Pyrazolo[3,4-Z>]pyridine compounds, and their use as PDE4 inhibitors

The present invention relates to pyrazolo[3,4-b]pyridine compounds or salts therof, processes for their preparation, intermediates usable in these processes, and pharmaceutical compositions containing the compounds or salts. The invention also relates to the use of the pyrazolo[3,4-b]pyridine compounds or salts thereof in therapy, for example as inhibitors of phosphodiesterase type IV (PDE4) and/or for the treatment and/or prophylaxis of inflammatory and/or allergic diseases such as chronic obstructive pulmonary disease (COPD), asthma, rheumatoid arthritis, allergic rhinitis, psoriasis or atopic dermatitis.

Background to the Invention

US 3,979,399, US 3,840,546, and US 3,966,746 (E.R.Squibb & Sons) disclose 4-amino derivatives ofpyrazolo[3,4-b]pyridine-5-carboxamides wherein the 4-amino group NR3R4 can be an acyclic amino group wherein R3 and R4 may each be hydrogen, lower alkyl (e.g. butyl), phenyl, etc.; NR3R4 can alternatively be a 3-6-membered heterocyclic group such as pyrrolidino, piperidino and piperazino. The compounds are disclosed as central nervous system depressants useful as ataractic, analgesic and hypotensive agents.

US 3,925,388, US 3,856,799, US 3,833,594 and US 3,755,340 (E.R.Squibb & Sons) disclose 4-amino derivatives of pyrazolo[3,4-b]pyridine-5-carboxylic acids and esters. The 4-amino group NR3R4 can be an acyclic amino group wherein R3 and R4 may each be hydrogen, lower alkyl (e.g. butyl), phenyl, etc.; NR3R4 can alternatively be a 5-6- membered heterocyclic group in which an additional nitrogen is present such as pyrrolidino, piperidino, pyrazolyl, pyrimidinyl, pyridazinyl or piperazinyl. The compounds are mentioned as being central nervous system depressants useful as ataractic agents or tranquilisers, as having antiinflammatory and analgesic properties. The compounds are mentioned as increasing the intracellular concentration of adenosine-3',5'- cyclic monophosphate and for alleviating the symptoms of asthma.

H. Hoehn et al., J. Heterocycl. Chem., 1972, 9(2), 235-253 discloses a series of IH- pyrazolo[3,4-b]pyridine-5-carboxylic acid derivatives with 4-hydroxy, 4-chloro, 4-alkoxy, 4-hydrazino, and 4-amino substituents. Ethyl 4-(n-butylamino)-l-ethyl-lH- ρyrazolo[3,4-b]-pyridine-5-carboxylate is disclosed therein; this compound is cartazolate.

The compound tracazolate, ethyl 4-(n-butylamino)-l-ethyl-6-methyl-lH-pyrazolo[3,4-b]- pyridine-5-carboxylate, is known as an anxiolytic agent (e.g. see J.B. Patel et al., Eur. J. Pharmacol., 1982, 78, 323). Other 1 -substituted 4-(NH2 or NH-alkyl)-lH-pyrazolo[3,4- b]-pyridine-5-carboxylic acid esters and amides are disclosed as potential anxiolytic agents in T.M. Bare et al., J. Med. Chem., 1989, 32, 2561-2573. CA 1003419, CH 553 799 and T.Denzel, Archiv der Pharmazie, 191A, 307(3), 177-186 disclose 4,5-disubstituted lH-pyrazolo[3,4-b]pyridines unsubstituted at the 1 -position.

Japanese laid-open patent application JP-2002-20386-A (Ono Yakuhin Kogyo KK) published on 23 January 2002 discloses pyrazolopyridine compounds of the following formula:

JP-2002-20386-A (Ono)

wherein R1 denotes 1) a group -OR6, 2) a group -SR7, 3) a C2-8 alkynyl group, 4) a nitro group, 5) a cyano group, 6) a C 1-8 alkyl group substituted by a hydroxy group or a C 1-8 alkoxy group, 7) a phenyl group, 8) a group -C(O)R8, 9) a group -SO2NR9R10, 10) a group -NR11SO2R12, 11) a group -NR13C(O)R14 or 12) a group -CH=NR15. R6 and R7 denote i) a hydrogen atom, ii) a C 1-8 alkyl group, iii) a C 1-8 alkyl group substituted by a Cl-8 alkoxy group, iv) a trihalomethyl group, v) a C3-7 cycloalkyl group, vi) a Cl-8 alkyl group substituted by a phenyl group or vii) a 3-15 membered mono-, di- or tricyclic hetero ring containing 1-4 nitrogen atoms, 1-3 oxygen atoms and/or 1-3 sulphur atoms. R2 denotes 1) a hydrogen atom or 2) a Cl-8 alkoxy group. R denotes 1) a hydrogen atom or 2) a Cl-8 alkyl group. R4 denotes 1) a hydrogen atom, 2) a Cl-8 alkyl group, 3) a C3-7 cycloalkyl group, 4) a Cl-8 alkyl group substituted by a C3-7 cycloalkyl group, 5) a phenyl group which may be substituted by 1-3 halogen atoms or 6) a 3-15 membered mono-, di- or tricyclic hetero ring containing 1-4 nitrogen atoms, 1-3 oxygen atoms and/or 1-3 sulphur atoms. R5 denotes 1) a hydrogen atom, 2) a Cl-8 alkyl group, 3) a C3- 7 cycloalkyl group, 4) a Cl-8 alkyl group substituted by a C3-7 cycloalkyl group or 5) a phenyl group which may be substituted by 1-3 substituents. In group R3, a hydrogen atom is preferred. In group R4 , methyl, ethyl, cyclopropyl, cyclobutyl or cyclopentyl are preferred. The compounds of JP-2002-20386-A are stated as having PDE4 inhibitory activity and as being useful in the prevention and/or treatment of inflammatory diseases and many other diseases.

l,3-Dimethyl-4-(arylamino)-pyrazolo[3,4-b]pyridines with a 5-C(O)NH2 substituent similar or identical to those in JP-2002-20386-A were disclosed as orally active PDE4 inhibitors by authors from Ono Pharmaceutical Co. in: H. Ochiai et al., Bioorg. Med. Chem. Lett, 5th January 2004 issue, vol. 14(1), pp. 29-32 (available on or before 4th December 2003 from the Web version of the journal: "articles in press"). Full papers on these and similar compounds as orally active PDE4 inhibitors are: H. Ochiai et al., Bioorg. Med. Chem. , 2004, 12(15), 4089-4100 (stated to have been available online 20 June 2004), and H. Ochiai et al., Chem. Pharm. Bull, 2004, 52(9), 1098-1104 (stated to have been published online 15 June 2004).

EP 0 076 035 Al (ICI Americas) discloses pyrazolo[3,4-b]pyridine derivatives as central nervous system depressants useful as tranquilisers or ataractic agents for the relief of anxiety and tension states.

J.W. Daly et al., Med. Chem. Res., 1994, 4, 293-306 and D. Shi et al., Drug Development Research, 1997, 42, 41-56 disclose a series of 4-(amino)substituted lH-pyrazolo[3,4- b]pyridine-5-carboxylic acid derivatives, including ethyl 4-cyclopentylamino-l-methyl- lH-pyrazolo[3,4-b]pyridine-5-carboxylate, and their affinities and antagonist activities at A^- and A2A"a(ienosine receptors, and the latter paper discloses their affinities at various binding sites of the GABA^-receptor channel. S. Schenone et al., Bioorg. Med. Chem. Lett., 2001, 11, 2529-2531, and F. Bondavalli et al., J. Med. Chem., 2002, vol. 45 (Issue 22, 24 October 2002, allegedly published on Web 09/24/2002), pp. 4875-4887 disclose a series of 4-amino-l-(2-chloro-2-phenylethyl)-lH-pyrazolo[3,4-b]pyridin e-5-carboxylic acid ethyl esters as Ai -adenosine receptor ligands.

WO 02/060900 A2 appears to disclose, as MCP-I antagonists for treatment of allergic, inflammatory or autoimmune disorders or diseases, a series of bicyclic heterocyclic compounds with a -C(O)-NR4-C(O)-NR5R6 substituent, including isoxazolo[5,4- b]pyridines and lH-pyrazolo[3,4-b]pyridines (named as pyrazolo[5,4-b]pyridines) with the -C(O)-NR4-C(O)-NR5R6 group as the 5-substituent and optionally substituted at the 1-, 3-, A-, and/or 6-positions. Bicyclic heterocyclic compounds with a-C(O)NH2

substituent instead of the -C(O)-NR4-C(O)-NR5R6 substituent are alleged to be disclosed in WO 02/060900 as intermediates in the synthesis of the -C(O)-NR4-C(O)-NR5R6 substituted compounds. See also WO 02/081463 Al for similar MCP-I antagonists.

WO 00/15222 (Bristol-Myers Squibb) discloses inter alia pyrazolo[3,4-b]pyridines having inter alia a C(O)-Xi group at the 5-position and a group Ε\ at the 4-position of the ring system. Amongst other things, Xi can for example be -OR9, -N(Rp)(RiQ) or -N(R5)(-A2"R2), and Ε\ can for example be -NH-Ai -cycloalkyl, -NH-Ai -substituted cycloalkyl, or -NH-Ai -heterocyclo; wherein Ai is an alkylene or substituted alkylene bridge of 1 to 10 carbons and A2 can for example be a direct bond or an alkylene or substituted alkylene bridge of 1 to 10 carbons. The compounds are disclosed as being useful as inhibitors of cGMP phosphodiesterase, especially PDE type V, and in the treatment of various cGMP-associated conditions such as erectile dysfunction. Compounds with a cycloalkyl or heterocyclo group directly attached to -NH- at the 4-position of the pyrazolo[3,4-b]pyridine ring system and/or having PDE4 inhibitory activity do not appear to be disclosed in WO 00/15222. G. Yu et. al., J. Med Chem., 2001, 44, 1025-1027 discloses some 4-[(3-chloro-4- methoxybenzyl)amino]-pyrazolopyridine-5-carboxamides as selective PDE5 inhibitors.

H. de Mello, A. Echevarria, et al., J Med. Chem., 2004, 47(22), 5427-5432, believed to have been published online on or before 21 September 2004, discloses 3-methyl or 3- phenyl 4-anilmo-lH-pyrazolo[3,4-b]pyridine 5-carboxylic esters as potential anti- Leishmania drugs.

Copending patent application PCT/EP2003/014867, filed on 19 December 2003 in the name of Glaxo Group Limited, published on 8 July 2004 as WO 2004/056823 Al , discloses and claims pyrazolo[3,4-b]pyridine compounds or salts thereof with a A- NR^R^a group (R-*a is preferably H) and with a group Het at the 5-position of the pyrazolo[3,4-b]pyridine, wherein Het is usually a 5-membered optionally substituted heteroaryl group. PCT/EP2003/014867 (WO 2004/056823 Al) also discloses the use of these compounds as PDE4 inhibitors and for the treatment and/or prophylaxis of inter alia COPD, asthma or allergic rhinitis.

Copending patent application PCT/EP03/11814, filed on 12 September 2003 in the name of Glaxo Group Limited, published on 25 March 2004 as WO 2004/024728 A2, discloses pyrazolo[3,4-b]pyridine compounds or salts thereof with a 4-NHR^ group and a 5- C(O)-X group, according to this formula (I):

HN' λ

(I) N' ^R2

R1' wherein: R1 is Ci_4alkyl, Ci_3fluoroalkyl, -CH2CH2OH or -CH2CH2CO2C i_2alkyl;

R^ is a hydrogen atom (H), methyl or Cifluoroalkyl;

R^ is optionally substituted C3_gcycloalkyl or optionally substituted mono-unsaturated-C5_7cycloalkenyl or an optionally substituted heterocyclic group of sub-formula (aa), (bb) or (cc);

or I (\ \ or

(aa) (bb) (cc) in which n* and n^ independently are 1 or 2; and in which Y is O, S, SO2, or NRlO; - -

where R10 is a hydrogen atom (H), Ci_4alkyl, Ci_2fluoroalkyl, CH2C(O)NH2, C(O)NH2, qO^Cj^alkyl, C(O)-C ifluoroalkyl or -C(O)-CH2O-C!.2alkyl;

/ — N 2/

H

or R3 is a bicyclic group (dd) or (ee): (dd)> VS

and wherein X is NR4R5 or OR5a.

In PCT/EP03/11814 (WO 2004/024728 A2), R4 is a hydrogen atom (H); C^alkyl; C1.

3fluoroalkyl; or C2_6alkyl substituted by one substituent R11.

In PCT/EP03/11814 (WO 2004/024728 A2), R5 can be: a hydrogen atom (H); C i .galkyl; Ci_8 fluoroalkyl; C3_gcycloalkyl optionally substituted by a Ci_2alkyl group;

-(CH2)n4-C3_gcycloalkyl optionally substituted, in the -(CH2)n4- moiety or in the

C3_gcycloalkyl moiety, by a Ci_2alkyl group, wherein n4 is 1, 2 or 3; C2_6alkyl substituted by one or two independent substituents R11J -(CH2)J11 ^C(O)R16;

-(CH2)n12-C(O)NR12R13; -CHR19-C(O)NR12R13; -(CH2)n12-C(O)OR16;

-(CH2)J112-C(O)OH; -CHR19-C(O)OR16; -CHR^-C(O)OH;

-(CH2)n12-SO2-NR12R13; -(CH^-SO^; or -(CH2)H12-CN; -(CH2)J113-Het; or optionally substituted phenyl.

Alternatively, in PCT/EP03/11814 (WO 2004/024728 A2), R5 can have the sub-formula (X)5 (YX (Yi) Or (Z):

"(CH2)n Av

-(CH2); F^_

(X) (y) (yυ (Z)

wherein in sub-formula (x), n = 0, 1 or 2; in sub-formula (y) and (yl), m = 1 or 2; and in sub-formula (z), r = 0, 1 or 2; and wherein in sub-formula (x) and (y) and (yl), none, one or two of A5 B5 D, E and F are independently nitrogen or nitrogen-oxide (N+-O") provided that no more than one of A, B, D, E and F is nitrogen-oxide, and the remaining of A, B, D, E and F are independently CH or CR^; and provided that when n is 0 in sub- formula (x) then one or two of A, B, D, E and F are independently nitrogen or nitrogen-oxide (N+-O") and no more than one of A, B, D, E and F is nitrogen-oxide;

In PCT/EP03/11814 (WO 2004/024728 A2), each R6, independently of any other R6 present, is: a halogen atom; C^galkyl; Ci_4fluoroalkyl; Ci_4alkoxy; Ci_2fluoroalkoxy; C3_6cycloalkyloxy; -C(O)R1Sa; -C(O)OR30; -S(O)2-R16a; R16^S(O)2-NR1Sa.;

R7R8N-S(O)2-; CI^aUCyI-C(O)-R15aN-S(O)2-; Ci_4aUcyl-S(O)-; Ph-S(O)-;

R7R8N-CO-; -NR15-C(O)R16; R7R8N; OH; Ci_4alkoxymethyl; C^alkoxyethyl;

C i _2alkyl-S(O)2-CH2-; R7R8N-S(O)2-CH2-; C i ^aUCyI-S(O)2-NR15a-CH2-;

-CH2-OH; -CH2CH2-OH; -CH2-NR7R8; -CH2-CH2-NR7R8; -CH2-C(O)OR30;

-CH2-C(O)-NR7R8; -CH2-NR15a-C(O)-Ci_3alkyl; -(CH^n^-Het1 where n14 is O or 1;

cyano (CN); Ar^b; or phenyl, pyridinyl or pyrimidinyl wherein the phenyl, pyridinyl or pyrimidinyl independently are optionally substituted by one or two of fluoro, chloro, Ci_2alkyl, Cifluoroalkyl, Ci_2alkoxy or Cifluoroalkoxy;

or two adjacent R6 taken together can be -0-(CMe2)-0- or -O-(CH2)n14^O-. where

n14a is 1 or 2.

The pyrazolo[3,4-b]pyridine compounds of formula (I) and salts thereof disclosed in PCT/EP03/11814 (WO 2004/024728 A2) are disclosed as being inhibitors of phosphodiesterase type IV (PDE4), and as being useful for the treatment and/or prophylaxis of a variety of diseases / conditions, especially inflammatory and/or allergic diseases in mammals such as humans, for example: asthma, chronic obstructive pulmonary disease (COPD) (e.g. chronic bronchitis and/or emphysema), atopic dermatitis, urticaria, allergic rhinitis, allergic conjunctivitis, vernal conjunctivitis, eosinophilic granuloma, psoriasis, rheumatoid arthritis, septic shock, ulcerative colitis, Crohn's disease, reperfusion injury of the myocardium and brain, chronic glomerulonephritis, endotoxic shock, adult respiratory distress syndrome, multiple sclerosis, cognitive impairment (e.g. in a neurological disorder), depression, or pain. PCT/EP03/11814 (WO 2004/024728 A2) states that the compounds of formula (I) and/or their pharmaceutical compositions may be administered by oral, parenteral, inhaled (topical to the lung), or nasal administration. However, the use of the pyrazolo[3,4- b]pyridine compounds by external topical administration is not disclosed.

PCT/EP03/11814 (WO 2004/024728 A2) does not disclose any specific pyrazolo[3,4- bjpyridine compounds having a 4-position group NHR3 in which R3 is an optionally substituted heterocyclic group of sub-formula (aa), (bb) or (cc) and in which Y is NR10 wherein R10 is C(O)NH2. Also, WO 2004/024728 A2 does not disclose any specific

pyrazolo[3,4-b]pyridine compounds having a 4-position group NHR3 in which R3 is a 4-(aminocarbonyl)cyclohexyl group. WO 2004/024728 has been reviewed, and WO 2004/056823 mentioned, inExpert Opin. Ther. Patents, 2005 (January edition), 15(1), 111-114.

The Invention

We have now found new pyrazolo[3,4-b]pyridine compounds, which compounds inhibit phosphodiesterase type IV (PDE4).

The present invention therefore provides a compound of formula (I) or a salt thereof (in particular, a pharmaceutically acceptable salt thereof):

NR4R5

(I)

wherein:

R1 is Ci_3alkyl, C^fluoroalkyl, or -CH2CH2OH;

R2 is a hydrogen atom (H), methyl or Cifluoroalkyl;

R3 is a 4-(aminocarbonyl)cyclohexyl (i.e. 4-(aminocarbonyl)cyclohexan-l-yl) group of sub-formula (aa), or an N-aminocarbonyl-piperidinyl or -pyrrolidinyl group of sub- formula (bb);

..

(aa) (bb)

wherein Y is NCONH2 and n1 is 0 or 1 ; - -

and wherein the cyclohexyl group of sub-formula (aa) or the piperidinyl or pyrrolidinyl groups of sub-formula (bb) are not further substitutedon any ring carbon; R4 is a hydrogen atom (H);

R.5 is a group of the sub-formula (x), (y), (yl) or (z):

-(CH2)n -G ^ ^B Q' -(CH2)r- F^_/D

(X) (y) (yi) (Z)

wherein in sub-formula (x), n = 0, 1 or 2; in sub-formula (y) and (yl), m = 1 or 2; and in sub-formula (z), r = 0, 1 or 2;

wherein in sub-formula (x) and (y) and (yl), none, one or two of A, B, D, E and F are nitrogen; and the remaining of A, B, D, E and F are independently CH or CR6;

wherein, each R6, independently of any other R6 present, is: a halogen atom; C^.galkyl (e.g. Ci_4alkyl or Ci_2alkyl); Ci_4fluoroalkyl (e.g. Ci_2fluoroalkyl); Cj^alkoxy (e.g. Ci_2alkoxy); C^fluoroalkoxy; C3_6cycloalkyloxy; -C(O)R16a; -C(O)OR30; -S(O)2-R16a (e.g. Ci_2alkylsulphonyL that is Ci_2alkyl-SO2-); R16^S(O)2-NR15a- (e.g. Ci_2alkyl-SO2-NH-); R7R8N-S(O)2-; C1^aUCyI-C(O)-R15aN-S(O)2-; Ci_4alkyl-S(O)-, Ph-S(O)-, R7R8N-CO-; -NR15-C(O)R16a; R7R8N; OH; Ci_4alkoxymethyl; Ci_4alloxyethyl; Ci_2alkyl-S(O)2-CH2-; R7R8N-S(O)2-CH2-; Ci_2alkyl-S(O)2-NR15a-CH2-; -CH2-OH; -CH2CH2-OH; -CH2-NR7R8; -CH2-CH2-NR7R8; -CH2-C(O)OR30; -CH2-C(O)-NR7R8; -CH2-NR15a-C(O)-Ci_3alkyl; -(CH^∏^-Het1 where n14 is O or 1; cyano (CN); Ar5b; or phenyl, pyridinyl or pyrimidinyl wherein the phenyl, pyridinyl or pyrimidinyl independently are optionally substituted by one or two of fluoro, chloro, Ci_2alkyl, Cifluoroalkyl, Ci_2alkoxy or Cifluoroalkoxy;

or where two adjacent R6 taken together are -O-(CMe2)-O- or -O-(CH2)n14-O- where n14 is 1 or 2;

wherein sub-formula (y) and (yl), independently, are optionally substituted by oxo (=0) at a ring carbon adjacent the 6-membered aromatic ring (for example, sub-formula (y) can optionally be m or ° , or sub-formula (yl) can optionally

be °

wherein in sub-formula (z), G is O or S or NR^ wherein R^ is a hydrogen atom (H), Ci_4alkyl or C^fluoroalkyl; none, one, two or three of J, L, M and Q are nitrogen; and the remaining of J, L, M and Q are independently CH or CR^ where R^, independently of any other R^ present, is as defined herein;

and wherein: R7 and R8 are independently a hydrogen atom (H); C^alkyl (e.g. Ci_2alkyl such as methyl); C3_gcycloalkyl; or phenyl optionally substituted by one or two substituents independently being: fluoro, chloro, Ci_2alkyl, Cifluoroalkyl, Ci_2alkoxy or Cifluoroalkoxy;

or R7 and R8 together are -(CH2)n6- or -C(O)-(CH2)n7- or -C(O)-(CH2)H1°-C(O)- or -(CH2)n8-χ7-(CH2)n9- or -C(O)-X7-(CH2)n10- in which: n6 is 3, 4, 5 or 6 (suitably n6 is 4 or 5), n7 is 2, 3, 4, or 5 (suitably n7 is 3 or 4), n8 and n^ and n1^ independently are 2 or 3 (suitably independently 2), and X7 is O or NR^;

R7a is a hydrogen atom (H) or C^alkyl (suitably H or Ci_2alkyl, more suitably H or methyl);

R8a is a hydrogen atom (H) or methyl (suitably H);

R14, independent of other R1^5 is a hydrogen atom (H); C^alkyl (e.g. Ci_2alkyl); Ci_2fiuoroalkyl (e.g. CF3); cyclopropyl; -C(O)-C i^alkyl (e.g. -C(O)Me); -C(O)NR7aR8a (e.g. -C(O)NH2); or -S(O)2-C i_4alkyl (e.g. -S(O)2Me) (preferably, Rl4 is: H, Ci_2alkyl, or -C(O)Me);

R!5, independent of other R^5 is a hydrogen atom (H); Cj_4alkyl (e.g. *Bu or Ci_2alkyl e.g. methyl); C3_gcycloalkyl; or phenyl optionally substituted by one or two of: a halogen 1 Q

atom, Ci_2alkyl, Cifluoroalkyl, Ci_2alkoxy or Cifluoroalkoxy (preferably R15 is H or Ci_2alkyl, more preferably H);

R.15a, independent of other R^^a, is a hydrogen atom (H) or Ci_4alkyl (e.g. H, tBu or

Ci _2alkyl such as methyl; preferably R^ ^a is JJ or C i _2alkyl, more preferably H);

Rl6ais: Cμgalkyl (e.g. Ci_4alkyl or C^alkyl); C3_6cycloalkyl (e.g. Cs.gcycloalkyl) optionally substituted by one oxo (=0), OH or Cj_2alkyl substituent (e.g. optionally substituted at the 3- or 4-position of a C5_5cycloalkyl ring; and/or preferably unsubstituted C3_6cycloalkyl); C3_6cycloalkyl-CH2- (e.g. C5_6cycloalkyl-CH2-); pyridinyl (e.g. ρyridin-2-yl) optionally substituted on a ring carbon atom by one of: a halogen atom, Ci_2alkyl, Cifluoroalkyl, Ci_2alkoxy or Cifluoroalkoxy;

Ar5c; phenyl optionally substituted by one or two substituents independently being: a halogen atom, Chalky!, Cifluoroalkyl, Ci_2alkoxy or Cifluoroalkoxy; benzyl optionally substituted on its ring by one or two substituents independently being: a halogen atom, Ci_2alkyl, Cifluoroalkyl, Ci_2alkoxy or Cifluoroalkoxy; or a A-, 5-, 6- or 7-membered saturated heterocyclic ring connected at a ring-carbon and containing one or two ring-hetero-atoms independently selected from O, S, and N; wherein any ring-nitrogens which are present are present as NR2^ where R2^ is H, Ci_2alkyl or -C(O)Me; and wherein the ring is optionally substituted at carbon by one Ci_2alkyl or oxo (=0) substituent, provided that any oxo (=0) substituent is substituted at a ring-carbon atom bonded to a ring-nitrogen;

R30, independent of other R^O, is a hydrogen atom (H), Ci_4alkyl or C3_6cycloalkyl;

Ar^b and Ar^c independently is/are a 5-membered aromatic heterocyclic ring containing one O, S or NRl 5a m the 5-membered ring, wherein the 5-membered ring can optionally additionally contain one or two N atoms, and wherein the heterocyclic ring is optionally substituted on a ring carbon atom by one of: a halogen atom, Ci_2alkyl, Cifluoroalkyl, -CH2OH, -CH2-OCi_2alkyl, OH (including the keto tautomer thereof) or - CH2-NR28R29 wherein R2^ and R2^ independently are H or methyl; and

Het* is a A-, 5-, 6- or 7-membered saturated heterocyclic ring connected at a ring-carbon and containing one or two ring-hetero-atoms independently selected from O, S, and N; wherein any ring-nitrogens which are present are present as NR-* 1 where R^ 1 is H, Ci_2alkyl or -C(O)Me; and wherein the ring is optionally substituted at carbon by one Ci_2alkyl or oxo (=0) substituent, provided that any oxo (=0) substituent is substituted at a ring-carbon atom bonded to a ring-nitrogen.

In compounds, for example in the compounds of formula (I), an "alkyl" group or moiety may be straight-chain or branched. Alkyl groups, for example Cj^galkyl or C^galkyl or Ci_4alkyl or Cj .3 alkyl or Ci_2alkyl, which may be employed include Cj.galkyl or Ci_4alkyl or C ^.3 alkyl or Ci_2alkyl such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, or n-hexyl or any branched isomers thereof such as isopropyl, t-butyl, sec-butyl, isobutyl, 3- methylbutan-2-yl, 2-ethylbutan-l-yl, or the like.

A corresponding meaning is intended for "alkoxy", "alkylene", and like terms derived from alkyl. For example, "alkoxy" such as Cj.galkoxy or Ci_4alkoxy or Ci_2alkoxy includes methoxy, ethoxy, propyloxy, and oxy derivatives of the alkyls listed above. "Alkylsulfonyl" such as Ci_4alkylsulfonyl includes methylsulfonyl (methanesulfonyl), ethylsulfonyl, and others derived from the alkyls listed above. "Alkylsulfonyloxy" such as Ci_4alkylsulfonyloxy includes methanesulfonyloxy (methylsulfonyloxy), ethanesulfonyloxy, et al.

"Cycloalkyl", for example C3_gcycloalkyl, includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. Preferably, a C3_gcycloalkyl group is C3_gcycloalkyl or C5_6cycloalkyl , that is contains a 3-6 membered or 5-6 membered carbocyclic ring.

"Fluoroalkyl" includes alkyl groups with one, two, three, four, five or more fluorine substituents, for example Ci_4fluoroalkyl or C \.3 fluoroalkyl or Ci_2fluoroalkyl such as monofluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl, 2,2,2-trifluoroethyl (CF3CH2-), 2,2-difluoroethyl (CHF2CH2-), 2-fluoroethyl (CH2FCH2-), etc. "Fluoroalkoxy" includes C \ _4fluoroalkoxy or C\ _2fluoroalkoxy such as trifluoromethoxy, pentafluoroethoxy, monofluoromethoxy, difluoromethoxy, etc. "Fluoroalkylsulfonyl" such as C^fluoroalkylsulfonyl includes trifluoromethanesulfonyl, pentafluoroethylsulfonyl, etc.

A halogen atom ("halo") present in compounds, for example in the compounds of formula (I), means a fluorine, chlorine, bromine or iodine atom ("fluoro", "chloro", "bromo" or "iodo"), for example fluoro, chloro or bromo.

When the specification states that atom or moiety A is "bonded" or "attached" to atom or moiety B, it means that atom/moiety A is directly bonded to atom/moiety B usually by means of a covalent bond or a double covalent bond, and excludes A being indirectly attached to B via one or more intermediate atoms/moieties (e.g. excludes A-C-B); unless it is clear from the context that another meaning is intended.

When R1 is Ci_3alkyl or Ci_3fluoroalkyl, it can be straight-chained or branched. Where

RI is Ci_3alkyl then it can for example be methyl, ethyl, n-propyl, or isopropyl. When

R1 is Cμ3fluoroalkyl, then R1 can for example be Cifluoroalkyl such as

monofluoromethyl, difluoromethyl, trifluoromethyl; or R* can be C2fluoroalkyl such as pentafluoroethyl or more preferably Cifluoroalkyl-CE^- such as 2,2,2-trifluoroethyl (CF3CH2-), 2,2-difluoroethyl (CHF2CH2-), or 2-fluoroethyl (CH2FCH2-).

R1 is Cχ_3alkyl (e.g. methyl, ethyl or n-propyl), Cifluoroalkyl or -CH2CH2OH. R1 can be Ci_3alkyl, Ci_2fluoroalkyl, or -CH2CH2OH. Preferably, R1 is C2_3alkyl (e.g. ethyl or n-propyl), C2fluoroalkyl (e.g. Cifluoroalkyl-CH2- such as CF3-CH2-) or -CH2CH2OH; in particular ethyl, n-propyl or -CH2CH2OH. More preferably, R1 is C2alkyl or C2fluoroalkyl. R* is most preferably ethyl.

Preferably, R^ is a hydrogen atom (H) or methyl, for example a hydrogen atom (H).

hi the group of sub-formula (bb), n* is preferably 1. Therefore, preferably, the group of sub-formula (bb), or more preferably R^, is:

N 1 NH,

When R3 is an N-aminocarbonyl-pyrrolidinyl group of sub-formula (bb), (i.e. wherein n* .0

N-

is 0), then preferably NHR^ is: ^- (wherein the -NH- connection point is underlined), and including mixtures of configurations wherein the illustrated configuration is the major component.

hi R^, the piperidinyl or pyrrolidinyl group of sub-formula (bb) is not substituted on a ring carbon. In R3, the cyclohexyl group of sub-formula (aa) has no further optional substituents (beyond 4-CONH2).

It will be appreciated that when R^ is the substituted cyclohexyl group of sub-formula (aa), then the substituent can be in the cis or trans configuration with respect to the -NH- group of formula (I) to which R^ is attached (bonded). The present invention covers each configuration as well as mixtures of configurations, in particular wherein the stated configuration is the major component. Therefore, the -C(O)NH2 substituent on the cyclohexyl group of sub-formula (aa) can for example be in the cis or trans configuration with respect to the -NH- group of formula (I) to which R^ is attached (bonded), including mixtures of configurations wherein the stated configuration is the major component.

Preferably, the aminocarbonyl substituent on the cyclohexyl group of sub-formula (aa) is in the cis configuration with respect to the -NH- group of formula (I) to which R^ is attached (bonded), i.e. preferably NHR^ is a czs-[4-(aminocarbonyl)cyclohexan-l- yl]amino group (including mixtures of configurations wherein the stated cis configuration is the major component).

Alternatively, NHR^ can be a racemic [4-(aminocarbonyl)cyclohexan-l-yl] amino group.

Preferably, R7 and/or R8 are independently a hydrogen atom (H); Ci_2alkyl such as methyl; C3_6cycloalkyl; or phenyl optionally substituted by one of: fluoro, chloro,

Ci_2alkyl, Cifluoroalkyl, Ci_2alkoxy or Cifluoroalkoxy; or R? and R8 together are

-(CH2)n6- or -(CH2)n8-χ7-(CH2)n9- wherein X7 is NR14 or preferably O.

When R7 is cycloalkyl or optionally substituted phenyl, then preferably R8 is neither cycloalkyl nor optionally substituted phenyl.

Most preferably, R7 and/or R8 independently are a hydrogen atom (H) or Ci_2alkyl. It is

preferable that R? is a hydrogen atom (H).

Preferably n^ is 4 or 5. Preferably n7 is 2, 3 or 4. Preferably, n8, τfi and/or n^ is/are independently 2.

Preferably, R7 and R8 independently are a hydrogen atom (H) or Ci_2alkyl; or R^ and

R8 together are -(CH2)n6- or -(CH2)n8-X7-(CH2)n9- wherein X7 is NR14 or preferably

O, n^ is 4 or 5, and n8 and n^ are both 2;

Preferably, R14 is H, C1^aIkVl, or -C(O)Me. Preferably, R15, independent of other R*5, is a hydrogen atom (H) or Ci_2alkyl.

Preferably, R15a is a hydrogen atom (H) or Ci_2alkyl.

Preferably, R^a is Ci_4alkyl, e.g. Ci_2alkyl such as methyl.

