Enzyme inhibitors

Abstract

Diketoacids of Formula A are useful as inhibitors of viral polymerases. In particular hepatitis C virus RNA dependent RNA polymerase (HCV RdRp), hepatitis B virus polymerase (HBV pol) and reverse transcriptase of human immunodeficiency virus (HIV RT): 1

Description

TECHNICAL FIELD

[0001] The present invention relates to compounds useful as enzyme inhibitors, in particular as inhibitors of enzymes involved in the transfer of phosphoryl groups and, especially as inhibitors of polymerases. The invention further relates to pharmaceutical compositions containing such compounds, and to their use in the treatment of viral infections.

[0002] Polymerases are the enzymes which catalyse the formation of phosphodiester bonds in RNA and DNA. They play an essential role in viral replication and, therefore, are an important target in the fight against viral diseases such as human immunodeficiency virus (HIV), hepatitis, and poliomyelitis.

BACKGROUND ART

[0003] U.S. Pat. No. 5,475,109 describes dioxobutanoic acids substituted with piperidine or similar N-substituted saturated cycloalkyls as inhibitors of the cap-dependent endonuclease of influenza virus.

DISCLOSURE OF THE INVENTION

[0004] The present inventors have discovered that a range of diketoacids have utility as enzyme inhibitors and, in particular, as polymerase inhibitors and more particularly as inhibitors of hepatitis C NS5 RNA-dependent RNA polymerase, HBV DNA-dependent RNA polymerase and HIV DNA- dependent DNA polymerase. Their investigations indicate that these compounds may act by interfering with the binding of phosporyl groups at the active site of the enzyme and may, therefore, have broad application in inhibiting enzymes involved in the transfer of phosphoryl groups.

[0005] According to a first aspect of the present invention there is provided a compound of formula A shown below. This compound is suitable for therapeutic use, for instance as an enzyme inhibitor. 2

[0006] Optionally, the compound may be in the form of a pharmaceutically acceptable salt or ester, which can be hydrolysed in vivo to the corresponding diketoacid.

[0007] In formula A, the group R is an organic moiety which contains from 2 to 24, preferably 4 to 20, most preferably 6 to 17 carbon atoms in total. R includes an optionally substituted cyclic or heterocyclic group in which the atom directly bonded to the adjacent carbonyl in the diketoacid is part of the ring structure. Preferably, this atom is a carbon atom.

[0008] The ring which is thus bonded to the carbonyl group is preferably a 3 to 8 membered ring, particularly a 4 to 6 membered ring.

[0009] Thus, for example, R may be selected from:

[0010] (i) optionally substituted aromatic groups, especially those including six membered rings, such as phenyl and naphthyl;

[0011] (ii) optionally substituted heteroaryl groups especially those including five and six membered rings such as thiophene, pyrrole, furan, imidazole, pyridyl, pyrimidyl, and pyridazyl; the heteroaryl ring may, optionally be fused to another ring;

[0012] (iii) optionally substituted cycloalkyl groups, especially those including five or six membered rings such as cyclopentyl, cyclohexyl and adamantyl;

[0013] (iv) optionally substituted cycloalkenyl groups, especially those including five or six numbered rings such as cyclohexenyl, cyclopentenyl;

[0014] (v) optionally substituted cyclic heteroalkyl groups, especially those including five or six numbered rings such as piperidyl, pyrrolidyl, tetrahydrofuranyl, and tetrahydropyranyl; in this class 4- piperidyl rings substituted with an aryl group at carbon 4 and on acyl or sulfonyl substituent at N1 are preferred.

[0015] In the case of optional substitution, one or more substituents may be present and a wide variety of substituents are possible. Preferred optional substituents for all compounds of the present invention are set out in the following list:

[0016] (a) —OH;

[0017] (b) —SH;

[0018] (c) - halogen, such as fluorine, chlorine or bromine,

[0019] (d) —CO2H;

[0020] (e) —CN;

[0021] (f) —NO2;

[0022] (g) —NR1R2 wherein each of R1 and R2 is selected from H and lower alkyl groups having 1 to 6 carbon atoms; or R1 and R2 together form a ring including 4 to 6 carbon atoms;

[0023] (h) —SO2NR1R2 where R1 and R2 are as defined above;

[0024] (i) —CONH2, —NHCO2H, or —NHCOCOOH;

[0025] (j) an alkyl (or alkenyl or alkynyl group) group having 1 to 12 (2 to 12) carbon atoms, preferably 1 to 7 (2 to 7) carbon atoms optionally substituted by any one or more of the groups (a)-(i) above and/or optionally interrupted by a group selected from —O—, —S—, —NR3—, 3

[0026]  —CO2—, —OCO—, —CONR3—, —NR3CONR3—, —SO2—, —NR3SO2—, and —SO2NR3—; where each R3 independently is H or lower alkyl of 1 to 6 carbon atoms;

