Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
ANTIVIRAL COMPOUNDS
Document Type and Number:
WIPO Patent Application WO/2017/001853
Kind Code:
A1
Abstract:
This invention relates to a series of tricyclic compounds comprising a pteridinone core linked to a third heterocycloalkyl ring. The compounds are useful in the treatment of Hepatitis B viral infections. The invention also relates to pharmaceutical compositions comprising these compounds and methods of using the compounds in treatment.

Inventors:
GREENHOUGH RICHARD (GB)
FINLAYSON JON (GB)
GIBSON KATIE (GB)
BUNT ADAM (GB)
Application Number:
PCT/GB2016/051964
Publication Date:
January 05, 2017
Filing Date:
June 30, 2016
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
REDX PHARMA PLC (GB)
International Classes:
C07D475/06; A61K31/525; A61P31/20
Other References:
PAUL A. ROETHLE ET AL: "Identification and Optimization of Pteridinone Toll-like Receptor 7 (TLR7) Agonists for the Oral Treatment of Viral Hepatitis", JOURNAL OF MEDICINAL CHEMISTRY, vol. 56, no. 18, 26 September 2013 (2013-09-26), American Chemical Society, Columbus, Ohio; US, pages 7324 - 7333, XP055226722, ISSN: 0022-2623, DOI: 10.1021/jm400815m
Attorney, Agent or Firm:
HGF LIMITED (LEEDS) (GB)
Download PDF:
Claims:
CLAIMS

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof:

wherein

-L1- is independently selected from: a bond, -0-, -NR6-, -C(O)-, -S(0)y- (where y is 0, 1 or 2), -C(0)0-, -OC(O)-, -C(0)NR6-, -NR6C(0)-, -S(0)2NR6- and -NR6S(0)2-;

=Y1 is independently selected from: =0 and =S;

X1 , X2 and X3 are each independently selected from a bond, a carbon atom, a nitrogen atom, -O- and -S(0)y-(where y is 0, 1 or 2); provided that no more than two of X1 , X2 and X3 are a bond and provided that no more than one of X1 , X2 and X3 is selected from a nitrogen atom, -O- and -S(0)y-;

R1 is independently selected from Ci-Cio-alkyl, Ci-Cio-haloalkyl, C2-Cio-alkenyl, C2-C10- alkynyl, C3-Cs-cycloalkyl, 4-12-heterocycloalkyl, aryl, heteroaryl and -d-Cs-alkylene-R7; wherein R7 is independently selected from C3-Cs-cycloalkyl, 4-12-heterocycloalkyl, aryl and heteroaryl, -0-Ci-C4alkyl, -S-Ci-C4alkyl and -NR6Ci-C4-alkyl;

R2 and R9 are each independently at each occurrence selected from: H, Ci-C4-alkyl, -C(O)- Ci-C4-alkyl and -S(0)2-Ci-C4-alkyl;

R3, R6, R8 and R11 are each independently at each occurrence selected from: H and C1-C4- alkyl;

R4 is independently at each occurrence selected from: halo, Ci-C6-alkyl, C2-C6-alkenyl, C2- Ce-alkynyl, C3-C6-cycloalkyl, Ci-C6-haloalkyl, nitro, cyano, NR8R9, S(0)2OR8, S(0)2R8, S(0)2NR8R8, CO2R8, C(0)R8, CONR8R8, SR8 and OR8; or two R4 groups attached to the same carbon atom, together with that carbon atom form a C=0 group;

-L2- is independently selected from: a bond, -(CR10R10)Z- (where z is 1 , 2 or 3), -0-, -S-, - NR6-, -C(O)-, -S(0)2- , -C(0)0-, -OC(O)-, -C(0)NR6-, -NR6C(0)-, -S(0)2NR6- and - NR6S(0)2-; wherein, if L2 is attached via X1 , X2 or X3 and the X1 , X2 or X3 to which L2 is attached is a nitrogen atom, -L2- is not -0-, -S-, -NR6-, -OC(O)-, -NR6C(0)- or -NR6S(0)2-;

-L3 is independently a bond or is -L4-L5-; -L4- is independently selected from: -(CR10R10)r- (where r is 1 , 2 or 3), aryl, heteroaryl, C3- Cs-cycloalkyl, 4-12-heterocycloalkyl;

-L5- is independently selected from: a bond, -0-, -S-, -N R6-, -C(O)-, -S(0)2-, -C(0)0-, - OC(O)-, -C(0)N R6-, -N R6C(0)-, -S(0)2N R6- and -N R6S(0)2-;

R5 is independently selected from: H, Ci-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, aryl, heteroaryl, C3-C8-cycloalkyl, 4-12-heterocycloalkyl, Ci-C6-haloalkyl and -(CR10R10)S-N R1 1 R12 (where s is 0, 1 , 2 or 3); wherein when -L2-, -L3- and -L4- are selected such that R5 is directly attached to a nitrogen or oxygen atom, R5 is not attached to the rest of the molecule through a nitrogen atom;

R10 is independently at each occurrence selected from: H, F and Ci-C4-alkyl;

R12 is independently selected from H, Ci-C6-alkyl. C2-C6-alkenyl, C2-C6-alkynyl, aryl, heteroaryl, C3-Cs-cycloalkyl, 4-12-heterocycloalkyl, -d-Cs-alkylene-R13; wherein R13 is independently selected from C3-Cs-cycloalkyl, 4-12-heterocycloalkyl, aryl and heteroaryl; or R1 1 and R12 together with the nitrogen to which they are attached form a 4-12 heterocycloalkyi group;

x is an integer selected from 0, 1 , 2, 3 and 4;

wherein any aryl, heteroaryl or heterocycloalkyi group may be monocyclic- or bicyclic; where a heterocycloalkyi group is bicyclic it comprises a 4-, 5-, 6- or 7-membered heterocycloalkyi ring fused to a ring selected from phenyl, 5- or 6- membered heteroaryl, C3-Cs-cycloalkyl, 4-, 5-, 6- or 7-membered heterocycloalkyi ring;

wherein each of the aforementioned alkyl, alkylene, alkenyl, alkynyl, haloalkyi, cycloalkyi, heterocycloalkyi, aryl (e.g. phenyl) and heteroaryl groups and rings is optionally substituted, where chemically possible, by 1 to 5 substituents which are each independently at each occurrence selected from the group consisting of: oxo, =N Ra, =NORa, halo, nitro, cyano, N RaRa, N RaS(0)2Ra, N RaC(0)Ra, N RaCON RaRa, N RaC02Ra, ORa, SRa, S(0)Ra, S(0)2ORa, S(0)2Ra, S(0)2N RaRa, C02Ra, C(0)Ra, CON RaRa, C1-C4- alkyl, C2-C4-alkenyl, C2-C4-alkynyl and Ci-C4-haloalkyl; wherein Ra is independently at each occurrence selected from H, Ci-C4-alkyl and Ci-C4-haloalkyl.

2. A compound of claim 1 , wherein R2 and R3 are each H.

3. A compound of claim 1 or claim 2, wherein =Y1 is =0.

4. A compound of any preceding claim, wherein -L1- is -0-.

5. A compound of any preceding claim, wherein R1 is independently selected from Ci- Cio-alkyl, C2-Cio-alkenyl, C2-Cio-alkynyl.

6. A compound of any preceding claim, wherein none of X1 , X2 and X3 is a bond.

7. A compound of any preceding claim, wherein X3 is nitrogen.

8. A compound of any preceding claim, wherein X2 is carbon.

9. A compound of any preceding claim, wherein X1 is carbon.

10. A compound of any preceding claim, wherein x is 0.

1 1. A compound of any preceding claim, wherein -L2-, -L3- and R5 are selected such that -L2-L3-R5 comprises at least one nitrogen atom.

12. A compound of claim 11 , wherein -L2-, -L3- and R5 are selected such that -L2-L3-R5 comprises at least one tertiary amine.

13. A compound of any preceding claim, wherein -L2-L3-R5 is preferably attached to the rest of the molecule at X3.

14. A compound of any preceding claim, wherein -L2- is C(O)-.

15. A compound of any preceding claim, wherein -L3- is -L4-L5-.

16. A compound of any preceding claim, wherein -L4- is selected from phenyl and 6- membered heteroaryl.

17. A compound claim 16, wherein -L5- is independently selected from: a bond, -0-, - S- and -NR6-.

18. A compound of any one of claims 1 to 14, wherein -L3- is a bond.

19. A compound of any preceding claim, wherein R5 is -(CR10R10)S-NR11 R12.

20. A compound of claim 19, wherein s is an integer selected from 1 , 2 and 3.

21. A compound of claim 19 or claim 20, wherein R11 and R12 together with the nitrogen to which they are attached form a 4-12 heterocycloalkyi group which may be monocyclic- or bicyclic; and where that heterocycloalkyi group is bicyclic it comprises a 4-, 5-, 6- or 7- membered heterocycloalkyi ring fused to a ring selected from phenyl, 5-or 6- membered heteroaryl, C3-C8 cycloalkyl, 4-, 5-, 6- or 7-membered heterocycloalkyi ring.

22. A compound of claim 21 , wherein NR11 R12 is selected from:

23. A compound of claim 1 , wherein the compound of formula (I) is selected from:

24. A compound of any preceding claim for medical use.

25. A compound of any one of claims 1 to 20 for use in promoting IFN-a production.

26. A compound of any one of claims 1 to 20 for use in treating a viral infection.

27. A compound for use of claim 23, wherein the viral infection is hepatitis B.

28. A compound of any one of claims 1 to 20 for use in treating cancer.

29. A compound of any one of claims 1 to 20 for use in treating an allergic disease.

30. A pharmaceutically acceptable composition comprising a compound of any one of claims 1 to 20 and at least one pharmaceutically acceptable excipient.

Description:
Antiviral Compounds

[0001] This invention relates to a series of tricyclic compounds comprising a pteridinone core linked to a third heterocycloalkyl ring. The compounds stimulate interferon-a (IFN-a) production useful in the treatment of Hepatitis B viral infections. The invention also relates to pharmaceutical compositions comprising these compounds and methods of using the compound in treatment.

BACKGROUND

[0002] Approximately 240 million people are thought to be chronically infected with hepatitis B virus (HBV) globally. Complications of chronic hepatitis B (CHB), including liver cirrhosis and hepatocellular carcinoma (HCC), are responsible for approximately 0.5-1 million deaths per year (EASL clinical practice guidelines 2012). Transmission can occur through a variety of routes, including perinatally or through contact with infected blood and bodily fluids. The risk of developing chronic HBV infection is highest in those who acquire the virus perinatally.

[0003] Although a prophylactic vaccine is available and effective in developed countries, uptake of the vaccine in developing countries is low and as a result the disease is still prevalent. Vaccination is also not useful as a treatment for established infection. The current mainstay of treatment for HBV are nucleoside/nucleotide analogues or interferon-a / pegylated interferon-a (IFN-a/peg IFN-a). Nucleoside/nucleotide analogues, can be effective at inhibiting viral replication, however not all patients respond and viral resistance is an emerging problem. IFN-a/peg IFN-a is indicated in patients with a very high viral load (chronic active infection), and although IFN-a/peg IFN-a displays a higher rate of seroconversion compared to nucleo(s)tide analogues, they are poorly tolerated and carry a risk of adverse events.

[0004] Therapeutic strategies aimed at modulating the host immune response have been trialled for various indications, including viral hepatitis, and show promising activity. Tolllike receptors (TLRs), which are expressed by immune cells, play an important role in innate immunity by recognising foreign antigens (such as viral DNA) and agonists of TLRs can directly stimulate the innate immune system to produce I FN-a to fight infection. In the context of viral hepatitis, where some individuals fail to mount sufficient innate and adaptive responses to clear infection, this approach offers hope of potentially achieving a cure.

[0005] Failure to clear the infection in the short term generally leads to a lifelong harbouring of the virus. The exact reasons why some individuals can clear infection and others do not are uncertain; however it has been observed that those who go on to develop CHB show weakened or defective immune responses, especially in terms of IFN- α production. Age and route of infection are key factors in determining how infection will progress, e.g. placental transmission from mother to child leads to chronic infection in >90% cases. Immune evasion strategies by the virus can also play a role in determining how the infection will progress. The chronicity of CHB and associated inflammation, albeit from a weakened immune system, eventually leads to liver damage in sufferers.

[0006] The most advanced TLR agonist program in development for HBV is Gilead's GS- 9620, which is currently in Phase II trials as an oral treatment for HBV infection. The drug has a reported high clearance rate. Oral administration and gut absorption results in direct impact at the site of infection, the liver, and reduces the risk of potential side effects due to systemic IFN-a or cytokine production. However, high inter-patient variability in uptake has been observed with GS-9620, which may be a result of its low permeability and because it is a potential substrate for efflux pumps within the gut. Data also show that GS-9620 may induce tumour necrosis factor alpha (TNF-a) production from human PBMCs (Turnas et al. Preclinical characterisation of GS-9620, a potent and selective oral TLR 7 agonist. Journal of Hepatology; 201 1 ; Volume 54, Supplement 1 , Pages S446-S447 abstract number 1 129). Excessive production of inflammatory cytokines, such as TNF-a, as a result of immunostimulation, has been shown to initiate a number of toxic effects in the clinic. Observed toxicities can include acute phase response, cell/tissue injury, coagulopathy, dyslipidaemia and cytopaenia, amongst others.

[0007] It is an aim of certain embodiments of the invention to provide compounds that are effective at treating hepatitis B. It is an aim of certain embodiments of the invention to provide compounds that are more effective at treating hepatitis B than prior art compounds.

[0008] It is an aim of certain embodiments of the invention to provide compounds that are active as promoters of IFN-a. It is an aim of certain embodiments of the invention to provide compounds that are more active as promoters of IFN-a than prior art compounds. It is an aim of certain embodiments of the invention to provide compounds that are more selective as promoters of IFN-a, for example relative to TNF-a. It is an aim of certain embodiments of the invention to provide compounds that are more selective as promoters of IFN-a, for example relative to TNF-a, than prior art compounds.

[0009] It is an aim of certain embodiments of the invention to provide compounds that have higher permeability than prior art compounds. It is an aim of certain embodiments of the invention to provide compounds that exhibit lower efflux in the gut than prior art compounds. [0010] Certain embodiments of the present invention satisfy some or all of the above aims.

