Patent Application
20060089375
The present invention relates to pyrazolopyridine compounds, processes for their preparation, intermediates usable in these processes, and pharmaceutical compositions containing the compounds. The invention also relates to the use of the pyrazolopyridine compounds in therapy, for example as inhibitors of phosphodiesterases and/or for the treatment and/or prophylaxis of inflammatory and/or allergic diseases such as chronic obstructive pulmonary disease (COPD), asthma, rheumatoid arthritis or allergic rhinitis.
U.S. Pat. No. 3,979,399, U.S. Pat. No. 3,840,546, and U.S. Pat. No. 3,966,746 (E.R.Squibb & Sons) disclose 4-amino derivatives of pyrazolo[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.
U.S. Pat. No. 3,925,388, U.S. Pat. No. 3,856,799, U.S. Pat. No. 3,833,594 and U.S. Pat. No. 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 1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid derivatives with 4-hydroxy, 4-chloro, 4-alkoxy, 4-hydrazino, and 4-amino substituents.
CA 1003419, CH 553 799 and T. Denzel, Archiv der Pharmazie, 1974, 301(3), 177-186 disclose 4,5-disubstituted 1H-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 Jan. 2002 discloses pyrazolopyridine compounds of the following formula:
EP 0 076 035 A1 (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.
The compound cartazolate, ethyl 4-(n-butylamino)-1-ethyl-1H-pyrazolo[3,4-b]-pyridine-5-carboxylate, is known. 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 1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid derivatives, including ethyl 4-cyclopentylamino-1-methyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate, and their affinities and antagonist activities at A1- and A2A-adenosine receptors, and the latter paper discloses their affinities at various binding sites of the GABAA-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 Oct. 2002, allegedly published on Web Sep. 24, 2002), pp. 4875-4887 disclose a series of 4-amino-1-(2-chloro-2-phenylethyl)-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid ethyl esters as A1-adenosine receptor ligands.
WO 02/060900 A2 appears to disclose, as MCP-1 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 1H-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-, 4-, 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.
It is desirable to find new compounds which bind to, and preferably inhibit, phosphodiesterase type IV (PDE4).
The present invention provides a compound of formula (I) or a salt thereof (in particular, a pharmaceutically acceptable salt thereof):
wherein:
Preferably, where X is OR5a, the compound is other than the compound wherein R1 is methyl, X is OEt, and R3 is cyclopentyl.
In one optional embodiment of the invention, R1 is C1-4alkyl or C1-2fluoroalkyl.
Alternatively or additionally, in one optional embodiment of the invention, R2 is a hydrogen atom (O).
Alternatively or additionally, in one optional embodiment of the invention, R3 is C3-8cycloalkyl or a heterocyclic group being
Alternatively or additionally, in one optional embodiment of the invention, R4 is hydrogen, C1-2alkyl or C1-2fluoroalkyl.
Alternatively or additionally, in one optional embodiment of the invention, R5 is hydrogen, C1-8alkyl, C1-8 fluoroalkyl, or C3-8cycloalkyl; or phenyl optionally substituted with one or two of: a halogen atom, C1-2alkyl, trifluoromethyl, C1-2alkoxy or trifluoromethoxy; or R5 has the sub-formula (x), (y) or (z):
In the alternative to the above R4 and/or R5 optional embodiments, in one optional embodiment of the invention, R4 and R5 taken together can be ——(CH2)p1— where p1=3, 4 or 5 (preferably p1=4 or 5).
In one optional embodiment of the invention, R3 is optionally substituted C3-8cycloalkyl or an optionally substituted heterocyclic group of sub-formula (aa), (bb) or (cc);
Alternatively or additionally to the above optional R3 definition, in one optional embodiment of the invention, X is NR4R5 or OR5a, in which:
In compounds, for example in the compounds of formula (I) (or formula (IA) or formula (IB), see later), an “alkyl” group or moiety may be straight-chain or branched. Alkyl groups, for example C1-8alkyl or C1-6alkyl or C1-4alkyl or C1-3alkyl or C1-2alkyl, which may be employed include C1-6alkyl or C1-4alkyl or C1-3alkyl or C1-2alkyl 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-1-yl, or the like.
A corresponding meaning is intended for “alkoxy”, “alkylene”, and like terms derived from alkyl. For example, “alkoxy” such as C1-6alkoxy or C1-4alkoxy or C1-2alkoxy includes methoxy, ethoxy, propyloxy, and oxy derivatives of the alkyls listed above. “Alkylsulfonyl” such as C1-4alkylsulfonyl includes methylsulfonyl (methanesulfonyl), ethylsulfonyl, and others derived from the alkyls listed above. “Alkylsulfonyloxy” such as C1-4alkylsulfonyloxy includes methanesulfonyloxy (methylsulfonyloxy), ethanesulfonyloxy, et al.
“Cycloalkyl”, for example C3-8cycloalkyl, includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. Preferably, a C3-8cycloalkyl group is C3-6cycloalkyl 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 C1-4fluoroalkyl or C1-3fluoroalkyl or C1-2fluoroalkyl such as monofluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl, 2,2,2-trifluoroethyl (CF3CH2—), 2,2-difluoroethyl (CHF2CH2—), 2-fluoroethyl (CH2FCH2—), etc. “Fluoroalkoxy” includes C1-4fluoroalkoxy or C1-2fluoroalkoxy such as trifluoromethoxy, pentafluoroethoxy, monofluoromethoxy, difluoromethoxy, etc. “Fluoroalkylsulfonyl” such as C1-4fluoroalkylsulfonyl includes trifluoromethanesulfonyl, pentafluoroethylsulfonyl, etc.
A halogen atom (“halo”) present in compounds, for example in the compounds of formula (I), can be a fluorine, chlorine, bromine or iodine atom (“fluoro”, “chloro”, “bromo” or “iodo”).
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 one or more covalent bonds, 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.
Preferably, R1 is C1-4alkyl (e.g. methyl, ethyl, n-propyl, isopropyl or n-butyl), C1-3fluoroalkyl or —CH2CH2OH; R1 is more preferably C1-3alkyl (e.g. methyl, ethyl or n-propyl), C1-2fluoroalkyl, or —CH2CH2OH; still more preferably C1-3alkyl, C2fluoroalkyl or —CH2CH2OH such as methyl, ethyl, n-propyl or —CH2CH2OH. Yet more preferably, R1 is C2-3alkyl (e.g. ethyl or n-propyl), C2fluoroalkyl (e.g. C1fluoroalkyl-CH2— such as CF3—CH2—) or —CH2CH2OH; in particular ethyl, n-propyl or —CH2CH2OH. R1 is most preferably ethyl.
Preferably, R2 is a hydrogen atom (H) or methyl, more preferably a hydrogen atom (H).
Preferably, in R3 there is one substituent or no substituent.
In one optional embodiment, R3 is the optionally substituted C3-8cycloalkyl or the optionally substituted heterocyclic group of sub-formula (aa), (bb) or (cc). In this embodiment, optionally, in R3, the C3-8cycloalkyl or the heterocyclic group of sub-formula (aa), (bb) or (cc) is optionally substituted with one or two substituents independently being (e.g. being) oxo (═O), OH, C1-2alkoxy, C1-2fluoroalkoxy (e.g. trifluoromethoxy), or C1-2alkyl; and wherein any OH, alkoxy or fluoroalkoxy substituent is not substituted at the R3 ring carbon attached (bonded) to the —NH— group of formula (I) and is not substituted at either R3 ring carbon bonded to the Y group of the heterocyclic group (aa), (bb) or (cc).
In one optional embodiment, where R3 is optionally substituted C3-8cycloalkyl, it is not optionally substituted C5cycloalkyl, i.e. not optionally substituted cyclopentyl. In this case, more preferably, R3 is optionally substituted C6-8cycloalkyl.
Where R3 is optionally substituted C3-8cycloalkyl, it is more preferably optionally substituted C6cycloalkyl (i.e. cyclohexyl); for example C6cycloalkyl optionally substituted with one or two substituents independently being (e.g. being) oxo (═O), OH, C1-2alkoxy, C2fluoroalkoxy (e.g. trifluoromethoxy), or C1-2alkyl, and wherein any OH, alkoxy or fluoroalkoxy substituent is not substituted at the R3 ring carbon attached (bonded) to the —NH— group of formula (I).
Where R3 is optionally substituted C3-8cycloalkyl, the one or two optional substituents preferably comprise (e.g. is or independently are (e.g. is or are)) oxo (═O); OH; C1alkoxy; C1fluoroalkoxy (e.g. trifluoromethoxy); NHR21 wherein R21 is a hydrogen atom (H) or C1-2 straight-chain alkyl; C1-2alkyl such as methyl; C1fluoroalkyl such as —CH2F or —CHF2; —CH2OH; —CH2NHR22 wherein R22 is H; —C(O)OR23 wherein R23 is H or methyl; —C(O)NHR24 wherein R24 is H or methyl; —C(O)R25 wherein R25 is methyl; fluoro; hydroxyimino (═N—OH); or (C1-2alkoxy)imino (═N—OR26 where R26 is C1-2alkyl); and wherein any OH, alkoxy, fluoroalkoxy or NHR21 substituent is not substituted at the R3 ring carbon attached (bonded) to the —NH— group of formula (I) and is not substituted at either R3 ring carbon bonded to the Y group of the heterocyclic group (aa), (bb) or (cc).
More preferably, where R3 is optionally substituted C3-8cycloalkyl, the one or two optional substituents comprise (e.g. is or independently are (e.g. is or are)) oxo (═O); OH; NHR21 wherein R21 is a hydrogen atom (H); C1-2alkyl such as methyl; C1fluoroalkyl such as —CH2F or —CHF2; —C(O)OR23 wherein R23 is H or methyl; —C(O)NHR24 wherein R24 is H or methyl; fluoro; hydroxyimino (═N—OH); or (C1-2alkoxy)imino (═N—OR26 where R26 is C1-2alkyl).
Still more preferably, where R3 is optionally substituted C3-8cycloalkyl, the one or two optional substituents comprise (e.g. is or independently are (e.g. is or are)) oxo (═O); OH; NHR21 wherein R21 is a hydrogen atom (H); methyl; —CH2F; —CHF2; —C(O)OR23 wherein R23 is H; fluoro; hydroxyimino (═N—OH); or (C1-2alkoxy)imino (═N—OR26 where R26 is C1-2alkyl). Yet more preferably, where R3 is optionally substituted C3-8cycloalkyl, the one or two optional substituents comprise (e.g. is or independently are (e.g. is or are)) oxo (═O); OH; methyl; fluoro; hydroxyimino (═N—OH); or (C1-2alkoxy)imino (═N—OR26 where R26 is C1-2alkyl).
Most preferably, where R3 is optionally substituted C3-8cycloalkyl, the one or two optional substituents comprise (e.g. is or independently are (e.g. is or are)) OH, oxo (═O) or oximo (═N—OH). For example, the one or two optional substituents can comprise (e.g. is or are) OH and/or oxo (═O).
Optionally, in R3, the C3-8cycloalkyl can be unsubstituted.
Where R3 is optionally substituted C3-8cycloalkyl, e.g. optionally substituted C5-8cycloalkyl such as optionally substituted C6cycloalkyl (optionally substituted cyclohexyl), the one or two optional substituents if present preferably comprise a substituent (for example is or are substituent(s)) at the 3-, 4- or 5-position(s) of the R3 cycloalkyl ring. (In this connection, the 1-position of the R3 cycloalkyl ring is deemed to be the connection point to the —NH— in formula (I)).
Where R3 is optionally substituted C3-8cycloalkyl, any OH, alkoxy, fluoroalkoxy, —CH2OH, —CH2CH2OH, —CH2NHR22, —C(O)OR23, —C(O)NHR24, —C(O)R25 or fluoro substituent (particularly any OH substituent) is more preferably at the the 3-, 4- or 5-position, e.g. 3- or 5-position, of the R3 cycloalkyl (e.g. C6-8cycloalkyl) ring. For example, any OH, alkoxy, fluoroalkoxy, —CH2OH, —CH2CH2OH, —CH2NHR22, —C(O)OR23, —C(O)NHR24, —C(O)R25 or fluoro substituent (particularly any OH substituent) can be at the 3-position of a R3 C5cycloalkyl (cyclopentyl) ring or at the 3-, 4- or 5-position, e.g. 3- or 5-position, of a R3 C6cycloalkyl (cyclohexyl) ring. (In this connection, and also below, the 1-position of the R3 cycloalkyl ring is deemed to be the connection point to the —NH— in formula (I)).
Where R3 is optionally substituted C3-8cycloalkyl, any NHR21 substituent is preferably at the 2-, 3-, 4- or 5-position, preferably the 2- or 3-position or more preferably the 3-position, of the R3 cycloalkyl (e.g. C6-8cycloalkyl e.g. cyclohexyl) ring.
Where R3 is optionally substituted C3-8cycloalkyl, any alkyl or fluoroalkyl substituent is preferably at the 1-, 2-, 3-, 4- or 5-position, more preferably the 1-, 2-, 3- or 5-position, still more preferably the 1- or 3-position, of the R3 cycloalkyl (e.g. C6-8cycloalkyl e.g. cyclohexyl) ring.
Where R3 is optionally substituted C3-8cycloalkyl, any oxo (═O), hydroxyimino (═N—OH); or (C1-4alkoxy)imino (═N—OR26) substituent is preferably at the 3- or 4-position, preferably at the 4-position, of the R3 cycloalkyl (e.g. C6-8cycloalkyl e.g. cyclohexyl) ring.
Where R3 is optionally substituted C3-8cycloalkyl, R3 is preferably cyclohexyl (i.e. unsubstituted), or cyclohexyl substituted by one oxo (═O), OH, NHR21, C1-2alkyl, C1-2fluoroalkyl, —CH2OH, —C(O)OR23, —C(O)NHR24, —C(O)R25, fluoro, hydroxyimino (═N—OH), (C1-4alkoxy)imino (═N—OR26) substituent, or cyclohexyl substituted by two fluoro substituents. More preferably, R3 is cyclohexyl (i.e. unsubstituted), or cyclohexyl substituted by one oxo (═O), OH, NHR21, C1-2alkyl, C1-2fluoroalkyl, —C(O)OR23, fluoro, hydroxyimino (═N—OH) or (C1-4alkoxy)imino (═N—OR26) substituent, or cyclohexyl substituted by two fluoro substituents. Still more preferably R3 is cyclohexyl (i.e. unsubstituted) or cyclohexyl substituted by one oxo (═O), hydroxyimino (═N—OH), C1-2alkyl or OH substituent. The optional substituent can be at the 3- or 4-position, e.g. 3-position, of the R3 cyclohexyl ring; more preferably any OH substituent is preferably at the 3-position of the R3 cyclohexyl ring, and/or any oxo (═O), hydroxyimino (═N—OH) or (C1-4alkoxy)imino (═N—OR26) substituent is preferably at the 4-position of the R3 cyclohexyl ring.
Where R3 is optionally substituted C6cycloalkyl, R3 can for example be 4-hydroxy-cyclohexyl (i.e. 4-hydroxycyclohexan-1-yl), but R3 is more preferably cyclohexyl (i.e. unsubstituted), 3-hydroxy-cyclohexyl (i.e. 3-hydroxycyclohexan-1-yl), 4-oxo-cyclohexyl (i.e. 4-oxocyclohexan-1-yl), 4-(hydroxyimino)cyclohexyl (i.e. 4-(hydroxyimino)cyclohexan-1-yl), 4-(C1-2alkoxyimino)cyclohexyl, 1-methylcyclohexyl or 3-methylcyclohexyl. Where R3 is optionally substituted C6cycloalkyl, R3 is most preferably cyclohexyl (i.e. unsubstituted), 4-oxo-cyclohexyl (i.e. 4-oxocyclohexan-1-yl) or 4-(hydroxyimino)cyclohexyl (i.e. 4-(hydroxyimino)cyclohexan-1-yl).
Where R3 is optionally substituted C5cycloalkyl (optionally substituted cyclopentyl), R3 can for example be cyclopentyl (i.e. unsubstituted) or 3-hydroxy-cyclopentyl.
Where R3 is optionally substituted mono-unsaturated-C5-7cycloalkenyl, preferably it is optionally substituted mono-unsaturated-C5-6cycloalkenyl, more preferably optionally substituted mono-unsaturated-C6cycloalkenyl (i.e. optionally substituted mono-unsaturated-cyclohexenyl=optionally substituted cyclohexenyl). Still more preferably, the R3 cyclohexenyl is optionally substituted cyclohex-3-en-1-yl.
Where R3 is optionally substituted mono-unsaturated-C5-7cycloalkenyl, preferably the R3 cycloalkenyl is optionally substituted with one or two substituents being fluoro or methyl provided that if there are two substituents then they are not both methyl. Preferably, the R3 cycloalkenyl is optionally substituted with one substituent being fluoro or C1-2alkyl (e.g. methyl); more preferably the R3 cycloalkenyl is substituted with one fluoro substituent or is unsubstituted. For R3 cycloalkenyl, the optional substituent(s) can be at the 1-, 2-, 3-, 4- or 5-position(s) of the cycloalkenyl ring.
Where R3 is the heterocyclic group of sub-formula (aa), (bb) or (cc), then Y is preferably O, S, SO2, NH or N—C(O)methyl, more preferably O, NH or N—C(O)methyl, still more preferably O or N—C(O)methyl, most preferably O. (When Y is NH or N—C(O)methyl, then R10 is H or C(O)methyl).
Preferably, R10 is a hydrogen atom (H), methyl, ethyl, C(O)NH2, C(O)methyl or C(O)—CF3. Optionally, R10 can be a hydrogen atom (H), methyl, ethyl, C(O)methyl or C(O)—CF3, more preferably H, C(O)methyl or C(O)—CF3, still more preferably H or C(O)methyl.
Where R3 is the heterocyclic group of sub-formula (aa), (bb) or (cc), then it is preferable that R3 is the heterocyclic group of sub-formula (aa) or (bb), more preferably of sub-formula (bb).
In sub-formula (bb), n1 is preferably 1. In sub-formula (cc), n2 is preferably 1. That is, six-membered rings are preferred in the R3 heterocyclic group.
Suitably, in R3, the heterocyclic group of sub-formula (aa), (bb) or (cc) is unsubstituted (In this connection, where Y is NR10, R10 is not classified as a substituent).
In the R3 heterocyclic group of sub-formula (aa), (bb) or (cc), the one or two optional substituents preferably comprise (e.g. is or independently are ((e.g. is or are)) OH; oxo (═O); C1-2alkyl (e.g. methyl) or C1-2fluoroalkyl (e.g. C1fluoroalkyl such as —CH2F or —CHF2). More preferably, in the R3 heterocyclic group of sub-formula (aa), (bb) or (cc), the one or two optional substituents comprise (e.g. is or independently are ((e.g. is or are)) OH and/or oxo; most preferably the one or two optional substituents comprise (e.g. is or are) oxo (═O). In the R3 heterocyclic group of sub-formula (aa), (bb) or (cc), any oxo (═O) substituents are preferably on a carbon atom bonded (adjacent) to X, and/or can be at the 2-, 3-, 4- or 5-position(s) of the R3 heterocyclic ring. (In this connection, the 1-position of the R3 heterocyclic ring is deemed to be the connection point to the —NH— in formula (I)). Preferably, only C1-2alkyl, C1-2fluoroalkyl, fluoro or oxo (═O) substitution or no substitution is allowed at each of the 2- and 6-positions of the R3 heterocyclic ring.
When R3 is the heterocyclic group of sub-formula (aa) and Y is NR10, then preferably R10 is not C(O)-Me. More preferably, when R3 is the heterocyclic group of sub-formula (aa) and Y is NR10, then R10 is preferably not C(O)R, i.e. or e.g. R10 is preferably not C(O)NH2, C(O)—C1-2alkyl or C(O)—C1fluoroalkyl. In one embodiment, Y is O, S, SO2 or NH when R3 is the heterocyclic-group of sub-formula (aa).
Optionally, according to one embodiment of the invention, NHR3 is not
More preferably, when R3 is the heterocyclic group of sub-formula (bb) and Y is NR10, and optionally when n1 is 1, then preferably R10 is not methyl. More preferably, when R3 is the heterocyclic group of sub-formula (bb) and Y is NR10, and optionally when n1 is 1, then R10 is preferably not alkyl or substituted alkyl, i.e. or e.g. R10 is preferably not C1-4alkyl (e.g. methyl or ethyl), C1-2fluoroalkyl or CH2C(O)NH2. In one embodiment, when R3 is the heterocyclic group of sub-formula (bb), Y is preferably O, S, SO2 or NR10, wherein R10 is H, C(O)NH2, C(O)—C1-2alkyl or C(O)—C1fluoroalkyl, or more preferably Y is H or C(O)Me. More preferably, for sub-formula (bb), Y is O or NR10.
Where R3 is a bicyclic group of sub-formula (dd) or (ee), preferably it is of sub-formula (ee). In sub-formula (ee), preferably Y1, Y2 and Y3 are all CH2.
Preferably, NHR3 is of sub-formula (a), (a1), (b), (c), (c1), (c2), (c3), (c4), (c5), (c6), (c7), (d), (e), (f), (g), (g1), (g2), (g3), (g4), (h), (i), (j), (k), (k1), (L), (m), (m1), (m2), (m3), (m4), (m5), (n), (o), (o1), (o2), (o3), (o4), (o5), (p), (p1), (p2), (p3), (p4), (p5), (p6), (p7), (p8) or (q):
In the sub-formulae (a) to (q) etc above, the —NH— connection point of the NHR3 group to the 4-position of the pyrazolopyridine of formula (I) is underlined.
Preferably, NHR3 is of sub-formula (c), (c1), (c2), (c3), (c4), (c5), (c6), (c7), (d), (e), (f), (g1), (g4), (h), (i), (j), (k), (k1), (L), (m), (m1), (m2), (m3), (m5), (n), (o), (o1), (o2), (o3), (o4), (o5), (p), (p2), (p3), (p5), (p6), (p7) or (q). More preferably, NHR3 is of sub-formula (c), (c1), (c4), (c5), (h), (i), (j), (k), (m1), (m2), (n), (o), (o2), (o3), (p2), (p5), (p6) or (q). Still more preferably, NHR3 is of sub-formula (c), (h), (k), (n), (o) or (o2); for example (c), (h), (o) or (o2). Most preferably, R3 is tetrahydro-2H-pyran-4-yl; that is NHR3 is most preferably of sub-formula (h), as shown above.
According to one embodiment, NHR3 is of sub-formula (a), (b), (c), (d), (e), (f), (g), (g1), (g2), (g3), (h), (i), (j), k), (L), (m), (m1), (n), (o), (o1), (p) or (q). In this embodiment, preferably, NHR3 is of sub-formula (c), (d), (e), (f), (g1), (h), (i), (j), (k), (m), (m1), (n), (o), (o1), (p), or (q); and more preferably in this embodiment, NHR3 is of sub-formula (c), (h), (i), (j), (k), (m1), (n), (o) or (q). Still more preferably in this embodiment, NHR3 is of sub-formula (c), (h), (k), (n) or (o). Most preferably, R3 is tetrahydro-2H-pyran-4-yl; that is NHR3 is most preferably of sub-formula (h), as shown above.