In one embodiment, R^ is a group of sub-formula (x) or (z). Preferably, R^ is a group of sub-formula (x).

In sub-formula (x), (y) and (yl), in particular in sub-formula (x), it is preferred that none, one or two of A, B, D, E and F are nitrogen; none, one, two or three of A, B, D, E and F are CR^; and the remaining of A, B, D, E and F are CH.. More preferably, none, one or two of A, B, D, E and F are nitrogen; none, one or two of A, B, D, E and F are CR^; and the remaining of A, B, D, E and F are CH.. Yet more preferably, none or one of A, B, D, E and F are nitrogen, and/or preferably none, one or two of A, B, D, E and F are CR^.

Preferably, sub-formula (x) is: benzyl; optionally substituted on the phenyl ring with one or two R^ substituents.

In one embodiment, sub-formula (yl) is:

or or wherein R^a is or independently are either R" as defined herein or preferably hydrogen.

Preferably, in sub-formula (z), none, one or two of J, L, M and Q are nitrogen.

hi sub-formula (x), (y), (yl) and/or (z), preferably, each R^, independently of any other R6 present, is a fluorine, chlorine, bromine or iodine atom, methyl, ethyl, n-propyl, isopropyl, C4alkyl, trifluoromethyl, -CH2OH, methoxy, ethoxy, Cifluoroalkoxy (e.g. trifluoromethoxy or difluoromethoxy), OH, Ci_3alkylS(O)2- (such as methylsulphonyl which is MeS(O)2-), Ci_3alkylS(O)2-NH- such as methyl-SC>2-NH-, Me2N-S(O)2", H2N-S(O)2-, -CONH2, -CONHMe, -CO2H, cyano (CN), NMe2, t-butoxymethyl, or

Ci_3alkylS(O)2-CH2- such as methyl-SO2-CH2-. More preferably, each R6,

independently of any other R^ present, is a fluorine, chlorine, bromine or iodine atom, methyl, ethyl, n-propyl, isopropyl, isobutyl, trifluoromethyl, -CH2OH, methoxy, ethoxy, Cifluoroalkoxy (e.g. trifluoromethoxy or difluoromethoxy), Ci_3alkylS(O)2- such as methylsulphonyl, Ci_3alkylS(O)2-NH- such as methyl-SO2-NH-, Me2N-S(O)2-5 H2N-S(O)2-, -CONH2, or Ci_3alkylS(O)2-CH2- such as methyl-SO2-CH2. Still more preferably, each R^, independently of any other R^ present, is a fluorine, chlorine or bromine atom, methyl, ethyl, n-propyl, isopropyl, trifluoromethyl, -CH2OH, methoxy, difluoromethoxy, methylsulphonyl, methyl-SO2-NH- or methyl-SO2-CH2-.

hi sub-formula (x), preferably, one, two or three R^ substituents are present in B, D and/or E; so that for example in sub-formula (x), one, two or three R^ substituents are present in the ortho- (2- and/or 6-) and/or meta- (3- and/or 5-) and/or para- (4-) positions with respect to the -(CH^)n- side-chain.

Preferably, R^ has the sub-formula (x), n is 1 and each of A, B, D, E and F is independently CH or CR^; that is R^ has the sub-formula (x) and is optionally substituted benzyl.

In one preferable embodiment, R^ has the sub-formula (x) and is: benzyl, (monoalkyl- phenyl)methyl, [mono(fluoroalkyl)-phenyl]methyl, (monohalo-phenyl)methyl, (monoalkoxy-phenyi)methyl, [mono(fluoroalkoxy)-phenyl]methyl, [mono(N,N- dimethylamino)-phenyl]methyl, [mono(methyl-SO2-NH-)-phenyl]methyl, [mono(methyl-Sθ2-)-phenyl]methyl, (dialkyl-phenyl)methyl, (monoalkyl-monohalo- phenyl)methyl, [mono(fluoroalkyl)-monohalo-phenyl]methyl, (dihalo-phenyl)methyl, (dihalo-monoalkyl-phenyl)methyl, [dihalo-mono(hydroxymethyl)-phenyl]methyl, or (dialkoxy-phenyl)methyl such as (3,4-dimethoxy-phenyl)methyl. The substituents can preferably be further defined, as defined in preferable embodiments herein.

In one preferable embodiment, R^ is of sub-formula (x) and is: (monoalkyl- phenyl)methyl, [mono(fluoroalkyl)-phenyl]methyl, (monohalo-phenyl)methyl, (monoalkoxy-phenyl)methyl, [mono(fluoroalkoxy)-phenyl]methyl, [mono(N,N- dimethylamino)-phenyl]methyl, (dialkyl-phenyl)methyl, (monoalkyl-monohalo- phenyl)methyl, (dihalo-phenyl)methyl or (dihalo-monoalkyl-phenyl)methyl or [dihalo- mono(hydroxymethyl)-phenyl]methyl. More preferably, in this embodiment, R^ is: - (monoCi_3alkyl-phenyl)methyl such as (4-Ci_3alkyl-phenyl)methyl; - (monoCifluoroalkyl-phenyl)methyl such as (4-Cifluoroalkyl-phenyl)methyl; - (monoC \ _2alkoxy-phenyl)methyl such as (4-C \ _2alkoxy-phenyl)methyl; - [mono(Cifluoroalkoxy)-phenyl]methyl such as (4-Cifluoroalkoxy-phenyl)methyl; - (diCi_2alkyl-phenyl)methyl or (dimethyl-phenyl)methyl such as (3,4-dimethyl- phenyl)methyl, (2,4-dimethyl-phenyl)methyl, (3,5-dimethyl-phenyl)methyl, (2,3- dimethyl-phenyl)methyl or (2,5-dimethyl-phenyl)methyl; more preferably (3,4-dimethyl- phenyl)methyl or (2,4-dimethyl-phenyl)methyl; Λ r - 16 -

- (monoCi_2alkyl-monohalo-phenyl)methyl or (monoC^alkyl-monochloro- phenyl)methyl such as (4-methyl-3-chloro-phenyl)methyl, (3-methyl-4-chloro-phenyl)methyl, (2-methyl-4-chloro-phenyl)methyl; - (dihalo-phenyl)methyl such as (2-chloro-4-fluorophenyl)methyl or (2,4-difluoro- phenyl)methyl or (4-bromo-2-fluorophenyl)methyl or preferably (4-chloro- 2-fluorophenyl)methyl; for example (dichloro-phenyl)methyl such as (3,4-dichloro- phenyl)methyl or (2,4-dichloro-phenyl)methyl or (2,6-dichloro-phenyl)methyl or preferably (2,3-dichloro-phenyl)methyl; - (dihalo-monoCi_2alkyl-phenyl)methyl e.g. (2,4-dichloro-6-methyl-phenyl)methyl; or - [dihalo-mono(hydroxymethyl)-phenyl]methyl such as [2,3-dichloro-6-(hydroxymethyl)- phenyljmethyl.

In an alternative preferable "biaryl" embodiment, R^ is of sub-formula (x) and is: benzyl optionally substituted on the phenyl ring with one or two (e.g. one) R^ substituents; wherein one of the R^ is: Ar^b5 or phenyl, pyridinyl or pyrimidinyl wherein the phenyl, pyridinyl or pyrimidinyl independently are optionally substituted by one or two of fluoro, chloro, Ci_2alkyl, Cifluoroalkyl, Ci_2alkoxy or Cjfluoroalkoxy; and wherein Ar^b is a

5-membered aromatic heterocyclic ring containing one O, S or NR^a in the 5-membered ring, wherein the 5-membered ring can optionally additionally contain one or two N atoms, and wherein the heterocyclic ring is optionally substituted on a ring carbon atom by one of: a halogen atom, Ci^alkyL Cifluoroalkyl, -CH2OH, -CH2-OCi_2alkyl, OH (including the keto tautomer thereof) OΓ-CH2-NR28R29 wnerein R^8 and R^9 independently are H or methyl.

In this "biaryl" embodiment, more preferably, R^ is of sub-formula (x) and is: benzyl optionally substituted on the phenyl ring with one or two (e.g. one) R^ substituents; wherein one of the R^ is: Ar^b, or phenyl or pyridinyl wherein the phenyl or pyridinyl independently are optionally substituted by one of fluoro, chloro, Ci_2alkyl,

Cjfluoroalkyl, Ci_2alkoxy or Cifluoroalkoxy; and wherein Ar^b is a 5-membered

aromatic heterocyclic ring containing one O, S or NR^a in the 5-membered ring, wherein the 5-membered ring can optionally additionally contain one N atom, and wherein the heterocyclic ring is optionally substituted on a ring carbon atom by one of: Ci_2alkyl or Cifluoroalkyl.

In this "biaryl" embodiment, when one R^ is optionally substituted phenyl or pyridinyl, it can in particular be phenyl or pyridinyl independently optionally substituted at the 3- or 4-position (e.g. 3-position) by one of fluoro, chloro, C^alkyl, Cifluoroalkyl, Ci_2alkoxy or Cifluoroalkoxy (for example by one of chloro, Cjalkyl, Cifluoroalkyl or - -

/r°\ Ii

Cialkoxy). Wlien one R6 is Ar513 then it can for example be or

In this "biaryl" embodiment, preferably, in sub-formula (x), A is CR^, wherein the R^ at position A is: Ar^, or phenyl, pyridinyl or pyrimidinyl wherein the phenyl, pyridinyl or pyrimidinyl independently are optionally substituted by one or two of fluoro, chloro, Chalky!, Cifluoroalkyl, C^alkoxy or Cifluoroalkoxy.

In this "biaryl" embodiment, NR4R5 can for example be:

NH

HN (connecting nitrogen underlined).

In another alternative embodiment, R^ has the sub-formula (z), and one or preferably none of J, L, M or Q is CR^, and/or R^ is a hydrogen atom (H) or methyl. Preferably r is - Io -

1. Preferably, for (z), R.6 is independently OH (including any keto tautomer thereof), or more preferably Ci_2alkyl (e.g. methyl) or Cifluoroalkyl.

In a preferred embodiment, the compound of formula (I) or the salt thereof is:

4-{[l-(aminocarbonyl)-4-piperidinyl]amino} -N- [(2,4-dimethylphenyl)methyl] -1-ethyl- lH-pyrazolo[3,4-b]pyridine-5-carboxamide,

4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4-dimeth ylphenyl)methyl]-l-ethyl- lH-pyrazolo[3,4-b]pyridine-5-carboxamide,

4-{ [1-(aminocarbonyl)-4-piperidinyl] amino} - 1-ethyl-N-{ [4-(methyloxy)phenyl]methyl}- 1H-pyrazolo[3 ,4-b]pyridine-5 -carboxamide, 4-{ [4-(aminocarbonyl)cyclohexyl] amino } -N- [(3 ,4-dimethylphenyl)methyl]- 1 -ethyl- IH- pyrazolo[3 ,4-δ]pyridine-5-carboxamide, 4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4-dimethylpheny l)methyl]-l-ethyl-lH- pyrazolo[3 ,4-δ]pyridine-5 -carboxamide, 4-{ [cz5r-4-(aminocarbonyl)cyclohexyl] amino} -N-[(3 ,4-dimethylphenyl)methyl]- 1-ethyl- lH-pyrazolo[3 ,4-δ]pyridine-5-carboxamide,

4-{[l-(aminocarbonyl)-4-piperidinyl]ammo}-N-[(2,4-difluor ophenyl)methyl]-l-ethyl- 1Η-pyrazolo[3 ,4-b]pyridine-5 -carboxamide, 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino }- 1 -ethyl-N- { [4- (trifluoromethyl)phenyl]methyl}-lΗ-pyrazolo[3,4-b]pyridine- 5-carboxamide, 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino }- 1 -ethyl-N- [(4-hydroxyphenyl)methyl]- 1 H- pyrazolo[3 ,4-b]pyridine-5 -carboxamide, 4- { [1-(aminocarbonyl)-4-piperidinyl] amino} - 1-ethyl-N-(phenylmethyl)-lH-pyrazolo [3 ,4- b]pyridine-5-carboxamide, 4-{ [1-(aminocarbonyl)-4-piperidinyl]amino}- 1 -ethyl-N- {[2- (methylsulfonyl)phenyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5- carboxamide, 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino} - 1-ethyl-N-[(3-hydroxyphenyl)methyl]- 1 H- pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2,3-dichlorop henyl)methyl]-l-ethyl- lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{[l-(ammocarbonyl)-4-piperidinyl]amino}-N-[(2,3-dimethylph enyl)methyl]-l-ethyl- lH-ρyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino} -N-(2-biphenylyhτiethyl)- 1 -ethyl- IH- pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-N-[(4-chloro-2-fl uorophenyl)methyl]-l- ethyl-lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{[l-(aminocarbonyl)-4-piperidmyl]amino} -N-({4- [(difluoromethyl)oxy]ρhenyl}methyl)-l-ethyl-lH-pyrazolo[3,4 -b]pyridme-5- carboxamide, 4-{[l-(aminocarbonyl)-4-piperidmyl]amino}-N-[(4-chloro-2-met hylphenyl)methyl]-l- ethyl-lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino }-N-[(4'-chloro-4-biphenylyl)methyl]- 1-ethyl- 1H-pyrazolo[3 ,4-b]pyridine-5 -carboxamide, 4- { [1-(aminocarbonyl)-4-piperidinyl] amino }- 1-ethyl-N-{ [4- (methylsulfonyl)phenyl]methyl}- 1 H-pyrazolo[3 ,4-b]pyridine-5-carboxamide, 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino }- 1 -ethyl-N- { [4'-(trifluoromethyl)-2- biphenylyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [1-(ammocarbonyl)-4-piperidinyl] amino }-N-[(4-chlorophenyl)methyl]- 1 -ethyl- 1 H- pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino} -N-(4-biphenylylmethyl)- 1 -ethyl- IH- pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{[1-(aminocarbonyl)-4-piperidinyl] amino} - 1 -ethyl-N- { [4'-(ethyloxy)-4- biphenylyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-N-(3-biphenylylme thyl)-l-ethyl-lH- pyrazolo[3 ,4-b]pyridine-5 -carboxamide, 4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1 -ethyl-N- { [4'-(trifluoromethyl)-4- biphenylyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino} - 1-ethyl-N-[(3'-methyl-2- biphenylyl)methyl]-lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino }- 1 -ethyl-N- { [3'-(methyloxy)-2- biphenylyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4- { [1-(aminocarbonyl)-4-piperidinyl] amino} -N-[(3'-chloro-2-biphenylyl)methyl]- 1-ethyl- lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino}-N-[(4'-chloro-2-biphenylyl)methyl]- 1-ethyl- 1 H-pyrazolo [3 ,4-b]pyridine-5 -carboxamide, 4-{[1-(aminocarbonyl)-4-piperidinyl]amino}- 1-ethyl-N-[(4'-methyl-2- biphenylyl)methyl]- 1 H-pyrazolo [3 ,4-b]pyridine-5 -carboxamide, 4- { [1-(aminocarbonyl)-4-piperidinyl] amino} - 1 -ethyl-N- {[4'-(methyloxy)-2- biphenylyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino} - 1 -ethyl-N- { [2-(3-furanyl)phenyl]methyl}- 1 H-pyrazolo [3 ,4-b]pyridine-5 -carboxamide, 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino} - 1-ethyl-N-({2-[6-(methyloxy)-3- pyridinyl]phenyl}methyl)-lH-pyrazolo[3,4-b]pyridine-5-carbox amide, 4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1 -ethyl-N- {[2-(3-thienyl)phenyl]methyl}- lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino }- 1 -ethyl-N- { [2-(4-methyl-3- thienyl)phenyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carboxam ide, 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino }- 1-ethyl-N-[(5-methyl-2-biphenylyl)methyl]- lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{[1-(aminocarbonyl)-4-piperidinyl] amino} - 1 -ethyl-N- { [4-methyl-2-(3- thienyl)phenyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carboxam ide, 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino }- 1 -ethyl-N- { [2-(3-furanyl)-4- methylphenyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carboxamid e, 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3'-chloro-5-m ethyl-2- biphenylyl)methyl]-l-ethyl-lH-pyrazolo[3,4-b]pyridine-5-carb oxamide, 4-{ [czs-4-(arninocarbonyl)cyclohexyl] amino }-N-[(2,4-dimethylphenyl)methyl]- 1-ethyl- lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{[trα7Z5-4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4-dimet hylphenyl)methyl]-l- ethyl-lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [cw-4-(aminocarbonyl)cyclohexyl] amino }- 1 -ethyl-N- { [4- (methyloxy)phenyl]methyl}-lH-pyrazolo[3,4-b]pyridme-5-carbox amide, 4-{[4-(aminocarbonyl)cyclohexyl]amino}-l-ethyl-N-{[4- (methylsulfonyl)phenyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5- carboxamide, 4-{ [4-(aminocarbonyl)cyclohexyl] amino }-N-[(2,3-dimethylphenyl)methyl]- 1 -ethyl- 1 H- pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [4-(aminocarbonyl)cyclohexyl] amino} -N-(2-biphenylylmethyl)- 1 -ethyl- 1 H- pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [4-(aminocarbonyl)cyclohexyl] amino }-N-[(4-chloro-2-fluorophenyl)methyl]- 1-ethyl- lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,3-dichloropheny l)methyl]-l-ethyl-lH- pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [4-(aminocarbonyl)cyclohexyl] amino} - 1 -ethyl-N- { [2- (methylsulfonyl)phenyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5- carboxamide, 4-{[4-(ammocarbonyl)cyclohexyl]amino}-N-[(4-chlorophenyl)met hyl]-l-ethyl-lH- pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [4-(aminocarbonyl)cyclohexyl] amino} - 1 -ethyl-N- { [4-(methyloxy)phenyl]methyl}- 1H-pyrazolo[3 ,4-b]pyridine-5-carboxamide, 4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-(4-biphenylyhnethyl )-l-ethyl-lH- pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [4-(aminocarbonyl)cyclohexyl] amino} - 1-ethyl-N-[(4-hydroxyphenyl)methyl]- 1 H- pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [4-(aminocarbonyl)cyclohexyl] amino }- 1-ethyl-N-(phenylmethyl)- 1 H-pyrazolo [3,4- b]pyridine-5-carboxamide, 4-{ [4-(aminocarbonyl)cyclohexyl] amino }-N-[(2,4-difluorophenyl)methyl]- 1 -ethyl- 1 H- pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-({4-[(difluoromethy l)oxy]phenyl}methyl)- l-ethyl-lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-(3-biphenylylmethyl )-l-ethyl-lH- pyrazolo[3 ,4-b]pyridine- 5 -carboxamide, 4-{[4-(aminocarbonyl)cyclohexyl]amino}-l-ethyl-N-[(4-fluorop henyl)methyl]-lH- pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{[4-(aminocarbonyl)cyclohexyl]aniino}-N-[(4-cliloro-2-meth ylphenyl)methyl]-l-ethyl- lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(4'-chloro-4-bipli enylyl)methyl]-l-etliyl- lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [4-(aminocarbonyl)cyclohexyl] amino} -N-{ [2,3-dichloro-6- (hydroxyiϊiethyl)plienyl]methyl}-l-ethyl-lH-pyrazolo[3,4-b] pyridine-5-carboxamide, 4-{ [4-(aminocarbonyl)cyclohexyl] amino} -N-{ [3 ,4-bis(methyloxy)phenyl]methyl}- 1- ethyl- lH-pyrazolo[3 ,4-b]pyridine-5-carboxamide, 4-{ [(3S)- 1-(aminocarbonyl)-3-pyrrolidinyl] amino }-N-[(2,4-dimethylphenyl)methyl]- 1- ethyl-lH-pyrazolo[3,4-b]pyridine-5-carboxamide, or 4-{ [(3S)- 1-(aminocarbonyl)-3-pyrrolidinyl] amino} -N-[(3 ,4-dimethylphenyl)methyl]- 1- ethyl-lH-pyrazolo[3,4-b]pyridine-5-carboxamide;

or a salt thereof, e.g. a pharmaceutically acceptable salt thereof.

According to one embodiment of the invention, the compound of formula (I) or the salt thereof is not 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4- dimethylphenyl)methyl]-l-ethyl-lH-pyrazolo[3,4-b]pyridine-5- carboxamide or a salt thereof.

A further aspect of the invention provides a compound of formula (IC) or a salt thereof (in particular, a pharmaceutically acceptable salt thereof):

3c .R" HN' O

NHR5c

<IC)

wherein: Rlc is ethyl or C2fluoroalkyl (preferably ethyl);

R2° is a hydrogen atom (H) or methyl (e.g. H);

NHR3C is a [l-(aminocarbonyl)-4-piperidinyl]amino group, a cw-[4-(aminocarbonyl)cyclohexan-l-yl]amino group (including mixtures of configurations wherein the stated cis configuration is the major component), or a racemic [4-(aminocarbonyl)cyclohexan-l-yl] amino group; andlMHR5c is of sub-formula (xxl), (xx2), (xx3) or (xx4):

NH

∏X1 X3V ∏X2

(xx1 ) (xx2) (xx3) (xx4)

wherein nxl and n*2 independently are 0 or 1, and Xj, X2, X3 and X4 independently are CH2 or O, provided that one or both ofXj and X2 are CH2, and provided that one or both of X3 and X4 are CB^-

NHR^c can for example be of sub-formula (xxl) or (xx2).

In one embodiment, in sub-formula (xxl), both ofX\ and X2 are CH2. hi one embodiment, in sub-formula (xx2), both ofX3 and X4 are CH2.

NHR^C can for example be: or

X O N NH,

R3° is preferably . That is, preferably NHR^ c is a [l-(aminocarbonyl)-4-piperidinyl] amino group.

The compound of formula (IC) or the salt thereof can be for example:

4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-N-(2,3-dihydro -l-benzofuran-5-ylmethyl)- l-ethyl-lH-pyrazolo[3,4-b]pyridine-5-carboxamide, 4-{ [4-(aminocarbonyl)cyclohexyl] amino} - 1-ethyl-N-(5 ,6,7,8-tetrahydro- 1- naphthalenylmethyl)-lH-pyrazolo[3,4-b]pyridine-5-carboxamide , or 4-{ [4-(aminocarbonyl)cyclohexyl] amino} -N-(2,3-dihydro- 1-benzofuran-5-ylmethyl)- 1- ethyl- 1H-pyrazolo[3 ,4-b]pyridine-5-carboxamide;

or a salt thereof, for example a pharmaceutically acceptable salt thereof. Salts, solvates, isomers, tautomeric forms, molecular weights, etc.

Because of their potential use in medicine, the salts of the compounds of formula (I) are preferably pharmaceutically acceptable. Suitable pharmaceutically acceptable salts may include acid or (where acidic groups are present within formula (I)) base addition salts.

A pharmaceutically acceptable acid addition salt is optionally formed by combination of a compound of formula (T) with a suitable inorganic or organic acid (such as hydrobromic, hydrochloric, sulfuric, nitric, phosphoric, succinic, maleic, formic, acetic, propionic, fumaric, citric, tartaric, lactic, benzoic, salicylic, glutamaic, aspartic, p-toluenesulfonic, benzenesulfonic, methanesulfonic, ethanesulfonic, naphthalenesulfonic such as 2- naphthalenesulfonic, or hexanoic acid), optionally in a suitable solvent such as an organic solvent, to give the salt which is usually isolated, for example by crystallisation and filtration.

A pharmaceutically acceptable acid addition salt of a compound of formula (I) optionally comprises or is for example a hydrobromide, hydrochloride, sulfate, nitrate, phosphate, succinate, maleate, formate, acetate, propionate, fumarate, citrate, tartrate, lactate, benzoate, salicylate, glutamate, aspartate, p-toluenesulfonate, benzenesulfonate, methanesulfonate, ethanesulfonate, naphthalenesulfonate (e.g. 2- naphthalenesulfonate) or hexanoate salt. A pharmaceutically acceptable acid addition salt of a compound of formula (I) can in particular comprise or be a hydrobromide, hydrochloride, sulfate, nitrate, phosphate, p-toluenesulfonate, benzenesulfonate, methanesulfonate, ethanesulfonate or naphthalenesulfonate (e.g. 2- naphthalenesulfonate) salt.

Where acidic groups are present within formula (I), a pharmaceutically acceptable base addition salt can be formed by reaction of a compound of formula (I) with a suitable inorganic or organic base (e.g. triethylamine, ethanolamine, triethanolamine, choline, arginine, lysine or histidine), optionally in a suitable solvent such as an organic solvent, to give the base addition salt which is usually isolated, for example by crystallisation and filtration.

Where acidic groups are present within formula (I), other pharmaceutically acceptable salts include pharmaceutically acceptable metal salts, for example pharmaceutically acceptable alkali-metal or alkaline-earth-metal salts such as sodium, potassium, calcium or magnesium salts; in particular pharmaceutically acceptable metal salts of one or more carboxylic acid moieties that may be present in the the compound of formula (I).

Other non-pharmaceutically acceptable salts, eg. oxalates, are in one less preferable embodiment used, for example in the isolation of compounds of the invention, and are included within the scope of this invention. The invention includes within its scope all possible stoichiometric and non-stoichiometric forms of the salts of the compounds of formula (I).

Also included within the scope of the invention are all solvates, hydrates and complexes of compounds and salts of the invention.

Certain groups, substituents, compounds or salts included in the present invention may be present as isomers. The present invention includes within its scope all such isomers, including racemates, enantiomers and mixtures thereof.

In the compounds or salts, pharmaceutical compositions, uses, methods of treatment/prophylaxis, methods of preparing, etc. according to the present invention, where a defined isomeric configuration e.g. stereochemical configuration is described or claimed, the invention includes a mixture comprising (a) a major component of the compound or salt which is in the described or claimed configuration, together with (b) one or more minor components of the compound or salt which is/are not in the described or claimed configuration. Preferably, in such a mixture, the major component of the compound or salt which is in the described or claimed configuration represents 70% or more, or 75% or more, more preferably 85% or more, still more preferably 90% or more, yet more preferably 95% or more, yet more preferably 98% or more, of the total amount of compound or salt present in the mixture on a molarity basis.

Certain of the groups, e.g. heteroaromatic ring systems, included in compounds of formula (I) or their salts may exist in one or more tautomeric forms. The present invention includes within its scope all such tautomeric forms, including mixtures.

Especially when intended for oral or external topical medicinal use, compound(s) of formula (I) can have a molecular weight of 1000 or less, for example 800 or less, in particular 650 or less or 600 or less. Molecular weight here refers to that of the unsolvated "free base" compound, that is excluding any molecular weight contributed by any addition salts, solvent (e.g. water) molecules, etc.

Synthetic Process Routes

The following processes can be used to make the compounds of formula (I) as hereinbefore defined. Most of the following synthetic processes are exemplified for compounds of Formula (I) wherein R.2 is a hydrogen atom (H). However, some or all of these processes can also be used with appropriate modification, e.g. of starting materials and reagents, for making compounds of Formula (I) wherein R.2 is methyl.

Process A - -

To form a compound of formula (I), a carboxylic acid of formula (II) can be converted into an activated compound of formula (III) wherein X^ = a leaving group substitutable by an amine (as defined below) and subsequently the activated compound can be reacted with an amine of formula NHR^R^:

HN

For example, the activated compound (the compound of formula (III)) can be the acid chloride. This can be formed from the carboxylic acid (II) e.g. by reaction with thionyl chloride, either in an organic solvent such as chloroform or without solvent. Alternatively, the activated compound (the compound of formula (III)) can be an activated ester wherein the leaving group X^ is

.N X2 = CH or N

The latter activated compound of formula (I∏) can be formed from the carboxylic acid (II) either: (a) by reaction of the carboxylic acid with a carbodiimide such as EDC (l-ethyl-3- (3'-dimethylaminopropyl)carbodiimide), or a salt thereof e.g. hydrochloride salt, preferably followed by reaction of the resulting product with 1-hydroxybenzotriazole (HOBT); reaction (a) being carried out usually in the presence of a solvent (e.g. anhydrous) such as dimethyl formamide (DMF) or acetonitrile, and/or usually in the presence of a base, e.g. tertiary organic amine base, such as diisopropylethylamine (ip^NEt = DIPEA), and/or usually at room temperature (e.g. about 20 to about 25 0C), and/or for example under anhydrous conditions; or: (b) by reaction with 2-(lH-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate (TBTU) or O-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) ,in the presence of a base, e.g. tertiary organic amine base, such as diisopropylethylamine (ip^NEt = DIPEA), and usually in the presence of a solvent such as dimethyl formamide (DMF) or acetonitrile and/or for example under anhydrous conditions and/or usually at room temperature (e.g. about 20 to about 25 0C). - -

Compounds of formula (II) can be prepared by hydrolysis of an ester of formula (IV) , wherein R7 is alkyl such as C^alkyl e.g. methyl or ethyl. This procedure can for example involve reaction of (IV) with either: (a) a base, e.g. alkali metal hydroxide such as sodium hydroxide, potassium hydroxide or lithium hydroxide, in a solvent e.g. an aqueous solvent (e.g. aqueous solvent comprising a mixture of water and a water-miscible organic solvent) such as aqueous ethanol or aqueous dioxane or (b) an acid such as hydrochloric acid in a solvent e.g. an aqueous solvent such as aqueous dioxane:

,R3 HN' O NaOH / EtOH N or HCI / Dioxane N

R M1

( IV ) ( H )

The invention also provides in one aspect a compound of formula (II), and in another aspect a compound of formula (IV), as defined herein.

When R3 is the N-aminocarbonyl-piperidinyl or -pyrrolidinyl group of sub-formula (bb), the compound of formula (IV) can be prepared by reacting a compound of formula (FVa) or a salt thereof (e.g. hydrochloride salt thereof) with a urea-forming reagent capable of converting the (4-piperidinyl)amino or (3-pyrrolidinyl)amino group in the compound of formula (IVa) into a [(l-aminocarbonyl)-4-piperidinyl]amino group or [(1- aminocarbonyl)-3 -pyrrolidinyl] amino group respectively:

(IVa)

The urea-forming reagent may be benzyl isocyanate (followed later by debenzylation e.g. reductive debenzylation), or preferably the urea-forming reagent is tri(Ci_4alkyl)silyl isocyanate such as a tri(Ci_2alkyl)silyl isocyanate, preferably trimethylsilyl isocyanate. The reaction of the compound (IVa) or salt thereof to the compound. (IV) can be carried out in the presence of a base such as N,N-diisopropylethylamine (ip^NEt = DIPEA), for example in more than one mole equivalent compared to the number of moles of (PVa) or salt. The reaction is optionally carried out at room temperature or by heating to reflux.

The reaction of the compound (PVa) or salt thereof to compound (PV) can be carried out in an organic solvent, the solvent preferably not being an aqueous-organic solvent system or mixture. The organic solvent can optionally be tetrahydrofuran (TPPF). However, although THF appears at first sight to work satisfactorily on an about 4g scale (see Intermediate 4, n1 = 1, R7 = ethyl, HCl salt of (PVa)), it appears at first sight that the yield might decrease when the reaction scaled up e.g. to about 33.5g (see first half of Intermediate 4A, n1 = 1, R7 = ethyl, HCl salt of (PVa)). It seems that the solubility of the HCl salt of (PVa) where n1 = 1, R7 = ethyl in THF is limited. Therefore, preferably the reaction of compound (IVa) or salt to compound (PV) is carried out in a solubilising organic solvent comprising (e.g. consisting essentially of or being) an organic solvent capable of dissolving the compound of formula (IVa) or salt thereof (whichever is used) to a substantially greater extent than THF. For example, the solubilising organic solvent can be dichloromethane or (probably) chloroform. Dichloromethane appears to work satisfactorily on a scale of about 33.5g, for n* = 1, R7 = ethyl and using the HCl salt of compound (PVa) (see e.g. second half of Intermediate 4A). (See also Intermediate 4B). In the reaction, the compound (PVa) or salt thereof is preferably substantially wholly in solution, rather than being at least partly in suspension, in the organic solvent.

