherein R 10 is (Cl-6)alkyl, (C 3 7 cycloalkyl, (C 1 -6)alkyl-(C 3 7 )cycloalkyl, Het, (C 16 alkyl)aryl or (Ci. 6 alkyl)Het, said alkyl, cycloalkyl, C.) 00 with R 1 60 d) SROB, SO 3 H, SO 2 N(R' 08 2 or SO 2 N(R1 0 8 )C(O)R 1 0 8 wherein each 11 1 08 is independently H, (C 16 )alkyl, (C 3 7 )cycloalkyl or (Cl-6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl. 6 alkyl)Het or both R 108 are covalently bonded together and to the nitrogen to which they are ri attached to form a 5, 6 or 7-membered saturated heterocycle, said *alkyl, cycloalkyl, aryl, Het, (C 1 -6alkyl)aryl or (C 16 alkyl)Het or heterocycle being optionally substituted with R 160 e) NR 1 11 R 1 12 wherein is H, (C 16 )alkyl, (C3. 7 )cycloalkyl or (Ci. 6 )alkyl-(C3.7)cycloalkyl, aryl, Het, (C 1 .6alkyl)aryl or (C 16 alkyi)I-et, and R' 12 is H, CN, (Cl. 6 )alkyl, (C 37 )CYCloalkyl or (Cl. 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 1 .6alkyl)aryl, (C 1 6 alkyl)Het COOR 1 5 or S0 2 114 15 wherein R"1 5 IS (Cl. 6 )alkyl, (C3. 7 )cycloalkyl, or (C 1 6 3)alkyl-(C3. 7 )CYCloalkyl1, aryl, Het, (Cl. 6 alkyl)aryl or (Cl. 6 alkyl)I-et, or both 1 1 '1 and R' 2 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (C 16 alkyl)Het, or heterocycle being optionally substituted with R 1 60 f) NR' 6 COW 17 wherein R 116 and R 11 7 is each H, (C 1 .6)alkyl, AC. 7 )cycloalkyl, (Cj- 6 )alkYl-(C 37 )CYCloalky, aryl, Het, (Cl 16 alkyl)aryl or (Cl. 6 alkyI)Het, said (C 1 .6)alkyl, (C 37 )CYcloalkyl, (C 1 6 )alkyl-(C 3 7 )CYCloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (C 16 alkyl)Het being optionally substituted with R 1 60 g) NR 118 C0NR 19 R 2 wherein R 118 R" 9 and R 12 is each H, (C 1 6 )alkyl, (C 37 )cycloalkyl, (Cl. 6 )alkyl-(C 37 )CYCloalkyl, aryl, Het, (C 1 6 alkyI)aryl or (Cl-6alkyi)Het, or R" is cova lently bonded to R 119 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or 1 1 and R 12 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (Cl. 6 )alkyl-(C 3 7 )CYCloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (Cl. 6 alkyl)Het or heterocycle being optionally substituted 76 CQ with R' 6 00 h) NR 121 COCOR 1 22 wherein R 121 and R 1 22 is each is H, (0 1 e6)alkyl, (C 37 )cycloalkyl, (CI- 6 )alkyl-(C3.7cycloalkyl, a 6- or 1O-membered aryl, Het, (C 16 alkyl)aryl or (C 1 6 alkyl)Het, said alky, cycloalkyl, alkyl- cycloalkyl, aryl, Het, (C 16 alkyI)aryl or (C 1 6 alkyl)Het being optionally substituted with R 1 60 123 12 ri or R'22 is OR 12 or N(R 2 2 wherein R 123 and each is independently H, (Cl. 6 alkyl), (C 3 7 )cycioalkyl, or (C 14 6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl.Balkyl)aryl or (Cl. 6 alkyl)Het, or R' 4 i OH or O(Cl, 6 alkyl) or both R 1 24 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl- cycloalkyl, aryl, Het, (C 14 6alkyI)aryl or (Cl-6alkyl)Het and heterocycle being optionally substituted with R 1 60 1) COR 1 27 wherein R 1 27 is H, (C 1 .6)alkyl, (C 37 )cycloalkyl or (C,-6)alkyl- (C 37 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl.6alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C 1 .6alkyl)aryI or (Cl. 6 alkyl)I-et being optionally substituted with R 16 0 J) tetrazole, COOR' 8 wherein R 1 2 1 is H, (C 1 .B)alkyl, (C3. 7 )cycloalkyl, or(Cl.,)alkyl-(C 3 q7)cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (C 1 6 alkyl)Het, said (C 1 -6)alkyI, (C3. 7 )cycloalkyl, or(C,. 6 )alkyl-(C3aq)cyclolkyl, aryl, Het, (Cl. 6 alkyl)aryl and (Cl 16 alkyl)Het being optionally substituted with R 1 60 and k) CONR 1 2 gR 30 wherein R 1 29 and R 1 30 are independently H, (C 1 6 )alkyl, (C 3 7 )cycloalkyl, (C 1 6 )alk yl.(C 37 )cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (Cl. 6 alkyl)Het, or both R 1 29 and R 1 30 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C 1 -6alkyl)aryI, (C 1 -6alkyl)Het and heterocycle being optionally substituted with R 1 0 wherein R 1 60 is defined as 1 or 2 substituents selected from: tetrazole, halogen, ON, C 16 alkyl, haloalkyl, COOR' 61 SO 3 H, SR 161 S0 2 R 161 OR'1 61 N(R 1 62 2 SO 2 N(R 1 62 2 or CON (R 16') 2 wherein R 1 6 1 and each R 1 62 is independently H, (C 1 6 )alkyl, (03. 7 )cycloalkyi or (C 16 )alkyl-(C 3 7 )cycloalkyl; or both R 162 are 77 C.) 00 are attached to form a 5, 6 or 7-membered saturated heterocycle; wherein said probe comprises a detectable label attached to any suitable position, whereby said probe binds to an HCV polymerase or an analog thereof and is capable of being displaced by an inhibitor thereof; 3. The method according to claim 2, wherein said probe has the following formula: R 5 N NN R 3 R 3 R 3 Ia lb or IC wherein R 1 is selected from the group consisting of: H or (C 1 6 )alkyl; R 2 is CON(R 22 2 wherein each R22is independently H, (C 16 )alkyl, (C3- 7 )cycloalkyl, (C 5 .4)cycloalkenyl, 6 or 1O-membered aryl or Het, or both R22are bonded together to form a 5, 6 or 7-membered saturated heterocycle with the nitrogen to which they are attached; or A 2 is selected from: H, halogen, (C 1 .6)alkyl, haloalkyl, (C 2 -6)alkenyl, (C 5 7 )cycloalkenyl, 6 or 1 0-membered aryl or Het; wherein each of said alkyl, haloalkyl, (C 24 6)alkenyl, (C 5 7 )cycloalkenyl, aryl or Het is optionally substituted with R 20 wherein R 20 is defined as: 1 to 4 substituents selected from: halogen, N0 2 cyano, azido, C(=NH)NH 2 C(=NH)NH(Cl.6)alkyl or C(=NH)NHCO(C1 6 )alkyl; or 1 to 4 substituents selected from: a) (C 1 6 alkyl or haloalkyl, (C 37 )cycloalkyl, (C 2 -6)alkenyl, (C 28 )alkynyl, (0.6) alkyl-(C 37 )cycloalkyl, all of which optionally substituted with 11315; b) 0 R 1 04wherein R 104 is H, (Cl. 6 alkyl), (C3- 7 )cycloalkyl, or (Cl-6)alkyl-(C3. 7 )cycloalkyl, aryl, Het, (Cl 16 alkyl)aryl or (Cl-6alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (Cl.ralkyl)aryl or (Cl. 6 alkyl)Het being optionally substituted with 78 Q c) 000R 05 wherein 11 1 05 is (C 16 )alkyl, (C3- 7 )cycloalkyl, (C 1 -6)alkyl-(C 3 00 7 )cycloalkyl, Het, (C 16 alkyl)aryl or (Cl.6alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C 1 -6alkyl)aryl or (Cl-ealkyl)Het being optionally substituted with R1 50 d) SR' 08 SO 3 H, S0 2 N(R' 08 2 or SO 2 N(R 1 08 )C(O)R' 0 8 wherein each Fil" is independently H, (Cl. 6 )alkyl, (C 3 7 )cycloalkyl or (Cl-6)alkyl-(C 37 )cycloalkyI, aryl, Het, (C 1 -6alkyl)aryl or (0 1 6 alkyl)H-et or both R 1 08 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7- membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (Cl. 6 alkyl)Het or heterocycle being optionally substituted with 10R50 e) NR 1 1 1 R 1 1 2 wherein R' 1 1 is H, (C 16 )alkyl, (C 3 7 )cycloalkyl or (Cl.6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl-6alkyl)aryl or (C 16 alkyl)Het, and R 1 1 2 is H, ON, (Cl. ,,)alkyl, (C 37 )cycloalkyl or (Cj-6)alkyl(C3. 7 )CYCloalkyl, aryl, H-et, (Cl-6alkyl)aryl, (C 1 6 alky)Het, COOR"' or SO 2 R1 15 wherein R 1 15 is (0 1 6 )alkyl, (P3. 7 )cycloalkyl, or (CI-6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl.6alkyl)aryl or (CI. 6 alkyl)Het, or both R 111 and R 11 2 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (CI-6alkyl)Het, or heterocycle being optionally substituted with R 150 f) NR 116 C0R 1 17 wherein R' 16 and R 1 1 7 is each H, (C 1 6 )alkyl, (C 37 )cycloalkyl, (C 16 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl-ealkyl)Het, said (Cl. 6 )alkyl, (C 3 7 )cycloalkyl, (C 16 )alkyi-(C 3 .7)cycloalkyl, aryl, Het, (Ci 6 alkyl)aryl or (Cl 4 alkyl)Het being optionally substituted with R' 50 g) NR 118 00NR 19 R 2 wherein R 118 R 119 and R 1 20 is each H, (Cl.6)alkyl, (03. 7 )cycloalkyl, (Cl-6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 16 alkyI)aryl or (Ci. 6 alkyl)Het, or R 1 "3 is covalently bonded to R'1 9 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R' and R 120 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (Cj. 6 )aikyI-(C 3 7 )cycloalkyl, aryl, Het, (C 1 5 alkyI)arl or (Ci. r 6 alkyl)Het or heterocycle being optionally substituted with RI 5 1; h) NR 121 C000R 22 wherein R 12 1 and R122 is each is H, (C, 8 6)alkyl, (C 3 7 )cycloalkyl, (CI- 6 )alkyl-(C3. 