BRIEF SUMMARY OF THE DISCLOSURE

[0011] In a first aspect of the invention there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof:

wherein

-L 1 - is independently selected from: a bond, -0-, -NR 6 -, -C(O)-, -S(0) y - (where y is 0, 1 or 2), -C(0)0-, -OC(O)-, -C(0)NR 6 -, -NR 6 C(0)-, -S(0) 2 NR 6 - and -NR 6 S(0) 2 -;

=Y 1 is independently selected from: =0 and =S;

X 1 , X 2 and X 3 are each independently selected from a bond, a carbon atom, a nitrogen atom, -O- and -S(0)y-(where y is 0, 1 or 2); provided that no more than two of X 1 , X 2 and X 3 are a bond and provided that no more than one of X 1 , X 2 and X 3 is selected from a nitrogen atom, -O- and -S(0)y-;

R 1 is independently selected from Ci-Cio-alkyl, Ci-Cio-haloalkyl, C2-Cio-alkenyl, C2-C10- alkynyl, C3-Cs-cycloalkyl, 4-12-heterocycloalkyl, aryl, heteroaryl and -d-Cs-alkylene-R 7 ; wherein R 7 is independently selected from C3-Cs-cycloalkyl, 4-12-heterocycloalkyl, aryl and heteroaryl, -0-Ci-C 4 alkyl, -S-Ci-C 4 alkyl and -NR 6 Ci-C 4 -alkyl;

R 2 and R 9 are each independently at each occurrence selected from: H , Ci-C4-alkyl, -C(O)- Ci-C 4 -alkyl and -S(0) 2 -Ci-C 4 -alkyl;

R 3 , R 6 , R 8 and R 11 are each independently at each occurrence selected from: H and C1-C4 -alkyl;

R 4 is independently at each occurrence selected from: halo, Ci-C6-alkyl, C2-C6-alkenyl, C2- Ce-alkynyl, C 3 -C 6 -cycloalkyl, Ci-C 6 -haloalkyl, nitro, cyano, NR 8 R 9 , S(0) 2 OR 8 , S(0) 2 R 8 , S(0) 2 NR 8 R 8 , CO2R 8 , C(0)R 8 , CONR 8 R 8 , SR 8 and OR 8 ; or two R 4 groups attached to the same carbon atom, together with that carbon atom form a C=0 group; -L 2 - is independently selected from: a bond, -(CR 10 R 10 ) Z - (where z is 1 , 2 or 3), -0-, -S-, - NR 6 -, -C(O)-, -S(0) 2 - , -C(0)0-, -OC(O)-, -C(0)NR 6 -, -NR 6 C(0)-, -S(0) 2 NR 6 - and - NR 6 S(0) 2 -; wherein, if L 2 is attached via X 1 , X 2 or X 3 and the X 1 , X 2 or X 3 to which L 2 is attached is a nitrogen atom, -L 2 - is not -0-, -S-, -NR 6 -, -OC(O)-, -NR 6 C(0)- or -NR 6 S(0) 2 -; -L 3 is independently a bond or is -L 4 -L 5 -;

-L 4 - is independently selected from: -(CR 10 R 10 ) r - (where r is 1 , 2 or 3), aryl, heteroaryl, C3- Cs-cycloalkyl, 4-i 2 -heterocycloalkyl;

-L 5 - is independently selected from: a bond, -0-, -S-, -NR 6 -, -C(O)-, -S(0) 2 -, -C(0)0-, - OC(O)-, -C(0)NR 6 -, -NR 6 C(0)-, -S(0) 2 NR 6 - and -NR 6 S(0) 2 -;

R 5 is independently selected from: H, Ci-C6-alkyl, C 2 -C6-alkenyl, C 2 -C6-alkynyl, aryl, heteroaryl, C 3 -C 8 -cycloalkyl, 4 -i 2 -heterocycloalkyl, Ci-C 6 -haloalkyl and -(CR 10 R 10 ) S -NR 11 R 12 (where s is 0, 1 , 2 or 3); wherein when -L 2 -, -L 3 - and -L 4 - are selected such that R 5 is directly attached to a nitrogen or oxygen atom, R 5 is not attached to the rest of the molecule through a nitrogen atom;

R 10 is independently at each occurrence selected from: H, F and Ci-C 4 -alkyl;

R 12 is independently selected from H, Ci-C6-alkyl, C 2 -C6-alkenyl, C 2 -C6-alkynyl, aryl, heteroaryl, C3-Cs-cycloalkyl, 4 -i 2 -heterocycloalkyl, -d-Cs-alkylene-R 13 ; wherein R 13 is independently selected from C3-Cs-cycloalkyl, 4 -i 2 -heterocycloalkyl, aryl and heteroaryl; or R 11 and R 12 together with the nitrogen to which they are attached form a 4 -i 2 heterocycloalkyi group;

x is an integer selected from 0, 1 , 2, 3 and 4;

wherein any aryl, heteroaryl or heterocycloalkyi group may be monocyclic- or bicyclic; where a heterocycloalkyi group is bicyclic it comprises a 4-, 5-, 6- or 7-membered heterocycloalkyi ring fused to a ring selected from phenyl, 5- or 6- membered heteroaryl, C3-Cs-cycloalkyl, 4-, 5-, 6- or 7-membered heterocycloalkyi ring;

wherein each of the aforementioned alkyl, alkylene, alkenyl, alkynyl, haloalkyi, cycloalkyi, heterocycloalkyi, aryl (e.g. phenyl) and heteroaryl groups and rings is optionally substituted, where chemically possible, by 1 to 5 substituents which are each independently at each occurrence selected from the group consisting of: oxo, =NR a , =NOR a , halo, nitro, cyano, NR a R a , NR a S(0) 2 R a , NR a C(0)R a , NR a CONR a R a , NR a C0 2 R a , OR a , SR a , S(0)R a , S(0) 2 OR a , S(0) 2 R a , S(0) 2 NR a R a , C0 2 R a , C(0)R a , CONR a R a , Ci-C 4 - alkyl, C 2 -C 4 -alkenyl, C 2 -C 4 -alkynyl and Ci-C 4 -haloalkyl; wherein R a is independently at each occurrence selected from H, Ci-C 4 -alkyl and Ci-C 4 -haloalkyl. [0012] For the absence of doubt, it is intended that R 4 and -L 2 - may be attached to any position, where chemically possible, on the 5-, 6- or 7-membered ring that comprises X 1 , X 2 and X 3 . Thus, where X 1 , X 2 or X 3 is a carbon atom or a nitrogen atom, that carbon atom or a nitrogen atom may be the point of attachment of one or more R 4 groups and/or -L 2 -. Otherwise, that carbon atom or nitrogen atom is attached to two or one hydrogen atoms respectively.

[0013] In an embodiment, the compound of formula (I) is a compound of formula (II):

wherein R 1 , R 4 , R 5 , -L 1 -, -L 2 -, -L 3 -, X 3 and x are as described above for formula (I).

[0014] In an embodiment, the compound of formula (I) is a compound of formula (III):

wherein R 1 , R 4 , R 10 , R 11 , R 12 , -L 1 -, -L 3 -, s and x are as described above for formula (I); wherein -L 2 - is independently selected from -C(O)- and -S(0)2-.

[0015] In an embodiment, the compound of formula (I) is a compound of formula (IV):

0 R 10 ) S NR 11 R 12 (IV) wherein R 1 , R 4 , R 10 , R 1 1 , R 12 , -L 5 -, x and s are as described above for formula (I); wherein - L 2 - is independently selected from -C(O)- and -S(0)2-; and wherein Z 1 , Z 2 , Z 3 , Z 4 and Z 5 are each carbon or nitrogen; wherein no more than two of Z 1 , Z 2 , Z 3 , Z 4 and Z 5 are nitrogen; R 14 is independently at each occurrence selected from the group consisting of: halo, nitro, cyano, NR a R a , NR a S(0) 2 R a , NR a C(0)R a , NR a CONR a R a , NR a C0 2 R a , OR a , SR a , S(0)R a , S(0) 2 OR a , S(0) 2 R a , S(0) 2 NR a R a , C0 2 R a , C(0)R a , CONR a R a , Ci-C 4 -alkyl, C 2 -C 4 - alkenyl, C 2 -C 4 -alkynyl and Ci-C 4 -haloalkyl; and a is an integer selected from 0, 1 , 2, 3 or 4. It may be that Z 1 , Z 2 , Z 3 , Z 4 and Z 5 are each carbon.

[0016] In an embodiment, the compound of formula (I) is a compound of formula (V):

10 R 10 ) S NR 11 R 12 (V) wherein R 1 , R 4 , R 10 , R 1 1 , R 12 , -L 5 -, x and s are as described above for formula (I); wherein - L 2 - is independently selected from -C(O)- and -S(0) 2 -; and wherein Z 1 , Z 2 , Z 4 , Z 5 , R 14 and a are as described above for formula (IV).

[0017] In an embodiment, the compound of formula (I) is a compound of formula (VI):

wherein R 1 , R 4 , R 10 , R 11 , R 12 , x and s are as described above for formula (I); wherein -L 2 - is independently selected from -C(O)- and -S(0) 2 -; and wherein R 14 and a are as described above for formula (IV).

[0018] In an embodiment, the compound of formula (I) is a compound of formula (VII): wherein R 1 , R 4 , R 10 , R 11 , R 12 , s and x are as described above for formula (I); wherein -L 2 - is independently selected from -C(O)- and -S(0)2-.

The following statements apply where chemically allowable to compounds of any of formulae (I) to (VII). These statements are independent and interchangeable. In other words, any of the features described in any one of the following statements may (where chemically allowable) be combined with the features described in one or more other statements below. In particular, where a compound is exemplified or illustrated in this specification, any two or more of the statements below which describe a feature of that compound, expressed at any level of generality, may be combined so as to represent subject matter which is contemplated as forming part of the disclosure of this invention in this specification.

[0019] It may be that at least one of R 2 and R 3 are H. Preferably, R 2 and R 3 are each H. Preferably, =Y 1 is =0.

[0020] -L 1 - may be selected from -0-, -NR 6 - and -S-. Preferably, -L 1 - is -0-.

[0021] It may be that R 1 is independently selected from Ci-Cio-alkyl, C2-Cio-alkenyl, C2- Cio-alkynyl. It may be that the groups of which R 1 is comprised are unsubstituted. Preferably, R 1 is unsubstituted Ci-Cio-alkyl. Thus, R 1 may be unsubstituted C2-C8 alkyl. R 1 may be unsubstituted C3-C5 alkyl . In certain specific embodiments, R 1 is n-butyl.

[0022] R 1 may be -Ci-C 5 -alkylene-0-Ci-C 4 alkyl.

[0023] It may be that X 3 is selected from a carbon atom and a nitrogen atom. It may be that X 3 is carbon. In certain preferred embodiments, X 3 is nitrogen.

[0024] It may be that X 2 is a bond. In certain preferred embodiments, X 2 is carbon.

[0025] It may be that X 1 is a bond. In certain preferred embodiments, X 1 is carbon.

[0026] Preferably, none of X 1 , X 2 and X 3 is a bond. The ring comprising X 1 , X 2 and X 3 is therefore preferably a 7-membered ring.

[0027] It may be that X 3 is nitrogen and X 1 and X 2 are each carbon. [0028] R 4 may independently at each occurrence be selected from halo, Ci-C6-alkyl, C2- Ce-alkenyl, C 2 -C 6 -alkynyl, C 3 -C 6 -cycloalkyl, Ci-Ce-haloalkyl, CO2R 8 , C(0)R 8 , CON R 8 R 8 ; or two R 4 groups attached to the same carbon atom, together with that carbon atom form a C=0 group. R 4 may at each occurrence be selected from Ci-C6-alkyl, e.g. methyl.

[0029] In certain particular embodiments, x is 0.

[0030] In certain particular embodiments x is 2. Where x is 2, it may be that both R 4 groups are attached to the same carbon atom. R 4 may at both occurrences be selected from Ci-C6-alkyl, e.g. methyl. In these embodiments, it may be that X 1 is a carbon atom and both R 4 groups are attached to X 1 .

[0031] It may be that -L 2 -, -L 3 - and R 5 are selected such that -L 2 -L 3 -R 5 comprises at least one (e.g. a single) nitrogen atom. Preferably, -L 2 -, -L 3 - and R 5 are selected such that -L 2 - L 3 -R 5 comprises at least one (e.g. a single) tertiary amine.

[0032] -L 2 -L 3 -R 5 is preferably attached to the rest of the molecule at X 3 . In these embodiments, X 3 will be selected from a carbon atom and a nitrogen atom. Where X 3 is nitrogen, -L 2 - may be selected from -C(O)- or -S(0)2-. -L 2 - may however be -(CR 10 R 10 ) Z - (e.g. -CR 10 CR 10 or -CH 2 -). In certain particular examples, -L 2 - is -C(O)-.

[0033] -L 3 - may be a bond. It may be that -L 3 - is a bond and R 5 is -(CR 10 R 10 ) S -N R 1 1 R 12 . In these embodiments, it may be that s is selected from 1 , 2 and 3. It may be that -L 2 - is selected from -C(O)- or -S(0)2-, that -L 3 - is a bond and s is selected from 1 , 2 and 3. In these embodiments, it may be that s is 1 or it may be that s is 2.

[0034] -L 3 - may be -L 4 -L 5 -.

[0035] -L 4 - may be independently selected from: -(CR 10 R 10 ) r - (where r is 1 , 2 or 3), aryl, heteroaryl, C3-Cs-cycloalkyl, 4-12-heterocycloalkyl. -L 4 - may be independently selected from aryl, heteroaryl, C3-Cs-cycloalkyl, 4-12-heterocycloalkyl. -L 4 - may be independently selected from aryl and heteroaryl, e.g. -L 4 - may be selected from phenyl and 6-membered heteroaryl. In certain particular embodiments, -L 4 - is phenyl. In other particular embodiments, -L 4 - is 6-membered heteroaryl, e.g. a pyridinyl.

[0036] -L 5 - may be independently selected from: a bond, -0-, -S-, -N R 6 -, -C(O)- and - S(0)2-. -L 5 - may be independently selected from: a bond, -0-, -S- and -N R 6 -. Alternatively, -L 5 - may be independently selected from -C(O)- and -S(0)2-. In certain particular embodiments, -L 5 - is a bond. In certain other embodiments, -L 5 - is -0-.

[0037] It may be that -L 4 - is independently selected from aryl and heteroaryl, -L 5 - is independently selected from: a bond, -0-, -S- and -N R 6 - and R 5 is -(CR 10 R 10 ) S -N R 1 1 R 1 2 . In these embodiments, it may be that s is selected from 1 , 2 and 3. It may also be that L 2 is selected from -C(O)- or -S(0) 2 -.

[0038] -L 3 -R 5 may be:

Z 2 , Z 3 , Z 4 and Z 5 are each carbon or nitrogen; wherein no more than two of Z Z 2 , Z 3 , Z 4 and Z 5 are nitrogen; R 14 is independently at each occurrence selected from the group consisting of: halo, nitro, cyano, NR a R a , NR a S(0)2R a , NR a C(0)R a , NR a CONR a R a , NR a C0 2 R a , OR a , SR a , S(0)R a , S(0) 2 OR a , S(0) 2 R a , S(0) 2 NR a R a , C0 2 R a , C(0)R a , CONR a R a , Ci-C 4 -alkyl, C 2 -C 4 -alkenyl, C 2 -C 4 -alkynyl and Ci- -haloalkyl; and a is an integer selected from 0, 1 , 2, 3 or 4. -L 3 -R 5 may be:

Z 2 , Z 3 , Z 4 and Z 5 are each carbon. -L 3 -R 5 may be:

. -L 5 - may be a bond, a may be 0.

[0039] It may be that R 5 is not H. Thus, R 5 may be independently selected from: Ci-ds- alkyl, C 2 -C6-alkenyl, C 2 -C6-alkynyl, aryl, heteroaryl, d-Cs-cycloalkyl, 4 -i 2 -heterocycloalkyl, d-Ce-haloalkyl and -(CR 10 R 10 ) S -NR 1 1 R 12 (where s is 0, 1 , 2 or 3);

[0040] It may be that R 5 is selected such that R 5 comprises at least one (e.g. a single) nitrogen atom. Preferably, R 5 is selected such that R 5 comprises at least one (e.g. a single) tertiary amine.

[0041] Preferably, R 5 is -(CR 10 R 10 ) S -NR 1 1 R 12 .

[0042] s may be selected from 1 , 2 and 3. Thus, it may be that s is selected from 1 and 2.

[0043] R 1 1 may be Ci-C -alkyl. R 12 may be selected from d-Ce-alkyl, C 2 -C 6 -alkenyl, C 2 - C6-alkynyl, aryl, heteroaryl, d-Cs-cycloalkyl, 4 -i 2 -heterocycloalkyl, -d-Cs-alkylene-R 13 . Thus, it may be that R 1 1 is Ci-C 4 -alkyl; and R 12 is selected from Ci-C6-alkyl d-d-alkenyl, C 2 -C6-alkynyl, aryl, heteroaryl, d-Cs-cycloalkyl, 4 -i 2 -heterocycloalkyl, -d-d-alkylene-R 13 ; or that R 11 and R 12 together with the nitrogen to which they are attached form a 4-12 heterocycloalkyi group which may be monocyclic- or bicyclic; where a heterocycloalkyi group is bicyclic it comprises a 4-, 5-, 6- or 7-membered heterocycloalkyi ring fused to a ring selected from phenyl, 5-or 6- membered heteroaryl, C3-C8 cycloalkyl, 4-, 5-, 6- or 7- membered heterocycloalkyi ring.