Compound (PVa) or the salt thereof can be prepared from compound (PVb), wherein Prot is a nitrogen protecting group such as (tert-butyloxy)carbonyl (Boc), by deprotection of the nitrogen protecting group. Boc removal can be effected by suitable acidic conditions, such as hydrogen chloride (e.g. 4M) in 1,4-dioxane:

Protv "N )„. Hipw∏i NH O ^\lH

(IVb) (IVa)

Compound (PVb), wherein R7 is ethyl and Prot is Boc, can be prepared according to a method, for example as described in Intermediate 2 or 2A herein, by reaction of a compound of formula (V) (illustrated below wherein R7 = ethyl) with 1,1-dimethylethyl 4-amino-l-piperidinecarboxylate (e.g. commercially available from AstaTech, Philadelphia, USA) or 1,1-dimethylethyl 3-amino-l-pyrrolidinecarboxylate (e.g. commercially available from Aldrich). The reaction is optionally carried out in the - -

presence of a base such as triethylamine or N,N-diisopropylethylamine (DIPEA), and/or in an organic solvent such as acetonitrile. The reaction may require heating e.g. to ca. 60- 1000C (e.g. ca. 80-900C), for example for about 16-18 hours:

Protv

Cl O ^NH Q

OR' N 'N

(V) (IVb) (R7 = ethyl) (in this case, R7 is Et, and Prat is Boc)

For one preparation of the compound of formula (V) wherein R7 is ethyl, see e.g. Intermediate 1 herein, and/or see Scheme 1 and compound 12 in G. Yu et. al., J. Med Chem., 2001, 44, 1025-1027. Therefore, a compound of formula (V) can be prepared by reaction of a compound of formula (VI) with, for example, diethylethoxymethylene malonate (where R^ = Et) with heating, followed by reaction with phosphorous oxychloride, again with heating (see for example Intermediate 1 hereinafter):

R7\ 1) o-co ,CO2R' Cl

Y OEt N N NH9 2) POCI3

R ■>1

( Vl ) ( V )

Where the desired amino pyrazole of formula (VI) is not commercially available, preparation can be achieved using methods described by Dorgan et. al. in J. Chem. Soc, Perkin Trans. 1, (4), 938-42; 1980, by reaction of cyanoethylhydrazine with a suitable aldehyde of formula R^OCHO in a solvent such as ethanol, with heating, followed by reduction with, for example sodium in a solvent such as t-butanol. R^O should be chosen so as to contain one less carbon atom than Rl, for example R^O = methyl will afford Rl = ethyl. I) R40CHO EtOH H2N' .W. ^CN /N ^NH2 2) Na / t-BuOH r-1

( Vl )

According to one alternative optional embodiment of Process A, a compound of formula (IV), wherein R7 is alkyl such as Ci_4alkyl e.g. methyl or ethyl, is optionally prepared according to a method, for example as described in Scheme 1 of Yu et. al., J. Med Chem., 2001, 44, 1025-1027, by reaction of a compound of formula (V) with an amine of formula R3NHz^. The reaction is optionally carried out in the presence of a base such as triethylamine or N,N-diisopropylethylarnine, and/or in an organic solvent such as ethanol, dioxane or acetonitrile. The reaction may require heating e.g. to ca. 60-1000C, for example ca. 80-900C:

R3NHc OR

(V) (IV)

In another alternative embodiment of Process A, the 4-chloro substituent in the compound of formula (V) can be replaced by a bromine or iodine atom, or by another suitable leaving group which is displaceable by an amine of formula R3NH2. The leaving group can, for example, be an alkoxy group -OR35 such as -OC1-4alkyl (in particular - OEt) or a group -O-S(O)2-R37, wherein R37 is Ci_galkyl (e.g. Cj^alkyl or Ci_2alkyl such as methyl), Ci.gfluoroalkyl (e.g. C^fluoroalkyl or Ci_2fhioroalkyl such as CF3 or C4F9), or phenyl wherein the phenyl is optionally substituted by one or two of independently Ci_2alkyl, halogen or Ci_2alkoxy (such as phenyl or 4-methyl-phenyl). The reaction may be carried out with or without solvent and may require heating.

Process B

Compounds of formula (I) can be prepared by reaction of a compound of formula (VII) with an amine of formula R3NH2 or a salt (e.g. HCl salt) thereof. The reaction is preferably carried out in the presence of a base, e.g. tertiary organic amine base, such as triethylamine or N,N-diisopropylethylamine (DIPEA), and/or in an organic solvent such as ethanol, tetrahydrofuran (THF), dioxane or acetonitrile. The reaction may require heating, e.g. to ca. 60-100 0C or ca. 80-90 0C, for example for 8-72 or 12-48 or 24-48 hours:

Cl O

R3NH, NR4R5 NR4R5

N' ~R"

( VII ) ( I )

For an example of a preparation of HCl salts of some amines of formula R^NH2, see e.g. Intermediates 12, 15, 19 and 20 herein.

Compounds of formula (VII) can be prepared in a two step procedure (e.g. see Bare et. al. inJ. Med. Chem. 1989, 32, 2561-2573). This process involves, first, reaction of a compound of formula (VIII) with thionyl chloride (or another agent suitable for forming an acid chloride from a carboxylic acid), either in an organic solvent such as chloroform or THF, or as a neat solution, preferably under substantially anhydrous conditions (e.g. under a nitrogen or argon atmosphere). This reaction may require heating (e.g. to reflux) and the thus-formed acid chloride intermediate may or may not be isolated. Step two involves reaction of the resulting acid chloride intermediate with an amine of formula R4R5NH, in an organic solvent such as THF or chloroform and may also involve the use of a base such as triethylamine or diisopropylethylamine (DIPEA), See, for example, Intermediate 7 (first step) and Intermediates 8, 9, and 10 (second step) herein:

Cl O I ) SOCI2 '/ NR4R5 N 2) NHR4R5 M N- r^2 N' "Ff

R L

( VIII ) ( VII )

Compounds of formula (VIII) can be prepared by hydrolysis of an ester of formula (V) according to the method described by Yu et. al. in J. Med Chem., 2001, 44, 1025-1027. This procedure preferably involves reaction with a base such as sodium hydroxide or potassium hydroxide in a solvent e.g. an aqueous solvent such as aqueous ethanol or aqueous dioxane:

Cl O Cl O NaOH EtOH

N-^N^R2

R /,

( VIII ) ( V )

In an alternative embodiment of Process B, the 4-chloro substituent in the compound of formula (VII) can be replaced by a bromine or iodine atom.

Process C: Conversion of one compound offormula (I) or salt thereof into another compound of formula (I) or salt thereof

One compound of formula (I) or salt thereof can be converted into another compound of formula (I) or salt thereof. This conversion can for example comprise or be one or more of the following processes Cl to C6 or C7:

Cl. An oxidation process. For example, the oxidation process can comprise or be oxidation of an alcohol to a ketone (e.g. using Jones reagent) or oxidation of an alcohol or a ketone to a carboxylic acid.

C2. A reduction process, for example reduction of a ketone or a carboxylic acid to an alcohol.

C3. Alkylation, for example alkylation of an amine or of a hydroxy group.

C4. Hydrolysis, e.g. hydrolysis of an ester to the corresponding carboxylic acid or salt thereof.

C5. Deprotection, e.g. deprotection (e.g. deacylation or t-butyloxycarbonyl (BOC) removal) of an amine group.

C6. Formation of an ester or amide, for example from the corresponding carboxylic acid.

Cl. A carbon-carbon bond coupling process, for example conversion of compounds of formula (Ib) to compounds of formula (Ia), for example as follows. Compounds of formula (I) which are of formula (Ia) are optionally prepared by reaction of a compound of formula (Ib), where HaI^ is a chlorine, bromine or iodine atom (preferably a bromine or iodine atom), with an aryl boronic acid (IX) or the corresponding boronate ester. The reaction can for example use a catalyst such as palladium tetrakis(triphenylphosphine) and a base such as sodium carbonate, in one or more solvents such as a mixture of dimethylformamide and water. The reaction may require heating, e.g. to about 15O0C, and the rate of reaction may perhaps be further enhanced by the use of a microwave reactor.

RV ArB-B(OH)2 NH O (IX)

( Ib ) ( Ia )

In this reaction of (Ib) to (Ia), Ar^ is typically one R^ which is: Ar^b, or phenyl or pyridinyl wherein the phenyl or pyridinyl independently are optionally substituted by one of fluoro, chloro, C^alkyl, Cifluoroalkyl, C^alkoxy or Cifluoroalkoxy; and wherein Ar^b is a 5-membered aromatic heterocyclic ring containing one O, S or NR^a in the 5- membered ring, wherein the 5-membered ring can optionally additionally contain one N atom, and wherein the heterocyclic ring is optionally substituted on a ring carbon atom by one of: C^alkyl or Cifluoroalkyl.

The present invention therefore also provides a method of preparing a compound of formula (I) or a salt thereof:

NR4R5 (I) N-N^R2

R1 wherein Rl, R^, R3, R4 and R5 are as defined herein, the method comprising :

(a) converting a compound of formula (ILA) into an activated compound of formula (IIIA) wherein χl = a leaving group substitutable by an amine: FT FT

(HA) 1^ (INA) and subsequent reaction of the activated compound of formula (IIIA) with an amine of formula R4R5NH; or

(b) reacting a compound of formula (VIIA) : Hal O

NR4R5

Rf

(VIIA) wherein Hal is a chlorine, bromine or iodine atom (such as a bromine atom or preferably a chlorine atom), with an amine of formula R^NH2 or a salt (e.g. HCl salt) thereof, or

(c) converting one compound of formula (I) or salt thereof into another compound of formula (I) or salt thereof (e.g. by a carbon-carbon bond coupling process, e.g. as described herein);

and, in the case of (a), (b) or (c), optionally converting the compound of formula (I) into a salt thereof e.g. a pharmaceutically acceptable salt thereof.

The optional or preferable features of steps (a), (b), (c) can be according to Processes A, B or C mentioned above.

The present invention also provides: (g) a method of preparing a pharmaceutically acceptable salt of a compound of formula (I) comprising conversion of the compound of formula (I) or a salt thereof into the desired pharmaceutically acceptable salt thereof.

The present invention also provides a compound of formula (I) or a salt thereof, prepared by a method as defined herein. Medical uses

The present invention also provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use as an active therapeutic substance in a mammal such as a human. The compound or salt can be for use in the treatment and/or prophylaxis of any of the diseases / conditions described herein (e.g. for use in the treatment and/or prophylaxis of an inflammatory and/or allergic disease in a mammal such as a human; or e.g. for use in the treatment and/or prophylaxis of cognitive impairment or depression in a mammal such as a human) and/or can be for use as a phosphodiesterase 4 (PDE4) inhibitor. "Therapy" may include treatment and/or prophylaxis.

The compound or salt can for example be for use in the treatment and/or prophylaxis of an inflammatory and/or allergic skin disease, such as atopic dermatitis or psoriasis, in a mammal such as a human.

Also provided is the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament (e.g. pharmaceutical composition) for the treatment and/or prophylaxis of any of the diseases / conditions described herein in a mammal such as a human, e.g. for the treatment and/or prophylaxis of an inflammatory and/or allergic disease in a mammal such as a human, or e.g. for the treatment and/or prophylaxis of cognitive impairment or depression in a mammal.

Also provided is a method of treatment and/or prophylaxis of any of the diseases / conditions described herein in a mammal (e.g. human) in need thereof, e.g. a method of treatment and/or prophylaxis of an inflammatory and/or allergic disease, cognitive impairment or depression in a mammal (e.g. human) in need thereof, which method comprises administering to the mammal (e.g. human) a therapeutically effective amount of a compound of formula (I) as herein defined or a pharmaceutically acceptable salt thereof.

Phosphodiesterase 4 inhibitors are thought to be useful in the treatment and/or prophylaxis of a variety of diseases / conditions, especially inflammatory and/or allergic diseases, in mammals such as humans, for example: asthma, chronic obstructive pulmonary disease (COPD) (e.g. chronic bronchitis and/or emphysema), atopic dermatitis, urticaria, allergic rhinitis, allergic conjunctivitis, vernal conjunctivitis, eosinophilic granuloma, psoriasis, rheumatoid arthritis, septic shock, ulcerative colitis, Crohn's disease, reperfusion injury of the myocardium and brain, chronic glomerulonephritis, endotoxic shock, adult respiratory distress syndrome, multiple sclerosis, cognitive impairment (e.g. in a neurological disorder such as Alzheimer's disease), depression, or pain (e.g. inflammatory pain). Ulcerative colitis and/or Crohn's disease are collectively often referred to as inflammatory bowel disease. In the treatment and/or prophylaxis, the inflammatory and/or allergic disease is preferably chronic obstructive pulmonary disease (COPD), asthma, rheumatoid arthritis, allergic rhinitis, psoriasis or atopic dermatitis in a mammal (e.g. human). More preferably, the treatment and/or prophylaxis is of COPD, asthma, psoriasis or atopic dermatitis in a mammal (e.g. human).

Most preferably, the treatment and/or prophylaxis is of atopic dermatitis in a mammal such as a human or pig, preferably in a human, in particular in a human aged 21 years or less, e.g. 18 years or less. For treatment and/or prophylaxis of atopic dermatitis in a mammal, external topical administration to the mammal of the compound of formula (I) or a pharmaceutically acceptable salt thereof (e.g. topical administration to the skin e,g. to skin affected by the atopic dermatitis) is preferably used. For treatment and/or prophylaxis of atopic dermatitis, inhaled administration is usually not suitable.

"Atopic dermatitis" has been proposed to include two general sub-classes: (1) an "allergic (extrinsic)" type of atopic dermatitis which generally occurs in the context of sensitization to environmental allergens and/or which is generally accompanied by elevated serum IgE levels; and (2) an "non-allergic (intrinsic)" type of atopic dermatitis generally with little or no detectable sensitization and/or generally with normal or low serum IgE levels (N. Novak et al, J Allergy Clin. Immunol, 2003, 112, 252-262; and T.C. Roos et al., Drugs, 2004, 64(23), 2639-2666, see e.g. pages 2640-2641). The compound of formula (I) or the pharmaceutically acceptable salt thereof can therefore be for the treatment and/or prophylaxis of allergic (extrinsic) atopic dermatitis and/or non- allergic (intrinsic) atopic dermatitis in a mammal (e.g. human or pig, preferably human).

"External topical" administration means topical administration to an external body part (i.e. excluding, for example, the lung or mouth, but including the lips), preferably excluding the eye.

"External topical" administration preferably is topical administration to the skin, for example to the skin of an arm, hand, leg, foot, head (e.g. face), neck and/or torso of a mammal such as a human. External topical administration can for example be to those parts of a mammal's skin affected by or susceptible to atopic dermatitis.

For the use of PDE4 inhibitors in atopic dermatitis, see for example: - J.M. Hanifin et al., "Type 4 phosphodiesterase inhibitors have clinical and in vitro anti¬ inflammatory effects in atopic dermatitis", J. Invest. Dermatol, 1996, 107(1), 51-56; which reports reductions of inflammatory parameters in atopic dermatitis patients treated with PDE4 inhibitor CP80,633 (0.5% ointment, twice daily topical application); - C.E.M. Griffiths et al., "Randomized comparison of the type 4 phosphodiesterase inhibitor cipamfylline cream, cream vehicle and hydrocortisone 17-butyrate cream for the treatment of atopic dermatitis", Br. J. Dermatol, 2002, 147(2), 299-307, which reports that cipamfylline (0.15%) cream is significantly more effective than vehicle, but significantly less effective than hydrocortisone 17-butyrate (0.1%) cream, in the treatment of atopic dermatitis patients; - T.C. Roos et al., "Recent advances in treatment strategies for atopic dermatitis", Drugs, 2004, 64(23), 2639-2666 (see e.g. page 2657 and refs. 201-209 therein); - A.M.Doherty, Current Opinion Chem. Biol, 1999, 3(4), 466-473 (e.g. see p. 470); and - HJ.Dyke et al., Expert Opinion Invest. Drugs, 2002, 11(1), 1-13 (e.g. see p.7 and refs. 74, 75 and 76 cited therein); and references cited in the above references.

For the use of the PDE4 inhibitors SB 207499 (cilomilast) and AWD 12-281 in mouse models of the allergic type of dermatitis, see W. Baumer et al., Eur. J. Pharmacol, 2002, 446, 195-200 and W. Baumer et al., J. Pharmacy Pharmacol, 2003, 55, 1107-1114.

PDE4 inhibitors are thought to be effective in the treatment of COPD. For example, see SX. Wolda, Emerging Drugs, 2000, 5(3), 309-319; Z. Huang et al., Current Opinion in Chemical Biology, 2001, 5: 432-438; HJ.Dyke et al., Expert Opinion on Investigational Drugs, January 2002, 11(1), 1-13; C.Burnouf et al., Current Pharmaceutical Design, 2002, 8(14), 1255-1296; A.M.Doherty, Current Opinion Chem. Biol, 1999, 3(4), 466- 473; A.M. Vignola, Respiratory Medicine, 2004, 98, 495-503; D. Spina, Drugs, 2003, 63(23), 2575-2594; and references cited in the aforementioned publications; and G. Krishna et al., Expert Opinion on Investigational Drugs, 2004, 13(3), 255-267 (see especially pp. 259-261 and refs. 102-111 and 201 therein).

The PDE4 inhibitor cilomilast (Ariflo ™) at 15 mg orally twice daily appears to improve forced expiratory volume in Is (FEV1) in COPD patients (C.H.Compton et al., The Lancet, 2001, vol.358, 265-270), and appears to have antiinflammatory effects in COPD patients (E.Gamble et al., Am. J. Respir. Crit. Care Med., 2003, 168, 976-982). On cilomilast, see also R.D. Border et al., Chest, 2003, vol. 124 Suppl. 4, p.170S (abstract) and J.D. Eddleston et al., Am. J. Respir. Crit. Care Med., 2001, 163, A277 (abstract). The PDE4 inhibitor roflumilast appears to show small improvements in FEV1 in COPD patients (see BJ. Lipworth, The Lancet, 2005, 365, 167-175, andrefs 49-50 therein).

COPD is often characterised by the presence of airflow obstruction due to chronic bronchitis and/or emphysema (e.g., see S.L. Wolda, Emerging Drugs, 2000, 5(3), 309- 319).

PDE4 inhibitors are thought to be effective in the treatment of asthma (e.g. see M.A.Giembycz, Drugs, Feb. 2000, 59(2), 193-212; Z. Huang et al., Current Opinion in Chemical Biology, 2001, 5: 432-438; HJ.Dyke et al., Expert Opinion on Investigational Drugs, January 2002, 11(1), 1-13; C.Burnouf et al., Current Pharmaceutical Design, 2002, 8(14), 1255-1296; A.M.Doherty, Current Opinion Chem. Biol., 1999, 3(4), 466- 473; and references cited in the aforementioned publications).

PDE4 inhibitors are thought to be effective in the treatment of allergic rhinitis (e.g. see B.M. Schmidt et al., J. Allergy & Clinical Immunology, 108(4), 2001, 530-536).

PDE4 inhibitors are thought to be effective in the treatment of rheumatoid arthritis and multiple sclerosis (e.g. see HJ.Dyke et al., Expert Opinion on Investigational Drugs, January 2002, 11(1), 1-13; C.Burnouf et al., Current Pharmaceutical Design, 2002, 8(14), 1255-1296; and A.M.Doherty, Current Opinion Chem. Biol., 1999, 3(4), 466-473; and references cited in these publications).

PDE4 inhibitors have been suggested as having analgesic properties and thus being effective in the treatment of pain (A.Kumar et al., Indian J. Exp. Biol., 2000, 38(1), 26- 30).

In the invention, the treatment and/or prophylaxis can be of cognitive impairment e.g. cognitive impairment in a neurological disorder such as Alzheimer's disease. For example, the treatment and/or prophylaxis can comprise cognitive enhancement e.g. in a neurological disorder. See for example: H.T.Zhang et al. in: Psychopharmacology, June 2000, 150(3), 311-316 andNeuropsychopharmacology, 2000, 23(2), 198-204; and T. Egawa et al., Japanese J. Pharmacol, 1997, 75(3), 275-81.

PDE4 inhibitors such as rolipram have been suggested as having antidepressant properties (e.g. J. Zhu et al., CNSDrug Reviews, 2001, 7(4), 387-398; O'Donnell, Expert Opinion on Investigational Drugs, 2000, 9(3), 621-625; and H.T. Zhang et al., Neuropsychopharmacology, October 2002, 27(4), 587-595; J. M. O'Donnell and H.-T. Zhang, Trends Pharmacol. ScL, March 2004, 25(3), 158-163; and T.E.Renau, Curr. Opinion Invest. Drugs, 2004, 5(1), 34-39).

PDE4 inhibition has been suggested for the treatment of inflammatory bowel disease (e.g. ulcerative colitis and/or Crohn's disease), see K.H.Banner and M.A.Trevethick, Trends Pharmacol. ScL, August 2004, 25(8), 430-436.

Pharmaceutical compositions, routes of administration, and dosing

For use in medicine, the compounds or salts of the present invention are usually administered as a pharmaceutical composition. The present invention therefore provides in a further aspect a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers and/or excipients.

The pharmaceutical composition can be for use in the treatment and/or prophylaxis of any of the conditions described herein, in particular atopic dermatitis in a mammal such as a human.

The invention also provides a method of preparing a pharmaceutical composition comprising a compound of formula (I), as herein defined, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers and/or excipients, he method comprising mixing the compound or salt with the one or more pharmaceutically acceptable carriers and/or excipients.

The invention also provides a pharmaceutical composition prepared by said method.

The compounds of formula (I) and/or the pharmaceutical composition may be administered, for example, by external topical (e.g. skin topical), oral, parenteral (e.g. intravenous, subcutaneous, or intramuscular), inhaled or nasal administration.

Accordingly, the pharmaceutical composition can be suitable for (e.g. adapted for) external topical (e.g. skin topical), oral, parenteral (e.g. intravenous, subcutaneous, or intramuscular), inhaled or nasal administration. The pharmaceutical composition is preferably suitable for inhaled administration or more preferably is suitable for external topical (e.g. skin topical) administration, e.g. to a mammal such as a human. Inhaled administration involves topical administration to the lung e.g. by aerosol or dry powder composition.

Although the compound of formula (I) or salt thereof may be administered orally, oral administration is not presently thought to be a preferred route of administration, at least for the compound 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4- dimethylphenyl)methyl]-l-ethyl-lHr-pyrazolo[3,4-&]pyridine-5 -carboxamide ("free base" form) (e.g. Example 2, 2A or 2B). This is because, without intending to be bound by this data, preliminary tests appear to indicate a low systemic exposure after oral administration of Example 2 or 2A ("free base" form) to rat(s), at a dose level of about lmg of the compound per kg bodyweight, when formulated in approximately [10% DMSO and 90% PEG200/water (70:30 PEG200 : water ratio)].

The pharmaceutical composition can optionally be in unit dose form. The unit dose form can for example be: (a) a rupturable or peel-openable sealed dose container containing a dry powder inhalable pharmaceutical composition (e.g. a plurality of which are usually disposed inside a suitable inhalation device); (b) a vial, ampoule or filled syringe for parenteral administration e.g. comprising a solution or suspension of the compound or ^

pharmaceutically acceptable salt in a suitable carrier such as an aqueous carrier or e.g. containing a lyophilised parenteral pharmaceutical composition (the vial or ampoule can optionally be manufactured using a blow-fill-seal process); or (c) (less preferred) a tablet or capsule for oral administration e.g. for oral administration to a human.

Alternatively, the composition can be in a form adapted for the administration of varying amounts of composition as desired by the user, such as a spreadable or sprayable external topical composition such as a cream, an ointment, a gel, or a liquid.

Pharmaceutical compositions suitable for external topical administration

The pharmaceutical composition of the invention is preferably suitable for (e.g. adapted for) external topical (e.g. skin topical) administration, for example to a mammal such as a human. More preferably, the pharmaceutical composition suitable for external topical administration is for the treatment and/or prophylaxis of atopic dermatitis in a mammal such as a human.

"External topical administration" is defined above under the "medical uses" section. External topical administration can for example be to those parts of the skin affected by or susceptible to the disease or condition e.g. atopic dermatitis, in particular in a mammal (e.g. human) suffering from or susceptible to atopic dermatitis.

An external-topical pharmaceutical composition, e.g. skin topical pharmaceutical composition, can for example be an ointment, a cream (usually an oil-in-water or water- in-oil pharmaceutical composition, usually an emulsion), an aqueous gel, or a microemulsion. The pharmaceutical composition can alternatively be a DMSO- containing solution such as a DMSO/acetone solution or DMSO/water solution (DMSO = dimethyl sulfoxide); a DMSO-containing solution can be used for experimental animal tests, but is not usually desirable for use in humans.

In the external-topical pharmaceutical composition, , e.g. an ointment or an oil-in-water or water-in-oil composition, the compound of formula (I) or the pharmaceutically acceptable salt thereof is suitably present in 0.05% to 10%, preferably 0.1% to 5%, more preferably 0.1% to 3%, still more preferably 0.2% to 3% (e.g. about 0.5% or about 2.5%), yet more preferably 0.2% to 1.5% (e.g. about 0.5%), by weight of the composition (w/w).

In one optional embodiment, the compound of formula (I) or the pharmaceutically acceptable salt thereof can optionally be in a particle-size-reduced form, for example obtained or obtainable by micronisation. This can be, for example, for use in a pharmaceutical composition suitable for (e.g. adapted for) external topical (e.g. skin topical) administration. See the Particle size reduction sub-section below, within the Inhalable pharmaceutical compositions section, for more details. Aqueous solubility: A preliminary screen aims to estimate roughly the aqueous solubility of compounds by (as an approximate summary): (i) creating a ca. 1OmM solution of the compound in DMSO, (ii) diluting a portion of this DMSO solution by mixing about 19 parts by volume of pH 7.4 aqueous phosphate buffered saline (PBS) buffer with 1 part by volume of the ca. 1OmM DMSO solution, (iii) "filtering" the mixture with the aid of centrifugation, and then (iv) measuring the concentration of the dissolved compound in the "filtrate". Although some DMSO (about 5% by volume) is present in this solubility screen "filtrate", the results from this preliminary screen for the compound 4-{[l- (aminocarbonyl)-4-piperidinyl] amino}-N-[(3 ,4-dimethylphenyl)methyl]- 1 -ethyl- IH- pyrazolo[354-6]pyridme-5-carboxamide ("free base" form) (e.g. Example 2 or 2A) (about 60 micrograms of the compound / ml) appear to suggest generally that this particular Example 2 or 2A compound (in the "free base" form) has a generally moderate aqueous solubility at about room temperature.

Lipophilicity: The compound 4-{[l-(ammocarbonyl)-4-piperidinyl]amino}-N-[(3,4- dimethylphenyl)methyl]-l-ethyl-lH-pyrazolo[3,4-b]pyridine-5- carboxamide ("free base" form) (e.g. Example 2 or 2A) is thought to have a clogP (calculated log of the octanol/water partition coeficient (P)) of approximately 2.5, suggesting moderate lipopliilicity. This particular compound (as the free base) is believed to have a measured logD (D = distribution coefficient, wherein log D is generally log P corrected for ionization) of approximately 3.4 at pΗ = 7.4, again suggesting modest / moderate lipophilicity.

Therefore, preferably, in a pharmaceutical composition suitable for external topical (e.g. skin topical) administration, the compound of formula (I) or salt thereof is 4-{[l- (aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4-dimethylphenyl) methyl]-l-ethyl-lH- pyrazolo[3,4-δ]pyridine-5-carboxamide or a pharmaceutically acceptable salt thereof, in particular 4-{[l-(ammocarbonyl)-4-piperidinyl]amino}-N-[(3,4-dimethylph enyl)methyl]- l-ethyl-lH-pyrazolo[3,4-ό]pyridine-5-carboxamide ("free base" form).

Solubilising and/or skin-penetration-enhancing agents: An external-topical pharmaceutical composition, e.g. an ointment or an oil-in-water cream or water-in-oil cream, preferably includes an agent which acts as a skin-penetration enhancer for and/or a solubiliser of the compound of formula (I) or the salt thereof. The skin-penetration- enhancing- and/or solubilising- agent can for example be propylene glycol, diethylene glycol monoethyl ether (e.g. TRANSCUTOL ) and/or caprylocaproyl macrogolglycerides (e.g. LABRASOL ™), preferably propylene glycol. The solubiliser and/or skin-penetration enhancer suitably does not comprise DMSO. The solubiliser and/or skin-penetration enhancer is preferably both a solubiliser and skin-penetration enhancer, and/or is suitably present in 0.5% to 50%, preferably 5% to 50%, more preferably 7% to 30%, still more preferably 7% to 25%, yet more preferably about 10% to about 20% (e.g. about 10% or about 20%), by weight of the composition (w/w). The skin-penetration enhancer is for delivery of the compound of formula (I) or salt thereof ("active agent" or "drug") through the skin. Solubilization of the drug also helps. The solubilising and/or skin-penetration-enhancing agents should ideally (a) be safe and/or tolerable, (b) have as low a potential for skin irritancy as possible consistent with being an effective skin penetration enhancer, and (c) be compatibile with the active pharmaceutical ingredient. Note that the agent preferably functions both as a solubilising agent and a skin-penetration-enhancing agent.

Surfactants: An external-topical pharmaceutical composition, e.g. an ointment or an oil- in-water cream or water-in-oil cream, preferably includes a surfactant (e.g. as an emulsifier), for example for achieving emulsification of compositions having two or more phases. The total surfactant content can for example be 0.3% to 20%, e.g. 0.5% to 15% or 0.5% to 12% or 0.5% to 10% or 1% to 12% or 3% to 10%, by weight of the composition (w/w). The surfactant can for example comprise one or more of the following: a polyoxyl Ci2_22alkyl emer (e<§- apolyoxyl Ci4_2oalkyl ether such as polyoxyl cetyl ether or polyoxyl stearyl ether) (e.g. present at 0.5% to 10% w/w, e.g. 2.5% to 10% w/w such as about 5% to about 8% w/w), glycerol monostearate (e.g. Arlacel 165 ™) (e.g. present at 0.5% to 10% w/w, e.g. about 2% w/w), sorbitan monostearate (e.g. Span 60 ™) (e.g. present at 0.05% to 10% w/w, e.g. about 1% w/w), cetyl alcohol and/or stearyl alcohol (e.g. wherein the total of any cetyl alcohol and any stearyl alcohol present is 0.1% to 15% w/w, e.g. 1% to 10% w/w such as about 2% to about 5% w/w), and sodium dodecyl sulphate (SDS) (e.g. present at 0.3% to 2% w/w such as about 1% w/w). Polyoxyl stearyl ether (steareth) can e.g. be polyoxyl 2 stearyl ether (steareth 2) or polyoxyl 21 stearyl ether (steareth 21).

DMSO-containing solutions: One possible external-topical pharmaceutical composition is a solution of the compound of formula (I) or the pharmaceutically acceptable salt thereof present at ca. 0.5% to ca. 2.5% w/w in a DMSO-containing solvent such as in DMSO/acetone or in DMSO/water; for example a solution of the compound or salt present at ca. 0.5% to ca. 2.5% w/w in DMSO/acetone (1:1). DMSO-containing solutions, often being capable of high skin penetration, are often good experimental pre¬ clinical formulations for use in animals, but their likely skin irritancy generally make them less suitable for use in humans such as patients, e.g. atopic dermatitis patients.

Ointments and creams (and oil phase): An external-topical pharmaceutical composition can be an ointment or an oil-in- water cream or water-in-oil cream. The ointment or cream typically contains an oil phase (oily ointment base). The oil phase (ointment base) typically comprises an oil and/or a fat, preferably of a consistency suitable for skin- spreadability.

Preferably, an oil comprising or being white soft paraffin (white petrolatum) and/or a mineral oil (such as liquid paraffin) can be used. (Mineral oil can also be used as a solubiliser and/or emollient). The white soft paraffin (white petrolatum) can be of various grades, for example (for Penreco supplier) Penreco Regent White grade, Penreco Snow White grade, or Penreco Ultima White grade, in particular high melting point white soft paraffin (e.g. of Penreco Ultima White grade). Microcrystalline wax or beeswax or beeswax substitute can be used as an oil / fat in the oil phase.

Alternatively or additionally, one or more fats like straight or branched chain mono- or di-alkyl esters such as isopropyl myristate, isopropyl palmitate, diisopropyl adipate, isocetyl stearate, isostearyl isostearate, decyl oleate, butyl stearate, 2-ethylhexyl palmitate, propylene glycol diester of coconut fatty acids, or a mixed ester of 2-ethyl hexanoic acid with a blend of cetyl or stearyl alcohols (e.g. known as Crodamol CAP) may be used in the oil phase (some of these are also solubilisers and/or surfactants). These may be used singly or in combination depending on the properties required.

The oil phase (oily ointment base) can for example be present at 25 to 85% w/w (e.g. 50 to 80% w/w) in an ointment (e.g. emulsion or homogeneous single phase), at 25 to 85% w/w (e.g. 35 to 70% w/w) in an water-in-oil cream (e.g. emulsion), or at 8 to 55% w/w (e.g. 10 to 45% w/w) in an water-in-oil cream (e.g. emulsion).

Exemplωγ ointments: An exemplary external-topical pharmaceutical composition is an ointment comprising: - the compound of formula (I) or pharmaceutically acceptable salt thereof present at 0.1% to 5% w/w (e.g. 0.1% to 3% w/w or 0.2% to 1.5% w/w); - an oil phase (oily ointment base) present at 25% to 99% w/w or 25% to 85% w/w or 50% to 80% w/w (for example, the oil phase can comprise white petrolatum present at 25 to 75% w/w or 45 to 75% w/w, and optionally also comprising mineral oil (e.g. as solubiliser and emollient) present at 2.5% to 15% w/w such as 4% to 12% w/w); - one or more surfactants (e.g. polyoxyl stearyl ether) present in total at 0.5% to 10% w/w or 3% to 10% w/w; and - one or more agents acting as a skin-penetration enhancer (preferably acting as both a solubiliser and skin-penetration enhancer and/or preferably hydrophilic such as propylene glycol) present in total at 0.5% to 50% w/w, such as 5% to 50% w/w or 7% to 30% w/w; and - optionally one or more antioxidants (e.g. butylated hydroxyanisole), e.g. present in total at 0.001 to 2% w/w such as 0.02 to 2% w/w; and - optionally one or more preservatives, e.g. present in total at 0.01 to 4% w/w such as 0.05 to 1% w/w (e.g. methylparaben present at 0.05 to 2% w/w and/or propylparaben present at 0.01 to 2% w/w).

The above exemplary composition, including the oil "phase" and the penetration enhancer, can optionally be a homogeneous single phase. However, in one embodiment of the above exemplary ointment composition, e.g. when using propylene glycol or another hydrophilic solubiliser and penetration enhancer, the oil phase (oily ointment base) and a hydrophilic phase containing the hydrophilic solubiliser and penetration enhancer (e.g. propylene-glycol-containing phase) have been emulsified to form an ointment emulsion.