According to another embodiment, NHR3 is of sub-formula (a), (b), (c), (d), (e), (f), (g), (h), (i), (j) or (k). In this embodiment, preferably, NHR3 is of sub-formula (c), (d), (e), (f), (h), (i), (j) or (k); and more preferably in this embodiment, NHR3 is of sub-formula (c), (h), (i), (j) or k). Most preferably, R3 is tetrahydro-2H-pyran-4-yl; that is NHR3 is most preferably of sub-formula (h), as shown above.
When NHR3 is of sub-formula (n), then preferably it is a cis-(3-hydroxycyclohex-1-yl)amino group, eg in any enantiomeric form or mixture of forms but preferably racemic.
Preferably, X is NR4R5.
Where R4 is C1-6alkyl, then preferably it is C1-4alkyl or C1-2alkyl. Where R4 is C1-3fluoroalkyl then preferably it is C1-2fluoroalkyl.
Most preferably, R4 is a hydrogen atom (H).
Where R4 is C2-6alkyl substituted by one substituent R1, then preferably R4 is C2-4alkyl (e.g. C2-3alkyl) substituted by one substituent R11. More preferably, R4 is —(CH2)n3—R11 wherein n3 is 2, 3 or 4. Still more preferably, n3 is 2 and/or R4 is —(CH2)n3—OH.
When R5 is C2-6alkyl substituted by one or two independent substituents R11, it is preferable that R5 is C2-4alkyl (e.g. C2-3alkyl) substituted by one or two independent substituents R11. When R5 is C2-6alkyl (e.g. C2-4alkyl or C2-3alkyl) substituted by one or two independent substituents R11, it is preferable that R5 is C2-6alkyl (e.g. C2-4alkyl or C2-3alkyl) substituted by one substituent R11. It is more preferable that R5 is —(CH2)n5—R11 wherein n5 is 2, 3 or 4. Preferably n5 is 2 or 3, more preferably 2.
Preferably, each substituent R11, independently of any other R11 substituent present, is: hydroxy (OH); C1-6alkoxy (e.g. C1-4alkoxy such as t-butyloxy, ethoxy or methoxy); phenyloxy; benzyloxy; —NR12R13; —NR15—C(O)R16; —NR15—C(O)—NH—R15; or —NR15—SO2R16 (more preferably C1-6alkoxy, —NR15—C(O)—NH—R15, or —NR15—SO2R16; most preferably —NR15—SO2R16). In all cases, any R1 substituent which is OH, alkoxy or —NR12R13 is not substituted at any carbon atom, of any R4 or R5 substituted alkyl, which is bonded to the nitrogen of NR4R5.
Where R5 is C1-8alkyl, then preferably it is C1-5alkyl or C1-3alkyl. Where R5 is C1-8fluoroalkyl then preferably it is C1-3fluoroalkyl or C1-2fluoroalkyl. Where R5 is C3-8cycloalkyl optionally substituted by a C1-2alkyl group, then preferably the C3-8cycloalkyl is not substituted at the ring-carbon bonded to the nitrogen of NR4R5. Where R5 is optionally substituted C3-8cycloalkyl, then more preferably it is C3-8cycloalkyl (i.e. unsubstituted).
When R5 is optionally substituted —(CH2)n4—C3-8cycloalkyl wherein n4 is 1, 2 or 3, then n4 is preferably 1 or 2 or more preferably 1, and/or preferably R5 is optionally substituted —(CH2)n4—C5-6cycloalkyl or optionally substituted —(CH2)n4—C6cycloalkyl. When R5 is optionally substituted —(CH2)n4—C3-8cycloalkyl, preferably it is not substituted. Most preferably R5 is (cyclohexyl)methyl-, that is —CH2-cyclohexyl.
When R19 is C1-4alkyl, then preferably it is isobutyl, sec-butyl, or C1-3alkyl such as methyl or isopropyl. When R19 is —(CH2)n20—OR20, then preferably n20 is 1 and/or preferably R20 is a hydrogen atom (H).
When R5 is —(CH2)n11—C(O)R16; —(CH2)n12—C(O)NR12R13; —CHR19—C(O)NR12R13; —(CH2)n12—C(O)OR16; —CHR19—C(O)OR16; —(CH2)n12—SO2—NR12R13; —(CH2)n12—SO2R16; or —(CH2)n12—CN; then in one embodiment of the invention R5 can be: —(CH2)n11—C(O)R16; —(CH2)n12—C(O)NR12R13; —(CH2)n12—C(O)OR16; —(CH2)n12—SO2—NR12R13; —(CH2)n12—SO2R16; or —(CH2)n12—CN.
When R5 is —(CH2)n11—C(O)R16; —(CH2)112—C(O)NR12R13; —(CH2)n2—C(O)OR16; —(CH2)n12—SO2—NR12R13; —(CH2)n12—SO2R16; or —(CH2)n112—CN; then R5 can for example be —(CH2)n11—C(O)R16; —(CH2)n12—C(O)NR12R13; or —(CH2)n12—CN; preferably —(CH2)n11—C(O)R16.
Preferably, n11 is 1, 2, 3 or 4; more preferably n11 is 1 or 2. Advantageously, n12 is 1 or 2.
When R5 is —(CH2)n13—Het, it is preferable that n13 is 0, 1 or 2, more preferably 0 or 1.
Preferably, Het is a 5- or 6-membered saturated or partly-saturated heterocyclic ring and/or preferably is a 4-, 5-, 6- or 7-membered saturated heterocyclic ring. Preferably, the heterocyclic ring Het contains one ring-hetero-atom selected from O, S and N.
Preferably, the carbon ring-atoms in Het are not substituted. Het is most preferably one of:
When R5 is optionally substituted phenyl, then preferably it is phenyl optionally substituted with one or two of the substituents defined herein.
When R5 is optionally substituted phenyl, then preferably R5 is phenyl optionally substituted with, independently, one, two or three (preferably one or two; or one) of: a halogen atom (preferably fluoro and/or chloro); C1-2alkyl; C1-2fluoroalkyl (e.g. trifluoromethyl); C1-2alkoxy (e.g. methoxy); trifluoromethoxy, C1-2alkylsulphonyl (C1-2alkyl-SO2—); C1-2alkyl-SO2—NH—; R7R8N—SO2—; R7R8N—CO—; —NR15—C(O)R16; R7R8N; OH; C1-2alkoxymethyl; C1-2alkyl-SO2—CH2—; cyano (CN); or phenyl optionally substituted by one of fluoro, C1-2alkyl, C1fluoroalkyl, C1-2alkoxy or C1fluoroalkoxy. More preferably R5 is phenyl optionally substituted with one or two (preferably one) of: a halogen atom, C1-2alkyl, trifluoromethyl, C1-2alkoxy, trifluoromethoxy, R7R8N—SO2—, R7R8N—CO—, or C1-2alkyl-SO2—CH2—. When R5 is optionally substituted phenyl, then preferably one or all of the one or two optional substituents are substituted at the meta-(3- and/or 5-) and/or para-(4-) position(s) of the phenyl ring with respect to the phenyl ring-carbon bonded to the nitrogen of NR4R5.
Preferably, R7 and/or R8 are independently a hydrogen atom (H); C1-2alkyl such as methyl; C3-6cycloalkyl; or phenyl optionally substituted by one of: fluoro, chloro, C1-2alkyl, C1fluoroalkyl, C1-2alkoxy or C fluoroalkoxy; or R7 and R8 together are —(CH2)n6— or —(CH2)n8—X7—(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 C1-2alkyl. It is preferable that R7 is a hydrogen atom (H).
Preferably n6 is 4 or 5. Preferably n7 is 2, 3 or 4. Preferably, n8, n9 and/or n10 is/are independently 2.
In general, it is preferable that R5 has the sub-formula (x) or (y) or (y1) or (z).
When R5 has the sub-formula (x) or (y) or (y1) or (z), then preferably R5 has the sub-formula (x) or (y) or (y1) or has the sub-formula (x) or (y) or (z). More preferably R5 has the sub-formula (x) or (y), most preferably (x). In one embodiment, R5 has the sub-formula (z).
Preferably, n is 1 or 2. More preferably, n=1. Preferably, m=1. Preferably, r=1 or 2, more preferably 1.
In sub-formula (x), (y) and/or (y1), 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 CR6; 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 CR6; and the remaining of A, B, D, E and F are CH.
In sub-formula (x), (y) and/or (y1), 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 CR6.
Preferably, sub-formula (x) is: benzyl; phenethyl (Ph-C2H4—); benzyl substituted on the phenyl ring with one or two R6 substituents; phenethyl (Ph-C2H4—) substituted on the phenyl ring with one or two R6 substituents; or one of the following:
, wherein R6a is either R6 as defined herein or preferably) hydrogen.
Most preferably, sub-formula (x) is benzyl or pyridinylmethyl [e.g. pyridin-4-ylmethyl
pyridin-3-ylmethyl, or preferably pyridin-2-ylmethyl
Preferably, sub-formula Cy) is:
Preferably, sub-formula (y1) is:
wherein R6a is or independently are either R6 as defined herein or preferably hydrogen.
Preferably, in sub-formula (z), none, one or two of J, L, M and Q are nitrogen.
In sub-formula (x), (y) and/or (z), preferably, each R6, independently of any other R6 present, is a fluorine, chlorine, bromine or iodine atom, methyl, ethyl, n-propyl, isopropyl, C4alkyl, trifluoromethyl, —CH2OH, methoxy, ethoxy, C1fluoroalkoxy (e.g. trifluoromethoxy or difluoromethoxy), OH, C1-3alkylS(O)2— (such as methylsulphonyl which is MeS(O)2—), C1-3alkylS(O)2—NH— such as methyl-SO2—NH—, Me2N—S(O)2—, H2N—S(O)2—, —CONH2, —CONHMe, —CO2H, cyano (CN), NMe2, t-butoxymethyl, or C1-3alkylS(O)2—CH2— such as methyl-SO2—CH2—. More preferably, each R6, independently of any other R6 present, is a fluorine, chlorine, bromine or iodine atom, methyl, ethyl, n-propyl, isopropyl, isobutyl, trifluoromethyl, —CH2OH, methoxy, ethoxy, C1fluoroalkoxy (e.g. trifluoromethoxy or difluoromethoxy), C1-3alkylS(O)2— such as methylsulphonyl, C1-3alkylS(O)2—NH— such as methyl-SO2—NH—, Me2N—S(O)2—, H2N—S(O)2—, —CONH2, or C1-3alkylS(O)2—CH2— such as methyl-SO2—CH2. Still more preferably, each R6, independently of any other R6 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—.
The above preferred R6 substituents are also, independently, the preferred phenyl optional and independent substituents for where R5 is optionally substituted phenyl.
In sub-formula (x) and/or (y), preferably, one, two or three R6 substituents are present in B, D and/or E; so that for example in sub-formula (x), one, two or three R6 substituents are present in the meta-(3- and/or 5-) and/or para-(4-) positions with respect to the —(CH2)n— side-chain.
Preferably, R5 has the sub-formula (x), n is 1 and none of A, B, D, E and F are nitrogen or nitrogen-oxide (N+—O−); and all of A, B, D, E and F are independently CH or CR6; that is R5 has the sub-formula (x) and is optionally substituted benzyl. In this embodiment, preferably, a R6 substituent is present at the 4-position with respect to the ——(CH2)n— side-chain (that is D is CR6: i.e. a R6 substituent is present in D); and/or preferably a R6 substituent is present at the 3- and/or 5-position with respect to the —(CH2)n— side-chain (that is B and/or E is CR6: i.e. one or two R6 substituents are present in B and/or E). For monosubstitution, i.e. where one of A, B, D, E and F is CR6, then the one R6 substituent is preferably present at the 4-position with respect to the ——(CH2)n— side-chain (i.e. D is CR6). Where there is disubstitution, that is where two of A, B, D, E and F are independently CR6, then 3,4-disubstitution (B+D or D+E are independently CR6), 2,4-disubstitution (A+D or D+F are independently CR6) or 2,3-disubstitution (A+B or E+F are independently CR6) is preferred.
In sub-formula (x) and/or (y), any optional R6 substituent can optionally be present only in B, D and/or E, so that in sub-formula (x) any optional R6 substituent is present only in the meta-(3- and/or 5-) and/or para-(4-) positions with respect to the ——(CH2)n— side-chain. Alternatively, in sub-formula (x), any optional R6 substituent can be present in the ortho-(2- and/or 6-) position with respect to the —(CH2)n— side-chain, either alone or in combination with one or more other optional R6 substituents.
Overall for R5, it is preferable that R5 is a hydrogen atom (H); C1-6alkyl (e.g. C1-2 or 3alkyl or C3-6alkyl); C1-4fluoroalkyl, C3-6cycloalkyl (e.g. C5-6cycloalkyl), (C5-6cycloalkyl)methyl-, phenyl optionally substituted with one or two of: a fluorine or chlorine atom, methyl, trifluoromethyl, methoxy or trifluoromethoxy; or R5 has the sub-formula (x), (y) or (z), for example as described above.
Still more preferably, R5 is a hydrogen atom (H), methyl, ethyl, n-propyl, iso-propyl, 2-ethylbutan-1-yl, cyclopentyl, cyclohexyl, (cyclohexyl)methyl-, optionally substituted phenyl e.g. fluorophenyl e.g. 4-fluorophenyl, optionally substituted benzyl, or optionally substituted pyridinylmethyl, or R5 has the sub-formula (z).
Optionally, R5 can be benzyl, pyridinylmethyl (e.g. pyridin-4-ylmethyl, pyridin-3-ylmethyl, or preferably pyridin-2-ylmethyl), or 4-fluorophenyl.
In one preferable embodiment, R5 has the sub-formula (x) and is: benzyl, (monoalkyl-phenyl)methyl, [mono(fluoroalkyl)-phenyl]methyl, (monohalo-phenyl)methyl, (monoalkoxy-phenyl)methyl, [mono(fluoroalkoxy)-phenyl]methyl, [mono(N,N-dimethylamino)-phenyl]methyl, [mono(methyl-SO2—NH—)-phenyl]methyl, [mono(methyl-SO2—)-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, R5 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, R5 is: -(monoC1-3alkyl-phenyl)methyl such as (4-C1-3alkyl-phenyl)methyl; -(monoC1fluoroalkyl-phenyl)methyl such as (4-C1fluoroalkyl-phenyl)methyl; -(monoC1-2alkoxy-phenyl)methyl such as (4-C1-2alkoxy-phenyl)methyl; -[mono(C1fluoroalkoxy)-phenyl]methyl such as (4-C1fluoroalkoxy-phenyl)methyl; -(diC1-2alkyl-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; -(monoC1-2alkyl-monohalo-phenyl)methyl or (monoC1-2alkyl-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-monoC1-2alkyl-phenyl)methyl e.g. (2,4-dichloro-6-methyl-phenyl)methyl; or -[dihalo-mono(hydroxymethyl)-phenyl]methyl such as [2,3-dichloro-6-(hydroxymethyl)-phenyl]methyl.
In an alternative preferable embodiment, R5 has the sub-formula (z), and one or preferably none of J, L, M or Q is CR6, and/or R9 is a hydrogen atom (H) or methyl. Preferably r is 1. Preferably, for (z), R6 is independently OH (including any keto tautomer thereof), or more preferably C1-2alkyl (e.g. methyl) or C1fluoroalkyl.
Preferably NR4R5 is not NH2. R5 is preferably not a hydrogen atom (H).
When R4 and R5 taken together are optionally substituted —(CH2)p1— or optionally substituted —C(O)—(CH2)p2— or —(CH2)p3—X5—(CH2)p4— or —C(O)—X5—(CH2)p5— or a partially unsaturated derivative of any of the foregoing, preferably R4 and R5 taken together are optionally substituted —(CH2)p1— or optionally substituted —C(O)—(CH2)p2— or —(CH2)p3—X5—(CH2)p4— or —C(O)—X5—(CH2)p5— (i.e. not a partially unsaturated derivative of any of these).
When R4 and R5 taken together are ——(CH2)p1— optionally substituted by R18, or —C(O)—(CH2)p2— optionally substituted by R18, or —(CH2)p3—X5—(CH2)p4—, NR4R5 can for example be
optionally substituted by R18, or
optionally substituted by R18, or
optionally substituted by R18, or
(i.e. R4 and R5 taken together are —(CH2)2—N(R17)—(CH2)2—), or
(i.e. R4 and R5 taken together are —(CH2)2—O—(CH2)2—).
Preferably, R17 is a hydrogen atom (H); C1-4alkyl (e.g. C1-2alkyl); C3-6cycloalkyl; —(CH2)p6—C(O)R16, or the optionally substituted phenyl or benzyl. More preferably, R17 is H; C1-2alkyl; —(CH2)p6—C(O)R16 or the optionally substituted phenyl.
When R4 and R5 taken together are —(CH2)p1— or —C(O)—(CH2)p2—, the NR4R5 heterocycle is preferably not substituted by R18.
When R4 and R5 taken together are —(CH2)p1— or —C(O)—(CH2)*2—, and if the NR4R5 heterocycle is substituted by R18, then optionally R18 is not substituted at a ring-carbon bonded to the NR4R5 ring-nitrogen.
When R4 and R5 taken together are ——(CH2)p1— or —C(O)—(CH2)p2— or —(CH2)p3—X5—(CH2)p4— or —C(O)—X5—(CH2)p5— or a partially unsaturated derivative of any of these, and wherein the NR4R5 heterocycle is fused to a phenyl ring optionally substituted on the phenyl by one or two of: a halogen atom, C1-2alkyl, C1fluoroalkyl, C1-2alkoxy or C1fluoroalkoxy; then in one embodiment of the invention NR4R5 is
wherein the phenyl is optionally substituted by one or two of: a halogen atom, C1-2alkyl, C1fluoroalkyl, C1-2alkoxy or C1fluoroalkoxy.
In one embodiment of the invention, NR7R8 and/or NR12R13 can for example independently be
(i.e. R12 and R13 together or R7 and R8 together are —(CH2)2—N(R14)—(CH2)2—), or (i.e. R12 and R13 together or R7 and R8 together are —(CH2)2—O—(CH2)2—), or NMe2.
Preferably, R15 is a hydrogen atom (H) or C1-4alkyl (e.g. tBu or C1-2alkyl e.g. methyl); more preferably, R15 is a hydrogen atom (H).
Preferably, however, R4 and R5 are not taken together, i.e. are not taken together to form the NR4R5 ring systems described herein.
(Similar preferances apply for R5a as for R5, except that R5a cannot be a hydrogen atom. Most preferably, R5a is ethyl.)
In an especially preferable embodiment, NR4R5 is the NR4R5 group as defined in any one of: Examples 21-98, 100-182, 187-188, 191-200, 201-203, 210-353, 355-651, 653-658, 660-664 and 665-686.
It is particularly preferred that the compound of formula (I) or the salt thereof is:
The structures of these specific compounds are given in Examples 1-98 hereinafter.
Alternatively, it is particularly preferred that the compound of formula (I) or the salt thereof is:
The structures of these specific compounds are given in Examples 100-201 hereinafter.
Alternatively, the compound of formula (I) or the salt thereof can be:
Alternatively, it is particularly preferred that the compound of formula (I) or the salt thereof is one of Examples 204 to 664 or one of Examples 665 to 686, as a compound or a salt thereof, e.g. a pharmaceutically acceptable salt thereof. The structures of these specific compounds are given in Examples 204 to 664 and Examples 665 to 686 hereinafter, and their names are given in the Examples section.
In one embodiment, is still further preferred that the compound of formula (I) or the salt thereof is a compound of Example 260, 261, 263, 266, 431, 493, 494, 518, 528, 584, 626, 643, 653, 679, 680, 681, 682, 683, 684, 685 or 686 (more preferably Example 260, 518, 653, 679, 680, 681 or 684), as defined by the structures and/or names described herein, or a salt thereof, e.g. a pharmaceutically acceptable salt thereof. The structures and names of these Examples are described in the Examples section. These Examples are thought to be suitable for inhaled administration.
In another embodiment, is still further preferred that the compound of formula (I) or the salt thereof is a compound of Example 21, 22, 83, 100, 109, 167, 172, 178 or 600, as defined by the structures and/or names described herein, or a salt thereof, e.g. a pharmaceutically acceptable salt thereof. The structures and names of these Examples are described in the Examples section. These Examples are thought to be suitable for oral administration.
A second aspect of the present invention provides a compound of formula (IA) or a salt thereof (in particular, a pharmaceutically acceptable salt thereof):
wherein:
In formula (IA), preferably, when R3 is the heterocyclic group being
and Y is NR10, then:
In formula (IA), preferably, where X is OR5a, the compound is other than the compound wherein R1 is methyl, X is OEt, and R3 is cyclopentyl.
In formula (IA), in sub-formula (x) and/or (y), 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 CR6; 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 or one or two of A, B, D, E and F are CR6; and the remaining of A, B, D, E and F are CH. In formula (IA), in sub-formula (x) and/or (y), preferably, none or one of A, B, D, E and F are nitrogen.
In formula (IA), preferably, sub-formula (x) is: benzyl; phenethyl (Ph-C2H4—); benzyl or phenethyl being substituted on the phenyl ring with a single R6 substituent, or one of the following:
wherein R6a is either R6 as defined herein or (preferably) hydrogen.
In formula (IA), preferably, sub-formula (y) is:
wherein R6a is either R6 as defined herein or preferably hydrogen.
Examples 1-99 are examples of compounds or salts of the second aspect of the invention (Formula (IA)).
A third aspect of the present invention provides a compound of formula (IB) or a salt thereof (in particular, a pharmaceutically acceptable salt thereof):
wherein:
In formula (IB), preferably, when R3 is the heterocyclic group of sub-formula (bb), n1 is 1, and Y is NR10, then:
In formula (IB), preferably, where X is OR5a, the compound is other than the compound wherein R1 is methyl, X is OEt, and R3 is cyclopentyl.
In formula (IB), where R3 is optionally substituted C3-8cycloalkyl, the one or two optional substituents preferably comprise (e.g. is or are) OH and/or oxo (═O). In formula (IB), in the R3 heterocyclic group of sub-formula (aa), (bb) or (cc), the one or two optional substituents preferably comprise (e.g. is or are) OH and/or oxo.
Examples 1-203 are examples of compounds or salts of the third aspect of the invention (Formula (IB)).
The preferred or optional features for the compound or salt of formula (IA) and for the compound or salt of formula (IB) are the same as or similar to the preferred or optional features for the compound or salt of formula (I), with all necessary changes (for example to the formula, to the R groups and/or to substituents) having been made. Generally, whenever formula (I) is mentioned herein, then in alternative embodiments the statement mentioning formula (I) applies to formula (IA) or formula (IB), with all necessary changes having been made.
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 can include acid or base addition salts. A pharmaceutically acceptable acid addition salt can be formed by reaction of a compound of formula (I) with a suitable inorganic or organic acid (such as hydrobromic, hydrochloric, sulfuric, nitric, phosphoric, succinic, maleic, acetic, fumaric, citric, tartaric, benzoic, p-toluenesulfonic, methanesulfonic or naphthalenesulfonic 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) can be for example a hydrobromide, hydrochloride, sulfate, nitrate, phosphate, succinate, maleate, acetate, fumarate, citrate, tartrate, benzoate, p-toluenesulfonate, methanesulfonate or naphthalenesulfonate salt. A pharmaceutically acceptable base addition salt can be formed by reaction of a compound of formula (I) with a suitable inorganic or organic base, 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. Other suitable 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, may be 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.
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 medicinal use, the compound of formula (I) can optionally 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 the invention:
Most of the following synthetic processes following are exemplified for compounds of Formula (I) wherein R2 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 R2 is other than H.