[0044] Where R 5 is -(CR 10 R 10 ) S -NR 11 R 12 it may be that R 11 and R 12 together with the nitrogen to which they are attached form a 4-12 heterocycloalkyi group which may be monocyclic- or bicyclic; where a heterocycloalkyi group is bicyclic it comprises a 4-, 5-, 6- or 7-membered heterocycloalkyi ring fused to a ring selected from phenyl, 5-or 6- membered heteroaryl, C3-C8 cycloalkyl, 4-, 5-, 6- or 7-membered heterocycloalkyi ring. It may be that R 11 and R 12 together with the nitrogen to which they are attached form a pyrrolidine ring which optionally forms part of a bicyclic heterocycloalkyi group.

[0045] Exemplary NR 11 R 12 groups include:

[0046] R 5 may be selected from heteroaryl or 4-12-heterocycloalkyl, wherein the heteroaryl group or heterocycloalkyi group comprise at least one ring nitrogen. Thus, R 5 may be imidazole, pyrrole or pyrazole, e.g. pyrazol-2-yl. Where R 5 is a heterocycloalkyi group comprising a heterocycloalkyi ring and a phenyl ring, it may be that the group is attached

to -L 2 - or -L 3 - via the phenyl ring. R 5 may also be In these embodiments, it may be that -L 3 - is a bond. It may also be that -L 2 - is selected from C(O)- and -S(0) 2 -.

[0047] Exemplary -L 2 -L 3 -R 5 groups include: 11

DETAILED DESCRIPTION

[0049] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

[0050] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0051] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Compounds

[0052] The term C m -C n refers to a group with m to n carbon atoms.

[0053] The term "alkyl" refers to a monovalent linear or branched hydrocarbon chain. For example, Ci-C6-alkyl may refer to methyl, ethyl, n-propyl, / ' so-propyl, n-butyl, sec-butyl, te/f-butyl, n-pentyl and n-hexyl. The alkyl groups may be unsubstituted or substituted by one or more substituents. Specific substituents for each alkyl group independently may be fluorine, OR a or NHR a .

[0054] The term "alkylene" refers to a bivalent linear hydrocarbon chain. For example, Ci-C3-alkylene may mean a methylene group (-CH2-), an ethylene group (-CH2CH2-) or a propylene group. The alkylene groups may be unsubstituted or substituted by one or more substituents. Specific substituents for each alkylene group independently may be C1-C4 alkyl, oxo or fluorine.

[0055] The term "haloalkyl" refers to a hydrocarbon chain substituted with at least one halogen atom independently chosen at each occurrence from: fluorine, chlorine, bromine and iodine. The halogen atom may be present at any position on the hydrocarbon chain. For example, Ci-C6-haloalkyl may refer to chloromethyl, fluoromethyl, trifluoromethyl, chloroethyl e.g. 1 -chloromethyl and 2-chloroethyl, trichloroethyl e.g. 1 ,2,2-trichloroethyl, 2,2,2-trichloroethyl, fluoroethyl e.g. 1 -fluoromethyl and 2-fluoroethyl, trifluoroethyl e.g. 1 ,2,2-trifluoroethyl and 2,2,2-trifluoroethyl, chloropropyl, trichloropropyl, fluoropropyl, trifluoropropyl. A halo alkyl group may be a fluoroalkyl group, i.e. a hydrocarbon chain substituted with at least one halogen atom.

[0056] The term "alkenyl" refers to a branched or linear hydrocarbon chain containing at least one double bond. The double bond(s) may be present as the E or Z isomer. The double bond may be at any possible position of the hydrocarbon chain. For example, "C2- C6-alkenyl" may refer to ethenyl, propenyl, butenyl, butadienyl, pentenyl, pentadienyl, hexenyl and hexadienyl. The alkenyl groups may be unsubstituted or substituted by one or more substituents. Specific substituents for any saturated carbon atom in each alkenyl group independently may be fluorine, OR a or NHR a .

[0057] The term "alkynyl" refers to a branched or linear hydrocarbon chain containing at least one triple bond. The triple bond may be at any possible position of the hydrocarbon chain. For example, "C2-C6-alkynyl" may refer to ethynyl, propynyl, butynyl, pentynyl and hexynyl. The alkynyl groups may be unsubstituted or substituted by one or more substituents. Specific substituents for any saturated carbon atom in each alkynyl group independently may be fluorine, OR a or NHR a .

[0058] The term "cycloalkyl" refers to a saturated hydrocarbon ring system containing 3, 4, 5 or 6 carbon atoms. For example, "C3-C6-cycloalkyl" may refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. The cycloalkyl groups may be unsubstituted or substituted by one or more substituents. Specific substituents for each cycloalkyl group independently may be Ci-C4-alkyl, oxo, fluorine, OR a or NHR a .

[0059] The term "aromatic" when applied to a substituent as a whole means a single ring or polycyclic ring system with 4n + 2 electrons in a conjugated π system within the ring or ring system where all atoms contributing to the conjugated π system are in the same plane.

[0060] The term "aryl" refers to an aromatic hydrocarbon ring system. The ring system has 4n +2 electrons in a conjugated π system within a ring where all atoms contributing to the conjugated π system are in the same plane. For example, the "aryl" may be phenyl and naphthyl. The aryl group may be unsubstituted or substituted by one or more substituents. Specific substituents for each aryl group independently may be Ci-C4-alkyl, Ci-C4-haloalkyl, cyano, halogen, OR a or NHR a .

[0061] The term "heteroaryl" may refer to any aromatic (i.e. a ring system containing (4n + 2) TT- electrons or n- electrons in the ττ-system) 5-10 membered ring system comprising from 1 to 4 heteroatoms independently selected from O, S and N (in other words from 1 to 4 of the atoms forming the ring system are selected from O, S and N). Thus, any heteroaryl groups may be independently selected from: 5 membered heteroaryl groups in which the heteroaromatic ring is substituted with 1-4 heteroatoms independently selected from O, S and N; and 6-membered heteroaryl groups in which the heteroaromatic ring is substituted with 1-3 (e.g.1-2) nitrogen atoms; 9-membered bicyclic heteroaryl groups in which the heteroaromatic system is substituted with 1-4 heteroatoms independently selected from O, S and N; 10-membered bicyclic heteroaryl groups in which the heteroaromatic system is substituted with 1-4 nitrogen atoms. Specifically, heteroaryl groups may be independently selected from: pyrrole, furan, thiophene, pyrazole, imidazole, oxazole, isoxazole, triazole, oxadiazole, thiadiazole, tetrazole; pyridine, pyridazine, pyrimidine, pyrazine, triazine, indole, isoindole, benzofuran, isobenzofuran, benzothiophene, indazole, benzimidazole, benzoxazole, benzthiazole, benzisoxazole, purine, quinoline, isoquinoline, cinnoline, quinazoline, quinoxaline, pteridine, phthalazine, naphthyridine. Heteroaryl groups may also be 6-membered heteroaryl groups in which the heteroaromatic ring is substituted with 1 heteroatomic group independently selected from O, S and NH and the ring also comprises a carbonyl group. Such groups include pyridones and pyranones. The heteroaryl system itself may be substituted with other groups. The heteroaryl group may be unsubstituted or substituted by one or more substituents. Specific substituents for each heteroaryl group independently may be C1-C4- alkyl, Ci-C4-haloalkyl, cyano, halogen, OR a or NHR a .

[0062] The term " m -nheterocycloalkyl" refers to a m to n membered monocyclic or bicyclic saturated or partially saturated group comprising 1 or 2 heteroatoms independently selected from O, S and N in the ring system (in other words 1 or 2 of the atoms forming the ring system are selected from O, S and N). Thus a Vi2heterocycloalkyl" is a 4 to 12 membered monocyclic or bicyclic saturated or partially saturated group comprising 1 or 2 heteroatoms independently selected from O, S and N in the ring system (in other words 1 or 2 of the atoms forming the ring system are selected from O, S and N). By partially saturated it is meant that the ring may comprise one or two double bonds. This applies particularly to monocyclic rings with from 5 to 8 members. The double bond will typically be between two carbon atoms but may be between a carbon atom and a nitrogen atom. Examples of heterocycloalkyl groups include; piperidine, piperazine, morpholine, thiomorpholine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, dihydrofuran, tetrahydropyran, dihydropyran, dioxane, azepine. Bicyclic systems may be spiro-fused, i.e. where the rings are linked to each other through a single carbon atom; vicinally fused, i.e. where the rings are linked to each other through two adjacent carbon or nitrogen atoms; or they may be share a bridgehead, i.e. the rings are linked to each other two non-adjacent carbon or nitrogen atoms. The heterocycloalkyl groups may be unsubstituted or substituted by one or more substituents. Specific substituents for any saturated carbon atom in each heterocycloalkyl group may independently be Ci-C4-alkyl, oxo, fluorine, OR a or NHR a .

[0063] Where the compound of formula (I) is an N-oxide, it will typically be a pyridine N- oxide, i.e. where the compound of formula (I) comprises a pyridine ring, the nitrogen of that pyridine may be N + -0 " . Alternatively, it may be that the compound of the invention is not an N-oxide.

[0064] Included within the scope of the present invention are all stereoisomers, geometric isomers and tautomeric forms of the compounds of the invention, including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof. Also included are acid addition or base salts wherein the counter ion is optically active, for example, d-lactate or l-lysine, or racemic, for example, dl-tartrate or dl-arginine.

[0065] Compounds of the invention containing one or more asymmetric carbon atoms can exist as two or more stereoisomers. Where a compound of the invention contains a double bond such as a C=C or C=N group, geometric cis/trans (or Z/E) isomers are possible. Specifically, the oxime groups present in certain compounds of the invention may be present as the E-oxime, as the Z-oxime or as a mixture of both in any proportion. Cis/trans isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallisation.

[0066] Where structurally isomeric forms of a compound are interconvertible via a low energy barrier, tautomeric isomerism ('tautomerism') can occur. This can take the form of proton tautomerism in compounds of the invention containing, for example, an imino, keto, or oxime group, or so-called valence tautomerism in compounds which contain an aromatic moiety.

[0067] Conventional techniques for the preparation/isolation of individual enantiomers when necessary include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). [0068] Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of the invention contains an acidic or basic moiety, a base or acid such as 1- phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted into the corresponding pure enantiomer(s) by means well known to a skilled person.

[0069] Chiral compounds of the invention (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically from 2% to 20%, and from 0 to 5% by volume of an alkylamine, typically 0.1 % diethylamine. Concentration of the eluate affords the enriched mixture.

[0070] When any racemate crystallises, crystals of two different types are possible. The first type is the racemic compound (true racemate) referred to above wherein one homogeneous form of crystal is produced containing both enantiomers in equimolar amounts. The second type is the racemic mixture or conglomerate wherein two forms of crystal are produced in equimolar amounts each comprising a single enantiomer.

[0071] While both of the crystal forms present in a racemic mixture have identical physical properties, they may have different physical properties compared to the true racemate. Racemic mixtures may be separated by conventional techniques known to those skilled in the art - see, for example, "Stereochemistry of Organic Compounds" by E. L. Eliel and S. H. Wilen (Wiley, 1994).

[0072] It follows that a single compound may exhibit more than one type of isomerism.

[0073] The present invention also includes the synthesis of all pharmaceutically acceptable isotopically-labelled compounds of formulae (I) to (VII) wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature.

[0074] Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as 2 H and 3 H, carbon, such as 11 C, 13 C and 14 C, chlorine, such as 36 CI, fluorine, such as 18 F, iodine, such as 123 l and 125 l, nitrogen, such as 13 N and 15 N, oxygen, such as 15 0, 17 0 and 18 0, phosphorus, such as 32 P, and sulfur, such as 35 S. [0075] Certain isotopically-labelled compounds, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e. 3 H, and carbon-14, i.e. 14 C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

[0076] Substitution with heavier isotopes such as deuterium, i.e. 2 H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

[0077] Substitution with positron emitting isotopes, such as 11 C, 18 F, 15 0 and 13 N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.

[0078] Isotopically-labelled compounds can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described using an appropriate isotopically-labelled reagent in place of the non-labelled reagent previously employed.

Uses, methods of treatment and pharmaceutical formulations

[0079] The compounds of the invention promote IFN-a production and will therefore be useful in the treatment of any indication which can be treated by stimulation of IFN-a. Stimulation of IFN-a boosts the immune system. It can be used to treat viral infections. The compounds of the invention are particularly useful in the treatment of Hepatitis B viral infections.

[0080] In an aspect of the invention is provided a compound for use in the preparation of a medicament. The medicament may be for use in the treatment of any of the diseases, infections and indications mentioned in this specification.

[0081] In another aspect of the invention is provided a compound of the invention for medical use. The compound may be used in the treatment of any of the diseases, infections and indications mentioned in this specification.

[0082] In another aspect of the invention is provided a method of treating a disease in a subject in need thereof. The disease may be any of the diseases, infections and indications mentioned in this specification. The method comprises administering a therapeutic amount of a compound of the invention to a subject in need thereof.

[0083] The invention provides a method of promoting IFN-a production in a subject in need thereof, the method comprising administering a therapeutic amount of a compound of the invention. [0084] The invention also provides a method of boosting the immune system in a subject in need thereof, the method comprising administering a therapeutic amount of a compound of the invention.

[0085] In another aspect of the invention is provided a pharmaceutical formulation comprising a compound of the invention and at least one pharmaceutically acceptable excipient.

[0086] The compounds of the invention can be used to treat viral infections. Illustrative viral infections include: HIV, Hepatitis B, Hepatitis C, herpes, norovirus, Epstein-Barr, chicken pox, smallpox, dengue fever, viral meningitis, cytomegalovirus, monkeypox, vaccinia, Ebola, Newcastle disease virus, Sendai virus, influenza, vesicular stomatis virus, west nile virus, herpes simplex viruses 1 and 2 and rotavirus. The compounds of the invention are particularly useful in the treatment of Hepatitis B infections.

[0087] The compounds of the invention can be used to treat cancer. In particular, IFN-a is used to treat cancer of the kidney, malignant melanoma, multiple myeloma, carcinoid tumours, lymphoma and leukaemia. Although IFN-a has been around for many years, we still do not know how it exerts its anticancer effects. According to in vitro studies, IFN-a modulates gene expression, promotes cell differentiation and apoptosis, directly inhibits cell growth and proliferation, restores regulation by the bone marrow microenvironment and induces an immunomodulatory response. Microarray analyses have shown that IFN-a IFN-a can induce expression of over 300 different genes. These genes encode apoptotic proteins (i.e., TRAIL, Fas, caspase-4, caspase-8 and XAF-1), anti-viral proteins (that is, PKR, 20 50 A oligoadenylate synthetase and Mx proteins), immunomodulatory proteins (that is, MHC I and II, LMP-2 and C1 inhibitor), host defense proteins (that is, PKR, IRF 1- 9, interleukin- 15 and interleukin-6) and transcription factors (that is, signal transducer and activator of transcription 1 , signal transducer and activator of transcription 2, ISGF3-g and IRF1-7).63 The precise function of many of the gene products induced by IFN-a remains unknown; however, several of the identified genes encode well-known pro-apoptotic proteins, including TRAIL/Apo2L and Fas/CD95.63 In CML progenitor cells, IFN-a enhances the expression of the Fas receptor, thereby increasing cell sensitivity to Fas ligand. In addition to activating apoptosis, IFN-a directly targets key regulators of the cell cycle, including retinoblastoma protein, cdc25A, cyclins (cyclin D3, cyclin E and cyclin A) and cyclindependent kinases (cdk4 and cdk6). Such targeting can block and/ or lengthen the cell cycle phases, allowing cells to differentiate or undergo apoptosis. The compounds of the invention have been shown to stimulate IFN-a production and it would thus be expected that they could be used in the treatment of cancer. [0088] The compounds of the invention may be useful in treatment of allergic diseases. Allergies are the result of aberrant immune reactivity to innocuous environmental proteins (allergens). A pivotal component of allergic pathogenesis is the generation of effector Type 2 helper T (Th2) cells, which secrete cytokines that drive the synthesis of allergen- specific Immunoglobulin E and activation of pro-inflammatory cells, leading to manifestation of symptoms. Most current therapies for allergic diseases alleviate symptoms by reducing either the activity of mediators of the allergic cascade or the degree of local inflammation. Though beneficial, this approach does not alter the underlying allergic immune pathology and therefore does not promote long-term disease remission. To this end, attention has been directed towards methods that promote the down- modulation of Th2 responsiveness to allergens by inducing counter-balancing Type 1 helper T (Th1)- type cytokines (i.e., interferons [IFNs] and interleukin [IL]-12) through manipulation of elements of host defense, such as the toll-like receptors (TLRs).