[0027] (k) an aryl or heteroaryl group having 2 to 10 carbon atoms optionally substituted with any one or more of groups (a) to (j) above;

[0028] (l) an aralkyl or heteroaralkyl group having 3 to 16 carbon atoms optionally substituted with any one or more of groups (a)-(j) above and/or in which the alkyl part of the group is optionally interrupted by a group selected from 4

[0029]  —O—, —S—, —NR3—, —CO2—, —OCO—, —CONR3—, —NR3CONR3—, —SO2—, —NR3SO2—, and —SO2NR3—; where R3 is as defined above;

[0030] (m) 5

[0031]  where R4 is an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or heteroaralkyl group as such groups are defined above at (j), (k) and (l);

[0032] (n) 6

[0033]  where R4 is as defined above;

[0034] (o) —OR4 where R4 is as defined above;

[0035] (p) 7

[0036]  where R4 is as defined above;

[0037] (q) —SO2R4 where R4 is as defined above;

[0038] (r) —NHR4 or —N(R4)2 where R4 is as defined above;

[0039] (s) —NHSO2R4 or —SO2NHR4, where R4 is as defined above;

[0040] (t) —SR4 and each of optional substituents (j) to (t) above may optionally itself be substituted by one or more groups selected from (j) to (t).

[0041] A preferred class of compounds of formula A is represented by formula E: 8

[0042] in which Ar is an optionally substituted aryl or heteroaryl group. Optional substituents may be selected from the list of preferred substituents set out above. Within this class of preferred compounds two especially preferred groups are set out below (formulas F and G) 9

[0043] R5, R6, R7 and R8 are, independently H or are selected from the optional substituents listed above and R7 and R8 taken together may form a 4 to 7, preferably 5 or 6 membered ring; and X is O, S, NH, or NR4 where R4 is as defined above.

[0044] In compounds of formula F, (which are optionally substituted phenyl diketoacids) ortho, meta and para substitution are possible.

[0045] In general, it is preferred that there is a single substituent, preferably at the position which is ortho- or meta- to the diketoacid group. Substitution at the meta-position is especially preferred. Where two substituents are present, then preferably the phenyldiketoacid is 2,5-substituted; 3,5-substitution is also possible, as is 2,4-substitution provided, in the latter case, that the substituent at the 4-position is relatively small (e.g. methyl). Disubstitution at the 2,3- and 2,6-positions is, in general, not preferred.

[0046] Preferred substituents, especially at the ortho and meta positions, are ether groups of formula (o) above (i.e. —OR4), hydroxyl, and —NHSO2R4. It is generally preferred that no more than one substituent be —OR 4 and/or —NHSO2R4.

[0047] Preferred examples of —OR4 groups which may be found at the ortho and meta positions and particularly at the meta position include:

[0048] —OCH2Ar or, less preferably —O(CH2)2Ar where Ar is an optionally substituted aryl or heteroaryl group and is particularly preferably an optionally substituted phenyl group. Examples of preferred substituents on the aryl group, and especially on the phenyl ring include halogens, especially fluorine and chlorine, and electron-withdrawing groups such as —CN, —CO2H, and —CF3 as well as ether and aryl groups;

[0049] —O—(CH2)3—CN; and

[0050] —O—(CH2)3—C≡CH.

[0051] Preferred sulfonamide groups which may be found at the ortho- and meta- positions, particularly at the meta- position are those of formula:

[0052] —NH—SO2—Ar, where Ar is an optionally substituted aryl or heteroaryl group, preferably an optionally substituted phenyl group. Preferred optional substituents for the aryl, preferably phenyl group, include: —CN; halogens, especially chlorine and fluorine, —CF3, lower (C1-6) alkyl (especially methyl), hydroxy-, ether, and —NO2 groups.

[0053] For both the —OCH2Ar and —NHSO2Ar substituted compounds, another preferred example of Ar is naphthyl.

[0054] Other preferred substituents at the ortho and meta positions are lower (eg C1-6) alkyl groups, especially C1-4 alkyl, such as methyl and ethyl, but in particular methyl, aralkyl groups, especially phenylmethyl groups, optionally substituted in the phenyl ring, especially by a halogen, and nitrogen-containing substituents such as primary, secondary or tertiary amine groups, optionally in protonated form, amide, urethane, or urea groups in each of which examples there is a nitrogen atom bonded to the phenyl ring.

[0055] One particularly preferred sub class of compounds of formula F is those in which each of R5 and R6 is selected from H, HO—, R4O—, and —NHSO2R4 provided that no more than one of R5 and R6 is R4O— or —NHSO2R4.

[0056] In compounds of formula G the diketoacid group may be at the 2- or 3- position of the ring. In many cases substitution at the 2-position is preferred.