Process A
Compounds of formula (I) where X═OR5a, can be prepared according to a method, for example as described by Yu et. al. in J. Med Chem., 2001, 44, 1025-1027, by reaction of a compound of formula (II) with an amine of formula R3NH2. The reaction is preferably carried out in the presence of a base such as triethylamine or N,N-diisopropylethylamine, and/or in an organic solvent such as ethanol, dioxane or acetonitrile. The reaction may require heating e.g. to ca. 60-100° C., for example ca. 80-90° C.:
Compounds of formula (II) are also described in the above reference and can be prepared by reaction of a compound of formula (III) with, for example, diethylethoxymethylene malonate (where R5a=Et) with heating, followed by reaction with phosphorous oxychloride, again with heating:
Where the desired amino pyrazole of formula (III) 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 R40CHO in a solvent such as ethanol, with heating, followed by reduction with, for example sodium in a solvent such as t-butanol. R40 should be chosen so as to contain one less carbon atom than R1, for example R40=methyl will afford R1=ethyl.
In an alternative embodiment of Process A, the 4-chloro substituent in the compound of formula (II) can be replaced by a halogen atom, such as a bromine atom or preferably a chlorine atom, in a compound of formula (IIA) as defined below. In this embodiment of Process A, the compound of formula (IIA) is reacted with the amine of formula R3NH2.
Process B
Compounds of formula (I) where X═NR4R5, can be prepared by reaction of a compound of formula (I) with an amine of formula R3NH2. The reaction is preferably carried out in the presence of a base, such as triethylamine or N,N-diisopropylethylamine, and/or in an organic solvent such as ethanol, THF, dioxane or acetonitrile. The reaction may require heating, e.g. to ca. 60-100° C. or ca. 80-90° C., for example for 8-48 or 12-24 hours:
Compounds of formula (IV) can be prepared in a two step procedure as described by Bare et. al. in J. Med. Chem. 1989, 32, 2561-2573. This process involves, first, reaction of a compound of formula (V) 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 TBF, or as a neat solution. This reaction may require heating and the thus-formed intermediate may or may not be isolated. Step two involves reaction 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 diisopropylethyl amine:
Compounds of formula (V) can be prepared by hydrolysis of an ester of formula (II) 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:
In an alternative embodiment of Process B, the 4-chloro substituent in the compound of formula (IV) can be replaced by a halogen atom, such as a bromine atom or preferably a chlorine atom, in a compound of formula (IVA) as defined below. In this embodiment of Process B, the compound of formula (IVA) is reacted with the amine of formula R3NH2.
Process C
Compounds of formula (I) can also be prepared according to a method, for example as described by Bare et. al. in J. Med. Chem. 1989, 32, 2561-2573, which involves reaction of a compound of formula (VI), in which —O—R35 is a leaving group displaceable by an amine, with an amine of formula R3NH2. The —O—R35 leaving group can be —O—C1-4alkyl (in particular —O-Et) or —O—S(O)2—R37, wherein R37 is C1-8alkyl (e.g. C1-4alkyl or C1-2alkyl such as methyl), C1-6fluoroalkyl (e.g. C1-4fluoroalkyl or C1-2fluoroalkyl such as CF3 or C4F9), or phenyl wherein the phenyl is optionally substituted by one or two of independently C1-2alkyl, halogen or C1-2alkoxy (such as phenyl or 4-methyl-phenyl). The reaction may be carried out with or without solvent and may require heating:
Compounds of formula (VI) (also described in the above reference) can be prepared by reaction of a compound of formula (VII) with a suitable alkylating agent of formula R1—X, where X is a leaving group such as halogen. The reaction is preferably carried out in the presence of a base such as potassium carbonate, in an anhydrous solvent such as DMF:
The preparation of compounds of formula VII, e.g. where OR35 is OEt, by oxidative cleavage of compounds of formula VIII is described by Bare et. al. in J. Med. Chem. 1989, 32, 2561-2573 (further referred to Zuleski et. al. in J. Drug. Metab. Dispos., 1985, 13, 139).
In another embodiment of Process C, the compound of formula (VI) can be replaced by a compound of formula (VIA), wherein X is NR4R5 or OR5a as defined herein:
In this embodiment of Process C, the compound of formula (VIA) is reacted with the amine of formula R3NH2.
Process D:
To form a compound of formula (I) wherein X═NR4R5, a compound of formula (I) but wherein X═OH (a carboxylic acid, the compound of formula (IX) as defined below) can be converted into an activated compound of formula (I) but wherein X=a leaving group X1 substitutable by an amine (a compound of formula (X) as defined below, wherein X1 is a leaving group substitutable by an amine); and subsequently the activated compound can be reacted with an amine of formula R4R5NH:
For example, the activated compound (the compound of formula (X)) can be the acid chloride i.e. an activated compound of formula (I) but wherein the leaving group X1═Cl. This can be formed from the carboxylic acid (X═OH, the compound of formula (DC)) e.g. by reaction with thionyl chloride, either in an organic solvent such as chloroform or without solvent. See for example Examples 81-85. Alternatively, the activated compound (the compound of formula (X)) can be an activated ester wherein the leaving group X1 is
The latter activated compound of formula (X) can be formed from the carboxylic acid (X═OH, the compound of formula (IX))
either:
The carboxylic acid wherein X═OH (the compound of formula (IX) below) is usually prepared by hydrolysis of the corresponding ester of formula (I) wherein X is OR5a. This ester can itself be prepared by any of Processes A, C, E or F as described herein.
Process D1
This is the same as Process D, but involves reaction of the activated compound of formula (X), wherein X1=a leaving group substitutable by an amine (for example a leaving group as defined herein), with an amine of formula R4R5NH.
Process E
Compounds of formula (I) can be prepared by reaction of a compound of formula (XI) with an alkylating agent of formula R1—X3, where X3 is a leaving group displaceable by the 1-position pyrazolopyridine nitrogen atom of the compound of formula (XI):
A suitable alkylating agent of formula R1—X3 can be used. For example, X3 can be a halogen atom such as a chlorine atom or more preferably a bromine or iodine atom, or X3 can be O—S(O)2—R36 wherein R36 is C1-8alkyl (e.g. C1-4alkyl or C1-2alkyl such as methyl), C1-6fluoroalkyl (e.g. C1-4fluoroalkyl or C1-2fluoroalkyl such as CF3 or C4F9), or phenyl wherein the phenyl is optionally substituted by one or two of independently C1-2alkyl, halogen or C1-2alkoxy (such as phenyl or 4-methyl-phenyl). The reaction is preferably carried out in the presence of a base; the base can for example comprise or be potassium carbonate, sodium carbonate, sodium hydride, potassium hydride, or a basic resin or polymer such as polymer-bound 2-tert-butylimino-2-diethylamino-1,3-dimethyl-perhydro-1,3,2-diazaphosphorine. The reaction is preferably carried out in the presence of a solvent, e.g. an organic solvent such as DMF; the solvent is preferably anhydrous. Examples of alkylation Process E include Examples 183, 185, 186 and 354.
For preferable methods of making compounds of formula (XI), see for example (Reference) Examples 19-20, and Intermediates 48 and 54A.
Process F: Conversion of One Compound of Formula (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 preferably comprises or is one or more of the following processes F1 to F10:
F1. An oxidation process. For example, the oxidation process can comprise or be oxidation of an alcohol to a ketone (e.g. using Jones reagent, e.g. see Example 205) or oxidation of an alcohol or a ketone to a carboxylic acid. The oxidation process can e.g. comprise or be conversion of a nitrogen-containing compound of formula (I) or salt thereof to the corresponding N-oxide (e.g. using meta-chloroperoxybenzoic acid), for example conversion of a pyridine-containing compound to the corresponding pyridine N-oxide (e.g. Examples 210-212).
F2. A reduction process, for example reduction of a ketone or a carboxylic acid to an alcohol.
F3. Acylation, for example acylation of an amine (e.g. Examples 329-349, Example 353) or hydroxy group.
F4. Alkylation, for example alkylation of an amine or of a hydroxy group.
F5. Hydrolysis, e.g. hydrolysis of an ester to the corresponding carboxylic acid or salt thereof (e.g. Examples 351, 488, 489, 650, 651).
F6. Deprotection, e.g. deprotection (e.g. deacylation or t-butyloxycarbonyl (BOC) removal) of an amine group (e.g. Examples 320, (321), and (352)).
F7. Formation of an ester or amide, for example from the corresponding carboxylic acid.
F8. Conversion of a ketone into the corresponding oxime (e.g. Examples 652, 653, 654 and 680-686).
F9. Sulfonylation, e.g. sulfonamide formation by reaction of an amine with a sulfonyl halide e.g. a sulfonyl chloride (e.g. Examples 322-328). and/or
F10. Beckmann rearrangement of one compound of formula (I) into another compound of formula (I), preferably using cyanuric chloride (2,4,6-trichloro-1,3,5-triazine) together with a formamide such as DMF, e.g. at room temperature (see L. D. Luca, J. Org. Chem., 2002, 67, 6272-6274). The Beckmann rearrangement can for example comprise conversion of a compound of formula (I) wherein NHR3 is of sub-formula (o2)
into a compound of formula (I) wherein NHR3 is of sub-formula
e.g. as illustrated in Examples 658 and 659.
The present invention therefore also provides a method of preparing a compound of formula (I) or a salt thereof:
In methods (d) and/or (d1), the activated compound of formula (X) wherein X1=a leaving group substitutable by an amine can be the acid chloride i.e. an activated compound of formula (X) wherein X1═Cl. Alternatively, the activated compound of formula (X) can be an activated ester wherein the leaving group X1 is
Preferred features of methods (a), (b), (c), (d), (d1) and (e), independently of each other, are as described above for Processes A, B, C, D, D1 and E, with all necessary changes being made.
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 (See for example Examples 490, 491, 518A, 593).
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) and/or for use as a phosphodiesterase inhibitor e.g. for use as a phosphodiesterase 4 (PDE4) inhibitor. “Therapy” may include treatment and/or prophylaxis.
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.
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 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. 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 or allergic rhinitis in a mammal (e.g. human). More preferably, the treatment and/or prophylaxis is of COPD or asthma in a mammal (e.g. human).
PDE4 inhibitors are thought to be effective in the treatment of asthma (e.g. see M. A. Giembycz, Drugs, February 2000, 59(2), 193-212; Z. Huang et al., Current Opinion in Chemical Biology, 2001, 5: 432-438; H. J. 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 refs cited therein).
PDE4 inhibitors are thought to be effective in the treatment of COPD (e.g. see S. L. Wolda, Emerging Drugs, 2000, 5(3), 309-319; Z. Huang et al., Current Opinion in Chemical Biology, 2001, 5: 432-438; H. J. 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 refs cited therein). COPD is often characterised by the presence of airflow obstruction due to chronic bronchitis and/or emphysema (S. L. Wolda, Emerging Drugs, 2000, 5(3), 309-319).
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 H. J. 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 refs cited therein). See e.g. A. M. Doherty, Current Opinion Chem. Biol., 1999, 3(4), 466-473 and refs cited therein for atopic dermatitis use.
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 and Neuropsychopharmacology, 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., CNS Drug 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).
Pharmaceutical Compositions and Dosing
For use in medicine, the compounds 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.
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,
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 oral, parenteral (e.g. intravenous, subcutaneous, or intramuscular), inhaled or nasal administration. Accordingly, the pharmaceutical composition is preferably suitable for oral, parenteral (e.g. intravenous, subcutaneous, or intramuscular), inhaled or nasal administration. More preferably, the pharmaceutical composition is suitable for inhaled or oral 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. Oral administration to a human is most preferred.
A pharmaceutical composition suitable 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 will 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.
A pharmaceutical composition suitable for oral administration being a tablet can comprise one or more pharmaceutically acceptable carriers and/or excipients suitable for preparing tablet formulations. Examples of such carriers include lactose and cellulose. The tablet can also or instead contain one or more pharmaceutically acceptable excipients, for example binding agents, lubricants such as magnesium stearate, and/or tablet disintegrants.
A pharmaceutical composition suitable for oral administration being a capsule can be prepared using encapsulation procedures. For example, pellets 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.
Preferably the composition is in unit dose form such as a tablet or capsule for oral administration, e.g. for oral administration to a human.
A parenteral composition can comprise a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil. Alternatively, the solution can be lyophilised; the lyophilised parenteral pharmaceutical composition can be reconstituted with a suitable solvent just prior to administration.
Compositions 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 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.
For pharmaceutical compositions suitable and/or adapted for inhaled administration, it is preferred that the compound or salt of formula (I) is in a particle-size-reduced form, and more preferably the size-reduced form is obtained or obtainable by micronisation. Micronisation usually involves subjecting the compound/salt to collisional and abrasional forces in a fast-flowing circular or spiral/vortex-shaped airstream often including a cyclone component. The preferable particle size (e.g. D50 value) of the size-reduced (e.g. micronised) compound or salt is about 0.5 to about 10 microns, e.g. about 1 to about 5 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 D10 of about 0.3 to about 3 microns (e.g. about 1 micron), and/or a D50 of about 1 to about 5 microns (e.g. about 2-5 or about 2-3 microns), and/or a D90 of about 2 to about 20 microns or about 3 to about 10 microns (e.g. about 5-8 or about 5-6 microns); for example as measured using laser diffraction. The laser diffraction measurement can use a dry method (suspension of compound/salt in airflow crosses laser beam) or a wet method [suspension of compound/salt in liquid dispersing medium, such as isooctane or (e.g. if compound soluble in isooctane) 0.1% Tween 80 in water, crosses 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.
An illustrative non-limiting example of a small-scale micronisation process is now given:
Micronisation Example: Micronisation of Example 518 or 518A
Equipment and Material
The Jetpharma MC1 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 an gasflow, a separate gas inlet for entry of gases, a gas outlet for exit of gases, and a collection vessel for collecting micronised material. The milling housing has two chambers: an outer annular chamber in gaseous connection with the gas inlet the chamber being for receiving pressurised gas (e.g. air or nitrogen), an 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 open into the inner chamber 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 is gaseous communication with the inner chamber via a nozzle directed tangentially to the inner chamber, within and near to the annular wall. Upper and lower broad-diameter exit vents in the central axis of the the inner milling chamber connect to (a) (lower exit) the collection vessel which has no air outlet, and (b) (upper exit) the gas outlet which leads to a collection bag, filter and a gas exhaust. Inside 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.
In 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 (eg air or nitrogen) the feed material is sucked into the gasstream thorough the compound inlet and accelerates it 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 center 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 thorugh the lower exit into the collection vessel, 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 venturi protrusion distance from input port is adjusted to 1.0 cm respectively (e.g. so that 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 (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 (see section on experimental run) is weighed into a plastic weigh boat.
The material is then fed into the micronizer using a vibrational spatula (e.g. V-shaped in cross-section) at a specified feed rate. The material feeding time and equipment pressures are monitored during the micronization process.
Upon completion of the micronising run, the nitrogen supply is shut off and the collection bag is tapped to allow particles to settle into the recovery/collection vessel at the bottom of the micronizer. The collection bag is removed and set aside. The micronised powder in the recovery vessel (collection vessel) and the cyclone (above the recovery vessel) are collected separately into different weighed+labelled collection vials. The weight of the micronised material is recorded. 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 by rinsing and wiping with suitable solvent and dried before subsequent runs are performed.
Preferred Experimental Parameters
Venturi outlet insertion depth: 10.0 mm
The above parameters can be varied using the skilled person's knowledge.
Results and/or Observations
% yield=[(Material from vessel+Material from cyclone)/Material input amount]×100
In general, very approximately 50-75% yields are achievable using this method. Procedure 1 has not been completed.
In Procedure 2, a 70.8% yield (0.6427 g) of Example 518 micronised material was obtained, including material from collection vessel and material from inside walls of cyclone.
Particle size analysis of Example 518 micronised material from Procedure 2, using laser diffraction measurement with Malvern Mastersizer longbed version, dispersing medium 0.1% Tween 80 in water, stir rate 1500 rpm, 3 mins sonification prior to final dispersion and analysis, 300 RF Reverse Fourier) lens, Fraunhofer calculation with Malvern software:
Alternative embodiment: Examples of the compounds/salts of the invention other than Examples 518 or 518A can be micronised.
For pharmaceutical compositions suitable and/or adapted for inhaled administration, it is preferred that the pharmaceutical composition is 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 performance modifier such as L-leucine, mannitol, trehalose and/or magnesium stearate. 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. Most importantly, it is preferable 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).
In the dry powder inhalable composition, preferably, the compound of formula (I) or salt thereof is 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 follows:
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. 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 ¾ 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™ 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.
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.
In the pharmaceutical composition, a or each dosage unit for oral or parenteral administration preferably contains from 0.01 to 3000 mg, more preferably 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 preferably contains from 0.001 to 50 mg, more preferably 0.01 to 5 mg, of a compound of the formula (I) or a pharmaceutically acceptable salt thereof, calculated as the free base.
A pharmaceutically acceptable compound or salt of the invention is preferably 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.
A pharmaceutically acceptable compound or salt of the invention is preferably 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 is preferably administered 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 or an anti-inflammatory agent.
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-adenoreceptor agonist, an anti-histamine, an anti-allergic, an anti-inflammatory agent or an antiinfective agent.
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). The combination with a β2-adrenoreceptor agonist can be as described in WO 00/12078.
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):
Preferred β2-adrenoreceptor agonists disclosed in WO 02/066422 include:
A preferred β2-adrenoreceptor agonist disclosed in WO 03/024439 is:
A combination of a compound of formula (I) or salt together with an anti-histamine is preferably 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 H1 antagonists such as cetirizine, loratadine (e.g. Clarityn™), desloratadine (e.g. Clarinex™) or fexofenadine (e.g. Allegra™).
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 M1, M2, M1/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 M1 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 A1 and U.S. 2002/052312 A1, 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 A1 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 suitable 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 drug (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, a CCR3 antagonist, or a 5-lipoxogenase inhibitor); or an antiinfective agent (e.g. an antibiotic or 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. Suitable CCR3 inhibitors include those disclosed in WO 02/26722.
In a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof together with an anti-inflammatory corticosteroid (which is preferably for treatment and/or prophylaxis of asthma, COPD or allergic rhinitis), then preferably the anti-inflammatory corticosteroid is fluticasone, fluticasone propionate (e.g. see U.S. Pat. No. 4,335,121), beclomethasone, beclomethasone 17-propionate ester, beclomethasone 17,21-dipropionate ester, dexamethasone or an ester thereof, mometasone or an ester thereof, ciclesonide, budesonide, flunisolide, or a compound as described in WO 02/12266 A1 (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 A1, then preferably it is Example 1 therein {which is 6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester}or Example 41 therein {which is 6α,9α-difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester}, or a pharmaceutically acceptable salt thereof. The anti-inflammatory corticosteroid is preferably for 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 (D) or a pharmaceutically acceptable salt thereof together with β2-adrenoreceptor agonist and an anti-inflammatory corticosteroid, for example as described in WO 03/030939 A1. 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 A1. 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 conveniently 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 may be administered either sequentially or simultaneously in separate or combined pharmaceutical composition.
In 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™) 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 Jan. 2003 (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 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 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.
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×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 was purified from bovine aorta 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 was purified from bovine retina 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 of PDE 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) was determined by Scintillation Proximity Assay (SPA) in 96-well format. Test compounds (preferably as a solution in DMSO, e.g. 0.5 to 1 microlitre (ul) volume) were preincubated at ambient temperature (room temperature, e.g. 19-23° C.) in Wallac Isoplates (code 1450-514) with PDE enzyme in 50 mM Tris-HCl buffer pH 7.5, 8.3 mM MgCl2, 1.7 mM EGTA, 0.05% (w/v) bovine serum albumin for 10-30 minutes (usually 30 minutes). The enzyme concentration was adjusted so that no more than 20% hydrolysis of the substrate defined below occurred 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) was added to give 0.05 uCi per well and ˜10 nM final concentration. For the PDE5 and PDE6 assays, [8-3H]Guanosine 3′,5′-cyclic phosphate (Amersham Pharmacia Biotech, code TRK.392) was added to give 0.05 uCi per well and ˜36 nM final concentration. Plates, e.g. containing approx. 100 ul volume of assay mixture, were 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) were added (−1 mg per well) to terminate the assay. Plates were 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 was measured using a WALLAC TRILUX 1450 Microbeta scintillation counter. For inhibition curves, 10 concentrations (1.5 nM-30 uM) of each compound were assayed. Curves were analysed using ActivityBase and XLfit (ID. Business Solutions Limited, 2 Ocean Court, Surrey Research Park, Guildford, Surrey GU2 7QB, United Kindgom) Results were 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 of PDE4B or PDE4D Activity: Fluorescence Polarisation (FP) Assay
The ability of compounds to inhibit catalytic activity at PDE4B (human recombinant) or PDE4D (human recombinant) was determined by IMAP Fluorescence Polarisation (PP) assay (IMAP Explorer kit, available from Molecular Devices Corporation, Sunnydale, Calif., USA; Molecular Devices code: R8062) in 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 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. 0.5 to 1 ul, of solution in DMSO) were 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 10 mM Tris-HCl buffer pH 7.2, 10 mM MgCl2, 0.1% (w/v) bovine serum albumin, and 0.05% NaN3 for 10-30 minutes. The enzyme level was set by experimentation so that reaction was linear throughout the incubation. Fluorescein adenosine 3′,5′-cyclic phosphate (from Molecular Devices Corporation, Molecular Devices code: R7091) was added to give about 40 nM final concentration (final assay volume usually ca. 25-40 ul). Plates were mixed on an orbital shaker for 10 seconds and incubated at ambient temperature for 40 minutes. IMAP binding reagent (as described above, from Molecular Devices Corporation, Molecular Devices code: R7207) was added (60 ul of a 1 in 400 dilution in binding buffer of the kit stock solution) to terminate the assay. Plates were allowed to stand at ambient temperature for 1 hour. The Fluorescence Polarisation (FP) ratio of parallel to perpendicular light was measured using an Analyst™ plate reader (from Molecular Devices Corporation). For inhibition curves, 10 concentrations (1.5 nM-30 uM) of each compound were assayed. Curves were analysed using ActivityBase and XLfit (ID Businesss Solutions Limited, 2 Ocean Court, Surrey Research Park, Guildford, Surrey GU2 7QB, United Kindgom). Results were expressed as pIC50 values.
In the FP assay, all reagents were dispensed using Multidrop™ (available from Thermo Labsystems Oy, Ratastie 2, PO Box 100, Vantaa 01620, Finland).
For a given PDE4 inhibitor, the PDE4B (or PDE4D) inhibition values measured using the SPA and FP assays can differ slightly. However, in a regression analysis of 100 test compounds, the pIC50 inhibition values measured using SPA and FP assays have been found generally to agree within 0.5 log units, for PDE4B and PDE4D (linear regression coefficient 0.966 for PDE4B and 0.971 for PDE4D; David R. Mobbs et al., “Comparison of the IMAP Fluorescence Polarisation Assay with the Scintillation Proximity Assay for Phosphodiesterase Activity”, poster to be presented at 2003 Molecular Devices UK & Europe User Meeting, 2 Oct. 2003, Down Hall, Harlow, Essex, United Kingdom).