7 )cycloalkyl, a 6- or 1 0-membered aryl, Het, 6 alkyl)aryl or (Cl. 6 alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, 79 Q (C1. 6 alkyl)aryl or (Cl. 6 alkyl)Het being optionally substituted with R 1 6 0 00or R 1 22is OR 123 or N(R 1 24 2 wherein R 12'and each R 1 24 is independently H, (Cl.ealkyl), (C 37 )cycloalkyl, or (G 1 6 )alkyI-(C3. 7 )CY~loaikyl, aryl, Het, (Cl. 6 alkyl)aryl or (C 1 6 alkyl)Het, or R 1 24 is OH or O(C 16 alkyl) or both R' 2 4 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C1. 6 alkyl)aryl or (0 1 8 alkyl)Het and heterocycle being optionally substituted with RI' 0 1) COR 2 wherein RIV is H, (Cl. 6 alkyl, (C 3 7 )cycloalkyl or (Cl. 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 14 6alky)aryl or (Cl. 4 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (C1. 6 alkyl)Het being optionally substituted with R1 50 j) CR1 28 wherein R 1 28 is H, (C 16 )alkyl, (C 37 )cycloalkyl, or(C, 4 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 1 6 alky)aryl or (C 1 .6alkyl)Het, said (C 16 )alkyl, (03. 7 )cycloalkyl, o r(Cl. 6 )alkyl-(C 37 )CYCloalkyl, aryl, Het, (CI 6 alkyl)aryl and (Cl. 6 alkyl)Het being optionally substituted with R1 50 k) CONR 1 29 R 1 30 wherein and R 13 are independently H, (Cl.r,)alkyl, (C 3 7 )cycloalkyl, (C 16 )alkyl-(C 37 )CYCloalkyl, aryl, Het, (C 16 alkyl)aryl or 6 alkyl)Het, or both R 1 29 and R 130 are Covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl, (Cl. 6 alkyl)Het and heterocycle being optionally substituted with R 1 1) aryl, Het, (Cl -6alkyl)aryl or (C1I-6alkyl)Het, all of which being optionally substituted with R 1 50 wherein R 1 50 is preferably: 1 to 3 substituents selected from: halogen, N0 2 cyano or azido; or 1 to 3 substituents selected from: a) (0146) alkyl or haloalkyl, (C 3 7 )cycloalkyl, (C 24 &)alkenyl, (C 24 8)alkynyl, (0146) alkyl-(C 37 )cycloalkyl, all of which optionally substituted with R1 60 b) OR104wherein R104 is H, (C 16 alkyl) or (C 3 7 )cycloalkyl, said alkyl or cycloalkyl optionally substituted with R' 60 d) SR' 08 SO 3 H, S0 2 N(R' 08 2 or SO 2 N(R 108 )C(O)R' 08 wherein each R' 0 8 is independently H, (Cl. 6 )alkyl, (C 37 )cycloalkyl or (Cl. 6 )alkyl-(C 3 7 )CYCloalkyl, aryl, Het, or both R1 08 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7- membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het and C0 heterocycle being optionally substituted with R 160 00 e) NR 1 12 wherein is H, (C 1 6 )alkyl, or (C 3 7 )CYCloalkyl, and R 112 is H, (C 1 6 )alkyl or (C 3 -7)cycloalkyl, C00R 15 or S0 2 R"5wherein 13 11 is (Cl.r,)alkyl or (C 37 )cycloalkyl, or both R 11 1 and R 112 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl and heterocycle being optionally substituted with R 160 f) N R' 16 00R 1 7 wherein R 1 16 and R 117 is each H, (Cl. 6 )alkyl or (C 3 7 )cycloalkyl said (C 16 )alkyl and (C3. 7 )CYCloalkyl being optionally substituted with R'1 60 g) NR 1 1 8 C0NR 1 9 R 120 wherein R' 1 8 R 1 1 9 and R 120 is each H, (C 1 6 )alkyl or (C 37 )cycloalkyl, or R"5 is covalently bonded to R 119 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R 119 and R 120 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, and heterocycle being optionally substituted with R 160 h) NR 1 21 COCOR 1 22 wherein R 121 is H, (Cl-6)alkyl or (C 3 7 )cycloalkyl, said alkyl and cycloalkyl being optionally substituted with R 160 or R'22 is OR 123 or N(R 124 2 wherein R 12 3 and each R 12 1 is independently H, (Cl 16 alkyl) or (C 37 )cycloalkyl, or both R 1 24 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl and heterocycle being optionally substituted with R 1 60 1) COR 1 27 wherein Rl27 is H, (C 16 )alkyl or (C 37 )cycloalkyl, said alkyl and cycloalkyl being optionally substituted with R 1 60 j) COOR 1 28 wherein R 1 28 is H, (C1.e)alkyl or (C 3 .7)cycloalkyl, said (Cl-6)alkyl and (C3. 7 )CYCloalkyl being optionally substituted with R 16 0 and k) CONR 1' 2 "R 130 wherein R 12' and R' 30 are independently H, (C 1 6 )alkyl or (C3- 7 )cycloalkyl, or both R 1 29 and R 1 30 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 81 C.) 00 heterocycle being optionally substituted with R 1 60 wherein R 1 60 is defined as 1 or 2 substituents selected from: halogen, CN, C,-6alkyl, haloalkyl, COOR 1 61 OR'1 61 N(R 1 62 2 SOANR 162 2 or CON(R 1 62 2 wherein R 1 61 and each R 1 1 2 is independently H, (Cl. 6 )alkyl, (C 37 )cycloalkyl or (C 1 -6)alkyl-(C 3 7 )cycloalkyl; or both R' 6 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7- membered saturated heterocycle; R 3 is selected from (C3- 7 )cycloalkyl, (C 6 10 )bicycloalkyl, 6- or 1 0-membered aryl, or Het; R 5 is wherein Z is OR 6 wherein R 6 is C1-6alkyl substituted with: 1 to 4 substituents selected from: OPO 3 H-1, NO 2 cyano, azido, C(=NH)NH 2 C(=NH)NH(CI-6)alkyl or C(=NH)NHCO(Ci. 6 )alkyl; or 1 to 4 substituents selected from: a) (01.6) alkyl or haloalkyl, (C 3 7 )cycloalkyl, C3.7 spirocycloalkyl optionally containing 1 or 2 heteroatorn, (C 26 )alkenyl, (C 2 -8)aikynyl, 6 alkyl-(C 3 7 )cycloalkyl, all of which optionally substituted with R 150 lb) OR'04 wherein R' 04 is 6 alkyl) substituted with (C 3 7 )cycloalkyl, or (CI- 6 )alkyl-(C3a.4cycloalkyl, aryl, Het, (C1. 6 alkyl)aryl or 6 alkyl)Het, said cycloalkyl, aryl, Het, (C1. 6 alkyl)aryl or (Cl. 6 alkyl)Het being optionally substituted with R 15 0 c) OCOR' 0 s wherein R' 05 is 6 )alkyl, (C 37 )cycloalkyl, (Cl. 6 )alkyl-(C3. 7 )cycloalkyl, Het, (Cl 16 alkyl)aryl or (Cj,,,alkyI)Het, said alkyl, cycloalkyl, aryl, Het, (C1. 6 alkyI)aryl or (Cl. 6 alkyl)Het being optionally substituted with R, 50 ;1 d) SR 1 0 8 SO 3 H, SO 2 N(R 08 2 or S0 2 N(R' 0 8 )C(O)R 1 08 wherein each R1 0 8 Is independently H, (C,.6)alkyl, (0 37 )cycloalkyl or (Cl-6)alkyl-(C3. 7 )CYCloalkyl, aryl, Het, 6 alkyl)aryl or (Cl, 6 alkyl)Het or both R108 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7- membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Cl. 82 C0 6 alkyi)aryl or (Cl-6alkyl)Het or heterocycle being optionally substituted with 00 R o e) NR R 12 wherein R 111 is (Ci 6 )alkyl substituted with R 150 (C 37 )cycloaikyl or (C 1 6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (C 1 -6alkyl)aryl or (Cl-6alkyl)Het, and R1 12 is CN, (Cl. 6 )alkyl substituted with R' 5 0 (C3- 7 )cycloalkyl or (Cl-6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl, (C 1 -6alkyl)Het, C00R 115 or S0 2 R 11 (Ni wherein R 1 5 Is (C 16 )alkyi, (C 37 )cycloaikyi, or (Cl.6)aikyi-(C 37 )cycioalkyi, aryl, Het, (C 16 alkyl)aryi or (Cl. 6 alkyi)Het, or both R' 11 and R 1 1 are covalently bonded together and to the nitrogen to which thiey are attached to form a 5, 6 or 7-membered saturated heterocycle, said cycloalkyl, aryl, Het, (C 1 6 aikyl)aryl or (Cl-6aikyl)Het, or heterocycle being optionally substituted with R 150 f) NR 116 00R' 17 wherein R' 1 6 and R'1 7 is each H, (C 16 )aikyl, (C 37 )cycloalkyi, (CI-6)akyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl-6alkyl)aryl or (C. 6 alky)Het, said (Cl. 6 )alkyi, (C 37 )cycloalkyl, (C 1 .6)alkyl-(C 3 7 )cyc:IoakIW, aryl, Het, (C 1 6 alkyl)aryl or (Cl-6alkyl)Het being optionally substituted with R'so; g) NR"lBCONR'1 9 R 2 wherein R" 18 R 119 and R' 2 is each H, (C 1 .O)alkyl, (C 3 7 )cycloalkyl, (C 16 )alkyl-(C 3 .7)cycloalkyI, aryl, Het, (Cl-6alkyl)ay or (Cl. 6 alkyl)I-et, or R 1 1 8 is covalently bonded to R1 19 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R 11 9 and R'1 2 are covaiently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyi, cycloalkyi, (C 16 )aikyl-(C 37 )cycioaikyi, aryl, Het, (C 16 alkyl)aryl or (Cl. 6 aikyl)Het or heterocycie being optionally substituted with R' 5 0 h) NR 121COCORI22 wherein R 1 21 and R 1 22 is each is H, (C 1 .6)alkyl, (C 3 7 )cycloalkyl, (Cj. 6 )alkyl-(C 3 7 )cycloalkyl, a 6- or lO-membered aryl, Het, (Cl. 6 3alkyl)aryl or (Cl. 6 alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloakyl, aryl, Het, (C 16 alkyl)aryl or (C 1 lkyl)Het being optionally substituted with R's 0 or R 1 22 is OR 123 or N(R 124 2 wherein R 1 2 3 and each R 1 2 1 is independently H, (C 1 6 alkyl), (C 3 .7)cycloalkyl, or (Cl- 6 )aikyl-(C 37 )cycioalkyl, aryl, Het, (Cl. 6 alkyi)aryi or (Cl. 6 alkyl)Het, or R 12 4 is OH or O(Cl-6alkyl) or both R 124 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Hetl, (C 1 6 alkyl)aryl or (Cl- 6 alkyl)Het and heterocycle being optionally substituted with R'1 50 1) C0R 127 wherein R'27 is H, (C 1 6 )alkyl, (C 37 )cycloalkyl or (Cl. 6 )alkyi-(C 3 83 CQ 7 )cycloalkyl, ary'l, Het, (C 16 alkyl)aryl or (Cl. 6 alkyl)Het, said alkyl, cycloalkyl, 00 aryl, Het, (Cl. 6 alkyl)aryl or (Cl. 6 alkyl)Het being optionally substituted with R 150 j) COOR'28 wherein R 128 is (0 1 6 )alkyl substituted with R' 5 0 (C 37 )cycloalkyl, or(Cj. 6 )alkyl-(C 37 )cClCoalkyl, aryl, Het, (C 1 6 alkyl)aryl or (Cl. 6 alkyl)Het, said (C 37 )cycloalkyl, or(0 1 6 )alkyl-(0 3 7 )cycloalkyl, aryl, Het, (0 1 6 alkyI)aryl and (Cl. 6 alkyl)Het being optionally substituted with R 150 12 13 1 k) CONR 29 R wherein R 12 and R' 30 are independently H, (Cl. 6 )alkyl, (03 7 )CYCloalkyl, (Cl. 6 )alkyl-(C3. 7 )cycloalkyl, aryl, Het, (0 16 alkyl)aryl or (C 1 12 130 6 alkyl)Het, or both R and R 0are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C 1 6 alky'l)aryl, (Cl. 6 alkyI)Het and heterocycle being optionally substituted with R' 50 1) aryl, Het, (Cl-ealkyl)aryl or (Cl. 6 alkyl)Het, all of which being optionally substituted with R' 50 wherein R' 50 is: 1 to 3 substituents selected from: halogen, N0 2 cyano, azido or 1 to 3 substituents selected from: a) (01.6) alkyl or haloalkyl, (C 3 7 )cycloalkyl, C3-7 spirocycloalkyl optionally containing 1 or 2 heteroatom, (C2. 6 )alkenyl, (CI-6) alkyl-(C. 7 )cycloalkyl, all of which optionally substituted with R 160 b) OR'04 wherein R 1 04 is H, (C 1 -6alkyl), (C3. 7 )cycloalkyl, or (Cl-6)alkyl- (C 3 7 )CYCloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl. 6 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (C 1 6 alkyl)Het being optionally substituted with Rlr; d) SO 3 H, SO 2 N(R'6 8 2 or SO 2 N(R 1 O 8 )C(O)R 1 08 wherein each R 1 0 8 is independently H, (C 1 6 ,)alkyl, (C3. 7 )cycloalkyl or (C 16 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (Cl-6alkyI)Het or both R1 08 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C 14 6a~kyl)aryl or (0 16 alkyI)Het or heterocycle being optionally substituted with R 1 6 0 e) NR 111 R 1 12 wherein 11 is H, (0 1 6 )alkyl, (C 3 7 )cycloalkyl or (Cl. 6 )alkyl-(0 37 )cycloalkyl, aryl, Het, (C 16 alkyI)aryl or (Cl- 6 alkyl)Het, and R' 12 is H, (0 1 6 )alkyl, (C 37 )cycloalkyl or (Cj.. 6 )alkyl-(C3. 7 )cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl, (C 1 -6alkyl)Het, COOR 15 or S0 2 111 15 wherein R 1 1 84 C.) 00 (Cl. 6 alkyl)aryl or (C 1 6 3alkyl)Het, or both and R 1 1 2 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Cl-6alkyl)aryl or (Cl. 6 alkyl)Het, or heterocycle being optionally substituted with R1 60 f) N R' O0R' 1 wherein R' 1 and R1" is each H, (C 1 -e)alkyl, (C 3 7 )cycloalkyl, (Cl. 6 )alkyl-(C 3 -7)cycloalkyl, aryl, Het, (Cl.6alkyl)aryl or (Cl. 6 alkyl)Het, said (C 1 -6)aI kyl, (C 37 )cycloalkyl, (Cl. 6 )alkyl-(C3. 7 )cycloalkyl, aryl, Het, (Cl-ralkyl)aryl or (Cl-6alkyl)Het being optionally substituted with g) NR' 1 8 00NR1 9 R 1 20 1 wherein R11 8 R119 and R1' 20 is each H, (C 1 6 )alkyl, (C 3 7 )cycloalkyl, (Cl. 6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (C I. 6 alkyl)aryl or (Cl-6alkyl)Het, or R11 9 and R 1 20 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (Ci. 6)alkyl-(C 37 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl- 6 alkyI)Het or heterocycle being optionally substituted with R 160 h) NR 1 21 COCOR 1 22 wherein R 1 2 is H, (C,-6)alkyI optionally substituted with R 160 or R122 is OR'23 or N(R 1 24 2 wherein R 1 23 and each R 1 24 1IS independently H, (Cl- 6 alkyl), (C 3 7 )cycloalkyl, or (Cl.6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C1. 6 alkyl)aryl or (Cj-6alkyl)Het, or R'1 24 is OH or O(C,-6alkyl) or both R 124 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl- cycloalkyl, aryl, Het, (C 16 alkyi)aryl or (C1. 6 alkyl)Het and heterocycle being optionally substituted with R'1 60 J) tetrazole, COOR 128 wherein R'n is H, (C 16 )alkyl, (C 37 )cycloalkyl, or(Cl. 6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (Cl-6alkyl)aryl or (C 1 6 alkyl)Het, said (C1. 6 )alkyl, (C 37 )CYCloalkyl, or(Cj,. 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl-6alkyl)aryl and (Cj-6alkyl)Het being optionally substituted with Rl' 60 and k) CON R'9R 1 3 1 wherein R 12' and R 1 3 are independently H, (Cl. 6 )alkyl, (C3- 7 )cycloalkyl, 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl. 0 6 alkyl)aryl or (Cl. 6 alkyl)Het, or both R 129 and R 130 are covalently 00 bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (Cl.ralkyl)aryi, (C 16 alkyl)Het and heterocycle being optionally substituted with R 160 wherein R' 6 is defined as 1 or 2 substituents selected from: citetrazole, halogen, ON, C 16 alkyl, haloalkyl, CO0R 6 SO 3 H, S0 2 R 161 OR 1 6 1 N(R 6 2 S0 2 N(R1 62 2 or CON(R 162 2 wherein R 1 61 and each R1 62 iS independently H, (C 14 6)alkyl, (03. 7 )cycloalkyl or (C 16 )alkyl-(C 37 )cycloalkyl; or both R 1 62 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or Z is N(R 6 8)R 6 wherein R"a is H or (C 16 alkyl) and R 6 is (C 1 .6)alkyl optionally substituted with: 1 to 4 substituents selected from: OPO 3 H, NO 2 cyano, azido, C(=NH)NH 2 C(=NH)NH(C1 .6)alkyl or C(=NH)NHCO(Cl. 6 )alkyl; or 1 to 4 substituentS selected from: a) (0146) alkyl substituted with Risca, haloalkyl, (C 3 7 )cycloalkyl, 03.7 spirocycloalkyl optionally containing 1 or 2 heteroatom, (C 26 )alkenyl, (02. E 8 )alkynyl, (016) alkyl-(Ga. 7 )cycloalkyl, all of which optionally substituted with R 150 wherein Rl"O' is the same as R1 50 but is not halogen, OR' o, COOR 5 b N(R110b 2 wherein Rl 5 0b is H or C 14 6alkyl; b) OR 1 04wherein R 1 04 is (C 14 6alkyl) substituted with R1 50 (C 3 7 )cycloalkyl, or (Cl. 6 )alkyl.(C 3 -7)cYcloalkyl, aryl, Het, (C 1 .6alkyl)aryl or (C 14 6alkyl)Het, said cycloalkyl, aryl, Het, (Cl-6alkyI)aryl or (Cl-6alkyl)Het being optionally substituted with R 150 c) 000R 1 05 wherein R 1 0 5 is (0 14 )alkyl, (0 3 7 )cycloalkyl, (C 14 O)alkyl-(C 3 7 )cycloalkyl, Het, (Cl.6alkyl)aryl or (C 1 .6alkyl)H-et, said alkyl, cycloalkyl, aryl, Het, (0 16 alky1)aryl or (Cl- 6 alkyl)Het being optionally substituted with R 150 d) SO 3 H, SO 2 N(R 1 06 2 or SO 2 N(R'08)C(O)R 08 wherein each R1'B is independently H, (0 1 6 )alkyl, (0 3 7 )cycloalkyl or (C 16 )alkyl-(C 3 7)cycloalkyl, aryl, 86 C0 Het, (C 16 alkyl)aryl or (C1. 