[0089] The compounds of the invention may be obtained, stored and/or administered in the form of a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, malic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulfonic, toluenesulfonic, benzenesulfonic, salicylic, sulfanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids. Suitable base salts are formed from bases which form nontoxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulfate and hemicalcium salts. Also included are acid addition or base salts wherein the counter ion is optically active, for example, d-lactate or l-lysine, or racemic, for example, dl-tartrate or dl-arginine.

[0090] Compounds of the invention may exist in a single crystal form or in a mixture of crystal forms or they may be amorphous. Thus, compounds of the invention intended for pharmaceutical use may be administered as crystalline or amorphous products. They may be obtained, for example, as solid plugs, powders, or films by methods such as precipitation, crystallization, freeze drying, spray drying, or evaporative drying. Microwave or radio frequency drying may be used for this purpose.

[0091] For the above-mentioned compounds of the invention the dosage administered will, of course, vary with the compound employed, the mode of administration, the treatment desired and the disorder indicated. For example, if the compound of the invention is administered orally, then the daily dosage of the compound of the invention may be in the range from 0.01 micrograms per kilogram body weight ( g/kg) to 100 milligrams per kilogram body weight (mg/kg).

[0092] A compound of the invention, or pharmaceutically acceptable salt thereof, may be used on their own but will generally be administered in the form of a pharmaceutical composition in which the compounds of the invention, or pharmaceutically acceptable salt thereof, is in association with a pharmaceutically acceptable adjuvant, diluent or carrier. Conventional procedures for the selection and preparation of suitable pharmaceutical formulations are described in, for example, "Pharmaceuticals - The Science of Dosage Form Designs", M. E. Aulton, Churchill Livingstone, 1988.

[0093] The compounds of the invention may be administered in combination with other active compounds (e.g. antifungal compounds, oncology compounds, antibacterial compounds, other modulators of the immune system) and, in particular, with antiviral compounds. The compound of the invention and the other active may be administered in different pharmaceutical formulations either simultaneously or sequentially with the other active. Alternatively, the compound of the invention and the other active may form part of the same pharmaceutical formulation.

[0094] Depending on the mode of administration of the compounds of the invention, the pharmaceutical composition which is used to administer the compounds of the invention will preferably comprise from 0.05 to 99 %w (per cent by weight) compounds of the invention, more preferably from 0.05 to 80 %w compounds of the invention, still more preferably from 0.10 to 70 %w compounds of the invention, and even more preferably from 0.10 to 50 %w compounds of the invention, all percentages by weight being based on total composition.

[0095] The pharmaceutical compositions may be administered topically (e.g. to the skin) in the form, e.g., of creams, gels, lotions, solutions, suspensions, or systemically, e.g. by oral administration in the form of tablets, capsules, syrups, powders, suspensions, solutions or granules; or by parenteral administration in the form of a sterile solution, suspension or emulsion for injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion); or by rectal administration in the form of suppositories; or by inhalation (i.e. in the form of an aerosol or by nebulisation). Typically, however, the compounds of the invention will be administered orally.

[0096] For oral administration the compounds of the invention may be admixed with an adjuvant or a carrier, for example, lactose, saccharose, sorbitol, mannitol; a starch, for example, potato starch, corn starch or amylopectin; a cellulose derivative; a binder, for example, gelatine or polyvinylpyrrolidone; and/or a lubricant, for example, magnesium stearate, calcium stearate, polyethylene glycol, a wax, paraffin, and the like, and then compressed into tablets. If coated tablets are required, the cores, prepared as described above, may be coated with a concentrated sugar solution which may contain, for example, gum arabic, gelatine, talcum and titanium dioxide. Alternatively, the tablet may be coated with a suitable polymer dissolved in a readily volatile organic solvent.

[0097] For the preparation of soft gelatine capsules, the compounds of the invention may be admixed with, for example, a vegetable oil or polyethylene glycol. Hard gelatine capsules may contain granules of the compound using either the above-mentioned excipients for tablets. Also liquid or semisolid formulations of the compound of the invention may be filled into hard gelatine capsules. Liquid preparations for oral application may be in the form of syrups or suspensions, for example, solutions containing the compound of the invention, the balance being sugar and a mixture of ethanol, water, glycerol and propylene glycol. Optionally such liquid preparations may contain colouring agents, flavouring agents, sweetening agents (such as saccharine), preservative agents and/or carboxymethylcellulose as a thickening agent or other excipients known to those skilled in the art.

[0098] For intravenous (parenteral) administration the compounds of the invention may be administered as a sterile aqueous or oily solution.

[0099] The size of the dose for therapeutic purposes of compounds of the invention will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well-known principles of medicine.

[00100] Dosage levels, dose frequency, and treatment durations of compounds of the invention are expected to differ depending on the formulation and clinical indication, age, and co-morbid medical conditions of the patient. The standard duration of treatment with compounds of the invention is expected to vary between one and seven days for most clinical indications. It may be necessary to extend the duration of treatment beyond seven days in instances of recurrent infections or infections associated with tissues or implanted materials to which there is poor blood supply including bones/joints, respiratory tract, endocardium, and dental tissues.

Synthesis

[00101] The skilled man will appreciate that adaptation of methods known in the art could be applied in the manufacture of the compounds used in the present invention. [00102] For example, the skilled person will be immediately familiar with standard textbooks such as "Comprehensive Organic Transformations - A Guide to Functional Group Transformations", RC Larock, Wiley- VCH (1999 or later editions), "March's Advanced Organic Chemistry - Reactions, Mechanisms and Structure", MB Smith, J. March, Wiley, (5th edition or later) "Advanced Organic Chemistry, Part B, Reactions and Synthesis", FA Carey, RJ Sundberg, Kluwer Academic/Plenum Publications, (2001 or later editions), "Organic Synthesis - The Disconnection Approach", S Warren (Wiley), (1982 or later editions), "Designing Organic Syntheses" S Warren (Wiley) (1983 or later editions), "Guidebook To Organic Synthesis" RK Mackie and DM Smith (Longman) (1982 or later editions), etc., and the references therein as a guide.

[00103] The skilled chemist will exercise his judgement and skill as to the most efficient sequence of reactions for synthesis of a given target compound and will employ protecting groups as necessary. This will depend inter alia on factors such as the nature of other functional groups present in a particular substrate. Clearly, the type of chemistry involved will influence the choice of reagent that is used in the said synthetic steps, the need, and type, of protecting groups that are employed, and the sequence for accomplishing the protection / deprotection steps. These and other reaction parameters will be evident to the skilled person by reference to standard textbooks and to the examples provided herein.

[00104] Sensitive functional groups may need to be protected and deprotected during synthesis of a compound of the invention. This may be achieved by conventional methods, for example as described in "Protective Groups in Organic Synthesis" by TW Greene and PGM Wuts, John Wiley & Sons Inc (1999), and references therein.

Certain compounds of the invention can be made according to or analogously to the methods described in the Examples below.

EXAMPLES

Example 1 - Synthesis of Tricyclic Compounds

Analytical Methods

NMR spectra were obtained on a LC Bruker AV400 using a 5 mm QNP probe (Method A) or Bruker AVIII 400 Nanobay using a 5 mm BBFQ with z-gradients (Method B). MS was carried out on a Waters Alliance ZQ MS (Methods A, B, D, E, F and G) or Acquity UPLC-SQD2#LCA081 (Method C) using H 2 0 and CH 3 CN mobile phase with pH modification as detailed under each method. Wavelengths were 254 and 210 nM.

Method A (Native pH) Column: Gemini NX C18, 5 μηι, 50 x 2 mm. Flow rate: 1.0 mL/min. Injection volume: 10 μΙ_ Mobile Phase A H 2 0

B CHsCN

Method B (Native pH)

Column: Waters XBridge C18, 5μηι, 50 x 2.1 mm. Flow rate: 0.8 mL/min. Injection volume: 10

Mobile Phase A H 2 0

B CHsCN

Time

A (%) B (%)

(min)

0 95 5

4 5 95

4.45 5 95

4.5 95 5

5 STOP Method C (Acidic pH)

Column: Acquity UPLC ® BEH C18 1.7 μητι, 50 x 2.1 mm. Flow rate: 0.6 mL/min. Injection volume: 2 μί.

Mobile Phase A H 2 0 + 0.1 % (v/v) formic acid

B CHsCN + 0.1 % (v/v) formic acid

Method D (Acidic pH)

Column: YMC-Triart C18 50 x 2 mm, 5 um. Flow rate: 0.8 mL/min. Injection volume: 5 μί.

Mobile Phase A H 2 0 B CHsCN C 50% H 2 0 / 50% CHsCN + 1.0% formic acid

Time

A (%) B (%) C (%)

(min)

0 95 0 5

4 0 95 5 4.4 0 95 5

4.5 95 5 0

4.5 STOP

Method E (Acidic pH)

Column: YMC Triart-C18 50 x 2 mm, 5 um Flow rate: 0.8 mL/min. Injection volume: 5 μΙ_ Mobile Phase A H 2 0 B CHsCN

C 50% H 2 0 / 50% CHsCN + 1 % formic acid

Method F (Basic pH)

Column: YMC-Triart C18 50 x 2 mm, 5 um. Flow rate: 0.8 mL/min. Injection volume: 5 μί. Mobile Phase A H 2 0

B CHsCN

C 50% H 2 0 / 50% CHsCN + 1.0% NH 3

Time

A (%) B (%) C (%)

(min) 0 95 0 5

4 0 95 5

4.4 0 95 5

4.5 95 5 0

4.5 STOP

Method G (Basic pH)

Column YMC Triart-C18 50 x 2 mm, 5 μηι Flow rate: 0.8 mL/min. Injection volume: 10 μΙ_ Mobile Phase A H 2 0 B CHsCN

C 50% H 2 0 / 50% CHsCN + 1.0% NH 3

Preparative HPLC was performed using column: XBridge™ prep C18 5 μΜ OBD 19 mm. Flow rate: 20 mL/min.

Method A

Waters 3100 Mass detector using H 2 0 and CH 3 CN + formic acid (0.05%-0.1 %) Method B

Waters 2767 Sample Manager (0.05% NH 3 ) Certain compounds of the invention can be synthesised according to general Scheme 1. Illustrative examples of reagents and conditions which are suitable for carrying out the reactions depicted in Scheme 1 can be found in the examples below.

Scheme 1

Depicted below are a selection of compounds of the invention:

8ai 8aii

Compounds 8ai, 8aiii, and 8bi-8bx and 8bxii-8bxv have been prepared according to Scheme 1 where n = 2 and 3 to incorporate a 6 and 7 membered heterocycle respectively.

Compounds in which -L 2 - is a sulfonyl group can be prepared analogously to Scheme 1 by reacting compounds of formula 6 with a sulfonyl chloride rather than an acylating agent. Thus, for example, compound 8bxi was prepared by adaptation of Scheme 1 involving reaction of 6 (n=3) with 2-chloroethanesulfonyl chloride.

Compound 8aii was prepared according to Scheme 2

Compound 8ci was prepared according to Scheme 6

Scheme 2

4,6-Dichloro-2-(methylsulfanyl)-5-nitropyrimidine 1 and 6-chloro-2-(methylsulfanyl)- 5-nitropyrimidin-4-amine 2

Compounds 1 and 2 of scheme 1 were prepared according to WO2013068438A1. 6-Chloro-2-methanesulfonyl-5-nitropyrimidin-4-amine 3 To a solution of 6-chloro-2-(methylsulfanyl)-5-nitropyrimidin-4-amine 2 (1.7 g, 7.8 mmol) in acetic acid (glacial) (7.5 mL, 131 mmol) was added sodium tungstate dihydrate (249 mg, 0.80 mmol) followed by H2O2 solution 30 wt% in water (2.90 mL, 94.5 mmol). The reaction mixture was stirred at room temperature for 1 h then cooled to 0 °C, filtered through filter paper and washed with cold EtOH (15 mL). The solid was dried to give 6-chloro-2- methanesulfonyl-5-nitropyrimidin-4-amine (1.18 g, 60%) as a pale yellow crystalline solid which was stored in a dessicator in vacuo; LC-MS (Method F) 253.3 [MH + ]; RT 1.94 min.

1 -tert-Butyl 3-ethyl 4-(6-amino-2-methanesulfonyl-5-nitropyrimidin-4-yl)piperazin e- 1 ,3-dicarboxylate 4a

A solution of 1 -te/f-butyl 3-ethyl piperazine-1 ,3-dicarboxylatedicarboxylate (487 mg, 1.88 mmol) (prepared according to J. Med. Chem. 201 1 , 40, 3793) in THF (3 mL) was added dropwise to a solution of 6-chloro-2-methanesulfonyl-5-nitropyrimidin-4-amine 3 (680 mg, 2.69 mmol) and Et 3 N (0.83 mL, 5.92 mmol) in THF (10 mL) at 0 °C and the reaction allowed to stir at this temperature for 1 h. The reaction mixture was diluted with water (20 mL) and EtOAc (20 mL) and the phases separated. The aqueous phase was extracted with EtOAc (30 mL) and the combined organic phases washed with brine (20 mL). The solvent was removed in vacuo to provide 1 -te/f-butyl 3-ethyl 4-(6-amino-2- methanesulfonyl-5-nitropyrimidin-4-yl)piperazine-1 ,3-dicarboxylate (800 mg, 62%) as a yellow crystalline solid. The material was used immediately in the subsequent step of the synthesis; 1 H NMR (Method A) (CD 3 OD): δ ppm 5.11 (br s, 1 H), 4.52-4.48 (m, 1 H), 4.23 (m, 1 H), 3.90 (m, 1 H), 3.52-3.35 (m, 4H), 3.23 (s, 3H), 1.45 (s, 9H), 1.29 (t, J = 7.0 Hz, 3H); LC-MS (Method F) 473.6 [MH " ]; RT 2.40 min.

1 -tert-Butyl 5-ethyl 4-(6-amino-2-methanesulfonyl-5-nitropyrimidin-4-yl)-1,4- diazepane-1 ,5-dicarboxylate 4b

A solution of 1 -te/f-butyl 5-ethyl 1 ,4-diazepane-1 ,5-dicarboxylate (2.16 g, 7.92 mmol) (prepared according to Bioorg. Med. Chem. Lett. 2005, 15, 1641) in THF (20 mL) was added dropwise to a solution of 6-chloro-2-methanesulfonyl-5-nitropyrimidin-4-amine 3 (2.00 g, 7.92 mmol) and Et 3 N (2.43 mL, 17.4 mmol) in THF (15 mL) at 0 °C and the reaction allowed to stir at this temperature for 1 h. The reaction mixture was diluted with water (20 mL) and EtOAc (20 mL) and the phases separated. The aqueous phase was extracted with EtOAc (30 mL) and the combined organic phases washed with brine (20 mL). The solvent was removed in vacuo to provide 1 -te/f-butyl 5-ethyl 4-(6-amino-2- methanesulfonyl-5-nitropyrimidin-4-yl)-1 ,4-diazepane-1 ,5-dicarboxylate (2.23 g, 58%) as a yellow crystalline solid. The material was used immediately in the subsequent step of the synthesis; LC-MS (Method F) 487.5 [MH " ]; RT 2.74 min.