Biological Data obtained for some of the Examples (PDE4B inhibitory activity, either as one reading or as an average of ca. 2-6 readings) are as follows, based on current measurements only. In each of the SPA and FP assays, absolute accuracy of measurement is not possible, and the readings given are accurate only up to about ±0.5 of a log unit, depending on the number of readings made and averaged:
Examples 1-201 were generally tested for PDE4B inhibition using the radioactive SPA assay. Of Examples 207-665, and 677-686, all or almost all (except perhaps for Examples 451, 631-632, 635, 652) were tested for PDE4B inhibition; and of these some were tested by the radioactive SPA assay, some were tested by the FP assay. Examples 1-201, 207-450, 452-630, 633-634, 636-651, 653-665, and 677-686, but excluding reference examples 19-20, have PDE4B inhibitory activities in the range of pIC50=about 6 (±about 0.5) to about 10.0 (±about 0.5). Examples 666-676 are predicted to have PDE4B inhibitory activities in the range of pIC50=about 6 (±about 0.5) to about 10.0 (±about 0.5).
The Examples wherein R3=cyclohexyl (NHR3=sub-formula (c)), tetrahydro-2H-pyran-4-yl (NHR3=group (h)), 4-oxocyclohexyl (NHR3=sub-formula (o)), or certain other types of substituted cyclohexyl or certain heterocycles, usually or often (especially with R1=ethyl) have a higher level of selectivity for PDE4B over PDE5, as measured in the above enzyme inhibition assays, compared to the selectivities of comparable Examples wherein R3=cyclopropyl (NHR3=sub-formula (b)). For example, based on current measurements only, and subject to cumulative assay inaccuracies:
The in vitro enzymatic PDE4B inhibition assay described above should be regarded as being the primary test of biological activity. However, additional in vivo biological tests, which are optional and which are not an essential measure of efficacy or side-effects, are described below.
In Vivo Assay 1. LPS-Induced Pulmonary Neutrophilia in Rats: Effect of Orally Administered PDE4 Inhibitors
Pulmonary neutrophil influx has been shown 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); 251s-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, N.C., USA) weighing approximately 300-400 grams are pretreated with either (a) test compound suspended in 0.5% methylcellulose (obtainable from Sigma-Aldrich, St Louis, Mo., USA) in water or (b) vehicle only, delivered orally in a dose volume of 10 ml/kg. Generally, dose response curves are generated using the following doses of PDE4 inhibitors: 10.0, 2.0, 0.4, 0.08 and 0.016 mg/kg. Thirty minutes following pretreatment, the rats are exposed to aerosolized LPS (Serotype E. Coli 026:B6 prepared by trichloroacetic acid extraction, obtainable from Sigma-Aldrich, St Louis, Mo., USA), generated from a nebulizer containing a 100 μg/ml LPS solution. Rats are exposed to the LPS aerosol at a rate of 4 L/min for 20 minutes. LPS exposure is carried out in a closed chamber with internal dimensions of 45 cm length×24 cm width×20 cm height. The nebulizer and exposure chamber are contained in a certified fume hood. At 4 hours-post LPS exposure the rats are euthanized by overdose with pentobarbital at 90 mg/kg, administered intraperitoneally. Bronchoalveolar lavage (BAL) is preformed 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.
Results: Based on current measurements, the compounds of Examples 22, 83 and 155, administered orally in the above procedure, exhibited neutrophilia-inhibition ED50 values in the range of about 0.5 mg/kg to about 2 mg/kg.
Alternative method and results: In an alternative embodiment of the procedure, single oral doses of 10 mg/kg or 1.0 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. In this embodiment, based on current measurements, the compounds of Examples 21, 100, 109, 167, 172 and 600, administered orally in the above procedure at a single dose of 1.0 mg/kg, exhibited percent neutrophilia-inhibition in the range of about 19% to about 69% (or in the range of about 32% to about 69% for Examples 21, 100, 109, 167 and 600).
Background: Selective PDE4 inhibitors have been shown 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 many species is emesis. Because many rat models of inflammation are well characterized, they have been 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 minimal-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.
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, Calif., 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 the compound/salt of the invention (different doses for different treatment groups) or with vehicle alone, at a dose volume of 2 ml/kg. In this oral dosing, the compound/salt is in the form of a suspension in 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 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.
Results: Using the above procedure, and according to current measurements, the compounds of Examples 22, 83 and 155 exhibited a Pica Response ED50 in the range of about 4.8 mg/kg to greater than or equal to about 40 mg/kg. It can be seen that these Pica Response ED50 doses are higher than the neutrophilia-inhibition ED50 values for these three Examples (see In Vivo Assay 1 above), so that a Therapeutic Index (TI) in rats of >2, as measured by Assays 1+2 and according to current measurements, appears at first sight to have been achieved for these three Examples.
The Therapeutic Index (TI) calculated this way is often significantly different to, and often higher than, the TI calculated in the ferret (see In vivo Assay 4 below).
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. I.t. 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, N.C., USA were housed in groups of 5 rats per cage, acclimatised after delivery for at least 7 days with bedding/nesting material regularly changed, fed on SDS diet R1 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. A 3-way sterile tap (Vycon Ref 876.00) was weighed, the drug blend or inhalation grade lactose (vehicle control) was then added to the tap, the tap closed to prevent loss of drug, and the tap was re-weighed to determine the weight of drug in the tap. After dosing, the tap was 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, was cut by engineering to approximately 132 mm (5.2 inches), a blunt end was made to prevent them damaging the rat's trachea, and the needle weighed prior to and after drug delivery to confirm that no drug was retained in the needles after dosing.
Device for wet suspension administration: This is the similar to the above but a blunt dosing needle, whose forward end was slightly angled to the needle axis, was used, with a flexible plastic portex canula inserted into the needle.
Drugs and Materials: Lipopolysaccharide (LPS) (Serotype:0127:B8) (L3129 Lot 61K4075) was dissolved in phosphate-buffered saline (PBS). PDE4 inhibitors are used in size-reduced (e.g. micronised) form, for example according to the Micronisation Example given above. For dry powder administration of the drug, the Dry Powder Formulation Example given above, comprising drug and inhalation-grade lactose, can be used. The inhalation-grade lactose usually used (Lot E98L4675 Batch 845120) has 10% fines (10% of material under 15 um particle size measured by Malvern particle size).
Wet suspensions of the drug can be prepared by adding the required volume of vehicle to the drug; the vehicle used being a mixture of saline/tween (0.2% tween 80). The wet suspension was sonicated for 10 minutes prior to use.
Preparation, and dosing with PDE 4 inhibitor: Rats were 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.minute−1) and oxygen (1 litre.minute−1). Once anaesthetised, the animals were placed onto a stainless steel i.t. dosing support table. They were positioned on their back at approximately a 35° angle. A light was angled against the outside of the throat to highlight the trachea. The mouth was 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 was introduced via a blunt metal dosing needle that had been carefully inserted into the rat trachea. The animals were intratracheally dosed with vehicle or PDE4 inhibitor via the dosing needle with a new internal canula used for each different drug group. The formulation was slowly (10 seconds) dosed into the trachea using a syringe attached to the dosing needle.
Dosing with a Dry Powder: The three-way tap device and needle were 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 2×4 ml of air 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. After dosing, the needle and tap 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 were placed into sealed Perspex containers and exposed to an aerosol of LPS (nebuliser concentration 150 μg.ml−1) for 15 minutes. Aerosols of LPS were generated by a nebuliser (DeVilbiss, USA) and this was directed into the Perspex exposure chamber. Following the 15-minute LPS-exposure period, the animals were returned to the holding cages and allowed free access to both food and water.
[In an alternative embodiment, the rats can exposed to LPS less than 2 hours after i.t. dosing. In another alternative embodiment, the rats can exposed to LPS more than 2 hours (e.g. ca. 4 or ca. 6 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 ravage: 4 hours after LPS exposure the animals were killed by overdose of sodium pentobarbitone (i.p.). The trachea was cannulated with polypropylene tubing and the lungs lavaged (washed out) with 3×5 mls of heparinised (25 units.ml−1) phosphate buffered saline (PBS).
Neutrophil cell counts: The Bronchoalveolar lavage (BAL) samples were centrifuged at 1300 rpm for 7 minutes. The supernatant was removed and the resulting cell pellet resuspended in 1 ml PBS. A cell slide of the resuspension fluid was prepared by placing 100 μl of resuspended BAL fluid into cytospin holders and then spun at 5000 rpm for 5 minutes. The slides were allowed to air dry and then stained with Leishmans stain (20 minutes) to allow differential cell counting. The total cells were also counted from the resuspension. From these two counts, the total numbers of neutrophils in the BAL were 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 PDE 4 Inhibitors in the Conscious Ferret
1.1 Materials
The following materials are used for these studies:
PDE4 inhibitors are prepared for oral (p.o.) administration by dissolving in a fixed volume (1 ml) of acetone and then adding cremophor to 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™ cat food given 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 1 ml/kg. Ferrets are fasted overnight but allowed free access to water. The animals are orally dosed with vehicle or PDE 4 inhibitor using a 15 cm 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 60-90 minutes after p.o. dosing.
1.3.2 Exposure to LPS
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 (30 μg/ml) for 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
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 20 ml heparinised (10 units/ml) phosphate buffered saline (PBS). The bronchoalveolar lavage (BAL) samples are centrifuged at 1300 rpm for 7 minutes. The supernatant is removed and the resulting cell pellet re-suspended in 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:
It is noted that the Therapeutic index (TI) calculated using this in vivo Assay 4 is often significantly different to, and often lower than, that calculated using the rat oral inflammation and pica feeding Assays 1+2.
The calculation of TI using the PDE4 inhibitor roflumilast in this Assay 4 is:
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.
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”.
Abbreviations used herein:
Machine Methods Used Herein:
The prep column used was a Supelcosil ABZplus (10 cm×2.12 cm) (usually 10 cm×2.12 cm×5 μm).
All reagents not detailed in the text below are commercially available from established suppliers such as Sigma-Aldrich. The addresses of the suppliers for some of the starting materials mentioned in the Intermediates and Examples below or the Assays above are as follows:
Ultrafine (UFC Ltd.), Synergy House, Guildhall Close, Manchester Science Park, Manchester M15 6SY, United Kingdom
Prepared from commercially available 5-amino-1-ethyl pyrazole as described by G. Yu et al. in J. Med. Chem., 2001, 44, 1025-1027:
Can be prepared by oxidative cleavage (SeO2) of 1-furanylmethyl derivative, as described by T. M. Bare et. al. In J. Med. Chem., 1989, 32, 2561-2573, (further ed to Zuleski, F. R., Kirkland, K. R., Melgar, M. D.; Malbica, J. Drug. Metab. Dispos. 1985, 13, 139)
A mixture of Intermediate 2 (0.47 g) and anhydrous potassium carbonate (0.83 g) (previously dried by heating at 100° C.) in anhydrous dimethylformamide (DMF) (4 ml) was treated with iodomethane (0.26 ml) and stirred vigorously for 3 h. The mixture was then filtered and the filtrate concentrated in vacuo to afford a residual oil, which was partitioned between dichloromethane (DCM) (25 ml) and water (25 ml). The layers were separated and the aqueous phase was extracted with further DCM (2×25 ml). The combined organic extracts were dried over anhydrous sodium sulphate and evaporated to an orange solid which was applied to an SPE cartridge (silica, 20 g). The cartridge was eluted sequentially with EtOAc:petrol (1:4, 1:2 and 1:1), then chloroform:methanol (49:1, 19:1 and 9:1). Fractions containing desired material were combined and concentrated in vacuo to afford Intermediate 3 (0.165 g). LCMS showed MH+=250; TRET=2.59 min.
A mixture of Intermediate 2 (0.47 g) and anhydrous potassium carbonate (0.83 g) (previously dried by heating at 100° C.) in anhydrous DMF (4 ml) was treated with benzyl bromide (0.72 g) then stirred vigorously and heated at 55° C. for 4.5 h. The mixture was allowed to cool, then filtered and the filtrate concentrated in vacuo to afford a residual oil, which was partitioned between DCM (25 ml) and water (25 ml). The layers were separated and the aqueous phase was extracted with further DCM (2×25 ml). The combined organic extracts were dried over anhydrous sodium sulphate and evaporated to a yellow oily solid which was dissolved in DCM and applied to an SPE cartridge (silica, 20 g). The cartridge was eluted with a gradient of EtOAc:petrol (1:4, 1:2 and 1:1) then chloroform:methanol (49:1, 19:1 and 9:1). Fractions containing desired material were combined and concentrated in vacuo to afford Intermediate 4 (0.33 g). LCMS showed MH+=326; TRET=3.24 min.
A mixture of 5-amino-1-phenyl pyrazole (2.0 g) and diethylethoxymethylene malonate (2.54 ml) was heated under Dean Stark conditions at 120° C. for 16 h. The solution was cooled, phosphorus oxychloride (16 ml) was then added and the mixture heated under reflux for a further 20 h. Excess phosphorus oxychloride was removed in vacuo and the residue partitioned between diethyl ether and water, proceeding with extreme caution on addition of water. The ethereal layer was washed with further water, then dried over magnesium sulphate and concentrated in vacuo to afford ethyl 4-chloro-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (2.09 g). LCMS showed MH+=302; TRET=3.80 min.
Prepared from commercially available N1-benzyl-4-aminopiperidine as described by Yamada et. al. In WO 00/42011:
Prepared from commercially available N-methyl-4-piperidone as described by C. M. Andersson et. al. in WO01/66521:
Commercially available from Combi-Blocks Inc., 7949 Silverton Avenue, Suite 915, San Diego, Calif. 92126, USA (CAS 38041-19-9)
Dibenzylamine (34.5 g) and acetic acid (6.7 ml) were added to a stirred solution of tetrahydro-4H-pyran-4-one (16.4 g, commercially available from e.g. Aldrich) in dichloromethane (260 ml) at 0° C. to 5° C. After 2.5 h at 0° C. to 5° C., sodium triacetoxyborohydride (38.9 g) was added portionwise, and the mixture was allowed to warm to room temperature. After stirring at room temperature overnight, the reaction mixture was washed successively with 2M-sodium hydroxide (200 ml and 50 ml), water (2×50 ml) and brine (50 ml), then dried and evaporated to give a yellow oil (45 g). This oil was stirred with methanol (50 ml) at 4° C. for 30 min to give the product as a white solid (21.5 g). LCMS showed MH+=282; TRET=1.98 min.
N,N-dibenzyltetrahydro-2H-pyran-4-amine (20.5 g) was dissolved in ethanol (210 ml) and hydrogenated over 10% palladium on carbon catalyst (4 g) at 100 psi for 72 h at room temperature. The reaction mixture was filtered and the filtrate was adjusted to pH 1 with 2M-hydrogen chloride in diethyl ether. Evaporation of solvents gave a solid which was triturated with diethyl ether to give the product as a white solid (9.23 g). 1H NMR (400 MHz in dr-DMSO, 27° C., δppm) 8.24 (br. s, 3H), 3.86 (dd, 12, 4 Hz, 2H), 3.31 (dt, 2, 12 Hz, 2H), 3.20 (m, 1H), 1.84 (m, 2H), 1.55 (dq, 4, 12 Hz, 2H).
Commercially available from Fluka Chemie AG, Germany (CAS 111769-27-8)
Commercially available from E. Merck, Germany; or from E. Merck (Merck Ltd), Hunter Boulevard, Magna Park Lutterworth, Leicestershire LE17 4XN, United Kingdom (CAS 104530-80-5)
Prepared from commercially available tetrahydrothiopyran-4-one as described by Subramanian et. al., J. Org. Chem., 1981, 46, 4376-4383. Subsequent preparation of the hydrochloride salt can be achieved by conventional means.
Prepared in an analogous manner to Intermediate 11 from commercially available tetrahydrothiophene-4-one. The oxime formation is described by Grigg et.al., Tetrahedron, 1991, 47, 4477-4494 and the oxime reduction by Unterhalt et. al., Arch. Pharm., 1990, 317-318.
Commercially available from Sigma Aldrich Library of Rare Chemicals (SALOR) (CAS-6338-70-1). Preparation of the hydrochloride salt of the amine can be achieved by conventional means.
Prepared in an analogous manner to Intermediate 11 from commercially available tetrahydrothiophene-4-one. Oxidation to 1,1-dioxo-tetrahydro-1λ6-thiopyran-4-one is described by Rule et. al., in J. Org. Chem., 1995, 60, 1665-1673. Oxime formation is described by Truce et.al., in J. Org. Chem., 1957, 617, 620 and oxime reduction by Barkenbus et. al., J. Am. Chem. Soc., 1955, 77, 3866. Subsequent preparation of the hydrochloride salt of the amine can be achieved by conventional means.
A solution of Intermediate 1 (3.5 g) in dioxane (28 ml) was treated with potassium hydroxide (6.3 g) as a solution in water (20 ml). The mixture was stirred for 2 h, then concentrated in vacuo, acidified to pH 3 with 2M aqueous hydrochloric acid and extracted with ethyl acetate. The layers were separated, the organic layer dried over sodium sulphate, then concentrated in vacuo to afford Intermediate 15 as a white solid (2.4 g). LCMS showed MH+=226; TRET=2.62 min.
That is, Intermediate 17 is:
Intermediate 15 (3.5 g) was dried over phosphorus pentoxide for 1 h, then treated with thionyl chloride (47 g). The mixture was stirred and heated at 75° C. for 1.3 h. Excess thionyl chloride was removed in vacuo and the residual oil azeotroped with dichloromethane (DCM) to afford Intermediate 16, presumed to be the acid chloride derivative of Intermediate 15, as a white solid (3.3 g).
Intermediate 16 (0.473 g) was dissolved in tetrahydrofuran (TBF) (4 ml) and treated with N,N-diisopropylethylamine (DIPEA) (0.509 ml), then with benzylamine (0.209 g) and the mixture stirred under nitrogen for 0.5 h. The mixture was concentrated in vacuo, then partitioned between dichloromethane and water. The layers were separated and the organics concentrated in vacuo to afford Intermediate 17 (0.574 g). LCMS showed MH+=315; TRET=2.90 min.
Similarly prepared were the following:
Acid chloride Intermediate 16 was synthesised from Intermediate 15 using the method shown above for Intermediate 17. Intermediate 16 (0.473 g) was dissolved in THF (4 ml) and treated with diisopropylethylamine (DIPEA) (0.509 ml), then with 4-(aminomethyl)pyridine (0.211 g) and the mixture stirred under nitrogen for 0.5 h. The mixture was concentrated in vacuo, then partitioned between DCM and water. The layers were separated and the organics concentrated in vacuo, then applied to an SPE cartridge (silica, 10 g) which was eluted with a gradient of cyclohexane:EtOAc (2:1 increasing stepwise up to 0:1), followed by MeOH:EtOAc (5:95, then 10:90). Fractions containing desired material were combined and concentrated in vacuo to afford Intermediate 22 (0.086 g). LCMS showed MH+=316; TRET=1.84 min.
Acid chloride Intermediate 16 was synthesised from Intermediate 15 using the method shown above for Intermediate 17. Intermediate 16 (0.473 g) was dissolved in TBF (4 ml) and treated with DIPEA (0.509 ml), then with n-propyl amine (0.115 g) and the mixture stirred under nitrogen for 0.5 h. A further portion of n-propyl amine (0.023 g) was then added and stirring continued for 18 h. The mixture was concentrated in vacuo, then partitioned between DCM and water. The layers were separated and the organics concentrated in vacuo to afford Intermediate 23 (0.405 g). LCMS showed MH+=267; TRET=2.54 min.
Acid chloride Intermediate 16 was synthesised from Intermediate 15 using the method shown above for Intermediate 17. Intermediate 16 (0.30 g) was dissolved in THF (3 ml) and treated with a 0.5M solution of ammonia in dioxane (4.92 ml). The mixture was stirred under nitrogen for 18 h. A further portion of 0.5M ammonia in dioxane (4.92 ml) was added and stirring continued for 72 h. The mixture was concentrated in vacuo and the residue partitioned between DCM and 2M sodium hydroxide solution. The layers were separated and the organics concentrated to afford Intermediate 24 (0.278 g). LCMS showed MH+=225; TRET=2.10 min.
A mixture of 5-amino-1-methylpyrazole (4.0 g) and diethylethoxymethylene malonate (9.16 ml) was heated at 150° C. under Dean Stark conditions for 5 h. Phosphorous oxychloride (55 ml) was carefully added to the mixture and the resulting solution heated at 130° C. under reflux for 18 h. The mixture was concentrated in vacuo, then the residual oil cooled in an ice bath and treated carefully with water (100 ml)(caution: exotherm). The resulting mixture was extracted with DCM (3×100 ml) and the combined organic extracts were dried over anhydrous sodium sulphate and concentrated in vacuo. The residual solid was purified by Biotage chromatography (silica, 90 g), eluting with Et2O:petrol (1:3). Fractions containing desired material were combined and concentrated in vacuo to afford Intermediate 25 (4.82 g). LCMS showed MH+=240; TRET=2.98 min
A solution of Intermediate 25 (4.0 g) in dioxane (30 ml) was treated with potassium hydroxide (7.54 g) as a solution in water (20 ml). The mixture was stirred for 16 h, then diluted with water (150 ml) and acidified to pH 3 with 5M aqueous hydrochloric acid. The mixture was stirred in an ice bath for 15 min, then collected by filtration, washed with ice-cold water and dried in vacuo over phosphorous pentoxide to afford Intermediate 26 as a white solid (2.83 g). LCMS showed MH+=212; TRET=2.26 min.
Intermediate 26 (2.5 g) (previously dried over phosphorus pentoxide) was treated with thionyl chloride (25 ml) and the mixture heated under reflux for 1 h. Excess thionyl chloride was removed in vacuo to afford Intermediate 27, presumed to be the acid chloride derivative of Intermediate 26, as a white solid (2.7 g).
Intermediate 27 (0.68 g) was dissolved in THF (10 ml) and treated with DIPEA (0.77 ml), then with benzyl amine (0.339 g) and the mixture stirred under nitrogen for 3 h. The mixture was concentrated in vacuo, then partitioned between DCM (20 ml) and water (10 ml). The layers were separated and the organics concentrated in vacuo to afford Intermediate 28 (0.90 g). LCMS showed MH+=301; TRET=2.72 min.
Similarly prepared were the following:
Acid chloride Intermediate 27 was synthesised from Intermediate 26 using the method shown above for Intermediate 28. Intermediate 27 (0.68 g) was then treated with a 0.5M solution of ammonia in dioxane (17.7 ml). Diisopropylethylamine (0.51 ml) was then added and the mixture stirred for 21 h. The mixture was then partitioned between DCM (100 ml) and water (30 ml). An insoluble solid was removed by filtration, washed with water (20 ml) and dried in vacuo over phosphorous pentoxide to afford Intermediate 31 (0.544 g). LCMS showed MH+=211; TRET=1.84 min.
Intermediate 1 (0.20 g) and triethylamine (0.55 ml) were suspended in ethanol (8 ml) and 4-aminotetrahydropyran (0.088 g) was added. The mixture was stirred under nitrogen, heated at 80° C. for 16 h, then concentrated in vacuo. The residue was partitioned between DCM and water. The layers were separated and the organic layer was loaded directly onto an SPE cartridge (silica, 5 g) which was eluted sequentially with; (i) DCM, (ii) DCM:Et2O (2:1), (iii) DCM:Et2O (1:1), (iv) Et2O and (v) EtOAc. Fractions containing desired material were combined and concentrated in vacuo to afford Intermediate 32 (0.21 g). LCMS showed MH+=319; TRET=2.93 min.
In an alternative embodiment, Intermediate 32 (=Example 3) can be made as described below under “Example 3”, in particular according to “Example 3, Method B” below.