6 alkyI)Het or both R' are covalently bonded 00 together and to the nitrogen to which they are attached to form a 5, 6 or 7- membered saturated heterocycle, said alkyl, cycloalkyl, ary, Het, (Cl. 6 aikyl)aryl or (C 1 -6aiky!)Het or heterocycle being optionally substituted with R 150 e) NR 111 R 11 wherein 11 1 11 is (Ci.e)alkyl substituted with R 150 (C3- 7 )cycloalkyl ri or (C 16 )alkyl-(C 37 )cycloalkyl, aryl, Het, (C 1 6 alkyl)arYl or (C 1 -6alkyl)Het, and R 1 1 is H, ON, (C 16 )alkyl, (C 37 )cycloalkyl or (Cl. 6 )alkyl-(C 3 7 )cycloalkyl, aryl, (Ni Het, (Cl. 6 alkyl)aryl, (Cl. 6 aikyl)Het or R 11 is H and R"1 2 is S0 2 R1 1 5 wherein R 1 1 5 is (C 1 8 )alkyl, (C 37 )cycloalkyl, or (C 1 -6)alkyl-(C 3 7 )CYCloalkyl, aryl, Het, (Cl-6alkyl)aryl or (Cl 16 alkyl)Het, or both and R' 12 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said cycloalkyl, aryl, Het, (Cl-6alkyl)aryl or (C 1 6 alkyl)Het, or heterocycle being optionally substituted with R1 50 f) NR 11 6 00R 1 17 wherein R 1 1 6 and R 11 7 is each H, (C 1 .6)alkyl, (C 3 7 )cycloalkyl, (C 1 .6)alkyI-(C 3 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl. 6 alkyi)Het, said (Cl. 6 )alkyl, (C 37 )cycloalkyl, (Ci 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Ci. 6 alkyl)aryl or (Cl. 6 alkyI)Het being optionally substituted with 111 5 g) NRI1BCONRlGR 2 wherein R 118 R 11 and R 12 is each H, (Cl-6)alkyl, (03. 7 )CYCloalkyl, (Cj. 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl. 6 alkyl)Het, or 11 11 8 is covalently bonded to R' 1 9 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or 1 1 and R'1 20 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (C 1 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl 16 alkyl)aryl or (Ci. 6 alkyl)Het or heterocycle being optionally substituted with R 15 0 h) NR 12ICOCOR 1 22 wherein R 1 21 and R 1 22 is each is H, (Cl. 6 )alkyl, (03. 7 )cycloalkyl, (C 1 6 )alkyl-(C 37 )cycloalkyl, a 6- or 1O-membered aryl, Het, (Ci. 6 alkyl)aryl or (Cl-6alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C 1 -6alky1aryl or (Ci- 6 a~kyl)Het being optionally substituted with R 15 0 or R 1 22 is OR 123 or N(R 1 24 2 wherein R 1 23 and each R 1 24 is independently H, (C 1 6 alkyl), (C3-7)cycloalkyl, or (Cl.6)alkyl.(C. 7 )CYCloalkyl, aryl, Het, (Ci. 6 alkyl)aryl or (C 16 alkyl)Het, or R 1 24 is OH or O(C 16 alkyl) or both R 12 4 are c-I 87 C.) 00 heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (Cl.6alkyl)Het and heterocycle being optionally substituted with R' 50 1) COR 1 27 wherein R 1 27 is H, (Cl. 6 )alkyl, (0 3 7 )cycioalkyl or (C 1 .6)alkyl-(C 3 IND 5 7 )cycloalkyl, aryl, Het, (C 16 alkyI)aryl or (0 1 6 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C 14 6alky)aryl or (Cl.6alkyI)Het being optionally substituted with R 1 c-ij)COOR' 2 wherein R 1 28 Is (C 1 -6)alkyl substituted with R 5 0 (C3. 7 )cycloalkyI, or(Cl.r 6 )aikyl-(C 37 )CyCloalkyl, aryl, Het, (C 16 alkyl)aryl or (C 16 alkyl)Het, said (C 37 )cycloalkyl, or(Cl-6)alkyl-(C3. 7 )cycloalkyl, aryl, Het, (C 1 6 alkyl)aryll and (C 1 6 alkyl)Het being optionally substituted with R 150 k) CONR'9R 130 wherein R 12'and R 13 are independently H, (Cl. 6 )alkyl, (0. 7 )cycloalkyl, (Cl- 6 )alkyi-(C 37 )CYCloalkyl, aryl, Het, (C 16 alkyI)aryl or (C 1 6 alkyl)Het, or both R 129 and R 1 30 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (Cl-6alkyl)aryl, (Ci. 6 alkyl)Het and heterocycle being optionally substituted with R 1 50 1) aryl, Het, (C 1 6 alkyl)aryl or (C 16 alkyl)Het, all of which being optionally substituted with R 150 and wherein R' 50 is selected from: 1 to 3 substituents selected from: halogen, NO 2 cyano, azido or 1 to 3 substituents selected from: a) (CI-6) alkyl or haloalkyl, (C 37 )cycloalkyl, 03.7 spirocycloalkyl optionally containing 1 or 2 heteroatom, (C 2 6 ,)alkenyl, (Ci.6) alkyl-(C 3 7 )cycloalky, all of which optionally substituted with b) OR' 04 wherein R 1 0' is H, (Cl. 6 alkyl), (C 37 )cycloalkyi, or (C 16 )alkyl- (C 3 7 )CYCloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl.6alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (C 16 alkyl)Het being optionally substituted with R' 60 d) SO 3 H, SO 2 N(R' 08 2 or SO 2 N(R 10 8 )C(O)R' 0 8 3 wherein each R' 1 8 is independently H, (Cl-6)alkyl, (C 37 )cycloalkyl or (Cl. 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (0 1 .6aikyl)Het or both R 1 0Os are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (C 16 alkyl)Het or 88 0heterocycle being optionally substituted with R 1 60 00 e) NR R' 12 wherein is H, (C 1 -6)alkyl, (C 3 7 )cycloalkyl or (C 1 6 )alkyi-(C 37 )cycloalkyl, aryl, Het, (C 16 alkyI)aryl or (Cl. 6 alkyl)Het, and R" 2 is H, (C 16 )alkyl, (C 3 7 )CYCloalkyl or (CI. 6 )alkyl-(0 3 .7)cycloalkyl, aryl, Het, (C 1 .Eialkyl)aryl, (Cl.,alkyl)Het, COOR' or SO 2 R"rawherein R 11 M ~is (Cl. 6 )alkyl, (C 3 7 )CYCloalkyl, or (CI-6)alkyl-(C3- 7 )CYCloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (Cl-6alkyl)Het, or both R' 11 and R 1 12 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Clealkyl)aryl or (Cl.6alkyI)Het, or heterocycle being optionally substituted with R 1 60 t) N R' 6 COR' 17 wherein R 16 and R 1 17 is each H, (Cl. 6 )alkyl, (C3- 7 )cycloalkyl, (Cl. 6 )alkyl-(C3. 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl. 6 alkyl)Het, said (C 16 )alkyl, (C3. 7 )cycloalkyl, (C 1 6 )alkyl-(C 3 7 )cycloalkyl, aryi, Het, (Cl-6alkyl)aryl or (C 14 6alkyl)Het being optionally substituted with R 1 60 g) NR 11 8 CONR' 19 R 1 20, wherein R 1 1 8 R' 1 9 and R 12 0 is each H, (Cl. 6 )alkyl, (C3. 7 )cycloalkyl, (Cl-6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl- ealkyl)aryl or (Cl. 6 alkyl)Het, or R 11 9 and R 120 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (C 1 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (C 1 6 alkyl)Het or heterocycle being optionally substituted with R 1 60 h) NR 121 COCOR 1 22wherein R 1 21 is H, (C 16 )alkyl optionally substituted with R 160 or R'22 is OR 123 or N(R 1 24 2 wherein R'~and each R 1 24 is independently H, (C 16 alkyl), (C 37 )cycloalk-yi, or (C 1 6 )alkyl-(C3- 7 )cycloalkyl, aryl, Het, (Cl-6alkyl)aryl or (C 1 6 alkyl)Het, or R 124 is OH or O(C 1 6 alkyl) or both R 1 2 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl- cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (C 1 .&alkyl)Het and heterocycle being optionally substituted with R 160 J) tetrazole, C00R 128 wherein R 128 is H, (C 1 6 )alkyl, (C3. 7 )CYCloalkyl, or(C 1 6 )alkyl-(C3. 7 )CYCloalkyl, aryl, Het, (C 1 .Balkyl)aryl or (C 16 alkyl)Het, 89 0 said (Cl. 6 )alkyl, (C 37 )cycloalkyl, or(Ci.6)alkyl-(C3. 7 )cycloalkyl, aryl, Het, 00 (Cl. 6 alkyl)aryl and (C 1 .ralkyl)Het being optionally substituted with R1 60 and k) C0NR 129 R 130 wherein R 1 2 9 and R 130 are independently H, (C 1 6 )alkyl, (C 3 7 )CYCloalkyl, (C 14 6)alkyl-(C 37 )cycloaikyl, aryl, Het, (C 1 6 alkyl)aryl or (C 1 -6alkyI)Het, or both R 129 and R 130 are covalently bonded together and to the nitrogen to which they are attached to. form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl, (Cl. 6 alkyl)Het and heterocycle being optionally substituted with R1 60 wherein R 1 60 is defined as 1 or 2 substituents selected from. tetrazole, halogen, CN, C 16 alkyl, haloalkyl, COOR 6 SO 3 H, S0 2 R 6 OR 161 N(R 6 2 SOANR 6 2 or CON(R 6 2 wherein R 1 6 1 and each R 1 62 is independently H, (C 1 -6)alkyl, (C 3 7 )cycloalkyl or (Cj. 6 )alkyl-'(C 3 7 )cycloalkyl; or both R 1 62 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R 6 is R 7 R 8 R 9 y 2 wherein, preferably, R 7 and Re are each independently H, (C 16 )alkyl, haloalkyl, (03. 7 )cycloalkyl, 6- or 1lO-membered aryl, Het, (C 1 6 )alkyl-aryl, (CI-6)alkyl-Het, wherein said alkyl, cycloalkyl, aryl, Het, (C 16 )alkyl-aryl, (Cl. 6 )alkyl-Het are optionally substituted with R 70 or R 7 and Re are covalently bonded together to form second (C 37 )cycloalkyl or a 4, 5- or 6-membered heterocycle having from 1 to 3 heteroatom selected from 0, N, and S; or when Z is N(R 6 either of R 7 or R 8 is covalently bonded to R 6a to form a nitrogen-containing 5-or 6-membered heterocycle; wherein is selected from: 1 to 4 substltuents selected from: halogen, N0 2 cyano, azldo; or C.) 00 a) (01.6) alkyl or haloalkyl, (C 3 7 )cycloalkyl, C 3 7 spirocycloalkyl optionally containing 1 or 2 heteroatom, (C 26 )alkenyl, (C 2 8 )aikynyl, (C 14 6) alkyl-(C3- 7 )cycloalkyl, all of which optionally substituted with R 150 b) OR' 0 wherein R 1 0 is H, (Cl.ralkyl), (C 3 7 )cycloalkyl, or (Cl-6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (CI-6alkyl)aryl or (Cl. 6 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (Cl-6alkyl)Het being optionally substituted with R 150 d) SO 2 N(R 1 08 2 or SO 2 N(R 1 08)C(O)R 10 8 wherein each R 1 0 8 is independently H, (Cl. 6 )alkyl, (C 3 7 )cycloalkyl or (Cl. 