General procedure A - nucleophillic displacement of sulfone

A solution of sulfone 4 (1.00 g, 2.04 mmol) in butan-1-ol (10 mL) was heated to 70 °C and stirred for 10 min. To the solution was added TFA (470 μί, 6.1 mmol) and the temperature increased to 100 °C. The solution was stirred at 100 °C for 4 h then allowed to cool to room temperature. To the solution was added Na2CC>3 (500 mg, 4.72 mmol) and the solvents removed in vacuo. The reaction mixture was diluted with water (20 mL), DCM (20 mL) and the phases separated. The aqueous phase was extracted with DCM (20 mL) and the combined organic phases washed with brine (20 mL). The solvent was removed in vacuo and the resulting residue purified by flash column chromatography eluting with 0-5% MeOH in DCM to give compound 5 (540 mg, 55%) as a yellow oil. tert-Butyl 4-(6-amino-2-butoxy-5-nitropyrimidin-4-yl)piperazine-1 -carboxylate 5a

Prepared from 4a according to General Procedure A LC-MS (Method F) 469.5 [MH + ]; RT 3.03 min

1 -tert-butyl 5-ethyl 4-(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-1 ,4-diazepane-1 , 5- dicarboxylate 5b

Prepared from 4b according General Procedure A

LC-MS (Method F) 483.5 [MH + ]; RT 3.50 min

General procedure B - removal of tert-butyl carbamate group

A solution of te/f-butyl carbamate 5 (1.00 g, 2.07 mmol) in DCM (10 mL) was treated with TFA (2 mL) and stirred for 10 h at room temperature. The solvents were removed in vacuo and the material taken up in MeOH (5 mL) and purified using a 20 g SCX cartridge. The column was washed through with MeOH followed by 3.5 M NH3 solution in MeOH to provide compound 6 (593 mg, 75%) as a yellow solid.

Ethyl 1 -(6-amino-2-butoxy-5-nitropyrimidin-4-yl)piperazine-2-carbox ylate 6a

Prepared from 5a according to General Procedure B

1 H NMR (CDC ): δ ppm 5.05 (m, 1 H), 4.35-4.12 (m, 4H), 3.68 (m, 2H), 3.26 (m, 1 H), 2.91 (m, 2H), 1.63 (m, 3H), 1.43 (m, 2H), 1.26 (m, 3H), 0.90 (t, J = 7.5 Hz, 3H); LC-MS (Method F) 369.5 [MH + ]; RT 2.05 min. Ethyl 1-(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-1,4-diazepane-2-c arboxylate 6b

Prepared from 5b according to General Procedure B

1 H NMR (Method A) (CDC ): δ ppm 4.73 (m, 1 H), 4.27-4.10 (m, 4H), 3.60 (m, 1 H), 3.42 (m, 1 H), 3.32 (m, 2H), 2.95 (m, 1 H), 2.81 (m, 1 H), 1.98 (m, 1 H), 1.86 (m, 1 H), 1.78-1.63 (m, 2H), 1.48-1.38 (m, 2H), 1.31-1.35 (m, 3H), 0.89 (t, J = 7.5 Hz, 3H); LC-MS (Method F) 383.5 [MH + ]; RT 2.55 min.

General procedure C - nitro reduction and lactamisation To a solution of the compound 7 (1.00 g, 2.05 mmol) in MeOH (10 mL) was added a catalytic amount of Raney ® -nickel in a slurry of water (-300 μΙ_). The reaction vessel was flushed with H2 and then stirred under a H2 atmosphere for 2 h. The mixture was filtered through Celite ® , eluting with 50% MeOH in DCM. The filtrate was concentrated in vacuo and when required purified by flash column chromatography to provide compound 8 (716 mg, 85%) as a cream solid. Occasionally, 1.0 M aqueous HCI (1.0 mL) was added to the filtrate prior to drying to provide the corresponding di-HCI salt.

4-Amino-2-butoxy-8-[2-(pyrrolidin-1-yl)acetyl]-5H, 6H, 6aH, 7H, 8H, 9H, 10H- piperazino[2,1-h]pteridin-6-one 8a i

Prepared from 7ai according to General Procedure C LC-MS (Method F) 404.5 [MH + ]; RT 1.98 min

Ethyl 1 -(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-4-[2-(pyrrolidin-1 - yl)acetyl]piperazine-2-carboxylate 7ai

A solution of 2-(pyrrolidin-1 -yl)acetic acid hydrochloride (185 mg, 1.12 mmol) in DCM (5 mL) was treated with Et 3 N (0.39 mL, 2.79 mmol), EDC.HCI (276 mg, 1.39 mmol) and HOBt hydrate (189 mg, 1.39 mmol). The solution was stirred for 5 min then ethyl 1-(6-amino-2- butoxy-5-nitropyrimidin-4-yl)-1 ,4-diazepane-2-carboxylate 6a (343 mg, 0.93 mmol) was added and the solution stirred overnight. The reaction was quenched by addition of a saturated aqueous solution of NH4CI (50 mL). The phases were separated and the aqueous phase extracted with DCM (2 x 40 mL). The combined organic phases were dried over MgSCU and the solvent removed in vacuo to provide ethyl 1-(6-amino-2-butoxy-5- nitropyrimidin-4-yl)-4-[2-(pyrrolidin-1-yl)acetyl]piperazine -2-carboxylate (455 mg, 99%) as a yellow oil; LC-MS (Method F) 480.5 [MH + ]; RT 2.38 min.

4-Amino-2-butoxy-8-{[3-(pyrrolidin-1-ylmethyl)phenyl]meth yl}- 5H, 6H, 6aH, 7H, 8H, 9H, 10H-piperazino[2, 1-h]pteridin-6-one 8aii Prepared from 7aii according to General Procedure C

1 H NMR (Method A) (CDC ): δ ppm 10.58 (s, 1 H), 7.34 (s, 1 H), 7.31-7.27 (m, 2H), 7.23 (m, 1 H), 5.17 (s, 2H), 4.50 (m, 1 H), 4.18 (t, J = 7.0 Hz, 2H), 4.08 (dd, J = 11.0, 3.0 Hz, 1 H), 3.75-3.69 (m, 2H), 3.62 (d, J = 13.0 Hz, 1 H), 3.53 (d, J = 13.0 Hz, 1 H), 3.30 (m, 1 H), 2.87- 2.81 (m, 2H), 2.65 (m, 4H), 2.22 (t, J = 1 1.0, 1 H), 2.08 (m, 1 H), 1.87-1.81 (m, 4H), 1.74- 1.67 (m, 2H), 1.48-1.39 (m, 2H), 0.93 (t, J = 7.5 Hz, 3H); LC-MS (Method F) 466.6 [MH + ]; RT 2.73 min.

Ethyl 1 -(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-4-{[3-(pyrrolidin- 1- ylmethyl)phenyl]methyl}piperazine-2-carboxylate 7aii

Prepared from XG according to General Procedure A LC-MS (Method F) 542.7 [MH + ]; RT 3.51 min

Ethyl 1 -(6-amino-2-methanesulfonyl-5-nitropyrimidin-4-yl)-4-{[3-(py rrolidin-1- ylmethyl)phenyl]methyl}piperazine-2-carboxylate XG A solution of ethyl 4-{[3-(pyrrolidin-1-ylmethyl)phenyl]methyl}piperazine-2-carb oxylate XF (0.50 g, 1.51 mmol) in THF (5 ml_) was added dropwise to a stirred solution of 6-chloro-2- methylsulfonyl-5-nitro-pyrimidin-4-amine 3 (419 mg, 1.66 mmol) and EtzN (0.46 ml_, 3.32 mmol) in THF (20 ml_) at 0 °C and the reaction mixture allowed to warm to room temperature. The solvent was removed in vacuo. Water (50 ml_) and EtOAc (50 ml_) was added to the resulting residue and the phases separated. The aqueous phase was extracted with EtOAc (50 ml_) and the combined organic phases was washed with brine (100 ml_) and dried over MgS0 4 . The solvent was removed in vacuo to provide ethyl 1-(6- amino-2-methanesulfonyl-5-nitropyrimidin-4-yl)-4-{[3-(pyrrol idin-1- ylmethyl)phenyl]methyl}piperazine-2-carboxylate (800 mg, 97%) as an orange oil which was used directly in the next step without further purification; LC-MS (Method F) 548.6 [MH + ]; RT 2.71 min.

Ethyl 4-{[3-(pyrrolidin-1-ylmethyl)phenyl]methyl}piperazine-2-carb oxylate XF

A stirred solution of ethyl piperazine-2-carboxylate dihydrochloride (1.43 g, 6.18 mmol) in MeOH (50 ml_) over 4A molecular sieves was treated with 3-(pyrrolidin-1- ylmethyl)benzaldehyde (1.17 g, 6.18 mmol) and 1.25 M hydrogen chloride in MeOH solution (0.05 ml_, 0.11 mmol). After stirring for 1 h NaCNBH 3 (427 mg, 6.80 mmol) was added and the reaction mixture allowed to stir for a further 1.5 h. TLC (3% MeOH in DCM) indicated complete consumption of starting material. The solvent was removed in vacuo. EtOAc (75 ml_) and a saturated aqueous solution of NH 4 CI (75 ml_) were added. The phases were separated and the aqueous phase basified to pH 1 1 with 2 M NaOH. The aqueous phase was extracted with EtOAc (2 x 100 ml_) and the combined organic phases washed with brine (100 ml_) then dried over MgS0 4 . The solvent was removed in vacuo to provide ethyl 4-{[3-(pyrrolidin-1-ylmethyl)phenyl]methyl}piperazine-2-carb oxylate (1.03 g, 50%) as a yellow oil; 1 H NMR (Method A) (CDC ): δ ppm 7.26-7.19 (m, 4H), 4.17 (q, J = 7.0 Hz, 2H), 3.62-3.58 (m, 2H), 3.56-3.53 (m, 2H), 3.46 (d, J = 13.0 Hz, 1 H), 3.05 (m, 1 H), 2.91-2.81 (m, 2H), 2.58 (m, 1 H), 2.50 (m, 4H), 2.35 (m, 1 H), 2.21 (m, 1 H), 1.78 (m, 4H), 1.24 (t, J = 7.0 Hz, 3H).

4-Amino-2-butoxy-5H, 6H, 6aH, 7H, 8H,9H, 10H-piperazino[2, 1-h]pteridin-6-one 8aiii Prepared from 6a according to General Procedure C

1 H NMR (Method A) (DMSO-cfe): δ ppm 9.63 (s, 1 H), 6.17 (s, 2H), 4.21 (d, J = 12.0 Hz, 1 H), 4.09 (t, J = 6.5 Hz, 2H), 3.76 (dd, J = 11.0, 3.5 Hz, 1 H), 3.23 (m, 1 H), 2.87 (d, J = 1 1.0 Hz, 1 H), 2.68-2.55 (m, 3H), 2.33 (m, 1 H), 1.66-1.53 (m, 2H), 1.43-1.30 (m, 2H), 0.90 (t, J = 7.5 Hz, 3H); LC-MS (Method F) 293.5 [MH + ]; RT 1.57 min. Methyl 4-{4-amino-2-butoxy-6-oxo-5H, 6H, 6aH, 7H, 8H, 91-1,1 OH, 11H-[1,4]diazepino[2, 1- h]pteridine-8-carbonyl}benzoate 8bi

Prepared from 7bi according to General Procedure C. Isolated as HCI salt

1 H NMR (Method A) (CD 3 OD): δ ppm 7.98 (m, 2H), 7.23 (m, 2H), 4.54 (m, 1 H), 4.42-4.36 (m, 3H), 3.96 (m, 1 H), 3.85 (s, 3H), 3.83-3.64 (m, 2H), 3.33 (m, 1 H), 3.03 (m, 1 H), 2.32 (m, 1 H), 1.94 (m, 1 H), 1.75-1.64 (m, 2H), 1.37 (m, 2H), 0.92-0.86 (m, 3H); LC-MS (method F) 469.5 [MH + ]; RT 2.41 min.

Ethyl 1-(6^mino-2-butoxy-5-nitropyn ' midin-4-yl)-4-[4-(methoxycarbonyl)benzoyl]-1,4- diazepane-2-carboxylate 7bi

A solution of 4-(methoxycarbonyl)benzoic acid (37 mg, 0.21 mmol) in DCM (5 mL) was treated with Et 3 N (0.09 mL, 0.63 mmol), EDC.HCI (60 mg, 0.31 mmol) and HOBt hydrate (42 mg, 0.31 mmol). The solution was stirred for 5 min followed by addition of ethyl 1 -(6- amino-2-butoxy-5-nitropyrimidin-4-yl)-1 ,4-diazepane-2-carboxylate 6b (80 mg, 0.21 mmol). The solution was allowed to stir overnight. The reaction was quenched by addition of a saturated aqueous solution of NH4CI (50 mL). The phases were separated and the aqueous phase extracted with DCM (2 x 40 mL). The combined organic phases were dried over MgS0 4 and the solvent removed in vacuo to provide ethyl 1-(6-amino-2-butoxy-5- nitro-pyrimidin-4-yl)-4-(4-methoxycarbonylbenzoyl)-1 ,4-diazepane-2-carboxylate (85mg, 75%) as a yellow oil; LC-MS (Method F) 545.5 [MH + ]; RT 2.98 min.

4-Amino-2-butoxy-8-[4-(pyrrolidin-1-ylmethyl)benzoyl]-5H, 6H, 6aH, 7H, 8H, 9H,10H, 11 H- [ 1,4]diazepino[2, 1 -h]pteridin-6-one 8bii

Prepared from 7bii according to General Procedure C

1 H NMR (CD3OD): δ ppm 7.57 (m, 2H), 7.28 (m, 2H), 4.56-4.44 (m, 5H), 4.32 (m, 1 H), 4.01 (m, 1 H), 3.85 (m, 1 H), 3.57 (m, 2H), 3.39 (m, 2H), 3.12 (m, 3H), 2.24 (m, 1 H), 2.10 (m, 2H), 1.96 (m, 2H), 1.89 (m, 2H), 1.70 (m, 2H), 1.57 (m, 1 H), 1.45-1.35 (m, 2H), 0.93-0.84 (m, 3H) [HCI salt]; LC-MS (Method F) 494.6 [MH + ]; RT 2.56 min. Ethyl 1 -(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-4-[4-(pyrrolidin-1 -ylmethyl)benzoyl]- 1 ,4-diazepane-2-carboxylate 7bii

A solution of 4-(pyrrolidin-1 -ylmethyl)benzoic acid (375 mg, 1.55 mmol) in DCM (5 mL) was treated with Et 3 N (0.65 mL, 4.66 mmol), EDC.HCI (446 mg, 2.33 mmol) and HOBt hydrate (314 mg, 2.33 mmol). The solution was stirred for 5 min followed by addition of ethyl 1 -(6- amino-2-butoxy-5-nitropyrimidin-4-yl)-1 ,4-diazepane-2-carboxylate 6b (594 mg, 1.55 mmol). The solution was allowed to stir overnight. The reaction was quenched by addition of a saturated aqueous solution of NH4CI (50 mL). The phases were separated and the aqueous phase extracted with DCM (2 x 40 mL). The combined organic phases were dried over MgSCU and the solvent removed in vacuo to provide ethyl 1 -(6-amino-2-butoxy-5- nitropyrimidin-4-yl)-4-[4-(pyrrolidin-1 -ylmethyl)benzoyl]-1 ,4-diazepane-2-carboxylate (552 mg, 62%) as a yellow oil; LC-MS (Method F) 570.6 [MH + ]; RT 3.18 min.