A solution of Intermediate 32 (Example 3) (0.21 g) in ethanol:water (95:5, 10 ml) was treated with sodium hydroxide (0.12 g). The mixture was heated at 50° C. for 8 h, then concentrated in vacuo, dissolved in water and acidified to pH 4 with acetic acid. The resultant white solid was removed by filtration and dried under vacuum to afford Intermediate 33 as an off-white solid (0.156 g). LCMS showed MH+=291; TRET=2.11 min.
An alternative preparation of Intermediate 33 is as follows:
A solution of Intermediate 32 (Example 3) (37.8 g) in ethanol:water (4:1, 375 ml) was treated with sodium hydroxide (18.9 g). The mixture was heated at 50° C. for 5 hours, then concentrated in vacuo, dissolved in water and acidified to pH 2 with aqueous hydrochloric acid (2M). The resultant white solid was removed by filtration and dried under vacuum to afford Intermediate 33 as an off-white solid (29.65 g). LCMS showed MH+=291; TRET=2.17 min.
Intermediate 1 (0.05 g) and (S)-(−)-3-aminotetrahydrofuran 4-toluenesulphonate (0.052 g) were suspended in ethanol (1 ml) and triethylamine (0.14 ml) was added. The mixture was stirred under nitrogen and heated at 80° C. for 24 h. After cooling to room temperature, ethanol was removed by evaporation under a stream of nitrogen and the residue partitioned between DCM (2 ml) and water (1.5 ml). The layers were separated and the organic layer concentrated to dryness. Purification was carried out using an SPE cartridge (silica, 5 g), eluting with a gradient of EtOAc:cyclohexane; (1:16 then, 1:8, 1:4, 1:2, 1:1 and 1:0). Fractions containing desired material were combined and concentrated in vacuo to afford Intermediate 34 (=Example 8) (0.052 g). LCMS showed MH+=305; TRET=2.70 min.
Similarly prepared were the following:
Intermediate 1 (0.05 g) and Intermediate 13 (0.027 g) were suspended in ethanol (1 ml) and triethylamine (0.14 ml) was added. The mixture was stirred under nitrogen and heated at 80° C. for 24 h. After cooling to room temperature, ethanol was removed by evaporation under a stream of nitrogen and the residue partitioned between DCM (2 ml) and water (1.5 ml). The layers were separated and the organic layer concentrated to dryness. Purification was carried out using an SPE cartridge (silica, 5 g), eluting with a gradient of EtOAc:cyclohexane; (1:8 then 1:4, 1:2, 1:1 and 1:0). Fractions containing desired material were combined and concentrated in vacuo to afford Intermediate 39 (=Example 13) (0.045 g) as a mixture of enantiomers. LCMS showed MH+=353; TRET=2.60 min.
Similarly prepared was the following:
A solution of Intermediate 34 (0.037 g) in ethanol:water (95:5, 3 ml) was treated with sodium hydroxide (0.019 g). The mixture was heated at 50° C. for 16 h, then concentrated in vacuo. The residue was dissolved in water (1.5 ml) and acidified to pH 4 with acetic acid. The resultant white solid precipitate was removed by filtration and dried under vacuum. The filtrate was extracted with ethyl acetate and the organic layer collected and concentrated in vacuo to afford a further portion of white solid. The two solids were combined to afford Intermediate 41 (0.033 g). LCMS showed MH+=277; TRET=2.05 Similarly prepared were the following:
Intermediate 2 (0.69 g) was suspended in cyclohexylamine (1.01 ml), and the mixture was heated at 90° C. for 3 h. The residual mixture was allowed to cool to room temperature and partitioned between chloroform (25 ml) and water (25 ml). The phases were separated and the organic phase was evaporated to dryness. The residue was triturated with Et2O (25 ml) and the insoluble solid was collected and dried to afford Intermediate 48 as a beige solid (0.58 g). LCMS showed MH+=289; TRET=2.91 min.
2M-Sodium hydroxide solution (0.5 ml) was added to a stirred suspension of Intermediate 48 (0.2 g) in dioxan (4 ml) and water (0.5 ml). After stirring overnight at room temperature, the reaction mixture was heated at 40° C. for 8 h. A further quantity of 2M-sodium hydroxide solution (1.5 ml) was added, and the reaction mixture was heated at 40° C. for 48 h. The reaction solution was concentrated, diluted with water (10 ml) and acidified with glacial acetic acid. The resulting precipitate was collected by filtration, washed with water and dried to give Intermediate 49 (0.18 g). LCMS showed MH+=261; TRET=2.09 min.
2M-Sodium hydroxide solution (0.7 ml) was added to a stirred suspension of Example 185 (0.23 g, described hereinafter) in ethanol (5 ml) and water (1.5 ml). After stirring overnight at room temperature, a further quantity of 2M-sodium hydroxide solution (0.7 ml) was added, and the reaction mixture was heated at 43° C. for 2.5 h. The reaction solution was concentrated, diluted with water (5 ml) and acidified with 2M-hydrochloric acid. The resulting precipitate was collected by filtration, washed with water and dried to give Intermediate 50 as a white solid (0.14 g). LCMS showed MH+=305; TRET=2.42 min.
A mixture of 5-amino-1-ethylpyrazole (1.614 g, 14.5 mmol) and diethyl 2-(1-ethoxyethylidene)malonate (3.68 g, 16.0 mmol, as described by P. P. T. Sah, J. Amer. Chem. Soc., 1931, 53, 1836) was heated at 150° C. under Dean Stark conditions for 5 hours. Phosphorous oxychloride (25 ml) was carefully added to the mixture and the resulting solution was heated at 130° C. under reflux for 18 hours. The mixture was concentrated in vacuo, then the residual oil was carefully added, with cooling, to water (100 ml). The resulting mixture was extracted with DCM (3×100 ml) and the combined organic extracts were dried over anhydrous sodium sulphate and concentrated in vacuo. The residual oil was purified by Biotage chromatography (silica, 90 g) eluting with ethyl acetate-petrol (1:19). Fractions containing the desired product were combined and concentrated in vacuo to afford Intermediate 51 (1.15 g). LCMS showed MH+=268; TRET=3.18 min.
2M-Sodium hydroxide solution (0.39 ml, 0.78 mmol) was added to Example 190 (0.128 g, 0.39 mmol, described hereinafter) in ethanol (1.5 ml), and the mixture was heated at 50° C. for 16 hours. The reaction mixture was concentrated, and the resulting aqueous solution was neutralised with 2M-hydrochloric acid to precipitate a solid which was collected by filtration. The filtrate was applied to an OASIS® hydrophilic-lipophilic balance (HLB) Extraction cartridge * (1 g) which was eluted with water followed by methanol. Evaporation of the methanol fraction gave a solid which was combined with the initial precipitated solid to afford Intermediate 52 (0.083 g) as a white solid, presumed to be the carboxylic acid.
* OASIS® A HLB Extraction cartridges are available from Waters Corporation, 34 Maple Street, Milford, Mass. 01757, USA. The cartridges include a column containing a copolymer sorbent having a HLB such that when an aqueous solution is eluted through the column, the solute is absorbed or adsorbed into or onto the sorbent, and such that when organic solvent (e.g. methanol) is eluted the solute is released as an organic (e.g. methanol) solution. This is a way to separate the solute from aqueous solvent.
2M-Sodium hydroxide solution (0.75 ml, 1.5 mmol) was added to Example 189 (0.248 g, 0.75 mmol, described hereinafter) in ethanol (2 ml), and the mixture was heated at reflux for 16 hours. The reaction mixture was concentrated, diluted with water (1 ml) and acidified with 2M-hydrochloric acid (0.75 ml) to precipitate a solid which was collected by filtration to afford Intermediate 53 (0.168 g). LCMS showed MH+=305; TRET=1.86 min.
A solution of hydrogen chloride in dioxan (0.5 ml, 2.0 mmol, 4M) was added to a stirred solution of tert-butyl 4-oxocyclohexylcarbamate (0.043 g, 0.20 mmol, commercially available from Astatech Inc., Philadelphia, USA) in dioxan (0.5 ml) and the mixture was stirred at room temperature. After 1 h, the reaction mixture was evaporated to give Intermediate 54 as a cream solid (34 mg). 1H NMR (400 MHz in d6-DMSO, 27° C., δppm) 8.09 (br. s, 3H), 3.51 (tt, 11, 3.5 Hz, 1H), 2.45 (m, 2H, partially obscured), 2.29 (m, 2H), 2.16 (m, 2H), 1.76 (m, 2H).
Benzylamine (0.16 ml) was added to a stirred mixture of Intermediate 49 (0.13 g), DIPEA (0.26 ml) and HATU (0.285 g) in DMF (3 ml). The resultant mixture was heated with stirring at 85° C. for 16 hours. Further portions of HATU (0.14 g), DIPEA (0.13 ml) and benzylamine (0.082 ml) were added and the mixture heated for 16 hours at 88° C. The resultant solution was concentrated, diluted with dichloromethane (20 ml) and washed with saturated sodium bicarbonate solution (20 ml), separated by hydrophobic frit and the organic layer concentrated. The residue was purified on a SPE cartridge (silica, 20 g) eluting with 60-80% ethyl acetate in cyclohexane. The residue was purified further on a SPE cartridge (Isolute SCX sulphonic acid cartridge, 5 g×2), eluting with methanol (2×20 ml) and 10% ammonia in methanol (4×20 ml); the basic fractions were combined and concentrated to give Intermediate 54A as a white solid (0.07 g). LCMS showed MH+=350; TRET=2.99 min.
That is, Intermediate 55 is:
Intermediate 15 (1.04 g) was treated with thionyl chloride (13.22 g). The mixture was stirred and heated at 75° C. for 2 h. Excess thionyl chloride was removed in vacuo and the residual oil azeotroped with toluene to afford Intermediate 16, presumed to be the acid chloride derivative of intermediate 15, as a cream solid (1.12 g).
Intermediate 16 (0.997 g) was dissolved in tetrahydrofuran (THF) (25 ml) and treated with N,N-diisopropylethylamine (1.07 ml) then with 1-[4-(methyloxy)phenyl]methanamine 4-methoxybenzylamine (0.54 ml) (obtainable from e.g. Aldrich, Acros, or Tetrahedron Lett., 2002, 43(48), 8735; or Meindl et al., J. Med. Chem., 1984, 27(9), 1111; or Organic Letters, 2002, 4(12), 2055) and the mixture was stirred for 3 h. The solution was concentrated in vacuo, then partitioned between DCM and water. The layers were separated and the organics concentrated in vacuo. The solid was then triturated in 1:1 ethyl acetate: cyclohexane to give Intermediate 55 (1.27 g). LCMS showed MH+=345, TRET=2.86 min.
Similarly prepared were the following:
A solution of sodium hydroxide (0.053 g, 1.32 mmol) in water (0.41 ml) was added to a stirred solution of Example 205 (0.1 g, 0.303 mmol) in ethanol (1 ml), and the resulting mixture was heated at 50° C. After 1 h, the cooled reaction mixture was adjusted to pH3 with 2M hydrochloric acid, and extracted with EtOAc (2×6 ml). The combined organic extracts were dried (Na2SO4) and evaporated to give Intermediate 58 (0.072 g) as a white solid. LCMS showed MH+=303; TRET=2.13 min.
An alternative preparation of Intermediate 58 is as follows:
A solution of sodium hydroxide (0.792 g, 19.8 mmol) in water (6 ml) was added to a stirred solution of Example 205 (1.487 g, 4.5 mmol) in ethanol (15 ml), and the resulting mixture was heated at 50° C. After 1 hour, the cooled reaction mixture was adjusted to pH4 with 2M hydrochloric acid, and extracted with EtOAc (3×30 ml). The combined organic extracts were dried (Na2SO4) and evaporated to give Intermediate 58 (1.188 g) as a white solid. LCMS showed MH+=303; TRET=2.12 min.
Intermediate 1 (0.76 g, 3.0 mmol)) was dissolved in acetonitrile (10 ml). Tetrahydro-2H-pyran-3-amine hydrochloride (0.5 g, 3.6 mmol, Anales De Quimica, 1988, 84, 148) and N,N-diisopropylethylamine (3.14 ml, 18.0 mmol) were added and the mixture was stirred at 85° C. for 24 h. After 24 h a further portion of tetrahydro-2H-pyran-3-amine hydrochloride (0.14 g, 1.02 mmol) was added and stirring was continued at 85° C. After a further 8 h, the mixture was concentrated in vacuo. The residue was partitioned between DCM (20 ml) and water (12 ml). The layers were separated and the aqueous layer was extracted with further DCM (12 ml). The combined organic extracts were dried (Na2SO4), and concentrated in vacuo to give a brown solid which was purified on a SPE cartridge (silica, 20 g) eluting with a gradient of ethyl acetate:cyclohexane (1:16, 1:8, 1:4, 1:2, 1:1, 1:0). Fractions containing the desired material were combined and evaporated to afford Intermediate 58A (0.89 g). LCMS showed MH+=319; TRET=2.92 min.
A solution of Intermediate 58A (0.89 g, 2.79 mmol) in ethanol (16.7 ml) was treated with sodium hydroxide (0.47 g, 11.7 mmol) as a solution in water (3.1 ml). The mixture was stirred at 50° C. After 12 h, the reaction mixture was concentrated in vacuo to give a residual oil which was dissolved in water (16 ml), then cooled and acidified to pH 3 with 2M hydrochloric acid. After stirring at 0° C. for 30 min, the resulting precipitate was collected by filtration, washed with cooled water (2 ml) and dried in vacuo to afford Intermediate 59 as a white solid (0.73 g). LCMS showed MH+=291; TRET=2.19 min.
Aqueous sodium hydroxide solution (8.55 ml, 2M) was added to a solution of Example 207 (1.55 g) in EtOH (13 ml). The mixture was heated at 50° C. for 18 h then neutralised using aqueous hydrochloric acid and evaporated in vacuo to afford a mixture of 1-ethyl-4-(4-piperidinylamino)-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid and 4-[(1-acetyl-4-piperidinyl)amino]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid Acetic acid (0.36 ml) was added to a stirred mixture of HATU (2.41 g) and N,N-diisopropylethylamine (2.21 ml) in N,N-dimethylformamide (65 ml). After stirring for 15 min the mixture was added to the mixture of 1-ethyl-4-(4-piperidinylamino)-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid and 4-[(1-acetyl-4-piperidinyl)amino]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid and the reaction stirred for 15 h. The reaction mixture was evaporated in vacuo and the residue purified by chromatography using Biotage (silica 90 g) eluting with DCM: MeOH (0%-5% MeOH) to afford Intermediate 60 (1.36 g) as a white solid. LCMS showed MH+=334; TRET=2.06 min.
A solution of Example 2 (5.37 g, 17 mmol) in ethanol (30 ml) was treated with a solution of sodium hydroxide (2.72 g, 68 mmol) in water (20 ml), and the resulting mixture was stirred at 50° C. for 3 h. The reaction mixture was concentrated in vacuo, dissolved in water (250 ml) and the cooled solution was acidified to pH 1 with 5M-hydrochloric acid. The resultant solid was collected by filtration and dried in vacuo to afford Intermediate 61 as a white solid (4.7 g). LCMS showed MH+=289; TRET=2.83 min.
(Diethylamino)sulphur trifluoride (DAST), (0.06 ml, 0.47 mmol), was added to a stirred solution of 1,1-dimethylethyl(4-oxocyclohexyl)carbamate, (250 mg, 1.17 mmol, commercially available from AstaTech Inc., Philadelphia, USA) in anhydrous dichloromethane (5 ml) and the mixture was stirred under nitrogen at 20° C. After 22 h, the reaction mixture was cooled to 0° C., treated with saturated sodium hydrogen carbonate solution (4 ml), and then allowed to warm to ambient temperature. The phases were separated by passage through a hydrophobic frit and the aqueous phase was further extracted with DCM (5 ml). The combined organic phases were concentrated in vacuo to give an orange solid (369 mg) which was further purified by chromatography using a SPE cartridge (silica, 10 g), eluting with DCM to afford Intermediate 62 (140 mg) containing 20% of 1,1-dimethylethyl (4-fluoro-3-cyclohexen-1-yl)carbamate. 1H NMR (400 MHz in CDCl3, 27° C., δppm) Minor component: δ5.11 (dm, 16 Hz, 1H), 4.56 (br, 1H), 3.80 (br, 1H) 2.45-1.45 (m's, 6H excess), 1.43 (s, 9H). Major component: δ4.43 (br, 1H), 3.58 (br, 1H), 2.45-1.45 (m's, 8H excess), 1.45 (s, 9H).
A solution of hydrogen chloride in dioxane (4M, 1.6 ml) was added at 20° C. to a stirred solution of Intermediate 62 (140 mg, 0.6 mmol), in dioxane (1.6 ml). After 3 h, the reaction mixture was concentrated in vacuo to afford intermediate 63 (96.5 mg) containing 4-fluoro-3-cyclohexen-1-amine. 1H NMR (400 MHz in d6-DMSO, 27° C., δppm) Minor component: δ8.22 (br, 3H excess), 5.18 (dm, 16 Hz, 1H), 3.28-3.13 (m, 1H excess), 2.41-1.53 (m's, 6H excess). Major component: δ8.22 (br, 3H excess), 3.28-3.13 (m, 1H excess), 2.41-1.53 (m's, 8H excess). Impurities are also present.
Intermediate 15 (0.06 g, 0.266 mmol) was treated with thionyl chloride (0.48 ml). The mixture was stirred and heated at 75° C. for 2 h. Excess thionyl chloride was removed in vacuo and the residual oil azeotroped with dichloromethane (DCM) to afford Intermediate 16, presumed to be the acid chloride derivative of Intermediate 15, as a white solid. Intermediate 16 was dissolved in anhydrous tetrahydrofuran (THF) (2 ml) and treated with N,N-diisopropylethylamine (DIPEA) (0.069 ml), then with methylamine (2M in tetrahydrofuran, 0.15 ml) and the mixture stirred under nitrogen for 16 h. A further 0.05 ml of methylamine (2M in THF) was added and the solution stirred for 2 h. The mixture was concentrated in vacuo, then partitioned between dichloromethane (2 ml) and aqueous sodium hydroxide solution (2M, 2 ml), then the organic layer washed with water (2 ml). The layers were separated and the organics concentrated in vacuo to afford Intermediate 64 (0.052 g). LCMS showed MH+=239; TRET=2.17 min.
A mixture of Intermediate 17 (2.0 g, 6.37 mmol), 1,1-dimethylethyl 4-amino-1-piperidinecarboxylate (2.04 g, 10.2 mmol) and N,N,-diisopropylethylamine (5.54 ml, 31.9 mmol) in MeCN (40 ml) was heated at 85° C. for 42 h. The reaction was evaporated and the residues partitioned between DCM and water. The organic phase was dried (MgSO4) then evaporated in vacuo. The residue was chromatographed on silica (Biotage, 90 g) eluting with cyclohexane:EtOAc (1:1) to give Intermediate 65 as a white solid (2.70 g). LCMS showed MH+=479; TRET=3.37 min.
A solution of 3-nitrobenzoyl chloride (2.0 g, 10.78 mmol) in DCM (20 ml) was added dropwise to a stirred mixture of N-methylcyclohexylamine (1.83 ml, 14.01 mmol), N,N,-diisopropylethylamine (3.76 ml, 21.56 mmol) and N,N-dimethylaminopyridine (0.01 g) in DCM at 20° C. The reaction mixture was stirred for 56 h then evaporated in vacuo. The residue was partitioned between ethyl acetate and water. The organic phase was washed with aqueous HCl then dried (MgSO4) and evaporated in vacuo. The residue was purified by chromatography on silica eluting with cyclohexane:EtOAc (9:1 followed by 2:1) to afford N-cyclohexyl-N-methyl-3-nitrobenzamide (1.40 g). MS showed MH+ 263.
A mixture of N-Cyclohexyl-N-methyl-3-nitrobenzamide (1.40 g, 5.35 mmol) and palladium on carbon (5%, 0.140 g) in ethanol (10 ml) was stirred under an atmosphere of hydrogen for 1 hour. The reaction mixture was filtered through Celite and the filtrate evaporated to afford Intermediate 67 as a brown solid (0.107 g). LCMS showed MH+=233; TRET=2.56 min.
A solution of ethyl isocyanate (2.31 g, 32.5 mmol) in DCM (40 ml) was added, dropwise over 15 min, to a vigorously stirred solution of 4-piperidone monohydrate hydrochloride (5.0 g, 32.5 mmol, commercially available from Aldrich) and sodium hydrogen carbonate (8.2 g, 97.5 mmol) in water (60 ml) at 0° C. The reaction mixture was stirred at room temperature for 20 h. Sodium chloride (7.0 g) was added to the reaction mixture and the organic phase was separated. The aqueous phase was extracted with further DCM (3×75 ml). The combined organic extracts were dried (Na2SO4) and evaporated in vacuo to give a white solid (4.0 g). Recrystallisation from ethyl acetate: cyclohexane (10:1) afforded Intermediate 68 as a white solid (2.3 g).
TLC (silica) gave Rf=0.24 (ethyl acetate). Anal. Found: C, 56.7; H, 8.3; N, 16.35. C8H14N2O2 requires C, 56.5; H, 8.3; N, 16.5.
A solution of Intermediate 68 (1.5 g, 8.8 mmol) and benzylamine (1.04 g, 9.7 mmol) in absolute ethanol (60 ml) was hydrogenated over pre-reduced 10% palladium on charcoal catalyst (0.6 g) in ethanol (20 ml) until the uptake of hydrogen had ceased (22 h). The reaction mixture was filtered through filter agent (Celite), and then through silica gel (100 ml) eluting with ethanol:0.88-ammonia (100:1) to give a black oil. The oil was dissolved in ethanol (30 ml) and treated with a solution of hydrogen chloride in ethanol (3M) until the solution was acidic. The solvent was evaporated and the residue was triturated with ethanol to afford Intermediate 69 as a white solid (1.09 g).
TLC (silica) gave Rf=0.73 (ethyl acetate:methanol, 10:1). Anal. Found: C, 45.9; H, 8.4; N, 19.8. C8H18ClN3O requires C, 46.3; H, 8.7; N, 20.2.
Cyclopropylamine (0.136 g, 2.39 mmol) and diisopropylethylamine (0.68 ml, 3.9 mmol) were added to a stirred solution of 4-[({[(1,1-dimethylethyl)oxy]carbonyl}amino)-methyl]benzoic acid (0.501 g, 2.0 mmol), EDC (0.612 g, 3.2 mmol) and HOBT (0.35 g, 2.6 mmol) in DMF (2 ml). The resulting mixture was stirred at room temperature overnight. Solvents were removed in vacuo, and the residue was dissolved in ethyl acetate (20 ml) and washed with 0.5M-hydrochloric acid (3×20 ml). The organic phase was dried (Na2SO4) and evaporated in vacuo to give the crude product which was purified by Biotage chromatography (silica) eluting with ethyl acetate:cyclohexane (1.3:1) to afford Intermediate 70 as a white solid (0.512 g). LCMS showed MH+=291; TRET=2.75 min.
Intermediate 70 (0.506 g, 1.74 mmol) was dissolved in a solution of hydrogen chloride in dioxan (20 ml, 4M) under nitrogen. After 1 h, methanol (3 ml) was added to the mixture and stirring was continued at room temperature overnight. Solvents were removed in vacuo to afford Intermediate 71 as a white solid (0.416 g). LCMS showed MH+=191; TRET=0.82 min.