[0057] Preferred examples of compounds of formula G are those in which the five membered aromatic ring, 10

[0058] is a pyrrole or thiophene ring. In the case of the pyrrole-substituted diketoacids, the groups R7 and R8 may both be hydrogen and in many cases that is preferred. If R7 and R8 correspond to substituent groups, then these may be at any of the positions not already occupied by the diketoacid group. Examples of possible substituents include alkyl (especially methyl), halogen, and aralkyl (especially benzyl) groups.

[0059] One embodiment of pyrrole substituted diketoacid is that in which the diketoacid group is at the 2- position of the ring and where the only other substituent in the ring is on the nitrogen atom. In this case, preferred examples of the substituent R4 present on the nitrogen atom, include alkyl, aryl or aralkyl groups, particularly aralkyl (such as benzyl) groups. Where an aryl or aralkyl group is present these are preferably substituted with halogen atoms, such as fluorine or chlorine, or by cyano-groups.

[0060] In the case of the thiophene-substituted diketoacids a wide range of substituents R7 and R8 may be employed in various positions as will be evident from the tables infra. Preferred thiophenes have an aralkyl (such as optionally substituted benzyl) or aryl (such as optionally substituted phenyl) substituent, e.g. at the 5-position of the thiophene ring.

[0061] Compounds containing furanyl rings may also be useful, especially for inhibiting HIV reverse transcriptase.

[0062] Preferred substituents are optionally substituted aryl groups (especially optionally substituted phenyl). Substitution is preferably at the 5-position of the ring.

[0063] The formulae of numerous preferred specific compounds of the present invention are presented later below.

[0064] The compounds of the present invention having formula A may be prepared by a process which comprises reaction of a compound of formula B with a dialkyloxalate of formula C followed by hydrolysis of the resulting diketo-ester of formula D: 11

[0065] where R′ is an alkyl group, typically having 1-6 carbon atoms. In the case where the target molecule is a pharmaceutically acceptable ester of the compound of formula A then R′ in formula C may be selected accordingly, and the step of hydrolysing the compound of formula D omitted, since in vivo hydrolysis can render the compounds active.

[0066] Preferred enzymes for inhibition by the compounds of the invention are those involved in phosphate transfer, in particular polymerases such as DNA polymerases, and RNA polymerases both of which may be either RNA dependent or DNA dependant. Compounds of the invention may particularly preferably be employed in the inhibition of viral enzymes. Examples of viral enzymes include RNA-dependent RNA polymerase and reverse transcriptases.

[0067] The compounds of the invention may be used as inhibitors of plant or animal (including human) viruses.

[0068] The viruses may be RNA viruses, which may, for example, be positive single stranded viruses of which polio virus, hepatitis C virus and encephalomyocarditis are examples, negative single stranded viruses such as orthomyxoviruses, and paramyxoviruses, and retroviruses of which HIV is a prominent example. Alternatively, the viruses may be DNA viruses, especially double stranded DNA viruses such as hepatitis B virus. In particular, compounds of the present invention may inhibit one or more of the following enzymes: hepatitis C virus RNA dependent RNA polymerase (HCV RdRp), hepatitis B virus polymerase (HBV pol) and reverse transcriptase of human immunodeficiency virus (HIV RT).

[0069] Especially preferred compounds of the invention will be suitable for use as HCV RdRp inhibitors.

[0070] Other classes of enzyme involved in phosphate transfer which may be susceptible to inhibition by compounds of the present invention include phosphatases, Rnases, integrases and ribozymes.

[0071] According to a further aspect of the invention there is provided the non-therapeutic use of compound of formula A or suitable salt or ester as an enzyme inhibitor, especially as an inhibitor of polymerases, especially viral polymerases. For instance, compounds of the invention may be of utility in agriculture and horticulture for treating plants infected with or susceptible to plant virus.

[0072] According to a further aspect of the invention there is provided the use of a compound of formula A or of a pharmaceutically acceptable salt or ester thereof in the manufacture of a medicament for treatment of a viral illness in a human or animal. For instance, the medicament may be used to treat viral illness by inhibiting one or more viral polymerase. Preferably the medicament is for treatment of hepatitis, such as hepatitis B or C, particularly hepatitis C, and human immunodeficiency virus.

[0073] A still further aspect of the invention provides a pharmaceutical composition comprising a compound of formula A, or a pharmaceutically acceptable salt or ester thereof and a pharmaceutically acceptable excipient, diluent or carrier. The composition may be in any suitable form, depending on the intended method of administration. It may for example be in the form of a tablet, capsule or liquid for oral administration, or of a solution or suspension for administration parenterally.

[0074] The pharmaceutical compositions optionally also include one or more other agents for the treatment of viral infections such as an antiviral agent, or an immunomodulatory agent such as α-, β-, or γ- interferon.