Ointment compositions having two phases can optionally be prepared using an emulsification process whereby the hydrophilic phase (e.g. propylene-glycol-containing phase) and oil phase are first prepared in separate vessels. The hydrophilic phase can optionally contain a penetration enhancer such as propylene glycol, and optionally some or all of the compound of formula (I) or salt thereof. The oil phase can optionally contain a surfactant. Temperatures of both phases are maintained at elevated temperatures, such as about 55-9O0C or preferably from above 70 to 90 0C, the oil phase temperature being sufficiently high (e.g. from above 70 to 90 0C) to melt the oil phase. While hot, one phase is added to another while mixing, e.g. using a high shear mixer, to effect emulsification, preferably keeping the temperature above 7O0C such as from above 70 to 90 0C. The resulting ointment emulsion is allowed to cool, e.g. to about 15-350C such as to about 18-3O0C, preferably while the agitation continues e.g. at lower speeds. The ointment emulsion can then optionally be dispensed from the manufacturing vessel and filled into primary packaging, for example tubes or sachets.

Optionally, an ointment can comprise a polyethylene glycol base, e.g. present at 25 to 98% w/w such as 50 to 95% w/w, instead of or as well as an oily ointment base.

Creams: An external-topical pharmaceutical composition can be a cream, e.g. a water-in- oil cream or an oil-in-water cream. Creams can sometimes be more fluid than ointments, can sometimes provide more moisture, and hence may in principle in certain cases allow for improved and/or good efficacy in patients with atopic dermatitis.

Water-in-oil creams: These usually have an increased aqueous content compared to ointments. Preferably, the water-in-oil cream is a water-in-oil cream emulsion. That is, preferably, in the water-in-oil cream, an oil phase and an aqueous phase have been emulsified to form a water-in-oil cream emulsion.

An exemplary external-topical pharmaceutical composition is a water-in-oil cream (e.g. cream emulsion) comprising: - the compound of formula (I) or pharmaceutically acceptable salt thereof present at 0.1% to 5% w/w (e.g. 0.1% to 3% w/w or 0.2% to 1.5% w/w); - an oil phase (oily ointment base) present at 25% to 85% w/w or 35% to 70% w/w (for example comprising white petrolatum present at 25% to 75% w/w or 30% to 65% w/w, and optionally also comprising mineral oil (e.g. as solubiliser and emollient) present at 2.5% to 15% w/w or 4% to 12% w/w); - water present in 2% to 30% w/w, e.g. 5% to 25% or 10% to 22% w/w; - one or more surfactants (e.g. polyoxyl stearyl ether) present in total at 0.5% to 12% w/w, such as 3% to 10% w/w; and - one or more agents acting as a skin-penetration enhancer (preferably acting as both a solubiliser and skin-penetration enhancer and/or preferably hydrophilic such as propylene glycol) present in total at 0.5% to 50% w/w, such as 5% to 50% w/w or 7% to 30% w/w; and - optionally one or more antioxidants (e.g. butylated hydroxyanisole), e.g. present in total at 0.001 to 2% w/w such as 0.02 to 2% w/w; and - optionally one or more preservatives, e.g. present in total at 0.01 to 4% w/w such as 0.05 to 1% w/w (e.g. methylparaben present at 0.05 to 2% w/w and/or propylparaben present at 0.01 to 2% w/w).

Oil-in-water creams: These usually have an increased aqueous content compared to ointments and water-in-oil creams. Preferably, the oil-in-water cream is a oil-in-water cream emulsion. That is, preferably, in the oil-in-water cream, an oil phase and an aqueous phase have been emulsified to form a oil-in-water cream emulsion.

Preferable oil-in-water creams are high-occlusion creams, wherein, after topical administration to the skin, moisture loss from the skin and/or from the cream is reduced or limited by means of sufficiently high coverage of the skin and/or by providing a sufficient barrier at the site of application.

Preferably, the oil-in-water cream contains one or more emollients (hydrating agents), such as silicones (e.g. dimethicone, e.g. dimethicone 360 or dimethicone 20), a high- viscosity wax such as microcrystalline wax, and/or mineral oil. A sufficiently high water content is also preferred, for example wherein the water is present in 15% to 60% w/w, 20% to 50% w/w, or 25% to 40% w/w.

An exemplary external-topical pharmaceutical composition is a oil-in-water cream (e.g. cream emulsion) comprising: - the compound of formula (I) or pharmaceutically acceptable salt thereof present at 0.1% to 5% w/w (e.g. 0.1% to 3% w/w or 0.2% to 1.5% w/w); - an oil phase (oily ointment base) containing one or more ingredients capable of acting as emollients, the oil phase being present at 5% to 60% w/w or preferably 20% to 60% w/w or 30% to 60% w/w or more preferably 30% to 55% w/w; - water present in 12% to 75% w/w or 15% to 75% w/w or 15% to 60% w/w, preferably 15% to 50% w/w or 20% to 40% w/w; - one or more surfactants present in total at 0.5% to 12% w/w, e.g. 3% to 10% w/w; and - one or more agents acting as a skin-penetration enhancer (preferably acting as both a solubiliser and skin-penetration enhancer and/or preferably hydrophilic such as propylene glycol) present in total at 0.5% to 50% w/w, preferably 5% to 50% w/w or 7% to 25% w/w; and - optionally one or more solubilisers (e.g. isopropyl myristate), e.g. present at 0.5% to 20% w/w, e.g. 3 to 12% w/w; and - -

- optionally one or more buffers (e.g. citric acid and/or dibasic sodium phosphate), e.g. present in total at 0.05 to 5% w/w.

hi the above exemplary oil-in-water cream composition, the oil phase preferably comprises mineral oil (e.g. as emollient and solubiliser) present at 15% to 50% w/w or 20% to 45% w/w, and/or comprises a high- viscosity wax such as microcrystalline wax (e.g. as emollient) present at 5% to 25% w/w such as 8% to 15% w/w, and/or comprises a silicone (such as dimethicone e.g. dimethicone 360 or dimethicone 20, e.g. as emollient) present at 0.5% to 20% such as 0.5% to 10% or 1% to 5% w/w.

In the above exemplary oil-in-water cream composition, the one or more surfactants preferably comprise: glycerol monostearate present at 0.5% to 10% w/w, and/or sorbitan monostearate present at 0.05% to 10% w/w, and/or [cetyl alcohol and/or stearyl alcohol] present in total at 0.1% to 15% or 1 to 10% w/w.

Cream emulsions, e.g. water-in-oil or oil-in-water cream emulsions, can be prepared by a process in which an aqueous phase is prepared, e.g. prepared before emulsification. The aqueous phase usually contains water and a solubiliser and/or skin-penetration enhancer such as propylene glycol, and optionally contains some or all of the compound of formula (I) or salt thereof, and/or optionally contains surfactant. The oil phase, e.g. containing white petrolatum and/or mineral oil, and/or optionally containing surfactant, can be prepared in a separate vessel. Temperatures of both phases are maintained at elevated temperatures, such as about 55-9O0C or preferably from above 70 to 90 0C, the oil phase temperature being sufficiently high (e.g. from above 70 to 90 0C) to melt the oil phase. While hot, one phase is added to another while mixing, e.g. using a high shear mixer, to effect emulsification, preferably keeping the temperature above 7O0C such as from above 70 to 90 0C. The resulting emulsion is allowed to cool, e.g. to about 15-350C such as to about 18-3O0C, preferably while the agitation continues e.g. at lower speeds. The cream emulsion can then optionally be dispensed from the manufacturing vessel and filled into primary packaging, for example tubes or sachets.

Typically, a pharmaceutical composition of the invention suitable for external topical administration can be administered once daily, twice daily or more than twice daily, to external body part(s), e.g. on the skin such as at a site of diseased skin, e.g. skin suffering from atopic dermatitis.

Inhalable and intranasal pharmaceutical compositions, and particle size reduction

Compositions suitable for (e.g. adapted for) nasal or inhaled administration may conveniently be formulated as aerosols, drops, gels or dry powders. - -

Aerosol formulations, e.g. for inhaled administration, can comprise a solution or fine suspension of the active substance in a pharmaceutically acceptable aqueous or non¬ aqueous solvent. Aerosol formulations can be presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device or inhaler. Alternatively the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve (metered dose inhaler) which is intended for disposal once the contents of the container have been exhausted.

Where the dosage form comprises an aerosol dispenser, it preferably contains a suitable propellant under pressure such as compressed air, carbon dioxide, or an organic propellant such as a chlorofluorocarbon (CFC) or more preferably hydrofluorocarbon (HFC). Suitable CFC propellants include dichlorodifluoromethane, trichlorofluoromethane and dichlorotetrafluoroethane. Suitable HFC propellants include 1, 1, 1,2,3,3, 3-heptafluoropropane and 1,1,1,2-tetrafluoroethane. The aerosol dosage forms can also take the form of a pump-atomiser.

Particle size reduction: For pharmaceutical compositions suitable for (e.g. adapted for) inhaled administration, it is preferred that the compound or salt of formula (I) is in a particle-size-reduced form. The size-reduced form can for example be obtained or obtainable by micronisation. Micronisation usually involves subjecting the compound/salt to collisional and/or abrasional forces in a fast-flowing circular or spiral/vortex-shaped airstream often including a cyclone component. The preferable particle size of the size-reduced (e.g. micronised) compound or salt is defined by a D50 value of about 0.5 to about 10 microns, e.g. about 1 to about 7 microns (e.g. as measured using laser diffraction). For example, it is preferable for the compound or salt of formula (I) to have a particle size defined by: a DlO of about 0.3 to about 3 microns (e.g. about 0.5 to about 2 microns, or about 1 micron), and/or a D50 of about 0.5 to about 10 microns or about 1 to about 7 microns (e.g. about 2 to about 5 microns or about 2 to about 4 microns), and/or a D90 of about 1 to about 30 microns or about 2 to about 20 microns or about 3 to about 15 microns (e.g. about 5 to about 15 microns or about 5 to about 10 microns); for example as measured using laser diffraction.

In particle size measurements, D90, D50 and DlO respectively mean that 90%, 50% and 10% of the material is less than the micron size specified. D50 is the median particle size. DV90, DV50 and DVlO respectively mean that 90%, 50% and 10% by volume of the material is less than the micron size specified. DM90, DM50 and DM10 respectively mean that 90%, 50% and 10% by weight of the material is less than the micron size specified.

Laser diffraction measurement of particle size can use a dry method (wherein a suspension of the compound/salt in an airflow crosses the laser beam) or a wet method [wherein a suspension of the compound/salt in a liquid dispersing medium, such as - -

isooctane or (e.g. if compound is soluble in isooctane) 0.1% Tween 80 in water, crosses the laser beam]. With laser diffraction, particle size is preferably calculated using the Fraunhofer calculation; and/or preferably a Malvern Mastersizer or Sympatec apparatus is used for measurement. For example, particle size measurement and/or analysis by laser diffraction can use any or all of (preferably all of) the following: a Malvern Mastersizer longbed version, a dispersing medium of 0.1% Tween 80 in water, a stir rate of ca. 1500 rpm, ca. 3 mins sonification prior to final dispersion and analysis, a 300 RF (Reverse Fourier) lens, and/or the Fraunhofer calculation with Malvern software.

For a small-scale non-limiting example of micronisation, see the Micronisation Example hereinafter.

Dry powder inhalable compositions: For pharmaceutical compositions suitable (e.g. adapted for) inhaled administration, the pharmaceutical composition may for example be a dry powder inhalable composition. Such a composition can comprise a powder base such as lactose or starch, the compound of formula (I) or salt thereof (preferably in particle-size-reduced form, e.g. in micronised form), and optionally a ternary agent such as L-leucine, mannitol, trehalose, magnesium stearate and/or cellobiose octaacetate (e.g. alpha-D-isomer of cellobiose octaacetate, e.g. available from Aldrich). For cellobiose octaacetate and storage stability, see WO 03/088943.

Preferably, the dry powder inhalable composition comprises a dry powder blend of lactose and the compound of formula (I) or salt thereof. The lactose is preferably lactose hydrate e.g. lactose monohydrate and/or is preferably inhalation-grade and/or fine-grade lactose. Preferably, the particle size of the lactose is defined by 90% or more (by weight or by volume) of the lactose particles being less than 1000 microns (micrometres) (e.g. 10-1000 microns e.g. 30-1000 microns) in diameter, and/or 50% or more of the lactose particles being less than 500 microns (e.g. 10-500 microns) in diameter. More preferably, the particle size of the lactose is defined by 90% or more of the lactose particles being less than 300 microns (e.g. 10-300 microns e.g. 50-300 microns) in diameter, and/or 50% or more of the lactose particles being less than 100 microns in diameter. Optionally, the particle size of the lactose is defined by 90% or more of the lactose particles being less than 100-200 microns in diameter, and/or 50% or more of the lactose particles being less than 40-70 microns in diameter. It is suitable that about 3 to about 30% (e.g. about 10%) (by weight or by volume) of the particles are less than 50 microns or less than 20 microns in diameter. For example, without limitation, a suitable inhalation-grade lactose is E9334 lactose (10% fines) (Borculo Domo Ingredients, Hanzeplein 25, 8017 JD Zwolle, Netherlands).

hi the dry powder inhalable composition, the compound of formula (I) or salt thereof can for example be present in about 0.1% to about 70% (e.g. about 1% to about 50%, e.g. about 5% to about 40%, e.g. about 20 to about 30%) by weight of the composition. - -

An illustrative non-limiting example of a dry powder inhalable composition is given in the Composition Examples below.

Dry powder inhalation devices: Optionally, in particular for dry powder inhalable compositions, a pharmaceutical composition for inhaled administration can be incorporated into a plurality of sealed dose containers (e.g. containing the dry powder composition) mounted longitudinally in a strip or ribbon inside a suitable inhalation device. The container is rupturable or peel-openable on demand and the dose, e.g. of the dry powder composition, can be administered by inhalation via a device such as the DISKUS ™ device, marketed by GlaxoSmithKline. The DISKUS ™ inhalation device is usually substantially as described in GB 2,242,134 A. In such device at least one container for the pharmaceutical composition in powder form (the at least one container preferably being a plurality of sealed dose containers mounted longitudinally in a strip or ribbon) is defined between two members peelably secured to one another; the device comprises: means defining an opening station for the said at least one container; means for peeling the members apart at the opening station to open the container; and an outlet, communicating with the opened container, through which a user can inhale the pharmaceutical composition in powder form from the opened container.

Pharmaceutical compositions for oral or parenteral administration

A pharmaceutical composition suitable for (e.g. adapted for) parenteral (e.g. intravenous, subcutaneous, or intramuscular) administration can comprise a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile parenterally acceptable aqueous carrier (e.g. sterile water) or parenterally acceptable oil. Alternatively, the solution can be lyophilised. A lyophilised pharmaceutical composition suitable for (e.g. adapted for) parenteral administration may, in use, optionally be reconstituted with a suitable solvent, e.g. sterile water or a sterile parenterally acceptable aqueous solution, just prior to administration.

A pharmaceutical composition for oral administration can be liquid or solid; for example it can be a syrup, suspension or emulsion, a tablet, a capsule or a lozenge.

A liquid formulation can generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable pharmaceutically acceptable liquid carrier(s), for example an aqueous solvent such as water, ethanol or glycerine, or a non¬ aqueous solvent, such as polyethylene glycol or an oil. The formulation may also contain a suspending agent, preservative, flavouring and/or colouring agent.

The composition can optionally be in unit dose form such as a tablet or capsule for oral administration, e.g. for oral administration to a human. ^

A pharmaceutical composition for oral administration being a tablet can comprise one or more pharmaceutically acceptable carriers and/or excipients suitable for preparing tablet formulations. The carrier can for example be or include lactose, cellulose (for example microcrystalline cellulose), or mannitol. The tablet can also or instead contain one or more pharmaceutically acceptable excipients, for example a binding agent such as hydroxypropylmethylcellulose or povidone (polyvinylpyrollidone), a lubricant e.g. an alkaline earth metal stearate such as magnesium stearate, and/or a tablet disintegrant such as sodium starch glycollate, croscarmellose sodium, or crospovidone (cross-linked polyvinylpyrollidone). The pharmaceutical composition being a tablet can be prepared by a method comprising the steps of: (i) mixing the compound of formula (I), as herein defined, or a pharmaceutically acceptable salt thereof, with the one or more pharmaceutically acceptable carriers and/or excipients, (ii) compressing the resulting mixture (which is usually in powder form) into tablets, and (iii) optionally coating the tablet with a tablet film-coating material.

A pharmaceutical composition for oral administration being a capsule can be prepared using encapsulation procedures. For example, pellets or powder containing the active ingredient can be prepared using a suitable pharmaceutically acceptable carrier and then filled into a hard gelatin capsule. Alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutically acceptable carrier, for example an aqueous gum or an oil and the dispersion or suspension then filled into a soft gelatin capsule.

Dosing regimens

In a pharmaceutical composition suitable for (e.g. adapted for) external topical administration, e.g. an ointment or an oil-in-water or water-in-oil composition, the compound of formula (I) or the pharmaceutically acceptable salt thereof can be present in 0.05% to 10%, preferably 0.1% to 5%, more preferably 0.1% to 3%, still more preferably 0.2% to 3% (e.g. about 0.5% or about 2.5%), yet more preferably 0.2% to 1.5% (e.g. about 0.5%), by weight of the composition. Typically, an external-topical pharmaceutical composition can be administered once daily, twice daily or more than twice daily, to external body part(s), e.g. to the skin such as at a site of diseased skin. The amount administered is usually such as substantially to cover the site(s) of diseased skin.

A pharmaceutical composition can optionally be in unit dose form. In the pharmaceutical composition, a or each dosage unit for oral or parenteral administration can for example contain from 0.01 to 3000 mg, for example 0.5 to 1000 mg, of a compound of the formula (I) or a pharmaceutically acceptable salt thereof, calculated as the free base. A or each dosage unit for nasal or inhaled administration can for example contains from 0.001 to 50 mg, e.g. 0.01 to 5 mg, of a compound of the formula (I) or a pharmaceutically acceptable salt thereof, calculated as the free base. When a parenteral or oral composition is used, pharmaceutically acceptable compound or salt of the invention can optionally be administered to a mammal (e.g. human) in a daily oral or parenteral dose of 0.001 mg to 50 mg per kg body weight per day (mg/kg/day), for example 0.01 to 20 mg/kg/day or 0.03 to 10 mg/kg/day or 0.1 to 2 mg/kg/day, of the compound of the formula (I) or a pharmaceutically acceptable salt thereof, calculated as the free base.

When an inhaled or nasal composition is used, a pharmaceutically acceptable compound or salt of the invention can optionally be administered to a mammal (e.g. human) in a daily nasal or inhaled dose of: 0.0001 to 5 mg/kg/day or 0.0001 to 1 mg/kg/day, e.g. 0.001 to 1 mg/kg/day or 0.001 to 0.3 mg/kg/day or 0.001 to 0.1 mg/kg/day or 0.005 to 0.3 mg/kg/day, of the compound of the formula (I) or a pharmaceutically acceptable salt thereof, calculated as the free base.

The pharmaceutically acceptable compounds or salts of the invention can optionally be administered to a human in a daily dose (for an adult patient) of, for example, an oral or parenteral dose of 0.01 mg to 3000 mg per day or 0.5 to 1000 mg per day e.g. 2 to 500 mg per day, or a nasal or inhaled dose of 0.001 to 300 mg per day or 0.001 to 50 mg per day or 0.01 to 30 mg per day or 0.01 to 5 mg per day or 0.02 to 2 mg per day, of the compound of the formula (I) or a pharmaceutically acceptable salt thereof, calculated as the free base.

Combinations

The compounds, salts and/or pharmaceutical compositions according to the invention may also be used in combination with another therapeutically active agent, for example, a β2 adrenoreceptor agonist, an anti-histamine, an anti-allergic, an anti-inflammatory agent, an antiinfective agent or an immunosuppressant.

The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof together with another therapeutically active agent, for example, a β2-adrenoreceptor agonist, an anti-histamine, an anti-allergic, an anti-inflammatory agent, an antiinfective agent or an immunosuppressant.

Preferably, the β2-adrenoreceptor agonist is salmeterol (e.g. as racemate or a single enantiomer such as the R-enantiomer), salbutamol, formoterol, salmefamol, fenoterol or terbutaline, or a salt thereof (e.g. pharmaceutically acceptable salt thereof), for example the xinafoate salt of salmeterol, the sulphate salt or free base of salbutamol or the fumarate salt of formoterol. Long-acting β2-adrenoreceptor agonists are preferred, especially those having a therapeutic effect over a 12-24 hour period such as salmeterol or formoterol. Preferably, the β2-adrenoreceptor agonist is for inhaled administration, e.g. once per day and/or for simultaneous inhaled administration; and more preferably the β2-adrenoreceptor agonist is in particle-size-reduced form e.g. as defined herein. Preferably, the β2-adrenoreceptor agonist combination is for treatment and/or prophylaxis of COPD or asthma. Salmeterol or a pharmaceutically acceptable salt thereof, e.g. salmeterol xinofoate, is preferably administered to humans at an inhaled dose of 25 to 50 micrograms twice per day (measured as the free base).

Preferred long acting β2-adrenoreceptor agonists include those described in WO 02/066422A, WO 03/024439, WO 02/070490 and WO 02/076933.

Especially preferred long-acting β2-adrenoreceptor agonists include compounds of formula(XX) (described in WO 02/066422): HOCH, T P11X

CHCH2NHCR14XR15X(CH2)m)^-O— (CH2)nχ— (\ ^j) (XX)

OH

or a salt or solvate thereof, wherein in formula (XX): mx is an integer of from 2 to 8; nx is an integer of from 3 to 11, with the proviso that mx + nx is 5 to 19, R11X is -XSO2NR16XR17X wherein X is -(CH2)P*- or C2-6 alkenylene; R16X and R17X are independently selected from hydrogen, C1-6alkyl, C3-7cycloalkyl, C(O)NR18XR19X, phenyl, and phenyl (C1-4alkyl)-, or R16X and R17X, together with the nitrogen to which they are bonded, form a 5-, 6-, or 7- membered nitrogen containing ring, and R16X and R17X are each optionally substituted by one or two groups selected from halo, C1-6alkyl, C1-6haloalkyl, C1-6alkoxy, hydroxy- substituted C1-6alkoxy, -CO2R18X, -SO2NR18XR19X, -CONR18XR19X, -NR18XC(O)R19X, or a 5-, 6- or 7-membered heterocylic ring; R18xand R19Xare independently selected from hydrogen, C1-6alkyl, C3-6cycloalkyl, phenyl, and phenyl (C1-4alkyl)-; and px is an integer of from 0 to 6, preferably from 0 to 4; R12X and R13X are independently selected from hydrogen, C1-6alkyl, C1-6alkoxy, halo, phenyl, and C1-6haloalkyl; and R14X and R15X are independently selected from hydrogen and C1-4alkyl with the proviso that the total number of carbon atoms in R14X and R15X is not more than 4.

Preferred β2-adrenoreceptor agonists disclosed in WO 02/066422 include: 3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)- phenyljethyl}amino)hexyl]oxy}butyl)benzenesulfonamide and - -

3-(3-{[7-({(2R)-2-hydroxy-2-[4-hydroxy-3-hydroxymethyl)ph enyl]ethyl}- amino)heptyl]oxy}propyl)benzenesulfonamide.

A preferred β2-adrenoreceptor agonist disclosed in WO 03/024439 is:

4-{(li?)-2-[(6-{2-[(2,6-dichlorobenzyl)oxy]ethoxy}hexyl)a mino]-l-liydroxyethyl}-2- (hydroxymethyl)phenol.

An anti-histamine usable in a combination of a compound of formula (I) or salt can for example be for oral administration (e.g. as a combined composition such as a combined tablet), and can be for treatment and/or prophylaxis of allergic rhinitis. Examples of anti¬ histamines include methapyrilene, or Hl antagonists such as cetirizine, loratadine (e.g. Clarityn TM)3 desloratadine (e.g. Clarinex TM) or fexofenadine (e.g. Allegra TM)

The invention also provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof together with an anticholinergic compound, e.g. a muscarinic (M) receptor antagonist in particular an M\, M2, Mj/M2, or M3 receptor antagonist, more preferably a M3 receptor antagonist, still more preferably a M3 receptor antagonist which selectively antagonises (e.g. antagonises 10 times or more strongly) the M3 receptor over the M\ and/or M2 receptor. For combinations of anticholinergic compounds / muscarinic (M) receptor antagonist with PDE4 inhibitors, see for example WO 03/011274 A2 and WO 02/069945 A2 / US 2002/0193393 Al and US 2002/052312 Al, and some or all of these publications give examples of anticholinergic compounds / muscarinic (M) receptor antagonists which may be used with the compounds of formula (I) or salts, and/or suitable pharmaceutical compositions. For example, the muscarinic receptor antagonist can comprise or be an ipratropium salt (e.g. ipratropium bromide), an oxitropium salt (e.g. oxitropium bromide), or more preferably a tiotropium salt (e.g. tiotropium bromide); see e.g. EP 418 716 Al for tiotropium.

The anticholinergic compound or muscarinic (M) receptor antagonist, e.g. M3 receptor antagonist, is preferably for inhaled administration, more preferably in particle-size- reduced form e.g. as defined herein. More preferably, both the muscarinic (M) receptor antagonist and the compound of formula (I) or the pharmaceutically acceptable salt thereof are for inhaled administration. Preferably, the anticholinergic compound or muscarinic receptor antagonist and the compound of formula (I) or salt are for simultaneous administration. The muscarinic receptor antagonist combination is preferably for treatment and/or prophylaxis of COPD.

Other possible combinations include, for example, a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof together with another anti¬ inflammatory agent such as an anti-inflammatory corticosteroid; or a non-steroidal anti- inflammatory drag (NSAID) such as a leukotriene antagonist (e.g. montelukast), an iNOS inhibitor, a tryptase inhibitor, a elastase inhibitor, a beta-2 integrin antagonist, a adenosine 2a agonist, or a 5-lipoxogenase inhibitor; or an antiinfective agent (e.g. an antibiotic or an antiviral). An iNOS inhibitor is preferably for oral administration. Suitable iNOS inhibitors (inducible nitric oxide synthase inhibitors) include those disclosed in WO 93/13055, WO 98/30537, WO 02/50021, WO 95/34534 and WO 99/62875.

Exemplarary combinations, in particular for external topical administration (e.g. versus atopic dermatitis), include, for example, a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof together with an immunosuppressant, e.g. a calcineurin inhibitor such as pimecrolimus or tacrolimus. The immunosuppressant can in particular be an externally-topically administrable immunosuppressant such as pimecrolimus (e.g. pimecrolimus at ca. 1% w/w concentration in a topical composition such as a cream, and/or e.g. Elidel ™) or tacrolimus (e.g. tacrolimus at from about 0.03% to about 0.1% w/w concentration in a topical composition such as an ointment, and/or e.g. Protopic ™). The externally- topically administrable immunosuppressant can be administered or administrable in a external-topical composition separately from the compound or salt of the invention, or it can be contained with the compound of formula (I) or pharmaceutically acceptable salt in a combined externally-topically-administrable composition.

For external topical administration, e.g. versus atopic dermatitis, a combination of the compound or salt of the invention together with an anti-infective agent can include an externally-topically-administrable antibacterial such as mupiricin or a salt (e.g. calcium salt) thereof (e.g. Bactroban ™), or an externally- topically-admmistrable pleuromutilin antibacterial. Alternatively or additionally, for external topical administration an externally-topically-administrable antifungal such as clortrimazole, clotrimazole or ketoconazole can be used.

For external topical administration, e.g. versus atopic dermatitis, a combination with an anti-itch compound may optionally be used.

In a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof together with an anti-inflammatory corticosteroid (which can for example be for treatment and/or prophylaxis of asthma, COPD or allergic rhinitis), then preferably the anti-inflammatory corticosteroid is fluticasone propionate (e.g. see US patent 4,335,121), beclomethasone 17-propionate ester, beclomethasone 17,21-dipropionate ester, dexamethasone or an ester thereof, mometasone or an ester thereof (e.g. mometasone furoate), ciclesonide, budesonide, flunisolide, or a compound as described in WO 02/12266 Al (e.g. as claimed in any of claims 1 to 22 therein), or a pharmaceutically acceptable salt of any of the above. If the anti-inflammatory corticosteroid is a compound as described in WO 02/12266 Al, then preferably it is Example 1 therein {which is 6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-l lβ-hydroxy-16α-methyl-3- oxo-androsta-l,4-diene-17β-carbothioic acid (S-fluoromethyl ester} or Example 41 therein {which is 6α,9α-difluoro-l lβ-hydroxy-16α-methyl-17α-[(4-methyl-l,3-thiazole-5- carbonytyoxyJ-S-oxo-androsta-l^-diene-iyβ-carbothioic acid S-fluoromethyl ester}, or a pharmaceutically acceptable salt thereof. The anti-inflammatory corticosteroid can for example be for external topical, intranasal or inhaled administration. Fluticasone propionate is preferred and is preferably for inhaled administration to a human either (a) at a dose of 250 micrograms once per day or (b) at a dose of 50 to 250 micrograms twice per day.

Also provided is a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof together with β2-adrenoreceptor agonist and an anti-inflammatory corticosteroid, for example as described in WO 03/030939 Al. Preferably this combination is for treatment and/or prophylaxis of asthma, COPD or allergic rhinitis. The β2-adrenoreceptor agonist and/or the anti-inflammatory corticosteroid can be as described above and/or as described in WO 03/030939 Al. Most preferably, in this "triple" combination, the β2-adrenoreceptor agonist is salmeterol or a pharmaceutically acceptable salt thereof (e.g. salmeterol xinafoate) and the anti- inflammatory corticosteroid is fluticasone propionate.

The combinations referred to above may be presented for use in the form of a pharmaceutical composition and thus a pharmaceutical composition comprising a combination as defined above together with one or more pharmaceutically acceptable carriers and/or excipients represent a further aspect of the invention.

The individual compounds of such combinations maybe administered either sequentially or simultaneously in separate or combined pharmaceutical composition.

m one embodiment, the combination as defined herein can be for simultaneous inhaled administration and is disposed in a combination inhalation device. Such a combination inhalation device is another aspect of the invention. Such a combination inhalation device can comprise a combined pharmaceutical composition for simultaneous inhaled administration (e.g. dry powder composition), the composition comprising all the individual compounds of the combination, and the composition being incorporated into a plurality of sealed dose containers mounted longitudinally in a strip or ribbon inside the inhalation device, the containers being rupturable or peel-openable on demand; for example such inhalation device can be substantially as described in GB 2,242,134 A (DISKUS TM) and/or as described above. Alternatively, the combination inhalation device can be such that the individual compounds of the combination are administrable simultaneously but are stored separately (or wholly or partly stored separately for triple combinations), e.g. in separate pharmaceutical compositions, for example as described in - -

PCT/EP03/00598 filed on 22 January 2003, published as WO 03/061743 (e.g. as described in the claims thereof e.g. claim 1).

The invention also provides a method of preparing a combination as defined herein, the method comprising either (a) preparing a separate pharmaceutical composition for administration of the individual compounds of the combination either sequentially or simultaneously, or (b) preparing a combined pharmaceutical composition for administration of the individual compounds of the combination simultaneously, wherein the pharmaceutical composition comprises the combination together with one or more pharmaceutically acceptable carriers and/or excipients.

The invention also provides a combination as defined herein, prepared by a method as defined herein. BIOLOGICAL TEST METHODS

PDE 3, PDE 4B, PDE 4D, PDE 5, PDE 6 Primary assay methods

The activity of the compounds or salts of the invention can be measured in the assay methods shown below. Preferred compounds of the invention are selective PDE4 inhibitors, i.e. they inhibit PDE4 (e.g. PDE4B and/or PDE4D, preferably PDE4B) more strongly than they inhibit PDE3 and/or more strongly than they inhibit PDE5 and/or more strongly than they inhibit PDE6 (though such selectivity is not essential to the invention).

Possible PDE enzyme sources and literature references

Human recombinant PDE4B, in particular the 2B splice variant thereof (HSPDE4B2B), is disclosed in WO 94/20079 and also M.M. McLaughlin et al., "A low Km, rolipram- sensitive, cAMP-specific phosphodiesterase from human brain: cloning and expression of cDNA, biochemical characterisation of recombinant protein, and tissue distribution of mRNA", J. Biol. Chem., 1993, 268, 6470-6476. For example, in Example 1 of WO 94/20079, human recombinant PDE4B is described as being expressed in the PDE- deficient yeast Saccharomyces cerevisiae strain GL62, e.g. after induction by addition of 150 uM CUSO4, and 100,000 x g supernatant fractions of yeast cell lysates are described for use in the harvesting of PDE4B enzyme.

Human recombinant PDE4D (HSPDE4D3A) is disclosed in P. A. Baecker et al., "Isolation of a cDNA encoding a human rolipram-sensitive cyclic AMP phoshodiesterase (PDE IVD)", Gene, 1994, 138, 253-256.

Human recombinant PDE5 is disclosed in K. Loughney et al., "Isolation and characterisation of cDNAs encoding PDE5A, a human cGMP-binding, cGMP-specific 3 ',5 '-cyclic nucleotide phosphodiesterase", Gene, 1998, 216, 139-147.

PDE3 can be purified from bovine aorta, e.g. as described by H. Coste and P. Grondin, "Characterisation of a novel potent and specific inhibitor of type V phosphodiesterase", Biochem. Pharmacol, 1995, 50, 1577-1585.