4-Amino-2-butoxy-5H, 6H, 6aH, 7H, 8H, 9H,10H, 11H-[1,4]diazepino[2, 1-h]pteridin-6-one 8/3/7/

Prepared from 6b according to General Procedure C

1 H NMR (CDC ): δ ppm 5.01 (s, 2H), 7.28 (m, 2H), 4.53 (m, 1 H), 4.21-4.07 (m, 3H), 3.50 (m, 1 H), 3.31 (m, 1 H), 3.06-2.98 (m, 3H), 2.75 (m, 1 H), 2.21 (m, 1 H), 1.47-1.37 (m, 3H), 1.26 (m, 1 H), 0.96-0.86 (t, J = 7.5 Hz, 3H); LC-MS (Method F) 307.5 [MH + ]; RT 1.66 min.

4-Amino-2-butoxy-8-[3-(pyrrolidin-1-yl)propanoyl]-5H, 6H, 6aH, 7H, 8H, 9H,10H, 11 H- [ 1,4]diazepino[2, 1 -h]pteridin-6-one 8biv

Prepared from 7biv according to General Procedure C

1 H NMR (DMSO-de): δ ppm 9.47 (s, 1 H), 5.92 (s, 2H), 4.28 (m, 1 H), 4.19-4.10 (m, 3H), 4.08-4.01 (m, 1 H), 3.85 (m, 1 H), 3.64 (m, 1 H), 3.49 (m, 1 H), 2.92-2.85 (m, 3H), 2.77-2.67 (m, 4H), 2.62-2.56 (m, 2H), 2.17 (m, 1 H), 1.88-1.74 (m, 5H), 1.68-1.61 (m, 2H), 1.44-1.36 (m, 2H), 0.93 (t, J = 7.5 Hz, 3H); LC-MS (Method G) 432.5 [MH + ]; RT 5.94 min. Ethyl 1-(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-4-[3-(pyrrolidi

diazepane-2-carboxylate 7biv

A solution of 3-(pyrrolidin-1-yl)propanoic acid (56 mg, 0.31 mmol) in DCM (8 mL) was treated with Et 3 N (0.11 mL, 0.78 mmol), EDC.HCI (75 mg, 0.39 mmol) and HOBt hydrate (53 mg, 0.39 mmol). The solution was stirred for 5 min followed by the addition of ethyl 1 - (6-amino-2-butoxy-5-nitropyrimidin-4-yl)-1 ,4-diazepane-2-carboxylate 6b (100 mg, 0.26 mmol). The solution was allowed to stir for 96 h. The reaction was quenched by addition of a saturated aqueous solution of NH 4 CI (50 mL). The phases were separated and the aqueous phase extracted with DCM (2 x 40 mL). The combined organic phases were dried over MgSCU and the solvent removed in vacuo to provide ethyl 1-(6-amino-2-butoxy-5- nitropyrimidin-4-yl)-4-[3-(pyrrolidin-1-yl)propanoyl]-1 ,4-diazepane-2-carboxylate (80 mg, 60%) as a yellow oil; 1 H NMR (Method A) (CDC ): δ ppm 4.69 (s, 2H), 4.35-4.01 (m, 5H), 3.79-3.43 (m, 3H), 3.30-3.17 (m, 2H), 2.93-2.59 (m, 6H), 2.59-2.44(m, 2H), 2.25 (m, 1 H), 1.91-1.70 (m, 3H), 1.70-1.49 (m, 2H), 1.42-1.25 (m, 2H), 1.24-1.10 (m, 3H), 0.87 (t, J = 7.5 Hz, 3H); LC-MS (Method F) 508.5 [MH + ]; RT 2.73 min.

8bv-ii was prepared from 8bv according to Scheme 3

8bv 8bv-i

8bv-ii

Scheme 3 4-Amino-2-butoxy-8-[2-(propan-2-yl)-1,2,3,4-tetrahydroisoqui noline-7-carbonyl]- 5H, 6H, 6aH, 7H, 8H, 9H,10H, 11H-[1,4]diazepino[2, 1-h]pteridin-6-one 8bv-ii

A solution of 4-amino-2-butoxy-8-(1 ,2,3,4-tetrahydroisoquinoline-7-carbonyl)- 5H,6H,6aH,7H,8H,9H, 10H, 11 H-[1 ,4]diazepino[2, 1-h]pteridin-6-one (61 mg, 0.13 mmol) 8bv-i in anhydrous DMF (2 mL) was treated with 2-iodopropane (0.01 mL, 0.14 mmol) and DIPEA (0.05 mL, 0.29 mmol) and left to stir at room temperature overnight. The reaction mixture was taken up in water (10 mL) and extracted with EtOAc (2 x 10 mL). The combined organic extracts were washed with brine (20 mL), dried over MgSCU and concentrated in vacuo. The resulting residue was purified by flash column chromatography eluting 0-20% MeOH in DCM to provide 4-amino-8-[7-(dimethylamino)-5,6,7,8- tetrahydronaphthalene-2-carbonyl]-2-propoxy-5H,6H, 6aH, 7H,8H, 9H, 1 OH, 11 H- [1 ,4]diazepino[2, 1-h]pteridin-6-one (14 mg, 21 %) as a pale yellow solid; 1 H NMR (Method A) (CDsOD): δ ppm 7.24 (s, 1 H), 7.02 (m, 1 H), 6.86 (m, 1 H), 4.56-4.42 (m, 2H), 4.39-4.29 (m, 2H), 4.00 (m, 1 H), 3.90-3.81 (m, 2H), 3.77-3.55 (m, 2H), 3.47-3.43 (m, 3H), 3.14- 3.07 (m, 2H), 3.05-3.02 (m, 2H), 2.99-2.94 (m, 2H), 1.99-1.91 (m, 1 H), 1.89- 1.77 (m, 2H), 1.62- 1.52 (m, 2H), 1.40 (m, 1 H), 1.30- 1.26 (m, 6H), 1.1 1-1.03 (m, 3H); LC-MS (Method F) 508.6 [MH + ]; RT 1.74 min.

4-Amino-2-butoxy-8-(1,2,3,4-tetrahydroisoquinoline-7-carb onyl)- 5H, 6H, 6aH, 7H, 8H, 9H,10H, 11H-[1,4]diazepino[2, 1-h]pteridin-6-one 8bv-i

A solution of terf-butyl 7-{4-amino-2-butoxy-6-oxo-5H,6H,6aH,7H,8H,9H, 10H, 11 H- [1 ,4]diazepino[2, 1-h]pteridine-8-carbonyl}-1 ,2,3,4-tetrahydroisoquinoline-2-carboxylate (75 mg, 0.13 mmol) 8bv in MeOH (3 mL) was treated with 1.0 M hydrogen chloride in 1 ,4 dioxane (0.33 mL, 1.33 mmol). The reaction was allowed to stir at room temperature for 24 h. The reaction was concentrated in vacuo to provide 4-amino-2-butoxy-8-(1 ,2,3,4- tetrahydroisoquinoline-7-carbonyl)-5H,6H,6aH,7H,8H,9H, 10H, 11 H -[1 ,4]diazepino[2, 1- h]pteridin-6-one (61 mg, 98%) as a pale yellow oil; LC-MS (Method F) 466.6 [MH + ]; RT 1.68 min. tert-Butyl 7-{4-amino-2-butoxy-6-oxo-5H, 6H, 6aH, 7H, 8H, 9H, 10H, 11 H- [1,4]diazepino[2,1-h]pteridine-8-carbonyl}-1,2,3,4-tetrahydr oisoquinoline-2- carboxylate 8bv

Prepared from 7bv according to General Procedure C tert-Butyl 7-[4-(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-5-(ethoxycarbo nyl)-1,4- diazepane-1-carbonyl]-1,2,3,4-tetrahydroisoquinoline-2-carbo xylate 7bv

A solution of 2-[(fe/f-butoxy)carbonyl]-1 ,2,3,4-tetrahydroisoquinoline-7-carboxylic acid (102 mg, 0.37 mmol) XC in DCM (10 ml_) was treated with Et 3 N (0.15 ml_, 1.10 mmol), EDC.HCI (105 mg, 0.55 mmol) and HOBt hydrate (74 mg, 0.55 mmol). The solution was stirred for 10 min followed by the addition of ethyl 4-(6-amino-2-butoxy-5-nitropyrimidin-4- yl)-1 ,4-diazepane-5-carboxylate 6b (140 mg, 0.37 mmol). The solution was allowed to stir overnight. The reaction was quenched by addition of saturated aqueous solution of NaHCC>3 (50 ml_). The phases were separated and the aqueous phase extracted with DCM (2 x 50 ml_). The combined organic phases were dried over MgSCU and the solvent removed in vacuo. The resulting residue was purified by flash column chromatography eluting with 0-100% EtOAc in hexane to provide te/f-butyl 7-[4-(6-amino-2-butoxy-5- nitropyrimidin-4-yl)-5-(ethoxycarbonyl)-1 ,4-diazepane-1-carbonyl]-1 , 2,3,4- tetrahydroisoquinoline-2-carboxylate (194 mg, 83%) as a yellow solid; LC-MS (Method F) 642.5 [MH + ]; RT 3.44 min.

XC was prepared from XA according to Scheme 4

XC

Scheme 4 2-[(tert-Butoxy)carbonyl]-1,2,3,4-tetrahydroisoquinoline-7-c arboxylic acidXC

A solution of 7-butyl 2-te/f-butyl 1 ,2,3,4-tetrahydroisoquinoline-2,7-dicarboxylate (155 mg, 0.46 mmol) XB in THF (5 mL) and water (5 mL) was treated with LiOH (22 mg, 0.93 mmol). The reaction was heated to reflux at 60 °C and allowed to stir for 18 h. The reaction mixture was diluted with water (50 mL) and washed with EtOAc (2 x 20 mL). The aqueous layer was acidified to pH 4 with glacial acetic acid and extracted with EtOAc (2 x 50 mL). The organic layer was washed with brine (20 mL), dried over MgS0 4 and concentrated in vacuo to provide 2-[(te/f-butoxy)carbonyl]-1 ,2,3,4-tetrahydroisoquinoline-7- carboxylic acid (104 mg, 81 %) as a pale yellow oil; LC-MS (Method F) 277.6 [MH + ]; RT 2.71 min.

7 -Butyl 2-tert-butyl 1,2,3,4-tetrahydroisoquinoline-2,7-dicarboxylate XB

A solution of te/f-butyl 7-bromo-1 ,2,3,4-tetrahydroisoquinoline-2-carboxylate (500 mg, 1.60 mmol) XA in 1 ,4-dioxane (5 mL) was treated with molybdenum hexacarbonyl (6:1) (21 1 mg, 0.80 mmol), acetoxy-[[2-(bis-o-tolylphosphanyl)phenyl]methyl]palladium (38 mg, 0.04 mmol), DMAP (391 mg, 3.2 mmol), DIPEA (0.06 mL, 0.32 mmol) and butan-1-ol (5 mL, 54.6 mmol). The resulting reaction mixture was irradiated in the microwave (Biotage Initiator) at 180 °C for 30 min. On cooling the reaction mixture was filtered through Celite® washing with THF (100 mL) and the filtrate concentrated in vacuo. The resulting residue was purified by flash column chromatography eluting 0-25% EtOAc in hexane to provide 7- butyl 2-te/f-butyl 1 ,2,3,4-tetrahydroisoquinoline-2,7-dicarboxylate (160 mg, 30%) as a pale yellow oil; 1 H NMR (Method A) (CDCb): δ ppm 7.75-7.74 (m, 1 H), 7.72 (m, 1 H), 4.26-4.24 (m, 2H), 3.61-3.57 (m, 3H), 2.83-2.79 (m, 2H), 1.72-1.65 (m, 2H), 1.43-1.42 (m, 9H), 1.22- 1.18 (m, 2H), 0.93-0.89 (m, 3H); LC-MS (Method F) 234.5 [MH + ]; RT 4.05 min.

tert-Butyl 7-bromo-1,2,3,4-tetrahydroisoquinoline-2-carboxylate XA

A solution of 7-bromo-1 ,2,3,4-tetrahydroisoquinoline (870 mg, 4.10 mmol) in THF (15 mL) and water (5 mL) was treated with EtzN (0.66 mL, 4.76 mmol) and di-te/f-butyl dicarbonate (940 mg, 4.31 mmol). The reaction was allowed to stir for 2 h at room temperature. The THF was removed in vacuo and the reaction mixture was taken up in water (100 mL) and extracted with EtOAc (100 mL). The organic layer was washed with a saturated NH 4 CI solution (20 mL), dried over MgS0 4 and the solvent removed in vacuo. The resulting residue was purified by flash column flash chromatography eluting 0-20% EtOAc in hexane to provide te/f-butyl 7-bromo-1 ,2,3,4-tetrahydroisoquinoline-2-carboxylate (1.21 g, 94%) as a yellow oil; 1 H NMR (Method A) (CDCb): δ ppm 7.21 (s, 1 H), 7.19 (m, 1 H), 6.93 (m, 1 H), 3.57-3.53 (m, 2H), 2.72-2.69 (m, 2H), 1.51-1.48 (m, 2H), 1.42-1.41 (m, 9H); LC-MS (Method F) 212.4 [MH + ]; RT 3.97 min.

4-Amino-2-butoxy-8-{6-[2-(pyrrolidin-1-yl)ethoxy]pyridine -3-carbonyl}- 5H, 6H, 6aH, 7H, 8H, 9H,10H, 11H-[1,4]diazepino[2, 1-h]pteridin-6-one 8bvi Prepared from 7bvi according to General Procedure C

1 H NMR (Method A) (CDCb): δ ppm 8.45 (s, 2H), 8.1 1 (s, 1 H), 7.61 (m, 1 H), 7.01 (m, 1 H), 4.83-4.75 (m, 2H), 4.48 (m, 1 H), 4.40-4.28 (m, 3H), 4.00 (m, 1 H), 3.83-3.59 (m, 4H), 3.52 (m, 4H), 3.11 (m, 1 H), 2.19- 2.15 (m, 4H), 1.99 (m, 1 H), 1.83-1.75 (m, 2H), 1.59-1.50 (m, 2H), 1.07-1.03 (m, 3H); LC-MS (Method F) 525.6 [MH + ]; RT 2.35 min. Ethyl 4-(6-amino-2-butoxy-5-nitropyn ' midin-4-yl)-1-{6-[2-(pyrrolidin-1- yl)ethoxy]pyridine-3-carbonyl}-1,4-diazepane-5-carboxylate 7bvi

A solution of 6-[2-(pyrrolidin-1 -yl)ethoxy]pyridine-3-carboxylic acid XE (185 mg, 0.78 mmol) in DCM (10 mL) was treated with Et 3 N (0.33 mL, 2.35 mmol), EDC.HCI (1 13 mg, 0.59 mmol) and HOBt hydrate (80 mg, 0.59 mmol). The solution was stirred for 5 min followed by addition of ethyl 4-(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-1 ,4-diazepane-5-carboxylate 6b (150 mg, 0.39 mmol). The solution was allowed to stir overnight. The reaction was quenched by addition of a saturated aqueous solution of NaHCC>3 (50 mL). The phases were separated and the aqueous phase extracted with DCM (2 x 50 mL). The combined organic phases were dried over MgS0 4 and the solvent removed in vacuo. The resulting residue was purified by flash column chromatography eluting 0-10% MeOH in DCM to provide ethyl 4-(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-1-{6-[2-(pyrrolid in-1- yl)ethoxy]pyridine-3-carbonyl}-1 ,4-diazepane-5-carboxylate (162 mg, 69%) as a pale yellow solid; 1 H NMR (Method A) (CDCb): δ ppm 8.18 (d, J = 2.5 Hz, 1 H), 7.57 (d, J = 8.5 Hz, 1 H), 6.80 (d, J = 8.5 Hz, 1 H), 4.98 (s, 1 H), 4.48 (t, J = 6.0 Hz, 2H), 4.23-4.18 (m, 2H), 4.16-4.10 (m, 2H), 3.69-3.59 (m, 2H), 3.55-3.47 (m, 2H), 2.92-2.88 (m, 2H), 2.65-2.59 (m, 4H), 2.20 (s, 1 H), 2.02 (s, 1 H), 1.84-1.79 (m, 4H), 1.72-1.65 (m, 2H), 1.61-1.54 (m, 4H), 1.49-1.40 (m, 2H), 1.29 (t, J = 7.0 Hz, 3H), 0.95 (t, J = 7.4 Hz, 3H); LC-MS (Method F) 601.7 [MH + ]; RT 2.98 min. XE was prepared from XD according to Scheme 5