[0075] A still further aspect of the invention provides a method of inhibiting an enzyme, especially a viral polymerase and/or of treating or preventing a viral illness, the method involving administering to a human or animal (preferably mammalian) subject suffering from the condition a therapeutically or prophylactically effective amount of the pharmaceutical composition described above or of a compound of formula A or salt or ester thereof. “Effective amount” means an amount sufficient to cause a benefit to the subject or at least to cause a change in the subject's condition.

[0076] The dosage rate at which the compound, salt or ester is administered will depend on the nature of the subject, the nature and severity of the condition, the administration method used, etc. Appropriate values are selectable by routine testing. The compound, salt or ester may be administered alone or in combination with other treatments, either simultaneously or sequentially. For instance, it may be administered in combination with effective amounts of antiviral agents, immunomodulators, anti-infectives, or vaccines known to those of ordinary skill in the art. It may be administered by any suitable route, including orally, intravenously, cutaneously, subcutaneously, etc. It may be administered directly to a suitable site or in a manner in which it targets a particular site, such as a certain type of cell. Suitable targeting methods are already known.

[0077] A further aspect of the invention provides a method of preparation of a pharmaceutical composition, involving admixing one or more compound of formula A or salt or ester thereof with one or more pharmaceutically acceptable adjuvants, diluents or carriers and/or with one or more other therapeutically or prophylactically active agents.

MODES FOR CARRYING OUT THE INVENTION

[0078] Embodiments of the invention are described below by the way of example only.

EXAMPLES

(1) Synthesis

[0079] The synthesis of the 2,4-dioxobutanoic acids consists of a Claisen condensation reaction between a methyl ketone substrate and diethyl oxalate in the presence of sodium ethoxide in tetrahydrofuran (Scheme 1A) and the subsequent hydrolysis of the ethyl ester with sodium hydroxide in methanol (Scheme 1B) 12

[0080] Reagents: (i) diethyl oxalate/NaOEt in THF 13

[0081] Reagents: (i) 5 eg. NaOH/MeOH

Exemplary Procedure for the Synthesis of the 2, 4-Dioxobutanoate Ethyl Esters

[0082] (Scheme 1A)

[0083] In a 50 ml round bottom flask with a stirring bar and under an inert atmosphere, the methyl ketone compound (1.0 mmole) in 10 ml of dry tetrahydrofuran (THF) is reacted with 2 equivalents of diethyl oxalate and 2 equivalents of sodium ethoxide (NaOEt) at ambient temperature for 3 hours. When reaction is completed, the reaction mixture is poured into a IN aqueous hydrochloric acid (HCl) and extracted with ethyl acetate (EtOAc). The organic phase is separated, washed first with water and then with brine. The organic layer is dried over sodium sulfate (Na2SO4), filtered and solvent is removed in vacuo leaving the desired dioxobutanoate ethyl ester in quantitative yield.

Exemplary Procedure for Hydrolysis of the Ethyl Ester

[0084] (Scheme 1B)

[0085] In a 50 ml round bottom flask with a stirring bar, the 2,4-dioxobutanoate ethyl ester compound (1.0 mmole) in 10 ml of methanol (MeOH) is reacted with 5 equivalents of sodium hydroxide (NaOH) at ambient temperature for 2 hours.

[0086] The methanol is removed in vacuo. The aqueous residue is washed with diethyl ether (Et2O). The aqueous fraction is acidified by addition of 1N aqueous hydrochloric acid solution (HCl) and the milky mixture is extracted with two portions of ethyl acetate (EtOAc). The combined organic fractions are washed with brine. The organic layer is dried over sodium sulfate (Na2SO4), filtered and solvent is removed in vacuo leaving the desired dioxobutanoic acid product.

[0087] Using this or analogous methods, compounds were produced as set out in the following Tables, which are categorised according to their “R” group.

[0088] The Tables include IC50 data and the methods for assay are explained after the Tables.

[0089] Notes to Table: NA=not active as an inhibitor at concentrations up to that stated.

[0090] ND=not done.

[0091] In the tables, where nitrogen atoms appear to be divalent, the presence of a hydrogen atom is implied.