PDE6 can be purified from bovine retina, e.g. as described by: P. Catty and P. Deterre, "Activation and solubilization of the retinal cGMP-specific phosphodiesterase by limited proteolysis", Eur. J. Biochem., 1991, 199, 263-269; A. Tar et al. "Purification of bovine retinal cGMP phosphodiesterase", Methods in Enzymology, 1994, 238, 3-12; and/or D. Srivastava et al. "Effects of magnesium on cyclic GMP hydrolysis by the bovine retinal rod cyclic GMP phosphodiesterase", Biochem. J, 1995, 308, 653-658. Inhibition ofPDE 3, PDE 4B, PDE 4D, PDE 5 or PDE 6 activity: radioactive Scintillation Proximity Assay (SPA)

The ability of compounds to inhibit catalytic activity at PDE4B or 4D (human recombinant), PDE3 (from bovine aorta), PDE5 (human recombinant) or PDE6 (from bovine retina) can optionally be determined by Scintillation Proximity Assay (SPA) in a 96-well format. Test compounds (as a solution in DMSO, preferably about 2 microlitre (ul) volume of DMSO solution) are preincubated at ambient temperature (room temperature, e.g. 19-230C) in Wallac Isoplates (code 1450-514) with PDE enzyme in 5OmM Tris-HCl buffer pH 7.5 , 8.3mM MgCl2, 1.7mM EGTA, 0.05% (w/v) bovine serum albumin for 10- 30 minutes (usually 30 minutes). The enzyme concentration is adjusted so that no more than 20% hydrolysis of the substrate defined below occurs in control wells without compound, during the incubation. For the PDE3, PDE4B and PDE4D assays, [5',8- 3H]Adenosine 3',5'-cyclic phosphate (Amersham Pharmacia Biotech, code TRK.559; or Amersham Biosciences UK Ltd, Pollards Wood, Chalfont St Giles, Buckinghamshire HP8 4SP, UK) is added to give 0.05uCi per well and about 1OnM final concentration. For the PDE5 and PDE6 assays, [8-3H]Guanosine 3 ',5 '-cyclic phosphate (Amersham Pharmacia Biotech, code TRK.392) is added to give 0.05uCi per well and about 36nM final concentration. Plates containing assay mixture, preferably approx. 100 ul volume of assay mixture, are mixed on an orbital shaker for 5 minutes and incubated at ambient temperature for 1 hour. Phosphodiesterase SPA beads (Amersham Pharmacia Biotech, code RPNQ 0150) are added (aboutlmg per well) to terminate the assay. Plates are sealed and shaken and allowed to stand at ambient temperature for 35 minutes to 1 hour (preferably 35 minutes) to allow the beads to settle. Bound radioactive product is measured using a WALLAC TRILUX 1450 Microbeta scintillation counter. For inhibition curves, 10 concentrations (e.g. 1.5nM - 3OuM) of each compound are assayed. Curves are analysed using ActivityBase and XLfit (ID Business Solutions Limited, 2 Ocean Court, Surrey Research Park, Guildford, Surrey GU2 7QB, United Kingdom) Results are expressed as pIC50 values.

In an alternative to the above radioactive SPA assay, PDE4B or PDE4D inhibition can be measured in the following Fluorescence Polarisation (FP) assay:

Inhibition ofPDE4B or PDE4D activity: Fluorescence Polarisation (FP) assay

The ability of compounds to inhibit catalytic activity at PDE4B (human recombinant) or PDE4D (human recombinant) can optionally be determined by EVIAP Fluorescence Polarisation (FP) assay (DVIAP Explorer kit, available from Molecular Devices Corporation, Sunnydale, CA, USA; Molecular Devices code: R8062) in a 384- well format. The IMAP FP assay is able to measure PDE activity in an homogenous, non- radioactive assay format. The FP assay uses the ability of immobilised trivalent metal cations, coated onto nanoparticles (tiny beads), to bind the phosphate group of Fl-AMP that is produced on the hydrolysis of fluorescein-labelled (Fl) cyclic adenosine mono¬ phosphate (Fl-cAMP) to the non-cyclic Fl-AMP form. Fl-cAMP substantially does not bind. Binding of Fl-AMP product to the beads (coated with the immobilised trivalent cations) slows the rotation of the bound Fl-AMP and leads to an increase in the fluorescence polarisation ratio of parallel to perpendicular light. Inhibition of the PDE reduces/inhibits this signal increase. Test compounds (small volume, e.g. ca. 0.5 to 1 microlitres (ul), preferably ca. 0.5 ul, of solution in DMSO) are preincubated at ambient temperature (room temperature, e.g. 19-23°C) in black 384-well microtitre plates (supplier: NUNC, code 262260) with PDE enzyme in 1OmM Tris-HCl buffer pH 7.2, 1OmM MgCl2, 0.1% (w/v) bovine serum albumin, and 0.05% NaN3 for 10-30 minutes. The enzyme level is set by experimentation so that reaction is linear throughout the incubation. Fluorescein adenosine 3 ',5 '-cyclic phosphate (from Molecular Devices Corporation, Molecular Devices code: R7091) is added to give about 4OnM final concentration (final assay volume usually ca. 20-40 ul, preferably ca. 20 ul). Plates are mixed on an orbital shaker for 10 seconds and incubated at ambient temperature for 40 minutes. BVIAP binding reagent (as described above, from Molecular Devices Corporation, Molecular Devices code: R7207) is added (60ul of a 1 in 400 dilution in binding buffer of the kit stock solution) to terminate the assay. Plates are allowed to stand at ambient temperature for 1 hour. The Fluorescence Polarisation (FP) ratio of parallel to perpendicular light is measured using an Analyst^ plate reader (from Molecular Devices Corporation). For inhibition curves, 10 concentrations (e.g. 1.5nM - 3OuM) of each compound are assayed. Curves are analysed using ActivityBase and XLfit (ID Business Solutions Limited, 2 Ocean Court, Surrey Research Park, Guildford, Surrey GU2 7QB, United Kingdom). Results are expressed as pIC50 values.

In the FP assay, all reagents are generally dispensed using MultidropTM (available from Thermo Labsystems Oy, Ratastie 2, PO Box 100, Vantaa 01620, Finland).

For a given PDE4 inhibitor, the PDE4BL(or PDE4D) inhibition values measured using the SPA and FP assays can differ slightly. However, in a regression analysis of 100 test compounds (not necessarily compounds of the invention), the PIC5Q inhibition values measured using SPA and FP assays have been found generally to agree within about 0.5 log units, for each of PDE4B and PDE4D (linear regression coefficient 0.966 for PDE4B and 0.971 for PDE4D; David R.Mobbs et al, "Comparison of the EVLAP Fluorescence Polarisation Assay with the Scintillation Proximity Assay for Phosphodiesterase Activity", poster presented at 2003 Molecular Devices UK & Europe User Meeting, 2nd October 2003, Down Hall, Harlow, Essex, United Kingdom).

Biological Data obtained for some of the Examples (PDE4B inhibitory activity, either as one reading (n = 1) or as an average of 2 or more readings (n = 2 or more)) are as follows, based on current measurements only, and using the above or similar or analogous assay methods. In each of the SPA and FP assays, absolute accuracy of measurement is not possible, and the readings given are generally accurate only up to about ± 0.5 of a log unit, depending on the number of readings made and averaged:

Example number PDE4B pICso (± about 0.5) 1, 2 9.1 to 9.2 3, 4, 5, 8 8.5 to 9.0 9, 46 6.7 to 6.8 10 to 22, 24, 26 8.6 to 9.9 23, 25, 27 to 32, 34 to 44 8.2 to 9.6 45, 47 8.3 to 8.9 48 to 68 7.9 to 8.95 69, 70 about 8.0 71, 72 7.0 to 7.5

Emesis: Some known PDE4 inhibitors can cause emesis and/or nausea to greater or lesser extents, e.g. after systemic exposure (e.g. see Z. Huang et al, Current Opinion in Chemical Biology, 2001, 5: 432-438, see especially pages 433-434 and refs cited therein). Therefore, it would be preferable, but not essential, if a PDE4 inhibitory compound or salt of the invention were to cause only limited or manageable emetic side-effects, e.g. after external topical, oral or parenteral administration. Emetic side-effects can for example be measured by the emeto genie potential of the compound or salt when administered to ferrets; for example one can measure the time to onset, extent, frequency and/or duration of vomiting, retching and/or writhing in ferrets after oral or parenteral administration of the compound or salt. See for example In vivo Assay 4 hereinafter for a measurement method for anti-inflammatory effect, emetic side-effects and therapeutic index (TI) in the ferret. See also for example A. Robichaud et al., "Emesis induced by inhibitors of [PDE IV] in the ferret", Neuropharmacology, 1999, 38, 289-297, erratum Neuropharmacology, 2001, 40, 465-465. However, optionally, emetic side-effects and therapeutic index (TI) after oral administration in rats can be conveniently measured by monitoring the pica feeding behaviour of rats after administration of the compound or salt of the invention (see In Vivo Assay 2 below).

Other side effects: Some known PDE4 inhibitors can cause other side effects such as headache and other central nervous sytem (CNS-) mediated side effects; and/or gastrointestinal (GI) tract disturbances. Therefore, it would be preferable but not essential if a particular PDE4 inhibitory compound or salt of the invention were to cause only limited or manageable side-effects in one or more of these side-effect categories. In Vivo Biological Assays

The in vitro enzymatic PDE4B inhibition assay(s) described above or generally similar assays should be regarded as being the primary test(s) of biological activity. However, some additional in vivo biological tests, which are optional, which are not an essential measure of activity, efficacy or side-effects, and which have not necessarily been carried out, are described below.

In Vivo Assay A: Activity of topically-applied compounds in apig model of atopic dermatitis: Effect of compounds, applied by skin topical administration, on the dinitrofluorobenzene (DNFB)-induced delayed type hypersensitivity (DTH) response inpigs

General Study Design:

The pig DTH (delayed type hypersensitivity) model of contact hypersensitivity utilizes the Th2-mediated inflammatory response in pig skin to mimic the pathology of atopic dermatitis in humans. The model measures the potential anti-inflammatory effect of compounds, topically-applied to the skin, on the acute DTH (delayed type hypersensitivity) response in castrated male Yorkshire pigs.

In general in the assay, pigs (domestic Yorkshire pigs, 15-18 kg at time of sensitization, castrated males) are first sensitized by topical application of ca. 10% (w/v) dinitrofluorobenzene (DNFB) dissolved in DMSO:acetone:olive oil (ca. 1:5:3) (ca. 40 mg DNFB, 400 microlitre solution total) to the ears (outer) and groin (inner). The pigs are then challenged 12 days later with ca. 0.6% (w/v) DNFB applied to randomized sites on the shaved back of the pigs (ca. 90 micrograms/site; sites are identified and numbered by grid made with marking pen).

On the day of challenge, the treatments are performed at the challenge sites at about 2 hours prior to and about 6 hours after challenge (for DMSO / acetone solutions/suspensions containing the PDE4 inhibitor, to maximize exposure to drug), or at about 30 minutes after and about 6 hours after challenge (for topical ointments or creams containing the PDE4 inhibitor, representing a more clinically relevant treatment protocol).

One day (about 24 hrs) after challenge, and optionally again at ca. 48 hrs post challenge, test sites are visually evaluated for intensity and extent of erythema by measuring the diameter of the reaction at its widest point and assigning scores of 0 to 4 for each of erythema intensity and erythema extent. Induration (a measure of swelling) is also scored 0 to 4. Scores for erythema intensity, erythema extent and induration are assigned according to the following criteria: Intensity of Erythema: 0=normal, l^minimal, barely visible, 2=mild, 3=moderate, 4=severe. Extent of Erythema (not raised): 0=no edema, l=macules of pin head size, 2=lentil sized macules, 3=confluent macules, 4==diffuse over entire site. Induration (palpable): 0=normal, l=nodules of pin head size, 2=doughy lentil sized nodules, 3=confluent firm nodules, 4=diffuse hard lesion. The summed visual score at ca. 24 hours includes the individual scores for erythema intensity, erythema extent, and induration; so the maximal summed score for each site would be 12. High summed scores can generally indicate a high inflammatory response. Visual scores are subject to some inaccuracy / error.

Differences in the summed score between adjacent control (placebo) and treatment sites on the grids are calculated. This difference value is then used to determine the percent inhibition compared to the summed score for the control (placebo) sites. The more negative the difference value, the greater the calculated inhibition. Percent inhibition of (percent inhibition compared to) the mean summed score can be calculated.

About 24 hours after challenge, treatment sites can optionally also be visually evaluated for lesion area.

Specific study design and results:

The anti-inflammatory effect of the compound 4-{[l-(aminocarbonyl)-4- piperidinyl] amino }-N-[(3 ,4-dimethylphenyl)methyl]- 1 -ethyl- 1H-pyrazolo[3 ,4-b]pyridine- 5-carboxamide ("Example 2 or 2A compound"), applied topically to the skin, on the acute DTH response in castrated male Yorkshire pigs is compared to that of another PDE4 inhibitor cipamfylline (BRL-61063) and the topical immunomodulator pimecrolimus.

In the above assay, the Example 2 or 2A compound is topically administered: either (A) at ca. 2.5% (w/v) concentration in a solution of ca. 10% DMSO / 90% acetone (ca. 50 microlitres or ca. 1.5 mg / site) at about 2 hours prior to and about 6 hours after the DNFB challenge; or (B) at ca. 0.5% (w/w) concentration in an ointment containing propylene glycol (PG)* (ca. 25 mg of formulation / site) at about 30 minutes after and about 6 hours after the DNFB challenge. (* The PG ointment used or usable with the Example 2 or 2 A compound generally contains inter alia approximately the following constituents [in % (w/w)]: ca. 69-70% white petrolatum, ca. 5% mineral oil, ca. 5% polyoxyl stearyl ether (e.g. Volpo S2) and ca. 20% propylene glycol. See for example Composition Example Cl hereinafter for a suitable ointment formulation.)

The Example 2 or 2A compound at ca. 0.5% (w/w) in PG ointment inhibits the mean summed score by about 13-14% compared to the placebo PG ointment (p<0.05 by ANOVA). The Example 2 or 2A compound administered at ca. 2.5% (w/v) in DMSO/acetone solution gives a reduction of about 21% in the mean summed score ^ - 62 -

compared to vehicle alone (p<0.05 by ANOVA). In comparison, cipamfylline at ca. 2.5% (w/v) in DMSO/acetone and cipamfylline at ca. 0.25% (w/w) in ointment reduces the summed scores by about 8% and about 6-7%, respectively (effects that are not statistically significant), whereas pimecrolimus applied in a 1% (w/w) cream formulation inhibits the summed scores by about 29% (p<0.05 by ANOVA).

Further, the skin adminitration of the Example 2 or 2A compound appears to decrease the lesion area when added in DMSO/acetone or PG ointment (results subject to some inaccuracy / error).

These results appear to demonstrate the potential anti-inflammatory activity of the Example 2 or 2A compound ("free base" form) in an acute pig DTH (delayed type hypersensitivity) model when the compound is topically administered to the skin in a suitable formulation.

In Vivo Assay 1. LPS-induced pulmonary neutrophilia in rats: effect of orally administered PDE4 inhibitors

Pulmonary neutrophil influx is thought to be a significant component to the family of pulmonary diseases like chronic obstructive pulmonary disease (COPD) which can involve chronic bronchitis and/or emphysema (G.F. Filley, Chest. 2000; 117(5); 25Is- 260s). The purpose of this neutrophilia model is to study the potentially anti¬ inflammatory effects in vivo of orally administered PDE4 inhibitors on neutrophilia induced by inhalation of aerosolized lipopolysaccharide (LPS), modelling the neutrophil inflammatory component(s) of COPD. See the literature section below for scientific background. Male Lewis rats (Charles River, Raleigh, NC, USA) weighing approximately 300- 400 grams are pretreated with either (a) test compound, for example suspended in about 0.5% methylcellulose (obtainable from Sigma-Aldrich, St Louis, MO, USA) in water or (b) vehicle only, delivered orally in a dose volume of ca. 10 ml/kg. Generally, dose response curves can for example be generated using the following approx. doses of PDE4 inhibitors: 2.0, 0.4, 0.08, 0.016 and 0.0032 mg/kg. About thirty minutes following pretreatment, the rats are exposed to aerosolized LPS (Serotype E. CoIi 026:B6 prepared by trichloroacetic acid extraction, obtainable from Sigma-Aldrich, St Louis, MO, USA), generated from a nebulizer containing a ca. 100 μg/ml LPS solution (ca. 100 ug/ml). Rats are exposed to the LPS aerosol at a rate of ca. 4 L/min for ca. 20 minutes. LPS exposure is carried out in a closed chamber with internal dimensions of roughly 45 cm length x 24 cm width x 20 cm height. The nebulizer and exposure chamber are contained in a certified fume hood. At about 4 hours-post LPS exposure the rats are euthanized by overdose with pentobarbital at ca. 90 mg/kg, administered intraperitoneally. Bronchoalveolar lavage (BAL) is performed through a 14 gauge blunt needle into the exposed trachea. Five, 5 ml washes are performed to collect a total of 25 ml of BAL fluid. Total cell counts and leukocyte differentials are performed on BAL fluid in order to calculate neutrophil influx into the lung. Percent neutrophil inhibition at each dose (cf. vehicle) is calculated and a variable slope, sigmoidal dose-response curve is generated, usually using Prism Graph-Pad. The dose-response curve is used to calculate an ED50 - -

value (in mg per kg of body weight) for inhibition by the PDE4 inhibitor of the LPS- induced neutrophilia. Alternative method : In an alternative simpler embodiment of the procedure, a single oral dose of 10 mg/kg or more usually 1.0 mg/kg or 0.3 mg/kg of the PDE4 inhibitor (or vehicle) is administered to the rats, and percent neutrophil inhibition is calculated and reported for that specific dose. Literature: Filley G.F. Comparison of the structural and inflammatory features of COPD and asthma. Chest. 2000; 117(5) 251s-260s. Howell RE, Jenkins LP, Fielding LE, and Grimes D. Inhibition of antigen- induced pulmonary eosinophilia and neutrophilia by selective inhibitors of phosphodiesterase types 3 and 4 in brown Norway rats. Pulmonary Pharmacology. 1995; 8: 83-89. Spond J, Chapman R, Fine J, Jones H, Kreutner W, Kung TT, Minnicozzi M. Comparison of PDE 4 inhibitors, Rolipram and SB 207499 (Ariflo™), in a rat model of pulmonary neutrophilia. Pulmonary Pharmacology and Therapeutics. 2001; 14: 157- 164. Underwood DC, Osborn RR, Bochnowicz S, Webb EF, Rieman DJ, Lee JC, Romanic AM, Adams JL, Hay DWP, and Griswold DE. SB 239063, ap38 MAPK inhibitor, reduces neutrophilia, inflammatory cytokines, MMP-9, and fibrosis in lung. Am J Physiol Lung Cell MoI Physiol. 2000; 279: L895-L902.

In Vivo Assay 2. Rat Pica Model of etnesis

Background: Selective PDE4 inhibitors are thought to inhibit inflammation in various in vitro and in vivo models by increasing intracellular levels of cAMP of many immune cells (e.g. lymphocytes, monocytes). However, a side effect of some PDE4 inhibitors in some species is emesis. Because many rat models of inflammation are well characterized, they can be used in procedures (see e.g. In Vivo Assay 1 above) to show beneficial anti-inflammatory effects of PDE 4 inhibitors. However rats have no emetic response (they have no vomit reflex), so that the relationship between beneficial anti¬ inflammatory effects of PDE 4 inhibitors and emesis is difficult to study directly in rats. However, in 1991, Takeda et al. (see Literature section below) demonstrated that the pica feeding response is analogous to emesis in rats. Pica feeding is a behavioural response to illness in rats wherein rats eat non-nutritive substances such as earth or in particular clay (e.g. kaolin) which may help to absorb toxins. Pica feeding can be induced by motion and chemicals (especially chemicals which are emetic in humans), and can be inhibited pharmacologically with drugs that inhibit emesis in humans. The Rat Pica Model, In Vivo Assay 2, can determine the level of pica response of rats to PDE 4 inhibition at pharmacologically relevant doses in parallel to in vivo anti-inflammatory Assays in (a separate set of) rats (e.g. In Vivo Assay 1 above). Anti-inflammatory and pica assays in the same species together can provide data on the "therapeutic index" (TI) in the rat of the compounds/salts of the invention. The Rat TI can for example be calculated as the ratio of a) the potentially-emetic Pica Response ED50 dose from Assay 2 to b) the rat anti-inflammatory ED50 dose (e.g. measured by rat neutrophilia-inhibition in eg In Vivo Assay 1), with larger TI ratios possibly indicating lower emesis at many anti-inflammatory doses. This might allow a choice of a non-emetic or low-emetic pharmaceutical dose of the compounds or salts of the invention which has an anti-inflammatory effect. It is recognised however that achieving a low-emetic PDE4 inhibitory compound is not essential to the invention. Procedure: On the first day of the experiment, the rats are housed individually in cages without bedding or "enrichment". The rats are kept off of the cage floor by a wire screen. Pre-weighed food cups containing standard rat chow and clay pellets are placed in the cage. The clay pellets, obtainable from Languna Clay Co, City of Industry, CA, USA, are the same size and shape as the food pellets. The rats are acclimated to the clay for 72 hours, during which time the cups and food and clay debris from the cage are weighed daily on an electronic balance capable of measuring to the nearest 0.1 grams. By the end of the 72 hour acclimation period the rats generally show no interest in the clay pellets. At the end of 72 hours the rats are placed in clean cages and the food cups weighed. Rats that are still consuming clay regularly are removed from the study. Immediately prior to the dark cycle (the time when the animals are active and should be eating) the animals are split into treatment groups and dosed orally with a dose of a compound or salt of the invention (different doses for different treatment groups) or with vehicle alone, at a dose volume of ca. 2 ml/kg. In this oral dosing, the compound/salt can for example be in the form of a suspension in about 0.5% methylcellulose (obtainable Sigma- Aldrich, St. Louis, MO, USA) in water. The food and clay cups and cage debris are weighed the following day and the total clay and food consumed that night by each individual animal is calculated. A dose response is calculated by first converting the data into quantal response, where animals are either positive or negative for the pica response. A rat is "pica positive" if it consumes greater than or equal to 0.3 grams of clay over the mean of its control group. The D50 value is usually calculated using logistic regression performed by the Statistica software statistical package. A Pica Response ED50 value in mg per kg of body weight can then be calculated. The Pica Response ED50 value can be compared to the neutrophilia-inliibition ED50 values for the same compound administered orally to the rat (measurable by In Vivo Assay 1 above), so that a Therapeutic Index (TI) in rats can be calculated thus:

Rat Therapeutic index (TI) (50/50) = Pica Response ED50 value rat neutrophilia-inhibition ED50 value

In general, the Therapeutic Index (TI) calculated this way is often substantially different to, and for example can often be substantially higher than, the TI (D20/D50) calculated in the ferret (see hi vivo Assay 4 below). Alternatively, e.g. for a simpler test, the In Vivo Assay 2 (pica) can use only a single oral dose of the test compound (e.g. 10 mg/kg orally). Literature: Beavo JA, Contini, M., Heaslip, RJ. Multiple cyclic nucleotide phosphodiesterases. MoIPharmacol. 1994; 46:399-405. Spond J, Chapman R, Fine J, Jones H, Kreutner W, Kung TT, Minnicozzi M. Comparison of PDE 4 inhibitors, Rolipram and SB 207499 (Ariflo™), in a rat model of pulmonary neutrophilia. Pulmonary Pharmacology and Therapeudtics. 2001; 14:157- 164. Takeda N, Hasegawa S, Morita M, and Matsunaga T. Pica in rats is analogous to emesis: an animal model in emesis research. Pharmacology, Biochemistry and Behavior. 1991; 45:817-821. Takeda N, Hasegawa S, Morita M, Horii A, Uno A, Yamatodani A and Matsunaga T. Neuropharmacological mechanisms of emesis. I . Effects of antiemetic drugs on motion- and apomorphine-induced pica in rats. Meth Find Exp CHn Pharmacol. 1995; 17(9) 589-596. Takeda N, Hasegawa S, Morita M, Horii A, Uno A, Yamatodani A and Matsunaga T. Neuropharmacological mechanisms of emesis. II . Effects of antiemetic drugs on cisplatin-induced pica in rats. Meth Find Exp CHn Pharmacol. 1995; 17(9) 647-652.

In Vivo Assay 3. LPS induced pulmonary neutrophilia in rats: effect of intratracheally administered PDE4 inhibitors This assay is an animal model of inflammation in the lung - specifically neutrophilia induced by lipopolysaccharide (LPS) - and allows the study of putative inhibition of such neutrophilia (anti-inflammatory effect) by intratracheally (i.t.) administered PDE4 inhibitors. The PDE4 inhibitors are preferably in dry powder or wet suspension form. Lt. administration is one model of inhaled administration, allowing topical delivery to the lung. Animals: Male CD (Sprague Dawley Derived) rats supplied by Charles River, Raleigh, NC, USA or Charles River, United Kingdom are housed in groups of 5 rats per cage, acclimatised after delivery for at least 5 days with bedding/nesting material regularly changed, fed on SDS diet Rl pelleted food given ad lib, and supplied with daily-changed pasteurised animal grade drinking water. Device for dry powder administration: Disposable 3-way tap between dosing needle and syringe. The intratracheal dosing device (a 3-way sterile tap, Vycon 876.00; or Penn Century dry powder insufflator, DP-4) is weighed, the drug blend or inhalation grade lactose (vehicle control) is then added to the tap, the tap is closed to prevent loss of drug, and the tap is re-weighed to determine the weight of drug in the tap. After dosing, the tap is weighed again to determine the weight of drug that had left the tap. The needle, a Sigma Z21934-7 syringe needle 19-gauge 152 mm (6 inches) long with luer hub, is cut by engineering to approximately 132 mm (5.2 inches), a blunt end is made to prevent them damaging the rat's trachea, and the needle is weighed prior to and after drug delivery to confirm that no drug is retained in the needles after dosing. Device for wet suspension administration: This is similar to the above but a blunt dosing needle, whose forward end is slightly angled to the needle axis, is used, with a flexible plastic portex canula inserted into the needle. Drugs and Materials: Lipopolysaccharide (LPS) (Serotype:0127:B8) is dissolved in phosphate-buffered saline (PBS). PDE4 inhibitors are preferably used in size-reduced (e.g. micronised) form, for example according to the Micronisation Example given herein. - OO -

For dry powder administration of the drug, the Dry Powder Formulation Example given herein, comprising drug and inhalation-grade lactose, can optionally be used. One suitable inhalation-grade lactose that can be used has 10% fines (10% of material under 15um (15 micron) particle size measured by Malvern particle size). Wet suspensions of the drug (aqueous) can be prepared by adding the required volume of vehicle to the drug; the vehicle used can for example be saline alone or a mixture of saline/tween (e.g. 0.2% tween 80). The wet suspension is usually sonicated for ca. 10 minutes prior to use. Preparation, and dosing with PDE 4 inhibitor: Rats are anaesthetised by placing the animals in a sealed Perspex chamber and exposing them to a gaseous mixture of isoflourane (4.5 %), nitrous oxide (3 litres.mmute"1) and oxygen (1 litre.minute"1). Once anaesthetised, the animals are placed onto a stainless steel i.t. dosing support table. They are positioned on their back at approximately a 35° angle. A light is angled against the outside of the throat to highlight the trachea. The mouth is opened and the opening of the upper airway visualised. The procedure varies for wet suspension and dry powder administration of PDE4 inhibitors as follows: Dosing with a Wet suspension: A portex cannula is introduced via a blunt metal dosing needle that has been carefully inserted into the rat trachea. The animals are intratracheally dosed with vehicle or PDE4 inhibitor via the dosing needle with a new internal canula used for each different drug group. The formulation is slowly (ca. 10 seconds) dosed into the trachea using a syringe attached to the dosing needle. Dosing with a Dry Powder: The intratracheal dosing device (a three-way sterile tap device, Vycon 876.00; or Perm Century dry powder insufflator, DP -4) and needle are inserted into the rat trachea up to a pre-determined point established to be located approximately 1 cm above the primary bifurcation. Another operator holds the needle at the specified position whilst 2 x 4ml of air (using 3-way tap device) is delivered through the three-way tap by depressing the syringes (ideally coinciding with the animal inspiring), aiming to expel the entire drug quantity from the tap. (Alternatively, 2 x 3ml of air is delevered using Perm Century dry powder insufflator device.). After dosing, the needle and tap or device are removed from the airway and the tap closed off to prevent any retained drug leaving the tap. After dosing with either wet suspension or dry powder, the animals are then removed from the table and observed constantly until they have recovered from the effects of anaesthesia. The animals are returned to the holding cages and given free access to food and water; they are observed and any unusual behavioural changes noted. Exposure to LPS: About 2 hours after i.t. dosing with vehicle control or the PDE4 inhibitor, the rats are placed into sealed Perspex containers and exposed to an aerosol of LPS (nebuliser concentration ca. 150 μg.ml"1 = 150 ug/ml) for ca. 15 minutes. Aerosols of LPS are generated by a nebuliser (DeVilbiss, USA) and this is directed into the Perspex exposure chamber. Following the 15-minute LPS-exposure period, the animals are returned to the holding cages and allowed free access to both food and water. - O l -

[In an alternative embodiment, the rats can be exposed to LPS less than 2 hours (e.g. about 30 minutes) after i.t. dosing. In another alternative embodiment, the rats can be exposed to LPS more than 2 hours (e.g. ca. 4 to ca. 24 hours) after i.t. dosing by vehicle or PDE4 inhibitor, to test whether or not the PDE4 inhibitor has a long duration of action (which is not essential).] Bronchoalveolar lavage: About 4 hours after LPS exposure the animals are killed by overdose of sodium pentobarbitone (Lp.). The trachea is cannulated with polypropylene tubing and the lungs are lavaged (washed out) with 3 x 5 mis of heparinised (25 units.ml"1) phosphate buffered saline (PBS). Neutrophil cell counts: The Bronchoalveolar lavage (BAL) samples are centrifuged at ca. 1300 rpm for ca. 7 minutes. The supernatant is removed and the resulting cell pellet resuspended in ca. 1 ml PBS. A cell slide of the resuspension fluid is prepared by placing ca. lOOμl (ca. 10OuI) of resuspended BAL fluid into cytospin holders and then is spun at ca. 5000 rpm for ca. 5 minutes. The slides are allowed to air dry and then stained with Leishmans stain (ca. 20 minutes) to allow differential cell counting. The total cells are also counted from the resuspension. From these two counts, the total numbers of neutrophils in the BAL are determined. For a measure of PDE4-inhibitor- induced inhibition of neutrophilia, a comparison of the neutrophil count in rats treated with vehicle and rats treated with PDE4 inhibitors is conducted. By varying the dose of the PDE4 inhibitor used in the dosing step (e.g. 0.2 or 0.1 mg of PDE4 inhibitor per kg of body weight, down to e.g. 0.01 mg/kg), a dose-response curve can be generated.

In Vivo Assay 4. Evaluation of Therapeutic Index of Orally-administered PDE 4 inhibitors in the conscious ferret 1.1 Materials The following materials can be used for these studies: PDE4 inhibitors are prepared for oral (p.o.) administration by dissolving in a fixed volume (ca. 1 ml) of acetone and then adding cremophor to ca. 20% of the final volume. Acetone is evaporated by directing a flow of nitrogen gas onto the solution. Once the acetone is removed, the solution is made up to final volume with distilled water. LPS is dissolved in phosphate buffered saline. 1.2 Animals Male ferrets (Mustela Pulorius Furo, weighing 1 - 2 kg) are transported and allowed to acclimatise for not less than 7 days. The diet comprises SDS diet C pelleted food given ad lib with Whiskers^M ca{ fooc[ g1Ven 3 times per week. The animals are supplied with pasteurised animal grade drinking water changed daily. 1.3 Experimental Protocol(s) 1.3.1 Dosing with PDE4 inhibitors PDE4 inhibitors are administered orally (p.o.), using a dose volume of ca. lml/kg. Ferrets are fasted overnight but allowed free access to water. The animals are orally dosed with vehicle or PDE 4 inhibitor using a ca. 15cm dosing needle that is passed down - -

the back of the throat into the oesophagus. After dosing, the animals are returned to holding cages fitted with perspex doors to allow observation, and given free access to water. The animals are constantly observed and any emetic episodes (retching and vomiting) or behavioural changes are recorded. The animals are allowed access to food ca. 60 - 90 minutes after p.o. dosing. 1.3.2 Exposure to LPS About thirty minutes after oral dosing with compound or vehicle control, the ferrets are placed into sealed perspex containers and exposed to an aerosol of LPS (ca. 30 μg/ml = ca. 30 ug/ml) for ca. 10 minutes. Aerosols of LPS are generated by a nebuliser (DeVilbiss, USA) and this is directed into the perspex exposure chamber. Following a 10-minute exposure period, the animals are returned to the holding cages and allowed free access to water, and at a later stage, food. General observation of the animals continues for a period of at least 2.5 hours post oral dosing. All emetic episodes and behavioural changes are recorded. 1.3.3 Bronchoalveolar lavage and cell counts About six hours after LPS exposure the animals are killed by overdose of sodium pentobarbitone administered intraperitoneally. The trachea is then cannulated with polypropylene tubing and the lungs lavaged twice with ca. 20 ml heparinised (10 units/ml) phosphate buffered saline (PBS). The bronchoalveolar lavage (BAL) samples are centrifuged at ca. 1300 rpm for ca. 7 minutes. The supernatant is removed and the resulting cell pellet re-suspended in ca. 1 ml PBS. A cell smear of re-suspended fluid is prepared and stained with Leishmans stain to allow differential cell counting. A total cell count is made using the remaining re-suspended sample. From this, the total number of neutrophils in the BAL sample is determined. 1.3.4 Pharmacodynamic readouts The following parameters are recorded: a) % inhibition of LPS-induced pulmonary neutrophilia to determine the dose of PDE4 inhibitor which gives 50% inhibition (D50). b) Emetic episodes - the number of vomits and retches are counted to determine the dose of PDE4 inhibitor that gives a 20% incidence of emesis (D20). c) A therapeutic index (TI), using this assay, is then calculated for each PDE4 inhibitor using the following equation:

Ferret Therapeutic index (TI) (D20/D50) = D20 incidence of emesis in ferret D50 inhibition of neutrophilia in ferret

It is noted that the Ferret Therapeutic index (TI) (D20/D50) calculated using this in vivo Assay 4 is often substantially different to, and for example is often substantially lower than, the Rat TI (50/50) calculated using the rat oral inflammation and pica feeding Assays 1+2. All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth. EXAMPLES

The various aspects of the invention will now be described by reference to the following examples. These examples are merely illustrative and are not to be construed as a limitation of the scope of the present invention. In this section, "Intermediates" represent syntheses of intermediate compounds intended for use in the synthesis of the "Examples". "Examples" are generally exemplary compounds or salts of the invention, for example a compound of formula (I) or a salt thereof. The "Composition Examples" are non-limiting illustrations of the pharmaceutical compositions of the invention.