Intermediate 33 (1.36 g, 4.7 mmol), EDC (1.26 g, 6.57 mmol) and HOBT (0.76 g, 5.62 mmol) were suspended in DMF (50 ml) and stirred vigorously at room temperature for 0.5 h, before adding 1,1-dimethylethyl 4-(aminomethyl)-1-piperidinecarboxylate (1.3 g, 6.07 mmol, commercially available from Maybridge Chemical Co. Ltd.,). After stirring at room temperature overnight, a further quantity of 1,1-dimethylethyl 4-(aminomethyl)-1-piperidinecarboxylate (1.01 g, 4.7 mmol) was added to the reaction mixture which was then heated at 50° C. After 6 h, diisopropylethylamine (0.25 ml, 1.44 mmol) was added, and the mixture was maintained at 50° C. for a further 6 h. Solvents were removed in vacuo and the residue was partitioned between DCM (100 ml) and water (10 ml). The phases were separated by passage through a hydrophobic frit, and the organic phase was evaporated in vacuo to give the crude product. Further purification using SPE cartridges (aminopropyl followed by silica) afford Intermediate 72 as a cream solid (1.24 g). LCMS showed MH+=487; TRET=2.97 min.
Intermediate 73 is used in situ in the general procedure for Examples 360-414.
Acetic anhydride (0.52 ml, 5.5 mmol) was added to a mixture of tert-butyl N-β-aminomethyl)benzyl] carbamate (1.1 g, 4.65 mmol commercially available from Astatech) and triethylamine (0.7 ml, 5 mmol) in THF (20 ml). The reaction mixture was stirred at 20° C. from 16 h then concentrated in vacuo. The residue was partitioned between EtOAc and water. The organic phase was dried (MgSO4) and evaporated in vacuo. The residue was chromatographed over silica eluting with hexanes:EtOAc (1:1) followed by EtOAc to afford Intermediate 74 (1.2 g) as a colourless oil. Anal. Found: C, 64.79; H, 7.93; N, 10.10. C15H22N2O3 requires C, 64.73; H, 7.97; N, 10.06. MS (M+Na)+ 301.
Hydrogen chloride in dioxane (4 ml, 4M) was added to a solution of Intermediate 74 (1.0 g, 3.6 mmol) in dioxane (10 ml) and the resultant mixture stirred for 6 hours at 20° C. The reaction was diluted with Et2O (20 ml) and filtered to afford Intermediate 75 (0.7 g) as a white solid. MS MH+ 179. 1H NMR (300 MHz in d6-DMSO, 27° C., δppm) δ 8.6-8.4 (br m, 3H), 7.38-7.26 (m, 3H), 7.22 (bm, 1H), 4.24 (d, J=5.7 Hz, 2H), 3.95 (dd, J=11.6, 5.7 Hz, 2H), 1.87 (s, 3H).
(cis-3-hydroxycyclohex-1-ylamino group, racemic)
A solution of Example 665 (0.681 g, 2.05 mmol) in ethanol (7 ml) was treated with a solution of sodium hydroxide (0.362 g, 9.05 mmol) in water (2.9 ml). The resulting mixture was stirred at 50° C. After 3 h, the reaction mixture was concentrated in vacuo to give a residual oil which was dissolved in water (3 ml), then cooled and acidified to pH 3 with 2M-hydrochloric acid. After stirring at 0° C. for 1 h, the resulting precipitate was collected by filtration, washed with cooled water (0.5 ml) and dried in vacuo to afford Intermediate 76 as a white solid (0.491 g). LCMS showed MH+=305; TRET=2.14 min.
That is, Example 1 is
Intermediate 1 (0.051 g) and cyclopentyl amine (0.019 g) were suspended in ethanol (2 ml) and triethylamine (0.14 ml) was added. The mixture was stirred under nitrogen and heated at 80° C. for 16 h. After cooling to room temperature, ethanol was removed by evaporation under a stream of nitrogen and the residue partitioned between dichloromethane (DCM) and water. The layers were separated and the organic layer was loaded directly onto an solid phase extraction (SPE) cartridge (silica, 5 g) which was eluted sequentially with; (i) DCM, (ii) DCM: Et2O (2:1), (iii) DCM:Et2O (1:1), (iv) Et2O, (v) EtOAc, (vi) MeOH. Fractions containing desired material were combined and concentrated in vacuo to afford Example 1 (0.074 g). LCMS showed MH+=303; TRET=3.45 min.
Similarly prepared were the following:
*For alternative synthesis of Example 5, see Example 207 hereinafter
Instead of the method shown above for Examples 1-5 (called Method A), the compound of Example 3 can also be made: either using the minor variation of Method A described in detail under “Intermediate 32” hereinabove, or using the following Method B: Example 3, Method B: Intermediate 1 (2.5 g) was dissolved in acetonitrile (15 ml). 4-Aminotetrahydropyran hydrochloride (1.1 g) and N,N-diisopropylethylamine (9.4 ml) were added and the mixture stirred under nitrogen at 85° C. for 16 h. A trace of starting material remained, so an additional portion of 4-aminotetrahydropyran hydrochloride (0.11 g) was added and stirring continued at 85° C. for a further 16 h. The mixture was then concentrated in vacuo. The residue was partitioned between DCM and water. The layers were separated and the organic layer was washed with further water (2×20 ml) then dried (Na2SO4) and concentrated in vacuo. The residue was further purified by chromatography using Biotage (silica, 90 g), eluting with cyclohexane:ethyl acetate to afford Example 3 (2.45 g). LCMS showed MH+=319; TRET=2.90 min.
Intermediate 3 (0.045 g) was placed in a Reactivial™ and treated with cyclopentyl amine (0.07 ml). The mixture was heated at 90° C. for 2 h, then allowed to cool to room temperature and partitioned between chloroform (2 ml) and water (1 ml). The layers were separated and the organic phase was evaporated to a brown solid, which was purified by mass directed autoprep HPLC, to afford Example 6 as a white solid (0.008 g). LCMS showed MH+=289; TRET=3.22 min.
Intermediate 3 (0.035 g) was placed in a Reactivial™ and treated with 4-amino tetrahydropyran (0.06 ml). The mixture was heated at 90° C. for 2 h, then allowed to cool to room temperature and partitioned between chloroform (2 ml) and water (1 ml). The layers were separated and the organic phase was concentrated, then applied to a preparative TLC plate (silica, 20 cm×20 cm×1 mm) which was eluted with ethyl acetate. The required band was removed from the plate and the silica washed with ethyl acetate (2×15 ml). Concentration of the ethyl acetate solution in vacuo afforded Example 7 as a white solid (0.008 g). LCMS showed MH+=305; TRET=2.67 min.
that is:
Intermediate 1 (0.05 g) and (S)-(−)-3-aminotetrahydrofuran 4-toluene sulphonate (0.052 g) were suspended in ethanol (1 ml) and triethylamine (0.14 ml) was added. The mixture was stirred under nitrogen and heated at 80° C. for 24 h. After cooling to room temperature, ethanol was removed by evaporation under a stream of nitrogen and the residue partitioned between DCM (2 ml) and water (1.5 ml). The layers were separated and the organic layer concentrated to dryness. Purification was carried out using an SPE cartridge (silica, 5 g), eluting with a gradient of EtOAc:cyclohexane; (1:16 then, 1:8, 1:4, 1:2, 1:1 and 1:0). Fractions containing desired material were combined and concentrated in vacuo to afford Example 8 (0.052 g). LCMS showed MH+=305; TRET=2.70 min.
Similarly prepared were the following:
Intermediate 1 (0.05 g) and Intermediate 13 (0.027 g) were suspended in ethanol (1 ml) and triethylamine (0.14 ml) was added. The mixture was stirred under nitrogen and heated at 80° C. for 24 h. After cooling to room temperature, ethanol was removed by evaporation under a stream of nitrogen and the residue partitioned between DCM (2 ml) and water (1.5 ml). The layers were separated and the organic layer concentrated to dryness. Purification was carried out using an SPE cartridge (silica, 5 g), eluting with a gradient of EtOAc:cyclohexane; (1:8 then 1:4, 1:2, 1:1 and 1:0). Fractions containing desired material were combined and concentrated in vacuo to afford Example 13 (0.045 g) as a mixture of enantiomers. LCMS showed MH+=353; TRET=2.60 min.
Similarly prepared was the following:
Intermediate 2 (0.035 g) was placed in a Reactivial™ and treated with cyclopentyl amine (0.05 ml). The mixture was heated at 90° C. for 1.5 h, then allowed to cool to room temperature and partitioned between chloroform (2 ml) and water (1 ml). The layers were separated and the organic phase was concentrated. The residual solid was triturated with Et2O and the insoluble off-white solid collected and air-dried to afford Example 19 (0.016 g). LCMS showed MH+=275; TRET=2.58 min.
that is:
Intermediate 2 (0.035 g) was placed in a Reactivial™ and treated with 4-aminotetrahydropyran (0.05 ml). The mixture was heated at 90° C. for 1.5 h, then allowed to cool to room temperature and partitioned between chloroform (2 ml) and water (1 ml). The layers were separated and the organic phase was concentrated. The crude product was purified by mass directed autoprep HPLC to afford Example 20 as an off-white solid (0.011 g). LCMS showed MH+=291; TRET=2.08 min.
Alternative Synthetic Method for Example 20:
Intermediate 2 (2 g) was suspended in 4-aminotetrahydropyran (2 g), and the mixture was heated at 90° C. for 6 h. The residual mixture was allowed to cool to room temperature and partitioned between chloroform (50 ml) and water (50 ml). The phases were separated and the organic phase was evaporated to dryness. The residue was triturated with Et2O (30 ml) and the insoluble solid was collected and dried to afford Example 20 as a cream solid (2.24 g). LCMS showed MH+=291; TRET=2.19 min.
that is, Example 21 is:
Three alternative methods, A, B and C, have been used to make Example 21, as follows:
A solution of the 4-chloro Intermediate 17 (0.031 g, 0.1 mmol) in ethanol (1.9 ml) was treated with triethylamine (0.07 ml, 0.5 mmol), followed by a 0.1M ethanolic solution of 4-aminotetrahydropyran (Intermediate 8, 1.1 ml of the 0.1M ethanolic solution=0.11 mmol). The mixture was heated at reflux (80° C.) for 18 h. A further portion of 4-amino-tetrahydropyran (0.01 ml of undiluted amine, not a solution thereof) was then added and heating continued for a further 24 h. Volatiles were removed in vacuo and the residue dissolved in dichloromethane (DCM), then applied to an solid phase extraction (SPE) cartridge (aminopropyl, 1 g) which was eluted first with DCM, then with methanol. Fractions containing desired material were concentrated in vacuo to afford Example 21 (0.004 g). LCMS showed MH+=380; TRET=2.92 min.
Intermediate 17 (0.031 g, 0.1 mmol) was dissolved in acetonitrile (1 ml). 4-Aminotetrahydropyran hydrochloride (Intermediate 8A, 0.015 g, 0.11 mmol) and N,N-diisopropylethylamine (0.08 ml, 0.5 mmol) were added and the mixture stirred under nitrogen at 85° C. for 16 h, then concentrated in vacuo. The residue was partitioned between dichloromethane (DCM) and water. The layers were separated and the organic layer was concentrated in vacuo to afford Example 21 (0.027 g). LCMS showed MH+=380; TRET=2.92 min.
This alternative route C to Example 21 involves formation of the ester of Example 3 Intermediate 32
using one of the methods described above, conversion of the ester of Example 3/Intermediate 32 into the carboxylic acid (Intermediate 33) using the method given above for Intermediate 33, and then amide bond formation to form Example 21 using the method of Examples 81-84 below.
The following compounds can be similarly prepared using one or more of Methods A, B or C above, preferably Method A or B:
that is, Example 39 is:
A solution of Intermediate 17 (0.031 g, 0.1 mmol) in ethanol (1 ml) was treated with triethylamine (0.07 ml, 0.5 mmol), followed by a 0.1M ethanolic solution of cyclopentyl amine (1.1 ml of the 0.1M ethanolic solution=0.11 mmol). The mixture was heated at reflux (80° C.) for 18 h. A further portion of cyclopentyl amine (0.009 ml of undiluted amine, not a solution thereof) was then added and heating continued for a further 24 h. Volatiles were removed in vacuo and the residue dissolved in DCM, then applied to an SPE cartridge (aminopropyl, 1 g) which was eluted first with DCM, then with methanol. The DCM fraction was concentrated in vacuo, then applied to an SPE cartridge (silica, 0.5 g) which was eluted sequentially with (i) DCM, (ii) Et2O, (iii) EtOAc and (iv) MeOH. Fractions containing desired material were combined to afford Example 39 (0.007 g). LCMS showed MH+=364; TRET=3.38 min.
Similarly prepared were the following:
that is, Example 57 is:
A solution of Intermediate 22 (0.03 g, ca. 0.1 mmol) in ethanol (1 ml) was treated with triethylamine (0.07 ml, 0.5 mmol), followed by a 0.1M ethanolic solution of Intermediate 6 (1.1 ml of the solution=0.11 mmol). The mixture was heated at reflux (80° C.) for 18 h. A further portion of Intermediate 6 (0.01 ml, undiluted) was then added and heating continued for a further 24 h. Volatiles were removed in vacuo and the residue dissolved in DCM, then applied to an SPE cartridge (aminopropyl, 1 g) which was eluted first with DCM, then with methanol.
The DCM fraction was concentrated in vacuo, then applied to an SPE cartridge (silica, 0.5 g) eluting with (I) DCM, (ii) EtOAc and (iii) a stepwise gradient of chloroform:methanol (from 99:1 up to 4:1). Fractions containing desired material were combined to afford Example 57 (0.003 g). LCMS showed MH+=422; TRET=2.1 min.
A solution of Intermediate 28 (0.03 g, 0.1 mmol) in ethanol (1 ml) was treated with a 0.1M ethanolic solution of cyclopentyl amine (1.1 ml of solution=0.11 mmol). Triethylamine (0.07 ml, 0.5 mmol) was then added and the mixture heated at reflux (85° C.), under nitrogen for 12 h. A further portion of cyclopentyl amine (0.009 ml, undiluted) was then added and heating continued for a further 36 h. The mixtures were concentrated in vacuo and the residue treated with chloroform. A small amount of insoluble material was collected by filtration, then the filtrate applied to an SPE cartridge (aminopropyl, 1 g) which was eluted first with DCM, then with methanol. Fractions containing desired material were combined to afford Example 61 (0.039 g). LCMS showed MH+=350; TRET=2.88 min.
Similarly prepared were the following:
that is, Example 74 is:
A solution of Intermediate 28 (0.03 g, 0.1 mmol) in ethanol (1 ml) was treated with a 0.1M ethanolic solution of Intermediate 6 (1.1 ml of solution=0.11 mmol). Triethylamine (0.07 ml, 0.5 mmol) was then added and the mixture heated at reflux (85° C.), under nitrogen for 12 h. A further portion of Intermediate 6 (0.1 mmol) was then added and heating continued for a further 36 h. The mixtures were concentrated in vacuo and the residue treated with chloroform. A small amount of insoluble material was collected by filtration, then the filtrate applied to an SPE cartridge (aminopropyl, 1 g) which was eluted first with DCM, then with methanol. Fractions containing desired material were combined and concentrated in vacuo. The residue was further purified by SPE (silica, 0.5 g) eluting with (i) DCM, (ii) chloroform, (iii) EtOAc and (iv) a stepwise gradient of chloroform:methanol (from 99:1 up to 4:1). Fractions containing desired material were combined to afford Example 74 (0.029 g). LCMS showed MH+=407; TRET=2.57 min.
To a stirred suspension of Intermediate 33 (0.025 g, ca. 0.08 to 0.09 mmol) in chloroform (2 ml) was added thionyl chloride (0.025 ml) and the mixture stirred at room temperature for 1 h. The mixture was cooled to 0° C. and methylamine added (2M solution in THF, 0.69 ml=1.38 mmol). After returning to room temperature the mixture was stirred for a further 1 h, then quenched by addition of water (4 ml) and the layers separated. The organic layer was concentrated then applied to an SPE cartridge (silica, 1 g) which was eluted with (i) DCM, (ii) Et2O (2:1), (iii) EtOAc, (iv) MeOH: EtOAc (1:9). Fractions containing desired material were combined to afford Example 81 (0.019 g). LCMS showed MH+=304; TRET=2.19 min.
Similarly prepared:
In an alternative embodiment to the process described for Examples 81-84 above, Example 83 can be made according to the following method:
A mixture of Intermediate 33 (3.0 g, 10.33 mmol), EDC (2.25 g, 11.7 mmol), and HOBT (1.68 g, 12.4 mmol) was stirred at room temperature for 1 hour. Ethylamine (6.2 ml, 12.4 mmol, 2M-solution in THF) was added, and stirring was continued at room temperature for 22 hours. The solvents were removed in vacuo, and the residual solid was dissolved in chloroform (250 ml) and washed successively with water (70 ml) and 5%-sodium hydrogen carbonate solution (70 ml). After drying over anhydrous sodium sulphate, the organic solution was evaporated in vacuo to give a pale orange solid (4.15 g). This solid was dissolved in a mixture of dichloromethane (5 ml) and chloroform (5 ml) and purified by-column chromatography (Biotage, silica, 100 g), eluting initially with EtOAc-cyclohexane (2:1) and finally with neat EtOAc. The product containing fractions were combined and evaporated to give Example 83 as a pale yellow solid (3.05 g). LCMS showed MH+=318; TRET=2.33 min. 1H NMR (400 MHz in d6-DMSO, 27° C., δppm) 9.76 (d, 1H) 8.35 (s, 1H) 7.94 (s, 1H) 5.99 (br m, 1H) 4.47 (q, 2H) 4.16-4.01 (m's, 3H) 3.62 (m, 2H) 3.48 (m, 2H) 2.13 (m, 2H) 1.77 (m, 2H) 1.49 (t, 3H) 1.28 (t, 3H).
That is, Example 85 is:
Intermediate 41 (0.017 g, 0.062 mmol) was dissolved in DMF (2 ml), then treated with HATU (0.023 g) followed by diisopropylethyl amine (0.021 ml) and the mixture stirred for 10 min. Benzylamine (0.007 ml) was then added and stirring continued for a further 64 h. The mixture was concentrated in vacuo and the residue dissolved in DCM (1.5 ml) then treated with saturated aqueous sodium bicarbonate solution (1.5 ml). This mixture was stirred for 30 min, then the layers were separated and the organic layer was applied to an SPE cartridge (silica, 1 g) which was eluted sequentially with a gradient of ethyl acetate: cyclohexane (1:4, then 1:2, 1:1, 2:1 and 1:0). Fractions containing desired material were concentrated in vacuo to afford Example 85 (0.017 g). LCMS showed MH+=366; TRET=2.80 min.
Similarly prepared were the following:
Intermediate 43 (0.019 g) was dissolved in DMF (2 ml), then treated with HATU (0.024 g) followed by diisopropylethyl amine (0.022 ml) and the mixture stirred for 10 min. Benzylamine (0.007 ml) was then added and stirring continued for a further 64 h. The mixture was concentrated in vacuo and the residue dissolved in DCM (1.5 ml) then treated with saturated aqueous sodium bicarbonate solution (1.5 ml). This mixture was stirred for 30 min, then the layers were separated and the organic layer applied to an SPE cartridge (silica, 1 g) which was eluted sequentially with a gradient of ethyl acetate: cyclohexane (1:4, then 1:2, 1:1 and 1:0). Fractions containing desired material were concentrated in vacuo to afford Example 91 (0.023 g). LCMS showed MH+=396; TRET=3.26 min.
that is, Example 92 is:
Intermediate 41 (0.017 g) was dissolved in DMF (2 ml), then treated with HATU (0.023 g) followed by diisopropylethyl amine (0.021 ml) and the mixture stirred for 10 min. 4-Fluoroaniline (0.006 ml) was then added and stirring continued for a further 64 h. The mixture was concentrated in vacuo and the residue dissolved in DCM (1.5 ml) then treated with saturated aqueous sodium bicarbonate solution (1.5 ml). This mixture was stirred for 30 min, then the layers were separated and the organic layer concentrated in vacuo. The crude mixture was purified by mass directed autoprep HPLC to afford Example 92 (0.013 g). LCMS showed MH+=370; TRET=2.91 min.
Similarly prepared were the following:
In all of Examples 22 to 98, where a 4-amino 5-carboxamide Example of the following Formula I has been synthesised from the 4-chloro derivative, then an alternative final-step synthesis is as follows:
An intermediate of Formula IV above (0.1 mmol) was dissolved in acetonitrile (1 ml). An amine of formula R3NH2 (0.11 mmol, 1.1 mole equivalents) and N,N-diisopropylethylamine (0.5 mmol, 5 mole equivalents) were added and the mixture stirred under nitrogen at 85° C. for 16 h. After concentration in vacuo, the residue was partitioned between dichloromethane (DCM) and water. The layers were separated and the organic layer was concentrated in vacuo to afford an Example of Formula I.
Intermediate 33 (0.048 mmol) was dissolved in DMF (0.5 ml), then treated with HATU (0.048 mmol) followed by diisopropylethyl amine (0.096 mmol) and the mixture stirred for 10 min. 4-Methylsulfonylbenzylamine (0.052 mmol, available from Acros Organics) was then added and stirring continued for a further 16 hours. The mixture was concentrated in vacuo. The crude mixture was purified by mass directed autoprep HPLC to afford Example 100 (0.013 g). LCMS showed MH+=458; TRET=2.22 min.
Similarly prepared, but replacing the 4-methylsulfonylbenzylamine with the same or similar number of moles of another amine R4R5NH, were the following compounds (Examples 102 to 182):
An alternative process for preparing Example 109 is given below: 1-Hydroxybenzotriazole (0.215 g, 1.59 mmol) and 1-[3-(dimethylamino)propyl]-3-ethyl-carbodiimide hydrochloride (0.357 g, 1.86 mmol) were added to a suspension of Intermediate 33 (0.384 g, 1.32 mmol) in DMF (10 ml). After stirring at room temperature for 30 minutes, (1,3-thiazol-2-ylmethyl)amine (0.182 g, 1.59 mmol) (commercially available from MicroChemistry Building Blocks (Russia) or Matrix Scientific (USA), or preparable as disclosed in Synthesis 1998, 641, or Tetrahedron 1995, 51, 12731) was added. The reaction was stirred for 18 hours and then partitioned between ether and water. The organic phase was washed with brine, dried (MgSO4) and evaporated in vacuo. The residue was purified by chromatography (Biotage, silica 90 g) eluting with cyclohexane: EtOAc followed by EtOAc. The material was triturated with cyclohexane and filtered to afford Example 109 (0.244 g) as a pale yellow solid. LCMS showed MH+ 387; TRET=2.49 min. 1H NMR (400 MHz in CDCl3, 8 ppm) δ 9.74 (d, 1H) 8.50 (s, 1H) 7.94 (s, 1H) 7.74 (d, 1H), 7.33 (d, 1H), 7.17 (m, 1H), 4.94 (d, 2H) 4.45 (q, 2H) 4.15-4.00 (m, 3H), 3.63 (m, 2H), 2.15 (m, 2H) 1.85-1.73 (m, 3H) 1.48 (t, 3H).