TABLE I
HCV-polymerase inhibitors: examples of 2,5-substituted
phenyldiketoacids
14
Ex. No.	R1	R2	IC 50 (μM)
1	15	16	5.6
2	17	18	3
3	19	20	27.9
4	21	22	8
5	23	24	17
6	25	26	18
7	27	28	2.92
8	29	30	44
9	31	32	51
10	33	34	20
11	35	36	7.08
12	37	38	16.7
13	39	40	2.6
14	41	42	26
15	43	44	83.5
16	45	46	4.3
17	47	48	11.6
18	49	50	2.2
19	51	52	11.9
20	53	54	0.38
21	55	56	0.955
22	57	58	19
23	59	60	0.94
24	61	62	19
25	63	64	28
26	65	66	26
27	67	68	2.84
28	69	70	6.2
29	71	72	3.9
30	73	74	15
31	75	76	18
32	77	78	6.1
33	79	80	18.2
34	81	82	9.6
35	83	84	6.1
36	85	86	1.6
37	87	88	18
38	89	90	16
39	91	92	22
40	93	94	8.3
41	95	96	28.9
42	97	98	16.6
43	99	100	20
44	101	102	18.5
45	103	104	12.9
46	105	106	30.1
47	107	108	20.7
48	109	110	22
49	111	112	32
50	113	114	7.8
51	115	116	1.9
52	117	118	10
53	119	120	0.115
54	121	122	2.3
55	123	124	10.8
56	125	126	23.6
57	127	128	2.1
58	129	130	13.6
59	131	132	25.3
60	133	134	40
61	135	136	31
62	137	138	10
63	139	140	1.7
64	141	142	0.23
65	143	144	45
66	145	146	11
67	147	148	16
68	149	150	30
69	151	152	14
70	153	154	9.2
71	155	156	10.6
72	157	158	0.48
73	159	160	5.6
74	161	162	3.6
75	163	164	19.2
76	165	166	50
77	167	168	4.8
78	169	170	0.67
79	171	172	6
80	173	174	3
81	175	176	1.4
82	177	178	19
83	179	180	9.4
84	181	182	0.95
85	183	184	13
86	185	186	2.05
87	187	188	2.3
88	189	190	0.7
89	191	192	3.3
90	193	194	1.8
91	195	196	6.2
92	197	198	1
93	199	200	1.9
94	201	202	5.8
95	203	204	0.48
96	205	206	50
97	207	208	2.8
98	209	210	1
99	211	212	0.6
100	213	214	7.8
101	215	216	7
102	217	218	1.5
103	219	220	6
104	221	222	50
105	223	224	13.7
106	225	226	6.8
107	227	228	0.14
108	229	230	6.9
109	231	232	0.17
110	233	234	30
111	235	236	0.12
112	237	238	1.33
113	239	240	0.1
114	241	242	0.5
115	243	244	3.7
116	245	246	0.3
117	247	248	0.14
118	249	250	0.2
119	251	252	0.049
120	253	254	0.36
121	255	256	4
122	257	258	2
123	259	260	0.29
124	261	262	28
125	263	264	0.17
126	265	266	0.056
127	267	268	0.3
128	269	270	24
129	271	272	1.6
130	273	274	0.14
131	275	276	0.78
132	277	278	0.67
133	279	280	3.2
134	281	282	23
135	283	284	21
136	285	286	0.2
137	287	288	0.9
138	289	290	1.1
139	291	292	1.4
140	293	294	1
141	295	296	0.56
142	297	298	0.4
143	299	300	0.45
144	301	302	14
145	303	304	1.2
146	305	306	15
147	307	308	1.3
148	309	310	0.26
149	311	312	0.55
150	313	314	2.3
151	315	316	0.5
152	317	318	20
153	319	320	19
154	321	322	30

[0092]

TABLE II
HCV-polymerase inhibitors: examples of 3,5-substituted phenyldiketoacids
323
Ex. No.	R1	R2	IC 50 (μM)
155	324	325	1.4
156	326	327	1.3
157	328	329	0.9
158	330	331	0.2
159	332	333	20
160	334	335	0.1

[0093]

TABLE III
HCV-polymerase inhibitors: examples of
2,4-substituted phenyldiketoacids
336
	Ex. No.	R1	R2	IC 50 (μM)
	161	337	338	2.8
	162	339	340	5.5
	163	341	342	26
	164	343	344	47
	165	345	346	2
	166	347	348	20
	167	349	350	0.6

[0094]

TABLE IV
HCV-polymerase inhibitors: examples of
2,3-substituted phenyldiketoacids
351
Ex. No.	R1	R2	IC 50 (μM)
168	352	353	18
169	354	355	>50
170	356	357	>50

[0095]

TABLE V
HCV-polymerase inhibitors: examples of
2,6-substituted phenyldiketoacids
358
Ex. No.	R1	R2	IC 50 (μM)
171	359	360	12
172	361	362	>50

[0096]

TABLE VIa
HCV-polymerase inhibitors: examples of
pyrrole-2-substituted diketoacids
363
Ex. No.	R1	IC 50 (μM)
173	364	21
174	365	13.4
175	366	25
176	367	29
177	368	25
178	369	17.9
179	370	12.8
180	371	93
181	372	30
182	373	30
183	374	32
184	375	6.7
185	376	6.3
186	377	24
187	378	36
188	379	12.7
189	380	28
190	381	18
191	382	10
192	383	8.2
193	384	12
194	385	16
195	386	11.1
196	387	15
197	388	11
198	389	7.9

[0097]