Abbreviations used herein:

DCM dichloromethane DIPEA diisopropylethyl amine (ip^NEt) DMF dimethyl formamide DMSO dimethyl sulfoxide EDC l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EtOAc ethyl acetate EtOH ethanol h hours HATU O-(7-Azabenzotriazol- 1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HCl hydrogen chloride or hydrochloric acid HOBT hydroxybenzotriazole = 1-hydroxybenzotriazole M molarity, or moles per litre MeCN acetonitrile MeOH methanol NaHCO3 sodium bicarbonate NaOH sodium hydroxide Na2SO4 sodium sulfate KOH potassium hydroxide THF tetrahydrofuran HPLC high performance liquid chromatography SPE solid phase extraction NMR nuclear magnetic resonance (in which: s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, m = multiplet, H = no. of protons) LCMS liquid chromatography/mass spectroscopy TLC thin layer chromatography h hours TRET retention time (generally from LCMS) Room (ambient) temperature this is usually in the range of about 20 to about 25 °C. General Experimental Details

Machine Methods generally used herein:

LCMS (liquid chromatography/mass spectroscopy) Waters ZQ mass spectrometer operating in positive ion electrospray mode, mass range 100-1000 amu. UV wavelength : 215-33OnM Column : 3.3cm x 4.6mm ID, 3μm ABZ+PLUS Flow Rate : 3ml/min Injection Volume : 5μl Solvent A : 95% acetonitrile + 0.05% formic acid Solvent B : 0.1% formic acid + lOmMolar ammonium acetate Gradient : 0% A/0.7min, 0-100% A/3.5min, 100% A/l.lmin, 100-0% A/0.2min It should be noted that retention times (TRET) quoted herein may vary slightly (+/- O.lmin.) when samples are run on different Waters machines, even when the same type of column and identical flow rates, injection volumes, solvents and gradients are used.

LCMS (liquid chromatography/mass spectroscopy) (for Intermediates 2 A, 3 A, 4 A and 5 A, and Example 2B only) Agilent 1100 mass spectrometer operating in positive ion electrospray mode, mass range 100-700 amu. UV wavelength : 214-254nM Column : 2.1cm x 50mm ID, 5μm Zorbax Flow Rate : lml/min Injection Volume : lμl Solvent A : water + 0.02% trifluoroacetic acid Solvent B : acetonitrile + 0.018% trifluoroacetic acid Gradient : 10-80% A/3.0min, 80% A/1.2min, 80-10% A/1.0min

Mass directed autoprep HPLC Prep column: a Supelcosil ABZplus (10cm x 2.12cm) (usually 10cm x 2.12cm x 5 μm). UV wavelength : 200-32OnM Flow : 20ml/min Injection Volume: ImI; or more preferably 0.5 ml Solvent A : 0.1% formic acid Solvent B : 95% acetonitrile + 5% formic acid; or more usually 99.95% acetonitrile + 0.05% formic acid Gradient : 100% A/lmin, 100-80% A/9min, 80-1% A/3.5min, 1% A/1.4min, 1- 100%A/0.1min Autoprep Gilson reverse-phase HPLC (for Example 2B) Prep column: YMC ODS-A (50mm x 50mm) UV wavelength : 215-254nM Flow : 70ml/min Injection Volume: 3ml Solvent A : water Solvent B : acetonitrile Gradient : 35-95% B/5min

Chiral Columns for Chromatographic Purification

ChiralPak AS columns can be obtained from: Chiral Technologies Europe Sari, Illkirch, France (Telephone: 0033(0)388795200; (cte@chiral.fr; www.chiral.fr).

Intermediates and Examples

Reagents not detailed in the text below are usually commercially available from chemicals suppliers, e.g. established suppliers such as Sigma- Aldrich. The addresses and/or contact details of the suppliers for some of the starting materials mentioned in the Intermediates and Examples below or the Assays above, or suppliers of chemicals in general, are as follows:

- Aldrich (catalogue name), Sigma-Aldrich Company Ltd., Dorset, United Kingdom, telephone: +44 1202 733114; Fax: +44 1202 715460; ukcustsv@eurnotes.sial.com: or - Aldrich (catalogue name), Sigma-Aldrich Corp., P.O. Box 14508, St. Louis, MO 63178- 9916, USA; telephone: 314-771-5765; fax: 314-771-5757; custserv@sial.com: or - Aldrich (catalogue name), Sigma-Aldrich Chemie GmbH, Munich, Germany; telephone: +49 89 6513 0; Fax: +49 89 6513 1169; deorders@eurnotes. sial. com. - Apin Chemicals Ltd; 3D Milton Park, Abingdon, Oxon, OX14 4RU, United Kingdom - AstaTech Inc., 8301 Torresdale Ave., 19C, Philadelphia, PA 19136, USA - Fluka Chemie AG, Industriestrasse 25, P.O. Box 260, CH-9471 Buchs, Switzerland - Interchim Intermediates (catalogue name), Interchim, 213 Avenue Kennedy, BP 1140, Montlucon, Cedex, 03103 , France - Lancaster Synthesis Ltd., Newgate, White Lund, Morecambe, Lancashire LA3 3DY, United Kingdom - Matrix Scientific, P.O. Box 25067, Columbia, SC 29224-5067, USA - Maybridge Chemical Company Ltd., Trevillett, Tintagel, Cornwall PL34 OHW, United Kingdom - Trans World Chemicals, Inc., 14674 Southlawn Lane, Rockville, MD 20850, USA - -

Table of Intermediates

Int No Name

1 Ethyl 4-chloro- 1 -ethyl- 1H-pyrazolo[3 ,4-b]pyridine-5-carboxylate

2 Ethyl 4-[(1-{ [(1 , 1-dimethylethyl)oxy]carbonyl}-4-piperidinyl)amino]- 1- ethyl-lH-pyrazolo[3,4-ό]pyridine-5-carboxylate 3 Ethyl l-ethyl-4-(4-piperidinylamino)-lΗ-pyrazolo[3,4-b]pyridine-5 - carboxylate hydrochloride 4 Ethyl 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino} - 1 -ethyl- 1H-pyrazolo[3 ,4- b]pyridine-5 -carboxylate 5 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino }- 1 -ethyl- 1H-pyrazolo[3,4- b]pyridine-5-carboxylic acid 6 4-chloro-l-emyl-lH-pyrazolo[3,4-δ]pyridine-5-carboxylic acid 7 4-chloro-l-ethyl-lH-pyrazolo[3,4-b]pyridine-5-carbonyl chloride 8 4-chloro-N-[(2,4-dimethylphenyl)methyl]-l-ethyl-lH-pyrazolo[ 3,4-b] pyridine-5-carboxamide 9 4-chloro-N-[(3,4-dimethylphenyl)methyl]-l-ethyl-lH-pyrazolo[ 3,4- b]pyridine-5-carboxamide * 10 4-chloro- 1-ethyl-N-{[4-(methyloxy)phenyl]methyl}- 1H-pyrazolo [3,4- b]pyridine-5-carboxamide 11 1 , 1-dimethylethyl [1-(aminocarbonyl)-4-piperidinyl] carbamate 12 4-amino- 1-piperidinecarboxamide hydrochloride 13 1 , 1 -dimethylethyl [4-(aminocarbonyl)cyclohexyl]carbamate 15 4-aminocyclohexanecarboxamide hydrochloride 17 1,1 -dimethylethyl [ds-4-(aminocarbonyl)cyclohexyl]-carbamate 18 1 , 1 -dimethylethyl [tra«>s-4-(aminocarbonyl)cyclohexyl] -carbamate 19 cw-4-aminocyclohexanecarboxamide hydrochloride 20 tr«ns-4-aminocyclohexanecarboxarnide hydrochloride 21 ethyl 4-{ [c/s-4-(aminocarbonyl)cyclohexyl] amino} - 1 -ethyl- IH- pyrazolo[3,4-Z)]pyridme-5-carboxylate 22 ethyl 4-{ [tra«>s-4-(aminocarbonyl)cyclohexyl] amino }- 1 -ethyl- IH- pyrazolo[3,4-ό]pyridine-5-carboxylate 23 4-{ [cw-4-(aminocarbonyl)cyclohexyl] amino} - 1 -ethyl- lH-pyrazolo[3,4- ό]pyridine-5-carboxylic acid 24 4-{[tran5-4-(aminocarbonyl)cyclohexyl]amino}-l-ethyl-lH-pyra zolo[3,4- δ]pyridine-5-carboxylic acid 25 ethyl 4-[((3S)- 1-{ [(1 , 1-dimethylethyl)oxy] carbonyl} -3-pyrrolidinyl)amino]- l-ethyl-lH-pyrazolo[3,4-ό]pyridine-5-carboxylate - -

26 ethyl 4-[((3R)- 1-{ [( 1 , 1-dimethylethyl)oxy]carbonyl}-3-pvrrolidinyl)amino] - l-ethyl-lH-pyrazolo[3,4-δ]pyridine-5-carboxylate 27 ethyl l-ethyl-4-[(3»S)-3-pyrrolidinylaniino]-lH'-pyrazolo[3,4-δ] pyridine-5- carboxylate hydrochloride 28 ethyl l-ethyl-4-[(3R)-3-pyrrolidinylamino]-lH-pyrazolo[3,4-δ]pyri dine-5- carboxylate hydrochloride 29 ethyl 4-{ [(3S)- 1-(aminocarbonyl)-3-pyrrolidinyl] amino }- 1 -ethyl- 1 H- pyrazolo[3,4-b]pyridine-5-carboxylate 30 ethyl 4-{[(3i?)-l-(aminocarbonyl)-3-pyrrolidinyl]arnmo}-l-ethyl-lH - pyrazolo[3 ,4-b]pyridine-5-carboxylate 31 4-{ [(3S)- 1-(aminocarbonyl)-3 -pyrrolidinyl] amino }- 1 -ethyl- lH-pyrazolo[3,4- &]pyridine-5-carboxylic acid 32 4-{ [(3R)- 1-(aminocarbonyl)-3 -pyrrolidinyl] amino }- 1 -ethyl- IH- pyrazolo[3,4-b]pyridine-5-carboxylic acid 33 4-[(l-{[(l,l-dimethylethyl)oxy]carbonyl}-4-piperidinyl)amino ]-l-ethyl-lH- pyrazolo[3,4-&]pyridine-5-carboxylic acid 34 _4- { [1-(aminocarbonyl)-4-piperidinyl] amino } -N- [(2-bromophenyl)methyl]- l-ethyl-lH-pyrazolo[3,4-&]pyridme-5-carboxamide 35 4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2-bromo-4- methylphenyl)methyl]-l-ethyl-lH-pyrazolo[3,4-b]pyridine-5-ca rboxamide

Intermediate 1 ; Ethyl 4-chloro-l-ethyl-lΗ-pyrazolo[3,4-b]pyridine-5-carboxylate

Intermediate 1 is thought to be capable of being prepared from commercially available 5- amino-1-ethyl pyrazole as described by G. Yu et. al. in J Med Chem., 2001, 44, 1025- 1027:

CO2Et NH, N Et

One specific method of preparing Intermediate 1 is as follows:

Intermediate 1 ; Ethyl 4-chloro-l-ethyl~lH-pyrazoIo[3,4-b]pyridine-5-carboxylate

Cl CO2Et A mixture of 5-amino-l -ethyl pyrazole (806g) (e.g. commercially available from Aldrich) and diethyl ethoxymethylenemalonate (1621 ml) (e.g. commercially available from Aldrich) was stirred and heated at 16O0C under nitrogen, in a 5 litre flask fitted with a Dean-Stark apparatus, for 1.5 h. Ethanol that distilled out of the reaction mixture (320ml) was collected in the Dean-Stark apparatus. The reaction mixture was stirred and heated at 16O0C, under nitrogen, for a further 6h, cooled to room temperature and divided into two batches (1200ml + 1000ml: "Batch 1" and "Batch 2"). The first (1200ml) batch ("Batch 1") was divided into two roughly equal portions. Phosphorus oxychloride (1.85 litres) was added to each portion. The reaction mixtures were then heated at reflux in two 51 flasks for 13h. Excess phosphorus oxychloride was distilled from both flasks under reduced pressure. The residues were cooled to room temperature, then the contents of both flasks were poured slowly onto one portion (10kg) of crushed ice. The mixture was stirred for 15 min and then extracted with diethyl ether (3 x 2.5 litres). The combined organics were washed with water (2 litres) and brine (2 x 2 litres), then dried over Na2SO4. Evaporation of the solvent afforded the crude Intermediate 1 as a brown oil (865g) which solidified immediately on cooling. An identical procedure was used to prepare a further 71Og of crude Intermediate 1 as a solid from "Batch 2" using 3.1 litres of phosphorus oxychloride; i.e. a total of 1575g of crude Intermediate 1 was isolated as a solid. This solid (43Og) was dissolved in hexane (4.3 litres, i.e. 10 vols.) by heating to 5O0C with stirring. Activated charcoal (64.5g) was added. The mixture was stirred at 5O0C for 1.0 h, then filtered through a celite bed. The celite bed was washed with hexane (2 x 430 ml). The combined filtrate and the washings were concentrated to about 950ml and left to stand at 10-150C overnight. The resultant suspension was filtered. The residual solid was washed with chilled hexane (3 x 215 ml slurry wash, plus 2 x 400 ml displacement wash) and dried to give Intermediate 1 (28Og) as a pale yellow solid. The combined mother liquor and the washings were concentrated to about 300 ml, then cooled and left to stand at 10-150C overnight to afford an additional 30g of Intermediate 1. LCMS showed MH+ = 254; TRET = 3.09 min.

Intermediate 2: Ethyl 4-[(l-{[(l,l-dimethyIethyI)oxy]carbonyl}-4- piperidiny^aminol-l-ethyl-lJy-pyrazoIoIS^-ftlpyridine-S-carb oxylate

J A solution of Intermediate 1 (0.5g, 2mmol), 1,1-dimethylethyl 4-amino-l- piperidinecarboxylate (0.59g, 2.9mmol) (e.g. available from AstaTech) and DIPEA (0.87ml, 5mmol, 2.5 equivalents) in MeCN (15ml) was heated at reflux for 18h. The reaction mixture was cooled. The solvent was removed under reduced pressure and the residue was partitioned between DCM (50ml) and saturated NaHCO3 solution (40ml). The organic fraction was collected through a hydrophobic frit and concentrated under reduced pressure. The residue was purified by passing through a lOOg silica cartridge, using a gradient of 0% to 100% EtOAc in cyclohexane as the eluent, and the fractions containing the product were concentrated under reduced pressure to yield Intermediate 2 as a solid (0.74g). LCMS showed MH+ = 418; TRET = 3.43min. - 7 -

Alternative optional synthesis: A solution of Intermediate 1 (2.3g) and 1,1-dimethylethyl 4-amino-l-piperidinecarboxylate (2g) in MeCN (50ml) and DIPEA (8.6ml) (optionally also with about 1.5 - 2 ml EtOH) is heated, for example at 85°C or 9O0C, for 16h. The solvents are removed under reduced pressure and the residue is partitioned between DCM (e.g. about 65ml) and water (e.g. about 30ml). The organic fraction is collected through a hydrophobic frit and the solvents are removed under reduced pressure to yield Intermediate 2.

Intermediate 2A: Ethyl 4-[(l-{[(l,l-dimethylethyl)oxy]carbonyl}-4- piperidinyl)amino]-l-ethyl~li?-pyrazolo[3,4-6]pyridine-5-car boxylate

A solution of Intermediate 1 (25g) in MeCN (565ml) was treated with solid 1,1- dimethylethyl 4-amino-l-piperidinecarboxylate (21.7g) and DIPEA (93.4ml, 69.3g). The reaction mixture was heated at 9O0C for 16h. After cooling the reaction mixture, the solvents were removed under reduced pressure and the residue was partitioned between DCM (1100ml) and water (800ml). The organic fraction was dried (MgSO4), was filtered and the solvents were removed under reduced pressure. The residue was subject to flash column chromatography (3:1 hexane / EtOAc) to yield Intermediate 2A as a yellow solid (39.54g). LCMS showed MH+ = 418; TRET = 3.13min.

IH NMR (400 MHz, chloroform-cO δ ppm 9.44 (d, J=7.83 Hz, 1 H), 8.89 (s, 1 H), 7.94 (s, 1 H), 4.49 (q, J-7.33 Hz, 2 H), 4.35 (q, J=7.16 Hz, 2 H), 3.95 - 4.11 (m, 3 H), 3.18 (t, J=10.86 Hz, 2 H), 2.10 - 2.20 (br d, 2 H), 1.62 - 1.73 (m, 2 H), 1.47 - 1.53 (m, 12 H), 1.41 (t, J=7.07 Hz, 3 H).

Intermediate 3: Ethyl l-ethyI-4-(4~piperidinyIamino)-lH-pyrazolo[3,4-b]pyridine-5- carboxylate hydrochloride

Xl H'

Intermediate 2 (4.Ig) was treated with 4.0M hydrogen chloride in 1, 4-dioxane (30ml) and the reaction mixture was stirred at 22°C for Ih. The solvents were removed to give Intermediate 3 as a white solid (4.Og). LCMS showed MH+ = 318; TRET = 2.1min. Intermediate 3A: Ethyl l-ethyl-4-(4-piperidinylamino)-lH-pyrazolo[3,4-A]pyridine- 5-carboxylate hydrochloride

Xl H'

Intermediate 2A (39.54g) was treated with 4M HCl in 1,4-dioxane (300ml) and the reaction mixture was stirred at room temperature for Ih. The solvents were removed and the residue subject to vacuum overnight, to give Intermediate 3 A as a white solid (34.2 Ig, some dioxane still present; from NMR theoretically 33.5g present excluding dioxane). LCMS showed MH+ = 318; TRET = 1.90min.

IH NMR (400 MHz, DMSO-J6) δ ppm 9.33 (d, J=8.08 Hz, 1 H), 9.21 (br s, 1 H), 9.10 (br s, 1 H), 8.74 (s, 1 H), 8.44 (s, 1 H), 4.43 (q, J=7.20 Hz, 2 H), 4.32 (q, J=7.09 Hz, 2 H), 3.20 - 3.35 (m, 4 H), 2.23 (d, J=I1.62 Hz, 2 H), 1.76 - 1.87 (m, 2 H), 1.38 (q, J=7.20 Hz, 3 H), 1.34 (q, J=7.09 Hz, 3 H).

Intermediate 4: Ethyl 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-l-ethyl-lH- pyrazolo[3,4-b]pyridine-5-carboxylate

H,N X N L Jv NH

A suspension of Intermediate 3 (4g) in THF (100ml) was treated with DIPEA (2.6ml) followed by trimethylsilyl isocyanate (1.99ml, 1.7g) and the solution was stirred at 22°C for 2h. The volatile solvents were removed under reduced pressure and the residue was partitioned between DCM (e.g. 50ml) and water (e.g. 25ml). The organic and aqueous layers were separated. The aqueous phase was extracted with DCM (e.g. 50ml). The organic layers were combined, separated from water by passing through a hydrophobic frit and concentrated under reduced pressure to yield Intermediate 4 as a solid (4g). LCMS showed MH+ = 361; TRET = 2.45min.

Intermediate 4A: Ethyl 4~{[l-(aminocarbonyl)-4-piperidmyl]amino}~l-ethyl-liϊ- pyrazolo[3,4-6]pyridine~5-carboxylate - -

H,N N

A suspension of Intermediate 3A (theoretical 33.5g excluding dioxane present) in THF (880ml) was treated with trimethylsilyl isocyanate (17.5ml, 14.9g) followed by DIPEA (22.6ml, 16.8g) and the solution was stirred at room temperature for 5h. LCMS indicated formation of only a small amount of product, so more DIPEA (22.6ml) was added and the mixture was stirred for an additional 24h. The THF was removed under reduced pressure. The residue was dissolved / diluted in DCM (1000ml), was washed with brine (200ml), was dried (MgSO4) and was evaporated under reduced pressure to give a residue which from LCMS appeared to contain a small amount of product.

The residue was dissolved in DCM (1000ml), was treated with trimethylsilyl isocyanate (17.5ml) followed by DIPEA (22.6ml) and the solution was stirred at room temperature for 48h. Trimethylsilyl isocyanate (17.5ml, 14.9g) followed by DIPEA (22.6ml, 16.8g) were again added and the solution was stirred at room temperature for an additional 48h. The volatile solvents were removed under reduced pressure and the residue was dissolved in DCM (600ml) and washed with brine (2 x 200ml). The organic layer was dried (MgSO4), was filtered and was concentrated under reduced pressure. The solid residue was stirred in diethyl ether (1000ml) for 2h until pulverized and was collected by filtration to yield Intermediate 4A as a light-yellow solid (24.56g). LCMS showed MH+ = 361; TRET = 2.19min. m.p. = 126-1270C.

IH NMR (400 MHz, chloroform-d) δ ppm 9.48 (d, J-7.83 Hz, 1 H), 8.90 (s, 1 H), 7.95 (s, 1 H), 4.65 (s, 2 H), 4.50 (q, J=7.24 Hz, 2 H), 4.35 (q, J=7.12 Hz, 2 H), 4.1 - 4.2 (m, 1 H), 3.87 - 3.93 (m, 2 H), 3.26 (ddd, J=13.58, 10.17, 3.03 Hz, 2 H), 2.16 - 2.23 (m, 2 H), 1.70 - 1.80 (m, 2 H), 1.51 (t, J=7.24 Hz, 3 H), 1.41 (t, J=7.12 Hz, 3 H).

Intermediate 4B: Ethyl 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}~l-ethyl-lH- pyrazolo[3,4-b]pyridine-5-carboxylate o H2N X κ

^ A suspension of ethyl l-ethyl-4-(4-piperidinylammo)-lH-pyrazolo[3,4-b]pyridine-5- carboxylate hydrochloride (13g, 36.77mmol) (e.g. Intermediate 3) in DCM (300ml) was treated with trimethylsilyl isocyanate (5g) followed by DIPEA (10ml) and was stirred at 22°C for 3h. The mixture was diluted with water, the organic layer was separated from the aqueous layer by passing through a hydrophobic frit, and the solvents were removed from the organic layer to give Intermediate 4B (1Og). LCMS showed MH+ = 361; TRET = 2.6min.

Intermediate s: 4-{[l-(aminocarbonyI)-4-piperidiny∏aminol-l-ethyI-lH- pyrazolo[3,4-b]pyridine-5-carboxyIic acid

H,N

A solution of Intermediate 4 (4g) in EtOH (50ml) was treated with a solution of NaOH (1.77g) in water (20ml) and the reaction mixture was heated at 6O0C for 5h. The solvents were removed and the residue was dissolved in water (ca. 8ml), the pH was adjusted to 3 (2M HCl) and the resultant precipitate was collected by filtration and dried at 6O0C under vacuum. LCMS indicated that partial hydrolysis of the urea portion had occurred. Therefore, the precipitate from the reaction was dissolved in EtOH (100ml), the solution was treated with trimethylsilyl isocyanate (3ml) and DIPEA (10ml) and then stirred at room temperature overnight. The solvents were removed, water was added to the residue, the pH was adjusted to 3 (2M HCl), the mixture was cooled to O0C for 30 minutes, and the resultant precipitate was collected by filtration and dried to give Intermediate 5 as a white solid (2.66g). LCMS showed MH+ = 333; TRET = 2.0min.

Intermediate 5A: 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-l-ethyI-l//- pyrazolo[3,4-6]pyridine-5-carboxyIic acid

H9N X N

A solution of Intermediate 4A (24.26g) in EtOH (360ml) and water (120ml) was treated with lithium hydroxide monohydrate (1 Ig) and the reaction mixture was stirred at room temperature overnight. The EtOH was removed under reduced pressure. Aqueous IN HCl solution (300ml) was added to the residue and the resultant precipitate was cooled in an ice bath for Ih, was collected by filtration, washed with cold water, dried in a vacuum dessicator overnight, and then further dried in a vacuum oven under reduced pressure at 6O0C overnight to give Intermediate 5A as a white solid (22.4g). LCMS showed MH+ = 333; TRET = 1.23min. m.p. = 204-2060C.

IH NMR (400 MHz, DMSO-J6) δ ppm 9.50 (d, J=8.O8 Hz, 1 H), 8.69 (s, 1 H), 8.25 (s, 1 H), 6.04 (br s, 2 H), 4.37 (q, J=7.20 Hz5 2 H), 4.14 - 4.24 (m, 1 H), 3.80 (d, J=13.64 Hz, 2 H), 3.15 (t, J=10.86 Hz, 2 H), 1.96 - 2.03 (m, 2 H), 1.39 - 1.47 (m, 2 H), 1.37 (t, J=7.20 Hz, 3 H).

Intermediate 6: 4-chloro-l-ethyl-lff-pyrazolo[3,4-&1pyridine-5-carboxylic acid

Cl

A solution of Intermediate 1 (2Og, 78.8mmol) in 1,4-dioxane (100ml) was treated with a solution of KOH (18g of pellets) in water (30ml) and the reaction mixture was stirred at room temperature for 24h. The solvent was evaporated and the residue was acidified to pH3 (2M hydrochloric acid). The resultant white precipitate was collected by filtration and dried under vacuum overnight to give Intermediate 6 as a white solid (16.9g). LCMS showed MH+ = 226; TRET = 2.61min.

Intermediate 7: 4-chloro-l-ethyI-lH-pyrazolo[3,4-blpyridine-5-carbonyl chloride

Cl

J

A solution of Intermediate 6 (17.8g, 78.8mmol) in thionyl chloride (SOCl2, 100ml) was heated at reflux under nitrogen for 3.5h. The solution was allowed to cool to room temperature overnight. The thionyl chloride was removed in vacuo, any remaining thionyl chloride was removed in vacuo by azeotropic distillation with toluene (ca. 30ml), and this was repeated to remove the thionyl chloride, to give Intermediate 7 as a beige solid (16.86g). LCMS (in MeOH, hence methyl ester) showed MH+ = 240 (MH+ for methyl ester); TRET = 2.88min.

Intermediate 8: 4-chloro-N-[(2,4-dimethylphenyI)methyl]-l-ethyl-lH-pyrazolo[ 3,4- b]pyridine-5-carboxamide A solution of Intermediate 7 (6g, 24.6mmol) and DIPEA (3.17g, 24.6mmol) in THF (60ml) was stirred for 20 min. A one-third aliquot (20ml) of the resultant solution was added to 2,4-dimethyl-benzylamine (1.1Ig, 8.2mmol) (e.g. available from Trans World Chemicals). The reaction mixture was stirred under nitrogen at room temperature for 24h. The solvents was removed in vacuo and the residue was partitioned between DCM (50ml) and 5% citric acid solution (50ml). The organic layer was separated using a hydrophobic frit, washed with 0.5M NaHCO3 solution (50ml), dried (Na2SO4), filtered and concentrated in vacuo to give Intermediate 8 as a white powder (1.6Ig). LCMS showed MH+ - 343; TRET = 3.22min.

The following Intermediate 10 can be prepared in a similar manner from Intermediate 7 and the appropriate amine reagent:

Amine reagent: Possible Source MH+ ion TRET of amine reagent (min)

Y Intermediate 10 Aldrich 345 2.9

Intermediate 9; 4-chloro-N-[(3,4-dimethylphenyI)methyl]-l-ethyl-lH-pyrazoIo[ 3,4- b]pyridine-5-carboxamide Cl

A solution of Intermediate 7 (6g) and DIPEA (3.17g) in THF (60ml) was stirred for 20 min. A one-third aliquot (20ml) of the resultant solution was added to 3,4-dimethyl- benzylamine (1.1 Ig) (e.g. available from Trans World Chemicals). The reaction mixture was stirred under nitrogen at room temperature for 24h, more THF (20ml) being added to aid dissolution of the reactants. The solvent was removed in vacuo and the residue was partitioned between DCM (50ml) and 5% citric acid solution (50ml). The organic layer was separated using a hydrophobic frit, washed with 0.5M NaHCO3 solution (50ml), dried (Na2SO4), filtered and concentrated in vacuo to give Intermediate 8 as a white powder (2.39g). LCMS showed MH+ = 343; TRET = 3.34min. - -

Intermediate 11: 1,1-dimethyIethyI [l-(aminocarbonyl)-4-piperidinyl]carbamate

o

A solution of 1,1-dimethylethyl 4-piperidinylcarbamate (0.35g) (e.g. AstaTech and/or Aldrich) in DCM (10ml) was treated with trimethylsilyl isocyanate (1.1ml, 0.86g). The reaction mixture was stirred at room temperature for 8h and then left to stand at room temperature over the weekend. The mixture was diluted with DCM (10ml) and washed with saturated NaHCO3 solution (20ml). The organic phase was separated and collected through a hydrophobic frit. The aqueous phase was extracted with DCM. The organics were combined and evaporated to dryness to give Intermediate 11 as a white foam (0.29g). 1H NMR (400MHz in CDCl3, 27°C, δ ppm) 4.45 (br. s, 3H), 3.90 (d, 2H), 3.65 (br. m, IH), 2.9-3.0 (dt, 2H), 1.95-2.0 (br. dd, 2H), 1.45 (s, 9H), 1.3-1.4 (dq, 2H).

Intermediate 12: 4-amino-l-piperidinecarboxamide hydrochloride

H2NΛN^

Intermediate 11 (0.29g) was treated with a 4M solution of hydrogen chloride in 1, 4- dioxane (5ml), and was stirred at room temperature for 4h. The reaction mixture was evaporated to dryness and co-evaporated with DCM to give a white foam. This was triturated with diethyl ether and a small amount (a few drops) of MeOH and the resulting white solid was filtered off and dried by suction to give Intermediate 12 as a white solid (0.27g, impurities present). 1H NMR (400MHz in d6-DMSO, 270C, δ ppm) 8.1 (br. s, 2H), 3.95 (d, 2H), 3.15 (m, IH), 2.7 (dt, 2H), 1.85 (dd, 2H), 1.35 (m, 2H); impurities present.

Intermediate 13: 1,1-dimethvIethyI [4-(aminocarbonyl)cycIohexyl]carbamate

,NH I n O A solution of 4-({[(l,l-dimethylethyl)oxy]carbonyl}amino)cyclohexanecarbox ylic acid (e.g. Fluka, Ig) in DMF (30ml) was treated with HATU (1.72g) and DIPEA (5.4ml). The reaction mixture was stirred at room temperature for 10 min. A 0.5M solution of ammonia in 1,4-dioxane (40ml) was added and the reaction mixture was stirred at room temperature for 72h. The solvents were evaporated and the residue was purified by loading the crude mixture onto a 5Og aminopropyl SPE cartridge and eluting with EtOAc (100ml), then MeOH (100ml). Intermediate 13 was isolated (by evaporation of the MeOH fraction) as a yellow oil (0.99g). LCMS showed MH+ = 242; TRET = 2.2min.

Intermediate 15: 4-aminocycIohexanecarboxamide hydrochloride

,Cl H'

4.0M Hydrogen chloride in 1,4-dioxane (14ml) was added to Intermediate 13 (0.99g) and the reaction mixture was stirred at room temperature for 30min. The solvent was evaporated to give Intermediate 15 as a yellow gum (1.03g). 1H NMR (400MHz in d6- DMSO, 270C, δppm) 7.9 (br. S, 2H), 3.9 (br. S, 2H), 3.10 (m, IH), 1.92 (m, 2H), 1.68 (m, 4H), 1.50 (m, 2H).

Intermediate 17: 1,1-dimethyIethyl [m-4-(aminocarbonyl)cyclohexyI]-carbamate

A solution of cz5-4-({[(l,l-dimethylethyl)oxy]carbonyl}amino)cyclohexane-c arboxylic acid (5.Og) (e.g. available from Fluka), EDC (5.9g) and HOBT (4.17g) was stirred for 20 min. Ammonia solution (Specific Gravity = 0.88; 8ml) was added. The reaction mixture was stirred at room temperature overnight, concentrated in vacuo and partitioned between DCM and saturated sodium bicarbonate solution. The aqueous phase was separated and washed with DCM. The combined organics were dried over MgSO4 and concentrated in vacuo to give Intermediate 17 (4.84g) as a white solid. LCMS showed MH+ = 243; TRET = 2.3min.

The following Intermediate 18 was prepared in a similar manner from trans-4-({[(l,l- dimethylethyl)oxy]carbonyl}amino)cyclohexanecarboxylic acid (e.g. available from Fluka): Intermediate 18; l.l-dimethvlethvl [frøns-4-(aminocarbonyl)cyclohexyl]-carbamate

O

LCMS showed MNH4+ = 260; TRET = 2.24min.

Intermediate 19: c/s-4-aminocvclohexanecarboxamide hydrochloride

.Cl H'

4.0M hydrogen chloride in dioxan (50ml) was added to a stirred solution of Intermediate 17 (4.84g) in dioxan (100ml). The reaction mixture was stirred for 1 hour at room temperature and then stood at O0C for 3 days. The reaction mixture was concentrated in vacuo to give Intermediate 19 (4.Ig) as a white solid. LCMS showed MH+ = 143; TRET = 0.31min.