In an alternative embodiment to the process described above for Examples 100-182, Example 167 can be made according to the following method:
A mixture of Intermediate 33 (0.498 g, 1.72 mmol), EDC (0.46 g, 2.41 mmol), and HOBT (0.278 g, 1.68 mmol) was stirred at room temperature for 0.25 hours. Veratrylamine (3,4-dimethoxybenzylamine, 0.31 ml, 2.05 mmol, obtainable from Aldrich or Synlett, 1999, 4, 409) was added, and stirring was continued at room temperature for 22 hours. The reaction mixture was partitioned between Et2O and water. The aqueous phase was extracted with Et2O and the combined organic phases washed with brine, dried (MgSO4) and evaporated in vacuo. The residue was purified by chromatography (Biotage, silica 40 g) eluting with EtOAc: cyclohexane (2:1). The material was further purified by SPE (SCX-2, 10 g) eluting with methanol then ammonia in methanol (0.5M). The ammonia methanol fractions were combined and evaporated in vacuo to afford Example 167 as a white foam (0.633 g). LCMS showed MH+=440; TRET=2.65 min. 1H NMR (400 MHz in CDCl3, 27° C., δppm) 9.78 (d, 1H) 8.37 (s, 1H) 7.94 (s, 1H) 6.94-6.82 (m, 3H) 6.29 (br m, 1H) 4.56 (d, 2H) 4.46 (q, 2H) 4.15-4.01 (m's, 3H) 3.89 (s, 6H) 3.63 (m, 2H) 2.15 (m, 2H) 1.78 (m, 2H) 1.49 (t, 3H).
The 1H NMR data for Example 178 (as prepared by the process described in Examples 100-182 above) was as follows:
1H NMR (400 MHz in CDCl3, δppm) δ 9.90 (m, 1H) 8.37 (s, 1H) 7.94 (s, 1H) 7.49 (s, 1H), 7.40 (s, 1H) 6.39 (m, 1H) 4.50-4.42 (m, 41) 4.15-4.00 (m, 3H) 3.89 (s, 3H), 3.63 (m, 21) 2.52 (m, 2H) 2.20-2.10 (m, 2H) 1.85-1.73 (m, 3H) 1.48 (t, 3H).
A vigorously stirred mixture of Intermediate 48 (40 mg), anhydrous potassium carbonate (57 mg) and ethyl 3-bromopropanoate (0.027 ml) in anhydrous DMF (1 ml) was heated at 65° C. overnight. The reaction mixture was concentrated, and the residue was partitioned between dichloromethane (5 ml) and water (5 ml). The phases were separated and the organic phase was evaporated to a residual oil which was purified by mass directed autoprep HPLC to afford Example 183 (5 mg). LCMS showed MH+=389; TRET=3.65 min.
Sodium hydride (0.067 g, 60% dispersion in oil) was added to a stirred solution of Example 20 (0.47 g) in DMF (19 ml), followed by n-propyl iodide (0.17 ml). The mixture was stirred at 23° C. for 16 hours, then concentrated, diluted with chloroform (30 ml) and washed with 1:1 water:brine solution (30 ml), separated and the organic layer concentrated. The residue was purified on a SPE catridge (silica, 10 g) eluting with 10 ml volumes of dichloromethane, 1:1 diethyl ether:cyclohexane, and diethyl ether. The combined 1:1 diethyl ether: cyclohexane, and diethyl ether, fractions were concentrated to give Example 185 as a clear gum (0.23 g). LCMS showed MH+=333; TRET=3.14 min.
2-Bromoethanol (0.008 ml) was added to a solution of Example 20 (0.03 g) in anhydrous DMF (1.5 ml), with 2-tert-butylimino-2-diethylamino-1,3-dimethyl-perhydro-1,3,2-diazaphosphorine (polymer bound, 2.3 mmol/g loading, 0.045 g). The mixture was shaken at 23° C. for 16 hours, then the solution drained from the resin, and the resin was washed with DMF. The combined organics were concentrated, and the residue purified on a SPE cartridge (silica, 1 g) eluting with 70-100% ethyl acetate in cyclohexane. The combined fractions were concentrated to give Example 186 as a white solid (0.011 g). LCMS showed MH+=335; TRET=2.47 min.
Intermediate 50 (0.03 g) was stirred in DMF (1 ml) with DIPEA (0.035 ml) and HATU (0.038 g) for 20 min. 4-(Methylsulfonyl)benzylamine hydrochloride (0.024 g) was added to the mixture and the solution was stirred for 8 hours at 23° C. The solution was concentrated and the residue dissolved in dichloromethane (6 ml) then washed with saturated sodium bicarbonate solution (6 ml) and 1:1 brine:water (6 ml), separated by hydrophobic frit. The organic layer was concentrated to give Example 187 as a white solid (0.039 g). LCMS showed MH+=472; TRET=2.67 min.
The synthetic method is as described in Example 187, except that in place of 4-(methylsulfonyl)benzylamine hydrochloride, 4-fluoroaniline (0.01 ml) was added to the mixture. The resultant product required further purification, which was performed by mass directed autoprep HPLC, giving Example 188 as a clear gum (0.03 g). LCMS showed MH+=398; TRET=3.13 min.
4-Aminotetrahydropyran hydrochloride (Intermediate 8A, 0.413 g, 3.0 mmol) was added to a mixture of Intermediate 51 (0.268 g, 11.0 mmol) and N,N-diisopropylethylamine (0.87 ml, 5.0 mmol) in acetonitrile (3 ml). The resulting mixture was heated at 85° C. for 24 hours. Volatiles were removed in vacuo and the residue was dissolved in chloroform (1.5 ml) and applied to a SPE cartridge (silica, 5 g). The cartridge was eluted successively with Et2O, EtOAc and EtOAc-MeOH (9/1). Fractions containing the desired product were combined and concentrated in vacuo to give the desired product contaminated with starting material (Intermediate 51). Further purification using a SPE cartridge (silica, 5 g) eluting with ethyl acetate-cyclohexane (1/3) afforded Example 189 (0.248 g). LCMS showed MH+=333; TRET=2.75 min.
Cyclohexylamine (0.149 g, 1.5 mmol) was added to a mixture of Intermediate 51 (0.201 g, 0.75 mmol) and N,N-diisopropylethylamine (0.65 ml, 3.73 mmol) in acetonitrile (3 ml). The resulting mixture was heated at 85° C. for 40 hours. Volatiles were removed in vacuo and the residue was dissolved in chloroform (1.5 ml) and applied to a SPE cartridge (silica, 5 g). The cartridge was eluted successively with Et2O, EtOAc and MeOH. Fractions containing the desired product were combined and concentrated in vacuo to afford Example 190 (0.128 g). LCMS showed MH+=331; TRET=3.64 min.
A mixture of Intermediate 52 (0.014 g, 0.046 mmol), HATU (0.018 g, 0.048 mmol) and DIPEA (0.022 ml, 0.125 mmol) in DMF (1 ml) was shaken at room temperature for 10 min. 1-[4-(Methylsulfonyl)phenyl]methanamine (0.009 g, 0.046 mmol) was then added, and the mixture was shaken for several minutes to give a solution. This solution was stored at room temperature for 16 hours. The solution was concentrated in vacuo, and the residue was dissolved in chloroform (0.5 ml) and applied to a SPE cartridge (aminopropyl, 0.5 g). The cartridge was eluted successively with chloroform (1.5 ml), EtOAc (1.5 ml) and EtOAc-MeOH (9:1, 1.5 ml). Fractions containing the desired product were concentrated in vacuo to afford Example 191 (0.005 g). LCMS showed MH+=470; TRET=2.54 min.
Example 192 was prepared from Intermediate 52 using a method analagous to Example 191. LCMS showed MH+=392: TRET=2.43.
Example 193 was prepared from Intermediate 52 using an analagous method to Example 191. LCMS showed MH+=396; TRET=2.6 min.
Example 194 was prepared from Intermediate 52 using an analagous method to Example 191. LCMS showed MH+=460; TRET=2.74 min.
Example 195 was prepared from Intermediate 52 using an analagous method to Example 191. LCMS showed MH+=418; TRET=2.55 min.
Example 196 was prepared from Intermediate 53 using an analagous method to Example 191. LCMS showed MH+=394; TRET=2.02 min.
3-Aminoazepan-2-one (0.043 g, 0.335 mmol, commercially available from Sigma-Aldrich Company Ltd) was added to a mixture of Intermediate 17 (0.021 g, 0.067 mmol) and DIPEA (0.058 ml, 0.335 mmol) in acetonitrile (0.5 ml). The resulting mixture was heated at 85° C. for 48 hours. Volatiles were removed in vacuo, and the residue was dissolved in chloroform (0.5 ml) and applied to a SPE cartridge (silica, 0.5 g) which was eluted successively with diethyl ether (1.5 ml), ethyl acetate (1.5 ml) and ethyl acetate-methanol (9/1, 1.5 ml). Fractions containing the desired material were concentrated in vacuo to afford Example 197 (0.009 g). LCMS showed MH+=407; TRET=2.81 min.
Similarly prepared, but replacing the 3-aminoazepan-2-one with the same or similar number of moles of another amine R3NH2 were the following compounds:
Intermediate 54 (0.048 g, 0.32 mmol) was added to a mixture of Intermediate 17 (0.050 g, 0.16 mmol) and DIPEA (0.17 ml, 0.98 mmol) in acetonitrile (3 ml). The resulting mixture was heated under reflux. After 12 hours, further quantities of Intermediate 54 (0.044 g, 0.29 mmol), DIPEA (0.17 ml, 0.98 mmol) and acetonitrile (1 ml) were added to reaction mixture which was maintained under reflux. After 36 hours, the reaction mixture was concentated in vacuo, and the residual oil was dissolved in dichloromethane (8 ml) and washed with 5% sodium bicarbonate solution (2 ml). Evaporation of the organic solution gave a viscous oil which was dissolved in dichloromethane (2 ml) and applied to a SPE cartridge (silica, 5 g). The cartridge was eluted successively with a gradient of ethyl acetate-cyclohexane (1:16, then 1:8, 1:4, 1:2, 1:1 and 1:0). Fractions containing the desired material were concentrated in vacuo to afford Example 201 (0.018 g). LCMS showed MH+=392; TRET=2.95 min.
Intermediate 33 (0.1 g, 0.34 mmol), EDC (0.066 g, 0.34 mmol) and HOBT (0.05 g, 0.37 mmol) were suspended in DMF (2 ml) and stirred at room temperature under nitrogen for 15 min. 2-aminopropan-1-ol (0.026 g, 0.34 mmol) and triethylamine (0.036 g, 0.36 mmol) were added and the mixture was stirred at room temperature under nitrogen for 6 hours. Solvents were removed in vacuo and the residue partitioned between DCM and water. The organic layer was concentrated and applied to an SPE cartridge (aminopropyl, 5 g), which was eluted with methanol. Concentration in vacuo afforded Example 202 (0.095 g). LCMS showed MH+=348, TRET=2.15 min.
Reaction scheme:
Intermediate 33 (0.1 g, 0.34 mmol), EDC (0.066 g, 0.34 mmol) and HOBT (0.05 g, 0.37 mmol) were suspended in DMF (2 ml) and stirred at room temperature under nitrogen for 15 mins. L-Serine methyl ester hydrochloride (0.054 g, 0.34 mmol) and triethylamine (0.036 g, 0.36 mmol) were added and the mixture stirred at room temperature under nitrogen for 18 hours. Solvents were removed in vacuo and the residue was partitioned between DCM and water. The organic layer was concentrated in vacuo and applied to an SPE cartridge (aminopropyl, 5 g), which was eluted with methanol. Concentration in vacuo afforded an impure residue which was further purified by SPE cartridge (silica, 5 g), eluting with ethyl acetate followed by 5% methanol/ethyl acetate. The desired fractions were concentrated in vacuo to afford Example 203 (0.055 g). LCMS showed MH+=393; TRET=2.22 min.
Intermediate 1 (1.5 g, 5.9 mmol) was dissolved in acetonitrile (80 ml). Trans-4-aminocyclohexanol (0.817 g, 7.1 mmol, commercially available from TCI-America; alternatively (e.g. as the HCl salt) from Aldrich) and diisopropylethylamine (6.18 ml, 35.5 mmol) were added and the mixture was stirred at 85° C. for 16 h. The mixture was concentrated in vacuo, and the residue was partitioned between DCM (120 ml) and water (30 ml). The phases were separated and the organic phase was dried (Na2SO4) and evaporated to give a pale yellow solid. The solid was dissolved in a mixture of DCM (10 ml) and chloroform (3 ml), and applied in equal portions to two SPE cartridges (silica, 20 g) which were eluted sequentially with a gradient of EtOAc:cyclohexane (1:16, then 1:8, 1:4, 1:2, 1:1 and 1:0). Fractions containing the desired material were combined and evaporated in vacuo to give Example 204 (1.893 g) as a white solid. LCMS showed MH+=333; TRET=2.79 min.
Example 204 (1.893 g, 5.7 mmol) was suspended in acetone (12 ml) and the stirred suspension was treated at 0° C. with Jones reagent (1.81 ml). After 30 min, a further quantity of Jones reagent (1.81 ml) was added to the reaction mixture which was maintained at 0° C. After a further 2 h, a final portion of Jones reagent (1.44 ml) was added to the reaction mixture, and stirring at 0° C. was continued for 1 h. Isopropanol (3.8 ml) was added to the reaction mixture, followed by water (15 ml). The resulting mixture was extracted with ethyl acetate (2×40 ml). The combined organic extracts were washed with water (8 ml), dried (Na2SO4) and evaporated to a grey solid. The solid was dissolved in DCM (10 ml) and applied in equal portions to two SPE cartridges (silica, 20 g) which were eluted sequentially with a gradient of ethyl acetate:cyclohexane (1:16, then 1:8, 1:4, 1:2, and 1:1). Fractions containing the desired material were combined and evaporated in vacuo to give Example 205 (1.893 g) as a white solid. LCMS showed MH+=331; TRET=2.84 min.
Intermediate 1 (2.58 g), Intermediate 6 (2.0 g) and N,N-diisopropylethylamine (8.9 ml) were dissolved in acetonitrile (98 ml). The reaction mixture was heated at 85° C. for 24 h then an additional portion of Intermediate 6 (0.18 g) was added and heating continued for a further 10 h. The reaction was concentrated in vacuo and the residues partitioned between DCM and water. The phases were separated and the organic phase evaporated in vacuo. The residue was purified by chromatography using Biotage (silica 90 g) eluting with DCM: MeOH (5%) to afford Example 207 (1.55 g) as a white solid. LCMS showed MH+ 360; TRET=2.71 min.
Example 209 was prepared from Intermediate 1 and (4-aminocyclohexyl)amine using an analogous method to that used for the preparation of Example 207. LCMS showed MH+=332; TRET=2.18 min
A solution of meta-chloroperoxybenzoic acid (45 mg, 0.26 mmol) in chloroform (1 ml) was added dropwise at 0° C. to a stirred solution of Example 138 (0.1 g, 0.26 mmol) in chloroform (1.5 ml). After 1.5 h at 0° C., a further quantity of meta-chloroperoxybenzoic acid (45 mg, 0.26 mmol) in chloroform (1 ml) was added, and stirring was continued at 0° C. for 1.5 h. A trace of starting material remained, so an additional quantity of meta-chloroperoxybenzoic acid (22 mg, 0.13 mmol) in chloroform (0.6 ml) was added. After 3.5 h at 0° C., 2M sodium carbonate solution (1 ml), was added to the reaction mixture.
The phases were separated by passage through a hydrophobic frit and the aqueous phase was extracted with more chloroform (2 ml). The combined organic extracts were evaporated to a residual foam which was purified by mass directed autoprep HPLC to afford Example 210 (44 mg). LCMS showed MH+=397; TRET=2.13 min.
Example 211 was prepared from Example 600 using an analogous method to that used for the preparation of Example 210. LCMS showed MH+=397; TRET=2.20 min
Example 212 was prepared from Example 33 using an analogous method to that used for the preparation of Example 210. LCMS showed MH+=397; TRET=2.13 min
General Procedure
Intermediate 17 (0.15 mmol) was treated with an aliquot of the amine (0.95 ml, equivalent to 0.19 mmol) from a stock solution in acetonitrile (0.2M) and N,N-diisopropylethylamine (0.24 mmol). The mixture was heated at reflux for 20 h then concentrated in vacuo. The residue was purified by SPE (silica) to give the desired product.
A preferred method for the preparation of Example 225 involving 1-methylcyclohexylamine and a longer reaction time is as follows:
A solution of Intermediate 17 (46 mg), 1-methylcyclohexylamine (26 mg) and diisopropylethylamine (94 mg) in acetonitrile (1 ml) was stirred and heated at reflux for 77 h. More 1-methylcyclohexylamine (102 mg), diisopropylethylamine (93 mg) and acetonitrile (1 ml) were added and the reaction mixture was heated at reflux for a further 68 h. The solution was cooled and concentrated in vacuo. The residue was triturated in ethyl acetate and filtered. The filtrate was purified by mass directed autoprep. HPLC to give Example 225 (19 mg). LCMS showed MH+=392; TRET=3.46 min.
Examples 231, 247 and 257, shown below and also involving 1-methylcyclohexylamine, can also preferably be prepared in a similar manner.
General Procedure
Immediate 55 (0.15 mmol) was treated with an aliquot of the amine (0.95 ml, equivalent to 0.19 mmol) from a stock solution in acetonitrile (0.2M) and N,N-diisopropylethylamine (0.24 mmol). The mixture was heated at reflux for 20 h then concentrated in vacuo. The residue was purified by SPE (silica) to give the desire product.
General Procedure
Intermediate 56 (0.15 mmol) was treated with an aliquot of the amine (0.95 ml, equivalent to 0.19 mmol) from a stock solution in acetonitrile (0.2M) and N,N-diisopropylethylamine (0.24 mmol). The mixture was heated at reflux for 20 h then concentrated in vacuo. The residue was purified by SPE (silica) to give the desire product.
General Procedure
Intermediate 57 (0.15 mmol) was treated with an aliquot of the amine (0.95 ml, equivalent to 0.19 mmol) from a stock solution in acetonitrile (0.2M) and N,N-diisopropylethylamine (0.24 mmol). The mixture was heated at reflux for 20 h then concentrated in vacuo. The residue was purified by SPE (silica) to give the desire product.
General Procedure
A mixture of Intermediate 58 (0.1 mmol), HATU (0.1 mmol) and DIPEA (0.4 mmol) in DMF (0.4 ml) was shaken at room temperature for 10 min. A solution of the amine (0.1 mmol) in DMF (0.2 ml) was then added and the mixture agitated for several minutes to give a solution. The solution was stored at room temperature for 16 hours then concentrated in vacuo. The residue was dissolved in chloroform (0.5 ml) and applied to a SPE cartridge (aminopropyl, 0.5 g). The cartridge was eluted successively with chloroform (1.5 ml), EtOAc (1.5 ml) and EtOAc:MeOH (9:1, 1.5 ml). Fractions containing the desired product were concentrated in vacuo and the residue purified by mass directed autoprep HPLC.
Alternative procedure for preparing Example 260:
A solution of Intermediate 58 (45 mg), HATU (63 mg) and DIPEA (39 mg) in acetonitrile (5 ml) was stirred for 10 min. A solution of 2,4-dimethylbenzylamine (24 mg) (available from Salor; or ICN Biomedicals, Inc.; or Synthesis, 1982, 12, 1036) in acetonitrile (1 ml) was added. The reaction mixture was stirred for 18 h. The solution was concentrated and the residue partitioned between ethyl acetate (25 ml) and 0.5M sodium bicarbonate (20 ml). The organic phase was separated, washed with water (20 ml), dried over Na2SO4 and concentrated to leave a gum which was applied to an SPE cartridge (5 g). The cartridge was eluted with ethyl acetate. Fractions containing the desired compound were combined and concentrated in vacuo to give Example 260 (32 mg). LC-MS showed MH+=420; TRET=3.16 min. δH (CDCl3): 1.49 (3H, t), 2.11 (2H, m), 2.33 (3H, s), 2.35 (3H, s), 2.40 (2H, m), 2.52 (2H, m), 2.61 (2H, m), 4.36 (1H, m), 4.47 (2H, q), 4.55 (2H, d), 6.14 (1H, t), 7.01+7.18 (2H, AA′BB′), 7.04 (1H, s), 8.01 (1H, s), 8.36 (1H, s), 9.96 (1H, d).
General Procedure
A mixture of Intermediate 59 (0.1 mmol), HATU (0.1 mmol) and DIPEA (0.4 mmol) in DMF (0.4 ml) was shaken at room temperature for 10 min. A solution of the amine (0.1 mmol) in DMF (0.2 ml) was then added and the mixture agitated for several minutes to give a solution. The solution was stored at room temperature for 16 hours then concentrated in vacuo. The residue was dissolved in chloroform (0.5 ml) and applied to a SPE cartridge (aminopropyl, 0.5 g). The cartridge was eluted successively with chloroform (1.5 ml), EtOAc (1.5 ml) and EtOAc:MeOH (9:1, 1.5 ml). Fractions containing the desired product were concentrated in vacuo and the residue purified by mass directed autoprep HPLC.
Diisopropylethylamine (0.113 ml, 0.65 mmol) was added to a stirred mixture of Intermediate 17 (40 mg, 0.13 mmol) and Intermediate 63 (45 mg, 0.26 mmol) in acetonitrile (2 ml). The mixture was stirred at 85° C. After 18 h, a further portion of Intermediate 63 (22.5 mg, 0.13 mmol) and diisopropylethylamine (0.113 ml, 0.65 mmol) was added to the reaction mixture and stirring was continued at 90° C. for 24 h. The mixture was then concentrated in vacuo and the residue was partitioned between DCM (20 ml) and water (5 ml). The phases were separated and the aqueous phase was extracted with further DCM (10 ml). The combined organic extracts were dried (Na2SO4) and evaporated in vacuo to give a brown oil (65 mg) which was partially purified on a SPE cartridge (silica, 10 g), eluting with ethyl acetate: petroleum ether (1:8; 1:4; 1:2; 1:1 and 1:0). The resulting two-component pale-brown oil (34 mg) was separated by mass directed auto prep HPLC to give Example 288 (19 mg) as a white foam (LCMS showed MH+=414; TRET=3.24 min) and Example 289 (9 mg) as a white solid (LCMS showed MH+=394; TRET=3.21 min).
General Procedure
A mixture of Intermediate 60 (0.1 mmol), HATU (0.1 mmol) and DIPEA (0.4 mmol) in DMF (0.4 ml) was shaken at room temperature for 10 min. A solution of the amine (0.1 mmol) in DMF (0.2 ml) was then added and the mixture agitated for several minutes to give a solution. The solution was stored at room temperature for 16 hours then concentrated in vacuo. The residue was dissolved in chloroform (0.5 ml) and applied to a SPE cartridge (aminopropyl, 0.5 g). The cartridge was eluted successively with chloroform (1.5 ml), EtOAc (1.5 ml) and EtOAc:MeOH (9:1, 1.5 ml). Fractions containing the desired product were concentrated in vacuo and the residue purified by mass directed autoprep HPLC.
A solution of hydrogen chloride in dioxan (30 ml, 4M, 0.12 mol) was added to a suspension of Example 126 (1.3 g, 2.75 mmol), in dioxan (10 ml) and the mixture was stirred at room temperature for 6 h. The reaction mixture was left to stand for 14 h, then the solution was evaporated, azeotroping with DCM to give a white solid the hydrochloride salt. The solid was suspended in ethyl acetate (50 ml) and washed with sodium hydroxide solution (2N, 50 ml). The organic layer was dried over Na2SO4 and concentrated in vacuo to give Example 318 as a white solid (995 mg). LCMS showed MH+=373; TRET=1.89 min.