TABLE VIb
HCV-polymerase inhibitors: examples of
thiophene-2-substituted diketoacids
390
Ex. No.	R1	IC 50 (μM)
191	391	10
192	392	8.2
193	393	12
194	394	16
195	395	11.1
196	396	15
197	397	11
198	398	7.9
199	399	17
200	400	8.2
201	401	20
202	402	68
203	403	19.8
204	404	11
205	405	74
206	406	65
207	407	9.9
208	408	11.6
209	409	12.6
210	410	27
211	411	82
212	412	7.5
213	413	5.9
214	414	17
215	415	15.3

[0098]

TABLE VIc
HCV-polymerase inhibitors: examples of
furan-2-substituted diketoacids
Ex.	R1	IC 50 (μM)
216	416	50
217	417	58
218	418	41.2

[0099]

TABLE VIIa
HCV-polymerase inhibitors: examples of
pyrrole-3-substituted diketoacids
419
Ex. No.	R1	IC 50 (μM)
219	420	23.7
220	421	4.6
221	422	20.6

[0100]

TABLE VIIb
HCV-polymerase inhibitors: examples of
thiophene-3-substituted diketoacids
423
Ex. No.	R1	IC 50 (μM)
222	424	4
223	425	27
224	426	50
225	427	167
226	428	17
227	429	15
228	430	17.8
229	431	80
230	432	8.6
231	433	9.4
232	434	11.8
233	435	9.2
234	436	14.5
235	437	7.5
236	438	26

[0101]

TABLE VIIc
HCV-polymerase inhibitors: examples of
furan-3-substituted diketoacids
439
Ex. No.	R1	IC 50 (μM)
237	440	14
238	441	47.5

[0102]

TABLE VIII
HCV-polymerase inhibitors: examples of alkyl-diketoacids
442
Ex. No.	R1	IC 50 (μM)
239	443	9.4
240	444	18
241	445	37
242	446	12.8
243	447	6.7
244	448	77
245	449	81.4
246	450	18
247	451	45
248	452	10
249	453	60
250	454	17
251	455	21
252	456	61
253	457	55
254	458	14
255	459	16.7
256	460	25
257	461	50

[0103]

TABLE IXa
most active HCV-inhibitors
462
Ex.
No.	R1	HCV	HIV	HBV
126	463	0.056	100	ND
160	464	0.1	NA	ND
113	465	0.1	90	ND
53	466	0.115	37	ND
111	467	0.12	80	ND
107	468	0.14	58	ND
117	469	0.14	100	ND
109	470	0.17	NA	ND
158	471	0.2	NA	ND
64	472	0.23	NA	ND
116	473	0.3	NA	ND
120	474	0.36	80	ND
20	475	0.38	27	ND
72	476	0.48	NA	ND
99	477	0.6	50	ND
78	478	0.67	35	ND
88	479	0.7	NA	ND
84	480	0.95	NA	ND
21	481	1	>50	ND
23	482	1	59	ND
112	483	1.33	90	ND
155	484	1.4	130	416
36	485	1.6	24	ND
90	486	1.8	NA	ND
165	487	2	NA	ND
18	488	2.2	30	ND
161	489	2.8	320	108
80	490	3	NA	ND
27	491	3	>50	ND
7	492	3.3	61	6
16	493	4.3	>100	ND
162	494	5.5	NA	ND
1	495	5.6	90	NA
103	496	6	NA	ND
243	497	6.7	26.8	ND
198	498	7.9	NA	ND
4	499	8	>100	ND
192	500	8.2	NA	ND
66	501	11	NA	ND
19	502	12	77	ND
179	503	12.8	NA	NA
190	504	18	NA	NA
24	505	19	71	ND
49	506	32	NA	ND

[0104]

TABLE IXb
most active HBV-Pol-inhibitors
507
Ex. No.	R1	HCV	HIV	HBV
206	508	65	NA	2
205	509	74	NA	3.3
225	510	167	86	4
202	511	70	>100	9
196	512	15	50	9

[0105]

TABLE IXc
most active HIV-RT-inhibitors
513
Ex. No.	R1	HCV	HIV	HBV
258	514	>100	3.6	NA
218	515	41.2	11.8	40
259	516	>100	16	NA
40	517	8.3	12	NA
20	518	0.38	27	ND
8	519	44	19	ND

2. Measurement of Inhibitory Activity

[0106] The effectiveness of the compounds set out above as polymerase inhibitors, stated above as IC50 values, was assessed in screening assays as follows.

[0107] In initial tests, the compounds were tested to see if they were effective as inhibitors of the RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV). The HCV NS5B protein is the viral RdRp; compounds capable of interfering with the activity of this enzyme are thus expected to block viral replication.