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Ci. 6 alkyl)aryl or (Cl. 6 alkyI)Het or both A 1 08 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C 14 6alkyl)aryl or (Ci. 6 alkyl)I-et or heterocycle being optionally substituted with e) NR 1 1 1 R' 12 wherein R 111 is H, (C 16 )alkyl, (C 3 7 )cycloalkyl or (C,-6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl.6alkyl)aryl or (Cl. 6 alkyl)Het, and 1131 2 Is H, CN, (Ci. 6 )alkyl, (C3. 7 )cycloalkyl or (C 1 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl.6alkyl)aryl, (Cl-6alky)Het, COOR 11 5 or S0 2 R' 15 wherein R"1 5 is (C 16 )alkyl, (C3. 7 )cycloalkyl, or (C 16 )alkyl-(C 37 )cycloalkyl, aryl, Het, (Cj.6alkyl)aryl or (Ci. 6 alkyl)Het, or both R 1 11 and R 1 12 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C 16 alkyl)ary! or (Cl 14 alkyl)Het, or heterocycle being optionally substituted with R1 50 f) NR' 16 00OR 1 7 wherein R11 6 and R1 7 is each H, (CI- 6 )alkyl, (C 3 7 )cycloalkyl, (CI-6)alkyl-(C 3 7 )CYCloalkyl, aryl, Het, (Cl. 6 alkyI)aryl or (C 1 .6alkyI)Het, said (Cl. 6 ,)alkyl, (C 3 7 )cycloalkyl, (Cl-6)alkyl-(C 37 )cycloalkyl, aryl, Het, (Cl-6alkyI)aryl or (Cl 16 alkyl)Het being optionally substituted with g) NR' 18 00NR 119 R 2 wherein R 1 18 RA" 9 and 131 0 is each H, (Cl-6)alkl, (03. 7 )cycloalkyi, (C 14 6)alkyl-(C 37 )cycloalkyl, aryl, Het, (C 1 .6alkyl)aryl or (Ci. 6 alkyl)Het, or R 1 1 is covalenty bonded to R 119 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R1 19 and R 1 20 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalk-yl, (Cl. 6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (Ci. 91 C.)0 0 6 alkyl)Het or heterocycle being optionally substituted with R 0 00 NR 1 21 CC0R 122 wherein R 1 21 Is H, (C 1 .6)alkyl, (C 37 )cycloalkyl, (Cl-6)alkyI- (C 37 )cycloalkyl, a 6- or 1O-membered aryl, Het, (C 1 .6alkyl)aryl or (Ci. 6 alkyl)Het, said alkyl, cycloalky, alky-cycloalkyl, aryl, Het, (Cl-6alkyl)aryl or (C 14 6alkyl)Het being optionally substituted with R 15 and R'22 is OR'1 23 or N(R 1 24 2 wherein R123 and each R 124 is independently H, (Cl. 6 alkyI), (C 37 )cycloalkyl, or (Cl 16 )alkyl.(C 37 )CYCloalkyl, aryl, Het, (Cl- 6 alkyl)aryl or (C 1 6 alkyl)Het, or R' 4 i OH or O(C 1 6 alkyl) or both R 12 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C 16 ralkyl)aryl or (Cl 1 ,alkyl)Het and heterocycle being optionally substituted with R 150 1) COR 1 27 wherein R'27 is H, (Cl. 6 )alkyl, (C3. 7 )cycloalkyl or (Cl.6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 1 .6alkyl)aryl or (Cl., 6 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (C 16 alkyl)Het being optionally substituted with R 150 j) COOR 1 28 wherein R 1 28 is H, (C 16 )alkyl, (C 37 )CYCloalkyl, or(C 1 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (C 1 6 alkyl)Het, said (C 16 )alkyl, (C 3 7 )cycloalkyl, or(C 1 6 )alkyl-(C. 7 )CYCloalkyl, aryl, Het, (C 1 .6alkyl)aryl and (Cl. 6 alkyl)Het being optionally substituted with R 1 50 k) CONR12 9 R 130 wherein R 12 9 and R 1 30 are independently H, (C 1 .B)alkyl, (C 3 7 )cycloalkyl, (Cl. 6 )alkyl-(C3. 7 )cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (C 1 6 alkyl)Het, or both R 12 and R1 30 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (Cl- 6 alkyl)aryl, (C 16 alkyl)Het and heterocycle being optionally substituted with R1 so; 1) aryl, Het, (Cl. 6 alkyl)aryl or (Cl -6alkyl)Het, all of which being optionally substituted with R's 50 wherein R 160 is selected from: -1 to 3 substituents selected from: halogen, NO 2 cyano, azido; or -1 to 3 substituents selected from: 8) (C1.6) alkyl or haloalkyl, (C 37 )cycloalkyl, 03.7 spirocycloalkyl optionally containing 1 or 2 heteroatom, (C 2 6 )alkenyl, (C 28 )alkynyl, all of which optionally substituted with R 1 60 b) OR 1 0 4 wherein R1 0 4 is H, (C 16 alkyl) or (C3- 7 )cycloalkyl, said alkyl and cycloalkyl being optionally substituted with R 1 92 0) d) SO 2 N(R' 08 2 wherein R 108 is H, (Cl 16 )alkyl or (C 37 )cycloalkyl, said 00 ~alkyl or cycloalkyll being optionally substituted with R 1 60 e) NR'R 112 wherein R' 11 Is H, (C 16 )alkyl or (C 3 7 )cycloalkyl, and R 1 1 2 is H, (C 16 )alkyl, (C 37 )cycloalkyl or (Cl 16 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl, (C 16 alkyl)Het, C00R 11 5 or S0 2 R' 1 5 wherein R 1 15 is (C 1 6 )alkyl, (0 3 7 )CYCloalkyl, or (C 16 )alkyl-(C 37 )CYCloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (Cl. 6 alkyl)Het, or both R 111 and R 112 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Cl-6alkyl)aryl or (Cl. 6 alkyl)Het, or heterocycle being optionally substituted with R16 0 f) N R 116 C0R 1 17 wherein R 116 and R1" is each H, (0 16 )alkyl or (03. 7 )cycloalkyl, said (CI. 6 )alkyl or (C 37 )cycloalkyl being optionally substituted with Rlr 60 g) NR 1 1 8 00NR 1 1 9 R 1 20 wherein R 1 1 8 R1 1 9 and R 1 20 is each H, (Cl. 6 )alkyl or (C 3 7 )cycloalkyl; or R 119 and are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl or heterocycle being optionally substituted with R 160 h) NR 1 21 0000R 1 2 2 wherein R121 is H, (C 1 .6)alkyl or (C3. 7 )CYCloalkyl, said alkyl or cycloalkyl being optionally substituted with R" 60 or R 1 22 is OR 123 or N(R 124 2 wherein R 1 23 and each R 1 24 is independently H, (C 16 alkyl) or (C 37 )cycloalkyl, or R 1 24 is OH or O(Cj- 6 alkyl) or both R 124 are covalently bonded together to form a 5, 6 or 7- membered saturated heterocycle, said alkyl, cycloalkyl and heterocycle being optionally substituted with R 1 6 c 0 Jtetrazole, C00R 12 1 wherein R'1 28 is H, (Cl. 6 )alkyl or (C 37 )cycloalkyl, said (C 1 .6)alkyl and (C3- 7 )cycloalkyl being optionally substituted with R 160 and k) C0NR 129 R 13 1 wherein R 1 29 and R 1 30 are independently H, (Cl- 6 )alkyl or (C 3 7 )cycloalkyl, or both R1 2 9 and R 1 3 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl and heterocycle being optionally substituted with R160; 93 0wherein R 160 is defined as 1 or 2 substituents selected from: 00 tetrazole, halogen, ON, C 1 6 alkyl, haloalkyl, COOR 1 OR 1 N(R 162 2 or CON(R 1 62 2 wherein R 1 61 and each R 162 is independently H or (Cl-6)alkyl; Re is H; or Re is covalently bonded to either of R 7 or Re to form a 5- or 6-membe red ri heterocycle; and 0 is a 6- or lO-membered aryl, Het, all of which being optionally substituted with: OH COOH 0 A) N NCOOH 0 CO or R 100 wherein R100 is: 1 to 4 substituents selected from: halogen, NO 2 cyano or azido; or 1 to 4 substituents selected from: a) (CI.6) alkyl or haloalkyl, (C 37 )cycloalkyl, (C 26 )alkenyl, (C 28 )alkynyl, (C 1 6 alkyl-(C 37 )cycloalkyl, all of which optionally substituted with 0' 0 b) OR' 04 wherein R 1 04 is H, (C 1 6 alkyl), (C 3 7 )cycloalkyl, or (C 1 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (Cl. 6 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (Cl. 6 alkyl)Het being optionally substituted with e) NOW 11 R 12 wherein R 11 is H, (C 16 )alkyl, (C 37 )CYCloalkyl or (Cl. 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (C 1 6 alkyl)Het, and R 1 1 2 is H, ON, (C 1 6 )alkyl, (C 3 7 )cycloalkyl or (C 1 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl, (Cl. 6 alkyl)Het COOR' 15 or SO 2 R" 5 wherein R 1 1 5 is (C 1 6 )alkyl, (C3. 7 )cycloalkyl, or (C 16 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cj- 6 alkyl)Het, or both R 11 and R' 12 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (C 1 6 alkyl)Het, or heterocycle being optionally substituted with R 1 50 f) NR" 6 00R" 7 wherein R1 16 and R 1 1 7 is each H, (C 16 )alkyl, (C 3 7 )cycloalky, (C 1 6 )alkyl-(C3. 