XD XE

Scheme 5

6-[2-(Pyrrolidin-1-yl)ethoxy]pyridine-3-carboxylic acid XE To a solution of 6-[2-(pyrrolidin-1-yl)ethoxy]pyridine-3-carbonitrile (0.18 g, 0.85 mmol) XD in EtOH:water (2 ml_:12 mL) was added NaOH (0.53 mL, 8.50 mmol) and the resultant solution was heated at reflux for 6 h. The reaction mixture was cooled to room temperature then EtOH was removed in vacuo. The remaining solution was acidified to pH 8 using aqueous HCI (1 M) and the solution was concentrated in vacuo to a white salt. The reaction mixture was re-dissolved in MeOH (10 mL) and the salt was filtered to provide 4- [2-(pyrrolidin-1-yl)ethoxy]benzoic acid (740 mg, 93%) as a white solid; 1 H NMR (Method A) (CDsOD): δ ppm 8.61 (m, 1 H), 8.09 (dd, J = 8.5, 2.5 Hz, 1 H), 6.71-6.68 (m, 1 H), 4.45 (dd, J = 6.5, 5.0 Hz, 2H), 3.11 (t, J = 5.5 Hz, 2H), 2.19-2.88 (m, 4H), 1.85 (q, J = 4.5, 4.0 Hz, 4H); LC-MS (Method F) 237.5 [MH + ]; RT 1.02 min. 6-(2-Pyrrolidin-1 -ylethoxy)pyridine-3-carbonitrile XD

1-(2-Hydroxyethyl)pyrrolidine (4.6 mL, 39.7 mmol) was slowly added to a suspension of NaH, (60% dispersed in mineral oil, 2.17 g, 54.1 mmol) in anhydrous DMF (50 mL) at 0 °C and then stirred for 30 min at room temperature. 6-Chloropyridine-3-carbonitrile (5.0 g, 36.1 mmol) in anhydrous DMF (10 mL) was added and the mixture was allowed to stir for 18 h. The reaction was quenched with 10% aqueous NaHCC (500 mL) then extracted with EtOAc (2 x 300 mL). The combined organic residues were washed with brine (300 mL), dried over MgSCU and concentrated in vacuo. The resulting residue was purified by flash column chromatography eluting with 0-10% MeOH in DCM to provide 6-(2-pyrrolidin- 1-ylethoxy)pyridine-3-carbonitrile (3.5 g, 45%) as a light orange solid; LC-MS (Method F) 218.5 [MH + ]; RT 1.89 min.

4-Amino-2-butoxy-8-[2-(1 ,2,3,4-tetrahydroisoquinolin-2-yl)acetyl]- 5H,6H,6aH,7H,8H,9H,10H,11 H-[1 ,4]diazepino[2,1 -h]pteridin-6-one 8bvii

Prepared from 7bvii according to General Procedure C LC-MS (Method D) 480.5 [MH + ]; 1.76 RT min

Ethyl 1 -(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-4-[2-(1 ,2,3,4-tetrahydroisoquinolin- 2-yl)acetyl]-1 ,4-diazepane-2-carboxylate 7bvii

A solution of 2-(1 ,2,3,4-tetrahydroisoquinolin-2-yl)acetic acid (55 mg, 0.29 mmol) (prepared as described in WO2004063198) , HOBt hydrate (45 mg, 0.34 mmol) , EDC.HCI (64 mg, 0.34 mmol) and Et 3 N (0.09 ml_, 0.67 mmol) in DCM (10 ml_) were stirred at room temperature for 10 min then ethyl 1-(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-1 ,4- diazepane-2-carboxylate 6b (86 mg, 0.22 mmol) was added and allowed to stir for a further 18 h. Reaction was incomplete so additional EDC.HCI (64 mg, 0.34 mmol) , HOBt hydrate (45 mg, 0.34 mmol), triethylamine (0.09 ml_, 0.67 mmol) and 2-(1 ,2,3,4- tetrahydroisoquinolin-2-yl)acetic acid (55 mg, 0.29 mmol) were added and allowed to stir for a further 20 h. The reaction mixture was diluted with DCM (50 ml_), washed with saturated NaHC03 solution (50 ml_), water (50 ml_), brine (50 ml_), dried over MgS0 4 and concentrated in vacuo. The resulting residue was purified by flash column chromatography eluting with 0-100% EtOAc in hexane to provide ethyl 1-(6-amino-2-butoxy-5- nitropyrimidin-4-yl)-4-[2-(1 ,2,3,4-tetrahydroisoquinolin-2-yl)acetyl]-1 ,4-diazepane-2- carboxylate (80 mg, 64%) as a yellow solid; LC-MS (Method F) 556.6 [MH + ]; 3.29 RT min.

4-Amino-8-(2-{3-azabicyclo[3.1.0]hexan-3-yl}acetyl)-2-but oxy- 5H, 6H, 6aH, 7H, 8H, 9H,10H, 11H-[1,4]diazepino[2, 1-h]pteridin-6-one 8bviii

Prepared from 7bviii according to General Procedure C

1 H NMR (Method A) (CD 3 OD): δ ppm 4.47-4.30 (m, 2H), 4.21 (m, 2H), 4.15 (m, 1 H), 3.86- 3.69 (m, 2H), 3.59-3.40 (m, 2H), 3.25-3.09 (m, 2H), 3.04-2.61 (m, 3H), 2.48-2.22 (m, 3H), 2.01-1.67 (m, 2H), 1.52-1.28 (m, 4H), 0.97 (t, J = 7.0 Hz, 3H), 0.66 (m, 1 H), 0.35 (m, 1 H); LC-MS (Method F) 430.5 [MH + ]; 2.37 RT min.

Ethyl 1 -(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-4-(2-{3-azabicyclo [3.1.0]hexan-3- yl}acetyl)-1,4-diazepane-2-carboxylate 7bviii

v

A solution of 2-{3-azabicyclo[3.1.0]hexan-3-yl}acetic acid hydrochloride (48 mg, 0.27 mmol) , HOBt hydrate (42 mg, 0.31 mmol) , EDC. HCI (60 mg, 0.31 mmol) and Et 3 N (0.12 mL, 0.84 mmol) in DCM (10 mL) was stirred at room temperature for 10 min then ethyl 1- (6-amino-2-butoxy-5-nitropyrimidin-4-yl)-1 ,4-diazepane-2-carboxylate 6b (80 mg, 0.21 mmol) was added and allowed to stir for a further 18 h. The reaction mixture was diluted with DCM (50 mL), washed with saturated NaHCC solution (50 mL), water (50 mL) and brine (50 mL), dried over MgSCU and concentrated in vacuo. The resulting residue was purified by flash column chromatography eluting with 10% MeOH in DCM to provide ethyl 1-(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-4-(2-{3-azabicycl o[3.1.0]hexan-3-yl}acetyl)-1 ,4- diazepane-2-carboxylate (72 mg, 68%) as a yellow solid; LC-MS (Method F) 506.6 [MH + ]; 3.04 RT min.

4-Amino-2-butoxy-8-[2-(pyrrolidin-1-yl)acetyl]-5H, 6H, 6aH, 7H, 8H, 9H,10H, 11 H- [ 1,4]diazepino[2, 1 -h]pteridin-6-one 8bix Prepared from 7bix according to General Procedure C

1 H NMR (Method A) (CDC ): δ ppm 10.58 (brs, 1 H), 5.32 (m, 2H), 4.49-4.07 (m, 5H), 3.92 (m, 1 H), 3.61-3.20 (m, 4H), 2.84-2.04 (m, 7H), 1.85-1.65 (m, 6H), 1.48-1.38 (m, 2H), 0.93 (m, 3H); LC-MS (Method G) 418.5 [MH + ]; RT 6.14 min.

Ethyl 1 -(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-4-[2-(pyrrolidin-1 -yl)acetyl]-1 ,4- diazepane-2-carboxylate 7bix

A solution of 2-(pyrrolidin-1 -yl)acetic acid (52 mg, 0.31 mmol) in DCM (5 mL)was treated with Et 3 N (0.11 mL, 0.78 mmol), EDC.HCI (75 mg, 0.39 mmol) and HOBt hydrate (53 mg, 0.39 mmol). The solution was stirred for 5 min followed by the addition of ethyl 1 -(6-amino- 2-butoxy-5-nitropyrimidin-4-yl)-1 ,4-diazepane-2-carboxylate 6b (100 mg, 0.26 mmol). The solution was allowed to stir overnight at room temperature. The reaction was quenched by addition of a saturated aqueous solution of NH4CI (50 mL). The phases were separated and the aqueous phase extracted with DCM (2 x 40 mL). The combined organic phases were dried over MgS0 4 and the solvent removed in vacuo to provide ethyl 1-(6-amino-2- butoxy-5-nitropyrimidin-4-yl)-4-[2-(pyrrolidin-1-yl)acetyl]- 1 ,4-diazepane-2-carboxylate (121 mg, 94%) as a yellow oil; LC-MS (Method F) 494.5 [MH + ]; RT 2.88 min.

4-Amino-2-butoxy-8-(1 H-pyrazole-3-carbonyl)-5H,6H,6aH,7H,8H,9H,10H,11 H- [1 ,4]diazepino[2,1 -h]pteridin-6-one 8bx

Prepared from 7bx according to General Procedure C

LC-MS (Method F) 401.5 [MH + ]; RT 1.83 min

Ethyl 1 -(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-4-(1 H-pyrazole-3-carbonyl)-1 ,4- diazepane-2-carboxylate 7bx

A solution of 1 H-pyrazole-3-carboxylic acid (23 mg, 0.21 mmol) in DCM (5 mL) was treated with Et 3 N(0.07 mL, 0.50 mmol), EDC.HCI (48 mg, 0.25 mmol) and HOBt hydrate (34 mg, 0.25 mmol). The solution was stirred for 5 min followed by the addition of ethyl 1-(6-amino- 2-butoxy-5-nitropyrimidin-4-yl)-1 ,4-diazepane-2-carboxylate 6b (64 mg, 0.17 mmol). The solution was allowed to stir overnight. The reaction was quenched by addition of a saturated aqueous solution of NH4CI (50 mL). The phases were separated and the aqueous phase extracted with DCM (2 x 40 mL). The combined organic phases were dried over MgS0 4 and the solvent removed in vacuo to provide ethyl 1-(6-amino-2-butoxy-5- nitropyrimidin-4-yl)-4-(1 H-pyrazole-3-carbonyl)-1 ,4-diazepane-2-carboxylate (80 mg, 100%) as a yellow oil; LC-MS (Method F) 447.5 [MH + ]; RT 2.21 min.

4-Amino-2-butoxy-8-[2-(pyrrolidin-1-yl)ethanesulfonyl]-5H , 6H, 6aH, 7H, 8H, 9H,10H, 11 H- [ 1,4]diazepino[2, 1 -h]pteridin-6-one 8bxi Prepared from 7bxi according to General Procedure C. Isolated as HCI salt

1 H NMR (CD3OD): δ ppm 4.63-4.51 (m, 4H), 4.03 (d, J = 6.5 Hz, 2H), 3.83-3.75 (m, 4H), 3.71-3.67 (m, 2H), 3.65-3.53 (m, 2H), 3.32 (m, 1 H), 3.27-3.20 (m, 2H), 2.37 (m, 1 H), 2.29- 2.20 (m, 2H), 2.16-2.08 (m, 2H), 2.01 (m, 1 H), 1.91-1.84 (m, 2H), 1.61-1.51 (m, 2H), 1.06 (t, J = 7.5 Hz, 3H); LC-MS (Method G) 468.5 [MH + ]; RT 6.47 min.

Ethyl 1-(6-amino-2-butoxy-5-nitropyrimidin^-yl)-4-[2-(pyrrolidin-1 -yl)etha

1 ,4-diazepane-2-carboxylate 7bxi

A solution of 2-chloroethanesulfonyl chloride (0.02 mL, 0.16 mmol) in DCM (0.2 mL) was added dropwise to a solution of ethyl 1-(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-1 ,4- diazepane-2-carboxylate 6b (50 mg, 0.13 mmol) and Et 3 N (0.03 mL, 0.20 mmol) in DCM (5 mL) at room temperature. The reaction mixture was left to stir for 2 h, at which time LC-MS showed formation of vinyl sulfonamide intermediate. Pyrrolidine (0.10 mL, 1.20 mmol) was added and the reaction left to stir for a further 1 h. The reaction was quenched with water (40 mL) and DCM (40 mL) was added. The phases were separated and the aqueous phase extracted with DCM (40 mL). The combined organic phases were washed with brine (80 mL), dried over MgS0 4 and the solvent was removed in vacuo. The resulting residue was purified by flash column chromatography eluting with 0-20% MeOH in DCM to provide ethyl 1-(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-4-[2-(pyrrolidin- 1-yl)ethanesulfonyl]-1 ,4- diazepane-2-carboxylate (50 mg, 70%) as a yellow oil; LC-MS (Method F) 544.5 [MH + ]; RT 3.01 min.

4-Amino-2-butoxy-8-[3-(pyrrolidin-1-ylmethyl)benzoyl]-5H, 6H, 6aH, 7H, 8H, 9H,10H, 11 H- [ 1,4]diazepino[2, 1 -h]pteridin-6-one 8bxii

Prepared from 7bxii according to General Procedure C 1 H NMR (Method A) (D 2 0): δ ppm 7.64 (m, 1 H), 7.56 (td, J = 8.0, 4.0 Hz, 1 H), 7.38 (m, 1 H), 7.13 (m, 1 H), 4.71-4.33 (m, 6H), 4.07-3.74 (m, 3H), 3.35 (m, 3H), 3.18-3.10 (m, 2H), 2.19-2.11 (m, 2H), 2.01-1.91 (m, 2H), 1.82-1.59 (m, 3H), 1.50-1.32 (m, 2H), 0.97-0.81 (m, 3H); LC-MS (Method G) 492.6 [MH + ]; RT 6.70 min. Ethyl 1-(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-4-[3-(pyrrolirt

1 ,4-diazepane-2-carboxylate 7bxii

A solution of 3-(pyrrolidin-1-ium-1-ylmethyl)benzoic acid chloride (63 mg, 0.26 mmol) in DCM (5ml_) was treated with Et 3 N (0.1 1 ml_, 0.78 mmol), EDC.HCI (75 mg, 0.39 mmol) and HOBt hydrate (53 mg, 0.39 mmol). The solution was stirred for 5 min followed by addition of ethyl 1-(6-amino-2-butoxy-5-nitro-pyrimidin-4-yl)-1 ,4-diazepane-2-carboxylate 6b (100 mg, 0.26 mmol). The solution was allowed to stir overnight at room temperature. The reaction was quenched by addition of a saturated aqueous solution of NH4CI (50 ml_). The phases were separated and the aqueous phase extracted with DCM (2 x 40 ml_). The combined organic phases were dried over MgS0 4 and the solvent removed in vacuo. The resulting residue was purified by flash column chromatography eluting with 10% MeOH in DCM to provide ethyl 1-(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-4-[3-(pyrrolidin- 1- ylmethyl)benzoyl]-1 ,4-diazepane-2-carboxylate (70 mg, 47%) as a yellow oil; LC-MS (Method F) 570.5 [MH + ]; RT 3.13 min.