The following Intermediate 20 was prepared in a similar manner from Intermediate 18:

Intermediate 20: fraws-^-aminocyclohexanecarboxamide hydrochloride

H,N^ ,O .Cl

NH2

LCMS showed MH+ = 143; TRET = 0.30min.

Intermediate 21: ethyl 4-(rαs-4-(aminocarbonvl)cvcIohexv∏aminoM-ethvl-lfi-r- pyrazolo[3,4-6]pyridine-5-carboxylate A solution of Intermediate 1 (2.Og), Intermediate 19 (1.55g) and DIPEA (6.9ml) in EtOH (140ml) was stirred and heated at reflux overnight. More Intermediate 19 (420mg) and DIPEA (3.5ml) were added. The reaction mixture was stirred and heated at reflux overnight, cooled and concentrated in vacuo. The residue was partitioned between DCM and saturated sodium bicarbonate solution. The organic phase was concentrated in vacuo. The residue was triturated in a mixture of DCM and cyclohexane to give a solid. The solid was filtered off and dried to give Intermediate 21 (2.16g) as a yellow solid. LCMS showed MH+ = 360; TRET = 2.56min.

The following Intermediate 22 was prepared in a similar manner from Intermediate 1 and Intermediate 20:

Intermediate 22: ethyl 4-{rfrafls-4-(aminocarbonyI)cvcIohexyl1aminoM-ethvI-li[jr- pyrazolo[3,4-£]pyridine-5-carboxylate

LCMS showed MH+ = 360; TRET = 2.84min.

Intermediate 23: 4-{ [c/s-4-(aminocarbonyl)cyclohexyl] amino}-l-ethyl-liZ- pyrazolo[3,4-#]pyridine-5-carboxy lie acid

A mixture of Intermediate 21 (1.54g) and sodium hydroxide (0.68g) in 95% aqueous EtOH (EtOH containing 5% water) (60ml) was stirred and heated at 50°C overnight. The solvent was removed in vacuo. The residue was dissolved in water. The solution was cooled to 0-5°C, with stirring, and acidified with 2M HCl. The resultant suspension was refrigerated for 3 days then filtered under suction. The residue was dried in vacuo to give Intermediate 23 (1.58g) as a yellow solid. LCMS showed MH+ = 332; TRET = 2.06min. - OO -

The following Intermediate 24 was prepared in an analogous manner from Intermediate 1 and Intermediate 22:

Intermediate 24: 4-{[fraws^4-(aminocarbonyI)cyclohexyl]amino}-l-ethyl-l/Z- pyrazolo[3,4-Λ]pyridine-5-carboxylic acid

LCMS showed MH+ = 332; TRET = 2.06min.

Intermediate 25: ethyl 4-[((3S)-l-{[(l,l-dimethylethyl)oxy]carbonyl}-3~ pyrrolidmyl)ammo]-l-ethyl-llf-pyrazolo[3,4-Z>]pyridine-5- carboxylate

A solution of Intermediate 1 (680mg), DIPEA (2.3ml) and 1,1-dimethylethyl (3S)-3- amino-1-pyrrolidinecarboxylate (500mg) (e.g. available from Aldrich) in MeCN (15ml) was stirred and heated at reflux for 16h. The solvent was evaporated and the residue was partitioned between DCM and water. The organic phase was isolated by passage through a hydrophobic frit. The solvent was evaporated and the residue was purified on a lOOg "flashmaster" cartridge (e.g. available from Jones Chromatography Ltd., United Kingdom), using a mixture of EtOAc and cyclohexane as the eluent, to give Intermediate 25 (720mg) as a solid. LCMS showed MH+ = 404; TRET = 3.20min.

The following Intermediate 26 was prepared in a similar manner from Intermediate 1 and 1,1-dimethylethyl (3R)-3 -amino- 1-pyrrolidinecarboxylate (e.g. available from Aldrich):

Intermediate 26: ethyl 4-[((3RVl-Wl J -dimethvlethvDoxvl carbon vIl-3- pyrrolidmyl)amino]-l-ethyl-ll/-pyrazolo[3,4-Z>]pyridine-5 -carboxyIate - -

LCMS showed MH+ = 404; TW = 3.20min.

Intermediate 27: ethyl l-ethyl-4-[(3S)-3-pyrrolidinylamino]-l/f-pyrazolo[3,4- 6]pyridine-5-carboxylate hydrochloride

Xl H'

Intermediate 25 (720mg) in a solution of 4.0M hydrogen chloride in dioxan (30ml) was stirred at room temperature for 3h. The solvent was evaporated to give Intermediate 27 (606mg) as a white solid. LCMS showed MH+ = 304; TRET = 2.00min.

The following Intermediate 28 was prepared in a similar manner from Intermediate 26:

Intermediate 28: ethyl l-ethyl-4- [(3R)-3-pyrrolidinylamino] -117-pyrazolo [3,4- £]pyridine-5-carboxylate hydrochloride

.Cl

LCMS showed MH+ = 304; TRET = 2.00min.

Intermediate 29: ethyl 4-{[(3S)-l-(aminocarbonyl)-3-pyrroIidinv∏aminol-l-ethyl- lfl-pyrazoloβ^-^pyridine-S-carboxylate - -

.0 H2N-f

A solution of Intermediate 27 (606mg) in DCM (30ml) was stirred and treated with DIPEA (1.15ml) followed by trimethylsilyl isocyanate (1.03ml). The reaction mixture was stirred at room temperature for 2h. The solution was washed with water. The aqueous phase was extracted with dichloromethane. The combined organics were passed through a hydrophobic frit and then concentrated to give Intermediate 29 (660mg) as a solid. LCMS showed MH+ = 347; TRET = 2.40min.

The following Intermediate 30 was prepared in a similar manner from Intermediate 28:

Intermediate 30; ethyl 4-{[(3ig)-l-(aminocarbonvI)-3-pvrroIidinvllaminol-l-ethvl- l/f-pyrazoloP^-^pyridine-S-carboxylate

O H2N^

LCMS showed MH+ = 347; TRET = 2.40min.

Intermediate 31 : 4-{f(3S)-l-(aminocarbonvl)-3-pvrrolidmv∏aininol-l-ethvI-lj H- pyrazoIo[3,4-#]pyridine-5-carboxylic acid

O H2N^f

A mixture of Intermediate 29 (660mg) and sodium hydroxide (300mg) in EtOH (15ml) and water (8ml) was stirred and heated at 60°C for 2h. The solvents were removed in vacuo. Water (8ml) was added to the residue and the resultant solution was acidified with 2M HCl. The resultant suspension was filtered under suction. The residue was dried in vacuo to give Intermediate 31 (270mg) as a solid. LCMS showed MH+ = 319; TRET = 1.90min.

The following Intermediate 32 was prepared in a similar manner from Intermediate 30:

Intermediate 32: 4-{r(3R)-l-(aminocarbonvlV3-pvrrolidiny∏aminol-l-ethvl-li3 r- pyrazolo[3,4-Z>]pyridine-5-carboxyIic acid

O H2N-^ N CL NH O

Jl I

LCMS showed MH+ = 319; TRET = 1.9OmJn.

Intermediate 33 : 4-f(l-{f(l,l-diinethvIethvl)oxvlcarbonvl}-4-piperidinvl)amin o1-l- ethyl-lif-pyrazolo[3,4-Z>]pyridine-5-carboxylic acid

O O N NH

A mixture of Intermediate 2 (750mg) and sodium hydroxide (290mg) in EtOH (20ml) and water (5ml) was stirred and heated at 50°C for 2.5h then cooled and concentrated under reduced pressure. A solution of the residue in water (20ml) was cooled to 0-5°C, with stirring, and acidified to pH=5 with 2M HCl solution. The resultant solid suspension was filtered. The solid residue was washed with water and dried to give Intermediate 33 (575mg) as a white solid. LCMS showed MH+ = 390; TRET = 2.86min.

Intermediate 34: 4-{fl-(aminocarbonvl)-4-piperidinvllamino>-iV-[(2- bromophenyI)methyl]-l-ethyl-li?-pyrazoIo[3,4-Λ]pyridine-5-c arboxamide H2N' ^N

A solution of Intermediate 5 (0.5g), EDC (0.32g), HOBT (0.22g) and DIPEA (0.65ml) in DMF (10ml) was stood at room temperature for lOmin then added to 2- bromobenzylamine hydrochloride (0.4Og) (e.g. Aldrich). The reaction mixture was stood at room temperature for 2Oh. The solvent was evaporated. The residue was partitioned between DCM (50ml) and saturated NaHCO3 solution. The organic phase was separated by passing through a hydrophobic frit and evaporated. The residual solid was triturated with a mixture of methanol and ether. The suspension was filtered and the residue was dried to give Intermediate 34 (0.52g) as a white solid. LCMS showed MH+ = 500 + 502; TRET = 2.70min.

The following Intermediate 35 was prepared in a similar manner from Intermediate 5 and and 2-bromo-4-methylbenzylamine:

Intermediate 35; 4-{ [l-(aminocarbonyl)-4-piperidinyl] amino}-iV-[(2-bromo-4- methylphenyI)methyl]-l-ethyHH-pyrazoIo[3,4-Λ]pyridine-5-car boxamide

H2N' ^N

LCMS showed MH+ = 514 + 516; TRET = 2.82min. - -

Table of Examples

Example Example Name Number I 4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2,4- dimethylphenyl)methyl]- 1 -ethyl- 1H-pyrazolo[3 ,4-b]pyridine-5- carboxatnide 2, 2A, 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4- dimethylphenyl)methyl]- 1 -ethyl- 1 H-pyrazolo [3 ,4-b]pyridine-5- carboxamide 2B 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-5-{[(3,4- dimethylphenyl)methyl]aminocarbonyl}- 1 -ethyl- lH-pyrazolo[3,4- δ]pyridine (same compound as Examples 2, 2A, alternative name) 2C 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino} -5-{ [(3 ,A- dimethylphenyl)methyl]aminocarbonyl}- 1 -ethyl- 1 H-pyrazolo [3,4- δ]pyridine hydrochloride 3 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino }- 1-ethyl-N-{[4- (methyloxy)phenyl]methyl}-lΗ-pyrazolo[3,4-b]pyridine-5-carb oxamide 4 4-{ [4-(aminocarbonyl)cyclohexyl]amino } -N- [(3 ,4-dimethylphenyl)methyl]- l-ethyl-lH-pyrazolo[3,4-έ]pyridine-5-carboxamide 5 4-{[4-(aminocarbonyl)cyclohexyl]amino}-iV-[(2,4-dimethylphen yl)methyl]- 1 -ethyl- lH-pyrazolo[3,4-£]pvridine-5-carboxamide 8 4-{ [cz5-4-(aminocarbonyl)cyclohexyl] amino } -N- [(3 ,A- dimethylphenyl)methyl]- 1 -ethyl- lH-pyrazolo[3 ,4-ό]pyridine-5- carboxamide 9 4-{[trα«5r-4-(aminocarbonyl)cyclohexyl]amino}-N-[(3,4- dimethylphenyl)methyl]- 1 -ethyl- lH-pyrazolo[3 ,4-b]pyridme-5- carboxamide 10 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino }-N-[(2,4- difluorophenyl)methyl]-l-ethyl-lΗ-pyrazolo[3,4-b]pyridine-5 -carboxamide II 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino} - 1 -ethyl-N- {[4- (trifluoromethyl)phenyl]methyl}-lH-pyrazolo[3 ,4-b]pyridine-5- carboxamide 12 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino} - 1-ethyl-N-[(4- hydroxyphenyl)methyl]- 1 H-pyrazolo [3 ,4-b]pyridine-5-carboxamide 13 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-l-ethyl-N-(phenyl methyl)- lH-pyrazolo[3,4-b]pyridine-5-carboxamide 14 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino }- 1 -ethyl-N- { [2- (methylsulfonyl)phenyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5- carboxamide - y2* ~

4-{ [1-(aminocarbonyl)-4-piperidinyl] amino} - 1-ethyl-N-[(3- hydroxyphenyl)methyl]- 1H-pyrazolo[3 ,4-b]pyridine-5-carboxamide 4-{[l-(ammocarbonyl)-4-piperidinyl]amino}-N-[(2,3- dichlorophenyl)niethyl]- 1 -ethyl- lH-pyrazolo[3 ,4-b]pyridine-5-carboxamide 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2,3- dimethylphenyl)methyl]-l-ethyl-lH-pyrazolo[3,4-b]pyridine-5- carboxamide 4-{[l-(aminocarbonyl)-4-piperidinyl]amiiio}-N-(2-biphenylylm ethyl)-l- etliyl-lH-pyrazolo[3,4-b]pyridine-5-carboxamide 4-{ [1-(aminocarbonyl)-4-piperidinyl]amino }-N-[(4-chloro-2- fluorophenyl)methyl]-l-ethyl-lH-pyrazolo[3,4-b]pyridine-5-ca rboxamide 4-{[l-(aniinocarbonyl)-4-piperidinyl]amino}-N-(2,3-diliydro- l-benzofuran- 5-ylmethyl)- 1 -ethyl- lH-pyrazolo[3 ,4-b]pyridine- 5 -carboxamide 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-N-({4- [(difluoromethyl)oxy]phenyl}methyl)-l-ethyl-lH-pyrazolo[3,4- b]pyridine- 5-carboxamide 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino} -N-[(4-chloro-2- methylphenyl)methyl]- 1 -ethyl- 1H-ρyrazolo[3 ,4-b]pyridine-5 -carboxamide 4-{[1-(aminocarbonyl)-4-piperidinyl] amino} -N-[(4'-chloro-4- biphenylyl)methyl]-l-ethyl-lH-pyrazolo[3,4-b]pyridine-5-carb oxamide 4-{[1-(aminocarbonyl)-4-piperidinyl]amino}- 1-ethyl-N-{[4- (methylsulfonyl)phenyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5- carboxamide 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-l-ethyl-N-{[4'- (trifluoromethyl)-2-biρhenylyl]methyl}-lH-pyrazolo[3,4-b]py ridine-5- carboxamide 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino }-N-[(4-chlorophenyl)methyl]- l-ethyl-lH-pyrazolo[3,4-b]pyridine-5-carboxamide 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-N-(4-biphenylylme thyl)-l- ethyl-lH-pyrazolo[3,4-b]pyridine-5-carboxamide 4-{[l-(aminocarbonyl)-4-piperidmyl]amino}-l-ethyl-N-{[4'-(et hyloxy)-4- biphenylyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carboxamide 4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-(3-biphenylyhne thyl)- 1- ethyl- 1 H-pyrazolo [3 ,4-b]pyridine-5 -carboxamide 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino} - 1 -ethyl-N- { [41- (trifluoromethyl)-4-biphenylyl]methyl}-lH-pyrazolo[3,4-b]pyr idine-5- carboxamide 4-{ [1-(arninocarbonyl)-4-piperidinyl] amino }- 1 -ethyl-N- [(3'-methyl-2- biphenylyl)methyl]- 1 H-pyrazolo [3 ,4-b]pyridine-5 -carboxamide 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-l-ethyl-N-{[3'-(m ethyloxy)-2- biphenylyl]methyl}-lH-pyrazolo[3 ,4-b]pyridine- 5 -carboxamide 4-{[1-(aminocarbonyl)-4-piperidinyl] amino} -N-[(3'-chloro-2- biphenylyl)methyl]-l-ethyl-lH-pyrazolo[3,4-b]pyridine-5-carb oxamide 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-N-[(4'-chloro-2- biphenylyl)methyl]-l-ethyl-lH-pyrazolo[3,4-b]pyridine-5-carb oxamide 4-{[l-(amiiiocarbonyl)-4-piperidinyl]ainino}-l-ethyl-N-[(4l- methyl-2- biphenylyl)methyl]-lH-pyrazolo[3 ,4-b]pyridine-5-carboxamide 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-l-ethyl-N-{[4l-(m ethyloxy)-2- biphenylyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carboxamide 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-l-ethyl-N-{[2-(3- furanyl)phenyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carboxam ide 4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-({2-[6- (methyloxy)-3-pyridinyl]phenyl}methyl)-lH-pyrazolo[3,4-b]pyr idine-5- carboxamide 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino }- 1-ethyl-N-{ [2-(3- thienyl)phenyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carboxam ide 4-{ [1-(aminocarbonyl)-4-piperidinyl] amino }- 1 -ethyl-N- { [2-(4-methyl-3- thienyl)pb.enyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carboxa mide 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-l-ethyl-N-[(5-met hyl-2- biphenylyl)methyl]- 1H-pyrazolo[3 ,4-b]pyridine-5-carboxamide 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-l-ethyl-N-{[4-met hyl-2-(3- thienyl)phenyl]metb.yl}-lH-pyrazolo[3,4-b]ρyridme-5-carboxa mide 4-{[1-(aminocarbonyl)-4-piperidinyl]amino}- 1 -ethyl-N- {[2-(3-furanyl)-4- methylρhenyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carboxami de 4-{[l-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3'-chloro-5-m ethyl-2- biphenylyl)methyl]-l-ethyl-lH-pyrazolo[3,4-b]pyridine-5-carb oxamide 4-{[cJs-4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4- dimethylphenyl)methyl]- 1 -ethyl- 1 H-pyrazolo [3 ,4-b]pyridine-5- carboxamide 4-{[trarø-4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4- dimethylphenyl)methyl]- 1 -ethyl- lH-pyrazolo[3,4-b]pyridine-5- carboxamide 4-{[cιs-4-(aminocarbonyl)cyclohexyl]amino}-l-ethyl-N-{[4- (methyloxy)phenyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carbo xamide 4-{[4-(aminocarbonyl)cyclohexyl]amino}- 1 -ethyl-N- {[4- (methylsulfonyl)phenyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5- carboxamide 4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,3-dimethylpheny l)methyl]- 1 -ethyl- 1 H-pyrazolo [3 ,4-b]pyridine-5 -carboxamide 4-{ [4-(aminocarbonyl)cyclohexyl] amino }-N-(2-biphenylylmethyl)- 1-ethyl- lH-pyrazolo[3,4-b]pyridine-5-carboxamide 4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(4-chloro-2- fluorophenyl)methyl]-l-ethyl-lH-pyrazolo[3,4-b]pyridine-5-ca rboxaniide 4-{ [4-(aminocarbonyl)cyclohexyl] amino }-N-[(2,3-dichlorophenyl)methyl]- l-ethyl-lH-pyrazolo[3,4-b]pyridine-5-carboxamide 4-{[4-(animocarbonyl)cyclohexyl]amino}-l-etliyl-N-{[2- (methylsulfonyl)phenyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5- carboxamide 4-{ [4-(aminocarbonyl)cyclohexyl] amino }-N-[(4-chlorophenyl)methyl]- 1- ethyl-lH-pyrazolo[3,4-b]pyridine-5-carboxamide 4-{ [4-(aminocarbonyl)cyclohexyl] amino }- 1-ethyl-N-{ [4- (methyloxy)phenyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carbo xamide 4-{ [4-(aminocarbonyl)cyclohexyl] amino} -N-(4-biphenylylmethyl)- 1-ethyl- 1H-pyrazolo[3 ,4-b]pyridine-5-carboxamide 4-{[4-(aminocarbonyl)cyclohexyl]amino}-l-ethyl-N-[(4- hydroxyphenyl)methyl]-lH-pyrazolo[3 ,4-b]pyridine-5-carboxamide 4-{[4-(aminocarbonyl)cycloh.exyl]aniino}-l-ethyl-N-(phenylme thyl)-lH- pyrazolo[3 ,4-b]pyridine-5 -carboxamide 4-{ [4-(aminocarbonyl)cyclohexyl] amino }-N-[(2,4-difluorophenyl)methyl]- l-ethyl-lH-pyrazolo[3,4-b]pyridine-5-carboxaniide 4-{ [4-(aminocarbonyl)cyclohexyl] amino }-N-({4- [(difluoromethyl)oxy]phenyl} methyl)- 1 -ethyl- 1H-pyrazolo[3 ,4-b]pyridine- 5 -carboxamide 4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-(2,3-dihydro-l-benz oruran-5- ylmethyl)- 1 -ethyl- 1 H-pyrazolo [3 ,4-b]pyridine-5 -carboxamide 4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-(3-biphenylyltnethy l)-l-ethyl- lH-pyrazolo[3,4-b]pyridine-5-carboxamide 4-{[4-(aminocarbonyl)cyclohexyl]amino}-l-ethyl-N-[(4- fluorophenyl)methyl]- lH-pyrazolo [3 ,4-b]pyridine-5-carboxamide 4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(4-chloro-2- methylphenyl)methyl]- 1 -ethyl- 1 H-pyrazolo[3 ,4-b]pyridine-5 -carboxamide 4-{ [4-(aminocarbonyl)cyclohexyl] amino }-N-[(4'-chloro-4- biphenylyl)methyl]-l-ethyl-lH-pyrazolo[3,4-b]pyridine-5-carb oxamide 4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-{[2,3-dichloro-6- (hydroxymethyl)phenyl]methyl}- 1 -ethyl- lH-pyrazolo[3 ,4-b]pyridine-5- carboxamide 4-{[4-(aminocarbonyl)cyclohexyl]amino}-l-ethyl-N-(5,6,7,8-te trahydro-l- naphthalenyhnethyl)-lH-pyrazolo[3,4-b]pyridine-5-carboxamide 4-{ [4-(aminocarbonyl)cyclohexyl] amino }-N-{[3,4- bis(methyloxy)phenyl]methyl}-l-ethyl-lH-pyrazolo[3,4-b]pyrid ine-5- carboxamide 4-{[(3S)-l-(aminocarbonyl)-3-pyrrolidinyl]amino}-N-[(2,4- dimethylphenyl)methyl]- 1 -ethyl- 1 H-pyrazolo [3 ,4-b]pyridme-5- carboxamide 4-{[(3S)-l-(aminocarbonyl)-3-pyrrolidinyl]amino}-N-[(3,4- dimethylphenyl)methyl]-l-ethyl-lH-pyrazolo[3,4-b]pyridine-5- carboxatnide 71 4-([(3R)- 1-(aminocarbonyl)-3-pyrrolidinyl] amino} -N-[(2,4- dimethylphenyl)methyl]- 1 -ethyl- 1H-pyrazolo[3 ,4-b]pyridine-5- carboxamide 72 4-{[(3R)-l-(aminocarbonyl)-3-pyrrolidinyl]amino}-N-[(3,4- dimethylphenyl)methyl]- 1 -ethyl- lH-pyrazolo[3 ,4-b]pyridine-5- carboxamide

Example 1 ; 4-{[l-(aminocarbonyl)-4-piperidinyl] amino} -N- [(2,4- dimethylphenyl)methyl] -l-ethyI-lH-pyrazolo[3,4-b]pyridine-5-carboxamide

NH O

J ^N

A solution of hiteraiediate 5 (0.066mmol) in DMF (ImI) was mixed with EDC (0.066mmol), HOBT (0.066mmol) and DIPEA (0.151mmol) followed by 2,4-dimethyl benzylamine (0.066mmol) (e.g. Trans World Chemicals Inc.). The reaction mixture was left to stand at room temperature overnight (e.g. for 16h). The DMF was evaporated and the residue was partitioned between DCM (5ml) and saturated aqueous NaHCO3 solution (2ml). The organic layer was collected through a hydrophobic frit and evaporated. The residue was purified by mass directed autoprep. HPLC to give the title compound Example 1 as a gum (7.9mg). LCMS showed MH+ = 450; TRET = 2.8min.

Example 2: 4-{[l-(aminocarbonyl)-4-piperidinyI]ammo}-N-[(3,4- dimethyIphenyI)methyl]-l-ethyl-lfi-pyrazolo[3,4-Λ]pyridine- 5-carboxamide

H2N N A solution of Intermediate 5 (lOOmg, 0.3 mmol) in dry DMF (e.g. can be about ImI) was treated with EDC (63mg, 0.33 mmol), HOBT (45mg, 0.33 mmol) and DIPEA (0.13ml, 0.75 mmol). 10 minutes later, 3,4-dimethyl-benzylamine (47 microlitres, 0.33 mmol) (e.g. available from Trans World Chemicals Inc.) was added and the resulting solution was left to stand at room temperature overnight. The DMF was removed by evaporation and the residue was partitioned between DCM and saturated aqueous NaHCO3 solution. The organic layer was collected through a hydrophobic frit and was concentrated in vacuo to dryness. The residue was purified by passing through a 2Og silica SPE cartridge, using firstly a gradient of EtOAc and cyclohexane (increasing concentration of EtOAc) and then a step gradient of EtOAc and methanol as the eluent. The product was eluted in the fraction containing 4 : 1 EtOAc : MeOH. The solvents were removed in vacuo to give a white solid (lOlmg). NMR showed the presence of EtOAc and DCM, so the solid was dried in vacuo at 4O0C to give Example 2 (80mg). LCMS showed MH+ = 450; TRET = 2.80min.

1H NMR (400MHz in d6-DMSO, 270C, δ ppm) 9.9 (d, IH), 8.93 (t, IH), 8.61 (s, IH), 8.19 (s, IH), about 7.08 (s, IH), 7.07 (d, IH), 7.02 (d, IH), 5.98 (s, 2H), 4.33 - 4.39 (m, 4H), 4.08 - 4.18 (br m, IH), 3.75 (dt, 2H), 3.13 (td, 3H), 2.19 (s, 3H), 2.18 (s, 3H), 1.92 - 2.00 (m, 2H), 1.33 - 1.42 (m, 5H). Plus some other peaks: possibly solvent.

A similar alternative method is: A solution of Intermediate 5 (0.066mmol) in DMF (ImI) is treated with EDC (0.066mmol), HOBT (0.066mmol) and DIPEA (0.151mmol) followed by 3,4-dimethylbenzylamine (0.066mmol). The reaction mixture is left to stand at 22°C for 16h. The DMF is evaporated and the residue is partitioned between DCM (5ml) and saturated aqueous NaHCO3 solution (2ml). The organic layer is collected through a hydrophobic frit and evaporated. The residue is purified by mass directed autoprep. HPLC to give the title compound.

Example 2A; 4-{[l-(aminocarbonyl)-4-piperidmyl]amino} -N- [(3,4- dimethyIphenyl)methyl]-l-ethyl-lH-pyrazoIo[3,4-A]pyridine-5- carboxamide O

H2N A.

A mixture of Intermediate 9 (27mg, O.Oδmmol), Intermediate 12 (16mg, 0.088mmol) and DIPEA (35 microlitres, 0.2mmol) in MeCN (2ml) was heated at reflux for 18h. More of Intermediate 12 (0.5 mole equivalents, ca. 0.04mmol, ca. 7mg) was added. The reaction mixture was heated at reflux for a further 24h, cooled to room temperature and the solvent removed in vacuo. The residue was partitioned between DCM and water. The organic phase was collected through a hydrophobic frit and evaporated to dryness. LCMS indicated that there were two products of the same molecular weight.

Therefore, the residue was purified by mass directed autopreparative HPLC to give the title compound as Example 2A (4.4mg); LCMS showed MH+ = 450 and TRET = 2.79min.

The other undesired product having the same molecular weight as Example 2A was also isolated from the mass directed autopreparative HPLC (0.6mg); and for this compound LCMS showed MH+ = 450 and TRET = 2.69min.

Example 2B: 4-{[l-(aminocarbonyl)-4-piperidinyllamino}-5-{[(3,4- dimethylpheny^methylJaminocarbonylJ-l-ethyl-l/T-pyrazoIoP^-� �lpyridine

H2N 1

A solution of Intermediate 5A (21.6g) in DMF (300ml) was treated with 3,4-dimethyl benzylamine (9.71ml, 9.23g), HOBT (9.66g) and DIPEA (25ml, 18.5g) followed by EDC (14. Ig). The reaction mixture was stirred at room temperature overnight. The DMF was evaporated under reduced pressure at 4O0C and the residue was partitioned between EtOAc (300ml) and water (200ml). The organic layer was separated and the aqueous phase was extracted with EtOAc (2 x 100ml). The combined organic extracts were washed with brine (200ml), were dried (MgSO4), were filtered and were evaporated under reduced pressure.

The residue was purified by flash column chromatography on silica gel (1500ml) using 95:5 DCM/MeOH as the eluting solvent. The purest fractions were collected and evaporated under reduced pressure. The residue was dissolved in EtOAc (500ml), was washed with 1 N NaOH solution (100ml), was dried (MgSO4), was filtered, was evaporated, and the residue was dried at 6O0C in a vacuum oven overnight to provide the title compound as a pale yellow solid (12.5g).

The remaining fractions from the flash column chromatography were collected and purified by autoprep. HPLC (Gilson reverse-phase HPLC, Solvent A water, Solvent B acetonitrile, see above for details). The water-acetonitrile fractions containing the product (UV detection) were combined, and the acetonitrile solvent was removed under reduced pressure. The remaining water was decanted off from the residue, and the residue was evaporated to dryness. The solid was collected and washed with ether to give the title compound as a pale yellow solid (6g).

The two batches of product were combined, were dissolved in MeOH and were evaporated under reduced pressure to provide the title compound Example 2B as a pale yellow solid.

LCMS showed MH+ = 450; TRET = 2.45min. m.p. = 152-1540C.

IH NMR (400 MHz3 DMSO-J6) δ ppm 10.01 (d, J=7.83 Hz, 1 H), 8.97 (t, J=5.81 Hz, 1 H), 8.63 (s, 1 H), 8.21 (s, 1 H), 7.02 - 7.10 (m, 3 H), 6.01 (br s, 2 H), 4.34 - 4.42 (m, 4 H), 4.10 - 4.20 (m, 1 H), 3.71 - 3.81 (m, 2 H), 3.14 (t, J=40.74 Hz, 2 H), 2.20 (s, 3H), 2.18 (s, 3 H), 1.92 - 2.02 (m, 2 H), 1.35 - 1.45 (m, 5 H). Plus peaks due to ether.

Example 2C: 4-{[l-(aminocarbonyl)-4-piperidmyl]ainiiio}-5-{[(3,4- dimethylphenyl)methyl]aminocarbonyl}-l-ethyl-lJH-pyrazolo[3, 4-Z>]pyridine hydrochloride

H2N X N' " " NH O N N H . HCI VI-"" N

A solution of Example 2B (15mg) in MeOH (0.5ml) was treated with a solution of IN hydrogen chloride in ether (10ml). The mixture was evaporated to provide the title compound (Example 2C) as a white solid (16mg). m.p. = 217-2180C (decomposition).

IH NMR (400 MHz, DMSO-J6) δ ppm 10.84 (s, 1 H), 9.48 (s, 1 H), 8.77 (s, 1 H), 8.46 (s, 1 H), 7.05 - 7.14 (m, 3 H), about 6.0 - 6.6 (br s, 2H), 4.53 (q, J=7.12 Hz, 2 H), 4.40 (d, J=5.56 Hz, 2 H), 4.27 (br s, 1 H), 3.76 (d, J=13.64 Hz, 2 H), 3.13 - 3.23 (t, J=10.95 Hz, 2 H), 2.21 (s, 3H), 2.19 (s, 3H), 1.94 - 2.03 (m, 2 H), 1.44 - 1.53 (m, 2 H), 1.40 (t, J=7.17 Hz, 3 H).

Example 3: 4-(ri-(ammocarbonvl)-4-piperidinv∏aminol-l-ethvl-N-{[4- (methyloxy)phenyl]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carbo xamide H2N 'N

A mixture of Intermediate 10 (27mg, 0.08mmol), Intermediate 12 (16mg, 0.088mmol) and DIPEA (35 microlitres, 0.2mmol) in MeCN (2ml) was heated at reflux for 18h. More of Intermediate 12 (0.5 mole equivalents, ca. 0.04mmol, ca. 7mg) was added. The reaction mixture was heated at reflux for a further 24h, cooled to room temperature and the solvent removed in vacuo. The residue was partitioned between DCM and water. The organic phase was collected through a hydrophobic frit and evaporated to dryness. LCMS indicated that there were two products of the same molecular weight.

Therefore, the residue was purified by mass directed autopreparative HPLC to give the title compound as Example 3 (4.2mg); LCMS showed MH+ = 452 and TRET = 2.57min.

The other product having the same molecular weight as Example 3 was also isolated from the mass directed autopreparative HPLC (1.4mg); and for this compound LCMS showed MH+ = 452 and TRET = 2.37min.

Alternative synthesis: A mixture of Intermediate 10 (27mg) and Intermediate 12 (16mg) in MeCN (2ml) is treated with DIPEA (35μL = 35 microlitres). The reaction mixture is heated at reflux for 72h. The solvent is evaporated and the residue is partitioned between DCM (5ml) and saturated aqueous NaHCO3 solution (2ml). The organic layer is collected through a hydrophobic frit and evaporated. The residue is purified by mass directed autoprep. HPLC to give Example 3. Examples 1 and 2 can also optionally be prepared using a similar procedure (e.g. see Example 2A).

Example 4; 4-{[4-(aminocarbonyl)cyclohexy∏amino}-iV-[(3,4- dimethylphenyl)methyl]-l-ethyl-liϊ-pyrazolo[3,4-6]pyridine- 5-carboxamide - -

A solution of Intermediate 9 (0.08mmol) in MeCN (ImI) was reacted with Intermediate 15 (0.088mmol) and DIPEA (0.2mmol). The reaction mixture was heated at reflux for 2Oh. The solvents were evaporated and the residue was partitioned between DCM (5ml) and water (2ml). The organic phase was collected through a hydrophobic frit and evaporated. The residue was purified by mass directed autoprep. HPLC to give Example 4 as a white solid (23mg). LCMS showed MH+ = 449; TRET = 2.8min.