7 )Cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (Cl. 6 alkyl)Het, said (Cl. v 6 )alkyl, (C 3 7 )CYCloailkyl, (C 1 -6)alkyl-(C 37 )CYCloalkyl, aryl, Het, (C 16 alkyI)aryl or (Cl. 6 alkyl)Het being optionally substituted with R 1 so; 94 12012 CQ g) NR1laCONR11 9 R 2 wherein R 119 and R' 2 is each H, (C. 6 )alkyl, (03- 00 7 )cycloalkyl, (C 1 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl 1 6alkyl)aryl or (Cl. 6 alkyl)Het, or R' 18 is covalently bonded to R' 1 9 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; INs or R 1 19 and R 1 20 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-memrbered saturated heterocycle; said alkyl, cycloalkyl, (Cl. 6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl. 6 alkyl)Het or heterocycle being optionally substituted with Rl-w,; h) NR'COCOR'22 wherein R 1 2 and R 1 is each is H, (Cl. 6 ,)alkyl, (C3. 7 )cycloalkyl, (Cl 1 e)alkyI-(C 37 )cycloalkyl, a 6- or 1lO-membered aryl, Het, (C. 6 alkyl)aryl or (Cl-6alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalky, aryl, Het, (C 16 alkyt)aryl or (C 1 .6alkyl)Het being optionally substituted with R 150 or R 122 is OR 123 or N(R 124 2 wherein R 123 and each R 124 is independently H, (Cl 16 alkyl), (C 3 7 )cycloalkyl, or (C 1 6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (Ci. 6 alkyl)aryl or (Cl. 6 alkyl)Het, or R 124 is OH or O(C 1 -6alkyl) or both R 124 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C 16 alkyI~aryl or (C 1 6 alkyl)Het and heterocycle being optionally substituted with R1 50 j) COOR 1 2 1 wherein R 12 8 is H, (Cl. 6 )alkyl, (C 37 )cycloalkyl, or(Cl. 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (0 1 6 alkyl)aryl or (Cl. 6 alkyl)Het, said (C 16 )alkyl, (C3. 7 )cycloalkyl, or(Cj- 6 )alkyl-(C 37 )cycloakyI, aryl, Het, (C 1 6 alkyl)aryl and (Ci. 6 alkyl)Het being optionally substituted with R1 50 k) CONR 129 R 1 30 wherein R 1 2 and R'1 30 are independently H, (Cl. 6 )alkyl, (C3. 7 )cycloalkyl, (CI- 6 )alkyl-(C 3 q7)cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl. 6 alkyl)Het, or both R 129 and R 1 30 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (0 1 6 alkyl)aryl, (C 1 6 alkyi)Het and heterocycle being optionally substituted with R' 50 1) aryl, Het, (C 16 alkyl)aryl or (Cl. 6 alkyl)Het, all of which being optionally substituted with R 150 wherein R 150 Is selected from: 1 to 3 substituents selected from: halogen, NO 2 cyano or azido; or I to 3 substituents selected from: a) (01.6) alkyl or haloalkyl, (C 3 7 )cycloalkyl, C3.7 spirocycloalkyl C.) 00 (Cl 1 6) alkyl-(C 37 )cycloalkyl, all of which optionally substituted with b) OR 1 04wherein R 104 is H, (Cl. 6 alkyl), (0 37 )cycloalkyl, or (C 16 )alkyl- (C 37 )cycloalkyl, aryl, Het, (Cl-Balkyl)aryi or (Cl. 6 alkyI)Het, said alkyl, cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl. 6 alkyI)Het being optionally substituted with R1 60 d) SO 3 H, SO 2 N(R 08 2 or SO 2 N(R' 08 )C(0)R' 08 wherein each R' is independently H, (Cl. 6 )alkyl, (C 37 )cycloalkyl or (C 1 6 )alkyl-(C 3 7 )CYCloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (Cl,,alkyl)Het or both R' 0 ,3 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (C 16 alkyl)Het or heterocycle being optionally substituted with R 160 e) NR' 11 R" 2 wherein R 111 is H, (C 16 )alkyl, (C 37 )cycloalkyl or (Ci. 6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (Cl 16 alkyl)aryl or (C 16 alkyl)Het, and R 1 12 is H, CN, (C 1 6 )alkyl, (C 3 47cycloalkyl or (Cl-6)alkyl-(C 37 )cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl, (Cl. 6 alkyl)Het or S0 2 R 115 wherein R' 1 5 is (Cl. 6 )alkyl, (C 3 7 )cycloalkyl, or (C 16 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl. 6 alkyI)aryl or (Cl-6alkyl)Het, or both R 11 and R' 12 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aryl, Het, (C 1 .6alkyl)aryl or (Cl-6alkyl)Het, or heterocycle being optionally substituted with R 160 Q) NR' 16 00R 17 wherein R' 16 and R' 17 is each H, (C 16 )alkyl, (C3. 7 )cycloalkyl, (Cl 16 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 1 -6alkyl)aryl or (Cl. 6 alkyl)Het, said (Cl-6)alkyl, (C 37 )cycloalkyl, (Cl. 6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (Cl-6alkyl)Het being optionally substituted with R 160 g) NR 118 00NR 1 1 9 R 2 wherein R' 18 R 11 and R 12 is each H, (Ci. 6 )alkyl, (C 37 )CYCloalkyl, (Cj. 8 )alkyl-(C 3 q)CyCloalkyl, aryl, Het, (C 1 6 alkyI)aryl or (C 1 .,,alkyl)Het, or R 118 is covalently bonded to R 119 and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; or R 119 and R 120 are covalently bonded together and to the nitrogen to 96 C.) 00 heterocycle; said alkyl, cycloalkyl, (C 16 )aikyl-(C 37 )cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (C,.6alkyl)Het or heterocycle being optionally substituted with R 1 60 h) NR 2 COCOR'22wherein R 12 Is H, (Cl-6).alkyl optionally (~KI substituted with R1 60 or R 122 is OR'1 23 or N(R 1 24 2 wherein R 1 23 and each R 1 is independently H, (0 1 6 aikyl), (C 37 )CYCloalkyl, or (C 1 -6)alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aqy or (C 1 .6alkyl)Het, or R 124 is OH or O(C 16 alkyl) or both R 12 4 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl- cycloalkyl, aryl, Het, (C 1 .6alkyl)aryl or (Cl. 6 alkyl)Het and heterocycle being optionally substituted with Rlr'; J) tetrazole, COOR 1 28 wherein R 1 28 is H, (Cl. 6 )alkyl, (C 37 )cycloalkyl, or(Cl. 6 )alkyI-(C3. 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl. 6 alkyl)Het, said (C 16 )alkyl, (C 37 )cycloalkyl, or(C 1 6 )alkyl-(C 3 q7)cycloalkyl, aryl, H-et, (Cl 16 alkyl)aryl and (Cl. 6 alky])Het being optionally substituted with R 1 60 and k) CONR R 29 R 1 30 wherein R 1 2 9 and R 1 30 are independently H, (C 1 6 )alkyl, (C3- 7 )cycloalkyl, (Cl. 6 )alkyl-(C3. 7 )cycloalkyl, aryl, H-et, (01. 6 alkyl)aryl or (C 1 6 alkyl)Het, or both R1 29 and R' 30 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, aikyl-cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl, (Cl. 6 alkyl)Het and heterocycle being optionally substituted with Rlr'; wherein R 16 0 is defined as 1 or 2 substituents selected from: tetrazole, halogen, ON, C 1 -6alkyl, haloalkyl, COOR 6 SO 3 H, SR 161 S0 2 R 1 6 1 OR 161 N(R 1 6 2 2 SO 2 N(R1 62 2 or CON(R' 62 2 wherein R 16 1 and each R 162 is independently H, (C,-6)alkyl, (C 3 7 )cycloalkyl or (C 1 6 )alkyl-(C 37 )cycloalkyl; or both R 1 62 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; 97 C.) 00 or a salt thereof; wherein said probe comprises a detectable label attached to any suitable position, whereby said probe binds to an HCV polymerase or an analog thereof and is capable of being displaced by an inhibitor thereof. 4. The method according to claim 3, wherein said probe is a compound having the following formula: 0 DN N N R' R 1 SO R 1 INIS (I Ib), NN( R 2 N N A N N H H Ri(l1c), H 1 0 R (ld) /H 0 o H t (lie) or wherein R1 is (C 5 6 ,)cycioalkyl; R 2 is phenyl, or Het both being optionally substituted with R 20 R 3 R 7 R8 11 9 R 100 and R' 50 are as defined according to claim 2; R" is OPO 3 H, N0 2 cyano, azido, C(=NH)NH 2 C(=NH)NH(Cl. 6 )alkyl or C(=NH)NHCO(C 1 6 )alkyl; or a) (01.6) alkyl substituted with R'1 5 0 a, haloalkyl, (0 37 )cycioakyl, C3.7 spirocycloalkyl optionally containing 1 or 2 heteroatom, (C 2 6 )alkenyl, (02. 8 )alkynyl, (0 1.6) alkyi-(C 3 7 )cycloaikyl, all of which optionally substituted with 111 5 D, wherein R 1 1 0 a is the same as R1 5 c) but is not halogen, ORl1b, CQOR I 5 b, N(R1 5b 2 wherein Rl 5 0b Is H or C 1 .6alkyl; b) 0 R1 4 wherein R' 04 is (C 1 -6alkyi) substituted with 1 50 (0 3 7 )cycioalkyl, or 98 O (Cl. 6 )alkyl-(C 37 )cycloalkyl, aryl, Het, (C 1 6 alKyI)aryl or (Cl. 6 alkyl)Het, said 00 cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl. 6 alkyl)Het being optionally substituted with c) OCOR' 05 s wherein R 1 0 5 is (Cl 16 )alkyl, (C 37 )cycloalkyl, (Cl. 6 )alkyl-(C3. 