4-Amino-2-butoxy-8-[2-(2,3-dihydro-1 H-isoindol-2-yl)acetyl]-1 H,6H,6aH, 7H,8H,9H, 10Η,11 H-[1 ,4]diazepino[2,1 -h]pteridin-6-one 8bxiii

Prepared from 7bxiii according to General Procedure C. Isolated as HCI salt.

1 H NMR (Method A) (CD 3 OD): δ ppm 7.48-7.46 (m, 4H), 5.53 (s, 4H), 5.15-4.96 (m, 2H), 4.79-4.63 (m, 4H), 4.60-4.51 (m, 4H), 4.25 (m, 1 H), 4.12 (m, 1 H), 3.69 (m, 1 H), 3.25 (m, 1 H), 2.29 (m, 1 H), 2.10 (m, 1 H), 1.90-1.82 (m, 2H), 1.60-1.50 (m, 2H), 1.08-1.03 (m, 3H) ; LC-MS (Method F) 466.5 [MH + ]; RT 2.43 min.

Ethyl 1-(6-amino-2-butoxy-5-nitropyn ' midin-4-yl)-4-[2-(2,3-dihydro-1H-isoi^ yl)acetyl]-1,4-diazepane-2-carboxylate 7bxiii

7bxiii

A solution of 2-(2,3-dihydro-1 H-isoindol-2-yl)acetic acid (93 mg, 0.52 mmol) in DCM (5 mL) was treated with Et 3 N (0.18 mL, 1.31 mmol), EDC.HCI (1 10 mg, 0.58 mmol) and HOBt hydrate (78 mg, 0.58 mmol). The solution was stirred for 5 min followed by the addition of ethyl 1-(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-1 ,4-diazepane-2-carboxylate 6b (100 mg, 0.26 mmol). The solution was allowed to stir overnight at room temperature. The reaction was quenched by addition of a saturated aqueous solution of NH 4 CI (50 mL). The phases were separated and the aqueous phase extracted with DCM (2 x 40 mL). The combined organic phases were dried over MgSCU and the solvent removed in vacuo to provide ethyl 1-(6-amino-2-butoxy-5-nitropyrimidin-4-yl)-4-[2-(2,3-dihydro -1 H-isoindol-2-yl)acetyl]-1 ,4- diazepane-2-carboxylate (100 mg, 70%) as a yellow oil; LC-MS (Method F) 542.5 [MH + ]; RT 3.08 min.

Methyl 3-{4-amino-2-butoxy-6-oxo-5H, 6H, 6aH, 7H, 8H, 9H,10H, 11H-[1,4]diazepino[2, 1- h]pteridine-8-carbonyl}benzoate 8bxiv Prepared from 7bxiv according to general procedure C

[HCI salt]; LC-MS (15 min base method) 469.4 [MH + ]; RT 6.01 min

Ethyl 1-(6-amino-2-butoxy-5-nitropyn ' midin-4-yl)-4-[3-(methoxycarbonyl)benzoyl]-1,4- diazepane-2-carboxylate 7bxiv

A solution of 3-(methoxycarbonyl)benzoic acid (38 mg, 0.21 mmol) in DCM (5 mL) was treated with Et 3 N (0.09 mL, 0.63 mmol), EDC.HCI (60 mg, 0.31 mmol) and HOBt hydrate (42 mg, 0.31 mmol). The solution was stirred for 5 min followed by the addition of ethyl 1- (6-amino-2-butoxy-5-nitropyrirnidin-4-yl)-1 ,4-diazepane-2-carboxylate 6b (80 mg, 0.21 mmol). The solution was allowed to stir overnight at room temperature. The reaction was quenched by addition of a saturated aqueous solution of NH4CI (50 mL). The phases were separated and the aqueous phase extracted with DCM (2 x 40 mL). The combined organic phases were dried over MgS0 4 and the solvent removed in vacuo to provide ethyl 1-(6- amino-2-butoxy-5-nitropyrimidin-4-yl)-4-[3-(methoxycarbonyl) benzoyl]-1 ,4-diazepane-2- carboxylate (85 mg, 75%) as a yellow oil; LC-MS (Method F) 545.5 [MH + ]; RT 2.99 min.

4-Amino-2-butoxy-8-(2-cyclopentylacetyl)-5H,6H,6aH,7H,8H, 9H,10H,11 H- [1 ,4]diazepino[2,1 -h]pteridin-6-one 8bxv Prepared from 7bxv according to General Procedure C. Isolated as HCI salt.

1 H NMR (Method A) (D 2 0): δ ppm 4.53-4.37 (m, 5H), 4.12 (m, 1 H), 3.70 (m, 1 H), 3.38 (m, 1 H), 3.33 (s, 1 H), 3.10 (m, 1 H), 2.40 (m, 1 H), 2.28 (dd, J = 15.5, 8.5 Hz, 1 H), 2.21-2.05 (m, 2H), 1.86-1.42 (m, 10H), 1.13 (m, 1 H), 0.97 (t, J = 7.5 Hz, 3H), 0.80 (m, 1 H); LC-MS (Method F) 417.5 [MH + ]; RT 6.99 min.

Ethyl 1-(6-amino-2-butoxy-5-nitropyn ' midin-4-yl)-4-(2-cyclopentylacetyl)-1,4- diazepane-2-carboxylate 7bxv

A solution of 2-cyclopentylacetic acid (30 mg, 0.23 mmol) in DCM (5 mL) was treated with Et 3 N (0.11 mL, 0.78 mmol), EDC.HCI (67 mg, 0.35 mmol) and HOBt hydrate (47 mg, 0.35 mmol). The solution was stirred for 5 min followed by the addition of ethyl 1-(6-amino-2- butoxy-5-nitropyrimidin-4-yl)-1 ,4-diazepane-2-carboxylate 6b (89 mg, 0.23 mmol). The solution was allowed to stir overnight at room temperature. The reaction was quenched by addition of a saturated aqueous solution of NH4CI (50 mL). The phases were separated and the aqueous phase extracted with DCM (2 x 40 mL). The combined organic phases were dried over MgS0 4 and the solvent removed in vacuo to provide ethyl 1-(6-amino-2- butoxy-5-nitropyrimidin-4-yl)-4-(2-cyclopentylacetyl)-1 ,4-diazepane-2-carboxylate (91 mg, 79%) as a yellow oil; LC-MS (Method F) 493.5 [MH + ]; RT 3.30 min. Compound 8ci was prepared according to Scheme 6

4c 5c

Scheme 6

(6aS,8R)^-Amino-2-butoxy^-hydroxy-5H,6H,6aHJH,8H,9H-pyrro lo[2,1-h]pteridin-6- one 8ci

Prepared from 5c according to General Procedure C

1 H NMR (Method A) (CD 3 OD): δ ppm 4.57 (t, J = 5.0 Hz, 1 H), 4.39 (dd, J = 6.0, 1 1.0 Hz, 1 H), 4.28 (t, J = 6.5 Hz, 2H), 3.93 (dd, J = 5.0, 12.0 Hz, 1 H), 3.55 (m, 1 H), 3.39 (s, 1 H), 2.35 (dd, J = 5.5, 13.0 Hz, 1 H), 2.19 (ddd, J = 5.0, 1 1.0, 13.0 Hz, 1 H), 1.80-1.73 (m, 2H), 1.58-1.47 (m, 2H), 1.03 (t, J = 7.5 Hz, 3H); LC-MS (Method G) 294.6 [MH + ]; RT 1.56 min.

Methyl (2S,4R)-1-(6^mino-2-butoxy-5-nitropyrimidin-4-yl)-4-hydroxyp yrrolidine-2- carboxylate 5c

Prepared from 4c according to General Procedure A

1 H NMR (Method A) (CDC ): δ ppm 5.00 (dd, J = 10.5, 7.0 Hz, 1 H), 4.62 (s, 1 H), 4.22 (dt, J = 10.5, 6.5 Hz, 1 H), 4.09 (m, 1 H), 3.89 (dd, J = 12.5, 4.0 Hz, 1 H), 3.32 (br s, 1 H), 2.88 (d, J = 12.5 Hz, 1 H), 2.41 (m, 1 H), 2.12 (ddd, J = 13.0, 10.0, 4.0 Hz, 1 H), 1.72-1.65 (m, 3H), 1.48-1.41 (m, 2H), 1.25 (t, J = 7.0 Hz, 1 H), 0.95 (t, J = 7.5 Hz, 3H); LC-MS (Method F) 356.4 [MH + ]; RT 1.96 min.

Methyl (2S,4R)-1-(6-amino-2-methanesulfonyl-5-nitropyrimidin-4-yl)- 4- hydroxypyrrolidine-2-carboxylate 4c

A solution of methyl (2S,4f?)-4-hydroxypyrrolidine-2-carboxylate (354 mg, 2.44 mmol) in THF (10 mL) was added dropwise to a solution of 6-chloro-2-methanesulfonyl-5- nitropyrimidin-4-amine 3 (617 mg, 2.44 mmol) and EtzN (0.75 mL, 5.37 mmol) in THF (15 mL) at 0 °C and the reaction stirred at this temperature for 1 h. The reaction mixture was diluted with water (20 mL) and EtOAc (20 mL) and the phases separated. The aqueous phase was extracted with EtOAc (30 ml_) and the combined organic phases washed with brine (20 ml_). The solvent was removed in vacuo to provide methyl (2S,4f?)-1 -(6-amino-2- methanesulfonyl-5-nitropyrimidin-4-yl)-4-hydroxypyrrolidine- 2-carboxylate (480 mg, 55%) as a yellow crystalline solid. The material was used immediately in the subsequent step of the synthesis; LC-MS (Method F) 462.3 [MH " ]; RT 2.74 min.

Example 2 - IFN-a stimulation

Stimulation of human PBMCs

[00105] Assays were performed to investigate cytokine production from human peripheral blood mononuclear cells (PBMCs) after being stimulated for 24 hours with compounds of the present invention. Cryo-preserved human PBMCs were thawed and seeded in 96 well plates at 5 x 10 5 cells/well in 225 μΙ_ RPMI 1640 media (supplemented with 0.5% foetal bovine serum, 2 mM sodium pyruvate and 2 mM Glutamax). The cells were then incubated for 1 h at 37°C and 5% CO2, following which 25 μΙ_ of compound was added. Compounds were diluted from a stock of 10 mM in 100% DMSO and tested in triplicate, covering a range of concentrations up to 10 μΜ. Cells and compound were incubated for a further 24 h at 37°C and 5% CO2. The supernatant was subsequently collected after centrifuging the plates for 10 minutes at 1200 rpm and then stored at -80°C until required for cytokine analysis.

Cytokine ELISAs

[00106] The supernatant was assayed for interferon alpha (IFN-a) and tumour necrosis factor alpha (TNF-a) in two separate ELISAs. MabTech's pan IFN-a kit was used to determine IFN-a levels. The kit will detect native and recombinant human IFN-a subtypes 1/13, 2, 4, 5, 6, 7, 8, 10, 14, 16 and 17. The assay was carried out following manufacturer's instructions. In brief, in a 96 well plate 100 μΙ_Λ/νβΙΙ of capture antibody was added and incubated overnight at 4°C. After washing 2 x with PBS, the plate was blocked with 200 μΙ_ incubation buffer for 1 h at room temperature. After washing 5 x with PBS-T (PBS containing 0.05% Tween20) 100 μΙ_ standard or sample was added for 2 h at room temperature. The PBS-T wash step was then repeated and 100 μΙ_Λ/νβΙΙ of detection antibody was added for 1 h at room temperature. Again, the PBS-T wash step was repeated, followed by the addition of 100 μΙ_Λ/νβΙΙ of Streptavidin-HRP. After a final PBS-T wash 100 μΙ_Λ/νβΙΙ of TMB substrate solution was added, then stopped after a suitable development time using 100 μΙ_Λ/νβΙΙ of 0.5 M HCI and the optical density was read at 450 nM.

[00107] The human TNF-a ELISA Ready-SET-Go! kit from eBioscience was used for TNF-a determination. The assay was carried out following manufacturer's instructions. In brief, in a 96 well plate 100 μΙ_Λ/νβΙΙ of capture antibody was added and incubated overnight at 4°C. After washing 5 x with wash buffer, the plate was blocked with 200 μΙ_ assay diluent for 1 h at room temperature. After washing 5 x with wash buffer, 100 μΙ_ standard or sample was added for 2 h at room temperature. The wash step was then repeated and 100 μΙ_Λ/νβΙΙ of detection antibody was added for 1 h at room temperature. Again, the wash step was repeated, followed by the addition of 100 μΙ_Λ/νβΙΙ of Avidin-HRP. After a final wash 100 μΙ_Λ/νβΙΙ of substrate solution was added, then stopped after a suitable development time using 50 μΙ_Λ/νβΙΙ of 0.5 M HCI and the optical density was read at 450 nM.

[00108] Dose response curves were generated and used to calculate the minimum effective concentration (MEC) for both IFN-a and TNF-a. The MEC was defined as the lowest concentration of compound required to stimulate cytokine production at least three fold greater than the background level.

[00109] The results are shown in Table 1 below.

Table 1 : A denotes an MEC of between 0.0001 and 1 ; B denotes an MEC of between 1 and 10 and C denotes an MEC of greater than 10. Grey shading indicates the cytokine level has not been determined.

Compound IFN-a MEC TNF-a MEC

(μΜ) (μΜ)

GS-9620 A A 8ai C

8aii C

8aiii C C

8bi C C

8bii A B

8biii C

8biv A C

8bv-ii A B

8bvi A B

8bvii A C

8bviii A B 8bix A C

8bx C

8bxi A B

8bxii A A

8bxiii A B

8bxiv C C

8bxv B C

8ci C

Thus, many of the compounds of the invention have been shown to be effective as IFN-a stimulators. Many of the compounds that are effective as IFN-a stimulators are considerably less effective as stimulators of TNF-a, in contrast to GS-9620.

Example 3 - Permeability

[00110] The passive permeability of the compounds of the present invention was determined using confluent MDR1-MDCKII cells growing on 24 well plates with transwell inserts.

[00111] Donor solutions (Hank's balanced salt solution [HBSS] with 10 mM HEPES, 10 μΜ compound and 100 μΜ Lucifer yellow) and receiver solution (HBSS buffer with 10 mM HEPES and 0.1 % DMSO) were prepared and pre-warmed to 37°C. Plates containing confluent MDR1-MDCKII cells with transepithelial electrical resistance (TEER) values found to be within 170-250 Ω were removed from the incubator and washed 3 x with HBSS buffer. On the final wash HBSS buffer was left covering the cells and the plate returned to the incubator at 37°C and 5% C0 2 for 20 minutes.

[00112] After the incubation period the plates were removed from the incubator and the apical compartment was separated and placed onto an empty 24 well plate. The remaining HBSS buffer was then aspirated from both the apical and basal compartments. Receiver solution was added to the relevant well within the relevant compartment, followed by donor solution to the corresponding opposite well/compartment (300 μΙ_ to the apical and 700 μΙ_ to the basal compartments). The whole plate was then re-assembled and placed back in the incubator at 37°C and 5% C02 for 120 minutes. Following the incubation period donor and receiver solutions were sampled, diluted as appropriate and analysed against a standard curve by LC/MS. Compounds were run in a cassette of 3. [00113] The results are shown in Table 2 below.

[00114] Table 2. MDCK permeability - A denotes a permeability of between 2 x 10 "6 and 5 x 10 "6 cm/sec; B denotes a permeability of between 0.5 x 10 "6 and 2 x 10 "6 cm/sec .and C denotes a permeability of between 0.01 x 10 "6 and 0.5 x 10 "6 cm/sec; MDCK efflux ratio - A denotes a ratio of less than 5; B denotes a ratio of between 5 and 25; and C denotes a ratio of greater than 25.

All of the compounds tested had both a higher permeability and a lower efflux ratio (meaning that they are less likely to be substrates for efflux pumps within the gut) than GS- 9620.




 
Previous Patent: LIQUID CONTAINER

Next Patent: CONTAINER