Test for Inhibition of Hepatatis C Virus RdRp

[0108] WO 96/37619 describes the production of recombinant HCV RdRp from insect cells infected with recombinant baculovirus encoding the enzyme. The purified enzyme was shown to possess in vitro RNA polymerase activity using RNA as template. The reference describes a polymerisation assay using poly (A) as a template and oligo(U) as a primer. Incorporation of tritiated UTP is quantified by measuring acid-insoluble radioactivity. The present inventors have employed this assay to screen the various compounds described above as inhibitors of HCV RdRp and other virally encoded polymerases.

[0109] Incorporation of radioactive UMP was measured as follows. The standard reaction (100 μl) was carried out in a buffer containing 20 mM tris/HCl pH 7.5, 5 mM MgCl2, 1 mM DTT,50 mM NaCl, 1 mM EDTA, 2 OU Rnasin (Promega), 0.05% Triton X-100, 1 μCi[3H] UTP (40 Ci/mmol,NEN), 10 μM UTP and 10 μg/ml poly (A). Oligo (U)12 (1 μg/ml, Genset) was added as a primer. The final NSSB enzyme concentration was 20 nM. After 1 hour incubation at 22° C. the reaction was stopped by adding 100 μl of 20% TCA and applying samples to DE81 filters. The filters were washed thoroughly with 5% TCA containing 1M Na2HPO4/NaH2PO4, pH 7.0, rinsed with water and then ethanol, air dried, and the filter-bound radioactivity was measured in the scintillation counter. By carrying out the reaction in the presence of various concentrations of each of the compounds set out above it was possible to determine IC50 values for each compound with the formula:

% residual activity=100/(1+[I]/IC50)s

[0110] where [I] is the inhibitor concentration and “s” is the slope of the inhibition curve.

Test for Inhibition of Hepatitis B Virus Polymerase

[0111] Analogous assays employed the polymerase of hepatitis B virus (HBV pol), obtained in the form of viral particles from the sera of HBV positive patients. These particles contain a polymerase bound to an incomplete double stranded DNA template. In the assay the incorporation of 32P-dNTP is measured as radioactivity incorporated in acid insoluble precipitate.

[0112] The standard reaction (100 μl) was carried out in a buffer containing 50 mM tris/HCl pH 7.5, 30 mM MgCl 2 , 1 mM DTT, 100 mM KCl, 0.02% Triton X-100, 1 μCi[32P] dCTP (300 Ci/mmol, NEN), 1 μM dATP, dTTP, dGTP. After 1 hour incubation at 37° C. the reaction was stopped by adding 100 μl of 20% TCA and applying samples to DE81 filters. The filters were processed and IC50 values calculated as described above.

Test for Inhibition of Human Immunodeficiency Virus-1 Reverse Transcriptase

[0113] Analogous assays employed the reverse transcriptase of HIV (HIV -1RT) from Boehringer Mannhium.

[0114] Incorporation of radioactive dTTP was measured as follows. The standard reaction (100 μl) was carried out in a buffer containing 50 mM tris/HCl pH 8.2, 2.5 mM MgCl 2, 1 mM DTT, 80 mM KCl, 5 mM EGTA, 0.05% Triton X-100, 1 μCi[3H] dTTP (40 Ci/mmol, NEN), 10 μM UTP and 10 μg/ml poly(A)/dT (from Pharmacia). The final HIV-1RT(enzyme concentration was 1 nM. After 1 hour incubation at 37° C. the reaction was stopped by adding 100 μl of 20% TCA and applying samples to DE81 filters. The filters were processed and IC50 values calculated as described above.

[0115] The results demonstrate that the compounds of the present invention are effective as inhibitors of viral polymerases at low micromolar concentrations.

[0116] It is apparent from the tables above that a compound of the present invention which is effective in the inhibition of one of the RNA dependent polymerases tested may not necessarily be as effective in inhibiting the other RNA dependent polymerases. The results shown in the tables above indicate a general trend, although this is not without exception. Generally, the most active inhibitors of HCV RdRp contained a phenyl ring attached to the diketoacid, whereas the HIV-RT inhibitors contained a furanyl group and those of HBV polymerase a thiophene group.

[0117] While not wishing to be bound by any particular theory, the present inventors hypothesize that the diketoacid fragment of the compounds of the present invention inhibits RNA dependent polymerase activity by providing an “active site anchor” and interacting with divalent metal cations (Mg2+, Mn2+) required for polymerase activity. The ring system found on the left hand side of the molecule can apparently be modified in order to build specificity towards a given polymerase.

Claims

1. The use of a compound of formula A, or of a pharmaceutically acceptable salt or ester thereof, wherein the group R is an organic moiety containing 2 to 24 carbon atoms which includes an optionally substituted cyclic or heterocyclic group, and wherein one of the atoms in the ring of the cyclic or heterocyclic group is directly bonded to the adjacent carbonyl in the diketoacid, in the manufacture of a medicament for treatment or prophylaxis of a viral illness in a human or animal by inhibition of a viral polymerase.