7 )CYCloalkyl, Het, (C 16 alkyl)aryl or (0 1 6 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (CI. 6 alkyl)Het being optionally substituted with R 150 r~l d) SO 3 H, SO 2 N(R 08 2 or SO 2 N(R 10 8 )C(O)Rl 08 wherein each R 1 0 is independently H, (Cl 16 )alkyl, (C 37 )cycloalkyl or (C 1 6 )alkyl.(C3. 7 )cycloalkyI, aryl, Het, (C 1 .6alkyl)aryl or (C 14 6alkyl)Het or both R' 0 '3 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7- membered saturatled heterocycle, said alkyl, cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (C 16 alkyl)Het or heterocycle being optionally substituted with e) NR'R 112 wherein R 11 1 is (Cl.r 6 )alkyl substituted with R 150 (C 37 )cycloalkyl or (C 16 )alkyl.(C. 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (C 16 alkyl)H-et, and R 1 1 2 is H, ON, (C 16 )alkyl, (C 3 7 )cycloalkyl or (Cl. 6 )alkyl-(C3- 7 )cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl, (C 1 -6alkyl)Het or R' 1 is H and R' 2 is S0 2 R 115 wherein R" 5 is (Cl*. 6 )alkyl, (C 37 )cycloalkyl, or (C 16 )alkyl.(C 3 7 )cycloalkyl, aryl, Het, (Cl 16 alkyl)aryl or (Cl.6alkyl)Het, or both R" 11 and R' are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said cycloalkyl, aryl, Het, (C0 16 alkyl)aryl or (0 1 6 alkyl)Het, or heterocycle being optionally substituted with R 1 50 f) NR 11 6 00R 1 17 wherein R 116 and R 1 17 is each H, (C 16 )alkyl, (C 37 )cycloalkyl, (C 1 6 )alkyl-(C3. 7 )cycloalkyl, aryl, Het, (Cl 16 alkyl)aryl or (C1.6alkyl)Het, said (Cl- 6 )alkyl, (C 37 )cycloalkyl, (C 1 6 )alkyl.(C3-7)cycloalkyl, aryl, Het, (C 1 .6alkyl)aryl or (C0 16 alkyl)Het being optionally substituted with R 150 g) NR 118 00NR 1 9 R 120 wherein R 1 1 8 R' 1 9 and R 120 is each H, (C 16 )alkyl, (C3. 7 )cycloalkyl, (C 16 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Ci.. 6 alkyl)Het, or R11 8 is covalently bonded to R 119 and to the nitrogen to which they are attached to form a 5, 6 or 7-membe red saturated heterocycle; or R'" 9 and R 12 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle; said alkyl, cycloalkyl, (Cl. 6 )alkyl.(C 3 7 )cycloalkyl, aryl, Het, (C 16 alkyl)aryl or (Cl. 99 C.) 00 h) NR 1 21 COCOR 1 22 wherein R 1 21 and R'1 22 is each is H, (C 16 )alkyl, (03. 7 )cycloalk'/l, (Cj.6)alkyl-(C 37 )cycloalkyl, a 6- or lO-membered aryl, Het, (C 1 6 alkyl)aryl or (Cl. 6 alkyl)Het, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (C 1 6 alkyl)Het being optionally substtuted with R 150 or R 1 2 i OR 12 or N(R' 24 2 wherein R 1 23 and each R 12 4 is independently H, (Cl- 6 alkyl), (C 3 7 )cycloalkyl, or (Cl. 6 )alkyl-(C3-7)cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (C 16 alkyl)Het, or R 1 24 is OH or O(Cl 16 alkyl) or both R 124 are covalently bonded together to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C 1 .6alkyI)aryl or (Cl. 6 alkyl)Het and heterocycle being optionally substituted with R 15 0 i) C0R 127 wherein R 127 is H, (Cl. 6 )alkyl, (C 3 7 )cycloalkyl or (C 16 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (Cl. 6 alkyl)aryl or (C 16 alkyl)Het, said alkyl, cycloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (Cl-6alkyl)Het being optionally substituted with R 1 j) COOR 1 28 wherein R 1 28 is H or (C 1 .r 6 )alkyl substituted with R 1 51, (03. 7 )cycloalkyl, or(Cl. 6 )alkyl-(C 37 )cyCloalkyl, aryl, Het, (C 1 6 alkyl)aryl or (Cl. 6 a1 kyl)Het, said (C 3 7 )cycloalkyl, or(C alkyl-(C 3 7 )cycloal kyl, aryl, Het, (Cl. 6 alkyI)aryl and (C 1 6 alkyl)Het being optionally -substituted with R 15 0 k) CONR 1 9R 130 wherein R 1 2 and R 13 are independently H, (C 14 6)alkyl, (03. 7 )cycloalkyl, (C 1 6 )alkyl-(C 3 7 )cycloalkyl, aryl, Het, (C 14 6alkyl)aryl or (Cl. 6 alkyI)Het, or both R'1 29 and R 1 30 are covalently bonded together and to the nitrogen to which they are attached to form a 5, 6 or 7-membered saturated heterocycle, said alkyl, cycloalkyl, alkyl-cycloalkyl, aryl, Het, (C 14 6alkyl)aryl, (Cl 6 alkyl)Het and heterocycle being optionally substituted with R' 50 1) aryl, Het, (Cl. 6 alkyl)aryl or (C 16 alkyl)Het, all of which being optionally substituted with R1 50 wherein R1 50 is as defined in claim 3; or a salt thereof; wherein said compound is optionally: a) marked with a radioactive isotope at any suitable position, b) linked to a detectable moiety by a suitable linker suitable position, except R' and R 3 or c) linked to an affinity tag at any suitable position, except R' and R 3 S100 0 5. The method according to claim 4, wherein said probe is a compound having 00 the following formula: O OH 0 H R N O OH N N l N 2 N C R 5 0 1 R (Ib), 3 H 2K1 N J SR 1 50 (lIc). wherein R' is (Cs 6 )cycloalkyl; R 2 is phenyl, or Het both being optionally substituted with R 20 R 3 and R 15 60 are as defined in claim 4; or a salt thereof; wherein said compound is optionally: a) marked with a radioactive isotope at any suitable position; b) linked to a detectable moiety by a suitable linker at any suitable position, except R 1 and R 3 or c) linked to an affinity tag at any suitable position, except R' and R 3 6. The method according to claim 2 wherein the detectable label selected from the group consisting of: a fluorescent label a radioactive atom, a chemiluminescent label, and a colorimetric label. 7. The method according to claim 6 wherein the label is a fluorescent label or chemiluminescent label.

8. The method according to claim 7, wherein the fluorescent label is selected from the group consisting of: fluorescein, Oregon green, dansyl, rhodamine, Texas- red, phycoerythrin and Eu 3

9. The method according to claim 8, wherein the fluorescent label is fluoresceln. 101 The method according to claim 2, wherein the detectable label is a fluorescent reporter/quencher pair.

11. The method according to claim 10, wherein the reporter/quencher pair is selected from the group consisting of: EDANS/DABCYL, tryptophan/2,4- dinitrophenyl, tryptophan/DANSYL, 7-methoxycoumarin/2,4-dinitrophenyl, 2- aminobenzoyl/2,4-dinitrophenyl and 2-aminobenzoyl/3-nitrotyrosine.

12. The method according to claim 6, wherein the radioactive atom is selected from 3 H, 1 4 C and 1251.

13. The method according to claim 2, wherein the probe is selected from:: )H (ii); 0 R 102 -900H 0^0 N SO O COOH (iv); N\ M COOH u 0 N 0 IN O -O OCOOH c-i r and F OH 0 (vi)

14. Use of a probe of formula I, according to claim 2, in the development of an assay for identifying inhibitors of HCV polymerase. A method for identifying compounds that inhibit HCV polymerase comprising the steps of: a) contacting said HCV polymerase or an analog thereof with a probe of formula I, according to claim 2, so as to form a complex having said probe bound to said polymerase; b) measuring the signal from said complex to establish a base line level; c) incubating the product of step a) with a test compound; and d) measuring the signal from said complex; and e) comparing the signal from step d) with the signal from step b); whereby a modulation in said signal is an indication that said test compound Inhibits said polymerase.

16. A method for identifying compounds capable of inhibiting HCV polymerase, 0 103 O comprising: 00 f) repeating steps to according to claim 15, in a high throughput screen.

17. The method according to claim 15, wherein the HCV polymerase is selected from the group consisting of: NS5B; NS5BA21; and NS5BA57 or analogs thereof.

18. The method according to claim 15, wherein the HCV polymerase is obtained Sfrom genotype HCV-la or HCV-1b strains optionally having a histidine tag at either the N- or C-terminal.