A solution of hydrogen chloride in dioxan (30 ml, 4M, 0.12 mol) was added to a suspension of Intermediate 72 (1.2 g, 2.5 mmol), in dioxan (10 ml) and the mixture was stirred at room temperature for 6 h. The reaction mixture was left to stand for 14 h, then the solution was evaporated, azeotroping with DCM to give a white solid (1.24 g). A portion of the solid (68 mg) was suspended in ethyl acetate and washed with 2M-sodium hydroxide solution. The organic layer was dried over Na2SO4 and concentrated in vacuo to afford Example 321 as a white solid (60 mg). LCMS showed MH+=387; TRET=1.92 min.
Triethylamine (0.023 ml, 0.16 mmol) was added to a solution of Example 320 (0.043 g, 0.115 mol) in DCM (1 ml). The mixture was cooled (ice/water bath for 10 min) and ethane sulfonyl chloride (0.014 ml, 0.138 mmol) was added. The resultant solution was stirred at room temperature for 18 h, then the solvent was removed with a steam of nitrogen. The residue was dissolved in dichloromethane (1.5 ml) and stirred with water (1.5 ml). The organic layer was separated and blown down with nitrogen, and applied to a SPE cartridge (silica, 2 g) eluting with 60%-100% ethyl acetate in cyclohexane. The desired fractions were concentrated in vacuo to afford Example 322 as a white solid (32 mg). LCMS showed MH+=465; TRET=2.52 min Similarly prepared were the following, using the same or a similar number of moles of reagents and the same or similar volumes of solvents:
Cyclopropane carboxylic acid (0.011 ml, 0.138 mmol), EDC (0.031 g, 0.161 mmol) and HOBT (0.019 g, 0.138 mmol) were suspended in DMF (2 ml) and stirred at room temperature for 1 h. Example 320 (0.043 g, 0.115 mmol) was added and the mixture was stirred at room temperature for 16 hours. Most of the solvent was removed using a stream of nitrogen and the residue was partitioned between DCM (3 ml) and water (3 ml). The organic layer was blown down with nitrogen and applied to a SPE cartridge (aminopropyl, 1 g), which was eluted with methanol. Concentration by blowing down with nitrogen afforded an impure residue which was further purified by SPE cartridge (silica, 1 g), eluting with 50-100% EtOAc in cyclohexane followed by 5% methanol in EtOAc. The desired fractions were concentrated in vacuo to afford Example 329 as a white solid (49 mg). LCMS showed MH+=441; TRET=2.23 min Similarly prepared, using the same or similar numbers of moles of reagents and volumes of solvents, and using Example 320 as the starting material to make Examples 330 to 343, but using Example 321 (similar number of moles) instead of Example 320 as the starting material to make Examples 344 to 349, were the following:
2M-Sodium hydroxide solution (0.5 ml) was added to a stirred suspension of Example 350 (0.12 g, 0.275 mmol) in methanol (3.5 ml) and water (0.8 ml). After stirring overnight at room temperature, the reaction solution was concentrated, diluted with water (3 ml) and acidified with 2M-hydrochloric acid. The resulting precipitate was collected by filtration, washed with water and dried to give Example 351, as a white solid (0.105 g). LCMS showed MH+=422; TRET=2.95 min.
Aqueous hydrochloric acid (20 ml, 5M) was added to a solution of Intermediate 65 (2.58 g, 5.40 mmol) in tetrahydrofuran (10 ml). The reaction mixture was stirred at 20° C. for 22 h then evaporated in vacuo. The residue was partitoned between DCM and water. The aqueous phase was basified with aqueous sodium hydroxide solution (2M) and extracted with diethyl ether. The organic phases was evaporated in vacuo to give Example 352 as a white solid (2.04 g). LCMS showed MH+=379; TRET=2.10 min.
Methoxyacetyl chloride (0.016 mg, 0.144 mmol) and triethylamine (0.02 mol, 0.144 mmol) were added to a solution of Example 352 (0.046 g, 0.122 mmol) in DCM in a Reactivial. The reaction was stirred for 22 h at 20° C. then diluted with DCM and washed with aqueous sodium hydrogen carbonate solution. The organic phase was separated and applied directly to a SPE cartridge (silica 2 g). The cartridge was eluted with DCM:MeOH (1% followed by 3%) to give Example 353 as a white solid (0.05 g). LCMS showed MH+=451; TRET=2.66 min.
Prepared in a similar manner to example 186 using Example 20 (0.03 g, 0.1 mmol), with isopropylbromide (10 uL, 0.11 mmol), a further 0.11 mmol of alkylating agent was added after 16 hours. The final compound was formed as a clear gum (16 mg). LCMS showed MH+=333; TRET=3.16 min.
Intermediate 64 (0.02 g, 0.084 mmol) and diisopropylethylamine (0.044 ml, 0.252 mmol) were suspended in N-methylpyrrolidinone (1 ml) and cyclohexylamine (0.012 ml, 0.1 mmol) was added. The mixture was heated at 85° C. with stirring in a Reactivial™ for 8 h, then concentrated in vacuo. The residue was partitioned between DCM (2 ml) and water (2 ml). The layers were separated and the organic layer was concentrated in vacuo, then purified by mass directed autoprep HPLC to afford Example 355 (0.012 g). LCMS showed MH+=302; TRET=2.85 min.
Example 356 was prepared from Intermediate 53 using an analogous method to Example 191. LCMS showed MH+=398; TRET=2.18 min.
Example 357 was prepared from Intermediate 53 using an analogous method to Example 191. LCMS showed MH+=472; TRET=2.15 min.
Example 358 was prepared from Intermediate 53 using an analogous method to Example 191. LCMS showed MH+=394; TRET=2.04 min.
General Procedure
Intermediate 33 (1.89 g) was treated with thionyl chloride (10 ml) and the mixture heated under reflux for 2 h. Excess thionyl chloride was removed in vacuo to afford Intermediate 73, presumed to be the acid chloride of Intermediate 33 as a cream solid. The solid was suspended in tetrahydrofaran (32.5 ml) and an aliquot of the suspension added to a mixture of the amine (0.1 mmol) and N,N-diisopropylethylamine (0.165-0.22 mmol) in TBF (0.5 ml). The reaction mixture was agitated for 24 h and the solvent removed in vacuo. The residue was purified by mass directed autoprep HPLC.
Example 414 was prepared from Intermediate 59 using the general method described for Examples 360-413 method. LCMS showed MH+=398; TRET=2.90 min.
General Procedure
A mixture of Intermediate 61 (0.1 mmol), HATU (0.1 mmol) and DIPEA (0.4 mmol) in DMF (0.4 ml) was shaken at room temperature for 10 min. A solution of the amine (0.1 mmol) in DMF (0.2 ml) was then added and the mixture agitated for several minutes to give a solution. The solution was stored at room temperature for 16 hours then concentrated in vacuo. The residue was dissolved in chloroform (0.5 ml) and applied to a SPE cartridge (aminopropyl, 0.5 g). The cartridge was eluted successively with chloroform (1.5 ml), EtOAc (1.5 ml) and EtOAc:MeOH (9:1, 1.5 ml). Fractions containing the desired product were concentrated in vacuo and the residue purified by mass directed autoprep HPLC.
2M-Sodium hydroxide solution (29 μL, 0.058 mmol) was added to a stirred solution of Example 470 (6 mg, 0.014 mmol) in methanol (28 μL) and water (2 μL). The resulting solution was stirred at 50° C. under nitrogen. After 16 h, the mixture was diluted with water (0.5 ml) and adjusted to pH 4 with acetic acid. The precipitated solid was collected by filtration and dried in vacuo to afford Example 488 as a white solid (4.5 mg). LCMS showed MH+=422; TRET=3.26 min.
2M-Sodium hydroxide solution (83 μL, 0.166 mmol) was added to a stirred solution of Example 468 (18 mg, 0.042 mmol) in methanol (88 μL) and water (5 μL). The resulting solution was stirred at 50° C. under nitrogen. After 16 h, a further quantity of 2M-sodium hydroxide solution (29 μL, 0.058 mmol) was added to the reaction mixture. After 24 h, the reaction mixture was diluted with water (0.5 ml) and adjusted to pH 4 with acetic acid. The mixture was extracted with ethyl acetate (2×0.5 ml), and the combined extracts were dried (Na2SO4) and evaporated in vacuo to give a solid (21 mg). This solid was purified on an SPE cartridge (silica, 1 g) eluting with ethyl acetate:cyclohexane (1:1) followed by methanol. Fractions containing the desired product were combined and concentrated to afford Example 489 as a white solid (4.6 mg). LCMS showed MH+=422; TRET=3.22 min.
A solution of Example 469 (71 mg, 0.17 mmol) in anhydrous THF (2 ml) was treated with hydrogen chloride in dioxane (4M, 0.3 ml). After standing at ambient temperature for 16 hours the resulting solid was collected by filtration and dried under vacuum to give Example 490 as rod like crystals (36 mg). LCMS showed MH+=404; TRET=3.60 min.
A solution of Example 469 (71 mg, 0.17 mmol) in anhydrous THF (2 ml) was treated with anhydrous methane sulphonic acid (11.4 μL, 0.17 mmol). After standing at ambient temperature for 16 hours the resulting solid was collected by filtration and dried under vacuum to give Example 491 as rod like crystals (23 mg). LCMS showed MH+=404; TRET=3.59 min.
General Procedure
A mixture of Intermediate 33 (0.1 mmol), HATU (0.1 mmol) and DIPEA (0.4 mmol) in DMF (0.4 ml) was shaken at room temperature for 10 min. A solution of the amine (0.1 mmol) in DMF (0.2 ml) was then added and the mixture agitated for several minutes to give a solution. The solution was stored at room temperature for 16 hours then concentrated in vacuo. The residue was dissolved in chloroform (0.5 ml) and applied to a SPE cartridge (aminopropyl, 0.5 g). The cartridge was eluted successively with chloroform (1.5 ml), EtOAc (1.5 ml) and EtOAc:MeOH (9:1, 1.5 ml). Fractions containing the desired product were concentrated in vacuo and the residue purified by mass directed autoprep HPLC.
An alternative process for preparing Example 518 is given below:
To a solution of Intermediate 33 (3.5 g, 12.07 mmol) in DMF (500 ml) was added HATU (4.5 g, 12.07 mmol) and the mixture stirred at room temperature for 30 min. 3,4-Dimethylbenzylamine (1.63 g, 12.07 mmol, obtainable from Matrix Scientific, Columbia, USA or by a process described in Chem. Ber., 1969, 102, 2770) was added followed by DIPEA (4.5 ml, 26.55 mmol) and the solution stirred at room temperature for 16 hours. The solvent was removed under reduced pressure and the residue partitioned between saturated aqueous NaHCO3 (200 ml) and ethyl acetate (250 ml), the aqueous phase re-extracted with ethyl acetate (2×200 ml), the organic extracts combined, dried (Na2SO4) and evaporated. The resultant viscous oil was recrystallised from hot ethyl acetate (ca. 100 ml) to give the title compound as a white crystalline solid (3.36 g, 80%). LCMS showed MH+=408; Tret=3.06 min. 8H P6 DMSO) 1.36 (3H, t), 1.51 (2H, m), 2.00 (2H, m), 2.18 (3H, s), 2.19 (3H, s), 2.50 (2H, m), 3.61 (2H, m), 3.83 (2H, m), 4.17 (1H, m), 4.36 (2H, q), 4.38 (2H, d), 7.02-7.09 (3H, m), 8.17 (1H, s), 8.62 (1H, s), 8.93 (1H, t), 9.96 (1H, d): δC (D6 DMSO) 14.65, 18.91, 19.33, 32.81, 41.06, 41.86, 48.57, 64.94, 101.69, 102.18, 124.44, 128.22, 129.24, 133.28, 134.31, 135.78, 136.91, 149.26, 149.59, 151.36, 168.81
A solution of Example 518 (1.3 g, 3.19 mmol) in anhydrous tetrahydrofuran (200 ml) was treated with a solution of hydrogen chloride in dioxane (4M, 8 ml) and the mixture stirred at ambient temperature for 16 hours. The resultant white precipitate was collected by filtration and recrystallised from hot methanol (100 ml) to give the title compound Example 518A as a white crystalline solid (1.12 g, 79%).
LCMS showed MH+=408; Tret=3.21 min. δH (D6 DMSO) 1.39 (3H, t), 1.59 (2H, m), 2.01 (2H, m), 2.19 (3H, s), 2.20 (3H, s), 3.64 (2H, t), 3.83 (2H, m), 4.28 (1H, m), 4.40 (2H, d), 4.50 (2H, q), 7.04-7.11 (3H, m), 9.40 (1H, s (br)), 10.72 (1H, s (br)).
2M-Sodium hydroxide solution (98 μL, 0.196 mmol) was added to a stirred solution of Example 593 (22 mg, 0.049 mmol) in methanol (104 μL) and water (6 μL). The resulting solution was stirred at 50° C. under nitrogen. After 16 h, the reaction mixture was diluted with water (0.5 ml) and adjusted to pH 4 with acetic acid. The mixture was extracted with ethyl acetate (2×0.5 ml), and the combined extracts were dried (Na2SO4) and evaporated in vacuo to give a solid (15 mg). This solid was suspended in water (0.5 ml) and treated with 2M-sodium hydroxide solution (15 μL). Evaporation of solvent in vacuo afforded Example 650 as a white solid (11 mg). LCMS showed MH+=425; TRET=2.69 min.
2M-Sodium hydroxide solution (98 μL, 0.196 mmol) was added to a stirred solution of Example 558 (22 mg, 0.049 mmol) in methanol (104 μL) and water (6 μL). The resulting solution was stirred at 50° C. under nitrogen. After 16 h, the reaction mixture was diluted with water (0.5 ml) and adjusted to pH 4 with acetic acid. The precipitated solid was collected by filtration and dried in vacuo to afford Example 651 as a white solid (15 mg). LCMS showed MH+=425; TRET=2.72 min.
A mixture of Example 205 (200 mg), hydroxylamine hydrochloride (50 mg) and anhydrous potassium carbonate (420 mg) in acetonitrile (10 ml) was stirred and heated at reflux for 17 hours. The solution was cooled and concentrated in vacuo. The residue was partitioned between EtOAc and water. The organic phase was separated, dried over Na2SO4 and concentrated in vacuo to give Example 652 as a white powder (203 mg). LCMS showed MH+=346; TRET=2.84 min.
A mixture of Example 263 (217 mg), hydroxylamine hydrochloride (43 mg) and anhydrous potassium carbonate (355 mg) in acetonitrile (10 ml) was stirred and heated at reflux for 17 hours. The solution was cooled and concentrated in vacuo. The residue was partitioned between EtOAc and water. The organic phase was separated, dried over Na2SO4 and concentrated in vacuo to give Example 653 as a yellow solid (186 mg). LCMS showed MH+=437; TRET=2.82 min. δH (CDCl3) 1.49 (3H, t), 1.80 (2H, m), 2.2-2.4 (4H, m), 2.54 (1H, m), 3.13 (1H, dt), 3.81 (3H, s), 4.13 (1H, m), 4.46 (2H, q), 4.54 (2H, d), 6.28 (1H, t), 6.90+7.28 (4H, AA′BB′), 7.98 (1H, s), 8.36 (1H, s), 9.84 (1H, d). Hydroxyl proton not visible.
The following examples were prepared by a similar procedure, e.g. using the same or a similar number of moles of reagents and the same or similar volumes of solvents:
See later for alternative preparation of Example 681.
A mixture of Example 263 (25 mg), ethoxyamine hydrochloride (R26ONH2 where R26=Et, 20 mg) and diisopropylethylamine (30 mg) in acetonitrile (3 ml) was stirred and heated at reflux for 3.25 hours. The solution was cooled and concentrated in vacuo. The residue was applied to an SPE cartridge (5 g). The cartridge was eluted with EtOAc. Fractions containing the desired product were concentrated in vacuo to give Example 655 as a colourless gum (20 mg). LCMS showed MH+=465; TRET=3.28 min.
The following examples were prepared by a similar procedure, e.g. using the same or a similar number of moles of reagents and the same or similar volumes of solvents:
A suspension of cyanuric chloride (2,4,6-trichloro-1,3,5-triazine) (I 50 mg) in DMF (0.2 ml) was stirred for 30 minutes at room temperature. The suspension was diluted to 7 ml with DMF, with stirring. A 11.0 ml portion of the resultant suspension was removed and added to Example 653 (52 mg). The resultant solution was stirred for 90 hours at room temperature, then concentrated in vacuo. The residue was partitioned between EtOAc and water. The organic phase was separated and washed consecutively with saturated sodium carbonate, 10% w/v citric acid and saturated brine, dried over Na2SO4 and concentrated in vacuo. The residue was applied to an SPE cartridge (2 g). The cartridge was eluted successively with EtOAc:cyclohexane (1:1), EtOAc and then a (100:8:1) mixture of dichloromethane, ethanol and ammonia. Fractions containing the desired product (eluted in the ammoniacal solution) were concentrated in vacuo to give Example 658 as a colourless oil (11 mg). LCMS showed MH+=437; TRET=2.50 min.
Example 659 was prepared from Example 652, using an identical procedure to that used for Example 658. LCMS showed MH+=346; TRET=2.56 min.
A solution of Example 258 (25 mg), butyraldehyde (5 mg) and glacial acetic acid (30 mg) in DCM (3 ml) was stirred for 10 min. Sodium triacetoxyborohydride (21 mg) was added. The reaction mixture was stirred for 1.5 hours. Sodium bicarbonate (1.0 Molar, 3 ml) was added dropwise, with stirring. After stirring for 5 min. the phases were separated. The organic phase was dried over Na2SO4 and applied to an SPE cartridge (5 g). The cartridge was eluted with a (100:8:1) mixture of dichloromethane, ethanol and ammonia. Fractions containing the desired product were concentrated in vacuo to give Example 660 as an amorphous, cream solid (19 mg). LCMS showed MH+=346; TRET=2.56 min.
General Procedure:
Intermediate 17 (0.16 mmol) in acetonitrile (1 ml) was treated with the R3NH2 amine (0.8 mmol) in acetonitrile (1 ml) and N,N-diisopropylethylamine (0.8 mmol). The mixture was heated at 50° C. for 18 h then concentrated in vacuo. The residue was diluted with water (3 ml) and extracted with dichloromethane (2×5 ml). The combined organic extracts were evaporated, and the residue was purified by mass directed autoprep HPLC to give the desired product containing formic acid. This material was dissolved in chloroform-methanol (10/1, 5.5 ml) and washed with 5% sodium hydrogen carbonate solution (1 ml) to give after evaporation of solvents the pure product.
** For NHR3 in Examples 214 and 661-663, NHR3 is the cys or trans isomer as shown. For Examples 662-664, NHR3 is the 3-amino- or 2-amino- cycohex-1-ylamino group in a racemic form.
3-Aminocyclohexanol (0.677 g, 5.9 mmol, as described in J. Chem. Soc., Perkin Trans 1, 1994, 537) in acetonitrile (10 ml) and ethanol (1 ml) was added at room temperature to a stirred solution of Intermediate 1 (1.24 g, 4.9 mmol) and diisopropylethylamine (4.26 ml, 24.5 mmol) in acetonitrile (25 ml). The resulting mixture was stirred at 85° C. for 17 h. The mixture was concentrated in vacuo, and the residue was partitioned between DCM (50 ml) and water (10 ml). The phases were separated and the organic phase was dried (Na2SO4) and evaporated to give an orange-brown oil. The oil was purified by Biotage chromatography (silica 100 g) eluting with 30-50% EtOAc in cyclohexane to give Example 665 as a white foam (0.681 g). LCMS showed MH+=333; TRET=2.76 min.
General Procedure:
A mixture of Intermediate 76 (0.1 mmol), HATU (0.1 mmol) and DIPEA (0.4 mmol) in DMF (0.5 ml) was shaken at room temperature for 10 min. A solution of the amine HNR4R5 (0.12 mmol) in DMF (0.5 ml) was then added and the mixture agitated for several minutes to give a solution. The solution was stored at room temperature for 16 h, then concentrated in vacuo. The residue was purified by mass directed autoprep HPLC.
Alternative procedure for preparing Example 260:
A solution of Intermediate 58 (45 mg), HATU (63 mg) and DIPEA (39 mg) in acetonitrile (5 ml) was stirred for 10 min. A solution of 2,4-dimethylbenzylamine (24 mg) (available from Salor; or ICN Biomedicals, Inc.; or Synthesis, 1982, 12, 1036) in acetonitrile (1 ml) was added. The reaction mixture was stirred for 18 h. The solution was concentrated and the residue partitioned between ethyl acetate (25 ml) and 0.5M sodium bicarbonate (20 ml). The organic phase was separated, washed with water (20 ml), dried over Na2SO4 and concentrated to leave a gum which was applied to an SPE cartridge (5 g). The cartridge was eluted with ethyl acetate. Fractions containing the desired compound were combined and concentrated in vacuo to give Example 260 (32 mg). LC-MS showed MH+=420; TRET=3.16 min. δH (CDCl3): 1.49 (3H, t), 2.11 (2H, m), 2.33 (3H, s), 2.35 (3H, s), 2.40 (2H, m), 2.52 (2H, m), 2.61 (2H, m), 4.36 (1H, m), 4.47 (2H, q), 4.55 (2H, d), 6.14 (1H, t), 7.01+7.18 (2H, AA′BB′), 7.04 (1H, s), 8.01 (1H, s), 8.36 (1H, s), 9.96 (1H, d).
The following Examples 677-679 were prepared in a similar manner to Example 260 (alternative procedure above), for example using the same or a similar number of moles of reagents and the same or similar volumes of solvents:
Examples 680-686 and their preparation are shown above together with Example 653.
Alternative Preparation of Example 681: N-[(3,4-dimethylphenyl)methyl]-1-ethyl-4-{[4-(hydroxyimino)cyclohexyl]amino}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
A mixture of Example 261 (35 mg), hydroxylamine hydrochloride (10 mg) and diisopropylethylamine (26 mg) in acetonitrile (4 ml) was stirred and heated at reflux for 2.5 hours. The solution was cooled and concentrated in vacuo. The residue was partitioned between EtOAc and water. The organic phase was separated, dried over Na2SO4 and concentrated in vacuo. The residue was applied to an SPE cartridge (10 g). The cartridge was eluted with EtOAc:cyclohexane (1:1) and then EtOAc. Fractions containing the desired compound were combined and concentrated in vacuo to give Example 681 as a white, amorphous solid (18 mg). LCMS showed MH+=435; TRET=3.08 min. δH (CDCl3) 1.49 (3H, t), 1.79 (2H, m), 2.24 (6H, s), 2.19-2.38 (4H, m), 2.56 (2H, dt), 4.13 (1H, m), 4.46 (2H, q), 4.53 (2H, d), 6.36 (1H, t), 7.09 (2H, t), 7.12 (1H, s), 7.98 (1H, s), 8.38 (1H, s), 9.79 (1H, d). Hydroxyl proton not visible.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0221455.9 | Sep 2002 | GB | national |
| 0230045.7 | Dec 2002 | GB | national |
| 0306595.0 | Mar 2003 | GB | national |
| 0308017.3 | Apr 2003 | GB | national |
| 0319708.4 | Aug 2003 | GB | national |
| 0321074.7 | Sep 2003 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP03/11814 | 9/12/2003 | WO | 9/27/2005 |