The following Example 5 was prepared from Intermediates 8 and 15 using a similar procedure:

Input aryl Input MH+ LC-MS Example chloride cyclohexylamine Ion retention Number time 5 Intermediate 8 Intermediate 15 449 2.9

Alternative Preparation of Example 4 and separation into cis and trans isomers (Examples 8 and 9)

4-{[4-(aminocarbonyl)cyclohexyl]amino}-7V-[(3,4-dimethylp henyl)methyl]-l-etIiyl- liϊ-pyrazoloβ^-^pyridme-S-carboxainide

H0N1

A solution of Intermediate 9 (75mg) in MeCN (5ml) was reacted with Intermediate 15 (42mg) and DIPEA (200μL). The reaction mixture was heated at reflux for 16h. The solvents were evaporated and the residue was partitioned between DCM (10ml) and water (5ml). The organic fraction was collected through a hydrophobic frit and evaporated. The residue was separated into its cis and trans isomers using a 25cm Chiralpak AS column, eluting with 40% EtOH : 60% heptane mixture to give the isomers. - 01 -

Example 8: 4-{[c/s-4-(aminocarbonyl)cyclohexyI]amino}-Λr-[(3,4- diinethylphenyl)methyl]-l-ethyl-liϊ-pyrazolo[3,4-Λ]pyridin e-5-carboxamide

First isomer to elute: isolated as a white solid (7.4mg). LCMS showed MH+ = 449; TRET = 2.8min. Confirmation of the czs-relationship between the substituents on the cyclohexane ring was demonstrated by NMR (ID TOCSY experiments).

Example 9: 4-{[tfrø«s-4-(aminocarbonyl)cyclohexyl]amino}-iV-[(3,4- dimethylphenyl)methyl]-l-ethyl-liϊ-pyrazolo[3,4-A]pyridine- 5-carboxamide

Second isomer to elute: isolated as a white solid (7.8mg). LCMS showed MH+ = 449; TRET = 2.8min. Confirmation of the trαns-relationship between the substituents of the cyclohexane ring was demonstrated by NMR (ID TOCSY experiments). - -

Examples 10 to 26

The following Examples 10 to 26 were prepared from Intermediate 5 and the appropriate amine reagent Ar-CH2NH2 using a procedure similar to that used in Example 1 or Example 2:

H2N X' N

NH

NHCH2Ar N N' 'N

NHCH7Ar One Possible LC-MS Example Source of amine MH+ retention Number (connecting nitrogen reagent time Ion underlined) H2NCH2Ar

10 Aldrich 458 2.7

HN

11 Aldrich 490 2.9 HN CF,

12 Apin 438 2.4 HN OH

13 Aldrich 422 2.43 HN

14 O2Me WO 2002/030930 500 2.49 A2 (2002) HN

15 OH Apin 438 2.43 HN

16 CI Aldrich 490 2.95 Cl HN - -

17 Trans World 450 2.83

HN

18 Trans World 498 2.99

19 Lancaster 474 2.85

HN Cl 20 Maybridge 464 2.6 International

21 Aldrich 488 2.78 HN OCHF,

22 Trans World 470 2.93

HN Cl

23 Interchim S. A. 532 3.26

24 Matrix 500 2.4 HN SO2Me

25 Not known 566 3.21

26 Aldrich 456 2.81 HN Cl Example 27; 4-{fl-(aminocarbonyI)-4-piperidinyl]amino}-N-(4-biphenyIyIme thyl)-l- ethyl-lH-pyrazolo[3,4-b]pyridine-5-carboxamide

H2N' ~N

A solution of Intermediate 5 (25mg), EDC (16mg), HOBT (llmg) and DIPEA (33ul) in DMF (ImI) was stood for lOmin then added to (4-biphenylylmethyl)amine acetate (Aldrich, 22mg). The reaction mixture was left to stand at room temperature for 4 days. The solvent was evaporated. The residue was partitioned between DCM (2ml) and saturated NaHCO3 solution. The organic phase was passed through a hydrophobic frit and concentrated. The residue was purified by mass directed autoprep HPLC to give Example 27 (12mg) as a white solid. LCMS showed MH+ = 498; TRET = 3.08min.

Examples 28 to 30

The following Examples 28 to 30 were prepared from Intermediate 5 and the appropriate amine reagent Ar-CH2NH2 using the above procedure of Example 27 or a similar procedure:

H0N N

NH O

NHCH2Ar N

J

NHCH7Ar One Possible LC-MS Example Source of amine MH+ retention Number (connecting nitrogen reagent time underlined) H2NCH2Ar 28 542 3.20 JHN^ ΪL JL

^"OEt

29 Trans World 498 3.06

30 — Ύ^ Interchim S. A. 566 3.33

C F3

ExaπiEle_3Jj_4-{[l-(aininocarbonyI)-4-piperidinyl]amino} -l-ethyl-N-[(3t-methyl-2- biphenylyl)methyl]-lH-pyrazolo[3,4-b]pyridine-5-carboxamide

A solution of Intermediate 34 (50mg), (3-methylplienyl) boronic acid_(20mg) (e.g. Aldrich), palladium tetrakis(triphenylphosphine) (6mg) and sodium carbonate (32mg) in a (3:1) mixture of DMF and water (ImI) was heated at 150° in a sealed vessel in a microwave reactor for lOmin. The reaction mixture was cooled to room temperature and the solvents were evaporated. The residue was partitioned between DCM (2ml) and water (2ml). The organic phase was separated by passing through a hydrophobic frit to give a solution containing a mixture of Example 31 [piperidinyl -NCONH2] and the [piperidinyl -NH] adduct. The solution was treated with DIPEA (26ul) and trimethylsilyl isocyanate (15ul). The solution was left to stand at room temperature overnight. The solvent was removed and the residue was purified by mass directed autoprep HPLC to give Example 31 (35mg) as a white solid. LCMS showed MH+ = 512; TRET = 3.02min.

Examples 32 to 44

The following Examples 32 to 44 were prepared from Intermediate 34 (Examples 32 to 40) or Intermediate 35 (Examples 41 to 44) and the appropriate boronic acid ArB(OH)2, using the above (Example 31) procedure or a similar procedure. H2N' 'N

NHCH2Ar

HNCH2Ar Boronic Acid LC-MS Example Reagent ArB(OH)2 MH+ retention Number (connecting time Ion nitrogen (with one possible underlined) source) 32 OMe OMe 528 2.9

B(OH)2 HN Aldrich

33 Cl .Cl 532 3.04

B(OH)2 HN Aldrich

34 Cl 532 3.06

B(OH)2 Aldrich

35 512 3.03

B(OH)2 Aldrich

36 OMe 528 2.91

B(OH)2 Aldrich o 488 2.88

B(OH)2 Aldrich OMe 529 2.82

1 B(OH)2 Matrix 504 2.97

B(OH)2 HN Aldrich

f 518 2.91

B(OH)2 HN Aldrich 512 2.97

B(OH)2 HN Aldrich

518 2.93

B(OH)2 HN Aldrich 502 2.85

B(OH)2 HN Aldrich Cl Cl 546 3.11

B(OH)2 HN Aldrich Example 45: 4-{fc/s-4-(aminocarbonyI)cycIohexyl]amino}-N-f(2,4- dimethylphenyl)methyl]-l-ethyl-lH-pyrazolo[3,4-b]pyridine-5- carboxamide

A solution of Intermediate 23 (30mg), EDC (19mg), HOBT (13.5mg) and DIPEA (39ul) in DMF (ImI) was stirred for 0.5h then added to 2,4-dimethylbenzylamine (e.g. Trans World Chemicals, Inc., 16mg). The reaction mixture was left to stand at room temperature overnight. The solvent was removed and the residue was partitioned between dichloromethane and 1.0M sodium bicarbonate solution. The organic phase was dried and concentrated. The residue was purified by mass directed autoprep. HPLC to give Example 45 (3.6mg). LCMS showed MH+ = 449; TRET = 2.78min.

The following Examples 46 and 47 were prepared in a similar manner from Intermediates 24 and 23 respectively:

Example 46: 4-{[^m/i5-4-(ammocarbonyI)cyclohexv∏amino}-N-r(2,4- dimethylphenyl) methyl] -1-ethyl-1H-pyrazolo[3 ,4-b]pyridine-5-carboxamide

From Intermediate 24 and 2,4-dimethylbenzylamine (e.g. Trans World Chemicals, Inc.). LCMS showed MH+ = 449; TRET = 2.82min. Example 47: 4-{fαs-4-(aminocarbonyI)cycIohexyIlamino}-l-ethyl-N-{[4- (methyloxy)phenyI]methyl}-lH-pyrazolo[3,4-b]pyridine-5-carbo xamide

OMe

From Intermediate 23 and 4-methoxybenzylamine (e.g. Aldrich). LCMS showed MH+ : 451; TREτ = 2.55min.

Examples 48 to 68

NHCH2Ar

General Procedure: A mixture of Intermediate 23 (O.lmmol), HATU (O.lmmol) and DIPEA (0.4mmol) in DMF (0.4ml) was shaken at room temperature for 10 min. A solution of the amine reagent ArCH2NH2 (O.lmmol) in DMF (0.2ml) was then added and the mixture was agitated for several minutes to produce a solution. The solution was stored at room temperature for 16-64 hours then concentrated in vacuo. The residue was dissolved in chloroform (0.5ml) and applied to a SPE cartridge (aminopropyl, 0.5g). The cartridge was eluted successively with chloroform (1.5ml), EtOAc (1.5ml) and EtOAc : MeOH (9:1, 1.5ml). Fractions containing the desired product were concentrated in vacuo and the residue purified by mass directed autoprep HPLC.

The following Examples 48 to 68 were prepared from Intermediate 23 and the appropriate amine reagent ArCH2NEΪ2 using this or a similar procedure. The Examples were believed to be isolated as a mixture ofcis and trans isomers at the cyclohexane ring, with the cis isomer believed to be predominating. - 1 -

NHCH2Ar

(believed to be mixture of CM and trans isomers at the cyclohexane ring, with the cis isomer believed to be predominating)

HNCH7Ar One Possible MH+ LC-MS Example Source of amine Ion retention Number (connecting nitrogen reagent time underlined) H2NCH2Ar

48 HN Matrix

SO2Me 499 2.52 49 Trans World HN

449 2.93 50 Trans World

497 3.1 51 Lancaster HN

Cl 473 2.95 52 Aldrich .Cl HN

489 3.04 53 O2Me WO 2002/030930 A2 (2002) HN

499 2.61 54 HN^V^ Aldrich

"Cl 455 2.92 55 HN Aldrich

OMe 451 2.71 - Ill -

Aldrich

497 3.18 HN Apin OH 437 2.5 HN Aldrich

421 2.71 Aldrich HN

457 2.81 Aldrich HN OCHFn 487 2.89 HN Maybridge International

463 2.71 Trans World

497 3.17 HN Aldrich

439 2.77 Trans World HN

Cl 469 3.03 Interchim S A

531 3.38 OH

HN

Cl Cl 519 2.88 67 JP 05194108 A2 (1993) HN^Vy

475 3.11 68 ^~\ ^s^ ^OMe Aldrich - Il I 481 2.6

Examples 69 and 70 O H2N^ N

NH O

NHCH2Ar N

General Procedure: A mixture of Intermediate 31 (30mg), HATU (120mg) and DIPEA (0.09ml) in acetonitrile (2ml) was added to the amine ArCH2NH2 (0.09mmol). The mixture was stood at room temperature for 16 hours. The solvent was evaporated. The residue was partitioned between DCM and saturated sodium bicarbonate solution. The organic phase was separated and evaporated. The residue was purified by mass directed autoprep. HPLC to obtain the desired product.

Examples 71 and 72

N N *N'

General Procedure: A mixture of Intermediate 32 (30mg), HATU (12Qmg) and DIPEA (0.09ml) in acetonitrile (2ml) was added to the amine ArCH2NH2 (0.09mmol). The mixture was stood at room temperature for 16 hours. The solvent was evaporated. The residue was partitioned between DCM and saturated sodium bicarbonate solution. The organic phase was separated and evaporated. The residue was purified by mass directed autoprep HPLC to obtain the desired product.

The following Tabulated Examples 69 and 70 (prepared from Intermediate 31 and appropriate amine reagent ArCH2NH2) and Examples 71 and 72 (prepared from Intermediate 32 and appropriate amine reagent ArCE^NH^) were synthesised using the procedure stated above for the relevant example number:

HNCH2Ar One Possible Source Example (connecting nitrogen of amine reagent MH+ LC-MS Number underlined) retention H2NCH2Ar Ion time

69 Trans World

436 2.65 70 Trans World

436 2.64 71 Trans World

436 2.6 72 Trans World

436 2.66 Pharmaceutical Composition Examples and Micronisation Examples

Examples of pharmaceutical compositions suitable for external topical administration

In each of the Composition Examples Cl, ClA, ClB, C2, C2A, C3, C3A, C4, and C5 to CIl disclosed below, the compound of formula (I) ("drug") in the composition for external topical administration can for example be 4-{[l-(aminocarbonyl)-4- piperidinyl]amino}-N-[(3,4-dimethylphenyl)methyl]-l-ethyl-lH "-pyrazolo[3,4-ό]pyridme- 5-carboxamide, for example the compound of Example 2, 2A or 2B ("free base" form).

Composition Examples Cl, ClA and ClB (Ointments)

Exemplary pharmaceutical compositions being ointments suitable for external topical administration are as follows:

Ingredients Function Example Cl Example ClA Alternative (+ preferable Ingredient Ingredient Example ClB: specifications *) Concen¬ Concen¬ Concentration tration tration range (% w/w) (% w/w) (% w/w) Compound of Active agent 0.5 0.5 0.1 to 3% formula (I) ("drug") PDE4 (e.g. 0.2 to 1.5%) (as free base) inhibitor White Petrolatum Wax / 69.65 69.16 25 to 75% (white soft paraffin) ointment (e.g. 45 to 75%) (e.g. USP) base Mineral Oil (e.g. Solubilizer / 4.975 5 2.5 to 15% USP or BP) emollient (e.g. 4 to 12%) Polyoxyl Stearyl Surfactant 4.975 5 0.5 to 10% Ether (e.g. (e.g. 3 to 10%) Volpo S-2 ™) Propylene Glycol Solubilizer / 19.90 20 0.5 to 50% (e.g. USP) Penetration (e.g. 5 to 50% or Enhancer 7 to 30%) Butylated Antioxidant 0 0.20 0%, Hydroxyanisole or 0.001 to 2% (e.g. NF or BP) (e.g. 0.02 to 2%) Methylparaben Preservative 0 0.11 0%, (e.g. NF) or 0.05 to 2% Propylparaben (e.g. Preservative 0 0.03 0%, NF) or 0.01 to 2% Total 100 100 * NF = National Formulary; USP = US Pharmacopeia; BP = British Pharmacopeia

Composition Example Cl can be prepared by the following method:

The white petrolatum (white soft paraffin) (14Og), the mineral oil (1Og), and the polyoxyl stearyl ether (e.g. Volpo S-2 ™) (1Og) are mixed together and melted using a hot water bath until all the ingredients are dissolved, to form an oil phase. The oil phase is heated to ca. 65-70 0C. The propylene glycol (4Og) is heated using a hot water bath to a temperature of ca. 65-70 0C, and is then added slowly to the oil phase under low homogenization stirring conditions (Polytron large shaft). The homogenization is then increased and the mixture is homogenized for 10 mins. The resulting formulation is then cooled to room temperature, using a cold water bath to facilitate cooling, to give an ointment formulation (ca. 20Og).

Approx. 5g of the ointment formulation in a vial is heated slowly in a hot water bath until the ointment reaches a temperature of approximately 40-45 0C. The compound of formula (I) ("drug", in free base form, 25.3 mg) is added slowly into the vial and then the resulting mixture is homogenized using a microhomogenizer for approx. 10 minutes, to give an ointment containing the drug (Composition Example Cl). The drug can for example be at least partly in suspension in the ointment.

Composition Example ClA (and/or alternative Composition Example ClB) can be prepared by the following method:

The ointment formulation is manufactured by first preparing the propylene glycol phase. The propylene glycol and the appropriate amount of the drug are mixed to provide an initial solution while stirring with a low shear propeller mixer. The antioxidant and preservatives are also included in the propylene glycol solution. The temperature of the propylene glycol solution is maintained at about 55-65 °C. Concurrently, all of the components of the oil phase (white petrolatum, polyoxyl stearyl ether and mineral oil) are added into a separate container and heated to about 75-85°C to melt and mix the components. The propylene glycol phase is then added into the oil phase while maintaining the temperature at above 7O0C (e.g. from above 7O0C to 9O0C, e.g. about 75-85°C) and mixing with a high shear homogenizer for a minimum of 15 minutes (e.g. 15-60 minutes or 15-30 minutes). The emulsification process can be carried out in conventional topical manufacturing equipment, such as a Lee Kettle or Malt-Mat, which allows for scraping of the materials from the sides of the vessel while the phases are being emulsified. Following the emulsification time of at least 15 minutes, the product is cooled to about 30°C, to form (semi-solidify) the ointment emulsion. During this cooling time, the homogenizer speed is reduced and low agitation is used. After the product has cooled, and the ointment is produced (Composition Example ClA or ClB), it is dispensed from - -

the manufacturing vessel into holding containers. It can then be packed into tubes, sachets or other suitable packaging components, as necessary.

Composition Examples C2 and C2A (Water-in-oil cream emulsion)

An exemplary external-topical pharmaceutical composition being a water-in-oil cream emulsion is as follows:

Ingredients Function Example C2: Alternative (+ preferable specifications: Ingredient Example C2A: NF = National Formulary; Concentration Concentration USP = US Pharmacopeia, etc) (% w/w) range (% w/w) Compound of formula (I) (as Active 0.5 0.1 to 3% free base) ("drug") (e.g. 0.2 to 1.5%) White Petrolatum (e.g. USP) Wax / ointment 41.34 25 to 75% base (e.g. 30 to 65%) Mineral Oil (e.g. USP or BP) Solubilizer / 10 2.5 to 15% emollient (e.g. 4 to 12%) Polyoxyl Stearyl Ether Surfactant 8 0.5 to 12% (e.g. 3 to 10%) Propylene Glycol USP Solubilizer 20 0.5 to 50% /Enhancer (e.g. 5 to 50%) Butylated Hydroxyanisole (e.g. Antioxidant 0.02 0.001 to 2% NF or BP) (e.g. 0.02 to 2%) Methylparaben (e.g. NF) Preservative 0.11 0.05 to 2%

Propylparaben (e.g. NF) Preservative 0.03 0.01 to 2%

Purified water (e.g. USP) Vehicle Qs 2 to 30% (20%) (e.g. 5 to 25%) Total 100

Composition Example C2 (and/or alternative embodiment Example C2A) can for example be prepared using a process similar to that described for Composition Example Cl.

For example, in one embodiment of the process, the water and propylene glycol can be mixed together (optionally with the surfactant, antioxidant and preservatives, and optionally with the drug) to form an aqueous phase. The oil phase containing the white petrolatum and mineral oil are prepared in a separate vessel. Temperatures of both the aqueous and oil phases are maintained at elevated temperatures, such as about 55-9O0C or preferably about [from above 70 to 90] 0C, the oil phase temperature being sufficiently high to melt the oil phase, and while hot, one phase is added to another while mixing using a high shear mixer to effect emulsification, preferably keeping the temperature above 7O0C such as from above 70 to 90 0C. The final emulsion is allowed to cool e.g. to about 18-350C such as about 3O0C, while the agitation continues at lower speeds. The ointment can then be dispensed from the manufacturing vessel and filled into the primary packaging, for example tubes or sachets.

Composition Examples C3 and C3A (Oil-in-water cream emulsion)

An exemplary external-topical pharmaceutical composition being a oil-in-water cream emulsion, and intended to be a high occlusion composition, is as follows:

Ingredients Function Example C3: Alternative (+optional specifications) Ingredient Example C3A: Concentration Concentration (% w/w) range (% w/w) Compound of formula (I) (as Active 0.5 0.1 to 3% free base) ("drug") (e.g. 0.2 to 1.5%) Mineral Oil (e.g. USP) Solubilizer 32.5 15 to 50% / emollient (e.g. 20 to 45%) Dimethicone (Silicone Fluid Emollient 2.5 0.5 to 20% 360) (e.g. NF) (e.g. 1 to 5%) Isopropyl Myristate (e.g. NF) Solubilizer 7.5 0.5 to 20% (e.g. 3 to 12%) Glycerol Monostearate (e.g. Surfactant 2 0.5 to 10% Arlacel 165 ™) Sorbitan Monostearate (e.g. Surfactant 1 0.05 to 10% Span 60 ™) Cetostearyl Alcohol (e.g. NF) Surfactant 2 0.1 to 15% (e.g. 1 to 10%) Microcrystalline Wax (Ross) Emollient/ 10 5 to 25% (e.g. NF) base (e.g. 8 to 15%) Propylene Glycol (e.g. USP) Solubilizer 10 0.5% to 50% /Enhancer (e.g. 7% to 25%) Citric Acid, Hydrous Granular Buffer 0.05 0.05 to 5% (e.g. USP) Sodium Phosphate, Dibasic (e.g. Buffer 0.06 0.05 to 5% USP) Imidurea (Germall 115 ™) (e.g. Preserva¬ 0.20 0.05 to 2% NF) tive Purified Water (e.g. USP) vehicle 32 15 to 60% (e.g. 20 to 50%) - -

Composition Example C3 (and/or alternative embodiment Example C3A) can for example be prepared using a process generally analogous to that described in Composition Example C2 above.

Composition Example C4 (cream emulsion)

Ingredients Concentration of Optional weight of ingredient (% w/w) ingredients (g) Oil Phase Mineral oil 29.86 29.90 Steareth 2 2 2.05 Cetostearyl Alcohol 2 2.01 Arlacel 165 ™ 2 2.10 (glycerol monostearate) Microcrystalline wax 10 10.12 isopropyl myristate (IPM) 7.5 7.87 Dimethicone 2.5 2.65

Aqueous Phase Propylene glycol 22 22.09 Purified water 22 22.34 Methylparaben 0.11 0.1142 Propylparaben 0.03 0.0354 (Optional Batch size: ca. 10Og).

Procedure Cream base without drug: The combined ingredients of the oil phase are melted in a hot water bath to a temperature of approx 60-700C. The combined ingredients of the aqueous phase are also heated in a hot water bath to a temperature of approx. 60-700C. The aqueous phase is then added slowly to the oil phase under low homogenization conditions and then is homogenized at a higher speed for approx. 10 mins under low heat in the water bath. With heating removed, the formulation is then stirred manually with a spatula while being allowed to cool, until room temperature is reached, giving a cream emulsion.

Cream containing drug: As a modification of the above procedure, after the aqueous phase is heated to a temperature of approx. 60-700C and before addition to the oil phase, the compound of formula (I) is added at 0.1% to 3% w/w or 0.2% to 1.5% w/w (e.g. 0.5% w/w) to the pre-formed hot aqueous phase. Then, the aqueous phase is added slowly to the oil phase under low homogenization conditions and then is homogenized at a higher speed for approx. 10 mins under low heat in the water bath. With heating removed, the formulation is then stirred (e.g. manually with a spatula) while being allowed to cool, until ca. 18-350C or ca. 18-3O0C (e.g. 3O0C or room temperature) is reached, giving a cream emulsion (Composition Example C4).

Composition Examples C5, C6, C7, C8, C9, ClO, and CIl (ointment compositions)

Exemplary ointments can be as follows:

Example C5 Ingedient Optional Example C6 Ingedient Optional Ingredients % w/w ingredients Ingredients % w/w ingredients weight (g) weight (g) Oil Phase Oil Phase

White 60 60.82 White 65 67.87 petrolatum petrolatum Mineral oil 5 5.04 Mineral oil 5 5.12 Steareth-2 5 5.09 Steareth-2 5 5.04 Beeswax 10 10.02 Beeswax 5 5.05 substitute substitute

Hydrophilic Hydrophilic phase phase Propylene 20 20.32 Propylene glycol 20 20.35 glycol Optional Batch size ca. lOOg Optional Batch size ca. lOOg

Example C7 Ingredients Optional Example C8 Ingredients Optional Ingredients % w/w ingredients Ingredients % w/w ingredients weight (g) weight (g) Oil Phase Oil Phase White 60 60.45 White 60 60.22 petrolatum petrolatum Mineral oil 10 10.04 Mineral oil 10 10.11 Steareth-2 5 5.03 Cetostearyl 5 5.08 alcohol Beeswax 5 5.07 Beeswax 5 5.09 substitute substitute

Hydrophilic Hydrophilic phase phase Propylene 20 20.28 Propylene 20 20.29 glycol glycol Optional Batch size ca. 10Og Optional Batch size ca. lOOg Example C9 Ingredients Optional Example ClO Ingredients Optional Ingredients % w/w ingredients Ingredients % w/w ingredients weight (g) weight (g) Oil Phase Oil Phase White 55 55.70 White 63 63.23 petrolatum petrolatum Mineral oil 15 15.11 Mineral oil 10 10.09 Cetostearyl 5 5.02 Steareth-2 5 5.03 alcohol Beeswax 5 5.07 Beeswax 2 2.03 substitute substitute

Hydrophilic Hydrophilic phase phase Propylene 20 20.08 Propylene 20 20.18 glycol glycol Optional Batch size ca. 10Og Optional Batch size ca. lOOg

Example CIl Ingredients Optional Ingredients % w/w ingredients weight (g) Oil phase White petrolatum 58 58.50 Mineral oil 15 15.12 Cetostearyl 5 5.01 alcohol Beeswax 2 2.00 substitute

Hydrophilic phase Propylene glycol 20 20.29 Optional Batch size ca. lOOg

Procedure for Composition Examples C5, C6, C7, C8, C9, ClO, and CIl (ointment base): The oil phase is melted in a hot water bath to a temperature of approx. 60-700C. The hydrophilic (propylene glycol) phase is also heated in a hot water bath to a temperature of approx. 60-700C. The hydrophilic phase is added slowly to the oil phase under low homogenization conditions and is then homogenized at a higher speed for approx. 10 mins under low heat in the water bath. With heating removed, the formulation is then stirred manually with a spatula while being allowed to cool, until room temperature is reached, giving an ointment.

Procedure for Composition Examples C5, C6, C7, C8, C9, ClO, and CIl (ointment containing drug): As a modification of the above procedure, after the hydrophilic (propylene glycol) phase is heated to a temperature of approx. 60-700C and before addition to the oil phase, the compound of formula (I) is added at 0.1% to 3% w/w or 0.2% to 1.5% w/w (e.g. 0.5% w/w) to the pre-formed hot hydrophilic phase. Then, the hydrophilic phase is added slowly to the oil phase under low homogenization conditions and then is homogenized at a higher speed for approx. 10 mins under low heat in the water bath. With heating removed, the formulation is then stirred (e.g. manually with a spatula) while being allowed to cool to ca. 15-350C or ca. 18-3O0C (e.g. to ca. 3O0C or room temperature), giving an ointment (Composition Examples C5, C6, C7, C8, C9, ClO, and CIl).

Micronisation Example;

• Purpose: To micronize a compound of formula (I) or a pharmaceutically acceptable salt thereof, e.g. in an amount of approximately 600-1000 mg thereof, using a Jetpharma MCl micronizer. • The parent (unmicronised) and micronised materials are analyzed for particle size by laser diffraction and crystallinity by PXRD.

Equipment and material

Equipment/material Description and specification Jetpharma MCl Micronizer Nitrogen supply: Air tank with 275psi rate tubing Analytical balance Sartorius Analytical Top loader balance Mettler PM400 Digital Caliper VWR Electronic caliper Materials to be micronised A compound of formula (I)

The Jetpharma MCl Micronizer comprises a horizontal disc-shaped milling housing having: a tubular compound inlet (e.g. angled at ca. 30 degrees to the horizontal) for entry of a suspension of unmicronised compound of formula (I) or salt in a gasflow, a separate gas inlet for entry of gases, a gas outlet for exit of gases, and a collection vessel (micronizer container) for collecting micronised material. The milling housing has two chambers: (a) an outer annular chamber in gaseous connection with the gas inlet, the chamber being for receiving pressurised gas (e.g. air or nitrogen), and (b) a disc-shaped inner milling chamber within and coaxial with the outer chamber for micronising the input compound / salt, the two chambers being separated by an annular wall. The annular wall (ring R) has a plurality of narrow-bored holes connecting the inner and outer - -

chambers and circumferentially-spaced-apart around the annular wall. The holes opening into the inner chamber are directed at an angle (directed part-way between radially and tangentially), and in use act as nozzles directing pressurised gas at high velocity from the outer chamber into the inner chamber and in an inwardly-spiral path (vortex) around the inner chamber (cyclone). The compound inlet is in gaseous communication with the inner chamber via a nozzle directed tangentially to the inner chamber, within and near to the annular wall / ring R. Upper and lower broad-diameter exit vents in the central axis of the inner milling chamber connect to (a) (lower exit) the collection vessel which has no air outlet, and (b) (upper exit) the gas outlet. Inside and coaxial with the tubular compound inlet and longitudinally-movable within it is positioned a venturi inlet (V) for entry of gases. The compound inlet also has a bifurcation connecting to an upwardly- directed material inlet port for inputting material. hi use, the narrow head of the venturi inlet (V) is preferably positioned below and slightly forward of the material inlet port so that when the venturi delivers pressurised gas (e.g. air or nitrogen) the feed material is sucked from the material inlet port into the gas stream through the compound inlet and is accelerated into the inner milling chamber tangentially at a subsonic speed. Inside the milling chamber the material is further accelerated to a supersonic speed by the hole/nozzle system around the ring (R ) (annular wall) of the milling chamber. The nozzles are slightly angled so that the acceleration pattern of the material is in the form of an inwardly-directed vortex or cyclone. The material inside the milling chamber circulates rapidly and particle collisions occur during the process, causing larger particles to fracture into smaller ones. "Centrifugal" acceleration in the vortex causes the larger particles to remain at the periphery of the inner chamber while progressively smaller particles move closer to the centre until they exit the milling chamber, generally through the lower exit, at low pressure and low velocity. The particles that exit the milling chamber are heavier than air and settle downward through the lower exit into the collection vessel (micronizer container), while the exhaust gas rises (together with a minority of small particles of micronised material) and escapes into the atmosphere at low pressure and low velocity.

Procedure: The micronizer is assembled. The narrow head of the venturi inlet is positioned below and slightly forward of the material inlet port and is measured with a micro-caliper to make sure that it is inserted correctly. The ring (R ) and venturi (V) pressures are adjusted according to the values specified in the experimental design (e.g. refer to experimental section below) by adjusting the valves on the pressure gauges on the micronizer. The setup is checked for leakage by observing if there is any fluctuation in the reading of the pressure gauges. Note that the venturi (V) pressure is kept at least 2 bars greater than the ring (R ) pressure to prevent regurgitation of material, e.g. outwardly from the material inlet port. Balance performance is checked with calibration weights. Specified amount of the parent material is fed into the input container of the micronizer using a spatula. The - -

input container plus material is weighed. The equipment pressure is monitored during the micronization process. Upon completion of the micronising run, the nitrogen supply, is shut off and the micronised material is allowed to settle into the micronizer container. The micronised powder in the micronizer container (collection vessel) and the cyclone (above the recovery vessel) are collected together into a pre-weighed and labelled collection vial. The weight of the micronised material is recorded. The input container is re- weighed in order to calculate the amount of input material by difference. The micronizer is disassembled and residual PDE4 compound on the micronizer inner surface is rinsed with 70/30 isopropyl alcohol / water and collected into a flask. The micronizer is then thoroughly cleaned in a Lancer washing machine and dried before subsequent runs are performed.

Optional Experimental Parameters Parent (unmicronised) material (Procedure 1): a compound of formula (I) or a pharmaceutically acceptable salt thereof Balance(s): Sartorius analytical

Material Ventun Intended Time Actual feed-rate Proc¬ input Pressure (V) / feed-rate needed to (g/min) edure amount (g) ring (R ) feed no. Pressure (bar) material (min+sec) 1 ca. 0.9 g V= 8 to 10 bar 180 to 200 procedure not R= 5.5 to 6 bar mg/min carried out

The above optional parameters can be varied using the skilled person's knowledge.

% yield = [(Material from vessel + Material from cyclone)/Material input amount] xlOO

Procedure 1 includes possible parameters and conditions and has not been carried out.

Inhalable Composition Example: Dry Powder Formulation Example - Dry powder Lactose Blend Preparation

Using a size-reduced e.g. micronised form of the compound of formula (I) or salt thereof (e.g. optionally as prepared in the Micronisation Example above), the dry powder blend is prepared by mixing the required amount of the compound/salt (e.g. 10 mg, 1% w/w) with inhalation-grade lactose containing 10% fines (e.g. 990 mg, 99% w/w) in a Teflon™ (polytetrafluoroethene) pot in a Mikro-dismembrator ball-mill (but without a ball bearing) at % speed (ca. 2000-2500 rpm) for about 4 hours at each blend concentration. The Mikro-dismembrator (available from B. Braun Biotech International, Schwarzenberger Weg 73-79, D-34212 Melsungen, Germany; www.bbraunbiotech.com) comprises a base with an upwardly-projecting and sidewardly-vibratable arm to which is attached the Teflon TM pOt. The vibration of the arm achieves blending. Other blends: 10% w/w compound/salt (50 mg) + 90% w/w lactose (450 mg, inhalation-grade lactose containing 10% fines). Serial dilution of the 1% w/w blend can achieve e.g. 0.1% and 0.3% w/w blends.