19. A kit for testing compounds potentially binding to HCV polymerase, said kit comprising the probe of formula according to claim 2, and instructions on how'to use said probe for identifying test compounds binding to said polymerase. A probe of formula I: S M R B L A is O, S, NR 3 or CR 3 B is NR 1 or CR'; with the proviso that, when A is CR 3 B is NR', and when A is O or S, B is CR'; represents either a single or a double bond; R' is selected from the group consisting of: (C4.7)cycloalkyl optionally substituted with alkyl); norbornane, 6- or 7-membered heterocycle having 1 to 4 heteroatoms selected from O, N, and S, all of which optionally substituted with 1 to 4 substituent ,selected from the group consisting of: halo, OH and C1-6 alkyl optionally substituted with hydroxy; R 2 is selected from the group consisting of: phenyl, pyridine-N-oxide, 5- or 6-membered aromatic heterocycle having 1 to 4 heteroatoms selected from O, N, and S, and 9- or 10-membered aromatic heterobicycle having 1 to 4 heteroatoms selected from O, N and S; 104 said phenyl, pyridine-N-oxide, aromatic heterocycle and aromatic C) heterobicycle being optionally substituted with from 1 to 4 substituents selected from the group consisting of: halogen, C1..6 haloalkyl, (Cl. 6 )alkyl, C 14 6 alkoxy, OH, amino optionally mono- or di-substituted with 01.6 alkyl; IND 5 R 3 is selected from the group consisting of: H, (C 16 )alkyl, (0146 alkyl)-(C 6 -1o 0 aryl), (01.6 6-membered heterocycle having 1 to 4 heteroatoms selected from 0, N, and S, and 5- or 6-membered heterocycle having 1 to 4 heteroatoms selected from 0, N and S, wherein said aryl and said heterocycle are optionally substituted with from 1 to 4 substituents selected from the group consisting of: COCH, COO(0 1 6 alkyl), halogen, and (01.6 alkyl); M is N, CR 4 a, or COR 4b, wherein R 43 is selected from the group consisting of: H, halogen, and (0146 alkyl); andR R 4 b is selected from the group consisting of: H and (Ci. 6 alkyl); K and L is each independently N or CR wherein R 6 is H, halo, C1..6 alkyl, OH, or Ci. 6 alkoxy; R 5 is wherein Y is 0 or S, and Z is NHR 5 3 or ORB6a; wherein: R 5 8 is selected from the group consisting of: H, (Ci. 6 )alky, (C 26 )alkenyl, (C, 3 .)cycloalkyl optionally substituted with C 16 alkyl or C 24 6alkenyl, (C 6 1 0 )aryl optionally substituted with C 1 -6alkyl or C 2 .6alkenyl, N{(Cl.6) alkyl) 2 NHCQO(C 14 )alkyl(C 6 1 0 )aryl, NHCO(Cr. 10 )aryl, or 6-atom heterocycle, having 1 to 4 heteroatoms selected from 0, N and S, and or 10-atom heterobicycle having i to 4 heteroatoms selected from 0, N and S; wherein said alkyl, alkenyl, cycloalkyl, aryl, heterocycle or heterobicycle are all optionally substituted with from 1 to 4 substituents selected from: OH, COOH, (C 1 6 )alkyl, (C 2 4alkenyl, (Cl-6)alkyl-hydroxy, COO(C 16 )alkyl, C3-7 cycloalkyl, benzyloxy, halogen, (C 2 -4alkeny-(C 1 6 )alkyl-COOH, coumarin, (Cl. 6 )alkyl-amino, NH(C 1 6 alkyl), C(halogen) 3 -C(O)N H(C.4)alkyl, and -C(O)NH(C 6 10 )aryl, 5- or 6-membered heterocycle having 1 to 4 heteroatoms selected from 0, N and S, 9- or lO-membered heterobicycle having 1 to 4 heteroatoms selected from 0, N and S, and 6- or lO-membered aryl; (Ni 105 C) wherein said alkyl, alkenyl, cycloalkyl, aryl, heterocycle and 00 heterobicycle are all optonally substituted with from 1 to 4 substituents selected from: halogen, OPO 3 H, sulfonamido, SO 3 H, SO 2 OH 3 -CONH 2 -COCH 3 (C 13 )alkyl, (02. 4 alkenyl)COOH, tetrazolyl, COOH, -OONH 2 triazolyl, OH, NO 2 NH 2 -0(01.6 alkyl)COOH, hydantoin, benzoyleneurea, (C 14 )alkoxy, cyano, azido,'-O-(C 14 6)alkyl COOH, -O-(Cl-6)alkyl COO-(C 14 )alkyl, NHCO-(Cl. 6 alkyl), -NHCOOOOH, -NHCOCONHOH,-NHCO0ONH 2 -NHOOCONHCH 3 -NHCO(0 1 6 )alkyl-COOH, .NHCOCONH(Cl. 6 )alkyl-COOH, -NHCO(C 3 7 )cycloalkyl-OOOH, -NHCONH(0 6 10 )aryi-COOH, NHCONH(C 610 )aryl-COO(Cl.6)alkyl, NHCONH(C 1 6 )alkyl- OOOH,- NHCONH(C 16 alkyl-000( 1 6 )alkyl, NHCONH(0 1 6 )alkyl-(C 2 .6)alkenyl-COOH, N H(Cl. 6 )alkY]-(0 6 1 o)aryI-O(0l- 6 )alkyl COOH, NH(Cj. 6 )alkyl-(0 6 .j 0 )aryl-COOH, -NHCH 2 000H, -N HCONH 2 -NHCO(C 1 6 )hydroxyalkyl OOOH, -OCO(Cl. 6 )hydroxyalkyl COOH, (0 3 6 )cycloalkyl COCH, 0 0 s -NHCN, -NHOHO, -NHSO 2 OH 3 -NHSO 2 OF 3 and -O(0 1 6 alkyI)-tetrazol; or Rs'is R9 0 wherein R 7and Re 8 are each independently H, alkyl), (03.7 cycloalky), (0146 alkyl)phenyl, (01.6 alkyl)-(Oaq7 cycloalkyl), (03.7 cycloalkyl)-( C 1. alkyl), (03.7 cycloalkyl)-(0 2 .4 alkenyl), (01.6 alkyl)-OH, phenyl, CH 2 biphenyl, 5- or 6-membered heterocycle having fromi to 4 heteroatoms selected from 0, N, and S, 9- or lO-membered heterobicycle having 1 to 4 heteroatoms selected from 0, N, and S, (01.6 alkyl)-5- or 6-membered heterocycle having fromi to 4 heteroatoms selected from 0, N, and S, or (0146 alkyl)-9- or lO-membered heterobicycle having 1 to 4 heteroatomns selected from 0, N, and S, or W 7 and Re are covalently bonded together to form (03.7 cycloalkyl), 5- or 106 C.) 00 or one of R 7 or Re is covalently bonded to R 9 to form a pyrrolidine; wherein said alkyl, cycloalkyl, heterocycle, heterobicycle, phenyl are optionally substituted with from 1 to 4 substituents selected from the group of: OH, COOH, (C 1 6 alkyl), alkenyl), CONH 2 NH- 2 NH(C 6 alkyl), N(C 1 6 alkyl) 2 NHCOCOOH, NHCOCON(Cl-6 alkyl) 2 NHCOCONH(Cl. 6 alkyl), SH, S(C 16 alkyl), NHC(=NH)NH 2 halogen, and COO(Cl 1 6 alkyI); Re is H or (01.6 alkyl); and Q is selected from the group consisting of: (Cl. 3 alkyl)CON Haryl, 6- or 1 O-membered aryl, biphenyl, 5- or 6-atom heterocycle having 1 to 4 heteroatoms selected from 0, N and S, 9- or lo-membered heterobicycle having 1 to 4 heteroatoms selected from 0, N and S; wherein said aryl, biphenyl, heterocycle and heterobicycle are all optionally substituted with from 1 to 4 substituents selected from: OH, COOH, COO(C 1 6 )alkyl, (Cl. 6 )alkyl, (Cl.6)alkylCOOH, (01.e alkyl)(C 2 4 alkynyl), (C 1 6 )alkyl-hydroxy, phenyl, benzyloxy, halogen, (C 2 4alkenyl, (C 2 -4alkenyl- (C 1 6 alkyl-COOH, 5- or 6-membered second heterocycle having 1 to 4 heteroatoms selected from 0, N and S, NH-5- or 6- membered second heterocycle having 1 to 4 heteroatoms selected from 0, N, and S, wherein said second heterocycle and phenyl being optionally substituted with from 1 to 4 substituents selected from: (01.6 alkyl), OF 3 OH, (C1.6alkyl) COOH, O(C 16 alkyl)COOH, (Cl-6alkyl) COO(Cl. 6 alkyl), CH 2 phenyl, 000(01.6 alkyl), (Cl.6alkyl)O(Cj.6aIkyl), COOH, NCH(C 1 6 alkyl) 2 NCO(C 1 6 alkyl), NH 2 NH(Cj.6 alkyl), halogen, N(Cl.6 alkyl) 2 and C2-.6 alkenyl-COOH halogen, OPO 3 H, benzyl, sulfonamido, SH, SOCH 3 SO 3 H, SO 2 CH 3 S(Cl6 alkyl)COOH, -CONH 2 -COOH 3 (0 1 3 )alkyl, (C 2 .4alkenyl)COOH wherein said alkenyl is optionally substituted with from 1 to 2 (01.6 alkyl) substituents, (C 2 .4alkenyl)COO(Cl.6alkyl), tetrazolyl, COOH, triazolyl, OH, NO 2 NH- 2 0(01.6 alkyl)COOH, hydantoin, benzoyleneurea, (C 14 )alkoxy, (Cl.4alkoxy(C 16 alkyl)COOH, cyano, azido, -0-(Cl.6)alkyl COOH, -O-(Cl. 6 )alkyl COO-(C 16 alkyl, -NHCOCOOH, -NHC000NHOH ,-NHCOCON H 2 -NHCOCONHCH 3 -NHCO(C 1 6 ))alkyl-COOH, -NHCOCON H(Cle)alkyl-COOH, -NHCO(C 37 )cycloalkyl-COOH, -NHCONH(0 610 )aryl-COOH, NHCONH(C 6 107 o 10 )aryl-COO(C 16 )alkyl NHCONH(C 1 6 )alkyl-COOH,- NHCONH(Cl. 6 )alkyl- 00 COO(Cl. 6 alkyl, NHCONH(Cl.6 )alkyl-(C 26 )alkenyl-COOH, NH(Cl 16 )alkyl-(C 6 10 )aryl-O(C 16 )alkyl COOH, NH(C 1 _6)aky-(C 6 1 o)aryl-COOH, -NHCH 2 000H, -NHCONH 2 -NHCO(C 14 6)hydroxyalkyl COOH, -OCO(Cl. 6 )hydroxyalkyl 5 COOH, (C 36 )cycloalkyl COOH, 0 0 -NHCN, -NHCHO, -NHSO 2 CH 3 -NHSO 2 CF 3 coumnarin, (C 1 .G)alkyl-amino, NH(C 1 -6alky) 2 C(halogen)3, -NH (C 24 )acyl, -NH(C 6 1 o)aroyl, -CONH (Cl. 6 alkyl), -CO(C 16 )alkyl-COOH, -CONH(C 1 6 )alkyl-COOH, -CO-NH-alanyl, -CON H(C 2 .4akyN(C.eakyl)2, -CONH(C 2 alkyl-Het, -CONH(C24) alkyl-(COOH)-Het, -CONH(C 1 2 alkyl) (OH)(Cj- 2 alkyl)OH, -CONH(Cl. 6 alkyl-COOH, -CONH(C 610 aryl), -CONH-Het, -CONH(C 6 1 o) aryl-COOH, -CONH(C 6 .iO) aryl-COO(Cl.6) alkyi, -CONH(C 1 -6) alkyl-COO(CI.6) alkyl, -CONH(C 6 1 0) aryl-(C 1 .6)alkyl-COOH, and -CONH (Cfrlo) aryl-(C 26 )alkenyl-COOH; or a salt thereof; said probe comprises a detectable label, whereby said probe binds to an HCV polymerase or an analog thereof and is capable of being displaced by an Inhibitor thereof.

21. A method for identifying compounds that inhibit HCV polymerase comprising the steps of: a) contacting said HCV polymerase or an analog thereof with a probe of formula I according to claim 20, so as to form a complex having said probe bound to said polymerase; b) measuring the signal from said complex to establish a base line level; c) incubating the product of step a) with a test compound; and d) measuring the signal from said complex; and e) comparing the signal from step d) with the signal from step b): whereby a modulation In said signal is an indication that said test compound Inhibits said polymerase.