2. The use according to claim 1 wherein the medicament is for the inhibition of a DNA polymerase or RNA polymerase.

3. The use according to claim 1 or claim 2 wherein the medicament is for treatment or prevention of infection by an RNA virus, such as a positive single-stranded virus, negative single stranded virus or retrovirus, or a DNA virus.

4. The use according to claim 3 wherein the virus is selected from polio virus, hepatitis C virus, encephalomyocarditis, orthomyxoviruses, paramyxoviruses, HIV, and hepatitis B.

5. The use according to claim 3 wherein the medicament is for the inhibition of hepatitis C virus RNA dependent RNA polymerase (HCV RdRp), hepatitis B virus polymerase (HBV pol) , or reverse transcriptase of human immunodeficiency virus (HIV RT).

6. The use according to any one of the following claims wherein the group R is selected from: (i) optionally substituted aromatic groups; (ii) optionally substituted heteroaryl groups; (iii) optionally substituted cycloalkyl groups; (iv) optionally substituted cycloalkenyl groups; and (v) optionally substituted cyclic heteroalkyl groups.

7. A compound of formula A, as set out in claim 1, or a pharmaceutically acceptable salt or ester thereof, for pharmaceutical use, wherein the group R is selected from: (i) optionally substituted aromatic groups; (ii) optionally substituted heteroaryl groups; (iii) optionally substituted cycloalkyl groups; (iv) optionally substituted cycloalkenyl groups; and (v) optionally substituted cyclic heteroalkyl groups, other than those containing a single nitrogen in the ring.

8. A compound, ester or salt according to claim 7 wherein the group R is an optionally substituted phenyl group of formula:

9. A compound, ester or salt according to claim 8 wherein the substituents R5 and R6 are independently selected from H—, —OH, —OR4, —NHSO2R4, lower alkyl, aralkyl, amino, amide, urethane or urea groups.

10. A compound, salt or ester according to claim 8 wherein the substituents R5 and R6 are independently selected from H—, —OH, —OR4, and —NHSO2R4.

11. A compound, salt or ester according to claim 9 or claim 10 containing only one substituent either of formula —OR4 or —NHSO2R4.

12. A compound, salt or ester of any one of claims 9 to 11 containing a group of formula —OR4 and/or —NHSO2R4 selected from: —OCH2Ar; —O(CH2)2Ar —O(CH2)3CN; —O(CH2)3C≡CH; and —NHSO2Ar; wherein Ar is an optionally substituted aryl or heteroaryl group.

13. A compound, salt or ester, according to any one of claims 8 to 12 having a single substituent at a position ortho- or meta- to the diketoacid group.

14. A compound, salt or ester according to any one of claims 8 to 12 having two substituents at the 2,5-; 3,5-; or 2,4-positions.

15. A compound, salt or ester according to claim 7 wherein the group of formula R has the formula:

16. A compound, salt or ester according to claim 15 which is a pyrrole-2-substituted diketoacid, a pyrrole-3-substituted diketoacid, a thiophene-2-substituted diketoacid, or a thiophene-3-substituted diketoacid.

17. A compound, salt or ester according to claim 16 which is a pyrrole substituted diketoacid in which each of R7 and R8 is hydrogen.

18. A compound, salt or ester according to any one of claims 15 to 17 which is a pyrrole substituted diketoacid having X=NR4 and wherein R4 is selected from optionally substituted or interrupted, alkyl aryl or aralkyl groups.

19. A compound, salt or ester according to claim 7 wherein R is selected from cyclopropyl-, cyclopentyl-, cyclohexyl-, cyclopentenyl-, cyclohexenyl and adamantyl groups, any of which may, optionally, be substituted.

20. A pharmaceutical composition comprising a compound, salt or ester of any one of claims 7 to 19 in combination with a pharmaceutically acceptable excipient, diluent or carrier.

21. Use, according to any one of claims 1 to 6, of a compound, salt or ester according to any one of claims 7 to 19.

22. A use according to any one of claims 1 to 6 or 21 wherein the medicament further comprises one or more other agents for the treatment of viral infections.

23. A method of inhibiting a viral polymerase and/or of treating or preventing a viral illness by inhibiting a viral polymmerase, the method comprising administering to a human or animal subject suffering from the condition a therapeutically or prophylactically effective amount of the compound of formula A set out in claim 1, or of a pharmaceutically acceptable salt or ester thereof.

24. A compound of formula A, as set out in claim 1 or a pharmaceutically acceptable salt or ester thereof wherein the group R is selected from: (i) optionally substituted aromatic groups; (ii) optionally substituted heteroaryl groups; (iii) optionally substituted cycloalkyl groups; (iv) optionally substituted cycloalkenyl groups; and (v) optionally substituted cyclic heteroalkyl groups, other than those containing a single nitrogen in the ring.